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目的 :探讨二甲双胍对人食管鳞癌KYSE450细胞增殖的抑制作用及可能的作用机制。方法 :常规培养人食管癌KYSE450细胞,经不同浓度的二甲双胍(5、10、20和40 mmol/L)处理后,MTT法检测对KYSE450细胞的增殖抑制率,FCM法检测细胞的凋亡率;分别采用半定量RT-PCR法和蛋白质印迹法检测4E结合蛋白1(4E-bing protein 1,4EBP1)和S6激酶1(S6 kinase 1,S6K1)mR NA及蛋白的表达水平。构建食管鳞癌KYSE450细胞裸鼠皮下移植瘤模型,并于建模成功7 d后分别予以连续腹腔注射二甲双胍或0.9%氯化钠溶液(对照组)15 d,每3 d 1次测量裸鼠移植瘤体积的大小;分别采用HE染色和免疫组织化学法检测,移植瘤组织中细胞学形态的改变及4EBP1和S6K1蛋白的表达水平。结果:不同浓度的二甲双胍(5、10、20和40 mmol/L)对KYSE450细胞的增殖抑制呈剂量、时间依赖性增强(P值均<0.000 1);5、10和20mmol/L二甲双胍作用48 h后KYSE450细胞的凋亡率呈浓度依赖性增强(P均值<0.05);随着浓度的增加或时间的延长二甲双胍组KYSE450细胞中4EBP1和S6K1 mRNA及蛋白的表达量均明显下降(P值均<0.05)。二甲双胍组裸鼠移植瘤的生长明显受到抑制(P<0.000 1),且移植瘤组织中4EBP1和S6K1蛋白的表达量明显降低(P值均<0.05)。结论 :二甲双胍可抑制人食管癌KYSE450的增殖,其机制可能与雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)下游通路中4EBP1和S6K1 mRNA和蛋白表达的下调有关。
Objective: To investigate the inhibitory effect of metformin on the proliferation of human esophageal squamous cell carcinoma KYSE450 cells and its possible mechanism. Methods: KYSE450 cells were cultured routinely. After treated with different concentrations of metformin (5, 10, 20 and 40 mmol / L), the proliferation inhibition rate of KYSE450 cells was detected by MTT assay and the apoptosis rate of KYSE450 cells was detected by FCM. Semi-quantitative RT-PCR and Western blotting were used to detect the mRNA and protein expression levels of 4E-bing protein 1, 4EBP1 and S6 kinase 1, S6K1, respectively. The nude mice model of esophageal squamous cell carcinoma KYSE450 was established by subcutaneous xenograft model. After 7 d of model establishment, methotrexate or 0.9% sodium chloride solution (control group) The size of the tumor volume were detected by HE staining and immunohistochemistry respectively. The morphological changes of the tumor tissue and the expression of 4EBP1 and S6K1 protein were observed. RESULTS: Metformin (5, 10, 20 and 40 mmol / L) inhibited the proliferation of KYSE450 cells in a dose-and time-dependent manner (P <0.0001). Metformin at concentrations of 5, 10 and 20 mmol / L (P <0.05). The mRNA and protein expressions of 4EBP1 and S6K1 in KYSE450 cells were significantly decreased with the increase of concentration or prolongation of time (P values <0.05). The growth of xenografted nude mice was significantly inhibited by metformin (P <0.000 1), and the expression of 4EBP1 and S6K1 in the xenografted tumor was significantly decreased (all P <0.05). CONCLUSION: Metformin inhibits the proliferation of human esophageal cancer KYSE450, and its mechanism may be related to the down-regulation of 4EBP1 and S6K1 mRNA and protein expression in downstream pathways of mammalian target of rapamycin (mTOR).