雌激素活化GPER介导的IL-6/STAT3通路促进乳腺癌细胞SKBR-3增殖作用

Estrogen activates GPER mediated IL-6 / STAT3 signaling pathway to enhance proliferation in breast cancer SKBR-3 cells

目的探讨雌激素活化膜性雌激素受体(G-protein coupled estrogen receptor,GPER)所介导的IL-6/STAT3炎症信号通路对乳腺癌SKBR-3细胞增殖能力的影响。方法用17-β雌二醇(E2)、GPER特异性激动剂(G1)、GPER特异性拮抗剂(G15)、IL-6中和抗体(Anti-IL-6)及STAT3特异性抑制剂JSI-124(cucurbitacin I)药物处理SKBR-3细胞后,分别得到对照组、E2处理组、G1处理组、E2+G15处理组、G1+G15处理组、E2+Anti-IL-6处理组、G1+Anti-IL-6处理组、E2+JSI-124处理组与G1+JSI-124处理组,用ELISA检测细胞培养液上清中IL-6的分泌量,CCK-8法检测细胞增殖能力的变化,Western blot检测细胞中p-STAT3与STAT3的蛋白表达水平。结果 E2和G1显著促进SKBR-3细胞上清中IL-6的分泌量,G15可显著阻断其分泌(P<0.05)。E2及G1药物处理细胞后增殖能力较对照组显著增强,相对细胞数分别为对照组的(1.68±0.13)倍与(1.74±0.21)倍,其促增殖作用被G15及IL-6中和抗体(Anti-IL-6)显著抑制(P<0.05)。E2及G1在不同时间点(1、3、6、12 h)均可显著促进细胞中p-STAT3的蛋白表达量,分别于12 h和3 h达到表达峰值,其蛋白相对表达量分别为对照组的(2.54±0.23)倍和(3.12±0.24)倍。G15、Anti-IL-6及JSI-124显著阻断以上变化(P<0.05)。JSI-124亦可明显抑制E2及G1所引起的促增殖效应(P<0.05)。结论雌激素活化膜性雌激素受体GPER促进乳腺癌SKBR-3细胞自分泌IL-6从而激活细胞中下游STAT3炎症信号通路,同时,GPER/IL-6/STAT3信号通路也介导了雌激素对细胞的增殖作用。

Objective To determine the effects of estrogen activation in the G-protein coupled estrogen receptor（ GPER） mediated-6 / STAT3（ GPER / IL-6 / STAT3） signaling pathway on the cell proliferation in breast cancer SKBR-3 cells. Methods 17-β-estradiol（ E2）,GPER specific agonist（ G1）,GPER specific antagonist（ G15）,human IL-6-neutralizing antibody（ anti-IL-6） and STAT3 signaling specific inhibitor（ cucurbitacin I, JSI-124） were used respectively to treat SKBR-3 cells. So the cells were accordingly assigned into the control group,E2 treatment group,G1 treatment group,E2＋ G15 treatment group,G1 ＋ G15 treatment group,E2＋ anti-IL-6 treatment group,G1 ＋ anti-IL-6 treatment group,E2＋ JSI-124 treatment group and G1 ＋ JSI-124 treatment group. The content of IL-6 in the supernatant was detected by ELISA. CCK-8 assay was used to test the change of proliferate ability,and Western blotting to the protein expression levels of p-STAT3 and STAT3. Results E2 and G1 significantly increased the content of IL-6 in the supernatant which could be blocked by GPER specific antagonist（ G15）（ P〈 0. 05）. After treating with E2 and G1 in the SKBR-3 cells,the cell proliferation was increased remarkably compared with control groups,with the relative cell amount of 1. 68 ± 0. 13 and 1. 74 ± 0. 21 times higher than those of the control groups（ P〈 0. 05）. However,the growth effects could be significantly blocked by G15 and anti-IL-6（ P〈 0. 05）.E2 and G1 enhanced the protein expression of p-STAT3 in 1,3,6,and 12 h after treatment,and the protein level reached the peak values at 12 h and 3 h respectively in E2 treatment group and G1 treatment group,with the relative protein expressions of 2. 54 ± 0. 23 and 3. 12 ± 0. 24 times higher than those of the control groups.G15,anti-IL-6 and JSI-124 blocked the above changes（ P〈 0. 05）. Furthermore,JSI-124 also inhibited the growth effects induced by E2 and G1 as well as G15 and anti-IL-6（ P〈 0. 05）. Conclusion Estrogen activates the membrane estrogen receptor GPER to enhance the autocrine secretion of IL-6 which activates its downstream STAT3 inflammatory signal pathway. Moreover,the GPER / IL-6 / STAT3 signal pathway mediates the cell proliferation induced by estrogen.