Cardiolipin is exclusively present in mitochondria and after being labeled with NAO and exhibits yellow fluorescence. When we analyzed our cells under the fluorescent Lomitapide Mesylate microscope, we observed that almost all the cells from control group were exhibiting yellow color. However, the yellow staining decreased and turned into green in capsaicin treated cells indicating drastic oxidation of cardiolipin. Nonetheless, catalase and EUK-134 completely prevented the oxidation of cardiolipin. These results were confirmed by flow cytometry where we observed that capsaicin causes cardiolipin oxidation in BxPC-3 cells as shown by a shift of NAO fluorescence towards left. We further used catalase and EUK-134 to see whether the oxidation of cardiolipin can be prevented. We found that addition of catalase or EUK-134 almost completely blocked the shift of NAO staining suggesting that the decrease of NAO fluorescence was due to oxidation of mitochondrial lipid cardiolipin by mitochondrial ROS. Mitochondria are a major physiological source of ROS, which are generated due to incomplete reduction of oxygen during normal mitochondrial respiration. Excessive ROS that are generated under certain pathological conditions acts as mediator of apoptotic signaling pathway. Under normal physiological conditions, mitochondria contain sufficient levels of antioxidants that prevent ROS generation and Gomisin-D oxidative damage. However, under circumstances in which excessive mitochondrial ROS are produced or when antioxidant levels are depleted, oxidative damage to mitochondria occurs. Our current results shows that capsaicin induced apoptosis in BxPC-3 and AsPC-1 cells but not in HPDE-6 cells was associated with ROS generation. The ROS generation by capsaicin was due to marked inhibition of mitochondrial electron transport chain complexes-I and III and downregulation of antioxidants such as GSH, catalase, SOD and 
GPx indicating the involvement of mitochondria. On the other hand, r0 cells derived from BxPC-3 cells, which lack normal oxidative phosphorylation were unable to cause ROS generation and were totally resistant to the apoptosis inducing effects of capsaicin. ROS once generated cause oxidation of critical redox sensitive proteins and lipids leading to mitochondrial damage. Our results clearly show that capsaicin treatment, cause massive oxidation of cardiolipin, which is specifically present in the mitochondria. Mitochondrial damage due to oxidation of cardiolipin has been documented in a recent study. Cytochrome c preferentially binds to cardiolipin and is liberated upon oxidation of cardiolipin. In agreement, our results show the release of cytochrome c into cytosol by capsaicin treatment, which could be due to cardiolipin oxidation. Our results also demonstrate massive depletion of ATP as evaluated by complex-V ATP synthase activity. ETC complex forms a transmembrane potential. ATP synthase uses potential energy stored in Dy to phosphorylate ADP. However, under certain pathological conditions, the Dy can collapse resulting in the release of molecules from the mitochondria into the cytosol. Our result do show decrease in Dy and release of cytochrome c into the cytosol in response to capsaicin treatment. Further, ATP production was shown to be highly sensitive to complex-III inhibition in a previous report. In agreement, our results also show a relationship between complex III inhibition and ATP depletion. Cellular redox homeostasis is maintained by a fine balance between antioxidants and pro-oxidants. Glutathione is a critical intracellular antioxidant responsible for maintaining redox balance. GSH can be oxidized to formGSSG and the ratio of GSH/GSSG is an indicator of oxidative stress in the cells. High concentrations of GSSG can oxidatively damage many critical enzymes. Our results reveal that capsaicin treatment caused time dependent increase in the levels of GSSG and decrease in GSH levels in BxPC3 cells. Similar observations were made in the tumors of capsaicin treated mice as compared to the tumors from control mice.