Study About The Mechanisms Of Apple Polyphenols Regulating The Proliferation And Migration Of Breast Cancer

Background:Breast cancer is one of the multiple tumors for women.At present,the incidence and related mortality of breast cancer are increasing worldwide.The spread of tumor cells and distant metastasis are the main causes of death for breast cancer patients.Therefore,control the metastasis of tumors has important implications for the improvement of the prognosis of breast cancer.The side-effect of chemotherapy drugs and the emergence of drug resistance severely limited their clinical application.Previous studies had shown that the individual component of apple polyphenols(AP),such as quercetin,epicatechin,anthocyanins,etc exhibited the inhibitory effects on breast cancer metastasis,but there had few studies exploring the relationship between AP and breast cancer metastasis,and the underlying mechanisms that AP regulating metastis of breast cancer also needs further investigatedObjective:To investigate the effects of AP on the proliferation and migration of highly invasive breast cancer cells MDA-MB-231 in vivo and in vitro,and the underlying mechanisms were also explored.Methods:1.Cell experimentMDA-MB-231 cells were cultured,and treated with 0,50,100,200,400,and 800 ?g/ml AP for 24,48,and 72 hours,respectively.Then,the viability of the cells was determined by trypan blue staining;the growth and proliferation of the cells were determined using the CCK8 kit;the migration of the cells was observed by the scratch experiment;and the change of the proteins and m RNA expression were determined by Western blot and q TR-PCR.UHRF1 was transfected into breast cancer cell line MDA-MB-231 by liposome transfection,and the stable transfected cell line MDA-MB-231/UHRF1 was screened by G418.The cells were treated with 0,50,100,200,400,and 800 ?g/ml of AP for 72 h.Trypan blue staining experiments,CCK8 experiments,scratch experiments,Western blot and q TR-PCR were performed to explore the role of UHRF1 in the regulation of breast cancer cell proliferation and migration mediated by AP2.Animal experimentBreast cancer model was prepared by MDA-MB-231 cells inoculation with 4 weeks old nude mice.Each animal was inoculated with 6×10 6 cells.As the tumors grew to 50 mm 3,all animals were randomly divided into control group(C),apple polyphenol low-dose group(L)(50 mg/100 g bw/d AP)and high-dose group(H)(200 mg/100 g bw/d AP).AP was solved in water,and mice in intervention groups were gavaged per day with AP,and control group was given the same volume of sterile water.Body weight and tumor volume was recorded biweekly.8 weeks later,the change of body weight and tumor volume,the expression of UHRF1 and CD34,the angiogenesis index,in tumor tissues were analyzed by immunohistochemistry.Results:1.Effect of AP on proliferation and migration of breast cancer cell MDA-MB-231 in vitro and the underlying mechanisms were exploredResults of Trypan blue experiment showed that 0~800?g/ml AP dose-dependently inhibited cell viability,while the inhibitory effect varied slightly for different treatment time with the same dose of AP treatment.Higher than 100 ?g/ml AP significantly inhibited the proliferation of cells based on the data of CCK8,and the inhibitory effects of AP was dose-and time-dependent.Results of scratch experiment showed the inhibitory effects of AP on the migration of breast cancer cells,especially the higher than 200?g/ml AP showed significant inhibitition of cell migration.Western blot results showed that AP dose-depenently inhibited the expression of tumor metastasis protein MMP2 mediated by downregulating the protein levels of ARK5,CX43,YB-1,GAB2 and GALNT14.The downregulated expression of UHRF1 and downstream targets DNMT3 A and DNMT3 B were also observed.Meanwhile,AP could negatively downregulate the m RNA levels of UHRF1,MMP2,DNMT3 A,DNMT3B,ARK5,CX43,YB-1,GAB2,and GALNT14 according to the qRT-PCR results.2.Effects of AP on growth and angiogenesis of tumor in nude mouse model of breast cancerDuring the treatment of AP,the body weight of mice in both the control group and the low-dose group continued to increase,which gradually decreased in the high-dose group,and the body weight in high-dose group was significantly lower than that in the control group and low-dose group.The tumor volume of mice in each group increased during the intervention,and the tumor volume increased by 666% in control group,676% in the low-dose group and 728% in high-dose group.At the end of the experiment,mice in the high-dose group had the smallest tumor volume,but the difference was insignificant.After the intervention of AP,the expression of CD34 in the tumor tissue of the high-dose group was significantly decreased,and the difference was statistically significant compared with the control group,but the difference between the high-dose and low-dose group was insignificant.Meanwhile,the difference in the UHRF1 expression in tumor tissues was insignificant among three groups.3.Effects and mechanisms of AP on the proliferation and migration of UHRF1-transfected breast cancer cells MDA-MB-231/UHRF1 in vitroTrypan blue test results showed the insignificant difference in the inhibitory rate of AP on MDA-MB-231,MDA-MB-231/pc and MDA-MB-231/UHRF1 cells as the dose of AP was lowere than 100 ?g/ml,while the higher dose of AP treatment showed significantly lower inhibition of cell viability of MDA-MB-231/UHRF1 cells.The results of CCK8 showed that the inhibitory effect of AP on cells growth and proliferation was positively with the dose of AP,no matter for MDA-MB-231,MDA-MB-231/pc or MDA-MB-231/UHRF1 cells,although the inhibitory effects of AP on the proliferation of breast cancer cells MDA-MB-231/UHRF1 was the lowest.The scratch experiment results showed that 200?g/ml AP could inhibit the migration of MDA-MB-231 and MDA-MB-231/PC cells but not MDA-MB-231/UHRF1 cells,while higher dose of AP(?400?g/ml)can effectively inhibit the migration of MDA-MB-231/UHRF1 cells.Western blot results displayed that AP can dose-dependently inhibit the expression of YB-1,GAB2,CX43,then downregulated the level of MMP2.The level of UHRF1 and its downstream target molecule DNMT3 B also decreased after AP treatmentment.qRT-PCR results demonstrated that the m RNA levels of UHRF1,DNMT3 B,MMP2,YB-1,GAB2,and CX43 decreased as the concentration of apple polyphenols increased.Conclusions:1.Apple polyphenols dose-dependently inhibited the proliferation and migration of breast cancer cells MDA-MB-231 by downregulating the protein and m RNA levels of tumor metastasis related genes(UHRF1,DNMT3 a,DNMT3b,MMP2,ARK5?CX43?YB-1?GAB2?GALNT14)in vitro.2.Apple polyphenols inhibited tumor growth and metastasis in nude mouse models of breast cancer by downregulating the expression levels of UHRF1 and CD34 in tumor tissues based on the data of immunohisto chemistry.