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Tumor Biology |
INRA, Laboratoire des Xénobiotiques, 31931 Toulouse Cedex, France [L. G-P., P. L., S. L., S. C., N. G., J. T.]; INSERM U326, 31059 Toulouse Cedex, France [F. T.]; INSERM U395, Service Commun d’Analyse et de Tri Cellulaire, 31024, Toulouse Cedex, France [G. C.]; and Laboratoire de Biologie Moléculaire des Eucaryotes, 31062, Toulouse Cedex, France [A. D., M-A. D.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSMeasurement of DNA, RNA,...Determination of Intracellular...Western Blot AnalysisStatistical MethodRESULTSDISCUSSIONREFERENCES |
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Here we investigated the effect of sulforaphane on the growth and viability of HT29 cells during their exponentially growing phase. We observed that sulforaphane induced a cell cycle arrest in a dose-dependent manner, followed by cell death. This sulforaphane-induced cell cycle arrest was correlated with an increased expression of cyclins A and B1. Moreover, we clearly demonstrated that sulforaphane induced cell death via an apoptotic process. Indeed, a large proportion of treated cells display the following: (a) translocation of phosphatidylserine from the inner layer to the outer layer of the plasma membrane; (b) typical chromatin condensation; and (c) ultrastructural modifications related to apoptotic cell death. We also showed that the expression of p53 was not changed in sulforaphane-treated cells. In contrast, whereas bcl-2 was not detected, we observed increased expression of the proapoptotic protein bax, the release of cytochrome c from the mitochondria to the cytosol, and the proteolytic cleavage of poly(ADP-ribose) polymerase. In conclusion, our results strongly suggest that in addition to the activation of detoxifying enzymes, induction of apoptosis is also involved in the sulforaphane-associated chemoprevention of cancer.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSMeasurement of DNA, RNA,...Determination of Intracellular...Western Blot AnalysisStatistical MethodRESULTSDISCUSSIONREFERENCES |
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The mechanisms by which isothiocyanates might exert their anticarcinogenic effects remain unclear. One proposed hypothesis involves the modulation of the metabolism of carcinogens (4 , 5) . The fate of chemical carcinogens in vivo is determined at least in part by the balance between phase I and phase II enzymes: phase I enzymes activate many carcinogens to highly reactive electrophilic metabolites capable of damaging DNA; phase II enzymes convert these reactive electrophiles to less toxic and more easily excretable products. It has been hypothesized that isothiocyanates may competitively inhibit enzymes such as cytochrome P-450 involved in the bioactivation of carcinogens (5) . The chemopreventive properties of isothiocyanates are also associated with the induction of phase II detoxifying enzymes including glutathione S-transferase, quinone reductase, epoxide hydrolase, and UDP-glucuronosyltransferase. Indeed, molecular studies have shown that isothiocyanates can induce phase II enzymes by stimulating transcription of their genes via a common antioxidant/electrophile enhancer element present in the upstream region of several phase II enzyme genes (5 , 6) .
However, some recent results suggest that the chemopreventive activities of isothiocyanates may involve other mechanisms as well. Indeed, isothiocyanates could also act at the DNA level or affect signal transduction pathways leading to growth arrest or cell death. Stoner et al. (7) have established that the inhibitory effects of PEITC2 on the production of esophagus tumors by N-nitrosobenzylmethylamine in rats paralleled the inhibition of the binding of the carcinogen to DNA and the formation of N-methylguanine and (O)-methylguanine. In addition, it was recently reported that PEITC could induce in vitro activation of c-Jun NH2-terminal kinase 1, an activation associated with the induction of apoptosis (8) . Moreover Huang et al. (9) have demonstrated that PEITC also induces p53 transactivation in a dose-and time-dependent fashion, a mechanism required for the PEITC-induced apoptosis and antitumor promotion effects of this compound in mouse epidermal cells.
Sulforaphane is an isothiocyanate that has been isolated from SAGA broccoli as the major phase II enzyme inducer present in organic solvent extracts of this vegetable (10) . Our interest on sulforaphane stemmed from the following observations: (a) it occurs naturally in widely consumed vegetables and at a particularly high concentration in broccoli (11) ; (b) it blocks chemical-initiated tumor formation in rats (11) ; (c) it is a very potent monofunctional inducer of phase II enzymes in both cultured cells and mouse tissues; and (d) it has recently been shown to inhibit at least one cytochrome P-450 (CYP2E1) involved in the activation of a variety of carcinogens (11 , 12) .
In this study, we have investigated whether sulforaphane has direct anticancer activities besides its blocking action on carcinogenesis. In a previous study (13) , we have shown that sulforaphane inhibits the reinitiation of growth and diminishes cellular viability in quiescent colon carcinoma cells (HT29) and had a lower toxicity on differentiated CaCo2 cells.
Here we show that in highly proliferative HT29 cells, sulforaphane induces a cell cycle arrest, followed by cell death. This arrest is correlated with an increased expression of cyclins A and B1. Moreover, we clearly demonstrate that sulforaphane induces cell death through an apoptotic pathway involving typical biochemical and ultrastructural modifications related to programmed cell death. We also demonstrate that the activation of the proapoptotic protein bax, but not p53, is required for sulforaphane-induced cell death. Finally, bax induction is correlated with cytochrome c release from the mitochondria to the cytosol and PARP cleavage. Our results strongly suggest that in addition to the activation of detoxifying enzyme activities, specific mechanisms such as apoptosis are also involved in the sulforaphane-associated chemoprevention of cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSMeasurement of DNA, RNA,...Determination of Intracellular...Western Blot AnalysisStatistical MethodRESULTSDISCUSSIONREFERENCES |
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Cell Culture
The HT29 cell line was established in permanent culture from a
human colon carcinoma by Dr. J. N. Fogh (Sloan Kettering Institute for
Cancer Research, Rye, NY; Ref. 15
). HT29 cells were purchased from
European Collection of Cell Culture (Salisbury, United Kingdom). Stock
cells were routinely cultured in DMEM containing 25 mM
glucose, 43 mM bicarbonate, 60 µM/ml
penicillin, and 100 µg/ml streptomycin at 37°C under an
air:CO2 (9:1) atmosphere supplemented with 5%
heat-inactivated FCS, and the medium was changed every 48 h. For
the experiments, HT29 cells were seeded at low density (5 x 104 cells/ml) in 35- or 120-mm diameter Primaria
dishes in standard medium containing 5% FCS. One day after seeding,
medium was changed, and HT29 cells were treated with sulforaphane. An
equivalent amount of the solvent (ethanol) was added to control cells
(0.2% final concentration).
Cell Viability Assay
Drug effect on cellular viability was evaluated using an assay
based on the cleavage of the yellow dye MTT to purple formazan crystals
by deshydrogenase activity in mitochondria, a conversion that occurs
only in living cells (16)
. At each time point, cells were
rinsed with phenol red-free RPMI 1640, and then they received MTT
diluted in RPMI 1640 for 4 h. The cells were then solubilized in
SDS/NaOH, and the optical density of the cellular homogenate was
measured at 570 and 690 nm.
Flow Cytometry Analysis
Drug effect on cell proliferation was evaluated by measuring the
distribution of the cells in the different phases of the cell cycle by
flow cytometry. This determination was based on the measurement of the
DNA content of nuclei labeled with propidium iodide according to the
method of Vindelov and Christensen (17)
, with slight
modifications. Cell suspensions from either control cultures or treated
cultures were prepared by trypsinization and washed twice with 0.9%
NaCl. Cells (1 x 106) were resuspended in
220 µl of solution A 3.4 mM trisodium citrate (pH 7.6),
0.1% NP40, 1.5 mM spermine tetrahydrochloride, and 0.5
mM Trisbase containing trypsin (30 mg/liter) for 10 min at
room temperature. Trypsin was then inhibited by the addition of 180
µl of solution A containing trypsin inhibitor (0.5 g/liter) and Rnase
A (100 mg/liter) for an additional 10 min. Finally, nuclei were labeled
by the addition of 180 µl of solution A containing PI (416 mg/liter)
and additional spermine tetrahydrochloride (1160 mg/liter). The
suspension was incubated overnight at 4°C to allow maximum labeling
of DNA. Cell cycle analysis was performed on a Coulter ELITE flow
cytometer through a 630 nm LP filter. Debris and doublets were
eliminated by gating on peak versus integrated signals, and
1.5 x 104 cells were collected per sample.
Calculations were performed with MULTICYCLE AV Software (Phoenix Flow
System).
Determination of Apoptosis
Analysis of Chromatin Condensation.
Cells were plated at low density (1.25 x 105 cells/well) on glass coverslides (Esco; 20 x 20 mm; Erie Scientific, Portsmouth, NH) in a 6-well
plate and treated with sulforaphane for 48 h. At the end of the
experiment, cells were washed twice with PBS at room temperature and
then fixed with ice-cold methanol/ethanol (v/v, 1/1) for 10 min at
-20°C. Fixed cells were rinsed with PBS and stained with Hoechst
33342 (10 µg/ml) in PBS (15 min at room temperature in the dark).
Finally, cells were washed three times with PBS and analyzed under a
fluorescence microscope with a UV light filter.
Detection of PS on the Outer Leaflet of Cells.
Analysis of the presence of PS on the outer leaflet of cell membrane
was performed using a double-labeling experiment with annexin
V-fluorescein and PI to discriminate apoptotic from necrotic cells. In
the presence of calcium, annexin V binds to PS, which are
translocated to the outer leaflet of the plasma membrane of apoptotic
cells. PI is a nonpermeant cell marker that is able to label the DNA of
cells with a permeable plasma membrane, i.e., necrotic or
lysed cells. Cells that stain only for annexin V are considered
apoptotic.
Cells were plated and treated as described above. After washing twice with PBS and once with the incubation buffer [10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, and 5 mM CaCl2], cells were incubated with annexin V-fluorescein and PI (1 µg/ml) for 10 min in the dark at room temperature. Cells were then washed two times with the incubation medium and fixed for 10 min in an ice-cold solution of methanol/ethanol (v/v, 1/1) at -20°C. Analysis was performed on a fluorescence microscope (488 nm excitation and a 515 nm longpass filter for detection).
Electron Microscopy Analysis.
HT29 cells were plated at a density of 7.5 x 105 cells/flask (Nunc, 80 cm2
) and treated with
control (ethanol) or sulforaphane-supplemented medium for 24, 48, or
72 h. At the end of each incubation time, cells were fixed for
2 h with 3% glutaraldehyde in 0.1 M sodium cacodylate
buffer (fixation buffer). After three washes in the same buffer, cells
were postfixed with 1% osmium tetroxide and then dehydrated in graded
ethanol. The 100% ethanol solution was then replaced by propylene
oxide and embedded in epon 812. Sections were stained with uranyl
acetate and lead citrate and then examined with a Jeol 1200EX electron
microscope.
For light microscopy observations, the epon-embedded semithin sections were stained with 1% methylene blue/azur II (v/v) for 10 min, rinsed with water, and observed using a Leitz Ortholux II microscope.
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Measurement of DNA, RNA, Protein, and Phospholipid Synthesis |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSMeasurement of DNA, RNA,...Determination of Intracellular...Western Blot AnalysisStatistical MethodRESULTSDISCUSSIONREFERENCES |
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Determination of Intracellular ATP Content |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSMeasurement of DNA, RNA,...Determination of Intracellular...Western Blot AnalysisStatistical MethodRESULTSDISCUSSIONREFERENCES |
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Cells were scraped in 0.6 M perchloric acid, and homogenates were rapidly neutralized with 0.75 M K2CO3 and centrifuged at 14,000 rpm for 1 min. The neutralized supernatants were filtered through 0.45-µm membrane filters before analysis. ATP standard solutions (concentrations between 7.81 and 125 µM) were prepared in a mixture of 0.6 M HClO4 0.75 M/K2CO3 (3.5:1.4, v/v) and filtered through 0.45-µm membrane filters.
Capillary zone electrophoresis was carried out on a HP 3DCE System (Hewlett-Packard Co., Wilmington, DE) with a built-in UV diode-array detector. Capillary zone electrophoresis separations were performed using a noncoated fused silica capillary equipped with a bubble detection cell to improve detection sensitivity (68.5 cm x 50/150 µm inner diameter; 60 cm, effective length; Hewlett Packard). Experiments were carried out in the cationic mode by applying a voltage of 30 kV. Hydrostatic injection was applied for 6 s at 50 mbar, followed by a 3-s flush with the buffer. Detection wavelength was 260 nm. The temperature was kept constant at 25°C. To improve the migration time and peak shape reproducibility, the system was rinsed between each run as follows: 3 min with 0.1 M NaOH, followed by 5 min with running buffer 50 mM disodium tetraborate (pH 9.18)].
The detector response linearity over the range has been determined (correlation coefficient, 0.99949). Ten preparations of ATP standard (125 µmol/liter) have been analyzed to determine the repeatability of the analysis (Relative Standard Deviation = 2.14%). The lower limit of detection was about 2 µmol/liter ATP (signal:noise ratio = 3).
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Western Blot Analysis |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSMeasurement of DNA, RNA,...Determination of Intracellular...Western Blot AnalysisStatistical MethodRESULTSDISCUSSIONREFERENCES |
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For cytochrome c and actin determination, 5 x 107 cells were suspended in 5 volumes of 20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, and 10 µM leupeptin containing 20 mM sucrose. After chilling on ice for 30 min, the cells were disrupted by stroking 40 times in a glass homogenizer. The nuclei were centrifuged at 1,000 x g for 10 min at 4°C. The resulting supernatant was centrifuged at 10,000 x g for 30 min to pellet mitochondria. A final centrifugation at 100,000 x g for 1 h at 4°C generated the cytoplasmic fraction.
Cell extracts or subfractions were then mixed with loading buffer 250 mM Tris (pH 8.8), 4% SDS, 16% glycerol, 8% 2-mercaptoethanol, and 0.1% bromophenol blue; fractionated by electrophoresis on SDS-polyacrylamide gels (7.5% for p53 and cylin A and B, 14% for bax, and cytochrome c); and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH) by electroblotting. After transfer, the filters were incubated in saturating buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4 · 2H2O, 1.4 mM KH2PO4, 0.05% Tween 20, and 3% nonfat dry milk) at 4°C overnight. Blots were subsequently incubated for 1.5 h at room temperature with the desired primary antibodies. After rinsing with saturating buffer, the filters were incubated with diluted enzyme-linked secondary antibody for 1.5 h. The proteins were then visualized with an enhanced chemiluminescence detection system (Pierce) according to the manufacturer’s instructions.
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Statistical Method |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSMeasurement of DNA, RNA,...Determination of Intracellular...Western Blot AnalysisStatistical MethodRESULTSDISCUSSIONREFERENCES |
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSMeasurement of DNA, RNA,...Determination of Intracellular...Western Blot AnalysisStatistical MethodRESULTSDISCUSSIONREFERENCES |
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. The inhibition of growth and the effect on the adherence were already
important at 15 µM (of the total cells present in the
plate, 46% were attached and 62% were floating), thus this
concentration was used in all further experiments. Viability was
determined by using the MTT assay on cells treated with the drug for up
to 96 h. As shown in Fig. 1b
, sulforaphane at 15
µM was able to inhibit cell growth and induce cell death.
Cell mortality was already 75% at 24 h and almost total at
96 h.
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, [H3]thymidine incorporation into DNA was rapidly
inhibited after sulforaphane treatment, decreasing to 63 ± 4% of control levels within 60 min. Protein synthesis (Fig. 2b)
and RNA (Fig. 2c)
synthesis were also
inhibited in a similar manner. In each case, the inhibitory effect of
sulforaphane was maintained over a 24-h period of treatment (data not
shown). However, the first significant inhibition (30%) was observed
at 30 min for DNA synthesis, whereas 60 min were needed for RNA and
protein synthesis. As estimated by [H3]choline
incorporation into phosphatidylcholine, the major class of phospholipid
(50% of total cellular phospholipids), phospholipid synthesis was not
altered by sulforaphane at up to 2 h of treatment (Fig. 2d)
. However, it decreased dramatically (down to 47%) after
3 h of treatment, indicating that a prolonged treatment affects
all the major biosynthetic pathways (data not shown).
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, no difference in ATP level could be seen after up to 8 h of
incubation between control and treated cells, ruling out ATP as the
initial trigger of the biochemical events.
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. At time 0, most cells (60%) were in S phase, due to the high
proliferative state of this cell line. Untreated control cells showed
the expected pattern for continuously growing cells. However, in the
presence of 15 µM sulforaphane, we observed a net
increase in the percentage of cells in the G2-M phase of
the cell cycle that was maintained throughout the overall time of
treatment (3 days). These results suggest that the G2-M
phase accumulation was caused not only by an increase in the duration
of the G2-M phase, but by a complete arrest of many cells
in the G2-M. The G2-M-phase accumulation was
accompanied by a decrease in the percentage of cells in S phase.
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shows that according to the blockage in the G2-M phase,
exponentially growing cells treated with 15 µM
sulforaphane expressed high levels of cyclin A and B1 as compared with
control cells.
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. Indeed, the number of cells with subdiploid DNA content increased with
time to reach 30% at 72 h (Fig. 5b)
. Moreover, about
half of the floating cells recovered in the culture medium were
subdiploid, with the other half being in the G2-M phase
(data not shown), strengthening the relationship between
G2-M-phase accumulation and apoptotic triggering. To
confirm the existence of an apoptotic process, we have analyzed the
presence of apoptosis by different techniques.
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Nuclear chromatin condensation is considered to be part of the cellular
events involved in apoptosis. Therefore, we have analyzed the
fluorescence of the nuclei of cells stained with the DNA-specific dye
Hoechst 33342. Pictures were taken after 48 h of treatment (Fig. 6)
. In untreated cells, we observed normal nuclei staining (Fig. 6F)
. By contrast, sulforaphane-treated cells (15
µM) displayed typical condensed chromatin and fragmented
nuclei (Fig. 6C)
. In addition, the dying cells appeared
rounded (Fig. 6A)
compared to control cells (Fig. 6D)
when observed using phase-contrast microscopy.
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, control
cells did not show fluorescence staining. In contrast, treated cells
displayed the binding of annexin V-fluorescein to the plasma membrane
after 48 h of treatment (Fig. 6B)
. To distinguish
between apoptotic and potential necrotic or lysed cells that may also
expose PS according to the loss of membrane integrity, we have
concomitantly used PI, a DNA dye that is excluded from cells with a
nonleaky plasma membrane (i.e., normal or apoptotic). We
never observed PI staining among the annexin V-positive cells (data not
shown), excluding the presence of necrotic or lysed cells in the
population.
Morphological and ultrastructural changes of HT29 cells exposed to 15
µM sulforaphane were examined at various times by
electron microscopy (Fig. 7)
or light microscopy (Fig. 8)
. In the healthy control cells (Figs. 7A
and 8A)
,
desmosomes keeping cells attached to each other in the organized cell
monolayer were clearly seen (arrow). Moreover, the structure
of the nucleus, as well as the size and shape of the mitochondria, was
normal. In contrast, in treated cells, characteristics of apoptosis
[namely, cell detachment, membrane blebbing (Fig. 8E)
, cell
shrinkage with a condensed cytoplasm, and vesicle formation (abundant
vacuoles with multivesicular bodies)] appeared (Fig. 7D)
.
The compaction and margination of nuclear chromatin into an amorphous
mass osmophilic is quite obvious (Fig. 7C
and Fig. 8,B–E
). We also observed swelling of the endoplasmic
reticulum cisternae (Fig. 7B)
. Moreover, the extent of
nuclear chromatin condensation could be correlated with changes in
mitochondria structure. At 24 h after the addition of
sulforaphane, the mitochondrial changes can be characterized with
interruption and/or absence of the cristae and with loss of matrix
density (Fig. 7E)
.
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. It is clear that HT29 cells do not undergo any change in p53 expression
at any time point or sulforaphane concentration studied. By contrast,
our results clearly show an increase in the bax protein content that
appears after a 24-h period of treatment with 5 and 15 µM
sulforaphane, suggesting that sulforaphane induces apoptosis in HT29
cells via a bax-dependent pathway. The antiapoptotic protein bcl-2 was
never detected under any conditions. We then examined whether the
expression of bax could promote the release of mitochondrial cytochrome
c into the cytosol. Fig. 9
also shows that as the level of
bax increased, cytochrome c could be detected in the
cytosol, whereas its level in the mitochondria decreased. Finally, the
caspase cascade was also induced by sulforaphane, as shown by the
proteolytic cleavage of PARP.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSMeasurement of DNA, RNA,...Determination of Intracellular...Western Blot AnalysisStatistical MethodRESULTSDISCUSSIONREFERENCES |
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Apoptosis is a crucial element in the behavior of mammalian cells in many different situations (20) . The apoptotic program is characterized by particular morphological features (21, 22, 23, 24) . In our model, we have clearly observed many of the typical structural and ultrastructural modifications that happen during the apoptotic pathway, which include cell shrinkage, translocation of PS to the outer layer of the plasma membrane, alteration of internal membrane of the mitochondria, and condensation of the cytoplasm or of nuclear chromatin. In contrast, we have never observed classical DNA fragmentation on sulforaphane treatment. This is not surprising because previous work (25) has shown that HT29 cells do not give rise to any of the DNA fragmentation patterns associated with programmed cell death when challenged with a variety of toxic stimuli.
Our results also clearly show that sulforaphane-induced HT29 cell death
is not associated with a change in p53 protein expression but is
accompanied by an overexpression of bax, one of the bcl-2
gene family, acting as a promoter of cell death. In contrast, the
antiapoptotic bcl-2 protein was not detected in our cells. bax is a
protein present predominantly in the cytosol (26)
, which
could partially translocate to the mitochondria on induction of
apoptosis (27
, 28) . This induces the opening of the
mitochondrial permeability transition pore, a critical event in the
loss of cell viability that mediates the release of cytochrome
c, finally activating the caspase cascade (28
, 29)
. In our model (15 µM sulforaphane),
overexpression of the bax protein was correlated with cytochrome
c release, suggesting it could trigger caspase-dependent
apoptosis. This was evidenced by the presence of a proteolytic fragment
of the caspase-3 substrate PARP in treated cells, suggesting that
caspase-3 was activated, as has been recently shown in
isothiocyanate-treated HeLa cells (30)
. Our conclusion is
strengthened by the observation of abnormalities in the mitochondria
ultrastructure identified by electron microscopy (Fig. 8)
because
altered mitochondrial functions have also been observed in other
apoptotic colon cancer cells (31)
. Surprisingly, the high
level of bax protein at 15 µM sulforaphane was not
associated with a high percentage of dead cells. However, the release
of cytochrome c in the cytosol and the cleavage of PARP were
also low at this concentration, suggesting a delay between the
induction of bax and the final processes of apoptosis.
Due to the lipophilic nature of isothiocyanates, inhibition of
phospholipid biosynthesis could have been a major event in the
induction of growth arrest and apoptosis, as already reported for other
lipidic compounds, i.e., hexadecylphosphocholine
(32)
,
1-0-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine
(33)
, or geranyl-geraniol (34)
. However, in
our case, inhibition of phosphatidylcholine (the major class of
phospholipid) biosynthesis was observed late (3 h) in contrast
to DNA, RNA, or protein synthesis (10 min; Fig. 2
), excluding an
apoptosis related to phospholipid metabolism.
Because apoptosis is the end point of at least some colonic epithelial
cell differentiation pathways, a process that results in apoptotic cell
death should also minimize the proliferative signal. Indeed, in our
model, apoptotic cell death is preceded by an arrest of the cell cycle
and an accumulation of cells in G2-M phase at the
expense of S phase (Figs. 1
and 5)
. This is also evidenced by
the increase in the level of cyclins B1 and A, proteins known to
regulate cdc2 kinase activity at G2-M phase
(35)
. Because overexpression of bax has already been
reported to induce the activation of cdk and caspase through the
degradation of the cdk inhibitor P27 (36)
, it will be of
interest to test whether the cdk inhibitors (p21, p27, and p16) are
implicated in the negative regulation of cell cycle progression by
sulforaphane in our model.
The resistance of cancer to therapy may be due in part to the high frequency of mutation in p53 that impairs p53-dependent apoptosis. Many anticancer agents such as doxorubicine, etoposide, or 5-fluorouracil induce apoptosis via a p53-dependent pathway (37) . The requirement of p53 tumor suppressor for efficient activation of apoptosis by these agents provides an attractive explanation for the poor efficacy of these drugs on p53 mutant tumors (38) . Thus, identifying chemotherapeutic agents that act independently of the p53 pathway is of major importance. Interestingly, the growth-inhibitory effect of sulforaphane was not apparently related to a change in p53 level. However, HT29 cells are known to present p53 mutations, and a strict p53-independent pathway in our model remains to be clearly demonstrated.
Although the presence of sulforaphane in the blood or intestine has not yet been quantified, consumption of 100 g of broccoli could release 40 µmol of sulforaphane, suggesting that local concentrations in the low micromolar range may be achieved in vivo (39, 40, 41) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at INRA, Laboratoire des Xénobiotiques, 180 Chemin de
Tournefeuille BP 3, 31931 Toulouse, Cedex 9, France. 
2 The abbreviations used are: PEITC, phenylethyl
isothiocyanate; PARP, poly(ADP-ribose) polymerase; PI, propidium
iodide; PS, phosphatidylserine; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; cdk,
cyclin-dependent kinase.
Received 7/13/99. Accepted 1/ 6/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSMeasurement of DNA, RNA,...Determination of Intracellular...Western Blot AnalysisStatistical MethodRESULTSDISCUSSIONREFERENCES |
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L. Mi, X. Wang, S. Govind, B. L. Hood, T. D. Veenstra, T. P. Conrads, D. T. Saha, R. Goldman, and F.-L. Chung The Role of Protein Binding in Induction of Apoptosis by Phenethyl Isothiocyanate and Sulforaphane in Human Non-Small Lung Cancer Cells Cancer Res., July 1, 2007; 67(13): 6409 - 6416. [Abstract] [Full Text] [PDF]
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 G. Pappa, J. Strathmann, M. Lowinger, H. Bartsch, and C. Gerhauser Quantitative combination effects between sulforaphane and 3,3'-diindolylmethane on proliferation of human colon cancer cells in vitro Carcinogenesis, July 1, 2007; 28(7): 1471 - 1477. [Abstract] [Full Text] [PDF]
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M. M.Y. Tin, C.-H. Cho, K. Chan, A. E. James, and J. K.S. Ko Astragalus saponins induce growth inhibition and apoptosis in human colon cancer cells and tumor xenograft Carcinogenesis, June 1, 2007; 28(6): 1347 - 1355. [Abstract] [Full Text] [PDF]
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A. Herman-Antosiewicz, H. Xiao, K. L. Lew, and S. V. Singh Induction of p21 protein protects against sulforaphane-induced mitotic arrest in LNCaP human prostate cancer cell line Mol. Cancer Ther., May 1, 2007; 6(5): 1673 - 1681. [Abstract] [Full Text] [PDF]
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A. Pledgie-Tracy, M. D. Sobolewski, and N. E. Davidson Sulforaphane induces cell type-specific apoptosis in human breast cancer cell lines Mol. Cancer Ther., March 1, 2007; 6(3): 1013 - 1021. [Abstract] [Full Text] [PDF]
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 M. C. Myzak, P. Tong, W.-M. Dashwood, R. H. Dashwood, and E. Ho Sulforaphane Retards the Growth of Human PC-3 Xenografts and Inhibits HDAC Activity in Human Subjects Experimental Biology and Medicine, February 1, 2007; 232(2): 227 - 234. [Abstract] [Full Text] [PDF]
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S. Choi, K. L. Lew, H. Xiao, A. Herman-Antosiewicz, D. Xiao, C. K. Brown, and S. V. Singh D,L-Sulforaphane-induced cell death in human prostate cancer cells is regulated by inhibitor of apoptosis family proteins and Apaf-1 Carcinogenesis, January 1, 2007; 28(1): 151 - 162. [Abstract] [Full Text] [PDF]
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 C. Zhou, E.-J. Poulton, F. Grun, T. K. Bammler, B. Blumberg, K. E. Thummel, and D. L. Eaton The Dietary Isothiocyanate Sulforaphane Is an Antagonist of the Human Steroid and Xenobiotic Nuclear Receptor Mol. Pharmacol., January 1, 2007; 71(1): 220 - 229. [Abstract] [Full Text] [PDF]
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N. M. Probst-Hensch, C.-L. Sun, D. V. D. Berg, M. Ceschi, W.-P. Koh, and M. C. Yu The effect of the cyclin D1 (CCND1) A870G polymorphism on colorectal cancer risk is modified by glutathione-S-transferase polymorphisms and isothiocyanate intake in the Singapore Chinese Health Study Carcinogenesis, December 1, 2006; 27(12): 2475 - 2482. [Abstract] [Full Text] [PDF]
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R. Hu, T. O. Khor, G. Shen, W.-S. Jeong, V. Hebbar, C. Chen, C. Xu, B. Reddy, K. Chada, and A.-N. T. Kong Cancer chemoprevention of intestinal polyposis in ApcMin/+ mice by sulforaphane, a natural product derived from cruciferous vegetable Carcinogenesis, October 1, 2006; 27(10): 2038 - 2046. [Abstract] [Full Text] [PDF]
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Y.-S. Keum, S. Yu, P. P.-J. Chang, X. Yuan, J.-H. Kim, C. Xu, J. Han, A. Agarwal, and A.-N. T. Kong Mechanism of Action of Sulforaphane: Inhibition of p38 Mitogen-Activated Protein Kinase Isoforms Contributing to the Induction of Antioxidant Response Element-Mediated Heme Oxygenase-1 in Human Hepatoma HepG2 Cells. Cancer Res., September 1, 2006; 66(17): 8804 - 8813. [Abstract] [Full Text] [PDF]
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T.-a. Matsui, Y. Sowa, T. Yoshida, H. Murata, M. Horinaka, M. Wakada, R. Nakanishi, T. Sakabe, T. Kubo, and T. Sakai Sulforaphane enhances TRAIL-induced apoptosis through the induction of DR5 expression in human osteosarcoma cells Carcinogenesis, September 1, 2006; 27(9): 1768 - 1777. [Abstract] [Full Text] [PDF]
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A. Herman-Antosiewicz, D. E. Johnson, and S. V. Singh Sulforaphane Causes Autophagy to Inhibit Release of Cytochrome c and Apoptosis in Human Prostate Cancer Cells Cancer Res., June 1, 2006; 66(11): 5828 - 5835. [Abstract] [Full Text] [PDF]
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M. C. Myzak, K. Hardin, R. Wang, R. H. Dashwood, and E. Ho Sulforaphane inhibits histone deacetylase activity in BPH-1, LnCaP and PC-3 prostate epithelial cells Carcinogenesis, April 1, 2006; 27(4): 811 - 819. [Abstract] [Full Text] [PDF]
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E. Bertl, H. Bartsch, and C. Gerhauser Inhibition of angiogenesis and endothelial cell functions are novel sulforaphane-mediated mechanisms in chemoprevention. Mol. Cancer Ther., March 1, 2006; 5(3): 575 - 585. [Abstract] [Full Text] [PDF]
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H. Kim, E. H. Kim, Y. W. Eom, W.-H. Kim, T. K. Kwon, S. J. Lee, and K. S. Choi Sulforaphane Sensitizes Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Resistant Hepatoma Cells to TRAIL-Induced Apoptosis through Reactive Oxygen Species-Mediated Up-regulation of DR5 Cancer Res., February 1, 2006; 66(3): 1740 - 1750. [Abstract] [Full Text] [PDF]
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 A. V Gasper, A. Al-janobi, J. A Smith, J. R Bacon, P. Fortun, C. Atherton, M. A Taylor, C. J Hawkey, D. A Barrett, and R. F Mithen Glutathione S-transferase M1 polymorphism and metabolism of sulforaphane from standard and high-glucosinolate broccoli Am. J. Clinical Nutrition, December 1, 2005; 82(6): 1283 - 1291. [Abstract] [Full Text] [PDF]
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C.-T. Yeh and G.-C. Yen Effect of sulforaphane on metallothionein expression and induction of apoptosis in human hepatoma HepG2 cells Carcinogenesis, December 1, 2005; 26(12): 2138 - 2148. [Abstract] [Full Text] [PDF]
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J.-C. Lee, C.-H. Lee, C.-L. Su, C.-W. Huang, H.-S. Liu, C.-N. Lin, and S.-J. Won Justicidin A decreases the level of cytosolic Ku70 leading to apoptosis in human colorectal cancer cells Carcinogenesis, October 1, 2005; 26(10): 1716 - 1730. [Abstract] [Full Text] [PDF]
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Y.-M. Yang, M. Jhanwar-Uniyal, J. Schwartz, C. C. Conaway, H. D. Halicka, F. Traganos, and F.-L. Chung N-Acetylcysteine Conjugate of Phenethyl Isothiocyanate Enhances Apoptosis in Growth-Stimulated Human Lung Cells Cancer Res., September 15, 2005; 65(18): 8538 - 8547. [Abstract] [Full Text] [PDF]
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C. C. Conaway, C.-X. Wang, B. Pittman, Y.-M. Yang, J. E. Schwartz, D. Tian, E. J. McIntee, S. S. Hecht, and F.-L. Chung Phenethyl Isothiocyanate and Sulforaphane and their N-Acetylcysteine Conjugates Inhibit Malignant Progression of Lung Adenomas Induced by Tobacco Carcinogens in A/J Mice Cancer Res., September 15, 2005; 65(18): 8548 - 8557. [Abstract] [Full Text] [PDF]
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L. Tang and Y. Zhang Mitochondria are the primary target in isothiocyanate-induced apoptosis in human bladder cancer cells Mol. Cancer Ther., August 1, 2005; 4(8): 1250 - 1259. [Abstract] [Full Text] [PDF]
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M. Traka, A. V. Gasper, J. A. Smith, C. J. Hawkey, Y. Bao, and R. F. Mithen Transcriptome Analysis of Human Colon Caco-2 Cells Exposed to Sulforaphane J. Nutr., August 1, 2005; 135(8): 1865 - 1872. [Abstract] [Full Text] [PDF]
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G. Brandi, G. F. Schiavano, N. Zaffaroni, C. De Marco, M. Paiardini, B. Cervasi, and M. Magnani Mechanisms of Action and Antiproliferative Properties of Brassica oleracea Juice in Human Breast Cancer Cell Lines J. Nutr., June 1, 2005; 135(6): 1503 - 1509. [Abstract] [Full Text] [PDF]
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S. V. Singh, S. K. Srivastava, S. Choi, K. L. Lew, J. Antosiewicz, D. Xiao, Y. Zeng, S. C. Watkins, C. S. Johnson, D. L. Trump, et al. Sulforaphane-induced Cell Death in Human Prostate Cancer Cells Is Initiated by Reactive Oxygen Species J. Biol. Chem., May 20, 2005; 280(20): 19911 - 19924. [Abstract] [Full Text] [PDF]
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S. Choi and S. V. Singh Bax and Bak Are Required for Apoptosis Induction by Sulforaphane, a Cruciferous Vegetable-Derived Cancer Chemopreventive Agent Cancer Res., March 1, 2005; 65(5): 2035 - 2043. [Abstract] [Full Text] [PDF]
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J. M. Visanji, S. J. Duthie, L. Pirie, D. G. Thompson, and P. J. Padfield Dietary Isothiocyanates Inhibit Caco-2 Cell Proliferation and Induce G2/M Phase Cell Cycle Arrest, DNA Damage, and G2/M Checkpoint Activation J. Nutr., November 1, 2004; 134(11): 3121 - 3126. [Abstract] [Full Text] [PDF]
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N.-A. Pham, J. W. Jacobberger, A. D. Schimmer, P. Cao, M. Gronda, and D. W. Hedley The dietary isothiocyanate sulforaphane targets pathways of apoptosis, cell cycle arrest, and oxidative stress in human pancreatic cancer cells and inhibits tumor growth in severe combined immunodeficient mice Mol. Cancer Ther., October 1, 2004; 3(10): 1239 - 1248. [Abstract] [Full Text] [PDF]
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E. Tseng, E. A. Scott-Ramsay, and M. E. Morris Dietary Organic Isothiocyanates Are Cytotoxic in Human Breast Cancer MCF-7 and Mammary Epithelial MCF-12A Cell Lines Experimental Biology and Medicine, September 1, 2004; 229(8): 835 - 842. [Abstract] [Full Text] [PDF]
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V. Svehlikova, S. Wang, J. Jakubikova, G. Williamson, R. Mithen, and Y. Bao Interactions between sulforaphane and apigenin in the induction of UGT1A1 and GSTA1 in CaCo-2 cells Carcinogenesis, September 1, 2004; 25(9): 1629 - 1637. [Abstract] [Full Text] [PDF]
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S. J. T. Jackson and K. W. Singletary Sulforaphane Inhibits Human MCF-7 Mammary Cancer Cell Mitotic Progression and Tubulin Polymerization J. Nutr., September 1, 2004; 134(9): 2229 - 2236. [Abstract] [Full Text] [PDF]
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M. C. Myzak, P. A. Karplus, F.-L. Chung, and R. H. Dashwood A Novel Mechanism of Chemoprotection by Sulforaphane: Inhibition of Histone Deacetylase Cancer Res., August 15, 2004; 64(16): 5767 - 5774. [Abstract] [Full Text] [PDF]
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T. K. Smith, E. K. Lund, M. L. Parker, R. G. Clarke, and I. T. Johnson Allyl-isothiocyanate causes mitotic block, loss of cell adhesion and disrupted cytoskeletal structure in HT29 cells Carcinogenesis, August 1, 2004; 25(8): 1409 - 1415. [Abstract] [Full Text] [PDF]
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 R. Hu, V. Hebbar, B.-R. Kim, C. Chen, B. Winnik, B. Buckley, P. Soteropoulos, P. Tolias, R. P. Hart, and A.-N. T. Kong In Vivo Pharmacokinetics and Regulation of Gene Expression Profiles by Isothiocyanate Sulforaphane in the Rat J. Pharmacol. Exp. Ther., July 1, 2004; 310(1): 263 - 271. [Abstract] [Full Text] [PDF]
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S. V. Singh, A. Herman-Antosiewicz, A. V. Singh, K. L. Lew, S. K. Srivastava, R. Kamath, K. D. Brown, L. Zhang, and R. Baskaran Sulforaphane-induced G2/M Phase Cell Cycle Arrest Involves Checkpoint Kinase 2-mediated Phosphorylation of Cell Division Cycle 25C J. Biol. Chem., June 11, 2004; 279(24): 25813 - 25822. [Abstract] [Full Text] [PDF]
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D. Xiao, C. S. Johnson, D. L. Trump, and S. V. Singh Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells Mol. Cancer Ther., May 1, 2004; 3(5): 567 - 576. [Abstract] [Full Text] [PDF]
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N. Miyoshi, K. Uchida, T. Osawa, and Y. Nakamura A Link between Benzyl Isothiocyanate-Induced Cell Cycle Arrest and Apoptosis: Involvement of Mitogen-Activated Protein Kinases in the Bcl-2 Phosphorylation Cancer Res., March 15, 2004; 64(6): 2134 - 2142. [Abstract] [Full Text] [PDF]
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 A.-S. Keck and J. W. Finley Cruciferous Vegetables: Cancer Protective Mechanisms of Glucosinolate Hydrolysis Products and Selenium Integr Cancer Ther, March 1, 2004; 3(1): 5 - 12. [Abstract] [PDF]
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S. J. T. Jackson and K. W. Singletary Sulforaphane: a naturally occurring mammary carcinoma mitotic inhibitor, which disrupts tubulin polymerization Carcinogenesis, February 1, 2004; 25(2): 219 - 227. [Abstract] [Full Text] [PDF]
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M. Paolini, P. Perocco, D. Canistro, L. Valgimigli, G. F. Pedulli, R. Iori, C. D. Croce, G. Cantelli-Forti, M. S. Legator, and S. Z. Abdel-Rahman Induction of cytochrome P450, generation of oxidative stress and in vitro cell-transforming and DNA-damaging activities by glucoraphanin, the bioprecursor of the chemopreventive agent sulforaphane found in broccoli Carcinogenesis, January 1, 2004; 25(1): 61 - 67. [Abstract] [Full Text] [PDF]
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A. V. Singh, D. Xiao, K. L. Lew, R. Dhir, and S. V. Singh Sulforaphane induces caspase-mediated apoptosis in cultured PC-3 human prostate cancer cells and retards growth of PC-3 xenografts in vivo Carcinogenesis, January 1, 2004; 25(1): 83 - 90. [Abstract] [Full Text] [PDF]
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B.-R. Kim, R. Hu, Y.-S. Keum, V. Hebbar, G. Shen, S. S. Nair, and A-N. T. Kong Effects of Glutathione on Antioxidant Response Element-Mediated Gene Expression and Apoptosis Elicited by Sulforaphane Cancer Res., November 1, 2003; 63(21): 7520 - 7525. [Abstract] [Full Text] [PDF]
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Y. Zhang, L. Tang, and V. Gonzalez Selected isothiocyanates rapidly induce growth inhibition of cancer cells Mol. Cancer Ther., October 1, 2003; 2(10): 1045 - 1052. [Abstract] [Full Text]
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S. K. Srivastava, D. Xiao, K. L. Lew, P. Hershberger, D. M. Kokkinakis, C. S. Johnson, D. L. Trump, and S. V. Singh Allyl isothiocyanate, a constituent of cruciferous vegetables, inhibits growth of PC-3 human prostate cancer xenografts in vivo Carcinogenesis, October 1, 2003; 24(10): 1665 - 1670. [Abstract] [Full Text] [PDF]
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J. H. Fowke, F.-L. Chung, F. Jin, D. Qi, Q. Cai, C. Conaway, J.-R. Cheng, X.-O. Shu, Y.-T. Gao, and W. Zheng Urinary Isothiocyanate Levels, Brassica, and Human Breast Cancer Cancer Res., July 15, 2003; 63(14): 3980 - 3986. [Abstract] [Full Text] [PDF]
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 S. Garattini New approaches to cancer therapy Ann. Onc., June 1, 2003; 14(6): 813 - 816. [Full Text] [PDF]
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D. Xiao, S. K. Srivastava, K. L. Lew, Y. Zeng, P. Hershberger, C. S. Johnson, D. L. Trump, and S. V. Singh Allyl isothiocyanate, a constituent of cruciferous vegetables, inhibits proliferation of human prostate cancer cells by causing G2/M arrest and inducing apoptosis Carcinogenesis, May 1, 2003; 24(5): 891 - 897. [Abstract] [Full Text] [PDF]
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T. K. Smith, R. Mithen, and I. T. Johnson Effects of Brassica vegetable juice on the induction of apoptosis and aberrant crypt foci in rat colonic mucosal crypts in vivo Carcinogenesis, March 1, 2003; 24(3): 491 - 495. [Abstract] [Full Text] [PDF]
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J. Zhang, V. Svehlikova, Y. Bao, A.F. Howie, G. J. Beckett, and G. Williamson Synergy between sulforaphane and selenium in the induction of thioredoxin reductase 1 requires both transcriptional and translational modulation Carcinogenesis, March 1, 2003; 24(3): 497 - 503. [Abstract] [Full Text] [PDF]
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K. R.K. Sticha, P. M.J. Kenney, G. Boysen, H. Liang, X. Su, M. Wang, P. Upadhyaya, and S. S. Hecht Effects of benzyl isothiocyanate and phenethyl isothiocyanate on DNA adduct formation by a mixture of benzo[a]pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mouse lung Carcinogenesis, September 1, 2002; 23(9): 1433 - 1439. [Abstract] [Full Text] [PDF]
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S. S. Hecht, P. Upadhyaya, M. Wang, R. L. Bliss, E. J. McIntee, and P. M.J. Kenney Inhibition of lung tumorigenesis in A/J mice by N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine and myo-inositol, individually and in combination Carcinogenesis, September 1, 2002; 23(9): 1455 - 1461. [Abstract] [Full Text] [PDF]
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G. P. Basten, Y. Bao, and G. Williamson Sulforaphane and its glutathione conjugate but not sulforaphane nitrile induce UDP-glucuronosyl transferase (UGT1A1) and glutathione transferase (GSTA1) in cultured cells Carcinogenesis, August 1, 2002; 23(8): 1399 - 1404. [Abstract] [Full Text] [PDF]
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C. Fimognari, M. Nusse, R. Cesari, R. Iori, G. Cantelli-Forti, and P. Hrelia Growth inhibition, cell-cycle arrest and apoptosis in human T-cell leukemia by the isothiocyanate sulforaphane Carcinogenesis, April 1, 2002; 23(4): 581 - 586. [Abstract] [Full Text] [PDF]
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Y. Nakamura, M. Kawakami, A. Yoshihiro, N. Miyoshi, H. Ohigashi, K. Kawai, T. Osawa, and K. Uchida Involvement of the Mitochondrial Death Pathway in Chemopreventive Benzyl Isothiocyanate-induced Apoptosis J. Biol. Chem., March 1, 2002; 277(10): 8492 - 8499. [Abstract] [Full Text] [PDF]
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Y.-M. Yang, C. C. Conaway, J. W. Chiao, C.-X. Wang, S. Amin, J. Whysner, W. Dai, J. Reinhardt, and F.-L. Chung Inhibition of Benzo(a)pyrene-induced Lung Tumorigenesis in A/J Mice by Dietary N-Acetylcysteine Conjugates of Benzyl and Phenethyl Isothiocyanates during the Postinitiation Phase Is Associated with Activation of Mitogen-activated Protein Kinases and p53 Activity and Induction of Apoptosis Cancer Res., January 1, 2002; 62(1): 2 - 7. [Abstract] [Full Text] [PDF]
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 N. Ahmad, V. M. Adhami, F. Afaq, D. K. Feyes, and H. Mukhtar Resveratrol Causes WAF-1/p21-mediated G1-phase Arrest of Cell Cycle and Induction of Apoptosis in Human Epidermoid Carcinoma A431 Cells Clin. Cancer Res., May 1, 2001; 7(5): 1466 - 1473. [Abstract] [Full Text]
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Y. Zhang Molecular mechanism of rapid cellular accumulation of anticarcinogenic isothiocyanates Carcinogenesis, March 1, 2001; 22(3): 425 - 431. [Abstract] [Full Text] [PDF]
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F.-L. Chung, C.C. Conaway, C.V. Rao, and B. S. Reddy Chemoprevention of colonic aberrant crypt foci in Fischer rats by sulforaphane and phenethyl isothiocyanate Carcinogenesis, December 1, 2000; 21(12): 2287 - 2291. [Abstract] [Full Text] [PDF]
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E. Heiss, C. Herhaus, K. Klimo, H. Bartsch, and C. Gerhauser Nuclear Factor kappa B Is a Molecular Target for Sulforaphane-mediated Anti-inflammatory Mechanisms J. Biol. Chem., August 17, 2001; 276(34): 32008 - 32015. [Abstract] [Full Text] [PDF]
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) was determined. Results are expressed
as the percentage of untreated cells. b, cell viability in
control (
) or in 15 µM sulforaphane-treated cells
(
) was measured every day using the MTT assay as described in
"Materials and Methods." Results are expressed as the percentage of
viable cells at the beginning of the experiment (time 0) and are the
mean ± SE of four separate experiments. When they do
not appear, error bars are smaller than the symbol size.
