FoxM1通过促进肿瘤干细胞化参与骨肉瘤细胞对顺铂的耐药

FoxM1 enhances resistance to cisplatin by promoting stemness in osteosarcoma cells

目的探讨FoxM1是否通过促进肿瘤干细胞化参与骨肉瘤细胞对顺铂的耐药。方法利用骨肉瘤亲本细胞MG-63,采用递增药物浓度筛选建立顺铂耐药细胞MG-63R;运用慢病毒转染FoxM1建立稳定过表达MG-63F细胞及空载体对照MG-63V细胞。MTT法检测细胞对顺铂药物的敏感性。Western blot法检测FoxM1及肿瘤干细胞相关标志物Sox-2、Oct-4、Nanog蛋白水平表达。无血清培养悬浮克隆球实验检测微球形成能力。检测FoxM1抑制剂Thiostrepton处理后对肿瘤干细胞相关标志物表达水平、微球形成能力和对顺铂药物敏感性的影响。结果在2μg/mL顺铂浓度下稳定生长和传代的耐药细胞MG-63R的半数有效抑制浓度IC50由亲本细胞中1.27上升为7.36,FoxM1蛋白水平在MG-63R中表达较MG-63明显增高;MG-63R中肿瘤干细胞相关标志物Sox-2、Oct-4、Nanog蛋白水平表达明显升高,同时平均悬浮克隆球数量明显增多。成功构建FoxM1过表达细胞MG-63F,其FoxM1蛋白表达水平较空载体对照MG-63V明显增高,肿瘤干细胞相关标志物也明显升高,平均悬浮克隆球数量由14增加至25,差异有统计学意义。MG-63F细胞对顺铂耐药性明显升高,IC50由对照组0.96升高为31.23;MG-63F细胞经4μmol/L Thiostrepton处理后,FoxM1蛋白表达明显降低,对顺铂的敏感性明显增加,同时肿瘤干细胞相关标志物表达明显降低,平均悬浮克隆球数目也明显减少。结论FoxM1过表达可能通过促进肿瘤干细胞化参与骨肉瘤细胞对顺铂的耐药,FoxM1抑制剂可能通过逆转肿瘤干细胞化而增强骨肉瘤耐药细胞对顺铂的敏感性。

Purpose To detect whether FoxM1 contributes to cisplatin resistance by promoting tumor stemness in osteosarcoma(OS).Methods Cisplatin-resistant MG-63R was induced by gradually increasing cisplatin concentration from its parental cell of MG-63.Stably over-expressed FoxM1 cell MG-63F and empty vector control cell MG-63V were established by lentiviral transfection.MTT assay was used to detect the drug sensitivity to cisplatin in OS cells.The expressions of FoxM1 and tumor stem cell-like markers Sox-2,Oct-4 and Nanog were detected by Western blot analysis.Microsphere formation ability was measured using a serum-free culture suspension cloned assay.The effect of FoxM1 inhibitor Thiostrepton on expression of tumor stem cell-like markers,microsphere formation ability and cisplatin drug sensitivity were detected.Results MG-63R were stably grew and passaged on a concentration of 2μg/mL cisplatin.Compared with the parental cells,the IC50 of MG-63R increased from 1.27 to 7.36,the expression of FoxM1 and tumor stem cell-like markers,Sox-2,Oct-4 and Nanog in MG-63R was significantly increased,and the average number of suspended microsphere was also significantly increased.Overexpressing FoxM1 cell MG-63F were successfully constructed and the expression level of FoxM1 was significantly increased.The expressions of tumor stem cell-like markers were significantly increased.The average number of microspheres was increased from 14 to 25.Compared with the control group,MG-63F was more resistant to cisplatin and the IC50 was increased from 0.96 to 31.23.After treatment with 4μmol/L thiostrepton in MG-63F,the expression of FoxM1 was significantly decreased and the sensitivity to cisplatin was significantly increased.Both the expression of tumor stem cell-like markers and the number of microspheres were significantly reduced.Conclusion Overexpression of FoxM1 may confer to cisplatin resistance by promoting tumor stemness in osteosarcoma cells.FoxM1 inhibition may sensitize osteosarcoma drug-resistant cells to cisplatin by reversing tumor stemness.