Notch信号通路在胰岛素调节子宫内膜癌细胞生长及凋亡中的作用

The role of Notch signaling pathway in insulin regulation of endometrial cancer cell growth and apoptosis

目的探讨抑制Notch信号传导对胰岛素诱导子宫内膜癌细胞增殖及相关凋亡蛋白表达水平的影响。方法将子宫内膜癌Ishikawa 3-H-12细胞行体外原代及传代培养,将培养好的细胞分为:对照组(加入磷酸盐缓冲液3mL)、胰岛素组(加入1×106 mol/L胰岛素单独刺激)及MW167组(应用不同剂量γ分泌酶抑制剂MW167预处理后再用胰岛素刺激),培养48h后应用噻唑蓝(MTT)比色法测定各组子宫内膜癌细胞生长抑制情况,应用蛋白质印迹法(Western blot)测定半胱氨酰天冬氨酸蛋白酶(Caspase)-3、Caspase-8及Notch1蛋白表达情况。结果胰岛素可促进子宫内膜癌细胞中Notch1蛋白表达,刺激48h后Notch1蛋白表达水平明显高于对照组(P<0.05),MW167可抑制胰岛素诱导Notch1蛋白表达,且抑制作用具有浓度依赖性。不同组别子宫内膜癌细胞在培养24、48、72h后570nm处吸光度(A570)值存在明显差异(P<0.05),其中胰岛素组各时间点A570值均高于对照组(P<0.05),胰岛素促子宫内膜癌细胞增殖在48h时达到最高水平。MW167以浓度及时间依赖的方式抑制胰岛素促子宫内膜癌细胞增殖,在48h时20μmol/L MW167可持久抑制胰岛素促子宫内膜癌细胞的增殖。胰岛素组各时间点Caspase-3、Caspase-8蛋白表达水平低于对照组(P<0.05),MW167以浓度及时间依赖的方式促进细胞Caspase-3、Caspase-8蛋白表达。结论 MW167可通过抑制Notch信号通路传导而抑制胰岛素促子宫内膜癌细胞增殖及促进相关凋亡蛋白表达,诱导子宫内膜癌细胞凋亡。

Objective To investigate the effects of Notch signaling pathway on proliferation of insulin-induced endometrial carcinoma cells and apoptosis related protein expression levels. Methods The endometrial carcinoma Ishikawa 3-H-12 cell line was primarily cultured and subcultured in vitro. Then, the cultured cells were divided into five groups： the control group （3 mL PBS was added into the group） ,the insulin group （cells were stimulated by 1 × 10 6 mol/L insulin） and MW167 groups （different doses of γ- secretase inhibitor MW167 pretreated with insulin stimulation）. After 48 h culturation, inhibition of endometrial carcinoma cell growth of each group was measured by MTT-colorimetric method, the apoptosis-related proteins （Caspase-3, Caspase-8） and Notchl protein expression levels of each group were determined by Western blot. Results Insulin can promote Notch1 protein expression in endometrial carcinoma cells, after 48 h insulin stimulation, the Notehl protein expression level was significantly higher than that in the control group （P〈0. 05）. MW167 can inhibit insulin-induced Notchl protein expression in a concentration-depend- ent inhibition manner. The absorbance at 570 nm （A570） of endometrial carcinoma cells cultured for 24,48 and 72 h in different groups were significantly different （P〈0. 05）. The A570 values in the insulin group at each time point were higher than those in the control group （P〈0, 05） ,and the insulin-induced endometrial carcinoma cell proliferation reached its highest level at 48 h. MW167 inhibited insulin-induced endometrial carcinoma cells proliferation in a concentration- and time-dependent manner, and 20 μmol/L MW167 persistently inhibited insulin-induced proliferation of endometrial carcinoma cells at 48 h. Western blot analysis showed that expression levels of Caspase-3 and Caspase-8 protein in the insulin group at each time point were lower than those in the control group （P〈0. 05） ,and MW167 promoted the expressions of Caspase-3 and Caspase-8 in a concentration-aM time-dependent manner. Conclusion MW167 can suppress the insulin-induced endometrial carcinoma cells proliferation and promote the expression of related apoptotic proteins by inhibiting Notch signaling pathway,and induce apoptosis of endometrial carcinoma cells.