The human intestinal cell line Caco-2 is a well-established model system to study cellular differentiation of human enterocytes of intestinal origin, because these cells have the capability to differentiate spontaneously into polarized cells with morphological and biochemical features of small intestinal enterocytes. Therefore, the cells are widely used as an in vitro model for the human intestinal barrier. In this study, a proteomic approach was used to identify the molecular marker of intestinal cellular differentiation. The proteome of proliferating Caco-2 cells was compared with that of fully differentiated cells. Two-dimensional gel analysis yielded 53 proteins that were differently regulated during the differentiation process. Pathway analysis conducted with those 34 proteins that were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis revealed subsets of proteins with common molecular and cellular function. It was shown that proteins involved in xenobiotic and drug metabolism as well as in lipid metabolism were upregulated upon cellular differentiation. In parallel, proteins associated with proliferation, cell growth and cancer were downregulated, reflecting the loss of the tumorigenic phenotype of the cells. Thus, the proteomic approach in combination with a literature-based pathway analysis yielded valuable information about the differentiation process of Caco-2 cells on the molecular level that contributes to the understanding of the development of colon cancer or inflammatory diseases such as ulcerative colitis -diseases associated with an imbalanced differentiation process of intestinal cells.

Furan fatty acids (furan-FA) are oxidative products of conjugated linoleic acids (CLA) and may therefore be ingested when CLA-containing food or food-additives are consumed. Due to the presence of a furan ring structure the question arises whether furan-FA may have toxic properties on enterocytes and liver cells. Here we show that furan-FA neither have toxic effects in human colon cancer cell line Caco-2 nor in human hepatoma cell line HepG2 at concentrations that could be relevant for humans. At concentrations up to 100 μM, all tested furan-FA isomers showed no pronounced cytotoxicity and did not affect cellular proliferation or apoptosis up to concentrations of 500 μM. In addition, furan-FA was neither genotoxic in the micronucleus test using Chinese hamster lung fibroblasts (V79) nor in the Ames test independent of the presence or absence of rat liver homogenate for enzymatic activation of the furan ring structure. A proteomic approach revealed that 48 proteins were differentially expressed when Caco-2 cells were incubated with up to 1 mM of 10,13-epoxy-10,12-octadecadienoic acid (10,12-furan-FA). Three of the 30 proteins that could be identified by MALDI-TOF analysis were upregulated and were associated with lipid droplet biogenesis. The remaining 27 proteins were downregulated and were considered to be associated with general cellular processes such as DNA replication and transcription, protein biosynthesis and protein processing, lipid and energy metabolism. From the proteomic data we conclude that furan-FA is predominantly stored in lipid droplets thereby downregulating cellular metabolic activity and driving the cells into a state of rest.

Furan fatty acids (furan-FA) can be formed by auto-oxidation of conjugated linoleic acids (CLA) and may therefore be ingested when CLA-containing foodstuff is consumed. Due to the presence of a furan ring structure, furan-FA may have toxic properties, however, these substances are toxicologically not well characterized so far. Here we show that 9,11-furan-FA, the oxidation product of the major CLA isomer cis-9,trans-11-CLA (c9,t11-CLA), is not toxic to human intestinal Caco-2 cells up to a level of 100 μM. Oil-Red-O staining indicated that 9,11-furan-FA as well as c9,t11-CLA and linoleic acid are taken up by the cells and stored in the form of triglycerides in lipid droplets. Chemical analysis of total cellular lipids revealed that 9,11-furan-FA is partially elongated probably by the enzymatic activity of cellular fatty acid elongases whereas c9,t11-CLA is partially converted to other isomers such as c9,c11-CLA or t9,t11-CLA. In the case of 9,11-furan-FA, there is no indication for any modification or activation of the furan ring system. From these results, we conclude that 9,11-furan-FA has no properties of toxicological relevance at least for Caco-2 cells which serve as a model for enterocytes of the human small intestine.