高、低浓度金雀异黄素对Ishikawa细胞生长及ERα/ERβ蛋白比值的影响

Influence of genistein in high and low concentration on growth and ERα/ERβ protein ratio of Ishikawa cells

目的研究高、低浓度金雀异黄素对雌激素受体(ER)阳性人子宫内膜癌Ishikawa细胞增殖、集落形成及ERα/ERβ蛋白表达比值影响的差异。方法以ER阳性人子宫内膜癌Ishikawa细胞为研究对象,正式实验前用Opti-MEM无酚红减血清培养基培养24 h,然后分为金雀异黄素低浓度组、高浓度组及溶剂对照组,依次加入由2%DCS-FBS的Opti-MEM无酚红减血清培养基配置所需浓度的金雀异黄素药液及含0. 5‰DMSO的培养液。应用CCK-8法检测高浓度(10、25、50μmoL/L)和低浓度(0. 001、0. 01、0. 1、1μmoL/L)的金雀异黄素干预Ishikawa细胞24、48、72 h对增殖的影响。平板集落形成实验检测高浓度(25、50μmoL/L)和低浓度(0. 01、0. 1μmoL/L)金雀异黄素干预1周对集落形成的影响。用Western blot法检测高浓度(25、50μmoL/L)和低浓度(0. 01、0. 1μmoL/L)金雀异黄素干预48 h后对ERα/ERβ蛋白表达比值的影响。结果低浓度(0. 001、0. 01、0. 1μmoL/L)范围内金雀异黄素促进Ishikawa细胞增殖,在干预48 h后最明显,0. 1μmoL/L浓度促增殖作用达到最强(P <0. 01);高浓度时(10、25、50μmoL/L)抑制细胞增殖(P <0. 05),且呈时间和浓度的依赖性,50μmoL/L浓度下干预72 h后抑制作用达到最强(P <0. 01)。低浓度金雀异黄素的集落形成率分别为14. 60%和15. 13%,和溶剂对照组(12. 20%)相比有统计学差异(P<0. 05);高浓度集落形成率分别为6. 07%、4. 27%,显著低于溶剂对照组(P <0. 01)。高浓度和低浓度下ERα蛋白表达均上调,且在0. 1μmoL/L时达到最高; ERβ蛋白表达均下降,在25、50μmoL/L时达到最低; ERα/ERβ蛋白比值均明显上调,在0. 1μmoL/L时达到最高。结论低浓度金雀异黄素具有促ER阳性Ishikawa细胞生长的作用,可能通过上调ERα/ERβ蛋白表达比值实现;高浓度金雀异黄素虽然也可以上调ERα/ERβ蛋白表达比值,但可能存在其他非ER依赖机制主导发挥抑制Ishikawa细胞生长的作用。

Objective To study the difference of genistein between high and low concentration on the proliferation,colony formation and protein expression ratio of ERα to ERβ( ERα/ERβ) in Ishikawa cells of human endometrial carcinoma with positive estrogen receptor( ER). Methods ER-positive Ishikawa cells of human endometrial carcinoma were taken as medium research objects. Before formal experiments,the cells were cultured in Opti-MEM phenol-free serummedium for 24 h,and then divided into low-dose genistein group,high-dose genistein group and solvent control group. The genistein solution and solvent control were prepared by the Opti-MEM phenol-free serum medium containing 2% DCS-FBS and were sequentially added to 3 groups. The influence of high-concentration genistein( 10 μmoL/L,25μmoL/L,50 μmoL/L) and low-concentration genistein( 0. 001 μmoL/L,0. 01 μmoL/L,0. 1 μmoL/L,1 μmoL/L) on proliferation of Ishikawa cells was detected by using cell counting kit-8( CCK-8) after intervening for 24 h,48 h and 72 h. The influence of high-concentration genistein( 25 μmoL/L,50μmoL/L) and low-concentration genistein( 0. 01 μmoL/L,0. 1 μmoL/L) on colony formation was detected by using colony formation assay after intervening for 1 week. The influence of high-concentration genistein( 25 μmoL/L,50 μmoL/L) and low-concentration genistein( 0. 01 μmoL/L,0. 1 μmoL/L)on protein expression ratio of ERα/ERβ by using Western blotting assay after intervening for 48 h.Results Low-concentration genistein( 0. 001 μmoL/L,0. 01 μmoL/L,0. 1 μmoL/L) promoted the proliferation of Ishikawa cells,which was the most significant after intervening for 48 h,and proliferation promoting effect was the highest when genistein concentration reached 0. 1 μmoL/L( P < 0. 01). Highconcentration genistein 10 μmoL/L,25 μmoL/L,50 μmoL/L inhibited the proliferation of Ishikawa cells and showed time-concentration dependence. The inhibitory effect reached the highest when genistein concentration was 50 μmoL/L after 72 h. The colony formation rate was 14. 60% and 15. 13%respectively in low-concentration genistein groups compared with solvent control group( 12. 20%,P <0. 05). The colony formation rate was 6. 07% and 4. 27% respectively in high-concentration genistein groups,which was significantly lower than that in solvent control group( P < 0. 01). The protein expression of ERα was up-regulated in high-concentration genistein groups and low-concentration genistein groups,and the expression reached the highest when genistein concentration was 0. 1 μmoL/L.The protein expression of ERβ was down-regulated,and the expression reached the lowest when genistein concentration was 25 μmoL/L or 50 μmoL/L. The protein expression ratio of ERα/ERβ was significantly up-regulated,and reached the highest when genistein concentration was 0. 1 μmoL/L. Conclusion Low-concentration genistein has effect of promoting Ishikawa cells with positive ER,which may be related to up-regulation of protein expression ratio of ERα/ERβ. High-concentration genistein can also upregulate protein expression ratio of ERα/ERβ,but may play the role of inhibiting growth of Ishikawa cells through non-ER dependent mechanism.