Effects Of Triazole Schiff Base Derivatives On The Differentiation And Apoptosis Of Human Hepatocarcinoma Cells

Recently, a lot of triazole-containing compounds has been reported that the compounds possessed potential anticancer activity. The mechanisms of these cytotoxic agents are not fully understood, as with many other agents, are likely to be inhibition of cell proliferation, cell cycle arrest, interfere with normal microtubule dynamics or induction of tumor cells apoptosis and differentiation. We also highlight the agents that may one day be translated into more effective cancer therapy.In the present study, a series of triazole derivatives was designed and synthesized by Dr Hu Guoqiang. Human hepatocarcinoma BEL-7402 cells were tested for its sensitivity to the triazole derivatives in order to explore the anticancer mechanism of triazole derivatives and to select new anti-cancer drugs. The results provide references for the development of the chemical which has the function of antineoplastic.Objective: 21 triazole schiff base derivatives as candidate agents was screened for find the compounds which have high inhibiting activity to proliferation of BEL-7402 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 triazole schiff base derivatives on the cell proliferation were examined by MTT assay. PI cytofluorescence staining and flow cytometric analysis was used to analysis cell cycle. The apoptotic cells were assayed by flow cytometry (FCM) with annexin V-FITC conjugated and propidium iodide (PI) staining. Cell apoptosis morphology was observed by fluorescence microscope techniques with Hoechst33258 labeling. The protein expressions of active caspase-3 in BEL-7402 cells were observed by immunohistochemical staining. Western blotting was used to assay changes of the proteins correlated cell apoptosis. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the changes of alpha fetoprotein (a-FP) and albumin (Alb) mRNA in order to explore differentiation state of BEL-7402 cells. The secretory Alb and a-FP of the cells was assayed by the radioimmunoassay. Colorimetric method was used to assay activity of superoxide dismutase (SOD), catalase (CAT) and alkaline phosphatase (ALP).Results: The results of drug screening indicated that LH-36, LH-37, LH-38 and LH-39 possessed high inhibiting activity to proliferation of BEL-7402 cells. The cell proliferation was inhibited by LH-37 and LH-38 at 0.1μmol/L~100μmol/L in dose and time dependent manners. The approximate the concentration of 50% growth inhibition (IC50) values of LH-37 and LH-38 for 48h was 302.7μmol/L and 4.61μmol/L, respectively. The agents induced BEL-7402 cell cycle arrest at G1/S stage. More apoptotic cells were observed in LH-37 and LH-38 treated the cells for 48h at the range of concentration from 0.1μmol/L to 100μmol/L compared to the controls, the difference was statistically significant. Treatment of BEL-7402 cells with different LH-37 and LH-38 concentration for 48 hours increased the percentage of active caspase-3 positive cells and protein expression of caspase-3, caspase-9 and P53 but did not increase protein expression of Bcl-2. Addition LH-37 and LH-38 increased mRNA expression of Alb, whereas the mRNA expression ofα-FP was decreased. After treated with LH-37 and LH-38, the secretory amount ofα-FP was reduced whereas Alb was increased. LH-37 and LH-38 markedly promoted the viability of SOD and ALP but decrease CAT in BEL-7402 cells.Conclusions:1. LH-37 and LH-38 possess high inhibiting activity to proliferation of BEL-7402 cells and can induce the cell cycle arrest. The cell proliferation is inhibited by LH-37 and LH-38 in dose and time dependent manners.2. At the approximate the concentration of IC50 values, LH-37 and LH-38 can induce apoptosis of human hepatocarcinoma BEL-7402 cells significantly. 3. At the lower concentration than IC50 values, LH-37 and LH-38 can induce differentiation of human hepatocarcinoma BEL-7402 cells.4. LH-37 and LH-38 can induce differentiation and apoptosis of human hepatocarcinoma BEL-7402 cells, which may be related to inhibition of CAT and increase of SOD activity. The result is increased the concentration of intracellular hydrogen peroxide (H2O2) and the oxidative damage of BEL-7402 cells.