We have previously shown that full-length SPARC and SPARC peptide FS-E, that corresponds to the highly conserved EGF-like module of the follistatin domain, potently inhibit angiogenesis and neuroblastoma tumor growth in preclinical models [10
]. The structure of the FS-E peptide is complex, and we have demonstrated that its anti-angiogenic function is conformation-dependent [26
]. In an effort to develop a therapeutic that may be suitable for clinical use, in this study we designed and synthesized two simply structured derivative peptides, FSEN and FSEC. Because proper structure is imperative to maintain activity of the FS-E peptide, peptides FSEN and FSEC were folded into their native conformation by linking the end cysteines during synthesis with disulfide bonds. This was possible due to the close proximity of the two central cysteines in the FS-E peptide [31
]. Linking cysteine 4 with cysteine 3 instead of cysteine 2 allowed us to design peptides that correspond to amino acid sequences in the N- and C-terminal loops without disturbing the native folding. We found that both FSEN and FSEC function as inhibitors of angiogenesis, although the FSEC peptide was more potent.
Due to potent anti-angiogenic activity in vitro
, a dose of 10 mg/kg was administered 5 days a week to investigate the effects of SPARC peptides on tumor progression in the preclinical model. This is a lower amount than the 10 to 100 mg/kg daily doses that have been used to test other anti-angiogenic peptides in preclinical studies [28
]. At this low dose, peptide FSEC significantly suppressed neuroblastoma tumor growth in experimental animals. Furthermore, consistent with properties of SPARC as an inhibitor of angiogenesis, the number of endothelial and perivascular cells was also significantly decreased in the peptide-treated animals, compared to controls.
Although the anti-tumor and anti-angiogenic effects of peptide FSEN were less potent, both SPARC peptides had profound effects on the architecture of tumor-induced blood vessels. In contrast to the structurally abnormal blood vessels that were seen in the control tumors, thin walled blood vessels were detected in the peptide-treated tumors, suggesting that treatment with FSEN and FSEC induced blood vessel normalization. Interestingly, hemorrhage was not detected in the peptide-treated tumors, whereas significant hemorrhage was detected in the control tumors.
Similar to the original SPARC peptide FS-E [26
], the proper structural conformation may be essential to maintain the anti-angiogenic activity of the FSEC and FSEN peptides. Disulfide bonds are likely to be unstable in the reducing environment in vivo
, leading to poor pharmacokinetic properties and decreased activity of the peptides. Stable analogs of disulfide bonds have been used to increase activity of other biologically active peptides [36
], and we plan to use this approach to produce more stable analogs of peptides FSEC and FSEN.
It is well established that cancer blood vessels are not structurally normal [37
]. In tumors, multiple layers of hypertrophic endothelial cells alternate with areas in which endothelial cell coverage is lacking. Corresponding abnormalities in the deposition of the basement membrane are also commonly observed. Perivascular smooth muscle cells, which provide both mechanical and physiological support for the endothelial monolayer in normal blood vessels, fail to co-localize with endothelial cells in neoplastic blood vessels. These abnormalities disrupt the integrity of the blood vessels, resulting in a heterogeneous blood supply of the tumor tissue, vessel leakiness, and hemorrhage. Our recent evaluation of blood vessel architecture in a series of neuroblastoma tumors demonstrated that structurally abnormal blood vessels are commonly seen in high-risk tumors, and the presence of MVP was statistically significantly associated with decreased survival [38
Normalization of neoplastic blood vessels has been demonstrated with other anti-angiogenic therapeutics [37
], and recently the extent of vascular normalization following treatment with an anti-VEGF therapy was shown to be predictive of outcome in patients with glioblastoma [39
]. Emerging evidence also indicates that by normalizing the abnormal structure and function of tumor vasculature, anti-angiogenic agents can alleviate hypoxia and increase the efficacy of conventional therapies [37
]. A recently completed phase I dose-escalation study of an anti-VEGF agent has provided evidence of both vascular normalization and sensitization of rectal tumors to radiation [40
]. Using a simple method to quantitatively assess blood vessel architecture, we show that treatment with either FSEC or FSEN similarly normalizes the tumor vasculature. Normalization of blood vessels may enhance drug delivery to the tumor tissue and improve efficacy of chemotherapeutic agents. A better understanding of the molecular and cellular basis of vascular normalization may ultimately lead to more effective strategies for combining chemotherapy and radiation with agents that are capable of inducing blood vessel normalization.