Inhibtory Effect And Mechanism Of Morroniside On Human Malignant Melanoma A375 Cells

Malignant melanoma is one of the most malignant tumors in human skin tumors,its motality rate accounted for 80% of patients for skin cancer,malignant melanoma easily deterioted and have a strong resistance to chemotherapy drugs.On the basis of previous research,the paper explored the inhibitory effect and mechanism of morroniside on human malignant melanoma A375 cells to provide a solid basis for the research and development of effective drugs against melanoma.Objective: To explore the inhibitory effect and mechanism of morroniside on human malignant melanoma A375 cells.Methods: 1.During the logarithmic growth phase,A375 cells were pre-incubated with drug for 24 hours,which were divided into the blank group,positive control group(cisplatin),medication group(intervation with different concentrations of morronside).2.The morphological changes of A375 cells treated with different concentrations of morronside for 24 hours were observed by inverted microscope.3.The proliferation rate of A375 cells treated with different concentrations of morronside for 24 hours was detected by MTT assay.4.The apoptosis rate of A375 cells treated with different concentrations of morronside for 24 hours was detected by Annexin V-FITC/PI double staining flow cytometry.5.The expression of MAPK signaling pathway protein in A375 cells treated with different concentrations of morronside for 24 hours was detected by Western blotting.6.The expression of Cyclin D1,P21,Bcl-2 family Bax and Bcl-2protein in A375 cells treated with different concentrations of morronside for 24 hours was detected by Western blotting,and investigate the effect of MAPK signaling pathway inhibitor on morronside function.7.The expression of Bcl-2 family Bax and Bcl-2 m RNA in A375 cells treated with different concentrations of morronside for 24 hours was detected by RT-qPCR.Results:1.The effect of morronside on the Morphology of A375 Cells:After the treatments with different concentrations of morronside for 24 hours in A375 cells,the cells were observed by inverted microscope.We could see more floating cells,more cell shrinkage,the larger gap,the decreased density and fewer adherent cells,The effect of that was in a dose dependent manner.2.The effect of morronside on proliferation of A375Cells:Morronside could significantly inhibit the proliferation of A375 cells in a dose dependent manner.3.The effect of morronside on the apoptosis rate of A375 Cells:After the treatments with different concentrations of morronside for 24 hours in A375 cells,The apoptosis rate of cells were dose-dependent.4.The effect of morronside on MAPK Signaling Pathway:After the treatments with different concentrations of morronside for24 hours in A375 cells,Western blotting analysis show the expression of ERK1/2,JNK,p-JNK,p38 MAPK and p-p38 MAPK protein were not significantly changed,while the expression of IL-1/2 protein was significantly increased(P<0.01).5.The effect of morronside on cyclin:After the treatments with different concentrations of morronside for24 hours in A375 cells,Western blotting analysis showed morronside down-regulated the expression of cyclin D1 protein and up-regulated the expression of P21 protein,the difference was statistically significant(P<0.01).After the treatments with MAPK inhibitor,ERK1/2 specific inhibitor U0126 could significantly up-regulated the expression of Cyclin D1 protein,down-regulated the expression of P21protein(P<0.01),However,after the treatments with p38 MAPK inhibitor SB203580,the expression of Cyclin D1 and P21 protein was not significantly changed.6.The effect of morronside on Bcl-2 family:A375 cells were treated with different concentrations of morronside for 24 hours,and detected by western blotting and RT-q PCR.morronside could significantly up-regulated the expression of Bax protein and m RNA(P<0.01),down-regulated the expression of Bcl-2 protein and m RNA(P<0.01),indicating that morronside can affect the expression of Bcl-2 and Bax from the protein and gene levels.After the treatments with MAPK inhibitor,ERK1/2specific inhibitor U0126 could significantly up-regulated the expression of Bcl-2protein(P<0.01),down-regulated the expression of Bax protein(P<0.01).However,after the treatments with p38 MAPK inhibitor SB203580,the expression of Bcl-2 and Bax protein was not significantly changed.Conclusion:1.Morronside could significantly inhibit the proliferation of A375 cells in a dose-dependent manner.2.Morronside could significantly promote the apoptosis of A375 cells in a dose-dependent manner.3.Morronside could block cell cycle and promote the expression of apoptosis gene to play an anti-tumor effect,its possible mechanism may be associated with activation of ERK/MAPK signaling pathway.