Specific structural determinants are responsible for the antioxidant activity and the cell cycle effects

53 Pages·2016·0.2 MB·English

JBC Papers in Press. Published on April 20, 2001 as Manuscript M101846200
Specific structural determinants are responsible for the antioxidant activity and
the cell cycle effects of resveratrol.
Stivala, L.A., Savio, M., Carafoli, F., Perucca, P., Bianchi, L., Maga, G.¶, Forti,
L.‡, Pagnoni, U.M. ‡, Albini, A.*, Prosperi, E.§ and Vannini, V.
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Dipartimento di Medicina Sperimentale, sez. Patologia Generale, *Dipartimento di Chimica d fro
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Organica, Università di Pavia; §Centro di Studio per l’Istochimica del CNR, ¶Istituto di http
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Genetica Biochimica ed Evoluzionistica IGBE-CNR, Pavia; Dipartimento di Chimica, w
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Università di Modena e Reggio Emilia, Italy. .o
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Correspondence address: A
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Dr. Lucia Anna Stivala ril 7
Dipartimento di Medicina Sperimentale , 2
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Sez. Patologia generale “C. Golgi” 19
Università di Pavia
Piazza Botta, 10 – 27100 PAVIA – ITALY
Tel.: [phone]
Fax: [phone]
E-mail: [email protected]
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural determinants of biological activity of resveratrol
Running Title: Structural determinants of biological activity of resveratrol
SUMMARY
Resveratrol (3,4’,5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and
wine, which shows antioxidant and antiproliferative activities. In this study we have
investigated whether these properties are dependent on similar or different structural
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determinants of the molecule. To this purpose, resveratrol derivatives, in which all or each w
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single hydroxylic function were selectively substituted with methyl groups, were synthesized. ed
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Analogues with the stilbenic double bond reduced or with the stereoisomery modified, were http
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also investigated. The antioxidant activity of these compounds was evaluated by measuring w
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the inhibition of citronellal thermo-oxidation, or the reduction of 2,2-diphenyl-1- .org
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picrylhydrazyl radical (DPPH). In addition, the protection against lipid peroxidation was u
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determined in rat liver microsomes, and in human primary cell cultures. The antiproliferative A
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activity was evaluated by a clonogenic assay, and by analysis of cell cycle progression and , 2
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DNA synthesis. The results showed that the hydroxyl group in 4’ position is not the sole
determinant for antioxidant activity. In contrast, the presence of 4’-OH together with
stereoisomery in the trans conformation (4’-hydroxystyryl moiety), was absolutely required
for inhibition of cell proliferation. Enzymatic assays in vitro demonstrated that inhibition of
DNA synthesis was induced by a direct interaction of resveratrol with DNA polymerases a
and d.
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Structural determinants of biological activity of resveratrol
INTRODUCTION
Resveratrol (3,4,5-trihydroxystilbene) is synthesized by several plants in response to adverse
conditions such as environmental stress or pathogenic attack. For this reason, it is classified as
a phytoalexin, a class of antibiotics of plant origin (1-3). Resveratrol has been found in a
multitude of dietary plants, such as peanuts, mulberries and in grape skin (4). Thus, relatively
high concentrations of this compound are present in grape juice and, especially, in red wine
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(5-8). Growing evidence suggest that resveratrol plays a role in the prevention of human o
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pathological processes, such as inflammation (9-11), atherosclerosis (12-14) and de
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carcinogenesis (4, 15, 16). The protective effect has been attributed to its antioxidant h
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properties (17-19), to an anticyclooxygenase activity (4, 20), and to a modulating activity of w
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lipid and lipoprotein metabolism (9, 21, 22). Resveratrol also inhibits platelet aggregation (13, .o
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23) and exhibits antiestrogenic activity (24, 25). However, these effects do not exhaustively g
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explain the antiproliferative and anticarcinogenic properties of resveratrol. The proliferation n A
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of various human malignant cell lines is slowed down by resveratrol (15, 16, 26). The , 2
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inhibition of cell growth, which has also been described in normal cells (27-29), is
accompanied by the accumulation of cells in S and G2 phases (16, 26, 30). Conflicting results
have been reported on the induction of apoptosis by resveratrol (26, 31, 32).
A number of antioxidants, such as vitamin E, N-acetylcysteine, flavonoids, and carotenoids
have been reported to interfere with cell cycle progression by inducing the expression of cdk
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Structural determinants of biological activity of resveratrol
inhibitors, like p21waf1cip1, p16ink4a, p27kip1 (33-36). In the case of resveratrol, such a
mechanism is still controversial (26, 27). The effects on cell cycle progression may be also
explained by the direct inhibition of ribonucleotide reductase (37) and DNA polymerase (38).
The structural determinants of these diverse properties of the resveratrol molecule are
obscure, but the number and position of the hydroxylic groups have been suggested to play an
important role in the antioxidant activity (14, 19, 39). The aim of this study was to extend
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these studies on the structural determinants of the activity of resveratrol, and in particular to ow
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establish whether the antioxidant and antiproliferative activities are dependent on i) the e
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stereoisomery, ii) the position of the different phenolic hydroxyl groups, and iii) the stilbenic h
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double bond of the molecule. For this purpose, the cis form (II) was obtained by UV w
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irradiation of trans-resveratrol; three different derivatives were synthesized in which the .o
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hydroxylic functions were selectively protected by methyl groups: 3,5-dihydroxy-4’- g
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methoxystilbene (III), 3,5-dimethoxy-4’-hydroxystilbene (IV) and 3,4,5-trimethoxystilbene A
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(V). Finally, the a,b-dihydro-3,4,5-trihydroxystilbene (VI) was obtained by reduction of the ril 7, 2
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stilbenic double bond.
The biological properties of trans-resveratrol were compared to those of the above
derivatives. In particular, the antioxidant activity was investigated in vitro by measuring the
inhibition of citronellal thermo-oxidation or the radical scavenging ability using the free
radical DPPH. The protection against lipid peroxidation induced by Fe/Ascorbate and tert-
butylhydroperoxide (TBHP) was also assessed in rat liver microsomes, or in cultured human
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fibroblasts, respectively. The effects on cell proliferation were studied by analyzing the cell
clonogenic efficiency and cell cycle progression. In addition, the recruitment of proliferating
cell nuclear antigen (PCNA) and replication protein A (RPA) to the DNA replication sites
were investigated. These proteins are required for the initiation and elongation steps of DNA
replication, respectively. Finally, the ability of resveratrol and its derivatives to inhibit
replicative DNA polymerases was also assessed with in vitro assays.
The results have shown that the hydroxyl group at the 4’ position is not the sole determinant D
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for the antioxidant activity. Similarly, the 4’ hydroxyl group is necessary for the ad
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antiproliferative activity and the DNA polymerase inhibition, but the trans conformation is m
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absolutely required for these effects. ://w
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Structural determinants of biological activity of resveratrol
EXPERIMENTAL PROCEDURES
Reagents
trans-resveratrol (99% purity) and DPPH were obtained from Sigma; aphidicolin was
obtained from Roche. Monoclonal antibodies anti-bromo-deoxy-uridine (BrdU clone
BU20) and anti PCNA (clone PC10) were obtained from Dako, while the anti-RPA (32 kDa
subunit) 9H8 monoclonal antibody was kindly provided by M.Wold (Iowa University, USA).
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The fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody was purchased from o
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Sigma. de
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[3H]-dATP (40 Ci/mmol) and [3H]-dTTP (40 Ci/mmol) were from Amersham. Activated http
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calf thymus DNA was prepared as described (40). Unlabelled dNTPs and Poly(dA) and w
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oligo(dT)12-18 homopolymers were from Pharmacia. Whatman was the supplier of the brg/
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GF/C filters. All other reagents were of analytical grade and purchased from Merck, Fluka es
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and Aldrich. pril 7
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Poly(dA)/oligo(dT)12-18 primer-template was prepared according to the manufacturer’s 19
protocol. Briefly, Poly(dA) template oligonucleotide was mixed with the complementary
oligo(dT)12-18 oligonucleotide in 10:1 molar ratio (w/w, nucleotides) in 20 mM Tris-HCl
(pH 8.0), containing 20 mM KCl and 1 mM EDTA, heated at 90 °C for 5 min then incubated
at 65 °C for two hours and slowly cooled at room temperature.
Calf thymus DNA polymerase a (pol a) and d (pol d) were purified as described (40). The pol
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Structural determinants of biological activity of resveratrol
d used in this study was 2,200 U/ml, (0.08 mg/ml). Pol a was 250 U/ml (0.2 mg/ml). 1 unit
of pol activity corresponds to the incorporation of 1 nmol of total dTMP into acid-
precipitable material in 60 min at 37 °C in a standard assay containing 0.5 µg (nucleotides) of
poly(dA)/oligo(dT)10:1 and 20 µM dTTP. Recombinant human wt PCNA was prepared as
described (41).
Resveratrol derivatives synthesis. D
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The cis form (II) was obtained by photoisomerization. The trans isomer (95 mg) was ad
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dissolved in 20 ml acetonitrile, flushed with nitrogen for 20 min, capped and irradiated by m
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means of 4 external 15 W phosphor coated lamps until the photostationary state (ca. 1 to 1) ://w
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was reached (40 min). Repeated separation by column chromatography eluting with c
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cyclohexane-ethyl acetate 1:1 gave a sample (20% yield) of 98% pure (HPLC) cis derivative. y g
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1H-NMR (CD3CN, 300 MHz): d 6.15 (dd, 1H, J 4,2 = J4,6 = 2.0 Hz, H-4); 6.25 (d, 2H, J2,4 = A
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J6,4 = 2.0 Hz, H-2 and H-6); 6.38 and 6.50 (AB system, 2H, J = 12.0 Hz, CH=CH); 6.62 (d, 01
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2H, J3’,2’ = J5’,6’ = 8.5 Hz, H-3’ and H-5’); 6.75 (bs, 2H, 2xOH); 6.90 (bs, 1H, OH); 7.13
(d, 2H, J2’,3’ = J6’,5’ = 8.5 Hz, H-2’ and H-6’).
trans-3,5-Dihydroxy-4’-methoxystilbene (4’-O-methylresveratrol) (compound III) was
obtained as previously described (44) by Wittig reaction between the phosphonium salt of the
commercially available 4-methoxybenzyl chloride and 3,5-bis-(tert-
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butyldimethylsilyloxy)benzaldehyde. Butyllithium (2.3 mL, 1.6 M in hexane, 3.6 mmol) was
added dropwise to a suspension of (4-methoxybenzyl)triphenylphosphonium chloride (3.6
mmol) in tetrahydrofuran (THF, 50 mL) at –15ºC. The resulting reddish solution was allowed
to warm at room temperature and stirred for 30 min. 3,5-bis-(tert-
butyldimethylsilyloxy)benzaldehyde (3.6 mmol) was then added and the reaction mixture was
stirred for 1 h, diluted with ice-cold water (2 x 25 mL) and extracted with ethyl acetate (3 x
30 mL). The organic extracts were washed with water and the solvent was removed under D
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reduced pressure to obtain a mixture of trans- and cis-3,5-di-(tert-butyldimethylsilyloxy)- ad
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4’-methoxystilbene, which were desilylated in THF at room temperature with m
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tetrabutylammonium fluoride. After addition of ethyl ether the solution was washed with ://w
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water and the solvent was removed under reduced pressure. The residue was filtered over c
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silica gel to obtain 3,5-dihydroxy-4’-methoxystilbene as a 2:1 mixture of the (trans/cis)- y g
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isomers in 75 % yield. After crystallization from CHCl3/pentane, the pure trans-isomer (III) n A
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was obtained (33% yield). 1H-NMR (Me2SO-d6, 200 MHz): d 3.78 (s, 3H, OCH 3); 6.14 , 201
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(dd, 1H, J4,2 = J4,6 = 2.05 Hz, H-4); 6.42 (d, 2H, J2,4 = J6,4 = 2.05 Hz, H-2 and H-6); 6.90
and 7.00 (AB system, 2H, J = 16.28 Hz, CH=CH); 6.94 (d, 2H, J3’,2’ = J5’,6’ = 8.77 Hz, H-
3’ and H-5’); 7.53 (d, 2H, J2’,3’ = J6’,5’ = 8.77 Hz, H-2’ and H-6’); 9.23 (s, 2H, 2 x OH).
trans-3,5-Dimethoxy-4’-hydroxystilbene (IV) was obtained by Perkin condensation
between 4-hydroxybenzaldehyde and 3,5-dimethoxyphenylacetic acid (Aldrich), as reported
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by Pezet and Pont (45). An alternative synthetic procedure was attempted, consisting in the
Wittig reaction between the phosphonium salt of 3,5-dimethoxybenzylbromide (synthesized
from the commercially available 3,5-dimethoxybenzyl alcohol) and 4-acetoxybenzaldehyde
on 3.6 mmol scale, as reported for the compound III. In this case, a 1:1 mixture of the
trans/cis-isomers was obtained in 48% yield. The 4’-hydroxyl function was deprotected by
treatment with K2CO3 in methanol at room temperature. After addition of ethyl acetate, the
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solution was washed with water and the solvent removed under reduced pressure: the residue ow
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was then chromatographed over silica gel using petroleum ether/diethyl ether gradient, giving e
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trans-3,5-dimethoxy-4-hydroxystilbene in 20% yield. 1H-NMR (Me2SO-d6, 200 MHz): d ttp
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3.78 (s, 6H, 2 x OCH3); 6.38 (dd, 1H, J4,2 = J4,6 = 2.22 Hz, H-4); 6.73 (d, 2H, J2,4 = J6,4 = .jb
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2.22 Hz, H-2 and H-6); 6.78 (d, 2H, J3,2 = J5,6 = 8.61 Hz, H-3 and H-5); 6.94 and 7.17 y g
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(AB system, 2H, J = 16.42 Hz, CH=CH); 7.43 (d, 2H, J2’,3’ = J6’,5’ = 8.61 Hz, H-2’ and H- n A
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6’); 9.56 (s, 1H, OH). , 2
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3,4’,5-trans-Trimethoxystilbene (derivative V) was synthesized by direct methylation,
refluxing a mixture of trans-resveratrol (0.44 mmol) and CH3I (9.24 mmol) in acetone (15
mL) in the presence of anhydrous potassium carbonate (6.6 mmol). After addition of ethyl
ether, the solution was washed with water and the solvent was removed under reduced
pressure: the residue was then chromatographed over silica gel using petroleum ether/diethyl
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ether gradient, giving trans-3,4,5-trimethoxystilbene in quantitative yield. 1H-NMR
(CDCl3, 200 MHz): d 3.87 (s, 9H, 3 x OCH 3); 6.42 (dd, 1H, J4,2 = J4,6 = 2.26 Hz, H-4); 6.69 (d, 2H,
J2,4 = J6,4 = 2.26 Hz, H-2 and H-6); 6.93 and 7.08 (AB system, 2H, J = 16.26 Hz, CH=CH);
6.934 (d, 2H, J3,2 = J5,6 = 8.71 Hz, H-3 and H-5); 7.48 (d, 2H, J2,3 = J6,5 = 8.71 Hz, H-2
and H-6).
a,b-Dihydro-3,4,5-trihydroxystilbene (a,b-dihydro-resveratrol) (derivative VI) was D
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obtained by catalytic hydrogenation of trans-resveratrol (0.15 mmol) with 10% Pd/C catalyst loa
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in methanol (5 mL) at room temperature and atmospheric pressure. The catalyst was filtered fro
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off through Celite and washed with methanol; the solvent was then removed under reduced ://w
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pressure and the residue chromatographed over silica gel (7:3 hexane-ethyl acetate) to obtain .jb
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a,b-dihydro-3,4,5-trihydroxystilbene (87% yield). 1H-NMR (Me2SO-d6, 200 MHz): d y gu
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2.55-2.77 (m, 4H, CH2CH2); 6.03 (dd, 1H, J4,2 = J4,6 = 2.11 Hz, H-4); 6.07(d, 2H, J2,4 = Ap
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J6,4 = 2.11 Hz, H-2 and H-6); 6.66 (d, 2H, J3,2 = J5,6 = 8.52 Hz, H-3 and H-5); 7.00 (d, 2H, 01
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J2’,3’ = J6’,5’ = 8.52 Hz, H-2’ and H-6’); 8.99 (s, 2H, 2 x OH); 9.08 (s, 1H, OH).
The products synthesized were further purified by crystallization to reach a final purity >
98%, as determined by HPLC.
Stock solutions of each substance were prepared in N, N-dimethylformamide (DMF) and
final dilution was performed in chlorobenzene for the in vitro oxidation test. For cell culture
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