Finally, we selected TCA for this study because of its solubility in the stomach, i

Finally, we selected TCA for this study because of its solubility in the stomach, i.e., TCA has pKa of 1 1.9 and GCA has pKa of 3.8 [37]. Some authors have reported various effects of bile acids not IRL-2500 only on cell death but also on cell proliferation. acids concentration of 14655 M and 18620 M [26]. The influence of tumor angiogenesis on cancer progression has been debated over the last decades. In the clinical setting, high rates of transforming growth VCA-2 factor (TGF)-1, vascular endothelial growth factor (VEGF), and Cox2 expression have been found to be associated with poor prognosis in patients with esophageal cancer [27]C[30]. To explore the role of angiogenesis on cancer progression induced by continuous TCA exposure, we analyzed protein and mRNA expression levels of angiogenic factors. We demonstrate that continuous TCA exposure promotes ESCC progression through reduced cell loss induced by TGF-1 and VEGF-mediated neovascularisation. Materials and Methods Cell Culture and TCA Treatment We used ESCC-DR cells that were established from a tumor induced in a rat model of gastroduodenal reflux [17]. The cells were grown and maintained in Dulbeccos altered Eagles medium (DMEM; Nacalai Tesque, Kyoto, Japan) supplemented with 1% antibioticCantimycotic answer (Gibco, NY, USA) and 10% fetal bovine serum (FBS; PAA Laboratories, Pasching, Austria) in a humidified incubator made up of 5% CO2 at 37C [20]. The cells were incubated in the growth medium made up of 2 mM taurocholic acid sodium salt hydrate (TCA, SIGMA, St. Louis, USA) for 2 months before analysis. These cells were termed tca. ESCC-DR IRL-2500 cells cultured in the growth medium without TCA over the same period were IRL-2500 used as a control in this study. Flow Cytometry for Cell Cycle Analysis The cells seeded in 75-cm2 flasks were exposed to 2 mM TCA or 300 M deoxycholic acid (DCA, Sigma) for 24 h. They were harvested, washed with PBS, and fixed with 70% ethanol at room heat for 30 min. The fixed cells were centrifuged and washed with PBS thrice. They were then resuspended in 0.5 mL of PBS containing 2 mg/mL RNase A (Sigma) at 37C for 30 min and stained with 50 g/mL propidium iodide (Nacalai Tesque) at 4C for 1 h. The cellular DNA content was measured using FACSCalibur (Becton Dickinson, NJ, USA). Cell Growth Assay An MTT assay was used to evaluate cell growth. The control and tca cells were seeded in 12-well plates (1104 cells/well). After 3, 24, 48, 72, or 96 h of incubation, medium made up of 0.25 mg/mL MTT was added to the calls (DOJINDO, Kumamoto, Japan). Formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm using an Infinite M200 microplate reader (TECAN, M?nnedorf, Switzerland). Preparation of Cell Lysate and Western Blotting The following primary antibodies were used to perform western blotting: Akt (pan) mouse mAb (cat. #2920, Cell Signlaing, MA, USA), Phospho-Akt (Ser473)(D9E)XP rabbit mAb (cat. #4060, Cell Signaling), p44/42 MAP Kinase (L34F12) mouse mAb (cat. #4696, Cell Signaling), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb (cat. #4370, Cell Signaling), Anti-Rat Cox2 Rabbit IgG Affinity Purify (cat. 18955, IBL, Gunma, Japan), and -actin (C4) mouse mAb (cat. sc-47778, Santa Cruz, CA, USA). Goat peroxidase-conjugated anti-rabbit IgG (cat. ab6721, Abcam, Cambridge, UK) and goat peroxidase-conjugated anti-mouse IgG (cat. AP124P, Millipore, MA, USA) were used as secondary antibodies. The cells were washed with PBS and lysed in lysis buffer [50 mM TrisCHCl, pH 7.4; 150 mM NaCl; 0.5 mM ethylenediaminetetraacetic acid (EDTA); 1% Nonidet P-40] made up of a mixture of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 g/mL leupeptin, 1 g/mL pepstatin A, and 0.09 U/mL aprotinin) and 1% phosphate inhibitor cocktail II (Sigma). After incubation at 4C for 30 min and mixing with a vortex mixer, the cell lysates were centrifuged at 12,000at 4C for 10 min. The supernatants were collected, and the protein content was quantified using the BCA protein assay reagent (Thermo Fisher Scientific, Waltham, MA, USA). PAGE was performed according to the manufacturers instructions (NuPAGE kit; Invitrogen, CA, USA). Protein samples were solubilized in NuPAGE LDS.