HSP the treatments, the cells were washed with cold PBS and immediately lysed with 1 ml lysis buffer, 50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X 100, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 mM sodium orthovanadate, 30 mM p nitrophenyl phosphate, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride,The cells were cultured in Lab Tek Chamber slides from Nalgene Nunc International and treated as described in the rowth assays?section. After 24 48 h of cell culture, the cells were washed in PBS and fixed either in 4% PBS paraformaldehyde for 5 min for the immunostaining of membrane associated antigens or in cold 1:1 acetone:methanol mixture in ice for 2 min for the immunostaining of cellular or nuclear antigens. Primary and secondary antibodies were used according to the manufacturer,s protocols. The quantification of TrkA, B, C and p75 NGFR as well as that of EGFR/ HER2 positive cells was performed by flow berberine cytometric analysis. The cells were trypsinised, centrifuged and left at 37C for 1 h in DMEM/10% FCS in polypropylene tubes in order to reconstitute the cellular external membrane.
The cells were washed in saline disufenton sodium buffer and 1×106 cells were treated with about 10 g/ml primary antibodies. After 1 h at 4C the cells were washed twice in PBS and FITC anti rabbit and anti mouse secondary antibodies were then added. After 30 min incubation at 4C, the cells were washed twice and resuspended in PBS at a density of 1×106 cells/ml before analysis using Cell Quest software. Statistical analysis. Data are expressed as the means SEM of at least three independent experiments. Statistical analysis was performed using an unpaired Student,s t test. The comparison on the growth factor receptor expression was performed using Fisher,s exact test. P values 0.05 and 0.01 were considered statistically significant.In addition, the effects with both the commercial AG879 and CEP701 were higher in PC3 TKI R when compared to the parental cells. The levels of the phosphorylated form of TrkA were higher in TKI R vs WT PC3 cells as shown in Fig. 6A and CEP701 was able to abrogate this activation status. In addition, we observed that NGF was able to activate Her2/ Neu and its phosphorylation was reduced by CEP701. EGF and NGF were able to induce Erk dabigatran activation both in PC3 WT and in TKI R cells. In Fig. 6D we show the Erk activation in TKI R cells.
As opposed to the parental cells, gefitinib treatment was not able to reduce Erk activation, whereas CEP701 or AG825 were able to significantly reduce Erk activity, supporting the idea that the basal and GF dependent increment in the MAPK activation was associated with gefitinib resistance. In addition, NGF was able to induce Her2 phosphorylation in a ligand independent manner suggesting that the cooperation between TrkA and Her2 is important in the progression of PCa and that a dual inhibition could represent an important mechanism for reducing epigenetic changes accompanying several phases of drug resistance.Clinical data have already demonstrated that the prevention of EGFR mediated signal transduction by the small molecule inhibitor, gefitinib, provides a promising new treatment option for a variety of cancer types. The data documented the existence of an intrinsic or de novo resistance to the drug.