Study On The Apoptosis And Associated Mechanisms Of Hepatoma Cells Induced By Bufalin

Study on the apoptosis and associated mechanisms ofhepatoma cells induced by bufalin1.IntroductionHepatocellular carcinoma (HCC) is one of the most common tumors in china, which is in third death line among the whole tumors. One hundred and twenty thousands people were died from HCC each year, and with forty percent of all the people died from HCC in the world. Traditional Chinese medicine (TCM) may have rising quality of life and prolonging survival time for HCC, but limited to kill HCC cells. The anti-carcinoma investigation of toxicity monome from TCM is one of the most active domains in anti-tumor investigation. To explore the anti-hepatoma investigation of toxicity monome from TCM will have far-reaching significance for development of anti-hepatoma drugs. Bufalin, one of the toxicity ligand isolated from Chansu, is belong to TopoⅡinhibitor and has anti-carcinoma effects. The anti-carcinoma investigation of bufalin on hematological neoplasm, carcinoma of prostate, gastric carcinoma have already been reported. and its anti-carcinoma mechanisms were involved in differentiation, apoptosis, expressions of associated gene, signal transduction and reverse drug resistence on tumor cells in vitro. Report has not been found yet for bufalin on apoptotic mechanisms to hepatoma cells and its anti-carcinoma effects in vivo. Thereby, To explore its anti-carcinoma effects in vivo and to induce apoptotic cells in situ, we established the orthotopic transplantation tumor model of human hepatocellular carcinoma(BEL-7402)in nude mice and orthotopic transplantation tumor model of hepatoma (H22) in mice. Furthermore, The expressions of associated apoptotic gene bcl-2/bcl-2 mRNA, bax/bax mRNAand apoptotic signal transduction pathway NF-κB/ p38MAPK in BEL-7402 cells by bufalin were determined. This study may provide an experimental basis on bufalin for HCC therapy.2.Objectives (1) To study the anti-carcinoma effects on orthotopic transplantation tumor model of human hepatocellular carcinomain nude mice and orthotopic transplantation tumor model of hepatoma in mice by bufalin and to examine the apoptosis in situ in vivo. (2) To conform apoptosis in human hepatoma cells BEL-7402 induced by bufalin and to study the influences on expressions of associated apoptotic gene (bcl-2/bcl-2 mRNA, bax/bax mRNA) and apoptotic signal transduction pathway (NF-κB/ p38MAPK) in BEL-7402 cells in the course of inducing apoptosis by bufalin. (3) To provide scientific experimental basis for bufalin to HCC therapy.3.Materials and methods PartI Study on the anti-carcinoma effects of orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice and orthotopic transplantation tumor model of hepatoma in mice by bufalin in vivo 1.To fixed on the dose of bufalin in vivo: 18 healthy male ICR mice were divided into high dose (2mg/kg), moderate dose (1.5mg/kg) and low dose of bufalin group(1mg/kg), randomly. Bufalin was njected imtraperitoneally at the three doses above for day1-10, respectively. Body weight, appetite and behavior of mice were observed, and the anti-carcinoma doses of bufalin for were fixed on 1.5mg/kg, 1mg/kg and 0.5mg/kg. 2.75 orthotopic transplantation tumor model of human hepatocellular carcinomain nude mice and 75 orthotopic transplantation tumor model of hepatoma in mice were divided into Bu1-3 groups, NS group and ADM group randomly. Tumor volumes were detected by cut the belly open before administration of the next day. Bufalin was injected imtraperitoneally at dose of 1.5mg/kg, 1mg/kg and 0.5mg/kg for day1-10, respectively. NS group were injected equal volume saline as above and Adriamycin was injected imtraperitoneally at dose 8.0mg/kg for day1. 10 mice in each group were killed at day2 after withdrawal and detected on morphological and ultrastructural changes in myocardium, brain, liver, kidneys and tumor tissues by phase contrast microscope and electron microscope. The apoptosis were examined by using electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP...