Abstract 3782: Cl-amidine, a PAD inhibitor, prevents UC and CRC in mice: Exploring novel mechanisms of miRNA and oxidative stress regulation

Abstract

Abstract Ulcerative Colitis (UC) is a chronic, relapsing inflammatory bowel disease that affects millions of patients worldwide. Due to the chronic inflammatory state of UC, which causes oxidative stress and leads to DNA damage, patients with UC are at a higher risk of developing colorectal cancer (CRC). Although the cause of UC is still unknown, the protein arginine deiminase (PAD) family of enzymes has been found to be a key component in many human inflammatory diseases and cancer, including UC and CRC. PADs are calcium dependent enzymes that post-translationally convert peptidyl-Arginine to peptidyl-Citrulline through ‘citrullination’. In hopes of treating UC and preventing CRC, we developed Chlor-amidine (Cl-amidine), a novel small molecule inhibitor of PADs. Cl-amidine irreversibly inhibits PADs by covalent modification of Cys645 at the active site of the PAD enzyme. Unlike current approved drugs for the treatment of UC, Cl-amidine shows no toxicity both in vitro and in vivo. Preliminary in vitro studies showed that Cl-amidine is able to suppress oxidative stress, DNA damage, and the inflammatory marker, iNOS. Correspondingly, we found similar results in our AOM/DSS mouse model of UC, as Cl-amidine suppressed colonic inflammation. In this UC model, Cl-amidine treatment also decreased reactive oxygen species (ROS) production by inflammatory cells and DNA damage in epithelial cells. We proceeded to look at the capability of Cl-amidine to prevent carcinogenesis in our AOM/DSS mouse model of UC-associated CRC and report that Cl-amidine treatment significantly decreased tumor incidence in this model. To further understand how Cl-amidine prevents carcinogenesis, we are currently exploring the effect of Cl-amidine on two separate mechanisms: 1) miRNA and 2) oxidative stress regulation. First, our recent studies have focused specifically on miR-16, a putative tumor suppressor, which is downregulated in cancers. Interestingly, we have found that Cl-amidine upregulates miR-16 expression in our in vitro and in vivo models of UC. We aim to determine if Cl-amidine acts through miR-16 to prevent UC and UC-associated CRC by suppressing DNA damage and/or by suppressing cell proliferation. The second mechanism that we are exploring involves the regulation of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase. In vitro results reveal that Cl-amidine increased protein levels of these enzymes. We plan to measure ROS levels in Cl-amidine treated cells that are introduced to SOD, GPx, and catalase inhibitors. This will provide evidence that Cl-amidine is working through these specific enzymes to reduce ROS activity. Based on the efficacy of Cl-amidine in our models of UC and CRC, and its potential multi-targeted mechanism of action; our research has revealed that Cl-amidine is a cutting-edge, prospective drug therapy for UC patients. Citation Format: Erin E. Witalison, Xiangli Cui, Paul R. Thompson, Lorne J. Hofseth. Cl-amidine, a PAD inhibitor, prevents UC and CRC in mice: Exploring novel mechanisms of miRNA and oxidative stress regulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3782. doi:10.1158/1538-7445.AM2014-3782