Figure 1.
Cytotoxicity and IC50 values of EGFR-TKIs in parental HCC827 and resistant cell lines.
HCC827, HCC827/GR and HCC827/ER2 cells were treated with the indicated concentrations of gefitinib and erlotinib for 72 h in medium containing 1% FBS. Cell viability and IC50 values were determined using the MTT assay.
Figure 2.
Expression of EGFR-related signals in HCC827 and resistant cell lines.
(A) Basal expression of EGFR and EGFR-related signalling molecules in HCC827, HCC827/GR and HCC827/ER cells were evaluated by Western blotting. The effects of gefitinib (B) and erlotinib (C) on EGFR-related signalling were also examined. HCC827, HCC827/GR and HCC827/ER cells were treated with 0.1 and 1 µM gefitinib and erlotinib for 72 h in medium containing 1% FBS. Protein (30 µg) from cell lysates was subjected to Western blot analysis for the indicated proteins.
Figure 3.
Loss of IGFBP-3 in resistant cell lines and culture medium.
IGFBP-3 mRNA (A) and secreted IGFBP-3 (B) were determined by RT-PCR and ELISA in HCC827, HCC827/GR and HCC827/ER. The ELISA was repeated three times and the error bars represent standard deviation (SD). *P<0.001 compared with HCC827 cells.
Figure 4.
Effects of increased IGFBP3 on the sensitivity to EGFR-TKIs.
Resistant cells were infected with Ad/IGFBP-3 at MOIs of 0 to 100 PFU/cell for 48 h and IGFBP-3 expression was determined by Western blotting (A) and ELISA (B). (C) HCC827/GR and HCC827/ER cells were treated with the indicated concentration of EGFR-TKIs for 72 h after infection with 100 MOI of Ad/IGFBP-3. (D) HCC827/GR and HCC827/ER cells were treated with 1 µM EGFR-TKIs and 1 µg/mL rh IGFBP-3 for 72 h. Results are representative of at least three independent experiments, and the error bars represent standard deviation (SD). (E and F) Cells were treated with drugs, rh IGFBP-3 or Ad/IGFBP-3 as in panel C and D. After 24 h, cells were harvested and the modulation of EGFR and IGF1R signalling in the indicated cell lines was detected by Western blotting. (G) Control and IGFBP-3 siRNAs (100 nM) were introduced into HCC827 cells, and IGFBP-3 suppression was confirmed by Western blotting. (H) Cell viability was measured using the MTT assay 72 h later.
Figure 5.
Serum IGFBP3 levels before EGFR-TKI treatment and after the development of resistance.
IGFBP-3 levels were determined by ELISA in the serum of patients with NSCLC. Acquired resistance developed in all patients who initially responded to EGFR-TKIs.