Inhibition of insulin-like growth factor signaling in ovarian carcinoma cells significantly potentiates paclitaxel cytotoxicity Free

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Objective: To evaluate the ability of a small molecule inhibitor of the insulin-like growth factor receptor 1 (IGF1R) tyrosine kinase, NVP-AEW541, to block insulin-like growth factor signaling in ovarian cancer cells and to enhance Taxol cytotoxicity.

Methods: Immunoprecipitation and western blot analyses were used to evaluate the ability of NVP-AEW541 to inhibit IGF1R autophosphorylation and downstream signaling in ovarian carcinoma cells (A2780, OVCAR8, HEY). To evaluate the interaction of NVP-AEW541 and Taxol, cells were treated with NVP-AEW541, Taxol, and combinations of both drugs. Taxol and NVP-AEW541 were combined in a variety of ways, utilizing fixed and non-fixed molar ratios; cytotoxicity experiments were performed in complete growth medium containing 10% serum. The fraction of viable cells, compared with untreated cells, was determined by protein staining using the sulforhodamine B (SRB) assay and by colorimetric measurement of cellular reductive capacity (Promega MTS Assay). Calcusyn software was used to generate dose-response curves and calculate the IC50 (dose which results in 50% inhibition of proliferation). The combination index (CI) method of Chou and Talalay was used for the determination of synergy, wherein CI values less than 1 indicate synergy.

Results: Western blot analyses demonstrate that NVP-AEW541 suppresses IGF1R autophosphorylation and downstream AKT phosphorylation resulting from IGF1 or IGF2 stimulation in ovarian carcinoma cells. The presence of NVP-AEW541 significantly potentiates suppression of proliferation by Taxol. In A2780 cells, 1 μM NVP-AEW541 is sufficient to decrease the Taxol IC50 to 0.48 ±0.06 nM, which is significantly lower than the IC50 of Taxol alone, 1.5 ±0.07 nM (p<0.001, two-tailed t-test). This concentration of NVP-AEW541 inhibits phosphorylation of IGF1R and downstream AKT phosphorylation in response to IGF1 or IGF2, while causing only mild suppression of proliferation as a single agent. For this combination in A2780 cells, the mean CI equals 0.65 ±0.05, indicating a strongly synergistic interaction. Combining Taxol and NVP-AEW541 at various fixed molar ratios also results in mean CI values less than 1. Significant potentiation of Taxol cytotoxicity by NVP-AEW541 is similarly observed in OVCAR8 and HEY cells.

Conclusions: NVP-AEW541 effectively blocks IGF signaling in ovarian cancer cells. Blockade of IGF signaling with NVP-AEW541 synergistically potentiates Taxol cytotoxicity in ovarian carcinoma cells.