Inhibition of neuroblastoma-induced angiogenesis by fenretinide

Abstract

Retinoids are a class of natural or synthetic compounds that participate in the control of cell proliferation, differentiation and fetal development. The synthetic retinoid fenretinide (HPR) inhibits carcinogenesis in various animal models. Retinoids have also been suggested to be effective inhibitors of angiogenesis. The effects of HPR on certain endothelial cell functions were investigated in vitro, and its effects on angiogenesis was studied in vivo, by using the chorioallantoic membrane (CAM) assay. HPR inhibited vascular endothelial growth factor-(VEGF-) and fibroblast growth factor-2-(FGF-2)-induced endothelial cell proliferation without affecting endothelial motility; moreover, HPR inhibited growth factor-induced angiogenesis in the CAM assay. Furthermore, a significant antiangiogenic potential of HPR has also been observed in neuroblastoma (NB) biopsy-induced angiogenesis in vivo. We previously demonstrated that supernatants derived from NB cell lines stimulated endothelial cell proliferation. In the present study, we found that this effect was abolished when NB cells were incubated in the presence of HPR. VEGF-and FGF-2-specific ELISA assays, performed on both NB cells derived from conditioned medium and cellular extracts, indicated no consistent effect of HPR on the level of these angiogenic cytokines. Moreover, RT-PCR analysis of VEGF and FGF-2 gene expression confirmed the above lack of effect. HPR was also able to significantly repress the spontaneous growth of endothelial cells, requiring at least 48 -72 hr of treatment with HPR, followed by a progressive accumulation of cells in G 1 at subsequent time points. Finally, immunohistochemistry experiments performed in the CAM assay demonstrated that endothelial staining of both VEGF receptor 2 and FGF-2 receptor-2 was reduced after implantation of HPR-loaded sponges, as compared to control CAMs. These data suggest that HPR exerts its antiangiogenic activity through both a direct effect on endothelial cell proliferative activity and an inhibitory effect on the responsivity of the endothelial cells to the proliferative stimuli mediated by angiogenic growth factors.

Ceramide Signaling in Fenretinide-induced Endothelial Cell Apoptosis

“…Decrease in capillary formation in the chick chorioallantoic membrane by 4-HPR has been demonstrated by two groups, indicating its in vivo antiangiogenic activity (14,15). However, only one of these groups found 4-HPR (2.5-5 M, 48 h) to be cytotoxic to endothelial cells (14), whereas the other did not detect cytotoxicity even after 72-h incubation with 10 M 4-HPR (15). In our experiments, 4-HPR was cytotoxic to the three types of endothelial cell preparations studied in tissue culture.…”

“…Our data now provide evidence for apoptosis by 4-HPR in cultured endothelial cells, achieved at concentrations similar to those that induce apoptosis in tumor cell lines (20,(37)(38)(39)(40). Decrease in capillary formation in the chick chorioallantoic membrane by 4-HPR has been demonstrated by two groups, indicating its in vivo antiangiogenic activity (14,15). However, only one of these groups found 4-HPR (2.5-5 M, 48 h) to be cytotoxic to endothelial cells (14), whereas the other did not detect cytotoxicity even after 72-h incubation with 10 M 4-HPR (15).…”

“…In addition, tumor necrosis factor-␣-and lipopolysaccharide-induced lactate dehydrogenase release (Sigma kit) were also similar in both the high and low passage human BMEC (data not shown). Experiments were performed using both low passage (passages [12][13][14][15][16][17][18][19][20][21][22][23][24][25] Apoptosis Assays-Apoptosis was assessed by staining ethanol-fixed, RNase-treated cells with propidium iodide (50 g/ml in phosphate-buffered saline containing 5 mM EDTA, 10 min on ice) and identifying cells with a sub-G 0 /G 1 DNA content, indicative of apoptosis, using a Coulter Epics ELITE flow cytometer (Coulter, Miami, FL). For morphological assessment of DNA condensation and/or apoptotic bodies, cells were grown on chamber slides and then incubated with the supravital DNA stain Hoechst 33342 (10 g/ml for 30 min at 37°C).…”

“…4-HPR has been shown to possess antiendothelial activity in tissue culture and the chick chorioallantoic membrane model (14,15). However, whereas Pienta et al (14) demonstrated that 4-HPR was cytotoxic to CPAE bovine artery endothelial cells, Ribatti et al (15) found that it inhibited proliferation but was not cytotoxic to human adrenal gland capillary endothelial cells. These data support an antiangiogenic role for 4-HPR, yet the molecular mechanism of the effects of 4-HPR in endothelial cells remains largely unknown.…”

“…In light of the antiendothelial effects of 4-HPR, its potential for antiangiogenic activity (14,15), and the role of ceramide as an important mediator of endothelial cell apoptosis (5,(22)(23)(24), we were interested in the role of ceramide signaling in medi-ating the antiendothelial effect of 4-HPR. In the work reported here we demonstrate that 4-HPR induces a caspase-dependent endothelial apoptosis.…”