二甲双胍与脂联素对子宫内膜癌细胞增殖的作用

目的：探讨二甲双胍对子宫内膜癌细胞脂联素受体的作用及联合使用二甲双胍和脂联素对子宫内膜癌细胞增殖的影响。方法：用CCK 8法检测不同浓度二甲双胍和脂联素对IK（ishikawa）和HEC--1B两种子宫内膜癌细胞系增殖的影响。用实时荧光定量RT-PCR（quantitative real time RT-PCR, qRT-PCR）法和Western blot法检测不同浓度二甲双胍作用下IK和HEC-1B细胞脂联素受体［包括脂联素受体1（adiponectin receptor 1，AdipoR1）和脂联素受体2（adiponectin receptor 2，AdipoR2）］的变化及磷酸腺苷活化的蛋白激酶（adenosine monophosphate-activated protein kinase，AMPK）通路抑制剂compound C对以上作用的影响。结果：（1）二甲双胍和脂联素均可抑制IK和HEC-1B细胞的生长增殖，并且都呈时间和浓度依赖性（P<0.05）；（2）二甲双胍和脂联素对IK和HEC-1B细胞有协同抑制增殖作用，IK细胞药物联合指数（combination index，CI）为0.906 34，HEC-1B细胞CI为0.827 65；（3）用5 mmol/L和10 mmol/L的二甲双胍分别刺激IK和HEC-1B细胞48 h，细胞AdipoR1和AdipoR2 mRNA的水平较对照组（0 mmol/L）显著增高（IK：AdipoR1：5 mmol/L和10 mmol/L组P均<0.001，AdipoR2：5 mmol/L组P<0.001；HEC-1B：AdipoR1：5 mmol/L组P<0.001，10 mmol/L组P=0.023，AdipoR2：5 mmol/L组P<0.001，10 mmol/L组P=0.024）， 联合compound C时，该作用被抑制，与对照组（0 mmol/L）相比AdipoR1和AdipoR2 mRNA水平差异无统计学意义（P>0.05）；（4）用5 mmol/L和10 mmol/L的二甲双胍分别刺激IK和HEC-1B细胞48 h，细胞AdipoR1和AdipoR2的蛋白水平较对照组（0 mmol/L）显著增高（IK：AdipoR1：5 mmol/L组P=0.04，10 mmol/L组P=0.033；AdipoR2：5 mmol/L组P=0.044，10 mmol/L组P=0.046。HEC-1B：AdipoR1：5 mmol/L组P=0.04，10 mmol/L组P=0.049；AdipoR2：5 mmol/L组P=0.043，10 mmol/L 组P=0.035）， 联合compound C时，该作用被抑制，与对照组（0 mmol/L)相比,AdipoR1和AdipoR2蛋白表达差异无统计学意义（P>0.05）。结论：二甲双胍和脂联素具有协同抑制子宫内膜癌细胞IK和HEC-1B增殖的作用，二甲双胍可上调子宫内膜癌细胞脂联素受体的表达，AMPK通路参与这一过程。

Effects of metformin and adiponectin on endometrial cancer cells growth

Objective：To determine the effect of metformin and adiponectin on the proliferation of EC cells and the relationship between metformin and adiponectin. Methods: The proliferation impact of different concentrations of metformin and adiponectin on two types of EC cells ishikawa (IK) and HEC-1B was confirmed by CCK-8 method. qRT-PCR and Western blot were used to detect the effect of different concentrations of metformin on the changes of adiponectin receptors (AdipoR1 and AdipoR2) of the EC cells both in mRNA and protein level and the role of compound C, an adenosine monophosphate-activated protein kinase (AMPK) inhibitor, on the above effects. Results: (1) Both metformin and adiponectin could significantly promote the proliferation of endometrial cancer (EC) cells in a time and concentration dependent manner (P<0.05).(2)Metformin and adiponectin had synergy anti-proliferative effect on EC cells and the combination index (CI) value of IK cells was 0.906 34 and of HEC-1B cells was 0.827 65.(3)qRT-PCR was used to detect the mRNA levels of AdipoR1 and AdipoR2 after 5 mmol/L and 10 mmol/L metformin, respectively, stimulating IK and HEC-1B cells for 48 hours and the mRNA expressions of AdipoR1 and AdipoR2 were significantly increased when compared with the control group (0 mmol/L)(IK: AdipoR1 of 5 mmol/L and 10 mmol/L group: P<0.001,AdipoR2 of 5 mmol/L group: P<0.001; HEC-1B: AdipoR1 of 5 mmol/L group: P<0.001, 10 mmol/L group: P=0.023, AdipoR2 of 5 mmol/L group: P<0.001, 10 mmol/L group: P=0.024). When combined with compound C, the RNA levels of AdipoR1 and AdipoR2 were not different compared with the control group (0 mmol/L, P>0.05). (4) Western blot was used to detect the protein levels of AdipoR1 and AdipoR2 after 5 mmol/L and 10 mmol/L metformin, stimulating IK and HEC-1B cells for 48 hours and the protein level was significantly increased when compared with the control group (0 mmol/L)(IK: AdipoR1 of 5 mmol/L group: P=0.04, 10 mmol/L group: P=0.033, AdipoR2 of 5 mmol/L group: P=0.044, 10 mmol/L group: P=0.046; HEC-1B: AdipoR1 of 5 mmol/L group: P=0.04, 10 mmol/L group: P=0.049, AdipoR2 of 5 mmol/L group: P=0.043, 10 mmol/L group: P=0.035). When combined with compound C,the protein levels of AdipoR1 and AdipoR2 were not different compared with the control group (0 mmol/L, P>0.05). Conclusion: We find that metformin and adiponectin have synergy anti-proliferative effect on EC cells. Besides, metformin can increase adiponectin receptors expressions of EC cells both in mRNA and protein levels and this effect is accomplished by the activation of AMPK signaling pathway.