Cancer Letters

Cancer Letters

Volume 194, Issue 1, 8 May 2003, Pages 55-66
Cancer Letters

Anti-tumor activity of the novel angiogenesis inhibitor anginex

https://doi.org/10.1016/S0304-3835(03)00015-6Get rights and content

Abstract

Anginex is a novel cytokine-like peptide with potent anti-angiogenic activity, which operates specifically against angiogenically-activated endothelial cells via prevention of cell adhesion/migration on the extracellular matrix and subsequent induction of apoptosis. Here, we demonstrate that anginex inhibits tumor growth in vivo in mouse xenograft models. In the MA148 ovarian carcinoma model, tumor growth was inhibited dose-dependently by up to 80% when systemically administered via osmotic mini-pumps starting at the time of tumor cell inoculation. The optimal dose was found to be 10 mg/kg per day. When tested against established tumors, mini-pump-administered anginex demonstrated essentially the same effectivity at this optimal dose, whereas once or twice-daily injections were only half as effective. When anginex was conjugated to human serum albumin, effectivity was significantly improved, most likely due to increased bioavailability of the conjugate. Immunohistochemical analysis of microvessel density indicated that the anti-tumor activity of anginex is mediated by angiogenesis inhibition. This was confirmed in an in vitro angiogenesis assay based on tube formation in a collagen gel. Animals demonstrated no signs of toxicity as judged by unaltered behavior, normal weight gain, blood markers and macro- and microscopic morphology of internal organs upon autopsy. Overall, these in vivo studies indicate that anginex is an effective anti-tumor agent.

Introduction

Angiogenesis or neovascularization is the process through which new blood vessels develop from pre-existing vasculature [1]. The growth of solid tumors is dependent on angiogenesis, as tumors generally cannot grow beyond the size of 1–2 mm in diameter without formation of new blood vessels to supply nutrition and oxygen and remove waste products. Most tumors release angiogenesis-regulating factors, and neovasculatization occurs only when there are more positive than negative regulators of angiogenesis [2], [3].

A highly vascularized tumor is correlated with a poor clinical prognosis, not only because of the potential for uncontrolled tumor growth, but also because of the increased access of the tumor to capillaries, which contributes to increased metastatic potential [4], [5]. Consequently, methods that inhibit angiogenic sprouting provide a unique opportunity to arrest tumor growth and prevent metastasis formation. Many endogenous and exogenous anti-angiogenic agents have been identified, such as angiostatin [6], endostatin [7], thrombospondin [8], TNP-470 [9], platelet factor-4 [10], thalidomide [11], squalamine [12], bactericidal/permeability-increasing protein [13] and carboxyamino-imidazole [14].

Although preclinical testing of anti-angiogenic agents shows promise, the need for more and better angiogenesis inhibitors is driven by the absence of any major clinical breakthroughs (e.g. SU5416, BB2516, AG3340, Bay 12-9566, IM-862 [15]). The most successful angiostatic agents have been those that directly act by inhibiting endothelial cell proliferation. Anginex, a designed β-sheet-forming cytokine-like 33-mer peptide, which potently inhibits multiple steps in the angiogenesis process, is a member of this class of angiogenesis inhibitors. A family of homologous βpep peptides was designed by using basic folding principles and incorporating short sequences from the β-sheets domains of α-chemokine IL-8 and bactericidal/permeability increasing (BPI) protein [16]. The angiostatic activity of anginex is based primarily on apoptosis-induction via prevention of adhesion on and migration through the extracellular matrix of angiogenically-activated endothelial cells [16].

The aim of the present study was to test and to optimize the in vivo efficacy of anginex as an anti-cancer agent in mouse models. Various treatment regiments, i.e. dose response, mode and frequency of administration, were investigated. For other angiogenesis inhibitors, the literature reports that continuous, systemic administration of, for example endostatin [17], results in more effective tumor growth suppression at significantly reduced doses compared with bolus administration. Continuous systemic administration of anginex in a 10 mg/kg per day dose provided optimal efficacy. Efficacy was significantly improved by conjugating anginex to human serum albumin to improve its bioavailability. These results demonstrate that anginex is a promising pharmacological anti-cancer agent that should be developed for clinical testing.

Section snippets

Cell culture

The human epithelial ovarian carcinoma cell line, MA148, was kindly provided by Prof. Dr Ramakrishnan. MA148 cells were cultured at 37 °C in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Cellgro, Mediatech, Washington, DC) and 1% penicillin/streptomycin (Cellgro, Mediatech). Cultures were grown in the presence of 5% CO2 and split 1:3 every 3 days. The human SK-OV-3 carcinoma cell line was kindly provided by Dr Rebecca Bagley (Genzyme).

Proliferation assay

Human

Anginex inhibits tumor growth dose dependently

The dose dependency of anginex was assessed in the MA148 human ovarian carcinoma xenograft model, whereby anginex was administered at 5, 10, and 20 mg/kg per day via osmotic mini-pumps. For this study, there were three control groups: one given PBS containing BSA and the other two given PBS containing homologous peptide βpep-28 (5 or 10 mg/kg per day). As a model for minimal residual disease, treatment was initiated immediately after inoculation of animals with tumor cells. Anginex was observed

Discussion

Anginex is a rationally designed, cytokine-like peptide of 33-mer, which has been shown in vitro to induce apoptosis specifically in angiogenically-activated endothelial cells (EC) by inhibiting EC from adhering to and migrating on the extracellular matrix [16]. The present paper reports the in vivo efficacy of this small peptide and its development as a therapeutic anti-cancer agent. We have shown here that anginex significantly inhibits the growth of ovarian carcinoma-derived tumors in

Acknowledgements

This research was supported by grants from the Department of Defense (DA/DAMD 17-99-1-9564) and the National Institutes of Health (R01 CA-96090) to K.H. Mayo.

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    Present address: Department of Chemistry, Mathematics and Physical Sciences, St. Francis University, Loretto, PA, USA.

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