Anti-angiogenesis Effect Of Arsenic Trioxide In Combination With TNP-470 On Hepatocellular Carcinoma

Objective: To illustrate the possible synergistic effect of Arsenic Trioxide (As2O3) and TNP-470 on anti-angiogenesis in hepatocellular carcinoma(HCC).Methods: Experiments in vitro and in vivo were done respectively. 1. In vitro studies. (1) Tetrazolium salt(sMTT)assay: human hepatocellular carcinoma cell line SMMC-7721 and human umbilical vein cell line ECV-304 were respectively inoculated in cultivating dishes, after cells adherencing, added As2O3 and TNP-470 (the final concentration of As2O3 0,1,2,4μg/ml combining with the final concentration of TNP-470 0,0.5,1,2μg/ml respectively, all 16 groups), then cell surviving fraction was analyzed by tetrazolium salts(MTT)assay. (2)Transmission election microscopy dectection: after cultivating As2O3 and TNP-470 for 48h, the cells were collected and morphological changes were observed by transmission election microscopy. (3)Immunohistochemistry: collecting the cells that were treated by As2O3 (2μg/ml) and TNP-470 (2μg/ml) for 48h, and detecting the expression of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor(EGFR) in SMMC-7721 and ECV-304. 2. In vivo studies. (1) Chick chorioallantoic membrane(CAM) method technology. One hundred well-developed eggs were divided into four groups randomly, twenty-five for each group (NS control group,20μg/ml As2O3 group,20μg/ml TNP-470 group,20μg/ml As2O3 and 20μg/ml TNP-470 group), then incubated with methyl cellulose discs as carriers and viewed the depressive effects of angiogenesis in CAM. (2) Thirty-two of tumor-bearing nude mice of the high metastatic human HCC LCI-D20 were induced and divided randomly into 4 groups(NS group, As2O3 group, TNP-470 group, As2O3 and TNP-470 group), 8 mice for each group. Administration of the drug started on the 9th day after the tumor's transplantation. As2O3(2.5mg/kg) were treated abdominally every day and TNP-470(30mg/kg) were treated subcutaneously every other day. Three weeks later, the mice were killed. The tumor mass were striped to weigh and detected by optical microscope and transmission electron microscope. The microvessel density(MVD) and the expression of VEGF,...