The Inhibition Effect And Mechanism Of Ursodeoxycholic Acid On Human Melanoma M14 Cells

Melanoma is one of the malignant tumor produced by skin and other sources of melanocytes.Ursodeoxycholic acid is one of the hydrophilic bile acids produced by intestinal bacteria,which has long been recognized as tumor suppressors of the liver and colon.However,it is unclear whether UDCA could inhibit human melanoma growth.In this study,MTT assay,AO/EB,Hoechst staining cell morphology observation,scratch assay,colony formation assay,flow cytometry,electron transmission microscopy and western blot were used to study the inhibitory effect of UDCA on melanocytes and its mechanism.Results showed that UDCA could significantly reduce the proliferation of melanoma cells(A375 and M14),but the inhibitory effect was not obvious to L02 and HaCaT in the same range of concentrations.In addition,UDCA could reduce the colony formation ability and migration ability of melanoma cells and cause the cells to exhibit apoptotic characteristic morphological phenomena such as cell rounding and nucleus chromatin condensation.FCM was used to detect the state of M14 cells treated with UDCA for 48 h further.After 48 h of UDCA induction,results showed that with the concentration of UDCA increasing,the number of M14 cells in G2/M phase increased significantly,the apoptosis rate increased significantly,the intracellular reactive oxygen species significantly increased,and the mitochondrial membrane potential significantly decreased.The mitochondrial morphology of M14 cells was observed by transmission electron microscopy.The results showed that as the concentration of UDCA increased,the mitochondria gradually became vacuoles.The results of western blot analysis demonstrated that under the effect of UDCA,the expression level of the oncogene Bax protein did not change significantly,the expression level of the tumor suppressor gene Bcl-2 protein was down-regulated,and the expression of cyclin-related proteins such as p53,p21,CDK1 and Cylin B1 was decreased.The expression levels of mitochondrial apoptosis pathway-related proteins such as cyt c,Apaf-1,cleaved-Caspase-3,cleaved-Caspase-9,and cleaved-PARP1 were up-regulated.Therefore,we hypothesized that UDCA could induce apoptosis of melanoma cell line M14 via a Caspase-dependent mitochondrial apoptotic pathway.UDCA could block the cell cycle in G2/M phase by affecting the expression of p21,p53,CDK1 and Cylin B1;By affecting the ratio of Bax/Bcl-2,the mitochondrial membrane potential decreases,membrane permeability increases,and Cyt c was released to the cytoplasm,which binds to cytosolic Apaf-1.The cascade between the factors amplifies the apoptotic effect,and the downstream apoptosis performer Caspase was activated,which causes apoptosis of M14 cells.This result provides a theoretical basis for future research on UDCA as a potential anti-melanoma drug.