二甲双胍对人肝癌细胞 HepG2增殖及脂肪酸合酶的影响

Effects of metformin on cel proliferation and fatty acid synthase in human hepatocel ular carcinoma cel line HepG2

目的二甲双胍治疗的糖尿病患者癌症发生风险较其他药物治疗者显著降低。探讨二甲双胍在人肝癌细胞HepG2中的抗肿瘤活性与脂肪酸合酶的关系。方法选取不同浓度(1、5、10、15 mmol/L)二甲双胍处理HepG2细胞24、48、72 h,用CCK-8法检测其对细胞增殖的影响。同时设阳性对照(紫杉醇10μg/m L),阴性对照(二甲双胍0 mmol/L)。设0、5、10、15 mmol/L二甲双胍处理72 h,用Western blot检测腺苷酸活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)、磷酸化AMPK(P-AMPK)、脂肪酸合酶(fatty acid synthase,FASN)、磷酸化哺乳动物雷帕霉素靶蛋白(P-m TOR)、磷酸化蛋白激酶B(P-Akt)蛋白表达,RT-PCR检测FASN mRNA的表达水平。结果 1、5、10、15 mmol/L二甲双胍干预24、48、72 h,分别随二甲双胍浓度与处理时间的增加,HepG2细胞的增殖抑制作用逐渐增强(P<0.05),且呈时间和浓度依赖性。其中10 mmol/L二甲双胍处理后增殖抑制率在72 h接近50%、15 mmol/L二甲双胍处理后增殖抑制率均>50%。0、5、10、15 mmol/L二甲双胍处理HepG2细胞72 h后,P-AMPK的蛋白表达随药物浓度增高而上调,P-m TOR、FASN的蛋白表达随药物浓度增高而下调,三者在10、15 mmol/L二甲双胍作用后的表达较阴性对照的差异均有统计学意义(P<0.05);而P-Akt蛋白表达在二甲双胍各浓度间差异无统计学意义(P>0.05)。FASN mRNA的表达随药物浓度增加呈下降趋势,5、10、15 mmol/L二甲双胍作用后表达量均较阴性对照显著下降(P<0.05)。结论二甲双胍的的抗肿瘤作用与其激活AMPK、抑制m TOR和FASN有关。二甲双胍虽然抑制了m TOR的活化,但没有造成Akt的反馈性激活。

Objective The cancer risk of patients with diabetes mellitus who are treated by metformin declines remarkably in comparison to patients receiving other drug therapies.The article was to investigate the relationship between antineopastic activity and fatty acid synthase （FASN） of metformin in human hepatocellular carcinoma cell（HCC） line HepG2. Methods HepG2 cells were treated with various concentrations of metformin（ 0, 1, 5, 10, 15 mmol/L） for 24, 48 and 72 h respectively and cell growth was assessed by CCK-8 assay.Positive control（paclitaxel 10μg/mL） and negative control（metformin 0mmol/L） were set up simultaneously.After being treated with doses of metformin（0, 5, 10,15mmol/L） for 72h, protein expression levels of AMPKα、P-AMPKα、FASN、P-mTOR and P-Akt were measured by western blotting analysis and FASN mRNA expression levels were measured by RT-PCR. Results Being treated with vari-ous doses of metformin（1, 5, 10, 15 mmol/L） for 24, 48 and 72 h, the growth of HepG2 cells were inhibited by metformin in dose-dependent and time-dependent manner（ P〈0.05） .The growth inhibition rate approached 50%after being treated with metformin for 72 h, and the growth inhibition rate were all greater than 50%after being treated with 15 mmol/L of metformin.At 72 h after the treatment of various do-ses of metformin（0, 5, 10, 15 mmol/L） on HepG2 cells, the protein expression of P-AMPK increased with the rise of metformin concentra-tion, while the protein expressions of P-mTOR and FASN decreased as the metformin concentration increased.Compared with negative control group, the protein expression levels of P-AMPKα, P-mTOR and FASN all changed significantly in 10 mmol/L group and 15 mmol/L group（P〈0.05）.However, there was no significant difference as to the protein expression levels of P-Akt in various metformin concen-trations（ P＆gt;0.05） .FASN mRNA expression levels decreased significantly in all metformin-treated groups（ P〈0.05） . Conclusion Met-formin actitiviates AMPK, inhibits mTOR and downregulates FASN, which are implicated in its antineopastic activity on HCC.Although metformin inhibits mTOR activation, it is not involved in Akt upregulation through a negative loop.