二甲双胍激活AMPK/p53调节尿素循环抑制结肠癌HCT116细胞增殖  

Metformin activates AMPK/p53 to regulate urea cycle and inhibits proliferation of colon cancer HCT116 cells

作　　者：张滔 胡玲 唐加峰 徐航 田宽[1,2] 黄世莹 杜玉梅 杨周倩 李静 陈地龙[3] 冉建华[1,2] ZHANG Tao;HU Ling;TANG Jia-feng;XU Hang;TIAN Kuan;HUANG Shi-ying;DU Yu-mei;YANG Zhou-qian;LI Jing;CHEN Di-long;RAN Jian-hua(Dept of Anatomy,Center of Neuroscience Research;Dept of Histology and Stem Cell Research,Dept of Histology and Embryology;Chongqing Three Gorges Medical College,Chongqing 404120,China;Dept of Public Health and Management,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学基础医学院解剖教研室神经科学研究中心,重庆400016 [2]重庆医科大学基础医学院干细胞与组织工程研究室,重庆400016 [3]重庆三峡医药高等专科学校,重庆404120 [4]重庆医科大学公共卫生与管理学院,重庆400016

出　　处：《中国药理学通报》2020年第10期1361-1367,共7页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81770738)。

摘　　要：目的探讨二甲双胍(metformin,Met)对HCT116细胞增殖的抑制作用及机制。方法采用CCK-8法检测细胞增殖情况;细胞克隆形成实验检测细胞克隆形成的能力;流式细胞术(flow cytometry,FCM)检测细胞周期和凋亡率;免疫荧光染色观察磷酸化腺苷酸激活蛋白激酶(p-AMPK)的定位情况,Western blot检测p-AMPK、AMPK、p53、p-p53、精氨酸酶1(ARG1)、鸟氨酸转氨甲酰酶(OTC)和鸟氨酸脱羧酶(ODC)的表达情况。结果 CCK-8结果显示:Met呈浓度和时间依赖性抑制细胞的增殖;FCM结果显示:Met能诱导细胞发生凋亡,将细胞周期阻滞于G1期;克隆形成实验显示:Met可以抑制细胞的克隆形成能力;经免疫荧光染色可知:Met处理组细胞胞质及胞核内p-AMPK的表达增加;Western blot结果显示:p-AMPK、p53、p-p53蛋白的表达上调,ARG1、OTC、ODC蛋白的表达下调。结论 Met可抑制结肠癌细胞HCT116的增殖,诱导G1期细胞周期阻滞,促进细胞凋亡;其可能的机制与激活AMPK、p53信号,抑制尿素循环ARG1、OTC和多胺生成限速酶ODC的表达有关。Aim To investigate the inhibitory effect of metformin(Met)on the proliferation of HCT116 cells and the mechanisms.Methods Cell proliferation was measured by CCK-8 assay.Cell proliferation ability after treatment with Met was examined by clonal formation assay;cell cycle and apoptosis were detected by flow cytometry.The localization of phosphorylated Adenosine 5′-monophosphate(AMP)-activated protein kinase(p-AMPK)in HCT116 cells was detected by immunofluorescence staining.The expression levels of AMPK,p-AMPK,p53,p-p53,arginase1(ARG1),ornithine transcarbamylase(OTC)and ornithine decarboxylase(ODC)proteins were detected by Western blot.Results CCK-8 results showed that the proliferation of HCT116 cells was increasingly inhibited by Met in a dose-and time-dependent manner.FCM showed that Met induced apoptosis and cell-cycle arrest in G1 phase in HCT116 cells.Clonal formation assay revealed that Met inhibited colony formation of HCT116 cells.According to the result of immunofluorescence staining,p-AMPK was highly expressed in cytoplasm and nucleus of HCT116 cells.Western blot results showed that the expression levels of p-AMPK,p53 and p-p53 proteins increased,while those of ARG1,OTC and ODC proteins decreased.Conclusions Met can inhibit the proliferation of colon cancer cells HCT116,induce cell cycle arrest at G1 phase and promote cell apoptosis,which may be related to activating AMPK and p53 signals,inhibiting ARG1 and OTC expression in urea cycle and resulting in decreasing of ODC expression.

关 键 词：二甲双胍 HCT116 P53 ARG1 OTC ODC 增殖 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R345.57[医药卫生—基础医学]