Date of Award

1-1-2013

Document Type

Open Access Dissertation

Department

College of Pharmacy

Sub-Department

Pharmaceutical Science

First Advisor

Lorne J Hofseth

Abstract

Inflammatory Bowel Disease (IBD) [Ulcerative colitis (UC) and Crohn's Disease (CD)] is a group of chronic disorders of unknown etiology characterized by inflammation in the gastrointestinal tract associated with a high colon cancer risk. UC is characterized by periods of active disease ("flare-ups"), followed by periods when the disease is inactive ("remission"). The end result is an abnormal immune response with repeated episodes of colonic inflammation. Conventional treatment of UC by aminosalicylates, TNFα inhibitors, and steroids have modest effects, and come with a high risk of side effects including dyspeptic symptoms, gastric ulceration and sometimes hospitalization and deaths. Because of higher acceptance, efficacy, minimal side effects and relatively low cost, patients (up to 50%) are using complementary and alternative medicines (CAMs). In this light, because of the anti-inflammatory and anti-oxidant properties of American Ginseng (AG), we tested the hypothesis that AG suppresses colitis and prevents colon cancer in mice.

AG (Panax quinquefolius), an obligate shade perennial native of North America has previously been shown by our lab to treat and prevent colitis and associated colon cancer. Here, to further delineate the putative active components of AG against colitis and colon cancer, we performed a bioassay-guided fractionation of AG using several polar (Water, Butanol, Dicholoromethane, Ethylacetate) and non-polar (Hexane) solvent extraction methods. Only the Hexane fraction of AG (HAG) was found to be potent anti-

oxidant and can drive inflammatory cell apoptosis and ameliorate colitis and associated colon cancer in an experimental mouse model (Chapter 2).

Inflammation induced Reactive Oxygen Species (ROS) and Nitric Oxide (NO) leads to p53 activation to eliminate damaged cells by apoptosis during colitis. HAG showed increased anti-inflammatory and pro-apoptotic properties in a mouse model of colitis (Chapter 2). From these observations, we tested the hypothesis that HAG might prevent colitis through p53-mediated apoptosis of inflammatory cells. Results are consistent with this hypothesis (Chapter 3).

MicroRNAs (miRNAs) have recently been shown to play a key role in inflammation and cancer. Alternatively, inflammation can modulate miRNA expression, which in turn regulates carcinogenesis. Because HAG suppresses colon inflammation and prevents colon cancer, we examined the effects of HAG in miRNA expression. miRNAs are the small non-coding RNA of approximately 22 nucleotides long that post-transcriptionally regulates the gene expression in plants and animals. Dysregulated microRNA (miR) expression has been observed in several disease conditions including colon cancer. Using global miR expression profiling, we observed increased miR-29b in colon cancer cells following exposure to HAG. Since miR-29b plays a role in regulating the migration of cancer cells, we hypothesized that HAG induces miR-29b expression to target matrix metalloproteinase-2 (MMP-2) thereby suppressing the migration and invasion of colon cancer cells. Results are consistent with this hypothesis. Our study supports the understanding that targeting MMP-2 by miR-29b is a mechanism by which HAG suppresses the migration and invasion of colon cancer cells (Chapter 4).

Finally to initiate further studies to identify the bioactive component/s present in HAG, preparative reverse-phase HPLC subfractionation was performed (Chapter 5). Subfractions F2 and F3 [both with a major polyacetylene content (Panaxynol, Panaxydol and Panaxydiol)] exhibited anti-inflammatory property, thereby supporting the notion that Polyacetylenes could be the bioactive compounds responsible for the anti-inflammatory and anti-cancer property of HAG. Future studies will confirm Polyacetylenes as bioactive compound of HAG in the suppression of colitis and prevention of colon cancer. In conclusion, data presented here have identified key components of AG and some mechanisms by which HAG suppresses colitis and prevents colon cancer in mice. These results support the possibility of testing individual components of AG in treating IBD in humans.

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