Cinnamaldehyde ofloxacin-3-ylhydrazone induces apoptosis of human hepatocarcinoma SMMC-7721 cells

This study is to observe the effect of N-(3-phenylallylidene)-6-fluoro-1, 8-(2, 1-propoxy)-7-(4- methylpiperazin-1-yl)-quinolin-4(1H)-one-3-carbonyl hyarazine (FQ16) on apoptosis of hepatocarcinoma SMMC-7721 cells in vitro.  With different concentrations of FQ16 at different times used to treat SMMC-7721 cells in vitro, the proliferation of the cells and the inhibition effect of FQ16 on the cell proliferation were     examined by MTT assay.  Cell apoptosis was determined by Hoechst 33258/PI fluorescence staining, TUNEL and agarose gel electrophoresis method.  The effect of FQ16 on topoisomerase II activity was measured by agarose gel electrophoresis using Plasmid pBR322 DNA as the substrate.  Mitochondrial membrane potential (MMP, △ψm) was measured by high content screening image system.  The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression changes of Bcl-2 mRNA and Bax mRNA.  The  caspase-9, caspase-8, caspase-3, p53, Bcl-2 and Bax protein expressions were detected by Western blotting  analysis.  The results showed that the cell proliferation was inhibited by FQ16 at 0.625 − 10 μmol·L⁻¹ in a time-dose dependent manner.  Treatment of SMMC-7721 cells with different concentrations of FQ16 for 24 h increased the percentage of the apoptosis cells obviously (P < 0.05), the typical ladder DNA in apoptotic cells and a concomitant dissipation of the mitochondrial membrane potential.  Compared with control group, FQ16 influenced obviously DNA topoisomerase II activity, stimulated DNA cleavage and inhibited DNA reunion  mediated by topoisomerase II.  In addition, FQ16 (3 − 7.39 μmol?L⁻¹) increased mRNA expression of Bax and protein expression of p53, Bax, caspase-9, caspase-3, separately, and induced cytosolic accumulation of activities caspase-9 and caspase-3, whereas the mRNA and protein expression of Bcl-2 decreased with no change of   caspase-8.  Therefore it can be concluded that the effects of inhibited topoisomerase II and mitochondrial-   dependent pathways were involved in FQ16 induction of apoptosis of SMMC-7721 cells.