Abstract 4443: Molecular mechanistic study of ASA404 (vadimezan)-induced endothelial cell death

Abstract

Abstract The tumor vasculature is a key component in maintaining tumor growth and regulating the tumor microenvironment through the supply of nutrients and oxygen. ASA404 (vadimezan, 5,6-dimethylxanthenone-4-acetic acid) is a flavonoid, non-tubulin-binding Tumor-Vascular Disrupting Agent (Tumor-VDA) that induces breakdown of the established tumor vasculature through the induction of tumor endothelial cell apoptosis, resulting in the inhibition of tumor blood supply, leading to tumor ischemia and extensive necrosis of the tumor core. In contrast to antiangiogenics, ASA404 has limited effects on angiogenesis at the tumor periphery and so the remaining peripheral tumor rim represents a source of viable cells for regrowth of the tumor upon cessation of ASA404 treatment. Preclinical and clinical studies have highlighted the effectiveness of combining chemotherapies, especially taxanes, with ASA404 for marked tumor inhibition. Currently, the benefits of adding ASA404 to the standard of care in first- and second-line NSCLC indications are being evaluated in the ATTRACT-1 and ATTRACT-2 Phase III trials respectively. Despite the prior clinical efficacy observed with ASA404, the detailed molecular mechanism by which ASA404 selectively targets the tumor vasculature still remains to be defined. To investigate this, we initiated studies to explore the function of ASA404 both in vitro using human endothelial cells (HUVEC) and in ASA404-treated MDA-MB-231 breast cancer xenografts examined ex vivo. Consistent with previous reports, we observed inhibition of HUVEC cell proliferation along with rapid cell death in vitro as assessed using FACS analysis and newly demonstrated the rapid induction of caspase 3 assessed by immunocytochemistry. Treatment of the MDA-MB-231 breast xenografts in vivo with ASA404 resulted in significant tumor growth inhibition, tumor necrosis and elevation of the hypoxia marker carbonic anhydrase 9 (CAIX). The nature of ASA404-induced endothelial cell death is likely to be due to apoptosis since rapid caspase 3 cleavage was observed in the endothelial cells both in vitro and in vivo. In addition, the mitochondrial potential of cultured HUVEC cells in vitro was found to be seriously compromised after 1-2 hours treatment with ASA404 as monitored by Mito-tracker staining. This was accompanied by cytochrome C release into the cytosol as evidenced by immunohistochemical co-localization of cytochrome C and mitochondria markers. We also detected a moderate, but concentration-dependent increase in ceramide levels in HUVEC cells following 2 hours of ASA404 treatment, which may serve as a link to the observed mitochondrial depolarization phenotype. Lastly, a pooled-based shRNA screen was also conducted to search for genes that could modulate ASA404 activity in HUVEC cells. Some of the candidates identified may provide new insights into the pathways that are critical in the ASA404 mechanism of action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4443.