Study On Effects Of Phytoestrogens And Their Mechanisms On MCF-7 Breast Cancer Cell Line

Phytoestrogens are plant-derived compounds with heterocyclic polyphenol structure,which can be mainly divided into isoflavones,coumarins and lignans three categories.Because of their similar structure with 17p-estradiol(E2),they competitively bind to estrogen receptor(ER)with endogenous estrogen.Most phytoestrogens show biphasic effect to estrogen-sensitive cells and play estrogenic or anti-estrogenic biological effect.The role of phytoestrogens in tumor prevention or promotion,as well as their molecular mechanism of action is not yet very clear.In order to investigate the effect of dietary phytoestrogens on breast tumor growth,vitro experiments were used to evaluate cell proliferative and apoptotic effect of three representative phytoestrogens named genistein(Gen),quercetin(Que)and coumestrol(Cou)by using estrogen-sensitive breast cancer cell line MCF-7.After taking factors of normal dietary intake amount and impact of endogenous estrogen into account,our study detected cell viability,cell proliferation rate and cell apoptosis rate of the three phytoestrogens.Besides,we made a deeper understanding on the molecular mechanisms of the phytoestrogens by investigating their estrogenic effect and related signaling pathways.In addition,by using implanted MCF-7 xenograft nude mice model,we studied breast tumor growth induced by phytoestrogens in vivo.It made our study on growth of MCF-7 cells and estrogenic effect of Gen,Que,Cou more comprehensive and systematic.The main contents are as follows:1.Estrogenic effect of Gen,Que,Cou in vitro.Based on the concentration of human daily intake,0.001-1 ?mol/L of Gen,Que,Cou were used to analyze estrogenic effect of phytoestrogens on ER-positive MCF-7 breast cancer cells.Cell viability was observed increase by MTT method.Besides,the results of flow cytometric analysis showed Gen,Que,Cou stimulated DNA synthesis and induced cell proliferation by reducing cell apoptosis rate and promoting more cells from G0/G1 phase to G2/M and S phases.In order to imitate endogenous hormone environment of female,10-10mol/L of E2 was combined with Gen,Que,Cou.The results of joint action showed Gen+E2?Que+E2?Cou+E2 promoted cell proliferation in the same manner as treated with Gen,Que,Cou alone.In addition,by using Western Blot assay,our study found ERa protein expression changed over time and reached its peak at 6 h.Fulvestrant(Ful),an antagonist of ER,inhibited cell proliferation induced by Gen+E2?Que+E2?Cou+E2.In conclusion,under endogenous hormone environment,Gen,Que,Cou could induce cell proliferation and exerted estrogen-like effect through ER signaling pathway.2.Select signaling pathways involved in estrogenic effect induced by Gen,Que,Cou.By using Western Blot assay detecting protein expression of p-Akt,p-ERK1/2,nuclear NF-?B p65 in time-controlled experiments,we found Gen+E2?Que+E2?Cou+E2 activated PI3K/Akt,ERK and NF-?B signal transduction pathways and reached the higest level at 6 h,6 h,24 h,respectively.Besides,LY294002,PD98059 and PDTC,which are inhibitors of PI3K/Akt,ERK and NF-?B pathways,could suppress cell proliferation induced by Gen+E2?Que+E2?Cou+E2.In conclusion,under endogenous hormone environment,PI3K/Akt,ERK and NF-?B were all involved in regulating MCF-7 cell proliferation.The three pathways might correlate with molecular mechanisms of estrogenic effect induced by Gen,Que,Cou.3.The relationship between ER,PI3K/Akt,ERK and NF-?B signal transduction pathways induced by Gen.Gen was a typical one of phytoestrogens.This study has taken it as an example to investigate relationship between ER,PI3K/Akt,ERK and NF-?B signal transduction pathways.By using Western Blot assay,we found the Ful,an ER antagonist,inhibited phosphorylation of Akt,which illustrated that PI3K/Akt might be downstream of ER pathway.Besides,LY294002,the inhibitor of PI3K,reduced phosphorylation of ERK1/2,which illustrated that ERK might be activated after PI3K/Akt pathway.Moreover,PD98059,inhibitor of ERK 1/2,suppressed NF-?B p65 expression in the nucleus,which meant the effect of NF-?B might only be exerted after ERK activation.In conclusion,under endogenous hormone environment,Gen activated ER,PI3K/Akt,ERK,NF-?B pathways one after another.It was a reflection of non-genomic effect mediated by ER,which also demonstrated the four signal pathways were all involved in estrogenic effect induced by phytoestrogens.4.Preliminary study about effect of Gen on implanted MCF-7 xenograft.Gen was a typical one of phytoestrogens.This study has taken it as an example to investigate relationship between phytoestrogens and tumor growth.Through implanting MCF-7 breast cancer cell line into mice,we established xenograft nude mice model.After observed and measured tumor size in the model,which has been intragastric administrated with Gen for 10 days,we found Gen promoted tumor growth in vivo.The results made additional explanations for cell experiments.In conclusion,Gen induced MCF-7 cell proliferation in vivo,which was consistent with our results in vitro.In summary,our study chose an estrogen-sensitive breast cancer cell line MCF-7,and 6-week nude mice as objects to investigate stimulatory effect of Gen,Que and Cou in vitro and in vivo.Furthermore,estrogenic effect and related signaling pathways induced by the three phytoestrogens were explored.The results would provide data base for carcinogenic risk assessment of phytoestrogens and theory guidance for people's diet.