Research Article The Natural Stilbenoid Piceatannol

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2013 Article ID 136539 8 pageshttpdxdoiorg1011552013136539

Research ArticleThe Natural Stilbenoid Piceatannol Decreases Activity andAccelerates Apoptosis of Human Neutrophils Involvement ofProtein Kinase C

Viera Jancinova1 Tomas Perecko1 Radomir Nosal1

Klara Svitekova2 and Katarina Drabikova1

1 Institute of Experimental Pharmacology and Toxicology Slovak Academy of Sciences Dubravska cesta 9 841 04 Bratislava Slovakia2National Transfusion Service Limbova 3 831 01 Bratislava Slovakia

Correspondence should be addressed to Viera Jancinova vierajancinovasavbask

Received 19 July 2013 Accepted 11 September 2013

Academic Editor Tullia Maraldi

Copyright copy 2013 Viera Jancinova et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Neutrophils are able to release cytotoxic substances and inflammatory mediators which along with their delayed apoptosis havea potential to maintain permanent inflammation Therefore treatment of diseases associated with chronic inflammation shouldbe focused on neutrophils formation of reactive oxygen species and apoptosis of these cells represent two promising targets forpharmacological intervention Piceatannol a naturally occurring stilbenoid has the ability to reduce the toxic action of neutrophilsThis substance decreased the amount of oxidants produced by neutrophils both extra- and intracellularly Radicals formed withinneutrophils (fulfilling a regulatory role) were reduced to a lesser extent than extracellular oxidants potentially dangerous for hosttissues Moreover piceatannol did not affect the phosphorylation of p40phoxmdasha component of NADPH oxidase responsible forthe assembly of functional oxidase in intracellular (granular) membranes The stilbenoid tested elevated the percentage of earlyapoptotic neutrophils inhibited the activity of protein kinase C (PKC)mdashthe main regulatory enzyme in neutrophils and reducedphosphorylation of PKC isoforms 120572 120573II and 120575 on their catalytic region The results indicated that piceatannol may be useful as acomplementary medicine in states associated with persisting neutrophil activation and with oxidative damage of tissues

1 Introduction

Piceatannol (trans-343101584051015840-tetrahydroxystilbene Figure 1) isa naturally occurring hydroxylated analogue of resveratroldisplaying remarkable antioxidative anticancer and anti-inflammatory properties The latter activity has been attrib-uted to the capacity of piceatannol to prevent activation oftranscription factors mRNA expression and production ofinflammatory mediators for example nitric oxide prostagl-andin E

2 interleukins monocyte chemotactic protein MCP-

1 cyclooxygenase-2 and tumour necrosis factor 120572 [1ndash4]Piceatannol as a potent spleen tyrosine kinase (Syk) inhibitorhas a great potential to suppress allergic and autoimmunedisorders by blocking immune receptor signalling in a varietyof inflammatory cells including neutrophils [5ndash9]

Neutrophils (neutrophilic polymorphonuclear leuko-cytes) represent the bodyrsquos primary line of defense against

invading pathogens Nevertheless these cells are able torelease cytotoxic substances and inflammatory mediatorswhich along with their delayed apoptosis have a potentialto maintain permanent inflammation [10 11] Thereforetreatment of diseases associated with chronic inflammationshould be focused onneutrophil functions formation of reac-tive oxygen species (ROS) and apoptosis of these cells repre-sent two promising targets for pharmacological intervention

Formation of ROS is initiated by the activation of phago-cyte NADPH oxidase (NOX2gp91phox) the first identifiedand the best studied member of the NOX family Duringactivation the cytosolic oxidase subunits p47phox p67phoxp40phox and Rac2 translocate to the plasma membraneand associate with the membrane-bound cytochrome b

558

complex Cytochrome b558

is formed by two subunitsmdashgp91phox (also known as NOX2) and by p22phox and this

2 Oxidative Medicine and Cellular Longevity

OH

OH

OH

HO

OH

OH

HO

trans-ResveratrolPiceatannol

Figure 1 Piceatannol (trans-343101584051015840-tetrahydroxystilbene) and its related compound trans-resveratrol (trans-43101584051015840-trihydroxystilbene)

heterodimer transfers electrons from NADPH to molecularoxygen Flavin adenine dinucleotide (FAD) and two hemegroups serve as the redox pathway which enables the transferof electrons across the membrane [12ndash14] If the assembly ofcatalytically active oxidase occurs on the plasma membranethe generated oxidants are liberated extracellularly or intophagosomesThese radicals are involved in the elimination ofpathogens however their overproductionmay result in tissuedamage ROS produced intracellularlymdashon membranes ofspecific granules participate in the initiation of neutrophilapoptosis [15 16] and they are considered key suppressorsof inflammation [17 18] Since the optimum therapy isexpected to minimise tissue damage without reduction ofthe physiological function of neutrophils separate analysis ofextra- and intracellular effects of antioxidants is of particularimportance

The production of ROS is limited by the programmeddeath of neutrophils Apoptosis represents a precisely reg-ulated process which includes release of proapoptotic pro-teins into the cytosol gradual activation of caspases DNAfragmentation chromatin condensation loss of membraneasymmetry and formation of apoptotic bodies Alterations inplasma membrane (eg externalisation of phosphatidylser-ine) facilitate the recognition and clearance of apoptoticneutrophils by macrophages resulting in safe removal ofthese cells from the site of inflammation Since the delayedapoptosis and impaired clearance of neutrophils aggravateand prolong tissue injury pharmacological interventionfocused on neutrophil apoptosis is studied as an originalapproach for the design of new anti-inflammatory strategies[16 19 20]

Modulation of protein kinase C activity represents aprospective method to regulate neutrophil functions Im-munochemical studies have shown that human neutrophilsexpress five PKC isoforms 120572 120573I 120573II 120575 and 120577 which partici-pate in NADPH oxidase activation as well as in proapoptoticand antiapoptotic signalling [21ndash25]

The present paper investigated the impact of piceatannolon the viability and oxidative burst of human neutrophilsWe analysed separately the effects of this stilbenoid on theconcentration of ROS produced by neutrophils extra- andintracellularly Protein kinase C activity was examined as anassumed target of piceatannol action and the phosphoryla-tion of PKC120572 PKC120573II and PKC120575 (the most abundant PKCisoforms in neutrophils) was assessed

2 Material and Methods

21 Chemicals Piceatannol was purchased from AcrosOrganics (Geel Belgium) Luminol isoluminol PMA (4120573-phorbol-12120573-myristate-13120572-acetate) Ca2+-ionophoreA23187superoxide dismutase dextran (average MW 464 000 kDa)zymosan (zymosan A from Saccharomyces cerevisiae) lu-ciferase from Photinus pyralis and D-luciferin sodium saltwere from Sigma-Aldrich Chemie (Deisenhofen Germany)HRP (horseradish peroxidase) and catalase were obtainedfrom Merck (Darmstadt Germany) and lymphoprep (den-sity 1077 gmL) was purchased from Nycomed PharmaAS (Oslo Norway) Propidium iodide and rh Annexin V-FITC (produced in E coli and conjugated with fluores-cein isothiocyanatemdashFITC) was received from eBioscience(Vienna Austria) and PKC kinase activity kit was from EnzoLife Sciences AG (Lausen Switzerland) Phosphospecificantibodies versus PKC isoforms and versus p40phox wereobtained from Cell Signaling Technology (Danvers MAUSA) Secondary anti-rabbit antibody and Lumigen Detec-tion Reagent were supplied by GE Healthcare Life Sciences(Little Chalfont UK)

This work was approved by the Local Ethic CommitteeInstitute of Experimental Pharmacology and ToxicologySlovak Academy of Sciences Bratislava

22 Blood Collection and Isolation of Human NeutrophilsFresh blood was obtained at the blood bank by venipuncturefromhealthymale donors (20ndash50 years)whohadnot receivedany medication for at least 7 days The samples were mixedwith 38 trisodium citrate in the ratio of 9mL of bloodto 1mL citrate Erythrocytes were allowed to sediment in1 dextran solution (1 timesg 25min 22∘C) and the suspensionof leukocytes and platelets in plasma (buffy coat) was usedfor flow cytometric analyses or for neutrophil isolationFor neutrophil isolation the buffy coat was centrifugedcells were resuspeded in phosphate-buffered saline andneutrophils were separated on Lymphoprep (500 timesg 30min22∘C) The contaminating erythrocytes were removed withhypotonic cold haemolysis and neutrophils were washedwithphosphate-buffered saline Neutrophil count was assessed byCoulter Counter (Coulter Electronics High Wycombe Eng-land) and adjusted to a final concentration of 104 cells1 120583LThe final suspension of neutrophils containedmore than 96

Oxidative Medicine and Cellular Longevity 3

of viable cells as evaluated by trypan blue exclusion and wasused maximally for 2 hmdashas long as control chemilumines-cence kept constant

23 Formation of ROS in Neutrophils Oxidative burst ofneutrophils was recorded on the basis of enhanced chemi-luminescence [26 27] in a microtitre plate computer-drivenluminometer LM-01T (Beckman Coulter Prague CzechRepublic) Chemiluminescence of human whole blood (250xdiluted) enhanced with luminol (250 120583molL) was stimu-lated with phorbol myristate acetate (PMA 005120583molL)opsonized zymosan (05mgmL) or Ca2+-ionophore A23187(1 120583molL) Chemiluminescence of isolated human neu-trophils (5times 105sample)was initiated by PMA(005120583molL)Oxidants released extracellularly were determined in thesystem containing isoluminol (5 120583molL) andHRP (8UmL)HRP was added to the system ensuring sufficient extra-cellular peroxidase concentration Intracellular chemilumi-nescence was enhanced with luminol (5120583molL) in thepresence of the extracellular scavengers superoxide dismutase(100 UmL) and catalase (2 000UmL) Concentration ofoxidants was evaluated on the basis of integral values ofchemiluminescence over 1 800 s (isolated neutrophils andA23187 stimulated whole blood) and over 3 600 s (wholeblood chemiluminescence initiated with PMA or zymosan)

24 Chemiluminescence of Cell-Free System The antioxida-tive activity of piceatannol was measured in cell-free systemcontaining piceatannol (001ndash100 120583molL) HRP (2UmL)luminol (10 120583molL) and hydrogen peroxide (100 120583molL)in 50 120583L aliquots Chemiluminescence was determined for10min at 37∘C [28]

25 Activity of Protein Kinase C Isolated neutrophils (5 times105 cells) were incubated with piceatannol (1ndash100120583molL) for30min at 37∘C and stimulated with PMA (015 120583molL) for3min Stimulation was stopped by the addition of a tenfoldvolume of ice-cold phosphate buffer After centrifugationneutrophils were broken by lysing solution and by sonicationcentrifuged and the supernatant was used for the determi-nation of protein kinase C activity using an enzyme-linkedimmunosorbent assay kit

26 Phosphorylation of p40phox and PKC120572 120573II 120575 Westernblot analysis was performed as previously described [29 30]Isolated human neutrophils (5 times 106 cells) were incubated at37∘C with piceatannol (10 or 100 120583molL 1min) stimulatedwith PMA (015120583molL 1min) and lysed by sonicationin a lysing solution containing protease and phosphataseinhibitors Debris from the lysed cells was pelleted by cen-trifugation and the supernatant was taken for blotting assayProtein concentration was measured using Bradford DyeReagent detection kit (Bio-RadHercules CAUSA) Proteins(20120583g per lane) were separated by SDS polyacrylamide gelelectrophoresis and transferred to Immobilon-P TransferMembrane From each membrane the area between 60 and100 kDa was detected with primary anti-phospho-PKC120572120573II(Thr638641) antibody (1 5 000) and with the secondary

antibody conjugated to HRP (1 5 000) Phosphorylated PKCisoforms 120572 and 120573II were visualised with Lumigen Detec-tion Reagent kit scanned and quantified densitometricallyusing ImageJ programme The membrane was then strippedusing stripping buffer (Re-blot Plus Mild Solution MilliporeTemecula CAUSA) and reprobedwith anti-phospho-PKC120575(Thr505) antibody (1 1 000) The membrane area between30ndash60 kDa was detected with regard to the presence of theinternal standard 120573-actin (120573-actin antibody 1 4 000) andthe phosphorylated subunit ofNADPHoxidase p40phox (anti-phospho- p40phox (Thr154) antibody 1 5 000)

27 Neutrophil Integrity Damaging effect of piceatannolon the integrity of plasma membranes was evaluated onthe basis of ATP liberation measured by the luciferin-luciferase chemiluminescence method [31] Suspension ofisolated neutrophils (3 times 104 cells) was incubated withpiceatannol (1ndash100 120583molL) for 15min at 37∘C Then themixture of luciferin (16 120583g) and luciferase (45 000 U) wasadded and chemiluminescence was recorded for 60 secondsChemiluminescence of ATP standards (1ndash500 nmolL) wasmeasured in each experiment and concentrations of ATPin samples were calculated from the calibration curve TotalATP content was assessed immediately after sonication ofneutrophils for 10 seconds

28 Analysis of Apoptosis Human plasma buffy coat (seeSection 22) was incubated with piceatannol (1ndash100 120583molL)for 10min at 37∘C The cells were double-stained with an-nexin-V conjugated with FITC (in dark at 4∘C for 10min) andwith propidium iodide and analysed by cytometer CytomicsFC 500 (Beckman Coulter Inc Brea CA USA) From thegranulocyte area 5 000 cells were gated and the percentage ofapoptotic (annexin positive and propidium iodide negative)dead (double positive) and viable cells (double negative) wasdetermined as described previously [32 33]

29 Data Analysis All values were given as the mean plusmn SEMand the statistical significance of differences between meanswas established by Studentrsquos 119905-test 119875 values below 005 wereconsidered to be statistically significant and were indicated inthe figures by lowast119875 le 005 and lowastlowast119875 le 001

3 Results

Piceatannol reduced the oxidative burst of human neu-trophils measured in whole blood (Table 1) It inhibitedchemiluminescence initiated by the stimulation of proteinkinase C increased calcium concentration and the activationof membrane receptors at the respective mean effective con-centrations of 065 plusmn 007 120583molL (PMA) 271 plusmn 041 120583molL(A23187) and 943 plusmn 053 120583molL (zymosan)

In isolated neutrophils stimulated with PMA extra-and intracellular chemiluminescencewas recorded separately(Table 2) Piceatannol decreased both the extracellular andintracellular chemiluminescence of neutrophils at the respec-tive mean effective concentrations 187 plusmn 035 120583molL and


Table 1 Dose-dependent inhibition of neutrophil oxidative burstin the presence of piceatannol Chemiluminescence measuredin whole blood was initiated with PMA (005120583molL) Ca2+-ionophore A23187 (1120583molL) or opsonized zymosan (05 gL) Theincubation of neutrophils with piceatannol was 60min (PMAzymosan) or 30min (A23187) depending on the kinetics of oxida-tive burst Mean plusmn SEM 119899 = 8 lowast119875 le 005 lowastlowast119875 le 001 versus Con-trol

Piceatannol(120583molL)

Inhibition of chemiluminescence ( of control)PMA A23187 Zymosan

001 9321 plusmn 264lowast9674 plusmn 141 9810 plusmn 110

01 8445 plusmn 199lowastlowast9164 plusmn 184

lowastlowast9863 plusmn 142



lowast



lowastlowast



lowastlowast

Table 2 Effect of piceatannol on extra- and intracellular chemi-luminescence of isolated human neutrophils stimulated with PMA(005 120583molL) and on the chemiluminescence produced by cell-free system The exposure to piceatannol was 30min (extra-intracellular) or 10min (cell-free system) Mean plusmn SEM 119899 = 3ndash8lowast119875 le 005 lowastlowast119875 le 001 versus Control


Inhibition of chemiluminescence ( of control)Extracellular Intracellular Cell-free system

001 9689 plusmn 405 10172 plusmn 434 9943 plusmn 119

01 9073 plusmn 580 10240 plusmn 216 9377 plusmn 055lowast


lowastlowast



lowastlowast



lowastlowast

1259plusmn096 120583molL in cell-free system the EC50of piceatan-

nol was 063 plusmn 001 120583molL The phosphorylation of p40phox(a component of NADPH oxidase essential for intracellularoxidant formation) was increased more than three timesafter PMA stimulation This increase was not modified bythe treatment of neutrophils with piceatannol (Figure 2)Considering the high efficiency of piceatannol in neutrophilsstimulated with PMA and its recorded intracellular activityin further experiments the effect of this phytochemical wasevaluated on PKC activity (Figure 3) The stimulation ofneutrophils with PMA increased protein kinase C activityby 50 piceatannol dose-dependently reduced this riseuntil the values of activity were comparable with thoseproduced by resting cells The phosphorylation of proteinkinases C 120572 120573II and 120575 (the most abundant PKC isoforms inneutrophils) was also decreased after piceatannol treatment(Figure 4) Phosphorylation of PKC120572 and 120573II was reducedin the presence of both concentrations used whereas inthe case of PKC120575 phosphorylation only 10 120583molL piceatan-nol was effective The observed inhibitory effects were notassociated with neutrophil damage as in the presence ofpiceatannol no increase in extracellular ATP concentrationwas recorded (Figure 5) Spontaneous ATP liberation fromisolated neutrophils was minimal approximately 5 of thetotal ATP content This amount remained unchanged or

0

100

200

300

400

500

PMA

PIC 10 100

( o

f con

trol)

Phos

phor

ylat

ion

ofp40phox

p40phox

120573-actin

minus

minus minus

+ + +

lowastlowast

Figure 2 Phosphorylation of p40phox in PMA-stimulated neu-trophils treatedwith 10 and 100120583molL piceatannol PhosphorylatedNADPH oxidase subunit was isolated by Western blotting anddetected with anti-phospho- p40phox (Thr154) antibody The valuesare presented as percentage of resting control Control value givenas optical density of p40phox band corrected to 120573-actin content was3813 plusmn 1067 Mean plusmn SEM 119899 = 6 lowastlowast119875 le 001

0

50

100

150

200

Control PMA 1 10 100

Prot

ein

kina

se C

activ

ity(

of c

ontro

l) lowastlowastlowast

lowast

PMA + piceatannol (120583molL)

Figure 3 Effect of piceatannol on PKC activity Neutrophils wereincubated with piceatannol (30min) and stimulated with PMA(3min) PKC activity was assessed by ELISA kit in the supernatant ofcell lysate The values are presented as percentage of resting control(PKC activity in absence of PMA) Control value given as kinaseactivity per 1mg of protein was 13 376 plusmn 2 417 Mean plusmn SEM 119899 = 5lowast

119875 le 005 lowastlowast119875 le 001 versus PMA

was slightly decreased after treatment of neutrophils withpiceatannol Spontaneous neutrophil apoptosis was acceler-ated by piceatannol as indicated by the significantly elevatednumber of apoptotic neutrophils (Figure 6) The percentageof dead (propidium iodide positive) neutrophils ranged from01 to 02 andwas not significantly increased in the presenceof piceatannol


0

100

200

300

400

500

600

PMA PIC

0

100

200

300

400(

of c

ontro

l)PK

C120572120573

II p

hosp

hory

latio

n

PKC120572 120573II

120573-actin

+ + +minus

minus minus 10 100

PMA PIC

+ + +minus

minus minus 10 100

( o

f con

trol)

PKC120575

phos

phor

ylat

ion

PKC120575

120573-actin

Figure 4 Phosphorylation of PKC120572 PKC120573II and PKC120575 in PMA-stimulated neutrophils treated with 10 and 100 120583molL piceatannol (PIC)Phosphorylated PKC isoforms were isolated byWestern blotting and detected with anti-phospho-PKC120572120573II (Thr638641) and anti-phospho-PKC120575 (Thr505) antibodies The values are presented as percentage of resting control Control values given as optical density of PKCbands corrected to 120573-actin content were 7807 plusmn 1786 (PKC120572 120573II) and 8484 plusmn 1880 (PKC120575) The representative blot manifests elevatedphosphorylation of PKC isoforms in neutrophils stimulated with PMA as well as the effect of piceatannol on this increase Mean plusmn SEM119899 = 4

0

50

100

150

200

250

0 1 10 100 Total

Extr

acel

lula

r ATP

(nm

olL

)

Piceatannol (120583molL)

lowastlowast lowastlowast

Figure 5 Integrity of neutrophils assessed on the basis of ATPliberation in the absence (0) and in the presence of piceatannol(1ndash100120583molL) Totalmdashamount of ATP determined immediatelyafter complete neutrophil destruction The given values representthe extracellular ATP concentration in samples containing 30 000neutrophils Mean plusmn SEM 119899 = 6 lowastlowast119875 le 001 versus Control (0)

4 Discussion

Novel strategies of anti-inflammatory therapy are basedupon pharmacological agents capable of enhancing theresolutionmdashthat is the termination of the beneficial inflam-mation before it may turn into an adverse chronic stage [3435] It is likely that several phytochemicals would act in thisway but this point has not been investigated In this paperthe natural stilbenoid piceatannol was analysed considering

0

20

40

60

80

100

120

0 1 10 100

Cel

l pop

ulat

ion

()

LiveApoptoticDead

921 930 926


298lowastlowast

699lowastlowast

Figure 6 Effect of piceatannol on neutrophil apoptosisThe lifespanof human neutrophils was recorded by flow cytometry usingdouble-staining with annexin-V (AN) and propidium iodide (PI)Of the population of 5 000 granulocytes the percentage of live(ANminusPIminus) apoptotic (AN+PIminus) and dead cells (AN+PI+) wascalculated The percentage of dead neutrophils (01-02) was notsignificantly increased in the presence of piceatannol Mean plusmn SEM119899 = 7 lowastlowast119875 le 001 versus Control (0)

its ability to affect activity and apoptosis of neutrophilsmdashtwoimportant inputs of resolution [19 20 36]

The incubation of human neutrophils with piceatan-nol resulted in decreased production of ROS Severalmechanismsmdashinhibition of Syk kinase ROS scavenging andthe interference with neutrophil activation through sup-pressed PKC activationmdashcould be involved in the reductionof the chemiluminescence signal Piceatannol widely used


as an inhibitor of Syk kinase [6 8 9] has the potential toblock oxidative burst at the receptor signaling level Howeverthis mechanism might not be essential with regard to thepronounced inhibition of chemiluminescence initiated withPMA or A23187 that is by receptor bypassing stimuli Themarked inhibition of the chemiluminescence produced bycell-free system suggested participation of antioxidant activ-ity which is closely involved in various effects of other naturalpolyphenols [37ndash41] Piceatannol was found to be a potentscavenger of hydroxyl radicals and superoxide anion [42]much more effective than resveratrol This high effectivenessresults from the presence of an additional hydroxyl groupin stilbene rings which makes the abstraction and transferof electrons easier and increases the stability of the resultingpiceatannol semiquinone radical [4] Due to the fact that theinhibition of chemiluminescence occurred in the presenceof each stimulus used the interference of piceatannol witha process involved in all mechanisms of initiation has beensuggested One of the potential candidates could be thesignalling enzyme protein kinase C Piceatannol inhibitedPKC activation initiated by PMA which was reflected inthe decreased activity of the enzyme as well as in thereduced phosphorylation of protein kinase isoenzymes 120572120573II and 120575 on their catalytic region Since PKC participatesin the activation of neutrophil NADPH oxidase [13 14 2225] its inhibition may result in reduced oxidant formationand thus explain the decreased chemiluminescence of neu-trophils treated with piceatannol The precise mechanism ofpiceatannol-mediated PKC inhibition is still not completelyclear Similar to other natural polyphenols it may involvethe competition for phorbol ester or calcium binding tothe regulatory domains of PKC [43 44] inhibition of PKCtranslocation from cytosol to membrane fraction [45] oxi-dation of thiol groups present within the catalytic domain ofPKC [46] or piceatannol-induced alterations in membraneordering [47]

Since activated neutrophils form and liberate ROS bothextra- and intracellularly [13 17 18] it was important toidentify which part of the chemiluminescence signal wasreduced in the presence of piceatannol This stilbenoid wasactive in both compartments however at different meaneffective concentrations minus1259 120583molL (intracellular) and187 120583molL (extracellular) It means that the radicals formedwithin neutrophils (fulfilling a regulatory role) were reducedto a lesser extent than extracellular oxidants potentially dan-gerous for host tissues Moreover piceatannol did not affectthe phosphorylation of p40phoxmdasha component of NADPHoxidase responsible for the assembly of functional oxidasein intracellular (granular) membranes [17 48 49] Finallythe phosphorylation of PKC120575 (responsible for intracellularoxidant production [50]) was affected by piceatannol to alesser extent than the phosphorylation of PKC120573 which isinvolved in the extracellular formation of oxidants [16] Yetthe interference of piceatannol with PKC120575 may not be adecisive mechanism involved in the inhibition of intracel-lular chemiluminescence as in the presence of 100120583molLpiceatannol the chemiluminescence was strongly reduceddespite the fact that the inhibition of PKC120575 phosphorylationwas minimal at this concentration

The more pronounced extracellular activity results fromthe structure of piceatannol Compared to resveratrol theadditional hydroxyl group makes the molecule of piceatan-nol more hydrophilic and less able to pervade biologicalmembranes [47] Thus piceatannol could minimise tissuedamagewith theminimal reduction of beneficial intracellularoxidants involved in the suppression of neutrophil proin-flammatory activity [17 18] and in the initiation of neutrophilapoptosis [15 16]

The observed reductions in chemiluminescence and inthe activity of protein kinase C were not associated withneutrophil damage because in the presence of piceatannolno increase in extracellular ATP concentration was recordedAs confirmed by flow cytometry this stilbene enhancedspontaneous apoptosis of neutrophils This was indicatedby an increased number of annexin-positive cells that iscells displaying a more pronounced externalisation of phos-phatidylserine The expression of phosphatidylserine on theexternal side of plasma membrane facilitates the recognitionof apoptotic neutrophils by macrophages and their saferemoval from the site of inflammation [15] The ability ofpiceatannol to increase apoptosis has been extensively stud-ied in cancer cells where it involves the increased activitiesof caspases activation of proapoptotic factors Bid Bax Bakor the inhibition of the antiapoptotic factor Bcl-xL [4] Inneutrophils all these mechanisms may be operative alongwith the repressed activation of the antiapoptotic enzymephosphoinositide-3-kinase [51 52]Moreover the acceleratedapoptosis observed in the presence of piceatannol mayresult from the inhibition of PKC120572 and PKC120575 as these PKCisoforms participate in antiapoptotic signalling in neutrophils[23 53] Increased neutrophil apoptosis was observed in thepresence of 100120583molL piceatannol that is in a concentrationseveral times higher than assumed piceatannol plasma levelsobtained by dietary intake Yet it should be taken into accountthat in comparison to in vivo conditions in these samplesneutrophil count was substantially higher and the exposureof the cells to piceatannol lasted only 30 minutes Similarlythe accelerated apoptosis of leukemic cells was detectedafter 24ndash48 h incubation with 10ndash60 120583molL piceatannol[54 55]

5 Conclusion

Piceatannol decreased the concentration of ROS producedby neutrophils and accelerated spontaneous apoptosis ofthese cells The observed effects classified piceatannol as apotentially useful complementary medicine in states asso-ciated with persistent activation of neutrophils oxidativedamage of tissues and persistent inflammation Howeverthe bioavailability and toxicity of this promising naturalsubstance is a decisive question requiring further studies

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Acknowledgments

The study was supported by the Slovak Research and Devel-opment Agency (APVV-0052-10 APVV-0315-07) and by theScientific Grant Agency VEGA (VEGA 2001013 VEGA2004511)

References

[1] B Djoko R Y Y Chiou J J Shee and Y W Liu ldquoChar-acterization of immunological activities of peanut stilbenoidsarachidin-1 piceatannol and resveratrol on lipopolysaccharide-induced inflammation of RAW 2647 macrophagesrdquo Journal ofAgricultural and Food Chemistry vol 55 no 6 pp 2376ndash23832007

[2] A Daikonya and S Kitanaka ldquoPolyphenols from Sophorayunnanensis and their inhibitory effects on nitric oxide produc-tionrdquo Chemical and Pharmaceutical Bulletin vol 59 no 12 pp1567ndash1569 2011

[3] T Szekeres P Saiko M Fritzer-Szekeres B Djavan and WJager ldquoChemopreventive effects of resveratrol and resveratrolderivativesrdquo Annals of the New York Academy of Sciences vol1215 no 1 pp 89ndash95 2011

[4] H PiotrowskaMKucinska andMMurias ldquoBiological activityof piceatannol leaving the shadow of resveratrolrdquo MutationResearch vol 750 no 1 pp 60ndash82 2012

[5] N Richard D Porath A Radspieler and J Schwager ldquoEffects ofresveratrol piceatannol triacetoxystilbene and genistein on theinflammatory response of human peripheral blood leukocytesrdquoMolecular Nutrition and Food Research vol 49 no 5 pp 431ndash442 2005

[6] J Schymeinsky A Mocsai and B Walzog ldquoNeutrophil activa-tion via 1205732 integrins (CD11CD18) molecular mechanisms andclinical implicationsrdquoThrombosis and Haemostasis vol 98 no2 pp 262ndash273 2007

[7] Y H Kim H S Kwon D H Kim et al ldquoPiceatannol a stilbenepresent in grapes attenuates dextran sulfate sodium-inducedcolitisrdquo International Immunopharmacology vol 8 no 12 pp1695ndash1702 2008

[8] J Ennaciri and D Girard ldquoIL-4R120572 a new member thatassociates with Syk kinase implication in IL-4-induced humanneutrophil functionsrdquo The Journal of Immunology vol 183 no8 pp 5261ndash5269 2009

[9] N Ozaki S Suzuki M Ishida et al ldquoSyk-dependent signalingpathways in neutrophils and macrophages are indispensable inthe pathogenesis of anti-collagen antibody-induced arthritisrdquoInternational Immunology vol 24 no 9 pp 539ndash550 2012

[10] R Cascao H S Rosario M M Souto-Carneiro and J E Fon-seca ldquoNeutrophils in rheumatoid arthritis more than simplefinal effectorsrdquoAutoimmunity Reviews vol 9 no 8 pp 531ndash5352010

[11] H L Wright R J Moots R C Bucknall and S W EdwardsldquoNeutrophil function in inflammation and inflammatory dis-easesrdquo Rheumatology vol 49 no 9 pp 1618ndash1631 2010

[12] K Bedard and K H Krause ldquoThe NOX family of ROS-generatingNADPHoxidases physiology and pathophysiologyrdquoPhysiological Reviews vol 87 no 1 pp 245ndash313 2007

[13] F R Sheppard M R Kelher E E Moore N J D McLaughlinA Banerjee and C C Silliman ldquoStructural organization ofthe neutrophil NADPH oxidase phosphorylation and translo-cation during priming and activationrdquo Journal of LeukocyteBiology vol 78 no 5 pp 1025ndash1042 2005

[14] J El-Benna P M C Dang and A Perianin ldquoTowards specificNADPH oxidase inhibition by small synthetic peptidesrdquo Cel-lular and Molecular Life Sciences vol 69 no 4 pp 2307ndash23142012

[15] H R Luo and F Loison ldquoConstitutive neutrophil apoptosismechanisms and regulationrdquo American Journal of Hematologyvol 83 no 4 pp 288ndash295 2008

[16] V Witko-Sarsat M Pederzoli-Ribeil E Hirsh S Sozzani andM A Cassatella ldquoRegulating neutrophil apoptosis new playersenter the gamerdquo Trends in Immunology vol 32 no 3 pp 117ndash124 2011

[17] J Bylund K L Brown C Movitz C Dahlgren and A Karls-son ldquoIntracellular generation of superoxide by the phagocyteNADPH oxidase how where and what forrdquo Free RadicalBiology and Medicine vol 49 no 12 pp 1834ndash1845 2010

[18] H Bjornsdottir D Granfeldt AWelin J Bylund and A Karls-son ldquoInhibition of phospholipase A

2abrogates intracellular

processing of NADPH-oxidase derived reactive oxygen speciesin human neutrophilsrdquo Experimental Cell Research vol 319 no5 pp 761ndash774 2013

[19] D El Kebir and J G Filep ldquoRole of neutrophil apoptosis in theresolution of inflammationrdquo TheScientificWorldJournal vol 10pp 1731ndash1748 2010

[20] S Fox A E Leitch R Duffin C Haslett and A G Rossi ldquoNeu-trophil apoptosis relevance to the innate immune response andinflammatory diseaserdquo Journal of Innate Immunity vol 2 no 3pp 216ndash227 2010

[21] P M C Dang A Fontayne J Hakim J El Benna andA Perianin ldquoProtein kinase C 120577 phosphorylates a subset ofselective sites of the NADPH oxidase component p47119901ℎ119900119909 andparticipates in formyl peptide-mediated neutrophil respiratoryburstrdquoThe Journal of Immunology vol 166 no 2 pp 1206ndash12132001

[22] A Fontayne P M C Dang M A Gougerot-Pocidalo and JEl Benna ldquoPhosphorylation of p47119901ℎ119900119909 sites by PKC 120572 120573II 120575and 120577 effect on binding to p22119901ℎ119900119909 and on NADPH oxidaseactivationrdquo Biochemistry vol 41 no 24 pp 7743ndash7750 2002

[23] L E Kilpatrick S Sun D M Mackie F Baik H Li and H MKorchak ldquoRegulation of TNF mediated antiapoptotic signalingin human neutrophils role of 120575-PKC and ERK12rdquo Journal ofLeukocyte Biology vol 80 no 6 pp 1512ndash1521 2006

[24] L E Kilpatrick S Sun H Li T C Vary and H M Kor-chak ldquoRegulation of TNF-induced oxygen radical productionin human neutrophils role of 120575-PKCrdquo Journal of LeukocyteBiology vol 87 no 1 pp 153ndash164 2010

[25] A Bertram andK Ley ldquoProtein kinase C isoforms in neutrophiladhesion and activationrdquo Archivum Immunologiae et TherapiaeExperimentalis vol 59 no 2 pp 79ndash87 2011

[26] T Perecko V Jancinova K Drabikova et al ldquoStructure-efficiency relationship in derivatives of stilbene Comparison ofresveratrol pinosylvin and pterostilbenerdquo NeuroendocrinologyLetters vol 29 no 5 pp 802ndash805 2008

[27] K Drabikova T Perecko R Nosalrsquo et al ldquoGlucomannanreduces neutrophil free radical production in vitro and in ratswith adjuvant arthritisrdquo Pharmacological Research vol 59 no6 pp 399ndash403 2009

[28] K Drabikova V Jancinova R Nosalrsquo et al ldquoInhibitory effectof stobadine on FMLP-induced chemiluminescence in humanwhole blood and isolated polymorphonuclear leukocytesrdquoLuminescence vol 22 pp 67ndash71 2007


[29] V Jancinova T Perecko R Nosalrsquo et al ldquoDecreased activity ofneutrophils in the presence of diferuloylmethane (curcumin)involves protein kinase C inhibitionrdquo European Journal ofPharmacology vol 612 no 1ndash3 pp 161ndash166 2009

[30] R Nosalrsquo T Perecko V Jancinova et al ldquoSuppression ofoxidative burst in human neutrophils with the naturally occur-ring serotonin derivative isomer from Leuzea carthamoidesrdquoNeuroendocrinology Letters vol 31 pp 69ndash72 2010

[31] V Jancinova T Perecko R Nosalrsquo et al ldquoThe natural stilbenoidpinosylvin and activated neutrophils effects on oxidative burstprotein kinase C apoptosis and efficiency in adjuvant arthritisrdquoActa Pharmacologica Sinica vol 33 pp 1285ndash1292 2012

[32] T Perecko K Drabikova L Rackova et al ldquoMolecular targetsof the natural antioxidant pterostilbene effect on protein kinaseC caspase-3 and apoptosis in human neutrophils in vitrordquoNeuroendocrinology Letters vol 31 pp 84ndash90 2010

[33] R Nosalrsquo T Perecko V Jancinova et al ldquoNaturally appearingN-feruloylserotonin isomers suppress oxidative burst of humanneutrophils at the protein kinase C levelrdquo PharmacologicalReports vol 63 pp 790ndash798 2011

[34] C N Serhan ldquoSystems approach to inflammation resolutionidentification of novel anti-inflammatory and pro-resolvingmediatorsrdquo Journal of Thrombosis and Haemostasis vol 7 no1 pp 44ndash48 2009

[35] P Kohli and B D Levy ldquoResolvins and protectins mediatingsolutions to inflammationrdquoBritish Journal of Pharmacology vol158 no 4 pp 960ndash971 2009

[36] J G Filep and D El Kebir ldquoNeutrophil apoptosis a target forenhancing the resolution of inflammationrdquo Journal of CellularBiochemistry vol 108 no 5 pp 1039ndash1046 2009

[37] A Augustyniak G Bartosz A Cipak et al ldquoNatural andsynthetic antioxidants an updated overviewrdquo Free RadicalResearch vol 44 no 10 pp 1216ndash1262 2010

[38] M Stefek ldquoNatural flavonoids as potential multifunctionalagents in prevention of diabetic cataractrdquo InterdisciplinaryToxicology vol 4 no 2 pp 69ndash77 2011

[39] K Bauerova S Ponist D Mihalova et al ldquoUtilization ofadjuvant arthritis model for evaluation of new approaches inrheumatoid arthritis therapy focused on regulation of immuneprocesses and oxidative stressrdquo Interdisciplinary Toxicology vol4 pp 33ndash39 2011

[40] M Cız M Pavelkova L Gallova J Kralova L Kubala and ALojek ldquoThe influence of wine polyphenols on reactive oxygenand nitrogen species production by murine macrophages RAW2647rdquo Physiological Research vol 57 no 3 pp 393ndash402 2008

[41] M Ciz P Denev M Kratchanova O Vasicek G Ambrozovaand A Lojek ldquoFlavonoids inhibit the respiratory burst ofneutrophils in mammalsrdquo Oxidative Medicine and CellularLongevity vol 2012 Article ID 181295 6 pages 2012

[42] L Camont F Collin M Couturier et al ldquoRadical-inducedoxidation of trans-resveratrolrdquo Biochimie vol 94 no 3 pp 741ndash747 2012

[43] S J Slater J L Seiz A C Cook B A Stagliano and C J BuzasldquoInhibition of protein kinase C by resveratrolrdquo Biochimica etBiophysica Acta vol 1637 no 1 pp 59ndash69 2003

[44] Y A Mahmmoud ldquoModulation of protein kinase C by cur-cumin inhibition and activation switched by calcium ionsrdquoBritish Journal of Pharmacology vol 150 no 2 pp 200ndash2082007

[45] Z Varga I Seres E Nagy et al ldquoStructure prerequisite forantioxidant activity of silybin in different biochemical systemsin vitrordquo Phytomedicine vol 13 no 1-2 pp 85ndash93 2006

[46] R Gopalakrishna and U Gundimeda ldquoAntioxidant regulationof protein kinase C in cancer preventionrdquo Journal of Nutritionvol 132 no 12 pp 3819Sndash3823S 2002

[47] O Wesołowska M Kuzdzał J Strancar and K MichalakldquoInteraction of the chemopreventive agent resveratrol and itsmetabolite piceatannol with model membranesrdquo Biochimica etBiophysica Acta vol 1788 no 9 pp 1851ndash1860 2009

[48] J D Matute A A Arias M C Dinauer and P J Patinoldquop40119901ℎ119900119909 the last NADPH oxidase subunitrdquo Blood CellsMolecules and Diseases vol 35 no 2 pp 291ndash302 2005

[49] J D Matute A A Arias N A M Wright et al ldquoA new geneticsubgroup of chronic granulomatous disease with autosomalrecessive mutations in p40119901ℎ119900119909 and selective defects in neu-trophil NADPH oxidase activityrdquo Blood vol 114 no 15 pp3309ndash3315 2009

[50] G E Brown M Q Stewart H Liu V L Ha and M B YaffeldquoA novel assay system implicates PtdIns(34)P2 PtdIns(3)P andPKC120575 in intracellular production of reactive oxygen species bythe NADPH oxidaserdquo Molecular Cell vol 11 no 1 pp 35ndash472003

[51] E M B Raeder P J Mansfield V Hinkovska-Galcheva J AShayman and L A Boxer ldquoSyk activation initiates downstreamsignaling events during human polymorphonuclear leukocytephagocytosisrdquo The Journal of Immunology vol 163 no 12 pp6785ndash6793 1999

[52] K H Choi J E Kim N R Song et al ldquoPhosphoinositide 3-kinase is a novel target of piceatannol for inhibiting PDGF-BB-induced proliferation and migration in human aortic smoothmuscle cellsrdquo Cardiovascular Research vol 85 no 4 pp 836ndash844 2010

[53] P R Webb K Q Wang D Scheel-Toellner J Pongracz MSalmon and J M Lord ldquoRegulation of neutrophil apoptosisa role for protein kinase C and phosphatidylinositol-3-kinaserdquoApoptosis vol 5 no 5 pp 451ndash458 2000

[54] Y H Kim C Park J O Lee et al ldquoInduction of apop-tosis by piceatannol in human leukemic U937 cells throughdown-regulation of Bcl-2 and activation of caspasesrdquo OncologyReports vol 19 no 4 pp 961ndash967 2008

[55] C H Kang D O Moon Y H Choi et al ldquoPiceatannolenhances TRAIL-induced apoptosis in human leukemia THP-1 cells through Sp1- and ERK-dependent DR5 up-regulationrdquoToxicology in Vitro vol 25 no 3 pp 605ndash612 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


OH

OH

OH

HO

OH

OH

HO

trans-ResveratrolPiceatannol

Figure 1 Piceatannol (trans-343101584051015840-tetrahydroxystilbene) and its related compound trans-resveratrol (trans-43101584051015840-trihydroxystilbene)

heterodimer transfers electrons from NADPH to molecularoxygen Flavin adenine dinucleotide (FAD) and two hemegroups serve as the redox pathway which enables the transferof electrons across the membrane [12ndash14] If the assembly ofcatalytically active oxidase occurs on the plasma membranethe generated oxidants are liberated extracellularly or intophagosomesThese radicals are involved in the elimination ofpathogens however their overproductionmay result in tissuedamage ROS produced intracellularlymdashon membranes ofspecific granules participate in the initiation of neutrophilapoptosis [15 16] and they are considered key suppressorsof inflammation [17 18] Since the optimum therapy isexpected to minimise tissue damage without reduction ofthe physiological function of neutrophils separate analysis ofextra- and intracellular effects of antioxidants is of particularimportance

The production of ROS is limited by the programmeddeath of neutrophils Apoptosis represents a precisely reg-ulated process which includes release of proapoptotic pro-teins into the cytosol gradual activation of caspases DNAfragmentation chromatin condensation loss of membraneasymmetry and formation of apoptotic bodies Alterations inplasma membrane (eg externalisation of phosphatidylser-ine) facilitate the recognition and clearance of apoptoticneutrophils by macrophages resulting in safe removal ofthese cells from the site of inflammation Since the delayedapoptosis and impaired clearance of neutrophils aggravateand prolong tissue injury pharmacological interventionfocused on neutrophil apoptosis is studied as an originalapproach for the design of new anti-inflammatory strategies[16 19 20]

Modulation of protein kinase C activity represents aprospective method to regulate neutrophil functions Im-munochemical studies have shown that human neutrophilsexpress five PKC isoforms 120572 120573I 120573II 120575 and 120577 which partici-pate in NADPH oxidase activation as well as in proapoptoticand antiapoptotic signalling [21ndash25]

The present paper investigated the impact of piceatannolon the viability and oxidative burst of human neutrophilsWe analysed separately the effects of this stilbenoid on theconcentration of ROS produced by neutrophils extra- andintracellularly Protein kinase C activity was examined as anassumed target of piceatannol action and the phosphoryla-tion of PKC120572 PKC120573II and PKC120575 (the most abundant PKCisoforms in neutrophils) was assessed

2 Material and Methods

21 Chemicals Piceatannol was purchased from AcrosOrganics (Geel Belgium) Luminol isoluminol PMA (4120573-phorbol-12120573-myristate-13120572-acetate) Ca2+-ionophoreA23187superoxide dismutase dextran (average MW 464 000 kDa)zymosan (zymosan A from Saccharomyces cerevisiae) lu-ciferase from Photinus pyralis and D-luciferin sodium saltwere from Sigma-Aldrich Chemie (Deisenhofen Germany)HRP (horseradish peroxidase) and catalase were obtainedfrom Merck (Darmstadt Germany) and lymphoprep (den-sity 1077 gmL) was purchased from Nycomed PharmaAS (Oslo Norway) Propidium iodide and rh Annexin V-FITC (produced in E coli and conjugated with fluores-cein isothiocyanatemdashFITC) was received from eBioscience(Vienna Austria) and PKC kinase activity kit was from EnzoLife Sciences AG (Lausen Switzerland) Phosphospecificantibodies versus PKC isoforms and versus p40phox wereobtained from Cell Signaling Technology (Danvers MAUSA) Secondary anti-rabbit antibody and Lumigen Detec-tion Reagent were supplied by GE Healthcare Life Sciences(Little Chalfont UK)

This work was approved by the Local Ethic CommitteeInstitute of Experimental Pharmacology and ToxicologySlovak Academy of Sciences Bratislava

22 Blood Collection and Isolation of Human NeutrophilsFresh blood was obtained at the blood bank by venipuncturefromhealthymale donors (20ndash50 years)whohadnot receivedany medication for at least 7 days The samples were mixedwith 38 trisodium citrate in the ratio of 9mL of bloodto 1mL citrate Erythrocytes were allowed to sediment in1 dextran solution (1 timesg 25min 22∘C) and the suspensionof leukocytes and platelets in plasma (buffy coat) was usedfor flow cytometric analyses or for neutrophil isolationFor neutrophil isolation the buffy coat was centrifugedcells were resuspeded in phosphate-buffered saline andneutrophils were separated on Lymphoprep (500 timesg 30min22∘C) The contaminating erythrocytes were removed withhypotonic cold haemolysis and neutrophils were washedwithphosphate-buffered saline Neutrophil count was assessed byCoulter Counter (Coulter Electronics High Wycombe Eng-land) and adjusted to a final concentration of 104 cells1 120583LThe final suspension of neutrophils containedmore than 96











3 Results












lowast



lowastlowast



lowastlowast







lowastlowast



lowastlowast



lowastlowast



0

100

200

300

400

500

PMA

PIC 10 100

( o

f con

trol)

Phos

phor

ylat

ion

ofp40phox

p40phox

120573-actin

minus

minus minus

+ + +

lowastlowast


0

50

100

150

200


Prot

ein

kina

se C

activ

ity(

of c

ontro


lowast






0

100

200

300

400

500

600

PMA PIC

0

100

200

300

400(

of c

ontro

l)PK

C120572120573

II p

hosp

hory

latio

n

PKC120572 120573II

120573-actin

+ + +minus

minus minus 10 100

PMA PIC

+ + +minus

minus minus 10 100

( o

f con

trol)

PKC120575

phos

phor

ylat

ion

PKC120575

120573-actin


0

50

100

150

200

250

0 1 10 100 Total

Extr

acel

lula

r ATP

(nm

olL

)




4 Discussion


0

20

40

60

80

100

120

0 1 10 100

Cel

l pop

ulat

ion

()

LiveApoptoticDead

921 930 926


298lowastlowast

699lowastlowast









5 Conclusion





Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























3 Results












lowast



lowastlowast



lowastlowast







lowastlowast



lowastlowast



lowastlowast



0

100

200

300

400

500

PMA

PIC 10 100

( o

f con

trol)

Phos

phor

ylat

ion

ofp40phox

p40phox

120573-actin

minus

minus minus

+ + +

lowastlowast


0

50

100

150

200


Prot

ein

kina

se C

activ

ity(

of c

ontro


lowast






0

100

200

300

400

500

600

PMA PIC

0

100

200

300

400(

of c

ontro

l)PK

C120572120573

II p

hosp

hory

latio

n

PKC120572 120573II

120573-actin

+ + +minus

minus minus 10 100

PMA PIC

+ + +minus

minus minus 10 100

( o

f con

trol)

PKC120575

phos

phor

ylat

ion

PKC120575

120573-actin


0

50

100

150

200

250

0 1 10 100 Total

Extr

acel

lula

r ATP

(nm

olL

)




4 Discussion


0

20

40

60

80

100

120

0 1 10 100

Cel

l pop

ulat

ion

()

LiveApoptoticDead

921 930 926


298lowastlowast

699lowastlowast









5 Conclusion





Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























lowast



lowastlowast



lowastlowast







lowastlowast



lowastlowast



lowastlowast



0

100

200

300

400

500

PMA

PIC 10 100

( o

f con

trol)

Phos

phor

ylat

ion

ofp40phox

p40phox

120573-actin

minus

minus minus

+ + +

lowastlowast


0

50

100

150

200


Prot

ein

kina

se C

activ

ity(

of c

ontro


lowast






0

100

200

300

400

500

600

PMA PIC

0

100

200

300

400(

of c

ontro

l)PK

C120572120573

II p

hosp

hory

latio

n

PKC120572 120573II

120573-actin

+ + +minus

minus minus 10 100

PMA PIC

+ + +minus

minus minus 10 100

( o

f con

trol)

PKC120575

phos

phor

ylat

ion

PKC120575

120573-actin


0

50

100

150

200

250

0 1 10 100 Total

Extr

acel

lula

r ATP

(nm

olL

)




4 Discussion


0

20

40

60

80

100

120

0 1 10 100

Cel

l pop

ulat

ion

()

LiveApoptoticDead

921 930 926


298lowastlowast

699lowastlowast









5 Conclusion





Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

100

200

300

400

500

600

PMA PIC

0

100

200

300

400(

of c

ontro

l)PK

C120572120573

II p

hosp

hory

latio

n

PKC120572 120573II

120573-actin

+ + +minus

minus minus 10 100

PMA PIC

+ + +minus

minus minus 10 100

( o

f con

trol)

PKC120575

phos

phor

ylat

ion

PKC120575

120573-actin


0

50

100

150

200

250

0 1 10 100 Total

Extr

acel

lula

r ATP

(nm

olL

)




4 Discussion


0

20

40

60

80

100

120

0 1 10 100

Cel

l pop

ulat

ion

()

LiveApoptoticDead

921 930 926


298lowastlowast

699lowastlowast









5 Conclusion





Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















5 Conclusion





Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article The Natural Stilbenoid Piceatannol