重楼皂苷I对结肠癌耐奥沙利铂细胞株的毒性研究

摘要

目的 探讨重楼皂苷Ⅰ对结肠癌耐奥沙利铂细胞的体外毒性及其机制.方法 复苏结肠癌SW480、LoVo细胞,采用大剂量冲击法诱导结肠癌耐奥沙利铂细胞,CCK-8实验检测其耐药性,Transwell细胞侵袭实验检测其侵袭性以鉴定其恶性生物学行为.CCK-8实验检测重楼皂苷Ⅰ对耐药细胞及其亲代细胞的毒性作用,Annexin V/PI染色流式细胞术检测重楼皂苷Ⅰ处理细胞后Annexin V/PI阳性细胞比例以检测其对细胞凋亡的作用.提取细胞总蛋白,Western blotting检测重楼皂苷I处理细胞后细胞中凋亡相关蛋白Bax和Caspass-3等蛋白的表达.结果 大剂量奥沙利铂冲击法成功诱导奥沙利铂耐药细胞LoVo/L-OHP、SW480/L-OHP.CCK-8细胞毒性实验显示,LoVo/L-OHP、SW480/L-OHP的IC50值比其亲代细胞提高5~25倍,Transwell细胞侵袭实验证实,耐药细胞比亲代细胞具有更强的侵袭性(P<0.019).CCK-8实验显示,重楼皂苷I对SW480、LoVo及其耐奥沙利铂细胞的IC50值相似.Annexin V/PI染色、流式细胞仪检测凋亡细胞比例显示,重楼皂苷I处理细胞后,Annexin V/PI阳性细胞率明显升高,Western blotting结果显示,重楼皂苷Ⅰ处理细胞后细胞中Bax和Caspass-3蛋白表达水平升高.这两个实验证实,重楼皂苷Ⅰ能够促进结肠癌细胞及其耐药细胞凋亡.结论 重楼皂苷I对结肠癌细胞及耐奥沙利铂细胞均有良好的抗肿瘤活性,其抗肿瘤作用是通过介导结肠癌细胞凋亡实现的.%Objective To investigate the anticancer activity of Polyphyllin Ⅰ on colon cancer cell lines resistant to oxaliplatin.Methods Polyphyllin Ⅰ was isolated from the rhizome of Sichuan polyphylla.The oxaliplation resistant cells (SW480/L-OHP, LoVo/L-OHP) were established by continuously exposing to high-dose concentration of oxaliplatin (L-OHP).The oxaliplation resistant cells and their parental cells were cultured and then treated with stepwise concentrations (0.1, 0.5, 1, 2, 4, 8 mg/L) of Polyphyllin I for 48 hours, respectively.The inhibiting rates of SW480, LoVo cells by Polyphyllin I were determined by CCK-8 assay.Transwell migration assays was used to assess cell invasion viability.Cells were stained with Annexin V/PI in the detection of apoptosis by flow cytometry.Total protein was extracted and Western blotting was used to detect the expressions of Bax and Caspass-3 after cultured with Polyphyllin I for 24 hours.Results LoVo-OHP, SW480/L-OHP were induced successfully by large dose of oxaliplatin method.CCK-8 cytotoxicity tests showed that IC50 values of LoVo-OHP and SW480/L-OHP were increased by 5-25 times than those of their parental cells.The proportion of Annexin V/PI positive cells elevated after treated with Polyphyllin Ⅰ.Polyphyllin Ⅰ up-regulated expressions of Bax and Caspass-3 Polyphylines Ⅰ could promote the apoptosis of colon cancer and resistant cells.Conclusion Polyphyllin I shows anticancer activity against oxaliplatin resistant colon cancer cell in vitro.