Nonetheless, to our knowledge, no study has examined the therapeutic effect of selleckchem everolimus on OSCC using in vitro and in vivo assays. We therefore conducted this study with three main aims. First, we examined the importance of mTOR activation in OSCC by determining the overall prevalence of p-mTOR expression in OSCC specimens and cell lines. Second, we evaluated the therapeutic effect of everolimus on OSCC cell lines by both in vitro and in vivo assays. Third, we specifically assessed the effect of everolimus in combination with cisplatin, which is one of the most frequently used chemotherapeutic drugs, on OSCC cells. Materials and methods Reagents and antibodies Everolimus was provided by Novartis Pharma AG (Basel, Switzerland) and formulated at 2% (w/v) in a microemulsion vehicle.

For in vivo analysis, everolimus was diluted to the appropriate concentration in double-distilled water just before administration by gavage. For in vitro analyses, everolimus was prepared in DMSO just before addition to cell cultures. Antibodies recognising mTOR, phospho-mTOR (Ser2448), p70s6k, phospho-p70s6k (Thr389), 4E-BP1, phospho-4E-BP1 (Thr70), and ��-actin were purchased from Cell Signaling Technology (Boston, MA, USA). Patients The present study involved 167 consecutive patients who underwent surgical resection of OSCC at the Kumamoto University Hospital from January 1996 to December 2007. None of these patients underwent endoscopic mucosal resection, palliative resection, preoperative chemotherapy, preoperative radiotherapy, or preoperative chemoradiotherapy.

This study was approved by the Institute Review Board of the Graduate School of Medical Science, Kumamoto University (Approval number: 236; 2 August 2008). Immunohistochemistry for p-mTOR The method of immunohistochemical staining for p-mTOR was described previously (Hirashima et al, 2010). Of the 167 tumours, 51 showed no p-mTOR expression, 84 showed weak expression, and 32 showed strong expression. As the aim of the immunohistochemistry in this study was to evaluate the prevalence of p-mTOR expression in OSCC tissues, both weak and strong p-mTOR expression were defined as positive. Cell culture Oesophageal squamous cell carcinoma cell lines (TE series) were obtained from the Cell Resource Center for Biomedical Research, Tohoku University. Cell cultures were grown in the recommended medium with 10% foetal bovine serum and incubated in 5% CO2 at 37��C.

Western blot analysis Cultured cells were harvested and lysed in lysis buffer (25m Tris-HCl (pH 7.4), 100m NaCl, 2m EDTA, 1% Triton X-100, leupeptin, 1m Na3VO4, and 1m PMSF) for 30min. Lysates were Carfilzomib centrifuged at 10000rpm for 5min at 4��C. Each protein sample (10��g) was mixed with 5 �� sample buffer containing 10% ��-mercaptoethanol and boiled for 5min.