基于雌激素受体信号通路雷公藤甲素抗乳腺癌作用及机制研究

Implication of triptolide inhibits 4T1 breast cancer cells proliferation by downregulating the phosphorylation of estrogen reporters

目的:研究雷公藤甲素体(TP)外对小鼠乳腺癌细胞系4T1的抑制作用,基于雌激素受体信号通路探讨TP抗肿瘤药效及其机制,为其临床治疗乳腺癌提供相关的实验依据。方法:MTT法检测TP对小鼠乳腺癌细胞4T1增殖的影响,采用流式细胞仪FITC-Annexin V/PI法检测TP诱导4T1细胞凋亡率;Western blot技术检测4T1细中胞雌激素受体信号通路相关蛋白ERα、p-ERα、ERβ、p-ERβ、ERK、p-ERK、MEK1/2、p-MEK1/2、p38-MAPK、p-p38 MAPK、SAPK/JNK、p-SAPK/JNK的表达。结果:TP浓度为100、10、1、0.1μmol/L时对4T1细胞均有增殖抑制作用,其中10、1、0.1μmol/L时能诱导4T1细胞凋亡,并能降低MEK1/2、p-MEK1/2、ERα、p-ERα、ERβ、p-ERβ的表达,但对ERK、p-ERK、p38 MAPK、p-p38 MAPK、SAPK/JNK、p-SAPK/JNK的表达没有影响。结论:一定浓度范围内,TP对小鼠乳腺癌细胞4T1具有增殖抑制作用,且其能诱导4T1细胞凋亡,可能与下调ERα、ERβ、MEK1/2的蛋白表达及其磷酸化有关。

Objective： In order to flndout the effective targets of triptolide, and provide experimental basis for its clinical treatment on breast cancer, the influence of triptolide on mice breast cancer cell line 4T1 in vitro was studied, as well as the possible mechanisms of inhibiting 4T1 ceils growth in vitro. Methods： Mice 4T1 breast cancer ceils were routinely cultured. The effect of triptolide on cell proliferation was determined by MTT assay for subsequent reasonable dose selecting in followed cell experiments; Flowcytometry combined with FITC-Annexin V/PI staining were used for detecting apoptosis rate of 4T1 cells induced by triptolide; Western blot technology was used for testing related protein expression of 4T1 cells including β-actin, MEK1/2, p-MEK1/2, ERα, p-ERα, ERβ, p-ERβ, ERK, p-ERk, p38 MAPK, p-p38 MAPK, SAPK/JNK, p-SAPK/JNK for preliminarily investigating its effect and mechanism for 4T1 cells in vitro of triptolide. Results： TP, which concentration is 100, 10, 1, 0.1μmol/L, all inhibited the proliferation of 4T1 cells by MTT assay and Crystal Violet Staining. TP which concentrations is 10, 1, 0. 1μmol/L could induce the apoptosis of 4T1 cells and reduce the expression of MEK1/2, p-MEK1/2, ERα, p-ERα, ERβ, p-ERβ, but there was no influence on the expression of ERK, p-ERK, p38 MAPK, p-p38 MAPK, SAPK/JNK, p-SAPK/JNK. Conclusion： Inhibitation of proliferation of mice 4T1 mammary tumor cell lines by triptolide, may be related with the effect of TP on inducing cell apoptosis in 4T1 cell. The anti-proliferation and induce apoptosis effects of triptolide in mice 4T1 breast cancer cells, may be caused by the down-regulation the total levels and phosphorylated levels of estrogen receptor and MEK in MAPK signal pathway.