Table 1.
IC50 of RTKIs for NSCLC cell lines with and without T790M mutation.
Fig 1.
Erlotinib and SU11724 synergism on H1975 cells.
H1975 cells were plated at 3000 cells per well in a 96 well plate and after 24 hours were treated with varying combinations of EGFR inhibitor erlotinib and c-Met inhibitor SU11274. After 72 hours of drug exposure MTT cell viability assay was performed. Synergistic inhibitory effects on H1975 cell growth inhibition were observed following combination treatment of erlotinib and SU11274 at concentrations of 1 μM and 3 μM (1:1 ratio of each drug). Drug synergism was calculated using Calcusyn v2.0 software and the CI values were below 1. ANOVA analysis was used to determine statistically significant differences between treatments (n = 3, p<0.01).
Fig 2.
Modulation of key Wnt and mTOR proteins in H2170 TKI-resistant cells.
H2170-P, H2170-ER and H2170-SR cells were plated in 35 mm dishes at 125,000 cells per dish and starved (RPMI 1640 with 0.5% BSA) for 24 hours before ligand (EGF and HGF) or/and drug (erlotinib and SU11274) treatments. After western blot analysis increased expression of Wnt and mTOR pathway related proteins in H2170-ER and H2170-SR cells were observed when compared to H2170-P cells with the exception of p-GSK-3β in H2170 SR cells. The fold changes were calculated using ImageJ software. (n = 3, p<0.05).
Fig 3.
Enhanced nuclear accumulation of active β-catenin in TKI-resistant H2170 cells.
H2170-P, H2170-ER and H2170-SR cells were plated in 8-well chamber slides at 20,000 cells per well and then starved overnight. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and then blocked with 5% Normal goat serum and 0.3% Triton X-100 before incubation with primary antibody. The cells were then incubated with Dylight 488 conjugated secondary antibody and then were observed under the fluorescence microscope. The green color in the image represents active β-Catenin staining in the cell and the purple color represents DAPI nuclear staining. Average intensity of active β-catenin staining was quantitatively measured using ImageJ software. We observed greater nuclear accumulation of active β-catenin in H2170-ER and H2170-SR cells, as compared to H2170-P cells (n = 3, p<0.01).
Fig 4.
Upregulation of the mTOR pathway in H1975 cells.
H1975 and H2170-P cells were plated at 125,000 cells per dish in 35 mm dishes and starved (RPMI 1640 with 0.5% BSA) for 24 hours before ligand (EGF and HGF) or/and drug (erlotinib and SU11274) treatments and analyzed using western blot. (A) Active β-catenin and GATA-6 were observed to be downregulated and p-ERK, p-mTOR, p-p70S6K, p-LRP5/6 and Axin1 were observed to be upregulated in H1975 cells when compared to the same treatments in H2170-P cells (n≥3, p<0.05). (B) Similarly active β-catenin and GATA-6 were observed to be downregulated while, p-GSK3β was not significantly modulated in H1975 cells when compared with H2170-P cells with same treatments. We also observed p-ERK, p-LRP5/6, p-mTOR, p-p70S6K, p-4E-BP1 and Axin1 were all upregulated in H1975 cells when compared to H2170-P cells with same treatments (n≥3, p<0.05).
Fig 5.
Downregulation of Wnt proteins and upregulation of mTOR proteins in H1975 EGFR-mutant cells compared H2170-ER and H2170-SR cells.
H1975, H2170-ER and H2170-SR cells were plated at 125,000 cells per dish in 35 mm dishes and starved (RPMI 1640 with 0.5% BSA) for 24 hours before ligand (EGF and HGF) or/and drug (erlotinib and SU11274) treatments and were analyzed using western blot. (A) GATA-6 and p-LRP5/6 were observed to be downregulated in H1975 cells when compared to H2170 ER cells in same treatments. However, p-ERK, p-mTOR and p-p70S6K were upregulated in H1975 cells when compared to H2170-ER cells in similar treatments (n≥3, p<0.05). (B) Active β-catenin and GATA-6 were observed to be downregulated in H1975 cells compared to same treatments in H2170-SR cells. We also observed that p-ERK, p-mTOR and p-p70S6K were upregulated in H1975 cells when compared to H2170-SR cells in same treatments. The fold changes were calculated using ImageJ software (n≥3, p<0.05).
Fig 6.
Modulations in gene expression of β-Catenin in H2170 and H1975 cells.
H2170 and H1975 cells were plated at 125,000 cells per 35 mm dishes and were allowed to adhere and grow for 24 hours. After which cells were starved (RPMI with 0.5% BSA) for 24 hours and then were processed for RNA collection. Real-Time PCR results show that β-Catenin is upregulated in H2170-ER and H2170-SR cells when compared to H2170-P cells. While, in H1975 (T790M EGFR mutated) cells we did not observe any significant modulation of β-Catenin when compared to H2170-P cells. Expression of each gene was analyzed in triplicate (n = 2, p<0.01).
Fig 7.
Inhibitory effect of XAV939 and Everolimus on H1975 cells alone and in combination with erlotinib.
H1975 cells were plated at 3000 cells per well in a 96 well plate and then treated with varying drug concentrations of XAV939, everolimus and erlotinib alone or in combinations for 72 hours. Cell viability was determined by measuring the absorbance colorimetrically after adding MTT dye. (A) IC50 for XAV939 alone in H1975 cells was greater than 10 μM, and similarly (B) the IC50 for everolimus alone in H1975 cells was greater than 10 μM. (C) Synergistic inhibition (approximately 53%) of H1975 cells was observed when everolimus and erlotinib were administered in combination. Drug synergism was calculated using Calcusyn v2.0 software. ANOVA analysis was used to determine differences between treatment conditions which were statistically significant (n = 3, p<0.01).