dalbinol通过ROS/Dvl/GSK-3β/β-catenin信号通路诱导人结肠癌细胞凋亡的作用机制研究

dalbinol induces apoptosis of human colon cancer cells through ROS / Dvl / GSK-3β/ β-catenin pathway

目的探讨天然产物dalbinol对人结肠癌HCT116细胞增殖和凋亡的影响及其作用机制。方法采用MTT法检测dalbinol对人结肠癌HCT116细胞的增殖抑制作用;利用Hoechst 33342荧光染色法观察细胞凋亡的形态学变化;应用流式细胞术检测细胞凋亡率和ROS水平;通过Western blot法检测Wnt/β-catenin信号通路及凋亡相关蛋白的表达情况。结果 dalbinol可抑制人结肠癌HCT116细胞的增殖,且呈剂量和时间依赖性,24、48和72 h的IC50分别为(4.8±0.53)、(2.5±0.43)和(0.6±0.22)μmol·L-1;Hoechst 33342染色观察到细胞皱缩、核固缩和染色质凝集等典型凋亡的形态学变化,同时dalbinol可剂量依赖性地提高细胞凋亡率,且能增加细胞内ROS水平;Western blot结果发现dalbinol通过下调抗凋亡蛋白Bcl-2和Mcl-1的表达水平,上调促凋亡蛋白Bax和Bim的表达,从而促进cleaved caspase-3和cleaved PARP活化裂解,进而诱导细胞凋亡;此外,dalbinol通过抑制Dvl-2和GSK3β(p S9)的蛋白表达,从而降低总的β-catenin和胞核β-catenin的表达,但胞质β-catenin无明显变化,最终下调Wnt/β-catenin通路下游靶蛋白c-Myc和Survivin的表达水平。结论dalbinol可抑制人结肠癌HCT116细胞增殖并诱导其凋亡,其分子机制可能与提高细胞内ROS水平和抑制Dvl/GSK-3β/β-catenin信号通路有关。

Aim To investigate the effects of dalbinol on proliferation and apoptosis of human colon cancer HCT116 cells and its mechanisms. Methods Antiproliferative effect of dalbinol was evaluated by MTT assay. The morphological changes of apoptosis were observed by Hoechst33342 staining. Apoptotic rate and ROS generation were analyzed by flow cytometry. The related proteins of Wnt / β-catenin pathway and the apoptosis-associated proteins expression were measured by Western blot. Results The growth of HCT116 treated with dalbinol was inhibited in a dose and time dependent manner with IC50（ 4. 8 ± 0. 53）,（ 2. 5 ±0. 43） and（ 0. 6 ± 0. 22） μmol·L^-1 at 24,48 and 72 h,respectively. Typical morphological changes of apoptosis such as cell shrinkage,karyopyknosis and nuclear condensation were observed by Hoechst33342 staining. Meanwhile,the apoptotic rate and intracellular ROS generation of dalbinol were both increased dose-dependently. Western blot results showed that dalbinol could activate the expression of cleaved Caspase-3 and cleaved PARP by decreasing anti-apoptotic protein levels such as Bcl-2 and Mcl-1 and increasing pro-apoptotic protein levels such as Bax and Bim,which induced further apoptosis. Moreover,dalbinol can reduce the protein expression of the total and nuclear β-catenin,but not cytoplasmic β-catenin by suppressing the protein expression of Dvl-2 and GSK-3β（ p S9）,as well as its target proteins c-Myc and Survivin. Conclusion dalbinol can induce apoptosis in colon cancer HCT116 cells by upregulating the intracellular ROS generation and suppressing Dvl / GSK-3β /β-catenin pathway.