Isothiocyanates have strong chemopreventive
properties against many carcinogen-induced cancers in experimental
animal models. Here, we report that phenylmethyl isocyacyanate
(PMITC) and phenylethyl isothio- cyanate (PEITC) induced sustained
c-Jun N-terminal kinase (JNK) activation in a
dose-dependent manner. The sustained JNK activation caused
by isothiocyanates was associated with apoptosis induction in various
cell types. An inhibitor of the caspase/interleukin-1
-converting enzyme blocked isothiocyanate-induced apoptosis without inhibiting the
JNK activation, which suggests that JNK activation by isothiocyanates is an event that is independent or upstream of the activation of
caspase/interleukin-1
-converting enzyme proteases. PEITC-induced apoptosis was suppressed by interfering with the JNK pathway with a
dominant-negative mutant of JNK1 or MEKK1 (JNK1(APF) and MEKK1(KR), respectively), implying that the JNK pathway is required for apoptotic signaling. Isothiocyanate-induced JNK activation was blocked by the
antioxidants 2-mercaptoethanol and
N-acetyl-L-cysteine, suggesting that the death
signaling was triggered by oxidative stress. Overexpression of Bcl-2
suppressed PEITC-induced JNK activation. In addition, Bcl-2 and
Bcl-xL suppressed PEITC-induced apoptosis, but failed to
protect cells from death induced by overexpression of activated JNK1.
These results suggest that Bcl-2 and Bcl-xL are upstream of
JNK. Taken together, our results indicate (i) that JNK mediates PMITC-
and PEITC-induced apoptosis and (ii) that PMITC and PEITC may have
chemotherapeutic functions besides their chemopreventive functions.
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INTRODUCTION |
Apoptosis plays important roles in developmental processes,
maintenance of homeostasis, and elimination of seriously damaged cells
(1, 2). The aberrant regulation of apoptosis has been observed in many
disorders such as neuronal diseases, AIDS, autoimmune diseases, and
cancers (2). In addition, many therapeutic agents eliminate tumor cells
by inducing apoptotic cell death (2). Therefore, understanding the
mechanism of apoptosis has important implications in the prevention and
treatment of many diseases.
Recent studies have identified c-Jun N-terminal kinases
(JNKs1; also named
stress-activated protein kinases) to be involved in cellular responses
to various extracellular stimuli (3). The JNK subfamily, including
JNK1, JNK2, and JNK3 in various isoforms, is a member of the
mitogen-activated protein kinase family (4). JNK activation requires
phosphorylation at a specific motif (TPY) by a dual-specificity kinase,
MKK4 (mitogen-activated protein kinase
kinase 4) (5-7). MKK4 itself is activated by
the upstream kinase MEKK1 (mitogen-activated protein
kinase/extracellular signal-regulated kinase
kinase kinase 1) (8). JNK can be
dephosphorylated and inactivated by dual-specificity phosphatases (9,
10). JNK phosphorylates transcription factors such as c-Jun, ATF-2, and Elk-1 and strongly augments their transcriptional activity (11-13). JNK activity is induced by mitogenic signals including growth factors
(14), oncogenic Ras (15), CD40 ligation (16, 17), and T-cell activation
signaling (18) as well as by environmental stresses such as protein
synthesis inhibitors (19), osmotic shock (20), pro-inflammatory
cytokines (21, 22), and shear stress (23). In addition, JNK activation
is required for apoptotic signaling induced by growth factor withdrawal
(24), UV-C (25, 26),
-radiation (26), ceramide (27), heat shock, and
DNA-damaging drugs (25). The general involvement of the JNK pathway in
cellular responses to various stimuli underscores its importance.
Furthermore, the mechanisms by which the JNK pathway is integrated into
the diverse cell signaling network are intriguing. Our previous results suggest that the duration of JNK activation determines cell
proliferation and apoptosis (26, 28).
Many isothiocyanates are effective chemopreventive agents against
carcinogen-induced cancers in experimental animals. Isothiocyanates inhibit cancer formation in various tissues such as rat lung, esophagus, mammary gland, liver, small intestine, colon, and bladder cancers (29-33). Isothiocyanates inhibit carcinogenesis caused by
different compounds, including nitrosamines and polycyclic aromatic
hydrocarbons (33). Previous studies suggested that isothiocyanates may
inhibit enzymes (e.g. cytochrome P-450 isoforms) that are
required for the bioactivation of carcinogens (34, 35). In addition,
isothiocyanates may increase the carcinogen excretion or detoxification
by inducing the phase II detoxifying enzymes, including glutathione
S-transferase (GST), quinone reductase, epoxide hydrolase,
and UDP-glucuronosyltransferase (35-37). Here, we report that
phenylmethyl isothiocyanate (PMITC; benzyl isothiocyanate) and
phenylethyl isothiocyanate (PEITC) are capable of inducing persistent
JNK activation in a dose-dependent manner. Our study indicates the involvement of JNK-mediated apoptosis in the
anticarcinogenic functions of isothiocyanates.
 |
MATERIALS AND METHODS |
Cells, Antibodies, Plasmids, and Reagents--
Human Jurkat
T-cells (clone J.LEI) were cultured as described (28). HeLa cells and
human embryonic kidney 293 cells were cultured in Dulbecco's modified
Eagle' medium supplemented with 10% fetal calf serum and
streptomycin/penicillin. Rabbit anti-JNK1 antiserum (Ab101) was
described previously (28). Anti-Bcl-2 (mouse monoclonal antibody 100),
anti-Bcl-xL (rabbit antibody S-18), anti-CPP32 (L-18), and
horseradish peroxidase-conjugated goat anti-mouse antibodies were
purchased from Santa Cruz. The horseradish peroxidase-conjugated goat
anti-rabbit antibody was obtained from Sigma. Plasmids GST-Jun-(1-79),
pCIneo-JNK1, pCMV-
MEKK1, pUna3-MEKK1(KR),
pcDNA3-Flag-JNK1(APF), pCMV-Flag-p38(AGF), and Raf-BXB301 were
described previously (26, 38, 39). Bcl-2- and
Bcl-xL-expressing vectors were obtained from Dr. D. Spencer (Baylor College of Medicine, Houston, TX). The
caspase/interleukin-1
-converting enzyme (ICE) inhibitor
Z-VAD-FK and anti-Fas antibody (CH-11) were purchased from
Kamiya Biomedical Co. Phenyl isothiocyanate (PITC), PMITC, and PEITC
were purchased from Fluka. Phenylpropyl isothiocyanate (PPITC), phorbol
12-myristate 13-acetate, ionomycin, and anisomycin were purchased from
Sigma.
DNA Fragmentation Assays--
106 cells were lysed
in 50 µl of 100 mM NaCl, 40 mM Tris-Cl (pH
7.4), and 20 mM EDTA containing 0.5% SDS. The lysate was
heated at 65 °C for 10 min for inactivation of nucleases and
digested with 0.5 mg/ml proteinase K at 50 °C for 2 h. The
lysate was then incubated with 0.2 mg/ml RNase A at 50 °C for 2 h. The DNA fragmentation was analyzed on a 1.8% agarose gel in the
presence of 0.5 µg/ml ethidium bromide.
Transient Transfection Cell Death/Protection Assays--
The
protection assay was performed as described (40) with modifications.
Briefly, 293 cells were plated 24 h before transfection at a
density of 1.5 × 105/35-mm well. Cells were
cotransfected with the pCMV-
gal plasmid encoding
-galactosidase
and plasmids for control vector, Bcl-2 or Bcl-xL,
or mutant kinases by a calcium phosphate precipitation protocol
(Specialty Media), with duplicates in each transfection. After removing
the transfection mixture, the cells were incubated in complete medium
for 12 h for recovery and then treated with or without drugs.
Cells were harvested 24 h post-treatment, washed, and fixed in 1%
paraformaldehyde in phosphate-buffer saline (PBS). The fixed cells were
washed once with PBS, resuspended in staining solution (PBS (pH 7.4), 1 mM MgCl2, 10 mM
K4(Fe(CN)4), 10 mM
K3(Fe(CN)4), 0.1% Triton X-100, and 1 mM X-gal) for 2-6 h, and then washed twice with PBS. The
ratio of
-galactosidase-expressing cells (blue color) was examined
with a hemocytometer. Cell survival was determined as follows: (% of
blue cells in treated group/% of blue cells in untreated group) × 100%.
To perform transient transfection/cell death assays, 293 cells were
transfected with empty vectors or JNK1- plus
MEKK1-expressing plasmids (in the absence or presence of Bcl-2 or Bcl-xL).
Cells were collected 48 h after transfection, fixed, and stained
as described above. Cell survival was determined as follows: (% of blue cells in the experimental group/% of blue cells in the control) × 100%.
Cell Extract Preparation and Immunocomplex Kinase
Assays--
Whole cell lysate was prepared by suspending 5 × 106 cells in 150 µl of lysis buffer (20 mM
HEPES (pH 7.9), 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 20% glycerol, 2 µg/ml
leupeptin, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl
fluoride, and 1 mM Na3VO4). The
cell lysates were kept on ice and vigorously vortexed every 5 min for
20 min. The lysate was cleared by centrifugation at 15,000 × g for 3 min, and the supernatant was stored at
80 °C. Kinase assays were carried out as described (41) with modifications. Endogenous JNK was precipitated by incubation with anti-JNK antiserum (Ab101) and protein A-agarose beads (Bio-Rad) in incubation buffer (20 mM HEPES (pH 7.4), 2 mM EGTA, 50 mM
glycerophosphate, 1% Triton X-100, 10% glycerol, 1 mM
dithiothreitol, 2 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na3VO4). The precipitates were washed twice
with incubation buffer, twice with LiCl buffer (500 mM
LiCl, 100 mM Tris-Cl (pH 7.6), and 0.1% Triton X-100), and
twice with kinase buffer (20 mM MOPS (pH 7.6), 2 mM EGTA, 10 mM MgCl2, 1 mM dithiothreitol, 0.1% Triton X-100, and 1 mM
Na3VO4) and then mixed with 5 µg of
GST-Jun-(1-79), 15 µM of ATP, and 10 µCi of
[
-32P]ATP in 30 µl of kinase buffer. The kinase
reaction was performed at 30 °C for 30 min and then terminated by
adding SDS sample buffer. The reaction mixtures were boiled and
analyzed by SDS-polyacrylamide gel electrophoresis and
autoradiography.
Western Blot Analysis--
Cells were lysed in lysis buffer (20 mM HEPES (pH 7.4), 2 mM EGTA, 50 mM
glycerophosphate, 1% Triton X-100, 10% glycerol, 1 mM
dithiothreitol, 2 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM
Na3VO4). The lysate was resolved by
SDS-polyacrylamide gel electrophoresis (12%) and then transferred to
polyvinylidene difluoride membrane. The membrane was incubated with
primary antibody (anti-CPP32, 1:200 dilution; anti-Bcl-2, 1:500; and
anti-Bcl-xL, 1:1000), washed, and blotted with horseradish
peroxidase-conjugated secondary antibody (1:1000 dilution). The
membrane was then developed in ECL reagent (Amersham Corp.) and exposed
to x-ray film.
 |
RESULTS |
Isothiocyanates Induce JNK Activation in a
Dose-dependent Manner--
Human leukemia Jurkat T-cells
were treated with different concentrations of PITC, PMITC, PEITC, and
PPITC. Cells were collected 2 h after treatment, and endogenous
JNK activity was determined by immunocomplex kinase assays. Among the
tested isothiocyanates, PMITC and PEITC induced strong JNK activation
at a concentration of 5 µM, and PITC induced mild JNK
induction at higher concentrations (50-100 µM). No
apparent JNK activation was observed with PPITC treatment at all
concentrations tested (Fig.
1A). In contrast to the JNK
activation, decreases in JNK activity (in comparison with the basal
levels in the untreated group) were observed in treatments with high
concentrations (>50 µM) of PMITC, PEITC, and PPITC (Fig.
1A). The decreases in JNK activity in treatments with high
doses of isothiocyanates may be due to the acute cytotoxicity of these
drugs. Jurkat cells swelled and lost the ability to exclude trypan blue
(Fig. 1B), and the protein recovery in the treated cell
lysate was significantly decreased after treatments with high doses of
PMITC, PEITC, and PPITC. These phenomena indicated that the integrity
of the cell membrane was lost and that the cells died by membrane
disintegration and cytolysis, which is reminiscent of necrosis.
However, >80% of the cells retained the ability to exclude trypan
blue 24 h post-treatment with different isothiocyanates at 5 µM (Fig. 1B).

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Fig. 1.
Dose response of Jurkat cells to
isothiocyanates. A, Jurkat cells were treated with different
isothiocyanates in various concentrations as indicated. Cell lysate was
collected 2 h after treatments, and endogenous JNK activity was
determined by immunocomplex kinase assays using GST-Jun-(1-79) as a
substrate. B, Jurkat cells were cultured in medium with or
without different doses of isothiocyanates for 24 h, and then the
percentage of dead cells was determined by the trypan blue exclusion
assay.
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PMITC and PEITC Induce Sustained JNK Activation and
Apoptosis--
To exclude the possibility that the differential
regulation of JNK by isothiocyanates is due to the selected observation
at the 2-h time point, we did a time course study of JNK activation with various isothiocyanates at either 5 µM (Fig.
2A) or 50 µM (Fig. 2B) in Jurkat T-cells. At a concentration of 5 µM, both PMITC and PEITC induced persistent JNK
activation. JNK activity increased at the 1-h time point, peaked around
2-4 h post-treatment, and gradually decreased, but remained higher
than basal levels even 12 h after treatment. In contrast, 5 µM PITC or PPITC failed to induce any JNK activation. At
50 µM, only PITC induced a slight JNK induction, whereas
PMITC, PEITC, and PPITC decreased the basal levels of JNK activity.
These results were consistent with the data in the dose-response
experiment (Fig. 1A), showing that isothiocyanates induced
JNK activation in a dose-dependent manner and also
revealing that PMITC and PEITC were strong JNK activators,
inducing sustained JNK activation at certain concentrations.

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Fig. 2.
Persistent JNK activation and apoptosis
induced by PMITC and PEITC. A and B, Jurkat cells
were treated with different isothiocyanates at either 5 µM (A) or 50 µM (B);
cells were collected at the indicated time points; and endogenous JNK
activity was determined by the immunocomplex assay. C,
Jurkat cells were treated with different isothiocyanates (5 µM) and collected at 12- and 24-h time points. Cellular
DNA was extracted and analyzed on a 1.8% agarose gel for DNA
fragmentation.
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In our previous study, persistent JNK activation was associated with
apoptosis induced by
-radiation and UV-C (26). Since most of the
early apoptotic cells maintain an intact cytoplasmic membrane, trypan
blue staining is not an appropriate criterion for apoptosis. Hence, a
DNA fragmentation assay was used to examine if the persistent JNK
activation induced by PMITC and PEITC was associated with apoptotic
cell death. Cellular DNA was extracted from Jurkat cells treated with
different isothiocyanates (5 µM) and analyzed for DNA
fragmentation. We found that only PMITC and PEITC, which induced
persistent JNK activation at 5 µM, caused chromosomal DNA
laddering at 12-24 h after treatment (Fig. 2C). This result
shows the correlation between persistent activation of JNK and
apoptosis induction by isothiocyanate treatments and the precedence of
JNK activation to DNA fragmentation.
The Caspase/ICE Protease Inhibitor Fails to Inhibit JNK Activation
by Isothiocyanates--
The caspase/ICE family of proteases are known
to be important apoptosis mediators (42); hence, we determined if
caspases/ICE-like proteases are involved in JNK-mediated apoptosis
induced by isothiocyanates. PMITC- and PEITC-induced DNA fragmentation
was completely inhibited by cotreatment with a caspase/ICE protease
inhibitor, Z-VAD-FK (Fig.
3A). Z-VAD-FK
blocked the cleavage of CPP32 (caspase 3) caused by anti-Fas treatment
(Fig. 3B), indicating that it is an effective caspase
inhibitor. In contrast to the inhibition of DNA fragmentation,
Z-VAD-FK did not abolish JNK activation induced by PMITC
(Fig. 3C) or PEITC (data not shown). The slight enhancement
of JNK activation by Z-VAD-FK was not reproducible in
repeated experiments, and therefore, was fluctuation of kinase assays.
These data indicate the requirement of caspases/ICE-like proteases in
isothiocyanate-induced apoptosis; however, JNK activation can occur in
the absence of caspase activity. Also, in light of the fact that JNK
activation occurred hours before the onset of DNA fragmentation during
the isothiocyanate treatments (Fig. 2), JNK activation is unlikely to
be a secondary effect of the cellular damage during apoptosis. These
data suggest that JNK activation initiates apoptotic signaling.

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Fig. 3.
Failure of Z-VAD-FK to suppress
isothiocyanate-induced JNK activation. A, Jurkat cells were
pretreated with the caspase/ICE inhibitor Z-VAD-FK (100 µM) for 3 h and treated with the different isothiocyanates at 5 µM for 24 h. The cells were
then harvested and assayed for DNA fragmentation. B, Jurkat
cells preincubated with Z-VAD-FK or with the solvent
(dimethyl sulfoxide) were treated with anti-Fas antibody (CH-11; 100 ng/ml) for 4 h. The cleavage of CPP32 was examined by the Western
blot analysis. C, Jurkat cells were pretreated with or
without the caspase/ICE inhibitor Z-VAD-FK (100 µM) for 3 h. These cells were then treated with 5 µM PMITC. Samples of cells were collected at the time
points indicated, and JNK activity was examined by immunocomplex
assays.
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PEITC Induces JNK Activation and Apoptosis in Various Cell
Types--
We next examined if the isothiocyanate induces persistent
JNK activation and apoptosis in different cell types. We found that different concentrations of PEITC were needed to induce JNK activation in distinct cell types; the effective concentration varied from 5 to 50 µM (data not shown). 20 µM PEITC induced
persistent JNK activation in HeLa and 293 cells (Fig.
4A). At the concentration that
induced sustained JNK induction, PEITC also caused apoptosis (Fig.
4B). On examination after Hoechst 33258 staining, the cell nuclei were condensed and fragmented after the PEITC treatment, in
comparison with the homogeneous nuclear staining of the untreated cells
(Fig. 4B). These data demonstrate that the induction of JNK
activation and apoptosis by PEITC can occur in various cell types.

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Fig. 4.
JNK induction and apoptosis induced by PEITC
in HeLa and 293 cells. A, HeLa and 293 cells were treated
with PEITC (20 µM), and the treated cells were harvested
at different time points as indicated. Endogenous JNK activity was
examined by immunocomplex assays. B, HeLa and 293 cells were
treated with or without PEITC (20 µM) for 24 h, and
then the cells were harvested, washed once with PBS, and fixed in 1%
paraformaldehyde in PBS. The fixed cells were incubated with Hoechst
33258 (2.5 µg/ml in PBS). Nuclear staining was examined with a
fluorescence microscope.
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Interfering with the JNK Pathway Suppresses PEITC-induced
Apoptosis--
Previously, we have shown that the JNK pathway is
involved in and required for radiation-induced apoptosis
(26). Other investigators also showed that the JNK cascade is required
for apoptosis induced by growth factor withdrawal or ceramide treatment
(24, 27). We then tested if interfering with the JNK pathway had a
suppressive effect on isothiocyanate-induced apoptosis. 293 cells were
transfected with pCMV-
gal with or without plasmids encoding a
dominant-negative kinase mutant, and each transfection was duplicated
for treatments with or without PEITC (20 µM). The cells
were harvested 24 h after treatment and stained with X-gal to
examine the
-galactosidase-expressing cells (blue in color). The
survival rate of transfected cells after the drug treatment was
determined as the percentage of blue cells in the treated group divided
by the percentage of blue cells in the untreated group. The
dominant-negative mutants of MEKK1 and JNK1 (MEKK1(KR) and JNK1(APF),
respectively) blocked PEITC-induced cell death in transfected 293 cells
(Fig. 5). In contrast, transfection of
wild-type JNK1, a dominant-negative Raf1 mutant (Raf-BXB301), or
dominant-negative p38 mitogen-activated protein kinase (p38(AGF)) did
not significantly affect apoptosis induced by PEITC. This result
indicates that interfering with the JNK pathway prevents the
PEITC-induced apoptosis, thereby suggesting that the JNK pathway is
required for isothiocyanate-induced apoptosis.

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Fig. 5.
Suppression of PEITC-induced apoptosis by
interfering with the JNK pathway. 293 cells were transfected in
duplicates with pCMV- gal (1 µg) and mutant kinase-expressing
vectors (3 µg for each) as indicated. Empty vectors were used to
bring the total transfected DNA to 7 µg. After transfection, the
cells were incubated in culture medium for 12 h and then treated
with or without PEITC (20 µM). Cells were harvested
24 h after treatment, and the -galactosidase-positive cells
(blue color) were examined by enzymatic staining using X-gal as a
substrate. Cell survival was determined as follows: (% of blue cells
in treated group/% of blue cells in untreated group) × 100%. The
data presented are the means ± S.D. of six experiments.
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Isothiocyanate-induced JNK Activation Is Inhibited by
Antioxidants--
JNK activity is regulated by the upstream kinase
cascade (MEKK1
MKK4
JNK, 3); however, it is not clear which
upstream signals regulate the JNK module after cells receive apoptotic stimuli. Isothiocyanates react with GSH and form dithiocarbamates (R-NH-C(=S)-SG) in the presence or absence of GST (43). Since apoptosis is activated by many oxidative agents (44), the
isothiocyanates may induce oxidative stress by reacting with and
depleting the intracellular GSH pool, which may then induce JNK
activation and apoptosis. We decided to test the roles of oxidation in
isothiocyanate-induced JNK activation using antioxidants. JNK
activation by PMITC and PEITC was inhibited by preincubation of the
Jurkat cells with antioxidants, 2-mercaptoethanol (10 mM,
1 h) or N-acetylcysteine (20 mM, 2 h)
(Fig. 6). The antioxidant treatments also
blocked
-radiation-induced JNK activation, but had no effect on JNK
induction by phorbol 12-myristate 13-acetate plus ionomycin or by
anisomycin (Fig. 6). The inhibition of JNK activation is not due to the
loss of viability of Jurkat cells after antioxidant treatment because >95% of the cells retained the ability to exclude trypan blue 6 h after antioxidant treatment (data not shown). These data indicate that the JNK activation in cells exposed to PMITC, PEITC, or
-radiation may be due to the induction of oxidative stresses. In
contrast, the induction of JNK by phorbol 12-myristate 13-acetate plus
ionomycin or by anisomycin may not be mediated through the
intracellular oxidative changes.

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Fig. 6.
Inhibition of JNK activation by
antioxidants. Jurkat cells were pretreated with or without
N-acetyl-L-cysteine (NAC; 20 mM) or 2-mercaptoethanol (2ME; 10 mM) and then treated with different stimuli: PMITC (5 µM, 2 h), PEITC (5 µM, 2 h),
-radiation (100 gray, 2 h), phorbol 12-myristate 13-acetate
(PMA; 50 ng/ml) plus ionomycin (1 µM), or
anisomycin (2 µM, 30 min). JNK activity was assayed by
immunocomplex kinase assays.
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Bcl-2 Suppresses PEITC-induced JNK Activation and
Apoptosis--
Bcl-2 family members are known to be important
apoptosis regulators (45). We used a transient transfection/cell
death protection assay to test if Bcl-2 or Bcl-xL can block
apoptosis induced by isothiocyanates. The levels of the Bcl-2
(Bcl-xL) protein were examined by Western blot analysis
(Fig. 7A), revealing the
production of transfected genes. The empty vector control group lost
40% of cells after the drug treatment. In comparison, transfection of
Bcl-2 or Bcl-xL prevented PEITC-induced apoptosis in most
of the transfected cells (Fig. 7A), indicating that Bcl-2
and Bcl-xL were capable of protecting cells from
PEITC-induced apoptosis. We also examined the PEITC-induced JNK
activation in 293 cells transfected with empty vector or Bcl-2-encoding
plasmids. Although only 40-50% of the cells were transfected, the
endogenous JNK activity induced by PEITC was evidently decreased in
Bcl-2-transfected cells (Fig. 7B). This result implicates
Bcl-2 as an upstream suppressor for JNK activation by apoptotic
stimuli.

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Fig. 7.
Suppression of PEITC-induced JNK activation
and apoptosis by Bcl-2. A, 293 cells were
transfected with pCMV- gal (1 µg) and control vector (3 µg) or
Bcl-2- or Bcl-xL-expressing plasmid (3 µg). Cells were
cultured in complete medium for 12 h after transfection and then
treated with or without PEITC (20 µM) for 24 h. Some
of the harvested cells were assayed for cell survival as described
under "Materials and Methods." The data presented are the
means ± S.D. of three experiments. The remaining cells were
lysed, and the expression of Bcl-2 and Bcl-xL was determined by Western blot
analysis. B, 293 cells were transfected with control vector
or Bcl-2-encoding plasmid (3 µg), and transfected cells were treated
for the indicated times with 20 µM PEITC 6 h after removing the transfection mixture. Endogenous JNK activity was examined
by immunocomplex kinase assays. C, 293 cells were
transfected with pCMV- gal (1 µg) plus different combinations of
plasmids as indicated (JNK1, 1.5 µg; MEKK1, 1.5 µg; and Bcl-2 or
Bcl-xL, 3 µg). Empty vector was added to normalize the
total DNA amount. Cells were collected 48 h after transfection,
fixed, stained, and examined as described under "Materials and
Methods." The cell survival data presented are the means of three
experiments. Western blotting for Bcl-2 and Bcl-xL was
performed as described under "Materials and
Methods."
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To further examine the molecular ordering between Bcl-2 or
Bcl-xL and the JNK pathway, we cotransfected Bcl-2 or
Bcl-xL with JNK1 plus constitutively active MEKK1
(
MEKK1) into 293 cells. The transfection of JNK1 plus
MEKK1 led
to persistent JNK activation and caused apoptosis independent of the
upstream signals (Fig. 7C). Neither Bcl-2 nor
Bcl-xL significantly affected the cell death caused by JNK1
plus
MEKK1, although the levels of Bcl-2 and Bcl-xL were
increased in the transfected cells (Fig. 7C). In comparison
to the Bcl-2- or Bcl-xL-transfected cells, the
lower levels of Bcl-2 and Bcl-xL detected in the cells
cotransfected with JNK1 plus
MEKK may be due to the loss of
transfected cells caused by apoptosis (Fig. 7C). This result
indicates that Bcl-2 and Bcl-xL failed to prevent cell
death induced by JNK activation; therefore, they may not be downstream
of JNK in the apoptotic signaling pathway. Taken together, our results
suggest that Bcl-2 and Bcl-xL are upstream, but not
downstream, of JNK in apoptotic signaling induced by isothiocyanates
(Fig. 8).
 |
DISCUSSION |
Activation of the JNK pathway has been shown to be a common
phenomenon in apoptotic cell death (24-27, 46); however, the importance of this activation seems to vary in apoptosis caused by
different agents. The JNK pathway is required for apoptosis induction
by growth factor withdrawal, heat shock, radiation, and ceramide
(24-27). In contrast, JNK may not be essential for receptor-mediated
apoptosis (e.g. Fas- and tumor necrosis factor-mediated apoptosis) (47, 48). In this study, we demonstrate the involvement of
JNK in isothiocyanate-induced apoptosis by proving that interfering with the JNK pathway suppressed isothiocyanate-induced cell death. We
observed that high doses of PMITC, PEITC, and PPITC (>50
µM) caused acute cell death in the absence of JNK
activation; however, the cell death resembled necrosis rather than
apoptosis. This result indicates that induction of JNK activity is not
a general event caused by stress during cell death, but rather it is a
specific phenomenon associated with apoptotic cell death. In addition, the failure of a caspase/ICE inhibitor to block JNK activation caused
by isothiocyanates indicates that JNK activation may initiate apoptotic
signaling and that it is not a secondary effect of cellular damage from
apoptotic cell death. However, we (26) and others (48) have shown that
JNK activation in Fas-mediated apoptosis can be suppressed by a
caspase/ICE inhibitor, which suggests that JNK induction could be
augmented by activation of caspases/ICE-like proteases. A recent report
indicates that CPP32, a caspase/ICE-like protease, is capable of
cleaving D4-GDI, a GDP dissociation inhibitor of the Ras-related Rho
family GTPase (49). The process of D4-GDI may irreversibly activate the
Rho family G proteins, which are activators of the JNK pathway (50,
51). Therefore, the activation of CPP32 may lead to the enhancement of
JNK activation. If this regulatory mechanism exists, it may work as a
signaling circuit to amplify the apoptotic signal, but it may not be
essential for Fas-mediated apoptosis.
Our result indicates that oxidative stress may initiate JNK activation
(Fig. 6). The anti-apoptotic regulator Bcl-2 may inhibit apoptosis by
suppressing the formation or the damaging effects of reactive oxygen
species (ROS) (52, 53), which are generated by many apoptotic agents
(44). In this report, we showed that Bcl-2 suppressed PEITC-induced JNK
activation. Bcl-2 or Bcl-xL blocked apoptosis caused by
PEITC, but failed to suppress apoptosis caused by overexpression of
activated JNK1. In addition, a recent report showed that Bcl-2 blocks
JNK activation induced by serum depletion or nerve growth factor
withdrawal in PC-12 cells (54). Taken together, Bcl-2 may be an
upstream suppressor of the JNK pathway acting to relieve the oxidative
stress and to prevent JNK induction and the initiation of apoptotic
signals (Fig. 8). The failure of Bcl-2 or Bcl-xL to block
cell death induced by JNK1 plus
MEKK1 suggests that Bcl-2 may not be
downstream of JNK. However, these data do not exclude the possibility
that JNK may directly or indirectly down-regulate the function of
Bcl-2, therefore causing cell death. The tumor suppressor p53, which is
essential for ionizing radiation-induced apoptosis (55), has been shown
to be a substrate of JNK (56). p53 is a positive regulator for the
expression of Bax (57), a counteracting molecule of Bcl-2 and a potent
apoptosis inducer (58). Although the exact physiological function of
this p53 phosphorylation is still unclear, p53 may be a mediator for
JNK-induced apoptosis.
Isothiocyanates are well known for their chemopreventive effects on
various carcinogens (29-33). Previous studies attributed this
anticarcinogenic property to their ability to affect the bioactivation,
detoxification, and excretion of carcinogens (34-37). Here, we show
that isothiocyanates induced apoptosis by activation of the JNK
pathway. We propose that isothiocyanate-mediated apoptosis may be one
possible mechanism to achieve the anticarcinogenic function.
Carcinogens usually cause genomic damages in the exposed cells. If the
damages are limited, the cells can repair those damages and maintain
normal functions. The cells that fail to repair the damages are
normally eliminated by apoptosis, which prevents the propagation of
genomic damages to progenitor cells. The cells with genomic damages
that escape cell death are prone to develop into cancerous cells. In
the presence of isothiocyanates, the cells will start the apoptosis
process because of an increase in oxidative stress and JNK activity.
The simultaneous or subsequent exposure to a carcinogen will trigger
the cell death progression, and the damaged cells will be eliminated by
apoptosis; therefore, fewer cells can survive and become cancer cells.
In addition, cancer cells usually have a higher metabolic rate and
generate higher levels of intracellular oxidants than normal cells. The ability of isothiocyanates alone to generate oxidative stress, activate
the JNK pathway, and induce apoptosis suggests that they may have a
therapeutic function in addition to their chemopreventive functions.
We thank M. C.-T. Hu, M. Karin, R. J. Davis, J. Bruder, D. Spencer, and D. Templeton for generous gifts;
members of the Tan laboratory for helpful discussions and critical
reading of this manuscript; A. Brown and S. Lee for technical
assistance; and M. Lowe for secretarial assistance.