山姜素抑制卵巢癌OVCAR-8细胞增殖迁移及其机制研究

Study of the inhibition effect of alpinetin on human ovarian cancer cell line OVCAR-8 and its mechanism

目的探讨山姜素对人卵巢癌OVCAR-8细胞的增殖、迁移的抑制作用及其可能机制。方法以DMSO为溶剂对照,使用不同浓度山姜素(25、50、100、200和400μmol/L)处理OVCAR-8不同时间(24、48和72h)后,采用CCK-8试剂盒检测细胞增殖活力;采用3D肿瘤微球形成实验检测50μmol/L山姜素处理7d后肿瘤微球形成能力;使用划痕实验检测100μmol/L山姜素处理24 h后对迁移能力;采用Western blot法检测不同浓度山姜素(50、100和200μmol/L,以DMSO为对照)作用48 h后OVCAR-8细胞STAT3信号通路及凋亡相关蛋白表达变化。结果 25~400μmol/L山姜素对OVCAR-8细胞增殖均有抑制作用,且呈剂量-时间依赖效应关系(均P<0.05);50μmol/L山姜素处理组肿瘤微球直径较DMSO组明显减小(P<0.05),而100μmol/L山姜素组划痕宽度明显宽于DMSO对照组(P<0.05);此外,50、100和200μmol/L山姜素处理组Bax、cleaved-caspase-3和-9的表达明显增加,Bcl-2、p-STAT3、c-myc、survivin蛋白表达明显减少,均呈一定的剂量依赖效应关系(均P<0.05)。而STAT3蛋白水平无明显变化(P>0.05)。结论山姜素对卵巢癌OVCAR-8细胞增殖和迁移具有抑制作用,且可能与STAT3信号通路抑制以及线粒体凋亡通路激活相关。

Objective To explore the effect of alpinetin on human ovarian cancer cell line OVCAR-8 and its mecha nism. Methods OVCAR-8 cells were treated with different concentrations of alpinetin（25, 50, 100, 200 and 400μmol/L,DMSO as control） for different hours（24, 48 and 72h）, then cell viabilities were detected by CCK-8 assays. 3D spheroid assay was used to measure the spheroid formation capacity of OVCAR-8 cell after treated with 50 μmol/L alpinetin（DMSO as control） for 7 days. Besides, wound healing assay was used to measure the migration capacity of OVCAR-8 cell after treated with 100 μmol/L alpinetin（DMSO as control） for 24h. In addition, Western blot was used to detect the protein levels of STAT3 signaling related and apoptosis related proteins in OVCAR-8 cells after alpinetin treatments（50, 100 and 200μmol/L, DMSO as control） for 48h. Results 25-400μmol/L alpinetin had remarkable inhibiting effects on OVCAR-8 cells and in a dose-and time-dependent manner（all P〈0.05）. After 7 days′ treatment, the diameter of spheroid in 50 μmol/L alpinetin group was significantly shorter than DMSO control group（P〈0.05）. Besides, the width of wound in 100 μmol/L alpinetin group was significantly longer than DMSO control group after treatment for 24h（P〈0.05）. In addition, the protein levels of Bcl-2, p-STAT3, c-myc and survivin were decreased in 50,100 and 200 μmol/L group of OVCAR-8 cells than DMSO control group after 48 exposure. On the contrary, the protein level of Bax, cleaved-caspase-3 and-9 was increased（all P〈0.05）, and there were no significantly changes in STAT3 levels（P〈0.05）. Conclusion Alpinetin can inhibit the proliferation and migration of OVCAR-8 cells,its mechanism may be related to the inhibition of STAT3 signaling pathway and the activation of mitochondrial apoptosis signaling.