高强度聚焦超声通过TRIF介导的ERK通路增强乳腺癌的顺铂化疗敏感性

目的　探究高强度聚焦超声（HIFU）通过β干扰素TIR结构域衔接蛋白（TRIF）介导的细胞外调节蛋白激酶（ERK）通路增强乳腺癌顺铂（DDP）化疗敏感性的作用机制。方法　依次增加DDP浓度间歇作用的方法诱导乳腺癌细胞（MDA-MB-231、MCF-7）/DDP耐药细胞；将MDA-MB-231/DDP、MCF-7/DDP细胞分为control组、HIFU组、HIFU+二甲基亚砜（DMSO）组、HIFU+漆黄素（fisetin）组。CCK-8检测细胞活力；集落形成实验检测细胞增殖情况；流式细胞术检测细胞周期及凋亡情况；Western blot检测细胞TRIF蛋白、耐药相关蛋白及ERK通路蛋白表达；体内成瘤实验检测肿瘤生长情况；免疫组化染色法检测肿瘤组织Ki-67的表达。结果　与亲本MDA-MB-231及MCF-7细胞相比，在相同浓度DDP处理下MDA-MB-231/DDP及MCF-7/DDP细胞活力更高，且半数抑制浓度（IC50）显著增加（P＜0.05），成功建立了稳定的DPP耐药细胞系；与control组相比，HIFU组MDA-MB-231/DDP及MCF-7/DDP细胞IC50、OD450值、克隆细胞数、S期细胞百分比、B淋巴细胞瘤-2基因（Bcl-2）、TRIF、p-糖蛋白（p-gp）、多药耐药基因1（MDR1）、p-ERK1/2/ERK1/2表达水平显著降低，G0/G1期细胞百分比、细胞凋亡率、Bcl-2关联X蛋白（Bax）表达水平显著增加（P＜0.05）；与HIFU组相比，HIFU+fisetin组细胞IC50、OD450值、克隆细胞数、S期细胞百分比、Bcl-2、TRIF、p-gp、MDR1、p-ERK1/2/ERK1/2表达水平显著增加，G0/G1期细胞百分比、细胞凋亡率、Bax表达水平显著降低（P＜0.05）。与control组相比，HIFU组肿瘤体积、肿瘤质量、TRIF及p-ERK1/2/ERK1/2表达水平、Ki-67阳性表达显著降低（P＜0.05）；与HIFU组相比，HIFU+fisetin组肿瘤体积、肿瘤质量、TRIF及p-ERK1/2/ERK1/2表达水平、Ki-67阳性表达显著增加（P＜0.05）。结论　HIFU通过抑制TRIF表达来抑制其介导的ERK通路，从而增强乳腺癌DDP化疗敏感性。

High-intensity focused ultrasound enhances cisplatin chemotherapy sensitivity of breast cancer through TRIF-mediated ERK pathway

Objective　To explore the mechanism of high-intensity focused ultrasound (HIFU) enhancing the chemosensitivity of breast cancer to cisplatin (DDP) through TIR-domain-containing adaptor inducing interferon-β (TRIF)-mediated extracellular regulated protein kinase (ERK) pathway. Methods　Breast cancer cells (MDA-MB-231, MCF-7)/DDP-resistant cells were induced by intermittent action of increasing DDP concentration. The MDA-MB-231/DDP and MCF-7/DDP cells were divided into the control group, the HIFU group, the HIFU+Dimethyl sulfoxide (DMSO) group and the HIFU+fisetin group. CCK-8 was used to detect cell viability. Colony formation test was used to detect cell proliferation. Flow cytometry was used to detect cell cycle and apoptosis. Western blot assay was used to detect the expression of cell TRIF protein, drug resistance-related protein and ERK pathway protein. In vivo tumor formation test was used to detect tumor growth. Immunohistochemical method was used to detect the expression of Ki-67 in tumor tissues. Results　Compared with the parental MDA-MB-231 and MCF-7 cells, the MDA-MB-231/DDP and MCF-7/DDP cells had higher viability under the same concentration of DDP treatment, and the half inhibitory concentration (IC50) was significantly increased (P＜0.05), indicating the successful establishment of a stable anti-DPP drug-resistant cell line. Compared with the control group, the IC50, OD450 values of MDA-MB-231/DDP and MCF-7/DDP cells, number of cloned cells, percentage of S-phase cells, expression levels of B cell lymphoma-2 (Bcl-2), TRIF, p-glycoprotein (p-gp), multidrug resistance 1 (MDR1) and p-ERK1/2/ERK1/2 were significantly reduced in the HIFU group. The percentage of cells in G0/G1 phase, the apoptosis rate and the expression level of Bcl-2-associated X protein (Bax) were significantly increased (P＜0.05). Compared with the HIFU group, the IC50, OD450 values of MDA-MB-231/DDP and MCF-7/DDP cells, number of cloned cells, percentage of S-phase cells, expression levels of Bcl-2, TRIF, p-gp, MDR1 and p-ERK1/2/ERK1/2 were significantly increased in the HIFU+fisetin group, the percentage of cells in G0/G1 phase, the apoptosis rate, and the expression level of Bax were significantly reduced (P＜0.05). Compared with the control group, the tumor volume, tumor weight, the expression levels of TRIF and p-ERK1/2/ERK1/2, and the positive expression of Ki-67 were significantly reduced in the HIFU group (P＜0.05). Compared with the HIFU group, the tumor volume, tumor weight, expression levels of TRIF and p-ERK1/2/ERK1/2, and the positive expression of Ki-67 were significantly increased in the HIFU+fisetin group (P＜0.05). Conclusion　HIFU inhibits the ERK pathway mediated by TRIF expression, thereby enhancing the sensitivity of breast cancer DDP chemotherapy.