Resveratrol, an antioxidant present in red wine, induces apoptosis in human promyelocytic leukemia (HL-60) cells

Cancer Letters 140 (1999) 1±10

Resveratrol, an antioxidant present in red wine, induces apoptosis in human promyelocytic leukemia (HL-60) cells Young-Joon Surh a,*, Yeon-Jin Hurh a, Jee-Young Kang a, Eunyong Lee a, Gu Kong b, Su Jeen Lee b a

Laboratory of Biochemistry and Molecular Toxicology, College of Pharmacy, Seoul National University, Shinlim-dong, Kwanak-gu, Seoul 151-742, South Korea b Department of Pathology, College of Medicine, Hanyang University, Seoul 133-791, South Korea Received 27 October 1998; received in revised form 6 January 1999; accepted 7 January 1999

Abstract Resveratrol, a triphenolic stilbene present in grapes and other plants, has striking antioxidant and anti-in¯ammatory activities which have been considered to be responsible for the bene®cial effects of red wine consumption on coronary heart disease. Recent studies reveal that resveratrol can inhibit each step of multistage carcinogenesis. However, the molecular mechanisms underlying anti-tumorigenic or chemopreventive activities of this phytochemical remain largely unknown. In the present work, we have found that resveratrol reduces viability and DNA synthesis capability of cultured human promyelocytic leukemia (HL60) cells. The growth inhibitory and antiproliferative properties of resveratrol appear to be attributable to its induction of apoptotic cell death as determined by morphological and ultrastructural changes, internucleosomal DNA fragmentation, and increased proportion of the subdiploid cell population. Resveratrol treatment resulted in a gradual decrease in the expression of anti-apoptotic Bcl-2. These results, together with previous ®ndings, suggest the cancer therapeutic as well as chemopreventive potential of resveratrol. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Resveratrol; Apoptosis; Programmed cell death; Chemoprevention; Human promyelocytic leukemia (HL-60) cells

1. Introduction A wide variety of naturally occurring phenolic substances have been reported to possess substantial anti-tumorigenic activities [1,2]. Resveratrol (3,5,4 0 trihydroxy-trans-stilbene), a phytoalexin present in grapes and other food products, has been suggested as a potential cancer chemopreventive agent based on its striking inhibitory effects on diverse cellular events

* Corresponding author. Tel.: 1 82-2-8807845; fax: 1 82-28749775. E-mail address: [email protected] (Y.J. Surh)

associated with tumor initiation, promotion, and progression [3]. The compound has strong antioxidative and anti-in¯ammatory activities which may contribute to its chemopreventive/chemoprotective properties [3±6]. Recent work by Uenobe and colleagues [7] demonstrated an antimutagenic activity of resveratrol against the food-borne heterocyclic amine, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) in bacteria. Resveratrol attenuated PC12 cell death induced by reactive oxygen species [8]. Synthetic resveratrol has also been found to inhibit growth of human breast epithelial cells in culture [9] and also proliferation of K-562 human erythroleukemia and P-815 murine mastocytoma cells, which

0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00039-7

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may be associated with suppression of ribonucleotide reductase [10]. Recently, considerable attention has focused on the sequence of events referred to as programmed cell death or apoptosis and the role that this process may play in the pathogenesis and treatment of various human diseases including cancer [11]. The maintenance of homeostasis in normal mammalian tissues re¯ects a critical balance between cell proliferation and cell death via apoptosis. In contrast, apoptosis may be inhibited or perturbed in tumors in which the rate of cell proliferation exceeds that of cell loss. If misregulation of apoptosis results in a failure of tissue size regulation, which eventually leads to the malignant transformation, apoptotic cell death could be induced to augment interventions designed to suppress or reverse the development of cancer. Indeed a variety of cytostatic/cytotoxic drugs have been reported to induce apoptosis in malignant cells in vitro [12,13]. It is hence conceivable that dietary and/or pharmacological manipulation of apoptosis may provide ef®cient and promising treatment strategies to protect against cancer [14]. The present study was designed to investigate the possibility that resveratrol could suppress cancer cell growth by inducing apoptotic death. 2. Materials and methods 2.1. Chemicals Resveratrol (,99% pure), 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT), sodium dodecylsulfate (SDS), and 4 0 ,6-diamidino-2phenylindole (DAPI) were purchased from Sigma Chemical Co. (St. Louis, MO). [methyl- 3H]Thymidine was obtained from Amersham (Arlington Heights, IL). 2.2. Cell culture Human promyelocytic leukemia (HL-60) cells were obtained from American Type Culture Collection (Rockville, MD) and maintained at 378C in a humidi®ed atmosphere of 95% air and 5% CO2 in RPMI-1640 (Celox, Hopkins, MN) supplemented with 0.5% (v/v) heat inactivated fetal bovine serum (Gibco BRL; Grand Island, NY), 5 ml/ml gentamicin, and 2 mM glutamine. Cell growth was measured by

the MTT colorimetric dye reduction method as described previously [15]. 2.3. Determination of DNA synthesis To assess the cell proliferation, tritiated thymidine incorporation was utilized as a measure of DNA synthesis. Exponentially growing cells were treated with [methyl- 3H]thymidine (sp. act., 54 Ci/mmol) at a ®nal concentration of 1 mCi/ml in the absence or presence of varying concentrations of resveratrol. The cells were incubated for 8 h, harvested by removing the incubation media, rinsed with phosphate-buffered saline (PBS) and 10% trichloroacetic acid, and treated with 0.5% SDS in 0.2 N NaOH at 378C for 30 min. The extent of [ 3H]thymidine incorporation was measured by liquid scintillation counting. 2.4. Morphological examination HL-60 cells (1 £ 106 cells/well) grown in 6-well plates were treated with 100 mM of resveratrol at 378C for 8 h. Morphological changes occurring in the cells were observed under a phase-contrast microscope and photographed. Ultrastructural examination was performed by conventional techniques using a Hitachi H-600 transmission microscope. 2.5. Nuclear staining with DAPI HL-60 cells (1 £ 106 cells/ml) were cultured in 6well dishes in RPMI-1640 medium containing 0.5% fetal bovine serum in the absence or presence of 100 mM resveratrol. After 7 h, a 1-ml aliquot was drawn from the suspension and centrifuged at 1200 rev./min for 2 min. To the cell pellet was added 4% neutral buffered formalin (100 ml). An aliquot (50 ml) was transferred to slides, which were left at room temperature for dryness. The ®xed cells were washed in PBS, air dried, and stained with the DNA-speci®c ¯uorochrome DAPI for 1 min. The adhered cells were washed with PBS, air dried, and mounted with 90% glycerol. The slides were observed under ¯uorescent microscopy. 2.6. Analysis of DNA fragmentation by agarose gel electrophoresis HL-60 cells at a density of 1 £ 106 cells/ml were treated with various concentrations of resveratrol for

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DNA was subjected to electrophoresis on 1.8% agarose gel at 5 V/cm and visualized under UV light after staining with ethidium bromide. The presence of apoptosis was indicated by the appearance of a ladder of oligonucleosomal DNA fragments which are approximately 180±200 bp multiples. An Eco RI± Hind III digest of lambda phage DNA was used as a molecular size standard. 2.7. Flow cytometry analysis HL-60 cells at a density of 5 £ 105 /ml were treated with resveratrol (100 mM) in dimethylsulfoxide (DMSO) or the solvent alone. After the allotted time in culture, cells were harvested by centrifugation, washed with PBS and resuspended at a density of 1 £ 106 cells/ml in 70% ethanol for 1 h. Cells were centrifuged, and the cell pellets were stained with DAPI. DNA content was then analyzed with a ¯ow cytometer (Fuji Partec Model PAS III). 2.8. Western blot analysis

Fig. 1. Effects of resveratrol treatment on viability (upper panel) and proliferation (lower panel) of HL-60 cells in culture. HL-60 cells were exposed to indicated amounts of resveratrol for 8 h as described in Section 2. The percent viability was determined by MTT dye reduction and each data point represents an average of two independent experiments. For the data on [ 3H]thymidine incorporation, values are the mean ^ SEM of triplicate samples analyzed in a typical experiment.

8 h. The cells were harvested by centrifugation at 1200 rev./min for 3 min, and washed with PBS. The pellet was lysed in a buffer containing 50 mM Tris± HCl, pH 8.0, 10 mM EDTA, 0.5% SDS, and 0.5 mg/ ml proteinase K (Sigma Chemical Co., St. Louis, MO) at 508C for 1 h. The mixtures were extracted with phenol±chloroform (1:1, v/v) and the aqueous phase was further extracted with chloroform/isoamyl alcohol (24:1, v/v). To the aqueous phase was added 4 M NaCl to make the ®nal concentration 0.5 M. DNA was precipitated with two volumes of ethanol. After recovery by centrifugation, the DNA pellet was washed with 70% ethanol, dried in air and suspended in TE buffer consisting of 10 mM Tris±HCl (pH 8.0) and 1 mM EDTA. After quantitation of DNA, 10 mg

After treatment with resveratrol (100 mM), about 1 £ 107 cells were washed twice with PBS and lysed in ice-cold 100 ml lysis buffer (1% Triton X-100, 0.15 M NaCl, 10 mM Tris±HCl, pH 7.4, 1 mM EDTA, 1 mM phenylmethylsulfonyl ¯uoride, 0.23 units/ml aprotinin, and 10 mM leupeptin) for 60 min. Lysates were centrifuged at 12 000 rev./min for 10 min, and aliquots of the supernatant containing 30±50 mg protein were boiled in SDS sample loading buffer for 5 min before electrophoresis on 12% SDS±polyacrylamide gel. After overnight transfer of SDS±polyacrylamide gel to nitrocellulose membrane, the blots were blocked with 5% non-fat dry milk in TBS-T buffer (50 mM Tris±HCl, pH 7.4, 0.15 mM NaCl, 0.1% Tween 20) for 2 h at room temperature and then washed in TBS-T buffer. Nitrocellulose membranes were incubated for 1 h at room temperature with a mouse Bcl-2 monoclonal antibody (Boehringer Mannheim, Germany) or rabbit polyclonal antibody against human Bax (Santa Cruz Biotechnology). Blots were rinsed with TBS-T and then incubated with a 1:1000 dilution of horseradishconjugated secondary antibody and again washed in TBS-T buffer for 10 min three times. The transferred proteins were visualized with an enhanced chemiluminescence (ECL) detection kit (Amersham Life

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Fig. 2. Morphological changes induced by resveratrol in HL-60 cells. Upper: phase-contrast photomicrographs of HL-60 cells treated with DMSO alone (A) or 100 mM resveratrol (B) for 8 h. Lower: ¯uorescence microscopic examination of untreated (C) HL-60 cells or those treated with 100 mM resveratrol (D) for 7 h followed by DAPI staining.

Sciences, Arlington Heights, IL), according to the manufacturer's instructions. 3. Results Treatment of HL-60 cells with resveratrol was accompanied by marked growth inhibition which was concentration dependent (Fig. 1, upper panel). The addition of resveratrol at 50 mM for 8 h caused about 45% reduction in the cell viability as determined by the conventional MTT assay. The effects of resveratrol on cell proliferation were assessed by measuring DNA synthesis using a radiolabeled precursor. In parallel with the suppression of cell viability, cell proliferation assessed in terms of nuclear incorporation of [ 3H]thymidine decreased

with increasing amounts of resveratrol added to the medium (Fig. 1, lower panel). To determine whether the growth inhibitory and antiproliferative effects of resveratrol are associated with programmed cell death or apoptosis, we initially examined the morphological changes of HL-60 cells treated with 100 mM resveratrol for 8 h. Cells exposed to resveratrol exhibit distinct morphological features of programmed cell death, such as cellular shrinkage and apoptotic body formation (Fig. 2B). Changes in nuclear morphology are also typical of apoptosis: staining of resveratroltreated cells with a DNA binding dye DAPI visualized the nuclei broken into chromatin-containing fragments of various sizes (Fig. 2D). Apoptotic cell death was further con®rmed by transmission electron microscopy, which revealed characteristic ultrastructural features of apoptosis. Thus, while control HL-60

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Fig. 3. Transmission electron microscopy of HL-60 cells treated with resveratrol (100 mM) for 8 h (£12 000). The section of ®xed cells reveals typical early nuclear features of apoptosis, including marginal condensation of nuclear chromatin, lysis of nucleolus, nuclear fragmentation, and swelling of cytoplasmic organelles.

cells showed intact cytoplasmic organelles and wellpreserved nuclear membranes as well as evenly distributed chromatin (data not shown), cells treated with resveratrol exhibited characteristic chromatin condensation at the periphery of the nucleus (Fig. 3). The moderate to severe swelling of intracellular organelles was also evident. In addition to morphological evaluation, apoptosis

induction by resveratrol was ascertained by using an assay developed to measure DNA fragmentation, a biochemical hallmark of programmed cell death. As illustrated in Fig. 4, agarose gel electrophoresis of DNA extracted from HL-60 cells treated with resveratrol at concentrations ranging from 10 to 200 mM for 8 h revealed a progressive increase in the non-random fragmentation into a ladder of 180-bp nucleosomes.

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Fig. 4. Detection of internucleosomal DNA fragmentation in HL-60 cells treated with 0 (lane 1), 10 (lane 2), 25 (lane 3), 50 (lane 4), 100 (lane 5), and 200 (lane 6) mM of resveratrol for 8 h. Nuclear DNA was extracted and analyzed by conventional agarose gel electrophoresis followed by ethidium bromide staining. The ®rst lane designated as `m' represents 1 kb ladder used as a molecular marker.

The induction of apoptosis in resveratrol-treated HL-60 cells was further veri®ed by ¯ow cytometric analysis of DNA content. Loss of DNA is a typical feature of apoptotic cells that occurs as a result of diffusion of degraded DNA out of ®xed cells after endonuclease cleavage. DNA content histograms obtained from DAPI-stained HL-60 cells reveals that the percentage of cell populations with reduced DNA content increased from 3% in untreated cells to 69% in cells exposed for 48 h to 100 mM resveratrol (Fig. 5). In parallel with progressive increases in the percentage of cells with reduced or subdiploid DNA content, there was a concomitant fall in the proportion of cells with diploid DNA. Diminished expression of the anti-apoptotic oncoprotein Bcl-2 has been observed in certain types cells undergoing apoptotic death [16,17]. In line with this notion, the levels of Bcl-2 decreased gradually following resveratrol treatment for up to 48 h (Fig. 6). 4. Discussion Chemoprevention, which refers to the use of nontoxic chemical substances to inhibit, delay and/or

reverse cellular events associated with carcinogenesis, is regarded as a promising alternative strategy to therapy for the management of cancer [18]. A vast variety of naturally occurring substances have been shown to protect against experimental carcinogenesis and it is becoming increasingly evident that certain phytochemicals, particularly those included in our daily diet have marked cancer chemopreventive properties [1,2]. Resveratrol is one such dietary chemopreventive phytochemical that has recently attracted considerable interest because of its remarkable multi-functional inhibitory effects on multi-stage carcinogenesis [3]. One of the plausible mechanisms that could account for the chemopreventive activity of resveratrol is its suppression of prostaglandin biosynthesis catalyzed cyclooxygenases. Prostaglandins are known to play pivotal roles in the pathogenesis of malignancy, particularly in colon carcinogenesis, and certain non-steroidal anti-in¯ammatory drugs (NSAIDs), such as aspirin, piroxicam, and sulindac, have protective effects on experimental carcinogenesis [19±22]. Furthermore, epidemiological data indicate a reduced risk of colorectal cancer among individuals who ingest aspirin or other NSAIDs on a regular basis [23]. Moreover, in patients with familial adenomatous polyposis, the NSAID sulindac caused marked regression of polyps and prevented their recurrence [24]. Suppression of prostaglandin biosynthesis through selective inhibition of cyclooxygenase is hence now regarded as an important cancer chemopreventive strategy. In line with this notion, resveratrol was shown to inhibit cyclooxygenase-1 (COX-1) activity in microsomes derived from sheep seminal vesicles [3]. More recently, Subbaramaiah and co-workers [25] reported that resveratrol inhibits the catalytic activity of cyclooxygenase-2 (COX-2) in human mammary epithelial cells in culture with and without phorbol-12-myristate-13acetate treatment. Likewise, human recombinant COX-2 expressed in baculovirus was inhibited by resveratrol [25]. Besides inhibiting the catalytic activity of COX-2, the compound also blocked phorbol ester-mediated induction of COX-2 mRNA in cultured human mammary epithelial cells via repression of AP-1-dependent transactivation [25]. Some anti-in¯ammatory chemopreventive agents have been found to suppress growth and proliferation of transformed or malignant cells through induction of

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Fig. 5. Flow cytometric analysis of DNA cleavage in resveratrol-treated cells. Cells were exposed to 100 mM resveratrol and stained with DAPI for DNA content analysis.

programmed death or apoptosis [26,27]. Although resveratrol exerts anti-in¯ammatory effects through selective suppression of COX-2, little information is available in the literature regarding its capability to induce apoptotic death. Mgbonyebi et al. [9] reported the growth inhibition by resveratrol of several types of human breast epithelial cells, which was independent of the estrogen receptor status of the cells. Although

Fig. 6. Effect of resveratrol treatment on Bcl-2 expression in HL-60 cells. The cells were treated with resveratrol for 0 (lane 1), 6 (lane 2), 24 (lane 3), and 48 (lane 4) h and total cell lysates were subjected to SDS±PAGE as described in Section 2.

the antiproliferative activity of resveratrol against cultured human breast epithelial cells was demonstrated [9], data on the mode of cell death induced by this phytochemical were not provided in this study. Our present investigation clearly demonstrates that resveratrol induces apoptosis in HL-60 cells in culture, which appears to account for its growth inhibitory and antiproliferative activities. The induction of apoptotic cell death was accompanied by characteristic morphological and ultrastructural changes, such as chromatin condensation, nuclear fragmentation, and collapse of the cell into apoptotic bodies. Internucleosomal DNA fragmentation as determined by agarose gel electrophoretic analysis also supports the progress of apoptosis in the resveratrol-treated cells. Flow cytometry allows a simultaneous estimation of cell cycle parameters and apoptosis. There is

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compelling evidence that apoptotic death induced by chemopreventive or chemotherapeutic agents is closely linked to perturbation of a speci®c phase of the cell cycle. For instance, epigallocatechin-3gallate, a major polyphenol present in green tea, arrests the cancer cells in the G0/G1 phase [28], while another chemopreventive phytochemical apigenin induces G2/M arrest in cultured keratinocytes, possibly through suppression of p34 cdc2 kinase activity and perturbation of cyclin B1 levels [29]. The effects of a given antiproliferative agent on cell cycle progression appear to depend on the concentration of the compound and/or the duration of the treatment. For some topoisomerase II inhibitors, such as teniposide, amsacrine and fostriecin, the accumulation of cells in the G2/M and S phases of the cell cycle was evident at relatively low concentrations, whereas, at higher concentrations, there was a dramatic increase in the proportion of cells with hypodiploid DNA content [30±32]. Treatment of the acute leukemic Tlymphocyte cell line CCRF-CEM.f2 cells for 4 h with the cytotoxic drug vincristine at concentrations ranging from 0.01 to 1 mM resulted in G2/M arrest [33]. After 18 h of drug exposure, however, the G1 peak disappeared and subpopulations of cells with apoptotic features were apparent [33]. In another study, treatment of HL-60 cells for 24 h with a cholestane glycoside from Ornithogalum saundersiae caused a decrease of G0/G1 cells and a concomitant increase of apoptotic cells [34]. Likewise, time course analysis of resveratrol-treated HL-60 cells by ¯ow cytometry in the present study demonstrated the reduced cell cycle distribution of the residual viable cells, especially those in the G0/G1 phase, with a progressive increase in the percentage of the subpopulation of cells with hypodiploid DNA. The gradual reduction in the proportion of unaffected cells in the G0/G1 phase with concomitant appearance of apoptotic cells after resveratrol treatment suggests that cells arrested in the G0/G1 phase were preferentially undergoing apoptosis in these cultures. While our manuscript was in preparation, Pervaiz and colleagues [35] reported that resveratrol induces apoptosis in both solid and non-solid cancer cells by triggering the CD95 signaling system. In this particular study, HL-60 cells maintained in RPMI-1640 supplemented with 10% fetal bovine serum were exposed to resveratrol at concentrations of up to 32

mM for 24 or 48 h. Treatment of HL-60 cells with 32 mM resveratrol resulted in more than 50% cell death 24 h after treatment, in comparison with less than 40% mortality observed with the same concentration of the compound in our study in which cells were maintained for a shorter period of time, i.e. 8 h in media with lower sera content. The growth of estrogendependent T47D breast carcinoma cells was also suppressed by treatment of 32-mM resveratrol [35]. The latter ®nding is contradictory to the result of previous work by Gehm et al. [36] who demonstrated stimulation of T47D cell proliferation by resveratrol at a concentration of 10 mM. The reason for this discrepancy remains unresolved. Apoptosis is a tightly regulated process, which involves changes in the expression of distinct genes. One of the major genes involved in regulating apoptosis is the protooncogene bcl-2 that encodes a 26-kDa mitochondria-associated protein. The bcl-2 gene product prolongs the cell survival by blocking apoptosis induced by a wide variety of stimuli and treatment [16,17]. Furthermore, bcl-2-transfected cells are often rescued from apoptotic death [37]. However, other studies have revealed that apoptosis is not accompanied by down-regulation of Bcl-2 [38,39]. The results of our present study indicate the apparent decrease in Bcl-2 expression in HL-60 cells with resveratrol treatment for up to 48 h. It becomes evident that cytoplasmic aspartatespeci®c cysteine proteases of the ICE/CED-3 family, collectively known as 'caspases', play an important role in driving apoptosis [40]. Some of these proteases, including CPP32/Yama/apopain (caspase3), can inactivate poly(ADP-ribose)polymerase (PARP) by cleaving this 116 kDa enzyme into a proteolytic 85 kDa fragment [27,36,41]. In agreement with this notion, treatment of HL-60 cells with resveratrol gave rise to proteolytic cleavage of a caspase substrate PARP while caspase inhibitors rescued the cells from resveratrol cytotoxicity [35]. Our data presented herein, together with previous reports by other investigators, suggest that resveratrol has chemotherapeutic as well as chemopreventive potential. Additional studies are required to evaluate the apoptosis-inducing activity of resveratrol in diverse cancer cell lines and also to ascertain if resveratrol can suppress, retard, or reverse the carcinogenic processes in vivo.

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