We first investigated histopathologic changes in the peritoneum and TGF-β1 concentrations in peritoneal lavage fluid. We then determined the effects of TGF-β1 on the function of human peritoneal mesothelial cells (HPMCs) and of microenvironment changes on the ability of gastric cancer cells to attach to mesothelial cells in the early stages of peritoneal dissemination. Materials and methods Reagent and Instrument Total Smad-2/3, phosphorylated-

Selleck RG7112 Smad2 and phosphorylated- Smad3 antibodies, as well as second antibodies were purchased from Santa Cruz Biotechnology Inc, USA. Calcein-AM was brought from CALBIOCHEM, UK. RGD (Arg-Gly-Asp), which is ATR inhibitor the cell binding domain of the ECM, were obtained from Sigma (Osaka, Japan). Dulbecco’s modified Eagle’s medium and fetal calf serum(FCS) were purchased from GIBCOBRL,

USA. Human TGF-β1 was obtained from Sigma, USA. human TGF-β1 ELISA kit (R&D, Minneapolis, MN, USA). Hematoxylin and eosin and Masson stain kit(Santa Cruz Biotechnology Inc, USA). Phasecontrast microscope (Japan Nikon). Spectrofluorometer (Japan Olympus, Japan) were employed. Other laboratory reagents were obtained from Sigma, USA. Cell line and culture A human peritoneal mesothelial cell line HMrSV5 was kindly provided by Prof. Youming Peng of the Second Hospital, Zhongnan University, Changsha, PR China and Prof. Pierre RONCO, Hospital TENON, Paris, France. This cell line was established after infection of a fully characterized primary culture of human peritoneal mesothelial cells with an amphotropic recombinant retrovirus that encodes SV40 large-T Ag under control of Moloney virus long terminal repeat. An undifferentiated human gastric carcinoma cell line, HGC-27, was obtained from the Cancer Research Institute of Beijing, Pregnenolone PR China, and HSC-39 cell line was derived from the ascites of a signet ring cell

gastric carcinoma, which was obtained from the Department of Medicine, Kyushu University, Japan. These cell lines were cultivated in T75 tissue culture flasks in DMEM supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 20 mM hydroxyethyl Vactosertib piperazine ethanesulfonic acid (HEPES). Cultures were grown at 37°C in a humidified 5% CO2 and 95% air incubator. Tissue samples Human peritoneum tissue samples were obtained from 36 gastric cancer patients and 6 benign disease patients who underwent surgery in the First Affiliated Hospital of China Medical University between March 2009 and October 2009. These tissue specimens were taken from the lower anterior abdominal wall. No patients had received any form of radiation or chemotherapy before surgery.