Molecular mechanisms of gossypol-induced apoptosis in chronic lymphocytic leukemia cells

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Gossypol, a natural product derived from cottonseed extracts, has shown antitumor activities in a broad range of solid and liquid tumors. This agent binds with high affinity to BH3 binding pocket of BCL-2 family members to attenuate the antiapoptotic functions of these proteins, leading to cell death. However, molecular mechanism of its action leading to cell death is not yet known. Since chronic lymphocytic leukemia (CLL) cells have high expression of antiapoptotic proteins, these lymphocytes were used as a model system. Leukemic lymphocytes were isolated from peripheral blood obtained from patients with CLL (n=35). These primary cells were used to determine the downstream effect of gossypol binding and its molecular mechanism of cell death. Gossypol induced apoptosis in a dose (3, 10, 30, 100 µM, n=4) and time- (4, 8, 24 h, n=20) dependent manner. There was significant amount of cell death with 30 µM gossypol at 24 hr (median; 50%; range 10% - 80%). Compared to gossypol treated cells, preincubation of CLL cells with pan-caspase inhibitor Z-VAD.fmk (50 µM; 2 hr) did not abrogate gossypol-induced cell death in 10 of 13 samples and had minor effect in 3 samples, suggesting that the cell death was caspase-independent. Among the early events, gossypol induced a loss of mitochondrial outer membrane potential (MOMP, n=15) as early as 4 h, which was not attenuated by preincubation with Z-VAD.fmk. Consistent with this mitochondrial event, there was an increase in the production of reactive oxygen species (ROS) at 4 h (n=15), which was partially reduced by antioxidant, N-acetyl cysteine but was not affected either by glutathione or by pan caspase inhibitor, Z-VAD.fmk. Because there was loss of MOMP, release of mitochondrial proapoptotic proteins such as apoptosis-inducing factor (AIF) and cytochrome C was investigated. Gossypol treatment (n = 5) caused cytochrome c release from mitochondria to cytosol and stimulated nuclear translocation of the AIF as early as 4 h. Concomitantly, gossypol triggered translocation of BAX from cytosol to mitochondria. In contrast, at this time, expression levels of antiapoptotic proteins (MCL-1, BCL-2, BCL-XL, n=8) did not decrease. However, at 24 h, there was a significant decrease in MCL-1 protein level but not BCL-2 and BCL-XL. Taken together, our results suggest that gossypol-induced apoptosis was caspase independent and was initiated by loss of MOMP and an increased ROS production. The loss of mitochondrial potential leads to cytochrome c release and AIF nuclear translocation. Additionally, cytosolic BAX was translocated to mitochondria. In conclusion, MOMP loss, ROS production, and release of proapoptotic proteins are the early events, while the decrease in antiapoptotic proteins are later events in the gossypol-induced apoptosis in CLL primary cells.