Repressive Effects Of Resveratrol On Proliferation And Invision In Different Breast Cancer Cells

ObjectiveBreast cancer is one of the highest incidence malignant tumor for women.Thegrowth of normal breast is regulated by the variety of hormone. The mammary glandcells express many kinds of hormone receptors, such as estrogen receptors,progesterone receptor, prolactin receptor and androgen receptor, etc. Estrogen is themain hormone influences the development of breast cancer. When breast cancer cellskeep all or most of the receptors, growth significantly affected by hormonal regulation,this kind is referred to as hormone dependent breast cancer.When breast cancer cellsare with few receptors or receptors are missing, the growth is not affected byhormones, this is called hormone dependent breast cancer. Estrogen Resistance oftenoccurs after hormone treatment of estrogen receptor positive breast cancer. Thisproblem is difficult to solve during the treatment of estrogen receptor positive breastcancer.Resveratrol is a kind of natural polyphenol compounds extracted from plants, hasmany biological functions. The main functions experiments have confirmed areprotection of heart and cerebral vessels, reduction of blood lipid, decrease of femaleosteoporosis, antitumor, etc. In recent years the antitumor functions of Resveratrol getmore and more attention of people.Through the experiment proved that Resveratrolcan inhibit tumor in multiple stages of tumor initiation, promotion and progRession， including tumor cell cycle, proliferation, migration, etc. Chemical structure ofResveratrol is very similar to synthetic estrogen diethylstilbestrol, and it hasestrogen-like effect. Experiments have suggested that in mice of high and lowestrogen levels, Res can play a distinct role of estrogen and anti-estrogen.This research mainly test the inhibition effects of Res on breast cancer cells withdifferent estrogen receptors expression, including proliferation, migration andinvasion in vitro, to explore the mechanism and the possibility of Res used in thetreatment of breast cancer.Methods and Results1. Effect of resveratrol on proliferation in breast cancer cell lines1.1The effects of resveratrol on the proliferation of DU4475、MDA-MB-231、MCF-7cells were analyzed with MTT assayDU4475、MDA-MB-231、MCF-7cells were grown in the presence of Res at a finalconcentration of6.25,12.5,25,50,100and200μM (3wells for each treatment).Cellulargrowth was assessed after24,48, and72hours by MTT assay.The results indicatedthat cell viability decreased with raised concentrations and prolonged treatment ofResveratrol. With raised concentrations and prolonged treatment of Resveratrol，theinhibition rate of DU4475and MDA-MB-231were higher than that of MCF-7.1.2The effects of resveratrol on cell cycle of DU4475、MDA-MB-231、MCF-7wereanalyzed using flow cytometryCell cycle profile was analyzed by flow cytometric analysis by DNA stainingwith PI.Breast cancer cells were treated for48hours with12.5μmol/L,25μmol/L,50μmol/L Res.The cell cycle of cells changed significantly: in contrast to control,Resveratrol caused an accumulation of cells in the S phase of the cell cycle.The ratioof S phase in MCF-7cells at three different concentrations of Resveratrol was lowerthan two ER negative cells.1.3The cell apoptosis of DU4475、MDA-MB-231and MCF-7was analyzed usingflow cytometry with Annexin-V and PI staining DU4475、 MDA-MB-231and MCF-7cells were treated for48hours with12.5μmol/L,25μmol/L,50μmol/L Resveratrol.We used flow cytometry to detect cellapoptosis rate. With raised concentrations of Resveratrol, both cell percentage ofliving cells were reduced, apoptic cells were increased gradually. The highestapoptosis rate in DU4475was39.69%, the highest apoptosis rate in MDA-MB-231was37.41%, that in MCF-7was only21.81%.Treat with50μM Resveratrol resultedin significantly higher apoptosis rate in DU4475、MDA-MB-231cells than MCF-7cell.2. The effects on activation of AKT and caspase-3proteins involved inbreast cancer cell proliferationMDA-MB-231and MCF-7cell were treated with a final concentration of12.5μmol/L,25μmol/L,50μmol/L Resveratrol for48h.We conducted western blot tocheck the proteins p-AKT and caspase-3of MDA-MB-231and MCF-7. The Resultsshowed that the level of phosphorylation of AKT was decreased, while the activationof caspase-3increased, the expression of cleaved-caspase-3increased.3. The effects on migration of MDA-MB-231,MCF-7and invasion ofMDA-MB-231cells3.1Wound healing assey was used to detect the effects of migration onMDA-MB-231and MCF-7After24h treatment of12.5μmol/L,50μmol/L,75μmol/L Resveratrol,the cellsmigration ability were inhibited.Resveratrol stimulated migration of bothMDA-MB-231and MCF-7in a concentration-dependent manner.3.2Transwell assays were used for the impact of Resveratrol on migration ofMDA-MB-231、MCF-7cellsTranswell assays were used to estimate the migration ability of breast caner cellstreated with Resveratrol for48h.The results suggest that Resveratrol could inhibit themigration ability of melanoma cells.With the increase of drug concentration, themigration ability of the MDA-MB-231、MCF-7cells gradually was weakened. 3.3Transwell assays were used for the impact of Resveratrol on invasion ofMDA-MB-231cellsIn upper chambers of transwell we put a matrigel layer, test MDA-MB-231cellsinvasion under25μmol/L Res treatment for48h.Cell number of invasion through thesubstrate and attached to the lower membrane was reduced in experimental groups.3.4RT-PCR detect matrix metalloproteinases-2(MMP-2) and matrixmetalloproteinases-9(MMP-9) expression levelAfter treatment of12.5μmol/L、50μmol/L Resveratrol for48h,RT-PCR is used todetect metalloproteinases-2(MMP-2) and matrix metalloproteinases-9(MMP-9)expression level of MDA-MB-231、MCF-7cells. The results show that, with theincrease of drug concentration, the expression of MMP-2and MMP-9graduallydecreased.ConclusionThis study discussed the related mechanisms of Resveratrol as a kind of anticanceragent inhibited breast cancer cells proliferation, promoted apoptosis and inhibedmetastasis of breast cancer cells.Altogether these experiments shows that Res is ableto significantly block cell proliferation,cell cycle and induce apoptosis in the breastcell lines DU4475, MDA-MB-231and MCF-7in dose dependent manner.ER negativebreast cancer cell lines are more sensitive to Resveratrol in cell proliferated inhibitionthan ER positive breast cancer cell line.In cell migration and invasion, Resveratrolhave obvious effects on both MCF-7and MDA-MB-231cells.