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Effect of vascular endothelia^l growth factor receptors inhibition on tumor angiogenesis in head and neck squamous cell carcinoma

Marta Miyazawa¹,², Zhihong Dong¹, Zhaocheng Zhang¹, Kathleen G. Neiva¹, Mabel M. Cordeiro¹, Denise T. Oliveira², Jacques E. Nör¹,³

¹Angiogenesis Research Laboratory; Department of Restorative Sciences, University of Michigan School of Dentistry; ²Department of Stomatology, Area of Pathology, Bauru School of Dentistry, University of São Paulo, Bauru, SP, Brazil; ³Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI, USA

Solid tumors require vascularization as a source of oxigen and nutrients to grow beyond a mass of approximately 1 mm in diameter. To acquire its own microvascular network, tumor cells start secreting pro-angiogenic factors through a process known as as angiogenic switch. There is convincing evidence that vascular endothelial growth factor (VEGF) is an important inducer of tumor angiogenesis. However, mechanism involved in the effect of anti-angiogenic drugs on the angiogenic switch are still unclear.

VEGF binding to their specific vascular endothelial growth factor receptors (VEGFR) , more specifically VEGFR2, induces phosphoinositide-3 kinase (PI3-K) activity and induces expression of anti-apoptotic protein Bcl-2 (REF). We have recenlty shown that Bcl-2 upregulation results in expression of pro-angiogenic chemokines CXCL-8 and CXCL-1, and enhancement of the angiogenic potential of endothelial cells (REF).

In 2000, Wood And collaborators presented a new anti-angiogenic drug called PTK787/ZK222584 (PTK/ZK; Novartis/Schering AG) with proved effect in vivo and in vitro in several tumors, such as prostate, kidney, tyroid, hypopharyngeal, liver, and oral câncer (REF). This drug is a potent VEGFR tyrosine kinase inhibitor without cytotoxic effects on cells that do not express VEGF receptors. When compared to other anti-angiogenic drugs, PTK/ZK is a small molecule inhibitor, it can be orally administered, and is well tolerated by patients. In addition, it appears that PTK/ZK does not affect physiological angiogenesis, such as wound healing.

To enhance the current understanding of the effect of PTK/ZK on VEGF signaling in tumor associated endothelial cells, in vitro and in vivo experiments were performed. Using an experimental model of human angiogenesis in immunodeficient mice, we treated animals that received co-implants of tumor cells (oral or laryngeal metastatic squamous cells carcinoma) and endothelial cells or implants of endothelial cells only with PTK/ZK for 21 days. Tumor progression was evaluated by in vivo bioluminescence imaging. After euthanasia of the mice, tumors were removed, fixed and paraffin embedded. 3 µm-thick sections were prepared, and immnohistochemistry with anti-factor VIII antibody was performed for microvessel density analysis. In addition, 7 µm-thick sections were used for tumor and endothelial cell RNA collection by laser microdissection. These RNA samples were analysed by RT-PCR and Real time PCR. in vitro studies co-cultures of tumor and endothelial cells were also used to evaluate Bcl-2, CXCL-8, and CXCL-1 expre
ssion by RT-PCR, Western Blot, and ELISA, and to confirm in vivo observations.

Here, we observed a significant and reproducible downregulation of Bcl-2 and CXCL-8 expression in endothelial cells exposed to PTK/ZK in vitro, and in tumor-associated endothelial cells of mice treated with the same drug in vivo. In addition, we observed a significant reduction of microvessel density in the tumors of animals treated with PTK/ZK.

These results demonstrate that the anti-angiogenic mechanism of PTK/ZK involves downregulation of Bcl-2 and the pro-angiogenic chemokine CXCL8 in tumor-associated endothelial cells. This response is correlated with a decrease in tumor microvessel density. These results suggest an effect of PTK/ZK on the angiogenic switch via regulation of Bcl-2 and pro-angiogenic chemokine expression. Our data also suggest PTK/ZK may be beneficial in the therapeutic scheme of patients with head and neck squamous cell carcinomas.

Acknowledgments

The authors thank Novartis/Schering AG for the PTK/ZK used in this study. This work was funded by project #1895/05-2 from CAPES (MM), and by grants R01-DE14601, R01-DE15948, R01-DE16586 from the NIH/NIDCR (JEN).

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