Proteomic characterization of the antiangiogenic activity of PF4-DLR in glioblastomas

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The prognosis of the most common glial tumors, the glioblastoma (GBM; World Health Organization grade IV), remains poor with 2-year survival rates at less than 20% despite significant advances in therapeutic options available to patients. The dependence of tumor growth and metastasis on angiogenesis has provided powerful rationale for anti-angiogenic approaches to cancer therapy (Folkman J. 1971; Carmeliet P. et al. 2000). One of the most important negative regulators of angiogenesis is platelet factor-4 (PF-4). In a previous study Hagedorn M. et al. demonstrated that PF4-DLR inhibits tumor growth in an intracranial glioma model. In this study we use a proteomic approach to better understand the molecular mechanisms involved in PF4-DLR response. Experiments were performed on gliomas treated for 10 and 20 days with PF4-DLR. Two–dimensional SDSpolyacrylamide gel elecrophoresis technique (2D-SDS-PAGE) was use to compare protein expression pattern between treated and untreated tumors. In tumors treated with PF4-DLR for 10 days we identified 24 proteins that were differentially expressed (p<0.05) in response to the treatment. In mice treated with PF4-DLR for 20 days we identified 30 proteins to be differentially expressed (p<0.05) subsequently to the treatment. A bioinformatics analysis of the identified proteins revealed the complexity of the pathway modulated by PF4-DLR treatment and suggest that two different protein populations is regulated by the treatment: one involved in growth inhibition mediated by PF4-DLR and one involved in tumor escape. Most of the proteins we have identified at 20 days of treatment seem to be functionally involved in regulating tumor growth and escape. Interestingly we detected in tumors from animals treated with PF4-DLR for 10 days a significant decrease of ILK levels compared with untreated tumors while in mice treated with PF4-DLR for 20 days we detected a significant increase of ILK levels compared with untreated tumors. We also analyzed the ILK levels in human low grade gliomas compared with human high grade gliomas. We found a significant increase of ILK in high grade gliomas compared to low grade. Consequently ILK can represent a new molecular target for gliomas therapy. Thus, as first approach, we have identified specific ILK siRNA that are able to reduce glioma cell proliferation. Our data taken together indicate that the combinatorial administration of compounds that simultaneously inhibit angiogenesis and tumor cell proliferation by targeting specific signaling pathways might results in a significant increase in the therapeutic efficacy.