Abstract 3782: Chloroquine induces defective autophagic flux and enhances pancreatic cancer cell death induced by pegylated-arginine deiminase

Abstract

Abstract We have previously shown that a majority of human pancreatic adenocarcinomas are deficient in argininosuccinate synthetase (ASS), a key enzyme in arginine synthesis and therefore sensitive to arginine deprivation by arginine deiminase (ADI). ASS deficient cell lines undergo cell death when deprived of arginine, but also undergo autophagy. The role of autophagy and its contribution to cell death is controversial. We hypothesized that autophagy is protective in the setting of arginine deprivation and that inhibition of autophagy by hydroxychloroquine (ChQ) would increase cell death. Methods: The human pancreatic cancer cell line MIA-PaCa2 was treated in vitro and in vivo with pegylated arginine deiminase (PEG-ADI) alone or in the presence of ChQ. Cell death was measured by propidium iodide-FACS and apoptosis was evaluated by Annexin V-PI flow cytometry. Caspase 3 cleavage was evaluated by western blotting and ELISA as was expression of LC3. In vivo experiments were conducted by creation of subcutaneous xenografts in athymic mice followed by treatment with intraperitoneal injections of PBS, PEG-ADI, ChQ and combination of PEG-ADI and ChQ. At sacrifice, tumors were removed for analysis. Tumor lysates were analyzed by western blot for caspase 3 and p62. Immunohistochemistry for activated caspase 3 and DNA fragmentation (TUNEL) were also performed. Results: In vitro treatment of MIA-PaCa2 with PEG-ADI induced caspase dependent cell death as well as autophagy at 72 hours. ChQ at low doses inhibited autophagy induced by PEG-ADI, but increased cell death. In vivo, addition of ChQ to a sub-therapeutic dose of PEG-ADI increased tumor suppression decreased autophagy and increased caspase 3 activation compared to either treatment alone. Conclusion: Arginine deprivation induces autophagy and cell death in cell lines deficient in ASS. Autophagy appears to play a protective role, and its inactivation by hydroxychloroquine leads to enhanced tumor suppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3782. doi:10.1158/1538-7445.AM2011-3782