Experimental Study On The Effect Of Tanshinone â…¡A On Human Breast Cancer Cell MDA-MB-231

Objective:Breast cancer is considered as one of the most common malignancy of women.Endocrine therapy is an important part of its comprehensive treatment,however, it isalmost invalid for estrogen receptor negative breast cancer patients. The aim of thisstudy is to investigate the effect of tanshinoneⅡA on proliferation inhibition andapoptosis of breast cancer cells MDA-MB-231in vitro, and to explore itsmechanism,and to provide a theoretical basis for its clinical application in estrogenreceptor negative breast cancer.Methods:MTT assay was used to measure the proliferation inhibitory effect of tanshinoneII A on human breast cancer cells MDA-MB-231and calculate the cell inhibition rate.Excel software was performed to draw the inhibition of cell growth curve andcalculate the semi-effect inhibitory concentration (IC50).Colony-forming unit assays were used to measure the proliferation inhibitoryeffect of tanshinoneⅡA on human breast cancer cells MDA-MB-231. The experim-ental group was treated with different dose of tanshinone II A (0.0625,0.125,0.250,0.500μg/ml), while the control group was not given any treatment. Foursamples were analyzed in each dose. Low magnification microscope was used toobserve clone formation and count each clone numbers.Inverted microscope was used to observe the morphology of cells treated bydifferent dose of tanshinone II A (0.0625,0.125,0.250,0.500,1.00μg/ml).ELISA was used to determine VEGF protein concentration in control group andserum-free culture medium experimental groups treated by different dose oftanshinone II A (0.0625,0.125,0.250,0.500μg/ml).Cell apoptosis of MDA-MB-231cells were analyzed by flow cytom etry(FCM)．The experiment was divided into control group and experimental groups,bydifferent dose of tanshinone II A (0.0625,0.125,0.250and0.500μg/ml). Three samples were analyzed in each experiment. The cells both of the experimental groupsand the control group were collected according to the reagent kit to fixate and stain.Cell apoptosis and cell cycle were analyzed by FCM.Diphenylamine Reaction method was used to determine the percent of DNAfragmentation of MDA-MB-231cells of control group and experimental groups.Results:1,A dose-and time-dependent inhibition of proliferation of MDA-MB-231cellswas observed (P<0.05),in experimental groups treated with tanshinone II A.Semieffect inhibitory concentration (IC50)was0.125μg/ml.2,Colony-forming unit assays suggested that the proliferation inhibitory effectof tanshinone II A on human breast cancer cells MDA-MB-231. The number of cloneformations of MDA-MB-231cell of control group is236±11, while the number ofclone formations of MDA-MB-231cell of experimental groups treated withtanshinone II A at concentrations of0.0625,0.125,0.250,0.500μg/ml were112±7,63±5,32±3,16±3seperatedly. Compared to the control group, the differenceswere statistically significant(P <0.05), and the inhibitory effect was dosedependent.3,MDA-MB-231cells treated by tanshinone II A displayed that cell shrinkedand became smaller,cell gap increased and the number of suspension cells increasedunder inverted microscope.With increasing concentration of tanshinone II A,theshape change was more obviously. And in the same perspective,the cell numberdecrease with the increased concentration of tanshinone II A.4,After treated with different concentrations of tanshinone IIA for48h, theVEGF protein concentration of MDA-MB-231cells in serum-free culture mediumwere significantly reduced.The inhibitory effect was dose-dependent.(P <0.05).5,Flow cytom etric(FCM) detection results showed that MDA-MB-23l cellapoptosis rate of the experimental groups increased gradually with the increasing oftanshinone II A concentration,and apoptosis rate was dose-dependent. Cell cycledistribution changed after the treatment of different concentrations of tanshinone IIAfor48h. Compared with the control group,G0/G1phase cell number of the experi-mental group increased significantly(P <0.05),while the S phase and G2/M phase cells number significantly reduced. Cell growth was arrest in G0/G1phase in a dose–dependent way. Propidium iodide stainied suggested that there was subdiploidapoptotic peak in front of the G1phase peak of MDA-MB-231cells.6,The percent of DNA fragmentation of MDA-MB-231cells treated bydifferent concentrations of tanshinone IIA increased significantly. Compared with thecontrol group the differences were statistically significant(P <0.05), and in adose-dependent manner (P <0.05).Conclusions:1,Tanshinone II A inhibites the proliferation of estrogen receptor negative breastcancer cells MDA-MB-231.2,Tanshinone II A mediates apoptosis on estrogen receptor negative breastcancer cells MDA-MB-231.3,Tanshinone II A inhibits VEGF expression on estrogen receptor negativebreast cancer cells MDA-MB-231.