Cytotoxicity of VEGF121/rGel on vascular endothelial cells resulting in inhibition of angiogenesis is mediated via VEGFR-2

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Abstract

Background

The fusion protein VEGF ₁₂₁/rGel composed of the growth factor VEGF ₁₂₁and the plant toxin gelonin targets the tumor neovasculature and exerts impressive anti-vascular effects. We have previously shown that VEGF ₁₂₁/rGel is cytotoxic to endothelial cells overexpressing VEGFR-2 but not to endothelial cells overexpressing VEGFR-1. In this study, we examined the basis for the specific toxicity of this construct and assessed its intracellular effects in vitro and in vivo.

Methods

We investigated the binding, cytotoxicity and internalization profile of VEGF ₁₂₁/rGel on endothelial cells expressing VEGFR-1 or VEGFR-2, identified its effects on angiogenesis models in vitro and ex vivo, and explored its intracellular effects on a number of molecular pathways using microarray analysis.

Results

Incubation of PAE/VEGFR-2 and PAE/VEGFR-1 cells with ¹²⁵I-VEGF ₁₂₁/rGel demonstrated binding specificity that was competed with unlabeled VEGF ₁₂₁/rGel but not with unlabeled gelonin. Assessment of the effect of VEGF ₁₂₁/rGel on blocking tube formation in vitro revealed a 100-fold difference in IC ₅₀levels between PAE/VEGFR-2 (1 nM) and PAE/VEGFR-1 (100 nM) cells. VEGF ₁₂₁/rGel entered PAE/VEGFR-2 cells within one hour of treatment but was not detected in PAE/VEGFR-1 cells up to 24 hours after treatment. In vascularization studies using chicken chorioallantoic membranes, 1 nM VEGF ₁₂₁/rGel completely inhibited bFGF-stimulated neovascular growth. The cytotoxic effects of VEGF ₁₂₁/rGel were not apoptotic since treated cells were TUNEL-negative with no evidence of PARP cleavage or alteration in the protein levels of select apoptotic markers. Microarray analysis of VEGF ₁₂₁/rGel-treated HUVECs revealed the upregulation of a unique "fingerprint" profile of 22 genes that control cell adhesion, apoptosis, transcription regulation, chemotaxis, and inflammatory response.

Conclusions

Taken together, these data confirm the selectivity of VEGF ₁₂₁/rGel for VEGFR-2-overexpressing endothelial cells and represent the first analysis of genes governing intoxication of mammalian endothelial cells by a gelonin-based targeted therapeutic agent.

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Most cited references 36

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Gabriele Bergers, Steven Song, Nicole P Meyer-Morse … (2003)

Functions of receptor tyrosine kinases implicated in angiogenesis were pharmacologically impaired in a mouse model of pancreatic islet cancer. An inhibitor targeting VEGFRs in endothelial cells (SU5416) is effective against early-stage angiogenic lesions, but not large, well-vascularized tumors. In contrast, a kinase inhibitor incorporating selectivity for PDGFRs (SU6668) is shown to block further growth of end-stage tumors, eliciting detachment of pericytes and disruption of tumor vascularity. Importantly, PDGFRs were expressed only in perivascular cells of this tumor type, suggesting that PDGFR(+) pericytes in tumors present a complimentary target to endothelial cells for efficacious antiangiogenic therapy. Therapeutic regimes combining the two kinase inhibitors (SU5416 and SU6668) were more efficacious against all stages of islet carcinogenesis than either single agent. Combination of the VEGFR inhibitor with another distinctive kinase inhibitor targeting PDGFR activity (Gleevec) was also able to regress late-stage tumors. Thus, combinatorial targeting of receptor tyrosine kinases shows promise for treating multiple stages in tumorigenesis, most notably the often-intractable late-stage solid tumor.

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Jie Ma, David Waxman (2008)

Angiogenesis is a hallmark of tumor development and metastasis and is now a validated target for cancer treatment. However, the survival benefits of antiangiogenic drugs have thus far been rather modest, stimulating interest in developing more effective ways to combine antiangiogenic drugs with established chemotherapies. This review discusses recent progress and emerging challenges in this field; interactions between antiangiogenic drugs and conventional chemotherapeutic agents are examined, and strategies for the optimization of combination therapies are discussed. Antiangiogenic drugs such as the anti-vascular endothelial growth factor antibody bevacizumab can induce a functional normalization of the tumor vasculature that is transient and can potentiate the activity of coadministered chemoradiotherapies. However, chronic angiogenesis inhibition typically reduces tumor uptake of coadministered chemotherapeutics, indicating a need to explore new approaches, including intermittent treatment schedules and provascular strategies to increase chemotherapeutic drug exposure. In cases where antiangiogenesis-induced tumor cell starvation augments the intrinsic cytotoxic effects of a conventional chemotherapeutic drug, combination therapy may increase antitumor activity despite a decrease in cytotoxic drug exposure. As new angiogenesis inhibitors enter the clinic, reliable surrogate markers are needed to monitor the progress of antiangiogenic therapies and to identify responsive patients. New targets for antiangiogenesis continue to be discovered, increasing the opportunities to interdict tumor angiogenesis and circumvent resistance mechanisms that may emerge with chronic use of these drugs.

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Phosphatidylinositol 3-kinase (PI 3-kinase) is a signaling molecule that controls numerous cellular properties and activities. The oncogene v-p3k is a homolog of the gene coding for the catalytic subunit of PI 3-kinase, p110alpha. P3k induces transformation of cells in culture, formation of hemangiosarcomas in young chickens, and myogenic differentiation in myoblasts. Here, we describe a role of PI 3-kinase in angiogenesis. Overexpression of the v-P3k protein or of cellular PI 3-kinase equipped with a myristylation signal, Myr-P3k, can induce angiogenesis in the chorioallantoic membrane (CAM) of the chicken embryo. This process is characterized by extensive sprouting of new blood vessels and enlargement of preexisting vessels. Overexpression of the myristylated form of the PI 3-kinase target Akt, Myr-Akt, also induces angiogenesis. Overexpression of the tumor suppressor PTEN or of dominant-negative constructs of PI 3-kinase inhibits angiogenesis in the yolk sac of chicken embryos, suggesting that PI 3-kinase and Akt signaling is required for normal embryonal angiogenesis. The levels of mRNA for vascular endothelial growth factor (VEGF) are elevated in cells expressing activated PI 3-kinase or Myr-Akt. VEGF mRNA levels are also increased by insulin treatment through the PI 3-kinase-dependent pathway. VEGF mRNA levels are decreased in cells treated with the PI 3-kinase inhibitor LY294002 and restored by overexpression of v-P3k or Myr-Akt. Overexpression of VEGF by the RCAS vector induces angiogenesis in chicken embryos. These results suggest that PI 3-kinase plays an important role in angiogenesis and regulates VEGF expression.

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Author and article information

Journal

Journal ID (nlm-ta): BMC Cancer

Title: BMC Cancer

Publisher: BioMed Central

ISSN (Electronic): 1471-2407

Publication date Collection: 2011

Publication date (Electronic): 17 August 2011

Volume: 11

Page: 358

Affiliations

[1 ]Departments of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA

[2 ]Departments of Neuro-Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA

[3 ]Departments of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA

[4 ]Departments of Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA

[5 ]Department of Medical Microbiology and Immunology, Southern Illinois University, School of Medicine, Springfield, IL, USA

[6 ]Department of Cardiology and Angiology, University Hospital Münster, Münster, Germany

[7 ]Simmons Comprehensive Cancer Center, The University of Texas Southwestern Medical Center, Dallas, TX, USA

[8 ]The University of Texas Southwestern Medical Center, Dallas, TX, USA

[9 ]GlycoFi/Merck & Co., Inc., Lebanon, NH, USA

Article

Publisher ID: 1471-2407-11-358

DOI: 10.1186/1471-2407-11-358

PMC ID: 3176242

PubMed ID: 21849059

SO-VID: c0573a16-9261-4d16-9bfc-9cd4b3d6361f

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.