低频低强度超声辐照微泡对三阴性乳腺癌MDA-MB 231/DOX细胞耐药性的影响

Effect of low-frequency and low-intensity ultrasound irradiation microbubbles on drug resistance of triple-negative breast cancer MDA-MB-231/DOX cells

目的探讨低频低强度超声辐照微泡(LILFU-MB)对三阴性乳腺癌MDA-MB-231/DOX细胞耐药性的影响。方法筛选LILFU-MB最佳非毒性辐照时间,计算细胞存活率;将MDA-MB-231/DOX细胞分为LILFU-MB辐照组和非LILFU-MB辐照组,绘制药物抑制拟合曲线图获得两组药物的半抑制浓度(IC_(50)),并筛选阿霉素最佳剂量。根据不同处理方式将细胞分为对照组、阿霉素组、LILFU-MB组、LILFU-MB+阿霉素组,观察细胞增殖活性,并计算细胞迁移率。使用倒置荧光显微镜观察细胞形态,并记录细胞核荧光强度及细胞核型;使用流式细胞仪检测细胞凋亡率;Westernblot检测细胞蛋白表达情况。结果LILFU-MB最佳非毒性辐照时间为30 s,该条件下细胞存活率为(92.33±2.58)%,故后续实验中LILFU-MB辐照时间均采用此条件。LILFU-MB辐照组中阿霉素对耐药细胞的IC_(50)低于非LILFU-MB辐照组(7.51μmol/Lvs.35.40μmol/L),后续实验采用35.40μmol/L为阿霉素最佳剂量。阿霉素组和LILFU-MB+阿霉素组细胞增殖活性均受到明显抑制,增殖速度减慢,其中LILFU-MB+阿霉素组抑制效果最明显。对照组、阿霉素组、LILFU-MB组、LILFU-MB+阿霉素组细胞迁移率分别为(56.79±1.11)%、(51.34±4.66)%、(46.09±9.42)%、(22.01±6.02)%,其中LILFU-MB+阿霉素组细胞迁移率低于其余各组,差异均有统计学意义(均P<0.01)。倒置荧光显微镜观察显示,LILFU-MB+阿霉素组中大部分细胞核明显皱缩和碎裂,胞核蓝染高亮,染色质呈浓缩和边缘化,表现出较强的细胞凋亡核型改变。流式细胞仪检测结果显示,对照组、阿霉素组、LILFU-MB组、LILFU-MB+阿霉素组细胞凋亡率分别为(13.30±0.66)%、(22.68±2.85)%、(20.60±3.72)%、(31.84±2.87)%,其中LILFU-MB+阿霉素组细胞凋亡率高于其余各组,差异均有统计学意义(均P<0.05)。LILFU-MB+阿霉素组P-gp、Bcl-2蛋白表达均显著下调,Cyt-c、Cl-caspase3蛋白表达均显著上调,与其余各组比较差异均有统计学意义(均P<0.05)。结论LILFU-MB能够显著抑制MDA-MB-231/DOX细胞增殖、迁移,并促进细胞凋亡,进而逆转三阴性乳腺癌MDA-MB-231/DOX细胞耐药性。

Objective To investigate the effect of low-frequency and low-intensity ultrasound irradiation microbubbles(LILFU-MB)on drug resistance of triple-negative breast cancer MDA-MB-231/DOX cells.Methods The best non-toxic irradiation time of LILFU-MB was screened and the cell survival rate was calculated.The MDA-MB-231/DOX cells were divided into LILFU-MB irradiation group and non LILFU-MB irradiation group.The drug inhibition fitting curve was drawn to obtain the half inhibitory concentration(IC_(50))of the two groups of drugs,the best dose of doxorubicin was screened.According to different treatment methods,the cells were divided into control group,doxorubicin group,LILFU-MB group,LILFU-MB+doxorubicin group,the cell proliferation activity was observed,and the cell migration rate was calculated.The morphology of the cells were observed by inverted fluorescence microscope,and the fluorescence intensity and karyotype of the cells were recorded.The apoptosis rate was detected by flow cytometry.The protein expression was detected by Western blot.Results The best non-toxic irradiation time of LILFU-MB was 30 s,and the cell survival rate was(92.33±2.58)%under this condition.This condition was used for the duration of LILFU-MB irradiation in subsequent experiments.The IC_(50) of doxorubicin on drug-resistant cells in the LILFU-MB irradiation group was lower than that in the non LILFU-MB irradiation group(7.51µmol/L vs.35.40µmol/L).The best dose of doxorubicine was 35.40µmol/L.The cell proliferation activity of doxorubicin group and LILFU-MB+doxorubicin group was significantly inhibited,and the proliferation rate was slowed down,and the inhibition effect of LILFU-MB+doxorubicin group was the most obvious.The cell migration rates of the control group,doxorubicin group,LILFU-MB group,LILFU-MB+doxorubicin group were(56.79±1.11)%,(51.34±4.66)%,(46.09±9.42)%,(22.01±6.02)%,respectively,and the cell migration rates of LILFU-MB+doxorubicin group were significantly different from that of other groups(all P<0.01).Inverted fluorescence microscope revealed that most of the nuclei in the LILFU-MB+doxorubicin group were obviously shrunk and fragmented,the blue staining of the nuclei was highlighted,the chromatin was concentrated and marginalized,and the strong apoptotic karyotype change was showed.The results of flow cytometry showed that the apoptosis rates of the control group,doxorubicin group,LILFU-MB group and LILFU-MB+doxorubicin group were(13.30±0.66)%,(22.68±2.85)%,(20.60±3.72)%,(31.84±2.87)%,respectively.The apoptosis rate of LILFU-MB+doxorubicin group was higher than that of the other groups,and the differences were statistically significant(all P<0.05).The expressions of P-gp protein and Bcl-2 in LILFU-MB+doxorubicin group were significantly down-regulated,while the expressions of Cyt-c and Cl-caspase3 protein were significantly up-regulated,and the differences were statistically significant compared with that of other groups(all P<0.05).Conclusion LILFU-MB can significantly inhibit the proliferation and migration of MDA-MB-231/DOX cells,promote cell apoptosis,and reverse the drug resistance of triple negative breast cancer MDA-MB-231/DOX cells.