Study On Mechanisms Of Apoptosis Liquiritigenin-induced In Human Hepatocellular Carcinoma SMMC-7721 Cells

Liver cancer is a kind of alimentary system cancers, the morbility of which is increasing in the world. The liver cancer development is not only related with the abnormal generation in cells but also with the disequilibrium in cell apoptosis. Emerging studies have suggested that flavonoids function their potent property of anti-tumor through cell growth inhibitory, induction of cell apoptosis and intervention effects on protien kinase in signal transduction pathway. Liquiritigenin is a flavanone extracted from licorice, possessing a wind range of physiology properties, however, few studies have been reported that liquiritigenin induces apoptosis in tumor cells. In the present study, we plan to investigate the growth inhibitory and pro-apoptotic effects on tumor cells of liquiritigenin; Molecular mechanism of liquiritigenin induce-apoptosis in SMMC-7721 cells was explored by detecting apoptosis-associated proteins and kinases, which was studied to clarify the mechanism of anti-tumor effects induced by liquiritigenin. Objective: The purpose of this study was to investigate the growth inhibitory effect of liquiritigenin on two kinds of tumor cells and explore apoptotic effects of SMMC-7721 cells induced by liquiritigenin. Methods: MTT assay was used to observe the effects on growth inhibition induced by liquiritigenin in Human colorectal cancer Lovo cells and Human hepatocellular carcinoma SMMC-7721 cells. The morphological changes effect of liquiritigenin -induced apoptosis was observed with Hoechst staining and transmission electron microscope, the percentage of apoptotic cells was analyzed by Annexin V-FITC/PI. Results: Liquiritigenin had the inhibitory effects on proliferation of Lovo cells after been treated with liquiritigenin for 24,48 and 72 hours at each concentration, and the high inhibition ratio were 15.4%,57.1% and 84.7%; The high inhibition ratio of liquiritigenin against SMMC-7721 cells was 85.6%, 94.2%,91.3% on 24,48 and 72 hours respectively, and it demonstrated obvious growth inhibitory effect in a dose dependent-manner. Using Hoechst 33258 staining and transmission electron microscope, morphological changes of SMMC-7721 cells induced by 0.4mM liquiritigenin for 72h were found, which exhibited characteristic features of apoptosis including chromatin condensation, nuclear fragmentation and vacuole. Liquiritigenin could induce apoptosis on SMMC-7721 cells in a time-and-dose dependent manner. At 24h, the apoptotic cells enhanced from 7.06% (control) to 23.52% at 0.4mM liquiritigenin. Conclusion: Liquiritigenin has unvarying anti-tumor effects on Human colorectal cancer Lovo cells and Human hepatocellular carcinoma SMMC-7721 cells. It could induce stronger inhibitory effect and apoptotic effect on SMMC-7721 cells.Section 2 Study on apoptosis mechanism of Human hepatocellular carcinoma SMMC-7721 cells induced by liquiritininObjective: The aim of our study was to explore the mechanism of liquiritigenin-induced apoptosis in human hepatocellular carcinoma SMMC-7721 cells. Methods: We used an oxidant-sensitive fluorescent probe, DCFH-DA to examine the production of reactive oxygen species, the mitochondrial membrane potential was investigated with Rh123 and the activity of glutathione (GSH), glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD) were detected by biochemical methods; The levels of p-53, bcl-2, survivin and caspase-3 proteins were detected by western blot. Results: The induction of apoptosis by 0.4mM liquiritigenin was accompanied with the production of reactive oxygen species (ROS), disruption of mitochondrial membrane potential and depletion of antioxidant enzymes. The significant ROS generation was firstly found at 3h and being time-dependent until 9h, while a time-dependent decrease in membrane potential occurred after liquiritigenin treatment and the significant loss appeared at 9h and 12h. Cells were pretreated with N-acetyl-cysteine (NAC), a free radical scavenger, which results provide the evidence that liquiritigenin inducing apoptosis and ROS production were suppressed. Further studies found that the level of p53 protein increased and the Bcl-2 protein decreased in time-dependent manner, the expression levels of survivin and pro-caspase-3 were down-regulated. In addition, a concomitant time-dependent increase in caspase-3 activity was observed by liquiritigenin treatment. Conclusion: These findings suggested that the liquiritigenin inducing apoptosis associated with liquiritigenin as a pro-oxidant in SMMC-7721 cells.