Effect of imatinib mesylate on neuroblastoma tumorigenesis and vascular endothelial growth factor expression.
BACKGROUND
Alternative treatment options are needed for advanced neuroblastoma patients because their prognosis remains poor after intensive chemotherapy. Neuroblastoma cells express platelet-derived growth factor (PDGF), stem cell factor (SCF), and vascular endothelial growth factor (VEGF) and their respective receptors, PDGFR, c-Kit, and Flk-1. We therefore evaluated the effects of imatinib mesylate (imatinib), a selective inhibitor of the tyrosine kinase activities of c-Kit and PDGFR, on the growth of neuroblastoma cells in vivo and in vitro.


METHODS
We tested seven human neuroblastoma cell lines for their sensitivity to imatinib. Cell viability was assessed by trypan blue dye exclusion. Apoptosis was evaluated by nuclear staining, flow cytometry, and western blotting. Protein assays included immunoprecipitation, western blotting, enzyme-linked immunosorbent assays, and immunohistochemistry. mRNA expression was assessed by northern blotting. We used a xenograft model in SCID mice (10 mice per group) to evaluate the effects of imatinib oral therapy (50 or 100 mg/kg every 12 hours for 14 days) on neuroblastoma tumor growth. All statistical tests were two-sided.


RESULTS
All seven neuroblastoma cell lines treated with imatinib displayed concentration-dependent decreases in cell viability, which coincided with an induction of apoptosis, and with ligand-stimulated phosphorylation of c-Kit and PDGFR. The imatinib concentrations that caused 50% inhibition of growth and 50% inhibition of ligand-induced phosphorylation of these receptors were 9-13 micro M and 0.1-0.5 microM, respectively. Expression of VEGF, but not phosphorylation of Flk-1, its receptor, was reduced in neuroblastoma cells treated with imatinib at 10 microM or higher. Mice treated with imatinib at 50 mg/kg or 100 mg/kg had statistically significantly smaller tumors than control mice treated with vehicle (mean tumor volume in mice treated with imatinib at 50 mg/kg = 1546 mm3, in control mice = 2954 mm3; difference = 1408 mm3, 95% confidence interval [CI] = 657 to 2159 mm3; P<.001; mean tumor volume in mice treated with imatinib at 100 mg/kg = 463 mm3; difference = 2491 mm3, 95% CI = 1740 to 3242 mm3; P<.001).


CONCLUSIONS
Imatinib inhibited the growth of neuroblastoma cells in vitro and in vivo. This inhibition was associated with suppression of PDGFR and c-Kit phosphorylation and inhibition of VEGF expression.
Beppu
Kiichiro
K
Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Jaboine
Jerry
J
Merchant
Melinda S
MS
Mackall
Crystal L
CL
Thiele
Carol J
CJ
D000284:Administration, Oral; D000704:Analysis of Variance; D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001549:Benzamides; D015152:Blotting, Northern; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D000068877:Imatinib Mesylate; D051379:Mice; D016513:Mice, SCID; D009447:Neuroblastoma / Q000188:drug therapy* / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010879:Piperazines / Q000008:administration & dosage / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D019009:Proto-Oncogene Proteins c-kit / Q000187:drug effects / Q000378:metabolism; D011743:Pyrimidines / Q000008:administration & dosage / Q000494:pharmacology*; D017479:Receptors, Platelet-Derived Growth Factor / Q000187:drug effects / Q000378:metabolism; D013194:Staining and Labeling; D014183:Transplantation, Heterologous; D042461:Vascular Endothelial Growth Factor A / Q000187:drug effects* / Q000378:metabolism*
D000970:Antineoplastic Agents; D001549:Benzamides; D004791:Enzyme Inhibitors; D010879:Piperazines; D011743:Pyrimidines; D042461:Vascular Endothelial Growth Factor A; D000068877:Imatinib Mesylate; D011505:Protein-Tyrosine Kinases; D019009:Proto-Oncogene Proteins c-kit; D017479:Receptors, Platelet-Derived Growth Factor