Abstract 4848: Pegylated arginine deiminase induces autophagy in canine melanoma and canine osteosarcoma
Authors: Rodriguez Carlos O. , Fascetti Andrea J. , Yu Zengshou , Shentu Shujun , Bold Richard J. , Kung Hsing-Jien , Stellrecht Christine M. 
Published: February 2011

Abstract
Arginine is a nonessential amino acid in adult mice, men and dogs, which undergoes both synthesis and degradation in complex series of compartmentalized steps at the cellular and systemic level. The urea cycle enzymes are responsible for this axis and is dependent on the levels of are argininosuccinate synthetase (ASS1) and argininosuccinate lyase (ASL). It has been shown that many human tumor types are auxotrophic for L-arginine typically due to very low or no specific activities of ASS1 and/or ASL and are thus sensitive to induction of cell death as a result of the depravation of this single nutrient. Pegylated arginine-deiminase (PEGADI) is a molecule with this mechanism of action and has entered phase I and II clinical trials in humans where it has proven its clinical utility in the treatment of hepatocellular carcinoma and melanoma.
The cancer-bearing dog serves an excellent spontaneous out-bred immune competent large animal model for numerous homologous neoplastic diseases in humans. The purpose of this study is to determine the status of arginine-synthetic enzymes and then evaluate the action of PEGADI in cultured canine cancer melanoma (12, 23, 36) and osteosarcoma (D17, 48-4, 29) cell lines. Using western blot analysis we found that each of these six canine cell lines expressed ASS1 and ASL. PEGADI depleted arginine in a time- and concentration-dependent manner in the cultured canine melanoma and osteosarcoma as measured by HPLC. Furthermore, the canine melanoma and osteosarcoma cells were very sensitive to this depletion. In particular, there was a dramatic loss of clonogenic survival in these cells treated with PEGADI (IC50 range: 0.03 uM at 96 h to 0.3 at 24 h). This sensitivity was apparently due to caspase independent cell killing as caspase 3, 8, or 9 were not activated. Changes in cell cycle distribution were quantitated with flow cytometry. Despite the lack of caspase activation, there was an increase in the number of cells in the Sub G1 population. To determine if PEGADI induced autophagy in these cells the conversion of microtubule-associated protein 1 light chain 3B (LC3B)-I to its lipidated form LC3B-II was assessed and showed there was a dose dependent induction of LC3B-II. Consistent with the induction of autophagy, results demonstrate an induction in the formation autophagolysosomes as determined by flow cytometery analysis of acridine orange staining of acidic vesicles, as well as fluorescent microscopy of treated cells stained with monodansylcadaverine. Our results support the notion that PEGADI may have clinical utility in the management of canine neoplasia. A clinical trial for this species is planned.
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 4848.