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Effect of Bortezomib on Human
Neuroblastoma Cell Growth, Apoptosis, and Angiogenesis
Chiara Brignole, Danilo
Marimpietri, Fabio Pastorino, Beatrice Nico,
Daniela Di Paolo, Michela Cioni, Federica
Piccardi, Michele Cilli, Annalisa Pezzolo,
Maria Valeria Corrias, Vito Pistoia, Domenico
Ribatti, Gabriella Pagnan, Mirco Ponzoni
Affiliations of authors: Laboratory
of Oncology, G. Gaslini Children's Hospital, Genoa, Italy (CB, DM, FP,
DDP, MC, AP, MVC, VP, GP, MP); Animal Research Facility, Istituto
Tumori, Genoa, Italy (FP, MC); Department of Human Anatomy and
Histology, University of Bari, Bari, Italy (BN, DR) -- Correspondence
to: Mirco Ponzoni, PhD, Differentiation Therapy Unit, Laboratory of
Oncology, G. Gaslini Children's Hospital, Largo G. Gaslini 5, 16147
Genoa, Italy (e-mail: mircoponzoni@ospedale-gaslini.ge.it
).
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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.
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