Chuanshu Huang, M.D., Ph.D.

Manganese induces tau hyperphosphorylation through the activation of ERK MAPK pathway in PC12 cells
Cai, Tongjian; Che, Honglei; Yao, Ting; Chen, Yaoming; Huang, Chuanshu; Zhang, Wenbin; Du, Kejun; Zhang, Jianbin; Cao, Yunxin; Chen, Jingyuan; Luo, Wenjing
2011 Jan;119(1):169-177, Toxicological sciences
Manganese has long been known to induce neurological degenerative disorders. Emerging evidence indicates that hyperphosphorylated tau is associated with neurodegenerative diseases, but whether such hyperphosphorylation plays a role in manganese-induced neurotoxicity remains unclear. To fill this gap, we investigated the effects of manganese on tau phosphorylation in PC12 cells. In our present research, treatment of cells with manganese increased the phosphorylation of tau at Ser199, Ser202, Ser396, and Ser404 as detected by Western blot. Moreover, this manganese-induced tau phosphorylation paralleled the activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK). The mitogen-activated protein kinase kinase-1 (MEK1) inhibitor PD98059, which inhibits the activation of ERK MAPK, partially attenuated manganese-induced tau hyperphosphorylation and cytotoxicity. Moreover, the activation of ERK MAPK was involved in the activation of glycogen synthase kinase-3beta (GSK-3beta) kinase, which also contributed to the hyperphosphorylation of tau and the cytotoxicity in PC12 cells induced by manganese. Taken together, we found for the first time that the exposure to manganese can cause the hyperphosphorylation of tau, which may be connected with the activation of ERK MAPK
— id: 121616, year: 2011, vol: 119, page: 169, stat: Journal Article,

A Cross-Talk Between NFAT and NF-kappaB Pathways is Crucial for Nickel-Induced COX-2 Expression in Beas-2B Cells
Cai, Tongjian; Li, Xueyong; Ding, Jin; Luo, Wenjing; Li, Jingxia; Huang, Chuanshu
2011 Jun 1;11(5):548-559, Current cancer drug targets
Cyclooxygenase-2 (COX-2) is a critical enzyme implicated in chronic inflammation-associated cancer development. Our studies have shown that the exposure of Beas-2B cells, a human bronchial epithelial cell line, to lung carcinogenic nickel compounds results in increased COX-2 expression. However, the signaling pathways leading to nickel-induced COX-2 expression are not well understood. In the current study, we found that the exposure of Beas-2B cells to nickel compounds resulted in the activation of both nuclear factor of activated T cell (NFAT) and nuclear factor-kappaB (NF-kappaB). The expression of COX-2 induced upon nickel exposure was inhibited by either a NFAT pharmacological inhibitor or the knockdown of NFAT3 by specific siRNA. We further found that the activation of NFAT and NF-kappaB was dependent on each other. Since our previous studies have shown that NF-kappaB activation is critical for nickel-induced COX-2 expression in Beas-2B cells exposed to nickel compounds under same experimental condition, we anticipate that there might be a cross-talk between the activation of NFAT and NF-kappaB for the COX-2 induction due to nickel exposure in Beas-2B cells. Furthermore, we showed that the scavenging of reactive oxygen species (ROS) by introduction of mitochondrial catalase inhibited the activation of both NFAT and NF-kappaB, and the induction of COX-2 due to nickel exposure. Taken together, our results defining the evidence showing a key role of the cross-talk between NFAT and NF-kappaB pathways in regulating nickel-induced COX-2 expression, further provide insight into the understanding of the molecular mechanisms linking nickel exposure to its lung carcinogenic effects
— id: 132877, year: 2011, vol: 11, page: 548, stat: Journal Article,

Arsenite stabilizes HIF-1alpha protein through p85alpha-mediated up-regulation of inducible Hsp70 protein expression
Guo, Wei; Yang, Zhuo; Xia, Qing; Liu, Jinyi; Yu, Yonghui; Li, Jingxia; Zuo, Zhenghong; Zhang, Dongyun; Li, Xueyong; Shi, Xianglin; Huang, Chuanshu
2011 Feb;68(3):475-488, Cellular & molecular life sciences: CMLS
Hypoxia-inducible factor-1alpha (HIF-1alpha) has been reported to regulate over 100 gene expressions in response to hypoxia and other stress conditions. In the present study, we found that arsenite could induce HIF-1alpha protein accumulation in both mouse epidermal Cl41 cells and mouse embryonic fibroblasts (MEFs). Knockout of p85alpha, a regulatory subunit of PI-3K, in MEFs (p85alpha(-/-)) dramatically decreased the arsenite-induced HIF-1alpha accumulation, indicating that p85alpha is crucial for arsenite effects on the stabilization of HIF-1alpha protein. Our further studies suggest that arsenite could induce inducible Hsp70 expression, and transfection of inducible Hsp70 into p85alpha(-/-) MEFs could restore HIF-1alpha protein accumulation. Moreover, the results using EMSA and Supershift assays indicate that p85alpha is crucial for arsenite-induced activation of the heat-shock transcription factor 1 (HSF-1), which is responsible for transcription of inducible Hsp70. Taken together, p85alpha-mediated HIF-1alpha stabilization upon arsenite exposure is specifically through HSF-1 activation and subsequent up-regulation of the inducible Hsp70 expression
— id: 120640, year: 2011, vol: 68, page: 475, stat: Journal Article,

Hydrogen peroxide contributes to the manganese superoxide dismutase promotion of migration and invasion in glioma cells
Li, Fei; Wang, Hui; Huang, Chuanshu; Lin, Jiangkai; Zhu, Gang; Hu, Rong; Feng, Hua
2011 Oct;45(10):1154-1161, Free radical research
Manganese superoxide dismutase (MnSOD) is over-expressed in most brain tumours, and high MnSOD expression is associated with poor prognosis. The mechanisms still remain largely unknown. In the present study, the elevation of hydrogen peroxide (H(2)O(2)) level and the enhancement of glioma migration/invasion by over-expression of MnSOD were demonstrated. Subsequent studies showed that over-expression of MnSOD significantly increased the activation of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinases (PI3Ks), including AKTs, s6-ribosomal protein, ERKs and JNKs. Over-expression of MnSOD was also associated with elevations of matrix metalloproteinases-1(MMP-1) and MMP-9 protein. The promotion of migration/invasion, activation of PI3Ks and MAPKs and up-regulation of MMPs were inhibited by the general reactive oxygen species scavenger N-acetyl-l-cysteine (NAC), over-expression of the H(2)O(2)-detoxifying enzyme mitochondrial catalase (mCat) and specific inhibitors of AKTs or ERKs. Collectively, our study indicated that H(2)O(2) would contribute to the MnSOD-promoted migration/invasion in glioma cells through activation of AKTs and ERKs. This study provided new molecular insights into the understanding of glioma migration and invasion
— id: 141192, year: 2011, vol: 45, page: 1154, stat: Journal Article,

The X-linked inhibitor of apoptosis protein (XIAP) mediates cancer cell motility Via RhoGDP dissociation inhibitor (RhoGDI)-dependent regulation of cytoskeleton
Liu J; Zhang D; Luo W; Yu Y; Yu J; Li J; Zhang X; Zhang B; Chen J; Wu XR; Rosas-Acosta G; Huang C
2011 May 6;286(18):15630-15640, Journal of biological chemistry
The X-linked inhibitor of apoptosis protein (XIAP) overexpression has been found to be associated with malignant cancer progression and aggression in individuals with many types of cancers. However, the molecular basis of XIAP in the regulation of cancer cell biological behavior remains largely unknown. In the current study, we found that the deficiency of XIAP expression in human cancer cells by approach of either knockout or knockdown led to a marked reduction of beta-Actin polymerization and cytoskeleton formation. Consistently, cell migration and invasion were also decreased in XIAP-deficient cells in comparison to those in the parental wild type cells. Subsequent studies demonstrated that the regulation of cell motility by XIAP depended on its interaction with RhoGDP dissociation inhibitor (RhoGDI) via XIAP RING domain. Furthermore, XIAP was found to negatively regulate RhoGDI SUMOylation, which might affect its activity in controlling cell motility. Collectively, our studies provide novel insights into the molecular mechanisms by which XIAP regulates cancer invasion, and offer further theoretical basis for setting XIAP as a potential prognostic marker and specific target for treatment of cancers with metastatic property
— id: 131663, year: 2011, vol: 286, page: 15630, stat: Journal Article,

p85alpha mediates p53 K370 acetylation by p300 and regulates its promoter-specific transactivity in the cellular UVB response
Song, L; Gao, M; Dong, W; Hu, M; Li, J; Shi, X; Hao, Y; Li, Y; Huang, C
2011 Mar 17;30(11):1360-1371, Oncogene
Inducible acetylation of p53 at lysine residues has a great impact on regulating the transactivation of this protein, which is associated with cell growth arrest and/or apoptosis under various stress conditions. However, the factor(s) for regulating p53 acetylation remains largely unknown. In the current study, we have shown that p85alpha, the regulatory subunit of phosphatidylinositol-3-kinase, has a critical role in mediating p53 acetylation and promoter-specific transactivation in the ultraviolet B (UVB) response. Depletion of p85alpha in mouse embryonic fibroblasts significantly impairs UVB-induced apoptosis, as well as p53 transactivation and acetylation at Lys370 (Lys373 of human p53); however, the accumulation, nuclear translocation and phosphorylation of p53 are not affected. Interestingly, p85alpha binds to p300, promotes the p300-p53 interaction and the subsequent recruitment of the p53/p300 complex to the promoter region of the specific p53 target gene in response to UVB irradiation. Moreover, ablation of p53 acetylation at Lys370 by site-directed mutagenesis dramatically suppresses UVB-induced expression of the specific p53-responsive gene as well as cell apoptosis. Therefore, we conclude that p85alpha is a novel regulator of p53-mediated response under certain stress conditions, and targeting the p85alpha-dependent p53 pathway may be promising for cancer therapy
— id: 127224, year: 2011, vol: 30, page: 1360, stat: Journal Article,

p27 suppresses arsenite-induced Hsp27/Hsp70 expression through inhibiting JNK2/c-Jun- and HSF-1-dependent pathways
Liu, Jinyi; Zhang, Dongyun; Mi, Xiaoyi; Xia, Qing; Yu, Yonghui; Zuo, Zhenghong; Guo, Wei; Zhao, Xuewei; Cao, Jia; Yang, Qing; Zhu, Angela; Yang, Wancai; Shi, Xianglin; Li, Jingxia; Huang, Chuanshu
2010 Aug 20;285(34):26058-26065, Journal of biological chemistry
p27 is an atypical tumor suppressor that can regulate the activity of cyclin-dependent kinases and G(0)-to-S phase transitions. More recent studies reveal that p27 may also exhibit its tumor-suppressive function through regulating many other essential cellular events. However, the molecular mechanisms underlying these anticancer effects of p27 are largely unknown. In this study, we found that depletion of p27 expression by either gene knock-out or knockdown approaches resulted in up-regulation of both Hsp27 and Hsp70 expression at mRNA- and promoter-derived transcription as well as protein levels upon arsenite exposure, indicating that p27 provides a negative signal for regulating the expression of Hsp27 and Hsp70. Consistently, arsenite-induced activation of JNK2/c-Jun and HSF-1 pathways was also markedly elevated in p27 knock-out (p27(-/-)) and knockdown (p27 shRNA) cells. Moreover, interference with the expression or function of JNK2, c-Jun, and HSF-1, but not JNK1, led to dramatic inhibition of arsenite-induced Hsp27 and Hsp70 expression. Collectively, our results demonstrate that p27 suppresses Hsp27 and Hsp70 expression at the transcriptional level specifically through JNK2/c-Jun- and HSF-1-dependent pathways upon arsenite exposure, which provides additional important molecular mechanisms for the tumor-suppressive function of p27
— id: 138091, year: 2010, vol: 285, page: 26058, stat: Journal Article,

Bid Mediates Anti-Apoptotic COX-2 Induction through the IKKbeta/NFkappaB Pathway Due to 5-MCDE Exposure
Luo, Wenjing; Li, Jingxia; Zhang, Dongyun; Cai, T; Song, Lun; Yin, Xiao-Ming; Desai, Dhimant; Amin, Shantu; Chen, Jingyuan; Huang, Chuanshu
2010 Feb 1;10(1):96-106, Current cancer drug targets
Although Bid has been considered as a cell apoptotic mediator, current studies suggest a possible role in cell survival in mouse embryonic fibroblasts (MEFs) response to low doses of anti-(+/-)-5- methylchrysene-1,2-diol-3,4-epoxide(</=0.25microM) (5-MCDE). We found that exposure of MEFs to 0.25 microM 5-MCDE resulted in a slight apoptotic induction, while this apoptotic response was substantially increased in the Bid knockout MEFs (Bid(-/-)), suggesting Bid-mediated anti-apoptotic function in this response. This notion was further supported by the findings that re-constitution expression of Bid into Bid(-/-) cells could inhibit the increased apoptosis. Further studies showed that Bid anti-apoptotic function was associated with its mediation of COX-2 expression, which was based on the results of the reduction of COX-2 expression in Bid(-/-) cells, restoration of low sensitivity to 5-MCDE apoptotic response by the introduction of Bid into Bid(-/-) cells and increased sensitivity of WT MEFs to 5-MCDE apoptosis by knockdown of COX-2 expression. Furthermore, Bid mediated COX-2 expression through the IKKbeta/NFkappaB pathway because the deficiency of Bid in Bid(-/-) MEFs resulted in blockade of IKK/NFkappaB activation and knockout of IKKbeta caused abrogation of COX-2 expression induced by 5-MCDE. Collectively, our results demonstrate that Bid is critical for COX-2 induction through the IKKbeta/NFkappaB pathway, which mediates its anti-apoptotic function, in cell response to low doses of 5-MCDE exposure
— id: 106267, year: 2010, vol: 10, page: 96, stat: Journal Article,

A novel role of IKKalpha in the mediation of UVB-induced G0/G1 cell cycle arrest response by suppressing Cyclin D1 expression
Song, Lun; Dong, Wen; Gao, Ming; Li, Jingxia; Hu, Meiru; Guo, Ning; Huang, Chuanshu
2010 Feb;1803(2):323-332, Biochimica & biophysica acta
Exposure to ultraviolet B (UVB) irradiation (290-320nm wavelength) from sunlight induces a variety of medical problems, including sunburn, immunosuppression and skin cancers. However, the molecular mechanisms related to UVB-induced cell damage and/or mutagenic effects have not been fully defined. Here, we demonstrate that one of the catalytic subunits of the IkappaB kinase complex (IKK), IKKalpha, plays a critical role in mediation of the UVB-induced G0/G1 cell cycle arrest response by suppressing Cyclin D1 expression. Notably, IKKa-dependent Cyclin D1 regulation is unrelated to IKKbeta/NF-kappaB activity. We further show that IKKalpha-dependent downregulation of Cyclin D1 expression in the UVB response results from the reduction of ERK1/2-dependent Cyclin D1 transcription coupled with an increase of p38 kinase-dependent Cyclin D1 proteolysis. Thus, our results have identified the novel role of IKKalpha in regulating cell cycle progression during the cellular UVB response. Targeting IKKalpha might be promising for the prevention of UVB-induced cell damage and tumorigenic effects
— id: 106215, year: 2010, vol: 1803, page: 323, stat: Journal Article,

JNK1 mediates degradation HIF-1alpha by a VHL-independent mechanism that involves the chaperones Hsp90/Hsp70
Zhang, Dongyun; Li, Jingxia; Costa, Max; Gao, Jimin; Huang, Chuanshu
2010 Jan 15;70(2):813-823, Cancer research
Hypoxia-inducible factor-1alpha (HIF-1alpha) is a master transcription factor that is critical for the regulation of a variety of cellular functions. HIF-1alpha is rapidly degraded under normoxic conditions by ubiquitin-mediated proteasome pathway controlled by the tumor suppressor von Hippel Lindau (VHL). Several recent studies reveal that heat-shock proteins (Hsp) can regulate HIF-1alpha protein degradation by a VHL-independent pathway. Here, we demonstrate that the stress kinase c-Jun NH(2)-terminal kinase 1 (JNK1) is required for Hsp-dependent regulation of HIF-1alpha. Stabilization of HIF-1alpha was impaired in JNK1-/- cells but could be rescued by JNK1 reconstitution under hypoxic conditions. These effects could be phenocopied in other cell settings by JNK1 silencing. Accordingly, HIF-1 transcriptional activity and target gene expression were dramatically reduced in JNK1-/- cells. Further, decreased levels of endogenous Hsp90/Hsp70 proteins in JNK1-/- cells affected the protective roles of these chaperones in stabilizing newly synthesized HIF-1alpha, whereas enforced expression of Hsp90/Hsp70 in JNK1-/- cells increased HIF-1alpha stability relative to parental control cells. Furthering this connection, we also found that defective expression of the Hsp90 acetyltransferase HDAC6 in JNK1-/- cells was associated with reduced Hsp90 chaperone activity. Taken together, our studies define a novel function for JNK1 in regulating HIF-1alpha turnover by a VHL-independent mechanism
— id: 106216, year: 2010, vol: 70, page: 813, stat: Journal Article,

Effects of nickel on cyclin expression, cell cycle progression and cell proliferation in human pulmonary cells
Ding, Jin; He, Guoping; Gong, Wenfeng; Wen, Wen; Sun, Wen; Ning, Beifang; Huang, Shanna; Wu, Kun; Huang, Chuanshu; Wu, Mengchao; Xie, Weifen; Wang, Hongyang
2009 Jun;18(6):1720-1729, Cancer epidemiology biomarkers & prevention
Frequent exposure to nickel compounds has been considered as one of the potential causes of human lung cancer. However, the molecular mechanism of nickel-induced lung carcinogenesis remains obscure. In the current study, slight S-phase increase, significant G(2)/M cell cycle arrest, and proliferation blockage were observed in human bronchial epithelial cells (Beas-2B) upon nickel exposure. Moreover, the induction of cyclin D1 and cyclin E by nickel was shown for the first time in human pulmonary cells, which may be involved in nickel-triggered G(1)/S transition and cell transformation. In addition, we verified that hypoxia-inducible factor-1alpha, an important transcription factor of nickel response, was not required for the cyclin D1 or cyclin E induction. The role of p53 in nickel-induced G(2)/M arrest was excluded, respecting that its protein level, ser(15) phosphorylation, and transcriptional activity were not changed in nickel response. Further study revealed that cyclin A was not activated in nickel response, and cyclin B1, which not only promotes G(2)/M transition but also prevents M-phase exit of cells if not degraded in time, was up-regulated by nickel through a manner independent of hypoxia-inducible factor. More importantly, our results verified that overexpressed cyclin B1, veiling the effect of cyclin D1 or cyclin E, mediated nickel-caused M-phase blockage and cell growth inhibition, which may render pulmonary cells more sensitive to DNA damage and facilitates cancer initiation. These results will not only deepen our understanding of the molecular mechanism involved in nickel carcinogenecity, but also lead to the further study on chemoprevention of nickel-associated human cancer
— id: 110423, year: 2009, vol: 18, page: 1720, stat: Journal Article,

TNF-alpha induction by nickel compounds is specific through ERKs/AP-1-dependent pathway in human bronchial epithelial cells
Ding, Jin; Huang, Yi; Ning, Beifang; Gong, Wenfeng; Li, Jingxia; Wang, Hongyang; Chen, Chang-Yan; Huang, Chuanshu
2009 Feb;9(1):81-90, Current cancer drug targets
The chronic lung inflammatory activity and carcinogenicity of nickel compounds have been well documented by previous studies from epidemiology both in vitro and in vivo. However, the molecular mechanism involved in nickel-induced chronic lung inflammation is much less understood. The current study demonstrates that exposure of human bronchial epithelial cells (Beas-2B) to nickel compounds results in the induction of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and transactivation of nuclear factor of activated T cells (NFAT), nuclear factor-kappaB (NF-kappaB), and activator protein-1 (AP-1). Further studies show that neither overexpression of IKKbeta-KM, a kinase inactive mutant of IKKbeta, nor the ectopic expression of a dominant negative mutant of NFAT could inhibit the TNF-alpha induction by nickel exposure. Overexpression of TAM67, a dominant-negative mutant of c-Jun, dramatically reduced the TNF-alpha induction, suggesting that AP-1 is a mediator of TNF-alpha induction in nickel responses. Our results show that ERKs are AP-1 upstream kinases responsible for TNF-alpha induction by nickel exposure; although JNKs, ERKs, and p38K were all activated in the Beas-2B cells exposed to nickel compounds. Our results demonstrate that inflammatory TNF-alpha could be induced by nickel exposure in Beas-2B cells specifically through an ERKs/AP-1-dependent pathway
— id: 97839, year: 2009, vol: 9, page: 81, stat: Journal Article,

PI3K/Akt/JNK/c-Jun signaling pathway is a mediator for arsenite-induced cyclin D1 expression and cell growth in human bronchial epithelial cells
Ding, Jin; Ning, Beifang; Huang, Yi; Zhang, Dongyun; Li, Jingxia; Chen, Chang-Yan; Huang, Chuanshu
2009 Jun;9(4):500-509, Current cancer drug targets
Arsenite exposure is associated with an increased risk of human lung cancer. However, the molecular mechanisms underlying the arsenite-induced human lung carcinogenesis remain elusive. In this study, we demonstrated that arsenite upregulates cyclin D1 expression/activity to promote the growth of human bronchial epithelial Beas-2B cells. In this process, the JNKs (c-Jun N-terminal kinases)/c-Jun cascade is elicited. The inhibition of JNKs or c-Jun by chemical or genetic inhibitors blocks the cyclin D1 induction mediated by arsenite. Furthermore, using a loss of function mutant of p85 (Deltap85, a subunit of PI3K) or dominant-negative Akt (DN-Akt), we showed that PI3K and Akt act as the upstream regulators of JNKs and c-Jun in arsenite-mediated growth promotion. Overall, our data suggest a pathway of PI-3K/Akt/JNK/c-Jun/cylin D1 signaling in response to arsenite in human bronchial epithelial cells
— id: 100190, year: 2009, vol: 9, page: 500, stat: Journal Article,

Diverse roles of GADD45alpha in stress signaling
Gao, Ming; Guo, Ning; Huang, Chuanshu; Song, Lun
2009 Aug;10(4):388-394, Current protein & peptide science
Mammalian cells are prone to tumorigenesis when suffering the genotoxic stresses, and the existence of the tumor suppressors validly decrease this possibility. Gadd45alpha is one of the growth arrest and DNA damage-inducible (Gadd) 45 gene family members and serves as a stress sensor and tumor suppressor under most stress conditions, which is evidenced by cell cycle arrest, DNA repair, senescence or apoptosis triggered by induction of GADD45alpha expression. However, some recent reports have challenged this notion by demonstrating the correlation of GADD45alpha expression to cell survival and even progression of certain tumor cells. Therefore, GADD45alpha seems to exert multiple roles in stress signaling and tumor development. Elucidation of the related mechanisms will be helpful for the establishment of novel tumor therapeutic strategy targeting GADD45alpha
— id: 106268, year: 2009, vol: 10, page: 388, stat: Journal Article,

Effect of microglia activation on dopaminergic neuronal injury induced by manganese, and its possible mechanism
Liu, Mingchao; Cai, Tongjian; Zhao, Fang; Zheng, Gang; Wang, Qiang; Chen, Yaoming; Huang, Chuanshu; Luo, Wenjing; Chen, Jingyuan
2009 Jul;16(1):42-49, Neurotoxicity research
Manganese (Mn) is an essential trace element. It is known to have various functions, such as participating in enzymatic synthesis, and promoting hematopoiesis. On the other hand, it can cause toxic injury upon excess intake. However, toxic effects and its mechanism on glial cells are unclear. In the present study, we demonstrated that MnCl(2) can activate microglia, and that this can cause dopaminergic neuronal injury. Investigation of the underlying mechanisms showed that inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) was induced and highly expressed following Mn treatment. Moreover, pretreatment with S-methylisothiourea (SMT. iNOS inhibitor), Mn-induced iNOS expression and dopaminergic neuronal injury were partly reverse. Pretreatment with minocycline (microglia activation inhibitor), Mn-induced activation of microglia and dopaminergic neuronal injury was partly reverse. Taken together, our results showed that Mn can cause microglia activation, which can up-regulate the level of IL-1beta, TNF-alpha and iNOS, and these inflammatory factors can cause dopaminergic neuronal injury. SMT and minocycline prevent Mn-induced dopaminergic neuronal injury
— id: 110421, year: 2009, vol: 16, page: 42, stat: Journal Article,

Soluble and insoluble nickel compounds exert a differential inhibitory effect on cell growth through IKKalpha-dependent cyclin D1 down-regulation
Ouyang, Weiming; Zhang, Dongyun; Li, Jingxia; Verma, Udit N; Costa, Max; Huang, Chuanshu
2009 Jan;218(1):205-214, Journal of cellular physiology
It is well-known that insoluble nickel compounds possess much more potent carcinogenic activities as compared with soluble nickel compounds. Although it is assumed that the different entry and clearance rate are responsible for the difference, the mechanisms underlying the different carcinogenic activities are still not well understood yet. In the present study, we found that exposure to soluble, but not insoluble nickel compounds, caused a significant inhibition of cell growth and G1/G0 cell cycle arrest, which was concomitant with a marked down-regulation of cylin D1, an essential nuclear protein for controlling G1/S transition, while both soluble and insoluble nickel compounds showed similar effects on NFkappaB activation, HIF-1alpha protein accumulation and TNF-alpha transcription and CAP43 protein expression at same doses range. The down-regulation of cyclin D1 is due to protein degradation rather than inhibition of transcription, because the nickel compounds treatment did not change cyclin D1 mRNA level, while MG132, the proteasome inhibitor, can rescue the degradation of cyclin D1 caused by soluble nickel compound. Moreover, the soluble nickel-induced cyclin D1 degradation is dependent on its Thr286 residue and requires IKKalpha, but not HIF-1alpha, which are both reported to be involved in cyclin D1 down-regulation. Taken together, we demonstrate that soluble, but not insoluble nickel compound, is able to cause cyclin D1 degradation and a cell growth arrest in an IKKalpha-dependent manner. Given the role of cyclin D1 and cell proliferation in carcinogenesis, we anticipate that the different effects of soluble and insoluble nickel compounds on cyclin D1 degradation and cell growth arrest may at least partially account for their different carcinogenic activities
— id: 93369, year: 2009, vol: 218, page: 205, stat: Journal Article,

PUMA is directly activated by NF-kappaB and contributes to TNF-alpha-induced apoptosis
Wang, P; Qiu, W; Dudgeon, C; Liu, H; Huang, C; Zambetti, G P; Yu, J; Zhang, L
2009 Sep;16(9):1192-1202, Cell death & differentiation
Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that has an important role in immunity and inflammation by inducing cellular responses such as apoptosis. The transcription factor nuclear factor-kappaB (NF-kappaB) can paradoxically suppress and promote apoptosis in response to TNF-alpha. In this study, we found that p53 upregulated modulator of apoptosis (PUMA), a p53 downstream target and a BH3-only Bcl-2 family member, is directly regulated by NF-kappaB in response to TNF-alpha. TNF-alpha treatment led to increases in PUMA mRNA and protein levels in human colon cancer cells. The induction of PUMA was p53 independent, and mediated by the p65 component of NF-kappaB through a kappaB site in the PUMA promoter. The apoptotic effect of PUMA induction by TNF-alpha was unmasked by depleting the antiapoptotic protein Bcl-X(L). In mice, PUMA was also induced by TNF-alpha in an NF-kappaB-dependent manner. TNF-alpha-induced apoptosis in a variety of tissues and cell types, including small intestinal epithelial cells, hepatocytes, and thymocytes, was markedly reduced in PUMA-deficient mice. Collectively, these results demonstrated that PUMA is a direct target of NF-kappaB and mediates TNF-alpha-induced apoptosis in vitro and in vivo
— id: 106254, year: 2009, vol: 16, page: 1192, stat: Journal Article,

Gadd45 proteins as critical signal transducers linking NF-kappaB to MAPK cascades
Yang, Z; Song, L; Huang, C
2009 Dec;9(8):915-930, Current cancer drug targets
The growth arrest and DNA damage-inducible 45 (Gadd45) proteins are a group of critical signal transducers that are involved in regulations of many cellular functions. Accumulated data indicate that all three Gadd45 proteins (i.e., Gadd45alpha, Gadd45beta, and Gadd45gamma) play essential roles in connecting an upstream sensor module, the transcription Nuclear Factor-kappaB (NF-kappaB), to a transcriptional regulating module, mitogen-activated protein kinase (MAPK). This NF-kappaB-Gadd45(s)-MAPK pathway responds to various kinds of extracellular stimuli and regulates such cell activities as growth arrest, differentiation, cell survival, and apoptosis. Defects in this pathway can also be related to oncogenesis. In the first part of this review, the functions of Gadd45 proteins, and briefly NF-kappaB and MAPK, are summarized. In the second part, the mechanisms by which Gadd45 proteins are regulated by NF-kappaB, and how they affect MAPK activation, are reviewed
— id: 105986, year: 2009, vol: 9, page: 915, stat: Journal Article,

The biological functions of NF-kappaB1 (p50) and its potential as an anti-cancer target
Yu, Yonghui; Wan, Yu; Huang, Chuanshu
2009 Jun;9(4):566-571, Current cancer drug targets
Nuclear factor-kappaB (NF-kappaB) is a key transcriptional factor family that consists of five members in mammalian cells, including NF-kappaB1 (p50), NF-kappaB2 (p52), RelA (p65), RelB and c-Rel. NF-kappaB is implicated in multiple physiological and pathological processes, including cell proliferation and differentiation, inflammatory and immune response, cell survival and apoptosis, cellular stress reactions and tumorigenesis. Recent studies by our group and others have highlighted the novel functions of the p50 protein. In this review, we will focus on the regulation and functions of NF-kappaB p50
— id: 110422, year: 2009, vol: 9, page: 566, stat: Journal Article,

A JNK1/AP-1-dependent, COX-2 induction is implicated in 12-O-tetradecanoylphorbol-13-acetate-induced cell transformation through regulating cell cycle progression. (vol 6, pg 165, 2008) [Correction]
Zhang, D; Huang, CS
2009 SEP ;7(9):1593-1593, Molecular cancer research
— id: 102960, year: 2009, vol: 7, page: 1593, stat: Journal Article,

c-Jun/AP-1 pathway-mediated cyclin D1 expression participates in low dose arsenite-induced transformation in mouse epidermal JB6 Cl41 cells
Zhang, Dongyun; Li, Jingxia; Gao, Jimin; Huang, Chuanshu
2009 Feb 15;235(1):18-24, Toxicology & applied pharmacology
Arsenic is a well-documented human carcinogen associated with skin carcinogenesis. Our previous work reveals that arsenite exposure is able to induce cell transformation in mouse epidermal cell JB6 Cl41 through the activation of ERK, rather than JNK pathway. Our current studies further evaluate downstream pathway in low dose arsenite-induced cell transformation in JB6 Cl41 cells. Our results showed that treatment of cells with low dose arsenite induced activation of c-Jun/AP-1 pathway, and ectopic expression of dominant negative mutant of c-Jun (TAM67) blocked arsenite-induced transformation. Furthermore, our data indicated that cyclin D1 was an important downstream molecule involved in c-Jun/AP-1-mediated cell transformation upon low dose arsenite exposure, because inhibition of cyclin D1 expression by its specific siRNA in the JB6 Cl41 cells resulted in impairment of anchorage-independent growth of cells induced by low dose arsenite. Collectively, our results demonstrate that c-Jun/AP-1-mediated cyclin D1 expression is at least one of the key events implicated in cell transformation upon low dose arsenite exposure
— id: 97450, year: 2009, vol: 235, page: 18, stat: Journal Article,

gamma-Aminobutyric acid(A) (GABA(A)) receptor regulates ERK1/2 phosphorylation in rat hippocampus in high doses of methyl tert-butyl ether (MTBE)-induced impairment of spatial memory
Zheng, Gang; Zhang, Wenbin; Zhang, Yun; Chen, Yaoming; Liu, Mingchao; Yao, Ting; Yang, Yanxia; Zhao, Fang; Li, Jingxia; Huang, Chuanshu; Luo, Wenjing; Chen, Jingyuan
2009 Apr 15;236(2):239-245, Toxicology & applied pharmacology
Experimental and occupational exposure to methyl tert-butyl ether (MTBE) has been reported to induce neurotoxicological and neurobehavioral effects, such as headache, nausea, dizziness, and disorientation, etc. However, the molecular mechanisms involved in MTBE-induced neurotoxicity are still not well understood. In the present study, we investigated the effects of MTBE on spatial memory and the expression and function of GABA(A) receptor in the hippocampus. Our results demonstrated that intraventricular injection of MTBE impaired the performance of the rats in a Morris water maze task, and significantly increased the expression of GABA(A) receptor alpha1 subunit in the hippocampus. The phosphorylation of ERK1/2 decreased after the MTBE injection. Furthermore, the decreased ability of learning and the reduction of phosphorylated ERK1/2 level of the MTBE-treated rats was partly reversed by bicuculline injected 30 min before the training. These results suggested that MTBE exposure could result in impaired spatial memory. GABA(A) receptor may play an important role in the MTBE-induced impairment of learning and memory by regulating the phosphorylation of ERK in the hippocampus
— id: 110424, year: 2009, vol: 236, page: 239, stat: Journal Article,

Benzo(a)pyrene-caused increased G(1)-S transition requires the activation of c-Jun through p53-dependent PI-3K/Akt/ERK pathway in human embryo lung fibroblasts
Jiao, Shi; Liu, Bingci; Gao, Ai; Ye, Meng; Jia, Xiaowei; Zhang, Fengmei; Liu, Haifeng; Shi, Xianglin; Huang, Chuanshu
2008 May 30;178(3):167-175, Toxicology letters
Benzo(a)pyrene (B(a)P) is a potent lung carcinogen mainly derived from tobacco smoking and environmental contamination, however, the molecular mechanisms by which it accelerates the cell cycle progression and induces the abnormal cell proliferation are still far away from understood. Our current analysis of human embryo lung fibroblasts (HELF) showed that B(a)P exposure was able to promote cell cycle G(1)-S phase transition. This effect was correlated with c-Jun activation because inhibition of c-Jun by its dominant negative mutant (TAM67) reversed B(a)P action on cell cycle with the down-regulation of expression of cyclin D1, pRb and E2F1. Further study found that overexpression of dominant negative mutants of, PI-3K or Akt, dramatically reduced B(a)P-induced the activation of c-Jun and extracellular signaling regulated kinase (ERK), but not c-Jun NH2 terminal kinase (JNK). Inhibition of p53 by either its inhibitor pifithrin-alpha or p53 siRNA markedly increased B(a)P-induced the activation of c-Jun, Akt and ERK in this context. Take together, our results indicate that c-Jun activation by p53-dependent PI-3K/Akt/ERK pathway is responsible for B(a)P-induced cell cycle alternations in human embryo lung fibroblasts
— id: 78680, year: 2008, vol: 178, page: 167, stat: Journal Article,

Differential effects of black raspberry and strawberry extracts on BaPDE-induced activation of transcription factors and their target genes
Li, Jingxia; Zhang, Dongyun; Stoner, Gary D; Huang, Chuanshu
2008 Apr;47(4):286-294, Molecular carcinogenesis
The chemopreventive properties of edible berries have been demonstrated both in vitro and in vivo, however, the specific molecular mechanisms underlying their anti-cancer effects are largely unknown. Our previous studies have shown that a methanol extract fraction of freeze-dried black raspberries inhibits benzoapyrene (BaP)-induced transformation of Syrian hamster embryo cells. This fraction also blocks activation of activator protein-1 (AP-1) and nuclear factor kappaB (NF-kappaB) induced by benzoapyrene diol-epoxide (BaPDE) in mouse epidermal JB6 Cl 41 cells. To determine if different berry types exhibit specific mechanisms for their anti-cancer effects, we compared the effects of extract fractions from both black raspberries and strawberries on BaPDE-induced activation of various signaling pathways in Cl 41 cells. Black raspberry fractions inhibited the activation of AP-1, NF-kappaB, and nuclear factor of activated T cells (NFAT) by BaPDE as well as their upstream PI-3K/Akt-p70(S6K) and mitogen-activated protein kinase pathways. In contrast, strawberry fractions inhibited NFAT activation, but did not inhibit the activation of AP-1, NF-kappaB or the PI-3K/Akt-p70(S6K) and mitogen-activated protein kinase pathways. Consistent with the effects on NFAT activation, tumor necrosis factor-alpha (TNF-alpha) induction by BaPDE was blocked by extract fractions of both black raspberries and strawberries, whereas vascular endothelial growth factor (VEGF) expression, which depends on AP-1 activation, was suppressed by black raspberry fractions but not strawberry fractions. These results suggest that black raspberry and strawberry components may target different signaling pathways in exerting their anti-carcinogenic effects
— id: 76764, year: 2008, vol: 47, page: 286, stat: Journal Article,

Anti-cancer effects of JKA97 are associated with its induction of cell apoptosis via a Bax-dependent and p53-independent pathway
Luo, Wenjing; Liu, Jinyi; Li, Jingxia; Zhang, Dongyun; Liu, Mingchao; Addo, James K; Patil, Shivaputra; Zhang, Lin; Yu, Jian; Buolamwini, John K; Chen, Jingyuan; Huang, Chuanshu
2008 Mar 28;283(13):8624-8633, Journal of biological chemistry
p53, one of the most commonly mutated genes in human cancers, is thought to be associated with cancer development. Hence, screening and identifying natural or synthetic compounds with anti-cancer activity via p53-independent pathway is one of the most challenging tasks for scientists in this field. Compound JKA97 (methoxy-1-styryl-9H-pyrid-[3,4-b]-indole) is a small molecule synthetic anti-cancer agent, with unknown mechanism(s). In this study we have demonstrated that the anti-cancer activity of JKA97 is associated with apoptotic induction via p53-independent mechanisms. We found that co-incubation of human colon cancer HCT116 cells with JKA97 inhibited HCT116 cell anchorage-independent growth in vitro and tumorigenicity in nude mice and also induced a cell apoptotic response, both in the cell culture model and in a tumorigenesis nude mouse model. Further studies showed that JKA97-induced apoptosis was dramatically impaired in Bax knock-out (Bax(-/-)) HCT116 cells, whereas the knock-out of p53 or PUMA did not show any inhibitory effects. The p53-independent apoptotic induction by JKA97 was confirmed in other colon cancer and hepatocarcinoma cell lines. In addition, our results showed an induction of Bax translocation and cytochrome c release from the mitochondria to the cytosol in HCT116 cells, demonstrating that the compound induces apoptosis through a Bax-initiated mitochondria-dependent pathway. These studies provide a molecular basis for the therapeutic application of JKA97 against human cancers with p53 mutations
— id: 79091, year: 2008, vol: 283, page: 8624, stat: Journal Article,

PI-3K/Akt pathway-dependent cyclin D1 expression is responsible for arsenite-induced human keratinocyte transformation
Ouyang, Weiming; Luo, Wenjing; Zhang, Dongyun; Jian, Jinlong; Ma, Qian; Li, Jingxia; Shi, Xianglin; Chen, Jingyuan; Gao, Jimin; Huang, Chuanshu
2008 Jan;116(1):1-6, Environmental health perspectives
BACKGROUND: Long-term exposure of arsenite leads to human skin cancer. However, the exact mechanisms of arsenite-induced human skin carcinogenesis remain to be defined. OBJECTIVES: In this study, we investigated the potential role of PI-3K/Akt/cyclin D1in the transformation of human keratinocytic cells upon arsenite exposure. METHODS: We used the soft agar assay to evaluate the cell transformation activity of arsenite exposure and the nude mice xenograft model to determine the tumorigenesis of arsenite-induced transformed cells. We used the dominant negative mutant and gene knockdown approaches to elucidate the signaling pathway involved in this process. RESULTS: Our results showed that repeated long-term exposure of HaCat cells to arsenite caused cell transformation, as indicated by anchorage-independent growth in soft agar. The tumorigenicity of these transformed cells was confirmed in nude mice. Treatment of cells with arsenite also induced significant activation of PI-3K and Akt, which was responsible for the anchorage-independent cell growth induced by arsenite exposure. Furthermore, our data also indicated that cyclin D1 is an important downstream molecule involved in PI-3K/Akt-mediated cell transformation upon arsenite exposure based on the facts that inhibition of cyclin D1 expression by dominant negative mutants of PI-3K, and Akt, or the knockdown of the cyclin D1 expression by its specific siRNA in the HaCat cells resulted in impairing of anchorage-independent growth of HaCat cells induced by arsenite. CONCLUSION: Our results demonstrate that PI-3K/Akt-mediated cyclin D1 expression is at least one key event implicated in the arsenite human skin carcinogenic effect
— id: 76459, year: 2008, vol: 116, page: 1, stat: Journal Article,

Downregulation of cyclin D1-CDK4 protein in human embryonic lung fibroblasts (HELF) induced by silica is mediated through the ERK and JNK pathway
Shen, Fuhai; Fan, Xueyun; Liu, Bingci; Jia, Xiaowei; Gao, Ai; Du, Hongju; Ye, Meng; You, Baorong; Huang, Chuanshu; Shi, Xianglin
2008 Oct;32(10):1284-1292, Cell biology international
Silica is a factor in the induction of acute injury and chronic pulmonary fibrosis. In 1996, silica was also listed as a human carcinogen by the International Agency for Research on Cancer (IARC). However, the molecular mechanisms involved in its pathologic effects are not well understood. We found that exposure of human embryonic lung fibroblasts (HELF) to crystalline silica for 2h decreased cyclin D1 and cyclin-dependent kinase 4 (CDK4) expression levels. Extracellular signal-regulated protein kinase (ERKs), c-Jun NH2-terminal amino kinase (JNKs), and p38 kinase, as well as their downstream transcription factor, AP-1, had different effects on the regulation of expression levels of cyclin D1 and CDK4 alterations induced by silica. Silica activates multiple signal transduction pathways involved in coordinating cellular responses to stress. We established the requirements for ERK and JNK, members of the mitogen-activated protein kinase (MAPK) family, in mediating G1 phase arrest of HELF induced by silica. Silica treatment activated ERK in a dose-dependent manner. AG126 (a chemical inhibitor of the ERK signaling pathway) and the dominant negative mutant of ERK2 (a molecular inhibitor of ERK2) prevented decreases in cyclin D1 and CDK4 expression levels. A chemical inhibitor of JNK, SP600125, prevented the decreased expression of both cyclin D1 and CDK4, whereas SB203580, a chemical inhibitor of p38, did not. Interestingly, curcumin prevented the decrease in DK4 expression, but not in cyclin D1. These results demonstrate that ERKs and JNKs are responsible for the decrease of cyclin D1 and CDK4 expression levels in HELF induced by silica. Activator protein-1 (AP-1) was responsible for the decrease of CDK4 expression level, but not that of cyclin D1. The findings help to explain the mechanisms of diseases induced by silica
— id: 110425, year: 2008, vol: 32, page: 1284, stat: Journal Article,

Both IKKalpha and IKKbeta are implicated in the arsenite-induced AP-1 transactivation correlating with cell apoptosis through NF-kappaB activity-independent manner
Song, Lun; Li, Jingxia; Hu, Meiru; Huang, Chuanshu
2008 Jul 1;314(11-12):2187-2198, Experimental cell research
Arsenite has been well-proved to act as both an environmental carcinogen as well as a tumor therapeutic agent. AP-1 is one of the transcription factors that can be induced upon arsenite stimulation. However, the study on the mechanism and the function of the arsenite-induced AP-1 transactivation remains far complete. Here we demonstrated that high dose of arsenite induced apoptotic response in mouse fibroblasts correlating with AP-1 transactivation, which events were mediated by both IKKalpha and IKKbeta, two major protein kinases responsible for NF-kappaB activation. In addition, the regulatory effect of IKKs on the arsenite-induced AP-1 activation was delivered by sequential induction of GADD45alpha expression and the activation of MAPKK (MKK3/4/6) and MAPK (JNK and p38K)-dependent pathways. We further provided evidence that p50, but not p65 subunit of NF-kappaB, was involved in GADD45alpha induction and the subsequent MAPKK/MAPK/AP-1 activation under arsenite exposure, while functional NF-kappaB induced by arsenite stimulation consisted of p65 but not of p50 subunit. Therefore, we concluded that both IKKalpha and IKKbeta can mediate arsenite-induced AP-1 transactivation through NF-kappaB activity-independent manner
— id: 80292, year: 2008, vol: 314, page: 2187, stat: Journal Article,

A JNK1/AP-1-dependent, COX-2 induction is implicated in 12-O-tetradecanoylphorbol-13-acetate-induced cell transformation through regulating cell cycle progression
Zhang, Dongyun; Li, Jingxia; Song, Lun; Ouyang, Weiming; Gao, Jimin; Huang, Chuanshu
2008 Jan;6(1):165-174, Molecular cancer research
Cyclooxygenase-2 (COX-2) is reported to be one of the early-response gene products induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the relevance of COX-2 in TPA-induced cell transformation and the underlying mechanisms remains to be explored. Initially, we verified COX-2 induction after TPA treatment in mouse embryonic fibroblasts (MEF) and mouse epidermal cells Cl 41. More importantly, introduction of COX-2 small interfering RNA in MEFs or Cl 41 cells suppressed the cell transformation caused by TPA treatment. This inhibition could be reversed by overexpression of human full-length COX-2, indicating that COX-2 is at least one of the critical molecules involved in TPA-induced cell transformation. We further showed that TPA-promoted cell cycle progression was partially suppressed by COX-2 small interfering RNA, indicating that COX-2 also participated in TPA-associated cell cycle progression. Investigation of the upstream signaling pathways revealed that c-Jun-NH(2)-kinase 1 (JNK1), but not JNK2, played important roles in COX-2 induction, because knockout of JNK1 gene rather than JNK2 gene markedly impaired COX-2 induction. Furthermore, inhibition of c-Jun/activator protein 1 pathway or JNKs/c-Jun pathway by overexpression of dominant negative mutants of c-Jun, or MKK4 and MKK7 together, resulted in impairment of COX-2 induction, suggesting that JNK1/c-Jun/activator protein 1 pathway is involved in TPA-associated COX-2 induction. In contrast, IKK/p65 nuclear factor-kappaB pathway was not implicated because knockout of IKKalpha, IKKbeta, or p65 gene did not affect COX-2 induction although nuclear factor-kappaB was activated by TPA. In addition, the TPA-promoted cell cycle progression was found impaired in JNK1-deficient, but not in JNK2-deficient, MEFs. Our results show that JNK1-associated COX-2 induction is implicated in TPA-associated cell transformation and cell cycle progression
— id: 76464, year: 2008, vol: 6, page: 165, stat: Journal Article,

Activation of both nuclear factor of activated T cells and inhibitor of nuclear factor-kappa B kinase beta-subunit-/nuclear factor-kappa B is critical for cyclooxygenase-2 induction by benzo[a]pyrene in human bronchial epithelial cells
Ding, Jin; Wu, Kangjian; Zhang, Dongyun; Luo, Wenjing; Li, Jingxia; Ouyang, Weiming; Song, Lun; Huang, Chuanshu
2007 Sep;98(9):1323-1329, Cancer Science
The carcinogenic effect of benzo[a]pyrene (B[a]P), presenting mainly in cigarette smoke and air pollution, has been well demonstrated both in vitro and in vivo. However, it is still not well understood how B[a]P facilitates pulmonary carcinogenesis. To explore this, we investigated the effect of B[a]P on the induction of cyclooxygenase-2 (COX-2), a critical enzyme implicated in inflammation and cancer development, as well as upstream signaling pathways leading to its expression in human bronchial epithelial cells (Beas-2B). We found that exposure of Beas-2B to B[a]P caused significant COX-2 induction at both the transcriptional and protein levels. B[a]P also switched on the nuclear factor of activated T cells (NFAT) and nuclear factor kappaB (NF-kappaB) signaling pathways. B[a]P-induced COX-2 expression was significantly blocked by inhibition of the NFAT pathway, and impairment of the NF-kappaB signaling pathway by ectopic expression of an inhibitor of nuclear factor-kappaB kinase beta-subunit (IKKbeta) kinase inactive mutant (IKKbeta-KM) also dramatically inhibited COX-2 induction. The IKKbeta/NF-kappaB-dependent COX-2 induction was further confirmed in mouse embryonic fibroblasts with IKKbeta deficiency (IKKbeta(-/-)) and in those that expressed reconstituted IKKbeta. However, activation of the NFAT and NF-kappaB signaling pathways by B[a]P were independent of each other, as blocking one signaling pathway didn't interrupt the activation of the other one. Mutation of either NFAT or NF-kappaB binding sites significantly blocked COX-2 promoter induction by B[a]P. Taken together, these data indicate that exposure of Beas-2B to B[a]P can upregulate COX-2 expression by increasing its transcription, which requires activation of both the NFAT and NF-kappaB signaling pathways
— id: 74406, year: 2007, vol: 98, page: 1323, stat: Journal Article,

[Benzo (a) pyrene-induced human embryo lung cell cycle alterations through positive regulation of mitogen-activated protein kinase signal pathways]
DU, Hong-ju; Tang, Ning; Liu, Bing-ci; Shi, Xiang-lin; Huang, Chuan-shu; Gao, Ai; Shen, Fu-hai; Ye, Meng; You, Bao-rong
2007 Jul;41(4):277-280, Zhonghua Yu Fang Yi Xue Za Zhi = Chinese journal of preventive medicine
OBJECTIVE: To study the effects of benzo(a)pyrene (BaP) on the cell cycle distribution and activities of mitogen-activated protein kinase (MAPK) signal molecules (ERK1/2, JNK1/2 and p38) in human embryo lung cells (HELF), and to investigate the relationship between alterations of MAPK protein phosphorylation and the cell cycle distributions. METHODS: The phosphorylation of MAPK were induced by exposing HELF cells to BaP at 0.1, 0.5, 2.5 and 12.5 micromol/L. The phosphorylation and protein expression levels of ERK1/2, JNK1/2 and p38 were determined through western-blotting assay. And the flow cytometry assay was used to measure the cell cycle effects in HELF cells after treatment with 2.5 micromol/L BaP for 24 h. RESULTS: The phosphorylation levels of ERK1/2, JNK1/2 and p38 were significantly increased through BaP exposure. In addition, the phosphorylation of these three MAPKs has similar alteration pattern. We found that exposure of cells to 2.5 microM of BaP for 24 h resulted in a decrease of G(0) and G(1) population by 11.9% (F = 41.38, P < 0.01) and an increase of S population by 17.2% (F = 68.13, P < 0.01). Three chemical inhibitors of MAPK (AG126, SP600125 and SB203580) could significantly inhibit the cell cycle alteration because of BaP treatment. CONCLUSION: ERK1/2, JNK1/2 and p38 could positively regulate the BaP independently induced cell cycle alterations
— id: 132264, year: 2007, vol: 41, page: 277, stat: Journal Article,

Phosphatidylinositol-3 kinase/Akt/p70S6K/AP-1 signaling pathway mediated benzo(a)pyrene-induced cell cycle alternation via cell cycle regulatory proteins in human embryo lung fibroblasts
Gao, Ai; Liu, Bingci; Shi, Xianglin; Jia, Xiaowei; Ye, Meng; Jiao, Shi; You, Baorong; Huang, Chuanshu
2007 Apr 5;170(1):30-41, Toxicology letters
Benzo(a)pyrene (B(a)P), a potent environmental procarcinogen, has been shown to cause cell cycle alternation. However, the mechanisms involved in this effect are not well understood yet. Our current results demonstrated that B(a)P exposure led to cell proliferation and a 33.5% increase in S phase cells as well as a 26.8% decrease in G1 phase cells in human embryo lung fibroblasts (HELFs). Those cell cycle alternations were accompanied with transactivation of activator protein-1 (AP-1) and phosphorylation of Akt and p70(S6K). These changes were blocked by overexpression of dominant negative mutants of phosphatidylinositol-3 kinase (Deltap85) or Akt (DN-Akt), respectively. Moreover, pretreatment of cells with rapamycin, a specific p70(S6K) inhibitor, inhibited B(a)P-induced AP-1 activation, cell cycle alteration and phosphorylation of p70(S6K), but had no effect on Akt phosphorylation. Our results, therefore, suggest that phosphatidylinositol-3 kinase (PI-3K)/Akt/p70(S6K)/AP-1 pathway participates in B(a)P-induced cell cycle alternations. Furthermore, we explored the effect of this pathway on cell cycle regulatory proteins. B(a)P markedly increases in the expression of cyclin D1 and E2F1 and phosphorylation of retinoblastoma protein (Rb). In addition, we found that inactivation of PI-3K, Akt or p70(S6K) could eliminate those effects on cell cycle regulatory proteins. Collectively, PI-3K/Akt/p70(S6K)/AP-1 pathway mediated B(a)P-induced alternation of cell cycle through regulation of cell cycle regulatory proteins such as cyclin D1, E2F1, and Rb in HELFs
— id: 106250, year: 2007, vol: 170, page: 30, stat: Journal Article,

Differential inhibition of UV-induced activation of NF kappa B and AP-1 by extracts from black raspberries, strawberries, and blueberries
Huang, Chuanshu; Zhang, Dongyun; Li, Jingxia; Tong, Qiangsong; Stoner, Gary D
2007 ;58(2):205-212, Nutrition & cancer
Recent studies from our laboratory have shown that the transactivation of nuclear factor kappa B (NF kappa B) and activator protein-1 (AP-1) plays an important mechanistic role in ultraviolet (UV)-induced skin carcinogenesis in mice. We also demonstrated that a methanol extract (ME) fraction from black raspberries (Rubus occidentalis) (RO; RO-ME) inhibits benzo[a]pyrene-7,8-diol-9,10-epoxide [B(a)PDE]-induced activation of NF kappa B and AP-1 in cultured mouse epidermal cells. In the present study, we determined if RO-ME might also inhibit the induction of NF kappa B and AP-1 in mouse epidermal cells exposed to mid UV radiation (UVB) and short UV radiation (UVC) and whether methanol fractions from strawberries and blueberries would also be effective. Our results showed that RO-ME inhibited UVB-induced activation of NF kappa B in mouse epidermal cells in a time- and dose-dependent manner; however, the methanol fractions from strawberries and blueberries were ineffective. Interestingly, none of the fractions from all 3 berry types inhibited UVB- or UVC-induced activation of AP-1, suggesting that inhibition of UV-induced signaling pathways is specific for black raspberries and NF kappa B. Cyanidin-3-rutinoside, an anthocyanin found in abundance in black raspberries and not in strawberries or high-bush blueberries, was found to contribute to the inhibition of UVB-induced activation of NF kappa B. These results suggest that berries differ in their ability to influence signaling pathways leading to activation of NF kappa B and AP-1 when using UV light as the inducer
— id: 73921, year: 2007, vol: 58, page: 205, stat: Journal Article,

Transription factors and their modulated genes as targets for chemoprevention
Huang, CS
2007 JUN ;7(4):303-303, Current cancer drug targets
— id: 73148, year: 2007, vol: 7, page: 303, stat: Journal Article,

Deferoxamine synergistically enhances iron-mediated AP-1 activation: a showcase of the interplay between extracellular-signal-regulated kinase and tyrosine phosphatase
Huang, Xi; Dai, Jisen; Huang, Chuanshu; Zhang, Qi; Bhanot, Opinder; Pelle, Edward
2007 Oct;41(10):1135-1142, Free radical research
Deferoxamine (DFO) is a drug widely used for iron overload treatment to reduce body iron burden. In the present study, it was shown in mouse epidermal JB6 cells that all iron compounds transiently induced extracellular signal-regulated kinases (ERK) phosphorylation, whereas DFO further enhanced ERK phosphorylation over long periods. The ERK phosphorylation by DFO treatment appears to be due to the inhibition of MAPK phosphatases (MKP) by DFO. The combined effects of iron-initiated MAPK activation and DFO-mediated MKP inhibition resulted in a synergistic enhancement on AP-1 activities. The results indicate that the interplay between MAPK and MKP is important in regulating the extent of AP-1 activation. It is known that administration of DFO in iron overload patients often results in allergic responses at the injection sites. The results suggest that this synergistic AP-1 activation might play a role in DFO-induced skin immune responses of iron overload patients
— id: 75378, year: 2007, vol: 41, page: 1135, stat: Journal Article,

Transcription factor NFAT, its role in cancer development, and as a potential target for chemoprevention
Lu, Haitian; Huan, Chuanshu
2007 Jun;7(4):343-353, Current cancer drug targets
The nuclear factor of activated T cells (NFAT) family proteins are transcription factors that regulate the expression of a variety of target genes with or without forming complexes with other transcription factors. Although NFAT proteins have been extensively investigated and characterized in immune systems, their role in carcinogenesis are far from being understood. We, to our knowledge, are first to determine the potential involvement of the NFAT pathway in cell responses to carcinogen exposure. Experimental evidence accumulated from our studies indicate the critical role of NFAT3 in some carcinogen-induced cell transformation and tumorigenicity. Moreover, NFAT proteins have been found to be involved in cell cycle regulation, cell differentiation, cell survival, angiogenesis, and tumor cell invasion and metastasis. In the meantime, NFAT inhibitors are being developed with the ultimate aim to specifically switch off NFAT signaling without side effects. This review comprehensively reviews the results from the most recent studies, and also discusses some difficulties in current studies. To validate whether NFAT can be a promising target for chemoprevention, more research has to be done to further detail the roles of NFAT and to differentiate the functions of different members of this protein family in future studies
— id: 75482, year: 2007, vol: 7, page: 343, stat: Journal Article,

Direct evidence for the critical role of NFAT3 in benzo[a]pyrene diol-epoxide-induced cell transformation through mediation of inflammatory cytokine TNF induction in mouse epidermal Cl41 cells
Ouyang, Weiming; Hu, Yu; Li, Jingxia; Ding, Min; Lu, Yongju; Zhang, Dongyun; Yan, Yan; Song, Lun; Qu, Qingshan; Desai, Dhimant; Amin, Shantu; Huang, Chuanshu
2007 Oct;28(10):2218-2226, Carcinogenesis
Nuclear factor of activated T cell (NFAT)-3 is a member of the transcription factor NFAT family, which has been demonstrated to be responsible for the up-regulation of the pro-inflammatory cytokine tumor necrosis factor (TNF) in the immune system. Our most recent studies have also shown that TNF is able to induce cell transformation in mouse epidermal Cl41 cells by induction of cyclooxygenase-2 (COX-2) expression. To provide direct evidence for NFAT3 in the environmental carcinogen-caused carcinogenic effect, (+/-)-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), an ultimate environmental carcinogen metabolized from benzo[a]pyrene, was utilized. We found that exposure of Cl41 cells to B[a]PDE was able to induce cell transformation in Cl41 cells, while specific knock-down of NFAT3 resulted in the dramatic inhibition of this cell transformation. The tumorigenicity of B[a]PDE-caused transformed cells was confirmed in nude mice, whereas the tumor formation of B[a]PDE-treated NFAT3 small interference RNA (siRNA) knock-down cells was significantly reduced. Further studies showed that the role of NFAT3 in B[a]PDE-caused cell transformation was mediated by up-regulation of its downstream targeted gene TNF. This conclusion was based on the findings that inhibition of NFAT3 activation by either FK506 or NFAT3 siRNA dramatically down-regulated the TNF induction upon B[a]PDE exposure, and that knock-down of TNF by its specific siRNA also led to abrogation of B[a]PDE-induced cell transformation in Cl41 cells and their tumorigenicity in nude mice. Collectively, these results provide direct evidence for the important role of NFAT3 activation in B[a]PDE-induced cell transformation by up-regulation of TNF expression in mouse epidermal Cl41 cells, further suggesting that B[a]PDE may exert its tumor promotion effect on skin carcinogenesis, at least partially, by inducing TNF expression
— id: 75360, year: 2007, vol: 28, page: 2218, stat: Journal Article,

PI-3K/Akt signal pathway plays a crucial role in arsenite-induced cell proliferation of human keratinocytes through induction of cyclin D1
Ouyang, Weiming; Li, Jingxia; Zhang, Dongyun; Jiang, Bing-Hua; Huang, Dr Chuanshu
2007 Jul 1;101(4):969-978, Journal of cellular biochemistry
Exposure of arsenite can induce hyperproliferation of skin cells, which is believed to play important roles in arsenite-induced carcinogenesis by affecting both promotion and progression stages. However, the signal pathways and target genes activated by arsenite exposure responsible for the proliferation remain to be defined. In the present study, we found that: (1) exposure of human keratinocytic HaCat cells to arsenite caused an increase in cell proliferation, which was significantly inhibited by pretreatment of wortmannin, a specific chemical inhibitor of PI-3K/Akt signal pathway; (2) arsenite exposure was also able to activate PI-3K/Akt signal pathway, which thereby induced the elevation of cyclin D1 expression level in both HaCat cells and human primary keratinocytes based on that inhibition of PI-3K/Akt pathway by either pretreatment of wortmannin or the transfection of their dominant mutants, significantly inhibited cyclin D1 expression upon arsenite exposure; (3) PI-3K/Akt pathway is implicated in arsenite-induced proliferation of HaCat cells through the induction of cyclin D1 because either knockdown of cyclin D1 by its siRNA or inhibition of PI-3K/Akt signal pathway by their dominant mutants markedly impaired the proliferation of HaCat cells induced by arsenite exposure. Taken together, we provide the direct evidence that PI-3K/Akt pathway plays a role in the regulation of cell proliferation through the induction of cyclin D1 in human keratinocytes upon arsenite treatment. Given the importance of aberrant cell proliferation in cell transformation, we propose that the activation of PI-3K/Akt pathway and cyclin D1 induction may be the important mediators of human skin carcinogenic effect of arsenite
— id: 73945, year: 2007, vol: 101, page: 969, stat: Journal Article,

Benzo[a]pyrene diol-epoxide (B[a]PDE) upregulates COX-2 expression through MAPKs/AP-1 and IKKbeta/NF-kappaB in mouse epidermal Cl41 cells
Ouyang, Weiming; Ma, Qian; Li, Jingxia; Zhang, Dongyun; Ding, Jin; Huang, Yi; Xing, Mingzhao M; Huang, Chuanshu
2007 Jan;46(1):32-41, Molecular carcinogenesis
Benzo[alpha]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), the major metabolite of benzo[a]pyrene (B[a]P), shows an ultimate complete carcinogen in various animals and is a causative agent for human cancers. However, its effects on the activation of signal pathways and the expression of genes involved in its carcinogenic effect remain largely unknown. In this study, the effects of B[a]PDE on induction of cyclooxygenase (COX)-2 and the signal pathways leading to the induction were investigated. Treatment of mouse epidermal Cl41 cells with B[a]PDE caused an increase in the expression of COX-2 at both transcription and protein levels, while its parental compound B[a]P did not show significant inductive effect. The COX-2 induction by B[a]PDE was dependent on the activation of mitogen-activated protein kinases (MAPK)s/activation protein (AP)-1 pathway, because inhibition of AP-1 by either overexpression of TAM67 (dominant negative mutant of c-jun), or pretreatment of cells with PD98059 (MEK1/2-ERKs pathway inhibitor) or SB202190 (p38K inhibitor), markedly inhibited B[a]PDE-induced COX-2 expression. In addition, impairment of NF-kappaB pathway by either NEMO-BDBP (an NF-kappaB specific inhibitor) or IkappaB kinase (IKK)beta-KM (dominant negative mutant of IKKbeta) also caused marked reduction of COX-2 induction by B[a]PDE. In contrast, inhibition of nuclear factor of activated T cells (NFAT) with FK506, did not show any effect on B[a]PDE-induced COX-2 expression. Collectively, these data indicate that exposure of Cl41 cells to B[a]PDE can induce COX-2 expression by increasing its transcription, which requires the activation of MAPKs/AP-1 and IKKbeta/NF-kappaB pathways, but not NFAT pathway. In view of the importance of COX-2 in carcinogenesis, we anticipate that the induction of COX-2 by B[a]PDE may coordinate its mutagenic effects to facilitate the development of skin cancer
— id: 70301, year: 2007, vol: 46, page: 32, stat: Journal Article,

Cyclooxygenase-2 induction by arsenite through the IKKbeta/NFkappaB pathway exerts an antiapoptotic effect in mouse epidermal Cl41 cells
Ouyang, Weiming; Zhang, Dongyun; Ma, Qian; Li, Jingxia; Huang, Chuanshu
2007 Apr;115(4):513-518, Environmental health perspectives
BACKGROUND: Arsenic contamination has become a major public health concern worldwide. Epidemiologic data show that long-term arsenic exposure results in the risk of skin cancer. However, the mechanisms underlying carcinogenic effects of arsenite on skin remain to be studied. OBJECTIVES: In the present study we evaluated cyclooxygenase-2 (COX-2) expression, the signaling pathways leading to COX-2 induction, and its antiapoptotic function in the response to arsenite exposure in mouse epidermal JB6 Cl41 cells. METHODS: We used the luciferase reporter assay and Western blots to determine COX-2 induction by arsenite. We utilized dominant negative mutant, genetic knockout, gene knockdown, and gene overexpression approaches to elucidate the signaling pathway involved in COX-2 induction and its protective effect on cell apoptosis. RESULTS: The induction of COX-2 by arsenite was inhibited in Cl41 cells transfected with IKKbeta-KM, a dominant mutant inhibitor of kbeta (Ikbeta) kinase (IKKbeta), and in IKKbeta-knockout (IKKbeta(-/-)) mouse embryonic fibroblasts (MEFs). IKKbeta/nuclear factor kappaB (NFkappaB) pathway-mediated COX-2 induction exerted an antiapoptotic effect on the cells exposed to arsenite because cell apoptosis was significantly enhanced in the Cl41 cells transfected with IKKbeta-KM or COX-2 small interference RNA (siCOX-2). In addition, IKKbeta(-/-) MEFs stably transfected with COX-2 showed more resistance to arsenite-induced apoptosis compared with the same control vector-transfected cells. CONCLUSIONS: These results demonstrate that arsenite exposure can induce COX-2 expression through the IKKbeta/NFkappaB pathway, which thereby exerts an antiapoptotic effect in response to arsenite. In light of the importance of apoptosis evasion during carcinogenesis, we anticipate that COX-2 induction may be at least partially responsible for the carcinogenic effect of arsenite on skin
— id: 72542, year: 2007, vol: 115, page: 513, stat: Journal Article,

[The activation of MAPK/cyclin D1-CDK4 signaling pathway caused by quartz]
Shen, Fu-Hai; Fan, Xue-Yun; Liu, Bing-Ci; Jia, Xiao-Wei; Gao, Ai; You, Bao-Rong; Ye, Meng; DU, Hong-Ju; Huang, Chuan-Shu; Shi, Xiang-Lin
2007 Jan;25(1):5-10, Zhonghua lao dong wei sheng zhi ye bing za zhi = Zhonghua laodong weisheng zhiyebing zazhi = Chinese journal of industrial hygiene & occupational diseases
OBJECTIVES: To study the phosphorylation level of mitogen activated protein kinase (MAPK) in human embryonic lung fibroblasts (HELF), and the expression level of cyclin D1-CDK4 protein in S-HELF and whether the expression level of cyclin D1-CDK4 protein mediated by MAPK/AP-1 signaling pathway in S-HELF. METHODS: Two kinds of treatment: (1) Cells were harvested after stimulation 2 h for the detection of cytokines. (2) Cells were stimulated by quartz for a long time (2 months) for transformation characters (S-HELF). The MAP kinase was detected by western blot. Cyclin D1 and CDK4 (cyclin dependent kinase 4) proteins was measured by immunocytochemistry. Flow cytometry was used to evaluate the alternation of cell cycle. RESULTS: Crystalline quartz could cause the phosphorylation level of ERKs, p38K, and JNKs in HELF increase. However, activated levels of ERKs and p46 of JNKs increased in S-HELF, and p38K activation decreased, and no effect on activation of p54 of JNKs, as compared with those in parental HELF. Cyclin D1 and CDK4 protein expression levels increased in S-HELF as compared with parental HELF. Inhibition of ERKs activation by AG126, AP-1 by curcumin, and JNKs by SP600125 could reduced the induction of cyclin D1 and CDK4, whereas inhibition of p38K by SB203580 did not show any inhibitory effects on S-HELF. CONCLUSIONS: The phosphorylation levels of ERK1/2, JNK1/2, and p38 increased in HELF exposed to quartz. The phosphorylation levels of ERK1/2 and JNK1 increased, but the phosphorylation level of p38 decreased in S-HELF. The expression level of cyclin D1-CDK4 protein increased in S-HELF. Overexpression of cyclin D1-CDK4 is due to the activation of ERKs, JNKs/AP-1 signaling pathway in S-HELF
— id: 132265, year: 2007, vol: 25, page: 5, stat: Journal Article,

p85alpha acts as a novel signal transducer for mediation of cellular apoptotic response to UV radiation
Song, Lun; Li, Jingxia; Ye, Jianping; Yu, Gang; Ding, Jin; Zhang, Dongyun; Ouyang, Weiming; Dong, Zigang; Kim, Sung O; Huang, Chuanshu
2007 Apr;27(7):2713-2731, Molecular & cellular biology
Apoptosis is an important cellular response to UV radiation (UVR), but the corresponding mechanisms remain largely unknown. Here we report that the p85alpha regulatory subunit of phosphatidylinositol 3-kinase (PI-3K) exerted a proapoptotic role in response to UVR through the induction of tumor necrosis factor alpha (TNF-alpha) gene expression. This special effect of p85alpha was unrelated to the PI-3K-dependent signaling pathway. Further evidence demonstrated that the inducible transcription factor NFAT3 was the major downstream target of p85alpha for the mediation of UVR-induced apoptosis and TNF-alpha gene transcription. p85alpha regulated UVR-induced NFAT3 activation by modulation of its nuclear translocation and DNA binding and the relevant transcriptional activities. Gel shift assays and site-directed mutagenesis allowed the identification of two regions in the TNF-alpha gene promoter that served as the NFAT3 recognition sequences. Chromatin immunoprecipitation assays further confirmed that the recruitment of NFAT3 to the endogenous TNF-alpha promoter was regulated by p85alpha upon UVR exposure. Finally, the knockdown of the NFAT3 level by its specific small interfering RNA decreased UVR-induced TNF-alpha gene transcription and cell apoptosis. The knockdown of endogenous p85alpha blocked NFAT activity and TNF-alpha gene transcription, as well as cell apoptosis. Thus, we demonstrated p85alpha-associated but PI-3K-independent cell death in response to UVR and identified a novel p85alpha/NFAT3/TNF-alpha signaling pathway for the mediation of cellular apoptotic responses under certain stress conditions such as UVR
— id: 71925, year: 2007, vol: 27, page: 2713, stat: Journal Article,

JNK1 is required for sulindac-mediated inhibition of cell proliferation and induction of apoptosis in vitro and in vivo
Song, Zibo; Tong, Chang; Liang, Jiao; Dockendorff, Ashley; Huang, Chuanshu; Augenlicht, Leonard H; Yang, Wancai
2007 Apr 10;560(2-3):95-100, European journal of pharmacology
Our previous studies demonstrated that sulindac, a non-steroidal anti-inflammatory drug, suppressed intestinal tumor formation in mouse, which is linked to the induction of wild-type p53-activated fragment 1 (p21WAF1, or p21). Here we showed that sulindac also required c-Jun N-terminal Kinase 1 (JNK1) to inhibit cell proliferation and induce apoptosis in vitro and in vivo. First, sulindac inhibited cell proliferation and induced apoptosis in colon cancer cell lines HCT116 with wild-type p21 or null p21, which were p21-dependent and were also associated with the induction of p21 and phosphorylation of JNK1. Second, sulindac increased apoptosis in JNK1(+/+) and JNK1(-/-) mouse embryonic fibroblast (MEF) cells, but, the increase of apoptosis in JNK1(+/+) cells was more than that in JNK1(-/-) cells. More interestingly, sulindac significantly inhibited cell proliferation in JNK1(+/+) cells, but the inhibition in JNK1(-/-) cells markedly decreased. Further studies indicated that JNK1 was dramatically induced by sulindac in the JNK1(+/+) cells which correlated with the induction of p21. However, the induction of p21 in JNK1(-/-) cells was less than that in JNK1(+/+) cells. Finally, we determined the expression of JNK1 in the intestinal mucosa of Apc(+/-), p21(+/+) mice, and found that sulindac significantly induced JNK1 phosphorylation, corresponding to the induction of p21, both in mRNA and protein levels. Our data indicates that sulindac-mediated proliferation inhibition and apoptosis induction were not only p21-dependent, but also required JNK1
— id: 106253, year: 2007, vol: 560, page: 95, stat: Journal Article,

Cancer prevention with freeze-dried berries and berry components
Stoner, Gary D; Wang, Li-Shu; Zikri, Nancy; Chen, Tong; Hecht, Stephen S; Huang, Chuanshu; Sardo, Christine; Lechner, John F
2007 Oct;17(5):403-410, Seminars in cancer biology
Our laboratory is developing a food-based approach to the prevention of esophageal and colon cancer utilizing freeze-dried berries and berry extracts. Dietary freeze-dried berries were shown to inhibit chemically induced cancer of the rodent esophagus by 30-60% and of the colon by up to 80%. The berries are effective at both the initiation and promotion/progression stages of tumor development. Berries inhibit tumor initiation events by influencing carcinogen metabolism, resulting in reduced levels of carcinogen-induced DNA damage. They inhibit promotion/progression events by reducing the growth rate of pre-malignant cells, promoting apoptosis, reducing parameters of tissue inflammation and inhibiting angiogenesis. On a molecular level, berries modulate the expression of genes involved with proliferation, apoptosis, inflammation and angiogenesis. We have recently initiated clinical trials; results from a toxicity study indicated that freeze-dried black raspberries are well tolerated in humans when administered orally for 7 days at a dose of 45 g per day. Several Phase IIa clinical trials are underway in patients at high risk for esophagus and colon cancer; i.e., Barrett's esophagus, esophageal dysplasia and colonic polyps, to determine if berries will modulate various histological and molecular biomarkers of development of these diseases
— id: 110426, year: 2007, vol: 17, page: 403, stat: Journal Article,

c-Jun NH2-terminal kinase 1 plays a critical role in intestinal homeostasis and tumor suppression
Tong, Chang; Yin, Zhinan; Song, Zibo; Dockendorff, Ashley; Huang, Chuanshu; Mariadason, John; Flavell, Richard A; Davis, Roger J; Augenlicht, Leonard H; Yang, Wancai
2007 Jul;171(1):297-303, American journal of pathology
The c-Jun NH(2)-terminal kinase (JNK) signal transduction pathway plays important roles in cellular processes and stress. However, the role of JNK1 in intestinal homeostasis and tumorigenesis is unknown. Therefore, we used a JNK1 knockout mouse model to characterize intestinal cell maturation and tumorigenesis. In addition, colon cancer cell lines were used to validate the role of JNK1 and to elucidate the underlying molecular mechanisms in vitro. To our surprise, we found that mice with targeted inactivation of JNK1 spontaneously developed intestinal tumors. The normal mucosa in JNK1-deficient mice showed decreased cell differentiation and increased cell proliferation. This tumorigenesis was closely linked to the down-regulation of p21(WAF1/cip1), a cyclin-dependent kinase inhibitor, in intestinal epithelial cells. Immunohistochemical staining showed that JNK1 was highly expressed in the differentiation compartment of the intestinal mucosa and that the expression of JNK1 was significantly decreased in both human colonic and mouse intestinal tumors. In the colon cancer cell lines, JNK1 expression was up-regulated during spontaneous differentiation, corresponding to the up-regulation of p21(WAF1/cip1). Moreover, butyrate-induced p21 expression was linked to phosphorylation of JNK1. Reduced JNK1 expression by small interfering RNA suppressed butyrate-induced apoptosis. We concluded that JNK1 plays a critical role in the regulation of homeostasis and in the suppression of tumor formation in the intestine, which was linked to the altered expression of p21(WAF1/cip1)
— id: 106252, year: 2007, vol: 171, page: 297, stat: Journal Article,

JNK1, but not JNK2, is required for COX-2 induction by nickel compounds
Zhang, Dongyun; Li, Jingxia; Wu, Kangjian; Ouyang, Weiming; Ding, Jin; Liu, Zheng-gang; Costa, Max; Huang, Chuanshu
2007 Apr;28(4):883-891, Carcinogenesis
Activation of the signaling pathways leading to gene expression regulation, is critical in the carcinogenic effects of nickel exposure. In the present study, we found nickel exposure could induce cyclooxygenase-2 (COX-2) expression at transcriptional and protein levels in both human bronchoepithelial cells (Beas-2B) and murine embryonic fibroblasts (MEFs). We further provided direct evidence for the specific involvement of the JNK1 signaling pathway in the COX-2 induction using specific gene knockout approaches. Our results demonstrated that COX2 induction by nickel was impaired in JNK1(-/-) MEFs, but not in JNK2(-/-) MEFs. Moreover, re-constitutional expression of JNK1 restored COX-2 induction, confirming the specific requirement of JNK1 in COX-2 induction. Further investigation revealed that JNK1 mediated the nickel-induced COX-2 expression in a c-Jun/AP-1-dependent manner. Ectopic expression of TAM67, a c-Jun dominant negative mutant, also suppressed the COX-2 induction. Our results demonstrate that the JNK1/c-Jun/AP-1 pathway, but not the JNK2 pathway, plays a critical role in nickel-induced COX-2 expression
— id: 68994, year: 2007, vol: 28, page: 883, stat: Journal Article,

High expression of nuclear factor of activated T cells in Chinese primary non-small cell lung cancer tissues
Zhang, K; Li, N; Chen, Z; Shao, K; Zhou, F; Zhang, C; Mu, X; Wan, J; Li, B; Feng, X; Shi, S; Xiong, M; Cao, K; Wang, X; Huang, C; He, J
2007 JUL-SEP ;22(3):221-225, International Journal of Biological Markers
Purpose: Nuclear factor of activated T cells (NFAT) has been reported to be involved in the development of various types of cancer including adenocarcinoma of the breast. This research was the first to investigate NFAT protein expression in primary non-small cell lung cancer (NSCLC) tissues from Chinese patients. Methods: NFAT protein expression was determined in 130 surgically resected primary NSCLC and matched normal tissues by immunohistochemical analysis. The association between NFAT expression and clinical categorical variables was further analyzed with the SPSS software. Results: We found that NFAT expression was much higher in 85 tumor tissues (65.4%) and lower in 45 tumor tissues (34.6%) compared with the matched normal tissues. Further statistical analysis by the chi-square test showed that high expression of NFAT proteins was significantly associated with tumor differentiation (p=0.045), invasion (p=0.031), histology (p<0.0001), tumor size (p=0.038) and cigarette smoking history (p=0.024). However, there was no correlation between the expression of NFAT proteins and pTNM classification, and no difference in 5-year survival rate between patients with high or low expression of NFAT proteins. Multivariate logistic regression analysis for the correlation between NFAT protein expression levels and various characteristics showed a significant association with histology (p=0.008, OR=0.273). Conclusion: Our results revealed that high NFAT expression was present in Chinese NSCLCs and that NFAT expression might be involved in the process of human lung cancer development
— id: 75201, year: 2007, vol: 22, page: 221, stat: Journal Article,

The PI3K/Akt pathway and its downstream transcriptional factors as targets for chemoprevention
Zhang, Xinhai; Jin, Boquan; Huang, Chuanshu
2007 Jun;7(4):305-316, Current cancer drug targets
The PI3K/Akt signalling pathway and its downstream transcription factors have been intensively studied for their role in cell proliferation, survival, cycle control, as well as other cellular functions. There is growing evidence showing that dysregulation of this pathway also plays an essential role in cancer development. The overexpression or permanent activation of RTKs and GPCRs, as well as the exposure to environmental carcinogens cause constant activation of PI3K/Akt. On the other hand, PI3K/Akt themselves can also become hyperactivated due to gene amplification or PTEN inactivation. Consequently, the targets downstream of PI3K/Akt can be abnormally activated, which promote proliferation and survival of cancer cells in carcinogenesis. Among these targets we find that the NFkappaB and AP-1 are the most interesting. Therefore, methods and compounds aiming to inhibit the altered components of this pathway can simultaneously prevent the proliferation of tumor cells and sensitize them toward apoptosis. To this regard, the natural compounds from vegetables and fruits with high affinity and non toxicity to target the PI3K/Akt pathway and prevent cancer are attractive
— id: 75481, year: 2007, vol: 7, page: 305, stat: Journal Article,

Effects of polycyclic aromatic hydrocarbons (PAHs) on vascular endothelial growth factor induction through phosphatidylinositol 3-kinase/AP-1-dependent, HIF-1alpha-independent pathway
Ding, Jin; Li, Jingxia; Chen, Jingyuan; Chen, Haobin; Ouyang, Weiming; Zhang, Ronghe; Xue, Caifang; Zhang, Dongyun; Amin, Shantu; Desai, Dhimant; Huang, Chuanshu
2006 Apr 7;281(14):9093-9100, Journal of biological chemistry
Previous studies have demonstrated that exposure to polycyclic aromatic hydrocarbons (PAHs) and its derivatives is associated with an increased risk of skin cancers, and the carcinogenic effect of PAHs is thought to involve both tumor initiation and promotion. Whereas PAH tumor initiation is well characterized, the mechanisms involved in the tumor promotion of PAHs remain elusive. In the present study, we investigated the effects of PAHs on vascular endothelial growth factor (VEGF) expression by comparison of its induction between the active metabolite and its parent compound (B[a]PDE versus B[a]P) or between active compound and its relatively inactive analog (5-MCDE versus CDE). We found that exposure of cells to (+/-)-anti-benzo-[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) or (+/-)-anti-5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE) led to marked induction of VEGF in Cl41 cells, whereas benzo[a]pyrene (B[a]P) or chrysene-1,2-diol-3,4-epoxide (CDE) did not exhibit significant inductive effects. Exposure of cells to B[a]PDE and 5-MCDE did not induce HIF-1alpha activation, whereas AP-1 was significantly activated. Moreover, overexpression of TAM67 (a dominant-negative mutant c-Jun) dramatically blocked that VEGF induction. Electrophoretic mobility shift assay showed that AP-1 was only able to specifically recognize and bind to its AP-1 potential binding site within -1136 and -1115 of the VEGF promoter region. Site-directed mutation of this AP-1 binding site eliminated the VEGF transcriptional activity induced by B[a]PDE, suggesting that the AP-1 binding site between -1136 and -1115 in the VEGF promoter region is critical for VEGF induction by B[a]PDE. In addition, overexpression of Deltap85 (a dominant-negative mutant PI-3K) impaired B[a]PDE- and 5-MCDE-induced VEGF induction. Considering our previous findings that PI-3K is an upstream mediator for c-Jun/AP-1 activation, we conclude that the VEGF induction by B[a]PDE and 5-MCDE is through PI-3K/AP-1-dependent and HIF-1alpha-independent pathways. These findings may help us to understand the mechanisms involved in PAH carcinogenic effects
— id: 64384, year: 2006, vol: 281, page: 9093, stat: Journal Article,

Cyclooxygenase-2 induction by arsenite is through a nuclear factor of activated T-cell-dependent pathway and plays an antiapoptotic role in Beas-2B cells
Ding, Jin; Li, Jingxia; Xue, Caifang; Wu, Kangjian; Ouyang, Weiming; Zhang, Dongyun; Yan, Yan; Huang, Chuanshu
2006 Aug 25;281(34):24405-24413, Journal of biological chemistry
Arsenite is a well known metalloid human carcinogen, and epidemiological evidence has demonstrated its association with the increased incidence of lung cancer. However, the mechanism involved in its lung carcinogenic effect remains obscure. The current study demonstrated that exposure of human bronchial epithelial cells (Beas-2B) to arsenite resulted in a marked induction of cyclooxygenase (COX)-2, an important mediator for inflammation and tumor promotion. Exposure of the Beas-2B cells to arsenite also led to significant transactivation of nuclear factor of activated T-cells (NFAT), but not activator protein-1 (AP-1) and NFkappaB, suggesting that NFAT, rather than AP-1 or NFkappaB, is implicated in the responses of Beas-2B cells to arsenite exposure. Furthermore, we found that inhibition of the NFAT pathway by either chemical inhibitors, dominant negative mutants of NFAT, or NFAT3 small interference RNA resulted in the impairment of COX-2 induction and caused cell apoptosis in Beas-2B cells exposed to arsenite. Site-directed mutation of two putative NFAT binding sites between-111 to +65 in the COX-2 promoter region eliminated the COX-2 transcriptional activity induced by arsenite, confirming that those two NFAT binding sites in the COX-2 promoter region are critical for COX-2 induction by arsenite. Moreover, knockdown of COX-2 expression by COX-2-specific small interference RNA also led to an increased cell apoptosis in Beas-2B cells upon arsenite exposure. Together, our results demonstrate that COX-2 induction by arsenite is through NFAT3-dependent and AP-1- or NFkappaB-independent pathways and plays a crucial role in antagonizing arsenite-induced cell apoptosis in human bronchial epithelial Beas-2B cells
— id: 69059, year: 2006, vol: 281, page: 24405, stat: Journal Article,

Involvement of nuclear factor of activated T cells 3 (NFAT3) in cyclin D1 induction by B[a]PDE or B[a]PDE and ionizing radiation in mouse epidermal Cl 41 cells
Ding, Jin; Zhang, Ronghe; Li, Jingxia; Xue, Caifang; Huang, Chuanshu
2006 Jul;287(1-2):117-125, Molecular & cellular biochemistry
The results from animal studies have shown that mouse skin is highly susceptible to both ionizing radiation and benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE). Previous studies have also indicated that cyclin D1 plays a crucial role in controlling cell proliferation and tumorigenesis. We, therefore, investigated here the effect of ionizing radiation and B[a]PDE on cyclin D1 transcription and potential involvement of NFAT3 in regulation of cyclin D1 transcription in mouse epidermal Cl 41 cells. We found that B[a]PDE exposure induced a high level of NFAT activation and cyclin D1 transcription in mouse epidermal Cl 41 cells. Ionizing radiation exhibited an enhancement for NFAT activation and cyclin D1 induction by B[a]PDE, even though ionizing radiation by itself had only a marginal effect. By stably knockdown of NFAT3 protein expression using specific NFAT3 small interfering RNA (siRNA), we found that cyclin D1 induction by B[a]PDE or B[a]PDE plus ionizing radiation was dramatically impaired. These results indicate that ionizing radiation is able to enhance cyclin D1 transcription induced by B[a]PDE, and NFAT3 is involved in the regulation of cyclin D1 transcription by B[a]PDE or B[a]PDE plus ionizing radiation
— id: 110431, year: 2006, vol: 287, page: 117, stat: Journal Article,

Nickel compounds render anti-apoptotic effect to human bronchial epithelial Beas-2B cells by induction of COX-2 through IKKbeta /p65-dependent, and IKKalpha - and p50-independent pathway
Ding, Jin; Zhang, Xinhai; Li, Jingxia; Song, Lun; Ouyang, Weiming; Zhang, Dongyun; Xue, Caifang; Costa, Max; Melendez, J Andres; Huang, Chuanshu
2006 Dec 22;281(51):39022-39032, Journal of biological chemistry
The carcinogenicity of nickel compounds has been well documented both in vivo and in vivo, however the molecular mechanisms by which nickel compounds cause cancers are far from understood yet. Since suppression of apoptosis is thought to contribute to carcinogenesis, current studies investigated the mechanisms implicated in nickel-induced anti-apoptotic effect in human bronchial epithelial (Beas-2B) cells. We found that exposure of Beas-2B cells to nickel compounds resulted in an increased COX-2 expression, and knockdown of COX-2 expression with its small interfering RNA (siCOX-2) resulted in the cell more sensitivity to nickel-triggered cell apoptosis, demonstrating that COX-2 induction renders the anti-apoptotic effect to Beas-2B cells. Overexpression of IKKb-KM, a kinase inactive mutant of IKKb, blocked NF-kB activation and COX-2 induction by nickel compounds, indicating that activated NF-kB may be a mediator for COX-2 induction. To further explore the implication of the NF-kB pathway in COX-2 induction and anti-apoptotic effect upon nickel exposure, mouse embryonic fibroblasts (MEFs) deficient with IKKb, IKKa, p65 and p50, were employed. Abortion of IKKb in IKKb-/- cells impaired COX-2 induction by nickel exposure, whereas knockout of IKKa only showed a marginal effect. Moreover, p65, and not p50 of NF-kB subunit, was critical for nickel-induced COX-2 expression. In addition, a deficiency of IKKb or p65 renders cells more sensitive to nickel-induced apoptosis as compared to those in wild type cells. Finally, it was shown that reactive oxygen species (ROS) were involved in both NFkB activation and COX-2 expression. Collectively, our results demonstrate that COX-2 induction by nickel compounds via IKKb/p65 NF-kB-dependent, and IKKa- and p50-independent pathway, plays a crucial role in antagonizing nickel-induced cell apoptosis in Beas-2B cells
— id: 68995, year: 2006, vol: 281, page: 39022, stat: Journal Article,

Involvement of protein kinase C in crystalline silica-induced activation of the MAP kinase and AP-1 pathway
Ding, Min; Huang, Chuanshu; Lu, Yongju; Bowman, Linda; Castranova, Vince; Vallyathan, Val
2006 Feb;290(2):L291-L297, American journal of physiology. Lung cellular & molecular physiology
Crystalline silica has long been well established as a fibrogenic agent, and recent evidence has implicated it as a potential human carcinogen. However, the mechanisms of silica-induced disease development and progression are not well understood. Our previous studies demonstrated that crystalline silica is able to activate activator protein-1 (AP-1) through mitogen-activated protein kinase (MAPK) pathways. The present study investigates the possible involvement of protein kinase C (PKC) in silica-induced activation of the MAPK/AP-1 signal transduction pathway. Treatment of mouse epidermal cells (JB6 cell line) with freshly fractured silica stimulated translocation of PKCalpha and PKCepsilon from the cytosol to the membrane and activated AP-1 transcription activity. Pretreatment of cells with PKC inhibitors, including RO-32-0432, calphostin C, and bisindolylmaleimide I, inhibited silica-induced AP-1 activation and phosphorylation of ERKs and p38 kinase. These inhibitory effects by PKC inhibitors were dose dependent. Furthermore, overexpression of dominant negative mutant (DNM) of PKCalpha or PKCepsilon markedly blocked AP-1 activation as well as phosphorylation of ERKs and p38 kinase induced by freshly fractured silica. These results demonstrate that PKCalpha and PKCepsilon are essential in silica-induced AP-1 activation through the MAP kinase (ERKs and p38 kinases) pathway
— id: 110434, year: 2006, vol: 290, page: L291, stat: Journal Article,

Benzo[a]pyrene-induced cell cycle progression is through ERKs/cyclin D1 pathway and requires the activation of JNKs and p38 mapk in human diploid lung fibroblasts
Du, Hong Ju; Tang, Ning; Liu, Bing Ci; You, Bao Rong; Shen, Fu Hai; Ye, Meng; Gao, Ai; Huang, Chuan shu
2006 Jul;287(1-2):79-89, Molecular & cellular biochemistry
Treatment of cells with carcinogen Benzo[a]pyrene (B[a]P) allows cells to evade G1 arrest and induces cells abnormal proliferation. However, the mechanisms of its action at cellular level are not well understood. To address this question, normal human embryo lung diploid fibroblasts (HELF) were selected in the present study. We found that exposure of cells with 2.5 microM of B[a]P for 24 h resulted in a decrease of G1 population by 11.9% (P < 0.05) and a increase of S population by 17.2% (P < 0.05). Treatment of cells with B[a]P also caused dose-related activation of MAPK and induction of cyclin D1 protein expression, whereas the CDK4 protein levels were not significantly affected by B[a]P. Overexpression of cyclin D1 protein stimulated by B[a]P was significantly inhibited by 50 microM AG126 (an inhibitor of ERK1/2), but not by 25 microM SP600125 (an inhibitor of JNK1/2) or 5 microM SB203580 (an inhibitor of p38 mapk), suggesting that B[a]P-induced cyclin D1 expression was only regulated by ERK1/2 pathway. However, AG126, SP600125 or SB203580 led to cell cycle significantly arrested in G1 phase, indicating that ERK1/2, JNK1/2 and p38 mapk pathways are all required for B[a]P-induced G1/S transition. In addition, HELF cells transfecting with antisense cyclin D1 cDNA or antisense CDK4 cDNA showed significantly G1 arrest after B[a]P stimulation. These results suggested that B[a]P exposure accelerated the G1-->S transition by activation of MAPK signaling pathways. Cyclin D1 and CDK4 are rate-limiting regulators of the G1-->S transition and expression of cyclin D1 is predominantly regulated by ERK1/2 pathway in HELF cells
— id: 132263, year: 2006, vol: 287, page: 79, stat: Journal Article,

[Vitamin C reverses benzo (a) pyrene-induced cell cycle changes by E2F pathway]
Gao, Ai; Liu, Bing-ci; Shen, Fu-hai; Du, Hong-ju; Huang, Chuan-shu; Jia, Xiao-wei; You, Bao-rong; Ye, Meng
2006 Mar;40(2):79-83, Zhonghua Yu Fang Yi Xue Za Zhi = Chinese journal of preventive medicine
OBJECTIVE: To study the role of E2F1/4 pathway in vitamin C reversing benzo (a) pyrene [B (a) P]-induced changes of cell cycle in human embryo lung fibroblasts (HELF) and the relationship between E2F1 and cyclin D1/CDK4. METHODS: The stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established to detect the relationship of signaling pathway. Cells were cultured and pretreated with vitamin C before stimulation with B (a) P for 24 hours. The expression levels of cyclin D1, CDK4, E2F1 and E2F4 were determined by Western blot and the band intensity was analysed as the relative value to control by using the Gel-Pro 3.0 software. Flow Cytometric Analysis was employed to detect the distributions of cell cycle. RESULTS: B (a) P significantly elevated the expression levels of cyclin D1, CDK4, E2F1 and E2F4 in HELF cells. Vitamin C decreased the expression levels of above proteins in B (a) P-stimulated HELF cells. The expression levels of these proteins in B (a) P-treated above transfectants were lower than those in B (a) P-treated HELF cells. The expression levels of above proteins with vitamin C combined with antisense cyclin D1 were decreased as compared to those with antisense cyclin D1 alone. B (a) P increased the percentage of S phase as compared to the controls [(41.1 +/- 0.2)% vs (33.5 +/- 3.2)%, P < 0.05]. Both vitamin C [(33.2 +/- 0.6)% vs (41.1 +/- 0.2)%, P < 0.05] and antisense cyclin D1 [(31.2 +/- 1.3)% vs (41.1 +/- 0.2)%, P < 0.05] suppressed the changes of cell cycle induced by B (a) P. Vitamin C combined with antisense CDK4 markedly suppressed B (a) P-induced changes of cell cycle as compared to those with antisense CDK4 alone. CONCLUSION: Vitamin C might reserve the B (a) P-induced changes of cell cycle via intracellular signaling pathway of cyclin D1-CDK4/E2F-1/4
— id: 132267, year: 2006, vol: 40, page: 79, stat: Journal Article,

Vitamin C inhibits benzo[a]pyrene-induced cell cycle changes partly via cyclin D1/E2F pathway in human embryo lung fibroblasts
Gao, Ai; Liu, Bing-Ci; Shit, Xiang-Lin; Huang, Chuan-Shu; Jia, Xiao-Wei; You, Bao-Rong; Ye, Meng; Shen, Fu-Hai; Du, Hong-Ju
2006 Jun;19(3):239-244, Biomedical & environmental sciences
OBJECTIVE: To study the molecular mechanism of the inhibitory effects of vitamin C on benzo[a]pyrene (B[a]P)-induced changes of cell cycle in human embryo lung fibroblast (HELF) cells. METHODS: The stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established. Cells were cultured and pretreated with vitamin C before stimulation with B[a]P for 24 h. The expression levels of cyclin D1, CDK4, E2F1, and E2F4 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle. RESULTS: B[a]P significantly elevated the expression levels of cyclin D1, E2F1, and E2F4 in HELF cells. Vitamin C decreased the expression levels of cyclin D1, E2F1, and E2F4 in B[a]P-stimulated HELF cells. Dose-dependent relationships were not found between the different concentrations of vitamin C (10, 100, 500, 1000, and 5000 micromol/L) and the expression levels of cyclin D1, E2F1, and E2F4 in HELF cells. The expression levels of cyclin D1, E2F1, and E2F4 in B[a]P-treated transfectants were lower than those in B[a]P-treated HELF cells. The expression levels of cyclin D1 and E2F4 treated with vitamin C and antisense cyclin D1 were decreased compared with those treated with antisense cyclin D1 alone. The effects of vitamin C combined with antisense CDK4 on the expression levels of cyclin D1 and E2F1/E2F4 were similar to those of antisense CDK4 alone. B[a]P progressed HELF cells from G1 to S phase. Both vitamin C and antisense cyclin D1 suppressed the changes of cell cycle progressed by B[a]P. However, antisense CDK4 did not attenuate the above changes. Vitamin C combined with antisense CDK4 markedly suppressed B[a]P-induced changes of cell cycle as compared with antisense CDK4. But the inhibitory effects of vitamin C combined with antisense cyclin D1 on B[a]P-induced changes of cell cycle were similar to those of vitamin C alone or antisense cyclin D1 alone. CONCLUSIONS: B[a]P progressed HELF cells from G1 to S phase via intracellular signaling pathway of cyclin D1/E2F. Vitamin C may modulate this signaling pathway to protect cells from injury caused by B[a]P
— id: 132266, year: 2006, vol: 19, page: 239, stat: Journal Article,

Identification of cyanidin glycosides as constituents of freeze-dried black raspberries which inhibit anti-benzo[a]pyrene-7,8-diol-9,10-epoxide induced NFkappaB and AP-1 activity
Hecht, Stephen S; Huang, Chuanshu; Stoner, Gary D; Li, Jingxia; Kenney, Patrick M J; Sturla, Shana J; Carmella, Steven G
2006 Aug;27(8):1617-1626, Carcinogenesis
Dietary freeze-dried black raspberries inhibit tumor induction by N-nitrosomethylbenzylamine in the rat esophagus, but the constituents responsible for this chemopreventive activity have not been identified. We fractionated freeze-dried black raspberries and used mouse epidermal JB6 Cl 41 cells stably transfected with either a nuclear factor kappa B (NFkappaB)- or an activator protein 1 (AP-1)-luciferase reporter, and treated with racemic anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), to assess the inhibitory effects of the fractions. The ethanol and water extracts of the freeze-dried black raspberries had inhibitory activity and these extracts were fractionated by HPLC to give several bioactive fractions. Further HPLC analysis yielded multiple subfractions, some of which inhibited BPDE-induced NFkappaB activity. Major constituents of the most active subfractions were identified by their spectral properties and in comparison with standards as cyanidin-3-O-glucoside, cyanidin 3-O-(2(G)-xylosylrutinoside) and cyanidin 3-O-rutinoside. Analysis of freeze-dried black raspberries indicated that these three components comprised approximately 3.4% of the material by dry weight. Consistent with these results, standard cyanidin-3-O-glucoside and cyanidin chloride were also good inhibitors of BPDE-induced NFkappaB activity. The results of this study demonstrate that cyanidin glycosides of freeze-dried black raspberries are bioactive compounds which could account for at least some of the chemopreventive activity observed in animal models
— id: 110432, year: 2006, vol: 27, page: 1617, stat: Journal Article,

Black raspberry extracts inhibit benzo(a)pyrene diol-epoxide-induced activator protein 1 activation and VEGF transcription by targeting the phosphotidylinositol 3-kinase/Akt pathway
Huang, Chuanshu; Li, Jingxia; Song, Lun; Zhang, Dongyun; Tong, Qiangsong; Ding, Min; Bowman, Linda; Aziz, Robeena; Stoner, Gary D
2006 Jan 1;66(1):581-587, Cancer research
Previous studies have shown that freeze-dried black raspberry extract fractions inhibit benzo(a)pyrene [B(a)P]-induced transformation of Syrian hamster embryo cells and benzo(a)pyrene diol-epoxide [B(a)PDE]-induced activator protein-1 (AP-1) activity in mouse epidermal Cl 41 cells. The phosphotidylinositol 3-kinase (PI-3K)/Akt pathway is critical for B(a)PDE-induced AP-1 activation in mouse epidermal Cl 41 cells. In the present study, we determined the potential involvement of PI-3K and its downstream kinases on the inhibition of AP-1 activation by black raspberry fractions, RO-FOO3, RO-FOO4, RO-ME, and RO-DM. In addition, we investigated the effects of these fractions on the expression of the AP-1 target genes, vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). Pretreatment of Cl 41 cells with fractions RO-F003 and RO-ME reduced activation of AP-1 and the expression of VEGF, but not iNOS. In contrast, fractions RO-F004 and RO-DM had no effect on AP-1 activation or the expression of either VEGF or iNOS. Consistent with inhibition of AP-1 activation, the RO-ME fraction markedly inhibited activation of PI-3K, Akt, and p70 S6 kinase (p70(S6k)). In addition, overexpression of the dominant negative PI-3K mutant delta p85 reduced the induction of VEGF by B(a)PDE. It is likely that the inhibitory effects of fractions RO-FOO3 and RO-ME on B(a)PDE-induced AP-1 activation and VEGF expression are mediated by inhibition of the PI-3K/Akt pathway. In view of the important roles of AP-1 and VEGF in tumor development, one mechanism for the chemopreventive activity of black raspberries may be inhibition of the PI-3K/Akt/AP-1/VEGF pathway
— id: 62607, year: 2006, vol: 66, page: 581, stat: Journal Article,

Essential role of ROS-mediated NFAT activation in TNF-alpha induction by crystalline silica exposure
Ke, Qingdong; Li, Jingxia; Ding, Jin; Ding, Min; Wang, Liying; Liu, Bingci; Costa, Max; Huang, Chuanshu
2006 Aug;291(2):L257-L264, American journal of physiology. Lung cellular & molecular physiology
Occupational exposure to crystalline silica has been associated with progressive pulmonary silicosis and lung cancer, but the underlying molecular mechanisms are not well understood. Previous studies have shown that crystalline silica exposure can generate reactive oxygen species (ROS) and induce the expression of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) in cells. TNF-alpha is believed to be critical in the development of silica-related diseases. Thus it will be of significance to understand the mechanisms of TNF-alpha induction by silica exposure. Given the fact that the transcription factor nuclear factor of activated T cells (NFAT) plays an important role in the regulation of TNF-alpha and can also be activated by ROS, in this study we investigated the potential role of ROS in silica-induced NFAT activity as well as TNF-alpha expression in Cl41 cells. The results showed that exposure of cells to silica led to NFAT transactivation and TNF-alpha induction, where superoxide anion radical (O(2)(-).), but not H(2)O(2), was involved. The knockdown of NFAT3 by its specific small interfering RNA significantly attenuated the silica-induced TNF-alpha transcription. This study demonstrated that silica was able to activate NFAT in an O(2)(-).-dependent manner, which was required for TNF-alpha induction
— id: 68743, year: 2006, vol: 291, page: L257, stat: Journal Article,

NFAT3 is required for EGF-induced COX-2 transcription, but neither iNOS transcription nor cell transformation in Cl 41 cells
Li, Jingxia; Lu, Haitian; Huang, Chuanshu
2006 Sep;289(1-2):73-82, Molecular & cellular biochemistry
Epidermal growth factor (EGF) has been reported to act as a tumor promoter in several tissues, such as skin, in association with the induction of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). However, molecular mechanisms involved in these regulations are not well defined. This study addressed a potential role of nuclear factor of activated T cells 3 (NFAT3) in EGF-induced COX-2 and iNOS transcription and cell transformation in mouse epidermal Cl 41 cells. We found that EGF markedly induced anchorage-independent growth (cell transformation) of Cl 41 cells, as well as COX-2 (> 6-fold) and iNOS (> 5-fold) promoter-dependent transcription. The EGF-induced COX-2 transcription was blocked by knockdown of NFAT3 with NFAT3 siRNA, whereas the transcription of iNOS and cell transformation induced by EGF were not affected. Although our recent studies supported that NFAT3 plays an essential role in chemical carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE)-induced cell transformation, the data presented here demonstrated that NFAT3 is required for EGF-induced COX-2 transcription, but neither iNOS transcription nor cell transformation, indicating that the role of NFAT3 in regulating cell transformation is carcinogen-specific
— id: 110429, year: 2006, vol: 289, page: 73, stat: Journal Article,

Knockdown of NFAT3 blocked TPA-induced COX-2 and iNOS expression, and enhanced cell transformation in Cl41 cells
Li, Jingxia; Song, Lun; Zhang, Dongyun; Wei, Lixin; Huang, Chuanshu
2006 Nov 1;99(4):1010-1020, Journal of cellular biochemistry
The nuclear factor of activated-T-cells (NFAT) family is a ubiquitous transcription factor that mediates regulation on various gene expressions. Recent studies indicate that NFAT may implicate in cancer process, mainly through its direct regulation on the cyclooxygenase-2 (COX-2) gene expression. There is also evidence suggesting another aspect of NFAT in tumor suppression. However, the according mechanism remains unknown. In this study, we used a small interfering RNA (siRNA) expression construct to study the role of NFAT3 in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation with the tumor promotion-sensitive mouse epidermal Cl41 cells. Our results showed that TPA was able to induce NFAT3 activation in Cl41 cells. Stable transfection of NFAT3 siRNA specifically reduced endogenous NFAT3 expression. At the same time, TPA-induced expression of both COX-2 and inducible nitric oxide synthase (iNOS) were blocked. However, anchorage-independent transformation in response to TPA was significantly enhanced in NFAT3 siRNA stable transfectants as compared with vector transfectants. Moreover, treatment with the iNOS specific inhibitor aminoguanidine (AG) also enhanced Cl41 cells transformation induced by TPA. As COX-2 expression is proved to be required for cell transformation in Cl41 cells in our recent studies, our results demonstrate that the inducible NFAT3-mediated iNOS upregulation represents a novel potent tumor-suppressing pathway and may contribute to the tumor suppressor functions of NFAT protein
— id: 69573, year: 2006, vol: 99, page: 1010, stat: Journal Article,

Phosphatidylinositol 3-kinase/Akt positively regulates Fas (CD95)-mediated apoptosis in epidermal Cl41 cells
Lu, Bin; Wang, Liying; Stehlik, Christian; Medan, Djordje; Huang, Chuanshu; Hu, Shuiying; Chen, Fei; Shi, Xianglin; Rojanasakul, Yon
2006 Jun 1;176(11):6785-6793, Journal of immunology
Fas (CD95)-mediated apoptosis is an essential mechanism for the maintenance of homeostasis, and disruption of this death pathway contributes to many human diseases. The cell survival protein kinase Akt/protein kinase B (PKB) is a known regulator of apoptosis, but its role in Fas-mediated cell death and its regulatory mechanisms are unclear. In this study, we show that stimulation of the Fas receptor by its ligand (FasL) induces rapid phosphorylation of Akt/PKB and a parallel increase in cell apoptosis in epidermal Cl41 cells. Inhibition of PI3K/Akt by dominant-negative overexpression of PI3K (Deltap85) and Akt (Akt-T308A/S473A) protects the cells from apoptosis, indicating an unexpected proapoptotic role of PI3K/Akt in the Fas signaling process. Treatment of the cells with pharmacological inhibitors of PI3K, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-1 (LY294002), similarly inhibits FasL-induced apoptosis and Akt/PKB phosphorylation, indicating that PI3K is an upstream mediator of Akt/PKB and is involved in Fas-mediated cell death. Electron spin resonance studies show that FasL treatment induces rapid generation of reactive oxygen species, and inhibition of ROS by antioxidants effectively inhibits Akt/PKB signaling, suggesting that FasL activation of Akt/PKB is redox sensitive. In cells transfected with dominant-negative PI3K/Akt, Fas expression is down-regulated, but FLIP expression is unaffected. Reporter gene and mRNA expression assays show that FasL activates fas transcriptional activity and this effect is inhibited by PI3K/Akt suppression. Together, our results indicate that the PI3K/Akt, in addition to its normal prosurvival role, also plays an apoptotic role in Fas-mediated cell death through a mechanism that involves transcriptional activation of Fas receptor
— id: 110430, year: 2006, vol: 176, page: 6785, stat: Journal Article,

Molecular mechanisms involved in chemoprevention of black raspberry extracts: from transcription factors to their target genes
Lu, Haitian; Li, Jingxia; Zhang, Dongyun; Stoner, Gary D; Huang, Chuanshu
2006 ;54(1):69-78, Nutrition & cancer
Berries have attracted attention for their chemopreventive activities in last a few years. Dietary freeze-dried blackberries have been shown to reduce esophagus and colon cancer development induced by chemical carcinogen in rodents. To elucidate molecular mechanisms involved in chemoprevention by berry extracts, we employed mouse epidermal Cl 41 cell line, a well-characterized in vitro model in tumor promotion studies. Pretreatment of Cl 41 cells with methanol-extracted blackberry fraction RO-ME resulted in a dramatical inhibition of B(a)PDE-induced activation of AP-1 and NFkB, and expression of VEGF and COX-2. The inhibitory effects of RO-ME on B(a)PDE-induced activation of AP-1 and NFkappaB appear to be mediated via inhibition of MAPKs and IkappaBalpha phosphorylation, respectively. In view of the important roles of AP-1, NFkappaB, VEGF and COX-2 in tumor promotion/progression, and VEGF and COX-2 are target of AP-1 and NFkappaB, we anticipate that the ability of black raspberries to inhibit tumor development may be mediated by impairing signal transduction pathways leading to activation of AP-1 and NFkappaB, subsequently resulting in down-regulation of VEGF and COX-2 expression. The RO-ME fraction appears to be the major fraction responsible for the inhibitory activity of black raspberries
— id: 69241, year: 2006, vol: 54, page: 69, stat: Journal Article,

Inflammation, a key event in cancer development
Lu, Haitian; Ouyang, Weiming; Huang, Chuanshu
2006 Apr;4(4):221-233, Molecular cancer research
Several recent studies have identified nuclear factor-kappaB as a key modulator in driving inflammation to cancers. Besides this transcription factor, essential in regulating inflammation and cancer development, an inflammatory microenvironment inhabiting various inflammatory cells and a network of signaling molecules are also indispensable for the malignant progression of transformed cells, which is attributed to the mutagenic predisposition of persistent infection-fighting agents at sites of chronic inflammation. As a subverted host response to inflammation-induced tumors, the inflammatory cells and regulators may facilitate angiogenesis and promote the growth, invasion, and metastasis of tumor cells. Thus far, research regarding inflammation-associated cancer development has focused on cytokines and chemokines as well as their downstream targets in linking inflammation and cancer. Moreover, other proteins with extensive roles in inflammation and cancer, such as signal transducers and activators of transcription, Nrf2, and nuclear factor of activated T cells, are also proposed to be promising targets for future studies. The elucidation of their specific effects and interactions will accelerate the development of novel therapeutic interventions against cancer development triggered by inflammation
— id: 64167, year: 2006, vol: 4, page: 221, stat: Journal Article,

Essential roles of PI-3K/Akt/IKKbeta/NFkappaB pathway in cyclin D1 induction by arsenite in JB6 Cl41 cells
Ouyang, Weiming; Li, Jingxia; Ma, Qian; Huang, Chuanshu
2006 Apr;27(4):864-873, Carcinogenesis
Skin is a major target of carcinogenic trivalent arsenic (arsenite, As3+). It has been thought that cell proliferation is one of the central events involved in the carcinogenic effect of arsenite. Cyclin D1, a nuclear protein playing a pivotal role in cell proliferation and cell cycle transition from G1 to S phases, has been reported to be induced in human fibroblast by arsenite via uncertain molecular mechanisms. In the present study, the potential roles of PI-3K/Akt/IKKbeta/NFkappaB signal pathway in cyclin D1 induction by arsenite were addressed in mouse epidermal Cl41 cells. We found that exposure of Cl41 cells to arsenite was able to induce cell proliferation, activate PI-3K-->Akt/p70(S6k) signal pathway and increase cyclin D1 expression at both transcription and protein levels. Pre-treatment of Cl41 cells with PI-3K inhibitor, wortmannin, significantly inhibited the phosphorylation of Akt and p70(S6k) and thereby dramatically impaired the cyclin D1 induction by arsenite, implicating the importance of the PI-3K signal pathway in the cyclin D1 induction by arsenite. Furthermore, inhibition of PI-3K/Akt by overexpression of Deltap85 or DN-Akt blocked arsenite-induced IKK phosphorylation, IkappaBalpha degradation and cyclin D1 expression, indicating that IKK/NFkappaB is the downstream transducer of arsenite-triggered PI-3K/Akt cascade. Moreover, inhibition of IKKbeta/NFkappaB signal pathway by overexpression of its dominant negative mutant, IKKbeta-KM, also significantly blocked arsenite-induced cyclin D1 expression. Overall, arsenite exposure triggered PI-3K/Akt/IKKbeta/NFkappaB signal cascade which in turn plays essential roles in inducing cyclin D1 expression
— id: 64132, year: 2006, vol: 27, page: 864, stat: Journal Article,

Overexpression of cyclin D1-CDK4 in silica-induced transformed cells is due to activation of ERKs, JNKs/AP-1 pathway
Shen, Fuhai; Fan, Xueyun; Liu, Bingci; Jia, Xiaowei; Du, Hongju; You, Baorong; Ye, Meng; Huang, Chuanshu; Shi, Xianglin
2006 Jan 25;160(3):185-195, Toxicology letters
Silica has been known to be a factor inducing acute injury and chronic pulmonary fibrosis. Silica has also been listed as a human carcinogen in 1996 by International Agency for Research on Cancer (IARC). However, the molecular mechanisms involved its pathologic effects are not well understood. In these studies, we found that exposure of human embryonic lung fibroblasts (HELF) to crystalline silica could cause increases in activation of extracellular signal-regulated kinases (ERKs), p38K, and c-Jun NH2-terminal amino kinases (JNKs), and HELF transformation. Interestingly, silica-induced transformation of HELF (S-HELF) led to increases in activated levels of ERKs and p46 of JNKs, and decrease in p38K activation, and no effect on activation of p54 of JNKs, as compared with those in parental HELF. Further studies showed that there are differential effects of ERKs, JNKs and p38K, as well as their downstream transcription factor AP-1, in regulation of expression of cyclin D1 and CDK4 and cell cycle alternations induced by silica. Cyclin D1 and CDK4 were increased in S-HELF as compared with those in HELF. Inhibition of ERKs activation by AG126, JNK by SP600125, and AP-1 by curcumin could reduced the induction of cyclin D1 and CDK4. There is no significant difference for cell cycle distribution between groups. These results demonstrate that ERKs and JNKs, but not p38K is responsible for induction of cyclin D1 and CDK4 in S-HELF, suggesting that overexpression of cyclin D1 and CDK4 caused by silica is mediated by ERK, JNK/AP-1signaling pathway
— id: 110435, year: 2006, vol: 160, page: 185, stat: Journal Article,

IKKbeta programs to turn on the GADD45alpha-MKK4-JNK apoptotic cascade specifically via p50 NF-kappaB in arsenite response
Song, Lun; Li, Jingxia; Zhang, Dongyun; Liu, Zheng-Gang; Ye, Jianping; Zhan, Qimin; Shen, Han-Ming; Whiteman, Matt; Huang, Chuanshu
2006 Nov 20;175(4):607-617, Journal of cell biology
Cross talk between NF-kappaB and c-Jun N-terminal kinases (JNKs) has been implicated in the cell life and death decision under various stresses. Functional suppression of JNK activation by NF-kappaB has recently been proposed as a key cellular survival mechanism and contributes to cancer cells escaping from apoptosis. We provide a novel scenario of the proapoptotic role of IkappaB kinase beta (IKKbeta)-NF-kappaB, which can act as the activator of the JNK pathway through the induction of GADD45alpha for triggering MKK4/JNK activation, in response to the stimulation of arsenite, a cancer therapeutic reagent. This effect of IKKbeta-NF-kappaB is dependent on p50 but not the p65/relA NF-kappaB subunit, which can increase the stability of GADD45alpha protein through suppressing its ubiquitination and proteasome-dependent degradation. IKKbeta-NF-kappaB can therefore either activate or suppress the JNK cascade and consequently mediate pro- or antiapoptotic effects, depending on the manner of its induction. Furthermore, the NF-kappaB p50 subunit can exert a novel regulatory function on protein modification independent of the classical NF-kappaB transcriptional activity
— id: 70092, year: 2006, vol: 175, page: 607, stat: Journal Article,

Hypoxia-induced mitogenic factor enhances angiogenesis by promoting proliferation and migration of endothelial cells
Tong, Qiangsong; Zheng, Liduan; Li, Bo; Wang, Danming; Huang, Chuanshu; Matuschak, George M; Li, Dechun
2006 Nov 1;312(18):3559-3569, Experimental cell research
Our previous studies have indicated that hypoxia-induced mitogenic factor (HIMF) has angiogenic properties in an in vivo matrigel plug model and HIMF upregulates expression of vascular endothelial growth factor (VEGF) in mouse lungs and cultured lung epithelial cells. However, whether HIMF exerts angiogenic effects through modulating endothelial cell function remains unknown. In this study, mouse aortic rings cultured with recombinant HIMF protein resulted in enhanced vascular sprouting and increased endothelial cell spreading as confirmed by Dil-Ac-LDL uptake, von Willebrand factor and CD31 staining. In cultured mouse endothelial cell line SVEC 4-10, HIMF dose-dependently enhanced cell proliferation, in vitro migration and tubulogenesis, which was not attenuated by SU1498, a VEGFR2/Flk-1 receptor tyrosine kinase inhibitor. Moreover, HIMF stimulation resulted in phosphorylation of Akt, p38 and ERK1/2 kinases in SVEC 4-10 cells. Treatment of mouse aortic rings and SVEC 4-10 cells with LY294002, but not SB203580, PD098059 or U0126, abolished HIMF-induced vascular sprouting and angiogenic responses. In addition, transfection of a dominant-negative mutant of phosphatidylinositol 3-kinase (PI-3K), Deltap85, blocked HIMF-induced phosphorylation of Akt, endothelial activation and tubulogenesis. These results indicate that HIMF enhances angiogenesis by promoting proliferation and migration of endothelial cells via activation of the PI-3K/Akt pathways
— id: 110427, year: 2006, vol: 312, page: 3559, stat: Journal Article,

VEGF is upregulated by hypoxia-induced mitogenic factor via the PI-3K/Akt-NF-kappaB signaling pathway
Tong, Qiangsong; Zheng, Liduan; Lin, Li; Li, Bo; Wang, Danming; Huang, Chuanshu; Li, Dechun
2006 ;7:37-37, Respiratory research
BACKGROUND: Hypoxia-induced mitogenic factor (HIMF) is developmentally regulated and plays an important role in lung pathogenesis. We initially found that HIMF promotes vascular tubule formation in a matrigel plug model. In this study, we investigated the mechanisms which HIMF enhances expression of vascular endothelial growth factor (VEGF) in lung tissues and epithelial cells. METHODS: Recombinant HIMF protein was intratracheally instilled into adult mouse lungs, VEGF expression was examined by immunohistochemical staining and Western blot. The promoter-luciferase reporter assay, RT-PCR, and Western blot were performed to examine the effects of HIMF on VEGF expression in mouse lung epithelial cell line MLE-12. The activation of NF-kappa B (NF-kappaB) and phosphorylation of Akt, IKK and IkappaBalpha were examined by luciferase assay and Western blot, respectively. RESULTS: Intratracheal instillation of HIMF protein resulted in significant increase of VEGF, mainly localized to airway epithelial and alveolar type II cells. Deletion of NF-kappaB binding sites within VEGF promoter abolished HIMF-induced VEGF expression in MLE-12 cells, suggesting that activation of NF-kappaB is essential for VEGF upregulation induced by HIMF. Stimulation of lung epithelial cells by HIMF resulted in phosphorylation of IKK and IkappaBalpha, leading to activation of NF-kappaB. In addition, HIMF strongly induced Akt phosphorylation, and suppression of Akt activation by specific inhibitors and dominant negative mutants for PI-3K, and IKK or IkappaBalpha blocked HIMF-induced NF-kappaB activation and attenuated HIMF-induced VEGF production. CONCLUSION: These results suggest that HIMF enhances VEGF production in mouse lung epithelial cells in a PI-3K/Akt-NF-kappaB signaling pathway-dependent manner, and may play critical roles in pulmonary angiogenesis
— id: 110433, year: 2006, vol: 7, page: 37, stat: Journal Article,

Participation of the PI-3K/Akt-NF-kappaB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells
Tong, Qiangsong; Zheng, Liduan; Lin, Li; Li, Bo; Wang, Danming; Huang, Chuanshu; Matuschak, George M; Li, Dechun
2006 ;7:101-101, Respiratory research
BACKGROUND: Hypoxia-induced mitogenic factor (HIMF), a lung-specific growth factor, promotes vascular tubule formation in a matrigel plug model. We initially found that HIMF enhances vascular endothelial growth factor (VEGF) expression in lung epithelial cells. In present work, we tested whether HIMF modulates expression of fetal liver kinase-1 (Flk-1) in endothelial cells, and dissected the possible signaling pathways that link HIMF to Flk-1 upregulation. METHODS: Recombinant HIMF protein was intratracheally instilled into adult mouse lungs, Flk-1 expression was examined by immunohistochemistry and Western blot. The promoter-luciferase reporter assay and real-time RT-PCR were performed to examine the effects of HIMF on Flk-1 expression in mouse endothelial cell line SVEC 4-10. The activation of NF-kappa B (NF-kappaB) and phosphorylation of Akt, IKK, and IkappaBalpha were examined by luciferase assay and Western blot, respectively. RESULTS: Intratracheal instillation of HIMF protein resulted in a significant increase of Flk-1 production in lung tissues. Stimulation of SVEC 4-10 cells by HIMF resulted in increased phosphorylation of IKK and IkappaBalpha, leading to activation of NF-kappaB. Blocking NF-kappaB signaling pathway by dominant-negative mutants of IKK and IkappaBalpha suppressed HIMF-induced Flk-1 upregulation. Mutation or deletion of NF-kappaB binding site within Flk-1 promoter also abolished HIMF-induced Flk-1 expression in SVEC 4-10 cells. Furthermore, HIMF strongly induced phosphorylation of Akt. A dominant-negative mutant of PI-3K, Deltap85, as well as PI-3K inhibitor LY294002, blocked HIMF-induced NF-kappaB activation and attenuated Flk-1 production. CONCLUSION: These results suggest that HIMF upregulates Flk-1 expression in endothelial cells in a PI-3K/Akt-NF-kappaB signaling pathway-dependent manner, and may play critical roles in pulmonary angiogenesis
— id: 68805, year: 2006, vol: 7, page: 101, stat: Journal Article,

NFAT3 is specifically required for TNF-alpha-induced cyclooxygenase-2 (COX-2) expression and transformation of Cl41 cells
Yan, Yan; Li, Jingxia; Ouyang, Weiming; Ma, Qian; Hu, Yu; Zhang, Dongyun; Ding, Jin; Qu, Qingshan; Subbaramaiah, Kotha; Huang, Chuanshu
2006 Jul 15;119(Pt 14):2985-2994, Journal of cell science
NFAT family is recognized as a transcription factor for inflammation regulation by inducing the expression of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), the key mediator of inflammation, which was reported to induce cell transformation in mouse epidermal Cl41 cells. In this study, we demonstrated that TNF-alpha was able to induce NFAT activation, as well as the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). The induction of COX-2 by TNF-alpha was abolished by knockdown of NFAT3 with its siRNA, while the induction of iNOS was not effected. Moreover, TNF-alpha-induced anchorage-independent cell growth was significantly inhibited by NFAT3 siRNA and cyclosporine A, a chemical inhibitor for the calcineurin/NFAT pathway, which suggests the importance of NFAT3 in regulating TNF-alpha-induced anchorage-independent cell growth. Consequently, impairment of COX-2 by its siRNA or selective inhibitor also inhibited TNF-alpha-induced anchorage-independent cell growth. Taken together, our results indicate that NFAT3 plays an important role in the regulation of TNF-alpha-induced anchorage-independent cell growth, at least partially, by inducing COX-2 expression in Cl41 cells. These findings suggest that NFAT3/cyclooxygenase-2 act as a link between inflammation and carcinogenesis by being involved in the tumor promotion stage
— id: 110428, year: 2006, vol: 119, page: 2985, stat: Journal Article,

Essential roles of ERKs and p38K in up-regulation of GST A1 expression by Maotai content in human hepatoma cell line Hep3B
Zhang, Dongyun; Lu, Haitian; Li, Jingxia; Shi, Xianglin; Huang, Chuanshu
2006 Dec;293(1-2):161-171, Molecular & cellular biochemistry
It is widely accepted that the consumption of alcohol may lead to hepatic injuries such as hepatic fibrosis and cirrhosis. However, consumption of Maotai, one of the famous liquors in China, is found to have no obvious relevance with hepatic injury as ordinary white wine does in both epidemiological and histopathological studies. Present study used human hepatoma cell line Hep3B to address the mechanisms involved in the resistance of alcohol-induced hepatic injury by Maotai liquor. We found that exposure of Hep3B cells to Maotai residue without ethanol (MRWE) resulted in the increased GST A1 anti-oxidant responsive element (ARE) transcriptional expression, while MRWE treatment did not affect Nrf-2-dependent transcriptional activity. Those findings were further confirmed at all time points and doses tested, suggesting that GST A1 transcription was regulated by MRWE via an Nrf-2-independent pathway. Consistent with GST A1 induction, the phosphorylation of c-Jun, extracellular signal-regulated kinases (ERKs) and p38 kinase (p38 K), were also observed in MRWE-treated Hep3B cells. Furthermore, pretreatment of cells with either PD98059 (an inhibitor specific for MEK1/2-ERKs pathway) or SB202190 (an inhibitor specific for p38 K) led to a significant decrease in the induction of GST A1 transcriptional expression by MRWE treatment. Our results indicate that certain content in MRWE is able to induce GST A1 ARE transcriptional expression, which may provide protective effects for hepatic cells by antagonizing the oxidative stress derived from ethanol via an ERKs- and p38 K-dependent pathway
— id: 71140, year: 2006, vol: 293, page: 161, stat: Journal Article,

Coordination of JNK1 and JNK2 is critical for GADD45alpha induction and its mediated cell apoptosis in arsenite responses
Zhang, Dongyun; Song, Lun; Li, Jingxia; Wu, Kangjian; Huang, Chuanshu
2006 Nov 10;281(45):34113-34123, Journal of biological chemistry
Arsenite is a well documented environmental pathogen, whereas it has also been applied as medication to treat various neoplasmas. The pathogenic and therapeutic effects of arsenite are associated with cellular apoptotic responses. However, the molecular mechanisms of arsenite-induced apoptosis are not very well understood. Our previous study has shown that arsenite exposure is able to activate JNKs, which subsequently mediate the apoptotic outcome. The present study further revealed that the coordination of JNK1 and JNK2 was critical for the arsenite-induced expression of GADD45alpha (growth arrest and DNA damage 45alpha), which in turn mediated the cellular apoptosis. The arsenite-induced apoptosis and GADD45alpha expression were significantly impaired in mouse embryonic fibroblasts deficient in either jnk1 (JNK1-/-) or jnk2 (JNK2-/-). Knockdown of GADD45alpha by its specific small interfering RNA also dramatically reduced the apoptotic responses, and overexpression of GADD45alpha in either JNK1-/- or JNK2-/- mouse embryonic fibroblasts partially resensitized the cell death. Furthermore, it was found that the regulation of GADD45alpha by JNK1 and JNK2 was achieved through mediating the activation of c-Jun, since in the JNK1-/- and JNK2-/- cells the c-Jun activation was impaired, and overexpression of the dominant negative mutant of c-Jun (TAM67) in wild type cells could also block GADD45alpha induction as well as cellular apoptosis. Our results demonstrate that the coordination of JNK1 and JNK2 is critical for c-Jun/GADD45alpha-mediated cellular apoptosis induced by arsenite
— id: 69592, year: 2006, vol: 281, page: 34113, stat: Journal Article,

Ionizing radiation synergistic induction of cyclooxygenase-2 with benzo[a]pyrene diol-epoxide through nuclear factor of activated T cells in mouse epidermal Cl 41 cells
Zhang, Ronghe; Li, Jingxia; Burns, Fredric J; Huang, Chuanshu
2006 Mar;15(3):721-727, Oncology reports
Carcinogenic effects of ionizing radiation and benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), a major metabolite of benzo[a]pyrene (B[a]P), have been well demonstrated both in vitro and in vivo. Two-stage carcinogenesis results indicate that mouse skin is highly susceptible to both ionizing radiation and benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), a major metabolite of benzo[a]pyrene (B[a]P). It is believed that signaling pathways leading to the regulation of gene expression play a significant role in the development of skin cancers. The NFAT family of proteins are important transcription factors involved in the regulation of various target genes, such as IL-1 and TNF-alpha, which play key roles in the regulation of inflammation and carcinogenesis. Thus, the effect of ionizing radiation and B[a]PDE on COX-2 induction and NFAT3 activation, and their relationship, was investigated in mouse epidermal Cl 41 cells. We found that B[a]PDE exposure induced a very high level of NFAT activation in mouse epidermal Cl 41 cells. Ionizing radiation exhibited a synergistic effect with B[a]PDE on NFAT activation and COX-2 induction, while ionizing radiation alone had no effect. By stably knocking down NFAT3 protein expression by means of the specific interfering RNA (siRNA) technique, we found that COX-2 induction by B[a]PDE and the synergistic effect of ionizing radiation with B[a]PDE was totally blocked. These results indicate that ionizing radiation acts synergistically with B[a]PDE on COX-2 induction, and the synergism is dependent on the NFAT3 pathway
— id: 63739, year: 2006, vol: 15, page: 721, stat: Journal Article,

Differential requirement of signal pathways for benzo[a]pyrene (B[a]P)-induced nitric oxide synthase (iNOS) in rat esophageal epithelial cells
Chen, Jingyuan; Yan, Yan; Li, Jingxia; Ma, Qian; Stoner, Gary D; Ye, Jianping; Huang, Chuanshu
2005 Jun;26(6):1035-1043, Carcinogenesis
Overexpression of inducible nitric oxide synthase (iNOS) has been reported in several human cancers, including esophageal squamous cell carcinoma (SCC). Benzo[a]pyrene (B[a]P), a polycyclic hydrocarbon carcinogen found in tobacco smoke and in the environment, induces cancer in multiple organ sites in animals and may be a causative agent for certain human cancers, such as esophageal cancer. In the present study, the effects of B[a]P on the induction of iNOS and the signaling pathways that lead to the induction were investigated in cultured rat esophageal epithelial (RE-149) cells. Treatment of RE-149 cells with B[a]P led to a marked increase in the expression of iNOS. The induction of iNOS by B[a]P was found to occur through an extracellular signal-regulated protein kinases (ERKs)-dependent pathway, since inhibition of ERKs by either pretreatment of RE-149 cells with PD98059, an inhibitor of ERKs upstream kinase MEK1/2, or overexpression of DN-ERK2, blocked the induction of iNOS by B[a]P. Furthermore, impairing nuclear factor-kappaB (NFkappaB) activation by either NEMO-BDBP, an NFkappaB specific inhibitor, or overexpression of DN-IkappaBalpha or IKK-KM markedly inhibited the expression of B[a]P-induced iNOS, suggesting that the NFkappaB pathway is also required for the induction of iNOS by B[a]P. In addition, treatment of RE-149 cells with either SB202190, a p38 kinase inhibitor, or c-JunN-terminal kinase inhibitor II, resulted in an increased induction of iNOS. Pretreatment of RE-149 cells with wortmannin, a PI-3K inhibitor, or with rapamycin, an mTOR/p70S6K pathway inhibitor, had no effect on the expression of iNOS. These results suggest that B[a]P initiates the signaling pathways leading to the induction of iNOS in cultured rat esophageal epithelial cells. In view of the potential role of iNOS in the development of esophageal SCC in humans, we speculate that the induction of iNOS by B[a]P may be one mechanism by which B[a]P could produce carcinogenic effects in the human esophagus
— id: 56052, year: 2005, vol: 26, page: 1035, stat: Journal Article,

Nickel carcinogenesis: epigenetics and hypoxia signaling
Costa, Max; Davidson, Todd L; Chen, Haobin; Ke, Qingdong; Zhang, Ping; Yan, Yan; Huang, Chuanshu; Kluz, Thomas
2005 Dec 30;592(1-2):79-88, Mutation research
Both water soluble and insoluble nickel compounds have been implicated in the etiology of human lung and nasal cancers. Water insoluble nickel compounds have been shown to enter cells by phagocytosis and are contained in cytoplasmic vacuoles, which are acidified thus accelerating the dissolution of soluble nickel from the particles. Using Newport Green, a dye that fluoresces when ionic nickel is bound, we have shown that following exposure (48-72 h) of human lung (A549) cells to NiS particles, most of the nickel is contained in the nucleus, while cells exposed to soluble NiCl2 exhibit most of the ions localized in the cytoplasm. This effect is consistent with previously published reports showing that short-term exposure of cells to crystalline nickel particles (1-3 days) is able to epigenetically silence target genes placed near heterochromatin, while similar short-term exposure to soluble nickel compounds are not able to induce silencing of genes placed near heterochromatin. However, a 3 week exposure of cells to soluble NiCl2 is also able to induce gene silencing. A similar effect was found in yeast cells where nickel was able to silence the URA-3 gene placed near (1.3 kb) a telomere silencing element, but not when the gene was placed farther away from the silencing element (2.0 kb). In addition to epigenetic effects, nickel compounds activate hypoxia signaling pathways. The mechanism of this effect involves the ability of either soluble or insoluble nickel compounds to block iron uptake leading to cellular iron depletion, directly affect iron containing enzymes, or both. This results in the inhibition of a variety of iron-dependent enzymes, such as aconitase and the HIF proline hydroxylases (PHD1-3). The inhibition of the HIF proline hydroxylases stabilizes the HIF protein and activates hypoxic signaling. Additional studies have shown that nickel and hypoxia decrease histone acetylation and increase the methylation of H3 lysine 9. These events are involved in gene silencing and hypoxia can also cause these effects in human cells. It is hypothesised that the state of hypoxia either by low oxygen tension or as a result of agents that signal hypoxia under normal oxygen tension (iron chelation, nickel and cobalt) results in low levels of acetyl CoA, which is a substrate for histone and other protein acetylation. This effect may in part be responsible for the gene silencing following nickel exposure and during hypoxia
— id: 67383, year: 2005, vol: 592, page: 79, stat: Journal Article,

ERKs activation and calcium signaling are both required for VEGF induction by vanadium in mouse epidermal Cl41 cells
Li, Jingxia; Tong, Qiangsong; Shi, Xianglin; Costa, Max; Huang, Chuanshu
2005 Nov;279(1-2):25-33, Molecular & cellular biochemistry
The previous studies have demonstrated that vanadium exposure can cause a variety of biological effects. However, the mechanisms involved in the biological effects caused by vanadium are not well understood. Our previous studies have shown that exposure of mouse epidermal Cl 41 cells to vanadate stimulated the phosphorylation of both ERKs and p38K, and calcium signaling leading NFAT activation. In view of the evidence that ERKs and p38 kinase contribute to VEGF induction, we investigated in the present study the potential roles of ERKs, p38K, and calcium signaling in VEGF induction caused by vanadium exposure. Exposure of Cl 41 cells to vanadium led to VEGF induction in both time- and dose-dependent manners. Pre-treatment of Cl 41 cells with PD98059, an inhibitor of MEK1/2-ERKs pathway, but not SB202190, an inhibitor for p38K pathway, resulted in a dramatic inhibition of VEGF induction by vanadium. More interesting, pre-treatment of Cl 41 cells with intracellular calcium chelator, but not calcium channel blocker, resulted in a dramatic decrease in VEGF induction by vanadium. However, both PI-3K inhibitors and overexpression of Deltap85, a dominant negative PI-3K mutant, resulted in only a marginal decrease in VEGF induction by vanadium. Moreover, mTOR, as a downstream molecule of PI-3K, did not attribute to VEGF induction by vanadium because rapamycin pre-treatment did not show any inhibitory effect on VEGF induction. These results indicate that ERKs and intracellular stored calcium release play a critical role in VEGF induction by vanadium. PI-3K is partially involved in VEGF induction by vanadium, while p38K and mTOR are not involved. Those results will help us to understand the molecular mechanisms involved in vanadium-induced biological effects
— id: 70870, year: 2005, vol: 279, page: 25, stat: Journal Article,

Carcinogenic effect of nickel compounds
Lu, Haitian; Shi, Xianglin; Costa, Max; Huang, Chuanshu
2005 Nov;279(1-2):45-67, Molecular & cellular biochemistry
Nickel is a widely distributed metal that is industrially applied in many forms. Accumulated epidemiological evidence confirms that exposures to nickel compounds are associated with increased nasal and lung cancer incidence, both in mostly occupational exposures. Although the molecular mechanisms by which nickel compounds cause cancer are still under intense investigation, the carcinogenic actions of nickel compounds are thought to involve oxidative stress, genomic DNA damage, epigenetic effects, and the regulation of gene expression by activation of certain transcription factors related to corresponding signal transduction pathways. The present review summarizes our current knowledge on the molecular mechanisms of nickel carcinogenesis, with special emphasis on the role of nickel induced reactive oxygen species (ROS) and signal transduction pathways
— id: 70872, year: 2005, vol: 279, page: 45, stat: Journal Article,

The role of epidermal growth factor receptor in ethanol-mediated inhibition of activator protein-1 transactivation
Ma, Cuiling; Bower, Kimberly A; Lin, Hong; Chen, Gang; Huang, Chuanshu; Shi, Xianglin; Luo, Jia
2005 Jun 15;69(12):1785-1794, Biochemical pharmacology
A potential mechanism underlying ethanol-induced alterations in gene expression is the disruption of transcription factor activity. Growth factor receptors, particularly receptor tyrosine kinases, play an important role in modulating many biological effects of ethanol. We demonstrated here that the expression of epidermal growth factor receptor (EGFR) mediated the effect of ethanol on the activity of transcription factor activator protein-1 (AP-1). Ethanol had little effect on AP-1 activity in the fibroblast cells devoid of EGFR (B82); however, it significantly suppressed AP-1 activity in B82 cells that were stably transfected with either a wild-type EGFR (B82L) or a kinase-deficient receptor (B82M721) in a concentration-dependent manner. EGF activated AP-1 only in B82L cells; the activation was mediated primarily by Akt and ERK. Ethanol inhibited EGF-induced EGFR autophosphorylation, phosphorylation of ERK as well as Akt and its substrate GSK-3beta, and subsequently blocked EGF-stimulated AP-1 activation in B82L cells. On the other hand, ethanol had little effect on EGF-stimulated JNK activation. Phorbol ester 12-O-teradecanoyl-phorbol-13-acetate (TPA) activated AP-1 in B82L and B82M721 cells, but not B82 cells. TPA-induced activation of ERK and PKCdelta was dependent on the expression of EGFR although the intrinsic kinase activity of EGFR was not required. In contrast, TPA-induced phosphorylation of p38 MAPK, JNKs and other PKC isoforms was independent of EGFR. Ethanol selectively inhibited TPA-induced phosphorylation of ERK and PKCdelta, and modestly suppressed TPA-stimulated AP-1 activation in B82L and B82M721 cells. Thus, EGFR plays a critical role in the interaction between ethanol and AP-1
— id: 110436, year: 2005, vol: 69, page: 1785, stat: Journal Article,

Essential role of PI-3K, ERKs and calcium signal pathways in nickel-induced VEGF expression
Ouyang, Weiming; Li, Jingxia; Shi, Xianglin; Costa, Max; Huang, Chuanshu
2005 Nov;279(1-2):35-43, Molecular & cellular biochemistry
Exposure to a highly nickel-polluted environment has the potential to cause a variety of adverse health effects, such as the respiratory tract cancers. Since numerous studies have demonstrated that nickel generally has weak mutagenic activity, research focus had turned to cell signalling activation leading to gene modulation and epigenetic changes as a plausible mechanism of carcinogenesis. Previous studies have revealed that nickel compounds can induce the expression of vascular endothelial growth factor (VEGF), which is a key mediator of angiogenesis both in physiological and pathologic conditions. In the present study, we investigated the potential roles of PI-3K, ERKs, p38 kinase and calcium signalling in VEGF induction by nickel in Cl 41 cells. Exposure of Cl 41 cells to nickel compounds led to VEGF induction in both time- and dose-dependent manners. Pre-treatment of Cl 41 cells with PI-3K inhibitor, wortmannin or Ly294002, resulted in a striking inhibition of VEGF induction by nickel compounds, implicating the role of PI-3K in the induction. However, mTOR, one of downstream molecules of PI-3K, may not contribute to the induction because pre-treatment of Cl 41 cells with its inhibitor, rapamycin, did not show obvious decrease in nickel-induced VEGF expression. Furthermore, pre-treatment of Cl 41 cells with MEK1/2-ERKs pathway inhibitor, PD98059, significantly inhibited VEGF induction by both NiCl2 and Ni3S2, whereas p38 kinase inhibitor, SB202190, did not impair the induction. Pre-treatment of Cl 41 cells with intracellular calcium chelator, but not calcium channel blocker, inhibited VEGF induction by nickel. Collectively these data demonstrate that PI-3K, ERKs and cytosolic calcium, but not p38 kinase, play essential roles in VEGF induction by nickel compounds
— id: 70871, year: 2005, vol: 279, page: 35, stat: Journal Article,

Cyclin D1 induction through IkappaB kinase beta/nuclear factor-kappaB pathway is responsible for arsenite-induced increased cell cycle G1-S phase transition in human keratinocytes
Ouyang, Weiming; Ma, Qian; Li, Jingxia; Zhang, Dongyun; Liu, Zheng-Gang; Rustgi, Anil K; Huang, Chuanshu
2005 Oct 15;65(20):9287-9293, Cancer research
Environmental and occupational exposure to arsenite is associated with an increased risk of human cancers, including skin, urinary bladder, and respiratory tract cancers. Although much evidence suggests that alterations in cell cycle machinery are implicated in the carcinogenic effect of arsenite, the molecular mechanisms underlying the cell cycle alterations are largely unknown. In the present study, we observed that exposure of human keratinocyte HaCat cells to arsenite resulted in the promotion of cell cycle progression, especially G(1)-S transition. Further studies found that arsenite exposure was able to induce cyclin D1 expression. The induction of cyclin D1 by arsenite required nuclear factor-kappaB (NF-kappaB) activation, because the inhibition of IkappaB phosphorylation by overexpression of the dominant-negative mutant, IKKbeta-KM, impaired arsenite-induced cyclin D1 expression and G1-S transition. The requirement of IkappaB kinase beta (IKKbeta) for cyclin D1 induction was further confirmed by the findings that arsenite-induced cyclin D1 expression was totally blocked in IKKbeta knockout (IKKbeta(-/-)) mouse embryo fibroblasts. In addition, knockdown of cyclin D1 expression using cyclin D1-specific small interference RNA significantly blocked arsenite-induced cell cycle progression in HaCat cells. Taken together, our results show that arsenite-induced cell cycle from G(1) to S phase transition is through IKKbeta/NF-kappaB/cyclin D1-dependent pathway
— id: 61269, year: 2005, vol: 65, page: 9287, stat: Journal Article,

Loss of tumor suppressor p53 decreases PTEN expression and enhances signaling pathways leading to activation of activator protein 1 and nuclear factor kappaB induced by UV radiation
Wang, Jian; Ouyang, Weiming; Li, Jingxia; Wei, Lixin; Ma, Qian; Zhang, Zhuo; Tong, Qiangsong; He, Jie; Huang, Chuanshu
2005 Aug 1;65(15):6601-6611, Cancer research
Transcription factor p53 and phosphatase PTEN are two tumor suppressors that play essential roles in suppression of carcinogenesis. However, the mechanisms by which p53 mediates anticancer activity and the relationship between p53 and PTEN are not well understood. In the present study, we found that pretreatment of mouse epidermal Cl41 cells with pifithrin-alpha, an inhibitor for p53-dependent transcriptional activation, resulted in a marked increase in UV-induced activation of activator protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB). Consistent with activation of AP-1 and NF-kappaB, pifithrin-alpha was also able to enhance the UV-induced phosphorylation of c-Jun-NH2-kinases (JNK) and p38 kinase, whereas it did not show any effect on phosphorylation of extracellular signal-regulated kinases. Furthermore, the UV-induced signal activation, including phosphorylation of JNK, p38 kinase, Akt, and p70S6K, was significantly enhanced in p53-deficient cells (p53-/-), which can be reversed by p53 reconstitution. In addition, knockdown of p53 expression by its small interfering RNA also caused the elevation of AP-1 activation and Akt phosphorylation induced by UV radiation. These results show that p53 has a suppressive activity on the cell signaling pathways leading to activation of AP-1 and NF-kappaB in cell response to UV radiation. More importantly, deficiency of p53 expression resulted in a decrease in PTEN protein expression, suggesting that p53 plays a critical role in the regulation of PTEN expression. In addition, overexpression of wild-type PTEN resulted in inhibition of UV-induced AP-1 activity. Because PTEN is a well-known phosphatase involved in the regulation of phosphatidylinositol 3-kinase (PI-3K)/Akt signaling pathway, taken together with the evidence that PI-3K/Akt plays an important role in the activation of AP-1 and NF-kappaB during tumor development, we anticipate that inhibition of AP-1 and NF-kappaB by tumor suppressor p53 seems to be mediated via PTEN, which may be a novel mechanism involved in anticancer activity of p53 protein
— id: 62323, year: 2005, vol: 65, page: 6601, stat: Journal Article,

Iron-induced interleukin-6 gene expression: possible mediation through the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways
Dai, Jisen; Huang, Chuanshu; Wu, Jing; Yang, Chengfeng; Frenkel, Krystyna; Huang, Xi
2004 Oct 15;203(1-3):199-209, Toxicology
Increased iron store in the body may increase the risk of many diseases such as cancer and inflammation. However, the precise pathogenic mechanism of iron has not yet been elucidated. In the present study, the early biological responses of cells to iron treatment were investigated in AP-1 luciferase reporter stably transfected mouse epidermal JB6 cells and primary rat hepatocytes. It was shown that water-soluble iron compounds, such as FeSO4 and Fe2(SO4)3, were more active in inducing AP-1 in JB6 cells than water-insoluble iron compounds, such as Fe2O3 and FeS. Iron stimulated mitogen-activated protein kinase (MAPK) family members of extracellular signal-regulated kinases (ERKs) and p38 MAPK but not c-jun NH2 terminal kinases (JNKs), both in JB6 cells and in primary rat hepatocytes, as determined by the phosphorylation assay. Interestingly, the increase in AP-1 luciferase activity by iron was inhibited by the pretreatment of the cells with PD98059, a specific MEK1 inhibitor, and SB202190, a p38 kinase inhibitor. Levels of interleukin-6 (IL-6), a pro-inflammatory cytokine, were increased in JB6 cells by iron in a dose-dependent manner. The increase in IL-6 and its mRNA by iron was also eliminated by the pretreatment of the cells with PD98059 and SB202190. Since the IL-6 promoter contains an AP-1 binding site, our studies indicate that the iron-induced IL-6 gene expression may be mediated through ERKs and p38 MAPK pathways, possibly one of the important mechanisms for the pathogenesis of iron overload
— id: 47848, year: 2004, vol: 203, page: 199, stat: Journal Article,

Inhibition of AP-1 and neoplastic transformation by fresh apple peel extract
Ding, Min; Lu, Yongju; Bowman, Linda; Huang, Chuanshu; Leonard, Stephen; Wang, Liying; Vallyathan, Val; Castranova, Vince; Shi, Xianglin
2004 Mar 12;279(11):10670-10676, Journal of biological chemistry
Consumption of fruits and vegetables has been associated with a low incidence of cancers and other chronic diseases. Previous studies suggested that fresh apples inhibit tumor cell proliferation. Here we report that oral administration of apple peel extracts decreased the number of nonmalignant and malignant skin tumors per mouse induced by 12-O-tetradecanolyphorbol-13-acetate (TPA) in 7,12-dimethylbenz(a)anthracene-initiated mouse skin. ESR analysis indicated that apple extract strongly scavenged hydroxyl (OH) and superoxide (O(2)(-)) radicals. Mechanistic studies showed that pretreatment with apple peel extract inhibited AP-1 transactivation induced by ultraviolet B irradiation or TPA in JB6 cells and AP-1-luciferase reporter transgenic mice. This inhibitory effect appears to be mediated by the inhibition of ERKs and JNK activity. The results provide the first evidence that an extract from fresh apple peel extract may inhibit tumor promoter-induced carcinogenesis and associated cell signaling, and suggest that the chemopreventive effects of fresh apple may be through its antioxidant properties by blocking reactive oxygen species-mediated AP-1-MAPK activation
— id: 42694, year: 2004, vol: 279, page: 10670, stat: Journal Article,

Molecular mechanisms of arsenic carcinogenesis
Huang, Chuanshu; Ke, Qingdong; Costa, Max; Shi, Xianglin
2004 Jan;255(1-2):57-66, Molecular & cellular biochemistry
Arsenic is a metalloid compound that is widely distributed in the environment. Human exposure of this compound has been associated with increased cancer incidence. Although the exact mechanisms remain to be investigated, numerous carcinogenic pathways have been proposed. Potential carcinogenic actions for arsenic include oxidative stress, genotoxic damage, DNA repair inhibition, epigenetic events, and activation of certain signal transduction pathways leading to abberrant gene expression. In this article, we summarize current knowledge on the molecular mechanisms of arsenic carcinogenesis with an emphasis on ROS and signal transduction pathways
— id: 42693, year: 2004, vol: 255, page: 57, stat: Journal Article,

Differential effects of polycyclic aromatic hydrocarbons on transactivation of AP-1 and NF-kappaB in mouse epidermal cl41 cells
Li, Jingxia; Chen, Haobin; Ke, Qingdong; Feng, Zhaohui; Tang, Moon-Shong; Liu, Bingci; Amin, Shantu; Costa, Max; Huang, Chuanshu
2004 Jun;40(2):104-115, Molecular carcinogenesis
Polycyclic aromatic hydrocarbons (PAHs) and their derivatives, such as benzo[a]pyrene (B[a]P), (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), and 5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE), are complete carcinogens. However, the tumor promotion effects of PAHs remain unclear. We therefore investigated the possible activation of activator protein-1 (AP-1) and nuclear factor-kappaB (NFkappaB) in mouse epidermal Cl41 cells after different PAHs treatments, including B[a]P, B[a]PDE, chrysene-1,2-diol-3,4-epoxid (CDE), and 5-MCDE. The results showed that B[a]PDE and 5-MCDE were able to activate AP-1 and NF-kappaB, whereas B[a]P showed only marginal effect on AP-1 activation, and B[a]P and CDE had no effect on NF-kappaB activation. Treatment with either B[a]PDE or 5-MCDE also resulted in mitogen-activated protein kinases (MAPKs) activation as well as inhibitory subunit kappa-B (IkappaBalpha) phosphorylation and degradation, whereas B[a]P and CDE had no effect. Pretreatment with PD98059, a specific inhibitor for extracellular signal-regulated protein kinases (ERKs) upstream kinase MEK1/2, or SB202190, a p38 kinase inhibitor, resulted in a dramatic inhibition of B[a]PDE-induced AP-1 transactivation. In addition, B[a]PDE-induced AP-1 activation was also inhibited by overexpressing a dominant negative mutant of JNK1 in the cells. All these suggest ERKs, c-jun N-terminal kinases (JNKs), and p38 kinase signal transduction pathways are required for AP-1 induction by B[a]PDE. Taken together, B[a]PDE and 5-MCDE are the active compounds of PAHs to initiate signaling pathways. Considering the important roles of AP-1 and NF-kappaB in tumor promotion, we speculated the activation of AP-1 and NF-kappaB by B[a]PDE and 5-MCDE may involve in their or their parent compounds' tumor promotion effects. This study may help in better understanding the tumor promotion effects of PAHs
— id: 42690, year: 2004, vol: 40, page: 104, stat: Journal Article,

PI-3K and Akt are mediators of AP-1 induction by 5-MCDE in mouse epidermal Cl41 cells
Li, Jingxia; Chen, Haobin; Tang, Moon-Shong; Shi, Xianglin; Amin, Shantu; Desai, Dhimant; Costa, Max; Huang, Chuanshu
2004 Apr 12;165(1):77-86, Journal of cell biology
5-Methylchrysene has been found to be a complete carcinogen in laboratory animals. However, the tumor promotion effects of (+/-)-anti-5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE) remain unclear. In the present work, we found that 5-MCDE induced marked activator protein-1 (AP-1) activation in Cl41 cells. 5-MCDE also induced a marked activation of phosphatidylinositol 3-kinase (PI-3K). Inhibition of PI-3K impaired 5-MCDE-induced AP-1 transactivation, suggesting that PI-3K is an upstream kinase involved in AP-1 activation by 5-MCDE. Furthermore, we found that Akt is a PI-3K downstream mediator for 5-MCDE-induced AP-1 transactivation, whereas another PI-3K downstream kinase, p70(S6K), was not involved in AP-1 activation by 5-MCDE. Moreover, inhibition of Akt activation blocked 5-MCDE-induced activation of extracellular signal-regulated protein kinases (ERKs) and c-Jun NH(2)-terminal kinases (JNKs), whereas it did not affect p38K activation. Consistently, overexpression of a dominant-negative mutant of ERK2 or JNK1 blocked the AP-1 activation by 5-MCDE. These results demonstrate that 5-MCDE is able to induce AP-1 activation, and the AP-1 induction is specifically through a PI-3K/Akt-dependent and p70(S6K)-independent pathway
— id: 42691, year: 2004, vol: 165, page: 77, stat: Journal Article,

Nickel compounds act through phosphatidylinositol-3-kinase/Akt-dependent, p70(S6k)-independent pathway to induce hypoxia inducible factor transactivation and Cap43 expression in mouse epidermal Cl41 cells
Li, Jingxia; Davidson, Gerard; Huang, Yi; Jiang, Bing-Hua; Shi, Xianglin; Costa, Max; Huang, Chuanshu
2004 Jan 1;64(1):94-101, Cancer research
Nickel compounds are a somewhat unique class of carcinogens. Previous studies have demonstrated that NiCl(2) exposure leads to marked induction of hypoxia inducible factor 1 (HIF-1) in human osteosarcoma and BALB/c 3T3 cells, a transcription factor that has been considered to play an important role in tumor promotion and progression. However, the signal transduction pathways leading to HIF-1 induction are not well understood. The present study indicated that exposure of mouse epidermal Cl41 cells to either Ni(3)S(2) or NiCl(2) resulted in activation of phosphatidylinositol 3-kinase (PI-3K), Akt, and p70 S6 kinase (p70(S6k)). Inhibition of PI-3K, Akt, and p70(S6k) by overexpression of a dominant-negative mutant of PI-3K (Deltap85) impaired nickel-induced HIF-1 transactivation. Furthermore, an overexpression of the dominant-negative Akt mutant (Akt-T308A/S473A) blocked nickel-induced Akt phosphorylation and HIF-1 transactivation, whereas inhibition of p70(S6k) activation by pretreatment of cells with rapamycin did not show significant inhibitory effects on HIF-1 transactivation induced by nickel compounds. Consistent with HIF-1 transactivation, inhibition of the PI-3K/Akt pathway by either overexpression of Deltap85 or Akt-T308A/S473A caused dramatic inhibition of Cap43 protein expression induced by nickel compounds, whereas pretreatment of cells with rapamycin did not exhibit inhibition of Cap43 induction. These results demonstrated that nickel compounds induce HIF-1 transactivation and Cap43 protein expression through a PI-3K/Akt-dependent and p70(S6k)-independent pathway. This study should help us understand the signal transduction pathways involved in the carcinogenic effects of nickel compounds
— id: 42615, year: 2004, vol: 64, page: 94, stat: Journal Article,

Activation of aPKC is required for vanadate-induced phosphorylation of protein kinase B (Akt), but not p70S6k in mouse epidermal JB6 cells
Li, Jingxia; Dokka, Sujatha; Wang, Liying; Shi, Xianglin; Castranova, Vincent; Yan, Yan; Costa, Max; Huang, Chuanshu
2004 Jan;255(1-2):217-225, Molecular & cellular biochemistry
Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms by which vanadate mediates adverse effects are not well understood. The present study investigated the vanadate-induced phosphorylation of Akt and p70S6K, two kinases known to be vital for cell survival, growth, transformation, and transition of the cell cycle in mammals. Exposure of mouse epidermal JB6 cells to vanadium led to phosphorylation of Akt and p70S6K in a time- and dose-dependent manner. Vanadium exposure also caused translocation of atypical isoforms of PKC (lambda, zeta) from the cytosol to the membrane, but had no effect on PKCalpha translocation, suggesting that the atypical PKCs (aPKC) were specifically involved in vanadium-induced cellular response. Importantly, overexpression of a dominant negative mutant PKClambda blocked Akt phosphorylation at Ser473 and Thr308, whereas it did not inhibit p70S6k phosphorylation at Thr389 and Thr421/Ser424, suggesting that aPKC activation is specifically involved in vanadium-induced activation of Akt, but not in activation of p70S6k. Furthermore, vanadium-induced p70S6k phosphorylation at Thr389 and Thr421/Ser424 and Akt phosphorylation at Thr308 occurred through a PI-3K-dependent pathway because a PI-3K dominant negative mutant inhibited induction as compared with vector control cells. These results indicate that there was a differential role of aPKC in vanadate-induced phosphorylation of Akt and p70S6k, suggesting that signal transduction pathways leading to the activation of Akt and p70S6k were different
— id: 42692, year: 2004, vol: 255, page: 217, stat: Journal Article,

A critical role of PI-3K/Akt/JNKs pathway in benzo[a]pyrene diol-epoxide (B[a]PDE)-induced AP-1 transactivation in mouse epidermal Cl41 cells
Li, Jingxia; Tang, Moon-shong; Liu, Bingci; Shi, Xianglin; Huang, Chuanshu
2004 May 13;23(22):3932-3944, Oncogene
Mouse skin tumorigenicity studies indicate that benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) contributes to carcinogenesis as both a tumor initiator and promoter. However, the mechanisms that mediate B[a]PDE tumor promotion effects remain unclear. Our results demonstrated that in mouse epidermal Cl41 cells, B[a]PDE treatment resulted in marked activation of AP-1 and its upstream MAPKs, including ERKs, JNKs and p38K. B[a]PDE exposure also led to activation of phosphotidylinositol 3-kinase (PI-3K), Akt and p70 S6 kinase (p70S6k). B[a]PDE-induced AP-1 transactivation was inhibited by pretreatment of cells with PI-3K inhibitors, wortmannin or Ly294002. In contrast, inhibition of p70S6k with rapamycin did not show any inhibitory effects. An overexpression of dominant-negative mutant of PI-3K, Deltap85, impaired B[a]PDE-induced activation of PI-3K, Akt and AP-1 transactivation. Furthermore, an overexpression of dominant-negative Akt mutant, Akt-T308A/S473A, blocked B[a]PDE-induced activation of Akt, AP-1 and JNKs, while it did not affect the activation of p70S6k, ERKs and p38 kinase. These results demonstrated that B[a]PDE was able to induce AP-1 transactivation and this AP-1 induction was specific through PI-3K/Akt/JNKs-dependent and p70S6k-independent pathways. This study also indicated that Akt-T308A/S473A blocks B[a]PDE-induced AP-1 activation specific through impairing JNK pathway. These findings will help us to understand the signal transduction pathways involved in the carcinogenic effects of B[a]PDE
— id: 42695, year: 2004, vol: 23, page: 3932, stat: Journal Article,

The role of phosphatidylinositol-3 kinase in vanadate-promoted S phase entry
Zhang, Zhuo; Gao, Ning; He, Hengjun; Huang, Chuanshu; Jiang, Bing-hua; Jia, Luo; Shi, Xianglin
2004 Jan;255(1-2):239-245, Molecular & cellular biochemistry
Phosphatidylinositil-3 kinase (PI3K) is a heterodimer of catalytic and regulatory subunits. It is involved in various signaling pathways and key functions of the cells. The present study investigated the role of PI3K in vanadate-induced alteration in cell cycle regulation in C141 mouse epidermal cells. Vanadate caused a time- and dose-dependent increase in PI3K activity and phosphorylation of p70 S6 kinase (p70S6K) at Thr421/Ser424 and Thr389 sites. The phosphorylation at these sites was inhibited by PI3K inhibitor, LY294002, and p70S6K mutation. Vanadate promoted S phase entry and this promotion was inhibited by LY294002 and rapmycin, a p70S6K inhibitor. Vanadate-induced enhancement in S phase entry was also inhibited in transfection with dominant negative p70S6K mutant cells. The results obtained show that vanadate is able to increase PI3K activity through phosphorylation. PI3K activated p70S6K, which phosphated protein S6, and promoted S phase entry
— id: 42696, year: 2004, vol: 255, page: 239, stat: Journal Article,

Vanadate activated Akt and promoted S phase entry
Zhang, Zhuo; Gao, Ning; He, Hengjun; Huang, Chuanshu; Luo, Jia; Shi, Xianglin
2004 Jan;255(1-2):227-237, Molecular & cellular biochemistry
Protein kinase B (PKB)/Akt and its upstream signal transducer, phosphatidylinosito-3 kinase (PI3K) play an essential role in control of transcription and translation, which impact cell growth, survival, and metabolism. Transcription factor E2F is a component of the downstream proliferative machinery regulated by Akt. Hyperphosphorylation of retinoblastoma protein (pRb), a pocket protein, leads to release of E2F1, resulting in transition from G1 to S phase. The present study shows that in normal C141 cells, vanadate treatment increased the percentage of cells at S phase and elevated cyclin E and cyclin A expression. Vanadate treatment triggered phosphorylation of pRb and release of E2F1. Furthermore, vanadate increased Akt kinase activity and caused its phosphorylation at Ser473 and Thr308. Inhibition of Akt by either inhibitors or transfected cells with dominant negative kinase mutant or dominant negative phosphorylation mutant decreased the percentage of the cells at the S phase induced by vanadate, and reduced both cyclin E and E2F1 expression and phosphorylation of pRb. The present study indicates that Akt plays an essential role in vanadate-induced increase in cell number at S phase and transition from G1 to S phase through E2F-pRb pathway
— id: 42697, year: 2004, vol: 255, page: 227, stat: Journal Article,

Deferoxamine synergistically enhancing iron-induced stimulation of activator protein-1 through increased ERKs phosphorylation
Dai, J; Huang, C; Huang, X
2003 MAR ;72(1):270-271, Toxicological sciences
— id: 38504, year: 2003, vol: 72, page: 270, stat: Journal Article,

Regulation of HIF transactivation and CAP43 expression by nickel compounds through PI-3K/AKT dependent, P70S6K-independent pathway
Davidson, G; Li, J; Huang, Y; Costa, M; Huang, C
2003 MAR ;72(1):106-106, Toxicological sciences
— id: 38494, year: 2003, vol: 72, page: 106, stat: Journal Article,

Deferoxamine synergistically enhances iron-induced stimulation of activator protein-1: Kinase activation versus phosphatase inhibition
Huang, X; Dai, JS; Zhang, Q; Huang, CS
2003 OCT ;35(11):S66-S66, Free radical biology & medicine
— id: 55392, year: 2003, vol: 35, page: S66, stat: Journal Article,

Differential requirement of EGF receptor and its tyrosine kinase for AP-1 transactivation induced by EGF and TPA
Li, Jingxia; Ma, Cuiling; Huang, Yi; Luo, Jia; Huang, Chuanshu
2003 Jan 16;22(2):211-219, Oncogene
The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including cell differentiation, proliferation, apoptosis and oncogenic transformation. It is thought that the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced AP-1 activity is because of the activation of the PKC/MAPK/AP-1 pathway, although the detailed molecular mechanism has not been fully characterized. The tyrosine kinases of epidermal growth factor receptor (EGFR) lie at the head of a complex of signal transduction cascade that modulates cell proliferation, survival, adhesion, migration and differentiation. Currently, little is known about whether EGFR or its tyrosine kinase is necessary for TPA-induced AP-1 activation. In the present study, we investigated this issue using a well-characterized mouse fibroblast B82 cell line, which is devoid of the EGFR, and its stable transfectants with either wild-type EGFR (B82L) or tyrosine kinase-deficient EGFR (mutation at Lys-721) (B82M721). We demonstrated that the TPA or epidermal growth factor (EGF) induced AP-1 activation in the B82L cells that express wild-type EGFR, but not in the B82 cell, whereas autophosphorylation at tyrosine(1173) of EGFR in B82L cells was only induced by EGF, but not TPA. The expression of tyrosine kinase-deficient EGFR (mutation at Lys-721) (B82M721) resulted in deficiency of AP-1 induction in cellular response to EGF, while TPA treatment led to fully AP-1 activation. Furthermore, the mutation at Lys-721 of EGFR resulted in impairing of EGFR autophosphorylation at tyrosine(1173) induced by EGF. Based on these results, we conclude that TPA-induced AP-1 activation requires the basal level-EGFR protein, but not EGFR tyrosine kinase and EGFR autophosphorylation at tyrosine(1173), whereas both EGFR tyrosine kinase and EGFR autophosphorylation at Y(1173) play a critical role in EGF-induced AP-1 activation
— id: 39329, year: 2003, vol: 22, page: 211, stat: Journal Article,

Activation of JNK by vanadate induces a Fas-associated death domain (FADD)-dependent death of cerebellar granule progenitors in vitro
Luo, Jia; Sun, Yanbo; Lin, Hong; Qian, Yong; Li, Zheng; Leonard, Stephen S; Huang, Chuanshu; Shi, Xianglin
2003 Feb 14;278(7):4542-4551, Journal of biological chemistry
Apoptosis is a highly regulated process that plays a critical role in neuronal development as well as the homeostasis of the adult nervous system. Vanadate, an environmental toxicant, causes developmental defects in the central nervous system. Here, we demonstrated that vanadate induced apoptosis in cultured cerebellar granule progenitors (CGPs). Treatment of cultured CGPs with vanadate activated ERKs and JNKs but not p38 MAPK and also induced c-Jun phosphorylation. In addition, vanadate induced FasL production, Fas (CD95) aggregation, and its association with the Fas-associated death domain (FADD), as well as the activation of caspase-8. Furthermore, vanadate generated reactive oxygen species (ROS) in CGPs; however, ROS was not involved in vanadate-mediated MAPK activation. Vanadate-induced FasL expression was ROS-dependent but JNK-independent. In contrast, vanadate-elicited Fas aggregation and Fas-FADD association, as well as caspase-8 activation, were dependent on JNK activation but were minimally regulated by ROS generation. The hydrogen peroxide scavenger, catalase, blocked vanadate-induced FasL expression and partially mitigated vanadate-induced cell death. On the other hand, dominant negative FADD and caspase-8 inhibitor completely eliminated vanadate-induced apoptosis. Thus, JNK signaling plays a major role in vanadate-mediated activation of the Fas-FADD-caspase-8 pathway that accounts for vanadate-induced apoptosis of CGPs
— id: 38410, year: 2003, vol: 278, page: 4542, stat: Journal Article,

Vanadium-induced apoptosis and pulmonary inflammation in mice: Role of reactive oxygen species
Wang, Liying; Medan, Djordje; Mercer, Robert; Overmiller, Dean; Leornard, Stephen; Castranova, Vincent; Shi, Xianglin; Ding, Min; Huang, Chuanshu; Rojanasakul, Yon
2003 Apr;195(1):99-107, Journal of cellular physiology
Pulmonary exposure to metals and metal-containing compounds is associated with pulmonary inflammation, cell death, and tissue injury. The present study uses a mouse model to investigate vanadium-induced apoptosis and lung inflammation, and the role of reactive oxygen species (ROS) in this process. Aspiration of the pentavalent form of vanadium, V (V), caused a rapid influx of polymorphonuclear leukocytes into the pulmonary airspace with a peak inflammatory response at 6 h post-exposure and resolution by 72 h. During this period, the number of apoptotic lung cells which were predominantly neutrophils increased considerably with a peak response at 24 h accompanied by no or minimum necrosis. After 24 h when the V (V)-induced inflammation was in the resolution phase, an increased influx of macrophages and engulfment of apoptotic bodies by these phagocytes was observed, supporting the role of macrophages in apoptotic cell clearance and resolution of V (V)-induced lung inflammation. Electron spin resonance (ESR) studies using lavaged alveolar macrophages showed the formation of ROS, including O(2)(*-), H(2)O(2), and (*)OH radicals which were confirmed by inhibition with free radical scavengers. The mechanism of ROS generation induced by V (V) involved the activation of an NADPH oxidase complex and the mitochondrial electron transport chain. The ROS scavenger, catalase (H(2)O(2) scavenger), effectively inhibited both lung cell apoptosis and the inflammatory response, whereas superoxide dismutase (SOD) (O(2)(*-) scavenger) and the metal chelator, deferoxamine (inhibitor of (*)OH generation by Fenton-like reactions) had lesser effects. These results indicate that multiple oxidative species are involved in V (V)-induced lung inflammation and apoptosis, and that H(2)O(2) plays a major role in this process
— id: 38408, year: 2003, vol: 195, page: 99, stat: Journal Article,

Role of reactive oxygen species and MAPKs in vanadate-induced G(2)/M phase arrest
Zhang, Zhuo; Leonard, Stephen S; Huang, Chuanshu; Vallyathan, Val; Castranova, Vince; Shi, Xianglin
2003 May 15;34(10):1333-1342, Free radical biology & medicine
Cell growth arrest is an important mechanism in maintaining genomic stability and integrity in response to environmental stress. Using the human lung alveolar epithelial cancer cell line A549, we investigated the role of reactive oxygen species (ROS), extracellular signal-regulated protein kinase (ERK), and p38 protein kinase in vanadate-induced cell growth arrest. Exposure of cells to vanadate led to cell growth arrest at the G(2)/M phase and caused upregulation of p21 and phospho-cdc2 and degradation of cdc25C in a time- and dose-dependent manner. Vanadate stimulated mitogen-activated protein kinases (MAPKs) family members, as determined by the phosphorylation of ERK and p38. PD98059, an inhibitor of ERK, and SB202190, an inhibitor of p38, inhibited vanadate-induced cell growth arrest, upregulation of p21 and cdc2, and degradation of cdc25C. In addition to hydroxyl radical ((*)OH) formation, cellular reduction of vanadate generated superoxide radical (O(2)(*)(-)) and hydrogen peroxide (H(2)O(2)), as determined by confocal microscopy using specific dyes. Generation of O(2)(*)(-) and H(2)O(2) was inhibited by specific antioxidant enzymes, superoxide dismutase (SOD) and catalase, respectively. ROS activate ERK and p38, which in turn upregulate p21 and cdc2 and cause degradation of cdc25C, leading to cell growth arrest at the G(2)/M phase. Specific ROS affect different MAPK family members and cell growth regulatory proteins with different potencies
— id: 38407, year: 2003, vol: 34, page: 1333, stat: Journal Article,

Differential role of hydrogen peroxide in UV-induced signal transduction
Ding, Min; Li, Jingxia; Leonard, Stephen S; Shi, Xianglin; Costa, Max; Castranova, Vincent; Vallyathan, Val; Huang, Chuanshu
2002 May-Jun;234-235(1-2):81-90, Molecular & cellular biochemistry
The present study investigated the differential requirement of ROS in UV-induced activation of these pathways. Exposure of the mouse epidermal C141 cells to UV radiation led to generation of ROS as measured by electron spin resonance (ESR) and by H2O2 and O2. fluorescence staining assay. Treatment of cells with UV radiation or H2O2 also markedly activated Erks, JNKs, p38 kinase and led to increases in phosphorylation of Akt and p70(S6k) in mouse epidermal JB6 cells. The scavenging of UV-generated H2O2 by N-acety-L-cyteine (NAC, a general antioxidant) or catalase (a specific H2O2 inhibitor) inhibited UV-induced activation of JNKs, p38 kinase, Akt and p70(S6k), while it did not show any inhibitory effects on Erks activation. Further, pretreatment of cells with sodium formate (an .OH radical scavenger) or superoxide dismutase (O2-. radical scavenger) did not inhibit any of these pathways. These results demonstrate that H2O2 generation is required for UV-induced phosphorylation of Akt and p70(S6k), and involved in activation of JNKs and p38 kinase, but not Erks
— id: 38402, year: 2002, vol: 234-235, page: 81, stat: Journal Article,

Inhibition of benzo(a)pyrene diol-epoxide-induced transactivation of activated protein 1 and nuclear factor kappaB by black raspberry extracts
Huang, Chuanshu; Huang, Yi; Li, Jingxia; Hu, Wenwei; Aziz, Robeena; Tang, Moon-shong; Sun, Nanjun; Cassady, John; Stoner, Gary D
2002 Dec 1;62(23):6857-6863, Cancer research
Freeze-dried black raspberries have been shown to inhibit the development of chemically induced esophageal and colon cancer in rodents.In addition, organic extracts of black raspberries inhibit benzo(a)pyrene (BaP)-induced cell transformation in vitro. The molecular mechanisms through which black raspberries inhibit carcinogenesis remain unclear. We investigated the effects of black raspberry extracts on transactivation of activated protein 1 (AP-1) and nuclear factor kappaB (NFkappaB) induced by BaP diol-epoxide (BPDE), the ultimate carcinogen of BaP, in mouse epidermal JB6 Cl 41 (Cl 41) cells. Black raspberries were extracted with methanol, and the methanol extract was partitioned and chromatographed into several fractions designated RU-F003, RU-F004, RU-DM, and RU-ME. Pretreatment of Cl 41 cells with RU-F003, RU-DM, or RU-ME resulted in an inhibition of BPDE-induced AP-1 and NFkappaB activities. The RU-ME fraction was the most potent inhibitor among the fractions tested. In contrast, fraction RU-F004 did not inhibit BPDE-induced AP-1 or NFkappaB activities in Cl 41 cells. The inhibitory effects of RU-ME on BPDE-induced activation of AP-1 and NFkappaB appear to be mediated via inhibition of mitogen activated protein kinase activation and inhibitory subunit kappaB phosphorylation, respectively. Pretreatment of cells with berry fractions did not result in an inhibition of BPDE binding to DNA; thus, this was not a mechanism of reduced AP-1 and NFkappaB activities. None of the fractions was found to affect p53-dependent transcription activity. In view of the important roles of AP-1 and NFkappaB in tumor promotion/progression, these results suggest that the ability of black raspberries to inhibit tumor development may be mediated by impairing signal transduction pathways leading to activation of AP-1 and NFkappaB. The RU-ME fraction appears to be the major fraction responsible for the inhibitory activity of black raspberries
— id: 38406, year: 2002, vol: 62, page: 6857, stat: Journal Article,

Ultraviolet-induced phosphorylation of p70(S6K) at Thr(389) and Thr(421)/Ser(424) involves hydrogen peroxide and mammalian target of rapamycin but not Akt and atypical protein kinase C
Huang, Chuanshu; Li, Jingxia; Ke, Qingdong; Leonard, Stephen S; Jiang, Bing-Hua; Zhong, Xiao-Song; Costa, Max; Castranova, Vincent; Shi, Xianglin
2002 Oct 15;62(20):5689-5697, Cancer research
The p70 S6 kinase (p70(S6k)) is a Ser/Thr kinase that plays an important role in cell growth, transformation, and the transition of the cell cycle in mammalian cells. Because UV radiation has been reported to induce activation of p70(S6k), which is believed to play some role in the carcinogenic effects of sun exposure, the present study investigated the signaling pathways involved in this activation induced by UV radiation in mouse epidermal JB6 Cl41 cells. Exposure of cells to UV radiation led to marked increases in p70(S6k) activity and phosphorylation at Thr(389) and Thr(421)/Ser(424). UV radiation also generated reactive oxygen species as measured by electron spin resonance and by H(2)O(2) and O( minus sign, dot below )(2) fluorescence staining assays in JB6 Cl 41 cells. The scavenging of UV-generated H(2)O(2) by N-acety-L-cyteine (a general antioxidant) or catalase (a specific H(2)O(2) scavenger) inhibited p70(S6k) phosphorylation at Thr(389) and Thr(421)/Ser(424), whereas pretreatment of cells with sodium formate (an.OH radical scavenger) or superoxide dismutase (an O( minus sign, dot below )(2) radical scavenger) did not show any inhibitory effects. Importantly, UV-induced increases in p70(S6k) phosphorylation at Thr(389) and Thr(421)/Ser(424) were dramatically inhibited by pretreatment of cells with rapamycin, LY294002, or PD98059, whereas overexpression of dominant-negative mutants of PKClambda/iota and Akt1 did not inhibit p70(S6k) phosphorylation at Thr(389) and Thr(421)/Ser(424). These results demonstrated that H(2)O(2), phosphatidylinositol 3-kinase, and mammalian target of rapamycin were important players for UV-induced p70(S6k) phosphorylation at Thr(389) and Thr(421)/Ser(424), whereas Akt and atypical protein kinase C were not involved in this activation. The role of H(2)O(2) in p70(S6k) phosphorylation at Thr(389) and Thr(421)/Ser(424) was further supported by the findings that treatment of cells with H(2)O(2) also caused p70(S6k) phosphorylation at Thr(389) and Thr(421)/Ser(424)
— id: 38401, year: 2002, vol: 62, page: 5689, stat: Journal Article,

Role of bioavailable iron in coal dust-induced activation of activator protein-1 and nuclear factor of activated T cells: difference between Pennsylvania and Utah coal dusts
Huang, Chuanshu; Li, Jingxia; Zhang, Qi; Huang, Xi
2002 Nov;27(5):568-574, American journal of respiratory cell & molecular biology
Activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) are two important transcription factors responsible for the regulation of cytokines, which are involved in cell proliferation and inflammation. Coal workers' pneumoconiosis (CWP) is an occupational lung disease that may be related to chronic inflammation caused by coal dust exposure. In the present study, we demonstrate that coal from the Pennsylvania (PA) coalmine region, which has a high prevalence of CWP, can activate both AP-1 and NFAT in JB6 mouse epidermal cells. In contrast, coal from the Utah (UT) coalmine region, which has a low prevalence of CWP, has no such effects. The PA coal stimulates mitogen-activated protein kinase (MAPK) family members of extracellular signal-regulated kinases (ERKs) and p38 MAPK but not c-Jun-NH(2)-terminal kinases, as determined by the phosphorylation assay. The increase in AP-1 by the PA coal was completely eliminated by the pretreatment of cells with PD98059, a specific MAPK kinase inhibitor, and SB202190, a p38 kinase inhibitor, further confirming that the PA coal-induced AP-1 activation is mediated through ERKs and p38 MAPK pathways. Deferoxamine (DFO), an iron chelator, synergistically enhanced the PA coal-induced AP-1 activity, but inhibited NFAT activity. For comparison, cells were treated with ferrous sulfate and/or DFO. We have found that iron transactivated both AP-1 and NFAT, and DFO further enhanced iron-induced AP-1 activation but inhibited NFAT. These results indicate that activation of AP-1 and NFAT by the PA coal is through bioavailable iron present in the coal. These data are in agreement with our previous findings that the prevalence of CWP correlates well with levels of bioavailable iron in coals from various mining regions
— id: 38471, year: 2002, vol: 27, page: 568, stat: Journal Article,

Activation of nuclear factor-kappaB and not activator protein-1 in cellular response to nickel compounds
Huang, Yi; Davidson, Gerard; Li, Jingxia; Yan, Yan; Chen, Fei; Costa, Max; Chen, Lung Chi; Huang, Chuanshu
2002 Oct;110 Suppl 5(4):835-839, Environmental health perspectives
The predominant exposure route for nickel compounds is by inhalation, and several studies have indicated the correlation between nickel exposure and respiratory cancers. The tumor-promoting effects of nickel compounds are thought to be associated with their transactivation of transcription factors. We have investigated the possible activation of activator protein-1 (AP-1) and nuclear factor KB (NF-kappaB) in mouse C141 epidermal cells and fibroblasts 3T3 and B82, and human bronchoepithelial BEAS-2B cells in response to nickel compound exposure. Our results show that NF-kappaB activity is induced by nickel exposure in 3T3 and BEAS-2B cells. Conversely, similar nickel treatment of these cells did not induce AP-1 activity, suggesting that nickel tumorigenesis occurs through NF-kappaB and not AP-1. We also investigated the role of NF-kappaB in the induction of Cap43 by nickel compounds using dominant negative mutant Ikappabeta kinase b-KM BEAS-2B transfectants
— id: 38400, year: 2002, vol: 110 Suppl 5, page: 835, stat: Journal Article,

Involvement of hydrogen peroxide in asbestos-induced NFAT activation
Li, Jingxia; Huang, Bihui; Shi, Xianglin; Castranova, Vincent; Vallyathan, Val; Huang, Chuanshu
2002 May-Jun;234-235(1-2):161-168, Molecular & cellular biochemistry
The present study investigated the role of reactive oxygen species (ROS) in activation of nuclear factor of activated T cells (NFAT), a pivotal transcription factor responsible for regulation of cytokines, by asbestos in mouse embryo fibroblast PW cells. Exposure of cells to asbestos led to the transactivation of NFAT in a time- and dose-dependent manner. Scavenging of asbestos-induced H2O2 with N-acety-L-cyteine (NAC, a general antioxidant) or catalase (a specific H2O2 inhibitor) resulted in inhibition of NFAT activation. In contrast, an increase in H2O2 generation by the addition of superoxide dismutase (SOD) slightly enhanced asbestos-induced NFAT activation. In addition, pretreatment of cells with sodium formate did not exhibit any inhibition of NFAT activity induced by asbestos. These results demonstrated that H2O2 appeared to play an important role in asbestos-induced NFAT transactivation. Furthermore, it was observed that incubation of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) not only resulted in NFAT activation by itself, but also enhanced asbestos-induced NFAT induction. Pretreatment of cells with cyclosporin A (CSA), a pharmacological inhibitor of the phosphatase calcineurin, blocked both asbestos- and TPA plus asbestos-induced NFAT activation. These data suggest that asbestos is able to induce NFAT activation through H2O2-dependent and CSA-sensitive pathways, which may be involved in asbestos-induced carcinogenesis
— id: 38414, year: 2002, vol: 234-235, page: 161, stat: Journal Article,

Regulation of Fas (CD95)-induced apoptosis by nuclear factor-kappaB and tumor necrosis factor-alpha in macrophages
Lu, Bin; Wang, Liying; Medan, Djordje; Toledo, David; Huang, Chuanshu; Chen, Fei; Shi, Xianglin; Rojanasakul, Yon
2002 Sep;283(3):C831-C838, American journal of physiology. Cell physiology
The APO-1/Fas ligand (FasL) and tumor necrosis factor-alpha (TNF-alpha) are two functionally related molecules that induce apoptosis of susceptible cells. Although the two molecules have been reported to induce apoptosis via distinct signaling pathways, we have shown that FasL can also upregulate the expression of TNF-alpha, raising the possibility that TNF-alpha may be involved in FasL-induced apoptosis. Because TNF-alpha gene expression is under the control of nuclear factor-kappaB (NF-kappaB), we investigated whether FasL can induce NF-kappaB activation and whether such activation plays a role in FasL-mediated cell death in macrophages. Gene transfection studies using NF-kappaB-dependent reporter plasmid showed that FasL did activate NF-kappaB promoter activity. Gel shift studies also revealed that FasL mobilized the p50/p65 heterodimeric form of NF-kappaB. Inhibition of NF-kappaB by a specific NF-kappaB inhibitor, caffeic acid phenylethyl ester, or by dominant expression of the NF-kappaB inhibitory subunit IkappaB caused an increase in FasL-induced apoptosis and a reduction in TNF-alpha expression. However, neutralization of TNF-alpha by specific anti-TNF-alpha antibody had no effect on FasL-induced apoptosis. These results indicate that FasL-mediated cell death in macrophages is regulated through NF-kappaB and is independent of TNF-alpha activation, suggesting the antiapoptotic role of NF-kappaB and a separate death signaling pathway mediated by FasL
— id: 38413, year: 2002, vol: 283, page: C831, stat: Journal Article,

Involvement of c-jun NH(2)-terminal kinases in resveratrol-induced activation of p53 and apoptosis
She, Qing-Bai; Huang, Chuanshu; Zhang, Yiguo; Dong, Zigang
2002 Apr;33(4):244-250, Molecular carcinogenesis
Resveratrol, a constituent of grapes and other foods, is one of the most promising agents for cancer prevention. In a previous study, we showed that the antitumor activity of resveratrol occurs through extracellular signal-regulated protein kinases (ERKs) and p38 kinase-mediated p53 activation. In this study, we also determined that c-jun NH(2)-terminal kinases (JNKs) are involved in resveratrol-induced p53 activation and induction of apoptosis. In the JB6 mouse epidermal cell line, resveratrol activated JNKs dose-dependently within a dose range of 10-40 microM, the same dosage responsible for the inhibition of tumor promoter-induced cell transformation. Stable expression of a dominant negative mutant of JNK1 or disruption of the Jnk1 or Jnk2 gene markedly inhibited resveratrol-induced p53-dependent transcription activity and induction of apoptosis. Furthermore, resveratrol-activated JNKs were shown to phosphorylate p53 in vitro, but this activity was repressed in the cells expressing a dominant negative mutant of JNK1 or in Jnk1 or Jnk2 knockout (Jnk1(-/-) or Jnk2(-/-)) cells. These data suggested that JNKs act as mediators of resveratrol-induced activation of p53 and apoptosis, which may occur partially through p53 phosphorylation
— id: 38422, year: 2002, vol: 33, page: 244, stat: Journal Article,

Role of neutrophil apoptosis in vanadium-induced pulmonary inflammation in mice
Wang, Liying; Medan, Djordje; Mercer, Robert; Shi, Xianglin; Huang, Chuanshu; Castranova, Vincent; Ding, Min; Rojanasakul, Yon
2002 ;21(4):343-350, Journal of environmental pathology, toxicology & oncology
Pulmonary exposure to airborne vanadium and vanadium-containing compounds is associated with acute pulmonary inflammation, characterized by a rapid influx of neutrophilic polymorphonuclear leukocytes with a peak response at 6 hours and resolution by 3 days. We hypothesized that neutrophil apoptosis is involved in the resolution of vanadium-induced lung inflammation. To test this hypothesis, mice were exposed to inspired vanadium or saline control and the bronchoalveolar lavage (BAL) cells were examined at various times for apoptosis using terminal deoxyribonucleotidyl transferase-mediated nick end labeling (TUNEL). Control mice showed only resident alveolar macrophages in the BAL with no evidence of apoptosis. In contrast, vanadium-treated mice showed clear apoptosis of BAL cells, which were predominantly neutrophils. The number of apoptotic cells gradually increased and reached a maximal level by 24 hours with subsequent decline. After 24 hours, when the vanadium-induced lung inflammation was in the resolution phase, we observed an increased number of alveolar macrophages in BAL and the engulfment of apoptotic bodies by these macrophages. At 72 hours, the total number of neutrophils in BAL fell to the baseline level, and the number of apoptotic cells was reduced. Clearance of the apoptotic product was demonstrated by the presence of apoptotic bodies in the cytoplasm of alveolar macrophages. We conclude that apoptosis of neutrophils and clearance by alveolar macrophages are important mechanisms in the resolution of vanadium-induced lung inflammation
— id: 38409, year: 2002, vol: 21, page: 343, stat: Journal Article,

Vanadate induces G2/M phase arrest in p53-deficient mouse embryo fibroblasts
Zhang, Zhuo; Chen, Fei; Huang, Chuanshu; Shi, Xianglin
2002 ;21(3):223-231, Journal of environmental pathology, toxicology & oncology
Vanadium compounds exert potent toxic and carcinogenic effects on a wide variety of biological systems. The mechanisms involved in their toxicity and carcinogenesis require investigation. Cell growth arrest and its regulation are important mechanisms in maintaining genomic stability and integrity in response to environmental stress. The p53 tumor suppressor plays a central role in the regulation of the normal cell cycle. To investigate the role of p53 in vanadate-induced cell growth arrest and its regulation, two cell lines--normal mouse embryo fibroblasts [p53(+/+)] and p53-deficient mouse embryo fibroblasts [p53(-/-)],--were used in this study. Flow cytometry was used to analyze cell growth arrest at G0/G1, S, or G2/M phase. Western blotting analysis was performed to determine several cell growth regulatory proteins. The results showed that in p53(-/-) cells vanadate induced G2/M phase arrest in a dose- and time-dependent manner without alteration of S phase. In p53(+/+) cells, vanadate treatment increased the S phase with no significant change in the G2/M phase. Furthermore, Western blotting results showed that in p53(-/-) cells vanadate caused cdc25C degradation and activation of phospho-cdc2 without alteration of the p21 level. In p53(+/+) cells, vanadate increased the expression of p21 and degraded cdc25A instead of cdc25C without any effect on cdc2. These results demonstrate that vanadate induced G2/M phase arrest in p53-deficient mouse embryo fibroblasts, and promoted S phase entry in p53 wild-type mouse embryo fibroblasts
— id: 38411, year: 2002, vol: 21, page: 223, stat: Journal Article,

MAPKs mediate S phase arrest induced by vanadate through a p53-dependent pathway in mouse epidermal C141 cells
Zhang, Zhuo; He, Hengjuan; Chen, Fei; Huang, Chuanshu; Shi, Xianglin
2002 Jul;15(7):950-956, Chemical research in toxicology
Mitogen-activated protein (MAP) kinases play an important role in mediation of the signal transduction pathway in cellular response to genotoxic stress. Cell growth arrest is considered as an early stage in response to the genotoxic stress. p53 is well-known as a tumor suppression gene involved in both cell growth arrest and apoptosis. The present study investigated the involvement of MAP kinases in vanadate-induced cell growth arrest and the relationship of p53. DNA content analysis showed that vanadate-induced S phase arrest is time- and dose-dependent in p53 wild-type C141 cells but not in p53-deficient C141 cells. Western blotting results indicated that vanadate caused an inactivation of p-cdk2 at Thr160, which is an important kinase for the progression of S phase, and an increase in expression of p21, which is a key for S phase arrest. In p53-deficient cells, vanadate did not induce any observable change in p21 or p-cdk2 level. In addition, vanadate up-regulated phospho-p38 and ERK, two members of MAP kinases. At the same time, vanadate increased the p53 activity as measured by luciferase assay. Addition of PD98059 and SB202190, inhibitors of ERK and p38, respectively, decreased vanadate-induced S phase arrest, reduced p21 levels, restored activation of p-cdk2, and decreased p53 activity. The study demonstrated that vanadate-induced S phase arrest is mediated by both ERK and p38 in a p53-dependent pathway
— id: 38415, year: 2002, vol: 15, page: 950, stat: Journal Article,

Vanadate-induced cell growth arrest is p53-dependent through activation of p21 in C141 cells
Zhang, Zhuo; Huang, Chuanshu; Li, Jinxia; Shi, Xianglin
2002 Apr 10;89(1-2):142-148, Journal of inorganic biochemistry
Vanadium is widely used in industry. It is a potent toxic agent and carcinogen. The mechanisms involved in its toxicity and carcinogenesis are still unclear. Improper cell growth is believed to be involved in cancer development. The present study investigated the regulation of p53 on vanadate-induced cell growth arrest using both p53 wild type C141 cells and p53 deficient embryo fibroblasts (p53 -/-). On vanadate stimulation, C141 cells exhibited a dose- and time-dependent S phase arrest as determined by DNA content analysis. In contrast, vanadate was unable to increase the percentage of S phase in p53 -/- cells. Luciferase assay showed that vanadate induced p53 activation in a dose- and time-dependent manner in p53 wild type C141 cells. Addition of pifithrin-alpha (PFT), a specific inhibitor of p53, reduced the activation of p53 with a concomitant decrease in growth arrest at S phase. Western blotting analysis demonstrated that vanadate caused a dose- and time-dependent increase of p21 level in C141 cells. Pretreatment of C141 cells with PFT decreased p21 expression induced by vanadate while the p21 expression did not vary in vanadate stimulated p53 -/- cells. The results obtained from the present study suggest that vanadate is able to induce S phase arrest through p53- and p21-dependent pathway
— id: 38416, year: 2002, vol: 89, page: 142, stat: Journal Article,

Epidermal growth factor up-regulates the transcription of mouse lon homology ATP-dependent protease through extracellular signal-regulated protein kinase- and phosphatidylinositol-3-kinase-dependent pathways
Zhu, Yunfeng; Wang, Mei; Lin, Hong; Huang, Chuanshu; Shi, Xianglin; Luo, Jia
2002 Oct 15;280(1):97-106, Experimental cell research
Epidermal growth factor (EGF) induces tumorigenic transformation of mouse epidermal cells (JB6 P(+)). We cloned a full-length EGF-responsive cDNA in JB6P(+) cells; EGF up-regulated mRNA expression of this gene 5- to 6-fold. The deduced amino acid sequence of this cDNA exhibited 84 and 96% homology with human and rat Lon homology ATP-dependent protease, respectively, and all conserved domains of Lon, such as ATPase/protease domains, are present in the mouse gene, indicating that this gene is mouse Lon. EGF increased the transcriptional rate without affecting the mRNA stability of m-Lon. The level of m-Lon in irreversibly transformed mouse epidermal cells (JB7) was 3.4-fold higher than that in parental JB6 P(+) cells. Similarly, human mammary epithelial cells overexpressing the proto-oncogene ErbB2 expressed significantly higher levels of Lon than normal mammary epithelial cells. EGF failed to regulate Lon expression in ERK-deficient JB6 P(-) cells or cells that expressed the dominant-negative p85 P13-K regulatory unit. Furthermore, selective chemical blockers for MEK1 and P13-K (PD98059 and LY294002) inhibited EGF-mediated induction. Mitochondria-localized Lon protease plays a critical role in the regulation of mitochondrial gene expression and genome integrity. Disruption of mitochondrial homeostasis is a general characteristic of tumorigenic transformation. Thus, the role of Lon in tumor promotion warrants further study
— id: 38412, year: 2002, vol: 280, page: 97, stat: Journal Article,

Role of N-acylethanolamines in cell signaling
Berdyshev EV; Schmid PC; Krebsbach RJ; Kuwae T; Huang C; Ma WY; Dong Z; Schmid HH
2001 ;88(2):207-214, World review of nutrition & dietetics
— id: 42699, year: 2001, vol: 88, page: 207, stat: Journal Article,

Cannabinoid-receptor-independent cell signalling by N-acylethanolamines
Berdyshev, E V; Schmid, P C; Krebsbach, R J; Hillard, C J; Huang, C; Chen, N; Dong, Z; Schmid, H H
2001 Nov 15;360(Pt 1):67-75, Biochemical journal
Anandamide and other polyunsaturated N-acylethanolamines (NAEs) exert biological activity by binding to cannabinoid receptors. These receptors are linked to G(i/o) proteins and their activation leads to extracellular-signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAP kinase) activation, inhibition of cAMP-dependent signalling and complex changes in the expression of various genes. Saturated and monounsaturated NAEs cannot bind to cannabinoid receptors and may thus mediate cell signalling through other targets. Here we report that both saturated/monounsaturated NAEs and anandamide (20:4(n-6) NAE) stimulate cannabinoid-receptor-independent ERK phosphorylation and activator protein-1 (AP-1)-dependent transcriptional activity in mouse epidermal JB6 cells. Using a clone of JB6 P(+) cells with an AP-1 collagen-luciferase reporter construct, we found that 16:0, 18:1(n-9), 18:1(n-7), 18:2(n-6) and 20:4(n-6) NAEs stimulated AP-1-dependent transcriptional activity up to 2-fold, with maximal stimulation at approx. 10-15 microM. Higher NAE concentrations had toxic effects mediated by alterations in mitochondrial energy metabolism. The AP-1 stimulation appeared to be mediated by ERK but not JNK or p38 signalling pathways, because all NAEs stimulated ERK1/ERK2 phosphorylation without having any effect on JNK or p38 kinases. Also, overexpression of dominant negative ERK1/ERK2 kinases completely abolished NAE-induced AP-1 activation. In contrast with 18:1(n-9) NAE and anandamide, the cannabinoid receptor agonist WIN 55,212-2 did not stimulate AP-1 activity and inhibited ERK phosphorylation. The NAE-mediated effects were not attenuated by pertussis toxin and appeared to be NAE-specific, as a close structural analogue, oleyl alcohol, failed to induce ERK phosphorylation. The data support our hypothesis that the major saturated and monounsaturated NAEs are signalling molecules acting through intracellular targets without participation of cannabinoid receptors
— id: 106269, year: 2001, vol: 360, page: 67, stat: Journal Article,

Induction of activator protein-1 through reactive oxygen species by crystalline silica in JB6 cells
Ding M; Shi X; Lu Y; Huang C; Leonard S; Roberts J; Antonini J; Castranova V; Vallyathan V
2001 Mar 23;276(12):9108-9114, Journal of biological chemistry
We reported previously that freshly fractured silica (FFSi) induces activator protein-1 (AP-1) activation through extracellular signal-regulated protein kinases (ERKs) and p38 kinase pathways. In the present study, the biologic activities of FFSi and aged silica (ASi) were compared by measuring their effects on the AP-1 activation and phosphorylation of ERKs and p38 kinase. The roles of reactive oxygen species (ROS) in this silica-induced AP-1 activation were also investigated. We found that FFSi-induced AP-1 activation was four times higher than that of ASi in JB6 cells. FFSi also caused greater phosphorylation of ERKs and p38 kinase than ASi. FFSi generated more ROS than ASi when incubated with the cells as measured by electron spin resonance (ESR). Studies using ROS-sensitive dyes and oxygen consumption support the conclusion that ROS are generated by silica-treated cells. N-Acetylcysteine (an antioxidant) and polyvinyl pyridine-N-oxide (an agent that binds to Si-OH groups on silica surfaces) decreased AP-1 activation and phosphorylation of ERKs and p38 kinase. Catalase inhibited phosphorylation of ERKs and p38 kinase, as well as AP-1 activation induced by FFSi, suggesting the involvement of H(2)O(2) in the mechanism of silica-induced AP-1 activation. Sodium formate (an ( small middle dot)OH scavenger) had no influence on silica-induced MAPKs or AP-1 activation. Superoxide dismutase enhanced both AP-1 and MAPKs activation, indicating that H(2)O(2), but not O(2), may play a critical role in silica-induced AP-1 activation. These studies indicate that freshly ground silica is more biologically active than aged silica and that ROS, in particular H(2)O(2), play a significant role in silica-induced AP-1 activation
— id: 38419, year: 2001, vol: 276, page: 9108, stat: Journal Article,

Effects of tea polyphenols on the signal transduction pathways
Dong Z; Nomura M; Huang C; Ma WY
2001 ;492(2):55-67, Advances in experimental medicine & biology
— id: 42700, year: 2001, vol: 492, page: 55, stat: Journal Article,

Transactivation of AP-1 in AP-1-luciferase reporter transgenic mice by arsenite and arsenate
Huang C; Bode AM; Chen NY; Ma WY; Li J; Nomura M; Dong Z
2001 Jan-Feb;21(1A):261-267, Anticancer research
Arsenic is a recognized carcinogen, which acts as a tumor promoter rather than as an initiator. Signal transduction pathways leading to activation of AP-1 and mitogen-activated protein kinases are proposed to be responsible for the tumor promotion activity by arsenic. Induction of AP-1 DNA binding activity and c-jun and c-fos expression was reported to be only observed in cells responding to arsenite, but not to arsenate. However, in this study, we found that both arsenite and arsenate could induce transactivation of AP-1 in mouse epidermal JB6 AP-1-luciferase reporter stable transfectants, P+1-1. This induction of AP-1 activity by arsenic appears to be through activation of mitogen-activated protein kinases and protein kinase C because increased AP-1 activity by arsenite could be blocked by either treatment of cells with PD98059 or overexpression of dominant negative protein kinase Ca. Furthermore, both arsenite and arsenate could induce transactivation of AP-1 in AP-1-luciferase reporter transgenic mice
— id: 42701, year: 2001, vol: 21, page: 261, stat: Journal Article,

Vanadium-induced nuclear factor of activated T cells activation through hydrogen peroxide
Huang C; Ding M; Li J; Leonard SS; Rojanasakul Y; Castranova V; Vallyathan V; Ju G; Shi X
2001 Jun 22;276(25):22397-22403, Journal of biological chemistry
The present study investigated the role of reactive oxygen species (ROS) in activation of nuclear factor of activated T cells (NFAT), a pivotal transcription factor responsible for regulation of cytokines, by vanadium in mouse embryo fibroblast PW cells or mouse epidermal Cl 41 cells. Exposure of cells to vanadium led to the transactivation of NFAT in a time- and dose-dependent manner. Scavenging of vanadium-induced H(2)O(2) with N-acety-L-cyteine (a general antioxidant) or catalase (a specific H(2)O(2) inhibitor) or the chelation of vanadate with deferoxamine, resulted in inhibition of NFAT activation. In contrast, an increase in H(2)O(2) generation by the addition of superoxide dismutase or NADPH enhanced vanadium-induced NFAT activation. This vanadate-mediated H(2)O(2) generation was verified by both electron spin resonance and fluorescence staining assay. These results demonstrate that H(2)O(2) plays an important role in vanadium-induced NFAT transactivation in two different cell types. Furthermore, pretreatment of cells with nifedipine, a calcium channel blocker, inhibited vanadium-induced NFAT activation, whereas and ionomycin, two calcium ionophores, had synergistic effects with vanadium for NFAT induction. Incubation of cells with cyclosporin A (CsA), a pharmacological inhibitor of the phosphatase calcineurin, blocked vanadium-induced NFAT activation. All data show that vanadium induces NFAT activation not only through a calcium-dependent and CsA-sensitive pathway but also involved H(2)O(2) generation, suggesting that H(2)O(2) may be involved in activation of calcium-calcineurin pathways for NFAT activation caused by vanadium exposure
— id: 21209, year: 2001, vol: 276, page: 22397, stat: Journal Article,

Hydrogen peroxide mediates activation of nuclear factor of activated T cells (NFAT) by nickel subsulfide
Huang C; Li J; Costa M; Zhang Z; Leonard SS; Castranova V; Vallyathan V; Ju G; Shi X
2001 Nov 15;61(22):8051-8057, Cancer research
Nickel compounds induce cell transformation in cell culture models and tumor formation in experimental animals. However, the molecular mechanisms by which nickel compounds induce tumors are not yet well understood. The present study found that exposure of cells to either Ni(3)S(2) or NiCl(2) could result in specific transactivation of nuclear factor of activated T cells (NFAT), although it did not show any activation of p53 or AP-1. Furthermore, nickel compounds were also able to cause generation of reactive oxygen species (ROS). The scavenging of nickel-induced H(2)O(2) with N-acety-L-cyteine (a general antioxidant) or catalase, or the chelation of nickel with deferoxamine, resulted in inhibition of NFAT activation. In contrast, pretreatment of cells with sodium formate (an .OH radical scavenger) or superoxide dismutase (an O(-.)(2) radical scavenger) did not show any inhibitory effects. These results demonstrate that nickel compounds are able to induce NFAT activation, and that the mechanism of NFAT activation seems to be mediated by the generation of H(2)O(2) by these metal compounds. This study should help us understand the signal transduction pathways involved in carcinogenic effects of these nickel compounds
— id: 26560, year: 2001, vol: 61, page: 8051, stat: Journal Article,

Transactivation of RARE and GRE in the cellular response to arsenic
Huang C; Li J; Ding M; Costa M; Castranova V; Vallyathan V; Ju G; Shi X
2001 Jun;222(1-2):119-125, Molecular & cellular biochemistry
Arsenic compounds are a somewhat unique class of metals, which have been considered as both carcinogens and chemotherapeutic agents for cancers. Tumor promotion effects of arsenic are believed to be associated with its transactivational activities on transcription factors, such as AP-1 and NFkappaB, while the induction of cell apoptosis and differentiation by arsenic is considered to be a mechanism for the chemotherapeutic effects of arsenic. Here, we found that exposure of cells to arsenite and arsenate leads to transactivation of retinoic acid response elements (RARE) and glucocorticoid response elements (GRE) in mouse epidermal JB6 cells. These inductions occur in a time-dependent manner. Furthermore, induction of RARE activity by arsenic was synergistically enhanced by co-treatment of cells with retinoic acid, while GRE activation by arsenic was not affected by combined treatment of cells with fluocinolone acetonide (FA). In consideration of the important role of RARE and GRE in induction of cell differentiation, we speculate that transactivation of RARE and GRE by arsenic may be involved in its induction of cell differentiation and anti-cancer activities in addition to its induction of apoptosis
— id: 38405, year: 2001, vol: 222, page: 119, stat: Journal Article,

UV Induces phosphorylation of protein kinase B (Akt) at Ser-473 and Thr-308 in mouse epidermal Cl 41 cells through hydrogen peroxide
Huang C; Li J; Ding M; Leonard SS; Wang L; Castranova V; Vallyathan V; Shi X
2001 Oct 26;276(43):40234-40240, Journal of biological chemistry
The exposure of mammalian cells to UV irradiation leads to the activation of transcription factors and protein kinases, which are believed to be responsible for the carcinogenic effects of excessive sun exposure. The present study investigated the effect of UV exposure on reactive oxygen species (ROS) generation and protein kinase B (Akt) phosphorylation in epidermal cells and determined if a relationship exists between these UV responses. Exposure of mouse epidermal JB6 Cl 41 cells to UV radiation led to specific phosphorylation of Akt at Ser-473 and Thr-308 in a time-dependent manner. This phosphorylation was confirmed by the observation that overexpression of Akt mutant, Akt-T308/S473A, attenuated phosphorylation of Akt at Ser-473 and Thr-308. UV radiation also generated ROS as measured by electron spin resonance (ESR) in JB6 Cl 41 cells. The generation of ROS by UV radiation was measured further by H(2)O(2) and O(-.2) fluorescence staining assays. The mechanism of ROS generation involved reduction of molecular oxygen to O(-.2), which generated H(2)O(2) through dismutation. H(2)O(2) produced .OH via a metal-independent pathway. The scavenging of UV-generated H(2)O(2) by N-acety-l-cyteine (NAC, a general antioxidant) or catalase (a specific H(2)O(2) inhibitor) inhibited Akt phosphorylation at Ser-473 and Thr-308, whereas the pretreatment of cells with sodium formate (an .OH radical scavenger) or superoxide dismutase (an O(-.2) radical scavenger) did not show any inhibitory effects. Furthermore, treatment of cells with H(2)O(2) increased UV-induced phosphorylation of Akt at Ser-473 and Thr-308. These results demonstrate that UV radiation generates a whole spectrum of ROS including O(-.2), .OH, and H(2)O(2) and induces phosphorylation of Akt at Ser-473. Among the various ROS, H(2)O(2) seems most potent in mediating UV-induced phosphorylation of Akt at Ser-473 and Thr-308. It is possible that Akt may play a role in the carcinogenesis effects by UV radiation
— id: 26594, year: 2001, vol: 276, page: 40234, stat: Journal Article,

Arsenic-induced NFkappaB transactivation through Erks- and JNKs-dependent pathways in mouse epidermal JB6 cells
Huang C; Li J; Ding M; Wang L; Shi X; Castranova V; Vallyathan V; Ju G; Costa M
2001 Jun;222(1-2):29-34, Molecular & cellular biochemistry
Tumor promoting effects of arsenic are believed to be associated with its transactivation activity on transcription factors, such as AP-1 and NFkappaB. However, the results from different groups studying the effects of arsenic on NFkappaB activation are contradictory in different cell models. Since arsenic is a strong skin carcinogen, we have investigated the activation of NFkappaB by arsenic in a mouse skin epidermal cell line, JB6 cells. Exposure of cells to arsenite or arsenate led to NFkappaB transactivation in mouse epidermal JB6 NFkappaB-luciferase reporter stable transfectants, C141 NFkappaB mass1. This induction of NFkappaB activity by arsenic was dose- and time-dependent. The transactivation of NFkappaB by arsenic appeared to be through activation of Erks and JNKs pathways because increased NFkappaB activity by arsenic could be dramatically inhibited by either pre-treatment of cells with PD98059 or overexpression of dominant negative JNK1. That Erks activation is required for arsenic-induced NFkappaB transactivation was further supported by the findings that arsenic-induced NFkappaB transactivation was impaired in JB6 30.7b cells, which were deficient in Erks
— id: 38403, year: 2001, vol: 222, page: 29, stat: Journal Article,

Involvement of sphingomyelinase in insulin-induced phosphatidylinositol 3-kinase activation
Huang C; Ma WY; Ding M; Li J; Shi X; Castranova V; Vallyathan V; Bode AM; Dong Z
2001 Apr;15(6):1113-1114, FASEB journal
— id: 38418, year: 2001, vol: 15, page: 1113, stat: Journal Article,

Involvement of Erks activation in cadmium-induced AP-1 transactivation in vitro and in vivo
Huang C; Zhang Q; Li J; Shi X; Castranova V; Ju G; Costa M; Dong Z
2001 Jun;222(1-2):141-147, Molecular & cellular biochemistry
Cadmium is a potent and effective carcinogen in rodents and has recently been accepted by IARC (International Agency for Research on Cancer) as a category I carcinogen. Cadmium-induced up-regulation of intracellular signaling pathways leading to increased mitogenesis is thought to be a major mechanism for the carcinogenic activity following chronic cadmium exposure. In the present study, we found that exposure of cells to cadmium induced significant activation of AP-1 and all three members of the MAP kinase family in mouse epidermal JB6 cells. The induction of AP-1 activity by cadmium appears to involve activation of Erks, since the induction of AP-1 activity by cadmium was blocked by pretreatment of cells with PD98058. Interestingly, the induction of AP-1 by cadmium was greatly enhanced by the chemical tumor promoter, TPA and the growth factor EGF, but not by ultraviolet C radiation. In vivo studies demonstrated that cadmium could also induce transactivation of AP-1 in AP-1-luciferase report transgenic mice. Considering the role of AP-1 activation in tumor promotion, the results presented in this study provide a possible molecular mechanism for cadmium-induced carcinogenesis
— id: 38404, year: 2001, vol: 222, page: 141, stat: Journal Article,

Vanadate-induced cell growth regulation and the role of reactive oxygen species
Zhang Z; Huang C; Li J; Leonard SS; Lanciotti R; Butterworth L; Shi X
2001 Aug 15;392(2):311-320, Archives of biochemistry & biophysics. ABB
While vanadium compounds are known as potent toxicants as well as carcinogens, the mechanisms of their toxic and carcinogenic actions remain to be investigated. It is believed that an improper cell growth regulation leads to cancer development. The present study examines the effects of vanadate on cell cycle control and involvement of reactive oxygen species (ROS) in these vanadate-mediated responses in a human lung epithelial cell line, A549. Under vanadate stimulation, A549 cells generated hydroxyl radical (*OH), as determined by electron spin resonance (ESR), and hydrogen peroxide (H2O2) and superoxide anion (O2*-), as detected by flow cytometry using specific dyes. The mechanism of ROS generation involved the reduction of molecular oxygen to O2*- by both a flavoenzyme-containing NADPH complex and the mitochondria electron transport chain. The O2*- in turn generated H2O2, which reacted with vanadium(IV) to generate *OH radical through a Fenton-type reaction (V(IV) + H2O2 --> V(V) +*OH + OH-). The ROS generated by vanadate induced G2/M phase arrest in a time- and dose-dependent manner as determined by measuring DNA content. Vanadate also increased p21 and Chk1 levels and reduced Cdc25C expression, leading to phosphorylation of Cdc2 and a slight increase in cyclin B1 expression as analyzed by Western blot. Catalase, a specific antioxidant for H2O2, decreased vanadate-induced expression of p21 and Chk1, reduced phosphorylation of Cdc2Tyr15, and decreased cyclin B1 levels. Superoxide dismutase, a scavenger of O2*-, or sodium formate, an inhibitor of *OH, had no significant effects. The results obtained from the present study demonstrate that among ROS, H2O2 is the species responsible for vanadate-induced G2/M phase arrest. Several regulatory pathways are involved: (1) activation of p21, (2) an increase of Chk1 expression and inhibition of Cdc25C, which results in phosphorylation of Cdc2 and possible inactivation of cyclin B1/Cdc2 complex
— id: 38417, year: 2001, vol: 392, page: 311, stat: Journal Article,

Activation of PKC is required for arsenite-induced signal transduction
Chen NY; Ma WY; Huang C; Ding M; Dong Z
2000 ;19(3):297-305, Journal of environmental pathology, toxicology & oncology
Trivalent arsenic (arsenite) is a human carcinogen. However, the molecular mechanism of arsenite-induced carcinogenesis is still not well understood. In this study, we found that arsenite induced translocation of PKCepsilon, PKCdelta, and PKCalpha from cytosol to membranes. Rottlerin, a selective inhibitor for PKCdelta, and safingol, a specific inhibitor for PKCalpha, both markedly inhibited arsenite-induced AP-1 activity. These inhibitory effects by rottlerin and safingol appeared to be dose dependent. Arsenite-induced phosphorylation of Erks was inhibited by rottlerin, while safingol inhibited arsenite-induced phosphorylation of JNKs and p38 kinases. Dominant negative mutant transfectant of PKCepsilon markedly blocked arsenite-induced AP-1 activity and the phosphorylation of Erks, JNKs, and p38 kinases. These data demonstrate that PKCdelta, PKCepsilon, and PKCalpha mediate arsenite-induced AP-1 activation in JB6 cells through different MAP kinase (Erks, JNKs, and p38 kinases) pathways
— id: 42702, year: 2000, vol: 19, page: 297, stat: Journal Article,

Free radical-mediated transgene inactivation of macrophages by endotoxin
Dokka S; Toledo D; Wang L; Shi X; Huang C; Leonard S; Rojanasakul Y
2000 Nov;279(5):L878-L883, American journal of physiology. Lung cellular & molecular physiology
Endotoxin, the lipopolysaccharide component of gram-negative bacteria, is a common contaminant of plasmid DNA preparations. The present study investigated the effect of endotoxin on gene transfection efficiency and the role of reactive oxygen species (ROS) in this process. Gene transfection studies were performed in various cell types with cytomegalovirus-luciferase as a reporter plasmid and cationic liposome as a transfecting agent. The presence of endotoxin in plasmid DNA preparations severely limited transgene expression in macrophages but had little or no effect in other cell types tested. This decreased transfection was dependent on ROS-mediated cellular toxicity induced by endotoxin. Neutralizing the endotoxin by the addition of polymyxin B effectively increased transfection efficiency and reduced toxicity. Electron spin resonance studies confirmed the formation of ROS in endotoxin-treated cells and their inhibition by free radical scavengers. The ROS scavenger N-t-butyl-alpha-phenylnitrone, the H(2)O(2) scavenger catalase, and the.OH scavenger sodium formate effectively inhibited endotoxin-induced effects, whereas the O(2)(-) scavenger superoxide dismutase had lesser effects. These results indicate that multiple oxidative species are involved in the transfection inactivation process and that.OH formed by H(2)O(2)-dependent, metal-catalyzed Fenton reaction play a major role in this process
— id: 38420, year: 2000, vol: 279, page: L878, stat: Journal Article,

Inhibition of atypical PKC blocks ultraviolet-induced AP-1 activation by specifically inhibiting ERKs activation
Huang C; Li J; Chen N; Ma W; Bowden GT; Dong Z
2000 Feb;27(2):65-75, Molecular carcinogenesis
Since ultraviolet (UV) radiation is a major etiologic factor in the development of human skin cancers, investigating the signal transduction pathways initiated by UV radiation may help with the understanding of the molecular mechanisms of UV-induced carcinogenesis. Our previous studies demonstrated that UV-induced activator protein-1 (AP-1) activation is blocked by dominant negative atypical PKCs (aPKCs). Here we investigated the role of aPKC in UV-induced activation of mitogen activated protein (MAP) kinase family members which are considered to be the mediators of AP-1 activation. We found that UV radiation led to translocation of protein kinase C (PKC) zeta and activation of MAP kinase family members as well as an increase of AP-1-dependent transcription activation at the same dose range. Pretreatment of cells or mouse skin with antisense oligonucleotides of PKCzeta impaired UV-induced activation of AP-1 in JB6 cells as well as in AP-1-luciferase transgenic mice. It also inhibited UV-induced activation of ERKs but not of JNK and p38 kinases in JB6 cells. In contrast, no significant inhibition of AP-1 activation and MAP kinase activation were observed in cells treated with sense oligonucleotides of PKCzeta. Furthermore, overexpression of a dominant negative mutant of PKClambda/iota specifically inhibited activation of extracellular signal-regulated protein kinases (ERKs) but not of c-jun N-terminal kinases (JNKs) nor p38 kinases induced by UV radiation. These results demonstrated that inhibition of aPKC impairs UV-induced AP-1 activation via suppression of ERKs activation but not of JNKs or p38 kinase activation
— id: 42720, year: 2000, vol: 27, page: 65, stat: Journal Article,

Involvement of nuclear factor of activated T cells activation in UV response. Evidence from cell culture and transgenic mice
Huang C; Mattjus P; Ma WY; Rincon M; Chen NY; Brown RE; Dong Z
2000 Mar 31;275(13):9143-9149, Journal of biological chemistry
Mammalian cells respond to UV radiation by signaling cascades leading to activation of transcription factors, such as activated protein 1, NFkappaB, and p53, a process known as the 'UV response.' Nuclear factor of activated T cells (NFAT) was first identified as an inducible nuclear factor in immune response and subsequently found to be expressed in other tissues and cells. To date, however, the regulation and function of NFAT in tissues and cells, other than the immune system, are not well understood. In this study, we demonstrate that UV radiation activates NFAT-dependent transcription through a calcium-dependent mechanism in mouse epidermal JB6 cell lines, as well as in the skin of NFAT-luciferase reporter transgenic mice. Exposure of JB6 cells to UV radiation leads to the transactivation of NFAT in a dose-dependent manner. A23187 had a synergistic effect with UV for NFAT induction, whereas pretreatment of cells with nifedipine, a calcium channel blocker, dramatically impaired the NFAT activity induced by either UV or UV plus A23187. Calcium-dependent activation of NFAT by UV was further confirmed by an in vivo study using NFAT-luciferase reporter transgenic mice. These results demonstrated that UV radiation is a strong activator for skin NFAT transactivation through calcium-dependent pathways, suggesting that NFAT activation may be a part of the UV response
— id: 34184, year: 2000, vol: 275, page: 9143, stat: Journal Article,

Vanadate induces p53 transactivation through hydrogen peroxide and causes apoptosis
Huang C; Zhang Z; Ding M; Li J; Ye J; Leonard SS; Shen HM; Butterworth L; Lu Y; Costa M; Rojanasakul Y; Castranova V; Vallyathan V; Shi X
2000 Oct 20;275(42):32516-32522, Journal of biological chemistry
Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms controlling vanadate-induced adverse effects remain to be elucidated. The present study investigated the vanadate-induced p53 activation and involvement of reactive oxygen species (ROS) in p53 activation as well as the role of p53 in apoptosis induction by vanadate. Exposure of mouse epidermal JB6 cells to vanadate led to transactivation of p53 activity in a time- and dose-dependent manner. It also caused mitochondrial damage, apoptosis, and generated ROS. Scavenging of vanadate-induced H(2)O(2) by N-acetyl-l-cysteine (a general antioxidant) or catalase (a specific H(2)O(2) inhibitor), or the chelation of vanadate by deferoxamine, resulted in inhibition of p53 activation and cell mitochondrial damage. In contract, an increase in H(2)O(2) generation in response to superoxide dismutase or NADPH enhanced these effects caused by vanadate. Furthermore, vanadate-induced apoptosis occurred in cells expressing wild-type p53 (p53+/+) but was very weak in p53-deficient (p53-/-) cells. These results demonstrate that vanadate induces p53 activation mainly through H(2)O(2) generation, and this activation is required for vanadate-induced apoptosis
— id: 34185, year: 2000, vol: 275, page: 32516, stat: Journal Article,

Activity of p44/42 MAP kinase in the caudal subnucleus of trigeminal spinal nucleus is increased following perioral noxious stimulation in the mouse
Huang, W J; Wang, B R; Yao, L B; Huang, C S; Wang, X; Zhang, P; Jiao, X Y; Duan, X L; Chen, B F; Ju, G
2000 Apr 7;861(1):181-185, Brain research
The extracellular signal-regulated protein kinase-1 and -2 (ERK1 and ERK2), also referred to as the p44/42 mitogen-activated protein kinase (p44/42 MAP kinase), plays an essential role in neuronal signal transduction, but its function involved in nociceptive response has not been deeply studied yet. Here we report immunohistochemical evidence that p44/42 MAPK might be critical in nociceptive response. We found that after formalin was injected into the perioral skin of the upper lip of mice, the number of activated p44/42 MAPK-like immunoreactive neurons was significantly increased in the laminae I and II of the caudal subnucleus of the trigeminal spinal nucleus (Sp5C). The positive neurons and fibers were mostly concentrated in the middle portion of Sp5C dorsoventrally, where the afferent fibers innervating the skin of the upper lip are terminated. The reactive products were localized in perikarya, dendrites, nuclei, and diffusely in the neuropil. The present result suggests that p44/42 MAPK may be important in the transmission and modulation of noxious information in Sp5C
— id: 106251, year: 2000, vol: 861, page: 181, stat: Journal Article,

Inhibition of ultraviolet B-induced AP-1 activation by theaflavins from black tea
Nomura M; Ma WY; Huang C; Yang CS; Bowden GT; Miyamoto K; Dong Z
2000 Jul;28(3):148-155, Molecular carcinogenesis
Theaflavins are believed to be key active components in black tea for chemoprevention of cancer. However, the molecular mechanisms underlying the inhibitory effects of theaflavins are not clear. With the JB6 mouse epidermal cell line, we investigated the effects of theaflavins on ultraviolet (UV) B radiation-induced activator protein-1 (AP-1)-dependent transcriptional activation and compared them with (-)-epigallocatechin-3-gallate (EGCG), a major green tea polyphenol that has cancer chemopreventative activity. Theaflavins and EGCG inhibited UVB-induced AP-1 activation in a concentration-dependent manner. The inhibitory effects of theaflavins were stronger than those of EGCG. We found that theaflavins significantly inhibited activation of extracellular signal-regulated protein kinases and c-jun NH(2)-terminal kinases. Because the transcription factor AP-1 is important in the process of tumor promotion, the inhibitory effect of these polyphenols on AP-1 activation may further explain the anti-tumor promotion action of these tea constituents. Mol. Carcinog. 28:148-155, 2000
— id: 42703, year: 2000, vol: 28, page: 148, stat: Journal Article,

Translocation of protein kinase Cepsilon and protein kinase Cdelta to membrane is required for ultraviolet B-induced activation of mitogen-activated protein kinases and apoptosis
Chen N; Ma W; Huang C; Dong Z
1999 May 28;274(22):15389-15394, Journal of biological chemistry
UV-induced signal transduction may be involved in tumor promotion and induction of apoptosis. The role of protein kinase C (PKC) in UVB-induced signal transduction is not well understood. This study showed that UVB markedly induced translocation of membrane-associated PKCepsilon and PKCdelta, but not PKCalpha, from cytosol to membrane. Dominant negative mutant (DNM) PKCepsilon or PKCdelta inhibited UVB-induced translocation of PKCepsilon and PKCdelta, respectively. UVB-induced activation of extracellular signal-regulated protein kinases (Erks) and c-Jun NH2-terminal kinases (JNKs) was strongly inhibited by DNM PKCepsilon and PKCdelta, whereas the DNM of PKCalpha was less effective on the UVB-induced phosphorylation of Erks and JNKs. Among the PKC inhibitors used only rottlerin, a selective inhibitor of PKCdelta, markedly inhibited the UVB-induced activation of Erks and JNKs, but not p38 kinases. Safingol, a selective inhibitor for PKCalpha, did not show any inhibitory effect on UVB-induced mitogen-activated protein kinase activation. GF109203X is a stronger inhibitor of classical PKC than novel PKC. Lower concentrations of GF109203X (<10 microM) had no effect on UVB-induced activation of Erks or JNKs. However, at higher concentrations (over 20 microM), GF109203X inhibited UVB-induced activation of JNKs, Erks, and even p38 kinases. Meanwhile, rottlerin and GF109203X markedly inhibited UVB-induced apoptosis of JB6 cells, whereas safingol had little inhibitory effect. DNM-Erk2 cells and PD98059, a selective inhibitor for mitogen-activated protein kinase/extracellular signal-regulated kinase 1 that directly activates Erks, inhibited UVB-induced apoptosis. DNM-JNK1 cells also blocked UVB-induced apoptosis, whereas SB202190, a specific inhibitor for p38 kinases, did not produce the inhibitory effect. These data demonstrate that PKCdelta and PKCepsilon, but not PKCalpha, mediate UVB-induced signal transduction and apoptosis in JB6 cells through activation of Erks and JNKs
— id: 42726, year: 1999, vol: 274, page: 15389, stat: Journal Article,

Inhibition of activator protein 1 activity and cell growth by purified green tea and black tea polyphenols in H-ras-transformed cells: structure-activity relationship and mechanisms involved
Chung JY; Huang C; Meng X; Dong Z; Yang CS
1999 Sep 15;59(18):4610-4617, Cancer research
ras gene mutation, which perpetually turns on the growth signal transduction pathway, occurs frequently in many cancer types. The mouse epidermal JB6 cell line has been transfected with a mutant H-ras gene to mimic carcinogenesis in vitro. These transformed cells (30.7b Ras 12) are able to grow in soft agar, exhibiting anchorage independence and high endogenous activator protein 1 (AP-1) activity, which can be detected by a stable AP-1 luciferase reporter. The present study investigated the ability of different pure green and black tea polyphenols to inhibit this ras signaling pathway. The major green tea polyphenols (catechins), (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin, (-)-epicatechin-3-gallate, (-)-epicatechin, and their epimers, and black tea polyphenols, theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'-digallate (TFdiG), were compared with respect to their ability to inhibit the growth of 30.7b Ras 12 cells and AP-1 activity. All of the tea polyphenols except (-)-epicatechin showed strong inhibition of cell growth and AP-1 activity. Among the catechins, both the galloyl structure on the B ring and the gallate moiety contributed to the growth inhibition and AP-1 activity; the galloyl structure appeared to have a stronger effect on the inhibitory action than the gallate moiety. The epimers of the catechins showed similar inhibitory effects on AP-1 activity. The addition of catalase to the incubation of the cells with EGCG or TFdiG did not prevent the inhibitory effect on AP-1 activity, suggesting that H2O2 does not play a significant role in the inhibition by tea polyphenols. Both EGCG and TFdiG inhibited the phosphorylation of p44/42 (extracellular signal-regulated kinase 1 and 2) and c-jun without affecting the levels of phosphorylated-c-jun-NH2-terminal kinase. TFdiG inhibited the phosphorylation of p38, but EGCG did not. EGCG lowered the level of c-jun, whereas TFdiG decreased the level of fra-1. These results suggest that tea polyphenols inhibited AP-1 activity and the mitogen-activated protein kinase pathway, which contributed to the growth inhibition; however, different mechanisms may be involved in the inhibition by catechins and theaflavins
— id: 42724, year: 1999, vol: 59, page: 4610, stat: Journal Article,

PI-3 kinase in signal transduction, cell transformation, and as a target for chemoprevention of cancer
Dong Z; Huang C; Ma WY
1999 Sep-Oct;19(5A):3743-3747, Anticancer research
Phosphatidylinositol-3 kinase (PI-3 K) plays a central role in a broad range of biological effects. However, little is known about its role in phorbol ester- or epidermal growth factor (EGF)-induced signal transduction to the transcriptional machinery of the nucleus and in tumor promoter-induced cell transformation. We have used JB6 cells to study the role of PI-3 K in 12-O-tetradecanoylphorbol-13-acetate (TPA)- or EGF-induced AP-1 activation and neoplastic cell transformation. We demonstrated that TPA, EGF and insulin induce PI-3 K activity in JB6 cells. The induced PI-3 K activity was blocked by a dominant negative mutant of PI-3 K, and by wortmannin or LY294002. Blocking of PI-3 K activity by these inhibitors also blocked TPA- or EGF-induced AP-1 activity and cell transformation. Furthermore, we have investigated the role of PKC and its isozymes in the synergistic induction of PI-3 K by TPA and insulin and found that bisindolylmaleimide, a PKC inhibitor, inhibits TPA-induced PI-3 K. Overexpression of a dominant negative PKC epsilon, but not dominant negative PKC alpha, blocks the TPA- or TPA plus insulin-induced PI-3 K activity. Inositol hexaphosphate (InsP6) is one of the most promising chemopreventive agents as demonstrated by Shamsuddin et al. and others. InsP6 profoundly inhibits EGF- or TPA-induced cell transformation and the signal transduction cascade to Erks and AP-1 activation. InsP6 also inhibits TPA- or EGF-induced PI-3 K activity in vivo and in vitro. These results suggest that the anticarcinogenesis action of InsP6 may be through inhibition of PI-3 K and inhibition of the AP-1 pathway. Because InsP6 is a naturally occurring compound with virtually no toxicity, and may be an effective anticarcinogenesis agent in humans, PI-3 K and AP-1 activities may be useful biomarkers for the effectiveness of InsP6 in clinical studies
— id: 42704, year: 1999, vol: 19, page: 3743, stat: Journal Article,

JNK activation is required for JB6 cell transformation induced by tumor necrosis factor-alpha but not by 12-O-tetradecanoylphorbol-13-acetate
Huang C; Li J; Ma WY; Dong Z
1999 Oct 15;274(42):29672-29676, Journal of biological chemistry
Signal transduction via mitogen-activated protein kinase pathways plays a key role in a variety of cellular responses, including cell proliferation, differentiation, tumor promotion, and cell death. c-Jun N-terminal kinases (JNKs) are identified as members of the mitogen-activated protein kinase family and are known to phosphorylate and activate several transcription factors, including c-Jun, ATF, and Elk-1. However, the role of JNK activation in tumor promotion is not yet defined. Because previous studies have indicated that exposure of JB6 Cl 41 cells to either 12-O-tetradecanoylphorbol-13-acetate (TPA) or tumor necrosis factor-alpha (TNF-alpha) results in cell transformation, we investigated the role of JNKs in this biological process by using dominant negative JNK(1) and the cell transformation model JB6 Cl 41 cells. Incubation of Cl 41 cells with TNF-alpha led to cell transformation and activation of JNKs. Introduction of the dominant negative mutant of JNK(1) into JB6 Cl 41 cells specifically inhibited TNF-alpha-induced activation of JNKs, but not Erks and p38 kinases. Most importantly, expressing dominant negative mutant JNK(1) inhibited TNF-alpha-induced cell transformation but not TPA-induced cell transformation. Our results directly demonstrated for the first time that JNK activation is required for TNF-alpha- but not TPA-induced cell transformation
— id: 34183, year: 1999, vol: 274, page: 29672, stat: Journal Article,

The extracellular-signal-regulated protein kinases (Erks) are required for UV-induced AP-1 activation in JB6 cells
Huang C; Ma WY; Dong Z
1999 May 6;18(18):2828-2835, Oncogene
Mitogen activated protein (MAP) kinase belongs to a large family of serine/threonine protein kinases, including extracellular-signal-regulated protein kinases (Erks), P38 kinase and c-Jun N-terminal kinases (JNKs). Although previous work has shown that both Erks and JNKs are activated in cells in response to ultraviolet (UV) irradiation, most studies have focused only on the role of JNKs in UV-induced AP-1 activation. Hence, the role of Erks in UV-induced AP-1 activity is not well defined. We here have investigated this issue by using MAP kinase kinase (MEK1) inhibitor PD098059 and a dominant negative Erk2, as well as wild-type Erk2, in a JB6 cell model. PD098059 inhibited UVB- or UVC-induced AP-1 activity and phosphorylation of MEK1 and Erks, but not JNKs, in JB6 Cl 41 cells. Overexpression of wild-type Erk2 in Cl 30.7b cells that contain small amounts of Erks caused a 46.6- or 138.1-fold increase of AP-1 activity by UVB and UVC, respectively; introduction of a dominant negative Erk2 into Cl 41 cells significantly blocked the UV-induced Erks activation as well as the AP-1 activation. In contrast, overexpression of wild-type Erk2 in Cl 30.7b cells and dominant negative Erk2 in Cl 41 cells did not show a marked influence on the phosphorylation of JNKs. These results demonstrate that activation of Erks, in addition to the previously reported JNKs, is required for UV-induced AP-1 activation
— id: 42706, year: 1999, vol: 18, page: 2828, stat: Journal Article,

Resveratrol suppresses cell transformation and induces apoptosis through a p53-dependent pathway
Huang C; Ma WY; Goranson A; Dong Z
1999 Feb;20(2):237-242, Carcinogenesis
Resveratrol, a plant constituent enriched in the skin of grapes, is one of the most promising agents for the prevention of cancer. However, the mechanism of the anti-carcinogenic activity of resveratrol is not well understood. Here we offer a possible explanation of its anti-cancer effect. Resveratrol suppresses tumor promoter-induced cell transformation and markedly induces apoptosis, transactivation of p53 activity and expression of p53 protein in the same cell line and at the same dosage. Also, resveratrol-induced apoptosis occurs only in cells expressing wild-type p53 (p53+/+), but not in p53-deficient (p53-/-) cells, while there is no difference in apoptosis induction between normal lymphoblasts and sphingomyelinase-deficient cell lines. These results demonstrate for the first time that resveratrol induces apoptosis through activation of p53 activity, suggesting that its anti-tumor activity may occur through the induction of apoptosis
— id: 42698, year: 1999, vol: 20, page: 237, stat: Journal Article,

Arsenic induces apoptosis through a c-Jun NH2-terminal kinase-dependent, p53-independent pathway
Huang C; Ma WY; Li J; Dong Z
1999 Jul 1;59(13):3053-3058, Cancer research
Arsenic has been used as an effective chemotherapy agent for some human cancers, such as acute promyelocytic leukemia. In this study, we found that arsenic induces activation of c-Jun NH2-terminal kinases (JNKs) at a similar dose range for induction of apoptosis in JB6 cells. In addition, we found that arsenic did not induce p53-dependent transactivation. Similarly, there was no difference in apoptosis induction between cells with p53 +/+ or p53 -/-. In contrast, arsenic-induced apoptosis was almost totally blocked by expression of a dominant-negative mutant of JNK1. These results suggest that the activation of JNKs is involved in arsenic-induced apoptosis of JB6 cells. Taken together with previous findings that p53 mutations are involved in approximately 50% of all human cancers and nearly all chemotherapeutic agents kill cancer cells mainly by apoptotic induction, we suggest that arsenic may be a useful agent for the treatment of cancers with p53 mutation
— id: 42705, year: 1999, vol: 59, page: 3053, stat: Journal Article,

Requirement of Erk, but not JNK, for arsenite-induced cell transformation
Huang C; Ma WY; Li J; Goranson A; Dong Z
1999 May 21;274(21):14595-14601, Journal of biological chemistry
Trivalent arsenic (arsenite, As3+) is a human carcinogen, which is associated with cancers of skin, lung, liver, and bladder. However, the mechanism by which arsenite causes cancer is not well understood. In this study, we found that exposure of Cl 41 cells, a well characterized mouse epidermal cell model for tumor promotion, to a low concentration of arsenite (<25 microM) induces cell transformation. Interestingly, arsenite induces Erk phosphorylation and increased Erk activity at doses ranging from 0.8 to 200 microM, while higher doses (more than 50 microM) are required for activation of JNK. Arsenite-induced Erk activation was markedly inhibited by introduction of dominant negative Erk2 into cells, while expression of dominant negative Erk2 did not show inhibition of JNK and MEK1/2. Furthermore, arsenite-induced cell transformation was blocked in cells expressing the dominant negative Erk2. In contrast, overexpression of dominant negative JNK1 was shown to increase cell transformation even though it inhibits arsenite-induced JNK activation. Our results not only show that arsenite induces Erk activation, but also for the first time demonstrates that activation of Erk, but not JNK, by arsenite is required for its effects on cell transformation
— id: 34182, year: 1999, vol: 274, page: 14595, stat: Journal Article,

p38 kinase mediates UV-induced phosphorylation of p53 protein at serine 389
Huang C; Ma WY; Maxiner A; Sun Y; Dong Z
1999 Apr 30;274(18):12229-12235, Journal of biological chemistry
The p53 tumor suppressor protein is a transcription factor that plays a key role in the process of apoptosis and the cell's defense against tumor development. Activation of p53 occurs, at least in part, by phosphorylation of its protein. Very recently it has been reported that UV induced a functional activation of p53 via phosphorylation at serine 389. Here, we report that the UV-induced phosphorylation of p53 at serine 389 is mediated by p38 kinase. UVC-induced phosphorylation of p53 at serine 389 was markedly impaired by either pretreatment of cells with p38 kinase inhibitor, SB202190, or stable expression of a dominant negative mutant of p38 kinase. In contrast, there was no inhibition observed in cells treated with specific MEK1 inhibitor, PD98059, or with stable expression of a dominant negative mutant of ERK2 or JNK1. Most importantly, p38 kinase could be co-immunoprecipitated with p53 by using antibodies against p53. Incubation of active p38 kinase with p53 protein caused the phosphorylation of p53 protein at serine 389 in vitro, while no phosphorylation of p53 at serine 389 was observed when p53 was incubated with activated JNK2 or ERK2. Furthermore, pretreatment of cells with SB202190 blocked the p53 DNA binding activity and p53-dependent transcription. These results strongly suggest that the p38 kinase is at least one of the most important mediators of p53 phosphorylation at serine 389 induced by UVC radiation
— id: 34181, year: 1999, vol: 274, page: 12229, stat: Journal Article,

The role of hydroxyl radical as a messenger in the activation of nuclear transcription factor NF-kappaB
Shi X; Dong Z; Huang C; Ma W; Liu K; Ye J; Chen F; Leonard SS; Ding M; Castranova V; Vallyathan V
1999 Apr;194(1-2):63-70, Molecular & cellular biochemistry
Although it is generally believed that reactive oxygen species activate NF-kappaB, a primary oxidative stress-responsive transcription factor, it is unclear which one among these species causes NF-kappaB activation. Our hypothesis is that hydroxyl radical (*OH) functions as a messenger for the activation of NF-kappaB. Jurkat cells, macrophages and JB6 cells were used to test this hypothesis. Cr(VI), silica and ZnO were used as sources of *OH radicals. None of these *OH generating systems involves exogenous H2O2. Cr(VI) expressed enhanced activity in induction of NF-kappaB in Jurkat cells. This activation of NF-kappaB was decreased by a metal chelator, diethylene triaminepentaacetic acid or a H2O2 scavenger, catalase, but was increased by superoxide dismutase. Mn(II), which reacts with Cr(IV) to inhibit this metal ion-mediated *OH generation, decreased the NF-kappaB activation. Sodium formate, an *OH radical scavenger, also inhibited the NF-kappaB activation. Electron spin resonance measurements show that Cr(VI) was reduced by Jurket cells to Cr(IV) and Cr(V). During the reduction process, molecular oxygen was reduced to O2 and then to H2O2, which reacted with Cr(IV) and Cr(V) to generate *OH radical. The *OH generation correlated with the Cr(VI)-induced NF-kappaB activation. Similarly, silica caused NF-kappaB activation in macrophages via the *OH radical-mediated reaction. This radical was generated via metal mediated reaction from H2O2, which was generated by the reduction of molecular oxygen via O2- as an intermediate during the silica-stimulated 'respirable burst'. Silica particles did not cause *OH generation either in Jurket or in JB6 cells and thus did not cause any observable NF-kappaB activation in these cells. ZnO induced NF-kappaB activation in JB6 cells through the generation of *OH resulting from light irradiation of ZnO which was measured by electron spin resonance. The results thus show that *OH radical functions as a messenger for NF-kappaB activation. Antioxidants, which scavenge *OH radical or its precursors, inhibit NF-kappaB activation. Metal chelators, which make metal ions incapable of generating *OH from H2O2, inhibit activation of this transcription factor
— id: 38421, year: 1999, vol: 194, page: 63, stat: Journal Article,

Inhibitory effects of perillyl alcohol on UVB-induced murine skin cancer and AP-1 transactivation
Barthelman M; Chen W; Gensler HL; Huang C; Dong Z; Bowden GT
1998 Feb 15;58(4):711-716, Cancer research
The monoterpene perillyl alcohol (POH) has proven efficacious against the formation and progression of a variety of cancers. In this study, we tested the ability of POH to inhibit photocarcinogenesis in a nonmelanoma model of mouse skin carcinogenesis and its ability to inhibit UVB-induced activator protein 1 (AP-1) transactivation in mouse skin and human keratinocytes. POH (10 mM) was applied topically to the ears and shaved dorsal surface of groups of 35 BALB/c mice throughout the experiment, during and after UVB treatment. Topical POH significantly inhibited tumor incidence and multiplicity, average tumor size, and the average tumor burden/mouse without any apparent toxicity. POH inhibited UVB-induced AP-1 transactivation in both cultured human keratinocytes and transgenic mice that stably express a luciferase reporter driven by AP-1 elements. The results suggest that POH might be used for chemoprevention of human skin cancer, and that inhibition of AP-1 activity is functionally related to inhibition of skin carcinogenesis
— id: 42721, year: 1998, vol: 58, page: 711, stat: Journal Article,

Increased synthesis of phosphocholine is required for UV-induced AP-1 activation
Dong Z; Huang C; Ma WY; Malewicz B; Baumann WJ; Kiss Z
1998 Oct 8;17(14):1845-1853, Oncogene
Exposure of mammalian cells to UV irradiation stimulates phosphatidylcholine hydrolysis and activates the transcription factor AP-1. Since phosphocholine (PCho), a phospholipid metabolite, is a potential regulator of mitogenesis and carcinogenesis, we examined the effect of UV exposure on the formation of PCho and the possible mediatory role of PCho in UVB-and UVC-induced activation of AP-1 in mouse JB6 epidermal cells. We found that both UVB and UVC irradiation resulted in increased PCho levels. Hemicholinium-3 (HC-3), an inhibitor of choline kinase, strongly inhibited UV-induced AP-1 activity. By contrast, relatively low levels of PCho (80 microM) or choline (20 microM) nearly doubled UV-induced AP-1 activity, while higher (2-20 mM) concentrations of PCho alone stimulated AP-1 activity 6-8-fold. Importantly, HC-3 inhibited only the stimulatory effect of choline, but not of PCho, on AP-1 activity. Of the mitogen-activated protein (MAP) kinases involved in the regulation of AP-1 activity, UVC stimulated the MAP kinase family ERK-1/ERK-2, JNK as well as p38 kinase activity. These UVC effects were all inhibited by HC-3. With UVB, by contrast, only the activation of ERK-1/ERK-2 was inhibited by HC-3. The data suggest that increased formation of PCho is required for UV-induced activation of AP-1 by an ERK-1/ERK-2-dependent mechanism
— id: 42707, year: 1998, vol: 17, page: 1845, stat: Journal Article,

Vanadium induces AP-1- and NFkappB-dependent transcription activity
Huang C; Chen N; Ma WY; Dong Z
1998 Oct;13(4):711-715, International journal of oncology
Vanadate has been reported to be involved in the causation of cancer. In this study, we found that both AP-1 and NFkappaB activities were increased after treatment with sodium vanadate in JB6 cells. Maximum induction of AP-1 and NFkappaB appeared at 48 to 72 h. Phosphorylations of Erks and p38 kinases were markedly increased at 100 microM of vanadate, while phosphorylation of JNKs was not affected. Vanadate also enhances the phosphorylation of IkappaBalpha. These results suggest that the activation of AP-1 and NFkappaB by vanadate may be mediated through enhancement of phosphorylation of Erk/p38 kinases and IkappaBalpha, respectively
— id: 42708, year: 1998, vol: 13, page: 711, stat: Journal Article,

Potentiation of insulin-induced phosphatidylinositol-3 kinase activity by phorbol ester is mediated by protein kinase C epsilon
Huang C; Ma WY; Dong Z
1998 Mar;10(3):185-190, Cellular signalling
Our previous results have demonstrated that phorbol 12-myristate 13-acetate (TPA) and insulin synergistically stimulate the activity of phosphatidylinositol-3 kinase (PI-3 kinase) and PI-3 kinase plays an important role in both of TPA-induced AP-1 activation and cell transformation in tumour promotion sensitive (P+) JB6 cells. In the present study, we investigated the role of PKC and its isozymes in the synergistic induction of PI-3 kinase by TPA and insulin. Bisindolylmaleimide inhibits TPA- and TPA+ insulin-induced PI-3 kinase activity. Pretreatment of cells for 24 h with TPA has significant inhibitory effects on TPA-induced PI-3 kinase activity and abolishes the synergistic effect of TPA and insulin-stimulated PI-3 kinase activity. Furthermore, overexpression of a dominant negative PKC epsilon, but not dominant negative PKC alpha, blocks the synergistic effect of TPA and insulin-induced PI-3 kinase activity. These results indicate that the potentiation effect of TPA on insulin-induced PI-3 kinase activity is specific through PKC epsilon in JB6 cells
— id: 42709, year: 1998, vol: 10, page: 185, stat: Journal Article,

Essential role of p53 in phenethyl isothiocyanate-induced apoptosis
Huang C; Ma WY; Li J; Hecht SS; Dong Z
1998 Sep 15;58(18):4102-4106, Cancer research
Phenethyl isothiocyanate (PEITC) is a natural product that is among the most effective cancer chemopreventive agents known. Mechanistic studies indicate that the chemopreventive activity of PEITC is associated with its favorable modification of carcinogen metabolism and its induction of apoptosis. Here, we found that PEITC blocks tumor promoter (12-O-tetradecanoylphorbol-13-acetate or epidermal growth factor)-induced cell transformation in mouse epidermal JB6 cells, and this inhibitory activity on cell transformation is correlated with induction of apoptosis. Most importantly, apoptosis induction by PEITC occurs through a p53-dependent pathway. This was demonstrated not only by results that PEITC induction of p53 protein expression and p53-dependent transactivation but also by PEITC-induced apoptosis in p53 +/+ cells but not in p53 -/- cells. In contrast, PEITC induced apoptosis in cells with both normal or deficient sphingomyelinase activity. Our results demonstrate for the first time that p53 elevation is required for PEITC-induced apoptosis, which may be involved in its cancer chemopreventive activity
— id: 34180, year: 1998, vol: 58, page: 4102, stat: Journal Article,

Shortage of mitogen-activated protein kinase is responsible for resistance to AP-1 transactivation and transformation in mouse JB6 cells
Huang C; Ma WY; Young MR; Colburn N; Dong Z
1998 Jan 6;95(1):156-161, Proceedings of the National Academy of Sciences of the United States of America
The JB6 mouse epidermal cell system, which includes tumor promotion-sensitive (P+) and tumor promotion-resistant (P-) cells, is a well-established and extensively used cell culture model for studying the mechanism of late-stage tumor promotion. Tumor promoters, such as 12-O-tetradecanoylphorbol 13-acetate (TPA) or epidermal growth factor (EGF), induce high levels of activator protein 1 (AP-1) activity and large, tumorigenic, anchorage-independent colonies in soft agar at a high frequency in JB6 P+ cells, but not in JB6 P- cells. We report here a molecular explanation for the defect in the AP-1 activation and promotion-resistant phenotype of P- cells. We demonstrate that the lack of AP-1 activation and cell transformation responses to TPA and EGF in P- cells appears attributable to the low level of mitogen-activated protein kinase (MAPK) (extracellular signal-regulated protein kinase, Erk) in these cells. TPA and EGF induce transactivation of AP-1 activity in P+ cells but not in P- cells. Nonphosphorylated forms and TPA- or EGF-induced phosphorylated forms of Erks (Erk1 and Erk2) in P- cells were much lower than those in P+ cells. Stable transfection of wild-type MAPK (Erk2) into P- cells restored its response to TPA and EGF for both AP-1 activation and cell transformation. These results suggest that the shortage of MAPK (Erk1 and Erk2) appears to be an important contributor to the tumor promotion-resistant phenotype in JB6 cells
— id: 34179, year: 1998, vol: 95, page: 156, stat: Journal Article,

Inhibition of ultraviolet C irradiation-induced AP-1 activity by aspirin is through inhibition of JNKs but not erks or P38 MAP kinase
Ma WY; Huang C; Dong Z
1998 Mar;12(3):565-568, International journal of oncology
The exposure of mammalian cells to ultraviolet (UV) irradiation leads to the activation of transcription factors, such as AP-1 and NFkB. We demonstrate that aspirin, a promising cancer chemopreventative agent, inhibited UVC-induced AP-1 activity in JB6 cells. In JB6 cells, UVC stimulated Erks, JNKs and P38 kinase activities; aspirin only inhibited activation of JNKs, but not the other MAP kinases. Since the transcription factor AP-1 is important for the process of tumor promotion, the inhibitory effect of aspirin on AP-1 activation suggests that it can be used as a chemopreventative agent against skin cancer
— id: 42710, year: 1998, vol: 12, page: 565, stat: Journal Article,

Expression of dominant negative Erk2 inhibits AP-1 transactivation and neoplastic transformation
Watts RG; Huang C; Young MR; Li JJ; Dong Z; Pennie WD; Colburn NH
1998 Dec 31;17(26):3493-3498, Oncogene
The mitogen activated protein (MAP) kinases or extracellular signal-regulated kinases (Erks) are activated in response to Ras expression or exposure to tumor promoters or to growth factors, and have been implicated in AP-1 transactivation in some models. We have shown that tumor promoter induced activation of the transcription factor AP-1 is required for induced neoplastic transformation in the Balb/C JB6 cell model. Jun and Fos family protein levels have been found not to be limiting for AP-1 response. The present study asks whether activation of Erks1 and 2 is required for AP-1 transactivation and transformation of JB6 cells and whether Erks might be targeted for cancer prevention. Expression of either of two different dominant negative kinase inactive Erk2 mutants in transformation sensitive (P+) JB6 cells substantially inhibited the tumor promoter induced activation of Erks1 and 2 and of AP-1 measured by a collagenase-luciferase reporter. Multiple mutant Erk2 expressing clonal lines were also rendered non-responsive to induced neoplastic transformation. These observations, together with our recent finding attributing AP-1 non-responsiveness to Erk deficiency in a clonal line of transformation resistant (P-) cells, argue for a requirement for Erks1 and/or 2 activation in AP-1 transactivation in the mouse JB6 neoplastic progression model, and suggest the utility of Erks as a prevention target
— id: 42728, year: 1998, vol: 17, page: 3493, stat: Journal Article,

Plasticity of GAP-43 innervation of the spleen during immune response in the mouse. Evidence for axonal sprouting and redistribution of the nerve fibers
Yang, H; Wang, L; Huang, C S; Ju, G
1998 Jan-Apr;5(1-2):53-60, Neuroimmunomodulation
The amount and distribution of growth-associated protein (GAP-43)-like immunoreactive nerve fibers in the spleen of normal and immunized BALB/c mice were studied using immunohistochemical methods. A significant increase in the amount, as well as redistribution and morphological changes, of the GAP-43-like immunoreactive nerve fibers occurred in PPD (purified protein derivative from tuberculin) immunized animals. In the control animals, the GAP-43-like immunoreactive nerve fibers were found mainly distributed in association with vascular plexuses, with minor extension into the parenchyma of the inner zone of the periarterial lymph sheath. In the immunized animals, in addition to denser vascular plexuses, more fibers appeared in the outer zone of the periarterial lymph sheath, the marginal zone, and the red pulp, all known to be the sites where immune responding lymphocytes are located. Furthermore, the nerve fibers tended to have more branches and bear richer varicosities. The results suggest that active nerve remolding takes place in the spleen during immune response, which may serve as a mechanism through which the nervous system regulates immune responses
— id: 106270, year: 1998, vol: 5, page: 53, stat: Journal Article,

Inhibition of activator protein 1 activity and neoplastic transformation by aspirin
Dong Z; Huang C; Brown RE; Ma WY
1997 Apr 11;272(15):9962-9970, Journal of biological chemistry
Aspirin, along with its analgesic-antipyretic uses, is now also being considered for prevention of cardiovascular disease, cancer, and treatment of human immunodeficiency virus infection. Although many of aspirin's pharmacological actions are related to its ability to inhibit prostaglandin biosynthesis, some of its beneficial therapeutic effects are not completely understood. Transcription factor activator protein 1 (AP-1) is critical for the induction of neoplastic transformation and induction of multiple genes involved in inflammation and infection. We have used the JB6 mouse epidermal cell lines, a system that has been used extensively as an in vitro model for the study of tumor promotion and anti-tumor promotion, to study the anti-carcinogenesis effect of aspirin at the molecular level. Aspirin and aspirin-like salicylates inhibited the activation of AP-1 in the same dose range as seen for the inhibition of tumor promoter-induced transformation. The inhibition of AP-1 and tumor promoter-induced transformation in JB6 cells occurs through a prostaglandin independent- and an Erk1- or Erk2-independent pathway. The mechanism of AP-1 and transformation inhibition in this cell culture model may involve the elevation of H+ concentration. The inhibition effects on the activation of AP-1 activity by aspirin and aspirin-like salicylates may further explain the anti-carcinogenesis mechanism of action of these drugs
— id: 42715, year: 1997, vol: 272, page: 9962, stat: Journal Article,

Inhibition of tumor promoter-induced activator protein 1 activation and cell transformation by tea polyphenols, (-)-epigallocatechin gallate, and theaflavins
Dong Z; Ma W; Huang C; Yang CS
1997 Oct 1;57(19):4414-4419, Cancer research
(-)-Epigallocatechin gallate (EGCG) and theaflavins are believed to be key active components in tea for the chemoprevention against cancer. However, the molecular mechanisms by which EGCG and theaflavins block carcinogenesis are not clear. We have used the JB6 mouse epidermal cell line, a system that has been used extensively as an in vitro model for tumor promotion studies, to examine the anti-tumor promotion effects of EGCG and theaflavins at the molecular level. EGCG and theaflavins inhibited epidermal growth factor- or 12-O-tetradecanoylphorbol-13-acetate-induced cell transformation in a dose-dependent manner. At the dose range (5-20 microM) that inhibited cell transformation, EGCG and theaflavins also inhibited AP-1-dependent transcriptional activity and DNA binding activity. The inhibition of AP-1 activation occurs through the inhibition of a c-Jun NH2-terminal kinase-dependent, but not an extracellular signal-regulated protein kinase (Erk) 1-dependent or Erk2-dependent, pathway. Because the transcription factor AP-1 is important for tumor promoter-induced neoplastic transformation, the inhibitory effects on AP-1 activation by EGCG and theaflavins may further explain the anti-tumor promotion action of these tea constituents
— id: 42725, year: 1997, vol: 57, page: 4414, stat: Journal Article,

Direct evidence for an important role of sphingomyelinase in ultraviolet-induced activation of c-Jun N-terminal kinase
Huang C; Ma W; Ding M; Bowden GT; Dong Z
1997 Oct 31;272(44):27753-27757, Journal of biological chemistry
Sphingomyelinase (SMase) and its product ceramide have recently attracted a great deal of attention because of their possible role in the signal transduction pathway. However, the role of sphingomyelinase in UV-induced c-June N-terminal kinase (JNK) activation is still unclear. Thus, we investigated this issue directly using a genetic SMase-deficient (2 approximately 3% residual acid SMase activity) lymphoblast cell line, MS1418. The results showed that while UV irradiation markedly induces JNK activation in a normal human lymphoblast cell line, JY, it induces only weak JNK activation in MS1418 cells. This difference of JNK response to UV irradiation between these two cell lines was further observed in time course and dose-response studies. In contrast, 12-O-tetradecanoylphorbol-13-acetate-induced JNK activation could be observed in both JY and MS1418 cells. Furthermore, significant JNK activation can be observed in MS1418 cells by exposure of the cells to SMase or C2-ceramide, whereas phospholipase A2 or phospholipase C did not show significant induction of JNK activity, and C2-dihydroceramide and sphingosine induce only much weaker JNK activation in MS1418 cells than that by C2-ceramide. These data demonstrated that SMase plays an essential role in UV-induced JNK activation
— id: 42722, year: 1997, vol: 272, page: 27753, stat: Journal Article,

Signal transduction through atypical PKCs, but not the EGF receptor, is necessary for UVC-induced AP-1 activation in immortal murine cells
Huang C; Ma W; Dong Z
1997 Apr 24;14(16):1945-1954, Oncogene
The exposure of mammalian cells to ultraviolet (u.v.) irradiation leads to activation of transcription factors, such as AP-1 and NFkappaB. It is postulated that the EGF receptor but not protein kinase C (PKC) is the major membrane mediator in UVC-induced signal transduction. We demonstrate here that the antisense oligonucleotides of PKC zeta and the dominant negative mutant of PKC lambda/iota as well as dominant negative PKC zeta markedly blocked UVC-induced AP-1 activity. In contrast, UVC-induced AP-1 activity in cells devoid of the EGF receptor (B82), is not significantly different from that of the stable transfectants with a kinase-deficient EGF receptor (B82M721), or wild-type EGF receptor (B82L). This was found at all UVC irradiation doses and time courses studied, while high levels of EGF-induced AP-1 activity were observed in B82L cells but not in B82 cells. This evidence strongly suggests that atypical PKCs, but not the EGF receptor, is necessary for UVC-induced AP-1 activation in JB6 and B82 cells
— id: 42727, year: 1997, vol: 14, page: 1945, stat: Journal Article,

Blocking activator protein-1 activity, but not activating retinoic acid response element, is required for the antitumor promotion effect of retinoic acid
Huang C; Ma WY; Dawson MI; Rincon M; Flavell RA; Dong Z
1997 May 27;94(11):5826-5830, Proceedings of the National Academy of Sciences of the United States of America
Retinoic acid is one of the most promising drugs for chemotherapy and chemoprevention of cancer. Either blocking activator protein-1 (AP-1) activity or activating retinoic acid response element (RARE) have been proposed to be responsible for its antitumor activity. However, evidence for this hypothesis is lacking in vivo studies. To address this issue, we used an AP-1-luciferase transgenic mouse as a carcinogenesis model and new synthetic retinoids that are either selective inhibitors of AP-1 activation or selective activators of the RARE. The results showed that the SR11302, an AP-1 inhibition-specific retinoid, and other AP-1 inhibitors such as trans-retinoic acid and fluocinolone acetonide, markedly inhibit both 12-O-tetradecanoylphorbol-13-acetate-induced papilloma formation and AP-1 activation in 7,12-dimethyl benz(a)anthracene-initiated mouse skin (P < 0.05). In contrast, repeated applications of SR11235, a retinoid with RARE transactivating activity, but devoid of AP-1 inhibiting effect, did not cause significant inhibition of papilloma formation and AP-1 activation (P > 0.05). These results provide the first in vivo evidence that the antitumor effect of retinoids is mediated by blocking AP-1 activity, but not by activation of RARE
— id: 42714, year: 1997, vol: 94, page: 5826, stat: Journal Article,

Inhibition of ultraviolet B-induced activator protein-1 (AP-1) activity by aspirin in AP-1-luciferase transgenic mice
Huang C; Ma WY; Hanenberger D; Cleary MP; Bowden GT; Dong Z
1997 Oct 17;272(42):26325-26331, Journal of biological chemistry
Aspirin is under consideration as a promising chemopreventative agent for human cancers. To study the usefulness of aspirin as a chemopreventative agent for UV-induced human skin cancer, we investigated the effect of aspirin on UVB-induced activator protein-1 (AP-1) activity. In the JB6 cell culture system, aspirin or sodium salicylate (SA) inhibited UVB-induced AP-1 activity in a dose-dependent manner; this inhibitory effect occurred only in cells pretreated with aspirin or SA before UVB irradiation but not cells treated with aspirin or SA after UVB irradiation. Furthermore, these inhibitory effects on UVB-induced AP-1 activity appeared to be mediated through blocking of activation of MAP kinase family members, including extracellular signal-regulated protein kinases, c-Jun N-terminal kinases, and p38. It was not due to absorption of UVB light by aspirin. In the skin of AP-1-luciferase transgenic mice, UVB irradiation induced a rapid increase in AP-1 activity, which reached the peak at 48 h post-UVB irradiation. The topical pretreatment of mouse skin with aspirin markedly blocked the UVB-induced AP-1 transactivation in vivo. These data provide the first evidence that aspirin and SA are inhibitors of UV-induced signal transduction and thus could be used as a chemopreventative agent for skin cancer
— id: 42712, year: 1997, vol: 272, page: 26325, stat: Journal Article,

Inositol hexaphosphate inhibits cell transformation and activator protein 1 activation by targeting phosphatidylinositol-3' kinase
Huang C; Ma WY; Hecht SS; Dong Z
1997 Jul 15;57(14):2873-2878, Cancer research
Inositol hexaphosphate (InsP6) is the most abundant inositol phosphate found in plants. In mammalian cells, the concentrations of InsP6 are between 10 and 100 microM. Previous work has indicated that InsP6 is an effective cancer chemopreventive and chemotherapeutic agent. However, the molecular mechanisms involved in the inhibition of carcinogenesis by InsP6 remain unclear. In this study, we investigated the influence of InsP6 on tumor promoter-induced cell transformation and signal transduction pathways leading to activator protein 1 activation, which is considered to play a crucial role in tumor promotion. InsP6 markedly blocks epidermal growth factor-induced phosphatidylinositol-3 (PI-3) kinase activity in a dose-dependent manner in JB6 cells and directly in vitro. Blocking PI-3 kinase activity by InsP6 profoundly impairs epidermal growth factor- or phorbol ester-induced JB6 cell transformation and extracellular signal-regulated protein kinases activation, as well as activator protein 1 activation. These results provide the first evidence that the molecular mechanism of InsP6 antitumor promotion effect targets and blocks PI-3 kinase activation and demonstrate that PI-3 kinase can serve as a molecular target for the development of cancer chemopreventive agents
— id: 42713, year: 1997, vol: 57, page: 2873, stat: Journal Article,

Proteinase inhibitors I and II from potatoes specifically block UV-induced activator protein-1 activation through a pathway that is independent of extracellular signal-regulated kinases, c-Jun N-terminal kinases, and P38 kinase
Huang C; Ma WY; Ryan CA; Dong Z
1997 Oct 28;94(22):11957-11962, Proceedings of the National Academy of Sciences of the United States of America
Solar UV irradiation is the causal factor for the increasing incidence of human skin carcinomas. The activation of the transcription factor activator protein-1 (AP-1) has been shown to be responsible for the tumor promoter action of UV light in mammalian cells. We demonstrate that proteinase inhibitor I (Inh I) and II (Inh II) from potato tubers, when applied to mouse epidermal JB6 cells, block UV-induced AP-1 activation. The inhibition appears to be specific for UV-induced signal transduction for AP-1 activation, because these inhibitors did not block UV-induced p53 activation nor did they exhibit any significant influence on epidermal growth factor-induced AP-1 transactivation. Furthermore, the inhibition of UV-induced AP-1 activity occurs through a pathway that is independent of extracellular signal-regulated kinases and c-Jun N-terminal kinases as well as P38 kinases. Considering the important role of AP-1 in tumor promotion, it is possible that blocking UV-induced AP-1 activity by Inh I or Inh II may be functionally linked to irradiation-induced cell transformation
— id: 42711, year: 1997, vol: 94, page: 11957, stat: Journal Article,

Phosphatidylinositol-3 kinase is necessary for 12-O-tetradecanoylphorbol-13-acetate-induced cell transformation and activated protein 1 activation
Huang C; Schmid PC; Ma WY; Schmid HH; Dong Z
1997 Feb 14;272(7):4187-4194, Journal of biological chemistry
Phorbol esters, which activate isoforms of protein kinase C, are general activators of the transcription factor activated protein 1 (AP-1). The pathway involved in this signal transduction is not very clear. Currently, little is known about whether phosphatidylinositol-3 (PI-3) kinase plays any role in phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced signal transduction. We demonstrate here that TPA not only has markedly synergistic effects on insulin-induced PI-3 kinase activity, but it also can induce PI-3 kinase activity and the PI-3 phosphates by itself. We also found that insulin, a PI-3 kinase activator, enhanced TPA-induced AP-1 trans-activation and transformation in JB6 promotion-sensitive cells. Furthermore, wortmannin and LY294002, two PI-3 kinase inhibitors, markedly decreased AP-1 activity induced by insulin, TPA, or TPA and insulin and inhibited JB6 promotion-sensitive cell transformation induced by TPA or TPA and insulin. Most importantly, constitutive overexpression of the dominant negative PI-3 kinase P85 mutants completely blocked insulin- or TPA-induced AP-1 trans-activation and TPA-induced cell transformation. All evidence from present studies suggests that PI-3 kinase acts as a mediator in TPA-induced AP-1 activation and transformation in JB6 cells
— id: 42716, year: 1997, vol: 272, page: 4187, stat: Journal Article,

Ultraviolet B-induced activated protein-1 activation does not require epidermal growth factor receptor but is blocked by a dominant negative PKClambda/iota
Huang C; Ma W; Bowden GT; Dong Z
1996 Dec 6;271(49):31262-31268, Journal of biological chemistry
The exposure of mammalian cells to UV irradiation leads to the activation of transcription factors such as activated protein-1 (AP-1) and NFkappaB. It is postulated that epidermal growth factor (EGF) receptor, but not protein kinase C (PKC), is the major membrane mediator in UV-induced signal transduction. Since UVB is responsible for most of the carcinogenic effects of sun exposure, we investigated the role of EGF receptors and PKC in UVB-induced AP-1 activation. Our results indicated that while the down-regulation of novel PKC (nPKC) and conventional PKC (cPKC) by pretreatment of cells with 12-O-tetradecanoyl phorbol-13-acetate cannot block UVB-induced AP-1 activity, it can block 12-O-tetradecanoyl phorbol-13-acetate-induced AP-1 activity. Further, the dominant negative mutant PKClambda/iota blocked UVB-induced AP-1 activity in all doses and time courses studied. In contrast, UVB-induced AP-1 activity from cells devoid of EGF receptor (B82) was not significantly different from that of the stable transfectants with a kinase-deficient EGF receptor (B82M721) or those with a wild-type EGF receptor (B82L) at all UVB irradiation doses and time courses studied. All of this evidence indicated that aPKC, but not EGF receptor, is involved in UVB-induced AP-1 activation
— id: 42723, year: 1996, vol: 271, page: 31262, stat: Journal Article,

Requirement for phosphatidylinositol 3-kinase in epidermal growth factor-induced AP-1 transactivation and transformation in JB6 P+ cells
Huang C; Ma WY; Dong Z
1996 Nov;16(11):6427-6435, Molecular & cellular biology
Phosphatidylinositol 3-kinase (PI 3-kinase) plays a role in a variety of biological processes, including regulation of gene expression, cell growth, and differentiation. However, little is known about its role in the cytoplasmic events involved in epidermal growth factor (EGF)-induced transduction of signals to the transcriptional machinery of the nucleus and in EGF-induced cell transformation. In this study, we examined whether PI 3-kinase is a mediator for the activation of AP-1 and neoplastic transformation by EGF in the murine epidermal cell line JB6. The results showed the following. (i) EGF not only induced a high level of PI 3-kinase activity by itself but also enhanced insulin-induced PI 3-kinase activity in JB6 P+ cells, the EGF-induced PI-3 kinase activity could be blocked by constitutive overexpression of a dominant negative P85 subunit of PI 3-kinase (deltaP85), and insulin could markedly promote EGF-induced AP-1 activity in a dose-dependent manner in JB6 P+ cells as well as promote EGF-induced JB6 P+ cell transformation. (ii) Inhibition of PI-3 kinase with wortmannin or LY294002 markedly decreased the AP-1 activity induced by insulin, EGF, or EGF and insulin in a dose-dependent manner, while wortmannin did not block UVB-induced AP-1 activity. (iii) AP-1 activation by insulin, EGF, or EGF and insulin could be completely inhibited by overexpression of deltaP85 in all the dose and time courses studied. (iv) Inhibitors of PI 3-kinase (wortmannin and LY294002) and stable overexpression of deltaP85 inhibited EGF-induced transformation but had no significant inhibitory effect on cell proliferation induced by EGF or EGF and insulin. These results demonstrate for the first time that PI 3-kinase appears to be required for EGF- or insulin-induced AP-1 transactivation and cell transformation but not cell proliferation in JB6 cells
— id: 42717, year: 1996, vol: 16, page: 6427, stat: Journal Article,

Inhibitory effects of ascorbic acid on AP-1 activity and transformation of JB6 cells
Huang, CS; Ma, WY; Dong, ZG
1996 FEB ;8(2):389-393, International journal of oncology
Ascorbic acid (vitamin C) has been reported as an anti-cancer agent. Previous in vitro studies using either primary cell cultures from cancer patients or tumor cell lines have indicated that different kinds of tumor cells may have different sensitivities to ascorbic acid for the inhibition of tumorigeneity or growth. Because the JB6 mouse epidermal cell system has been used extensively as an in vitro model for the study of tumor promotion and progression, we assessed the effects of ascorbic acid on transformation in JB6 cell variants. The results show that ascorbic acid could inhibit 5.5% to 97.1% of transformation of JB6 P+ cell Cl 41-19 induced by TPA, EGF or EGF + insulin, but has no effect on anchorage-independent growth of JB6 transformed cell A33. Since our previous results indicated that induced AP-1 activity is required for tumor promoter induced-transformation, we tested whether inhibition of tumor promoter-induced transformation by ascorbic acid is through an AP-1 inhibition mechanism. Our results indicated that ascorbic acid inhibited AP-I activity at the same dose range for inhibition of transformation. These results demonstrated that ascorbic acid has inhibitory effects on JB6 cell tumor promoter-induced transformation, but no influence on tumor cell phenotype expression and provided the basic knowledge for understanding of vitamin C action on tumor prevention
— id: 106289, year: 1996, vol: 8, page: 389, stat: Journal Article,

Hemorrhagic fever with renal syndrome: relationship between pathogenesis and cellular immunity
Huang, C; Jin, B; Wang, M; Li, E; Sun, C
1994 Apr;169(4):868-870, Journal of infectious diseases
After phenotype analysis of peripheral blood mononuclear cells (PBMC), soluble interleukin-2 receptor (sIL-2R) levels in plasma or sera from patients with hemorrhagic fever with renal syndrome (HFRS) were measured. The results showed the ratio of activated antigen (CD25, TLiSA1, CD71, and Ia)-positive lymphocytes of PBMC in the acute phase of HFRS was higher than that in convalescent phase. Moreover, there was much higher expression of heteromorphologic lymphocytes than of small lymphocytes. Decreases in T lymphocytes and CD4:CD8 ratios were seen with increases in B lymphocyte ratios and interferon-gamma (IFN-gamma) expression on PBMC surfaces in the acute phase of HFRS. IFN-gamma-positive lymphocytes included CD4, CD8, and CD56 subsets. sIL-2R levels were much higher in sera and plasma in the acute phase, especially the oliguric phase. These findings suggest that patients with HFRS are in a state of high-level cellular immune response, which may be involved in the development of inflammation and pathologic lesions
— id: 106249, year: 1994, vol: 169, page: 868, stat: Journal Article,

The effects of hybridoma growth factor in conditioned media upon the growth, cloning, and antibody production of heterohybridoma cell lines
Zhu, Y; Jin, B; Sun, C; Huang, C; Liu, X
1993 Jan;4(1):31-35, Human antibodies & hybridomas
Interleukin 6 (IL-6)/hybridoma growth factor (HGF) has been shown to be the requirement for growth of murine hybridomas in vivo or in vitro. In this paper, two kinds of conditioned media (CM) from the culture supernatants of a human fibroblast cell line CRL1506 and a cloned Epstein-Barr virus (EBV) transformed human lymphoblastoid cell line (LCL) N23 were found to have IL-6 activity by strongly promoting the growth, antibody secretion (increase of one- to three-fold), and cloning efficiencies of heterohybridomas secreting human monoclonal anti-hemorrhagic fever with renal syndrome virus antibodies and LCL. Since these CM contained no detectable IL-2, and IL-4 had no effects on the growth of the cell lines, IL-6 was considered to be the main active component of the CM responsible for promoting hybridoma growth. This effect was further confirmed by IL-6-dependent cell line 7TD1 bioassay (IL-6 activity in the CM ranging from 1,000 to 10,000 units/ml). Moreover, we successfully established four EBV-transformed lymphoblastoid cell lines at single-cell level by adding an equal volume of CRL1506-CM to 10% FCS-RPMI1640 in limiting dilution. Finally, it is worth noting that the sensitivity of the heterohybridomas to the two kinds of CM was not the same and was not consistent with that of their parental myeloma cell lines. Thus, it suggests that the CM might contain more than one factor, and the choice of proper conditioned media should be very useful for human monoclonal antibody production
— id: 106248, year: 1993, vol: 4, page: 31, stat: Journal Article,

Activation of corticotropin-releasing factor-containing neurons in the paraventricular nucleus of the hypothalamus by interleukin-1 in the rat
Ju, G; Zhang, X; Jin, B Q; Huang, C S
1991 Nov 11;132(2):151-154, Neuroscience letters
Interleukin-1 (IL-1) alpha was injected into the lateral ventricle of the rat. An antiserum against Fos oncoprotein was used to detect the activated neurons immunohistochemically. A large number of neurons in the parvocellular paraventricular nucleus of the hypothalamus, in an area corresponding to the location of the corticotropin-releasing factor (CRF)-containing neurons, were strongly Fos-like immunoreactive (LI). Double immunostaining for Fos and CRF revealed that many of the Fos-LI cells were CRF-LI. Furthermore, the CRF-like immunoreactivity was greatly enhanced in the rats injected with IL-1 alpha, indicating an increase in CRF synthesis
— id: 106247, year: 1991, vol: 132, page: 151, stat: Journal Article,