Induction of Apoptosis by 3,4′-Dimethoxy-5-hydroxystilbene in Human Promyeloid Leukemic HL-60 Cells

• Abstract
• Introduction
• Materials and Methods
• Materials
• Cell culture
• Cell viability assay
• DNA fragmentation
• FACS analysis
• Preparation of mitochondrial and cytosolic extracts
• Western blot analysis
• Results
• Discussion
• References

Abstract

3, 4′-Dimethoxy-5-hydroxystilbene (DMHS) is a hydroxystilbene compound obtained by methylation and acid hydrolysis of piceid (resveratrol-3-O-glucoside) from Polygonum cuspidatum. Herein, we report that DMHS induces programmed cell death or apoptosis in human promyelocytic leukemic HL-60 cells. We found that treatment of HL-60 cells with DMHS suppressed the cell growth in a concentration-dependent manner with an IC₅₀ value of 25 μM. DMHS increased internucleosomal DNA fragmentation in a time-dependent manner. The cell death by DMHS was partially prevented by the caspase inhibitor, zVAD-fmk. DMHS caused activation of caspases such as caspase-3, -8, and -9. Immunoblot experiments revealed that DMHS-induced apoptosis was associated with the induction of Bax expression. The release of cytochrome c from mitochondria into the cytosol was increased in response to DMHS. Taken together, our present results indicated that DMHS leads to apoptotic cell death in HL-60 cells through increased Bax expression and release of cytochrome c into cytosol and may be considered as a good candidate for a cancer chemopreventive agent in humans.

Introduction

Resveratrol (trans-3,4′,5-trihydroxystilbene), a hydroxystilbene compound occurring naturally in grapes and a variety of medicinal plants, was considered as a chemopreventive agent because it has anticarcinogenic and anti-inflammatory activities [1]. It decreased the development of preneoplastic lesions in carcinogen-treated mouse mammary glands and inhibited tumorigenesis in a two-stage mouse skin cancer model. Resveratrol suppressed prostaglandin synthesis via inhibition of cyclooxygenases 1 and 2 [1], [2]. It induced quinone reductase in mouse hepatoma cells and promoted HL-60 cell differentiation. The chemopreventive effect of resveratrol has been related to its ability to induce apoptosis [3], [4], [5].

Apoptosis is described by its morphological characteristics, including plasma membrane blebbing, cell shirinkage, nuclear condensation, chromosomal DNA fragmentation, and formation of apoptotic bodies [6]. It plays an important role as a protective mechanism in the organism by removing damaged cells or over-proliferating cells due to an improper mitotic stimulus. However, inappropriate regulation of apoptosis may cause many serious disorders such as neural degeneration, AIDS, autoimmune disease, and cancers. Many anticancer drugs or cancer chemopreventive agents may act though the induction of apoptosis to prevent tumor promotion and progression. Apoptosis is mediated by activation of caspases, a family of cysteine proteases [7]. Caspases are synthesized as relatively inactive precursor forms, and an apoptotic signal converts the precursors to active enzymes. Once activated, caspases cleave a variety of intracellular polypeptides, including major structural elements of the cytoplasm and nucleus, components of the DNA repair machinery, and a number of protein kinases [8]. Resveratrol-induced apoptosis is mediated via the activation of caspase-3 and p53-dependent pathways [3], [4]. Clement et al. [4] demonstrated that resveratrol inhibited the growth of T47D cells by inducing CD95/Fas-mediated apoptosis. However, Fas-independent apoptosis by resveratrol in THP-1 human monocytic leukemia cells was also reported [5].

To find new cancer chemopreventive agents from various hydroxystilbene compounds having a similar chemical structure to resveratrol, we examined the cytotoxic effects of hydroxystilbenes in human promyelocytic leukemia HL-60 cells. Of four compounds tested, 3,4′-dimethoxy-5-hydroxystilbene (DMHS) exhibited a strong inhibition of HL-60 cell growth. The biochemical mechanism of cytotoxic signaling in response to DMHS was studied and we suggest that DMHS induces apoptosis through a caspase-dependent signal pathway.

Materials and Methods

Materials

Hydroxystilbenes used were isolated from herbal extracts or obtained by chemical modification (Table [1]) [9]. Rhapontigenin (1), 3,5-dihydroxy-4′-methoxystilbene (2) were purified from Rheum undulatum L. Oxyresveratrol (3) was isolated from Morus alba L. Resveratrol was isolated from Veratrium album var. grandiflorum Maxim. Piceid was isolated from Polygonum cuspidatum Sieb. et Zucc., and DMHS (4) was obtained by methylation of piceid followed by the acid hydrolysis. Anti-human caspase-8, anti-human caspase-9, anti-human Bax, anti-human Bcl-2, and anti-human β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-poly(ADP-ribose) polymerase (PARP) antibody was from Roche Molecular Biochemicals (Mannheim, Germany). Anti-cytochrome c antibody was purchased from Amersham Pharmacia Biotech. (Piscataway, NJ). ZVAD-fmk was obtained from Enzyme Systems Products (Livermore, CA).

Cell culture

The human promyelocytic leukemia HL-60 cells were grown in RPMI 1640 medium supplemented with 10 % fetal bovine serum, 100 units penicillin/ml, 100 μg streptomycin/ml, and 2 mM L-glutamine at 37 ^oC in 5 % CO₂ humidified incubator. All experiments were carried out at 37 ^oC and 5 % CO₂ on confluent cells in the medium.

Cell viability assay

Cells were plated at a density of 5 × 10⁴ cells/well into 96-well plates. All hydroxystilbenes were dissolved in DMSO. The final concentration of DMSO in the culture medium was controlled to less than 0.1 %. Following 24 h of incubation with the chemicals, the cell viability was assayed with the MTT colorimetric dye reduction method.

DNA fragmentation

Cells were collected by centrifugation at 2,000 × g for 5 min and then washed twice with ice-cold PBS (pH 7.4). The cell pellets were resuspended in 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM EDTA, 10 % (w/v) SDS, and 0.5 mg/ml proteinase K. Proteolytic digestion was allowed to proceed at 50 ^oC for 12 - 16 h. The samples were extracted with phenol/chloroform and DNA was precipitated with 100 % ethanol and 7.5 M ammonium acetate. The isolated DNA fragments were separated by 1 % (w/v) agarose gel electrophoresis, stained with 0.5 μg/ml ethidium bromide, and analyzed under ultraviolet light.

FACS analysis

Cells were harvested by centrifugation at 2,000 × g for 5 min, washed, and resuspended in 0.2 ml phosphate-buffered saline (PBS). Cells were fixed in 70 % ethanol. After fixation, medium was removed by centrifugation and 0.4 ml PBS was added. Cells were stained with propidium iodide (40 μg/ml) in the presence of DNase-free RNase A (0.1 mg/ml) for 30 min. Stained cells were analyzed by flow cytometry with the excitation at 448 nm.

Preparation of mitochondrial and cytosolic extracts

Cells were harvested by centrifugation at 2,000 × g for 5 min, washed twice with ice-cold PBS, and resuspended in buffer A [20 mM Hepes buffer (pH 7.5) containing 10 mM KCl, 1.5 mM MgCl₂, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, 250 mM sucrose, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 μg/ml chymostatin]. Cells were disrupted by homogenization. Homogenates were centrifuged at 1,000 × g for 10 min at 4 °C. After centrifugation, supernatants were further centrifuged at 10,000 × g for 20 min at 4 °C. The resulting supernatants were stored at -20 °C. The 1,000 × g pellets were resuspended in buffer A and centrifuged at 10,000 × g for 20 min at 4 °C. Mitochondrial pellets were resuspended in 50 μl of ice-cold 10 mM Tris-acetate buffer (pH 8.0) containing 0.5 % NP-40 and 5 mM CaCl₂ and stored at -20 °C.

Western blot analysis

Protein samples were fractionated by SDS-PAGE and the separated proteins were transferred onto a nitrocellulose membrane. The membrane was stained with Ponceau S to confirm equal loading and transfer of proteins. Membranes were blocked with 5 % (w/v) non-fat dry milk in 20 mM Tris-HCl (pH 7.4) containing 8 mg/ml NaCl and 0.05 % (w/v) Tween 20 (TBS) at room temperature overnight and incubated with primary antibodies at room temperature for 2 h. The membranes were washed three times with TBS and blotted with secondary antibodies conjugated with horse-radish peroxidase at room temperature for 1 h, followed by three washes in TBS. Immunoreactive proteins were visualized by the enhanced chemiluminescence (ECL) procedure according to the manufacturer′s protocol (Amersham Pharmacia).

Results

Previous studies have shown that resveratrol causes cell growth inhibition and apoptosis in cultured tumor cells [3], [4], [5]. Here we selected four resveratrol analogs to find new cancer chemopreventive agents: rhapontigenin, oxyresveratrol, DMHS, and 3,5-dihydroxy-4′-methoxystilbene. The structures of these hydroxystilbenes are illustrated in Table [1]. Their cytotoxic effects on cell viability in HL-60 cells were determined by a MTT assay (Fig. [1]). Of four compounds tested, DMHS exhibited a significant inhibition of HL-60 cell growth with an IC₅₀ of 25 μM (Fig. [1]). However, oxyresveratrol, rhapontigenin, or 3,5-dihydroxy-4′-methoxystilbene had little inhibitory effects on HL-60 cell proliferation. DMHS inhibited HL-60 cell growth in a time-dependent manner (Fig. [2] A). To characterize whether the cell death induced by DMHS was mechanism-dependent, DNA fragmentation by DMHS in HL-60 cells was determined. Within 24 h of DMHS treatment, HL-60 cells exhibited significant amounts of DNA ladder formation (Fig. [2] B). Flow cytometric analysis of propidium iodide-stained cells also showed the induction of apoptosis by DMHS treatment (Fig. [2] C). These results suggested that DMHS induced apoptotic cell death in HL-60 cells.

Activation of caspases may play a central role in the execution stage of apoptosis. We asked whether caspase-dependent signal pathways are involved in the apoptotic cell death induced by DMHS in HL-60 cells. Treatment of HL-60 cells with 50 μM DMHS for 24 h induced the cleavages of PARP, procaspase-8, and -9 (Fig. [3]). Moreover, 50 μM of zVAD-fmk, a caspase inhibitor, blocked the cell death induced by DMHS (Fig. [4]).

To determine the mechanism of apoptotic cell death by DMHS we measured the change in proapoptotic Bax protein expression in HL-60 cells. Treatment of HL-60 cells with 1, 5, 10, 25, or 50 μM DMHS increased Bax expression in a concentration-dependent manner (Fig. [5] A). However, no significant change in β-actin expression was observed during DMHS treatment. Because increased Bax expression triggers cytochrome c release from mitochondrial membrane into cytosol, the effect of DMHS on the accumulation of cytochrome c in the cytosol was examined. Treatment of HL-60 cells with DMHS caused a significant release of cytochrome c from mitochondria into the cytosol (Fig. [5] B). Cytochrome c release was strongly increased at 24 h after cells were exposed to DMHS (Fig. [5] C). These results showed that increased Bax expression and cytochrome c release may be important in the biochemical mechanism by which DMHS induced apoptosis in HL-60 cells.

Discussion

Recently increasing interest has been paid to resveratrol as a cancer chemopreventive agent. Several mechanisms of the anticarcinogenic effect of resveratrol were investigated. Resveratrol induces apoptosis and arrests the cell cycle at the S/G₂ phase transition [10]. Resveratrol also induces quinone reductase, inhibits cyclooxygenase-1 and -2, and promotes cell differentiation [1], [2]. Moreover, resveratrol was shown to inhibit human cytochrome P450 1A1 in vitro and in cultured hepatoma cells [11], [12].

In this study, we have examined the cytotoxic effect of four hydroxystilbenes obtained from natural medicinal plants in HL-60 cells to find a good chemopreventive agent from resveratrol analogs. Of all the compounds screened, DMHS was found to be strong for inhibiting HL-60 cell growth measured by MTT assay with IC₅₀ values of 25 μM. DMHS was 2-fold more cytotoxic than resveratrol in HL-60 because the IC₅₀ value of resveratrol is about 50 μM [13]. However, rhapontigenin, oxyresveratrol, and 3,5-dihydroxy-4′-methoxystilbene had low inhibitory effects on cell proliferation of HL-60 cells. To investigate the mechanism of the cytotoxic effect by DMHS, we determined whether the programmed cell death was involved in cell death by DMHS. DMHS caused a concentration- and time-dependent inhibition of cell proliferation and the cells revealed the characteristics of apoptosis by DMHS. Treatment of HL-60 cells with a caspase inhibitor zVAD-fmk in the presence of DMHS prevented the suppression of cell proliferation by DMHS. These data suggested that caspase-dependent pathways were involved in the mechanism of DMHS-mediated apoptosis in HL-60 cells.

Caspases become activated by cleavage via upstream proteases in an intracellular cascade. Studies in cell-free systems and in transformed yeast cells have demonstrated that certain caspases are capable of efficient activation of other caspases [14]. For example, caspase-8 cleaves procaspases-3, -4, -7, and -9. Caspase-9 can activate caspases-3 and -7. Ligand-induced activation of membrane death receptors such as CD95/Fas/Apo-1, type 1 tumor necrosis factor α (TNFα) receptor (TNFR1), death receptor 3 (DR3), DR4, and DR5 triggers caspase-8 activation and induces cell death by acting as scaffolds for caspase activation [8]. The observation that procaspase-8 is cleaved to active caspase-8 by DMHS treatment suggests that the upstream part of procaspase-8 may be involved in the signal pathway. It was reported that anticancer drugs could induce apoptosis by activating the CD95 receptor system through inducing synthesis of CD95 ligand and the receptor [15]. The CD95 is also suggested as the selective target of human tumor cells by resveratrol [4]. Resveratrol treatment enhanced CD95 ligand expression on HL-60 cells and induced CD95 signaling-dependent cell death. We propose that DMHS may induce expression of CD95 and/or its ligand and therewith activate the CD95 pathway.

The Bcl-2 family members such as Bax, Bcl-2, and Bcl-x_L localize to the mitochondrial membrane. Bax is found as intracellular membrane-associated proteins of the outer mitochondrial membrane, endoplasmic reticulum, and nuclear envelope [16]. Oligomeric Bax was shown to form channels in lipid membranes and the role of Bax as a pro-death protein appears to be elicited through an intrinsic pore-forming activity, thus leading to release of cytochrome c from the mitochondrial intermembrane into the cytosol. The induction of apoptosis in tumor cells has been proposed to result from the inability of Bcl-2 to heterodimerize with Bax. Bax overexpression increases the sensitivity of cells to anticancer drugs due to the lack of free Bcl-2 in the cell. Our results showed that the induction of apoptosis by DMHS was accompanied by the overexpression of Bax. It was found that Bax could directly induce the release of cytochrome c from isolated mitochondria. In contrast, Bcl-2 prevents the release of cytochrome c not only in cells undergoing apoptosis but also when coadded with Bax to isolated mitochondria. A high ratio of Bax to Bcl-2 can release cytochrome c from mitochondria to cytosol. Apaf-1, a cytoplasmic protein that shares limited homology with the C. elegans ced-4 apoptosis regulator, binds the released cytochrome c and undergoes a conformational change that allows binding of procaspase-9 [17]. Apaf-1:procaspase 9 complexes cleave and activate procaspase-9. Activated caspase-9 also activates downstream caspase-3, leading to the cleavage of PARP, fodrin and lamin and apoptotic nuclear morphology. DMHS induced the cleavage of procaspase-9 and PARP, indicating the activation of caspases. Several studies suggest that ceramide acts as a lipid second messenger of apoptosis signaling in various cell lines [18]. TNFR1, CD95, γ-radiation, or anticancer drugs such as daunorubicin, vincristine, and cytosine arabinoside can induce the accumulation of ceramide. Although we did not measure the changes of intracellular ceramide in this study, the possibility of an effect of ceramide in DMHS-induced apoptosis remains to be investigated.

Recent in vivo and in vitro studies indicate that various potent chemopreventive agents such as sulindac, isothiocyanates, and N-acetylcysteine induce apoptosis in human tumor cell lines. These compounds are considered for use as anticarcinogenic agents because many anticancer drugs are known to induce apoptosis in tumor cells. The ability of DMHS to induce apoptosis in HL-60 implies the possibility to develop this compound as new chemopreventive/anticarcinogenic agents. Recently we studied that DMHS showed strong inhibition of human cytochrome P450 1A1 and 1B1 [19]. Because these P450 s are primarily involved in the metabolic activation of procarcinogens, DMHS may have strong cancer chemopreventive activity.

In summary, based on the results shown in the present study, we report for the first time that DMHS obtained from Polygonum cuspidatum induces apoptosis in HL-60 cells via a caspase-dependent mechanism and may be considered as a good candidate for a cancer chemopreventive agent.

References

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Prof. Dr. Young Jin Chun

College of Pharmacy

Chungang University

221 Huksuk-dong

Dongjak-gu

Seoul 156-756

Korea

Email: yjchun@chungang.edu

Fax: +82 2 825 5616

References

• 1 Jang M, Cai L, Udeani G O, Slowing K V, Thomas C F, Beecher C WW, Fong H HS, Farnsworth N R, Kinghorn A D, Mehta R G, Moon R C, Pezzuto J M. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes.  Science. 1997;  275 218-20
  CrossrefPubMedSearch in Google Scholar
• 2 Subbaramaiah K, Chung W J, Michaluart P, Telang N, Tanabe T, Inoue H, Jang M, Pezzuto J M, Dannenberg A J. Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells.  Journal of Biological Chemistry. 1998;  273 21 875-82
  PubMedSearch in Google Scholar
• 3 Huang C, Ma W Y, Goranson A, Dong Z. Resveratrol suppresses cell transformation and induces apoptosis through a p53-dependent pathway.  Carcinogenesis. 1999;  20 237-42
  CrossrefPubMedSearch in Google Scholar
• 4 Clement M V, Hirpara J L, Chawdhury S H, Pervaiz S. Chemopreventive agent resveratrol, a natural product derived from grapes, triggers CD95 signaling-dependent apoptosis in human tumor cells.  Blood. 1998;  92 996-1002
  CrossrefPubMedSearch in Google Scholar
• 5 Tsan M -F, White J E, Maheshwari J G, Bremner T A, Sacco J. Resveratrol induces Fas signaling-independent apoptosis in THP-1 human monocytic leukemia cells.  British Journal of Haematology. 2000;  109 405-12
  CrossrefPubMedSearch in Google Scholar
• 6 Wyllie A H. Apoptosis: an overview.  British Medical Bulletin. 1997;  53 451-65
  CrossrefPubMedSearch in Google Scholar
• 7 Earnshaw W C, Martins L M, Kaufmann S H. Mammalian caspases: structure, activation, substrates, and functions during apoptosis.  Annual Review of Biochemistry. 1999;  68 383-424
  CrossrefPubMedSearch in Google Scholar
• 8 Nagata S. Apoptosis by death factor.  Cell. 1997;  88 355-65
  CrossrefPubMedSearch in Google Scholar
• 9 Ryu S Y, Han Y N, Han B H. Monoamine oxidase-a inhibitors from medicinal plants.  Archives of Pharmacentical Research. 1988;  11 230-9
  PubMedSearch in Google Scholar
• 10 Hsieh T, Juan G, Darzynkiewicz Z, Wu J M. Resveratrol increases nitric oxide synthase, induces accumulation of p53 and p21(WAF1/CIP1), and suppresses cultured bovine pulmonary artery endothelial cell proliferation by perturbing progression through S and G2.  Cancer Research. 1999;  59 2596-601
  PubMedSearch in Google Scholar
• 11 Chun Y J, Kim M Y, Guengerich F P. Resveratrol is a selective human cytochrome P450 1A1 inhibitor.  Biochemical and Biophysical Research Communications. 1999;  262 20-4
  CrossrefPubMedSearch in Google Scholar
• 12 Ciolino H P, Yeh G C. Inhibition of aryl hydrocarbon-induced cytochrome P-450 1A1 enzyme activity and CYP1A1 expression by resveratrol.  Molecular Pharmacology. 1999;  56 760-7
  PubMedSearch in Google Scholar
• 13 Surh Y -J, Hurh Y -J, Kang J -Y, Lee E, Kong G, Lee S J. Resveratrol, an antioxidant present in red wine, induces apoptosis in human promyelocytic leukemia (HL-60) cells.  Cancer Letters. 1999;  140 1-10
  CrossrefPubMedSearch in Google Scholar
• 14 Kang J J, Schaber M D, Srinivasula S M, Alnemri E S, Litwack G, Hall D J, Bjornsti M A. Cascades of mammalian caspase activation in the yeast Saccharomyces cerevisiae .  Journal of Biological Chemistry. 1999;  274 3189-98
  CrossrefPubMedSearch in Google Scholar
• 15 Friesen C, Herr I, Krammer P H, Debatin K -M. Involvement of the CD95 (APO-1/FAS) receptor/ligand system in drug-induced apoptosis in leukemia cells.  Nature Medicine. 1996;  2 574-7
  CrossrefPubMedSearch in Google Scholar
• 16 Krajewski S, Tanaka S, Takayama S, Schibler M J, Fenton W, Reed J C. Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes.  Cancer Research. 1993;  53 4701-14
  PubMedSearch in Google Scholar
• 17 Liu X, Kim C N, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c.  Cell. 1996;  86 147-57
  CrossrefPubMedSearch in Google Scholar
• 18 Hannun Y A, Obeid L M. Ceramide: an intracellular signal for apoptosis.  Trends in Biochemical Science. 1995;  20 73-7
  CrossrefPubMedSearch in Google Scholar
• 19 Chun Y J, Ryu S Y, Jeong T C, Kim M Y. Mechanism-based inhibition of human cytochrome P450 1A1 by rhapontigenin.  Drug Metabolism and Disposition. 2001;  29 389-93
  PubMedSearch in Google Scholar

Prof. Dr. Young Jin Chun

College of Pharmacy

Chungang University

221 Huksuk-dong

Dongjak-gu

Seoul 156-756

Korea

Email: yjchun@chungang.edu

Fax: +82 2 825 5616