目的:检测大豆异黄酮代谢产物雌马酚对大肠癌细胞增殖的影响,并探讨其分子作用机制.方法:培养大肠癌细胞 DLD1、HCT15、COLO205、LOVO和SW480,用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT]法检测雌马酚对大肠癌细胞增殖的影响,用逆转录PCR和蛋白免疫印迹法分别检测雌激素受体和核因子红系2相关因子2[nuclear factor (erythroid-derived 2)-like 2,Nrf2]表达状况.用MTT法检测雌激素受体抑制剂和激动剂对大肠癌细胞增殖的影响.结果:DLD1、HCT15、COLO205、LOVO和SW480均表达雌激素受体(estrogen receptor, ER)β,但只有DLD1和HCT15有ERα弱表达.MTT试验显示不同剂量雌马酚(0、0.5、1、5、10 μmol/L)不仅能够显著抑制ERα和ERβ均表达的大肠癌细胞HCT-15的增生,且呈现明显的剂量效应关系,而且能够抑制只有ERβ表达的大肠癌细胞LOVO和SW480的增生,且呈现明显的剂量效应关系.此外,逆转录PCR发现不同剂量雌马酚刺激后,HCT-15的雌激素受体ERα和ERβ表达明显增加,呈剂量效应关系.蛋白免疫印迹法进一步证实雌激素受体ERα和ERβ蛋白表达随雌马酚剂量的增加而增加,蛋白免疫印迹法发现Nrf2随干预剂量的增加而表达明显增加.采用ERβ激动剂、雌激素受体抑制剂和特异性雌激素受体ERα激动剂对SW480、LOVO、HCT-15细胞刺激后,只发现不同剂量ERβ激动剂能够显著抑制大肠癌细胞SW480、LOVO、HCT-15的增生,且呈明显的剂量效应关系,但是,雌激素受体抑制剂和特异性雌激素受体ERα激动剂对SW480、LOVO和HCT-15细胞没有呈现显著的影响.结论:雌马酚能够通过雌激素样作用和抗氧化活性抑制大肠癌细胞的增殖.
Objective:To investigate the effect of equol on the proliferation of colom cancer cells and to explore the mechanisms.Methods: Colon cancer cells (DLD1,HCT15,COLO205,LOVO,SW480) were incubated, the cell proliferation was identified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay.Reverse transcription PCR and Western blot were used to measure the mRNA and the protein expression of estrogen receptor and nuclear factor (erythroid-derived 2)-like 2 (Nrf2)in the colon cancer cells, respectively.Moreover, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay was used to investigate the effect of estrogen receptor(ER) inhibitor,ERα agonist, and estrogen receptor ERβagonist on the cell proliferation.Results: ERα was faintly expressed in the DLD-1 and HCT-15 cells.However, ERβ expression in DLD1, HCT15, COLO205, LOVO, and SW480 colon cancer cells.Different concentrations of equol (0, 0.5, 1, 5, 10 μmol/L) significantly inhibited the growth of HCT-15 cell with the expression of ERα and ERβ.More-over, different concentrations of equol (0, 0.5, 1, 5, 10 μmol/L) significantly inhibited the growth of LOVO, and SW480 cells with the ERβ expression in a dose-dependent manner as demonstrated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay.mRNA expressions of ERα and ERβ in HCT-15 were stimulated significantly.Western blotting proved that the protein expressions of ERα and ERβ increased with the increasing of equol dose.Moreover we found significant difference of Nrf2 protein expression in HCT-15 cell stimulated by different concentrationss of equol.After the similation of estrogen receptor inhibitor, ERα agonist, or ERβ agonist, we found that only dif-ferent concentrations of ERβ agonist(0, 1, 10, 100, 1 000, 10 000 nmol/L) significantly inhibited the growth of HCT-15, LOVO, and SW480 in adose-dependent manner.Estrogen receptor inhibitor and ERα agonistdid not present significant effect on the cell proliferation of HCT-15, LOVO, and SW480.Conclusion: Equol inhibited the colon cancer cell proliferation by its estrogenic activities and antioxidant activities.