AC-93253 iodide, a novel Src inhibitor, suppresses NSCLC progression by modulating multiple Src-related signaling pathways

Abstract

Background: The tyrosine kinase Src is involved in the progression of many cancers. Moreover, inhibiting Src activity has been shown to obstruct several signaling pathways regulated by the EGFR. Thus, Src is a valuable target molecule in drug development. The purpose of this study was to identify compounds that directly or indirectly modulate Src to suppress lung cancer cell growth and motility and to investigate the molecular mechanisms underlying the effects of these compounds.

Methods: Human non-small cell lung cancer (NSCLC) cell lines (PC9, PC9/gef, A549, and H1975) with different EGFR statuses were tested by cytotoxicity and proliferation assays after AC-93253 iodide treatment. Src and Src-related protein expression in AC-93253 iodide-treated PC9, PC9/gef, and A549 cells were assessed by western blotting. The effects of AC-93253 iodide on cancer cell colony formation, invasion, and migration were assessed in PC9 and PC9/gef cells. The synergistic effects of gefitinib and AC-93253 iodide were evaluated by combination index (CI)-isobologram analysis in gefitinib-resistant cell lines. The efficacy of AC-93253 iodide in vivo was determined using nude mice treated with either the compound or the vehicle.

Results: Among the compounds, AC-93253 iodide exhibited the most potent dose-independent inhibitory effects on the activity of Src as well as on that of the Src-related proteins EGFR, STAT3, and FAK. Furthermore, AC-93253 iodide significantly suppressed cancer cell proliferation, colony formation, invasion, and migration in vitro and tumor growth in vivo. AC-93253 iodide sensitized tumor cells to gefitinib treatment regardless of whether the cells were gefitinib-sensitive (PC9) or resistant (H1975 and PC9/gef), indicating that it may exert synergistic effects when used in combination with established therapeutic agents. Our findings also suggested that the inhibitory effects of AC-93253 iodide on lung cancer progression may be attributable to its ability to modulate multiple proteins, including Src, PI3K, JNK, Paxillin, p130cas, MEK, ERK, and EGFR.

Conclusions: Our data suggest that AC-93253 iodide inhibits NSCLC cell growth and motility by regulating multiple Src-related pathways. Our findings may facilitate the development of therapeutic strategies and anti-tumor drugs that may be useful for treating lung cancer in the future.

Keywords: AC-93253 iodide; EGFR; Gefitinib; NSCLC; Src.

Fig. 1

Effects of the candidate compounds on Src and EGFR expression and cell viability in different cell lines. a Src and EGFR expression and phosphorylation in H358 and PC9 cells treated with the candidate compounds for 24 h and analyzed by western blotting. Veh (vehicle) represents 0.1% DMSO; Da represents dasatinib, a positive control. GAPDH served as a loading control. Protein expression was quantified by ImageJ software (NIH), and the results are shown directly below the gel graph. b AC-93253 iodide cytotoxicity determined by cell viability assays in NSCLC cell lines with different EGFR statuses. The results are presented as percentages of the vehicle control (0 μM, 0.1% DMSO). The IC₅₀ of the designated time point is indicated at the bottom of each bar chart. Each experiment was performed independently and in triplicate. *P < 0.05 compared with the vehicle control

Fig. 2

Effects of AC-93253 iodide on the expression and phosphorylation of Src and related proteins. Lung cancer PC9 cells (a) and PC9/gef cells (b) were treated with the designated concentrations of AC-93253 iodide for 24, 48, and 72 h. Protein expression was quantified by ImageJ software (NIH), and the results are shown directly below the gel graph. 0 nM: 0.1% DMSO. The Src, EGFR, STAT3, and FAK expression and phosphorylation levels were measured by immunoblot analysis using the indicated antibodies. GAPDH was used as an internal control. Each experiment was performed independently and was repeated three times

Fig. 3

Suppressive effects of AC-93253 iodide on tumor cell growth. a AC-93253 iodide reduced proliferation in PC9, PC9/gef, A549, and H1975 cells as determined by PrestoBlue cell viability assays at the indicated time points. b AC-93253 iodide inhibited clonogenicity, as determined by colony formation assay. Upper panel: anchorage-dependent cell growth, colonies with diameters ≥ 0.3 mm were counted; lower panel: anchorage-independent cell growth, colony diameter ≥ 0.5 mm. Each experiment was performed independently and in triplicate; 0 nM: 0.1% DMSO. c Tumourigenesis assay. The indicated number of live PC9/gef cells was subcutaneously injected into mice divided into vehicle-treated (n = 8) and drug-treated groups (n = 6). Tumor volumes were measured every 3–4 days. d AC-93253 iodide decreased tumor weights, which were presented as the mean ± standard deviation. e pSrc and Src expression levels and distributions in murine tumor tissues were determined by immunohistochemical staining and observed using a light microscope (×400 magnification). Vehicle represents 0.1% DMSO, and AC-93253 represents 0.25 mg/kg of the compound. The scale bars represent 20 μm. *P < 0.05 compared with the vehicle control (0 nM, 0.1% DMSO)

Fig. 4

The impact of AC-93253 iodide on cell motility and drug synergism. a Inhibitory effects of AC-93253 iodide on cancer cell invasion as determined by the transwell assay with Matrigel. b Inhibitory effects of AC-93253 iodide on cancer cell migration as determined by the transwell assay without Matrigel. c Synergistic effects of AC-93253 iodide and gefitinib on gefitinib-resistant lung adenocarcinoma cells determined by cell viability assay. The indicated combinations of AC-93253 iodide and gefitinib were used to treat lung adenocarcinoma A549, PC9/gef, and H1975 cells for 72 h. The CI was calculated using the data and CalcuSyn software. Each experiment was performed independently and was repeated three times. *P < 0.05 compared with control (0 nM; 0.1% DMSO)

Fig. 5

Expression of Src-related proteins in lung cancer cell lines treated with AC-93253 iodide. Lung cancer PC9, PC9/gef, and A549 cells were treated with AC-93253 iodide at the indicated concentrations for 72 h and then analyzed on western blots; 0 nM represents 0.1% DMSO. GAPDH was the loading control. a PI3K, JNK, Paxillin, and p130cas expression and phosphorylation levels. b MEK and ERK expression and phosphorylation levels. Protein expression was quantified by ImageJ software (NIH), and the results are shown directly below the gel graph. Each experiment was performed independently and in triplicate

Fig. 6

AC-93253 iodide inhibited Src and Src-related gene transcription and induced protein degradation. a AC-93253 iodide induced increases in protein degradation. Western blot analyses of Src, EGFR, STAT3, and FAK expression levels in PC9 cells treated with or without the protein synthesis inhibitor CHX and/or AC-93253 iodide for 4, 8, and 12 h. b AC-93253 iodide induced increases in ubiquitination, as determined by western blotting. The proteasome inhibitor MG132 and/or AC-93253 iodide were administered to PC9 cells for 72 h. GAPDH served as a loading control. c Repressive effects of AC-93253 iodide on Src, EGFR, STAT3, and FAK transcription as determined by real-time RT-PCR in PC9 cells. Relative gene expression levels were calculated using the comparative CT method (2^–ΔΔCT). TBP: internal control. Each experiment was performed independently and in triplicate. *P < 0.05 compared with control (0 nM: 0.1% DMSO). d Enhanced ubiquitination of Src-related proteins induced by AC-93253 iodide. PC9 cell lysates with or without AC-93253 treatment were immunoprecipitated by the indicated antibodies and then analyzed by western blotting with anti-ubiquitin. GAPDH served as the internal control. Con represents 0 nM (0.1% DMSO)