Our early observations also found that expression of Nrf2 was up-regulated in gallbladder cancer (GC) tissues and served as an independent prognostic factor [18]. Propofol has antioxidant properties partly through up-regulation of HO-1, a downstream target gene of Nrf2. We tested the hypothesis that propofol activates Nrf2, hence it affects the progression of cancer. The aims of the current study were to evaluate effects of propofol on the behavior of human GC cells

and role of Nrf2 in these effects. Materials and methods Cell culture and reagents Gallbladder carcinoma cells (GBC-SD) were obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Cells were cultured in RPMI 1640 media (Sigma, St. Louis, USA), supplemented with 10% fetal bovine serum and 100 units/mL of penicillin and streptomycin at 37°C in a humidified 5% CO2. Propofol selleck products was purchased from Aldrich (Milwaukee, WI). Propofol was diluted in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA) for in vitro assays. Cell growth assay The cells were seeded at a density of 5 × 103 cells/well in 96-well plates at a Selleck Tubastatin A final volume of 180 μL in incubation, at 37°C, with 5% CO2. After different time incubation, 20 μL of 5 mg/mL solution of MTT

(Sigma, St. Louis, MO, USA) in 1× PBS was added to each well. The plates were then incubated for 4 h at 37°C. The reaction was then solubilized in 100% DMSO, 20 μl/ well, and shaken for 15 min. Absorbance of each well was measured on a multidetection microplate reader (BMG LABTECH, Durham, NC, USA) at a wavelength of 570 nm. Apoptosis analysis The cells were washed twice with cold 10 mM 1× PBS and

resuspended in 1× binding buffer (BD Biosciences, San Jose, CA, USA). Apoptosis in GC cells was quantified by staining with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) [annexin V-Phycoerythrin (PE) and 7-amino-actinomycin D (7-AAD) for apoptosis analysis for cells transfected by ShRNA vectors with the GFP fluorescence] The samples were Orotidine 5′-phosphate decarboxylase analyzed using flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA). Cell invasion assay For invasion assay, the membrane invasion culture system (transwell membranes of 6.5 mm diameter and 8 μm pore size; Costar) was used according to the standard protocol. Briefly, harvested cells (1 × 105) resuspended in 100 μL of serum free RPMI 1640 were added into the upper compartment of the chamber. A total of 1000 μL conditioned RPMI 1640 medium with 20% (v/v) fetal bovine serum was used as a source of chemoattractant and placed in bottom compartment of chamber. After 48 hours, the noninvasive cells on the upper surface of the membrane were removed with a cotton swab. The transformed cells that migrated through the Matrigel matrix and stuck to the lower surface of the membrane were fixed with 4% paraformaldehyde, stained with 1% crystal purple.