| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics |
B/IB Signaling Pathway May Contribute to the Mediation of Paclitaxel-induced Apoptosis in Solid Tumor Cells1
Departments of Pathology and Laboratory Medicine [Y. H., K. R. J., W. F.] and Microbiology and Immunology [J. S. N.], Medical University of South Carolina, Charleston, South Carolina 29425
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B-
, the cytoplasmic inhibitor of transcription
factor nuclear factor-B (NF-B), which in turn promoted the
nuclear translocation of NF-B and its DNA binding activity. In
contrast, we found that glucocorticoids could antagonize
paclitaxel-mediated NF-B nuclear translocation and activation
through induction of IB- protein synthesis. Northern blotting
analyses demonstrated that the steady-state level of IB- mRNA was
not affected by paclitaxel, which suggests that the down-regulation of
IB- by paclitaxel is attributable to protein degradation rather
than suppression of transcription. Furthermore, through transfection
assays, we demonstrated that tumor cells stably transfected with
antisense IB- expression vectors remarkably increased their
sensitivity to paclitaxel-induced apoptosis. Finally, we found that a
key subunit of IB kinase (IKK) complex, IKKß, was up-regulated by
paclitaxel, which implies that paclitaxel might down-regulate IB-
through modulation of IKKß activity. All of these results suggest
that the NF-B/IB- signaling pathway may contribute to the
mediation of paclitaxel-induced cell death in solid tumor cells. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In recent years, several laboratories demonstrated that paclitaxel, at clinically relevant concentrations, is able to induce internucleosomal DNA fragmentation and other typical morphological features of apoptosis in a number of solid tumor cells (8, 9, 10, 11, 12) . These results clearly indicated that paclitaxel, in addition to its classical activity against microtubule, also possesses cell-killing activity by induction of apoptosis. However, it is currently unclear whether this finding suggests a novel mechanism of action for paclitaxel against tumor cells or just represents an end product of the well-known action of paclitaxel on microtubule and cell cycle. Recent studies in this laboratory revealed that glucocorticoids selectively inhibit paclitaxel-induced apoptotic cell death in a number of solid tumor cells but did not affect the ability of paclitaxel to induce microtubule bundling and mitotic arrest (10 , 13 , 14) . This selective inhibition by glucocorticoids on paclitaxel cytotoxicity implies that apoptotic cell death induced by paclitaxel might occur via a pathway independent of mitotic arrest and has provided us with a unique model system to investigate the molecular basis of paclitaxel induced apoptotic cell death.
The inhibitory action of glucocorticoids on paclitaxel-induced
apoptosis without affecting mitotic arrest has suggested two
possibilities: (a) glucocorticoids may specifically disrupt
the downstream events of mitotic arrest; or (b)
paclitaxel-induced apoptosis may occur via a separate pathway that can
be blocked by glucocorticoids. No matter which pathway is correct,
glucocorticoids are hypothesized to interfere with the action of
paclitaxel through regulation of gene expression, particularly for
those genes or proteins whose activation or altered expression is
potentially involved in the apoptotic pathway. Recently, a number of
apoptosis-associated genes or regulatory proteins have been reported to
be activated or regulated by paclitaxel. These include genes that act
primarily to suppress apoptosis, such as the bcl-2 gene
family (9
, 15
, 16) , and genes that may act as effectors of
apoptosis, such as the interleukin-1ß converting enzymes family of
proteases (17)
, and genes that may act as mediators of
signal transduction, such as p21waf, TNF-,
c-raf-1, and BID (18
, 19)
. Although the
discrete roles of these altered genes in paclitaxel-induced apoptosis
remain unclear, studies have reported that paclitaxel-altered gene
expression might be independent of microtubule stabilization (11
, 20)
. Therefore, it is possible that paclitaxel induces apoptosis
via a gene-directed process, i.e., paclitaxel may directly
induce or modulate gene expression, which, in turn, triggers the
apoptotic process.
NF-B,3
a member of Rel transcription factor family, and its
specific intracellular inhibitor IB- participate in the mediation
of many biological activities including inflammation, immune response,
cell proliferation, and apoptotic cell death (21)
. NF-B
normally resides in the cytoplasm as an inactivated form by forming a
complex with IB-. Upon certain stimulations, IB- is rapidly
phosphorylated and degraded, allowing NF-B to translocate to the
nucleus, where it participates in transcriptional regulation of
numerous genes (21
, 22)
. In recent years, increasing
evidence indicates that activation of NF-B plays an important role
in coordinating the control of apoptotic cell death (23)
.
In this study, through analyzing the possible modulation of
paclitaxel and glucocorticoids on the expression of
apoptosis-associated proteins in several paclitaxel-sensitive tumor
cell lines, we reported that paclitaxel profoundly down-regulated
IB-, which in turn promoted the nuclear translocation and DNA
binding activities of NF-B. In contrast, paclitaxel-induced
IB- degradation and the activation of NF-B were blocked if
tumor cells were coadministered with glucocorticoids. Transfection
assays demonstrated that induction of antisense IB- cDNA into
BCap37 cells remarkably increased the sensitivity of tumor cells to
paclitaxel-induced apoptosis. Moreover, one subunit of IB kinase
complex IKK ß was revealed to be up-regulated by paclitaxel. These
findings suggest the possible involvement of NF-B/IB- in the
mediation of paclitaxel-induced apoptotic cell death.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Western Blotting.
After exposure to paclitaxel with or without pretreatment of
glucocorticoids (10-7 M TA, 24 h
prior to paclitaxel treatment) in a time or dose course, cells were
harvested by trypsinization and washed with PBS. Cellular protein was
isolated using protein extraction buffer containing 150 mM
NaCl, 10 mM Tris (pH 7.2), 5 mM EDTA, 0.1%
TritonX-100, 5% glycerol, and 2% SDS. Protein concentrations were
measured via Biuret and Lowry assay. Equal amounts (50 µg/lane) of
protein were fractionated on 12.5% SDS-PAGE gels and transferred to
PVDF membranes. The membranes were incubated with anti-p53, bcl-2, bax,
c-myc, c-raf, IB-, IKK , and IKK ß
primary antibodies (1:3000; Santa Cruz Biotechnology, Inc.). After
washing with PBS, the membranes were incubated with
peroxidase-conjugated goat antimouse or goat antirabbit secondary
antibodies (1:4000; ImmunoResearch), followed by enhanced
chemiluminescent staining (ECL system; Amersham). ß-actin protein was
used to normalize protein loading.
Northern Blotting.
BCap37 cells were treated with 100 nM paclitaxel with or
without pretreatment of glucocorticoids (10-7
M TA, 24 h prior to paclitaxel treatment) for 12, 24,
and 48 h. Total RNA was isolated, and 20 µg were fractionated in
1% agarose-formaldehyde gel, transferred to nitrocellulose membrane,
and UV cross-linked. The membrane was probed with
[32P]UTP-labeled antisense IB- RNA probes
generated from the subcloned IB- cDNA fragments in pCDNA3
vectors. The membrane was then washed and autoradiographed. The same
membrane was stripped and reprobed with human antisense ß-actin RNA
probes to normalize RNA loading.
Immunofluorescence Assays.
BCap37 cells were cultured in 35-mm dishes and treated with 100
nM paclitaxel with or without pretreatment of
glucocorticoids (10-7 M TA). After
24 h, the dishes were washed with PBS and fixed with 3.7%
formaldehyde in PBS for 30 min. The cells were then incubated in 0.1%
saponin and 4 mg/ml normal goat globulin with anti-NF-B (p65)
antibodies (1:200; Santa Cruz Biotechnology, Inc.) for 30 min at room
temperature. After washing with PBS, cells were incubated with
affinity-purified, rhodamine-conjugated mouse antirabbit IgG (1:4000;
Jackson Immuno Research, West Grove, PA). The dishes were viewed and
photographed with a Zeiss Axioplan epifluorescence microscope equipped
with a rhodamine filter set.
Nuclear Extraction and EMSAs.
The promoter and enhancer regions of TNF- genomic DNA
containing NF-B binding sites were cloned by PCR from BCap37 genomic
DNA and subcloned into the pCRII vector (Invitrogen, CA).
[32P]CTP-labeled double-stranded
oligonucleotides containing NF-B consensus B enhancer sequence
(5'-CAGTGGGGTCTGTGAATTCCCGGGGGTGATTTCA-3')
(24
, 25) were prepared by PCR and purified on a 5%
polyacrylamide gel, excised, and eluted by shaking in 1 ml of high salt
buffer [10 mM Tris (pH 7.5), 1 mM EDTA, and
0.5 M NaCl] overnight at 37°C. After phenol extraction
and precipitation with ethanol, 4000 cpm of radiolabeled probe was used
for each reaction. BCap37 cells were treated with 100 nM
paclitaxel with or without pretreatment of 10-7
M glucocorticoids for 12, 24, and 48 h. Cells were
harvested and resuspended in 800 µl of hypotonic lysis buffer [10
mM HEPES (pH 7.8), 10 mM KCl, 2 mM
MgCl2, 1 mM DTT, 0.1 mM
EDTA, and 0.1 mM phenylmethylsulfonyl fluoride] and
incubated on ice for 15 min. Then, 50 µl 10% NP-40 were added, and
cells were vigorously mixed and centrifuged. The nuclei pellets were
suspended in 50 µl of buffer containing 50 mM KCl, 300
mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1
mM phenylmethylsulfonyl fluoride, and 10% glycerol (v/v)
and mixed for 20 min and centrifuged to produce supernatant containing
nuclear proteins. EMSA binding reaction mixture contained 1 µg of
protein of nuclear extract, 2 µg of poly(deoxyinosinic-deoxycytidylic
acid) (Sigma Co.), and [32P]-labeled probe
(4000 cpm) in binding buffer [10 mM HEPES (pH 7.9), 50
mM NaCl, 1 mM EDTA, 1 mM DTT, 10%
glycerol, and 0.2 mg/ml albumin]. The reaction was incubated for 30
min at room temperature before separation on a 5% acrylamide gel,
followed by autoradiography.
Isolation of Intact IB- cDNA Clone and Construction of
Expression Vectors.
Total RNA was isolated from BCap37 cells and transcribed into cDNAs by
RT-PCR reaction. RT-PCR was performed using a pair of primers IB-5'
(5'-CTCGTCCGCGCCATGTTC-3') and IB-3' (5'-CTTTGCACTCATAACGTCAGA-3')
designed according to the published IB- cDNA sequence
(26)
. The PCR products were inserted into pCR II vectors
(Invitrogen) and sequenced. Sense and antisense IB- expression
vectors were constructed, respectively, by using unique restriction
sites available within the pCR II vector. Full-length cDNAs were
excised from pCR II vectors and inserted into the high-level pcDNA3
mammalian expression vector system (Invitrogen) in either sense or
antisense orientations. All constructs were confirmed by DNA
sequencing.
Stable Transfection and Selection of Transfected Cells.
Transfections were performed by lipofectin (Life Technologies, Inc.) as
recommended by the manufacturer. Briefly, BCap37 cells were washed
twice with Opti-MEM reduced serum medium, and 3 ml of the same medium
were added to the cells. Plasmid DNA (2 µg per 6-cm plate) containing
either sense or antisense IB- inserts was mixed with lipofectin
before addition to the tumor cells. After transfection, stable
transfectants were selected by incubating the cells in the medium
containing 500 µg/ml Genectin (G418). Surviving colonies were picked
2 weeks later. Single colonies were amplified and continually grew
in medium containing G418. Cells from each individual colony were
examined for sense and antisense IB- expression by Western
blotting assays. Positive colonies were maintained in culture medium
with G418 for additional experiments. All transfectants were routinely
cultured in RPMI 1640 containing 10% FCS and 1%
penicillin-streptomycin.
Determination of Internucleosomal DNA Cleavage.
Internucleosomal DNA fragmentation was analyzed by a modification
of methods described previously (10)
. After BCap37
transfectants were exposed to paclitaxel at different concentrations
(1, 10, and 50 nM) for 48 h, cells were harvested,
counted, and washed with PBS at 4°C. Cells were then suspended in
lysis solution (5 mM Tris-HCl, 20 mM EDTA, and
0.5% Triton X-100) for 20 min on ice. The remaining steps for DNA
fragmentation were performed as described previously (10)
.
DNA samples were analyzed by electrophoresis in a 1.2% agarose slab
gel containing 0.2 µg/ml ethidium bromide and visualized under UV
illumination.
Flow Cytometry Analysis.
Cell sample preparation and PI staining for flow cytometry
analysis were performed according to the method described by Nicoletti
et al. (27)
. BCap37 cells transfected with
empty expression vector pcDNA3 (control), IB- sense cDNA
(IB--SEN8), and IB- antisense cDNA (IB--ANT5) were
treated with paclitaxel in different concentrations (1, 10, and 50
nM) for 24 and 48 h, respectively. Cells
were then harvested by trypsinization and washed twice with PBS,
followed by fixing in 1% formaldehyde and dehydrating in 70% ethanol
diluted in PBS. Cells were then incubated in PBS containing 100 µg/ml
RNase and 40 µg/ml PI at 37°C for 1 h before flow cytometry
analysis. Cell cycle distribution was determined using a Coulter Epics
V instrument (Coulter Corp.) with an argon laser set to excite
at 488 nm. The results were analyzed using Elite 4.0 software (Phoenix
Flow System, San Diego, CA).
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B-.B-/NF-B,
c-myc, c-raf-1, and others (15
, 18
, 19)
. To determine whether any of these proteins are involved in
the mediation of paclitaxel-induced cell death, we have first examined
their expressions in paclitaxel-sensitive BCap37 cells under treatment
of paclitaxel at the clinically relevant concentration of 100
nM with or without pretreatment of
glucocorticoids (10-7 M
TA, 24 h prior to paclitaxel treatment). By using Western
blotting, we determined that expressions of p53, bax, and
c-myc were basically not regulated by either paclitaxel or
glucocorticoids. As reported previously (14
, 28)
,
paclitaxel was found to cause bcl-2 and c-raf-1
phosphorylation, but neither bcl-2 nor c-raf-1 was affected
by glucocorticoids. Through this screening, however, we discovered that
treatment of BCap37 cells with paclitaxel led to a significant decrease
in protein level of IB-. Conversely, pretreatment of cells with
glucocorticoids remarkably enhanced the protein levels of IB- in
BCap37 cells (Fig. 1A
). To confirm the down-regulation of IB- by
paclitaxel, we examined two more paclitaxel-sensitive tumor cell lines,
human ovarian tumor cell line OV2008 and human epidermoid tumor cell
line KB. The results indicated that paclitaxel-induced down-regulation
of IB- also occurred in these two tumor cell lines (Fig. 1B
). To further characterize whether paclitaxel-induced
down-regulation of IB- occurs at lower concentrations of
paclitaxel, we examined the expression of IB- in BCap37 cells
treated with different concentrations of paclitaxel (1
nM to 100 nM) for 24 h. As shown in Fig. 1C
, the down-regulation of IB- was
observed at concentrations as low as 1 nM and
greater. This result implied that paclitaxel-induced down-regulation of
IB- might be independent of microtubule stabilization because
previous studies from this laboratory and others have revealed that
microtubule stabilization was not detected at such low concentrations
of paclitaxel treatment (1–10 nM; Refs.
9
, 11
, 13,
and 28, 29, 30
).
|
B- by Paclitaxel Is Caused by Protein
Degradation.B- and how glucocorticoids block this procedure, Northern
blotting was performed to determine the IB- mRNA level in BCap37
cells under the treatment of paclitaxel with or without pretreatment of
glucocorticoids. As depicted in Fig. 2
, the transcription of IB- (the steady state level of mRNA) in
BCap37 cells was not affected by 100 nM paclitaxel for 12,
24, and 48 h. It suggests that paclitaxel-induced down-regulation
of IB- may be caused by protein degradation rather than the
repression of transcription. However, we found that pretreatment of
cells with glucocorticoids (10-7 M
TA) significantly induced IB- transcription. This result
indicates that paclitaxel and glucocorticoids possess opposite
regulatory effects on IB- at different levels.
|
B.B- is the specific cytoplasmic inhibitor of NF-B. To address
whether the opposite regulatory effects on IB- by paclitaxel and
glucocorticoids were manifested by alternations in the nuclear
translocation of NF-B, we performed immunofluorescence assays to
localize NF-B by using rabbit polyclonal anti-NF-B (p65)
antibody, followed by rhodamine-labeled goat antirabbit IgG. As shown
in Fig. 3
, typical fields of stained BCap37 cells showed the exclusive
cytoplasmic localization of NF-B protein (p65) in nonstimulated
(Fig. 3A
) or glucocorticoid-treated
(10-7 M TA) BCap37 cells
(Fig. 3B
). After cells were treated with 100
nM paclitaxel for 24 h, most NF-B protein
translocated into the nucleus (Fig. 3C
). However, if the
cells were pretreated with glucocorticoids (10-7
M TA) 24 h prior to paclitaxel treatment,
paclitaxel-promoted nuclear translocation of NF-B was clearly
inhibited (Fig. 3D
).
|
B by
EMSAs assay. Nuclear extracts from untreated, paclitaxel-treated,
or paclitaxel-plus-glucocorticoid-treated BCap37 cells were incubated
with the [32P]CTP-labeled NF-B probes
containing a typical NF-B binding motif from the promoter region of
TNF-. As shown in Fig. 4
, the increased level of DNA-binding activity was detected after cells
were exposed to 100 nM paclitaxel for 12, 24, and 48 h
(Lanes 2–5). Conversely, when glucocorticoids
(10-7 M TA) were
administrated 24 h before paclitaxel treatment, elevated
DNA-binding activity of NF-B by paclitaxel was markedly inhibited
(Lanes 6–9). Lane 1 is free probe only.
|
B- Sensitizes Paclitaxel-induced Apoptosis.B/IB signaling cascade is involved in
the mediation of paclitaxel-induced apoptosis, IB- cDNAs were
inserted into pcDNA3 expression vectors in either sense or antisense
orientations and introduced into wild-type BCap37 cells. Three pairs of
positive colonies transfected with either sense or antisense IB-
expression vectors were selected for a series of experiments including
DNA fragmentation and flow cytometric assays. Analytic data from all
these three pairs of transfectants showed similar results. Here,
analysis of one pair of transfected cell lines, colonies IB--SEN8
(sense IB- transfection) and IB--ANT5 (antisense IB-
transfection) is presented from Figs. 5
6
7
. Fig. 5
indicates that the protein level of IB- was
significantly increased in IB--SEN8 cells. However, IB- was
still down-regulated in the presence of paclitaxel, suggesting that
paclitaxel-induced down-regulation also occurred for exogenous
IB-. For IB--ANT5 cells, the endogenous expression of
IB- was markedly blocked by antisense IB- mRNA.
Interestingly, we noticed that glucocorticoids could still
significantly induce IB- expression, even in cells transfected
with antisense IB-. We suspected that the induction of
glucocorticoids on IB- expression might be dominant so that the
exogenous antisense IB- mRNA was unable to significantly alter
the protein level of IB- when cells were treated with
glucocorticoids. By using these transfectants, we performed DNA
fragmentation and flow cytometry assays to determine their sensitivity
to paclitaxel-induced apoptosis. The experimental results indicated
that BCap37 cells transfected with antisense IB- cDNA exhibited a
marked increase in their sensitivity to paclitaxel-induced apoptosis as
compared with the empty vector or sense IB- transfectants. Fig. 6
illustrates these findings in which we see a dose-dependent DNA
fragmentation response when these cells were treated with various
concentrations of paclitaxel (1–50 nM) for 48 h. The
results indicated that the concentration of 10 nM or
greater was required for induction of the typical DNA ladders in the
cells transfected with the empty vector (Fig. 6
, Lane 3) or
sense IB- (Fig. 6
, Lane 7). However, DNA fragments
were observed in antisense IB- transfectants treated with 1
nM paclitaxel (Fig. 6
, Lane 10).
Subsequently, apoptosis and cell cycle distributions were further
analyzed by flow cytometry assays. In Fig. 7A
, cells transfected with empty vector or sense IB-
cDNA were induced to undergo apoptosis by 50
nM paclitaxel for 24 h treatment
(pre-G1 peak AP represents the apoptotic cells).
However, the cells transfected with antisense IB- were found to
significantly increase their sensitivity to paclitaxel-induced
apoptosis. The apoptotic peak was observed even in the group treated
with 10 nM paclitaxel. When cells were exposed to
paclitaxel for 48 h, 1 nM paclitaxel was
able to induce apoptosis in antisense IB- transfectants. However,
at this time point, 10 nM or greater
concentrations of paclitaxel were required to cause apoptotic cell
death in sense IB- or empty vector transfectants (Fig. 7B
). These results indicate that BCap37 cells transfected
with antisense IB- are more sensitive to paclitaxel-induced
apoptosis.
|
|
|
B kinases (consisting of IKK
and ß subunits) are responsible for IB protein degradation and
NF-B activation (31, 32, 33)
. To determine the possible
involvement of the IB kinase complex in paclitaxel-mediated
down-regulation of IB-, we have examined whether the expressions
of IKK or IKKß were affected by paclitaxel. The results of Western
blotting as shown in Fig. 8
indicated that neither paclitaxel nor glucocorticoids altered the
expression of IKK. However, paclitaxel markedly increased the
protein level of IKKß but glucocorticoids did not affect the
up-regulation of IKKß by paclitaxel. This finding raised the
possibility that the primary target of paclitaxel in the regulation of
the IB/NF-B pathway might be IKKß.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B- protein was down-regulated by paclitaxel
at the clinically relevant concentration 100 nM in several
paclitaxel-sensitive cell lines (Fig. 1
, A and
1B). Conversely, glucocorticoids
(10-7 M TA) were found to
possess an inverse regulatory effect on IB- through inducing
IB- expression (Fig. 1A
). The inverse regulatory
effect of paclitaxel and glucocorticoids on IB- implies that
IB- might be an important mediator involved in the inhibitory
action of glucocorticoids on paclitaxel-induced apoptosis. Further
studies revealed that down-regulation of IB- was also observed
when BCap37 cells were treated with lower concentrations of paclitaxel
(1 or 10 nM; Fig. 1C
). Because
microtubule stabilization is usually not detectable at such low
concentrations of paclitaxel treatment (28, 29, 30)
, this
result implies that paclitaxel-induced down-regulation of IB- may
occur independently of microtubule stabilization.
IB-, the specific cytoplasmic inhibitory protein of transcription
factor NF-B, normally forms a complex with NF-B in the cytoplasm
of nonstimulated cells. In various cell lines, the endogenous IB-
is rapidly degraded as a consequence of stimulation by proinflammatory
cytokinases, viral infection, oxidants, phorbol esters, and UV
irradiation (34
, 35)
. As a result, NF-B translocates to
the nucleus, where it participates in the regulation of numerous gene
transcriptions (36
, 37)
. Therefore, it is generally
believed that IB- degradation is the critical step for activation
of NF-B (38, 39, 40)
. Northern blotting assay in this study
indicated that a steady-state level of IB- mRNA was not affected
by paclitaxel treatment (Fig. 2
). This result suggests that decreased
levels of IB- protein may be caused by protein degradation rather
than transcriptional repression, although decreased rates of
translation might be a possibility. From the same experiment, we also
found that pretreatment with glucocorticoids induced a significant
increase in IB- mRNA levels. Further analyses revealed that the
degradation of IB-, in turn, promotes the nuclear translocation
and DNA-binding activity of NF-B. However, this paclitaxel-induced
NF-B activation was markedly blocked if cells were pretreated with
glucocorticoids (Figs. 3
4
). These results suggest that paclitaxel
and glucocorticoids regulate the NF-B/IB signaling pathway at
different levels.
The phenomenon of paclitaxel-induced IB- degradation and
NF-B activation raised a question as to the possible role of
activation of IB/NF-B on the paclitaxel-induced apoptosis. In
recent years, NF-B has been believed to play an important role in
coordinating the control of apoptotic cell death. However, the exact
mechanism of NF-B in the modulation of apoptosis is not entirely
clear. Some laboratories have reported that activation of NF-B is
able to either promote or prevent apoptosis, depending on different
stimuli and different cell types (22
, 41
, 42)
. For
example, Grimm et al. (23)
reported that serum
starvation activated NF-B and induced human embryonic kidney cells
into apoptosis. Qin et at (43)
found that
NF-B activation contributed to the excitotoxin-induced death of
striatal neurons. However, somewhat inconsistent results have also been
presented by Beg and Baltimore (44)
that NF-B
activation generally inhibits apoptosis in embryonic fibroblasts. In
our case, if the NF-B/IB signaling pathway is indeed involved in
the mediation of the cell-killing activity of paclitaxel, the
activation of NF-B is assumed to promote apoptosis in
paclitaxel-sensitive tumor cells. To test this hypothesis, we carried
out transfection assays. The results indicated that BCap37 cells
transfected with antisense IB- significantly increased their
sensitivity to paclitaxel-induced apoptosis (Figs. 6
7
). This
finding indicates that under certain conditions, paclitaxel-activated
NF-B activity may act as a signal transducer and gene activator in
the induction of apoptosis. In addition, recent studies have revealed
that many potential target genes for NF-B can be induced during the
apoptotic process. These target genes, including the so called "death
genes" like FAS/APO-1 ligand, c-myc, p53,
ICE, and others, have been reported to be activated or regulated
by low concentrations of paclitaxel (17
, 31
, 41
, 45)
. In
another study, we analyzed and compared the alteration of
NF-B/IB- in some paclitaxel-resistant tumor cell lines
including human breast tumor MCF7 cells and rat prostate tumor R3227
cells. The analytic results indicated that paclitaxel-induced
degradation of IB- and consequent elevated DNA-binding activity
of NF-B did not occur in these two tumor cells (data not shown).
This finding provided another piece of evidence that activated
NF-B/IB signaling pathway is required to execute the apoptotic
program.
On the basis of these observations, if paclitaxel-induced activation of
NF-B is independent of microtubule bundling and
G2-M phase arrest, the question then becomes:
What is the possible primary upstream target of paclitaxel that
mediates the degradation of IB- and the activation of the NF-B
signaling cascade? Because IB- showed its phosphorylated form in
paclitaxel-treated and untreated BCap37 cells from the Western blotting
results (Fig. 1
), it is highly possible that the key player in this
cascade of events is the kinase responsible for the phosphorylation and
degradation of IB-. Recent studies have identified a high
molecular weight complex of IB kinases (IKK and IKKß) that play
a key role in IB protein phosphorylation and degradation in some
cell lines (46)
. Hence, it would be interesting to examine
whether the IKK complex participates in the mediation of
paclitaxel-induced IB- degradation. By Western blotting, we
examined the possible influence of paclitaxel and glucocorticoids on
protein expression of both IKK and IKKß in BCap37 cells. Our
results indicated that the protein level of IKKß was remarkably
increased by paclitaxel, whereas IKK was essentially not affected
(Fig. 8
). This result is in agreement with the recent reports by other
laboratories that IKKß, and not IKK, was responsible for
cytokine-induced activation of NF-B (34)
. From the same
experiment, we also found that glucocorticoids did not interfere with
the action of paclitaxel on IKKß (Fig. 8
), which suggests that
glucocorticoids might mediate the NF-B/IB cascade by stimulating
IB- gene transcription, not by modulating
its upstream regulatory factor(s). Therefore, it is possible that
IKKß is the primary target of paclitaxel and may play a critical role
in the mediation of the activation of IB/NF-B cascade and the
induction of apoptosis.
In summary, we have reported that paclitaxel may induce apoptotic
cell death through activation of the NF-B/IB- signaling
pathway. On the basis of our experimental results and current data on
the activation of NF-B by paclitaxel, we would hypothesize the
following pathway by which NF-B/IB- mediates
paclitaxel-induced apoptosis and the inhibitory action of
glucocorticoids (Fig. 9
). Briefly, exposure of tumor cells to paclitaxel leads to the enhanced
expression of IKKß, which causes the degradation of IB- and the
disassociation of NF-B/IB- complex. The released cytoplasmic
NF-B then translocates into nucleus, where it functions as an
important transcription factor to regulate the apoptosis-associated
gene expressions. Conversely, glucocorticoids may inhibit
paclitaxel-induced apoptosis through induction of IB- protein
synthesis, which antagonizes paclitaxel-mediated NF-B nuclear
translocation and activation. These results suggest that the
NF-B/IB- signaling pathway may contribute to the mediation of
paclitaxel-induced cell death in solid tumor cells.
|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was supported by NIH Grants CA 71851
and CA 82440 (to W. F.) and the Health Science Foundation of Medical
University of South Carolina. 
2 To whom requests for reprints should be
addressed, at Department of Pathology and Laboratory Medicine, Medical
University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425.
Phone: (843) 792-5108; Fax: (843) 792-7762; E-mail: fanw{at}musc.edu
3 <.>The abbreviations used are:
NF-B, nuclear factor-B; IB-, inhibitor B ; TA,
triamcinolone acetonide; TNF, tumor necrosis factor; PI, propidium
iodide; EMSA, electrophoretic mobility shift assay; PVDF,
polyvinylidene difluoride.
Received 1/18/00. Accepted 6/16/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B and its inhibitor IB-. Proc. Natl. Acad. Sci. USA, 90: 2532-2536, 1993.B: regulation by distinct protein subunits. Biochim Biophys. Acta, 1072: 63-80, 1991.[Medline]
B induced upon apoptosis. J. Cell Biol., 134: 13-23, 1996. transcription in macrophages: involvement of four B-like motifs and of constitutive and inducible forms of NF-B. Mol. Cell. Biol., 10: 1498-1506, 1990.B-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor gene in primary macrophages. J. Exp. Med., 171: 35-47, 1990.B-like activity. Cell, 65: 1281-1289, 1991.[Medline]
B Rel family of transcription factors. Mol. Cell. Biol., 14: 1039-1044, 1994.B kinase: kinsmen with different crafts. Science (Washington DC), 284: 271-273, 1999.B kinase activity through IKKß subunit phosphorylation. Science (Washington DC), 284: 309-313, 1999.B. Int. J. Biochem. Cell Biol., 27: 865-879, 1995.[Medline]
B transcription factor. Cell, 53: 211-217, 1988.[Medline]
B is regulated by the I-B-associated PKAc subunit through a cyclic AMP-independent mechanism. Cell, 89: 413-424, 1997.[Medline]
B- in mediation of immunosuppression by glucocorticoids. Science (Washington DC), 270: 283-286, 1995. and interleukin-1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor B. Proc. Natl. Acad. Sci. USA, 86: 2336-2340, 1989.B transcription factor and HIV-1. EMBO J., 10: 2247-2258, 1991.[Medline]
B translocation in murine macrophages. Infect. Immun., 64: 878-884, 1996.[Abstract]
B activation of p53: a potential mechanism for suppressing cell growth in response to stress. J. Biol. Chem., 269: 20067-20074, 1994.B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. J. Biol. Chem., 270: 14255-14258, 1995.B contributes to excitotoxin-induced apoptosis in rat striatum. Mol. Pharmacol., 53: 33-42, 1998.B in preventing TNF--induced cell death. Science (Washington DC), 274: 782-784, 1996.B Kinase complex (IKK) contains two kinase subunits, IKK and IKKß, necessary for IB phosphorylation and NF-B activation. Cell, 91: 243-252, 1997.[Medline]
This article has been cited by other articles:
|
|
 |
|
  S. D. Conzen Minireview: Nuclear Receptors and Breast Cancer Mol. Endocrinol., October 1, 2008; 22(10): 2215 - 2228. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Doublier, C. Riganti, C. Voena, C. Costamagna, E. Aldieri, G. Pescarmona, D. Ghigo, and A. Bosia RhoA Silencing Reverts the Resistance to Doxorubicin in Human Colon Cancer Cells Mol. Cancer Res., October 1, 2008; 6(10): 1607 - 1620. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. H. Lee, Y. Zhu, K. M Govindasamy, and G. Gopalan Downregulation of Aurora-A overrides estrogen-mediated growth and chemoresistance in breast cancer cells Endocr. Relat. Cancer, September 1, 2008; 15(3): 765 - 775. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. H. Lee, S. M. Son, D. J. Son, S. M. Kim, T. J. Kim, S. Song, D. C. Moon, H. W. Lee, J. C. Ryu, D.-Y. Yoon, et al. Epothilones induce human colon cancer SW620 cell apoptosis via the tubulin polymerization independent activation of the nuclear factor-{kappa}B/I{kappa}B kinase signal pathway Mol. Cancer Ther., October 1, 2007; 6(10): 2786 - 2797. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Furuya, T. Ozaki, T. Hanamoto, M. Hosoda, S. Hayashi, P. A. Barker, K. Takano, M. Matsumoto, and A. Nakagawara Stabilization of p73 by Nuclear I{kappa}B Kinase-{alpha} Mediates Cisplatin-induced Apoptosis J. Biol. Chem., June 22, 2007; 282(25): 18365 - 18378. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Sui, Y. Huang, B. H. Park, N. E. Davidson, and W. Fan Estrogen Receptor {alpha} Mediates Breast Cancer Cell Resistance to Paclitaxel through Inhibition of Apoptotic Cell Death Cancer Res., June 1, 2007; 67(11): 5337 - 5344. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Jin, X. Liu, Z. Zhou, P. Yue, R. Lotan, F. R. Khuri, L. W.K. Chung, and S.-Y. Sun Activation of Nuclear Factor-{kappa}B Contributes to Induction of Death Receptors and Apoptosis by the Synthetic Retinoid CD437 in DU145 Human Prostate Cancer Cells Cancer Res., July 15, 2005; 65(14): 6354 - 6363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. V. Bava, V. T. Puliappadamba, A. Deepti, A. Nair, D. Karunagaran, and R. J. Anto Sensitization of Taxol-induced Apoptosis by Curcumin Involves Down-regulation of Nuclear Factor-{kappa}B and the Serine/Threonine Kinase Akt and Is Independent of Tubulin Polymerization J. Biol. Chem., February 25, 2005; 280(8): 6301 - 6308. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Wu, T. Pew, M. Zou, D. Pang, and S. D. Conzen Glucocorticoid Receptor-induced MAPK Phosphatase-1 (MPK-1) Expression Inhibits Paclitaxel-associated MAPK Activation and Contributes to Breast Cancer Cell Survival J. Biol. Chem., February 11, 2005; 280(6): 4117 - 4124. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mabuchi, M. Ohmichi, Y. Nishio, T. Hayasaka, A. Kimura, T. Ohta, J. Kawagoe, K. Takahashi, N. Yada-Hashimoto, H. Seino-Noda, et al. Inhibition of Inhibitor of Nuclear Factor-{kappa}B Phosphorylation Increases the Efficacy of Paclitaxel in in Vitro and in Vivo Ovarian Cancer Models Clin. Cancer Res., November 15, 2004; 10(22): 7645 - 7654. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ikezoe, Y. Hisatake, T. Takeuchi, Y. Ohtsuki, Y. Yang, J. W. Said, H. Taguchi, and H. P. Koeffler HIV-1 Protease Inhibitor, Ritonavir: A Potent Inhibitor of CYP3A4, Enhanced the Anticancer Effects of Docetaxel in Androgen-Independent Prostate Cancer Cells In vitro and In vivo Cancer Res., October 15, 2004; 64(20): 7426 - 7431. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Kanthou, O. Greco, A. Stratford, I. Cook, R. Knight, O. Benzakour, and G. Tozer The Tubulin-Binding Agent Combretastatin A-4-Phosphate Arrests Endothelial Cells in Mitosis and Induces Mitotic Cell Death Am. J. Pathol., October 1, 2004; 165(4): 1401 - 1411. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. N. Papandreou and C. J. Logothetis Bortezomib as a Potential Treatment for Prostate Cancer Cancer Res., August 1, 2004; 64(15): 5036 - 5043. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sui, J. M. Dziadyk, X. Zhu, and W. Fan Cell Cycle-Dependent Antagonistic Interactions between Paclitaxel and {gamma}-Radiation in Combination Therapy Clin. Cancer Res., July 15, 2004; 10(14): 4848 - 4857. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Mabuchi, M. Ohmichi, Y. Nishio, T. Hayasaka, A. Kimura, T. Ohta, M. Saito, J. Kawagoe, K. Takahashi, N. Yada-Hashimoto, et al. Inhibition of NF{kappa}B Increases the Efficacy of Cisplatin in in Vitro and in Vivo Ovarian Cancer Models J. Biol. Chem., May 28, 2004; 279(22): 23477 - 23485. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Wu, S. Chaudhuri, D. R. Brickley, D. Pang, T. Karrison, and S. D. Conzen Microarray Analysis Reveals Glucocorticoid-Regulated Survival Genes That Are Associated With Inhibition of Apoptosis in Breast Epithelial Cells Cancer Res., March 1, 2004; 64(5): 1757 - 1764. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Huang, Y. Fang, J. Wu, J. M. Dziadyk, X. Zhu, M. Sui, and W. Fan Regulation of Vinca alkaloid-induced apoptosis by NF-{kappa}B/I{kappa}B pathway in human tumor cells Mol. Cancer Ther., March 1, 2004; 3(3): 271 - 277. [Abstract] [Full Text]
|
|
|
|
|
|
P. Mistry, K. Deacon, S. Mistry, J. Blank, and R. Patel NF-{kappa}B Promotes Survival during Mitotic Cell Cycle Arrest J. Biol. Chem., January 9, 2004; 279(2): 1482 - 1490. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. H. Yang, M. H. Ma, R. A. Vescio, and J. R. Berenson Overcoming Drug Resistance in Multiple Myeloma: The Emergence of Therapeutic Approaches to Induce Apoptosis J. Clin. Oncol., November 15, 2003; 21(22): 4239 - 4247. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. M. Mitchell and R. Z. Orlowski Correspondence re: Somasundaram et al., Dietary Curcumin Inhibits Chemotherapy-induced Apoptosis in Models of Human Breast Cancer. Cancer Res., 62: 3868-3875, 2002. Cancer Res., August 15, 2003; 63(16): 5165 - 5167. [Full Text] [PDF]
|
|
|
|
|
|
M. H. Ma, H. H. Yang, K. Parker, S. Manyak, J. M. Friedman, C. Altamirano, Z.-q. Wu, M. J. Borad, M. Frantzen, E. Roussos, et al. The Proteasome Inhibitor PS-341 Markedly Enhances Sensitivity of Multiple Myeloma Tumor Cells to Chemotherapeutic Agents Clin. Cancer Res., March 1, 2003; 9(3): 1136 - 1144. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Han and N. Sidell RU486-Induced Growth Inhibition of Human Endometrial Cells Involves the Nuclear Factor-{kappa}B Signaling Pathway J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 713 - 719. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Lee, W. Yang, K.-H. Lan, S. Sellappan, K. Klos, G. Hortobagyi, M.-C. Hung, and D. Yu Enhanced Sensitization to Taxol-induced Apoptosis by Herceptin Pretreatment in ErbB2-overexpressing Breast Cancer Cells Cancer Res., October 15, 2002; 62(20): 5703 - 5710. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Somasundaram, N. A. Edmund, D. T. Moore, G. W. Small, Y. Y. Shi, and R. Z. Orlowski Dietary Curcumin Inhibits Chemotherapy-induced Apoptosis in Models of Human Breast Cancer Cancer Res., July 1, 2002; 62(13): 3868 - 3875. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Huang and W. Fan Ikappa B Kinase Activation Is Involved in Regulation of Paclitaxel-Induced Apoptosis in Human Tumor Cell Lines Mol. Pharmacol., January 1, 2002; 61(1): 105 - 113. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Brandes, Z. P. Lin, S. R. Patierno, and K. A. Kennedy Reversal of Physiological Stress-Induced Resistance to Topoisomerase II Inhibitors Using an Inducible Phosphorylation Site-Deficient Mutant of Ikappa Balpha Mol. Pharmacol., September 1, 2001; 60(3): 559 - 567. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M. McDaid and S. B. Horwitz Selective Potentiation of Paclitaxel (Taxol)-Induced Cell Death by Mitogen-Activated Protein Kinase Kinase Inhibition in Human Cancer Cell Lines Mol. Pharmacol., August 1, 2001; 60(2): 290 - 301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-i. Okano and A. K. Rustgi Paclitaxel Induces Prolonged Activation of the Ras/MEK/ERK Pathway Independently of Activating the Programmed Cell Death Machinery J. Biol. Chem., May 25, 2001; 276(22): 19555 - 19564. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
