These cell lines also appeared to vary in PEITC sensitivity

as seen from the proliferation study. Further, we showed that treatment with PEITC leads to loss of microtubular filaments with the subsequent formation of apoptotic bodies in transfected Kato-III cells, suggesting this mechanism as an important element in induction of cell cycle arrest and apoptosis. Collectively, our findings contribute to an increased understanding of the mechanisms underlying the PEITC-induced growth inhibition in gastric cancer cells and consequently prevention of gastric cancer induced by naturally occurring ITCs. Non-adherent human gastric cancer Pirfenidone chemical structure cell line Kato-III[19] was purchased from LGC (U.S.). Adherent human gastric cancer cell line MKN74 was kindly provided by Timothy Wang. Non-adherent Kato-III cells and monolayer culture of MKN74 were maintained in coated 75 cm2 cultivation vessels with RPMI-1640 (Sigma, Oslo, Norway) and Dulbecco’s modified Eagle’s medium (DMEM; Sigma), respectively, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin

cocktail (Sigma) at 37°C and 5% CO2 in a humidified incubator. Cells (5 × 103) were seeded out MG-132 solubility dmso in the wells of a 96-well plate before cultivating over night in the case for Kato-III or over two nights for MKN74. The cells were then treated with solvent (0.1% dimethylsulfoxide [DMSO]) or PEITC (Sigma). At ended treatments, cells were added 100 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) in a solution of 0.5 mg/mL followed by incubation for 4 h in the growth incubator. The volume was then halfed, and the remaining suspensions were mixed with 100 μL acidified isopropanol for 20–25 min at room temperature before absorbance STK38 at 595 nm was measured using a spectrophotometric plate reader. Using a 6-well plate, 0.5 × 106 cells were

seeded out before incubating either over night (Kato-III) or over two nights (MKN74) before treated with solvent (0.1% DMSO) or PEITC. After 24 h treatment, cultures were investigated using a contrast microscope. Using a 24-well plate, 0.1 × 106 MKN74 cells were seeded out followed by incubating over two nights. In the centrum of a confluent cell culture, a wound was made by denuding an area using a custom modified silicon cell scrape. The cultures were then washed with serum-free DMEM to remove non-confluent cell debris before fresh medium with vehicle control or PEITC was added to the cultures. Migration was monitored by capturing pictures through a contrast microscope. For cell cycle analysis, 5 × 105 cells were incubated in 25 cm2 flasks either over night (Kato-III) or over two nights (MKN74) before treatment. Following treatment, cells were harvested and then resuspended and incubated with −20°C chilled pure methanol for 15 min at −20°C.