Original article

B. OLAS*, B. WACHOWICZ, E. BALDa, R. GLOWACKIa


THE PROTECTIVE EFFECTS OF RESVERATROL AGAINST CHANGES IN BLOOD PLATELET THIOLS INDUCED BY PLATINUM COMPOUNDS


Department of General Biochemistry, University of Lodz, Lodz, Poland;
aDepartment of Environmental Chemistry, University of Lodz, Lodz, Poland


  Cisplatin (cis-diamminedichloroplatinum II, cisPt) is especially useful in the treatment of epithelial malignancies, however, the use of cisplatin is accompanied by several toxicities including haematological toxicity. Contrary to cisplatin, selenium-cisplatin conjugate ((NH3)2Pt(SeO3); Se-Pt) has only a slight toxicity effect on blood platelet function. In the mechanism of platinum compounds action on platelets thiols are involved. The aim of the present studies was to examine in vitro how trans-resveratrol (trans-3,4',5-trihydroxystilbene) acts on the levels of platelet glutathione (GSH) and other thiol-containing compounds and how, as an antioxidant, protecs blood platelets against the oxidative stress caused by platinum compounds (cisPt and Se-Pt). To analyse the level of thiols in human blood platelets treated with platinum compounds and with resveratrol the classical technique HPLC has been used. Blood platelets isolated by differential centrifugation of human blood were incubated (30 min, 37°C) with cisPt or Se-Pt at dose of 10 µg/ml that inhibits platelet function and with resveratrol (25 µg/ml). The obtained results indicate that platinum compounds caused in platelets a decrease of both, reduced glutathione (GSH) and free thiols of cysteine (CSH) and cysteinylglycine (CGSH). The pool of these compounds in unreduced form was increased. Platinum compounds caused the reduction of platelet protein thiols. Resveratrol (after 30 min action) at the concentration of 25 µg/ml partly reduced the platinum compounds induced decrease of platelet thiols, particularly thiols in acid-soluble fraction.

Key words: blood platelets; glutathione; thiols; resveratrol; cisplatin



INTRODUCTION

Blood platelets, the smallest, anucleated blood cells are not only involved in haemostasis, they also play an important role in pathomechanism of altered haemostasis in cancer patients. Platelets from patients with cancer exhibit impaired adhesion, aggregation and altered responses to different agonists (1, 2). Clinical observations suggest that anticancer drugs could contribute to the thrombotic complications in patients treated with anticancer drugs. Our earlier results indicate that cisplatin (cis-diamminedichloroplatinum II, cisPt; the most effective anticancer compound among all platinum-based drugs) (3) can affect blood platelet function (4-9). Selenium-cisplatin conjugate ((NH3)2Pt(SeO3); Se-Pt) had less toxic effect on these cells (9, 10). In earlier studies we reported that the intracellular level of glutathione (GSH) plays an important role in the action of cisplatin on blood platelets (11). Glutathione has a variety of physiologically important functions in cellular defense and metabolism, including modulation of thiol-disulfide status of cellular proteins, protection of cells from oxidative stress, detoxication of electrophilic compounds, or synthesis and transport of biologically active, endogenous substances. Moreover, GSH interacts with a wide range of drugs. Our previous studies show that after incubation of blood platelets with cisplatin the amount of GSH decreased, and the complex of cisplatin with GSH (GS-cisPt) was formed (via reaction catalysed by glutathione S-transferases) (4, 11-13). We suggest that not only cisplatin alone but also the GS-cisPt complex formed in blood platelets may be responsible for the toxic effects of cisplatin in haemostasis and in suppressing platelet functions (5, 11, 12). Therefore the aim of our study was to investigate in vitro by means of HPLC method the levels not only glutathione, but also cysteine (CSH) and cysteinylglycine (CGSH) in reduced and unreduced form after the action of platinum compounds on blood platelets and in the presence of antioxidant.

Protein thiols are also able to maintain the redox status of cells due to their cysteine content (14-16). Concentration of thiol groups in proteins is much greater than that of GSH. Protein -SH groups can be present as free thiols, disulfides and mixed disulfides when conjugated with glutathione, cysteine, homocysteine and c-glutamylcysteine (GSH is a dominant ligand). Oxidation and reduction of the thiols and disulfide form of protein is a dynamic, reversible process that occurs under physiological conditions in cells. The oxidation of protein thiols to mixed disulfides is an early cellular response to oxidative stress (16-19). There is a good evidence that free radicals and oxidative stress contributes to the pathology of cancers. Therefore we measured the level of protein thiols in platelets during oxidative stress caused by platinum compounds (cisPt and Se-Pt). In human diet there are many nutritive and non-nutritive compounds present which have antioxidative action and may have anti-cancer effects. Fruits, teas, vegetables and red wines are rich in antioxidants: ascorbate, tocopherols, different phenols including resveratrol. Resveratrol (trans-3,4',5-trihydroxystilbene) has multifunctional biological effects, including anti-platelet, anti-cancer and antioxidant action (20-29). Resveratrol is present naturally in grapes, fruits and in a variety of medicinal plants. In the present study, we investigated the influence of trans-resveratrol (the concentration used in our studies corresponds to the reference of physiological range of resveratrol in plasma) on changes of different thiols (GSH, CSH, CGSH) and protein thiol level induced by platinum compounds.


MATERIALS AND METHODS

Materials

Cisplatin and resveratrol were purchased from Sigma (St Louis, MO). Conjugate of selenium with cisplatin ((NH3)2Pt(SeO3)) synthetised in the Institute of Pur Chemicals, Lachema, Brno (Batch no 290592) was a gift obtained from Prof. V. Kleinwachter (Institute of Biophysics, Czech Academy of Sciences, Brno). Stock solutions of resveratrol was made in 50% dimethylsulfoxide (DMSO) at the concentration of 5 mg/ml and kept frozen. All other reagents were of analytical grade and were provided by commercial suppliers.

Isolation of blood platelets

Human blood was collected into ACD solution (citric acid/ citrate/dextrose; 5:1 v/v) and platelets were isolated by differential centrifugation of blood as described by Wachowicz and Kustroń (6). The final platelet concentration was about 4x108 platelets/ml. The platelets were counted by the photometric method according to Walkowiak et al. (30). The entire platelets washing procedure was performed in plastic tubes and carried out at room temperature. Washed human platelet suspensions in the modified Tyrode's Ca+2/Mg+2 free buffer (15 mM Tris-HCl, 140 mM NaCl, 10 mM glucose, pH 7.4), were incubated with resveratrol and/or platinum compounds (cisPt, Se-Pt) at 37°C for 30 minutes. To frozen control or tested compounds-treated platelets (1 ml of platelet suspension) 1 ml of protein precipitating solution was added (30% NaCl, 0.85% H3PO4, 0.2% EDTA). Acid-soluble (glutathione) and acid-insoluble (proteins) platelet fractions were separated according to Ando and Steiner (31, 32) and then the amount of free -SH groups in the both fractions was estimated by HPLC (33) or with 5,5'-dithio-bis(2-nitro-benzoic acid) (DTNB) (31, 32), respectively.

Determination of thiols

The classical technique HPLC has been used to analysis of thiols from human blood platelets treated with resveratrol and/or platinum compounds. HPLC analyse was performed with a Hewlett-Packard 1100 Series system according to Głowacki et al. (33).

Determination of free -SH groups with DTNB was performed in acid-insoluble (proteins) platelet fraction. To the pellet (the acid-precipitable fraction) 5 ml of H2O and 3 ml of 10% SDS were added. After solubilization, 0.5 ml of samples were taken and free -SH groups were determined (31, 32). A standard -SH curve was prepared for GSH.

The thiol values in this study were expressed as means ± SD. The statistically significant differences between means were assessed by applying the paired Student's t-test.


RESULTS

Using HPLC method we determined in human blood platelets the levels of different thiols: glutathione, cysteine and cysteinylglycine in both, reduced and unreduced forms. Incubation of human blood platelets with platinum compounds (cisPt and Se-Pt) resulted in a decreased concentration of thiols in both, acid-soluble (p<0.05) (Fig. 1-3) and acid-insoluble fraction (p<0.05) (Fig. 4). After 30 min incubation of blood platelets with cisPt (10 µg/ml) the amount of glutathione was decreased to about 1.6 ± nmol/ml (by 63.9 ± 5.7 %) (p<0.05) (Fig. 1), whereas in platelets treated by Se-Pt the decrease of glutathione was only by 38.8 ± 5.8 % (p<0.05) (Fig. 1). In the case of blood platelets incubated with cisPt the amounts of cysteine and cysteinylglycine in reduced form decreased by about 63.1 % and 90.2 %, respectively (Fig. 2, 3). The Se-Pt compound at the same concentration (10 µg/ml) depleted the level of glutathione only by about 50 % (p<0.05) (Fig. 2, 3). After incubation of blood platelets with platinum compounds (cisPt and Se-Pt) the amount of all tested thiols (glutathione, cysteine and cysteinylglycine) in unreduced form in acid-soluble fraction significantly increased (p<0.05) (Fig. 1-3). Platinum compounds changed also in blood platelets the GSH/GSSG ratio and ratios of other thiols (cysteine and cysteinylglycine) (Tab. 1). In the acid-insoluble platelet fraction (proteins) the level of free -SH groups after incubation of platelets with platinum compounds was also markedly reduced (p<0.05) (Fig. 4).

Fig. 1. The effects of resveratrol (25 µg/ml) and platinum compounds (cisPt and Se-Pt, 10 µg/ml) (30 min, 37°C) on the level of reduced and unreduced form of glutathione in platelet acid-soluble fraction (n=3, p<0.05, *p>0.05).

Fig. 2. The effects of resveratrol (25 µg/ml) and platinum compounds (cisPt and Se-Pt, 10 µg/ml) (30 min, 37°C) on the level of reduced and unreduced form of cysteine in platelet acid-soluble fraction (n=3, p<0.05, *p>0.05).

Fig. 3. The effects of resveratrol (25 µg/ml) and platinum compounds (cisPt and Se-Pt, 10 µg/ml) (30 min, 37°C) on the level of reduced and unreduced form of cysteinylglycine in platelet acid-soluble fraction (n=3, p<0.05, *p>0.05).

Fig. 4. The concentration of -SH groups in platelet acid-unsoluble fraction (protein) after incubation of platelets with resveratrol (25 µg/ml) and platinum compounds (cisPt and Se-Pt, 10 µg/ml) (30 min, 37°C) (n=3-6, p<0.05, *p>0.05).

Our studies demonstrate that resveratrol (after 30 min action) at the concentration of 25 µg/ml had no effects on the amounts of thiols in control platelet acid-soluble and in acid-insoluble fraction (Fig. 1-4), but in these cells resveratrol distinctly reduced the changes in redox state induced by platinum compounds (Fig. 1-4, Tab.1).

Table 1. Changes of the GSH/GSSG ratio and other thiols (cysteine and cysteinylglycine) in blood platelets treated with resveratrol (25 µg/ml) and platinum compounds (cisPt and Se-Pt; 10 µg/m, 30 min, 37°C) (n=3, p<0.05, *p>0.05).


DISCUSSION

Thiol homeostasis determines critical aspects of cell functions (16, 34-36). In cancer patients the decreased content of reduced thiol groups and reduced glutathione in plasma and red blood cells was described (37). The decrease in blood -SH group content observed in cancer patients may be the consequence of peroxidation and oxidation process induced by free radicals. In cancer, the observed decrease of thiols appears to be also consistent with the decreased levels of vitamin A and C in blood (37). There are not many data relating thiol metabolism in blood platelets. In blood platelet function thiol-disulfide reactions with redox control are required for platelet responses. Protein disulfide isomerase was found on the platelet surface, where it appears to play an important role in the platelet aggregation and secretion. This enzyme and thiols are also involved in platelet adhesion (38). Our earlier results suggest that the possible correlation between oxidative stress induced by platinum compounds and the level of thiols in platelet exists (4, 11). In blood platelets exposed to cisplatin, the GS-cisPt complex is formed as a major metabolite and is more active than cisPt alone (4, 5, 11, 12). In the present study, we observed that after incubation of platelets with cisplatin at the concentration of 10 µg/ml (the concentration used in our experiments is consistent with cisplatin plasma concentrations recorded in cancer patients) not only the level of GSH distinctly decreased (probably by forming GS-cisPt complex and by oxidation of GSH), but the amount of cysteine and cysteinylglycine in reduced form measured by HPLC was also significantly decreased; the level of these thiols in unreduced form was augmented (Fig. 1-3). Oxidative stress induced by platinum compounds in platelets seems to be associated with the oxidation of thiols to disulfides. In platelet protein fraction the level of free -SH groups, after incubation with cisPt or Se-Pt was distinctly decreased (Fig. 4). These results suggest that not only GSH, cysteine or cysteinylglycine but also platelet protein-bound thiols are highly unstable and may be oxidized under oxidative stress condition induced by platinum compounds.

There are many nutritive and non-nutritive compounds present in the diet which may affect platelet function in various ways, and may reduce the toxic effects of different drugs. Nowadays there is growing interest of compounds with antiplatelet and antioxidative activity. Among them poliphenols, vitamins and minerals could be considered as a tool in prevention of oxidative changes in platelets. Resveratrol is a phenolic antioxidant found in grapes and other food products (22). Our earlier observations showed that resveratrol inhibits different steps of blood platelet activation (platelet adhesion to collagen and fibrinogen, aggregation, secretion) and has antioxidative action on platelets (26-29). Moreover, resveratrol has anti-proliferative effect and has a wide range of potential targets; it inhibits ribonucleotide reductase, DNA polymerase, cyclooxygenase activities and cell cycle progression (23). Results of our present study suggest that resveratrol protects against changes in thiol metabolism in blood platelets induced by platinum compounds (Fig. 1-4). Arts et al. (39) demonstrated that tea polyphenols react with proteins and this interaction reduces the antioxidant capacity of flavonoids, but in our experiments the interaction of resveratrol with different thiols and -SH groups of proteins does not exist (Fig. 1-4). Kim et al. (40) showed that flawonoids of Inula Britannica reduced in neurons the diminution of GSH induced by receptor stimulation, but did not influence the synthesis of GSH. These flavonoids may facilitate GSH redox system in glutamate-injured cortical cells through preserving reductase GSSG and glutathione peroxidase. Van Acker et al. (41) demonstrated that a new synthetic flavonoid (3,7-disubstituted-2(3',4'-dihydroxyphenyl) is a potent protector against doxorubicin-induced cardiotoxicity.

Selenium (Se) as a component of glutathione peroxidases family is a part of the body's antioxidant defence mechanism. Organic and inorganic forms of Se are an important class of chemopreventive compounds. Our results showed that selenium is antioxidant with antiplatelet activity and has a protective effect against cisPt-induced inhibition of platelet function (9, 10, 42). Selenium-cisplatin conjugate has also less toxic effects on blood platelet functions in the relation to cisPt (9, 10). In the present study, we observed that in comparison with cisPt, Se-Pt compound in less extent changed the level of platelet thiols in both reduced and unreduced forms, and resveratrol has a protective effect on the action of platinum compounds (Fig. 1-4).

In conclusion, the observed behaviour of thiol groups in blood platelets shows that oxidation-reduction process may play an important role in biological function of these cells. Potential therapeutic strategies should be taken into account in the preventing of cellular thiols changes induced by some anti-cancer drugs (including platinum compounds). In agreement with this hypothesis, the compounds present in human diet (resveratrol and selenium compounds) may have a protective action and may minimise the toxicity and side effects of the chemotherapeutic agent without affecting antitumor activity. Here, we showed that in blood platelets resveratrol has the protective effects on the changes in different thiols (glutathione, cysteine and cysteinylglycine and thiol groups in proteins) induced by platinum compounds.

Acknowledgements: Supported by grant 505/448 from University of Lodz.

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R e c e i v e d : November 26, 2003
A c c e p t e d : May 7, 2004

Author’s address: Dr. B. Olas, Dept. General Biochemistry University of Lodz, 90-237 Lodz, Banacha 12/16, Poland, Tel: (+48) 6354484, Fax: (+48) 6354484
e-mail: olasb@biol.uni.lodz.pl