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 ((NH
3)
2Pt(SeO
3);
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 ((NH
3)
2Pt(SeO
3))
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 4x10
8 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% H
3PO
4,
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 H
2O
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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