| Introduction:Hepatocarcinoma is one of the most commonly seen tumors in China. It is highly malignant. These patients of hepatocarcinoma have poor prognosis. These patients often died within 4~6 months without treatment. Resection,radiotherapy,chemotherapy and intervention are common therapic methods of hepatocarcinoma. At present, resection offers the best chance for cure of these patients. Unfortunately, it is always metaphase or afternoon when the hepatocarcinoma is diagnosed. These patients had resectable tumors is less than 20%. Drugs most in use are adriamycin,fluracil,PDD and so on. But the effects of these drugs are not satisfactory. So, lowly-toxicity and highly-effect drugs have been explored.Triptolide(Molecular formula,C20H24O6 ; molecular weight,360.4)is a diterpenoid triepoxide derived from the Chinese herb Tripterygium wilfordii Hook f. It has been demonstrated to be effective in Patients with a variety of inflammatory and autoimmune disease , such as rheumatoid arthritis,nephritis and lupus erythematosus. Recent studies showed that TPL possessed extensive and efficient anti-tumors properties. Through 30 years research , it was found that TPL can inhibit the growth of more than 60 tumors ,such as leukemia,lymphoma,breast cancer,osteosarcoma,stomach carcinoma,pancreatic cancer,hepatocellular carcinoma,cervix cancer,ovarian cancer,lung cancer,colon cancer,melanocarcinoma. TPL can inhibit the growth of hepatocellular carcinoma cell. Refering to the records of past years, TPL can inhibit the growth of SMMC-7721,Hepg2 and induce the apoptosis. But there were no study in TPL to hepatocellular carcinoma cells Bel-7402 in vitro and in vivo.Objectives: We chose Bel-7402,Hepg2 cells and xenografts of Bel-7402 in nude mice as objective and tried to reveal the hepatocarcinoma effects in vitro and in vivo and investigate the probable mechanism, provided theory for clinic.Methods:1.MTT and colony forming assay were used to detect the effect of TPL(5～80ng/ml) on the cell proliferation of Bel-7402 and Hepg2.2.AO-EB (acridine orange-thidium bromide ) staining,TUNEL assay,DNA fragmentation and transmission electron microscope (TEM) were used to detect the effect of TPL(5～80ng/ml) on the cell apoptotic of Bel-7402 and Hepg2.3. Reverse transcriptase-polymerase chain reaction analysis (RT-PCR) were used to examine the level of bcl-2,c-myc,bax mRNA. The mRNA was abstracted from Bel-7402 cells which treated by TPL(20,40,80ng/ml).4. The graph of tumor growth and the inhibitory rate of tumor weight were used to evaluate the effect of TPL(100,200,400μg/kg) on the xenograft. The xenograft models were established by injecting the Bel-7402 cells into nude mice. When the diameters of tumors were 0.5 centimeters, these mice bearing growing tumors were divided into six groups. There were 2% propylene glycolsaline (NS) negative control group,5-FU (30mg/kg) positive control and three treatment groups in which the dose of TPL in every animal was 400μg/kg,200μg/kg,100μg/kg. Reagents were administered via injection into the caudal veins every two days for seven times. We observed the length (a) and width (b) regularly, account volume of tumors with the formula V=ab2/2 and paint graph of tumor growth. We also observed the weight and appetite of nude mice regularly. These mice were executed after 24 hours of the last treatment. Tumors were peeled off fully and quantified. The changes of tumor weights were used to evaluate the effects of TPL on xenografts growth of Bel-7402 cells. The apoptosis of tumor cell was tested by transmission electron microscope assay. The blood was gained to detect the cell numbers, liver and renal function. The important organs were detected by histology assay to evaluate the toxic action of TPL to nude mice.Results: 1. TPL evidently inhibit proliferation of Bel-7402 and Hepg2 cells in a time dependent and dose dependent manner. The IC50 values were 42.82ng/ml and 7.32ng/ml respectively after 48 hours treatment.2. TPL could induce apoptosis in Bel-7402 and Hepg2 cells: AO-EB assay showed that TPL can induce apoptosis of Bel-7402 and Hepg2 cells. Transmission electron microscope also revealed that TPL can induce apoptosis of Bel-7402 cells. TUNEL assay showed that TPL induced apoptosis of Bel-7402 and Hepg2 cells in a dose-dependent manner. The apoptotic percentage of Bel-7402 cells treated by TPL (5,10,20,40,80ng/ml) were 9.2%,19.8%,30.2%,52.0% and 64.6% respectively. The apoptotic percentage of Hepg2 cells treated by TPL (5,10,20,40,80ng/ml) were 15.0%,31.6%,49.2%,58.7% and 69.1% respectively. DNA LADDER displayed typical echelon stripe.3. TPL could regulate the expression of apoptosis-related genes: RT-PCR analysis showed that the expression of c-myc,bcl-2 were down-regulated in Bel-7402 cells treated with TPL after 48 hours, whereas the expression of bax were up-regulated. These changes were in a dose dependent manner.4. In vivo TPL could inhibit xenograft growth of Bel-7402 cells. The inhibition rate Were 27.5%(p﹥0.05),50.0% (p﹤0.01)and 69.5%( p﹤0.01) in the group which TPL was injected to each mouse with a dose of 100μg/kg,200μg/kg and 400μg/kg every two days,the group of 400μg/kg TPL was superior to the group of 200μg/kg TPL(p﹤0.05). These showed that the inhibition was in a dose dependent manner. the group of 400μg/kg TPL was superior to the group of 5-FU(p﹤0.01).Transmission electron microscope showed there were more apoptotic cells in TPL groups. The mean weights of mice were uniform in TPL groups and negative control group(p﹥0.05). The blood cells count and liver and renal function of nude mice treated with TPL was not different to the nude mice of negative control group also. The pathologic examination of heart,liver,spleen,lung and kidney showed there were no pathological changes.Conclusion:1. TPL evidently inhibit proliferation of Bel-7402 and Hepg2 cells in a time dependent and dose dependent manner.2. TPL induce apoptosis of Bel-7402 and Hepg2 cells in a dose dependent manner.3. The mechanism that TPL induce Bel-7402 apoptosis was possibly related to down-regulated c-myc,bcl-2 and up-regulated bax.4. TPL could inhibit xenograft growth of Bel-7402 cells in vivo in a dose dependent manner and it is superior to 5-FU.
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