Combination simvastatin and metformin induces G1-phase cell cycle arrest and Ripk1- and Ripk3-dependent necrosis in C4-2B osseous metastatic castration-resistant prostate cancer cells.
Combination simvastatin and metformin induces G1-phase cell cycle arrest and Ripk1- and Ripk3-dependent necrosis in C4-2B osseous metastatic castration-resistant prostate cancer cells.
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DOI：
10.1038/cddis.2014.500
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发表时间：
2014-11-20
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9
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去势抵抗性前列腺癌（CRPC）细胞获得对化疗和凋亡的抗性，部分原因是由于有氧糖酵解和生物质产生的增强，称为瓦尔堡效应。我们先前证明辛伐他汀（SIM）和二甲双胍（MET）组合改善C4-2B细胞的关键瓦尔堡效应相关代谢畸变，协同并显著降低CRPC细胞活力和转移特性，对正常前列腺上皮细胞的影响最小，并在转移性CRPC的原位模型中抑制原发性前列腺肿瘤生长、转移和生化失败，比多西他赛化疗更有效。已经报道了SIM或MET单独处理激活的几种细胞死亡模式;然而，在转移性CRPC细胞中SIM和MET联合处理诱导的细胞死亡过程仍然未知。这必须在将SIM和MET组合推进转移性CRPC临床试验之前确定。用4 μM SIM和2 mM MET（SIM+MET）组合处理C4-2B细胞导致显著的G1期细胞周期停滞，并在24小时内降低S期DNA复制细胞的百分比;停滞在整个96小时处理期间持续。SIM+MET处理导致C4-2B细胞中自噬通量增强72-96 h，通过增加的LC 3B-II（用溶酶体抑制剂氯喹进一步增强）和减少的Sequestosome-1蛋白表达、显著增加的酸性囊泡细胞器阳性细胞百分比以及通过透射电子显微镜评估的自噬结构积累增加来确定。然而，氯喹不能拯救CRPC细胞活力，消除自噬性细胞死亡;相反，C4-2B细胞上调自噬以试图耐受化疗。相反，SIM+MET处理导致Ripk 1和Ripk 3依赖性坏死，通过碘化丙啶-膜联蛋白V流式细胞术测定，Ripk 1和Ripk 3蛋白表达增加，坏死体形成，HMGB-1细胞外释放，以及坏死诱导和坏死抑制素-1和Ripk 3靶向siRNA的活力拯救。SIM+MET的坏死诱导能力可能使这些药物成为对细胞凋亡和化疗耐药的转移性CRPC细胞的高效治疗方法。
Castration-resistant prostate cancer (CRPC) cells acquire resistance to chemotherapy and apoptosis, in part, due to enhanced aerobic glycolysis and biomass production, known as the 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 4 μM SIM and 2 mM MET (SIM+MET) led to significant G1-phase cell cycle arrest and decrease in the percentage of DNA-replicating cells in the S-phase by 24 h; arrest was sustained throughout the 96-h treatment. SIM+MET treatment led to enhanced autophagic flux in C4-2B cells by 72–96 h, ascertained by increased LC3B-II (further enhanced with lysosomal inhibitor chloroquine) and reduced Sequestosome-1 protein expression, significantly increased percentage of acidic vesicular organelle-positive cells, and increased autophagic structure accumulation assessed by transmission electron microscopy. Chloroquine, however, could not rescue CRPC cell viability, eliminating autophagic 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 necrosis by 48–96 h, determined by propidium iodide-Annexin V flow cytometry, increase in Ripk1 and Ripk3 protein expression, necrosome formation, HMGB-1 extracellular release, and necrotic induction and viability rescue with necrostatin-1 and Ripk3-targeting siRNA. The necrosis-inducing capacity of SIM+MET may make these drugs a highly-effective treatment for apoptosis- and chemotherapy-resistant metastatic CRPC cells.