Effect of VEGFR Inhibition on Tumor Angiogenesis

Objectives: The Vascular Endothelial Growth Factor (VEGF) has been considered the most important mediator in inducing tumor angiogenesis. It binds to specfic receptors known as VEGF Receptors (VEGFR). An important pathway for angiogenesis is VEGF binding to VEGFR-2 and through PI3-K stimulates expression of anti-apoptotic protein Bcl-2, inducing expression of pro-angiogenic cytokines CXCL-8 and CXCL-1, resulting in endothelial cell proliferation. A recently developed anti-angiogenic drug is PTK787/ZK222584 (PTK/ZK), a potent tyrosine kinase VEGFR inhibitor, without aparent cytotoxic effect on cells without expression of these receptors.

Methods: For the better understanding of this mechanism and the angiogenic VEGF/VEGFR pathway of PTK/ZK in squamous cell carcinoma (SCC), it was developed in vitro and in vivo studies evaluating Bcl-2, CXCL-8 and CXCL-1 expression. A human angiogenesis experimental model was used in immunodeficient mice that received co-implants of tumor and endothelial cells or endothelial cells only, treated with PTK/ZK orally administered during 21 days. Tumor progression was evaluated by bioluminescence imaging. After mice sacrifice, the implants were paraffin embedded and 3µm sections were obtained for microvessel density analisis and 7µm sections for tumor and endothelial cells RNA retrieving by laser microdissection and analyzed by RT-PCR and Real time PCR. In vitro studies with co-culture of tumor and endothelial cells were analyzed by RT-PCR, Western Blot and ELISA to evaluate Bcl-2, CXCL-8 and CXCL-1 expression.

Results: The in vitro and in vivo results showed important downregulation of Bcl-2 as well as CXCL-8 expression in endothelial cells when treated with PTK/ZK. In addition, it was observed a significant reduction of microvessel density in implants of animals treated with the drug.

Conclusions: These results suggest that VEGFR inhibition with the anti-angiogenic drug PTK/ZK on endothelial cells seems to occur by downregulation of anti-apoptotic Bcl-2 expression leading to downregulation of pro-angiogenic CXCL-8, resulting in less tumor growth.