Mechanism Of Miconazole Inducing Apoptosis And Increasing Sensitivity Of Cisplatin In Lung Squamous Cell Carcinoma Cells By Inhibiting PI3K/Akt/NOX4 Pathway

Object:Chemotherapy and radiotherapy are important methods for the treatment of tumors at this stage.However,these two methods will not only kill tumor cells during the treatment process,but also cause damage to normal human cells,resulting in a decline in the patient’s immune function,so cancer patients are susceptible to fungal infections.Recent studies have shown that a variety of azole antifungal drugs can inhibit tumor cells viability.However,the anti-tumor of azole antifungal drugs on lung squamous cell carcinoma and its effect on the efficacy of cisplatin have not been reported yet.The purpose of this study is to screen out the best drugs for inhibiting lung squamous cell carcinoma from the azole antifungal drugs,to further study its anti-lung squamous cell carcinoma activity in vivo and in vitro,and to explore its related mechanism,so as to provide experimental basis for clinical selection for lung squamous cell carcinoma treatment.Methods:（1）MTT assay:MTT method was used to measure the effect of different azole antifungal agents on the cells viability of H520,and to screen out the drug with the best inhibitory effect on cell viability（2）Hoechst cell nuclear staining:Hoechst staining method was used to measure the effect of different contains of miconazole on the apoptosis of H520 and H226 cells.（3）Flow cytometry:The effect of different concentrations of miconazole on apoptosis and reactive oxygen species of lung squamous cell carcinoma H520 and H226 cells were detected by flow cytometry.Explore the effect of hydrogen peroxide and miconazole on the production of reactive oxygen species and cell apoptosis.（4）Real-time Quantitative polymerase chain reaction（RT-q PCR）assay:RT-q PCR method was used to detect the effect of different concentrations of miconazole on the expression of NOX4 m RNA in lung squamous cell carcinoma H520 and H226 cells.（5）After the addition of NOX4 inhibitor GKT137831,flow cytometry was used to detect the changes of apoptosis and the production of reactive oxygen species in lung squamous cell carcinoma H520 and H226 cells induced by miconazole.（6）Western blot assay:Western blot method was used to explore the effects of certain concentration range of miconazole on the protein expression of phosphorylated Akt（p-Akt）and NOX4 protein in lung squamous cell carcinoma H520 and H226 cells.After using PI3K/Akt inhibitor LY294002,western blot was used to detect the effect of the inhibitor combined with miconazole on the expression of NOX4 protein.（7）The MTT method and flow cytometry were used to detect the effect of miconazole and cisplatin on the cytotoxicity of lung squamous cell carcinoma H520 and H226 cells.（8）Xenograft studies:The tumor inhibition effect of miconazole and its combination with cisplatin was verified by constructing tumor model in nude mice.Immunohistochemical method was used to detect the expression of p-Akt and NOX4 in tumor tissues.Results:（1）Among the selected azole antifungals,14 drugs have inhibitory effects on the viability of lung squamous cell carcinoma H520cells,and the half inhibitory concentration(IC₅₀)of miconazole is the lowest.（2）Miconazole can reduce the production of reactive oxygen species and induce apoptosis in lung squamous cell carcinoma H520 and H226 cells.（3）Miconazole inhibits p-Akt expression and reduces NOX4m RNA and protein expression.There was no significant difference in NOX4 protein expression between PI3K/Akt inhibitor LY294002 and miconazole comblin LY294002.（4）In lung squamous cell carcinoma H520 and H226 cells,miconazole can increase the sensitivity of cisplatin.（5）Miconazole can inhibit tumor growth in nude mice.Miconazole can inhibit tumor growth in nude mice.The results of immunohistochemistry showed that miconazole can reduce the expression of NOX4 and p-Akt in tumors of nude mice.Conclusion:Miconazole can inhibit the expression of NOX4mediated by PI3K/Akt,induce apoptosis of lung squamous cell carcinoma cells and enhance the inhibitory effect of cisplatin on lung squamous cell carcinoma cells.