Jagat J. Mukherjee

Inhibition of benzopyrene-diol-epoxide (BPDE)-induced bax and caspase-9 by cadmium: role of mitogen activated protein kinase.

Cadmium, a major metal constituent of tobacco smoke, elicits synergistic enhancement of cell transformation when combined with benzo[a]pyrene (BP) or other polynuclear aromatic hydrocarbons (PAHs). The mechanism underlying this synergism is not clearly understood. Present study demonstrates that (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), an ultimate carcinogen of BP, induces apoptosis in human leukemic HL-60 cells and others, and cadmium at non-cytotoxic concentration inhibits BPDE-induced apoptosis. We observed that BPDE treatment also activates all three MAP kinases e.g. ERK1/2, p38 and JNK in HL-60 cells, and inhibition of BPDE-induced apoptosis by cadmium is associated with down-regulation of pro-apoptotic bax induction/caspase-9 activation and up-regulation of ERK phosphorylation, whereas p38 MAP kinase and c-Jun phosphorylation (indicative of JNK activation) remain unaffected. Inhibition of ERKs by prior treatment of cells with 10muM U0126 relieves cadmium-mediated inhibition of apoptosis/bax induction/caspase-9 activation. Our results suggest that cadmium inhibits BPDE-induced apoptosis by modulating apoptotic signaling through up-regulation of ERK, which is known to promote cell survival.

DNA synthesis inhibition in response to benzo[a]pyrene dihydrodiol epoxide is associated with attenuation of p34cdc2: Role of p53

Our previous findings demonstrated that DNA damage by polynuclear aromatic hydrocarbons (PAHs) triggers a cellular protective response of growth inhibition (G1-S cell cycle arrest and inhibition of DNA synthesis) in human fibroblasts associated with accumulation of p53 protein, a growth-inhibitory transcription factor. Here, we report that BPDE (the ultimate carcinogenic metabolite of the PAH benzo[a]pyrene) treatment triggers a variable extent of inhibition of DNA synthesis/cell growth, which does not correspond to the extent of increased p53 accumulation. BPDE treatment of cells significantly attenuates expression of p(34)cdc2, a cell cycle activating protein. Although the role of cdc2 down-regulation in inhibition of cell cycle progression is well known, cdc2 down-regulation in response to cellular insult by PAHs has not been reported. Unlike p53 accumulation, there is a correspondence between DNA synthesis/cell growth inhibition and cdc2 down-regulation by BPDE. BPDE-induced cdc2 down-regulation is p53 dependent, although there is no correspondence between p53 accumulation and cdc2 down-regulation. BPDE-induced cdc2 down-regulation corresponded with accumulation of the cell cycle inhibitor protein p21 (transactivation product of p53). DNA synthesis/cell growth inhibition in response to DNA-damaging PAHs may involve down-regulation of cdc2 protein mediated by p53 activation (transactivation ability), and the extent of p53 accumulation is not the sole determining factor in this regard.

Phenolic fraction of tobacco smoke condensate potentiates benzo[a]pyerene diol epoxide-induced cell transformation: role of protein kinase C.

In this study we separated weakly acidic phenolic components from other neutral, acidic and basic components of tobacco smoke condensate (TSC) and observed that phenolic fraction of TSC significantly increased the number of colonies of promotion-sensitive JB6 Cl41 cells that showed anchorage-independent growth on soft agar in response to BPDE (an ultimate carcinogen produced by metabolic activation of the PAH benzo[a]pyrene). Anchorage-independent cell growth is indicative of cell transformation resulting in acquisition of tumorigenic potential. In order to understand the underlying mechanism by which TSC phenolic fraction potentiates BPDE-induced tumorigenicity, we examined its effect on the activation of two transcription factors AP-1 and NF-kappaB which are known to be influenced by established tumor promoter TPA. BPDE treatment caused induction of both AP-1 and NF-kappaB activity as determined by luciferase reporter assay and only NF-kappaB induction in response to BPDE was significantly attenuated by TSC phenolic fraction whereas AP-1 induction remains unaltered. Attenuation of NF-kappaB activation by TSC phenolic fraction was associated with significant decrease of intracellular PKC substrate phosphorylation in BPDE treated cells. Non-specific PKC inhibitors staurosporine and bisindolylmaleimide II as well as inhibitors specific to conventional PKCs (Go6976) and PKC-delta (rottlerin) attenuated NF-kappaB activation in BPDE treated cells to a varying degree indicating a possible link between PKC down-regulation and the attenuation of NF-kappaB activity by TSC phenolic fraction. Treatment of cells with PKC inhibitors also potentiated anchorage-independent growth of BPDE treated cells on soft agar. Our data suggest a possible role of PKC down-regulation in potentiation of BPDE-induced tumorogenicity by TSC phenolic fraction.

Abstract 1581: Ethanol overrides DNA synthesis inhibition in response to PAHs (tobacco smoke carcinogens): a mechanistic explanation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA It is a well-established fact that alcohol intake with smoking presents a potential health risk to human. Mechanistic understanding in this regard is not clear. We previously reported that DNA damage by tobacco smoke carcinogens polynuclear aromatic hydrocarbons (PAHs) is responded by cells through induction of cell growth inhibition in different cell lines. Here we observed that ethanol (EtOH) treatment at physiologically relevant concentration (60 mM) increases DNA synthesis in DNA damaging carcinogen (BPDE) treated cells. BPDE treatment of cells elicits G1-S cell cycle arrest, but EtOH does not have any significant modulating effect on G1-S arrest indicating increased DNA synthesis by EtOH is due to modulation of some other signaling event(s). EtOH co-treatment/pretreatment is unable to modulate BPDE-induced p53 accumulation and p53 phosphorylation at ser15 and ser20 indicating increased DNA synthesis by EtOH is p53 independent. We also observed that EtOH co-treatment/pretreatment potentiates BPDE-induced phosphorylation and activation of ERKs only and not p38 MAPK. Interestingly, treatment of cells with MEK1 inhibitor (PD098059) significantly reduced ethanols ability to increase DNA synthesis in BPDE treated cells. This finding indicates that the ability of EtOH to potentiate BPDE-induced ERK activation may potentiate PAH-induced tumorigenesis. Note: This abstract was not presented at the meeting. Citation Format: Jagat J. Mukherjee, Subodh Kumar. Ethanol overrides DNA synthesis inhibition in response to PAHs (tobacco smoke carcinogens): a mechanistic explanation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1581. doi:10.1158/1538-7445.AM2014-1581

Abstract 3586: Benzopyrene diol epoxide attenuates p34cdc2 and inhibits cell growth: role of p53.

Our previous findings demonstrated that DNA damage by polynuclear aromatic hydrocarbons (PAHs) triggers cellular protective response of cell growth inhibition (G1-S cell cycle arrest and inhibition of DNA synthesis) in human fibroblast associated with accumulation of p53 protein, a known cell growth inhibitory transcription factor. Here we report that in different cell lines BPDE treatment (ultimate carcinogenic metabolite of the PAH benzo[a]pyrene) triggers variable extent of cell growth inhibition which do not correspond to the extent of increased p53 accumulation as we observed. We also observed that BPDE treatment of cells significantly down-regulates expression of p34cdc2, a known cell cycle activating protein. Although the role of cdc2 down-regulation in inhibition of cell cycle progression is well known, to the best of our knowledge cdc2 down-regulation in response to cellular insult by PAHs has not been reported. Unlike p53 accumulation there is correspondence between extent of cell growth inhibition and the extent of cdc2 down-regulation by BPDE in different cell lines. Interestingly, BPDE-induced cdc2 down-regulation is observed to be p53 dependent although there is lack of correspondence between the extent of p53 accumulation and cdc2 down-regulation. However, extent of BPDE induced cdc2 down-regulation was observed to have correspondence with the extent of accumulation of cell cycle inhibitor protein p21 (transactivation product of p53) in different cell lines. These findings may have an implication that cell growth inhibition in response to DNA damaging PAHs may involve down-regulation of cdc2 protein mediated by p53 activation (transactivation ability), and the extent of p53 accumulation is not the determining factor in this regard. Citation Format: Jagat J. Mukherjee, Subodh Kumar. Benzopyrene diol epoxide attenuates p34cdc2 and inhibits cell growth: role of p53. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3586. doi:10.1158/1538-7445.AM2013-3586

Abstract 5551: Potentiation of PAH-induced cell transformation by phenolic fraction of tobacco smoke: A mechanistic insight

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL It is reported that the known content of carcinogenic polynuclear aromatic hydrocarbons (PAHs) in the tobacco smoke condensate (TSC) alone cannot account for the observed carcinogenicity of the TSC tested on mouse skin (Orris, L. et.al., 1958. J. Natl. Cancer Inst., 21: 557-561). The phenolic fraction of TSC (TSCPhFr) which is devoid of PAHs exhibits strong tumor-promoting activity in mouse skin initiated with PAHs (Roe et. al., 1959. Brit. J. Cancer., 13: 623-633). The mechanism(s) underlying the tumor promoting effect of TSCPhFr is not known. In the present communication we report that (±)-anti-BP-7,8-diol-9,10-epoxide (BPDE), an ultimate carcinogenic metabolite of tobacco smoke carcinogen benzo[a]pyrene, elicits both apoptosis induction and cell transformation in promotion sensitive mouse epidermal JB6 cell which has been extensively used for tumor promotion studies. We observed that TSCPhFr significantly potentiates BPDE-induced cell transformation associated with decreased apoptosis response indicating its tumor promoting potential. Increased cell transformation and decreased apoptosis by TSCPhFr was associated with attenuation of BPDE-induced p53 accumulation and NF-κB activation. We examined whether attenuation of p53 and NF-κB responses by TSCPhFr has any role in this regard. Chemical inhibition of p53 in JB6 cells using known p53 inhibitor pifithrin alpha showed potentiation of BPDE-induced p53 accumulation instead of down-regulation as expected. Since pifithrin alphas role in p53 inhibition is controversial as reported in many studies, we used genomic inhibitor (p53 siRNA) to down-regulate p53 response. We observed that either JB6 cells transfected with p53 siRNA or p53 deficient HCT116 cells (HCT116p53-/−) shows significantly less apoptosis induction and more colony formation on soft agar by BPDE compared to control cells. In p53 impaired both cell lines (which are observed to have faster growth rate compared to normal cells) TSCPhFr has practically negligible effect on apoptosis induction and soft agar colony formation in response to BPDE. Interestingly treatment of cells with IKK-NBD peptide NF-κB-specific inhibitor) has variable effects on BPDE-induced apoptosis induction and cell transformation. Our observations indicate that attenuation of BPDE-induced p53 response by TSCPhFr has a role in apoptosis inhibition and increased cell transformation. These findings have implication with regard to the underlying mechanism of tumor promoting activity of TSCPhFr in PAH-induced carcinogenesis. Although p53 mediated NF-κB activation is involved in DNA damage-induced apoptosis induction as reported by others (Nature 2000, 404:892-897), the role of NF-κB in PAH-induced apoptosis and cell transformation is not clear (supported by NCI grant 1R15CA125630). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5551. doi:10.1158/1538-7445.AM2011-5551