二甲双胍增强胆管癌细胞对吉西他滨敏感性机制的研究

The mechanism of metformin enhanceing the sensitivity of bile duct cancer cells to gemcitabine

目的探讨二甲双胍通过抑制丙酮酸激酶M2(PKM2)促进线粒体凋亡增强胆管癌细胞对吉西他滨敏感性的作用及机制。方法将二甲双胍及吉西他滨和PKM2激动剂ML265不同组合后作用于人胆管癌细胞HCC9810和RBE:(1)HCC9810/RBE+溶剂组:HCC9810/RBE细胞在含有终浓度5%二甲基亚砜(DMSO)的培养基中培养;(2)HCC9810/RBE+吉西他滨组:HCC9810/RBE细胞在含有终浓度为100 nmol/L吉西他滨(溶于5%DMSO)的培养基中培养;(3)HCC9810/RBE+二甲双胍+吉西他滨组:HCC9810/RBE细胞在含有终浓度为10 mmol/L二甲双胍和100 nmol/L吉西他滨(溶于5%DMSO)的培养基中培养;(4)HCC9810/RBE+二甲双胍+吉西他滨+ML265组:HCC9810/RBE细胞在含有终浓度为10 mmol/L二甲双胍、100 nmol/L吉西他滨和100 nmol/L ML265(溶于5%DMSO)的培养基中培养。48 h后采用噻唑蓝染色法检测各组细胞存活率,qRT-PCR法检测各组细胞PKM2 mRNA表达水平,乳酸定量检测试剂盒检测各组细胞培养基乳酸含量,乳酸脱氢酶(LDH)定量检测试剂盒检测各组细胞LDH活性;线粒体膜电位检测染色法检测各组细胞线粒体凋亡情况。结果与HCC9810/RBE+溶剂组比较,HCC9810/RBE+吉西他滨组、HCC9810/RBE+二甲双胍+吉西他滨组、HCC9810/RBE+二甲双胍+吉西他滨+ML265组细胞存活率均降低而线粒体凋亡均增加,HCC9810/RBE+二甲双胍+吉西他滨组、HCC9810/RBE+二甲双胍+吉西他滨+ML265组细胞培养基乳酸含量、PKM2 mRNA表达水平均降低,细胞LDH活性均升高,差异均有统计学意义(均P<0.05);与HCC9810/RBE+吉西他滨组比较,HCC9810/RBE+二甲双胍+吉西他滨组细胞存活率降低,HCC9810/RBE+二甲双胍+吉西他滨组、HCC9810/RBE+二甲双胍+吉西他滨+ML265组细胞培养基乳酸含量、PKM2 mRNA表达水平均降低而线粒体凋亡均增加、细胞LDH活性均增加,差异均有统计学意义(均P<0.05);与HCC9810/RBE+二甲双胍+吉西他滨组细胞比较,HCC9810/RBE+二甲双胍+吉西他滨+ML265组细胞存活率、PKM2 mRNA表达水平均升高而线粒体凋亡、细胞LDH活性均降低,差异均有统计学意义(均P<0.05)。结论二甲双胍可能通过抑制胆管癌细胞PKM2的表达、激活线粒体凋亡来增强胆管癌细胞对吉西他滨的敏感性。

Objective To investigate the effect and mechanism of metformin on enhancing the sensitivity of bile duct cancer cells to gemcitabine by inhibiting pyruvate kinase M2(PKM2)and promoting mitochondrial apoptosis.Methods HCC9810 and RBE were treated with metformin,gemcitabine and PKM2 agonist ML265 in different combinations:(1)HCC9810/RBE+solvent group:HCC9810/RBE cells were cultured in medium containing a final concentration of 5%dimethyl sulfoxide(DMSO),(2)HCC9810/RBE+gemcitabine group:HCC9810/RBE cells were cultured in medium containing a final concentration of 100 nmol/L gemcitabine(dissolved in 5%DMSO),(3)HCC9810/RBE+metformin+gemcitabine group:HCC9810/RBE cells were cultured in medium containing a final concentration of 10 mmol/L metformin and 100 nmol/L gemcitabine(dissolved in 5%DMSO),(4)HCC9810/RBE+metformin+gemcitabine+ML265 group:HCC9810/RBE cells were cultured in a medium cont aining a final concentration of 10 mmol/L metformin,100 nmol/L gemcitabine and 100 nmol/L ML265(dissolved in 5%DMSO).After 48 h,the cells survival rate was detected by MTT assay,the expression of PKM2 mRNA was detected by qRT-PCR,the content of lactic acid was detected by lactic acid quantitative detection kit,the activity of lactate dehydrogenase(LDH)was detected by LDH quantitative detection kit,and the mitochondrial apoptosis was detected by mitochondrial membrane potential(JC-1)staining.Results Compared with HCC9810/RBE+solvent group,the cells survival rate was significantly decreased,and the mitochondrial apoptosis was increased in HCC9810/RBE+gemcitabine group,HCC9810/RBE+metformin+gemcitabine group,HCC9810/RBE+metformin+gemcitabine+ML265 group,while the content of lactic acid and the expression of PKM2 mRNA were significantly decreased,and the activity of LDH was increased in HCC9810/RBE+metformin+gemcitabine group,HCC9810/RBE+metformin+gemcitabine+ML265 group(all P<0.05).Compared with HCC9810/RBE+gemcitabine group,the cells survival rate was significantly decreased in HCC9810/RBE+metformin+gemcitabine group,and the content of lactic acid and the expression of PKM2 mRNA were significantly decreased while the mitochondrial apoptosis and the activity of LDH were increased in HCC9810/RBE+metformin+gemcitabine group,HCC9810/RBE+metformin+gemcitabine+ML265 group(all P<0.05).Compared with HCC9810/RBE+metformin+gemcitabine group,the cells survival rate and the expression of PKM2 mRNA were significantly increased in HCC9810/RBE+metformin+gemcitabine+ML265 group,while the mitochondrial apoptosis,and the activity of LDH were significantly decreased(all P<0.05).Conclusion Metformin may enhance the sensitivity of bile duct cancer cells to gemcitabine by inhibiting PKM2 expression and activating mitochondrial apoptosis.