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Figure 1. Effect of pemetrexed on cell viability, cell cycle, and apoptosis in lung cancer cell lines. (A) A549, NCI-H460, NCI-H2228 EML4-ALK V3, and NCI-H3122 EML4-ALK V1 cells were evaluated for their basal expression of ALK protein by Western blotting; β-actin served as the loading control. (B) Cell viability assay. A549, NCI-H460, NCI-H2228, and NCI-H3122 cells were treated with the indicated concentrations of PEM for 3 days. (C) Cell cycle analysis by PI staining and flow cytometry. A total of 1 × 106 cells were seeded in 60-mm plates and treated with 0, 0.5, 5, 10, 50, 100, and 200 nM of PEM for 24 h. Data are presented as histograms (black, Sub-G1; gray, G0/G1 phase; white, S phase, and dark gray, G2/M phase). (D) Caspase 3/7 activity was quantified 24 h after PEM treatment in A549, NCI-H460, NCI-H2228, and NCI-H3122 cells. Means with different letters (a, b, c, and d) indicate statistically significant differences (p < 0.05). (E) γ–H2AX, Caspase-9, PARP, cyclin B, and TS expression in A549, NCI-H460, NCI-H2228, and NCI-H3122 cells, as determined by Western blotting; β-actin served as the loading control. (F) Expression of ALK was evaluated using Western blotting in NCI-H2228 and NCI-H3122 with or without siALK; β-Actin served as the loading control. (G) Cell viability assay. NCI-H2228 and NCI-H3122 cells with or without siALK were treated with the indicated concentrations of PEM for 3 days.
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Figure 2. Development of parental and PEM-acquired resistant cell lines and evaluation of PEM sensitivity in A549, NCI-H460, and NCI-H3122 cells. (A) A549, NCI-H460, and NCI-H3122 cells were treated with or without 200 nM of PEM for 15 months. (B) Cell viability assays. A549 P, A549 R, NCI-H460 P, NCI-H460 R, NCI-H3122 P, and NCI-H3122 R cells were treated with the indicated concentrations of PEM for 3 days (***, p < 0.001 versus corresponding control). (C) Colony formation assays were conducted in A549 P, A549 R, NCI-H460 P, NCI-H460 R, NCI-H3122 P, and NCI-H3122 R cell lines. Means with different letters (a, b, and c) indicate statistically significant differences (p < 0.05; N.S., not significant).
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Figure 3. The PEM-acquired resistant NSCLC cell harboring EML4-ALK rearrangement model displays overexpression of the RTK signaling pathway. (A,B) RTK and EGFR phosphorylation arrays that were altered in NCI-H3122 R compared to the parental cell line. The densitometric ratio of duplicate spots for activated RTK and EGFR proteins to the internal loading controls on the RTK phosphorylation and EGFR phosphorylation array was calculated using Image J software (**, p < 0.01; ***, p < 0.001; N.S. not significant). (C,D) Western blotting analysis of A549 P, A549 R, NCI-H460 P, NCI-H460 R, NCI-H3122 P, and NCI-H3122 R cells for phospho-EGFR, EGFR, phospho-HER2, and HER2 expression was performed. GAPDH served as the loading control (SEM, serum-free media; GM; growth media). (E) EGFR and HER2 mRNA expression were determined in NCI-H3122 P and NCI-H3122 R cells by qRT-PCR. Fold change in EGFR, HER2, and HER3 mRNA in NCI-H3122 R cells was compared to that in parental cells (***, p < 0.001; N.S. not significant). (F) Western blotting analysis of NCI-H3122 P and NCI-H3122 R cells for p-AKT S473, AKT, p-MEK S217/221, p-MEK S298, and MEK. β-Actin served as the loading control (SEM, serum-free media; GM; growth media).
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Figure 4. Knockdown of EGFR-HER2 exerts a pro-apoptotic effect in NCI-H3122 R cells. (A) NCI-H3122 R cells were transfected with siEGFR and/or siHER2 and were incubated for 24 h. Cells were harvested and the expression of EGFR and HER2 was evaluated with Western blotting. β-Actin served as the loading control. (B) Apoptosis was evaluated using flow cytometry of Annexin V-PI double-stained NCI-H3122 R cells after transfection with siEGFR and/or siHER2 for 24 h. The Y-axis represents the PI-labeled population, whereas the X-axis represents the Annexin V positive cells. The left lower gating (Annexin V-, PI-) indicates normal cells, whereas the right lower gating (Annexin V+, PI-) and the right upper gating (Annexin V+, PI+) are the early and late apoptotic cells, respectively. (C) Data are presented as histograms. Means with different letters (a and b) indicate statistically significant differences (p < 0.05; N.S., not significant). (D) Caspase 3/7 activity was quantified 24 h after transfection with siEGFR and/or siHER2 in NCI-H3122 R cells. Means with different letters (a and b) indicate statistically significant differences (p < 0.05; N.S., not significant).
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Figure 5. RTK inhibition sensitizes the acquired PEM-resistant model of NSCLC. (A) NCI-H3122 P and NCI-H3122 R cells were treated with the indicated concentrations of PEM for 3 days. (B) Apoptosis was evaluated by flow cytometry of Annexin V-PI double-stained NCI-H3122 R cells after treatment with gefitinib or afatinib for 24 h. The Y-axis represents the PI-labeled population, whereas the X-axis represents the Annexin V positive cells. The left lower gating (Annexin V-, PI-) indicates normal cells, whereas the right lower gating (Annexin V+, PI−) and the right upper gating (Annexin V+, PI+) are the early and late apoptotic cells, respectively. (C) Data are presented as histograms (*** p < 0.001). (D) Caspase 3/7 activity was quantified 24 h after gefitinib or afatinib treatment in NCI-H3122 R cells. Means with different letters (a, b, c, and d) indicate statistically significant differences (p < 0.05; N.S., not significant).
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Figure 6. RTK inhibition abrogates RTK expression in NCI-H3122 R cells. (A) NCI-H3122 R cells were treated with afatinib or gefitinib. The expression levels of phospho-EGFR, EGFR, phospho-AKT S473, AKT, phospho-HER2, HER2, phospho-MEK S217/221, phospho-MEK S298, and MEK were assessed by Western blotting in NCI-H3122 R cells. β-Actin served as the loading control. (B) Basal expression of HIF-1α was evaluated with Western blotting. GAPDH served as the loading control (SEM, serum-free media; GM; growth media). (C) NCI-H3122 R cells were treated with afatinib of gefitinib. The expression levels of HIF-1α were evaluated with Western blotting in NCI-H3122 R cells. GAPDH served as the loading control. (D) Colony-forming assays were performed in NCI-H3122 R cells. Means with different letters (a, b, and c) indicate statistically significant differences (p < 0.05; N.S., not significant).
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