Abstract
Gossypol, a cottonseed extract derivative, acts as a BH3-mimetic, binding to the BH3 pocket of antiapoptotic proteins and displacing pro-death partners to induce apoptosis. However, knowledge on the molecular underpinnings of its downstream effects is limited. Since chronic lymphocytic leukemia (CLL) cells express high levels of antiapoptotic proteins that act as a survival mechanism for these replicationally quiescent lymphocytes, we investigated whether gossypol induces apoptosis in these cells and what mechanism underlies gossypol-mediated cytotoxicity. Gossypol induced cell death in a concentration- and time-dependent manner; 24-hour incubation with 30 microM gossypol resulted in 50% cell death (median; range, 10%-80%; n = 47) that was not abrogated by pan-specific caspase inhibitor. Starting at 4 hours, the mitochondrial outer membrane was significantly permeabilized (median, 77%; range, 54%-93%; n = 15). Mitochondrial outer membrane permeabiliztaion (MOMP) was concurrent with increased production of reactive oxygen species (ROS); however, antioxidants did not abrogate gossypol-induced cell death. Mitochondrial membrane permeabilization was also associated with loss of intracellular adenosine triphosphate (ATP), activation of BAX, and release of cytochrome c and apoptosis-inducing factor (AIF), which was translocated to the nucleus. Blocking AIF translocation resulted in a decreased apoptosis, suggesting that AIF contributes to gossypol-mediated cytotoxicity in CLL lymphocytes.