去甲斑蝥素通过调节Hedgehog信号通路对卵巢癌耐药细胞株肿瘤干细胞活性的影响  

Effects of norcantharidin on cancer stem cell activity of drug resistant ovarian cancer cells via Hedgehog signaling pathway

作　　者：钱俏红[1] 陈瑞芳[1] 李晶[1] 安彩萍[1] 朱琰[1] 曹琦 QIAN Qiao-hong;CHEN Rui-fang;LI Jing;AN Cai-ping;ZHU Yan;CAO Qi(Obstetrics and Gynecology Hospital,Fudan University,Shanghai 200011,China)

机构地区：[1]复旦大学附属妇产科医院,上海200011 [2]复旦大学附属妇产科医院中西医结合科,上海200011

出　　处：《中华中医药杂志》2021年第5期2926-2930,共5页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：上海市自然科学基金项目(No.16ZR1404000)。

摘　　要：目的:观察去甲斑蝥素(NCTD)对两种不同的卵巢癌耐药细胞株中肿瘤干细胞活性的影响,探讨NCTD抑制卵巢癌耐药细胞株细胞增殖的可能机制。方法:采用低浓度加量持续诱导法构建卵巢癌顺铂和紫杉醇耐药细胞株,分别命名为A278Cis顺铂耐药株和SKOV3^(Pac)紫杉醇耐药株。采用CCK-8活性实验检测NCTD对卵巢癌耐药细胞株细胞增殖的影响。采用流式分选技术分离卵巢癌耐药细胞株中CD44^(+)/CD24^(-)肿瘤干细胞样细胞,分别用顺铂、紫杉醇及NCTD处理,使用流式细胞技术检测顺铂、紫杉醇及NCTD处理对卵巢癌亲本细胞及耐药株中干细胞样细胞凋亡的影响。顺铂、紫杉醇及NCTD分别处理卵巢癌耐药细胞株,Western Blot检测肿瘤干细胞标志物Nanog、Sox2和Bmi1的表达情况以及Hedgehog信号通路分子Ptch-1和Shh的表达情况。结果:CCK-8活性检测显示NCTD抑制卵巢癌耐药细胞株A2780^(Cis)和KOV3^(Pac)的细胞增殖,并呈剂量和时间依赖性。细胞凋亡检测显示NCTD、顺铂和紫杉醇对A2780^(Cis)顺铂耐药株和SKOV3^(Pac)紫杉醇耐药株中CD44^(+)/CD24^(-)肿瘤干细胞样细胞均能产生诱导凋亡作用,其中NCTD诱导凋亡作用最为显著。Western Blot检测显示NCTD可下调A278Cis顺铂耐药株和SKOV3^(Pac)紫杉醇耐药株干细胞标志物Nanog、Sox2和Bmi1的表达。与顺铂和紫杉醇比较,NCTD处理组Hedgehog信号通路分子Ptch-1的蛋白表达水平上升,Shh的蛋白表达水平下降。结论:NCTD可有效抑制卵巢癌耐药细胞株的增殖,诱导卵巢癌耐药株中肿瘤干细胞样细胞的凋亡,其作用机制可能与调节Hedgehog信号通路有关。Objective:To observe the effect of norcantharidin(NCTD)on cancer stem cell activity of two drug resistant ovarian cancer cells and explore the possible mechanism.Methods:Cisplatin or paclitaxel resistant ovarian cancer cells were established by exposure human ovrian cancer cell line,A2780 and SKOV3,to increasing concentrations of these two drugs,named as A2780^(Cis) cisplatin resistant cells or SKOV3^(Pac) paclitaxel resistant cells respectively.CCK-8 was used to determine the effects of cisplatin,paclitaxel and NCTD on the viability of A2780 and SKOV3 cells as well as their drug resistant counterparts.Flow separation technology was used to separate CD44^(+)/CD24^(-)stem-like cells of drug resistant cells.Flow cytometry analysis was used to determine the apoptosis of CD44^(+)/CD24^(-)stem-like cells treated by cisplatin,paclitaxel or NCTD.Western Blot was used to detect the expression of Nanog,Sox2,Bmil,Ptch-1 and Shh of A2780^(Cis) and SKOV3^(Pac) treated by NCTD.Results:CCK-8 activity assay showed that NCTD treatment inhibited the proliferation of ovarian cancer drug resistant cells A2780^(Cis) and SKOV3^(Pac) in a dose and time dependent manner.Flow cytometry analysis showed that cisplatin,paclitaxel and NCTD could induce the apoptosis of CD44^(+)/CD24^(-)stem-like cells of A2780^(Cis) cisplatin resistant cells and SKOV3^(Pac) paclitaxel resistant cells,while NCTD had the most significant effect.The expression of stem-features Nanog,Sox2 and Bmil were decreased by NCTD in A2780^(Cis) cisplatin resistant cells and SKOV3^(Pac) paclitaxel resistant cells.The expression of Ptch-1 was increased and the expression of Shh was decreased by NCTD in A2780^(Cis) cisplatin resistant cells and SKOV3^(Pac) paclitaxel resistant cells.Conclusion:NCTD can inhibit proliferation of ovarian cancer drug resistant cells and induce apoptosis of CD44^(+)/CD24^(-)stem-like cells cancer cells,via the possible mechanism of inhibiting Hedgehog pathway.

关 键 词：去甲斑蝥素 卵巢癌 化疗耐药 肿瘤干细胞 HEDGEHOG信号通路 

分 类 号：R285[医药卫生—中药学]