From the Division of Pulmonary and Critical Care
Medicine, Stanford University Medical Center,
Stanford, California 94305-5236, ¶ Transgenic Oncogenesis Group,
Laboratory of Cell Regulation and Carcinogenesis, National Institutes
of Health, Bethesda, Maryland 20892, and
Divison of
Pulmonology, Department of Internal Medicine, Dankook University
Hospital, Cheonan, Chungnam 330-180, Korea
Received for publication, October 24, 2000
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ABSTRACT |
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Triptolide (PG490), a diterpene triepoxide, is a
potent immunosuppressive agent extracted from the Chinese herb
Tripterygium wilfordii. We have previously shown that
triptolide blocks NF- The p53 tumor suppressor protein plays a critical role in
regulating cell cycle checkpoints and apoptosis (reviewed in Ref. 1).
Various post-translational modifications and protein-protein interactions stabilize the level of the p53 protein (1). For example,
DNA-dependent protein kinase and mutated in ataxia
telangiectasia phosphorylate p53 at serine 15 activating p53 following
DNA damage, but Mdm2, through association with p53, negatively
regulates p53 by translocating p53 from the nucleus to the cytosol and
mediating the ubiquitin/proteosome degradation of p53 (2, 3). A less well characterized mechanism is translational regulation of p53 (4).
Translational regulation of p53 expression is controlled by a negative
autoregulatory feedback (4, 5). The p53 protein interacts with its
3'-untranslated region (UTR)1
by binding to a 330-nucleotide region at the distal end of the 3'-UTR.
For example, following The increase in p53 protein in response to various stresses is an
important regulator of cell cycle and apoptosis (1). In
transcription-dependent p53 activation, p53 functions as a site-specific transcription factor that induces p53-inducible genes
such as p21cip1/waf1, bax, gadd45, and
mdm2. This in turn initiates the program of growth arrest or
apoptosis in a stress-specific and/or cell type-specific manner
(reviewed in Ref. 8). p53 also acts as a transcriptional repressor by
inhibiting the expression of genes such as c-fos, DNA
polymerase The p53 gene is inactivated in ~50% of tumors. The effect of p53
inactivation on the response of tumors to chemotherapy or radiation is
conflicting with some studies showing enhanced sensitivity and others
showing increased resistance to the same compounds. Two recent studies
have shown that p53-mediated induction of p21 inhibits the apoptotic
response by inducing growth arrest (13, 14). Bunz et al.
(13), for example, show that p53- or p21-deficient cells are more
sensitive to adriamycin (doxorubicin)-induced apoptosis than wild-type
cells because they do not induce p21 in response to doxorubicin.
Therefore, a compound that blocks chemotherapy-mediated growth arrest
may accelerate and enhance apoptosis.
A crude extract from the Chinese herb, Tripterygium
wilfordii, also called T2, has been used as an immunosuppressant
for the treatment of inflammatory diseases such as rheumatoid
arthritis. One purified component of T2, the diterpene triepoxide
triptolide is immunosuppressive and cytotoxic to tumor cells, and a
recent study showed that triptolide inhibits cytokine-mediated
activation of NF- In this study, we show that triptolide and doxorubicin act in synergy
to kill tumor cells. Interestingly, we find that triptolide induces
translation and phosphorylation of p53, but triptolide-modified p53 is
transcriptionally inactive. Tritpolide also blocks doxorubicin-mediated induction of p21 and doxorubicin-mediated growth arrest. Our results suggest that triptolide enhances doxorubicin-mediated apoptosis, at
least in part, by blocking p21-mediated growth arrest.
Reagents--
PG490 (triptolide, MW 360) was obtained from
Pharmagenesis (Palo Alto, CA). A549 (non-small cell lung cancer) and
HT1080 (fibrosarcoma) cell lines were from ATCC. Mouse embryonic
fibroblasts (p53 +/+ and p53 Cell Culture and Plasmids--
A549 and HT1080 cells were
cultured in the appropriate media with 10% fetal calf serum
supplemented with L-glutamine, penicillin, and
streptomycin. p53 wild-type (+/+) and null (
The full-length coding region of human p21 cDNA was
amplified by RT-PCR from HT1080 cells with oligo primers
5'-GGATCCGCCACCATGTCAGAACCGGCTGGGG-3' and
5'-GTCGACTCACTTGTCATCGTCGTCCTTGTAGTCCTCGAGGGGCTTCCTCTTGGAGAAGATCAG-3'. The 3' primer was manipulated to add an in-frame FLAG-tag
sequence before the stop codon. Subsequently, the cDNA fragments
were cloned into the pIND, ecdysone-inducible vector (Invitrogen,
Carlsbad, CA). The full-length sequence of human p21 coding region was
confirmed by DNA sequencing. The cDNA of Cell Viability Assay--
HT-1080 cells were seeded into 6-well
plates at 2 × 105 per well the day before the
treatment. For the inducible expression of exogenous p21 and LacZ, the
stable transfectants were cultured in the presence of 5 µM ponasterone A for 16 h. Three fields from each
well were carefully selected, marked, and counted to ensure a similar
cell number greater than 300 cells per field before the treatment. The
cells were untreated or treated with doxorubicin, triptolide, or the
combination of both at indicated dosages for 8 h at 37 °C.
Subsequently, the medium was replaced with new media plus 5 µM ponasterone A. After 16 h incubation at 37 °C,
the number of viable cells within the same fields were determined by
trypan blue exclusion with a 2% trypan blue solution. Cell death was confirmed as apoptotic by annexin V/propidium iodide (PI) staining followed by FACS analysis as described previously (19).
Northern Blot Analysis--
RNA was prepared from HT1080 cells
using RNeasy Mini Kit from Qiagen Inc. (Valencia, CA). cDNAs for
Northern blot analysis for p21 and p53 were prepared using
RT-PCR with 2 µg of total RNA. The following primer pairs were used:
p53, 5'-AGTCAGATCCTAGCGTCGAG-3' and 5'-TCTTCTTTGGCTGGGGAGAG-3'; p21,
5'-AGTGGGGCATCATCAAAAAC-3' and 5'-GACTCCTTGTTCCGCTGCTAATC-3'; and
glyceraldehyde-3-phosphate dehydrogenase,
5'-CCCATCACCATCTTCCAG-3' and 5'-ATGACCTTGCCCACAGCC-3'. Northern
blot analysis was performed as described previously (18).
Electromobility Shift Assay (EMSA)--
HT1080 cells were
treated as shown. The EMSA was performed as described previously using
an end-labeled 32P-p53 consensus binding site (Santa Cruz
Biotechnology, Santa Cruz, CA) (18).
Immunoblotting--
Cells were harvested at the conditions and
times indicated and lysed using HNET buffer (50 mM HEPES,
pH 7.5, 100 mM NaCl, 1 mM EGTA, and 1% Triton
X-100) supplemented with 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors
mixture (Roche Molecular Biochemicals). 100 µg of protein was loaded
on 10% SDS-PAGE followed by transferring to polyvinylidene difluoride membrane. Immunoblotting was performed as described previously using a
p53 mouse monoclonal antibody from Oncogene Research Products (19). To
measure p53 half-life, cycloheximide (30 µg/ml) was added to HT1080
cells 30 min after the addition of triptolide and harvested at
the times shown for immunoblot analysis of p53. Immunoblot analysis
using other antibodies was performed as described above. The band
intensity was measured by NIH Image 1.62.
Cells Cycle Analysis--
HT1080 cells (2 × 106) were treated with triptolide (20 ng/ml) and/or
doxorubicin (100 nM) for 16 h. Cells were then
harvested and washed with cold PBS. The cells were resuspended gently
in 5 ml of 100% ethanol and fixed at 25 °C for 1 h. After
washing with PBS, the cells were incubated with DNase-free RNase A (200 µg/ml) at 37 °C for 1 h and washed with PBS. Propidium iodide (10 µg/ml) was added, and the cells were incubated at 37 °C for 5 min. Cells were separated by sonicating at 20% output level for
15 s using a VirSonic 50 sonicator (Vitis Inc., NY). The samples were then sorted by FACS, and cell cycle analysis was done with FlowJo
(version 3.0.3) (Tree Start, Inc, San Carlos, CA).
Metabolic Labeling of HT1080 Cells--
Cells were grown to 80%
confluence followed by pretreatment with triptolide (20 ng/ml) for
6 h in the appropriate medium. Cells were washed twice with short
term labeling medium (RPMI with 5% dialyzed fetal calf serum
supplemented with L-glutamine, penicillin, and
streptomycin). To deplete intracellular pools of methionine, short term
labeling medium was added for 15 min at 37 °C and then replaced by
short term labeling medium containing 0.1 mCi/ml
[35S]methionine (Amersham Pharmacia Biotech). Cells were
labeled for 30 min at 37 °C and washed with ice-cold PBS before
harvesting for immunoprecipitation. The cells were lysed using RIPA
buffer supplemented with protease inhibitors and immunoprecipitated
using an agarose-conjugated p53 mAb (Ab-6, Oncogene Research Products) followed by 10% SDS-PAGE. The intensity of labeled p53 protein was
measured by NIH Image 1.62.
In Vivo [32P]Orthophosphate
Labeling--
Subconfluent HT1080 cells were pretreated with 20 ng/ml
triptolide for 2 h in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum and antibiotics. These cells
were washed twice with 37 °C labeling medium (Dulbecco's modified
Eagle's medium, 10% fetal bovine serum dialyzed against
phosphate-free) lacking sodium phosphate. Cells were then labeled for
an additional 1 h with the labeling medium containing 1 mCi/ml
[32P]orthophosphate and 20 ng/ml triptolide. The labeling
medium was removed, and the cells were washed three times with cold
Tris-buffered saline (TBS). The cells were scraped in 1 ml of lysis
buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.2, 2 mM EDTA, 50 mM sodium fluoride, 0.2 mM sodium vanadate, and 100 units/ml aprotinin) and kept on ice for 10 min. Lysates were passed through 27-gauge needles and spun
at 11,000 × g for 10 min, and 100 µg of the
supernatants were subjected to immunoprecipitation with p53 mAb (Ab-6)
conjugated with agarose for 4 h. Samples were then spun at 2500 rpm at 4 °C for 5 min and washed three times with the lysis buffer.
The pellets were boiled in SDS sample buffer and analyzed on 10%
SDS-PAGE. The bands were visualized and quantified by Optiquant PhosphorImager.
Triptolide Enhances Chemotherapy-induced Apoptosis--
Triptolide
is a diterpene triepoxide extracted from a Chinese herb with potent
immunosuppressive effects (15-17). We have recently shown that
triptolide sensitizes several solid tumor cell lines to TNF- Triptolide Induces p53 but Inhibits p21 Expression--
p53
mediates cell death responses to cytotoxic stimuli such as hypoxia,
irradiation, and DNA-damaging chemotherapeutic agents. Since triptolide
alone is cytotoxic and it cooperates with DNA-damaging chemotherapeutic
agents, we hypothesized that triptolide-induced apoptosis may be
mediated by p53. In HT1080 cells that contain wild-type p53, triptolide
(20 ng/ml) increased p53 steady-state protein levels 4-fold for 9 h, and triptolide (5 ng/ml) induced a 2.4-fold increase in p53 (Fig.
2A). Doxorubicin induced a
4.9-fold increase in p53, and the combination of doxorubicin plus
triptolide induced a 4-fold increase in p53 protein (Fig.
2A). In A549 cells, the combination of triptolide (20 ng/ml)
plus doxorubicin (100 nM) at 24 h showed greater than
5-fold increase in p53 (data not shown). We next examined if the
increase in the p53 protein level was due to an increase in the p53
mRNA. The level of p53 mRNA was not affected by triptolide
(data not shown). These data suggest, therefore, that triptolide
induces post-transcriptional accumulation of p53.
A current model of p53-mediated apoptosis is that upon cellular
stresses (such as DNA damage), p53 is stabilized, and this increases
expression of genes such as mdm2, bax, p21cip1/waf1,
and gadd45. Recent studies show that doxorubicin and
Triptolide (20 ng/ml) reduced basal p21 levels by 50% despite inducing
p53 (Fig. 2A). Doxorubicin induced a 14.5-fold increase in
p21 that was completely blocked by triptolide (20 ng/ml), and triptolide (5 ng/ml) reduced doxorubicin-mediated induction of p53
by 58%.
We planned to examine the effect of triptolide on p21 expression in p53
wild-type and null MEFs, but p21 basal expression is almost absent in
p53 Triptolide Inhibits p21 mRNA Expression--
Triptolide (20 ng/ml) also completely blocked doxorubicin-mediated induction of p21
mRNA (Fig. 3A). Triptolide
or doxorubicin did not affect p21 expression in the p53 mutant HT29
colon cancer cell line (data not shown). Triptolide also blocked
doxorubicin-mediated induction of mdm2 mRNA, but
it did not affect gadd45 or map4 mRNA expression (Fig. 3B). These data show that triptolide alone
inhibits transcription of p21, and it blocks doxorubicin-mediated
transcriptional induction of p21 despite increasing p53 levels.
Triptolide does not inhibit DNA binding of p53--
Triptolide
induces p53 but inhibits p21 expression. Also, triptolide blocks
doxorubicin-mediated induction of p21. We then performed EMSA to
determine whether triptolide, alone or in combination with doxorubicin,
inhibits DNA binding of p53 to a p53 consensus binding site in the p21
promoter. Triptolide alone slightly enhanced DNA binding of p53, and it
did not block doxorubicin-mediated induction of DNA binding (Fig.
4). These data suggest triptolide represses expression of p21 by blocking transactivation but not DNA
binding of p53.
Triptolide Inhibits p21-mediated Growth Arrest--
We then
evaluated the effect of triptolide alone and in combination with
chemotherapy on cell cycle progression. Triptolide (20 ng/ml) alone
increased the number of cells in S phase from 23.7% in unstimulated
cells to 46% in triptolide-treated cells (Fig.
5). Doxorubicin induced accumulation of
cells in G2/M from 11 to 49.8%, but triptolide inhibited
doxorubicin-mediated G2/M accumulation from 49.8 to 22.6%
(Fig. 5). A recent study showed that potent inhibition of tumor
survival is achieved by combining drugs with different cell cycle
checkpoints (20). Li et al. (20) show that Overexpression of p21 Inhibits Cytotoxic Synergy between Triptolide
and Doxorubicin--
To determine whether triptolide-mediated
inhibition of p21 is involved in the cytotoxic synergy between
triptolide and doxorubicin, we overexpressed p21 in HT1080 cells using
a ponasterone-inducible p21 vector (pIND-p21). The addition of
ponasterone A (5 µM) strongly induced exogenous p21
expression that was slightly reduced by triptolide plus doxorubicin
(Fig. 6A). The combination of
triptolide (5 ng/ml) and doxorubicin (100 nM) reduced cell
viability to 30% in the induced vector control, and viability
increased to 58% following the induction of exogenous p21 (Fig.
6B).
Triptolide Induces Translation of p53--
To determine the
mechanism by which triptolide induces p53, we examined the effect of
triptolide on p53 protein stability and translation. To examine the
effect on stability, we examined levels of p53 in the presence of
cycloheximide (30 µg/ml) in HT1080 cells, a dose that blocks
translation. In cells that were pretreated with triptolide (20 ng/ml)
for 0.5 h prior to the addition of cycloheximide, there was a
slight increase in p53 stability at 30 min, but there was no difference
from untreated cells at 60 min (Fig.
7A). These data suggested that
the increased steady-state level of the p53 protein in response to
triptolide did not result from an increase in the half-life of the p53
protein. We then examined if triptolide induces translation of p53 by
in vivo [35S]methionine metabolic labeling of
HT1080 cells. We found, interestingly, that triptolide induced a
4.9-fold increase in p53 translation (Fig. 7B). Thus,
triptolide-induced p53 accumulation is mediated by an increase in p53
translation.
Triptolide Induces Phosphorylation of p53--
Many studies have
shown that phosphorylation of p53 in response to DNA damage regulates
p53-mediated apoptosis and transcriptional activity (21-23).
Therefore, we examined if p53 undergoes hyperphosphorylation upon
triptolide treatment in HT1080 cells. There was a 2-3-fold increase in
phosphorylated p53 in triptolide-treated cells compared with p53 from
the untreated cells (Fig. 8). Western
blot analyses of the samples show equivalent levels of
immunoprecipitated p53 in both samples (Fig. 8). These data show that
triptolide induces phosphorylation of p53 at 3 h that is prior to
the triptolide-mediated induction of p53 protein expression.
We have recently shown that triptolide cooperates with TNF- A recent study showed that doxorubicin-mediated activation of p53
induces p21 in tumor cells which inhibits apoptosis by inducing growth
arrest. Bunz et al. (13) show that inhibition of
p21-mediated growth arrest sensitizes these tumor cells to
doxorubicin-mediated apoptosis (13, 14). We show here that triptolide
inhibits p21-mediated accumulation of cells in G2/M and
induces accumulation of cells in S phase. Also, in combination with
doxorubicin triptolide enhances apoptosis in p53 wild-type tumor
cells, and overexpression of p21 inhibits the cytotoxic synergy between
triptolide and doxorubicin. Triptolide, however, does not repress p21
expression in p53 mutant tumor cells, but it does induce accumulation
of p53 mutant cells in S phase.2 A possible explanation for
the triptolide-mediated accumulation of cells in late G1/S
would be inhibition of cyclin-dependent kinase-2 (cdk-2)
activity, but triptolide does not affect cdk-2 activity in HT1080
cells.3 Therefore, triptolide
may directly inhibit cyclins required for G1/S transition
such as cyclin A, cyclin D, and cyclin E or block cdk-4 or cdk-6
activity. Li et al. (20), for example, recently showed that
cells treated with drugs that activate different cell cycle
checkpoints, We observed that triptolide induces translation of p53. A recent study
demonstrated that p53 translation is inhibited in the presence of
increased levels of p53 protein by binding of p53 protein to its
corresponding mRNA in the 5'-untranslated region which inhibits
further translation of p53 (24). Also, there are unidentified 3'-UTR
binding factor(s) that are essential for regulating translation of p53
(24). Thus, we are examining if triptolide directly binds to p53 or
modifies factors that regulate translation of p53. We are also in the
process of determining if triptolide enhances the redistribution of p53
messages to polysomes as seen in DNA damage induced by B activation and sensitizes tumor necrosis
factor (TNF-
)-resistant tumor cell lines to TNF-
-induced
apoptosis. We show here that triptolide enhances chemotherapy-induced
apoptosis. In triptolide-treated cells, the expression of p53 increased
but the transcriptional function of p53 was inhibited, and we observed
a down-regulation of p21waf1/cip1, a p53-responsive gene. The
increase in levels of the p53 protein was mediated by enhanced
translation of the p53 protein. Additionally, triptolide induced
accumulation of cells in S phase and blocked doxorubicin-mediated
accumulation of cells in G2/M and doxorubicin-mediated induction of p21. Our data suggest that triptolide, by blocking p21-mediated growth arrest, enhances apoptosis in tumor cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-irradiation, there is enhanced translation
of p53 mRNA without an increase in p53 stability. This is mediated,
at least in part, by the increased binding of p53 mRNAs to
polysomes (6, 7).
, microtubule-associated protein 4, insulin-like growth
factor-1 receptor, presenilin-1, RelA, and bcl-2
(reviewed in Refs. 8-10). However, the exact mechanism(s) by which p53
mediates apoptosis is still elusive. Also, recent studies suggest that the transactivation function of p53 is not required for
p53-dependent apoptosis and that transrepression may
play a role in inducing apoptosis (8, 10-12). For example, Bcl-2,
adenovirus E1B 19K, and the tumor suppressor WT1 inhibit apoptosis and
suppress p53-dependent transcriptional repression but not
transactivation (10). In addition, inhibition of p53-mediated apoptosis
has been demonstrated through overexpression of MAP4 that supports a
potential role for p53-dependent transrepression in
mediating apoptosis (9).
B in immune cells (15-17). Recently, our
laboratory has shown that triptolide sensitizes tumor cells to
TNF-
-induced apoptosis through inhibition of NF-
B (18). We also
found that triptolide, alone, induces apoptosis in solid tumor
cells. To elucidate further the mechanism of triptolide-induced
apoptosis, we investigated a potential role for the p53 pathway.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
) cell lines were provided by Dr.
Amato J. Giaccia (Stanford University). Doxorubicin, cycloheximide, and
the anti-FLAG (M2) antibody were obtained from Sigma. Antibodies for
p53, p21waf1/cip1 and protein phosphatase-1 were from Calbiochem.
/
) mouse embryonic fibroblasts (MEFs) transfected with the E1A/Ras were grown in Dulbecco's modified Eagle's medium containing 15% fetal calf serum supplemented with L-glutamine, penicillin, and streptomycin.
-galactosidase (LacZ)
was cloned into the same vector as a control. Transfection into HT1080
cells was performed with the LipofectAMINE Plus kit (Life Technologies, Inc.) according to the manufacturer's protocol. Briefly, HT-1080 cells
at 70% confluence were cotransfected with 1 µg of pINDp21FLAG or
pINDLacZ-FLAG plus 1 µg of pVgRXR (Invitrogen, Carsbad, CA) in 6-well
plates. After incubation for 3 h at 37 °C, the medium was
replaced with fresh media. Then 48 h after transfection, cells from each well were transferred into three 10-cm culture dishes. After
overnight culture, stable transfectants were selected by adding 600 µg/ml of zeocin (Invitrogen) and 800 µg/ml G-418 (Life Technologies). The selection was carried on for 2 weeks. Individual clones were isolated and tested for protein expression induced by the
addition of 5 µM ponasterone A (Invitrogen).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-induced
apoptosis through inhibition of NF-
B. To examine if
triptolide also sensitizes tumor cells to chemotherapy, we examined if
triptolide enhances cell death induced by doxorubicin, a topoisomerase
II inhibitor. After 24 h of treatment, doxorubicin (100 nM) or triptolide (5 ng/ml) alone did not reduce HT1080
cell viability (Fig. 1). The combination
of triptolide (5 ng/ml) plus doxorubicin reduced cell viability by 65%
(Fig. 1). Triptolide alone at 20 ng/ml reduced HT1080 cell viability by
84% (Fig. 1). Cytotoxic synergy between triptolide and doxorubicin was
also observed in A549 lung cancer cells, and triptolide also enhanced cell death by carboplatinum, another topoisomerase II inhibitor, in
A549 and HT1080 cells (data not shown). Doxorubicin did not induce
NF-
B transcriptional activity in HT1080 cells so that triptolide is
not enhancing doxorubicin-mediated apoptosis through inhibition of
NF-
B (data not shown).
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Fig. 1.
Triptolide acts in synergy with
doxorubicin. HT1080 cells were treated with doxorubicin (100 nM) and/or triptolide (5 ng/ml or 20 ng/ml) for 8 h
and then removed. Cell viability was analyzed at 24 h by trypan
blue exclusion. Data represent mean of triplicates from two
experiments ± S.D.
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Fig. 2.
Triptolide induces p53 but inhibits p21
expression. A, subconfluent HT1080 tumor cell lines
were treated as shown. After 9 h, total cellular lysate was
harvested followed by immunoblot analysis with a p53 mAb. The blot was
then stripped and reprobed with a p21 mAb. B, MEFs (p53 +/+
and p53 /
) were immunoblotted with a p21 mAb. The gel was scanned,
and the p53 bands were measured using a densitometry program. Protein
phosphatase-1 (PP1) is used as a loading control.
-irradiation-mediated activation of p53 in p53 wild-type cells
induces p21 and causes growth arrest which inhibits apoptosis (13,
14). To determine whether triptolide enhances doxorubicin-mediated
apoptosis by blocking p21-mediated growth arrest, we examined the
effect of triptolide on p21 expression.
/
MEFs, and it is not inducible by doxorubicin (Fig.
2B and data not shown).
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Fig. 3.
Triptolide inhibits p21 mRNA
expression. A, total RNA was harvested from HT1080
cells after the indicated treatments followed by Northern blot analysis
with a 32P-radiolabeled p21 cDNA probe. B,
RT-PCR analysis of p21, mdm2, p53,
map4, and gadd45 mRNA expression is shown
following treatment with triptolide (20 ng/ml) for the indicated times.
mRNA expression is expressed relative to glyceraldehyde
phosphate-3-dehydrogenase (GAPDH).
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Fig. 4.
Triptolide does not affect DNA binding of
p53. EMSA in HT1080 cells were treated as shown. 100×
corresponds to a 100-fold excess of unlabeled p53 oligonucleotide
probe, which is a consensus p53-binding site. The * denotes a
nonspecific band.
-lapachone
inhibits taxol-mediated G2/M arrest which leads to enhanced
apoptosis through generation of conflicting signals regarding cell
cycle progression versus arrest. Since p21 mediates
G2/M arrest in p53 wild-type cells in response to chemotherapy, our data suggest that triptolide inhibits
doxorubicin-mediated G2/M arrest by blocking induction of
p21.
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Fig. 5.
Triptolide inhibits doxorubicin-mediated
G2/M arrest. HT1080 cells were treated as shown and
harvested 16 h later for cell cycle analysis with PI staining and
data analysis with the FlowJo program (Stanford FACS facility).
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Fig. 6.
Overexpression of p21 inhibits cytotoxic
synergy between triptolide and doxorubicin. A,
ponasterone A was added for 16 h to HT1080 cells followed by the
addition of triptolide or doxorubicin alone or in combination for
8 h. Cells were then harvested for Western blot analysis with an
anti-FLAG antibody. Protein phosphatase-1 (PP1) is used as a
loading control. B, HT1080 cells were treated as described
above, and cell viability was determined by trypan blue exclusion at
24 h. pIND-LacZ (vector control, uninduced); LacZ(I) (vector
control, induced); pIND-p21 (p21 expression vector, uninduced);
pIND-p21(I) (p21 expression vector, induced).
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Fig. 7.
Triptolide induces translation of p53.
A, prior to cycloheximide (30 µg/ml) addition, HT1080
cells were pretreated with 20 ng/ml triptolide for 0.5 h. At the
times indicated, cells were collected, and total lysate was prepared.
35 µg of total protein was used for immunoblot analysis with a p53
antibody. The gel was scanned, and the p53 bands were measured using a
densitometry program. The values are an average of three
experiments ± S.D. B, in vivo metabolic
labeling was performed using [35S]methionine. HT1080
cells were pretreated with 20 ng/ml triptolide for 6 h followed by
the addition of 0.1 mCi/ml [35S]methionine for 30 min.
Cells were collected and lysed for immunoprecipitation with a p53 mAb
and 10% SDS-PAGE.
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Fig. 8.
Triptolide induces phosphorylation of
p53. HT1080 cells were labeled for 3 h with
[32P]orthophosphate followed by immunoprecipitation
(IP) with an agarose-conjugated p53 mAb. The samples were
boiled in SDS sample buffer and analyzed by 10% SDS-PAGE followed by
transfer to polyvinylidene difluoride membrane. The bands were
visualized and quantified by Optiquant PhosphorImager. Western blot
(WB) analysis on the same membrane was then done with a p53
polyclonal antibody.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
to
enhance apoptosis in solid tumor cells (18). We found that triptolide
sensitizes tumor cells to TNF-
by blocking TNF-
-mediated activation of NF-
B. Triptolide, however, blocked transactivation but
not DNA binding of NF-
B. Triptolide is an oxygenated diterpene purified from the Chinese herb T. wilfordii with
anti-inflammatory and tumoricidal properties in vitro and
in vivo (15-17). Triptolide with its three expoxides may
act as an electrophile possibly inactivating target molecules through
alkylation and reaction with exposed cysteines. We are presently
looking for targets of triptolide in tumor cells. Here we show that
triptolide enhances chemotherapy-induced cell death in p53 wild-type
cells. Triptolide induced p53 protein expression but inhibits basal p21
expression and doxorubicin-mediated induction of p21. We observed that
p21 levels are almost undetectable in p53
/
cells suggesting that
p53 is also required for basal p21 (Fig. 2B). These data
likely reflect the inhibitory effect of triptolide on transcriptional
activity but not DNA binding of p53 which is analogous to triptolide
blocking transactivation and not DNA binding of NF-
B. Triptolide,
however, is not a general transcriptional inhibitor because it does not
inhibit growth arrest and DNA damage-inducible (gadd45)
elongation factor-
(EF-1
) or glyceraldehyde-3-phosphate
dehydrogenase expression (GAPDH) (Fig.
3B).2
Additionally, we observed that triptolide induces phosphorylation of
p53, but this is the first example of a modification of p53 that
inhibits p21 expression. We are presently mapping the site(s) of p53
that is phosphorylated by triptolide and that may provide insight into
the mechanism of triptolide-mediated transcriptional repression of p21.
-lapachone and taxol, produce conflicting signals that
enhance tumor cell apoptosis (20). We show here that not only do
triptolide and doxorubicin affect different cell cycle checkpoints but
triptolide blocks p21-mediated G2/M arrest which likely
enhances apoptosis by blocking growth arrest. Topoisomerase I
inhibitors such as
-lapachone and CPT-1I also induce accumulation of
cells in S phase in a p53-independent manner. Triptolide, however, does
not affect topoisomerase I
activity.4 Our data presented
here suggest that triptolide enhances doxorubicin-mediated apoptosis,
at least in part, by blocking p21-mediated G2/M arrest.
-irradiation.
In addition, a recent study showed that N-acetylcysteine
induces apoptosis in tumor cells via a
p53-translational-dependent mechanism involving the
cellular redox potential (25). Since triptolide shows enhanced cytotoxicity in combination with DNA-damaging agents, it may also interfere with DNA repair. It has been reported (26) that casein kinase
II phosphorylates p53 at serine 386 which causes p53-mediated repression. The proline-rich region of the p53 protein has been shown
to be important in chromatin remodeling and is required to overcome
p53-mediated transcriptional repression (27). The cytotoxic activity of
triptolide alone and its ability to cooperate with other cytotoxic
agents may represent a novel method to enhance cytolysis of solid tumor
cells in vivo. In support of this observation, we have found
that PG490-88, a water-soluble derivative of triptolide, cooperates
with chemotherapy to cause tumor regression in a tumor xenograft
model.5
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ACKNOWLEDGEMENTS |
---|
We thank Amato Giaccia and Rudy Alarcon
(Stanford University) for p53 /
cells and for helpful discussions;
James Ford (Stanford University) for helpful discussions; and E. S. Lennox and John Fidler (Pharmagenesis) for providing PG490
(triptolide) and for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by a grant from the California Breast Cancer Research Program and a grant from Pharmagenesis.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
** To whom correspondence should be addressed: Stanford University Medical Center, Division of Pulmonary and Critical Care Medicine, 300 Pasteur Dr., Stanford, CA 94305-5236. Tel.: 650-725-9536; Fax: 650-725-5489; E-mail: grosen@leland.stanford.edu.
Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M009713200
2 J. J. Kang and G. D. Rosen, unpublished results.
3 C. Chung and G. D. Rosen, unpublished results.
4 M. Gao and G. D. Rosen, unpublished results.
5 J. M. Fidler and G. D. Rosen, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
UTR, untranslated
region;
TNF-, tumor necrosis factor-
;
PAGE, polyacrylamide gel
electrophoresis;
RT-PCR, reverse transcriptase-polymerase chain
reaction;
MAP4, microtubule-associated protein 4;
EMSA, electromobility
shift assay;
PBS, phosphate-buffered saline;
mAb, monoclonal antibody;
FACS, fluorescence-activated cell sorter;
MEFs, mouse embryonic
fibroblasts.
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