Effects Of Quinolone Derivatives On The Apoptosis Of Human Hepatocarcinoma Cells In Vitro

Objective 19 Quinolone derivatives as candidate agents was screened for find the compounds which have high inhibiting activity to proliferation of SMMC-7721 cells, and studied the antineoplastic mechanism of the compounds in order to provide references for the development of new anticancer drugs.Methods The proliferation of the cells and the inhibition effect of Quinolone derivatives on the cell proliferation were examined by MTT assay. PI fluorescence staining and flow cytometric analysis was used to analysis cell cycle. Cell apoptosis was observed by fluorescence microscope techniques with Hoechst 33258 associated with propidium iodide (PI) staining and TUNEL assay. DNA ladder was observed through agarose gel electrophoresis. The effect of FQ16 on TOPOⅡactivity was measured by agarose gel electrophoresis using Plasmid pBR322 DNA as the substrate. Mitochondrial membrane potential(△ψm) were measured by High content screening. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression changes of p53 mRNA,Bcl-2 mRNA and Bax mRNA. Western blotting was used to assay changes of the proteins correlated cell apoptosis.Results The results of drug screening indicated that FQ1,FQ4and FQ16 possessed high inhibiting activity to proliferation of SMMC-7721 cells. The cell proliferation was inhibited by FQ16 at 0.625μmol·L-1~10μmol·L-1 in dose and time dependent manners. The approximate the concentration of 50% growth inhibition (IC50) values of FQ16 for 24h was 4.48μmol·L-1.The agents induced SMMC-7721 cell cycle arrest at G2/M stage. More apoptotic cells were observed in FQ16 treated the cells for 24h compared to the controls, the difference was statistically significant and the typical ladder DNA in apoptotic cells were detected by agarose gel electrophoresis.Compared with control group, FQ16 influenced obviously DNA topoisomeraseⅡactivity, stimulated DNA cleavage and inhibited DNA reunion mediated by topoisomeraseⅡ. FQ16 influenced obviously a concomitant dissipation of the mitochondrial membrane potential of SMMC-7721 cell, the cells treated with 3,4.48,7.39μmol·L-1 FQ16 respectively for 24h, it compared with the control group to reduce(9.52±5.62)%,(37.32±5.52)%,(45.59±3.17)%.In addition FQ16 (3.0μmol·L-1~7.39μmol·L-1) increased mRNA expression of p53 and Bax. Treatment of SMMC-7721 cells with different FQ16 concentration for 24 hours increased the protein expression of caspase-3, caspase-9, Bax, and p53, induced cytosolic accumulation of activities Caspase-9 and Caspase-3, whereas the mRNA and protein expression of Bcl-2 was decreased, but did not increase protein expression of caspase-8.Conclusions1. FQ16 possess high inhibiting activity to proliferation of SMMC-7721 cells and can induce the cell cycle arrest. The cell proliferation is inhibited by FQ16 in dose and time dependent manners.2. At the approximate the concentration of IC50 values, FQ16 can induce apoptosis of human hepatocarcinoma SMMC-7721 cells significantly.3. FQ16 can induce apoptosis of human hepatocarcinoma SMMC-7721 cells, by inhibited topoisomeraseⅡand via mitochondrial-dependent pathways.