Abstract 746: Bioactivation of caffeic acid phenethyl ester prodrug by tyrosinase inhibits human glutathione S-transferase and cause selective toxicity to melanoma cells

Abstract

Abstract Glutathione S-transferase (GST) plays a major role in detoxification of anti-cancer drugs and hence diminishing their toxicity towards cancer cells. In the current study, we investigated Caffeic Acid Phenethyl Ester (CAPE) as selective cytotoxic agent and GST inhibitor in melanoma cells. Tyrosinase is found abundantly in melanoma cells, was used as a primary molecular target to bioactivate CAPE to an o-quinone, which could react with glutathione to form CAPE glutathione conjugate. The inhibition of GST was investigated using CDNB method. LC/MS/MS was used to determine the formation of CAPE glutathione conjugate by tyrosinase. MTT assay and Annexin V apoptosis assay were used in the investigation of biochemical mechanism of CAPE toxicity. A number of modulators were used to investigate the role of GST and glutathione synthesis in melanoma cell lines. At 30 min incubation, CAPE was metabolized 65% by tyrosinase as measured by glutathione depletion. LC/MS/MS studies revealed that the major product formed as the result of CAPE bioactivation by tyrosinase in the presence of the glutathione was a CAPE-glutathione conjugate. In the presence of tyrosinase, CAPE 10 and 25µM showed 47% and 90% of GST inhibition whereas in the absence of tyrosinase CAPE did not show significant GST inhibition. Both pre-formed CAPE-glutathione conjugate and CAPE-quinone demonstrated 95% GST inhibition, whereas CAPE alone did not show any GST inhibition. It was also found that CAPE was not a substrate for GST. Our results showed that CAPE 15µM was selectively toxic towards five melanoma cell lines in comparison to four non-melanoma cells lines. For instance, CAPE 15µM showed 90% apoptotic cell death in melanoma cells. Furthermore, the role of intracellular glutathione was investigated. BSO, a GSH biosynthesis inhibitor, increased the CAPE toxicity by 26%, whereas GSH and N-acetyl cysteine marginally prevented CAPE toxicity towards melanoma cells for 10%. It was also shown that CAPE lead to significant intracellular glutathione depletion, intracellular GST inhibition and ROS formation in melanoma cells. On the other hand, although 4-hydroxyanisole, L-tyrosine and caffeic acid, were found to be metabolized by tyrosinase to form quinones and glutathione conjugates none of these compounds inhibited GST in the presence and absence of tyrosinase indicating that CAPE was the only compound from this series that selectively inhibited GST in the presence of tyrosinase. In summary, our results suggests CAPE was metabolized by tyrosinase to form a quinone and CAPE glutathione conjugate, which played a major role in inhibition of GST. In addition, the biochemical mechanisms of CAPE toxicity revealed that quinone formation, intracellular glutathione depletion and ROS formation played a role in CAPE selective toxicity towards melanoma cells. 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 746.