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Protection by L-fucose and fucose-rich polysaccharides against ROS-produced cell death in presence of ascorbate
Biomedicine & Pharmacotherapy 57 (2003) 130–133 www.elsevier.com/locate/biopha
Dossier : Oxidative stress pathologies and antioxidants
Protection by L-fucose and fucose-rich polysaccharides against ROS-produced cell death in presence of ascorbate G. Péterszegi a,b, A.M. Robert a,b, L. Robert a,b,* a
Laboratoire de Recherche en Ophtalmologie, Faculté de Médecine Broussais-Hôtel Dieu, Université Pierre et Marie Curie (Paris 6), 1, place Parvis Notre Dame, 75181 Paris cedex 4, France b Pavillon Claude Galien, Hôpital Émile Roux, 94456 Limeil-Brévannes, France Received 16 January 2003
Abstract It was shown previously, that millimolar concentrations of ascorbate have cytotoxic and anti-proliferative effects (Eur. J. Clin. Invest. 32 (2002) 372). With increasing concentrations of ascorbate an increasing number of fibroblasts was detached from the culture dish and shown to be lysed by the effect of ascorbate-induced generation of reactive oxygen species (ROS-s). It also could be shown, that this cytotoxic effect is partly due to the dose-dependent inhibition by ascorbate of fibronectin biosynthesis. Superoxide dismutase (SOD) and catalase were shown to salvage cells from ROS-induced cell-death by preventing the inhibition of fibronectin biosynthesis. We used this model system to test the cyto-protective effect of L-fucose and fucose-rich oligo- and polysaccharides (FROP-s). It appeared that relatively low concentrations of L-fucose and FROP-3 (Biomed. Pharmacother. in press) could efficiently protect fibroblasts from the ascorbate-induced cell-death. These novel pharmacological properties of L-fucose and FROP-3 might well be related to their accelerating effect of wound healing. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: L-Fucose; Fucose-rich polysaccharide (FROP); Free radicals; Reactive oxygen species (ROS); Cytotoxicity; Superoxide dismutase (SOD)
1. Introduction Production of free radicals, as superoxide, oxygen radicals, nitric oxide and hydrogen peroxide (commonly designated as reactive oxygen species, ROS) was shown to play an important role in tissue damage and loss of function in a number of tissues and organs [2,3,9]. Although the organisms possess efficient free radical scavenging mechanisms and anti-ROS defence systems, in a number of circumstances the balance between ROS-production and elimination is displaced in favour of the damaging actions of ROS-s. This is the case in a number of inflammatory processes as well as in age-associated diseases as pulmonary emphysema or atherosclerosis. It is, therefore, of interest to develop efficient and pharmacologically meaningful protective mechanisms. We carried out over the last years a number of studies on L-fucose and fucose-rich oligo- and polysaccharides (FROP-s, for details of preparation and chemical properties see [8]) exhibiting interesting pharmacological effects in * Corresponding author. E-mail address: [email protected] (L. Robert). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 10.1016/S0753-3322(03)00019-2
several model systems related to age-dependent damaging mechanisms. Most important of the previously demonstrated properties of L-fucose and FROP-s are the down-regulation of MMP-2 and MMP-9 expression [6] and stimulation of cell-proliferation of human fibroblasts [8]. We now demonstrate a novel interesting property of L-fucose and FROP-s as efficient protective agents against the free radical and more generally ROS-induced cell damage. For these experiments we used a model system consisting in the addition of millimolar concentrations of Na-ascorbate to human fibroblast cultures [7]. We could show that the progressive detachment of cells and cell-death observed in these conditions is the result of an ascorbate-induced dose-dependent inhibition of fibronectin biosynthesis [7]. Ultrastructural studies showed that cell-death observed in these experiments was of a necrotic type, the results of lesions of the cell-membrane [7]. This system proved useful for the evaluation of the efficiency of free-radical scavenging and anti-ROS-systems. In our original studies we could show that catalase and Superoxide dismutase (SOD) efficiently protected human skin fibroblasts from ascorbate-induced detachment and celldeath, other scavenging agents as desferrioxamine, ergothio-
G. Péterszegi et al. / Biomedicine & Pharmacotherapy 57 (2003) 130–133
nein and vitamin E were inefficient. Catalase and SOD prevented also efficiently the inhibition of fibronectin biosynthesis by ascorbate. This effect appears as the most plausible explanation of the efficient control by these enzymes of the ascorbate induced cell lesions. Therefore, we used this method to test the protective effect of L-fucose and FROP-3 against ascorbate produced, ROS-induced celldeath.
2. Materials and methods 2.1. Culture conditions Human skin fibroblasts were cultured from skin explants of healthy individual donors as previously described [4,5], with 1:2 split ratio and used at the eighth passage. Sodium ascorbate (Sigma) was added to cultures at increasing concentrations, from 5.0 µM to 5.0 mM for 72 h. Cell proliferation and its arrest were quantified by [3H]thymidine incorporation [7], cell-detachment by counting non viable cells, which take up vital dye (Trypan blue) and by the MTT method [1]. L-Fucose was obtained from Sigma. FROP-3 was prepared from Fucogel® (Solabia), a high molecular weight polymer (polysaccharide) extracted from a non-pathogenic strain of bacteria, by controlled partial degradation with an endo-glycosidase preparation. Its chemical characterisation was described in detail [8]. L-Fucose or the FROP-3 preparation were added simultaneously with Na ascorbate to fibroblast cultures and incubated for 72 h. The use of SOD and catalase as model scavengers was described [7]. Catalase (Sigma) was from bovine liver, SOD (Sigma) from bovine erythrocytes. Scavengers were also used for an incubation of 72 h. Every experiment was carried out with six independent parallel cultures. The biosynthetic activityof cells was tested by [3H]-proline incorporation. L-[2,3-3H]-proline (Amersham, 370 kBq/ml, 10 Ci/ml) added to the fibroblast cultures for incorporation, as described in [4].
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3. Results Fig. 1 shows cell-detachment followed by increasing celldeath produced by increasing Na-ascorbate concentrations added to the human skin fibroblast cultures [7]. We choose the concentration of 500 µg/ml (2.5 mM) of Na ascorbate for our test system. Fig. 2 shows the disinhibition of fibronectin biosynthesis by 50 and 100 U/ml SOD and by 1000 U/ml catalase. Fig. 2a refers to radioactive fibronectin recovered from the cell-bound fraction, and Fig. 2b to fibronectin recovered from the culture medium (excreted fraction). In presence of catalase (both in cell-bound and in excreted fractions) and of SOD (in the excreted fraction), incorporation was even higher in newly synthesised fibronectin as compared to the control. Table 1 shows the comparative efficiencyof L-fucose at concentrations ranging from 1 to 10 µg/ml on fibronectin biosynthesis in presence of 500 µg/ml (2.5 mM) of Na ascorbate, and of FROP-3 from 1 to 500 µg/ml concentrations (the average molecular weight of this oligo- and polysaccharidic preparation is about 10 kDa (see [7] for details) on fibronectin biosynthesis in presence of the same concentration of ascorbate. The results are expressed as % of increase of the fibronectin biosynthesis determined by [3H]-proline incorporation in immunoprecipitable fibronectin, compared to the value found in presence of 500 µg/ml ascorbate alone. L-Fucose increased incorporation in the cell-bound fraction of fibronectin by 42–63% at 1 and 10 µg/ml, and by 123% and 143% for the same concentrations in excreted fibronectin. In presence of FROP-3 we demonstrated a dosedependent increase of incorporation of the label in neosynthesised fibronectin, increasing from 8% at 0.1 µg/ml to 60% at 10 µg/ml in the cell-bound fraction, and by 27–135%
2.2. Fibronectin biosynthesis As shown previously [7], the cytotoxic effect of millimolar concentrations of ascorbate is essentially due to the strong inhibition of fibronectin biosynthesis, as well as to its excretion into the pericellular matrix. This was tested as previously described, by measuring the incorporation of 3H-proline in immunoprecipitable fibronectin. We compared the disinhibition of fibronectin biosynthesis in presence of 500 µg/ml ascorbate by L-fucose and FROP-3 to that of catalase and SOD, shown previously to efficiently counter ascorbate produced inhibition of its biosynthesis. The significance of the average values was tested with the distribution-free Mann-Whitney U-test.
Fig. 1. Cell detachment and cell-death induced by increasing concentrations of Na-ascorbate added to the fibroblast cultures. Abscissa: ascorbate concentration added to human fibroblasts. Ordinates: cell detachment, % of total cell count, determined by vital dye uptake (for other details see [7]). Significance vs. control indicated by asterisks: *P < 0.05; * *P < 0.01; * * *P < 0.001.
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G. Péterszegi et al. / Biomedicine & Pharmacotherapy 57 (2003) 130–133
a
b Fig. 2. Inhibition of fibronectin biosynthesis by ascorbate (500 µg/ml) and protection by SOD (50 and 100 U/ml) and catalase (1000 U/ml). (a) Cell-bound fibronectin. (b) Excreted fibronectin. Significance compared to control without ascorbate, or to the effect of ascorbate alone, as indicated on Fig. 1.
Table 1 Effect of L-fucose and the FROP-3 preparation on ascorbate (500 µg/ml) induced inhibition of fibronectin biosynthesis measured by [3H]-proline incorporation in immunoprecipitable fibronectin. Values correspond to % increase of incorporation in presence of L-fucose or FROP-3 at the indicated concentrations, as compared to the action of ascorbate alone. Experiments concerned both cell-bound and excreted fibronectin Substance added and concentration (µg/ml) l-Fucose, 1.0 l-Fucose, 10.0 FROP-3, 0.1 FROP-3, 1.0 FROP-3, 5.0 FROP-3, 10.0 FROP-3, 50.0
Increase in [3H]-proline incorporation in fibronectin as compared to ascorbate (500 µg/ml) inhibited control (% increase) Cell-bound fraction Excreted fraction 42.2 123 63.6 143 8.2 27 20 69 45 135 60.0 102 51 97
(maximal at 5 µg/ml) for the excreted fraction, comparable to the protection found for L-fucose at the same concentration. At higher concentrations, above 5–10 µg/ml, the protective effect of FROP-3 reached a plateau for the cell-bound fraction, but decreased slightly to 97% in the excreted fraction at 50 µg/ml. Further increase of FROP-3 concentration did not increase its efficiency. The reason and mechanism of this bell-shaped dose-effect curve of FROP-3, as far as the excreted fraction of neosynthesised fibronectin is concerned, remains to be determined.
4. Discussion As shown by the above detailed results, both L-fucose and FROP-s efficiently protected fibroblasts of human dermis against the Na ascorbate induced decrease of fibronectin
G. Péterszegi et al. / Biomedicine & Pharmacotherapy 57 (2003) 130–133
biosynthesis, resulting in cell-detachment and necrotic celldeath [7]. Although the mechanism of this protection remains to be elucidated, it might be related to the partially hydrophobic nature of fucose, because of the presence of a methyl group attached to carbon-5 [8]. This would facilitate interaction with the cell-membrane, where the toxic effect was shown to occur [7]. Ultrastructural studies showed discontinuities in the cell-membrane of ascorbate treated cells [7]. Another possibility would be the interaction of fucose and FROP-3, which contains fucose end-groups [8], with the fucose/ mannose and/or the elastin/laminine receptor [10]. It is interesting to notice, that the protective effect of L-fucose and FROP-3 is more important for the excreted fraction of fibronectin, than for the cell-bound fraction. It can and does exceed 100% for the excreted fraction, but remained below 100% for the cell-associated fraction. This might indicate an effect of L-fucose and FROP-3 not only on the biosynthesis of fibronectin, but also on its excretion. The effect of ascorbate was most efficiently counteracted by catalase (Fig. 2), suggesting that H2O2 might be the most active ROS-product involved in the necrotic cell lesion [7]. The mechanism of action of L-fucose and FROP-3 might concern the ROS-produced inhibition of active excretion of neosynthesised fibronectin as well as the regulation of its biosynthesis itself. The demonstration of the intranuclear penetration of FROP-3 [8] suggests a possible action at the level of the regulation of expression of the fibronectin coding genes, and/or its modulation. Whatever the details of these mechanisms, these results further confirm the pharmacological value of L-fucose and FROP-3 in age-related tissue-alterations attributed to increased ROS-production. Acknowledgements Supported by Institut DERM, Paris, and NATURA, Sao Paolo, Brazil. The hospitality and helpful discussions with
133
Professor Gilles RENARD are thankfully acknowledged. Helpful discussions with Mr. Philippe Pommez, Jean-Luc Gesztesi, Eduardo Luppi and Pierre Fodor from NATURA Sao Paolo, Brazil, are gratefully acknowledged.
References [1]
Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of tetrazolium-based semiautomated colorimetric essay: assessment of chemosensitivity testing. Cancer Res 1987;47(4): 936–42.
[2]
Chuang CC, editor. Reactive oxygen species. From radiation to molecular biology, vol. 899. New York: The New York Academy of Sciences; 2000 Ann New York Acad Sci.
[3]
Emerit I, Chance B, editors. Free radicals and aging. Basel, Boston, Berlin: Birkhäuser Verlag; 1992.
[4]
Fodil-Bourahla I, Drubaix I, Robert L, Labat-Robert J. Effect of in vitro aging on the modulation of protein and fibronectin biosynthesis by the elastin-laminin receptor in human skin fibroblasts. Gerontology 1999;45:23–30.
[5]
Fodil-Bourahla I, Drubaix I, Robert L. Effect of in vitro aging on the biosynthesis of glycosaminoglycans by human skin fibroblasts. Modulation by the elastin-laminin receptor. Mech Ageing Dev 1999; 106:241–60.
[6]
Isnard N, Péterszegi G, Robert AM, Robert L. Regulation of elastasetype endopeptidase activity, MMP-2 and MMP-9 expression and activation in human dermal fibroblasts by fucose and fucose-rich polysaccharides. Biomed Pharmacother 2002;56:258–64.
[7]
Péterszegi G, Dagonet FB, Labat-Robert J, Robert L. Inhibition of cell proliferation and fibronectin biosynthesis by Na ascorbate. Eur J Clin Invest 2002;32:372–80.
[8]
Péterszegi G, Isnard N, Robert AM, Robert L. Studies on skin aging. Preparation and properties of fucose-rich oligo- and polysaccharides (FROP-s). Effect on fibroblast proliferation and survival. Biomed Pharmacother 2003 (in press).
[9]
Rice-Evans CA, Burdon RH, editors. Free radical damage and its control. Amsterdam, London, New York, Tokyo: Elsevier; 1994.
[10] Robert L. Mechanisms of aging of the extracellular matrix. Role of the elastin-laminin receptor. Novartis price lecture. Gerontology 1998;44: 307–17.
Dossier : Oxidative stress pathologies and antioxidants
Protection by L-fucose and fucose-rich polysaccharides against ROS-produced cell death in presence of ascorbate G. Péterszegi a,b, A.M. Robert a,b, L. Robert a,b,* a
Laboratoire de Recherche en Ophtalmologie, Faculté de Médecine Broussais-Hôtel Dieu, Université Pierre et Marie Curie (Paris 6), 1, place Parvis Notre Dame, 75181 Paris cedex 4, France b Pavillon Claude Galien, Hôpital Émile Roux, 94456 Limeil-Brévannes, France Received 16 January 2003
Abstract It was shown previously, that millimolar concentrations of ascorbate have cytotoxic and anti-proliferative effects (Eur. J. Clin. Invest. 32 (2002) 372). With increasing concentrations of ascorbate an increasing number of fibroblasts was detached from the culture dish and shown to be lysed by the effect of ascorbate-induced generation of reactive oxygen species (ROS-s). It also could be shown, that this cytotoxic effect is partly due to the dose-dependent inhibition by ascorbate of fibronectin biosynthesis. Superoxide dismutase (SOD) and catalase were shown to salvage cells from ROS-induced cell-death by preventing the inhibition of fibronectin biosynthesis. We used this model system to test the cyto-protective effect of L-fucose and fucose-rich oligo- and polysaccharides (FROP-s). It appeared that relatively low concentrations of L-fucose and FROP-3 (Biomed. Pharmacother. in press) could efficiently protect fibroblasts from the ascorbate-induced cell-death. These novel pharmacological properties of L-fucose and FROP-3 might well be related to their accelerating effect of wound healing. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: L-Fucose; Fucose-rich polysaccharide (FROP); Free radicals; Reactive oxygen species (ROS); Cytotoxicity; Superoxide dismutase (SOD)
1. Introduction Production of free radicals, as superoxide, oxygen radicals, nitric oxide and hydrogen peroxide (commonly designated as reactive oxygen species, ROS) was shown to play an important role in tissue damage and loss of function in a number of tissues and organs [2,3,9]. Although the organisms possess efficient free radical scavenging mechanisms and anti-ROS defence systems, in a number of circumstances the balance between ROS-production and elimination is displaced in favour of the damaging actions of ROS-s. This is the case in a number of inflammatory processes as well as in age-associated diseases as pulmonary emphysema or atherosclerosis. It is, therefore, of interest to develop efficient and pharmacologically meaningful protective mechanisms. We carried out over the last years a number of studies on L-fucose and fucose-rich oligo- and polysaccharides (FROP-s, for details of preparation and chemical properties see [8]) exhibiting interesting pharmacological effects in * Corresponding author. E-mail address: [email protected] (L. Robert). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 10.1016/S0753-3322(03)00019-2
several model systems related to age-dependent damaging mechanisms. Most important of the previously demonstrated properties of L-fucose and FROP-s are the down-regulation of MMP-2 and MMP-9 expression [6] and stimulation of cell-proliferation of human fibroblasts [8]. We now demonstrate a novel interesting property of L-fucose and FROP-s as efficient protective agents against the free radical and more generally ROS-induced cell damage. For these experiments we used a model system consisting in the addition of millimolar concentrations of Na-ascorbate to human fibroblast cultures [7]. We could show that the progressive detachment of cells and cell-death observed in these conditions is the result of an ascorbate-induced dose-dependent inhibition of fibronectin biosynthesis [7]. Ultrastructural studies showed that cell-death observed in these experiments was of a necrotic type, the results of lesions of the cell-membrane [7]. This system proved useful for the evaluation of the efficiency of free-radical scavenging and anti-ROS-systems. In our original studies we could show that catalase and Superoxide dismutase (SOD) efficiently protected human skin fibroblasts from ascorbate-induced detachment and celldeath, other scavenging agents as desferrioxamine, ergothio-
G. Péterszegi et al. / Biomedicine & Pharmacotherapy 57 (2003) 130–133
nein and vitamin E were inefficient. Catalase and SOD prevented also efficiently the inhibition of fibronectin biosynthesis by ascorbate. This effect appears as the most plausible explanation of the efficient control by these enzymes of the ascorbate induced cell lesions. Therefore, we used this method to test the protective effect of L-fucose and FROP-3 against ascorbate produced, ROS-induced celldeath.
2. Materials and methods 2.1. Culture conditions Human skin fibroblasts were cultured from skin explants of healthy individual donors as previously described [4,5], with 1:2 split ratio and used at the eighth passage. Sodium ascorbate (Sigma) was added to cultures at increasing concentrations, from 5.0 µM to 5.0 mM for 72 h. Cell proliferation and its arrest were quantified by [3H]thymidine incorporation [7], cell-detachment by counting non viable cells, which take up vital dye (Trypan blue) and by the MTT method [1]. L-Fucose was obtained from Sigma. FROP-3 was prepared from Fucogel® (Solabia), a high molecular weight polymer (polysaccharide) extracted from a non-pathogenic strain of bacteria, by controlled partial degradation with an endo-glycosidase preparation. Its chemical characterisation was described in detail [8]. L-Fucose or the FROP-3 preparation were added simultaneously with Na ascorbate to fibroblast cultures and incubated for 72 h. The use of SOD and catalase as model scavengers was described [7]. Catalase (Sigma) was from bovine liver, SOD (Sigma) from bovine erythrocytes. Scavengers were also used for an incubation of 72 h. Every experiment was carried out with six independent parallel cultures. The biosynthetic activityof cells was tested by [3H]-proline incorporation. L-[2,3-3H]-proline (Amersham, 370 kBq/ml, 10 Ci/ml) added to the fibroblast cultures for incorporation, as described in [4].
131
3. Results Fig. 1 shows cell-detachment followed by increasing celldeath produced by increasing Na-ascorbate concentrations added to the human skin fibroblast cultures [7]. We choose the concentration of 500 µg/ml (2.5 mM) of Na ascorbate for our test system. Fig. 2 shows the disinhibition of fibronectin biosynthesis by 50 and 100 U/ml SOD and by 1000 U/ml catalase. Fig. 2a refers to radioactive fibronectin recovered from the cell-bound fraction, and Fig. 2b to fibronectin recovered from the culture medium (excreted fraction). In presence of catalase (both in cell-bound and in excreted fractions) and of SOD (in the excreted fraction), incorporation was even higher in newly synthesised fibronectin as compared to the control. Table 1 shows the comparative efficiencyof L-fucose at concentrations ranging from 1 to 10 µg/ml on fibronectin biosynthesis in presence of 500 µg/ml (2.5 mM) of Na ascorbate, and of FROP-3 from 1 to 500 µg/ml concentrations (the average molecular weight of this oligo- and polysaccharidic preparation is about 10 kDa (see [7] for details) on fibronectin biosynthesis in presence of the same concentration of ascorbate. The results are expressed as % of increase of the fibronectin biosynthesis determined by [3H]-proline incorporation in immunoprecipitable fibronectin, compared to the value found in presence of 500 µg/ml ascorbate alone. L-Fucose increased incorporation in the cell-bound fraction of fibronectin by 42–63% at 1 and 10 µg/ml, and by 123% and 143% for the same concentrations in excreted fibronectin. In presence of FROP-3 we demonstrated a dosedependent increase of incorporation of the label in neosynthesised fibronectin, increasing from 8% at 0.1 µg/ml to 60% at 10 µg/ml in the cell-bound fraction, and by 27–135%
2.2. Fibronectin biosynthesis As shown previously [7], the cytotoxic effect of millimolar concentrations of ascorbate is essentially due to the strong inhibition of fibronectin biosynthesis, as well as to its excretion into the pericellular matrix. This was tested as previously described, by measuring the incorporation of 3H-proline in immunoprecipitable fibronectin. We compared the disinhibition of fibronectin biosynthesis in presence of 500 µg/ml ascorbate by L-fucose and FROP-3 to that of catalase and SOD, shown previously to efficiently counter ascorbate produced inhibition of its biosynthesis. The significance of the average values was tested with the distribution-free Mann-Whitney U-test.
Fig. 1. Cell detachment and cell-death induced by increasing concentrations of Na-ascorbate added to the fibroblast cultures. Abscissa: ascorbate concentration added to human fibroblasts. Ordinates: cell detachment, % of total cell count, determined by vital dye uptake (for other details see [7]). Significance vs. control indicated by asterisks: *P < 0.05; * *P < 0.01; * * *P < 0.001.
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G. Péterszegi et al. / Biomedicine & Pharmacotherapy 57 (2003) 130–133
a
b Fig. 2. Inhibition of fibronectin biosynthesis by ascorbate (500 µg/ml) and protection by SOD (50 and 100 U/ml) and catalase (1000 U/ml). (a) Cell-bound fibronectin. (b) Excreted fibronectin. Significance compared to control without ascorbate, or to the effect of ascorbate alone, as indicated on Fig. 1.
Table 1 Effect of L-fucose and the FROP-3 preparation on ascorbate (500 µg/ml) induced inhibition of fibronectin biosynthesis measured by [3H]-proline incorporation in immunoprecipitable fibronectin. Values correspond to % increase of incorporation in presence of L-fucose or FROP-3 at the indicated concentrations, as compared to the action of ascorbate alone. Experiments concerned both cell-bound and excreted fibronectin Substance added and concentration (µg/ml) l-Fucose, 1.0 l-Fucose, 10.0 FROP-3, 0.1 FROP-3, 1.0 FROP-3, 5.0 FROP-3, 10.0 FROP-3, 50.0
Increase in [3H]-proline incorporation in fibronectin as compared to ascorbate (500 µg/ml) inhibited control (% increase) Cell-bound fraction Excreted fraction 42.2 123 63.6 143 8.2 27 20 69 45 135 60.0 102 51 97
(maximal at 5 µg/ml) for the excreted fraction, comparable to the protection found for L-fucose at the same concentration. At higher concentrations, above 5–10 µg/ml, the protective effect of FROP-3 reached a plateau for the cell-bound fraction, but decreased slightly to 97% in the excreted fraction at 50 µg/ml. Further increase of FROP-3 concentration did not increase its efficiency. The reason and mechanism of this bell-shaped dose-effect curve of FROP-3, as far as the excreted fraction of neosynthesised fibronectin is concerned, remains to be determined.
4. Discussion As shown by the above detailed results, both L-fucose and FROP-s efficiently protected fibroblasts of human dermis against the Na ascorbate induced decrease of fibronectin
G. Péterszegi et al. / Biomedicine & Pharmacotherapy 57 (2003) 130–133
biosynthesis, resulting in cell-detachment and necrotic celldeath [7]. Although the mechanism of this protection remains to be elucidated, it might be related to the partially hydrophobic nature of fucose, because of the presence of a methyl group attached to carbon-5 [8]. This would facilitate interaction with the cell-membrane, where the toxic effect was shown to occur [7]. Ultrastructural studies showed discontinuities in the cell-membrane of ascorbate treated cells [7]. Another possibility would be the interaction of fucose and FROP-3, which contains fucose end-groups [8], with the fucose/ mannose and/or the elastin/laminine receptor [10]. It is interesting to notice, that the protective effect of L-fucose and FROP-3 is more important for the excreted fraction of fibronectin, than for the cell-bound fraction. It can and does exceed 100% for the excreted fraction, but remained below 100% for the cell-associated fraction. This might indicate an effect of L-fucose and FROP-3 not only on the biosynthesis of fibronectin, but also on its excretion. The effect of ascorbate was most efficiently counteracted by catalase (Fig. 2), suggesting that H2O2 might be the most active ROS-product involved in the necrotic cell lesion [7]. The mechanism of action of L-fucose and FROP-3 might concern the ROS-produced inhibition of active excretion of neosynthesised fibronectin as well as the regulation of its biosynthesis itself. The demonstration of the intranuclear penetration of FROP-3 [8] suggests a possible action at the level of the regulation of expression of the fibronectin coding genes, and/or its modulation. Whatever the details of these mechanisms, these results further confirm the pharmacological value of L-fucose and FROP-3 in age-related tissue-alterations attributed to increased ROS-production. Acknowledgements Supported by Institut DERM, Paris, and NATURA, Sao Paolo, Brazil. The hospitality and helpful discussions with
133
Professor Gilles RENARD are thankfully acknowledged. Helpful discussions with Mr. Philippe Pommez, Jean-Luc Gesztesi, Eduardo Luppi and Pierre Fodor from NATURA Sao Paolo, Brazil, are gratefully acknowledged.
References [1]
Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of tetrazolium-based semiautomated colorimetric essay: assessment of chemosensitivity testing. Cancer Res 1987;47(4): 936–42.
[2]
Chuang CC, editor. Reactive oxygen species. From radiation to molecular biology, vol. 899. New York: The New York Academy of Sciences; 2000 Ann New York Acad Sci.
[3]
Emerit I, Chance B, editors. Free radicals and aging. Basel, Boston, Berlin: Birkhäuser Verlag; 1992.
[4]
Fodil-Bourahla I, Drubaix I, Robert L, Labat-Robert J. Effect of in vitro aging on the modulation of protein and fibronectin biosynthesis by the elastin-laminin receptor in human skin fibroblasts. Gerontology 1999;45:23–30.
[5]
Fodil-Bourahla I, Drubaix I, Robert L. Effect of in vitro aging on the biosynthesis of glycosaminoglycans by human skin fibroblasts. Modulation by the elastin-laminin receptor. Mech Ageing Dev 1999; 106:241–60.
[6]
Isnard N, Péterszegi G, Robert AM, Robert L. Regulation of elastasetype endopeptidase activity, MMP-2 and MMP-9 expression and activation in human dermal fibroblasts by fucose and fucose-rich polysaccharides. Biomed Pharmacother 2002;56:258–64.
[7]
Péterszegi G, Dagonet FB, Labat-Robert J, Robert L. Inhibition of cell proliferation and fibronectin biosynthesis by Na ascorbate. Eur J Clin Invest 2002;32:372–80.
[8]
Péterszegi G, Isnard N, Robert AM, Robert L. Studies on skin aging. Preparation and properties of fucose-rich oligo- and polysaccharides (FROP-s). Effect on fibroblast proliferation and survival. Biomed Pharmacother 2003 (in press).
[9]
Rice-Evans CA, Burdon RH, editors. Free radical damage and its control. Amsterdam, London, New York, Tokyo: Elsevier; 1994.
[10] Robert L. Mechanisms of aging of the extracellular matrix. Role of the elastin-laminin receptor. Novartis price lecture. Gerontology 1998;44: 307–17.