Limited chemotherapeutic options exist for metastatic castration-resistant prostate cancer (CRPC). We have previously demonstrated that 1:500 combination simvastatin (SIM) and metformin (MET) synergistically inhibits cell viability, invasion, migration, and adhesion-independent cell growth of the C4-2B3 and C4-2B4 in vitro model of osseous metastatic CRPC, and inhibits primary ventral prostate tumor growth, incidence of cachexia and metastasis to femurs and spinal column, and prevents biochemical failure in an orthotopic C4-2B4 mouse model of osseous metastatic CRPC, more effectively than docetaxel, the standard-of-care chemotherapy for metastatic CRPC. Autophagy, apoptosis, and necrosis have all been reported as mechanisms by which SIM or MET induce cell death in prostate cancer cells; however, the mechanism of cell death remains unclear in combination. Therefore, the purpose of this study was to determine the mechanism of cell death associated with the synergistic effect of SIM/MET combination chemotherapy in CRPC cells. Treatment of C4-2B3 and C4-2B4 cells, in vitro models of osseous metastatic CRPC, with 4µM SIM and 2mM MET combination treatment led to an earlier (24h treatment) and more pronounced (>90%) G1-phase cell cycle arrest, which was sustained through 96h treatment. 4µM SIM and 2mM MET treatment, individually or in combination, for up to 96h, does not induce cell death by apoptosis in C4-2B3 and C4-2B4 cells, as determined by <3% of cells staining PI(-)/AV(+), <2% of cells staining Apo-BrdU(+), and no presence of activated cleaved caspase-3 protein expression, in comparison to 2µM camptothecin positive control. Treatment of C4-2B3 and C4-2B4 cells with 1:500 SIM and MET combination led to autophagy by 48h, sustained through 96h, ascertained by significantly increased LC3B-II/LC3B-I ratio, quantity of acidic vesicular organelles, and number of autophagic structures, both in the presence and absence of lysosomal inhibitor chloroquine, compared to untreated CRPC cells. SIM/MET-induced autophagy led to secondary necrosis by 96h which was determined by PI-Annexin V flow cytometry; 67% of C4-2B3 and 64% of C4-2B4 osseous metastatic CRPC cells were PI(+)/AV(-) or PI(+)/AV(+) necrotic following 96h of SIM and MET combination treatment. The caspase-independent necrotic cell death was confirmed by receptor interacting protein kinase-1 and -3 (RIP1 and RIP3) protein expression following 96h SIM/MET combination treatment, in the presence or absence of necrostatin-1 inhibitor. Taken together, combination SIM and MET causes pronounced G1-phase cell cycle arrest in osseous metastatic CRPC cells by 24h, which culminates in cell death by autophagy and secondary necrosis by 48-96h of treatment.
Citation Format: Melissa A. Babcook, R. Michael Sramkoski, Christine Z. Oak, Sanjay Gupta. Combination simvastatin and metformin induces cell death by autophagy and secondary necrosis in osseous metastatic castration-resistant prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 804. doi:10.1158/1538-7445.AM2014-804