冬凌草甲素干预SHH信号通路抑制人结肠癌细胞SW1116增殖的实验探讨

Oridonin can inhibit SW1116 human colon cancer cell proliferation by the intervention of SHH pathway

目的研究冬凌草甲素对结肠癌细胞SW1116增殖的影响及机制。方法用不同浓度的冬凌草甲素处理SW1116细胞,MTT测定细胞增殖情况,计算其半数抑制浓度。SW1116细胞分为对照组、冬凌草甲素组、Cyclopamine组、联合组,对照组细胞培养液中不添加任何药物,冬凌草甲素组细胞培养液中添加70μmol/L的冬凌草甲素,Cyclopamine组细胞培养液中添加10μmol/L的SHH信号通路抑制剂Cyclopamine,联合组细胞培养液中添加10μmol/L的SHH信号通路抑制剂Cyclopamine和70μmol/L的冬凌草甲素,MTT检测细胞增殖,细胞克隆实验检测克隆形成能力,流式细胞术检测细胞凋亡,Western blot检测细胞中音猬因子(SHH)、平滑蛋白(Smo)、锌指转录因子1(Gli-1)、剪切的Caspase-3(Cleaved Caspase-3)、B细胞淋巴瘤/白血病-2(Bcl-2)蛋白水平。结果冬凌草甲素处理后的SW1116细胞增殖能力明显降低,其半数抑制浓度为(69.65±6.32)μmol/L。冬凌草甲素处理后的SW1116细胞增殖、克隆形成能力明显降低,细胞凋亡率明显升高,细胞中SHH、Smo、Gli-1蛋白表达水平降低,Cleaved Caspase-3蛋白表达水平升高,Bcl-2蛋白表达水平降低,与未用冬凌草甲素处理的细胞比较,差异有统计学意义(P<0.05)。SHH信号通路抑制剂处理也可以抑制SW1116细胞增殖和克隆形成,诱导SW1116细胞凋亡,促进细胞中Cleaved Caspase-3蛋白表达,减少细胞中Bcl-2蛋白表达。联合组的SW1116细胞增殖能力和克隆形成能力下降更多,细胞凋亡增加更多,细胞中Cleaved Caspase-3蛋白水平更高,Bcl-2蛋白水平更低,与单纯冬凌草甲素处理的SW1116细胞比较,差异有统计学意义(P<0.05)。结论冬凌草甲素通过抑制SHH信号通路抑制结肠癌细胞增殖和克隆能力。

Objective Study on the effect of oridonin on colon cancer SW1116 cell proliferation and its mechanism. Methods SW1116 cells were treated with different concentrations of oridonin, the proliferation of cells was measured by MTT, the half inhibitory concentration was calculated. SW1116 cells were divided into control group, oridonin group, Cyclopamine group and combined group. The control group did not add any drugs in the cell culture medium. Oridonin was added to the cell culture medium with 70 μmol/L of oridonin. In group Cyclopamine, 10 μmol/L SHH signal pathway inhibitor Cyclopamine was added to the cell culture medium.The cell culture medium of the combined group was added with 10 μmol/L SHH signal pathway inhibitor Cyclopamine and 70 μmol/L of oridonin, MTT was used to detect cell proliferation, cell cloning test was used to detect the ability of clone formation. Cell apoptosis was detected by flow cytometry. Western blot was performed to detect the levels of SHH, Smo, Gli-1, Cleaved Caspase-3 and Bcl-2 protein in the cells. Results The ability of oridonin on proliferation of SW1116 cells was reduced after treatment, the median inhibitory concentration was （69.65± 6.32） mol/L. Oridonin SW1116 cell proliferation and hormone treatment after the colony formation ability decreased significantly, the rate of apoptosis was significantly increased. The expression level of SHH, Smo and Gli-1 in cells decreased, the expression level of Cleaved Caspase-3 protein elevated, the expression level of Bcl-2 protein decreased with oridonin treated cells, the difference was statistically significant （ P 〈0.05）. SHH signaling pathway inhibitor treatment also inhibited the proliferation and cloning of SW1116 cells, inducing apoptosis of SW1116 cells, promoted the expression of Cleaved Caspase-3 protein in cells, reduced the expression of Bcl-2 protein in cells, oridonin and SHH signaling pathway inhibitor treatment after SW1116 cell proliferation and colony formation ability decreased more, cell apoptosis increased more, the level of Cleaved Caspase-3 protein in the cells was higher, the level of Bcl-2 protein was lower, compared with oridonin treated SW1116 cells, the difference was statistically significant （ P 〈0.05）. Conclusion Oridonin can inhibit SHH signaling pathway to inhibit colon cancer cell proliferation and clone ability .