| Objective: Melanoma is characterized by low morbidity and high mortality,and the global incidence has been increased in recent years.For early melanoma,most patients can obtain a better prognosis through surgical resection.Because the incidence of melanoma is hidden and the invasive ability is strong,metastasis has occurred when it is found,so it can only be treated with radiotherapy and chemotherapy,but the effect is poor.In recent years,with the development of molecular targeted therapy and immunotherapy,the prognosis of melanoma has been greatly improved,but the emergence of drug resistance makes the treatment of melanoma face new challenges.Therefore,the development of anti-melanoma drugs with high efficiency and low toxicity is an urgent problem to be solved in clinic.In recent years,it has been found that flavonoids,such as apigenin and luteolin,play a prominent role in anti-cancer,which can significantly inhibit melanoma cell growth,proliferation,migration,invasion,capillarity and induce apoptosis,without obvious side effects.Morusin is a kind of flavonoids found in mulberry bark in nature.Previous studies have confirmed that it has obvious inhibitory effect on tumors such as gastric cancer and prostate cancer.However,so far,there has been no report on the effect of morusin on melanoma.Therefore,this study explored the effect and mechanism of morusin on melanoma cells in vivo and in vitro,in order to provide a theoretical basis for exploring new anti-melanoma drugs.Methods:1.Different doses of morusin(0.001 μM,0.01 μM,0.1 μM,0 μM,1 μM,10 μM,20 μM,40 μM,60 μM,80 μM,160 μM)were treated with melanoma cell lines A375 and MV3 cells for 24 h.The IC50 of melanoma cells was determined by MTT assay.2.Cell counting and MTT assay were used to analyze the effect of different doses of morusin on the proliferation of melanoma cells.The inhibition of morusin on the cell proliferation of A375 and MV3 cells was analyzed by Brd U staining.3.The PI staining flow cytometry and Western blot were used to detect the effect of morusin on the cell cycle of melanoma.4.Flow cytometry and Western blot were used to detect the effect of morusin on the apoptosis of melanoma cells.5.The effects of morusin on the migration and invasion ability of melanoma cells were detected by wound-healing assay,transwell assay and Western blot assay.6.Soft agar assay and in vivo tumor formation experiments in mice verified the effect of morusin on the tumor formation of melanoma cell lines A375 and MV3 cells.7.After knocking down p53,the effects of morusin on the cell proliferation,cell cycle,apoptosis,migration and invasion of melanoma A375 cells were detected.Results:1.MTT assay showed that the IC50 of morusin in A375 and MV3 cells were 4.634 μM and 9.7 μM.2.Cell counting and MTT assay results showed that morusin inhibited the proliferation of melanoma cells.After treating with different doses of morusin,the morphology of cells changed significantly,and the number of cells decreased significantly(P<0.05).The Brd U staining assay showed that compared with the DMSO group,the percentage of Brd U-positive cells decreased significantly after 5 μM treatment of A375 cells and 10 μM treatment of MV3 cells for 24 h(P<0.05).3.The cell cycle was detected by PI staining flow cytometry.The results showed that compared with the control group,the percentage of cells in G2/M phase increased significantly after 5 μM treatment of A375 cells and 10 μM treatment of MV3 cells for 24 h.It is statistically significant(P<0.05).Western blot showed that compared with the control group,the protein expression levels of p53 and p21 in the morusin treatment group were at a concentration(2 μM,5 μM,10 μM for A375 cells,and 5 μM,10 μM,15 μM for MV3 cells)and time(0 h,12 h,24 h,36 h)-dependent increase(P<0.05),the protein expression levels of Cyclin B1 and CDK1 decreased in a concentration and time correlation.4.Flow cytometry analysis showed that compared with the control group,the morusin treatment group(2 μM,5 μM treatment of A375 cells,5 μM,10μM treatment of MV3 cells)showed obvious apoptosis in A375 and MV3cells(P < 0.05).Western blot was used to detect the expression of apoptosis-related proteins,the results showed that in A375 and MV3 cells,compared with DMSO group,the PARP of the morusin group was down-regulated,while Cleaved-PARP(C-PARP)and Cleaved-Caspase3(C-Caspase3)were up-regulated,and there was a significant dependence on dose(A375 cells treatment doses were 2 μM,5 μM,10 μM,MV3 cells treatment doses were 5 μM,10 μM,15 μM)and time(0 h,12 h,24 h,36 h)(P<0.05).5.The results of the wound-healing assay and the transwell assay showed that compared with the control group,the migration and invasion ability of melanoma cells A375 and MV3 was significantly reduced after morusin treatment,and showed a time-dependent change.Western blot to detect the expression of migration and invasion-related proteins showed that in A375 and MV3 cells,compared with the DMSO group,Vimentin was down-regulated and E-Cadherin was up-regulated in the morusin-treated group,and there was a significant dependence on dose(2,5,10 μΜ for A375 and 5,10,15 μΜ for MV3)and time(0,12,24 and 36h)(P<0.05).6.Soft agar assay showed that compared with the control group,the size of single clones in the morusin treatment groups were significantly smaller,and the number of clones formed was significantly reduced(P<0.05).The results of NOD/SCID mouse model of xenotransplantation of melanoma cells in vivo showed that the tumor volume and weight of morusin treatment group were significantly lower than the control group(P<0.05).7.After knocking down p53 showed that compared with DMSO and the morusin group,the knockdown p53 group partially restored the effects of morusin on the cell proliferation,cell cycle,apoptosis,migration and invasion of A375 cells.Conclusion:Morusin,which inhibits cell proliferation,induces cell cycle arrest at G2/M phase,promotes apoptosis,and inhibits cell migration and invasion by increasing the expression of the p53.These results indicate that morusin,as a flavonoid,has great potential clinical significance in the anti-melanoma and can be used as an effective candidate drug for the treatment of melanoma. |
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