Quinacrine induces cytochrome c-dependent apoptotic signaling in human cervical carcinoma cells

Quinacrine (QU), a phospholipase-A2 (PLA-2) inhibitor has been used clinica lly as a chemotherapeutic adjuvant To understand the mechanisms leading to its chemotherapeutic effect, we have investigated QU-induced apoptotic sign aling pathways in human cervical squamous carcinoma HeLa cells. In this stu dy, we found that QU induced cytochrome c-dependent apoptotic signaling. Th e release of pro-apoptotic cytochrome c was QU concentration- and time-depe ndent and preceded activation of caspase-9 and -3. Flow cytometric FACScan analysis using fluorescence intensities of DiOC(6) demonstrated that QU-ind uced cytochrome c release was independent of mitochondrial permeability tra nsition (Mm), since the concentrations of QU that induced cytochrome c rele ase did not alter mitochondrial membrane potential (triangle Psi (m)). More over, kinetic analysis of caspase activities showed that cytochrome c relea se led to the activation of caspase-9 and downstream death effector, caspas e-3. Caspase-3 inhibitor (Ac-DEVD-CHO) partially blocked QU-induced apoptos is, suggesting the importance of caspase-3 in this apoptotic signaling mech anism. Supplementation with arachidonic acid (AA) sustained caspase-3 activ ation induced by QU. Using inhibitors against cellular arachidonate metabol ism of lipooxygenase (Nordihydroxyguaiaretic Acid, NDGA) and cyclooxygenase (5,8,11,14-Eicosatetraynoic Acid, ETYA) demonstrated that QU-induced apopt otic signaling may be dependent on its role as a PLA-2 inhibitor. Interesti ngly, NDGA attenuated QU-induced cytochrome c release, caspase activity as well as apoptotic cell death. The blockade of cytochrome c release by NDGA was much more effective than that attained with cyclosporin A (CsA), a MPT inhibitor. ETYA was not effective in blocking cytochrome c release, except under very high concentrations. Caspase inhibitor z-VAD blocked the release of cytochrome c suggesting that this signaling event is caspase dependent and caspase-8 activation may be upstream of the mitochondrial events. In su mmary, we report that QU induced cytochrome c-dependent apoptotic signaling cascade, which may be dependent on its role as a PLA-2 inhibitor. This apo ptotic mechanism induced by QU may contribute to its known chemotherapeutic effects.