Hesperetin Inhibits The Invasion And Metastasis Of Cervical Cancer Cells Through TGF-β1/Smad Signaling Pathway Modulated The Epithelial-to-Mesenchymal Transition

Objective This study we established an Epithelial-to-Mesenchymal Transition(EMT)model in vitro by TGF-β1 stimulation of CaSki and HeLa 299 cells,which in order to investigate the effect of hesperetin(HES)on the invasion and metastasis of cervical cancer cells modulated the EMT,and then clarify the molecular mechanism.Methods 1.Investigate the effects of hesperetin and TGF-β1 on the invasion and metastasis in cervical cancer cells.First,we established an EMT model in vitro by TGF-β1 stimulation of CaSki and HeLa299 cells,then cells divided into four groups randomly,Control group,10ng/mL TGF-β1group,10ng/mL TGF-β1+HES10?M/L group,10ng/mL TGF-β1+HES20?M/L group.(1)MTT assay was used to measure cell viability.A.Cervical cancer cells treated with hesperetin in different concentrations after 24,48 and 72h;B.Cervical cancer cells treated with TGF-β1and hesperetin after 24,48 and 72h;(2)Cell morphological assessment was used to observe cell morphology changes.A.Cervical cancer cells treated with 10ng/mL TGF-β1 after 0,24,48,72 and 96h;B.Cervical cancer cells treated with TGF-β1 and hesperetin after different time;(3)Wound healing and transwell migration assay were used to investigate the effect of hesperetin and TGF-β1 on the migration ability of CaSki and HeLa 299 cells;(4)Transwell invasion assay was used to detect the effect of hesperetin and TGF-β1 on the invasion ability of CaSki and HeLa 299 cells.2.Explore the molecular mechanism of inhibitory effect of hesperitin on invasion andmetastasis in cervical cancer cells.First,we established an EMT model in vitro by TGF-β1 stimulation of CaSki and HeLa299 cells,then cells divided into four groups randomly,Control group,10ng/mL TGF-β1group,10ng/mL TGF-β1+HES10?M/L group,10ng/mL TGF-β1+HES20?M/L group.(1)Fluorescence intensity of E-cadherin and N-cadherin in the cytoplasm of cervical cancer cells was detected by immunofluorescence;(2)Western blot was used to detect the expression levels of E-cadherin,N-cadherin,Snail,Twist2,ZEB,Laminin R,Fibronectin 1,MMP2,MMP9,Galectin 1,Pard 6A,Pard 3,?PKC,TGF-β Receptor II,TGF-β Receptor I,P-TGF-βReceptor I,P-Smad2/3,Smad4 proteins.Results 1.(1)A.Compared with the control group,the viability of CaSki and HeLa 299 cells decrease in a dose-dependent manner after hesperetin treated 24,48 and 72h(P<0.05);B.TGF-β1 can promote the proliferation of CaSki and HeLa 299 cells with time-dependently(P<0.05).However,the viability of cells was decreased after treated with hesperetin(P<0.01).(2)A.10ng/mL TGF-β1 was applied to CaSki and HeLa 299 cells in different time,the morphology of cells changed from the phenotype of epithelial cells to mesenchymal cells;B.TGF-β1 can promote the morphology of CaSki and HeLa 299 cells into the phenotype of mesenchymal cells,however,after treated with hesperetin the phenotype was reversed.(3)TGF-β1 could enhance the migration of CaSki and HeLa 299 cells(P<0.05).A.In wound-healing assay,the healing ability of CaSki and HeLa 299 was dramatically decreased by hesperetin co-treatment cells with the concentration dependent(P<0.01,P<0.05);B.In transwell migration assay,the migration activity of CaSki and HeLa 299 was dramatically decreased by hesperetin co-treatment cells with the concentration dependent(P<0.05,P<0.01).(4)TGF-β1 could promoted the invasion ability of CaSki and HeLa 299 cells(P<0.05),the increased number of migrated cells in transwells assay,which was dramatically decreased by hesperetin co-treatment(P<0.001,P<0.001).2.(1)Compared with the control group,the fluorescence intensity of E-cadherin in thecervical cancer cells membrane decreased,but the fluorescence intensity of N-cadherin increased obviously at 10ng/mL TGF-β1 group.But when hesperetin was added,the expression of E-cadherin and N-cadherin approached gradually to the control group.(2)A.Compared with the control group,at 10ng/mL TGF-β1 group,the protein expression of E-cadherin in the cervical cancer cells decreased(P<0.05),but the expression of N-cadherin increased(P<0.05).However,the expression reversed by hesperetin co-treatment with the concentration dependently(P<0.05,P<0.05).B.Compared with the control group,the protein expression of Snail,Twist2,ZEB in the cervical cancer cells increased obviously at 10ng/mL TGF-β1 group(P<0.05).However,hesperetin co-treatment cells could decreased the expression of them in various degrees(P<0.01).C.Compared with the control group,the protein expression of Laminin R、Fibronectin1、Galectin 1、MMP2、MMP9 in the cervical cancer cells increased obviously at 10ng/mL TGF-β1 group(P<0.05).However,hesperetin co-treatment cells could decreased the expression of them invarious degrees(P<0.01).D.Compared with the control group,the protein expression of Pard 3 in CaSki cells increased at10ng/mL TGF-β1 group(P<0.05),20?M/L hesperetin co-treatment cells could decreased the expression(P<0.05);the protein expression of Pard 6A,Pard 3,? PKC in HeLa 299 cells same at 10ng/mL TGF-β1 group.E.Compared with the control group,the protein expression of TGF-β Receptor II and P-TGF-β Receptor I in the cervical cancer cells increased obviously at 10ng/mL TGF-β1 group(P<0.05);the protein expression of TGF-β Receptor I,P-Smad2/3and Smad4 decreased(P<0.05).However,hesperetin co-treatment cells could reversed the expression of them in various degrees(P<0.01).Conclusion 1.Hesperetin can modulate TGF-β1-induced EMT in cervical cancer cells and inhibit cells proliferation,migration and invasion.2.Hesperetin inhibits the invasion and metastasis of cervical cancer cells through TGF-β1/Smad signaling pathway modulated the EMT.