The PVDF membranes were probed with antibodies at optimal concentrations according to the manufacturer��s instructions. The specific signals were detected using the WesternBreeze chemiluminescence kit (Alkaline phosphatase such information conjugated secondary antibody) (Invitrogen, UK). Results were visualized using the GenGnome5 imaging system (Syngene, UK). Statistical analysis The unpaired two-tailed Student��s t-test was used for comparing mean values between two groups. Data are presented as mean �� SD. P < 0.05 was considered statistically significant. Results IGF-IR expression in pancreatic cancer cells We have reported recently the cell surface expression levels of HER family members on seven human pancreatic cancer cell lines and found all seven cancer cell lines to be positive for both EGFR and HER-2 , negative for HER-4 while expressing extremely low or undetectable levels of HER-3 [19].
Here, we determined the expression levels of IGF-IR in the same panel of pancreatic cancer cell lines using flow cytometry. All pancreatic tumour cell lines were found to be positive for IGF-IR, with MFIs ranging from 4.2 (FA6) to 22.7 (PT45) (adjusted to negative control) (Figure 1). In the majority of the pancreatic cancer cell lines examined, the IGF-IR expression levels were similar to the IGF-IR expression level in the control MCF-7 breast tumour cell line (MFI = 19.6) (Figure 1). Figure 1 Expression of IGF-IR in human pancreatic tumour cell lines assessed by Flow Cytometry as described in the Materials and methods. Results are expressed as Mean Fluorescent Intensity (MFI) values.
Breast cancer cell line MCF-7 was used as a positive control. … Growth response of human pancreatic cancer cell lines to treatment with HER family growth factors, IGF-I, IGF-II and insulin We determined the growth response of human pancreatic cancer cell lines to treatment with EGFR ligands (EGF, TGF��, AR, Epigen), HER-3 and HER-4 ligand NRG-1, EGFR and HER-4 ligands ( HB-EGF, Epiregulin and BTC) , IGF-IR ligands (IGF-I and IGF-II) and insulin at a concentration of 40 nM for 72 h using the SRB assay (Figure 2). For this assay, cells were grown in medium containing 2% FBS as in growth inhibition studies with other agents. We have shown previously that, at nM concentrations, EGFR ligands inhibit the growth of EGFR overexpressing tumour cell lines in vitro[24].
To confirm the bioactivity of exogenous HER ligands, we examined their effects on the growth of EGFR overexpressing HN5 cells. All HER ligands, except NRG-1, inhibited the growth of HN5 cells in vitro (Figure 2). In addition, with the exception of BxPC3 and AsPc-1 cell lines which exhibited significant growth response to NRG-1 (BxPc3: 36% increase Anacetrapib compared to the control, p<0.01, AsPc-1: 19% increase compared to the control, p<0.