幽门螺旋杆菌空泡毒素对结肠癌细胞自噬及凋亡的影响

Effect of helicobacter pylori vacuolating cytotoxin on autophagy and apoptosis in colorectal cancer

目的采用幽门螺旋杆菌空泡毒素干预结肠癌SW480细胞进行体外实验,观察及研究其对结肠癌细胞自噬及凋亡的影响。方法采取MTT法测定不同浓度幽门螺旋杆菌空泡毒素在不同作用时间内对结肠癌SW480细胞增殖的影响。荧光显微镜下观察幽门螺旋杆菌空泡毒素诱导结肠癌SW480细胞产生的自噬荧光颗粒。Western blotting法检测自噬相关蛋白LC3-Ⅱ/Ⅰ和Beclin-1的表达。流式细胞术检测经幽门螺旋杆菌空泡毒素处理过的结肠癌SW480细胞凋亡率的变化。结果幽门螺旋杆菌空泡毒素可促进SW480细胞增殖,且呈现浓度依赖性。经幽门螺旋杆菌空泡毒素处理的SW480细胞增殖,12 h出现细胞内自噬荧光颗粒,且随浓度增加,荧光颗粒数目增加。Western blotting分析显示,与对照组相比,经幽门螺旋杆菌空泡毒素作用后Beclin-1及细胞微管相关蛋白1轻链3(LC3)-II/I比值明显升高,且二者的升高呈浓度依赖性。浓度为20 mg/mL的幽门螺旋杆菌空泡毒素可抑制SW480细胞的凋亡。加入幽门螺旋杆菌空泡毒素组细胞凋亡率为3.63%±0.86%,低于SW480细胞正常生长组的9.72%±0.65%;而3-MA+幽门螺旋杆菌空泡毒素组细胞凋亡率为11.26%±1.32%,明显高于幽门螺旋杆菌空泡毒素组的3.63%±0.86%。结论幽门螺旋杆菌空泡毒素有助于结肠癌SW480细胞的体外生存,促进自噬,抑制细胞凋亡。幽门螺旋杆菌感染很可能是诱发结肠癌的重要因素之一。

Objective To study the effect of helicobacter pylori vacuolating cytotoxin on autophagy and apoptosis in human colorectal cancer SW480 cells in vitro. Methods MTT assay were used to examine the effect of different concentrations of helicobacter pylori vacuolating cytotoxin on the proliferation of colorectal cancer SW480 cells at different times. The autophagy of colorectal cancer SW480 cells which induced by helicobacter pylori vacuolating cytotoxin were observed under fluorescence microscopy. Western blotting method was used to detect autophagy related proteins LC3-Ⅱ/Ⅰand Beclin-1 expression. Flow cytometry was used to detect the apoptosis rate of colorectal cancer SW480 cells which treated with helicobacter pylori vacuolating cytotoxin.Results Helicobacter pylori vacuolating cytotoxin promoted the proliferation of colorectal cancer SW480 cells and this effect was dependent on concentration. Colorectal cancer SW480 cells proliferated with the treat of helicobacter pylori vacuolating cytotoxin,and intracellular fluorescence autophagy particles appeared 12 hour later. The number of fluorescence particles increased with the concentration. The western blot showed that compared with the control group,both beclin1 and the ratio of cellular microtubule-associated protein 1-light chain 3(LC3)-II/I was significantly increased after treated by helicobacter pylori vacuolating cytotoxin. Helicobacter pylori vacuolating cytotoxin at a concentration of 20 mg/mL inhibited the apoptosis rate of SW480 cells. The apoptosis rate was(3.63±0.86)% in the helicobacter pylori vacuolating cytotoxin group which was lower than those in the SW480 cells normal growth group(9.72±0.65)%(P<0.05). The apoptosis rate in the 3-MA+ helicobacter pylori vacuolar toxin group was(11.26±1.32)%,significantly higher than those in the helicobacter pylori vacuolating cytotoxin group(3.63±0.86)%(P<0.05).Conclusion Helicobacter pylori vacuolating cytotoxin has benefit to the survival of colorectal cancer SW480 cells in vitro,which can promote autophagy and inhibit apoptosis. Helicobacter pylori infection is probably one of important risk factors for colorectal cancer.