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Effects of some aldehydes on brain microtubular protein
Chem.-Biol. Interactions, 78 (1991) 183--191 Elsevier Scientific Publishers Ireland Ltd.
183
EFFECTS OF SOME ALDEHYDES ON BRAIN MICROTUBULAR PROTEIN
ANTONELLA MIGLIETTA, ANTONELLA OLIVERO, ELENA GADONI and LUDOVICA GABRIEL
Dipartimento di Medicina ed Oncologia Sperimentale, Sezione di Patologia Generale, Corso Raffaello 30, 10125 Torino (Italy) (Received May llth, 1990) (Revision received December 20th, 1990) (Accepted December 20tb, 1990)
SUMMARY
4-Hydroxynonenal is one of the main breakdown products of lipid peroxidation. It has an antiproliferative effect, which may partly be the consequence of an interaction with cytoskeletal structures. Its effects on microtubular protein are compared with those of homologous aldehydes with the same number of carbon atoms, and with that of benzaldehyde. Unlike the other aliphatic aldehydes, this latter aldehyde does not impair microtubular functions at every concentration in the range. Nonanal has the greatest effect on tubulin polymerization, whereas it only slightly impairs colchicine binding activity. 2-Nonenal and 4-hydroxynonenal have less inhibiting effect on tubulin polymerization; their effect on colchicine binding activity is dose-dependent. The targets of 4-hydroxynonenal on tubulin are --SH groups; the action mechanism of other aldehydes has not yet been identified.
Key words: Aldehydes -- Tubulin -- Polymerization -- Colchicine binding ---SH groups
INTRODUCTION
Aldehydes have different biochemical and biological properties, and play several roles in metabolic pathways. Some aldehydes are cytotoxic [1] and exert an inhibitory effect on different cell functions [2]. The general action mechanism of aldehydes is to attack both the sulphydryl and the amino groups of Correspondence to: Antonella Miglietta, Dipartimento di Medicina ed Oncologia Sperimentale, Sezione di Patologia Generale, Corso Raffaello 30, 10125 Torino, Italy. 0009-2797/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
184 biomolecules. They influence cell division and tumour growth by deactivating different enzymes and interfering with the metabolism and the biosynthesis of proteins and nucleic acids. Among aldehydes with a carbon-carbon double bond, 4-hydroxyalkenals are particularly reactive, impairing cellular and subcellular functions by attacking sulphydryl groups of biological molecules. 4-Hydroxyalkenals, and in particular 4-hydroxynonenal [3], derive from the breakdown of polyunsatured fatty acids during lipid peroxidation. The latter aldehyde inhibits several enzymes [2,4,5], and affects the proliferation of tumour cells [6]. Another hydroxyalkenal, 4-hydroxypentenal, markedly depresses the growth of Yoshida ascites hepatoma AH-130 [7], as well as that of several transplantable tumours. Various aldehydes have antimicrotubular activity, among them pentanal and hexanal [8], acetaldehyde [9] or the antiproliferative aldehyde methylglyoxal [10]. The antiproliferative effect of various aldehydes, and in particular of the hydroxyalkenals [11], may thus be related to a damage to microtubules, which play an important role in cell division and proliferation [12]. It appeared interesting to evaluate whether the carcinostatic effect of aliphatic aldehydes could be due to a direct interaction with tubulin, and to investigate a possible structure-activity relationship. In this research we studied the effect on microtubular protein of aldehydes having the same chain length, in order to clarify whether the different action mechanism may be related to the hydrophilic, lipophilic or polar characteristics of the molecules. We chose 4-hydroxynonenal, the most representative aldehyde produced in lipid peroxidation, and two homologous aldehydes with the same number of carbon atoms, 2-nonenal and nonanal; furthermore, an aromatic aldehyde, benzaldehyde, was also studied, to compare its effect with that of the aliphatic aldehydes. MATERIALS AND METHODS
Materials 4-Hydroxynonenal and 2-nonenal were kindly given by Dr. Esterbauer, Institut fur Biochemie, Universitat, Graz. Calf brains were obtained from a local slaughterhouse. [3H]Colchicine (spec. act., 4.9 Ci/mmol) was obtained from the Radiochemical Centre, Amersham, U.K. Electrophoresis reagents were from Bio-Rad. All other chemicals were reagent grade from Merck or Sigma.
Microtubular protein purification Calf brains were used within 2 h of the animals' death. Microtubular protein was prepared by two cycles of polymerization and depolymerization following the Shelanski et al. [13] glycerol method for the first cycle, while the second cycle was carried out without glycerol. Twice-cycled microtubular proteins were stored at - 8 0 ° C in a reassembly buffer (0.1 M MES pH 6.6, 1 mM EGTA, 0.5 mM MgC12).
Microtubule assembly The polymerization reaction was monitored at 350 nm following Gaskin et al.
185 [14] by measuring the increase in absorbance in a spectrophotometer connected to a constant-temperature circulating water bath set at 37°C. The polymerization reaction was initiated by adding 1 mM GTP.
Colchicine binding Colchicine binding was assayed following Borisy's method [15]. Tubulin samples were incubated at 37°C for 60 min with [3H]colchicine, filtered through four Whatman DE 81 DEAE-cellulose filters and washed with diluted reassembly buffer. Radioactivity was measured by liquid scintillation counting.
Sulphydryl titration --SH group titration was carried out following Ellman's DTNB method [16] in samples containing 2 t~M tubulin in a final volume of 1 ml. The absorbance was measured at 412 nm against a blank without tubulin.
Protein concentrations Protein concentrations were determined by Hartree's method [17] using bovine serum albumin as a standard.
Polyacrylamide gel electrophoresis The purity of microtubular protein was assessed by sodium dodecylsulphate polyacrylamide gel electrophoresis on 8% slab gels, by Laemmli's method [18]. RESULTS Even at 1 mM concentration of benzaldehyde, neither the rate nor the extent of microtubule assembly are markedly modified (Fig. 1A): tubulin polymerization is not therefore impaired. Microtubule assembly is, however, inhibited by the C-9 aliphatic aldehydes, in proportion to the concentrations used. Nonanal has the strongest inhibitory effect: at concentrations as low as 25 ~M it exerts a strong inhibitory effect (Fig. 1B). 2-Nonenal begins to inhibit assembly only above 0.1 mM (Fig. 1C), and 4-hydroxynonenal above 0.5 mM (Fig. 1D). Nonanal has the same inhibiting effect on microtubular protein at half the concentration, whereas the unsaturated aldehydes have a greater effect on diluted protein, above all at the intermediate concentration (Fig. 2). 4-Hydroxynonenal is the only aldehyde which reduces titratable sulphydryl groups of tubulin, the other aldehydes being ineffective in this respect (Table I). The effect of hydroxyalkenal on tubulin polymerization is prevented in a dose-related manner by the addition of cysteine (Fig. 3);/~-mercaptoethanol, on the other hand, does not represent a protective drug. The amount of colchicine bound by tubulin slightly increases after incubation with benzaldehyde, whereas it decreases after incubation with aliphatic aldehydes (Fig. 4). The unsaturated aldehydes proved the most effective, reducing the colchicine-binding activity of tubulin proportionally to the doses used, whereas nonanal has a weak inhibitory effect, and benzaldehyde has none.
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Fig~ 1~ Effect of aldehydes on polymerization (A, benzaldehyde; B, nonanal; C, 2-nonenal; I), 4-hydroxynonenal). Microtubular protein (2 mg/ml) was incubated for 30 rain at 37°C with different concentrations of the aldehydes (a = 0, b = 0.1, e = 0.5, d = 1 mM in panels A, C and D; b = 0.01, c = 0.025, d = 0.1, e = 0.5, f = 1 mM in panel B). Polymerization was initiated by adding 1 m l GTP and the increase in turbidity was monitored continuously by the change in absorbanee at 350 nm.
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concentration (mM) Fig. 2. Relation between the extent of microtubular protein polymerization (--, 2 mg/ml, - -, 1 mg/ml) and increasing doses of aldehydes ( u , benzaldehyde; e , nonanal; v, 2-nonenal; A, 4-hydroxynonenal). Absorbance values were taken after 30-min incubation and percentage values calculated with respect to the average of control samples, taken as 100%.
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Fig. 3. Percentage polymerization of microtubula~ protein with 1 mM 4-hydroxynonenal in presence of cysteine or ~$-mercaptoethanol. Data are the average of percentage values :e S.E.M. of the extent of polymerization vs. a control of microtubule polymerization.
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concentration (mM) Fig. 4. Colchicine binding activity of bovine brain microtubular protein after incubation with different aldehydes at various concentrations for 20 min at 20°C. Samples were further incubated for 60 min at 37°C with [3H]colchicine. Values are means ± S.D. of six determinations; the statistical significance of the differences was evaluated by variance analysis: *P < 0.025, **P < 0.01, ***P < 0.005. (B, benzaldehyde; N, nonanal; NE, 2-nonenal; HNE, 4-hydroxynonenal).
DISCUSSION
Aldehydes have different cell targets, and their molecular interactions with biological molecules lead to antiproliferative action and antitumour effect. Some aldehydes depress the growth of experimental tumours, such as Yoshida or Ehrlich ascites [6,7]. 4-Hydroxyalkenals inhibit both alpha and beta DNA polymerases [19] and block DNA synthesis [6]. Furthermore, these compounds
TABLE I EFFECT OF ALDEHYDES ON TITRATABLE SULPHYDRYL GROUPS OF MICROTUBULAR PROTEIN Microtubular protein was incubated with different concentrations of the aldehydes for 30 rain at 37°C and sulphydryls were analysed with DTNB. Experiments were carried out at least twice; percentage values are referred to controls taken as 100%. Aldehydes (mM)
% nmol --SH/mg PT 0.1
4-Hydroxynonenal 2-Nonenal Nonanal Benzaldehyde
85 106 106 109
0.5 74 100 ---
1 64 94 114 105
189 interfere with processes related to cellular movements, as they can modulate the chemotactic and chemokinetic activity of polymorphonuclear leucocytes [20]. Similarly, 2-alkenals have been found to be chemotactic, whereas alkanals are ineffective in this respect [21]. 4-Hydroxynonenal is one of the major breakdown products of lipid peroxidation [3], and it is the most studied of the hydroxyalkenals. Its antiproliferative action might also be related to an antimicrotubular effect, since microtubules are involved in processes connected with cell replication and movement. The action displayed by 4-hydroxynonenal on microtubular protein polymerization is weak compared to that of the saturated aldehyde nonanal, which is effective at much lower concentrations than the hydroxylated alkenal. Nonanal decreases both the rate and the extent of polymerization, whereas the unsaturated aldehydes have more effect on the extent than they do on the rate, 2-nonenal appearing more effective than 4-hydroxynonenal. Since dilution of microtubular protein does not alter the percentage values of polymerization in the presence of nonanal, with the same effect even when the aldehyde/tubulin ratio changes, this aldehyde does not seem to influence the critical concentration. The other aldehydes behave differently: the critical concentration appears to increase in the presence of increasing doses of either. A different mechanism of interaction may thus be supposed, with a stoichiometric rate for 2-nonenal and 4-hydroxynonenal. Colchicine binding activity of tubulin has been also studied, in order to verify whether molecule integrity is impaired. There, too, the behaviour of nonanal differs in comparison to the alkenals; the latter show a dose-related inhibitory effect, while nonanal is less active. The structural analogue of 4-hydroxynonenal, 2-nonenal, inhibits tubulin polymerization and colchicine binding to about the same extent, suggesting that the --CH = CH--CHO group reacts with tubulin in a particular manner, different from the saturated aldehyde nonanal. The reaction of 4-hydroxynonenal and of its homologue, 2-nonenal, may be competitive at the colchicine binding site of tubulin, or it may determine conformational changes to the protein, making it unable to bind the alkaloid. This aldehyde has also been observed to interact with tubulin on intact cells: microtubule networks disappeared after 3T3 cells treatment with 4-hydroxynonenal, similarly to what has been observed after treatment with colchicine [22]. As also shown in previous studies, 4-hydroxynonenal inhibits tubulin polymerization by blocking its --SH groups, which are fundamental for the assembly mechanism [23,24]. Tubulin is the probable target of 4-hydroxynonenal, since polymerization is inhibited even when MAPs, which facilitate this process, are removed. In the presence of a thiol compound such as cysteine, the polymerization reaction is restored, while 13-mercaptoethanol is not able to protect tubulin against the damaging effects of the hydroxyalkenal, perhaps because its affinity with the aldehyde is less. On the contrary, the mechanism by which aldehydes other than the hydroxyalkenals react with tubulin does not seem to involve functional --SH groups of tubulin, since the number of these groups is not significantly different from untreated samples in the presence of hydroxyalkenals. On the other hand, some saturated aldehydes [8], and the antiproliferative aldehyde methylglyoxal [11], also alter some properties of microtubular protein with a different mechanism from hydroxyalkenals, as thiol
190
groups are not involved. In some circumstances, such as after repeated exposures to acetaldehyde, the protein forms an adduct with the molecule, with a cumulative effect [25]. In the reaction of acetaldehyde with tubulin, adducts are formed due to the interaction of e-amino group of lysine, which is a nucleophilic group, with the electrophilic carbonyl carbon of the aldehyde [26]. Since lysine residues in the tubulin a-chain are essential for microtubule assembly [27], and since nonanal does not react with sulphydryl groups of tubulin, a reaction between the saturated aldehyde and the amino groups of lysine may be presumed. The different behaviour of nonanal in comparison with the other aldehydes could also suggest an interaction with microtubule associated proteins (MAPs) rather than with tubulin. In fact, nonanal determines an increase in the lag phase which precedes polymerization and, according to Roobol et al. [28], this effect could be related to a reaction with MAPs. In addition, since benzaldehyde is not able to interfere with tubulin functionality and integrity, the presence of an aliphatic chain is probably important for the reaction of the aldehydic group with the functional sites of tubulin, and further studies would discover whether the different kinds of interaction can modulate the in vivo assembly and disassembly of microtubular system, and thus play a role in cell proliferation. REFERENCES 1 2
3
4
5
6
7
8 9 10
11 12
E. Schauenstein, H. Esterbauer and H. Zollner, in: J.R. Lagnado (Ed.), Aldehydes in Biological Systems, Pion Limited, London, 1977, pp. 1--205. M.U. Dianzani, Biochemical effects of saturated and unsaturated aldehydes, in: D.C.H. McBrien and T.F. Slater (Eds.), Free Radicals, Lipid Peroxidation and Cancer, Academic Press, London, 1982, pp. 129--158. H. Esterbauer, K.H. Cheesman, M.U. Dianzani, G. Poll and T.F. Slater, Separation and characterization of the aldehydic products of lipid peroxidation by ADP-Fe 2÷ in rat liver microsomes, Biochem. J., 208 (1982) 129--140. A. Benedetti, M. Comporti and H. Esterbauer, Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids, Biochim. Biophys. Acta, 620 (1980) 281--296. L. Paradisi, C. Panagini, M. Parola, G. Barrera and M.U. Dianzani, Effects of 4-hydroxynonenal on adenylate cyclase and 5'-nucleotidase activities in rat liver plasma membranes, Chem.-Biol. Interact., 53 (1985) 209--217. S. Hauptlorenz, H. Esterbauer, W. Moll, R. Pumpel, E. Schauenstein and B. Puschendorf, Effect of the lipid peroxidation product 4-hydroxynonenal and related aldehydes on proliferation and viability of cultured ascites tumor cells, Biochem. Pharmacol, 34 (1985) 3803--3809. L. Tessitore, G. Bonelli, P. Costelli, L. Matera, A. Pileri, F.M. Baccino and M.U. Dianzani, Effect of two aliphatic aldehydes, methylglyoxal and 4-hydroxypentenal, on the growth of Yoshida ascites hepatoma AH-130, Chem.-Biol. Interact., 70 (1989) 227--240. A. Miglietta, L. Gabriel and E. Gadoni, Microtubular protein impairment by pentanal and hexanal, Cell Biochem. Funct., 5 (1987) 189--194. R.B. Jennett, D.J. Tuma and M.F. Sorrell, Effect of ethanol and its metabolites on microtubule formation, Pharmacology, 21 (1980) 363--368. A. Miglietta and L. Gabriel, Methylglyoxal-tubulin interaction: studies on the aldehyde effects on hepatoma, liver and purified microtubular protein, Res. Commun. Chem. Pathol. Pharmacol., 51 (1986) 245--260. L. Gabriel, A. Miglietta and M.U. Dianzani, 4-Hydroxyalkenals interaction with purified microtubular protein, Chem.-Biol. Interact., 56 (1985) 201--212. D.H. Carney and W.C. Thompson, Role of cytoplasmic microtubules in regulation of cell pro-
191
13 14 15 16 ~7 18 19
20
21
22 23 24 25
26
27 28
liferation, in: H.M. Zimmerman (Ed.), Progress in Neuropathology, Vol. 6, Raven Press, New York, 1986, pp. 91--117. M.L. Shelanski, F. Gaskin and C.R. Cantor, Microtubule assembly in the absence of added nucleotides, Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 765--768. F. Gaskin, C.R. Cantor and M.L. Shelanski, Turbidimetric studies of the 'in vitro' assembly and disassembly of porcine neurotubules, J. Mol. Biol., 89 (1974) 737--758. G.G. Borisy, A rapid method for quantitative determination of microtubule protein using DEAE-Cellulose filters, Anal. Biochem., 50 (1972) 373--385. G.L. Ellman, Tissue sulphydryl groups, Arch. Biochem. Biophys., 82 (1959) 70--77. E.F. Hartree, Determination of proteins: a modification of the Lowry method that gives a linear photometric response, Anal. Biochem., 48 (1972) 422--427. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680--685. E. Wawra, H. Zollner, R.J. Schaur, H.M. Tillian and E. Schauenstein, The inhibitory effect of 4-hydroxy-nonenal on DNA-polymerases alpha and beta from rat liver and rapidly dividing Yoshida ascites hepatoma, Cell Biochem. Funct., 4 (1986) 31--36. M. Curzio, H. Esterbauer, C. Di Mauro, G. Cecchini and M.U. Dianzani, Chemotactic activity of the lipid peroxidation product 4-hydroxynonenal and homologous hydroxyalkenals, Biol. Chem. Hoppe-Seyler, 367 (1986) 321--329. M. Curzio, H. Esterbauer, G. Poll, F. Biasi, G. Cecchini, C. Di Mauro, N. Cappello and M.U. Dianzani, Possible role of aldehydic lipid peroxidation products as chemoattractants, Int. J. Tiss. Reac., 9 (1987) 295--306. A. Olivero, A. Miglietta, E. Gadoni and L. Gabriel, 4-Hydroxynonenal interacts with tubulin by reacting with its functional --SH groups, Cell Biochem. Funct., 8 (1990) 99--105. M.G. Mellon and L.I. Rebhun, Sulphydryls and the in vitro polymerization of tubulin, J. Cell Biol., 70 (1976) 226--238. R. Kuriyama and H. Sakai, Role of tubulin --SH groups on polymerization of microtubules, J. Biochem., 76 {1974) 651--654. G. McKinnon, J. de Jersey, B. Shanley and L. Ward, The reaction of acetaldehyde with brain microtubular proteins: formation of stable adducts and inhibition of polymerization, Neurosci. Lett., 79 (1987) 163--168. R.B. Jennett, M.F. Sorrell, E.L. Johnson and D.J. Tuma, Covalent binding of acetaldehyde to tubulin: evidence for preferential binding to the a-chain, Arch. Biochem. Biophys., 256 (1987) 10--18. G. Sherman, T.L. Rosenberry and H. Sternlicht, Identification of lysine residues essential for microtubule assembly, J. Biol. Chem., 258 (1983) 2148--2156. A. Roobol, K. Gull and C.I. Pogson, Inhibition by griseofulvin of microtubule assembly in vitro, FEBS Lett., 67 (1976) 248--251.
183
EFFECTS OF SOME ALDEHYDES ON BRAIN MICROTUBULAR PROTEIN
ANTONELLA MIGLIETTA, ANTONELLA OLIVERO, ELENA GADONI and LUDOVICA GABRIEL
Dipartimento di Medicina ed Oncologia Sperimentale, Sezione di Patologia Generale, Corso Raffaello 30, 10125 Torino (Italy) (Received May llth, 1990) (Revision received December 20th, 1990) (Accepted December 20tb, 1990)
SUMMARY
4-Hydroxynonenal is one of the main breakdown products of lipid peroxidation. It has an antiproliferative effect, which may partly be the consequence of an interaction with cytoskeletal structures. Its effects on microtubular protein are compared with those of homologous aldehydes with the same number of carbon atoms, and with that of benzaldehyde. Unlike the other aliphatic aldehydes, this latter aldehyde does not impair microtubular functions at every concentration in the range. Nonanal has the greatest effect on tubulin polymerization, whereas it only slightly impairs colchicine binding activity. 2-Nonenal and 4-hydroxynonenal have less inhibiting effect on tubulin polymerization; their effect on colchicine binding activity is dose-dependent. The targets of 4-hydroxynonenal on tubulin are --SH groups; the action mechanism of other aldehydes has not yet been identified.
Key words: Aldehydes -- Tubulin -- Polymerization -- Colchicine binding ---SH groups
INTRODUCTION
Aldehydes have different biochemical and biological properties, and play several roles in metabolic pathways. Some aldehydes are cytotoxic [1] and exert an inhibitory effect on different cell functions [2]. The general action mechanism of aldehydes is to attack both the sulphydryl and the amino groups of Correspondence to: Antonella Miglietta, Dipartimento di Medicina ed Oncologia Sperimentale, Sezione di Patologia Generale, Corso Raffaello 30, 10125 Torino, Italy. 0009-2797/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
184 biomolecules. They influence cell division and tumour growth by deactivating different enzymes and interfering with the metabolism and the biosynthesis of proteins and nucleic acids. Among aldehydes with a carbon-carbon double bond, 4-hydroxyalkenals are particularly reactive, impairing cellular and subcellular functions by attacking sulphydryl groups of biological molecules. 4-Hydroxyalkenals, and in particular 4-hydroxynonenal [3], derive from the breakdown of polyunsatured fatty acids during lipid peroxidation. The latter aldehyde inhibits several enzymes [2,4,5], and affects the proliferation of tumour cells [6]. Another hydroxyalkenal, 4-hydroxypentenal, markedly depresses the growth of Yoshida ascites hepatoma AH-130 [7], as well as that of several transplantable tumours. Various aldehydes have antimicrotubular activity, among them pentanal and hexanal [8], acetaldehyde [9] or the antiproliferative aldehyde methylglyoxal [10]. The antiproliferative effect of various aldehydes, and in particular of the hydroxyalkenals [11], may thus be related to a damage to microtubules, which play an important role in cell division and proliferation [12]. It appeared interesting to evaluate whether the carcinostatic effect of aliphatic aldehydes could be due to a direct interaction with tubulin, and to investigate a possible structure-activity relationship. In this research we studied the effect on microtubular protein of aldehydes having the same chain length, in order to clarify whether the different action mechanism may be related to the hydrophilic, lipophilic or polar characteristics of the molecules. We chose 4-hydroxynonenal, the most representative aldehyde produced in lipid peroxidation, and two homologous aldehydes with the same number of carbon atoms, 2-nonenal and nonanal; furthermore, an aromatic aldehyde, benzaldehyde, was also studied, to compare its effect with that of the aliphatic aldehydes. MATERIALS AND METHODS
Materials 4-Hydroxynonenal and 2-nonenal were kindly given by Dr. Esterbauer, Institut fur Biochemie, Universitat, Graz. Calf brains were obtained from a local slaughterhouse. [3H]Colchicine (spec. act., 4.9 Ci/mmol) was obtained from the Radiochemical Centre, Amersham, U.K. Electrophoresis reagents were from Bio-Rad. All other chemicals were reagent grade from Merck or Sigma.
Microtubular protein purification Calf brains were used within 2 h of the animals' death. Microtubular protein was prepared by two cycles of polymerization and depolymerization following the Shelanski et al. [13] glycerol method for the first cycle, while the second cycle was carried out without glycerol. Twice-cycled microtubular proteins were stored at - 8 0 ° C in a reassembly buffer (0.1 M MES pH 6.6, 1 mM EGTA, 0.5 mM MgC12).
Microtubule assembly The polymerization reaction was monitored at 350 nm following Gaskin et al.
185 [14] by measuring the increase in absorbance in a spectrophotometer connected to a constant-temperature circulating water bath set at 37°C. The polymerization reaction was initiated by adding 1 mM GTP.
Colchicine binding Colchicine binding was assayed following Borisy's method [15]. Tubulin samples were incubated at 37°C for 60 min with [3H]colchicine, filtered through four Whatman DE 81 DEAE-cellulose filters and washed with diluted reassembly buffer. Radioactivity was measured by liquid scintillation counting.
Sulphydryl titration --SH group titration was carried out following Ellman's DTNB method [16] in samples containing 2 t~M tubulin in a final volume of 1 ml. The absorbance was measured at 412 nm against a blank without tubulin.
Protein concentrations Protein concentrations were determined by Hartree's method [17] using bovine serum albumin as a standard.
Polyacrylamide gel electrophoresis The purity of microtubular protein was assessed by sodium dodecylsulphate polyacrylamide gel electrophoresis on 8% slab gels, by Laemmli's method [18]. RESULTS Even at 1 mM concentration of benzaldehyde, neither the rate nor the extent of microtubule assembly are markedly modified (Fig. 1A): tubulin polymerization is not therefore impaired. Microtubule assembly is, however, inhibited by the C-9 aliphatic aldehydes, in proportion to the concentrations used. Nonanal has the strongest inhibitory effect: at concentrations as low as 25 ~M it exerts a strong inhibitory effect (Fig. 1B). 2-Nonenal begins to inhibit assembly only above 0.1 mM (Fig. 1C), and 4-hydroxynonenal above 0.5 mM (Fig. 1D). Nonanal has the same inhibiting effect on microtubular protein at half the concentration, whereas the unsaturated aldehydes have a greater effect on diluted protein, above all at the intermediate concentration (Fig. 2). 4-Hydroxynonenal is the only aldehyde which reduces titratable sulphydryl groups of tubulin, the other aldehydes being ineffective in this respect (Table I). The effect of hydroxyalkenal on tubulin polymerization is prevented in a dose-related manner by the addition of cysteine (Fig. 3);/~-mercaptoethanol, on the other hand, does not represent a protective drug. The amount of colchicine bound by tubulin slightly increases after incubation with benzaldehyde, whereas it decreases after incubation with aliphatic aldehydes (Fig. 4). The unsaturated aldehydes proved the most effective, reducing the colchicine-binding activity of tubulin proportionally to the doses used, whereas nonanal has a weak inhibitory effect, and benzaldehyde has none.
0
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0.3
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Fig~ 1~ Effect of aldehydes on polymerization (A, benzaldehyde; B, nonanal; C, 2-nonenal; I), 4-hydroxynonenal). Microtubular protein (2 mg/ml) was incubated for 30 rain at 37°C with different concentrations of the aldehydes (a = 0, b = 0.1, e = 0.5, d = 1 mM in panels A, C and D; b = 0.01, c = 0.025, d = 0.1, e = 0.5, f = 1 mM in panel B). Polymerization was initiated by adding 1 m l GTP and the increase in turbidity was monitored continuously by the change in absorbanee at 350 nm.
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concentration (mM) Fig. 2. Relation between the extent of microtubular protein polymerization (--, 2 mg/ml, - -, 1 mg/ml) and increasing doses of aldehydes ( u , benzaldehyde; e , nonanal; v, 2-nonenal; A, 4-hydroxynonenal). Absorbance values were taken after 30-min incubation and percentage values calculated with respect to the average of control samples, taken as 100%.
100
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Fig. 3. Percentage polymerization of microtubula~ protein with 1 mM 4-hydroxynonenal in presence of cysteine or ~$-mercaptoethanol. Data are the average of percentage values :e S.E.M. of the extent of polymerization vs. a control of microtubule polymerization.
188 ~0
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concentration (mM) Fig. 4. Colchicine binding activity of bovine brain microtubular protein after incubation with different aldehydes at various concentrations for 20 min at 20°C. Samples were further incubated for 60 min at 37°C with [3H]colchicine. Values are means ± S.D. of six determinations; the statistical significance of the differences was evaluated by variance analysis: *P < 0.025, **P < 0.01, ***P < 0.005. (B, benzaldehyde; N, nonanal; NE, 2-nonenal; HNE, 4-hydroxynonenal).
DISCUSSION
Aldehydes have different cell targets, and their molecular interactions with biological molecules lead to antiproliferative action and antitumour effect. Some aldehydes depress the growth of experimental tumours, such as Yoshida or Ehrlich ascites [6,7]. 4-Hydroxyalkenals inhibit both alpha and beta DNA polymerases [19] and block DNA synthesis [6]. Furthermore, these compounds
TABLE I EFFECT OF ALDEHYDES ON TITRATABLE SULPHYDRYL GROUPS OF MICROTUBULAR PROTEIN Microtubular protein was incubated with different concentrations of the aldehydes for 30 rain at 37°C and sulphydryls were analysed with DTNB. Experiments were carried out at least twice; percentage values are referred to controls taken as 100%. Aldehydes (mM)
% nmol --SH/mg PT 0.1
4-Hydroxynonenal 2-Nonenal Nonanal Benzaldehyde
85 106 106 109
0.5 74 100 ---
1 64 94 114 105
189 interfere with processes related to cellular movements, as they can modulate the chemotactic and chemokinetic activity of polymorphonuclear leucocytes [20]. Similarly, 2-alkenals have been found to be chemotactic, whereas alkanals are ineffective in this respect [21]. 4-Hydroxynonenal is one of the major breakdown products of lipid peroxidation [3], and it is the most studied of the hydroxyalkenals. Its antiproliferative action might also be related to an antimicrotubular effect, since microtubules are involved in processes connected with cell replication and movement. The action displayed by 4-hydroxynonenal on microtubular protein polymerization is weak compared to that of the saturated aldehyde nonanal, which is effective at much lower concentrations than the hydroxylated alkenal. Nonanal decreases both the rate and the extent of polymerization, whereas the unsaturated aldehydes have more effect on the extent than they do on the rate, 2-nonenal appearing more effective than 4-hydroxynonenal. Since dilution of microtubular protein does not alter the percentage values of polymerization in the presence of nonanal, with the same effect even when the aldehyde/tubulin ratio changes, this aldehyde does not seem to influence the critical concentration. The other aldehydes behave differently: the critical concentration appears to increase in the presence of increasing doses of either. A different mechanism of interaction may thus be supposed, with a stoichiometric rate for 2-nonenal and 4-hydroxynonenal. Colchicine binding activity of tubulin has been also studied, in order to verify whether molecule integrity is impaired. There, too, the behaviour of nonanal differs in comparison to the alkenals; the latter show a dose-related inhibitory effect, while nonanal is less active. The structural analogue of 4-hydroxynonenal, 2-nonenal, inhibits tubulin polymerization and colchicine binding to about the same extent, suggesting that the --CH = CH--CHO group reacts with tubulin in a particular manner, different from the saturated aldehyde nonanal. The reaction of 4-hydroxynonenal and of its homologue, 2-nonenal, may be competitive at the colchicine binding site of tubulin, or it may determine conformational changes to the protein, making it unable to bind the alkaloid. This aldehyde has also been observed to interact with tubulin on intact cells: microtubule networks disappeared after 3T3 cells treatment with 4-hydroxynonenal, similarly to what has been observed after treatment with colchicine [22]. As also shown in previous studies, 4-hydroxynonenal inhibits tubulin polymerization by blocking its --SH groups, which are fundamental for the assembly mechanism [23,24]. Tubulin is the probable target of 4-hydroxynonenal, since polymerization is inhibited even when MAPs, which facilitate this process, are removed. In the presence of a thiol compound such as cysteine, the polymerization reaction is restored, while 13-mercaptoethanol is not able to protect tubulin against the damaging effects of the hydroxyalkenal, perhaps because its affinity with the aldehyde is less. On the contrary, the mechanism by which aldehydes other than the hydroxyalkenals react with tubulin does not seem to involve functional --SH groups of tubulin, since the number of these groups is not significantly different from untreated samples in the presence of hydroxyalkenals. On the other hand, some saturated aldehydes [8], and the antiproliferative aldehyde methylglyoxal [11], also alter some properties of microtubular protein with a different mechanism from hydroxyalkenals, as thiol
190
groups are not involved. In some circumstances, such as after repeated exposures to acetaldehyde, the protein forms an adduct with the molecule, with a cumulative effect [25]. In the reaction of acetaldehyde with tubulin, adducts are formed due to the interaction of e-amino group of lysine, which is a nucleophilic group, with the electrophilic carbonyl carbon of the aldehyde [26]. Since lysine residues in the tubulin a-chain are essential for microtubule assembly [27], and since nonanal does not react with sulphydryl groups of tubulin, a reaction between the saturated aldehyde and the amino groups of lysine may be presumed. The different behaviour of nonanal in comparison with the other aldehydes could also suggest an interaction with microtubule associated proteins (MAPs) rather than with tubulin. In fact, nonanal determines an increase in the lag phase which precedes polymerization and, according to Roobol et al. [28], this effect could be related to a reaction with MAPs. In addition, since benzaldehyde is not able to interfere with tubulin functionality and integrity, the presence of an aliphatic chain is probably important for the reaction of the aldehydic group with the functional sites of tubulin, and further studies would discover whether the different kinds of interaction can modulate the in vivo assembly and disassembly of microtubular system, and thus play a role in cell proliferation. REFERENCES 1 2
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