Chemicals and reagents
STB-HO (particled Mica; Korea Patent Registration; 10–0454200) was supplied from Seobong Biobestech Company (Seoul, Republic of Korea). SW620, HCT116 and HCT15 human colorectal adenocarcinoma cells from the American Type Culture Collection (ATCC, Manassas, VA, USA) were maintained in RPMI 1640 supplemented with fetal bovine serum (FBS), liquid gentamicin reagent solution, penicillin and streptomycin (PEST), and trypsin EDTA were purchased from Gibco (Carlsbad, CA, USA). Human umbilical vein endothelial cells (HUVECs) cells from the American Type Culture Collection (Manassas, VA, USA) were maintained in M199 supplemented with 20% fetal bovine serum (FBS), liquid gentamicin reagent solution, penicillin and streptomycin (PEST), 3 ng/ml bFGF, 5 units/ml heparin. Enhanced chemiluminescence (ECL) Western blotting detection reagents and Hyperfilm ECL were from Amersham-Pharmacia Korea (Seoul, Korea). Anti-rabbit IgG heavy and light chain-specific (rabbit, mouse) peroxidase conjugates and antibody against p21, p27, p53, pp53, cyclin D1, pAKT, AKT, PI3K and PCNA were purchased from Cell signaling technology (Denver, MA, USA). Antibodies of VEGFR2 and pVEGFR2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). β-actin was purchased from Sigma Chemical Co. (St. Louis, MO, USA). VEGF and MMP-9 ELISA kit were purchased from Invitrogen (Carlsbad, CA, USA). Human recombinant VEGF was purchased from R&D systems (Minneapolis, MN, USA). Cell Proliferation ELISA kit was purchased from ROCHE (F. Hoffmann-La Roche Ltd, Switzerland). All other reagents used were purchased from Sigma Chemical (St Louis, MO, USA).
SW620(ATCC CCL-227™), HCT116(ATCC CCL-247™) and HCT15 cells(ATCC CCL-225™) were seeded onto 100 mm Falcon plates at 2 × 106 cells/mL in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin. 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. 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 (FBS), 3 ng/ml bFGF, 5units/ml heparin, 100 units/ml antibiotic-antimycotic solution (complete M199) 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 of STB-HO was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) 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 concentrations of STB-HO (0, 15.63, 31.25, 62.5,125, 250, 500, or 1000 μg/ml) for 48 h. After indicated incubation times, MTT (1 mg/ml) (Sigma Chemical Co., St. Louis, MO) solution was added for 2 h and MTT lysis buffer (20% SDS and 50% dimethylformamide) was then added for overnight. Optical density (OD) was measured using a microplate reader (TECAN, Austria) at 570 nm. Cell viability was calculated as a percentage of viable cells in STB-HO treated group versus untreated control by following equation.
Cell proliferation in HCT116 cells with STB-HO was evaluated as described by using Cell proliferation ELISA kit (Roche, Swiss) according to the manufacturer’s instructions. Briefly, after 48 h treatment of STB-HO, the cells were added by 10 μl/well of bromodeoxyuridine (BrdU) solution and reincubated for 2 h at 37°C. Then, BrdU solution was removed and 200 μl of FixDenat was added to each well. After incubation for 30 min at room temperature, FixDenat solution was removed and 100 μl of anti-BrdU-POD working solution was added to each well. After washing with PBS three times, 100 μl of substrate solution was added to each well and the optical density was measured at 450 nm using microplate reader (Molecular Devices Co., Sunnyvale, CA, USA). All samples were prepared in triplicates and the assay was repeated at least three times.
Cell cycle analysis
HCT116 cells were treated with STB-HO (250 and 500 μg/ml) for 24, 48 and 72 h. The cells were fixed in 75% ethanol at -20°C and treated with RNase A (10 mg/ml) for 1 h at 37°C, stained with propidium iodide (PI) (50 μg/ml) and analyzed for the DNA content by FACSCalibur (Becton–Dickinson, Franklin Lakes, NJ, USA) using CellQuest Software (BD Bio-sciences, San Jose, CA, USA).
Cells (5×106 cells) treated with STB-HO were lyzed by using lysis buffer (50 mM Tris–HCl, pH 7.4, 300 mM NaCl, 0.5% Triton X-100, 0.1% SDS, 5 mM EDTA, and protease inhibitor cocktail). The extracts were incubated on ice for 30 min, and then centrifuged at 13,000×g for 30 min at 4°C and the supernatants were collected for western blotting. Protein concentrations were determined by Bradford assay (Bio-Rad), and equal amounts of proteins (30 μg) were separated by electrophoresis sodium dodesyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Amersham Biosciences, Piscataway, NJ, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 for 2 h at room temperature. The membranes were probed overnight at 4°C with mouse anti-human β-actin (1:1000; Sigma Aldrich, St. Louis, MO, USA), anti-human pAKT, AKT, p21, p27, p53, pp53, cyclin D1, PCNA and PI3K (1:1000; Cell signaling, Danvers, MA, USA), anti-human VEGFR2 and pVEGFR2 (1:500; Santa Cruz Biotechnology, CA, USA) followed by washing and incubation with HRP conjugated secondary antibody (AbD Serotec, Raleigh, NC, USA). Immunoreactive bands were visualized using the ECL system (Amersham-Pharmacia, Seoul, Korea).
Measurement of VEGF and MMP-9 production by ELISA
VEGF and MMP-9 levels in HCT116 cells treated with STB-HO were measured using VEGF and MMP-9 ELISA kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, the culture supernatants was added onto a 96-well microplate, and incubated for 2 h at room temperature. The plate was then washed four times with washing buffer and 100 μl of biotin conjugate was placed to each well for 1 h at room temperature. After washing four times with washing buffer, 100 μl of the stabilized chromogen was placed to each well and incubated for 30 min at room temperature in dark. Finally, 100 μl of stop solution was added to each well and the optical density was measured at 450 nm using microplate reader (Molecular Devices Co., Sunnyvale, CA, USA).
HCT116 xenograft model
Four-week-old female BALB/c athymic nude mice (18 ± 3 g) were purchased from Chung-Ang Laboratory Animals (Seoul, Korea) and housed in animal facility at 22 ± 3°C and 60 ± 10% humidity with light-controlled (12 h, 07:00–19:00) environment. All materials including bedding and feed were sterilely cleaned by UV rays for 30 min before treatment to the mice. The animal study was conducted under the guidelines approved by Institutional Animal Care and use Committee, Kyung Hee University [KHUASP(SE)-11-005] as previously described with minor modifications . Briefly, 2 × 10 6 of HCT116 cells were mixed with Matrigel (Becton Dickinson, 50% in 100 μl) and injected subcutaneously into the right flank of 6-week-old male BALB/c athymic nude mice (Chung-Ang Laboratory Animals, Seoul, Korea)) for 3 groups (Control and two STB-HO treated groups). After 1 week adaptation, the animals were assigned to four groups (n = 6): negative control (vehicle (saline) + HCT116 inoculation), STB-HO50 (50 mg/kg+ HCT116 inoculation), and STB-HO100 (100 mg/kg+ HCT116 inoculation). Everyday STB-HO dissolved in saline was orally treated to the athymic nude mice for 41 days during experiment period. Tumor size was monitored twice a week with a caliper, and tumor volume was also calculated as described . At the end of animal study, tumors were dissected, weighed and photographed.
Data were shown as means ± SE. Significant differences were evaluated using Student’s t-test and a Turkey-Kramer multiple-comparison post test.