Suppression of malignant phenotype of human neuroblastoma by thrombospondin-1 peptide in combination with valproic acid

B1

Background: More effective treatment strategies are desperately needed for children with high-risk neuroblastoma (NB). Anti-angiogenic agents and histone deacetylase (HDAC) inhibitors are capable of suppressing the growth of many types of adult cancer, but little is known about the efficacy of these agents in pediatric malignancies. In the present study, we investigated the in vitro and in vivo effects of the angiogenesis inhibitor, thrombospondin-1 (TSP-1) peptide (ABT-510), in combination with the HDAC inhibitor, valproic acid (VPA) on NB proliferation, angiogenesis, and tumor growth.Methods: Two MYCN-amplified NB cell lines (SMS-KCNR and NMB) were used to test ABT-510 and VPA anti-tumor efficacy, alone or combination. ABT-510, is a modified nonapeptide based on a heptapeptide sequence from the second type-I repeat of TSP-1. MTT assays, TUNEL assays and flow cytometry were used to measure proliferation, apoptosis, and cell cycle respectively. Nude mice bearing subcutaneous NB tumors were treated one time per day with ABT-510 (IP, 40mg/kg) or VPA (IP, 400mg/kg) alone or in combination, once tumors were palpable or tumor size reached a volume larger than 380 mm³. Mice with less advanced tumors or with larger tumors at the start of treatment were sacrificed after 20 or 10 days, respectively. Tumors were excised and analyzed for apoptosis, proliferation, and angiogenesis.Results: Our studies showed that VPA at a minimum concentration of 1 mM inhibited NB cell proliferation. Cell cycle arrest and induction of apoptosis were seen at a minimum concentration of 2.5 mM VPA. However, ABT-510 at a maximum concentration of 1 μM had no effect. VPA concentrations ranging from 10 nM to 1 mM and ABT-510 ranging from 0.1 nM to 100 nM markedly inhibited endothelial cell migration. NB xenograft growth was inhibited with either single agent therapy, and enhanced effects were seen with combination therapy. After 20 days of combination therapy, tumor volume was reduced by 91% (p<0.001) in the SMS-KCNR model, and by 87% in the NMB xenografts (p=0.029) compared to controls. Tumor growth was also inhibited by these agents when treatment was started after the tumors were >380 mm³ in size. The increase (>3 fold) in tumor volumes was reduced by 33% and 44%, over 10 days of treatment respectively (p<0.01 and p<0.001), while combination therapy immediately halted all further growth of the established NB xenografts (p<0.001). Tumor cell proliferation and microvessel density were significantly lower in xenografts treated with ABT-510 plus VPA compared to tumors treated with single-agents or controls. The number of structurally abnormal vessels with vasculature endothelial proliferation and the degree of hemorrhage was significantly decreased in the treated tumors compared to controls (p<0.001). The majority of vessels in treated tumors were thin-walled or capillary-like.Conclusion: Our results indicate that ABT-510 combined with VPA may be an effective anti-angiogenic treatment strategy for children with high-risk NB.