雌激素激活GPR30/ERK通路促进三阴性乳腺癌MDA-MB-468细胞迁移及侵袭

Estrogen enhances migration and invasion in triple-negative breast cancer MDA-MB-468 cells via activating GPR30/ERK signaling pathway

目的探讨雌激素/GPR30/ERK信号通路的激活对三阴性乳腺癌(triple-negative breast cancer,TNBC)MDAMB-468细胞迁移及侵袭的影响。方法免疫荧光及Western blot检测MDA-MB-468细胞中雌激素受体GPR30的蛋白表达量及细胞内定位,Western blot检测药物处理后细胞中磷酸化细胞外信号调节激酶(phospho-extracellular regulate kinase,p-ERK)的蛋白表达变化,细胞划痕实验及Transwell小室实验分别检测细胞的迁移及侵袭能力的改变。结果雌激素受体GPR30在TNBC细胞系MDA-MB-468中高表达,且主要位于细胞胞浆部位。17-β雌二醇(E2)和GPR30特异性激动剂(G1)可以显著活化细胞中GPR30/ERK信号通路(P<0.05),上调p-ERK蛋白水平,其相对表达量分别是空白对照组的(2.07±0.11)倍和(1.98±0.06)倍。E2及G1药物处理后的细胞迁移能力得到显著提高(P<0.05),24 h后迁移入划痕区域的相对细胞数分别为空白对照组的(2.10±0.20)倍和(2.14±0.34)倍。细胞侵袭实验也可得到类似结果。GPR30特异性拮抗剂(G15)以及ERK特异性抑制剂(U0126)均可显著抑制E2和G1引发的以上改变(P<0.05)。结论雌激素通过活化TNBC细胞MDA-MB-468中GPR30/ERK信号通路,促进细胞迁移及侵袭的恶性潜能。靶向雌激素/GPR30/ERK通路可能成为TNBC的有效治疗手段。

Objective To explore the effects of activated estrogen/GPR30/ERK signaling on the migration and invasion of triple-negative breast cancer（ TNBC） cell line MDA-MB-468.Methods Immunofluorescent assay and Western blotting were used to test the expression and localization of estrogen receptor GPR30 in MDA-MB-468 cells.The expression level of phospho-extracellular regulate kinase（ p-ERK）was detected by Western blotting.The changes of cell migration and invasion ability were examined by woundhealing assay and Transwell assay,respectively.Results Estrogen receptor GPR30 was detected with high expression level in MDA-MB-468 cells and was mostly expressed in the cytoplasm.After treating with 17-βestradiol（ E2） and GPR30 specific agonist（ G1）,the GPR30 /ERK signaling was remarkably activated.The relative protein expressions of p-ERK in the E2 and G1 treatment groups were（ 2.07 ± 0.11） and（ 1.98 ±0.06） times higher than those of the control group（ P〈0.05）,respectively.Moreover,E2 and G1 significantly increased the ability of cell migration.The relative migrated cell numbers in the E2 and G1 treatment groups were 2.10 ± 0.20 and 2.14 ± 0.34 times higher than those of the control group（ P〈0.05）,respectively.Transwell assays indicated the similar results as wound-healing assays.Interestingly,these changes induced by E2 and G1 could be significantly blocked by GPR30 specific antagonist（ G15） and ERK specific inhibitor（ U0126）（ P〈0.05）.Conclusion Estrogen increases the ability of cell migration and invasion through activating GPR30 /ERK signaling in TNBC cells.Inhibition of estrogen /GPR30 /ERK signaling represents a novel targeted therapy in TNBC.