Metformin suppresses hepatocellular carcinoma cell growth through induction of cell cycle G1/G0 phase arrest and p21CIP and p27KIP expression and downregulation of cyclin D1 in vitro and in vivo
Metformin suppresses hepatocellular carcinoma cell growth through induction of cell cycle G1/G0 phase arrest and p21CIP and p27KIP expression and downregulation of cyclin D1 in vitro and in vivo
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DOI：
10.3892/or.2013.2718
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发表时间：
2013-11-01
期刊：
影响因子：
4.2
通讯作者：
Jing, Xubin
中科院分区：
文献类型：
--
作者：
Cai, Xianbin;Hu, Xi;Jing, Xubin
二甲双胍被用作治疗2型糖尿病的一线药物，有报道称它对预防和控制几种类型的癌症有用。本研究探讨二甲双胍对肝细胞癌的治疗作用。培养人肝癌细胞系HepG2和PLC/PRF/5，分别用一磷酸腺苷(AMP)激活蛋白激酶激活剂二甲双胍或5-氨基咪唑-4-甲酰胺核糖核苷(AICAR)处理。用四甲基偶氮唑盐比色法和流式细胞仪分别检测细胞活力和细胞周期分布的变化。荧光染色检测细胞凋亡率。6~8周龄BALB/c-nu小鼠皮下注射PLC/PRF/5细胞建立肝癌模型。1周后，给小鼠灌胃二甲双胍或赋形剂。采用免疫组织化学方法检测移植瘤组织中细胞周期蛋白D1、p21(CIP)和p27(KIP)的表达。分别从体外和体内实验中提取肝癌细胞和组织，并进行蛋白质提取和蛋白质印迹。我们发现，与AICAR治疗类似，二甲双胍治疗以剂量依赖的方式降低了肝癌细胞的存活率。此外，二甲双胍还可诱导肝癌细胞周期停滞于G1/G0期，并诱导细胞发生凋亡。用二甲双胍灌胃治疗小鼠PLC/PRF/5细胞移植瘤模型，结果表明，二甲双胍不仅能抑制肿瘤的发展，而且能降低肿瘤的发病率。在体外和体内实验中，二甲双胍上调了p21(CIP)和p27(KIP)的表达，下调了细胞周期蛋白D1的水平。二甲双胍治疗还上调了异种移植组织中磷酸化AMPK蛋白的表达。这些发现表明，二甲双胍作为一种新的治疗和预防肝癌的策略值得进一步评估。
Metformin is used as a first-line therapy for type 2 diabetes, with reports of its usefulness for the prevention and control of several types of cancers. This study investigated the effects of metformin on hepatocellular carcinoma (HCC). The human HCC cell lines HepG2 and PLC/PRF/5 were cultured and treated with metformin or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an activator of adenosine monophosphate (AMP)-activated protein kinase. Changes in cell viability and cell cycle distribution were evaluated by MTT and flow cytometry, respectively. Apoptosis was assessed by fluorescent-dye staining. An HCC model was established in 6-to 8-week-old BALB/c-nu mice by subcutaneous injection of PLC/PRF/5 cells. After 1 week, mice were treated intra-gastrically with metformin or vehicle. Tumor xenograft tissues were examined using immunohistochemistry for evaluation of the the expression of cyclin D1, p21(CIP) and p27(KIP). HCC cells and tissues from the in vitro and in vivo experiments, respectively, were subjected to protein extraction and western blotting. We found that metformin treatment reduced HCC cell viability in a dose-dependent manner similar to AICAR treatment. In addition, metformin treatment induced HCC cell cycle arrest at G1/G0 phase and apoptosis. Intragastric treatment of the mouse PLC/PRF/5 cell xenograft model with metformin showed that metformin not only blocked tumor progression, but also reduced tumor morbidity. Treatment with metformin upregulated the expression of p21(CIP) and p27(KIP), but downregulated cyclin D1 levels, both in vitro and in vivo. Metformin treatment also upregulated the expression of phosphorylated AMPK protein in xenograft tissues. These findings indicate that metformin warrants further evaluation as a novel therapeutic and preventive strategy against HCC.