title: Quinacrine induces cytochrome c-dependent apoptotic signaling in human cervical carcinoma cells.
abstract: Quinacrine (QU), a phospholipase-A2 (PLA-2) inhibitor has been used clinically as a chemotherapeutic adjuvant. To understand the mechanisms leading to its chemotherapeutic effect, we have investigated QU-induced apoptotic signaling pathways in human cervical squamous carcinoma HeLa cells. In this study, we found that QU induced cytochrome c-dependent apoptotic signaling. The release of pro-apoptotic cytochrome c was QU concentration- and time-dependent, and preceded activation of caspase-9 and -3. Flow cytometric FACScan analysis using fluorescence intensities of DiOC6 demonstrated that QU-induced cytochrome c release was independent of mitochondrial permeability transition (MPT), since the concentrations of QU that induced cytochrome c release did not alter mitochondrial membrane potential (delta pai(m)). Moreover, kinetic analysis of caspase activities showed that cytochrome c release led to the activation of caspase-9 and downstream death effector, caspase-3. Caspase-3 inhibitor (Ac-DEVD-CHO) partially blocked QU-induced apoptosis, suggesting the importance of caspase-3 in this apoptotic signaling mechanism. Supplementation with arachidonic acid (AA) sustained caspase-3 activation induced by QU. Using inhibitors against cellular arachidonate metabolism of lipooxygenase (Nordihydroxyguaiaretic Acid, NDGA) and cyclooxygenase (5,8,11,14-Eicosatetraynoic Acid, ETYA) demonstrated that QU-induced apoptotic signaling may be dependent on its role as a PLA-2 inhibitor. Interestingly, 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 summary, we report that QU induced cytochrome c-dependent apoptotic signaling cascade, which may be dependent on its role as a PLA-2 inhibitor. This apoptotic mechanism induced by QU may contribute to its known chemotherapeutic effects.
authors.affiliation: Department of Pharmaceutics and Pharmacodynamics, Center for Pharmaceutical Biotechnology, College of Pharmacy, Rutgers University, Piscataway, NJ 08854, USA.
authors.forename: A A
authors.initials: AA
authors.lastname: Fasanmade
authors.forename: E D
authors.initials: ED
authors.lastname: Owuor
authors.forename: R P
authors.initials: RP
authors.lastname: Ee
authors.forename: D
authors.initials: D
authors.lastname: Qato
authors.forename: M
authors.initials: M
authors.lastname: Heller
authors.forename: A N
authors.initials: AN
authors.lastname: Kong
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D003574:Cytochrome c Group / Q000502:physiology*; D004347:Drug Interactions; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D054467:Phospholipases A2; D011796:Quinacrine / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D013050:Spectrometry, Fluorescence; D002583:Uterine Cervical Neoplasms / Q000473:pathology*
chemical_list: D000962:Antimalarials; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D010741:Phospholipases A; D054467:Phospholipases A2; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D011796:Quinacrine