Inhibitory Effect Of IBUD-1 On HepG2 And Its Mechanism

IBUD-1is a new synthesized compound with anti-inflammatory activity based on the combination principles. This paper studied its anti-cancer activity on HepG2cells and possible mechanisms.1. Cytotoxicity effect of IBUD-1on HepG2cellsWe used the MTT assay to investigate the effect of IBUD-1on HepG2cells. The result showed that when HepG2cells were exposed to various concentrations of IBUD-1and for different incubation times, the cellular cytotoxicities were significantly increased in a time-and dose-dependent manner. The morphology of treated cells became rounded, detached from the culture flask and showed membrane blebbing under the inverted light microscope, while the untreated cells retained the normal size and shape.2. Effect of IBUD-1on HepG2cells apoptosisFlow cytometry analysis was used to quantify apoptotic cells after treatment with IBUD-1. The percentage of apoptotic cells was increased from16.82%to21.02%and29.4%after24h of treatment with25,50and100μM IBUD-1, compared with the control2.5%. These results suggested that apoptosis ratio of IBUD-1was increased with the drug concentrations.Western blot analysis was performed to investigate the expression of cell apoptosis related proteins. With the increase of treated IBUD-1concentrations, the procaspase-3, p-p38, p-Erkl/2and Bcl-2were decreased significantly, while the p38, Erk1/2and Bax had no change. The IBUD-1could decrease the expression of COX-2in the dose-dependent manner. Which indicates that the IBUD-1induced HepG2cells apoptosis through regulation of the expression and activity of these porteins.3. Effect of IBUD-1on the cell cycle distributionHepG2cells were treated with IBUD-1for24h, and the distribution of cells in various compartments of the cell cycle was analyzed by flow cytometry. These results provided evidence that the observed growth-inhibitory effect of IBUD-1on Hep G2cells was partly due to the cell cycle arrest. In control cultures,15.87%and12.32%of cells were in S and G2/M phase at24h. However, in IBUD-1-treated cells, the number of cells in S phase (50μM,12.37%;100μM,23.25%) and G2/M phase (50μM,15.99%;100μM,17.79%) were significantly increased. In addition, the percentage of cells in GO/Gl (50μM, 71.64%;100μM,58.96%) were remarkably decreased.Western blot analysis was performed to investigate the expression of cell cycle regulator proteins. The result showed that the p21protein level was increased after24h incubation with IBUD-1in a dose-dependent manner. Simultaneously, a dose-dependent down-regulation of cyclinA and cyclin B1and up-regulation of pCdc2was observed. The expression of Cdc2had no change. Which indicates that the IBUD-1induced HepG2cell cycle distribution through the regulation of the expression and activity of these porteins.In conclusion, our findings strongly indicate the inhibitory effect of IBUD-1on the proliferation of the human hepatocellular carcinoma (HepG2) cells. The mechanisms through which IBUD-1act are at least from:induction of apoptosis, COX-2inhibition and cell cycle redistribution. The above changes of proteins are mediated, at least in part, by the activation of ERK1/2and p38MAP kinases.