Analysis of these data using Calcusyn software to graphically quantitate combined drug effects also confirmed a synergistic effect of mitotane and NVP-AEW541 in vitro (supplementary Fig. 3). Figure 6 IGF-1R antagonists enhance the inhibitory effects of mitotane. A, RL251 (left kinase inhibitor Sorafenib panel) and H295 (right panel) cells were incubated in triplicate with a combination of mitotane and increasing concentrations of NVP-AEW541. Proliferation was assessed with … To evaluate this effect in vivo, H295 xenografted nude mice were treated with mitotane or IMC-A12 either as single agents or in combination (n = 20 tumors per treatment group) (Fig. 6B6B).). Treatment with antibody alone resulted in a 51% reduction in tumor size (P < 0.001), comparable with the 55% reduction observed in the monotherapy experiment detailed in Fig.
5A5A.. Mitotane treatment as a single agent was not as effective as IMC-A12 but resulted in a 25% decrease in tumor volume (P = 0.035). The combination of IMC-A12 and mitotane resulted in a 70% decrease in tumor burden over untreated controls (P < 0.001). Although the combined treatment induced more than an additive effect predicted with mitotane and IMC-A12 (as calculated at d 21 of treatment), this difference was not sufficient to demonstrate synergy between these two compounds (P = 0.22, two sided F test). IMC-A12, mitotane, and their combination were well tolerated in treated mice with no substantial adverse effects or weight changes observed between groups (data not shown). After 21 d, xenograft specimens were collected for histochemical analysis (Fig. 6C6C).
). Interestingly, hematoxylin and eosin staining revealed treatment results in a clear decrease in vascularity compared with control sections. We investigated this further by immunohistochemically evaluating microvessel density using a FITC-conjugated lectin molecule that binds specifically to endothelial cells. Dense areas of lectin-positive endothelial cells are observed throughout control xenograft sections, but IMC-A12-treated xenograft sections display a moderate decrease in endothelial cell density. Moreover, the greatest histologic decrease was observed in xenografts treated with the combination of IMC-A12 and mitotane. We hypothesized IGF inhibition may reduce the angiogenic potential imparted by the expression of IGF-II via induction of vascular endothelial growth factor (VEGF) expression, seen in other systems (19,20).
Therefore, we generated cDNA from xenografts for quantitative RT-PCR to assess human VEGF expression using PCR primers Brefeldin_A designed to recognize all four isoforms of VEGF transcripts (21) (Fig. 6D6D).). Results demonstrated a 1.6-fold decrease in human VEGF expression in IMC-A12-treated samples (P = 0.044) and a 2.2-fold decrease when antibody was combined with mitotane (P = 0.008). Mitotane treatment alone resulted in a small and statistically insignificant decrease (P = 0.46).