Setsuko Kaneko

Expression of angiogenic factors and tumor progression in human neuroblastoma.

Abstract.Purpose: The growth and metastasis of malignant tumors is largely dependent on angiogenesis. Angiogenic factors produced by tumor cells are known to promote tumor angiogenesis. The aim of this study was to investigate which angiogenic factor is the most important in the progression of neuroblastoma (NB). Procedure: The relative expression levels of vascular endothelial growth factor-A (VEGF-A), VEGF-C, basic fibroblast growth factor (bFGF), and platelet-derived endothelial growth factor (PD-ECGF/TP) were studied in 28 NB tumor specimens by real-time quantitative reverse transcriptase/polymerase chain reaction (RT-PCR). The relationships between the expression of these four angiogenic factors and stage, patient age, primary site, MYCN copy number, and lymph node metastasis were analyzed. Results: High VEGF-A expression was correlated with stage 4 disease (blood-borne metastasis). No relationship between VEGF-A expression and age, primary site, MYCN copy number, or lymph node metastasis was found. The expression of VEGF-C, bFGF, or PD-ECGF/TP showed no correlation with stage, age, primary site, MYCN copy number, or lymph node metastasis. Conclusions: Our findings suggest that VEGF-A, but not VEGF-C, bFGF, or PD-ECGF/TP, may be associated with progression of NB. VEGF-A could be a target for antiangiogenic therapy for disseminated NB.

Continuous versus intermittent administration of human endostatin in xenografted human neuroblastoma

PURPOSE The authors examined whether recombinant human endostatin (rhEndostatin), an antiangiogenic agent, is effective against a human neuroblastoma cell line (designated TNB9) using a human neuroblastoma xenograft model and investigated whether continuous infusion is more effective than intermittent administration. METHODS In the first experiment, when tumors on the back of nude mice reached a weight of 90 to 95 mg, rhEndostatin, 10 mg/kg/d mouse weight, was administered subcutaneously to the mice (n = 5) every day for 10 consecutive days. In the second experiment, the same daily dose of rhEndostatin was administered continuously to the TNB9-bearing mice (n = 6) via subcutaneous infusion pumps for 3 consecutive days with total dose being 30% of that in the first experiment. Nestin and factor VIII expression levels were studied immunohistochemically to elucidate whether histologic evidence of the effects of rhEndostatin was present on day 4 in the second experiment. RESULTS In the first experiment, relative tumor weight in treated mice (n = 5) was significantly less than that in controls (n = 12) on day 2 only after treatment initiation (P <.05). The maximum inhibition rate (MIR) of TNB9 xenograft growth by rhEndostatin was 46.4%, indicating lack of efficacy. In the second experiment, the effects of rhEndostatin were much more marked than those in the first experiment, with an MIR of 60.7%. The mean relative tumor weight in the treated group (n = 6) in the second experiment was significantly less than that in controls (n = 10) on days 2, 4, and 6 (P <.01) as well as on days 8 and 10 (P <.05). Nestin staining in the endothelium of control tumors (n = 2) was marked, whereas it showed a loss of fibrillar structure in rhEndostatin-treated tumors (n = 2). The number of vessels immunostained with antifactor VIII antibody was markedly reduced in tumors (n = 2) from rhEndostatin-treated mice compared with that in tumors from control animals (n = 2). CONCLUSIONS Continuous administration of rhEndostatin resulted in more significant tumor regression than intermittent administration of the agent in the same model. This indicates that rhEndostatin, if administered in continuous fashion, could become an effective agent for treating patients with neuroblastoma in the future.

Epithelial-mesenchymal transition-related gene expression as a new prognostic marker for neuroblastoma.

Neuroblastoma (NB) is a highly metastatic tumor in children. The epithelial-mesenchymal transition (EMT) is an important mechanism for both the initiation of tumor invasion and subsequent metastasis. This study investigated the role of EMT in the progression of NB. Using EMT assays on samples from 11 tumors, we identified 14 genes that were either differentially expressed between tumors of different stages or highly upregulated in NB. Quantitative RT-PCR of these genes was conducted in 96 NB tumors and their expression levels were compared between stages and between tumors with the presence and absence of MYCN amplification. The association of survival rate with differential gene expression was investigated. Expression of KRT19 was significantly decreased in stage 3 or 4 NB as well as stage 4S NB compared with stage 1 or 2 NB. Expression levels of KRT19 and ERBB3 were significantly low, and expression levels of TWST1 and TCF3 were high in MYCN-amplified NB. The patients with low expression of KRT19 or ERBB3 showed significantly worse overall survival. Furthermore, the correlation between high invasive ability and low expression of KRT19 and ERBB3 was suggested in vitro using six NB cell lines. The authors conclude that downregulation of KRT19 is highly associated with tumor progression in NB and metastasis in localized primary NB and that low expression of ERBB3 is also associated with progression of NB.

Prolonged low-dose administration of the cyclooxygenase-2 inhibitor celecoxib enhances the antitumor activity of irinotecan against neuroblastoma xenografts

Cyclooxygenase (COX)‐2 is overexpressed in many human tumors including neuroblastoma (NB) and promotes tumor progression. We evaluated the antitumor effect of irinotecan (CPT‐11) treatment combined with prolonged very low‐dose administration of celecoxib, a selective COX‐2 inhibitor, against three human NB xenografts, TNB9, TS‐N‐2nu, and TS‐N‐5nu. In addition, the effects of the celecoxib‐combined treatment were examined on tumor cell proliferation, apoptosis, angiogenesis, and expression of vascular endothelial growth factor and apoptosis‐related proteins in xenografts. Celecoxib administered daily at 5 mg/kg body weight/day could not prevent the growth of any of the NB xenografts. However, the combination of daily low‐dose CPT‐11 (5.9 mg/kg body weight/day) and simultaneous very low‐dose celecoxib resulted in highly significant suppression of tumor growth in all three xenografts (P < 0.001) compared not only with low‐dose CPT‐11 therapy alone but also with the combination therapy of intermittent conventional‐dose CPT‐11 (59 mg/kg body weight) and celecoxib accompanied by decreased proliferation and increased induction of apoptosis in tumor cells. Induction of apoptosis by CPT‐11 with and without celecoxib was associated with the up‐regulation of Bax expression and the down‐regulation of Bcl‐2 expression. The enhanced antitumor effect of the combination of the two drugs against the NB xenografts might be partially COX‐2‐independent and was probably mediated through multiple factors including diminished expression of VEGF and activation of the caspase‐dependent mitochondrial apoptosis pathway. These findings demonstrate that prolonged low‐dose CPT‐11 treatment combined with very low‐dose celecoxib shows promising antitumor activity through the blockage of multiple critical targets related to NB tumor cell survival and proliferation. (Cancer Sci 2009; 00: 000–000)

Expression of mRNAs for PACAP and its receptor in human neuroblastomas and their relationship to catecholamine synthesis.

PURPOSE Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal peptide family, induces the expression of catecholamine-synthesizing enzymes in adrenal medullary cells. In addition, PACAP and its receptor have been detected in human neuroblastoma tissues and cell lines, though it is not yet known whether PACAP enhances the expression of genes encoding catecholamine-synthesizing enzymes. To address this question, we analyzed PACAP, PACAP receptor and tyrosine hydroxylase (TH) mRNAs in neuroblastomas. METHODS The levels of mRNA for PACAP and vasoactive intestinal peptide (VIP), as well as their receptors and the mRNA for TH were measured by RT-PCR or real-time PCR analysis. RESULTS VPAC1R mRNA was detected in all of 16 tissues and 3 cell lines that were examined, while VPAC2R mRNA was detected in 5 of 16 (31%) tissue and 2 of 3 cell lines. PAC1R mRNA was detected in 6 out of 16 (38%) tissues and none of 3 cell lines. mRNA expression of PACAP and TH were detected in many tissues (10/16 and 16/16, respectively). However, neither in tissues nor cell lines did PACAP mRNA expression correlate with TH mRNA expression. CONCLUSION Our findings suggest that PACAP is not involved in the regulation of expression of TH in neuroblastomas.

Vascular endothelial growth factor expression is closely related to irinotecan-mediated inhibition of tumor growth and angiogenesis in neuroblastoma xenografts

In the present study, irinotecan (CPT‐11) was highly effective not only against the chemosensitive neuroblastoma (NB) xenografts SK‐N‐ASnu and TNB9, but also against the multidrug‐resistant NB xenograft TS‐N‐2nu. SK‐N‐ASnu and TNB9 were significantly more responsive to low‐dose daily CPT‐11 treatment than to intermittent administration of one‐third of the median lethal dose. For TS‐N‐2nu, there was no significant difference in tumor growth inhibition between the two treatment schedules. Treatment with CPT‐11 alone could not completely abolish tumor growth in mice. For TNB9, tumor regrowth seemed to result from an inability to regress host vessels in the stroma during treatment and an inability to suppress host‐derived vascular endothelial growth factor (VEGF) expression throughout therapy. In the multidrug‐resistant TS‐N‐2nu, VEGF was not suppressed by low‐dose therapy with CPT‐11, and neurofilament‐positive tumor cells escaped from apoptosis and were growth arrested at G0/G1 phase. These findings suggest a mechanism for the incomplete responsiveness of TS‐N‐2nu to CPT‐11. Our data demonstrate that diminished VEGF gene and protein expression is closely correlated with tumor growth inhibition and inhibition of angiogenesis by CPT‐11 in NB xenografts. Our results further suggest that a persistent blocker of stroma‐derived VEGF will need to be combined with CPT‐11 to completely inhibit the growth of chemosensitive NB, and that administration of CPT‐11 at higher doses will be required to inhibit the growth of multidrug‐resistant NB. (Cancer Sci 2008; 99: 1209–1217)