 |
INTRODUCTION |
Despite the development of new chemotherapeutic agents and
aggressive treatment of solid tumors there has been sporadic
improvement in long term survival. We have focused our efforts on
enhancing cytotoxicity in solid tumor cell lines with members of the
tumor necrosis factor (TNF)1
family. Tumor necrosis factor-
(TNF-
), for example, shows broad cytotoxicity against many tumor cell lines but its clinical use is
limited because it induces a profound inflammatory response through
activation of NF-
B. Moreover, activation of NF-
B inhibits TNF-
-induced apoptosis. We, therefore, set out to identify compounds that would sensitize tumor cells to TNF-
through inhibition of NF-
B.
Extracts of traditional Chinese herbs have been used for many years in
China to treat a variety of inflammatory conditions such as rheumatoid
arthritis. An alcohol extract of the Chinese herb Tripterygium
Wilfordii hook called T2 has potent antiinflammatory properties and has
been suggested to be effective for the treatment of arthritis (1). The
purified component of T2, which possesses immunosuppressive activity is
the diterpene triepoxide, triptolide. Interestingly, triptolide also
possesses anticancer activity. For example, it shows potent
antileukemic activity in animal models, it inhibits proliferation of
tumor cell lines, and it shows antitumor activity in a murine breast
tumor model (2, 3).
NF-
B is the prototype of a family of dimeric transcription factors
that have Rel regions, which bind to DNA, interact with each other, and
bind the I
B inhibitors (reviewed in Ref. 4). NF-
B regulates the
expression of many biologically important genes such as those encoding
inflammatory cytokines, interferons, growth factors, cell adhesion
molecules, and viruses. A recent role for NF-
B in the control of
apoptosis has been demonstrated based on the observation that mice
lacking RelA (p65) die during embryogenesis from massive liver cell
apoptosis (5). Several studies have now demonstrated an essential role
for NF-
B in preventing apoptosis induced by TNF-
and chemotherapy
(6-8). In these studies, cells were made sensitive to TNF-
and
chemotherapy-induced apoptosis through inhibition of NF-
B activity.
Also, a recent study demonstrated that the downstream effectors of
NF-
B activation, which include TRAF-1 (TNFR-associated factor 1),
TRAF2, and c-IAP1, also known as hiap-2, and c-IAP2, also known as
hiap-1, were required to suppress TNF-
-induced apoptosis (9). These
studies clearly demonstrate that activation of NF-
B suppresses
apoptosis in tumor cells and tumor cells are sensitized to
TNF-
-induced apoptosis through inhibition of NF-
B.
Here we show that purified triptolide sensitizes several solid tumor
cell lines to TNF-
-induced apoptosis through inhibition of NF-
B.
Additionally, triptolide alone induces apoptosis in tumor cells.
Interestingly, triptolide inhibited transcriptional activation of
NF-
B but not DNA binding of NF-
B. Also, triptolide blocked
induction of c-IAP2 and c-IAP1 by TNF-
. We propose that the ability
of triptolide to augment TNF-
-induced cytotoxicity while
simultaneously inhibiting activation of NF-
B may enhance the
cytotoxic potential of TNF-
and limit its proinflammatory effects
in vivo.
 |
MATERIALS AND METHODS |
Source of Triptolide--
PG490 (triptolide, molecular weight,
360) was obtained from Pharmagenesis (Palo Alto, CA). The material was
composed of white to off-white crystals, had a melting point of
226-240 °C, conformed to a standard triptolide preparation by
proton nuclear magnetic resonance (10), and was 97% pure by reverse
phase high pressure liquid chromatography evaluation using
acetonitrile:methanol:water (18:9:73).2
Cells and Transfections--
A549 (nonsmall cell lung cancer)
and HT1080 (fibrosarcoma) cell lines were purchased from ATCC. An MCF-7
(breast cancer) cell subline was provided by Dr. Ron Weigel (Stanford
University). Cells were cultured in the appropriate media with 10%
fetal calf serum supplemented with L-glutamine, penicillin,
and streptomycin. TNF-
was obtained from R&D Systems (Minneapolis,
MN) and phorbol myristyl acetate from Sigma. Cells were left untreated
or stimulated with PG490 (20 ng/ml) and/or TNF-
(10 ng/ml), and
6 h after the addition of TNF-
, cells were harvested for
analysis of luciferase activity according to the manufacuter's
protocol (Promega Corp., Madison, WI). In experiments with PG490 and
TNF-
, cells were pretreated with PG490 for 4 h before the
addition of TNF-
. Luciferase activity was measured in samples that
contained equal protein concentration with a Luminometer (Analytical
Luminescence Laboratory, San Diego, CA).
Plasmids--
An oligonucleotide containing the IgG
-NF-
B
site (sequence 5'-GGGGACTTTCC-3') was placed upstream of a minimal
interleukin-8 promoter (position
67 to +44) in a luciferase reporter
gene construct and stably tranfected into A549, HT180, and MCF-7 cells.
Resistant clones were pooled after selection in 400-600 µg/ml G418.
The I
B
super-repressor construct was provided by Dean Ballard,
Vanderbilt University, Nashville, TN (11). The Gal4 DNA binding domain (amino acids 1-147), Gal4-p65 TA1, and Gal4-p65 TA2 constructs were
provided by Albert Baldwin, University of North Carolina and have been
described previously (12). The Gal4-luciferase construct was provided
by Jeff Riegel (Stanford University) and contains 5× Gal4 binding
sites upstream of a minimal interleukin-2 promoter with a luciferase
reporter gene.
Cell Death Reagents and Assays--
Cell viability was measured
by a 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide
assay. Briefly, untreated cells or cells treated with PG490 and/or
TNF-
in a 96-well plate were harvested at the indicated times
followed by the addition of 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl
tetrazolium bromide to the cells. Cells were then solubilized with 0.1 N acidified CH3Cl-HCL. The 96-well plate was
read at a wavelength of 590 nm on an iEMS Labsystems plate reader.
Induction of cell death by TNF-
, PG490, and PG490 plus TNF-
was
confirmed as apoptotic by Annexin staining followed by
fluorescence-activated cell sorter analysis as described previously
(13).
Electrophoretic Mobility Shift Assay--
A549, HT1080, and
MCF-7 cells were treated under the following conditions: (1)
unstimulated; (2) PG490 (20 ng/ml) for 5 h; (3) TNF-
(10 ng/ml)
for 1 h; (4) PG490 pretreatment for 4 h followed by TNF-
for 1 h. Nuclear extracts were prepared using a protocol described
previously (14). The electrophoretic mobility shift assay was performed
as described previously using a Klenow-labeled 32P-IgG
NF-
B site as a probe (15). Supershift studies were done with a p65
monoclonal antibody (Santa Cruz Biochemical, Santa Cruz, CA).
Coimmunoprecipitation Studies--
To detect the association of
p65 with cAMP response element-binding protein (CBP) in untreated A549
cells or A549 cells treated with PG490 (20 ng/ml) and/or TNF-
(10 ng/ml), cellular protein was extracted for immunoprecipitation as
described previously (13). Cellular protein was immunoprecipitated with
a CBP mouse polyclonal antibody (Santa Cruz Biotechnology) followed by
Western blot analysis with a p65 mouse monoclonal antibody (BIOMOL,
Plymouth Meeting, PA) using a Western blot protocol described
previously (13).
Northern Blot Analysis and RT-PCR--
RNA was harvested from
A549 cells with RNA STAT-60, a solution containing guanidine
isothiocyanate and phenol (Tel-Test "B", Friendswood, TX), and
Northern blot analysis was done as described previously (16). The
c-IAP1 and c-IAP2 cDNAs were provided by Dr. David Goeddel
(Tularik, South San Francisco, CA). A plasmid that contains a 115-base
pair fragment from the 28 S ribosomal RNA cDNA was purchased from
Ambion (Austin, TX), excised with KpnI and XbaI
and used in Northern blot analysis to demonstrate equal loading of RNA.
For RT-PCR RNA was isolated from MCF-7 cells using RNA STA-60 and
subjected to RT-PCR with oligonucleotide primers for c-IAP2.
 |
RESULTS |
PG490 Induces Apoptosis in Solid Tumor Cells and Sensitizes Tumor
Cells to TNF-
-induced Apoptosis--
In an effort to sensitize
tumor cells to TNF-
, we sought out compounds that would sensitize
tumor cells to TNF-
through inhibition of NF-
B. PG490
(triptolide) is a diterpene epoxide derived from a traditional Chinese
herb that possesses potent immunosuppressive and anticancer activity
in vitro (1, 2, 17-19). Triptolide was shown to inhibit
induction of cytokine expression in T cells (17). Cytokine expression
in T cells depends, at least in part, on activation of NF-kB. NF-
B
is, therefore, a putative target for triptolide. We examined,
therefore, whether PG490 inhibits TNF-
-mediated activation of
NF-
B and sensitizes tumor cells to TNF-
-induced apoptosis.
PG490 alone decreased cell viability by 40-70% of cells in several
solid tumor cell lines at dosages between 5-20 ng/ml (Table I). Cell death was confirmed as apoptotic
by Annexin staining followed by fluorescence-activated cell sorter
analysis (data not shown). Cell death was maximal at 48 h after
the addition of PG490. Several tumor cells lines such as HT1080 cells
are resistant to TNF-
because of NF-
B activation following
stimulation with TNF-
. We also found that A549 lung cancer cells are
resistant to TNF-
-induced apoptosis (Table I). MCF-7 breast cancer
cells show intermediate sensitivity to TNF-
with TNF-
inducing
cell death in approximately 30% of cells (Table I). A recent study confirms differences in the sensitivity of MCF-7 sublines to TNF-
(20). PG490 alone (20 ng/ml) induced cell death in approximately 30%
of cells in the A549 cell line but the combination of PG490 and
TNF-
-induced cell death in over 80% of cells (Table I). In contrast
to A549 cells, PG490 alone (20 ng/ml) induced cell death in 70-75% of
cells in the MCF-7 cell line and 50-55% of cells in the HT1080 cell
line (Table I). PG490 at a concentration of 5 ng/ml induced cell death
in approximately 35% of MCF-7 cells and the combination of PG490 (5 ng/ml) plus TNF-
induced cell death in approximately 80% of MCF-7
cells (Table I). In HT1080 cells the combination of PG490 (20 ng/ml)
plus TNF-
induced cell death in more than 99% of cells (Table
I).
View this table:
[in this window]
[in a new window]
|
Table I
Effect of PG490 and TNF- on tumor cell viability
Cell lines were incubated with PG490 and/or TNF- for 48 h
followed by analysis of cell viability by an MTT assay. In experiments
with PG490 plus TNF- cells were pretreated with PG490 for 4 h
before the addition of TNF- . Values are mean of three
experiments ± S.D.
|
|
In view of recent studies, which show that inhibition of NF-
B
following TNF-
stimulation augments TNF-
-induced apoptosis in
tumor cells, we examined whether PG490 inhibits TNF-
-mediated activation of NF-
B in HT1080, A549, and MCF-7 cells. We found that
PG490 inhibited TNF-
-induced activation of an IgG
NF-
B luciferase reporter gene construct in all three cell lines but PG490
did not affect basal NF-
B activity (Fig.
1). PG490 alone did not affect basal
NF-
B activity in A549 and HT1080 cells but it slightly induced
NF-
B transcriptional activity in MCF-7 cells. We have observed that
PG490 inhibits expression of the I
B
protein in MCF-7 cells
leading to increased binding of NF-
B, which may explain the increase
in NF-
B transcriptional activity (data not shown). We confirmed,
through overexpression of an I
B
super-repressor construct, that
inhibition of NF-
B sensitizes HT1080 cells to TNF-
as was
described previously (7) (data not shown). We also found that
inhibition of NF-
B with MG132 (3 µM), a proteasome inhibitor, or with a calpain inhibitor sensitizes A549 and MCF-7 cells
to TNF-
-induced apoptosis (data not shown).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 1.
PG490 blocks induction of NF-kB
transcriptional activity by TNF- in tumor cell
lines. A549, HT1080, and MCF-7 cells that stably express an IgG
NF- B luciferase reporter gene construct were pretreated with PG490
(20 ng/ml) for 5 h followed by the addition of TNF- (10 ng/ml)
for 6 h. The cells were then harvested for analysis of luciferase
activity using equal amounts of protein for each sample. The fold
luciferase activation was calculated relative to a normalized value of
one given to control (untreated) cells. Data represent the mean
luciferase value from triplicates in one experiment, which was used to
calculate the mean ± S.D. from two experiments.
|
|
PG490 Does not Inhibit DNA Binding of NF-
B--
To determine
whether PG490 inhibits activation of NF-
B through the inhibition of
DNA binding of NF-
B, we examined the effect of PG490 on
TNF-
-induced binding of NF-
B by electrophoretic mobility shift
assay. TNF-
induced binding of NF-
B in A549 cells, and an
antibody to p65 (Rel A) (Santa Cruz Biotechnology) supershifted the
complex demonstrating that p65 is part of the NF-
B complex induced
by TNF-
(Fig. 2). PG490 did not affect
the intensity of the NF-
B complex induced by TNF-
or its
migration in A549 cells (Fig. 2). PG490 alone did not induce binding of
NF-
B; antibodies to p65 also supershifted a specific complex in
PG490 plus TNF-
-treated cells (Fig. 2). PG490 also did not affect
TNF-
-induced binding of NF-
B in HT1080 or MCF-7 cells (data not
shown). Our results suggest, therefore, that PG490 inhibits
transactivation but not DNA binding of NF-
B.

View larger version (65K):
[in this window]
[in a new window]
|
Fig. 2.
PG490 does not inhibit the
TNF- -mediated induction of
NF- B DNA binding activity. Nuclear
extracts were prepared and analyzed in an electrophoretic mobility
shift assay in A549 cells with a radiolabeled IgG NF- B probe.
Equal amounts (10 µg) of nuclear protein was loaded in each lane. In
lanes 3 and 7, a rabbit polyclonal antibody to
p65 (Santa Cruz Biotechnology) was added to the nuclear extract 10 min
before the addition of radiolabeled probe. In lane 4, an
100× excess of unlabeled IgG oligonucleotide was added 5 min before
the addition of radiolabeled probe.
|
|
PG490 Inhibits Transcriptional Activation of p65--
Recent
studies show that phosphorylation of p65 is important for
transcriptional activation of NF-
B (21, 22). For example, Zhong
et al. (21) demonstrated that lipopolysaccharide induces phosphorylation of p65 on serine 276, which increases p65-mediated transactivation. Also, Wang et al. (22) showed that TNF-
induces phosphorylation of p65 on serine 529, in the C-terminal region of p65. They demonstrate that inducible phosphorylation of p65 does not
affect nuclear translocation or DNA binding activity of NF-
B but it
increases transcriptional activity. Because PG490 blocks
transactivation of NF-
B but not DNA binding we hypothesized that
PG490 would inhibit transactivation of p65. A plasmid encoding a fusion
protein of the transactivating domains of p65,
Gal4-p65521-551 (Gal4-p65 TA1) or
Gal4-p65268-551 (Gal4-p65 TA2), with the DNA binding
domain of the yeast transcription factor Gal4, was transfected into
A549 cells along with a luciferase reporter containing upstream Gal4
binding sites. The TA1 domain is contained within the TA2 construct. We
found that PG490 blocked transcriptional activity of the TA1 domain of
p65 by 20-25% and of the TA2 domain by over 50% (Fig.
3). TNF-
did not stimulate transcriptional activity of the TA1 or TA2 domain even when the A549
cells were grown in medium containing 0.1% serum, which reduced basal
NF-
B activity (data not shown). This lack of response to TNF-
may
be explained by our observation that A549 cells contain significant
basal NF-
B activity (see Fig. 4).
Nonetheless, PG490 significantly blocked transcriptional activity of
the transactivating domains of p65.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 3.
PG490 inhibits transcriptional activity of
p65. A vector encoding a fusion protein between the DNA binding
domain of Gal4 and either the TA1 (Gal4-p65 TA1) or TA2 (GAL4-p65 TA2,
which contains TA1) activation domains of p65 or Gal4 was cotransfected
with a 5XGal4-luciferase reporter gene construct into A549 cells and
placed in 0.1% serum-containing medium 3 h after transfection.
After 36 h, cells were pretreated with PG490 (20 ng/ml) for 5 h followed by the addition of TNF- (10 ng/ml) for 6 h. Cells
were then harvested for analysis of luciferase activity using equal
amounts of protein.
|
|

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 4.
PG490 does not affect the interaction of p65
with CBP. A549 cells were treated as shown and as described in the
legend for Fig. 1, and cellular extract was immunoprecipitated with a
CBP rabbit polyclonal antibody (Santa Cruz Biotechnology) followed by
Western blot analysis with a p65 mouse monoclonal antibody
(BIOMOL).
|
|
PG490 Does not Affect the Interaction of p65 with CBP--
Recent
studies suggest that CBP/p300 are transcriptional coactivators of the
p65 subunit of NF-
B (21, 23). The interaction of p65 with CBP/p300
requires phosphorylation of p65 at serine 276 (21). p65 then interacts
with CBP/p300 in the nucleus, which enhances
NF-
B-dependent transcription (21). We examined whether PG490 inhibits transactivation of NF-
B by blocking the interaction of p65 with CBP/p300. We found that p65 associated with CBP/p300 in
unstimulated A549 cells, and this complex was induced by TNF-
(Fig.
4). PG490, however, did not affect the intensity or migration of the
p65·CBP complex in TNF-
-treated A549 cells (Fig. 4). PG490 alone,
in fact, increased the association of CBP with p65 in unstimulated A549
cells (Fig. 4).
PG490 Suppresses the Induction of c-IAP2 and c-IAP1 by
TNF-
--
Recent studies demonstrate that members of the inhibitor
of apoptosis family such as c-IAP1 (hiap-2) and c-IAP2 (hiap-1)
suppress TNF-
-mediated cell death (9, 24). We hypothesized that
PG490 would block induction of c-IAP2 and c-IAP1 by TNF-
. TNF-
induced a 6-fold increase in c-IAP2 mRNA and a 4-fold increase in
c-IAP1 mRNA in A549 cells, which was almost completely blocked by
PG490 (Fig. 5, A and
B). TNF-
also induced a 4-fold increase in c-IAP2 mRNA in MCF-7 cells, which was blocked by PG490 (Fig.
5C). These results suggest that PG490 sensitizes tumor cells
to TNF-
-induced apoptosis, at least in part, by suppressing the
induction of c-IAP2 and c-IAP1.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 5.
PG490 blocks
TNF- -mediated induction of c-IAP2 (hiap-1) and
c-IAP1 (hiap-2) mRNA. Northern blot analysis in A549 cells of
c-IAP2 (A) with 10 mg of total RNA per lane probed with a
full-length 32P-labeled c-IAP2 probe. The blot was then
stripped and reprobed with a full-length 32P-labeled c-IAP1
probe (B). Then, the blot was restripped and probed with a
115-base pair 32P-labeled 28 S ribosomal cDNA probe to
demonstrate equal loading of RNA. Panel C shows RT-PCR with
RNA from MCF-7 cells using c-IAP2 oligonucleotide primers.
|
|
 |
DISCUSSION |
Over 50-70% of solid tumors harbor mutations in p53, which
confers relative chemoresistance. TNF family members such as TNF-
, Fas, and TNF-related apoptosis-inducing ligand, also known as Apo2L,
induce apoptosis in tumor cells regardless of the p53 phenotype. Unfortunately, TNF-
-induced apoptosis is limited by activation of
NF-
B. Additionally, activation of NF-
B induces the release of
pro-inflammatory cytokines, which damage the host. We set out to
identify a compound that sensitizes tumor cells to TNF-
-induced apoptosis through inhibition of NF-
B. PG490 (triptolide) is an oxygenated diterpene derived from a traditional Chinese herb that has
been used as an immunosuppressant in China for the treatment of
rheumatoid arthritis. There are also studies that show PG490 is
cytotoxic in leukemia and breast cancer cell lines. We show here that
PG490 cooperates with TNF-
to induce apoptosis in diverse solid
tumor cell lines.
PG490 alone induces apoptosis in 30-70% of cells in the A549, HT1080,
and MCF-7 cell lines. We have found that other tumor cell lines are
even more sensitive to PG490-induced apoptosis (data not shown). We are
presently investigating the mechanism of PG490-induced apoptosis. We
also show that PG490 almost completely suppresses TNF-
-induced
activation of NF-
B and sensitizes tumor cell lines to
TNF-
-induced apoptosis. Recent studies suggest that c-IAP1 (hiap-2)
and c-IAP2 (hiap-1) mediate, at least in part, the resistance of some
tumor cells to TNF-
-induced apoptosis. We found that TNF-
induced
c-IAP1 and c-IAP2 and that PG490 blocked TNF-
-mediated induction of
c-IAP2 and c-IAP1 (Fig. 5).
PG490 did not affect the DNA binding of NF-
B but it blocked
transactivation of NF-
B. Recent studies suggest that transactivation of NF-
B is enhanced through the phosphorylation of p65 at Ser-276 and Ser-529 (21, 22). Lipopolysaccharide induces phosphorylation of p65
at Ser-276, which promotes the interaction of p65 with CBP/p300 in the
nucleus (21). This interaction enhances transactivation of NF-
B. A
recent study showed that TNF-
induces phosphorylation of p65 at
Ser-529 in the C-terminal region of p65 (22). This phosphorylation of
p65 at Ser-529 also enhances transactivation of NF-
B but a
transcriptional coactivator that interacts with p65 phosphorylated at
Ser-529 has, as yet, not been identified. We show that PG490 inhibits
transactivation of both the TA1 and TA2 regions of p65 (Fig. 3). PG490
did not, however, affect the interaction of p65 with CBP in TNF-
treated cells (Fig. 4). Our results suggest, therefore, that PG490 may
block the phosphorylation of NF-
B at Ser-529 and/or the association
of p65 with an, as yet, unidentified transcriptional cofactor in the
nucleus. We are presently examining the effect of PG490 on the
phosphorylation of p65. Our data, nonetheless, suggests that PG490
inhibits activation of NF-
B by a novel mechanism.
In A549 cells we observed some basal NF-
B activity reflected by the
interaction of p65 with CBP/p300 in unstimulated cells (Fig. 4). Also,
the IgG NF-
B luciferase reporter construct was active in
unstimulated cells (data not shown). Basal NF-
B activity in A549
cells may explain why TNF-
did not induce Gal4-p65 TA1 transcriptional activity and it suggests that phosphorylation of p65 in
A549 cells is partly constitutive. In support of this possibility, a
recent study showed that TNF-
only weakly induces phosphorylation of
p65 in COS cells, because phosphorylation of p65 is largely
constitutive (22).
The identification of compounds that are both cytotoxic and block
activation of NF-
B may enhance the cytotoxicity of TNF family
members in vivo. We are presently examining the effect of
PG490 in combination with TNF-
and in combination with chemotherapy in tumor xenograft models in vivo.