| Primary hepatic carcinoma is one of the most common malignancies in China, comprehensive treatment is the major method for inoperative hepatic carcinoma. Chemotherapy has been one of the important adjuvant treatment in hepatocellular carcinoma, it plays an important role in the comprehensive treatment of hepatocellular carcinoma. Conventional chemotheraptic drugs don't often have good therapeutic effects in hepatocellular carcinoma because of high recurrence rate, low susceptibility and multidrug resistance; so it is important for us to find a new effective chemotheraptic drug. In recent years, more and more attention was paid to arsenious acid(arsenical compound) because it had a significant therapeutic effect and had no evident bone marrow suppression in the treatment of acute promyelocytic leukemia; researches about its related mechanisms showed that it was the main mechanism that arsenious acid could inhibit proliferation, induce differentiation and apoptosis in leukemic cells. Furthermore, the effects of arsenious acid could be observed in various cell lines of either myeloid or solid origins. However, to date, the effects of arsenious acid on human hepatocellular cells and its mechanisms still remained unclear. In this study, using human hepatocellular cell line BEL-7402 as a model, we would provide a few certain proofs for clinical application of arsenious acid in the treatment of hepatocellular carcinoma by investigating its effects on proliferation, differentiation and apoptosis in BEL-7402 cells and its possible mechanisms in vitro.Objective To investigate the effects of arsenious acid on human hepatocellular cellline BEL-7402 and its related mechanisms.Methods Part I Study of the effects of arsenious acid on proliferation and differentiation in human hepatocellular cells and its related mechanismsThe cell culture in vitro, MTT assay was used to study the change of cell growth curve, the method of trypan blue exclusion was used to observe the alteration of TD, colony-forming rate of BEL-7402 cells was studied by colony-forming assay. The production of AFP and the specific activities of GGT, LDH were measured by automated chemiluminescence system and biochemical method respectively. The morphologic changes of cell differentiation were observed by HE staining and transmission electron microscope respectively. The expression of proliferating cell nuclear antigen (PCNA), c-Myc, human telomerase reverse transcriptase (hTERT) protein was determined by immunohistochemical analysis.Part II Study of the effects of arsenious acid on apoptosis in human hepatocellular cells and its related mechanismsBEL-7402 HCC cell line was used in this study. Cell apoptosis was assessed by morphology, DNA gel electrophoresis, and flow cytometry. The morphological changes of cell apoptosis were observed by HE staining and transmission electron microscope respectively. Mitochondrial transmembrane potentials were evaluated by using Mitochondrial Membrane Sensor Kit. Wild-type p53 <. bcl-2, bax gene expression were measured at the mRNA and protein levels by RT-PCR, strepvavidin-peroxidase(S-P) immunohistochemical method respectively.Results (1) Arsenious acid could inhibit the growth and proliferation of BEL-7402 cells in a dose and time dependent manner. After treatment with arsenious acid, BEL-7402 cells' inhibitive rate increased(2.0,4.0, 8.0 u mol/L arsenious acid treatment for 24, 48,72h; the inhibitive rate was 27.13, 42.65, 47.74%; 35.52, 53.24, 60.41%; 50.61, 65.88,70.81% respectively; PO.05 vs control), TD delayed gradually(2.0, 4.0, 8.0 u mol/L arsenious acid treatment for 48h; TD was 24.68 + 3.99; 32.42+4.46; 36.95 + 8.91 respectively; P<0.05 and PO.01 vs control) while the colony-forming rate decreased(0, 8.0 u mol/L arsenious acid treatment for 7d; the colony-forming rate was 46.00, 18.80% respectively; PO.05). After treatment of 8.0 u mol/L arsenious acid, the amount of AFP production and the specific activities of GGT\ LDH were significantly decreased, morphologic changes of cell differentiation occurred in some of the cells observed by HE staining and transmission electron microscope respectively. In arsenious acid treated BEL-7402 cells, PCNA, c-Myc, hTERT expressions were significantly down-regulated at protein level.(2) BEL-7402 cells were arrested at G2/M phase with arsenious acid treatment and apoptosis induced at different arsenic acid concentrations, as shown by cell cycle and nuclear DNA contents, morphology, DNA gel electrophoresis. AA-induced apoptosis was accompanied by mitochondrial transmembrane potentials collapse, which was time-dependent(P<0.01). Wild-type p53 mRNAand protein expression was not detected in BEL-7402 cells, but BEL-7402 cells showed evident expression of bcl-2> bax mRNA and protein. After treatment with arsenious acid, bcl-2> bax gene expression were decreased and increased respectively (PO.01), and the bcl-2/bax ratio was decreased.Conclusions (1) In vitro, arsenious acid could significantly inhibit the proliferation of human hepatocarcinoma cells in a dose and time dependent manner, and it also could induce cell differentiation. Down-regulation the expression of PCNA, c-Myc, hTERT protein might be the mechanism.(2) In vitro, arsenious acid could significantly induce the apoptosis of human hepatocarcinoma cells and arrest the cells at G2/M phase in a dose and time dependent manner.
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