In Vitro Synergistic Antitumor Effect Of Niclosamide Ethanolamine Combined With Icotinib On HCC827 Cell

Objective:To investigate the effect of the combination of niclosamide ethanolamine and icotinib hydrochloride on the proliferation inhibition and apoptosis promotion of human lung adenocarcinoma HCC827 cells in vitro,and to explore whether the combination of the two drugs has synergistic anti-tumor activity,so as to provide theoretical basis for the selection of potential combination drugs for the clinical treatment of non-small cell lung cancer.Methods:1.Prepare the reserve solution of icotinib hydrochloride,use gradient dilution method to configure different concentrations of icotinib hydrochloride,take unadded HCC827 cells as the control group,use CCK-8 method to detect cell activity,calculate the cell activity of each group of icotinib hydrochloride,and determine half of the inhibitory concentration of icotinib hydrochloride.2.Preparation of niclosamide ethanolamine mother liquor,using the gradient dilution method from different concentrations of niclosamide ethanolamine solution,with no dosing HCC827 cells as a control group,with CCK-8 method to detect cell activity,calculation of niclosamide ethanolamine dosage groups each cell vitality,calculation of niclosamide ethanolamine,half inhibitory concentration of niclosamide ethanolamine combination of concentration to provide the reference.3.According to the determined IC50 of clonitramine ethanolamine,three concentrations,0.1,0.5 and 1.0?M,were fixed to increase the concentration of icotinib hydrochloride,and the inhibition of HCC827 cell proliferation was investigated.When the concentration of icotinib hydrochloride ranged from 0.01?M to 25?M,the inhibition curve of the combination of the two drugs on the proliferation of HCC827 cells was plotted,and the inhibition level of the cells was compared with that of icotinib hydrochloride alone.4.To test whether the two drugs have synergistic effect: set different groups: control group,icotinib hydrochloride 0.1?M group,icotinib hydrochloride 0.5?M group,chloraminosilethanolamine 0.25?M,chloraminosilethanolamine 0.25?M + icotinib hydrochloride 0.1?M,chloraminosilethanolamine 0.25?M + icotinib hydrochloride0.5?M group.5.Assay:(1)Annexinv-fitc /PI double staining combined with flow cytometry to detect cell apoptosis.(2)The cloning formation experiment calculated the cloning formation rate.(3)Count the number of cell migrations in the Transwell cell.Results:1.The IC50 of icotinib hydrochloride on HCC827 cells was 0.61?M under 48 h treatment.At the same concentration,the inhibitory effect on cell proliferation became stronger and stronger with the prolonging of the action time of icotinib hydrochloride.2.There was a dose-dependent correlation between the proliferation inhibition of niclosamide on HCC827 cells.After the concentration reached 2.5?M,the proliferation inhibition reached the saturation state.By calculation,the IC50 of niclosamide on HCC827 cells was 0.46?M under the action of niclosamide for 48 h.Compared with icotinib hydrochloride alone,the inhibition of cell proliferation was more obvious in the icotinib hydrochloride group.Icotinib hydrochloride in the 1.0?M group had the most obvious inhibitory effect on cells,and the cell growth could be significantly inhibited at very low concentration.3.The single-drug treatment of HCC827 cells with icotinib hydrochloride mainly showed an increase in the proportion of cells undergoing late apoptosis and a significant increase in the proportion of necrotic cells.The apoptosis of HCC827 was dose-dependent with icotinib hydrochloride.The monotherapy of HCC827 cells with niclosamide also promoted the apoptosis of HCC827 cells.In the combined application group,compared with the single application group,the proportion of late apoptosis and necrosis of HCC827 cells increased,while the proportion of early apoptosis did not change significantly.4.The HCC827 cells without drug treatment formed obvious cell cloning.After treatment with either icotinib hydrochloride or niclosamide,the cloning of HCC827 cells was greatly reduced and the population growth was significantly inhibited.In the combined treatment group of niclosamide and icotinib hydrochloride,almost no clones were formed in HCC827 cells.After the treatment of HCC827 cells with different concentrations for 72 h,the invasiveness of HCC827 cells was significantly reduced compared with the control group,and the number of cells in the combination group with icotinib hydrochloride and niclosamide was less than that in the combination group with icotinib hydrochloride and niclosamide monotherapy.Conclusion:1.Icotinib hydrochloride inhibits the proliferation of HCC827 cell line in human lung cancer,and its inhibition is concentration-dependent.2.Clonitramine ethanolamine inhibits the proliferation of human lung cancer HCC827 cell,and its inhibition is concentration-dependent.3.Niclosamide combined with icotinib hydrochloride can synergistic promote the apoptosis of HCC827 cells.4.Niclosamide combined with icotinib hydrochloride can synergistic inhibit cell cloning and cell invasion of HCC827 cells.