4羟基他莫昔芬应激MCF-7乳腺癌细胞上清对巨噬细胞极化的影响

Effects of 4-hydroxytamoxifen Induced MCF-7 Breast Cancer Cells Supernatant on Macrophages Polarization

目的探讨4羟基他莫昔芬(4-hydroxytamoxifen,4-OHTAM)刺激MCF-7乳腺癌细胞后上清对巨噬细胞极化的影响。方法实验组分别用不同浓度(2.5、5、10、15、20μmol/L)的4-OHTAM刺激MCF-7乳腺癌细胞2 h和5 h,提取细胞上清液,对照组未经4-OHTAM处理;Western blot检测MCF-7细胞中应激蛋白C/EBP家族同源蛋白(C/EBP-homologous protein,Chop)及葡萄糖调节蛋白78(glucose regulated protein 78,Grp78)的表达水平。并将上清与佛波酯(phorbol 12-myristate 13-acetate,PMA)诱导6 h的人白血病单核细胞株THP-1共同培养66 h后,获得贴壁巨噬细胞。酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测巨噬细胞上清中白细胞介素10(interleukin 10,IL-10)及白细胞介素1受体拮抗剂(interleukin 1 receptor antagonist,IL-1Ra)的水平;Western blot检测巨噬细胞IL-1Ra及CD206的表达水平。结果5μmol/L的4-OHTAM刺激MCF-7细胞5 h后,实验组中应激蛋白Chop和Grp78表达水平明显上调(P<0.05);巨噬细胞上清中细胞因子IL-10和IL-1Ra的水平以及细胞中CD206和IL-1Ra的蛋白表达水平在4-OHTAM浓度为5μmol/L时水平最高(P<0.05)。结论5μmol/L的4-OHTAM刺激MCF-7细胞5 h后,MCF-7细胞发生了细胞应激,并且释放的细胞因子可以诱导巨噬细胞向M2型极化转变,这种极化方向的改变可能对乳腺癌的发生发展及4-OHTAM耐药有一定影响。

Objective To investigate the effect of 4-hydroxytamoxifen(4-OHTAM)induced MCF-7 breast cancer cells supernatant on macrophage polarization.Methods MCF-7 breast cancer cells were stimulated with 4-OHTAM at different concentrations(2.5,5,10,15,20μmol/L)for 2 and 5 hours in the experimental group.The supernatant of each group was extracted,and the control group was not treated with 4-OHTAM.The expression levels of stress protein C/EBP family homologous protein(Chop)and glucose regulated protein 78(Grp78)in MCF-7 cells were detected by Western blot assay.The supernatant was co-cultured with human leukemia monocytic cell line THP-1 induced by phorbol 12-myristate 13-acetate(PMA)for 6-hour,and then adherent macrophages were obtained after 66-hour.The levels of interleukin 10(IL-10)and interleukin 1 receptor antagonist(IL-1 Ra)in the supernatant of macrophages were detected by enzyme linked immunosorbent assay(ELISA),and the expression levels of IL-1 Ra and CD206 in macrophages were detected by Western blot.Results After 5-hour stimulation of MCF-7 cells with 5μmol/L 4-OHTAM,the expression levels of stress proteins Chop and Grp78 were significantly increased in the experimental group(P<0.05).The levels of cytokines IL-10 and IL-1 Ra in macrophages supernatant and the protein expression levels of CD206 and IL-1 Ra in macrophages were highest at 4-OHTAM concentration of 5μmol/L(P<0.05).Conclusion After 5μmol/L of 4-OHTAM stimulated MCF-7 cells for 5-hour,MCF-7 cells develop cell stress,and the released cytokines can induce macrophage to M2 polarization,and this polarization direction may have a certain influence on the occurrence and development of breast cancer and 4-OHTAM resistance.