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自噬和凋亡是乳腺癌细胞数量和活力减少的重要因素,低频超声对肿瘤细胞的作用引起了研究者们的广泛兴趣,但是低频超声对乳腺癌细胞自噬和凋亡的作用尚不清楚。该文采用功率0.5 W/cm2的1 MHz低频超声联合微泡造影剂,作用于人乳腺癌细胞株MDA-MB-231 60s后,吖啶橙染色,电镜观察自噬的数量,Western blot检测微管相关蛋白轻链3-II(microtubule associated protein1 light chain 3-II,LC3-II)、自噬相关基因5(autophagy related 5,ATG5)和SQSTM1(sequestosome 1)/p62蛋白质水平变化,分别分析自噬的水平。Western blot检测caspase-3,膜联蛋白V/PI染色和DAPI染色方法分析MDA-MB-231细胞凋亡水平。ATG5 si RNA转染细胞可抑制自噬,caspase抑制剂Z-VAD-FMK可抑制凋亡,使用CCK-8分析自噬和凋亡对MDA-MB-231细胞的作用。结果显示,低频超声联合微泡造影剂促使LC3-II和ATG5蛋白质表达水平显著升高,而使SQSTM1/p62蛋白质表达水平显著下降(P<0.05)。透射电镜和共聚焦观察发现,MDA-MB-231细胞自噬体数量增加。低频超声联合微泡造影剂促使caspase-3蛋白质表达水平升高,凋亡率增加。抑制自噬和凋亡后均明显缓解低频超声联合微泡造影剂对细胞活力的抑制作用(P<0.05)。该研究结果说明,低频超声联合微泡造影剂通过激活自噬和凋亡抑制乳腺癌细胞株MDA-MB-231的增殖活力。
Autophagy and apoptosis are important factors in reducing the number and viability of breast cancer cells. The effect of low-frequency ultrasound on tumor cells has attracted a great deal of researchers’ interest, but the role of low-frequency ultrasound in the autophagy and apoptosis of breast cancer cells is unclear . In this study, 1 MHz low-frequency ultrasound combined with microbubble contrast agent with a power of 0.5 W / cm2 was used to detect the number of autophagy after treated with human breast cancer cell line MDA-MB-231 for 60s by acridine orange staining. The changes of protein levels of microtubule associated protein1 light chain 3-II (LC3-II), autophagy related 5 (ATG5) and SQSTM1 (sequestosome 1) / p62 were analyzed respectively The level of bite Western blot was used to detect the apoptosis of MDA-MB-231 cells by caspase-3, Annexin V / PI staining and DAPI staining. ATG5 si RNA transfected cells can inhibit autophagy, caspase inhibitor Z-VAD-FMK can inhibit apoptosis, the use of CCK-8 analysis of autophagy and apoptosis on MDA-MB-231 cells. The results showed that low-frequency ultrasound combined with microbubble contrast agent promoted the expression of LC3-II and ATG5 protein significantly increased, while the SQSTM1 / p62 protein expression decreased significantly (P <0.05). Transmission electron microscopy and confocal observation found that MDA-MB-231 cells increased the number of autophagosomes. Low-frequency ultrasound combined with microbubble contrast agent promoted caspase-3 protein expression and increased apoptosis rate. Inhibition of autophagy and apoptosis were significantly alleviated by low-frequency ultrasound combined with microbubble contrast agent on cell viability (P <0.05). The results show that low-frequency ultrasound combined with microbubble contrast agent inhibits the proliferation activity of breast cancer cell line MDA-MB-231 by activating autophagy and apoptosis.