西黄丸逆转三阴性乳腺癌多药耐药的作用机制研究

A study on the mechanism of Xihuang Pill reversing multidrug resistance of triple-negative breast cancer

目的:探索西黄丸对三阴乳腺癌耐药株MDA-MB-231/ADM多药耐药的逆转作用及其机制。方法:细胞实验:采用MTT法检测不同浓度西黄丸作用于MDA-MB-231、MDA-MB-231/ADM细胞48 h后肿瘤细胞的生长抑制率,以生长抑制率低于20%的药物浓度作为非细胞毒性浓度;MTT法检测非细胞毒性浓度西黄丸对MDA-MB-231/ADM细胞多药耐药的逆转作用,计算耐药倍数;采用Western Blot法检测西黄丸对MDA-MB-231/ADM细胞P-gp、P-Akt、AKt、PTEN蛋白的表达的影响,RT-PCR法检测西黄丸干预后的MDA-MB-231/ADM细胞耐药基因的表达。结果:西黄丸对MDA-MB-231及MDA-MB-231/ADM细胞均有抑制增殖作用,且呈剂量依赖性,并选取4mg/ml和6mg/ml作为西黄丸的非细胞毒性浓度;非细胞毒性浓度的西黄丸联合ADM作用于MDA-MB-231/ADM细胞后,能明显降低ADM对MDA-MB-231/ADM的IC50值,西黄丸在4mg/ml和6mg/ml浓度下,逆转倍数分别为1.82和2.19倍;Western-blot结果显示,西黄丸能降低MDA-MB-231/ADM细胞中P-gp蛋白表达及PI3K/Akt信号通路中的p-AKt蛋白表达,增高PTEN蛋白水平表达,总Akt蛋白表达量的变化不明显。RT-PCR结果显示,西黄丸能明显降低耐药基因P-gp及MRP1的表达。结论:西黄丸能有效逆转三阴乳腺癌多药耐药株MDA-MB-231/ADM的耐药性,从而增强耐药株对化疗药物ADM的敏感性,与化疗药物ADM联用后具有化疗增效作用,其机制可能是通过抑制PI3K/AKT信号通路发挥作用。

Objective： To investigate the reversal effect of Xihuang Pill on multidrug resistance and its signal transduction mechanism of triple-negative breast cancer. Methods： Cells were treated with different concentrations of Xihuang Pill and LY294002. MTT assay was used to determine inhibitory rates of drugs to triple-negative breast cell lines MBA-MB-231 and MBA-MB-231/ADM after 48 hours＇ treatment. Drug had no cytotoxicity when its inhibitory rate to cells was below 20%. Western Blot was applied to analyze protein expressions of P-gp,P-Akt,Akt and PTEN in transplanted tumors of nude mice. Expression levels of multiple drug resistance 1（ MDR1） and MRP1 mRNA in cells MBA-MB-231/ADM were measured by real-time fluorescent quantitative PCR. Results： Xihuang Pill inhibited proliferations of MDA-MB-231 and MDA-MB-231/ADM cells in a dose-dependent matter,and 4 mg/ml and 6 mg/ml Xihuang Pill were non-cytotoxic; after the non-cytotoxic concentration of Xihuang Pill combined with ADM treated MDA-MB-231/ADM cells,there was a significant decrease of IC50 of ADM（ p 0. 05）. When Xihuang Pill was at concentrations of 4 mg/ml and 6 mg/ml,reverse ratios were 1. 82 and 2. 19 respectively; Western-blot results showed that Xihuang Pill reduced P-gp protein expression in the transplanted tumor and down-regulated P-Akt protein expression in PI3 K/AKT signal pathway,up-regulated PTEN protein expression. RT-PCR results showed that Xihuang Pill reduced P-gp and MRP1 mRNA of the MDA-MB-231/ADM. Conclusion： Xihuang Pill could effectively reverse the multidrug resistance of MDA-MB-231/ADM cells,enhance its sensitivity to chemotherapy drugs,have a synergistic effect of chemotherapy,and the mechanism may be related to inhibiting the PI3 K/AKT signal pathway.