Resveratrol (3,5,4Ј-trihydroxystilbene) is a natural polyphenol present in a variety of medicinal plants and in grapes. 1 Its function is to protect plants against pathogenic attack and environmental stress. 2,3 Because of its production by grape skin, significant amounts are found in red wines. 4 First evidence of the beneficial effects of resveratrol for human health was shown by its ability to protect against cardiovascular diseases. Resveratrol exhibits strong antioxydant activity 5,6 and inhibits platelet aggregation. 7,8 More recently, numerous cancer chemopreventive properties of resveratrol have been demonstrated . 9 -12 Ptero-stilbene, a derivative of resveratrol that is methylated at positions 3 and 5, exhibits greater antifungal activity than resveratrol. 13,14 However, this molecule is only present in very low concentrations in grapes and seems to be a minor effector of the plant defensive response. It has been shown that methylated derivatives of flavonoïds exhibit higher antiproliferative potency on cancer cells than their hydroxylated counterparts. 15 This ability may be related to the increased lipophilic properties of the methoxyflavonoids and their increased uptake through the cell membrane. On this basis we have developed a methylated derivative of resveratrol (R3: Z-3,5,4Ј-trimethoxystilbene; Fig. 1) with the aim to evaluate its antiproliferative effects on the human colon cancer cell line Caco-2.Previously resveratrol was shown to inhibit the proliferation of Caco-2 cells through the accumulation of cells in the S phase of the cell cycle and by inhibiting polyamine biosynthesis. 16 The effects of resveratrol were not cytotoxic but mainly cytostatic and reversible. In the present study, we show that R3 exerts a 100-fold higher inhibitory effect on colon cancer cell growth. The growth supressive effects induced by R3 were totally different from those observed with resveratrol and were related to a selective blockade of cells at the G2/M phase of the cell cycle, and to the disruption of the microtubule network. MATERIAL AND METHODS Synthesis of (Z)-3,5,4.ЈЈ-trimethoxystilbene (R3)The compound was synthesized by a Wittig-Horner reaction from 4-methoxybenzyl-diethyl phosphonate and 3,5-dimethoxybenzaldehyde 17,18 (Fig. 2). All reagents were commercially available and used without further purification.Melting points were measured without correction in capillary tubes on a Büchi apparatus.1 H and 13 C NMR spectra were recorded on a Bruker AC 200 MHz spectrometer. Mass spectra were established on a Fisons spectrometer at 70 eV chamber voltage, using a direct inlet tube. Silica gel 60 (40 -63 mesh, Merck Eurolab SA, Strasbourg, France) and thin layer chromatography with aluminium sheets 20 ϫ 20 cm 60 F 254 (Merck Eurolab SA, Strasbourg, France) were used. Dimethylformamide was distilled over 0.4 nm molecular sieves under reduced pressure prior to its use; 4-Methoxybenzyl alcohol (1) was purchased from Acros (Noisy le Grand, France), 3,5-dimethoxybenzaldehyde from Avocado (La Tour du Pin, France) an...
Self-renewing tissues such as the intestine contain progenitor proliferating cells which subsequently differentiate. Cell proliferation and differentiation involve gene regulation processes which take place in the nucleus. A human intestinal epithelial cell line model (Caco2/TC7) which reproduces these dynamic processes has been used to perform proteomic studies on nuclear proteins. Nuclei from Caco2/TC7 cells at proliferative and differentiated stages were purified by subcellular fractionation. After two-dimensional gel electrophoresis separation and ruthenium staining, 400 protein spots were detected by image analysis. Eighty-five spots corresponding to 60 different proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry in nuclei from proliferative cells. Comparison of nuclear proteomes from proliferative or differentiated cells by differential display resulted in the identification of differentially expressed proteins such as nucleolin, hnRNP A2/B1 and hnRNP A1. By using Western blot analysis, we found that the expression and number of specific isoforms of these nuclear proteins decreased in differentiated cells. Immunocytochemistry experiments also showed that in proliferative cells nucleolin was distributed in nucleoli-like bodies. In contrast, hnRNPs A2/B1 and A1 were dispersed throughout the nucleus. This study of the nuclear proteome from intestinal epithelial cells represents the first step towards the establishment of a protein database which will be a valuable resource in future studies on the differential expression of nuclear proteins in response to physiological, pharmacological and pathological modulations.
Intestinal epithelial cells are characterized by continuous renewal and differentiation events, which may be influenced by the basement membrane, and in particular laminins, which are major components of this specialized extracellular matrix. The function and signaling pathways of laminins in these processes are still poorly documented. In this study, we investigated the possible role and the subcellular localization of nucleolin, a nuclear shuttling protein, in relation to differentiation of human intestinal epithelial Caco2/TC7 cells triggered by exogenous laminin-1. Immunofluorescence and Western blot analysis indicated that laminin-1 induced early differentiation of the cells concomitantly to a decrease in nuclear nucleolin and its a cell surface location. We also showed that both effects of laminin-1 on Caco2/TC7 cells--induction of the differentiation marker sucrase-isomaltase and redistribution of nucleolin--could be mediated by a beta1-integrin dependent cascade that implicated activation of the p38 MAPK pathway. In addition, knock-down of nucleolin expression by the small interfering RNA strategy mimicked the effect of laminin-1 as it resulted in the induction of cell polarization and differentiation. Thus, our study suggests that changes in the subcellular distribution and expression level of nucleolin play an important role in intestinal cell differentiation and relay the signaling pathway induced by laminin-1.
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