Research On Estrogenic Activities Of Quercetin And Psoralen In Human Breast Cancer Cells

BackgroundEstrogens are a class of steroid hormones that play a critical role in the development of the normal breast and in the genesis of breast cancer.Estrogens mediate their cellular responses through two distinct intracellular receptors,estrogen receptor-alpha(ERα)and estrogen receptor-beta(ERβ),which are encoded by separate genes.With the decline of ovarian function and the falling down of the internal level of estrogen in postmenopausal women,the incidence of osteoporosis,cardiovascular disease and dementia rise up.Such menopausal symptoms can be alleviated by estrogen replacement therapy(HRT).But on the other hand, estrogen also contributes to the development of hypertension,oedema and some estrogendependent cancers such as breast cancer and endometrial cancer.Therefore,a substitute of estrogen was gone in for seeking,which possess most positive functions of estrogen with less negative effects.The substitute are called selective estrogen receptor modulators (SERMs),which has two-ways regulation effects.The estrogenic or antiestrogenic effects of SERMs depend on the original level of internal estrogen.Phytoestrogens are a group of selective estrogen receptor modulators,which are active plant substances with a similar chemical structure to estradiol.Many kinds of Chinese medicine have been widely applied in female health protection and treatment of gynecological diseases.Phytoestrogens in these Chinese medicine account for their pharmacological effects.Our topic is based on the research that the phytoestrogenic effects of several active components in chinese medicine by reportor gene technique,which had shown that fructus psoraleae and semen cuscutae exerted estrogenic activity.In this research,we investigated the estrogenic effects and molecular biologic mechanism of psoralen and quercetin,which were active compounts in fructus psoraleae and semen cuscutae.The study purpose is seeking for the monomer compounds that have estrogenic activity from chinese medicine of estrogenic effects,which have be screened in our departed studies.MethodIn this research,two kinds of human breast cancer cells were selected as model,including ER-positive MCF-7 and T47D cells and ER-negative MDA-MB-231 cells.17β-estradiol(E₂)and phytoestrogen genistein as reference.To observe and evaluate the phytoestrogenic effects and their mechanism at the protein and the gene level of quercetin and psoralen,techniques of flow cytometry, reverse transcription polymerase chain reactions(RT-PCR),protein immunoblotting and gene chip were applied.Result1 Experimental study on the effect of quercetin and psoralen on human breast cancer cells proliferation.1.1 Effect of quercetin and psoralen on breast cancer cells proliferation as measured by MTT assay.1.1.1 Effect of quercetin and psoralen on ER-positive breast cancer cellsTwo ER-positive cell lines(MCF-7 and T47D)were exposed to genistein,quercetin and psoralen at concentrations ranging from 1 to 50μM for 24h,48h,72h,respectively.Cells were also treated with 10^-3μM E₂ to confirm their known responses to estrogenic stimulation under our experimental conditions(controls).Genistein,at concentrations ranging from 1 to 50μM for 24h,48h,72h,enhanced the proliferation of MCF-7 and T47D.Quercetin,at concentrations ranging from 10 to 50μM for 24h,at concentrations of 1μM for 48h,72h,enhanced the proliferation of MCF-7.At concentrations ranging from 10 to 40μM for 24h, 48h,72h,enhanced the proliferation of T47D.Psoralen,at concentrations ranging from 1 to 40μM for 24h,48h,72h,enhanced the proliferation of MCF-7.At concentrations ranging from 10 to 40μM for 24h,48h,72h,enhanced the proliferation of T47D.To further investigate whether the proliferation effect was required the presence of ER,genistein, quercetin and psoralen at concentrations ranging from 1 to 50μM with pure estrogen receptor antagonist ICI182,780 were treated MCF-7 cells.Genistein as well as quercetin,psoralen had no effect on the growth of these cells at doses influencing MCF-7 cells.1.1.2 Effect of quercetin and psoralen on ER-negative breast cancer cells Genistein,quercetin and psoralen showed no significant effect on the proliferation of ER-negative cell lines MDA-MB-231 in the concentration range tested.1.2 Effect of quercetin and psoralen on the cell cycle of MCF-7,T47D as measured by flow cytometerMCF-7,T-47D cells were treated with 10μM Genistein,10μM quercetin and 10μM psoralen for 48h respectively.The cells were impulsed from G1 to S phase,proliferation indexes were increased,DNA synthesis were also inhanced.The above function on boosting proliferation could be inhibited by adding estrogen receptor antagonism ICI182,780.2 Effects of quercetin and psoralen on the levels of ERαand ERβ 2.1 Effects of quercetin and psoralen on ERα,ERβtranscript levels in T47D cellsT-47D cells were treated with 10μM Genistein,10μM quercetin and 10μM psoralen for 48h respectively.Total RNA was isolated and the level of ERαand ERβtranscripts were examined by RT-PCR analysis.Compared to the control group,genistein,quercetin and psoralen up-regulated expression of ERαtranscripts,without any effect on ERβmRNA levels.2.2 Effects of quercetin and psoralen on ERα,ERβprotein levels in MCF-7,T47D cellsMCF-7 and T47D breast cancer cells were treated with 10μM Genistein,10μM quercetin and 10μM psoralen for 48h respectively.Total cell extracts examined for the levels of ERαand ERβprotein by western blot analysis.The results with MCF-7 and T47D cells show that they up-regulated ERαprotein levels without altering ERβlevels.The above up-regulation on ERαprotein could be inhibited by adding estrogen receptor antagonism ICI182,780.3 Effects of genistein and psoralen on gene expression in T47D breast cancer cells using an oligo microarrayT47D breast cancer cells were treated with 10^-3μM E₂,10μM genistein,10μM psoralen for 48h respectively.Oligo GEArray human breast cancer and estrogen receptor signaling microarray profiles are used for the characterization of Genistein,psoralen to examine genetic responses,to predict effects.Cell cycle-related protein genes such as CCNE1(Cyclin E1),CCNE2(Cyclin E2), CCNA2(Cyclin A2)were found to be significantly up-regulated by genistein,psoralen as compared to vehicle-treated control cells.ESR1(ERα)gene and classic estrogen-responsive genes such as TFF1(also known as pS2),PGR,BCL2,ERBB2,C3,CCND1,TEF,IGFBP2,IGFBP5,GATA3 were up-regulated induced at 10μM genistein or 10μM psoralen.Conclusion1 Quercetin and psoralen at concentrations ranging from 10 to 50μM stimulated proliferation of ER-positive cells MCF-7 and T47D,and proliferation effects of quercetin and psoralen were antagonized by pure estrogen receptor antagonist ICI182,780.In contrast,quercetin and psoralen showed no significant effect on the proliferation of ER-negative cell lines MDA-MB-231 in the concentration range tested.The results indicate that quercetin and psoralen have estrogenic activity that should be ER-dependent.2 Genistein,quercetin and psoralen up-regulated expression of ERαtranscripts,without any effects on ERβmRNA levels,and up-regulated ERαprotein levels without altering ERβlevels.The results indicate that the y may exert estrogenic activity to ER-positive cells proliferation through inter action with ERαexpression. 3 Smilar to genistein,psoralen has estrogenic effect,up-regulation of ERαgene and classic estrogen-responsive genes,which indicate that psoralen belong to the phytoestrogen.Promoting cells proliferation was induced by over expression of cell cycle protein that regulated CDK to accelerate S phase.