Abstract 4096: TRAIL enhances cytotoxicity of arginine depletion therapy in argininosuccinate synthetase-negative melanoma cells through interruption of autophagy via activation of caspases (2011)

Abstract Melanomas which do not express argininosuccinate synthetase (ASS) cannot make arginine from citrulline; thus arginine depletion caused by pegylated arginine deiminase (ADI-PEG20, provided by Polaris Inc.) results in growth inhibition and eventually leads to cell death. However, autophagy does take place during arginine deprivation which prevents cell death. During this period, certain melanoma cells can turn on the ASS gene and hence become resistant to this form of treatment. In order to increase the effectiveness of ADI-PEG20 therapy, we have previously found that combining tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with ADI-PEG20 can accelerate cell death (apoptosis) process and hence greatly enhance cytotoxicity (Biochem. Biophys. Res. Commun. 2010; 394(3):760-6). We have found that the addition of TRAIL increases formation of tBid. In this study, we found that with the combination treatment, autophagy-related protein Atg5 and Beclin-1 are cleaved which suggests that autophagic process is interrupted and apoptosis can take place. In order to investigate whether caspase activation plays a role in Atg5 and Beclin-1 cleavage, we have used specific caspase inhibitors (all at 25 nM) to address this question. A375 (ASS non-inducible) and A2058 (ASS inducible) cell lines were used for this study. About 70-80 % cell death occurs at 24 h after combination treatment, while only 10-20% cell death occurs when treated with TRAIL alone. Almost no cell death is apparent when treated with arginine-free medium alone at this time point. Adding caspase-8 (Z-IETD-FMK), caspase-9 (Z-LEHD-FMK) or caspase-10 (Z-AEVD-FMK) single inhibitor can almost completely rescue cell death on combination treatment while caspase-6 inhibitor (Z-VEID-FMK) has less rescuing effect (about 10-30% cell death). Inhibitors for caspase-3 (Z-DEVD-FMK) or 3/7 (Ac-DNLD-CHO) have least effect (more than 50% cell death). These data confirm that the enhancement effect of the combination needs the participation of both the extrinsic and intrinsic pathways of apoptosis. The cleavage of Beclin-1 and Atg5 are also least affected when caspase-3 or 3/7 inhibitor is applied, but blocked or reduced when single inhibitor to caspase-6, 8, 10 or 9 is added. There is little difference between the ASS inducible or non-inducible cell lines. This finding suggests that the combination treatment turns off the autophagic process during arginine deprivation, possibly through caspase activation and allows apoptotic process to take place. Overall, our findings indicate that autophagy is a pro-survival mechanism in melanoma cells under nutritional stress, and interfering with this pathway may achieve greater efficacy with ADI-PEG20 treatment. (Supported by NIH grant 1R01CA109578) 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 4096. doi:10.1158/1538-7445.AM2011-4096