Abstract 1850: Benzyl isothiocyanate causes proteasome-mediated degradation of aryl hydrocarbon receptor in Hepa1c1c7 cells

Abstract

Abstract The biotransformation and detoxification of polycyclic aromatic carcinogens is dependent upon the activity of CYP1A1 and NQO1. Transcriptional activation of these enzymes is regulated by the aryl hydrocarbon receptor (AhR) and Nuclear Factor-Erythroid 2-Related Factor. These transcription factors reside in the cytoplasm and when activated, shed their respective chaperones and translocate into the nucleus where they interact with nuclear proteins involved with transcription of genes possessing xenobiotic (XRE) and antioxidant (ARE) response elements. We have previously reported that the phytochemical benzyl isothiocyanate (BITC) induces ligand-independent nuclear translocation of the AhR in Hepa1c1c7 cells and also induces AhR heterodimerization with ARNT. This suggests that BITC might bind the AhR and affect enzyme activities through transcriptional regulation of XRE-responsive genes. We also have observed that BITC causes rapid degradation of the AhR both alone and in combination with known receptor ligands. In the current study, we investigated whether BITC causes proteasomal degradation of the AhR and whether this degradation occurs through increased ubiqitination of the receptor. This is important because it provides a potential mechanism by which BITC affects AhR turnover and consequently its regulation of phase I and II enzymes. Hepa1 cells were treated overnight with DMSO (control), 10 µM BITC, 10 µM β-napthoflavone (BNF) or a combination of BITC and BNF both at 10 µM. AhR was immunoprecipitated and detected using western blot. AhR was degraded in samples treated with BITC alone compared to control. Additionally, BITC enhanced degradation of AhR in samples treated with both BITC and BNF compared to BNF alone. Western blots re-probed with anti-ubiquitin showed increased ubiquitination of proteins in BITC and BNF-treated samples compared to control. In additional experiments, cells were treated for 16 hours with DMSO, 10 µM BITC, 10 µM BNF or a combination of BNF (10 µM) and increasing concentrations of BITC (1-10 µM). Sub-cellular fractionation of samples treated with a combination of BITC and BNF showed a dose-dependent increase in the amount of insoluble AhR compared to either BITC or BNF alone. AhR degradation was blocked in cells pre-treated with the proteasome inhibitor MG132 but not in cells pre-treated with broad spectrum protease inhibitors or PMSF. This suggests that degradation is specific to the proteasome rather than other proteases such as serine proteases and calpains. Collectively these results show that BITC degrades the AhR and this is mediated by ubiquitin and the proteasome. AhR degradation might affect transcription of XRE responsive genes and therefore carcinogen activation. These results also provide a potential mechanism for BITC as a cancer chemopreventive agent. (Support: NIH MBRS/GM028248; RCMI RR033034) 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 1850. doi:10.1158/1538-7445.AM2011-1850