However, little is known about the situation for biliary tract cancer (BTC), unless a rare tumor with a grim prognosis and limited treatment options. Two recent studies showed expression of IGF-1R and its ligands in gallbladder carcinoma (GBC)[40] and cholangiocarcinoma (CC) specimens[41]. Therefore, the objectives of the current study were to investigate IGF-1R expression in BTC cell lines and to evaluate the efficacy of in vitro treatment with selective IGF-1R inhibitor NVP-AEW541 alone or in combination with gemcitabine, 5-fluorouracil (5-FU) or Polo-like kinase 1 inhibitor BI 2536, which is currently being investigated in phase II studies including our hospital for the treatment of solid tumors[42].

MATERIALS AND METHODS Drugs and cells Seven BTC cell lines; five extrahepatic CC cell lines (EGI-1, TFK-1, CC-SW-1, CC-LP-1, and SK-ChA-1)[43-47] and two GBC cell lines (Mz-ChA-1, Mz-ChA-2)[46], were examined. All cell lines were cultured in a 37��C incubator with 5%-10% CO2 in appropriate media, which were changed every 3 d. NVP-AEW541 (targeting IGF-1R) was obtained from Novartis (Basel, Switzerland), dissolved in dimethyl sulfoxide (DMSO) (as 10 mmol/L stock) and stored at -20��C according to manufacturer��s instructions. BI 2536 (targeting Plk-1) was kindly provided by Boehringer (Ingelheim, Germany). Gemcitabine and 5-FU (diluted in 0.9% NaCl) were provided by our hospital pharmacy. Inhibition of cell growth Cytotoxic effects of drugs alone and in combination were determined by automated cell counting (Casy Cell Counter Model TT; Innovatis AG, Reutlingen, Germany) according to manufacturer��s instructions.

Briefly, 2 �� 105 cells were seeded in duplicates in T25 flasks with media containing the designated drugs or vehicle control followed by incubation for 3 or 6 d. For the 6 d experiment, medium was changed after 3 d and treatment repeated. At the end of incubation, cells were trypsinized, washed, and analyzed in triplicates by automated cell counting. Immunoblotting Cell culture monolayers were washed with ice-cold PBS and lysed in flask with a buffer containing Tris-HCl (50 mmol/L, pH 7.4), NP-40 (10 g/L), NaCl (200 mmol/L), sodium-orthovanadate (200 mmol/L), 2-glycerophosphate (1 mmol/L), sodium fluoride (20 mmol/L), DTT (10 mmol/L), PMSF (200 mmol/L) and 0.2% proteinase inhibitor cocktail (Sigma-Aldrich, Munich, Germany) on ice for 30 min. The lysate was then centrifuged at 13 000 r/min for 15 min and proteins in supernatant were quantified by Bradford protein assay (Bio-Rad, Munich, Germany) and stored AV-951 at -80��C.