4,5,7-三羟基异黄酮对绒癌耐药细胞株JAR/MTX增殖、凋亡和侵袭的影响及相关机制

Effects of genistein on proliferation,apoptosis and invasiveness in methotrexate-resistant human choriocarcinoma JAR/MTX cells and it's mechanism

目的:研究4,5,7-三羟基异黄酮(genistein)对绒癌耐药细胞株JAR/MTX增殖、凋亡和侵袭能力的影响,并探讨其影响侵袭的相关机制。方法:分别采用MTT法、Annexin-Ⅴ和PI双染法、transwell小室法,检测不同浓度genistein作用于JAR/MTX细胞72 h后细胞增殖、凋亡和侵袭能力的变化。采用RT-PCR技术检测雌激素受体(ER)以及与侵袭有关的MTA3、Snail基因mRNA的表达,采用蛋白印迹法和明胶酶谱法检测E-钙黏附蛋白(E-cadherin)和基质金属蛋白酶2(MMP-2)和9(MMP-9)蛋白的表达。结果:Genistein能抑制JAR/MTX细胞的增殖和侵袭能力,并呈剂量依赖关系。小剂量genistein(10μmol/L)仅能引起少部分JAR/MTX细胞凋亡,而25、50、100μmol/L genistein则引起大量细胞凋亡和坏死。JAR/MTX细胞经genistein作用后,ERβ和MTA3的mRNA表达量多于对照组,Snail基因的mRNA表达量少于对照组。E-cadherin的蛋白表达大于对照组,MMP-2和MMP-9的蛋白表达量少于对照组。结论:Genistein能抑制JAR/MTX细胞的增殖,诱导细胞凋亡和坏死,通过上调E-cadherin和下调MMP-2以及MMP-9的表达、抑制JAR/MTX细胞的侵袭能力,MTA3→Snail→E-cadherin通路可能是genistein抑制JAR/MTX细胞侵袭的信号通路之一。

AIM： To study the effects of genistein on JAR/MTX cell proliferation, apoptosis and invasion and it mechanism in vitro. METHODS： MTT assay, Annexin - Ⅴ and propidium iodide label analysis and invasion assay were used to determine the effects of genistein on proliferation, apoptosis and invasiveness in JAR/MTX methotrexate - resistant human choriocarcinoma cells. RT - PCR was used to estimate the relative mRNA amounts of estrogen receptor（ER）, MTA3 and snail in the cells. Western blotting and gelatin zymography assay were used to estimate the relative protein amounts of MMP - 2, MMP- 9 and E - cadherin in the cells. RESULTS： After treatment of genistein, the proliferation and invasiveness of JAR/MTX cells were decreased significantly in a dose -dependent manner. 10 μmol/L genistein induced apaptosis, whereas 25, 50, 100 μmol/L genistein induced apoptosis and necrosis significantly. Genistein led to an increase in ERβ, MTA3 mRNA and E - cadherin protein expression, and decreases in the amounts for snail mRNA and MMP - 2 and MMP - 9 protein expression of JAR/MTX cells. CONCLUSIONS： Genistein inhibits the cell proliferation by inducing cell apoptosis and necrosis. Genistein also may inhibit JAR/MTX cell invasion in part through the upregulation of E - cadherin and downregnlation of MMP - 2 and MMP - 9. The signal transduction pathway of invasion suppression induced by genistein in JAR/MTX cells may be as follows： MTA3→snail→E- cadherin.