Differentiation- and polarization-dependent zinc tolerance in Caco-2 cells.

PURPOSE: Enterocytes are feasibly confronted with enormous zinc concentrations especially as a result of oral zinc supplementation. In the present study, we investigated the mechanisms underlying the exceptional ability to withstand this usually toxic load using the enterocytic cell line Caco-2. METHODS: By MTT test analysis, we compared zinc tolerance in undifferentiated Caco-2 cells (udCaco-2) and differentiated Caco-2 cells (dCaco-2). By RT-PCR, we compared the respective baseline expression levels of certain zinc transporters and metallothioneins (MTs) as well as the regulation of these components in response to high zinc concentrations. Moreover, using dCaco-2 cells cultured on porous membranes, we explored zinc tolerance in dependence of the side of zinc administration: apical versus basolateral. RESULTS: dCaco-2 cells tolerate significantly higher levels of zinc compared to udCaco-2 cells. This adaptation was accompanied by upregulated ZnT-1 and downregulated ZIP1 levels. The expression of metallothioneins MT1A and MT1X was also significantly downregulated during differentiation. Moreover, apparent from profound differences in zinc tolerance between apical and basolateral zinc application, polarization was concluded to have substantial impact on cellular zinc tolerance. CONCLUSIONS: The profound increase in zinc tolerance we found in differentiated enterocyte-type Caco-2 cells and in particular, the impact of polarization are likely to reflect the physiologically indispensable capability of enterocytes to cope with remarkable concentrations of intestinal zinc.

Lactoferrin induces growth arrest and nuclear accumulation of Smad-2 in HeLa cells.

Lactoferrin (Lf) is a multifunctional glycoprotein. Due to its anti-inflammatory and anti-cancer properties and the resulting therapeutical potential, lactoferrin is at present focus of a variety of research areas. The regulation of cell growth represents one of the prominent performances of lactoferrin. In this study we found lactoferrin to inhibit proliferation of the human epithelial cancer cell line HeLa. The extent of this growth inhibition was comparable to the one induced by the transforming-growth-factor-beta-1 (TGFbeta1). In contrast to other cell lines where lactoferrin stimulates growth, lactoferrin failed to activate the MAP kinases ERK1/2 or p38 in HeLa cells. However, by immunocytochemistry and cell fractionation experiments, we found that lactoferrin is capable of activating the TGFbeta/Smad-2 pathway. The nuclear accumulation of Smad-2 induced by Lf was comparable in magnitude to the one induced by TGFbeta1. We therefore conclude that the canonical TGFbeta1 pathway is a feasible route for lactoferrin to transduce its antiproliferative effect in HeLa cells, when MAPkinase activation is absent.

Evaluation of chondroitin sulfate bioactivity in hippocampal neurones and the astrocyte cell line U373: influence of position of sulfate groups and charge density.

Chondroitin sulfates are linear polysaccharides of alternating glucuronic acid and N-acetylgalactosamine, sulfated in varying positions. They form the extracellular framework providing the information for the structural establishment of tissues in multicellular organisms. Growth cones of neurones modulate their outgrowth according to signals received from proteoglycans. The exact molecular structures behind these functions are not fully understood, but structural details of the carbohydrate backbone are crucial. In this report we have employed quantitative cytometry on hippocampal neurite outgrowth in the presence of chondroitin sulfate added in solution to determine the influence of the position and density of the sulfate groups of the N-acetyl-D-galactosamine-residues of chondroitin sulfates. It is of profound interest whether externally added chondroitin sulfates can compete with core protein bound chondroitin sulfate to modulate the effects of tissue-synthesized matrix. In series of microscopic images 3 parameters of neuritic outgrowth activity, neurite length, number of neurites and fasciculation (thickness of neurites) are analyzed at concentrations occurring in intact tissues. Fasciculation increased and number of neurites decreased with high di-sulfation. No significant differences on process length reduction were found between the isotypes. Specificity of effects found is emphasized, as no influence on cell proliferation with U373 human astrocyte cell line is detectable, while neurones clearly are inhibited. The IC30 and IC50 values of chondroitin sulfates isoforms are presented for neurones. The data indicate that the soluble fragments from chondroitin sulfate are actively modulating cell development. Besides dosage, sulfation density and position are relevant for effects of chondroitin sulfate in neuronal regenerative activity.