阿帕替尼与白蛋白结合型紫杉醇在MDA-MB-231乳腺癌细胞系中的协同抗癌作用

目的阐明阿帕替尼 (apatinib)和白蛋白结合型紫杉醇 (nab-Paclitaxel)诱导MDA-MB-231乳腺癌细胞凋亡的分子机制。方法本研究以MDA-MB-231乳腺癌细胞为研究对象,并以apatinib和nab-Paclitaxel处理细胞后分组:0.1 %DMSO处理为阴性对照组;10 μmol/L apatinib处理组 (APA组);经5、10、15、20 nmol/L nab-Paclitaxel 处理组 (Nab-p 5组、Nab-p 10组、Nab-p 15组和Nab-p 20组);以及10 μmol/L apatinib分别与5、10、15、20 nmol/L nab-Paclitaxel 联合处理组 (APA+Nab-p 5组、APA+Nab-p 10组、APA+Nab-p 15组和APA+Nab-p 20组)。使用乳酸脱氢酶释放测定法测定apatinib和nab-Paclitaxel对MDA-MB-231细胞诱导的细胞毒活性,结合流式细胞术分析不同处理组细胞凋亡情况,通过JC-1染色法测定不同干预方式对MDA-MB-231细胞线粒体膜电位变化 (ΔΨ_m)的影响,借助FAM-FLICA荧光成像检测caspase-8和caspase-9 活性,通过彗星试验评估apatinib和nab-Paclitaxel对非致瘤上皮细胞株MCF-10A细胞DNA损伤的影响。两组之间独立样本t检验或单向ANOVA进行比较,多组之间使用Tukey事后检验。结果细胞毒性检测结果显示,与阴性对照组相比,Nab-p 20组MDA-MB-231细胞杀伤率在24 h时接近90 %;与单药处理组 (Nab-p 5组和Nab-p 10组)相比,APA+Nab-p 5组和APA+Nab-p 10组联合处理24 h和48 h后,分别检测到约85 %和95 %细胞死亡,差异均具有统计学意义 (P 均 < 0.001)。流式细胞术统计结果显示,与Nab-p5组和Nab-p10组相比,APA+Nab-p 5组和APA+Nab-p 10组24 h时MDA-MB-231细胞凋亡率(31.8 %±1.48 %、33.25 %±1.77 %比76.11 %±1.14 %、89.4 %±1.07%)升高 (P 均 < 0.05)。线粒体膜电位检测结果表明,与对照组相比,在单药处理组中,仅APA组和Nab-p 10组24 h时去极化细胞 (12.35 %±1.05%比78.33%±1.11%、46.74%±1.75%)增多;在联用处理组中,APA+Nab-p 5组和APA+Nab-p 10组24 h时去极化细胞 (68.47%±1.94%比90.03%±1.79%)增多,差异均有统计学意义 (P 均 < 0.05)。FAM-FLICA荧光成像结果显示,相较于单一处理组,apatinib和nab-Paclitaxel联用处理组的caspase-8和caspase-9蛋白高度活化。结合彗星试验分析,apatinib和nab-Paclitaxel 干预对MCF-10A非致瘤上皮细胞株DNA完整性没有显著影响。结论 apatinib/nab-Paclitaxel联用通过内源性的线粒体功能扰动和外源性的caspase激活诱导三阴性乳腺癌细胞MDA-MB-231凋亡,发挥协同抗癌作用。

Synergistic anticancer effects of apatinib and nab-paclitaxel in MDA-MB-231 breast cancer cell line

Objective To elucidate the molecular mechanisms of apatinib and nab-Paclitaxel in inducing apoptosis of MDA-MB-231 breast cancer cells.Methods MDA-MB-231 cells were treated with apatinib and nab-Paclitaxel alone or together and named:control group (0.1 % DMSO treatment);10 μmol/L apatinib treatment group (APA group);5,10,15,20 nmol/ L nab-Paclitaxel treatment group (Nab-p 5 groups,Nab-p 10 groups,Nab-p 15 groups and Nab-p 20 groups)and 10 μmol/L apatinib combined with 5,10,15,20 nmol/L nab-Paclitaxel treatment group (APA Nab-p 5 groups,APA Nab-p 10 groups,APA Nab-p 15 groups and APA Nab-p 20 groups).The cytotoxic activity induced by apatinib and albumin-bound paclitaxel on MDA-MB-231 cells was determined by a lactate dehydrogenase release assay.Flow cytometry was used to quantify apoptosis in different treatment groups.Effects of different intervention methods on mitochondrial membrane potential change (ΔΨm) of MDA-MB-231 cells were examined by JC-1 staining.Caspase-8 and Caspase-9 activities were detected by FAM-FLICA fluorescence imaging.The effects of apatinib and albumin-bound paclitaxel on DNA damage in non-tumorigenic epithelial cell line MCF-10A were evaluated by comet assay.Student’s t test or one-way ANOVA was used for comparison between two groups,and Tukey post-test was used for comparison within multiple groups.Results Cytotoxicity test results showed that the MDA-MB-231 cell death rate in the Nab-p 20 group was close to 90 % at 24 h,and the difference was extremely significant compared with the control group (P < 0.001).After treatment for 24 h and 48 h,about 85 % and 95 % of cell deaths were detected in the APA+Nab-p 5 group and APA+Nab-p 10 group respectively,which showed a significantly difference compared with the single-drug treatment group (Nab-p 5 group and Nab-p 10 group) (P < 0.001).The results of flow cytometry showed that the apoptotic rate of APA+Nab-p5 (76.11±1.14)and APA+Nab-p10 (89.4±1.07)groups were significantly increased (P < 0.05)compared with the Nab-p5 (31.8±1.48) and Nab-p10(33.25±1.77)groups.The results of mitochondrial membrane potential test showed that only depolarized cells in APA group (78.33±1.11) and Nab-p 10 group (46.74±1.75) was increased significantly at 24 h (all P < 0.01)compared with the control group (12.35±1.05),in the single-drug treatment group,while in the combined treatment group,the depolarised cells in the APA+Nab-p 5 (68.47±1.94)and APA+Nab-p 10 group(90.03±1.79) also increased significantly (P < 0.05).FAM-FLICA fluorescence imaging results showed that compared with the single treatment group,the caspase-8 and caspase-9 proteins in the combination group of apatinib and nab-Paclitaxel were highly activated.Comet assay analysis showed that the intervention of apatinib and nab-Paclitaxel did not significantly affect the DNA integrity of MCF-10A non-tumorigenic epithelial cell line.Conclusion Combined apatinib with nab-paclitaxel induces apoptosis through intrinsic mitochondrial function perturbation and extrinsic caspase activation in triple-negative breast cancer cells MDA-MB-231,and may exhibit a synergistic anticancer effect.