Abstract 3241: The selective inhibition of GST by CAPE enhances the cytotoxicity of doxorubicin and camptotechin in SK-MEL-28 melanoma cells

Abstract

Abstract Glutathione S-transferase (GST) and multidrug resistance proteins (MRPs) play major roles in the detoxification of anti-melanoma drugs; hence diminishing their toxicity towards melanoma cells. In this study, we investigated i) the selective inhibition of GST by Caffeic Acid Phenethyl Ester (CAPE) in presence of tyrosinase, ii) the role of MRP in CAPE induced toxicity, and iii) the effect of CAPE in combination with probenecid, doxorubicin and camptotechin. The inhibition of GST was investigated using CDNB method. LC/MS/MS was used to identify a CAPE glutathione conjugate formation by tyrosinase. MTT assay, Annexin V apoptosis assay and TMRM florescence method were used in the investigation of biochemical mechanism of CAPE efficacy in human SK-MEL-28 melanoma cells. At 30 min incubation, CAPE was bioactivated 65% by tyrosinase as measured by glutathione depletion. LC/MS/MS analyses identified CAPE-glutathione conjugate m/z [MH]+ 590 as the major metabolite formed as the result of CAPE bioactivation by tyrosinase. In the presence of tyrosinase, CAPE 10-25 µM showed 47-90% GST inhibition whereas in the absence of tyrosinase, CAPE 10-25 µM led to minimal GST inhibition. Both CAPE-SG conjugate and CAPE-quinone (25 µM) demonstrated ≥90% GST inhibition via reversible and irreversible competitive mechanisms with Ki of 5 µM with respect to GSH and with Ki of 6 µM with respect to CDNB, respectively. CAPE, a non-substrate for GST, only at concentrations >50 µM acted as a weak reversible non-competitive and competitive GST inhibitor with Ki of 277 µM with respect to GSH and with Ki of 234 µM with respect to CDNB, respectively. Moreover, computational auto docking analyses suggest that CAPE binds to the GST catalytic active site. CAPE (15-100 µM) showed 61-86% GST inhibition in human SK-MEL-28 cell homogenate. Furthermore, probenecid, a MRP inhibitor, increased CAPE (15, 30 µM) cell toxicity by 27-29%, apoptotic cell death by 10-14%, and decreased mitochondrial membrane potential by 27-40% in SK-MEL-28 melanoma cells, suggesting that the inhibition of MRP increases CAPE induced toxicity. The combination of CAPE (5 µM) with doxorubicin (0.1 µM) demonstrated synergistic effects as the combination index was 0.53 (<1). Similarly, CAPE (7.5 µM) showed synergistic effects with camptotechin (0.05 µM) with a combination index of 0.75. In summary, our results suggest that CAPE was bioactivated by tyrosinase to form a CAPE-quinone and CAPE glutathione conjugate, which played a major role in the inhibition of GST. Our study also elucidates the role of MRP in the biochemical mechanism of CAPE induced toxicity in melanoma cells. For the first time, we showed that CAPE in the presence of tyrosinase acts as a selective inhibitor of GST. CAPE therapy in combination with probencid, doxorubicin and camptotechin has synergistic effects in human SK-MEL-28 melanoma cells. 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 3241. doi:10.1158/1538-7445.AM2011-3241