Abstract 16: Combination simvastatin and metformin induces G1-phase cell cycle arrest and Ripk1- and Ripk3-dependent necroptosis in C4-2B osseous metastatic castration-resistant prostate cancer cells
Authors: Babcook Melissa A. , Sramkoski R. Michael , Fujioka Hisashi , Daneshgari Firouz , Almasan Alexandru , Shukla Sanjeev , Gupta Sanjay 
Published: August 2015

Abstract
Castration-resistant prostate cancer (CRPC) cells acquire resistance to chemotherapy and apoptosis in part due to enhanced aerobic glycolysis and biomass production, known as Warburg effect. We previously demonstrated that combination simvastatin (SIM) and metformin (MET) ameliorates critical Warburg effect-related metabolic aberrations of C4-2B cells, synergistically and significantly decreases CRPC cell viability and metastatic properties, with minimal effect on normal prostate epithelial cells, and inhibits primary prostate tumor growth, metastasis, and biochemical failure in an orthotopic model of metastatic CRPC, more effectively than docetaxel chemotherapy. Several modes of cell death activated by individual treatment of SIM or MET have been reported; however, the cell death process induced by combination SIM and MET treatment in metastatic CRPC cells remains unknown. This must be determined prior to advancing combination SIM and MET to clinical trial for metastatic CRPC. Treatment of C4-2B cells with combination 4μM SIM and 2mM MET (SIM + MET) led to significant G1-phase cell cycle arrest and decrease in percentage of DNA-replicating cells in S-phase by 24h; arrest was sustained throughout 96h treatment. SIM + MET treatment led to enhanced autophagic flux in C4-2B cells by 72-96h, ascertained by increased LC3B-II (further enhanced with lysosomal inhibitor chloroquine) and reduced Sequestosome-1 protein expression, significantly increased percentage acidic vesicular organelle-positive cells, and increased autophagic structure accumulation assessed by transmission electron microscopy. Chloroquine, however, could not rescue CRPC cell viability, eliminating autophagy as a mechanism of cell death; rather, autophagy was upregulated by C4-2B cells in attempt to withstand chemotherapy. Instead, SIM + MET treatment led to Ripk1- and Ripk3-dependent necroptosis by 48-96h, determined by propidium iodide-Annexin V flow cytometry, increase in Ripk1 and Ripk3 protein expression, necrosome formation, HMGB-1 extracellular release, and necroptotic-induction and viability rescue with necrostatin-1 (Ripk1 activity inhibitor) and Ripk3-targeting siRNA. The necroptosis-inducing capacity of SIM + MET may reinvent these drugs as highly-effective treatment for apoptosis- and chemotherapy-resistant metastatic CRPC cells.
Citation Format: Melissa A. Babcook, R. Michael Sramkoski, Hisashi Fujioka, Firouz Daneshgari, Alexandru Almasan, Sanjeev Shukla, Sanjay Gupta. Combination simvastatin and metformin induces G1-phase cell cycle arrest and Ripk1- and Ripk3-dependent necroptosis in C4-2B osseous metastatic castration-resistant prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 16. doi:10.1158/1538-7445.AM2015-16