AsPc-1 and Capan-1 cell lines were kindly STI 571 provided by Dr. Charlotte Edling (Blizard Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry). All cell lines were cultured routinely at 37��C in a humidified atmosphere (5% CO2) in either DMEM (Sigma �C Aldrich, UK) (Miapaca-2, Panc-1, HN5 and MCF-7) or RPMI-1640 medium (Sigma �C Aldrich, UK) (BxPC3, PT45, AsPc-1, Capan-1 and FA6) supplemented with 10% Foetal Bovine Serum (PAA, UK), antibiotics penicillin (50 units/mL), streptomycin (0.05 mg/mL) and neomycin (0.1 mg/mL) as described previously [19]. RPMI-1640 medium was supplemented with 2mM Glutamine (Sigma – Aldrich, UK). Antibodies and other reagents MAb ICR62 (IgG2b) was raised against the external domain of the EGFR on the breast cancer cell line MDA-MB468 as described previously [21].
The primary mouse anti-IGF-IR antibody used in this study for flow cytometry was purchased from R&D Systems (Abingdon, UK). Secondary FITC-conjugated rabbit anti-mouse mAb STAR9B was obtained from AbD Serotec (Kidlington, UK) while gemcitabine was acquired from Healthcare at Home (UK). PI3K inhibitor LY294002 and MAPKK/MEK inhibitor U0126 were purchased from Cell signaling (UK). The anti-IGF-IR TKI NVP-AEW541 and pan-HER inhibitor afatinib were kindly provided by Novartis (Basel, Switzerland) and Boehringer Ingelheim respectively (Vienna, Austria) [20,22]. Mouse antibodies against HER-2, HER-3, HER-4, p-IGF-IR (Tyr1165/1166) and anti-IGF-IR rabbit antibody were obtained from Santa Cruz, UK.
Mouse antibody against ��-actin was purchased from Cell Signalling, UK, while mouse anti-EGFR antibody from Sigma-Aldrich, UK. Rabbit antibodies against AKT, MAPK, phospho-MAPK (Thr202/Tyr204), p-HER-3 (Tyr1289), p-HER-2 (Tyr1221/1222) and phospho EGFR (Tyr1086) were purchased from Cell Signalling,UK while anti-phospho AKT (S473) rabbit antibody was obtained from Biosource, UK. Determination of cell surface expression of growth factor receptors The cell surface expression of IGF-IR was assessed by flow cytometry as described previously [19]. Briefly, about 1 million cells were incubated for 1 hour by rotation at 4��C, with the primary antibody or control medium alone. Cancer cells were then washed three times by centrifugation and incubated for 1 hour by rotation at 4��C with FITC-conjugated rabbit anti-mouse IgG STAR9B (AbD Serotec, UK).
A minimum of 10.000 events were recorded following excitation with an argon laser at 488 nm using the FL-1 detector (525 nm) of a BD FACsCalibur flow cytometer (Becton Dickinson Ltd, UK). Mean fluorescence intensity values were calculated using the CellQuest Pro software Carfilzomib (Becton Dickinson Ltd, UK) and compared with those of negative controls (no primary antibody). Cell growth studies The effect of the various agents, on the growth of human cancer cell lines was investigated using the Sulforhodamine B (SRB; Sigma �C Aldrich, UK) colorimetric assay as described previously [19].