重楼皂苷Ⅰ对结肠癌耐奥沙利铂细胞株的毒性研究

Study on toxicity of Polyphyllin Ⅰ on colon cancer cell lines resistant to oxaliplatin

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/LOHP。CCK-8细胞毒性实验显示,LoVo/L-OHP、SW480/L-OHP的IC_(50)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IC_(50)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 concen- trations （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 via- bility. 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 cy- totoxicity 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 [ could promote the apoptosis of colon cancer and re- sistant cells. Conclusion Polyphyllin I shows anticancer activity against oxaliplatin resistant colon cancer cell in vitro.