| BACKGROUNDHepatocellular carcinoma (HCC) is the leading cause of of morbility and mortality of malignant tumors nationwide, which is frequently associated with metastasis in early stage and has a high rate of recurrence postoperation or intervention treatment and seriously threat our health and lives. However, So far, the long-term prognosis of patients undergoing potentially curative hepatic resection is still poor, with reported 3-year survival rates ranging from 40%to 50%and the overall 5-year survival rate for HCC patients less than 5%. The development and progression of HCC is a complicated process involving multiple genes and multiple steps in human bodies. This poor prognosis mainly contributes to the high rate of intrahepatic neighboring and distant metastasis after resection or transplantation. To date, the metastatic mechanisms and the regulated mechanisms of tumors are not known clearly, so it is very important for understanding the mechanisms of invasion and metastasis and searching effective approaches to prevent the recurrence and metastasis of HCC is of ever-increasingly important.The major therapeutics of HCC is surgery, traditional chemotherapy and radiotherapy. Traditional tumor therapies show high side-effects due to their non-specificity, which not only kill tumor cells but do harm to normal tissue. Nowaday, trends in anticancer drugs are personalized usage of chemotherapeutic agents and discovery on molecular targeted ones, tumor targeted therapy become promising strategies and increasingly important for cancer therapy, however, those roles are limited in clinic by severe side effects and huge costs.It's an important task for us to find out some drugs that possess higher anticancer activity and lower toxicity and are even cheaper. Traditional Chinese medicine (TCM) may be an useful model for scientific research because of its standardized system of therapies and long-time practices. Chinese medicine provides a rich pool of novel and efficacious agents for treating a variety of cancer. So, the discovery, isolation, identification and study of new effective anticancer constituents of Chinese medicine and investigation its mechanism using molecular biochemistry methods are the major issue and a long-term task for the biomedical research and are also ascendant aspects of drug-development in our country.Gecko is tradition medicine,which treats many difficulty miscellaneous disease,especial maligmant tumors through literature retrieval. Gecko is an anticancer drug avaible cheaply, efficient and safe .OBJECTIVETo study the antitumor effect and mechanism of Gecko ethanol extract (GEE) on H22 hepatoma transplanted subcutaneously in Kunming mice in vivo and human Hepatocellular carcinoma-HepG2 cells in vitro.METHODSIn the study, GEE is preparated in the laboratory by oneself. HepG2 cells were treated with GEE at different concentration ( 0.125, 0.25, 0.50, 1.0, 2.0, 4.0, 8.0 mg/ml) in vitro, additionally, HepG2 cells were established with positive group ( adriamycin, ADM 0.005mg/ml ) and control group. MTT assay was applied to detect and analyze the proliferation inhibitory effect of the GEE on HepG2 cell and to calculate inhibitory concentration and get cell growth curve. The change of cell morphology was observed through inverted microscope. Morphological chang of apoptotic cell was observed by Hoechst33258 fluorescence staining. Cobalt chloride was used to mimic the cell hypoxic microenvirovement, The protein expression of hypoxia-inducible factor-1a( HIF-1α) in HepG2 cells induced by cobalt chloride in vitro was studied by SP(streptavidin-horseradis hperoxidase) immunohistochemistry.The transplant tumor model of H22 hepatocarcinoma in mice was established. Fifty mice were randomly divided into five groups, H22-bearing mice were given GEE via intraperitoneal injection, the adriamycin group, high, middle, low dose GEE groups and the control group,they were treated respectively with intraperitoneal injection of high, middle, low dose GEE(1.60, 0.80, 0.40g/kg/d) and adriamycin 5μg/g every day(3 times totally), and equal volume of saline, the medication was given for 15 days totally. Fifty mice were killed by cervical dislocation on the 16th day. the inhibitory effect of GEE on tumor growth was examined on transplanted model of mice H22. the tumor inhibitory rate and the levels of serum IL-12/IL-18 of mice were detected. The tumor weight, tumor index, the count of peripheral white blood cells, index of thymus and spleen were calculated. Statistical AnalysisStatistical treatment with SPSS 13.0 software was applied to deal with all the experimental data, data were analyzed by t-test between groups and one-way ANOVA followed by a Student-Newman-Keuls means comparison test (data from in vitro and in vivo samples were analyzed with one repeated measure) means were considered significantly different if P<0.05.RESULTSGEE inhibited the human hepatocarcinoma HepG2 cells growth, induced HepG2 cell apoptosis in vitro. MTT assay showed that with concentrations from 0.125 mg/ml to 8mg/ml, GEE can significantly inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner (P<0.01). After HepG2 cells were dealed with different concentration of GEE in 24~72hours, the OD values of the treatment groups decreased to some extent compared with the control group. GEE can significantly inhibit cell proliferation and induce apoptosis in HepG2 cells, GEE can also down-regulate the expression of HIF-1αin HepG2 cells (P<0.01). GEE possessed markedly inhibitory activity on the growth of H22 tumor in mice, the dosage of GEE were 1.60, 0.80 and 0.40 mg/kg, their inhibitory rate was 55.25%, 47.89% and 15.16%respectively in H22 mice. Compared with model group or positive group, The weight of thymus and spleen had a significant difference, The numbers of peripheral blood white blood cells and the levels of serum IL-12/IL-18 increased significantly.CONCLUSION1. GEE can significantly inhibit tumor growth in vitro and in vivo, their anticancer activatity presents a time-and dose-dependent manner.2. GEE has a significant immunoregulation effect in vivo.3. GEE can induce apoptosis and down-regulate markedly the expression of HIF-1αin hepatic carcinoma HepG2 cells. 4. This work contributes to disclose the global profile of antitumor effect and mechanism of GEE human HepG2 in vitro and in vivo tumor-bearing H22 mice. |
