The Antitumor Effects And Its Mechanism Of Baicalein On Melanoma

Objective:To explore the anticancer effects and mechanisms of baicalein(BAI)on melanoma cells A375 and B16,Methods:Human melanoma A375 and murine melanoma B16 cells were treated with different concentrations of BAI,MTT assay was used to detected cell proliferation;The plate colony formation assay was used to detect cell colony formation ability;The cell migration was detected by wound healing and transwell migration assay;Hoechst33342 staining were used to examine the formation of apoptotic bodies;Annexin V-FITC/PI double staining flow cytometry was used to test cell apoptosis;Immunofluorescence assay was used to detect the expression of autophagosome-labeled protein LC3;The expression level of apoptosis associated proteins,autophagosome marker protein LC3,EMT markers,key members of Akt and Erkl/2 signaling pathway-associated proteins were tested by Western blot.Results:1.MTT assay showed that:Compared with the control group,A375 and B16 cells were treated with 0,5,10,25 and 50?mol/L BAI for 24h,48h and 72h,the proliferation of A3 75 and B16 cells were inhibited in a time-and dose-dependent manner(P<0.05 or P<0.01);2.Colony formation assay showed that A375 and B16 cells were treated with 0,5,10,25?mmol/L BAI for two weeks,compared with the control group,the colony forming ability of A375 and B16 cells in the BAI treatment group was supressed in a dose-dependent manner(P<0.05 orP<0.01);3.The results of the Wound healing test indicated that 0,5,10,25?mol/L BAI treatment of melanoma A375 and B16 cells for 24h,48h,72h,compared with control group,the horizontal migration ability of A3 75 and B16 cells was supressed in a dose-dependent manner(P<0.01);4.Transwell migration assay demonstrated that the longitudinal migration ability of A375 and B16 cells was supressed compared with the control group after treatment of A375 and B16 cells with BAI at 0,5,10,25?mol/L for 48h(P<0.06 or P<0.01);5.Hoechst33342 staining results also showed:after treatment of A375 and B16 cells with BAI at 0,5,10,25?mol/L for 48h,the number of apoptotic bodies was up-regulated and concentration-dependent(P<0.01);6.The results of flow cytometry demonstrated that after treatment with 25?mol/L BAI in A375 and B16 cells,the apoptosis rate was higher than the control group(P<0.01);7.Cellular immunofluorescence staining results showed that melanoma cells A3 75 and B16 treated with 25?mol/L BAI for 48h,the expression of LC3 was up-regulated;8.The results of Western blot demonstrated that after treatment of A3 75 and B16 cells with BAI at 0,5,10,25?mol/L for 48h,the expression of apoptosis-related proteins Cleaved caspase 9,Cleaved caspase 3,and Bax were up-regulated and Bcl-2 was down-regulated in the BAI-treated group compared with the control group(P<0.05 or P<0.01);the expression of autophagosome-labeled protein LC3-II was up-regulated(P<0.01);the expression of EMT marker protein E-Cadherin was up-regulation,Vimentin,Snail,MMP9 and MMP2 were down-regulation(P<0.05 or P<0.01);The expression of Akt singling pathway associated proteins,p-Akt and p-4EBP1 were inhibited.Key members of Erkl/2 signaling pathway,p-Erkl/2 were downregulated(P<0.05 or P<0.01).No significant changes in all the total proteins.Conclusion:1.BAI can suppress the cell proliferation,induce apoptosis,autophagy and inhibit the occurrence of EMT of melanoma A375 and B16 cells.2.BAI inhibits melanoma is associated with regulation apoptosis-related proteins,LC3 protein and downregulation of Akt and Erkl/2 signaling pathway protein levels.