Date of Award

6-30-2016

Document Type

Open Access Dissertation

Department

College of Pharmacy

First Advisor

Lorne Hofseth

Abstract

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that affects the quality of life of millions of patients worldwide. This disease is associated with inflammation and ulceration of the colonic epithelium, leading to an increased risk for the development of UC-associated colorectal cancer (CRC). Current UC medications are designed to manage the symptoms and induce remission; however, several challenges are faced with current treatment options. 5-aminosalicyclic acid and corticosteroids have few side effects, but have limited efficacy and often the disease becomes refractory. Biologics are introduced after initial treatments fail, but serious side effects are often associated with these medications. In the hope of developing a safe and effective UC therapeutic, we are testing a small molecule inhibitor of Protein Arginine Deiminases (PADs), which are implicated in many inflammatory diseases, like Alzheimer’s disease, Multiple Sclerosis, Rheumatoid Arthritis, and UC.

PADs catalyze the conversion of peptidyl-Arginine to peptidyl-Citrulline through a process termed ‘citrullination’. Chlor-amidine (Cl-amidine) is designed to irreversibly inhibit PADs through covalent modification at the active site of the enzymes. Our initial studies have determined that Cl-amidine is generally nontoxic in cell and animal models. Next, we tested the efficacy of Cl-amidine in the HCT116 colon cancer cell line and the Azoxymethane (AOM)/Dextran Sulfate Sodium (DSS) murine model of UC. We showed that Cl-amidine effectively reduced inflammation, oxidative stress produced by activated leukocytes, and DNA damage. Based on these findings, we hypothesized that Cl-amidine could prevent the progression to UC-associated CRC. In vitro studies indicated that Cl-amidine may act as a tumor suppressor by upregulating miR-16, a putative tumor suppressor miRNA with cell cycle targets, and causing G1 cell cycle arrest. Our results showed that in the AOM/DSS murine model of UC-associated CRC, Cl-amidine significantly inhibited tumorigenesis, further supporting our hypothesis.

For a mechanistic study of Cl-amidine, we explored the idea that Cl-amidine may be preventing the citrullination and/or hyperactivity of DNA methyltransferases (DNMTs). Hypermethylation is reported to cause methylation-mediated gene silencing; therefore, we postulated that PADs may be citrullinating DNMTs and causing hypermethylation of genes regulating tumor suppressor miRNAs. We verified that DNMTs can be citrullinated and that the inhibition of DNMTs or PADs restores miR-16 levels to comparable levels. Cl-amidine also reduced the methylation of the miR-16 promoter as effectively as DNMT inhibition. Ultimately, the objective of our research is to provide evidence of nontoxic and more successful treatment options for UC.

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