COMMENTARY

Resveratrol induces FasL-related apoptosis through Cdc42 activation ofASK1/JNK-dependent signaling pathway in human leukemia HL-60 cells

Jen-Liang Su1,*, Ming-Tsan Lin2,*, Chih-Chen Hong3,Cheng-Chi Chang1, Shine-Gwo Shiah4, Cheng-Wen Wu4,Szu-Ta Chen5, Yat-Pang Chau6 and Min-Liang Kuo1,7

1Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology,College of Medicine, National Taiwan University, Taipei, Taiwan,2Department of Surgery, National Taiwan University Hospital, Taipei,Taiwan, 3Division of Cancer Research, National Health Research Institutes,Taipei 115, Taiwan, 4President’s Laboratory, National Health ResearchInstitute, Taipei 115, Taiwan, 5Department of Pediatrics, National TaiwanUniversity Hospital, Taipei, Taiwan and 6Institute of Anatomy, School ofMedicine, National Yang-Ming University, Taipei, Taiwan

7To whom correspondence should be addressedEmail: toxkml@ha.mc.ntu.edu.tw

Trans-resveratrol, a phytoalexin found at high levels ingrapes and in grape products such as red wine, has beenshown to prevent carcinogenesis or antitumor growth inmurine models. Here we dissect the detailed signaling path-way involved in resveratrol-induced apoptosis. Our datashowed that treatment with resveratrol-induced activationof apoptosis signal-regulating kinase 1, a mitogen-activatedprotein kinase kinase kinase, in turn, activated thedownstream kinases c-Jun N-terminal kinase and p38mitogen-activated protein kinase, but not extracellularsignal-regulated kinase. Transfection with a dominant-negative c-Jun N-terminal kinase expression vectorreducedFasLexpressionandDNAfragmentationinducedbyresveratrol. However, inhibition of p38 mitogen-activatedprotein kinase activity by treatment with SB203580 (p38mitogen-activated protein kinase specific inhibitor) or ex-pression of mutant p38 mitogen-activated protein kinaseexpression vector did not alter the apoptosis and FasLexpression in response to resveratrol. Furthermore, geneticinhibition of apoptosis signal-regulating kinase 1 signalinginhibited not only the activation of c-Jun N-terminalkinase, but also the expression of FasL and apoptosis.Similarly, over-expression of wild-type apoptosis signal-regulating kinase 1 strengthened the resveratrol-inducedc-Jun N-terminal kinase activation, FasL expression andsubsequent apoptosis. These results suggest the possibleinvolvement of apoptosis signal-regulating kinase 1/c-JunN-terminal kinase signaling in the regulation of FasLexpression and subsequent apoptosis induced by resvera-trol in HL-60 cells. Resveratrol also activated the smallGTP-binding protein Cdc42, rather than other memberssuch as RhoA or Rac1. Expression of a mutant Cdc42(N17 Cdc42) dramatically reduced resveratrol-inducedc-Jun N-terminal kinase activity, FasL expression and

apoptotic cell death. These results showed that resveratrolinduced apoptosis through the Cdc42/apoptosis signal-regulating kinase 1/c-Jun N-terminal kinase/FasL signal-ing cascade in HL-60 cells.

Introduction

A new dimension in the management of neoplasia was theincreasing awareness that chemoprevention, which refers tothe administration of chemical agents to prevent the eventsassociated with carcinogenesis (1) and this could be the mostdirect way to reduce mortality and morbidity. A large number ofchemopreventive and chemotherapeutic agents have been dis-covered from natural products and these provide a promisingstrategy to fight against cancer by inducing apoptosis in malig-nant cells (2,3). Resveratrol (trans-3,40,5-trihydroxystilbene) isa phytoalexin found in grapes, fruits, red wines and root extractsof the weed Polygonum cuspidatum and has been an importantconstituent of Japanese and Chinese folk medicine; it hasbecome one of the most promising chemopreventive agentsagainst cancer (4,5). Resveratrol had been shown to exhibitcancer chemopreventive effects in different systems based onits striking inhibition of a variety of diverse cellular eventsassociated with tumor initiation, promotion and progression(6). In addition, resveratrol also suppressed the expression ofinducible nitrogen oxide synthetase and cyclooxygenase-2 andthis was likely to contribute to both its anti-inflammatory andanti-oncogenic mechanisms (7--9). The antitumor activity ofresveratrol has been indicated by its apoptosis-inducing abilityin numerous cell types (4--6). Resveratrol induces apoptosis inhuman leukemia cells as demonstrated by DNA fragmentation,an increasedproportionof sub-diploids in the cell population, anda dose-dependent increase in cleavage of the caspase substratepoly (ADP-ribose) polymerase (PARP) (4). Recent evidenceemphasized the importance of up-regulating the Fas/FasL(CD95/CD95L) system for the control of apoptosis and a num-ber of cytotoxic drugs up-regulate such expression leading toFas/FasL-mediated signal transduction, activation of caspases,and ultimately, cell death (10). Induction of the Fas/FasL systemwas also shown to be a critical mechanism of resveratrol-induced cell death in HL-60 cells, as well as T47D breastcarcinoma cells (4). Despite these important advances, themolecular mechanism(s) by which resveratrol exerts its anti-cancer effect has not yet been elucidated.Response to numerous types of extracellular signals were

mediated by mitogen-activating protein kinases (MAPKs),which were members of the serine/threonine kinase family.Several studies revealed that c-Jun N-terminal kinase (JNK)1/stress-activated protein kinase (SAPK) and/or p38 MAP kinaseactivation were involved in apoptosis induced by a variety ofdifferent stimuli, such as g-radiation-induced apoptosis inJurkat T-cells (11), glutamate-induced apoptosis in rat cere-bellar granule cells (12), mIgM-induced apoptosis in human B

Abbreviations: ASK1, apoptosis signal-regulating kinase 1; DTT, dithiothre-itol; ERK, extracellular signal-regulated kinase;GST, glutathioneS-transferase;MAPKs, mitogen-activating protein kinases; PBS, phosphate-buffered saline;SAPK, stress-activated protein kinase; SDS--PAGE, sodium dodecyl-sulfate--polyacrylamide gel electrophoresis; TNF, tumor necrosis factor.

�J.-L.Su and M.-T.Lin contributed equally to this article.

Carcinogenesis vol.26 no.1 # Oxford University Press 2005; all rights reserved. 1

Carcinogenesis vol.26 no.1 pp.1--10, 2005doi:10.1093/carcin/bgh220

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lymphocytes (13) or apoptosis induced by genotoxic stresses,such as ultraviolet, X-ray, H2O2, heat shock (14--18) and cellsurface receptor Fas (19,20). Various well-known chemother-apeutic drugs, such as adriamycin, vinblastine, VP-16 and CPTwere also capable of activating JNK. These drugs were criticalin triggering apoptosis in different cell lines (21--24). Thissuggests that the JNK signaling cascade may be the dominantparticipant in apoptosis. Apoptosis signal-regulating kinase 1(ASK1), a member of MAPK kinase kinase group, wasreported to activate two different subgroups of MAPK kinase,SEK1/MKK4 and MKK3/MKK6, which in turn, activate JNK/SAPK and p38 MAPK, respectively (25). Over-expression ofASK1 in epithelial cells cultured in a low serum environment-induced apoptosis. In ovarian cancer cells, expression of adominant-negative mutant of ASK1 or JNK1 inhibited micro-tubule-interfering agent-induced apoptosis, suggesting thatASK1 plays a role in the mechanism of stress-induced apo-ptosis (26,27). ASK1 was activated upon treatment with tumornecrosis factor (TNF)-a or agonistic anti-Fas antibody, and adominant-negative mutant of ASK1 reduced TNF-a and Fas-induced JNK activation and apoptosis, suggesting that ASK1was a pivotal component in cytokine-induced apoptosis(28,29).Thus, we examined the signaling pathway involved in

resveratrol-induced apoptosis in human leukemic HL-60cells. Our data showed that resveratrol induced the activationof the small GTP-binding protein, Cdc42 and, in turn, theapoptotic signaling pathway ASK1/JNK1/FasL.

Materials and methods

Cell culture and chemicals

HL-60 cells, a human promyelocytic leukemia cell line, were obtained fromthe American Type Culture Collection. Cells were maintained in a humidified5% CO2 atmosphere and cultured in Roswell Park Memorial Institute (RPMI)medium 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine and100 U/ml penicillin and streptomycin. Resveratrol, propidium iodide andSB203580 (specific inhibitor of p38 MAP kinase) were obtained from SigmaChemical (St Louis, MO). A stock solution (100 mM) of resveratrol wasprepared in dimethyl sulfoxide (DMSO) and stored at �20�C. For treatment,the resveratrol was diluted in RPMI 1640 and added to cultures to give thedesired final concentrations. Untreated cultures received the same amount ofthe carrier solvent DMSO.

Quantification of apoptosis by flow cytometry

Cells were harvested and washed with phosphate-buffered saline (PBS), andhypodiploid cells were analyzed by flow cytometry as described previously(30). Briefly, 1 � 106 cells were washed with PBS and re-suspended in 500 mlof a buffer (0.5%Triton X-100/PBS/0.05%RNase A) and incubated for 30min.Finally, 0.5 ml of propidium iodide solution (50 mg/ml) was added. Cells werethen left on ice for 15--30 min. Fluorescence emitted from propidium iodi-de--DNA complexes was quantified after laser excitation of the fluorescent dyeby fluorescence-activated cell sorting flow cytometry (Becton Dickinson,Mountain View, CA). Finally, the extent of apoptosis was determined bymeasuring DNA content of the cells below the G0/G1 peak.

Cell transfection and establishing stable expressing cell lines

Transfection was created by electroporation (model T800, BTX, San Diego,CA) of HL-60 cells with expression vectors for wild-type (WT)-ASK1 anddominant-negative ASK1 (pcDNA3-ASK1-HA and pcDNA3 dominant-negative ASK1-HA, respectively, a gift from Dr H.Ichijo). The dominant-negative expression vector of JNK/SAPK (pSR-APF) was kindly provided byDr G.-L.Johnson (27). Briefly, cells were suspended in 1 ml of HEPES-buffered saline containing plasmid DNA, which then received electric treat-ment as follows: electric amplitude 900 V, pulse width 99 ms. After 10 min onice, the cells were transferred to fresh complete medium and cultured for 48 h.The cells were re-plated in cell culture medium containing 800 mg/ml G418.G418-resistant clones were selected and then expanded for further studies.

DNA fragmentation assay

Cells were collected, washed with PBS twice and then lysed in 100 ml of lysisbuffer [50 mM Tris (pH 8.0), 10 mM EDTA, 0.5% sodium lauryl sarkosinateand 1 mg/ml proteinase K] for 3 h at 56�C and treated with 0.5 mg/ml RNase Afor an additional 1 h at 56�C. DNA was extracted by the phenol/chloroform/isoamyl alcohol (25/24/1) method before loading. Loading buffer [50 mMTris,10 mMEDTA, 1% (w/v) lowmelting point agarose, 0.25% (w/v) bromophenolblue] and samples were loaded onto a pre-solidified, 2% (w/v) agarose gelcontaining 0.1 mg/ml ethidium bromide. Agarose gels were electrophoresed at50 V for 90 min in TBE buffer. Gels were observed and photographed underultraviolet light.

Western blot analysis

Western blotting was performed according to a previously described method(24). Briefly, cell lysates were prepared, electrotransferred and then immuno-blotted with anti-phospho-JNK1, anti-phospho-p38 anti-phospho-extracellularsignal-regulated kinase (ERK)1/2, anti-JNK1, anti-p38, anti-ERK1, anti-CD95L and anti-CD95 antibodies (Santa Cruz Biotechnology, Santa Cruz,CA). Detection was performed using ECL western blotting reagent andchemiluminescence exposed onto Kodak X-Omat film.

Immunofluorescence for CD95L

Immunofluorescence staining for CD95L was performed as described pre-viously (4). Briefly, HL-60 cells were treated with resveratrol (20 mM),washed twice with 1% bovine serum albumin in PBS, and fixed in 1 ml of4% paraformaldehyde for 15 min on ice. After a wash with PBS, cells werepermeabilized in ethanol for 1 h on ice, washed with PBS, and incubated with1 mg of anti-CD95L for 30 min on ice. Following another wash with PBSþ 1%FBS, samples were exposed to fluorescein isothiocyanate-conjugated goatanti-mouse IgG for 30 min, washed and re-suspended in 0.5 ml of 2% para-formaldehyde. Mouse IgG1k was used as the isotype control. Viable cellswere analyzed by flow cytometric forward side scatter analysis, and a totalof 10 000 events were analyzed using an emission wavelength of 525 nm.

Immunoprecipitation and kinase activity assays

Cell lysis and immune complex kinase assays were performed as describedpreviously (24). Briefly, cells were washed twice with ice cold PBS and lysedin buffer containing 20 mM HEPES (pH 7.4), 50 mM b-glycerophosphate, 1%Triton X-100, 10% glycerol, 2 mM EGTA, 1 mM dithiothreitol (DTT), 10 mMsodium fluoride, 1 mM sodium orthovanadate, 1 mg/ml leupeptin, 1 mg/mlaprotinin and 1 mM phenylmethylsulfonyl fluoride. The soluble extracts wereprepared by centrifugation at 14 500 r.p.m. for 15 min at 4�C. Followingnormalization of the protein concentration, equal amounts of protein wereincubated with protein A--Sepharose and anti-JNK1 or anti-p38 antibody for3 h at 4�C. The immune complexes were washed twice with lysis buffer andthen once with kinase assay buffer (20 mM MOPS, pH 7.2, 2 mM EGTA,20 mM MgCl2, 1 mM DTT and 0.1% Triton X-100), following which, theywere re-suspended in 20 ml of kinase assay buffer containing 5 mCi of[32P]ATP, 30 mM cold ATP and 2 mg of substrate and incubated for 20 minat 30�C. Reactions were terminated by the addition of the sodium dodecylsul-fate (SDS) sample buffer and boiling for 5 min. The phosphorylated proteinswere resolved by SDS--polyacrylamide gel electrophoresis (PAGE) and visua-lized by autoradiography. Glutathione S-transferase (GST)-c-jun (amino acids1--169) was used as the substrate for JNK1 and GST-ATF-2 was used as thesubstrate for p38.

Rho family pull-down assay

The Rho family pull-down assay was performed as described by Ren et al. (31)and Zondag et al. (32). Briefly, cells were plated in 10-cm dishes, serum-starved for 12 h, and lysed in 50 mM Tris--Cl, pH 7.2, 500 mM NaCl, 1% (v/v)Triton X-100, 5 mM MgCl2, 1 mM DTT and protease inhibitors. One-tenth ofthe cell lysates were subjected to immunoblotting. The remaining cell lysatewas mixed with 10 mg of bacterially expressed GST-rhotekin (murine aminoacids 7--89, for RhoA-GTP) and GST-PAK (rat PAK amino acids 1--252, forRac1-GTP and Cdc42-GTP) bound to glutathione--Sepharose and incubated at4�C with tumbling for 30 min. Beads were collected by centrifugation andwashed twice in 50 mM Tris, pH 7.2, 150 mM NaCl, 1% (v/v) Triton X-100,5 mMMgCl2 and 1 mM DTT before addition of Laemmli buffer and analyzedby western blotting with anti-RhoA, anti-Rac1 and anti-Cdc42 antibodies.

Separation of particulate and cytosolic fractions

Cells were plated in 10-cm dishes, serum-starved for 12 h, and lysed by freeze-thawing in ice cold 400 ml of lysis buffer [50 mM HEPES (pH 7.5), 50 mMNaCl, 1 mM MgCl2, 2 mM EDTA, 10 mM NaF, 1 mM phenylmethylsulfonylfluoride,10 mg/ml aprotinin and 10 mg/ml leupeptin] and centrifuged at 14 500r.p.m. for 20 min at 4�C, and the supernatant was collected as the cytosolicfraction. Pellets were re-suspended, and membrane proteins were lysed in250 ml of lysis buffer containing 2% Triton X-100. The homogenate was

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centrifuged at 100 000 r.p.m. for 30 min at 4�C. The supernatant was collectedand is referred to here as the particulate fraction. Cytosolic and particulatefraction proteins were separated by SDS--PAGE.

Results

Resveratrol increases FasL expression and MAPKs activity inHL-60 cells

It has been shown that resveratrol induces apoptosis viathe activation of the Fas/FasL-dependent pathway in HL-60cells, but the mechanism(s) are not clear (4). To elucidate themechanism(s) of Fas/FasL induction in response to resveratrol,we examined the MAP kinases, which play a critical role in theapoptosis-related signaling pathway. Time-dependent studiesrevealed that resveratrol enhanced FasL expression, but notFas in HL-60 cells and this was detectable at 1 h as well asbeing sustained to 24 h (Figure 1A). Incubation of HL-60 cellswith resveratrol (20 mM) led to phosphorylation of JNK up to24 h (8.6-fold induction compared with control) (Figure 1B,upper panel). Activation of JNK was detected at 1 h andincreased for up to 24 h upon resveratrol treatment. Resvera-trol also induced phosphorylation of p38, and phosphorylatedp38 MAPK remained elevated for up to 24 h (Figure 1B) (4.1-fold induction compared with control). Western blot analysis

showed that the activation of JNK and p38 was not due toenhanced expression of the JNK and p38 proteins (Figure 1B).However, under the same treatment, we did not detect anysignificant difference in ERK1/2 activation, with or withoutresveratrol treatment (Figure 1B, lower panel). These observa-tions suggest that JNK and p38, but not ERK1/2, are persist-ently activated during resveratrol-induced FasL expression.

JNK kinase activity is required for resveratrol-induced FasLexpression and subsequent apoptosis

To analyze the involvement of JNK and p38 MAPK in FasLinduction by resveratrol, we used a genetic approach to inhibitJNK activity by establishing a HL-60 cell line constitutivelyexpressing a dominant-negative JNK (HL-60/DN-JNK).Resveratrol-induced JNK activation was effectively blockedin HL-60/DN-JNK cells, but not in the control vector expres-sing cells (HL-60/vector), according to immunocomplexkinase assay results (Figure 2A). Blocking the JNK pathwayby DN-JNK significantly diminished not only the resveratrol-induced up-regulation of FasL (~75% less), but also DNAfragmentation (Figure 2B). To confirm the above observations,we used another well-established method to quantify apo-ptosis. Detection of the number of hypodiploid cells (apoptoticcells), which are stained less intensely with propidium iodide,

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Fig. 1. Effects of resveratrol on FasL expression and MAPKs activation in human HL-60 cells. (A) FasL expression was induced by resveratrol. HL-60 cellswere treated with 20mM resveratrol for indicated times, after which cells were harvested and the cytosolic fraction analyzed for FasL expression (upper blot) or forFas protein level (bottom panel). The protein levels were determined by western blot as described in the Materials and methods. (B) Western blot analysis of thephosphorylation of MAPKs with or without resveratrol. Equal amounts of cell lysates (75 mg) were resolved by SDS--PAGE, transferred to nitrocellulose, andprobed with specific phosphorylated MAPKs antibodies and anti-MAPKs antibodies. Results are representative of at least three independent experiments.Numbers below lanes show folds of protein expression level.

Resveratrol induces apoptosis through Cdc42/ASK1/JNK/FasL signaling pathway

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can be unequivocally measured from the peak in the flowcytometry sub-G1 region. Figure 2C indicates that treatmentwith resveratrol for 24 h induced dramatic apoptotic cell deathin vector control cells and the dominant-negative mutant ofJNK (DN-JNK) reduced450% apoptosis compared with con-trol cells (resveratrol-induced apoptotic cells was from 53% incontrol cells to 26% in HL-60/DN-JNK cells). These resultssuggest that the JNK kinase cascade is required for resveratrol-mediated FasL expression and apoptotic cell death for humanleukemic HL-60 cells.Activation of p38MAPK is involved inFas/FasL signaling and

cell death (33--35) but the role of p38 MAPK in resveratrol-induced FasL expression and subsequent apoptosis in HL-60cells is not clear. To address this issue, HL-60 cells were pre-treated with the specific p38 MAPK inhibitor SB203580 with orwithout resveratrol, and then FasL expression and apoptotic cellnumbers were analyzed by flow cytometry. Figure 3A revealsthat 20mMSB203580 significantly reduced the increased levelsof phosphorylated p38MAPK induced by resveratrol. However,there was no significant difference in FasL protein expression(Figure 3A, lower panel) and subsequent apoptosis (Figure 3B)in the same treatment. To further confirm this observation, weused another approach to test the role of p38MAPKby establish-ing a HL-60 cell line expressing dominant-negative p38(p38AF). These results support those obtained previously andresveratrol-induced up-regulation of FasL protein (Figure 3C,upper panel) and the number of apoptotic cells (Figure 3D) were

not changed by expression of p38AF. However, the failure todecrease resveratrol-induced FasL expression and apoptosis byp38AF was not due to a low level of inhibition efficiency,because resveratrol-induced activation of p38 MAPK was dra-matically reduced in p38AF expressed cells as measured by aimmunocomplex kinase assay (Figure 3C, lower panel). Theseresults suggest the possible involvement of JNK, but not p38MAPK, in the regulation of FasL expression and apoptosis inleukemic cancer cells induced by resveratrol.

ASK1 acts as an up-stream regulator of resveratrol-inducedapoptosis

ASK1 is necessary for cytotoxic stress-induced apoptosis andrelays its signals to stress-activated MAPKs such as JNK andp38 (36,37). To examine the role of ASK1 in resveratrol-induced apoptotic process, the kinase-inactive ASK1 mutant(ASK1-KM) was transfected into HL-60 cells (HL-60/ASK1-KM) and the JNK activity was assessed. Data from theimmune complex kinase assay confirmed that over-expressionof ASK1-KM significantly decreased resveratrol-induced JNKactivation and FasL expression (Figure 4A). Similarly, HL-60/ASK1-KM cells exhibited more resistance to resveratrol-induction of DNA fragmented hypodiploid cells than the HL-60/vector control cells (Figure 4B). Furthermore, expressionof wild-type ASK1 (ASK1-WT) strengthened resveratrol-induced JNK activation and FasL expression (Figure 4C).The increase in apoptotic cells was also enhanced by 45%

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Fig. 2. Activation of JNK is required for resveratrol-induced FasL expression and apoptosis. (A) The DN-JNK expressed cells (HL-60/DN-JNK) and vectorcontrol cells were treated with 20 mM resveratrol for 12 h, after which cells were harvested and the kinases activities were determined by immune complexkinase assay (top), and the protein levels were determined by western blot (bottom). GST-c-jun, myelin basic protein was used as a substrate for JNK1. Arepresentative kinase activity assay out of the three is shown. (B) HL-60/DN-JNK and vector control cells were treated with resveratrol for 24 h and analysis of theexpression of FasL (upper panel) and DNA fragmentation (lower panel) by western blot and DNA fragmentation assay as described in the Materials and methods.(C) Inhibition of resveratrol-induced apoptosis by DN-JNK. Cells were obtained that stably expressed DN-JNK or empty vector and then these cells wereincubated with 20 mM resveratrol for 24 h. FACScan was performed as an apoptosis assay and described in the Materials and methods. Columns, means of threeindependent experiments; bars, SD. Asterisk denotes a statistically significant decrease compared with values of vector control (*P 5 0.05).

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under the expression of the ASK1-WT in response to resvera-trol for 12 h (Figure 4D). Together, these findings providerealistic evidence that ASK1 was involved in resveratrol-induced JNK activation, FasL expression and subsequentapoptosis.

Cdc42 is involved in resveratrol-induced signaling cascadeand apoptosis

Many researchers have reported that MAP kinase pathways areactivated via small GTP-binding proteins in response to dif-ferent stimulations (38,39). Because of these previous studies,we attempted to demonstrate whether the Rho family of smallGTP-binding proteins was involved in resveratrol-inducedapoptotic signaling in HL-60 cells. The effects on Rho familyprotein activity were assayed using GST pull-down assaysafter treatment with resveratrol for indicated times. Our resultsshow that resveratrol activated Cdc42, rather than the twoother Rho family small GTPases, RhoA and Rac1 in HL-60cells in a time-dependent manner (Figure 5A, upper panel).

Resveratrol-induced Cdc42 activation was noticed at 30 minand peaked at 60 min. As shown in Figure 5A (lower panel),Cdc42 was translocated from the cytosol to the membrane andtranslocation of Cdc42 was initially detected at 5 min, becameevident at 30 min and maximal at 60 min after stimulation ofHL-60 with resveratrol. To further confirm that activatedCdc42 played a critical role in resveratrol-induced apoptosisin HL-60 cells, we used a series of dominant-negative mutantsof the various proteins involved. Transient transfection withthe dominant-negative Cdc42 expression vector (N17Cdc42)dramatically decreased resveratrol-induced apoptosis(Figure 5B, upper panel). Transient expression of dominant-negative RhoA or Rac1 (N19RhoA or N17Rac1) did notreduce the number of apoptotic cells in response to resveratrol(Figure 5B, upper panel). However, the failure of inhibition ofresveratrol-induced apoptosis by N19RhoA and N17Rac1 wasnot due to the under-expression of these two mutant constructs,because the expression level of these three myc-taggeddominant-negative mutants were similar by detection of

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Fig. 3. Effects of p38 MAPK on resveratrol-induced FasL expression and apoptosis. (A) HL-60 cells were treated with resveratrol (20 mM) combined with orwithout 20 mM SB203580 for 12 or 24 h and analysis of the expression of p38 MAPK and phosphorylated p38 MAPK and FasL by western blot. (B) HL-60 cellswere treated with resveratrol for 24 h and a FACScan performed as an apoptosis assay as described in the Materials and methods. Columns, means of threeindependent experiments; bars, SD. Asterisk denotes a statistically significant difference compared with values of control (�P5 0.05). (C) HL-60 cells weretransfected with empty vector or p38AF and treated with resveratrol (20 mM) for 12 and 24 h to analyse the p38 MAP kinase activity and FasL expression,respectively. The expression of FasL (upper panel) was detected by western blot. The kinases activities were determined by immune complex kinase assay(bottom) as described in the Materials and methods. (D) HL-60/p38AF and vector control cells were treated with resveratrol for 24 h and an analysis of thehypodiploid cells (apoptotic cells) by the flow cytometry analysis of propidium iodide-stained cells was carried out. Data are representative of threeindependent experiments. Columns, means of three independent experiments; bars, SD. Asterisk denotes a statistically significant difference compared withvalues of control (�P5 0.05).

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the expression of myc protein by western blot (Figure 5B,lower panel). More importantly, inhibition of Cdc42 reducedJNK kinase activity and also the expression of FasL and thelevels that result were similar to those detected before stimula-tion with resveratrol (Figure 5C). This suggests that Cdc42controls resveratrol-induced activation of JNK kinase,

FasL expression and subsequent apoptosis. To the best of ourknowledge, this is the first time that Cdc42 has been shown toplay a role in resveratrol-induced apoptosis in HL-60 cells andwe have shown that the expression of FasL and subsequentapoptosis are regulated via Cdc42/ASK1/JNK signalingcascade.

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Fig. 4. Effects of kinase-inactive ASK1 mutant (ASK1-KM) on resveratrol-induced JNK activity. (A) HL-60/ASK1-KM and vector control cells were treatedwith resveratrol for 12 and 24 h to analyze JNK kinase activity and FasL expression. (B) Inhibition of resveratrol-induced DNA fragmentation (upper panel) andhypodiploid cells (apoptotic cells) (lower panel) by expression of ASK1-KM. Transfected ASK1-KM and control vector HL-60 cells were incubated with20 mM resveratrol for 24 h. The apoptosis assay was performed by DNA fragmentation analysis using 1.8% agarose gel electrophoresis and flow cytometryanalysis of propidium iodide-stained cells. Columns, means of three independent experiments; bars, SD. Asterisk denotes a statistically significant decreasecompared with values of vector control (*P5 0.05). (C) HL-60/ASK1-WT and vector control cells were treated with resveratrol (20 mM) for 12 and 24 h for JNKkinase activity assay and FasL expression, respectively. The JNK kinases activities were determined by immune complex kinase assay (upper panel) and the FasLexpression were determined by western blot (lower panel). A representative data out of three is shown. (D) HL-60 cells were transfected with vector orASK1-WT and treated with or without resveratrol 20 mM for 24 h. The apoptosis assay was performed by flow cytometry analysis of propidium iodide-stainedcells. Columns, means of three independent experiments; bars, SD. Asterisks denote a statistically significant decrease compared with values of vectorcontrol (*P50.05).

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Discussion

Recent reports have shown that resveratrol was a potent cancerchemopreventive agent in assays representing the three majorstages of carcinogenesis (4). Resveratrol is a potent chemopre-vention agent that prevents, inhibits or reverses carcinogenesisby either reducing angiogenesis or inducing cancer cell apo-ptosis (40--42). In a recent study, we indicated that resveratrolinhibits VEGF-induced angiogenesis through interruption ofSrc-dependent VE-cadherin phosphorylation (43). Althoughtreatment with resveratrol is an appropriate strategy for che-moprevention, the mechanism is, as yet, unclear. In this studywe verified that the activation of a novel signaling pathwayfrom the Cdc42 small GTP-binding protein to subsequentASK1-mediated JNK activation is a potential requirement forresveratrol-induced elevation of FasL expression and apo-ptosis in human leukemic cancer cells. Activation of thiscascade seems to be necessary for resveratrol-induced celldeath, since genetic inhibition of Cdc42, JNK or ASK1 pre-vents the induction of apoptosis.Fas is a well characterized member of the death receptor

family. Engagement of Fas by a Fas-ligand led to the formationof a protein complex known as death-inducing signaling com-plex and permit acute execution of apoptosis by caspase-8activation (44). The Fas-binding protein, Daxx, binds to theN-terminal region of ASK1 and, thereby, activated JNK,

which may sensitize the cell to apoptosis (29). The results ofour study showed that a signaling cascade of Cdc42/ASK1/JNK/FasL in response to resveratrol-induced apoptosis.Therefore, we suggest that resveratrol-induced FasL expres-sion through the Cdc42/ASK1/JNK signaling pathway andincreased FasL may activate the same pathway, enhancingFasL expression and/or inducing apoptosis. This accumulatesmore evidence to support such an auto-enhancementhypothesis.In the present study, inhibition of FasL expression by genetic

inhibition of JNK, but not p38, indicates that resveratrol-induced activation of these two kinases may have differenteffects on FasL expression and apoptosis. Other studies(45,46) have shown that a single inhibition of JNK or p38MAPK does not prevent programmed cell death induced bysome stresses, such as Fas or ceramide. Nonetheless, combinedinhibition of JNK and p38 MAPK significantly reduced apo-ptosis; the activation of both kinases seems to be required forinduction of apoptosis in Jurkat cells (11). Likewise, JNKactivation was essential for the induction of apoptosis afterionizing radiation, ultraviolet light exposure, heat shock,TNFa or H2O2 (14--18). In some cell types, blockage of JNKactivity might be sufficient to prevent apoptosis by ceramide,Fas or stress, whereas in other cells, e.g. Jurkat cells, inhibitionof both JNK and p38MAPKwas required to prevent apoptosis.JNK and p38 MAPKs did not seem to be equally important in

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Fig. 5. Cdc42 activity is essential for the apoptosis induced by resveratrol. (A) HL-60 cells were treated with or without 20 mM resveratrol for indicated times,and the activity of small GTP-binding proteins were determined by GST pull-down assays described in the Materials and methods and the translocation ofCdc42 protein was assayed by western blot. (B) HL-60 cells were transfected with vector or myc-tagged dominant-negative mutants of Rac1 (N17 Rac1), Cdc42(N17 Cdc42) and RhoA (N19 Rho A), and analysis the expression level of these mutants by western blot with anti-myc antibody. The transfected cells then treatedwith resveratrol (20 mM) for 24 h. An apoptosis assay was performed for the detection of the hypodiploid cells (apoptotic cells) by flow cytometry. Columns,means of three independent experiments; bars, SD. Asterisk denotes a statistically significant decrease compared with values of vector control (�P50.05).(C) HL-60 cells were transfected with vector or dominant-negative mutants of Cdc42 (N17 Cdc42) and treated with resveratrol (20 mM) for 12 and 24 h for JNKkinase activity assay and FasL expression, respectively. The JNK kinases activity was determined by immune complex kinase assay (upper panel) and theFasL expression were determined by western blot (lower panel). Data are representative of three independent experiments.

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all forms of apoptosis, since inhibition of both kinases did notprevent apoptosis in Jurkat cells incubated with thapsigargine,a microsomal Ca2þ ATPase inhibitor. Resveratrol alsoinduced apoptosis through these two kinases in different celltypes (4,40,41). However, in this study, JNK-DN-transfectedHL-60 cells were almost completely resistant to induction ofFasL and apoptosis by resveratrol (Figure 2). In contrast,inhibition of resveratrol-induced p38 MAPK activity by treat-ment with SB203580 or transfection with the mutant p38expression vector (p38AF) did not decrease the induction ofFasL (Figure 3A and C), but slightly increased apoptosis in thepresence of resveratrol (Figure 3B). According to the aboveobservation, we presume that resveratrol-induced FasL expres-sion is p38-independent and the slight difference in apoptosismay be due to inhibition of p38-dependent survival signals,such as those from ERK1/2 (47--49). The ASK1/p38 MAPKsignal cascade is also reported to be involved in the differen-tiation and survival of other cells (50). Based on our observa-tions, we suggest that resveratrol-induced FasL expression andapoptosis are mediated through the JNK pathway, rather thanthe p38 MAPK pathway.Over-expression of wild-type or constitutively active ASK1

induced apoptosis in various cells through mitochondria-dependent caspase activation (51,52), and ASK1 was requiredfor apoptosis induced by oxidative stress, TNF and endoplas-mic reticulum stress (26,28,53). Several lines of evidencesuggest that ASK1 has diverse functions in deciding cell fate,such as differentiation and survival. We showed that resvera-trol activated ASK1/JNK signaling and this activation wasnecessary for FasL induction and apoptotic cell death(Figure 4). On the other hand, resveratrol-activated ASK1 alsoinduced p38 MAPK activity in HL-60 cells (data not shown),but there was no significant difference in the resveratrol-induced apoptosis with or without inhibition of p38 MAPK(Figure 3). Thus, we suggest that resveratrol-activated ASK1triggers phosphorylation of the downstream MAPK members,JNK and p38 MAPK, in individual ways. Resveratrol-inducedASK1/JNK and ASK1/p38 MAPK pathways also had differenteffects on FasL expression and apoptosis. We propose thatthese differences might be due to the different transcriptionfactors activated by JNK and p38 MAPK, inducing expressionof different genes. Our data confirm that ASK1 appears to be apivotal component not only in stress-induced cell death, butalso in a broad range of biological activities.Small GTP-binding proteins of the Rho family have been

traditionally linked to the reorganization of the actin-basedcytoskeleton (54). More recent evidence has revealed thatRho-related GTPase can also regulate gene expressionoften through the activation of kinase cascades leading toenhanced activity of SAPKs, including JNK and p38 MAPkinase (38,39,55). The ability to stimulate SAPK also sug-gests that these Rho GTP-binding proteins can promote theinitiation of the apoptotic programs mediated by JNK andp38, thus resulting in cell death. To the best of our knowl-edge, there are currently no reports on Rho family smallGTP-binding protein being involved in resveratrol-inducedapoptosis. In the present study, we showed that resveratrol acti-vated Cdc42 but not other Rho family members (Figure 5A).Inhibition of Cdc42 also significantly reduced resveratrol-induced apoptosis in HL-60 cells (Figure 5B). Supportively,previous studies have reported that expression of the activeform of Cdc42 in Jurkat T lymphocytes (56) as well as in ratsympathetic neurons (57) induce an apoptotic response. Cdc42

regulates signaling by binding to downstream effector proteins(58) mainly through its CRIB domain (59). Reports have shownthat Cdc42 and its downstream effectors, PAKs and JNK/SAPK,are involved in stress-activated apoptosis (60). In the presentstudy, ASK1 may be the novel potent downstream events or thephosphorylation targets of Cdc42 that promote resveratrol-induced apoptosis. Cdc42 has been reported to recruit PAK1and phosphorylates JNK and thus induce subsequent apoptosis(60). In this study, the questions of whether Cdc42 can directlyinteract with ASK1 or JNKmolecules need further investigation.Other naturally occurring polyphenol agents, such as gar-

cinol, induced Rho family GTPase-related apoptosis throughD4-GDI, a hematopoietic cell-abundant regulator of Rhofamily GTPases in HL-60 cells (61). Similarly, resveratrol,just like garcinol, has antioxidant and free radical scavengingeffects and antibacterial/antifungal activities. In contrast tothis, we found that Cdc42 played a crucial role in resveratrol-induced apoptosis in the current study. Because of its criticalrole as a modulator of Cdc42, resveratrol might, in turn, have asignificant impact on the mechanisms that induce cytoskeletaland morphological changes in apoptotic cells.In conclusion, we present the first evidence demonstrating

that the cancer-chemopreventive agent resveratrol induces anovel Cdc42-dependent apoptosis program. We delineated theresveratrol-induced apoptosis-signaling pathway in whichCdc42 is initially activated and which, in turn, activated theASK1/JNK pathway that triggered the expression of FasL andresulted in apoptosis of HL-60 cells (Figure 6).

Fig. 6. Resveratrol induced apoptosis via the novel signaling cascadeCdc42/ASK1/JNK/FasL. Resveratrol induced Cdc42 expression, andactivates ASK1/JNK pathway that triggers the expression of FasL andinduces apoptosis of HL-60 cells.

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Received April 8, 2004; revised and accepted June 15, 2004

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