The Molecular Mechanism Of Icariin On Cell Differentiation And Cell Cycle Arrest In Mouse Melanoma B16 Cells

Objective:The study aimed to investigate the effects of Icariin(ICA)on cell cycle and cell differentiation in mouse melanoma B16 cells and further explore the related mechanism.Methods:B16 cell proliferation inhibition rate was detected by MTT assay;the morphologic changes of B16 cells treated with different concentrations of ICA were observed under microscope;the cell colony formation rate was measured by plate colony formation assay;capability of icariin-treated B16 cell’s migration was detected by scratch wound assay and transwell migration assay;the effect of ICA on the content of melanin and tyrosinase activity in B16 cells was detected by spectrophotometry;the effect on cell cycle of icariin-treated B16 cells was examined by flow cytometry;the mRNA levels of Tyr,Trp 1 and Trp 2 were determined by PT-qPCR;the protein levels of differentiation-/cell cycle-related molecules(MITF,Cyclin A,CDK2 and p21)and MAPK signaling molecules(Erkl/2,p-Erkl/2,p38,p-p38,JNK and p-JNK)were determined by western blot.Results:ICA inhibited B16 cells proliferation in a concentration-and time-dependent manner;with the increase of drug concentration,the cells gradually increased into spindle shape,presented dendritic network structure and reduced,compared with the control group,ICA inhibited the migration of B16 cells in a concentration-dependent manner.Moreover,the melanin content was increased and the tyrosinase(Tyr)activity was enhanced in a concentration dependent manner after ICA treatment in B16 cells;furthermore,the mRNA levels of Tyr,Trp 1 and Trp 2 and the protein level of MITF were increased.In addition,according to flow cytometry,after treated with different concentrations of ICA,the percentage of G0/G1 phase cells was increased,and the percentage of G2/M phase and S phase cells was decreased,ICA-treatment led to G0/G1 phase arrest in B16 cells;and the protein levels of Cyclin A,CDK2 and p21 were decreased.And the protein levels of Erkl/2,p-Erkl/2,p38,p-p38 and p-JNK were decreased.Conclusion:The results showed that ICA could inhibit the MAPK pathway related protein Erkl/2,p-Erkl/2,p38,p-p38 and p-JNK expression,increased the protein level of MITF and the mRNA levels of Tyr,Trp 1 and Trp 2,and then increased the melanin content and enhanced the tyrosinase activity.Moreover,ICA could inhibit the expression of cell cycle related proteins Cyclin A,CDK2 and p21,led to G0/G1 phase arrest in B16 cells.So,ICA induces cell differentiation and cell cycle arrest in mouse melanoma B16 cells.