Investigation of apoptotic effects of za in prostate cancer stem cells induced by nitric oxide

Cancer stem cells (CSCs) are a subset of tumor cells responsible for tumor initiation and exert a high self-renewal potential. In addition to their role in tumor recurrence, they also exhibit increased resistance to chemotherapy and radiation. Evidence shows that nitric oxide (NO)-donors such as DETA NONOate possess chemo-sensitizing effects. Our previous observations of apoptotic effects of third generation bisphosphonate zoledronic acid (ZA) in human prostate DU-145 CSCs through over-expression of some pro-apoptotic and under-expression of some anti-apoptotic proteins prompted us to investigate the impact of DETA NONOate on cell survival and ZA-induced apoptosis in prostate cancer. CSCs and non-CSCs isolated from human prostate cancer DU-145 cell line were first treated with 500 microM of DETA NONOate for 24 hours and then with IC50 doses of ZA for an additional 72 hours. Cell viability tests and Annexin V assays were performed by MuseTM cell analyzer. Expressions of 84 apoptosis-related genes were analyzed by qRT-PCR array. Sequential treatment with DETA NONOate demonstrated significantly greater cytotoxicity through decreased cell viability and enhanced levels of total apoptosis in prostate DU-145 CSCs compared to our previous results of treatment with ZA only. Expressions of inhibitor of apoptosis protein (IAP) family BIRC2 and BIRC3 genes decreased significantly in CSCs pretreated with DETA NONOate compared with those treated with ZA alone. These effects of DETA NONOate were not observed in non-CSCs. Our findings indicate that combined treatment with ZA and DETA NONOate has more cytotoxic and apoptotic effect on CSCs than non-CSCs and that nitric oxide sensitizes CSCs to ZA-induced apoptosis through down-regulation of IAP proteins BIRC2 and BIRC3. Therefore, sequential treatment with nitric oxide donor DETA NONOate and drugs specifically targeting key survival pathways in prostate CSCs warrant testing in prospective clinical trials for treatment of aggressive prostate cancer.