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Effect of Silybin on Phorbol Myristate Actetate-Induced Protein Kinase C Translocation, NADPH Oxidase Activity and Apoptosis in Human Neutrophils

Posted on: Thursday, 22 April 2004, 06:00 CDT

Summary

Mechanism of the action of silybin (1) and its derivatives (2- 4), possessing different lipid solubility in PMA-stimulated neutrophils was evaluated. Silybin (1) inhibited the calcium, phosphatidylserine- and diacylglycerol-dependent protein kinase C translocation and the NADPH oxidase activity in PMA-stimulated neutrophils and resulted in decreased apoptosis. Furthermore, silybin (1) inhibited xanthine oxidase activity and hem-mediated oxidative degradation of low-density lipoprotein, as well. Its derivatives (2-4), possessing different lipid-solubility, affected all the studied parameters. The lipid solubility of silybin (1) was enhanced by methylation (5'7'4'' trimethylsilybin: 2), whereas a decrease in lipid-solubility by acetylation of compound 2 (5',7,'4''- trimethylsilybin-acetate: 3) or all the hydroxyl groups of silybin (peracetyl-silybin: 4) attenuated the antioxidant capacity by decreasing the inhibition in PKC translocation and NADPH oxidase activation. All the derivatives of silybin (2-4) showed no inhibition in cell free systems; e.g. did not alter the xanthine oxidase activity and the hem-mediated oxidative degradation of LDL. In conclusion, the antioxidant activity of (1) might be due to its ability to inhibit PKC translocation and NADPH oxidase activation in PMA-stimulated neutrophils. The increase of lipid solubility of silybin (1) supports its penetration through cell membrane and enhances its inhibitory effects. This structural modification of (1) might have pharmacological consequences.

Key words: oxidative stress, silybin, antioxidants, protein kinase C, NADPH oxidase, apoptosis, neutrophil

Abbreviations: PKC - protein kinase C; NADPH - nicotinamide adenine dinucleotide phosphate; PMA - phorbol myristate acetate; Ca - calcium; PS - phosphatidylserine; DAG - diacylglycerol; HBSS - Hanks' balanced salt solution; PMSF - phenylmethylsulphonyl fluoride; EDTA - ethylenediaminetetraacetic acid; EGTA - ethylene glycolbis(2-aminoethylether)-N,N,N',N'-tetraacetic acid

Introduction

It is generally accepted that free radicals leading to oxidative stress play an important role in pathomechanism of various diseases such as atherosclerosis, alcoholic liver cirrhosis and cancer etc. (Freeman and Crapo, 1982; Maxwell and Lip, 1997; Halliwell and Gutteridge, 1999). The oxidative stress is initiated by reactive oxygen species (ROS) such as Superoxide anion (O2^sup -^), perhydroxyl (HOO*) and hydroxyl (HO*) radicals. Propagation cycle of lipid peroxidation is broken by either enzymatic inactivation of ROS or non-enzymatic reactions due to the intervention of free radical scavengers and antioxidants. Recently, several experiments have demonstrated anti-inflammatory, anticancer and antioxidant properties of naturally occurring flavonoids. For example, falvanolignans such as silymarin, isolated from the seeds of the violet-flowered varieties of Silybum marianum, possess anticancer (Zi et al. 1998; Ahmad et al. 1998; Agarwal, 2000) and radical scavenging activities (Gyorgy et al. 1992; Pietrangelo et al. 1995.) and suppress several processes and apoptosis (Manna et al. 1999; Singh et al. 2002.).

One source of ROS production in physiological circumstances is stimulated neutrophils which play a central role in host defence and are requited in vast numbers to sites of infections where they phagocytose and kill invading bacterial pathogens. Previously, it was demonstrated that silybin (1) inhibits Superoxid anion (O2^sup - ^) formation and H^sub 2^O^sub 2^ release in PMA-stimulated human neutrophils (Varga et al. 2001). Structural modification of silybin (1) by methylation and acetylation of its free hydroxy groups resulted in different lipid soluble derivativers (2-4). Methylated products (5,7,4''-trimethylsilybin: 2) with elevated lipid solubility enhanced the inhibitory capacity of silybin measured either in O2^sup -^ or H^sub 2^O^sub 2^ release in PMA-stimulated neutrophils (Varga et al. 2001).

Considering that the O2^sup -^ production induced by PMA involves the activation and translocation of protein kinase C family (PKCs) which resulted in the NADPH oxidase activation in neutrophils (Nixon and McPhail, 1999; Vancurova et al. 2001), the aims of present study were to examine the effects of 1-4 on PKC translocation [calcium (Ca^sup 2+^), diacylglycerol (DAG) and phosphatidylserine (PS) dependent PKCs] and NADPH oxidase activity in PMA-stimulated neutrophils. Furthermore, effects of structural modification of silybin (1) in oxidative processes induced by xanthine/xanthine oxidase (measured by uric acid and O2^sup -^ production) and hem- mediated oxidative degradation of LDL were also examined.

Materials and Methods

Materials

Silybin (1) was purchased from Sigma (St. Luis, Mo, USA). Its derivatives: 5,7,4''-trimethylsi!ybin (2), 5,7,4''-trimethylsilybin acetate (3), and peracetylsilybin (4) were prepared as published elsewhere (Varga et al. 2001). Structures of compounds arc presented on Fig. 1. Flavonolignan stock solutions and diluted working solutions were prepared in DMSO. From diluted working solutions, 5 l was added to the cell suspension to achieve the required concentrations. In all experiments, the same volume of DMSO was added to the control cells.

Neutrophils were separated from heparinized blood of healthy volunteers by gradient centrifugation on Histopaque 1077 (Sigma Co., St. Luis, MO, USA) according to the method of Boyum (Boyum, 1968). A neutrophil purity over 95% and cell viability greater than 95% were microscopically ascertained by Giemsa staining and trypan blue exclusion, respectively.

PKC translocation was determined in the cytosol of the PMA- activated neutrophils using a non-radioactive Elisa kit from Calbiochem (Calbiochem-Novabiochem Co. St Diego, CA, USA). Cell free cytosol was prepared from neutrophils (5 10^sup 6^ cells/ml) preincubated with silybin (1) and its analogues (2-4) (concentration range of 2-50 M) for 15 min at 37 C in HBSS. Afterwards, neutrophils were stimulated with phorbol myristate acetate (PMA at 10^sup -7^ M, Sigma, St. Luis, Mo. USA) for 3 min at 37 C. The reaction was stopped by 10 volume of ice-cold phosphate buffer. After centrifugation, cells were resuspended in the sample preparation buffer (part of the kit containing 50 mM Tris.HCl, 50 mM [beta]- mercaptoethanol, 10 mM EGTA, 5 mM EDTA, 1 mM PMSF, 10 mM benzamidine, pH = 7.5) and were disrupted. The cell lysate was centrifugea (4 C, for 1 h) and the supernatant was used immediately in the kit. PKC activity was determined in the presence of calcium, phosphatidylserine and ATP (final concentrations in reaction mixture: 25 mM Tris.HCI (pH = 7.0), 0.3 mM MgCl^sub 2^, 0.1 mM ATP, 2 mM CaCl^sub 2^, 50 g/ml phosphatidylserine, 0.5 mM EDTA, 1 mM EGTA, 5 mM [beta]-mercaptoethanol). all experiments were performed in duplicates in neutrophils of three different donors. Protein content of cytosol fraction was determined by the method of Lowry (Lowry et al. 1951).

NADPH oxidase activity was determined in PMA-stimulated neutrophil lysate (Jones and Hancock, 1994). Cell suspension in HBSS (1 10^sup 7^ cells/ml) was stimulated with PMA (10^sup -7^ M) for 3 min at 37 C and cells were disrupted and were kept on ice. NADPH oxidase activity was determined by measuring reduction rate of Cytochrome C (Sigma, St. Luis, CA, USA) at 550 nm after addition of NADPH (Sigma, St. Luis, CA, USA). Absorbance at 542 nm was used for baseline correction. Flavonolignans were added to cell lysate at final concentrations of 5-100 M and incubated for 10 min at room temperature before addition of NADPH. Changes in the absorbance of Cytochrome C were recorded for 5 min at room temperature. Superoxide dismutase was added to determine the specificity of the reaction. In separate experiments, proteolytic enzyme inhibitors (final concentrations: 1 mM PMSF, 5 mM [beta]-mercaptoethanol, 10 mM benzamidine) were added to the reaction buffer prior to the addition of flavonolignans and NADPH. all experiments were performed in triplicates. Results are expressed as mean SD of three independent experiments.

Fig. 1. Structure of silybin and its derivatives [(1): silybin; (2): 5,7,4''-trimethylsilybin; (3): 5,7,4''-trimethylsilybin- acetate; (4): peracetylsilybin].

Apoptosis PMA-stimulated cell suspension was incubated in the presence and absence of flavonolignans for 4 h at 37 C. At the end of incubation, acridine orange (10 g/ml) was added (5 min incubation) and analysed by fluorescence microscopy. Cells with fragmented nuclei and/or condensed chromatin were scored as apoptotic (Durrieu et al. 1999). At least 100 cells were counted for each sample. Data show results of three independent experiments.

Xanthine oxidase activity was determined in 50 mM phosphate buffer (pH = 7.8) using xanthine as substrate (at a final concentration of 60 M) and xanthine oxidase as was described by Sheu et al. (Sheu et al. 1998) in the presence and absence of flavonolignans (concentrations range of 5-200 M). Formation of uric acid was followed at 295 nm with a spectrophotometer (Hewlet Packard type: 8453, USA). In another sets of experiments, Cytochrome C was also added to the reaction mixture, and reduction of Cytochrome C was followed as described above. all experiments were performed in triplica\tes. Results are expressed as mean SD of three independent experiments.

Hem-mediated oxidative degradation of LDL was measured as described previously (Ujhelyi et al. 1998). Namely, LDL was separated from the plasma of healthy donors by gradient centrifugation using 1210 g/l KBr and single spin ultracentrifugation (50.2 Ti rotor, Beckman Instruments, 300000 g, 4 C, 3 hours. Silybin (1) and its structural analogues (2-4) were added to diluted LDL samples (200 mg/1) at final concentration range of 5-50 M. Samples were preincubated for 15 min at room temperature. Oxidative degradation of LDL was induced by the addition of hem (5 mol/l) and hydrogen-peroxide (150 mol/l) at 37 C in HEPES buffer (10 mmol/1, pH 7.4). Hem degradation which is inversed with the conjugated dien formation (e.g. lipid peroxidation) was monitored at 405 nm with a computer-assisted ELISA reader. The oxidative resistance of LDL was characterized by delta T at V^sub max^ in second, the time period until the maximal velocity of hem disappearance in the propagation phase (Ujhelyi et al. 1998). all determinations were performed in triplicates. Results are expressed as mean SD of three independent experiments.

Spectral analysis

Neutrophils (10^sup 7^ cells/ml) were preincubated with 50 M of flavonolignans (for 15 min at 37 C, in HBSS). Cells were lysed by addition of Triton X 100 (Sigma, St Luis, CA, USA) and after centrifugation (2000 rpm/min, 20 min, 4 C), spectra of cell lysates were recorded between 260-400 nm using an UV-VIS spectrophotometer (Hewlet Packard, type: 8453, USA).

Statistical analysis

ANOVA statistical program was used. The means SD of the results were compared using paired or non paired student t test. Statistical significance was determined by p < 0.05.

Results

Silybin (1) and its structural analogues (2-4) inhibited the translocation of the Ca^sup 2+^, DAG, and PS dependent PKC(s) in PMA- stimulated neutrophils from cytosol to membrane fraction almost regardless the concentration (Fig. 2). 5,7,4''-trimethylsilybin (2) was the most effective in the inhibition of PKC translocation. Total inhibition of PKC(s) translocation was achieved at 10 M concentration of silybin (1) and 7.5 M concentration of 5,7,4''- trimethylsilybin (2). However, acetylated analogues of silybin (3 and 4), were not so effective even at concentrations of 50 M. None of the tested molecules affected the Ca^sup 2+^-independent PKC (e.g. PKC-delta) activity (data not shown).

Silybin (1) and its derivatives (2-4) inhibited the NADPH oxidase activity in PMA-stimulated cell lysate by a concentration dependent manner (Fig. 3a). The most distinct inhibition was observed when cell lysate was preincubated with 5,7,4''-trimethylsilybin (2) possessing high lipid solubility. 50% inhibition of NADPH oxidase activity was achieved when using 5 M of 5,7,4''-trimethylsilybin (2) and 10 M of silybin (1) at final concentrations. However, when the reaction mixture was supplemented with enzyme inhibitors, silybin derivatives (2, 3, and 4) lost totally their inhibitory effects on PMA-stimulated NADPH oxidase activation (Fig. 3b).

These results suggest that after penetration into the cells, silybin derivatives (2-4) underwent demethylation and/or deacetylation to result in the formation of silybin (1) itself. After preincubation with 1-4 the UV-spectral analysis of neutrophils confirmed this hypothesis. Thus, the characteristic UV maximum of silybin (1) at 330 nm could be detected in each cases as follows: (2): 120%, (3): 81% and (4): 74% (expressed as % of absorbance measured in cells preincubated with the same concentration of silybin). These results confirmed it is the silybin (1) itself that is responsible for the antioxidant activity.

Fig. 2. Concentration-dependent effect of silybin (1) and its derivatives (2-4) on calcium, phosphatidylserine- and diacylglycerol- dependent PKC translocation in PMA-stimulated human neutrophils.

Silybin (1) and its methylated analogue, 5,7,4''- trimethylsilybin (2), significantly inhibited the PMA-induced apoptosis in healthy human neutrophils (Fig. 4). However, acetylated derivatives of silybin (3) or (4) did not modify the PMA-induced apoptosis in neutrophils similarly to other measured parameters.

When O2^sup -^ production was induced in xanthine/xanthine oxidase system, only silybin (1) inhibited uric acid formation (Fig. 5a) with an IC^sub 50^ of 32.2 M. Silybin (1) affected both v^sub max^ (1.231 mol/min vs. 1.465 mol/min) and K^sub M^ (60.4 M vs. 9.3 M) as assessed by Lineweaver-Burk plots suggesting a competitive- non-competitive mechanism. Neither methylated (2) nor acetylated derivatives (3, 4) affected uric acid formation at any concentrations tested. In a parallel experiment, Cytochrome C was also added to the reaction mixture, and both uric acid and Cytochrome C reduction were followed (Fig. 5b). It was found that inhibition in uric acid formation occurred parallel with inhibition of Cytochrome C reduction (e.g. with O2^sup -^ formation) suggesting that the observed effects are due to the inhibition of xanthine oxidase activity rather than the scavenging of O2^sup -^ radicals.

Fig. 3. Concentration dependent effect of silybin (1) and its derivatives (2-4) on NADPH oxidase activity in PMA-stimulated cell lysate. A: cell lysate without enzyme inhibitors. B: cell lysate containing enzyme inhibitors and flavonolignans at 25 M concentrations (details in Material and Methods).

Similar results were found when the effects of silybin (1) and its derivatives (2, 3, and 4) were studied on the hem-mediated oxidative degradation of the LDL. A dramatic increase in oxidative resistance of LDL was observed when LDL was preincubated with silybin (1) which was not found for its derivatives (2, 3, and 4) (Fig. 6).

Fig. 4. Effect of silybin (1) and its derivatives (2-4) on the apoptotic cell number in PMA-stimulated human neutrophils.

Fig. 5. Effects of silybin (1) and its derivatives (2-4) on xanthine oxidase activity A: Concentration dependent effects on uric acid formation in xanthine/xanthine oxidase system. B: Formation of uric acid and reduction of Cytochrome C in the presence of silybin (1) at 10 M concentration.

Fig. 6. Effect of silybin (1) and its derivatives (2-4) on oxidative resistance of human low-density lipoprotein.

Discussion

Inhibitory effects of flavonolignans, flavonoids and flavonols on Superoxide generation in neutrophils induced by chemotactic peptide or phorbol esters have been demonstrated (Lu et al. 2001; Selloum et al. 2001; Varga et al. 2001). However, the mechanism of the inhibitory effect of those molecules was not clear.

We demonstrated that the inhibitory effect of silybin (1) on O2^sup -^ production in PMA-stimulated human neutrophils is due to the inhibition of the translocation of Ca^sup 2+^, DAG, and PS- dependent PKC(s) from cytosol to the membrane fraction. Parallel to the inhibition of PKCs translocation, a decrease in NADPH oxidase activity, which is responsible for O2^sup -^ production, was observed. We also demonstrated that the increase in the lipid solubility of silybin (1) enhanced the inhibition of PKC(s) translocation and NADPH oxidase activity and resulted in a more effective inhibition in all the studied parameters. In contrast, a decrease of its lipid solubility (1[arrow right]3 or 1[arrow right]4) attenuated all inhibitory effect in PMA-stimulated neutrophils (e.g. inhibition of PKC translocation, NADPH oxidase activity). Furthermore, all silybin derivatives (2-4) totally lost their inhibitory effects on the PMA-stimulated NADPH oxidase activation when the reaction mixture of cell lysate was supplemented with enzyme inhibitors. On the basis of these results, it might be supposed that the silybin derivatives (2-4) underwent a demethylation and/or deacetylation to result in silybin (1) after entering into the cell or counteracting with non-specific esterases in cell lysate. The UV analysis of neutrophil lysate confirmed this hypothesis. Thus, the characteristic absorbance maximum of silybin (1) at 330 nm could be detected in all cases. These results confirm that it is the silybin (1) itself that is responsible for the antioxidant activity and an increase in its lipid solubility supports its penetration through cell membranes. It has to be noted, that in inhibition of O2^sup -^ and H^sub 2^O^sub 2^ production by PMA stimulated neutrophils, peracethyl sylibin (4) proved to be more efficient (Varga et al. 2001) than in inhibition of enzymes (PKC and NADPH oxidase) responsible for formation of O2^sup -^ production. To explore this contradiction more experiment has to be performed.

Nevertheless, various preventive antioxidant agents inhibit PKC- dependent cellular responses and apoptosis (Forrest et al. 1994; Vancurova et al. 2001), and hence PKC is a logical candidate for redox modification by oxidants and antioxidants that may to some extent determine their cancer-promoting, anticancer and antioxidant activities (Gopalakrishna and Jaken, 2000), therapeutic strategies to resolve chronic diseases could usefully target neutrophil apoptosis or PKC signalling pathways (Nixon and McPhail, 1999; Webb et al. 2000).

Neutrophils acts as a first line in defending against bacterial infection and accelerated apoptosis of neutrophils may represent a mechanism for increased incidence of certain clinical diseases. Consequently, any compounds which arrest or delay apoptosis of neutrophils as demonstrated for silybin (1) and its highly lipid soluble derivative (2) would provide more functional neutrophils in these diseases.

Dietary polyphenolic flavonoids and isoflavones are being studied extensively as cancer-preventive and antioxidant agents. Very recently, it was demonstrated that a series of plant extracts used in the Mediterranean and Chinese medicine (Schinella et al. 2002) and silymarin, an extract of Silybum marianum seeds containing silybin (1), inhibite\d xanthine oxidase activity (Sheu et al. 1998). It was shown that it was only the silybin (1) that inhibited uric acid formation in xanthine/xanthine oxidase system with an IC50 of 32.2 M and prevented LDL from hem-mediated oxidative degradation. Silymarin, as a mixture of flavonolignans containing silybin (1), had IC^sub 50^ of 27.58 M for inhibition of xanthine oxidase (Sheu et al. 1998). Considering these observations, one can conclude that silybin (1) is only partially responsible for silymarin induced xanthine oxidase inhibition and other constituents such as silydianin and silychristine might have also such an effect. Finally, inhibition of O2^sup -^ anion production induced by xanthine oxidase paralleled to the inhibition of the enzyme activity (measured by uric acid formation) suggest that the decreased O2^sup - ^ formation might be due to the inhibition of xanthine oxidase activity rather than the scavenge O2^sup -^ radical.

Finally, silybin (1) enhanced the oxidative resistance of LDL. A dramatic increase in the oxidative resistance was observed when LDL was preincubated with silybin (1). The effective concentration of silybin (1) was 25 M which is in good agreement with other findings. Recently, it was published that silymarin (50-200 mol/l) prolonged the lag times of both LDL autooxidation and oxidation by copper with >50%, as assessed recording of diene formation (Locher et al. 1998). Our results suggest that silybin (1) is also a very powerful agent in prevention of LDL oxidation induced by physiological activators (such as hem).

In summary, we investigated the effect of silybin (1) on cell signalling in PMA-stimulated human neutrophils. It was demonstrated that silybin (1) inhibited the translocation of Ca^sup 2+^, PS and DAG-dependent PKC from cytosol to membrane fraction upon PMA- stimulation in the function of its concentration. The membrane- bound NADPH-oxidase activity was also suppressed in the PMA- stimulated cell lysate. Structural modification of silybin (1) resulted in different lipid-soluble molecules affected all the studied parameters: 5,7,4'-trimethylsilybin (2), possessing enhanced lipid-solubility showed improved activity compared to silybin (1), whereas the decreased lipid-solubility of silybin analogues (3 and 4) attenuated theirs inhibitory effects on those enzymes. Inhibition of PKC(s) translocation and NADPH oxidase activity resulted in a decreased apoptosis in the PMA-stimulated human neutrophils. Finally, it was also demonstrated that silybin (1) inhibited xanthine oxidase activity and increased the oxidative resistance of LDL induced by hem. Therefore, silybin (1) may serve as a novel tool in the prevention and therapy of atherosclerosis and other diseases representing enhanced oxidative stress and its increased lipid solubility might support transport of silybin through cell membrane and improves its antioxidant capacity.

Acknowledgement

Research work was sponsored by National Scientific Research Foundation of Hungarian Academy of Sciences (Grant number: OTKA T 29090). The authors thanks to Ms. Gyongyi Sallai for her technical assistance.

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Zs. Varga1, L. Ujhelyi1, A. Kiss1, J. Balla1, A. Czompa2, and S. Antus2

1 Ist Department of Medicine, Medical and Health Science Centre,

2 Department of Organic Chemistry, University of Debrecen, Debrecen, Hungary

Address

Zsuzsa Varga PhD, CSc, First Department of Medicine, Medical and Health Science Center, P. O. Box 19, H-4012 Debrecen, Hungary

Tel: (36)-52-411-600/5833;Fax: (36)-52-414-951; e-mail: vargazs@ibel.dote.hu

Copyright Urban & Fischer Verlag Feb 2004

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