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Epidemiologic studies have provided solid evidence for the neurotoxic effect of lead for decades of years. In view of the fact that children are more vulnerable to the neurotoxicity of lead, lead exposure has been an urgent public health concern. The modes of action of lead neurotoxic effects include disturbance of neurotransmitter storage and release, damage of mitochondria, as well as induction of apoptosis in neurons, cerebrovascular endothelial cells, astroglia and oligodendroglia. Our studies here, from a novel point of view, demonstrates that lead specifically caused induction of COX-2, a well known inflammatory mediator in neurons and glia cells. Furthermore, we revealed that COX-2 was induced by lead in a transcription-dependent manner, which relayed on transcription factor NFAT, rather than AP-1 and NFκB, in glial cells. Considering the important functions of COX-2 in mediation of inflammation reaction and oxidative stress, our studies here provide a mechanistic insight into the understanding of lead-associated inflammatory neurotoxicity effect via activation of pro-inflammatory NFAT3/COX-2 axis., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: Hexavalent chromium [Cr(VI)] is recognized as a human carcinogen via inhalation. However, the molecular mechanisms by which Cr(VI) causes cancers are not well understood., Objectives: We evaluated cyclooxygenase-2 (COX-2) expression and the signaling pathway leading to this induction due to Cr(VI) exposure in cultured cells., Methods: We used the luciferase reporter assay and Western blotting to determine COX-2 induction by Cr(VI). We used dominant negative mutant, genetic knockout, gene knockdown, and chromatin immunoprecipitation approaches to elucidate the signaling pathway leading to COX-2 induction., Results: We found that Cr(VI) exposure induced COX-2 expression in both normal human bronchial epithelial cells and mouse embryonic fibroblasts in a concentration- and time-dependent manner. Deletion of IKKβ [inhibitor of transcription factor NFκB (IκB) kinase β; an upstream kinase responsible for nuclear factor κB (NFκB) activation] or overexpression of TAM67 (a dominant-negative mutant of c-Jun) dramatically inhibited the COX-2 induction due to Cr(VI), suggesting that both NFκB and c-Jun/AP-1 pathways were required for Cr(VI)-induced COX-2 expression. Our results show that p65 and c-Jun are two major components involved in NFκB and AP-1 activation, respectively. Moreover, our studies suggest crosstalk between NFκB and c-Jun/AP-1 pathways in cellular response to Cr(VI) exposure for COX-2 induction., Conclusion: We demonstrate for the first time that Cr(VI) is able to induce COX-2 expression via an NFκB/c-Jun/AP-1-dependent pathway. Our results provide novel insight into the molecular mechanisms linking Cr(VI) exposure to lung inflammation and carcinogenesis.

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-κB (NF-κB). 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-κB was dependent on each other. Since our previous studies have shown that NF-κB 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-κB 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-κB, 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-κB 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.

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(

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.

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.

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., (Copyright 2006 Wiley-Liss, Inc.)

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.

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.

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.

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.

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.

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., (Copyright 2004 Wiley-Liss, Inc.)

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.

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.

Highlights: [•] As3+ induces p27 in IKKβ−/− cells. [•] IKKβ−/−/p65 impedes p27 induction. [•] GSK3β participates. [ABSTRACT FROM AUTHOR]