Inhibition Of Ornithine Decarboxylase Alters The Roscovitine-Induced Mitochondrial-Mediated Apoptosis In MCF-7 Breast Cancer Cells

Abstract

Polyamines (PAs) are small aliphatic amines that play a major role in multicellular functions. The PA levels are controlled by ornithine decarboxylase (ODC), the rate limiting enzyme of PA biosynthesis. alpha-difluoromethylornithine (DFMO) is the ODC inhibitor, which has been shown to act as an antiproliferative agent in human cancer cells by irreversibly inhibiting ODC, which is overexpressed in breast cancer cells. Roscovitine (ROSC; CYC202), a selective cyclin-dependent kinase inhibitor, induces cell cycle arrest and concomitantly apoptosis in tumor cells. In this study, we aimed to investigate the possible role of PAs in ROSC-induced apoptosis in estrogen-dependent MCF-7 breast cancer cells. Cell viability was assessed following the exposure of MCF-7 cells to DFMO and/or ROSC by MTT cell viability assay. To evaluate the drug-induced apoptotic events, DNA fragmentation by Cell Death ELISA assay and 4,6-diamidino-2-phenylindole staining, were utilized. The disruption of mitochondrial membrane potential, caspase-9 and PARP cleavage was also determined in order to investigate the role of mitochondrial-mediated apoptosis. The modulation of Bcl-2 family members was also evaluated using the immunoblotting technique. Drug-induced reactive oxygen species was determined by a fluorometer following 2',7'-dichlorofluorescein diacetate staining. We found that ROSC induced apoptosis in a dose- and caspase-dependent manner. The ODC specific inhibitor, DFMO, altered the apoptotic effects of ROSC by increasing the generation of reactive oxygen species, decreasing the PA intracellular pool and modulating pro-apoptotic and anti-apoptotic Bcl-2 family members. All these findings suggest that ODC may be a critical target for evaluating the PA metabolic pathway as a therapeutic target in ROSC-induced mitochondrial-mediated apoptotis in estrogen-dependent MCF-7 breast cancer cells.