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