Effects Of N-(5-fluorobenzylidene Rhodanine) Ciprofloxacin Amide Derivativeon To Proliferation,Apoptosis And Autophagy Of Human Hepatocarcinoma Cells

Objective: To study the effects of N-（p-fluorobenzylidene rhodanine）Ciprofloxacin amide derivative 1-cyclopropyl-6-fluoro-7-（4-methyl-piperazin-1-yl）-N-[5-（4-fluorobenzylidene）rhodanine-3-yl]-quinolin-4（1H）-one-3-carboxamide（HGQ-15）on proliferation,apoptosis and autophagy of human hepatocarcinoma SMMC-7721 cells in vitro.To investigate the relationship between the autophagy and the apoptosis.Methods: Human hepatocarcinoma SMMC-7721 cells were treated with different concentrations of HGQ-15.Cell viability was analyzed by MTT assay.Cell apoptosis was determined by flow cytometry（FCM）with annexin V-FITC conjugated propidium iodide（PI）staining and TUNEL assay.The effect of gene silencing on Beclin-1 and the effect of the authophagy-specific inhibitor chloroquine（CQ）on cell proliferation and apoptosis were respectively analyzed by MTT assay and FCM.The autophagy marker protein microtubule associated protein 1 light chain 3（LC3）expression was detected by indirect immunofluorescence assay.The expression and ratio of LC3Ⅰ/LC3Ⅱwere observed by Western blot analysis.Results: SMMC-7721 cells were treated with various concentrations of HGQ-15 for 24,48 and 72 h,resulting in a significant decrease in cell viability（P<0.05,p<0.01）in a dose-and time-dependent manner.The IC₅₀ value for 24,48 and 72 h treatment was 4.531μmol/L（r² = 0.8742）,4.063μmol/L（r² = 0.8573）,3.894μmol/L（r² =0.9590）,respectively.After treatment with various concentrations of HGQ-15 for 24 h,TUNEL and FCM with annexin V-FITC conjugated PI staining assay were used to confirm the apoptotic effect of HGQ-15.SMMC-7721 cells were treated with HGQ-15 of 0,1.25 and 2.5μmol/L respectively for 24 h,and the percentage of apoptotic cells were 3.01%,35.68% and 57.28% respectively.Compared with the control group（0μmol/L HGQ-15）,the apoptosis rate of the HGQ-15 treatment group increased significantly（P<0.01）.The expressions of autophagy marker LC3 in the cytosol were evaluated by an indirect immunofluorescence assay.When SMMC-7721 cells were treated with HGQ-15 for 24 h,the LC3 fluorescence intensite in cytosol increased significantly.The results of Western blot analysis showed that HGQ-15 increased protein expression of autophagy-related proteins LC3-Ⅱ in SMMC-7721 cells.These results indicated that HGQ-15 induced autophagy in SMMC-7721 cells.Treatment of SMMC-7721 cells with Beclin-1 siRNA for 24 h decreased the expressions of Beclin1 mRNA and protein expression of LC3 in SMMC-7721 cells significantly.These results indicated that the gene silencing of Beclin-1 could inhibit the autophagy in SMMC-7721 cells significantly.Treatment of SMMC-7721 cells with Beclin-1 siRNA alone had no significant effect on the proliferation and apoptosis rate of the cells,while the inhibition of autophagy pathway by Beclin-1 siRNA enhanced the proliferation inhibition effect of HGQ-15 on the cells and increased the apoptosis rate induced by HGQ-15.The autophagy inhibitors further decreased the viability of cells treated by HGQ-15 and increased the percentage of the apoptosis cells.The autophagy inhibitor CQ（62.5μmol/L）alone had no significant effect on the proliferation and apoptosis rate of SMMC-7721 cells,but could enhance the proliferation inhibition effect of HGQ-15 on SMMC-7721 cells and increase the apoptosis rate induced by HGQ-15.Conclusion:1.N-（5-fluorobenzylidene rhodanine）ciprofloxacin amide derivativeon（HGQ-15）inhibits the proliferation of human hepatocarcinoma SMMC-7721 cells.2.HGQ-15 induces apoptosis and autophagy of SMMC-7721 cells.3.The autophagy induced by HGQ-15 has protective effect on SMMC-7721 cells and can significantly reduce the apoptosis rate of SMMC-7721 cells induced by HGQ-15.