阿帕替尼联合多西紫杉醇对卵巢癌干细胞的影响及其作用机制

Effect of Apatinib Combined with Docetaxel on Ovarian Cancer Stem Cells and Its Mechanism

目的探讨阿帕替尼联合多西紫杉醇对卵巢癌干细胞的影响及其作用机制。方法人卵巢浆液性乳头状腺癌细胞(SKOV3)复苏及传代后提取、培养卵巢癌干细胞样细胞并进行鉴定,用四甲基偶氮唑盐比色法检测不同浓度(5、10、20、40、80μg/m L)阿帕替尼对卵巢癌干细胞增殖的抑制作用,并将卵巢癌干细胞分为对照组、阿帕替尼组、多西紫杉醇组及联合组,用相应培养基及药物进行培养后,采用流式细胞术检测阿帕替尼及多西紫杉醇对卵巢癌干细胞凋亡的影响,Transwell细胞迁移实验检测阿帕替尼及多西紫杉醇对卵巢癌干细胞侵袭、迁移的影响,采用实时定量逆转录聚合酶链反应与Western blotting法检测阿帕替尼及多西紫杉醇对卵巢癌干细胞上皮钙黏素(E-cadhefin)、波形蛋白(Vimentin)、Twist信使RNA(mRNA)及其蛋白表达的影响。结果 SKOV3干细胞相关标志基因Oct-4、CD133及Nanog的表达水平均显著高于卵巢癌SKOV3贴壁细胞[(6.44±0.27)比(1.52±0.24)、(5.37±0.39)比(1.23±0.34)、(10.02±0.49)比(1.30±0.37)](P <0.05)。阿帕替尼浓度在5~80μg/m L时,随着阿帕替尼浓度的增加,卵巢癌干细胞增殖抑制率明显增高;与对照组比较,阿帕替尼组、多西紫杉醇组及联合组卵巢癌干细胞凋亡率均明显升高[(21.97±2.21)%、(21.70±2.26)%、(59.56±3.68)%比(5.58±1.55)%](P <0.05),以联合组卵巢癌干细胞凋亡率升高最显著(P <0.05);与对照组比较,阿帕替尼组、多西紫杉醇组、联合组卵巢癌干细胞穿膜细胞数均明显减少(P <0.05),且联合组卵巢癌干细胞穿膜细胞数减少更显著(P <0.05);与对照组比较,阿帕替尼组、多西紫杉醇组、联合组卵巢癌干细胞E-cadhefin mRNA及其蛋白表达水平均显著升高,Vimentin与Twist mRNA及其蛋白表达水平均显著降低(P <0.05),以联合组变化最显著(P <0.05)。结论阿帕替尼与多西紫杉醇联合应用可显著抑制卵巢癌干细胞的增殖、侵袭及迁移,促进卵巢癌干细胞的凋亡,其抑制卵巢癌干细胞侵袭及迁移的作用机制可能与其能够上调E-cadhefin的表达、下调Vimentin及Twist的表达相关。

Objective To investigate the effect and mechanism of apatinib combined with docetaxel on ovarian cancer stem cells.Methods After resuscitation and passage of human ovarian serous papillary adenocarcinoma cells(SKOV3),were extracted,cultured for ovarian cancer stem cell-like cells and identifie.The inhibitory effects of different concentrations(5,10,20,40,80μg/mL)of apatinib on the proliferation of ovarian cancer stem cells were detected by MTT.And the ovarian cancer stem cells were divided into a control group,an apatinib group,a docetaxel group and a combination group.After cultured with corresponding media and drugs,the effects of apatinib and docetaxel on the apoptosis of ovarian cancer stem cells were detected by flow cytometry;the effects of apatinib and docetaxel on the invasion and migration of ovarian cancer stem cells were detected by Transwell cell migration assay;the effect of apatinib and docetaxel on the expression of E-cadherin,Vimentin,Twist messenger RNA(mRNA)and proteins of ovarian cancer stem cells was investigated by real-time quantitative reverse transcription polymerase chain reaction and Western blotting.Results The expression levels of SKOV3 stem cell related marker genes Oct-4,CD133 and Nanog were significantly higher than those of ovarian cancer SKOV3 adherent cells[(6.44±0.27)vs(1.52±0.24),(5.37±0.39)vs(1.23±0.34),(10.02±0.49)vs(1.30±0.37)](P<0.05).When the concentration of apatinib was within the range of 5-80μg/mL,the inhibition rate of ovarian cancer stem cells increased significantly with the increase of the concentration.Compared with the control group,the apoptosis rate of ovarian cancer stem cells was significantly increased in the apatinib group,docetaxel group and the combination group[(21.97±2.21)%,(21.70±2.26)%,(59.56±3.68)%vs(5.58±1.55)%](P<0.05),and the highest apoptosis rate was seen in the combination group(P<0.05).Compared with the control group,the number of ovarian cancer stem cell penetrating cells in the apatinib group,docetaxel group and the combination group were significantly decreased(P<0.05),and the number in the combination group was more significantly decreased(P<0.05).Compared with the control group,the E-cadhefin mRNA and protein expression levels of ovarian cancer stem cells in the apatinib group,docetaxel group and the combination group were significantly increased,while the mRNA and protein expression levels of Vimentin and Twist were significantly decreased(P<0.05),especially significantly in the combination group(P<0.05).Conclusion Apatinib combined with docetaxel can significantly inhibit the proliferation,invasion and migration of ovarian cancer stem cells,and promote the apoptosis of ovarian cancer stem cells.The mechanism of its inhibitory effect on the invasion and migration of ovarian cancer stem cells may be related to the up-regulation of E-cadherin expression and the down-regulation of Vimentin and Twist.