Antiangiogenic systemic gene therapy combined with doxorubicin administration induced caspase 8 and 9-mediated apoptosis in endothelial cells and an anti-metastasis effect

Ad-PPE-Fas-c is an adenovector that expresses Fas-c under the control of the modified pre-proendothelin-1 (PPE-1) promoter. Fas-c is a chimeric death receptor containing the extracellular portion of tumour necrosis factor 1 receptor (TNFR1) and the transmembrane and intracellular portion of Fas. We recently demonstrated that Ad-PPE-Fas-c induced Fas-receptor-mediated endothelial cell apoptosis. Previously, doxorubicin was shown to enhance Fas-receptor clustering and the induction of its cascade. Therefore, the goal of this work was to test whether doxorubicin augments the capacity of Ad-PPE-Fas-c to induce endothelial cell apoptosis and to elucidate whether either the death-receptor-mediated apoptotic cascade or the mitochondria-associated apoptotic cascade is involved in the combined treatment effect. We found that a combined treatment of Ad-PPE-Fas-c and doxorubicin synergistically induced a reduction in endothelial cell viability and apoptosis. z-IETD-FMK, a caspase-8 inhibitor, and z-LEHD-FMK, a caspase-9 inhibitor, significantly decreased apoptosis induced by the combined treatment. Systemically administered combined therapy significantly reduced the lung metastases burden (70%) in mice as compared to each treatment alone. Thus, a combined treatment of Ad-PPE-Fas-c gene therapy and chemotherapy may be effective in the treatment of metastatic diseases and both the Fas cascade and the mitochondria-associated cascade are essential for this effect.