The toxin is an acidic enzyme that exhibits high enzymatic activity (4,884.53 U/mg/min), has isoelectric point of 3.9 and molecular mass of 60.36 kDa, and represents 0.3% of the venom proteins. cell viability of HepG2 cells in monoculture at all concentrations tested. In co-culture, 1.00 and 5.00 g/mL induced cytotoxicity ( 0.05). BjussuLAAO-II increased the methylation of and decreased the methylation of in monoculture and in both cell-culture models ( 0.05). A 839977 Conclusion: Data showed BjussuLAAO-II induced cytotoxicity and altered DNA methylation of the promoter regions of cell-cycle genes in HepG2 cells in monoculture and co-culture models. We suggested the analysis of DNA methylation profile of as a potential biomarker of the cell cycle effects of BjussuLAAO-II in malignancy cells. The tumor microenvironment should be considered to comprise a part of biotechnological strategies during the development of snake-toxin-based novel drugs. snake venom, in human hepatocellular carcinoma (HepG2) cells in monoculture and in co-culture with an endothelial cell collection (HUVEC). Methods Toxin BjussuLAAO-II was isolated from snake venom according to the process explained by Carone RCBTB1 et al. . The toxin is an acidic enzyme that exhibits high enzymatic activity (4,884.53 U/mg/min), has isoelectric point of 3.9 and molecular mass of 60.36 kDa, and represents 0.3% of the venom proteins. Before performing the biological assays, LAAO enzymatic activity was determined by a spectrophotometric assay using L-leucine as a substrate . The isolated and purified protein was stored at 4C. A 839977 The vehicle employed to dilute the protein was phosphate buffered saline (PBS, pH 7.4). Cell lines and culture conditions Human hepatocarcinoma cells (HepG2 – catalog #HB8065) and human umbilical-vein endothelial cells (HUVEC – catalog #CRL-1730) were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). The cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 1% antibiotic-antimycotic answer (5 mg/mL penicillin, 5 mg/mL streptomycin, and 10 mg/mL neomycin), and 0.024% (w/v) NaHCO3, in a CO2 incubator with 5% atmosphere, at 37 C and relative humidity of 96%. The media were changed every 2-3 days; when the cultures experienced reached confluency, the cells were washed twice in PBS, detached with Trypsin/EDTA (0.25%), centrifuged at 174 x for 5 min and sub-cultured. All the experiments were conducted between the third and the eighth cell passage and they were cultured as reported by Bal-Price and Coecke . Co-culture system Thincert? (Greiner Bio-one, Kremsmnster, Austria) cell-culture inserts with 0.4 m porous polycarbonate membranes in 6-well plates were used in cellular co-culture systems. HepG2 cells (2105 cells/well) were grown adhering to the bottom of the well whereas HUVEC cells (1104 cells/well) were grown in the upper compartment [20-23]. The Millicell ERS? volt-ohm meter (Merck-Millipore, Burlington, Massachusetts, USA) was employed to monitor electrical resistivity of HUVEC cells. The inserts whose transepithelial electrical resistance was greater than or equal to 750 /cm2 were considered confluent; when this value was reached, HepG2 cells were seeded underneath the well in co-culture plates. Experiments in co-culture systems followed the same protocols explained for monoculture systems. MTT assay A 839977 Cell viability was decided using the MTT assay, as reported by Mosmann . In monoculture systems, HepG2 and HUVEC (1104 cells/well) were seeded in 96-well plates. In co-culture systems, 6-well plates were used and HepG2 were seeded in the lower (4105 cells/well) and HUVEC (1104 cells/well) A 839977 was placed in upper compartments. In both systems, cells were incubated for 24 h and treated with BjussuLAAO-II (0.25; 0.50; 1.00 and 5.00 g/mL), PBS (negative control) or.