基于IL-6/STAT3信号通路研究冬凌草甲素对乳腺癌细胞的作用及机制

Effect and mechanism of oridonin on breast cancer cells based on IL-6/STAT3 signaling pathway

目的基于白细胞介素-6(interleukin-6,IL-6)/信号传导及转录激活蛋白3(signal transducer and activator of transcription3,STAT3)信号通路研究冬凌草甲素对乳腺癌细胞增殖、自噬和凋亡的影响,并进一步探究冬凌草甲素对乳腺癌细胞增殖和凋亡相关蛋白表达的影响。方法通过MTT法检测冬凌草甲素对MDA-MB-231、MDA-MB-453、MDA-MB-468乳腺癌细胞增殖的影响;采用GFP-LC3荧光斑点检测冬凌草甲素对MDA-MB-468细胞自噬的影响;采用DAPI染色法观察冬凌草甲素对MDA-MB-468细胞生长抑制的影响;采用流式细胞仪测定冬凌草甲素对MDA-MB-468细胞凋亡的影响;利用稳定转染STAT3-luciferase报告基因质粒的HepG2-STAT1/3-Luc细胞,检测冬凌草甲素对IL-6/STAT3信号通路的作用;采用Western blotting检测冬凌草甲素对STAT3磷酸化的影响以及对细胞增殖及凋亡相关蛋白表达的影响。结果冬凌草甲素抑制乳腺癌细胞增殖,诱导乳腺癌细胞自噬和凋亡作用,呈剂量相关性;冬凌草甲素对IL-6/STAT3信号通路和STAT3磷酸化均有明显的抑制作用;冬凌草甲素能够上调乳腺癌细胞p53、剪切型多聚腺苷二磷酸核糖聚合酶(cleaved poly ADP-ribose polymerase,cleaved PARP)、剪切型半胱氨酸天冬氨酸蛋白酶-3(cleaved cystein-asparate protease-3,cleaved Caspase-3)以及Caspase-9蛋白表达。结论冬凌草甲素通过抑制STAT3磷酸化而抑制IL-6/STAT3信号通路的激活,并能上调p53、cleaved PARP、cleaved Caspase-3及cleaved Caspase-9蛋白表达,进而抑制乳腺癌细胞增殖,诱导乳腺癌细胞的自噬和凋亡。

Objective To study the effect of oridonin on proliferation and autophagy of breast cancer cells based on interleukin-6(IL-6)/signal transducer and activator of transcription 3(STAT3)signaling pathway,and further explore the effect of oridonin on proliferation and apoptosis-related protein expressions of breast cancer cells.Methods Effect of oridonin on proliferation of MDAMB-231,MDA-MB-453 and MDA-MB-468 breast cancer cells was detected by MTT method;Effect of oridonin on autophagy of MDA-MB-468 cells was detected by GFP-LC3 fluorescent spotsin;Effect of oridonin on nuclear morphology of MDA-MB-468 cells was observed by DAPI staining;Effect of oridonin on apoptosis of MDA-MB-468 cells was determined by flow cytometry;Effect of oridonin on IL-6/STAT3 signaling pathway was detected by HepG2-STAT1/3-Luc cells stably transfected with STAT3-luciferase reporter gene plasmid;Effect of oridonin on phosphorylation of STAT3 and cell proliferation and apoptosis-related protein expressions was detected by Western blotting.Results Oridonin inhibited the proliferation of breast cancer cells,induced autophagy and apoptosis of breast cancer cells in a dose-dependent manner;Oridonin significantly inhibited IL-6/STAT3 signaling pathway and STAT3phosphorylation;Oridonin up-regulated p53,cleaved poly ADP-ribose polymerase(cleaved PARP),cleaved caspase-aspartic protease-3(cleaved Caspase-3)and Caspase-9 protein expressions in breast cancer cells.Conclusion Oridonin may inhibit the activation of IL-6/STAT3 signaling pathway by inhibiting the phosphorylation of STAT3,and up-regulate protein expressions of p53,cleaved PARP,cleaved Caspase-3 and cleaved Caspase-9,thereby inhibiting the proliferation of breast cancer cells,inducing autophagy and apoptosis in breast cancer cells.