The number of TUNEL positive cells was significantly lower in the BYHWD group t

Methods Chemicals and reagents STB HO was supplied from Seobong Biobestech Com pany, SW620, HCT116 and HCT15 human colorectal adenocarcinoma cells from the American Type Culture Collection were maintained in オーダー KU-0063794 RPMI 1640 supplemented with fetal bovine serum, liquid gentamicin reagent solu tion, penicillin and streptomycin, and trypsin EDTA were purchased from Gibco, Human umbilical vein endothelial cells cells from the American Type Culture Collection were maintained in M199 supplemented with 20% fetal bovine serum, liquid gentamicin reagent solution, penicillin and streptomycin, 3 ng ml bFGF, 5 units ml heparin.<br><br> Enhanced chemiluminescence Western blotting detection reagents and Hyperfilm ECL were from Amersham Pharmacia Korea, Anti rabbit IgG heavy and light chain specific peroxidase conjugates and antibody against p21, p27, p53, pp53, cyclin D1, pAKT, AKT, PI3K and PCNA were purchased from オーダー Lenalidomide Cell signaling technology, Antibodies of VEGFR2 and pVE GFR2 were purchased from Santa Cruz Biotechnology, B actin was purchased from Sigma Chemical Co, VEGF and MMP 9 ELISA kit were purchased from Invitrogen, Human recombinant VEGF was purchased from R D systems, Cell Proliferation ELISA kit was purchased from ROCHE, All other reagents used were purchased from Sigma Chemical, Cell culture SW620, HCT116 and HCT15 cells were seeded onto 100 mm Falcon plates at 2 × 106 cells mL in RPMI 1640 supplemented with 10% FBS and 1% penicillin strepto mycin. The cells were cultured at 37 C in a humidified atmosphere containing 5% CO2 to 60 80% confluence and then used for Western blot analysis.<br><br> STB HO was treated to various human colon cancer cells for 24, 48, 72 and 96 h. HUVECs were maintained in M199 plus 20% heat inactivated fetal bovine serum, 3 ng ml bFGF, 5units LY294002 154447-36-6 ml heparin, 100 units ml antibiotic antimycotic so lution in 0. 1% gelatin coated flasks and incubated at 37 C in a humidified atmosphere containing 5% CO2. Once confluent, the cells were detached by trypsin EDTA solution and used in experiments from the third to the sixth passages. Cytotoxicity assay Cytotoxicity of STB HO was evaluated by 3 2,5 diphenyl tetrazolium brom ide assay. Briefly, HUVECs were seeded onto 0. 1% gelatin coated 96 well microplates at a density of 5×103 cells per well and treated with various concen trations of STB HO for 48 h.<br><br> After indicated incubation times, MTT solution was added for 2 h and MTT lysis buffer was then added for overnight. Optical density was mea sured using a microplate reader at 570 nm. Cell viability was calculated as a percentage of viable cells in STB HO treated group versus untreated Cell proliferation in HCT116 cells with STB HO was evaluated as described by using Cell proliferation ELISA kit according to the manufacturers instructions. Briefly, after 48 h treatment of STB HO, the cells were added by 10 ul well of bromodeoxyuridine solution and reincubated for 2 h at 37 C. Then, BrdU solution was removed and 200 ul of FixDenat was added to each well. After incubation for 30 min at room temperature, FixDenat solution was removed and 100 ul of anti BrdU POD working solution was added to each well. After washing with PBS three times, 100 ul of sub strate solution was added to each well and the optical density was measured at 450 nm using microplate reader, All sam ples were prepared in triplicates and the assay was re peated at least three times.