RESUMO
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.
Assuntos
Diferenciação Celular , Enterócitos/efeitos dos fármacos , Zinco/metabolismo , Células CACO-2 , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Regulação para Baixo , Enterócitos/citologia , Humanos , Mucosa Intestinal/metabolismo , Metalotioneína/genética , Metalotioneína/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima , Zinco/análiseRESUMO
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.
Assuntos
Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Lactoferrina/farmacologia , Proteína Smad2/metabolismo , Animais , Bovinos , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Humanos , Cinética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transporte Proteico/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Recent evidence suggests that scavenger receptor, class B, type I (SR-BI) plays a physiological role in VLDL metabolism. SR-BI was reported to mediate beta-VLDL uptake; however, cellular details of this process are not well characterized. In the present study we show that SR-BI delivers cholesterol derived from beta-VLDL to LDL receptor negative SR-BI over-expressing Chinese Hamster Ovarian cells (ldlA7-SRBI). Cell association of beta-VLDL was approximately 3 times higher after SR-BI over-expression, which was competed by beta-VLDL, but only to a lesser extent by HDL and LDL. Almost all of the associated beta-VLDL was located intracellularly, and therefore could not be released by a 50-fold excess of unlabeled beta-VLDL. beta-VLDL was degraded at a rate of 6 ng beta-VLDL/mg cell protein and hour. In contrast to ldlA7 cells, beta-VLDL association was competed by LDL in cells with a functional LDL receptor like CHO and HepG2 cells, indicating a strong impact of the LDL receptor in beta-VLDL uptake. beta-VLDL degradation was similar to ldlA7-SRBI cells. When beta-VLDL uptake was followed using fluorescence microscopy, beta-VLDL showed a different uptake pattern in SR-BI over-expressing cells, ldlA7-SRBI, compared to LDL receptor containing cells, CHO and HepG2.
