Study On The Correlation Between ROS And Apoptosis Of MCF-7 Cells Induced By Formononetin

Formononetin(FMN)is a herbal isoflavone derined from the roots of herbs such as Trifolium pratense,Astragalus membranaceus,Black cohosh.It reported that formononetin possess mlti pharmacological effects including anti-inflammation,anti-tumor,cardioprotective activies,vasorelaxant,antiviral,neuroprotective activies.it has anti-tumor research potential especially.However,research on the correlation between ROS and apoptosis of tumor cells induced by formononetin has not been reported.Therefore,this paper take human breast cancer MCF-7 cells cultured in vitro to discuss the mechanism to provide theoretical basis for the research and development of anti-tumor drugs.Method:(1)The antiproliferative effect of formononetin on MCF-7 cells was tested by the MTT assay.(2)Annexin V-FITC/propidium iodide(PI)double staining was used to detect the cell apoptosis rates.(3)The reactive oxygen species(ROS)in MCF-7 cells was detected by FCM.(4)Rhodamine 123 probe staining was used to detect the effect of formononetin and the combination of formononetin and ROS inhibitor NAC on mitochondrial membrane potential in MCF-7 cells.(5)Western blot was used to analyze the effects of formononetin and the combination of formononetin and NAC on the expression of bcl-2,Bax,caspase-3,P-JNK and JNK.(6)Spectrophotometry was used to measure activities of catalase(CAT),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in MCF-7 cells.Results:(1)formononetin could obviously inhibit the proliferation of MCF-7 cells.The proliferation inhibition rate increased with the increased formononetin dose.,IC50 for 48 h was 79.1?M.the concentrations of formononetin in the low medium and high dose group were 39.5?M,79.0?M and 158.0?M.2.4?M epirubicin was set as the positive control group.final concentration of 10mmol/L NAC combined with 79.0 ?M formononetin was set as the inhibitory group,compared with the medium dose group.(2)The apoptosis rates were:blank control group was(2.78±0.62)%,positive control group was(34.85±3.89)%.low dose group was(5.43=0.32)%.the medium dose group was(17.46 ± 1.69)%,the high dose group was(39.55±4.31)%,and the inhibitor group was(4.20 ±0.87)%.The apoptosis rates increased with the increased formonetin concentration,which were significantly different from the blank control group,and the apoptosis rate of the inhibitor group was significantly lower than the medium dose group.It can be speculated that the apoptosis of MCF-7 cells linduced by formononetin is related to the increased ROS level.(3)ROS levels were increased with the increased formononetin concentration.The ROS level of the inhibitor group was significantly lower than that in the medium dose group.It indicated that formononetin could increase the ROS level in MCF-7 cells.(4)The mitochondrial membrane potential(MMP)decreased gradually with the increased formononetin dose,which was significantly different from the control group(P<0.01).MMP of the inhibitor group was significantly higher than that in the medium dose group.It indicated that formononetin could decrease the mitochondrial membrane potential of MCF-7 cells.(5)Western blot results showd that with the increase of formononetin dose,the expression of Bcl-2 protein was increased and the expression of Bax,cleaved caspase-3,P-JNK protein was decreased.The expression ofbcl-2 protein in the inhibitor group was significantly higher than that in the medium dose group,and the expression of Bax,cleaved caspase-3,P-JNK was significantly lower than that in the medium dose group.It indicated that formononetin could down-regulate Bcl-2 protein and up-regulate Bax,Cleaved caspase-3,P-JNK protein expression,which was related to the increase of ROS level(6)T-SOD,GSH-Px and CAT activity in MCF-7 cells decreased with the increased dose of formononetin,suggesting that formononetin can inhibit the activity of antioxidant enzymes in MCF-7 cells.Conclusion:formononetin induced MCF-7 cell apoptosis,which is associated with increased ROS.By inhibiting the activity of antioxidant enzymes and elevating ROS level of MCF-7 cells,.JNK signaling pathway was activated,then the expression of Bax protein was up-regulated and the expression of Bcl-2 protein was down-regulated,which resulted in decline in mitochondrial membrane potential.afterwards,caspase-3 was activated to execute apoptosis.