Abstract 2557: GSTP1 is a determinant of temozolomiode resistance in human glioblastoma cells

Abstract

Abstract Temozolomide (TMZ) is a methylating agent used in first-line treatment of malignant gliomas and melanomas. Unfortunately, its use is limited by multifaceted resistance mechanisms. Although, the glutathione S-transferase P1 (GSTP1) protein metabolizes several alkylating and free radical generating anticancer agents, its role in TMZ metabolism and/or tumor resistance to TMZ are not known. In this study, we investigated the potential role of GSTP1-based metabolic detoxification of TMZ in glioma resistance to TMZ. The ability of GSTP1 to metabolize TMZ was assessed indirectly by measuring changes in the level of residual (free, un-metabolized) TMZ in a cell-free system containing TMZ, reduced glutathione (GSH) and human placental GSTP1 protein. We observed a time- and both GSH and GSTP1 concentration-dependent decrease in residual TMZ levels in reaction mixtures containing GSTP1 and after a 6 hr incubation, the level of free TMZ in GSTP1-containing reaction mixtures was decreased to 40% of controls without GSTP1. Presence of GSH alone resulted in only a 10% decrease in residual TMZ. Using three GSTP1 expressing glioma cell lines, viz, MGR1, MGR3 and U87, we examined the effect of siRNA-mediated knockdown of GSTP1 expression on tumor survival to TMZ and the level of TMZ-induced apoptosis using caspase-3/7 activation as endpoint. The TMZ IC50 values for MGR1, MGR3 and U87 of 1.31, 0.91 and 1.06 mM, respectively, were reduced significantly to 0.78, 0.6 and 0.69 mM at 48hrs following GSTP1 knockdown. Similarly, siRNA-mediated knockdown of GSTP1 increased TMZ-induced caspase-3/7 activation, significantly, in all three cell lines when exposed to TMZ for 48 hrs. Experiments evaluating the effect of combined knockdown of GSTP1 and O6-methylguanine-DNA methyltransferase (MGMT) on cellular sensitivity of TMZ suggest synergism between the two strategies. Overall, these results provide support for a pivotal role that GSTP1 may play in glioma cell resistance to TMZ-based chemotherapy and the potential of targeting GSTP1 as part of strategies to reverse this resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2557.