雌激素受体拮抗剂对人子宫内膜样癌Ishikawa细胞增殖的影响及可能的作用机制

Effect of estrogen receptor antagonist on proliferation of human endometrioid carcinoma Ishikawa cells and its possible mechanism

目的 :探讨雌激素受体(estrogen receptor,ER)拮抗剂[他莫昔芬(tamoxifen,TAM)和氟维司群(fulvestrant或ICI-182780)]对高分化人子宫内膜样癌Ishikawa细胞增殖的影响,以及可能的作用机制。方法 :体外培养Ishikawa细胞,实验分为6组:TAM、ICI-182780、β-雌二醇(β-estradiol)单药组以及TAM、ICI-182780联合β-雌二醇组,以未用任何药物处理的Ishikawa细胞作为对照组。采用MTT法检测细胞的增殖情况,分别在光学显微镜和电子显微镜下观察细胞形态的变化,实时荧光定量PCR法和蛋白质印迹法检测细胞中p57^(kip2)以及cyclin E m RNA和蛋白的表达情况。结果 :与对照组相比,TAM组细胞的增殖能力稍有增强(P>0.05),其余各组细胞的增殖能力均有减弱(P值均<0.05);与β-雌二醇组相比,TAM组、β-雌二醇+TAM组细胞的增殖能力增强,而ICI-182780组、β-雌二醇+ICI-182780组细胞的增殖能力减弱(P值均<0.05)。与对照组相比,β-雌二醇+ICI-182780组细胞密度明显减少,其余各组细胞则无明显变化;各实验组细胞表面微绒毛均有减少,TAM组和β-雌二醇组中仅有个别细胞出现内质网轻度肿胀,ICI-182780组出现部分细胞内质网中度肿胀,β-雌二醇+TAM组和β-雌二醇+ICI-182780组中多数细胞内质网和线粒体明显肿胀,自噬现象增多。与对照组相比,TAM组中p57^(kip2) m RNA和蛋白的表达水平均有所下调,cyclin E m RNA和蛋白的表达水平则被上调,在其余各组中p57^(kip2)m RNA和蛋白的表达水平均被有所上调,cyclin E m RNA和蛋白的表达水平均被下调;与β-雌二醇组相比,在ICI-182780组和β-雌二醇+ICI-182780组中p57^(kip2) m RNA和蛋白的表达水平均有上调,cyclin E m RNA和蛋白的表达水平均为下调,在TAM组和β-雌二醇+TAM组中p57^(kip2) m RNA和蛋白的表达水平均为下调,cyclin E m RNA和蛋白的表达水平则为上调。结论:ICI-182780可抑制Ishikawa细胞的增殖,而TAM则微弱促进Ishikawa细胞增殖,ICI-182780和TAM对子宫内膜样癌的作用并不完全一致。这可能与ICI-182780能上调抑癌基因p57^(kip2)表达,下调癌基因cyclin E表达;TAM则是微弱下调p57^(kip2)表达,上调cyclin E表达有关。ICI-182780可能比TAM更适合子宫内膜样癌患者的内分泌个体化治疗。

Objective： To investigate the effects of estrogen receptor（ER） antagonists tamoxifen（TAM） and fulvestrant（ICI-182780） on the proliferation of highly differentiated human endometrioid carcinoma Ishikawa cells, and to explore the possible mechanism.Methods： Ishikawa cells were cultured in vitro, and treated with ER antagonist TAM alone, ICI-182780 alone, β-estradiol alone, TAM combined with β-estradiol, ICI-182780 combined with β-estradiol, respectively; while Ishikawa cells were not treated with any drugs as the control. The cell proliferation was detected by MTT method. The changes of cell morphology were observed under a light microscope and an electron microscope, respectively. The expression levels of p57 ^（kip2） and cyclin E m RNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting, respectively.Results： Compared with the control group, the proliferation ability of Ishikawa cells in TAM group was lightly enhanced（P〈0.05）, while those in the other groups were decreased（all P〈0.05）. Compared with β-estradiol group, the proliferation abilities of Ishikawa cells in TAM group and β-estradiol＋TAM group were increased（both P〈0.05）, while those in ICI-182780 group and β-estradiol＋ICI-182780 group were weakened（both P〈0.05）. Compared with the control group, the cell density in β-estradiol＋ICI-182780 group was reduced, while there was no obvious change in the other groups; the microvilli on the cell surface were reduced in each experimental group. The slight swelling of endoplasmic reticulum was seen in TAM group and β-estradiol group, and the moderate swelling of partial endoplasmic reticulum was seen in ICI-182780 group, while the most of endoplasmic reticulum and mitochondria had significant swelling in β-estradiol＋TAM group and β-estradiol＋ICI-182780 group and the autophagy increased. Compared with the control group, the expressions of p57 ^（kip2） m RNA and protein were decreased, while the expressions of cyclin E m RNA and protein were increased in the TAM group; but in other groups, the expressions of p57 ^（kip2） m RNA and protein were increased, while the expressions of cyclin E m RNA and protein were decreased. Compared with β-estradiol group, the expressions of p57 ^（kip2） m RNA and protein were increased in ICI-182780 group and β-estradiol＋ICI-182780 group, while the expressions of cyclin E m RNA and protein were decreased; but in TAM group and β-estradiol＋TAM group, the expressions of p57 ^（kip2） m RNA and protein were decreased, while the expressions of cyclin E m RNA and protein were increased.Conclusion： ICI-182780 can restrain the proliferation of Ishikawa cells, while TAM slightly promotes the proliferation of Ishikawa cells. The effects of ICI-182780 and TAM on endometrioid carcinoma are not completely consistent. This may be related to ICI-182780 can up-regulate the expression of the anti-oncogene p57 ^（kip2） and down-regulate the expression of the oncogene cyclin E, TAM can slightly down-regulate the expression of p57 ^（kip2） and up-regulate the expression of cyclin E. ICI-182780 may be more suitable for individual endocrine therapy of the patients with endometrioid carcinoma than TAM.