Table 1.
IC50 of RTKIs and Combinations for Parental and Resistant NSCLC cell lines.
Figure 1.
MTT assay showing differential response between parental and resistant NSCLC cell lines.
H2170 and H358 cells were treated with tivantinib (0.01–0.4 µM) for 24 hours, tivantinib was removed, and cells were incubated for 72 hours, after which MTT viability assay was performed. SR H2170 cells showed a 3.2-fold decrease in sensitivity to the anti-proliferative effect of tivantinib at 0.1 µM tivantinib compared with parental cells. A 3.7-fold decrease in growth inhibition was also observed in SR H358 cells with 0.2 µM tivantinib compared to parental cells. Data shown are representative of three independent experiments showing similar results (n = 6, p<0.01).
Figure 2.
Differences in protein expression between parental and erlotinib resistant cell lines by western blotting.
A. EGFR is autophosphorylated in ER H2170 and downregulated in H358-E4 resistant cell lines. p-mTOR (S2448) and its downstream signaling protein phospho-p70S6K (T389) are upregulated in both resistant cell lines. H2170 and H358 parental and resistant cell lines were starved overnight in 0.5% BSA and then treated with or without 7.0 µM of erlotinib for 24 hours and cells were stimulated with 10 ng/mL of EGF for 2.5 minutes. Higher concentrations of erlotinib were used since these NSCLC cell lines have no EGFR TK mutation. Autophosphorylation of EGFR on Y1068 was seen in the absence of EGF in ER H2170 cells which was not seen in ER H358-E4 cells. Upregulation of p-mTOR and its downstream protein phosho-p70S6K (T389) is seen in H2170 resistant lines +/− erlotinib. ER H2170 cells show increased EGFR phosphorylation +/− EGF. Upregulation of p-ERK (2–5-fold) was also seen in ER H2170 and H358 cells in +/− erlotinib B. To confirm autophosphorylation of EGFR, cells were plated on chamber slides, allowed to adhere for 24 hours and then starved overnight. Cells were then treated with +/− EGF for 15 minutes, fixed with acetone: methanol and visualized with p-EGFR (Y1068) primary antibody and anti rabbit DyLight secondary antibody (Thermo Fisher Scientific) (green) or Hoechst dye for nuclear staining (blue) on a Zeiss Axio Observer Z1 fluorescent microscope. Graph showing relative average total cell fluorescence units per 8 microscopic fields. There was a 3.8-fold increase in fluorescence when comparing parental to resistant cells in the absence of EGF in H2170 cells.
Figure 3.
Differential expression of mTOR pathway proteins in parental and SU11274 resistant H2170 and H358 cell lines by western blotting.
Cells were starved overnight and then treated with or without 8.0 µM SU11274 for 24 hours. Cells were stimulated with 40 ng/mL of HGF for 2.5 minutes after which western blot analysis was performed. Downregulation of p-c-Met (Y1003) was seen in both cell lines. Upregulation of p-p70S6kinase (S371) was observed in SR H2170 cells. Upregulation of p-4E-BP1 (T37/46) was also observed in both cells lines +/− SU11274.
Figure 4.
Differential expression of ERK/Wnt pathway proteins in parental and SU11274/Erlotinib resistant H2170 cells by western blotting.
A. In SR H2170 cells, HGF induced pronounced p-ERK signaling compared to parental cells. Cells were starved for 48 hours and then stimulated with 40 ng/mL of HGF. Western blotting in SR H2170 indicated that, HGF activated p-ERK (T202/Y204) remained high for 120 minutes compared to parental lines. Basal levels of active β-catenin were also 2-fold higher and remained high (3.6-fold) for 120 minutes after HGF treatment in SR H2170 cells compared to those in parental cells over 60 minutes incubation. These experiments were done in triplicate. Relative densitometry of p-ERK/β-actin in SR H2170 cells was depicted which is an average of three independent experiments (n = 3, p<0.01). B. Regulation of proteins in the Wnt signaling pathway after treatment of H2170 with SU11274. Upregulation of pLRP6 (2 to 3.0-fold) and β-catenin (3 to 8.0-fold) were seen in resistant H2170 cells in the presence or absence of SU11274. C. Regulation of proteins in the Wnt signaling pathway after treatment of ER H2170 cells with erlotinib. Upregulation of LRP6 (2 to 5-fold), and Axin1 (2 to 3.5-fold) were seen in resistant H2170 cells in the presence or absence of erlotinib.
Figure 5.
Growth of combination resistant (CR) cell lines is inhibited significantly by adding everolimus and XAV939 in the presence of SU11274 and erlotinib.
Cells were treated for 96% growth inhibition was observed when everolimus was used with both SU11274 and erlotinib. B. Parental H2170 cells show little or no inhibition when given increasing concentrations of XAV939. Conversely, CR H2170 cells when treated with XAV939, were inhibited in a dose responsive manner. H2170 CR cells showing 40% inhibition to Wnt antagonist XAV939 (10 µM) alone, showed an 85% inhibition with triple combination of XAV939, SU11274 and erlotinib (p<0.01). Each experiment for each treatment condition was repeated three times.