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Vol. 53, Issue 3, 402-407, March 1998
B
Institute of Pharmacology, Toxicology, and Pharmacy, Königinstrasse 16, D-80539 Munich, Germany (V.M.D., A.M.V.) and Department of Medicine II, Klinikum Grosshadern, University of Munich, D-81377 Munich, Germany (A.L.G.)
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Summary |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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The pharmacological role of garlic in prevention and treatment of
cancer has received increasing attention, but thorough investigations into the molecular mechanisms of action of garlic compounds are rare.
The present study demonstrates that ajoene, a major compound of garlic
induces apoptosis in human leukemic cells, but not in peripheral
mononuclear blood cells of healthy donors. The effect was dose and time
dependent. Apoptosis was judged by three criteria, morphology of cells,
quantification of subdiploid DNA content by flow cytometry, and
detection of DNA fragmentation by gel electrophoresis. Ajoene increased
the production of intracellular peroxide in a dose- and time-dependent
fashion, which could be partially blocked by preincubation of the human
leukemic cells with the antioxidant N-acetylcysteine.
Interestingly, N-acetylcysteine-treated cells showed a
50% loss of ajoene-induced apoptosis. Moreover, ajoene was
demonstrated to activate nuclear translocation of the transcription factor nuclear factor 
B, an effect that was abrogated in
N-acetylcysteine-loaded cells. These results suggested
that ajoene might induce apoptosis in human leukemic cells via
stimulation of peroxide production and activation of nuclear factor
B. This is a novel aspect in the biological profile of this garlic
compound and an important step in elucidating the underlying molecular
mechanisms of its antitumor action.
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Introduction |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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The
role of dietary compounds as drugs in cancer prevention and treatment
is widely discussed (Pezzuto, 1993
; Agarwal, 1996; Koch and Lawson,
1996; Milner, 1996). In this regard the potential chemopreventive
effect of garlic (Allium sativum) was subject of various
clinical trials (Dorant et al., 1993; Steinmetz et al., 1994; Agarwal, 1996; Dorant et al., 1996; Koch and
Lawson, 1996; Lea, 1996; Milner, 1996). The results, however, were
quite contradictory depending on the type of tumor examined and the garlic preparation used (Dorant et al., 1993). Because crude
garlic extracts contain numerous pharmacologically active substances. including organosulfur compounds with varying stability and biological activity (Milner, 1996), more detailed studies of the effects of
chemically defined garlic compounds on tumor genesis are needed. Some
garlic constituents have been shown to alter the activation of several
carcinogens and to cause growth inhibition and/or death of tumor cells
(Takeyama et al., 1993; Hatono et al., 1996; Koch and Lawson, 1996; Singh et al., 1996). However, the
molecular mechanisms underlying the tumor cytotoxicity of garlic
substances are poorly defined. The cytotoxicity of most classical
antitumor drugs is thought to be mediated by their ability to induce
apoptosis (Sen and D'Incalci, 1992). Apoptosis is a form of
physiological cell death, characterized by chromatin condensation,
cytoplasmatic blebbing, and DNA fragmentation (Wyllie et
al., 1980). Apoptosis can be initiated by alterations in signaling
pathways (Jones et al., 1989; Hsu et al., 1996)
or by oxidative stress mediated by the generation of ROS (Buttke and
Sandstrom, 1994). It has been shown that oxidative stress activates the
transcription factor NF-B and that activation of NF-B is involved
in inducing the apoptotic cell death in some cells (Grimm et
al., 1996).
The aim of the present study was first to examine whether ajoene
[(E,Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide]
(Fig. 1), a major compound of crushed
garlic (Agarwal, 1996), was able to induce apoptosis in the human
promyelocytic leukemia cell line HL-60. This cell line provides a valid
model system for testing antileukemic or general antitumoral compounds
(Suh et al., 1995). Second, we investigated the mechanisms
underlying apoptosis induction. We examined the generation of ROS by
ajoene and the effect of ajoene on the activation of the transcription
factor NF-B. Apoptosis was assessed by morphological analysis of
cells as well as characterization and quantification of DNA degradation
by flow cytometry and gel electrophoresis. The ROS formation of HL-60
cells exposed to ajoene was monitored by oxidation of the dye DHR-123.
Activation of NF-B was examined by its DNA-binding activity using
EMSA.
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Materials and Methods |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Cell cultures. The human promyelocytic leukemia HL-60 cell line and in addition the human colon adenocarcinoma DLD-1 cell line, human squamous carcinoma cells (A431), and human neuroblastoma cells (SH-SY5Y) were cultured (37° and 5% CO2) in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 units/ml penicillin/100 µg/ml streptomycin, and L-glutamine (2 mM) (all from GIBCO/BRL, Eggenstein, Germany). For experiments 1-5 × 105 cells/well were seeded (1-ml, 24-well plates; Peske, Aindling-Pichl, Germany) and grown overnight.
Human PMBC were recovered from heparin-anticoagulated blood of healthy volunteers by centrifugation with Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) following the manufacturer's instructions and cultivated as described for the HL-60 cells. In some experiments, PMBC were stimulated with phytohemagglutinin (1 µg/ml) for either 24 or 48 hr. In addition, PMBC of a patient (male, 54 years old) with a chronic myelogenous leukemia undergoing a myeloid blast crisis were purified and cultured as described above. Blood cell differentiation analysis of the patient revealed 70% myeloblastocytes. General viability of cultured cells was determined by either trypan-blue exclusion or by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan (Mosmann, 1983).
Ajoene (GBF, Braunschweig, Germany), which was a mixture of
cis- and trans-ajoene, was dissolved in 10%
dimethylsulfoxide and further diluted in PBS. Ajoene was added to cells
in various concentrations (1-80 µM) for various periods
of time as indicated in Results. Final dimethylsulfoxide concentration
was less than 0.1% and has been tested not to interfere with the test
systems employed.
Apoptosis. Apoptosis was examined by cell morphology, DNA gel electrophoresis, and flow cytometry. Cells after treatment with ajoene were cytospun, fixed in methanol/acetone (1:1) (4°, 5 min), and stained with Wright solution (Merck, Darmstadt, Germany) for light microscopic determination of morphological changes.
Apoptotic cells were detected by flow cytometry using PI (Nicoletti et al., 1991). Briefly, after incubation with test
substances, cells were harvested and fixed in 70% ethanol for 2 hr at
20°. Carcinoma cells had to be detached by trypsinization. Cells
were washed and resuspended in PBS (0.2 ml) containing 0.5 mg/ml RNase and 0.1 mg/ml PI (both Sigma, Deisenhofen, Germany). Samples were kept
in the dark for 30 min. Cytometry was performed using the FACScan flow
cytometer (Becton Dickinson, Heidelberg, Germany) equipped with a
488-nm argon-ion laser. DNA content per cell was estimated by
collecting PI-fluorescence through 585-nm filter (FL-2). Only those
events with detectable and linear FL-2 area versus width were included
in analysis. In addition forward and side scatter of cells were
recorded to observe morphological changes of cells. Ten thousand events
were collected per sample. Experiments were performed in triplicate and
repeated at least three times. All analyses were performed with the
Cell Quest software (Becton Dickinson).
DNA isolation and electrophoresis was performed (Tuosto et
al., 1994). After centrifugation (200 × g, 5 min), cell pellets (containing about 106 cells) were
resuspended in 1 ml of lysis buffer (10 mM EDTA, 50 mM Tris·HCl, pH 8.0) containing 0.5% sodium dodecyl
sulfate and 0.5 mg/ml RNase A (Sigma) and incubated at 37° for 1 hr
followed by treatment with 0.5 mg/ml proteinase K (Sigma) for another
1h at 50°. The lysate was extracted twice with phenol/chloroform (1:1) and the DNA precipitated with ethanol. After centrifugation, pellets were dissolved in Tris/EDTA buffer (10 mM
Tris·HCl, pH 8.0, and 1 mM EDTA). DNA was analyzed after
separation by gel electrophoresis (1.8% agarose) and staining by
ethidium bromide. Experiments with HL-60 and PBMC were repeated at
least three times, experiments with carcinoma cells at least twice.
Measurement of ROS generation.
Production of ROS (peroxide)
by HL-60 was measured by flow cytometry as previously described
(Vollmar et al., 1997). Briefly, HL-60 cells suspended in 5 mM saline buffered with HEPES (2 × 106/ml) were
placed in polypropylene tubes (200 µl; Becton-Dickinson) and loaded
for 15 min with 0.4 mM DHR-123 (Molecular Probes, Eugene, OR) (dissolved in N,N-dimethylformamide at a
stock concentration of 43.3 mM) and thereafter washed with
HEPES-buffered saline. DHR-containing cells were stimulated with ajoene
(1-20 µM) for various times (5-40 min). To estimate
intracellular peroxide production, fluorescence intensity (FL1, 530 nm)
of 10,000 cells was recorded. Cells incubated with DHR only were
employed to monitor basal peroxide synthesis. Fluorescence intensity
was obtained as histogram statistics. Triplicates of three independent
experiments were performed.
Detection of activation of NF-B by EMSA.
Nuclear extracts
were prepared as described previously (Schreiber et al.,
1989). In brief, cells (106 per tube) were washed with PBS
and resuspended in 400 µl of ice-cold buffer A [10 mM
HEPES, pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM DTT; 0.5 mM
phenylmethylsulfonyl fluoride], kept on ice for 15 min, followed by
addition of 25 µl of 10% Nonidet NP-40. Tubes were vigorously
vortexed for 10 sec and the homogenate centrifuged (30 sec, 10,000 × g). The pellet was resuspended in 50 µl of buffer B (20 mM HEPES, pH 7.9; 0.4 M NaCl; 1 mM
EDTA; 1 mM EGTA; 1 mM DTT; 1 mM
phenylmethylsulfonyl fluoride) and vigorously rocked for 15 min (4°).
The nuclear extract was centrifuged (5 min, 10,000 × g), and after determination of protein concentration (method of Lowry), aliquots were either frozen at 70° or immediately used
for EMSA as previously described (Boese et al., 1996).
Briefly, an oligonucleotide containing the most common NF-B
consensus sequence (22mer; Promega, Heidelberg, Germany) was
end-labeled with [
-32P]ATP (300 Ci/mmol;
Hartman, Braunschweig, Germany) using the T4 polynucleotide kinase
(Promega). Binding reactions were performed incubating 50,000-200,000
cpm of 32P-labeled DNA with nuclear protein
extract (10 µg of protein) in a final volume of 15 µl of buffer [5
mM HEPES, pH 7.5, 100 mM NaCl, 1 mM
DTT, 5% glycerol, 1 mM EDTA, 1 µg poly dI-dC (Promega)] for 30 min (22°). The mixture was electrophoresed on a 4.5%
nondenaturing polyacrylamide gel (100 V) and the gel was exposed to
X-ray film overnight. Three independent experiments were performed.
Films were evaluated by densitometry (EASY plus system; Herolab,
Wiesloch, Germany).
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Results |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Induction of apoptosis by ajoene. Ajoene, whose chemical structure is shown in Fig. 1, induced morphological changes that are characteristic of apoptosis in HL-60 cells. Whereas untreated cells exhibit typical nonadherent, fairly round morphology, cells exposed to ajoene (10 µM, 20 hr) frequently display condensation of chromatin and appearance of apoptotic bodies (data not shown).
Flow cytometric analysis of HL-60 cells exposed to ajoene (10 µM, 20 hr) confirmed the morphological observations. Ajoene-treated cells (Fig. 2B) contained a population of cells with higher side scattering of cells than untreated HL-60 cells, in accord with the different nucleus/cytoplasm consistency of apoptotic cells (Tuosto et al., 1994) (Fig. 2A). The DNA fluorescence histograms of
PI-stained cells showed the low DNA stainability of the ajoene-treated, apoptotic cells (Fig. 2D), which resulted in a distinct, quantifiable region below the G1 peak. In contrast, the
G1 peak predominates in control cells (Fig. 2C).
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). Quantification of dose dependency was
done by monitoring the amount of nuclei with subdiploid DNA content by
flow cytometry (Fig. 3B). Ajoene in concentrations higher than 5 µM incubated for 20 hr increased the percentage of
apoptotic cells significantly, up to 60% (40 µM). Time
dependency of this effect was assessed by exposing cells to 10 µM ajoene for the periods of time indicated (Fig. 3C). As
early as 6hr after ajoene addition, apoptotic cells could be detected
by flow cytometry. Maximal apoptotic cells detection was reached after
24hr and declined after 30-hr and 48-hr incubation times.
Interestingly, ajoene dose dependently induced apoptosis in PBMC of a
chronic leukemic patient (Fig. 4). On the
basis of blood cell differentiation, the patient was diagnosed as
suffering from a myeloid blast crisis with the percentage of
myeloblasts at 70%. Fig. 4A shows the appearance of the characteristic
DNA ladder in the patient's ajoene-exposed PBMC. Quantification of
apoptotic cells by flow cytometry (Fig. 4B) revealed similar potency in ajoene with respect to inducing apoptosis in patient leukemia cells as
observed in HL-60 cells.
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Induction of peroxide production by ajoene. An important mechanism by which compounds induce apoptosis is through generation of ROS, predominantly peroxides. To assess intracellular peroxide production, we measured oxidation of DHR-123 in HL-60 cells by flow cytometry.
Treatment of HL-60 cells with ajoene caused a dose- and time-dependent increase in peroxide production. Ajoene (5 µM) increased ROS production by 35% after 5 min (Fig. 5A), with production reaching a plateau after 30 min, followed by a slight decrease after 60 min (data not shown). Preincubation of cells with the antioxidant NAC inhibited ajoene-induced ROS production by 50% (Fig. 5B). As a control for the reaction, we added catalase, which was unable to permeate into the cell in our experimental condition. Catalase did not prevent the ajoene-induced increase in peroxide production.
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Activation of NF-B by ajoene.
Induction of apoptosis and
peroxide production by ajoene may be linked through activation of
NF-B, which is known to be induced by oxidative stress and to be
involved in signaling of apoptotic processes (Grimm et al.,
1996). As demonstrated by EMSA (Fig. 7),
ajoene (10 µM, 3 hr) was able to activate the nuclear
translocation of NF-B, and importantly, cells pretreated with 15 mM NAC displayed significantly (i.e., 50-65%) less
NF-B activation when exposed to ajoene than cells treated with
ajoene only.
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Discussion |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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The present study elucidates the biological effect of ajoene, a
major component of garlic, on human leukemia cells and supports the
notion of garlic as a chemopreventive or chemotherapeutic drug. We
showed that ajoene induces apoptosis in a human promyeloleukemic cell
line (HL-60) as well as in peripheral blood cells of a chronic leukemic
patient suffering from a myeloid blast crisis. In contrast, ajoene does
not induce apoptosis in proliferating as well as nonproliferating PMBC
of healthy human donors. We demonstrated further that ajoene stimulates
reactive oxygen production in HL-60 cells and activates the nuclear
translocation of the transcription factor NF-B in these cells.
Apoptosis is a highly controlled form of cell death and plays an
important role in maintaining normal tissue homeostasis as well as in
the development of various diseases including cancer (Fisher, 1994;
McConkey et al., 1996). Recently, interest has focused on
the manipulation of apoptotic processes in the treatment and prevention
of cancer. Thus, much effort has been directed toward the search for
compounds that influence apoptosis and their mechanism of action. The
signaling cascade leading to programmed cell death seems to involve ROS
as second messengers (Khan and Wilson, 1995). This is in agreement with
the fact that Bcl-2, a protooncogene product that has been shown to
protect cells from apoptosis, is suggested to interfere with signal
transduction events caused by oxidative stress. In fact, the
antiapoptotic effect of Bcl-2 has been at least partially explained by
its antioxidant properties (Khan and Wilson, 1995; Grimm et
al., 1996). Today, a large body of evidence suggests that
oxidative stress induces apoptosis and that a concomitant increase of
hydrogen peroxide might function as a cellular messenger (Khan and
Wilson, 1995; Grimm et al., 1996).
Substances such as ajoene that alter the redox status of the cell may be potent tools for corresponding mechanistic investigations.
Observations that NF-B, a transcription factor known to respond to
oxidative stress, is activated by certain apoptotic stimuli and is
down-regulated by Bcl-2 (Grimm et al., 1996) led to the speculation that this transcription factor may mediate aspects of
programmed cell death.
Substances causing oxidative stress may induce cell apoptosis via
activation of NF-B. Our findings that ajoene not only induces apoptosis but also stimulates production of ROS and NF-B activation in HL-60 cells would fit into the above proposed model. At this time we
cannot prove a functional association of the three observations. However, the fact that incubation of cells loaded with the antioxidant N-acetylcysteine resulted in decreased apoptosis and ROS
formation as well as NF-B activation suggests a correlation of these
events. Ajoene may serve as a valuable tool for investigating the
mechanisms underlying apoptotic processes.
The ability of ajoene to induce programmed cell death in leukemic cells but not in peripheral blood cells of healthy subjects represents a novel and important aspect of the discussion of the antitumoral effects of garlic compounds. The observation that a variety of carcinoma cell lines exposed to ajoene were not affected by apoptosis may suggest that the apoptotic activity of ajoene is specific to leukemic cells. Clearly, the significance of these findings in a broader context has to be proven by further studies employing various other tumor cells as well as established xenograft tumor models.
The data presented here, however, provide evidence for the antileukemic activity of the garlic compound ajoene and thus support the contention of a benefit of garlic intake in cancer prevention and treatment.
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Acknowledgments |
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We are grateful to Prof. Dr. Wagner, University of Munich, for providing ajoene and for his helpful discussions. We thank Dr. Ulla Knaus (Scripps Clinic, La Jolla, CA) for correcting the style of the manuscript. We thank Miss U. Rüberg and Miss C. Seidler for their excellent technical assistance.
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Footnotes |
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Received July 17, 1997; Accepted November 11, 1997
This work was supported by Deutsche Forschungsgemeinschaft Grant Vo 376/6-2.
Send reprint requests to: Angelika M. Vollmar, Ph.D., Institute of Pharmacology, Toxicology and Pharmacy, Koniginstr. 16, D-80539 Munich, Germany. E-mail: vollmar{at}pharmtox.vetmed.uni-muenchen.de
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Abbreviations |
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ROS, reactive oxygen species;
DHR, dihydrorhodamine;
EGTA, ethylene glycol bis(
-aminoethyl
ether)-N, N, N
,
N-tetraacetic acid;
EMSA, electrophoretic mobility
shift assay;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
HL, human leukemic cells;
NF, nuclear factor;
NAC, N-acetylcysteine;
PI, propidium iodide;
PBS, phosphate-buffered saline;
PMBC, peripheral mononuclear blood cells.
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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