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 3 H-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 twosided. Results: Bortezomib inhibited proliferation and colony formation of neuroblastoma cell lines in a time- and dosedependent manner. The mean bortezomib concentration that caused 50% inhibition of growth was 6.1 nM (95% confi dence interval [CI] = 0.9 to 11.3 nM) at 72 hours. Bortezomibtreated neuroblastoma cells were arrested at G 2 /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 fi vefold with respect to untreated cells). Similar results were obtained for primary neuroblastoma cells exposed to bortezomib. Bortezomib inhibited angiogenesis in CAMs stimulated by con ditioned medium from neuroblastoma cell lines, by neuro blastoma xenografts, and by primary neuroblastoma biopsy specimens (microvessel area: 2.9 × 10 − 2 mm 2 , 95% CI = 1.8 × 10 − 2 to 3.8 × 10 − 2 mm 2 in CAMs treated with biopsy specimens alone and 1.3 × 10 − 2 mm 2 , 95% CI = 1 × 10 − 2 to 1.5 × 10 − 2 mm 2 in CAMs treated with biopsy specimens plus bortezomib, P = .024). In both mouse models, mice treated with bortezomib lived statistically signifi cantly 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 fi ndings provide the rationale for further clinical investigation of bortezomib in pediatric neuroblastoma.
Effect of bortezomib on human neuroblastoma cell growth, apoptosis, and angiogenesis
NICO, Beatrice;RIBATTI, Domenico;
2006-01-01
Abstract
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 3 H-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 twosided. Results: Bortezomib inhibited proliferation and colony formation of neuroblastoma cell lines in a time- and dosedependent manner. The mean bortezomib concentration that caused 50% inhibition of growth was 6.1 nM (95% confi dence interval [CI] = 0.9 to 11.3 nM) at 72 hours. Bortezomibtreated neuroblastoma cells were arrested at G 2 /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 fi vefold with respect to untreated cells). Similar results were obtained for primary neuroblastoma cells exposed to bortezomib. Bortezomib inhibited angiogenesis in CAMs stimulated by con ditioned medium from neuroblastoma cell lines, by neuro blastoma xenografts, and by primary neuroblastoma biopsy specimens (microvessel area: 2.9 × 10 − 2 mm 2 , 95% CI = 1.8 × 10 − 2 to 3.8 × 10 − 2 mm 2 in CAMs treated with biopsy specimens alone and 1.3 × 10 − 2 mm 2 , 95% CI = 1 × 10 − 2 to 1.5 × 10 − 2 mm 2 in CAMs treated with biopsy specimens plus bortezomib, P = .024). In both mouse models, mice treated with bortezomib lived statistically signifi cantly 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 fi ndings provide the rationale for further clinical investigation of bortezomib in pediatric neuroblastoma.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
