低频超声联合微泡对比剂通过PI3K/AKT通路对乳腺癌耐药株MCF-7/ADR多药耐药的逆转机制研究

目的 探索低频超声联合微泡（LFUSMB）通过磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（AKT）通路对乳腺癌 耐药株MCF-7/阿霉素（ADR）多药耐药的逆转效果及机制。方法 CCK-8法筛选LFUSMB作用时间并测定ADR对 MCF-7/ADR 细胞的半抑制浓度（IC50）。将 MCF-7/ADR 细胞分组为：对照（C）组，采用 MCF 细胞培养基正常培养； ADR组，采用加入ADR（终质量浓度20 mg/L）的MCF细胞培养基培养；LFUSMB+ADR组，采用加入ADR（终质量浓度 20 mg/L）的MCF细胞培养基培养并经LFUSMB处理30 s；LFUSMB+ADR+740 YP（PI3K/AKT通路活化剂）组，采用加 入 ADR（终质量浓度 20 mg/L）和 740 YP（终质量浓度 50 mg/L）的 MCF 细胞培养基培养并经 LFUSMB 处理 30 s。 Annexin V-FIFC/PI 法检测 MCF-7/ADR 细胞凋亡率，Western blot 检测 P 糖蛋白（P-gp）、抗多药耐药蛋白（MRP）1、 MRP2、乳腺癌抗性蛋白（BCRP/ABCG2）、PI3K、AKT、p-PI3K、p-AKT蛋白表达。结果 采用LFUSMB干预30 s进行 后续研究。LFUSMB 30 s组ADR对MCF/ADR细胞的IC50低于未超声处理组，相对耐药逆转倍数约为8.20倍。ADR 组细胞凋亡率较C组增高（P＜0.05），MRP1、MRP2、P-gp、BCRP/ABCG2、p-PI3K/PI3K和p-AKT/AKT 蛋白水平无明 显变化；LFUSMB+ADR组细胞凋亡率较ADR组增高，MRP1、MRP2、P-gp、BCRP/ABCG2、p-PI3K/PI3K和p-AKT/AKT 蛋白水平显著下调（P＜0.05）。与 LFUSMB+ADR 组比较，LFUSMB+ADR+740 YP 组细胞凋亡率显著降低，MRP1、 MRP2、P-gp、BCRP/ABCG2 蛋白水平显著增高（P＜0.05）。结论 LFUSMB 可通过抑制 PI3K/AKT 通路活化，下调 MRP1等腺苷三磷酸结合盒（ABC）家族蛋白表达，逆转乳腺癌MCF-7/ADR细胞耐药性。

Low-frequency ultrasound combined with microbubble contrast agent reversed the multidrug resistance of breast cancer resistant strain MCF-7/ADR through PI3K/AKT pathway

Objective To explore the reversal effect and mechanism of low-frequency ultrasound combined with microbubbles (LFUSMB) on the multidrug resistance of breast cancer resistant strains MCF-7/Adriamycin (ADR) through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. Methods The CCK-8 method was used to screen the action time of LFUSMB and determine the half inhibitory concentration (IC50) of ADR on MCF-7/ADR cells. The MCF-7/ADR cells were divided into: control group (normally cultured with MCF cell culture medium), ADR group (used MCF cell culture medium with ADR, final concentration 20 mg/L), LFUSMB+ADR group (cultured with MCF cell culture medium with ADR, final concentration 20 mg/L and treated with LFUSMB for 30 s) and LFUSMB+ADR+740 YP (PI3K/AKT pathway activator) group (MCF cell culture medium added with ADR, final concentration 20 mg/L and 740 YP, final concentration 50 mg/L, and were cultured and treated with LFUSMB for 30 s). Annexin V-FIFC/PI method was used to detect the apoptosis rate of MCF-7/ADR cells. Western blot assay was used to detect the expression of glycoprotein (P-gp), multidrug resistance protein (MRP) 2, breast cancer resistance protein (BCRP/ABCG2), PI3K, AKT, p-PI3K, p-AKT and MRP1 proteins. Results The LFUSMB intervention for 30 s was used for the follow-up study. The IC50 of ADR to MCF/ ADR cells was lower in the LFUSMB 30 s group than that of the non-ultrasound group, and the relative drug resistance reversal ratio was approximately 8.20 times. The apoptosis rate of MCF-7/ADR cells was significantly higher in the ADR group than that in the control group (P＜0.05). There were no significantly differences in the protein levels of MRP1, MRP2, P-gp, BCRP, p-PI3K/PI3K and p-AKT/AKT between the two groups. The apoptosis rate of MCF-7/ADR cells was higher in the LFUSMB+ADR group than that in the ADR group (P＜0.05), and the protein levels of MRP1, MRP2, P-gp, BCRP, p PI3K/PI3K and p-AKT/AKT were significantly lower than those in the ADR group (P＜0.05). Compared with the LFUSMB+ ADR group, the cell apoptosis rate was significantly decreased in the LFUSMB+ADR+740 YP group (P＜0.05), and the protein levels of MRP1, MRP2, P-gp and BCRP/ABCG2 were significantly increased (P＜0.05). Conclusion LFUSMB can reverse the drug resistance of breast cancer MCF-7/ADR cells by inhibiting the activation of PI3K/AKT pathway and down regulating the expression of ATP-binding cassette (ABC) family proteins.