Abstract 2012: MAP kinase activation and suppression of anti-apoptotic Bcl-2 family members are associated with induction of apoptotic death in melanoma cells following down-regulation of glutathione S-transferase P1

Abstract

Abstract Introduction. The multifunctional protein, glutathione S-transferase P1, GSTP1, is frequently overexpressed in malignant melanoma and has been associated with increased tumor growth and resistance to therapy and poor overall patient survival. A major cellular function of GSTP1 is as a regulator of cell signaling mediated through its ability to interact with and regulate key signaling proteins, notably, the MAP kinases. In a previous study, we showed that RNA interference-mediated GSTP1 downregulation in GSTP1-overexpressing melanoma cells decreased cell growth and increased the levels of both spontaneous and drug-induced apoptosis. Here, we examined the molecular mechanisms underlying these observations, particularly, those involving alterations in MAP kinase signaling and pro-and anti-apoptotic members of the Bcl2 family of proteins. Methods. Exponentially growing cells of two human melanoma cell lines A375 and DM6 were infected with a lentiviral vector carrying GSTP1-targeted short hairpin RNA (shRNA) targeting. A heterogeneous stable population was isolated by puromycin selection. Alternatively, GSTP1 was knocked down by transfection of the cells with GSTP1-targeted siRNA. The cells were confirmed as GSTP1-ve by PCR and western blotting. At various time-points post GSTP1 suppression, the cells were analyzed for the level of apoptosis by flow cytometry (FCM) and by measuring the level of caspase 3/7 mediated cleavage of the peptide substrate, Ac-DEVD-AFC. Extracts were prepared from control and GSTP1-knockdown cells and analyzed for total and phosphorylated MAP kinases (P38, ERK, and JNK) and for proteins of the Bcl-2 family. Results. There was a near-complete suppression of GSTP1 transcripts and protein in both melanoma cell lines following shRNA-lentiviral infection or at 72 hrs post-siRNA transfection. Over 7 days, there was a steady increase in the level of apoptosis in the cells with GSTP1 suppression, as evidenced by an increase in the pre-G1 population by FCM or in caspase 3/7 activity. This was associated with a significant increase in the level of phosphorylation of the MAP kinases, Erk1/2 and JNK1/2. Following GSTP1 suppression, we also observed a significant decrease in the pro-survival anti-apoptotic Bcl-2 proteins Mcl-1, Bcl-XL, and Bcl-2 with little to no change in pro-apoptotic Bcl2 proteins Bad, Bim and Bid. Conclusion. These results suggest that GSTP1 is obligate for growth and survival of of GSTP1-expressing melanoma cells and that suppression of GSTP1 expression in these cells induces an apoptotic response that involves activation of the MAP Kinases, ERK and JNK, and suppression of anti-apoptotic Bcl-2 proteins. Supported by NIH grants RO1 CA 153050, RO1CA127872, RO1 CA 112519, P50CA108786 and P30-CA14236. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2012. doi:1538-7445.AM2012-2012