大麻二酚对人子宫内膜癌细胞活性的影响

Effect of cannabidiol on the activity of human endometrial cancer cells

目的 研究大麻二酚对子宫内膜癌Ishikawa细胞的活性、增殖、迁移生物学作用以及促凋亡的作用机制。方法 用0,5,10,20μmol·L^(-1)的大麻二酚处理Ishikawa细胞24 h。用细胞计数法-8(CCK-8)、集落形成实验和Transwell实验分别测定细胞增殖和迁移情况;用Hoechst 33258染色及AnnexinⅤ-FITC/PI双重染色方法检测Ishilawa细胞凋亡情况;用DCFH-DA荧光染料对细胞活性氧进行检测;以蛋白质印迹(Western blot)法检测B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、细胞色素C(Cyto C)抗体、活化的胱天蛋白酶-3(cleaved caspase 3)的蛋白表达情况。结果 CCK-8检测显示,5,10,20μmol·L^(-1)大麻二酚组的细胞增殖率分别为(82.38±9.63)%,(47.38±5.32)%,(36.06±4.14)%,具有浓度依赖性。与对照组相比,5,10,20μmol·L^(-1)大麻二酚组细胞克隆形成率分别为(78.22±8.87)%,(42.69±4.70)%,(31.18±3.69)%,表明大麻二酚对Ishikawa细胞具有显著的抑制Ishikawa细胞克隆的作用。5,10,20μmol·L^(-1)大麻二酚组细胞的迁移率分别为(78.62±4.25)%,(47.29±5.22)%,(36.24±3.98)%,提示大麻二酚对Ishikawa细胞迁移具有抑制作用。5,10,20μmol·L^(-1)大麻二酚组细胞凋亡率分别为(12.26±4.78)%,(19.94±5.95)%,(33.79±3.68)%,提示大麻二酚显著诱导了细胞凋亡。随着大麻二酚浓度的增加,大麻二酚显著促凋亡蛋白Bax的表达水平,抑制凋亡Bcl-2蛋白的水平,同时细胞色素C和cleaved caspase 3也有显著增加。结论 大麻二酚通过增加活性氧水平和线粒体凋亡途径抑制细胞活性和迁移并促进其凋亡。

Objective To explore the biological effect of cannabidiol on the cell viability,proliferation and migration of endometrial carcinoma Ishikawa cells, as well as the mechanism of promoting apoptosis.Methods Ishikawa cells were treated with 0,5,10 and 20 μmol·L^(-1)cannabidiol for 24 h. Cell proliferation and migration were measured by cell counting kit-8 (CCK-8) ,colony formation assay and Transwell assay,respectively. Apoptosis of Ishilawa cells were detected by Hoechst33258 staining and Annexin Ⅴ-FITC / PI double staining methods;DCFH-DA fluorescent dye was used to detect reactive oxygen species in cells. Western blot was used to detect the expression of B lymphocytoma-2 (Bcl-2) ,Bcl-2 associated X protein (Bax) ,cytochrome C(Cyto C) ,cleaved caspase 3 protein expression. Results CCK-8 test showed that the cell proliferation rate of 5 ,1 0 ,2 0 μmol · L^(-1)cannabidiol groups were (82. 38±9. 63) % ,(47. 38±5. 32) % ,(36. 06±4. 14) % ,respectively,which were concentration-dependent. Compared with the control group,the cell clone formation rate of the 5,10,and 20μmol·L^(-1)cannabidiol groups were (78. 22±8. 87) % ,(42. 69±4. 70) % ,(31. 18±3. 69) % ,indicating that cannabidiol has a significant inhibitory effect on Ishikawa cell cloning. The migration rates of Ishikawa cells in 5,10,and 20 μmol · L^(-1)cannabidiol groups were (78. 62±4. 25) % ,(47. 29±5. 22)% ,and (36. 24±3. 98)% ,respectively,suggesting that cannabidiol has an inhibitory effect on the migration of Ishikawa cells. The apoptosis rates of Ishikawa cells in 5,10,and 20 μmol·L;cannabidiol groups were (12. 26±4. 78)% ,(19. 94±5. 95)% ,and(33. 79±3. 68)% ,respectively,suggesting that cannabidiol significantly induced apoptosis. With the increase of the concentration of cannabidiol,cannabidiol treatment significantly increased the expression level of pro-apoptotic protein Bax and inhibited the level of apoptotic Bcl-2 protein, cytochrome C and cleaved caspase 3 also increased significantly. Conclusion Cannabidiol inhibits cell activity and migration and promotes cell apoptosis by increasing ROS level and mitochondrial apoptosis pathway.