Ursodeoxycholic acid differentially affects three types of sphingomyelinase in human colon cancer Caco 2 cells

doi:10.1016/j.canlet.2005.04.016

Cancer Letters

Volume 235, Issue 1, 8 April 2006, Pages 141-146

https://doi.org/10.1016/j.canlet.2005.04.016 Get rights and content

Abstracts

We investigated the effects of UDCA on sphingomyelinase (SMase) in Caco 2 cells cultured in monolayer and polarized conditions. Alkaline SMase activity was high in polarized cells whereas, acid and neutral SMase activities were high in monolayer cells. In polarized cells, UDCA increased alkaline SMase expression and caspase 3 activity but had no effect on acid and neutral SMases. In monolayer cells, UDCA reduced both acid and neutral SMase activities, inhibited cell proliferation, but had little effect on alkaline SMase and caspase 3 activities. In conclusion, UDCA differentially affects SMase activity, cell proliferation, and apoptosis in colonic cells depending on the cell conditions.

Introduction

Ursodeoxycholic acid (UDCA) is a type of bile salt naturally occurring in human bile in a relatively small amount. A novel chemopreventive effect of UDCA against colonic carcinogenesis was first identified by Earnest et al. [1] in an animal study and confirmed by others in both animal and human studies [2], [3]. The mechanism underlying the anticancer effects of UDCA is not fully understood, and multiple molecules that are effected by UDCA have been reported, including Cox 2, phospholipase A, cycline D, E-cadherin, and Ras [2], [4], [5].

Sphingomyelin (SM) metabolism generates multiple lipid messengers such as ceramide, sphingosine and sphingosine-1-phosphate, which regulate cell proliferation and apoptosis [6], [7]. A particular link between SM and colon cancer has been indicated. SM was found to be accumulated and ceramide reduced in precancer lesions and cancer tissues in either animal models or human beings [8], [9]. Supplement of SM and other sphingolipids in the diet could inhibit the formations of aberrant crypt foci and carcinomas in animals treated with a chemical carcinogen [10]. Since SM and glycosphingolipids cannot be absorbed intact [11], the hydrolysis of sphingolipids in the gut may be a prerequisite for their anticancer effects. In the intestinal tract, there are at least three types of SMase called acid, neutral and alkaline SMase. The acid and neutral SMases are common enzymes whereas, alkaline SMase is specifically present in the intestinal mucosa as an ecto enzyme with the highest hydrolytic capacity among these SMases [12], [13], [14]. The alkaline SMase has been shown to be significantly decreased in both precancer lesions such as ulcerative colitis [15] and in colonic adenocarcinoma [16]. A mutation of the enzyme caused by an alternative splicing, which inactivates the enzyme has been identified in HT 29 cells but not in Caco-2 cells [17]. Caco-2 cells cultured in monolayer conditions express little alkaline SMase and the expression increases significantly in polarized conditions undergoing differentiation [17].

We reported previously that, feeding rats UDCA significantly increased alkaline SMase activity in the colonic mucosa in positive correlation with the activity of caspase 3, a key enzyme for apoptosis [18]. In the present study, we examined the direct effect of UDCA on three types of SMase in Caco-2 cells cultured in both monolayer and polarized conditions.

Section snippets

Materials

UDCA (purity>98%) was provided by Dr Falk Pharma GmbH (Freiburg, German). SM was purified from bovine milk and labelled with [¹⁴C–CH₃] choline ([¹⁴C-SM]) at Astra Zeneca (Stockholm, Sweden). Anti-human alkaline SMase antibody was developed in AgriSera AB (Vännäs, Sweden) using purified human alkaline SMase as an antigen [13]. All cell culture mediums and other chemicals used were purchased from Sigma Co. (Stockholm, Sweden).

Cell cultures

The Caco 2 cells were cultured in both monolayer and polarized

Effects of UDCA on alkaline SMase

As shown in the top panel of Fig. 1, alkaline SMase activity was much lower in monolayer cells than in polarized cells. UDCA increased the activity of alkaline SMase in polarized cells dose-dependently, with 75% increase obtained by 0.5 mM UDCA. UDCA at concentrations less than 0.5 mM had no effect on monolayer cells, and 1 mM UDCA only induced a slight increase of alkaline SMase activity. The increased activity in polarised cells may duo to an enhanced expression, as Western blotting showed that

Discussion

The present work demonstrates that the activities of SMases in the colon varied significantly with the conditions of the cells. Alkaline SMase activity was high in polarized cells whereas, acid and neutral SMase activities were high in monolayer cells. UDCA affected SMase activities, cell proliferation, and apoptosis, depending on the cell conditions, stimulating alkaline SMase expression and apoptosis in polarized cells, and inhibiting acid and neutral SMase activities and cell proliferation

Acknowledgements

The work was supported by Swedish Cancer Society, Swedish Research Council, Albert Påhlsson Foundation, Crafoord Foundation, Gunnar Nilsson Cancer Foundation, Lund University Hospital Research Foundation in Sweden and grants from Dr Falk Pharma GmbH, Freiburg, in Germany.

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Sphingolipids are abundant in the microvillar membrane of intestinal epithelial cells where they are essential for structural integrity and may act as receptors for toxins, virus and bacteria. Metabolism of dietary and membrane sphingolipids in the intestine generates ceramide, sphingosine, sphingosine-1-phosphate, and ceramide-1-phosphate, via the action of alkaline sphingomyelinase, neutral ceramidase, sphingosine-1-kinase, and ceramide-1-kinase. These intermediary metabolites act as bioactive lipid messengers, influencing numerous cellular functions including growth, differentiation and apoptosis of both epithelial and immunocompetent cells in the gastrointestinal tract, and also the progress of inflammation and responsiveness of the mucosal cells to pathogens. This review summarizes background and recent progress in the metabolism of dietary and endogenous sphingolipids in the gut and its pathophysiological implications.
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Alkaline sphingomyelinase (alk-SMase) is present in the intestinal tract and additionally human bile. It hydrolyses sphingomyelin in both intestinal lumen and the mucosal membrane in a specific bile salt dependent manner. The enzyme was discovered 36 years ago but got real attention only in the last decade, when sphingomyelin metabolism was realized to be a source of multiple lipid messengers, and when dietary sphingomyelin was found to inhibit colonic tumorigenesis in animals. The enzyme shares no structural similarity with other SMases and belongs to the nucleotide pyrophosphatase/phosphodiesterase family. The enzyme is of specific properties, such as bile salt dependency, trypsin resistance, high stability, and tissue specific expression. In the colon, the enzyme may play antiproliferative and antiinflammatory roles through generating ceramide, reducing the formation of lysophosphatidic acid, and inactivating platelet-activating factor. The enzyme is down regulated in human long-standing ulcerative colitis and colonic adenocarcinoma, and mutation of the enzyme has been found in colon cancer cells. In the small intestine, alk-SMase is the key enzyme for sphingomyelin digestion. The hydrolysis of sphingomyelin may affect the cholesterol uptake and have impact on sphingomyelin levels in plasma lipoproteins. The review summarizes the new information of alk-SMase from biochemical, cell and molecular biological studies in the last decade and evaluates its potential implications in development of colon cancer, inflammatory bowel diseases, and atherosclerosis.

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Ursodeoxycholic acid differentially affects three types of sphingomyelinase in human colon cancer Caco 2 cells

Abstracts

Introduction

Section snippets

Materials

Cell cultures

Effects of UDCA on alkaline SMase

Discussion

Acknowledgements

Cancer Lett.

Gastroenterology

Prog. Lipid Res.

J. Nutr.

Biochim. Biophys. Acta

J. Lipid Res.

J. Biol. Chem.

Methods Enzymol.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Chemoprevention of azoxymethane-induced colonic carcinogenesis by supplemental dietary ursodeoxycholic acid

Cancer Res.

Ursodeoxycholic acid and F(6)–D(3) inhibit aberrant crypt proliferation in the rat azoxymethane model of colon cancer: roles of cyclin D1 and E-cadherin

Cancer Epidemiol. Biomarkers Prev.