Fig 1.
Cell inhibition growth analysis of ERL-resistant NSCLC cell lines.
A) Representative dose-effect curve plots of HCC827 and HCC4006 parental cell lines to the indicated TKIs. Cell viability was determined by MTT assays. The results are expressed as the percentage of cell viability in drug-treated cultures relative to DMSO-treated control samples; B) Dose effect curve plots of derived ERL-resistant HCC827 and HCC4006 cell lines. The results are expressed as described above. Data (mean ± s.e.m) in A and B are representative of more than three independent experiments.
Table 1.
IC50 values of TKIs against parental and ERL-resistant NSCLC cell lines.
Fig 2.
The ERL-resistant HCC827 cells lines are tumorigenic.
A) Soft agar colonies were counted by ImageJ software. The percentage of colony forming efficiency (CFE) of parental (P) and ERL-resistant cell lines, as mean ± s.e.m., is shown. Histograms in gray and white colors indicate respectively vehicle (DMSO, 0.01%) and ERL (erlotinib 1 μM) cell treatments; B) Tumor volume of xenograft nodes, calculated as described in material and methods, are shown as mean ± s.e.m; C) Times to four (4x) and six fold (6x) increases from initial tumor volumes (day 3) are shown respectively by white and grey bars. P-values < 0.0001 are indicated.
Fig 3.
Analysis of the EGFR gene in the RA2 ERL-resistant cell line.
A) Analysis of EGFR exon 19 nucleotides sequence. The pherogram of the parental cell line with peaks corresponding to the EGFR mutated sequence (ΔE746-A750) and the pherogram of the RA2 resistant cells with peaks corresponding to the mutated and wild type (WT) EGFR nucleotides sequence are shown. B) qPCR analysis. Relative EGFR gene copy number (GCN) in genomic DNA, normalized to the Rnase P gene, is expressed relative to the levels in parental cell lines (P) set as 1 (mean ± SD of triplicate determinations). Results are representative of those obtained from 2 independent analysis.
Table 2.
Analysis of EGFR and KRAS gene mutations.
Fig 4.
Erlotinib impairs EGFR and ERK1/2 phosphorylation in ERL-resistant cell lines.
A) Representative western blots with EGFR and ERK1/2 antibodies and B) pEGFR (Y1068) and pERK1/2 (T202/Y204) antibodies in the indicated parental and ERL-resistant cell lines treated with EGF (100 μg/ml), ERL (Erlotinib, 100 nM) or vehicle (DMSO) at different time points (8’, 30’, 1hr, 3hrs). Densitometric analyses of band signals were normalized with GAPDH, the number indicates the signals quantification at 30’ upon ERL-treatment. For RA2 cell line double amount of total cell lysate was loaded to analyze EGFR expression.
Fig 5.
HER2/HER3 and AXL expression and phosphorylation analysis.
A) Representative western blots of total cell lysates of HCC827 and HCC4006 parental cell lines (P) and their derived ERL-resistant cell lines. Arrows indicate the expected molecular weight size. Total cell lysates loaded were 40 μg for AXL and pAXL analyses and 25 μg for the others. B) qPCR analysis of AXL mRNA normalized to rp-L31 mRNA and expressed relative to the levels in parental cell lines set as 1 (mean ± SD of triplicate determinations). Western blots and qPCR data are representative of those obtained respectively from 3 and 2 independent analysis. C) Dose-effect curves were calculated using CompuSyn software and plotting the entered Fa values against the entered dose values. For combination treatments, the combined drugs dose was entered. Each data point represents the mean of 3 replicates.
Table 3.
Dm50 of single agent and drugs combination for parental and ERL-resistant NSCLC cell lines.
Fig 6.
MET analysis in ERL-resistant cell lines.
A) qPCR analysis of gene copy numbers of MET; B) western blots of total cell lysates with the antibodies indicated of parental (P) and ERL-resistant cell lines; C) qPCR analysis of MET mRNA expression in parental (P) and ERL-resistant cell lines. MET gene and mRNA in A) and C) are normalized to RNaseP gene and rp-L31 mRNA respectively and both are expressed relative to the levels in parental cell lines set as 1 (mean ± SD of triplicate determinations). qPCR data are representative of those obtained from 2 independent analysis; D) Confocal microscopy analysis of MET receptor (green) expression in xenograft nodes of mice subcutaneously injected with the parental HCC827 cells and the ERL-resistant RA1, RB1 and RA2 cell lines. Images show representative xy-plane maximum projection of the specimens. Scale bars correspond to 15 μm.
Fig 7.
Synergistic effects of Erlotinib and MET inhibitors in ERL-resistant NSCLC cell lines.
Dose-effect curves were calculated using CompuSyn plotting the entered Fa values against the entered dose values. For combination treatments, the combined drugs dose were entered. Each data point represents the mean of 3 replicates. Combination index (CI) values were generated by non-linear regression method. Trendlines indicate CI values at any given effect (Fa, fraction affected, %inhibition), actual data points are also shown. CI = 1, additivity; CI >1, antagonism; CI<1, synergy.
Table 4.
Dm50 of single agent and drugs combination for parental and ERL-resistant NSCLC cell lines.
Fig 8.
Biochemical analysis of the effects of Erlotinib and MET inhibitors in ERL-resistant NSCLC cell lines.
Western blotting of 8% SDS-PAGE of parental and ERL-resistant HCC827 cells treated with ERL (erlotinib, 1 μM), SU (SU11274, 4 μM) for 3 hrs. In (a) are shown representative western blots with antibodies specific for phospho-MET (Y1234/1235), phospho-EGFR (Y1068), phosho-HER3 (Y1289) and phospho-AKT (S473); in (b) western blots for MET, EGFR, HER3 and AKT proteins.