Mechanism Study Of Niclosamide Combined With Quinacrine In Treating Melanoma

Objective:Melanoma is a malignant tumor that occurs in the skin,mucosa,and uvea with a high metastatic rate and high mortality.Melanoma is prone to widespread metastasis in the early stages of the disease.Despite some breakthroughs in the treatment of melanoma in the past decade,there are still problems such as side effects of drugs,drug resistance,and low effectiveness.A major feature of tumors is a change in glucose metabolism,mainly manifested by the conversion of oxidative phosphorylation to aerobic glycolysis in normal cells.That is,under sufficient oxygen conditions,tumor cells transport glucose into cells through glucose transporters on the cell membrane for glycolysis,providing energy and raw materials for their proliferation and biosynthesis.Due to the low productivity of aerobic glycolysis,tumor cells adapt to the enormous energy demand of their rapid proliferation by increasing glucose uptake.Studies have shown that the consumption and uptake rate of glucose by tumor cells are higher than those of normal cells,and glucose deprivation is more likely to have a toxic effect on tumor cells than normal cells.The classic anti worm drug niclosamide and the anti-malaria drug quinacline can both exert anti-cancer effects,and some studies have shown that the combination of the two drugs can produce significant anti melanoma effects,but the relevant mechanism is not yet clear.This article aims to clarify the specific mechanism of quinacrine combined with niclosamide in inhibiting the proliferation of melanoma cells and the impact of drug combination on cell glucose metabolism,providing new ideas and theoretical support for the treatment of melanoma Methods:Melanoma cell lines B16 and B16-F10 were used as research objects in this experiment.1.Using CCK8,scratch test,and invasion test to detect the effects of drug combination(quinacrine combined with niclosamide)and glucose free culture medium on the proliferation,migration and invasion ability of melanoma cells.2.Using qRT-PCR and WBexperiments to detect the phosphorylation level of Akt pathway in melanoma cells treated with drug combination and glucose free medium for 24 hours.3.Using CCK8,scratch test,and invasion test to detect the proliferation,migration,and invasion ability of melanoma cells after administration of Akt pathway inhibitor LY294002.4.Using qRT-PCR and WB assay to detect the expression of glucose metabolism related proteins GLUT1,hexokinase 2,and proliferation related protein cyclin D1 in melanoma cells after drug combination or glucose deprivation.Transfecting specific RNA into melanoma cells to reduce HK2 expression.The glucose uptake ability of melanoma cells after drug combination treatment was tested by glucose uptake test.5.Reduce the expression of HK2 in melanoma cells by transfecting specific RNA,and detect the proliferation and motility of melanoma cells after knockdown of HK2 using CCK8,scratch test,migration test,and invasion test.6.The regulatory relationship between transcription factor E2F3 and proliferation related protein cyclin D1 was determined through luciferase assay and wb assay,and the effect of drug combination on the proliferation of melanoma cells after overexpression of E2F3 was tested through CCK8.7.The subcutaneous tumor formation model was established by injecting mouse melanoma cell B16 into the back of C57BL/6 mice.Immunohistochemical and H.E.staining experiments were used to detect the anti melanoma effect and mechanism of the drug combination in vivo.Results:1.The results of CCK8,scratch test and invasion test show that the drug combination and glucose free culture medium can significantly inhibit the proliferation and movement of melanoma cells after 24 hours of treatment.It is suggested that drug combination has anti melanoma effects and that glucose plays a significant role in the proliferation and movement of melanoma.2.The results of qRT-PCR and WB experiments showed that the phosphorylation level of Akt decreased after treatment of melanoma cells with drug combination and glucose free medium.And glucose uptake experiment showed that drug combination can inhibit the glucose uptake ability of melanoma cells.It is suggested that the drug combination and glucose free culture medium both play an anti melanoma role by inhibiting the Akt survival pathway3.The results of CCK8,scratch test and invasion test showed that the Akt pathway inhibitor LY294002 significantly decreased the proliferation and motility of melanoma cells after treatment.4.The results of qRT-PCR and WB experiments showed that the expression of glucose metabolism related proteins GLUT1,HK2,and cell proliferation related protein cyclin D1 in melanoma cells decreased at both transcription and translation levels after drug combination and glucose deprivation.It is suggested that drug combination can reduce glucose entry into cells by inhibiting GLUT1 expression,resulting in glucose deprivation.5.The results of CCK8,scratch test and invasion test showed that after inhibiting the expression of HK2 in melanoma cells,the proliferation and motor ability of the cells decreased.6.The results of luciferase and WB experiments showed that there was a positive regulatory relationship between transcription factor E2F3 and cyclin D1.7.The animal experiment results showed that the tumor volume in the back of mice in the quinacrine combined with niclosamide treatment group was significantly smaller than that in the control group.The results of H.E.staining showed that compared with the control group,the morphology of the main organ cells of the mice in the combined treatment group was normal and not damaged.Immunohistochemical results showed that the expression of GLUT1,Akt,HK2,and cyclin D1 decreased significantly during the combined treatment.Conclusion:Through this study,we proved that the combination of quinacrine and niclosamide can synergistically inhibit the proliferation,migration and invasion of melanoma.We also revealed that the anti melanoma effect is related to glucose deprivation.The specific mechanism is that the combination of drugs inhibits the proliferation of melanoma cells through the Akt/HK2/cyclin D1 axis mediated by glucose deprivation,which provides a new method and new idea for the treatment of melanoma.