VEGF在二甲双胍诱导HepG2细胞凋亡的作用

Role of VEGF in metformin induced apoptosis of HepG2 cells

目的初步探讨二甲双胍(metformin,MET)诱导人肝癌HepG2细胞凋亡的分子机制.方法将不同浓度的MET(0-20 mmol/L)作用于HepG2细胞24 h或10 mmol/LMET作用于HepG2细胞不同时间(0-48 h),采用MTT法测定MET抑制细胞增殖效应.将HepG2细胞暴露于不同浓度的MET(0-20 mmol/L)作用24 h或10 mmol/L MET不同时间(0-48 h),用Annexin V-FITC/PI流式双染来测定其细胞凋亡率;用RT-PCR检测不同浓度MET作用于HepG2细胞或相同浓度作用于HepG2细胞不同时间后血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的变化,以了解MET诱导HepG2细胞凋亡与VEGF的关系.结果MET对HepG2细胞生长有明显的抑制作用.不同浓度MET(0、5、10、15、20 mmol/L)处理HepG2细胞24 h后,其相对细胞活力分别为100%、80.56%±0.72%、71.06%±0.70%、64.73%±0.35%、54.73%±0.40%,呈现浓度依赖性;10 mmol/L MET作用于HepG2细胞0、12、24、36、48 h后,其相对相对细胞活力分别为100%、83.40%±0.70%、69.86%±0.45%、60.40%±0.88%、50.70%±0.45%,呈现时间依赖性.不同浓度MET(0、5、10、15、20 mmol/L)处理HepG2细胞24 h后,Annexin V-FITC/PI流式双染提示细胞凋亡明显增加,其凋亡率分别为2.78%±0.68%、9.33%±0.22%、17.13%±0.10%、21.61%±0.20%、25.26%±1.09%,呈现浓度依赖性;10 mmol/LMET作用于HepG2细胞12、24、36、48 h后,Annexin V-FITC/PI流式双染提示细胞凋亡明显增加,其凋亡率分别为2.05%±0.04%、8.10%±0.08%、16.53%±0.93%、20.95%±0.16%、25.65%±0.44%,呈现时间依赖性.随着MET浓度的升高或作用时间延长,VEGF的表达均减少,呈现剂量或时间依赖性.结论二甲双胍可以通过诱导HepG2细胞凋亡来抑制其增殖,其过程可能与抑制VEGF的表达有关.

AIM To investigate the molecular mechanism of metformin induced apoptosis of HepG2 cells.METHODS HepG2 cells were treated with different concentrations （0-20 mmol/L） of metformin （MET） for 24 h or 10 mmol/L MET for different times （0-48 h）, and the effect of MET on cell proliferation was measured by MTT assay. Annexin V-FITC/PI flow cytometry was used to determine the apoptosis rate. RT-PCR was used to analyze the expression of vascular endothelial growth factor （VEGF） in HepG2 cells treated with MET.RESULTS MET had an obvious inhibitory effect on HepG2 cell proliferation. After treatment with 0, 5, 10, 15, and 20 mmol/L MET for 24 h, the relative cell viability rates of HepG2 cells were 100%, 80.56% ± 0.72%, 71.06% ± 0.70%, 64.73% ± 0.35%, and 54.73% ± 0.40%, respectively, showing a dose-dependent manner. After treatment with 10 mmol/L MET for 0, 12, 24, 36, and 48 h, the relative cell viability rates of HepG2 cells were 100%, 83.40% ±_ 0.70%, 69.86% ± 0.45%, 60.40% ± 0.88%, and 50.70% ± 0.45%, respectively, showing a time-dependent manner. Annexin V-FITC/PI flow cytometry revealed that the apoptosis rates of HepG2 cell were increased after treatment with 0, 5, 10, 15, and 20 mmol/L MET for 24 h, and the apoptosis rates were 2.78% ± 0.68%, 9.33% ± 0.22%, 17.13% ± 0.10%, 21.61% ± 0.20%, and 25.26% ± 1.09%, respectively, showing a dose-dependent manner. The apoptosis rates of HepG2 cells were increased after treatment with 10 mrnol/L for 0, 12, 24, 36, 48 h, and the apoptosis rates were 2.05% ± 0.04%, 8.10% ± 0.08%, 16.53% ± 0.93%, 20.95% ± 0.16%, and 25.65% ± 0.44%, showing a time-dependent manner. The expression of VEGF decreased after treatment with different concentrations of MET for 24 h or 10 mmol/L MET for different times, showing a dose- and time-dependent manner.CONCLUSION MET can inhibit HepG2 cell proliferation via inducing apoptosis, which may involve the expression of VEGF.