Effect of bortezomib on human neuroblastoma cell growth, apoptosis, and angiogenesis.
BACKGROUND
Bortezomib is a selective and reversible inhibitor of the 26S proteasome that shows potent antitumor activity in vitro and in vivo against several human cancers of adulthood. No data are available on bortezomib activity against human pediatric neuroblastoma.


METHODS
Ten neuroblastoma cell lines and suspensions of primary neuroblastoma cells from three patients were tested for sensitivity to bortezomib. Colony formation, cell proliferation, cell cycle progression, and apoptosis were evaluated by a clonogenic assay and by measuring 3H-thymidine incorporation, bromodeoxyuridine uptake, DNA fragmentation, and phosphatidylserine exposure and propidium iodide staining, respectively. Angiogenesis was assessed by the chick embryo chorioallantoic membrane (CAM) assay. Two mouse xenograft models that mimic the growth and spread of neuroblastoma in humans were used to examine in vivo sensitivity of neuroblastoma to bortezomib. All statistical tests were two-sided.


RESULTS
Bortezomib inhibited proliferation and colony formation of neuroblastoma cell lines in a time- and dose-dependent manner. The mean bortezomib concentration that caused 50% inhibition of growth was 6.1 nM (95% confidence interval [CI] = 0.9 to 11.3 nM) at 72 hours. Bortezomib-treated neuroblastoma cells were arrested at G2/M and underwent apoptosis (mean percentage of apoptotic cells in four neuroblastoma cell lines treated with 20 nM bortezomib for 24 hours ranged from 20% to 35%, and caspases were activated by two- to fivefold with respect to untreated cells). Similar results were obtained for primary neuroblastoma cells exposed to bortezomib. Bortezomib inhibited angiogenesis in CAMs stimulated by conditioned medium from neuroblastoma cell lines, by neuroblastoma xenografts, and by primary neuroblastoma biopsy specimens (microvessel area: 2.9 x 10(-2) mm2, 95% CI = 1.8 x 10(-2) to 3.8 x 10(-2) mm2 in CAMs treated with biopsy specimens alone and 1.3 x 10(-2) mm2, 95% CI = 1 x 10(-2) to 1.5 x 10(-2) mm2 in CAMs treated with biopsy specimens plus bortezomib, P = .024). In both mouse models, mice treated with bortezomib lived statistically significantly longer than control mice (mean survival time in the pseudometastatic model: 74.2 versus 50.3 days, P<.001; mean survival time in the orthotopic model: 72.3 versus 50.6 days, P<.001).


CONCLUSIONS
Bortezomib is an effective inhibitor of neuroblastoma cell growth and angiogenesis. These findings provide the rationale for further clinical investigation of bortezomib in pediatric neuroblastoma.
Brignole
Chiara
C
Laboratory of Oncology, G. Gaslini Children's Hospital, Largo G. Gaslini 5, 16147 Genoa, Italy.
Marimpietri
Danilo
D
Pastorino
Fabio
F
Nico
Beatrice
B
Di Paolo
Daniela
D
Cioni
Michela
M
Piccardi
Federica
F
Cilli
Michele
M
Pezzolo
Annalisa
A
Corrias
Maria Valeria
MV
Pistoia
Vito
V
Ribatti
Domenico
D
Pagnan
Gabriella
G
Ponzoni
Mirco
M
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D001897:Boronic Acids; D000069286:Bortezomib; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008940:Mitotic Index; D009389:Neovascularization, Pathologic / Q000517:prevention & control*; D009447:Neuroblastoma / Q000098:blood supply / Q000188:drug therapy* / Q000473:pathology; D011480:Protease Inhibitors / Q000494:pharmacology*; D011719:Pyrazines; D014183:Transplantation, Heterologous; D014410:Tumor Stem Cell Assay
D000970:Antineoplastic Agents; D001897:Boronic Acids; D011480:Protease Inhibitors; D011719:Pyrazines; D000069286:Bortezomib