Experimental Studies of Ursolic Acid and Alkaline Sphingomyelinase in Colon Cancer and Colitis

Abstract: Sphingomyelin (SM) is a sphingolipid present in all eukaryotic cell membranes. Hydrolysis of SM by sphingomyelinase (SMase) generates ceramide, which has effects on cell-cycle regulation and apoptosis. Alkaline sphingomyelinase (Alk-SMase) is the main SM-hydrolysing enzyme in the human intestine, and earlier studies have shown that it has anti-proliferative properties on colon cancer cells, and that its activity is decreased in colon tumors as well as in ulcerative colitis. Ursolic acid (UA) is a plant-derived pentacyclic triterpenoid with anti-cancer and anti-inflammatory properties, whose effects on instestinal disorders has not previously been studied. The aim of the studies in this thesis was to examine the effects of UA on experimental colon cancer and its relation to gut sphingolipid metabolism, and finally to generate recombinant human Alk-SMase and use it as a topical treatment in a rat colitis model. When treating human colon cancer cells (HT-29) with UA, we found that UA dose-dependently decreased cell proliferation and induced apoptosis, accompanied by activation of caspase 3, 8 and 9. UA also selectively increased the activity of intestinal alkaline sphingomyelinase before the activation of caspases, suggesting this to be an early event in UA-mediated apoptosis. In an in vitro study, we saw that UA as well as four other pentacyclic triterpenoids specifically enhanced the activity of purified rat intestinal Alk-SMase in a dose-dependent manner. In the third study, using an Azoxymethane (AOM) rat cancer model, we examined the effect of orally administered UA on the formation of aberrant crypt foci (ACF) and intestinal SMase activity. UA significantly reduced the incidence of ACF containing three or more crypts when administered during the initiation phase. AOM reduced mucosal Alk-SMase activity, and the inhibitory effects could not be prevented by UA. In both AOM-treated and normal rats, UA increased the activity of colonic neutral SMase markedly and that of acid SMase activity mildly, all of which indicates that UA has chemopreventive effects in the initiation phase of colon cancer associated with changes in SM metabolism. In the fourth and final study, we, for the first time, report a method to express a biologically active human Alk-SMase from Pichia pastoris yeast cells. We then used the expressed recombinant enzyme to treat a dextran sulfate sodium rat acute colitis model, and found that intrarectal instillation of Alk-SMase once daily for 1 week significantly reduced the inflammation score and protected the colonic epithelium from inflammatory destruction. We also found a tendency towards decreased tumor necrosis factor (TNF)-alpha expression in the Alk-SMase-treated group. To summarize, these studies show evidence that UA and alk-SMase have chemopreventive effects against colon cancer and inflammation, and provide evidence indicating a link between the effects of UA and sphingolipid metabolism. The last study also demostrates an effective method to generate recombinant alk-SMase for potential applications of the enzyme against colonic tumor and inflammation.