Figure 4.

(A) Halofuginone (an inhibitor of glutamyl-prolyl-tRNA synthetase) and CX-5461 (an inhibitor of rRNA synthesis) significantly suppress growth of neuroblastoma cells in vitro. Four neuroblastoma cell lines with various genetic profiles were treated with these small molecule inhibitors at the concentrations indicted for 48 hours. IMR5 is MYCN amplified and TP53 normal; Kelly is MYCN amplified and TP53 mutated; SY5Y is non-MYCN amplified and TP53 normal; SKNAS is non-MYCN amplified and TP53 mutated. At the end of the 48 hour-incubation, cell were subjected the MTS assay to determine growth effect of the compounds on the neuroblastoma cells. (B) Halofuginone and CX-5461 down-regulate MYC and MYCN expression in neuroblastoma cells at low to submicromolar concentrations. Western blot assay employing anti-pan MYC monoclonal antibody (NCM II 143) was used to determine the effect of the compounds on MYC and MYCN protein expression in neuroblastoma cells. Beta-actin (β-ACT) was used as a protein loading control. (C) The preclinical efficacy of Halofuginone on growth of Kelly orthotopic xenografts. One million Kelly cells per mouse were implanted into the left adrenal grand of nude mice. When the tumors reached > 100 mm3, silk films loaded with 20 μg Halofuginone (HF; n = 5) or PBS/DMSO (CT; n = 5) were surgically inserted into the tumors. Growth of tumors was monitored by high frequency ultrasound using a VisualSonics Vevo 2100 sonographic probe (Toronto, Ontario, Canada), and the tumor volume was measured using the 3-D reconstruction tool (Vevo Software v1.6.0, Toronto, Ontario, Canada). Statistical analysis was done using a Student t-test. (D) The effect of Halofuginone on MYCN expression in Kelly xenografts. Kelly xenografts treated with the HF silk films (HF) or control silk films (CT) were stained with H&E (H&E HF vs. H&E CT) or processed for MYCN immunohistochemistry MYCN HF vs. MYCN CT). Xenografts were resected at 17 days after the initiation of HF treatment (20 μg).