Abstract B26: Targeting glucose and glutamine metabolism in melanoma cells influences Noxa and Mcl-1 levels as well as susceptibility to TRAIL-induced apoptosis

Abstract

Abstract Melanoma cells are notoriously resistant to conventional therapeutics (some of which trigger the intrinsic mitochondrial-based apoptotic pathway), whilst some melanoma cells are also resistant to extrinsic death ligand-mediated apoptosis. Emerging studies focused on metabolomics of cancer cells revealed that metabolic alterations in tumors could be exploited as novel targets for cancer therapy. Many tumor cells not only display a high rate of glucose uptake and glycolysis, but also exhibit increased glutamine consumption. In this study, apoptotic susceptibility to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2L) was investigated in four human melanoma cell lines in the presence of a metabolic inhibitor of glucose (2-deoxyglucose, 2DG, 10 mM) or glutamine (aminooxyacetate; AOA; 1mM, inhibitor of glutamate dependent transaminase). 2DG or AOA alone did not trigger significant apoptosis measured by Annexin V-PI staining and FACS analysis in these melanoma cell lines within a period of 24 hrs. However, the combined treatment with either 2DG plus TRAIL (100ng/ml), or AOA plus TRAIL, significantly induced apoptosis in 3 of 4 melanoma cell lines, compared to a single treatment protocol. Furthermore, the co-treatment selectively enhanced TRAIL mediated cytotoxicity only in melanoma cells, but not in normal human melanocytes or skin derived dermal fibroblasts. To more firmly establish the vital links between inhibition of glucose or glutamine metabolism and TRAIL efficacy, melanoma cells were also cultured with medium depleted of either glucose or glutamine for 1 day followed by adding TRAIL for another 24 hrs. Withdrawal of either glucose or glutamine by itself induced low level of spontaneous apoptosis in both melanoma cell lines tested. Similar to the treatment with 2DG or AOA, withdrawal of glucose or glutamine also sensitized both melanoma cell lines to TRAIL mediated apoptosis. The apoptosis induced by co-treatment with 2DG or AOA plus TRAIL was markedly blocked by a pan caspase inhibitor (Z-VAD), implicating a caspase-dependency. Western blotting analysis confirmed cotreatments effectively induced caspases 3 cleavage and Bid degradation in melanoma cells. Additionally 2DG or AOA plus TRAIL also resulted in loss of mitochondrial membrane potential (reduced Rhodamine123 uptake). Furthermore, both 2DG and AOA changed the balance between anti- and pro- apoptotic proteins of Bcl-2 family members. AOA was a stronger inducer compared to 2DG in enhancing BH3-only protein Noxa levels. 2DG was more effective in reducing Bcl-XL levels, while AOA more strongly inhibited Mcl-1 expression. Taken together, the findings of this study described a novel strategy to overcome the apoptotic resistance of melanoma cells by potentially activating both TRAIL-based, and mitochondrial-based, apoptosis by targeting either glucose or glutamine metabolic pathways for future melanoma therapeutic trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B26.