Treatment of biliary tract cancer with NVP-AEW541: mechanisms of action and resistance

Abstract

Aim: To investigate in vitro treatment with NVP-AEW541, a small molecule inhibitor of insulin-like growth factor-1 receptor (IGF-1R), in biliary tract cancer (BTC), since this disease is associated with a poor prognosis due to wide resistance to chemotherapeutic agents and radiotherapy.

Methods: Cell growth inhibition by NVP-AEW541 was studied in vitro in 7 human BTC cell lines by automated cell counting. In addition, the anti-tumoral mechanism of NVP-AEW541 was studied by Western blotting, cell cycle analysis and reverse transcription-polymerase chain reaction (RT-PCR). Anti-tumoral drug effect in combination with gemcitabine, 5-fluorouracil (5-FU) and Polo-like kinase 1 inhibitor BI2536 was also studied.

Results: In vitro treatment with NVP-AEW541 suppressed growth in all human BTC cell lines, however response was lower in gallbladder cancer. Treatment with NVP-AEW541 was associated with dephosphorylation of IGF-1R and AKT. In contrast, phosphorylation of p42/p44 and Stat3 and expression of Bcl-xL were inconsistently downregulated. In addition, treated cells showed cell cycle arrest at the G1/S-checkpoint and an increase in sub-G1 peak. Moreover, IGF-1R and its ligands IGF-1 and IGF-2 were co-expressed in RT-PCR, suggesting an autocrine loop of tumor cell activation. Combined with gemcitabine, NVP-AEW541 exerted synergistic effects, particularly at low concentrations, while effects of combination with 5-FU or BI 2536 were only additive.

Conclusion: Our findings suggest that NVP-AEW541 is active against BTC in vitro and potentiates the efficacy of gemcitabine.

Figure 1

In vitro cell growth inhibition. A: Treatment of seven human biliary tract cancer cell lines with NVP-AEW541 for 3 d (n = 3); B: 6 d incubation (n = 3); C: Incubation of selected cell lines EGI-1 and Mz-ChA-1 with calculated IC₅₀ for 24-96 h (n = 3). Error bars represent SD.

Figure 2

Mechanism of NVP-AEW541 drug action. A: Protein expression of IGF-1R in tested human biliary tract cancer cell lines was determined by immunoblot; B: p-IGF-1R, IGF-1R, p-AKT, AKT, p-p42/44, p42/44, p-Stat-3, Stat-3, and Bcl-xL protein levels were examined by immunoblot for selected cell lines EGI-1 and Mz-ChA-1. After 24 h of starvation, cell lines EGI-1 and Mz-ChA-1 were treated with NVP-AEW541 (12 h prior to lysis) and stimulated with ligand IGF-1 (30 min prior to lysis). β-actin served as loading control in all experiments. Densitometry was performed to analyze results which are shown relative to lane 2 as standard.

Figure 3

Cell cycle analysis. A: In vitro treatment of selected cell line EGI-1 with NVP-AEW541 for 36 h (n = 3); B: In vitro treatment of selected cell line Mz-ChA-1 (n = 3). Cells were stained with propidium iodide and analyzed by flow cytometry. ModFitLT 2.0 software was used to determine cell cycle proportions according to cellular DNA content; doublets were excluded by gating for width of fluorescence signal (FL2-W) (^aP < 0.05 for G0/G1 fraction, treated cells vs control); C: Cells having less than single DNA content (sub-G1 fraction) were presumed to be apoptotic (n = 3) (^aP < 0.05, treated cells vs control; error bars represent SD).

Figure 4

Influence of NVP-AEW541 (3 d treatment with calculated IC₅₀) on mRNA expression of IGF-1R ligands IGF-1 and IGF-2 in two of the tested cell lines. Ratio of IGF mRNA expression in treated vs untreated cell lines is shown (n = 3).

Figure 5

In vitro treatment with drug combinations of NVP-AEW541 and gemcitabine, 5-fluorouracil (5-FU) or BI 2536. Selected cell lines EGI-1 and Mz-ChA-1 were incubated with increasing concentrations of gemcitabine (A, B), 5-FU (C, D) or BI 2536 (E, F) alone (black circles) and in combination with fixed IC₂₀ concentration of NVP-AEW541 (black triangles). The shaded area represents a possible drug synergism calculated according to the model of Berenbaum[50]; error bars represent SD (n = 3).