NT157 exerts antineoplastic activity by targeting JNK and AXL signaling in lung cancer cells

Abstract

Combination therapies or multi-targeted drugs have been pointed out as an option to prevent the emergence of resistant clones, which could make long-term treatment more effective and translate into better clinical outcomes for cancer patients. The NT157 compound is a synthetic tyrphostin that leads to long-term inhibition of IGF1R/IRS1-2-, STAT3- and AXL-mediated signaling pathways. Given the importance of these signaling pathways for the development and progression of lung cancer, this disease becomes an interesting model for generating preclinical evidence on the cellular and molecular mechanisms underlying the antineoplastic activity of NT157. In lung cancer cells, exposure to NT157 decreased, in a dose-dependent manner, cell viability, clonogenicity, cell cycle progression and migration, and induced apoptosis (p < 0.05). In the molecular scenario, NT157 reduced expression of IRS1 and AXL and phosphorylation of p38 MAPK, AKT, and 4EBP1. Besides, NT157 decreased expression of oncogenes BCL2, CCND1, MYB, and MYC and increased genes related to cellular stress and apoptosis, JUN, BBC3, CDKN1A, CDKN1B, FOS, and EGR1 (p < 0.05), favoring a tumor-suppressive cell signaling network in the context of lung cancer. Of note, JNK was identified as a key kinase for NT157-induced IRS1 and IRS2 phosphorylation, revealing a novel axis involved in the mechanism of action of the drug. NT157 also presented potentiating effects on EGFR inhibitors in lung cancer cells. In conclusion, our preclinical findings highlight NT157 as a putative prototype of a multitarget drug that may contribute to the antineoplastic arsenal against lung cancer.

Figure 1

NT157 reduces cell viability, clonogenicity and induces apoptosis in lung cancer cells. (a) Dose- and time-response cytotoxicity was evaluated by the sulforhodamine B (SRB) assay. H1299 and H460 cells were treated with vehicle (Ø) or different concentrations of NT157 (1.6, 3.2, 6.4, 12.5, 25, 50, and 100 µM) for 24, 48, and 72 h. Values are expressed as the percentage of viable cells for each condition relative to vehicle-treated cells. Results are shown as mean ± SD of at least 3 independent experiments. (b) Colony formation of the cells treated with vehicle or NT157 (1.6, 3.2, 6.4, and 12.5 µM) and for 7 days. The bar graph represents the mean ± SD of the relative number of colonies (% of control). ***p < 0.001; ANOVA and Bonferroni post-test. (c) Apoptosis was evaluated through annexin V/PI staining and flow cytometry. H1299 and H460 cells were treated with vehicle or NT157 (6.4 and 12.5 μM). Representative dot plots are shown for each condition; the upper and lower right quadrants (Q2 plus Q3) cumulatively contain the apoptotic population (annexin V + cells). **p < 0.01, ***p < 0.001; ANOVA and Bonferroni post-test. (d) Mitochondrial membrane potential analysis was evaluated using the JC-1 staining method and flow cytometry. Lung cancer cells were treated with vehicle or NT157 (6.4 and 12.5 μM). Note that NT157 increased the percentage of cells with disrupted mitochondrial membrane. Representative dot plots are shown for each condition; the gate FL-2 contains cells with intact mitochondria and the gate FL-2/FL-1 contains cells with damaged mitochondria. ***p < 0.001; ANOVA and Bonferroni post-test.

Figure 2

NT157 disrupts the cell cycle in lung cancer cells. (a) Cell cycle progression was determined by PI staining and flow cytometry in H1299 and H460 cells treated with vehicle or NT157 (6.4 and 12.5 μM). A representative histogram for each condition is illustrated. Bar graphs represent the mean ± SD of the percent of cells in subG1, G0/G1, S, G2/M, and > 4 N cells. Note that NT157 increased cell population at subG1 for both cell lines. In H1299 and H460 cells, NT157 increased cell population at S and G2/M phases, respectively. *p < 0.05, **p < 0.01 and ***p < 0.001; ANOVA and Bonferroni post-test. (b) Immunofluorescence analysis of H1299 and H460 cells treated with vehicle or NT157 (6.4 and 12.5 μM), displaying α-tubulin (green) and DAPI (blue) staining. The white arrows indicate pyknotic nuclei. Scale bar 100 µm.

Figure 3

NT157 reduces cell migration on H1299 cells. (a) Wound healing assay was performed in H1299 cells treated with vehicle or NT157 (6.4 and 12.5 μM). Images of the cells were obtained at 6 and 12 h. Note that H1299 cells treated with NT157 had a lower wound closure when compared to control. The bar graph represents the mean ± SD of the relative wound closure (% of control). *p < 0.05, **p < 0.01 and ***p < 0.001; ANOVA and Bonferroni post-test. (b) Migration assay of a spheroid model derived from H1299 cells treated with vehicle or NT157 (6.4 and 12.5 μM) for 12 h. The final area of the NT157-treated spheroids was smaller than the vehicle-treated spheroids, indicating that the migration was inhibited. Boxplot graph represents the fold-change of the initial migration area. *p < 0.05, **p < 0.01 and ***p < 0.001; ANOVA and Bonferroni post-test.

Figure 4

NT157 modulates IGF1R/IRS, JNK, and AXL signaling in lung cancer cells. (a) Western blot analysis for p-IRS1^Ser636/639, IRS1, IRS2, p-IGF1R^Tyr1136, IGF1R, p-STAT3^Tyr705, STAT3, p-AXL^Tyr702, AXL, p-SAPK/JNK^Thr183/185, SAPK/JNK, p-p38 MAPK^Thr180/182, p38 MAPK, p-ERK1/2^{Thr202/Tyr204}, ERK1/2, p-AKT^Ser473, AKT, p-4EBP1^Thr70, 4EBP1, PARP1, and γH2AX in total cell extracts from H1299 and H460 cells treated with vehicle or NT157 (3.2, 6.4, and 12.5 μM) for 24 h. (b) Western blot analysis of indicated protein involved in IRS1/2-JNK axis for H1299 and H460 cells treated with vehicle, SP600125 (20 μM) and/or NT157 (12.5 μM) for 6 h. Note that NT157 induces IRS1 and IRS2 phosphorylation, which is prevented by SP600125. Expression of p-c-JUN^Ser63/73 was used as a control for SP600125 efficacy. Membranes were reprobed with the antibody for the detection of the respective total protein or α-tubulin, and developed with the SuperSignal™ West Dura Extended Duration Substrate system using a G:BOX Chemi XX6 gel doc imaging system. (c) H1299 and H460 cells were treated with vehicle (Ø) or SP600125 20 µM for 1 h before the addition of NT157 at 12.5 µM for 6 h and replacement by drug-free medium. Colony formation was evaluated after 7 days of incubation. The bar graph represents the mean ± SD of the relative number of colonies (% of control). *p < 0.05 for SP600125-treated and/or NT157-treated cells vs. vehicle cells, ^#p < 0.05 for SP600125-treated or NT157-treated cells vs. combination treatment at the corresponding doses, ANOVA and Bonferroni post-test.

Figure 5

NT157 decreases the expression of oncogenes and induces expression of cellular stress- and apoptosis-related genes in lung cancer cells. (a) Heatmap of the gene expression in H1299 and H460 cells treated with vehicle or NT157 (6.4 and 12.5 μM). The data are represented as the fold-change of vehicle-treated cells, and downregulated and upregulated genes are shown by blue and red colors, respectively. (b) The bar graphs represent the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001, ANOVA and Bonferroni post-test.

Figure 6

NT157 and gefitinib have potentiating antineoplastic effects on lung cancer cells. (a) The heatmap indicates the cell viability of H1299 and H460 cells treated with graded concentrations of NT157 (0.4, 0.8, 1.6, 3.2, and 6.4 μM) and gefitinib (3.2, 6.4, 12.5, 25, and 50 μM) alone or in combination with each other for 48 h, as indicated. Increased cell viability is indicated by green color, while reduced cell viability is indicated by red color. (b) Dose–response cytotoxicity for NT157 in combination with gefitinib (25 or 50 μM) for H1299 and H460 cells. Values are expressed as the percentage of viable cells for each condition relative to untreated controls. Results are shown as the mean of at least three independent experiments. (c) Western blot analysis of p-AXL^Tyr702, AXL, p-SAPK/JNK^Thr183/185, SAPK/JNK in H1299 and H460 cells treated with vehicle, NT157 (12.5 μM) and/or gefitinib (25 μM) for 24 h. Membranes were reprobed with the antibody for the detection of the respective total protein or α-tubulin, and developed with the SuperSignal™ West Dura Extended Duration Substrate system using a G:BOX Chemi XX6 gel doc imaging system.