| ObjectiveEpidemiological investigation showed that breast cancer is the most common cancer in Europe and north America women;at the same time,the incidence of breast cancer is rising year by year in Asia,especially in China and India.So the new efficient therapy for breast should be focused.Promisingly,tumor necrosis factor-related apoptosis-inducing ligand(TRAIL),a member of the tumor necrosis factor super family,cause extensive attention due to its remarkable ability of inducing apoptosis in malignant human cells while leaving normal cells unscathed.However,it was recently reported that more than 50%tumor cells including MCF-7 cells were risistant to TRAIL,One of the probable mechanisms was that tumor cells developed resistance to the TRAIL-induced apoptosis.Fortunately,soybean isoflavones(SI)could exert not able anti-tumor effects by inducing tumor cells apoptosis.So in the present study,the effects and its potential mechanism of genistein combining with tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)on the proliferation and apoptosis in breast cancer cell line MCF-7 were explored.MethodsFirstly,MCF-7 cells were treated with 1.25,2.5,5,10,20,40μg/ml genistein and 1,10,100,1000ng/ml TRAIL,respectively.Cell proliferations were detected by MTT assay.After the caculation of coefficient of drug interaction(CDI)based on above result,20μg/ml genistein and 100ng/ml TRAIL were selected for the following experiments.Secondly,MCF-7 cells were divided into four groups:control,genistein(the final concentration of 20μg/ml),TRAIL(the final concentration of 100ng/ml),and,combination group.And theen,the four group cells were incubated with different treatments for 48h.Finally,MCF-7 cells apoptosis and mitochondria membrane potential were detected by flow cytometry(FCM).Caspase-3,8,9 activities were measured through immunofluorescence.The content of NF-κB was tested by enzyme-linked immunosorbent assay(ELISA).And the expression of Bax and Bcl-2 protein were analysiz by western blotting.Results1.The changes in MCF-7 cell proliferation:The inhibiting rate of cell proliferation was significantly different among each genistein group cells and negative control cells.There was a linear positive correlation between cell inhibiting rate and genistein concentration at range of 2.5μg/ml to 40μg/ml.50%inhibitory concentrations(IC50)of genistein was 20μg/ml.The inhibiting rate slightly rose in TRAIL-treated cells when TRAIL concentration exceeded 100ng/ml,but there was no dose-dependent manner any more.So 20μg/ml Gen and 100ng/ml TRAIL were identifid for the following tests.2.Morphological changes of MCF-7 cells:The control group cells grew in good condition;Most of TRAIL group cells grew well but some vacuoles were observed in a few cytoplasm;in Gen group,adherent cells reduced rapidly and shrinked,and a number of cells floated in the medium;few combination group cells attached on the wall,which losing their original shape,full of vacuoles in cytoplasm.Medium was filled with floating cells.3.Results of the apoptosis rate:Apoptosis appeared in all treated MCF-7 cells.The apoptotic rate of TRAIL-treated alone cells was 12.61±1.53%,which increased rapidly to 42.20±1.35%after combinating genistein treatment.4.The changes of △Ψm in MCF-7 cells:△Ψm of combination group cells(68.35±13.73)was lower significantly comparing with that of TRAIL group cells(826.38±7.47).5.The activity of caspase-3,8,9:The levels of caspase-3,8,9 in combination group cells(44.000±0.445,13.76±0.38 and 24.64±0.1 μmol/L/hr/mgprotein)were significant higher than that in TRAIL group cells(17.32±0.88,10.14±0.20 and 9.26±0.04 μmol/L/hr/mg protein.)6.The expression of NF-κB in MCF-7 cell:The expression of NF-κB in TRAIL group was 343.333±8.064 pg/ml.After combination with Gen treatment,the activity of NF-κB was inhibited intensively(177.453±25.389pg/ml).7.The levels of Bax and Bcl-2 protein in MCF-7 cells:The expression of Bcl-2,Bax proteins and the ratio of Bax/Bcl-2 in TRAIL-treated cells were 0.90±0.06,0.96±0.08 and 1.06±0.09,seperately.After combining Gen use,down-regulating of Bcl-2(0.63±0.06),up-regulating of Bax(1.18±0.03)were observed in cells.ConclusionGen could inhibit MCF-7 cells proliferation in the dose-dependent manner at and above the concentration of 2.5μg/ml.While.TRAIL alone slightly inhibited the growth of breast cancer cells only in case that the concentration was more than 100ng/ml.Furthurmore,unlike the inhibiting effects of Gen,no dose-dependent maner was found in TRAIL-treated cells.It was suggested that MCF-7 cells were not sensitive to TRAIL.To evalute the combination effects of Gen and TRAIL,CDI value was caculated.Highest inhibition rate and lowest CDI value were observed in cells treated with 20μg/ml Gen and 100ng/ml TRAIL,implying that this kind of combination could obviously inhibite cells proliferation.Gen could synergize TRAIL to inhibite cell proliferation and induce apoptosis,thus enhancing sensitive of MCF-7 cells to TRAIL.The mechanism might be that genistein has a synergic effect with TRAIL on magnifying mitochondria-mediated apoptosis signal,activating caspase-3,8,9,inhibiting the formation of NF-κB,up-regulating Bax expression and down-regulating Bcl-2 protein expression,all of which resulting promotion of apoptosis in MCF-7 cells. |
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