Nuclear factor-kappaB/IkappaB signaling pathway may contribute to the mediation of paclitaxel-induced apoptosis in solid tumor cells.
ABSTRACT Paclitaxel (Taxol), a naturally occurring antimitotic agent, has shown significant cell-killing activity in a variety of tumor cells through induction of apoptosis. The mechanism by which paclitaxel induces cell death is not entirely clear. Recent studies in our laboratory demonstrated that glucocorticoids selectively inhibited paclitaxel-induced apoptosis without affecting the ability of paclitaxel to induce microtubule bundling and mitotic arrest. This finding suggests that apoptotic cell death induced by paclitaxel may occur via a pathway independent of mitotic arrest. In the current study, through analyses of a number of apoptosis-associated genes or regulatory proteins, we discovered that paclitaxel significantly down-regulated IkappaB-alpha, the cytoplasmic inhibitor of transcription factor nuclear factor-kappaB (NF-kappaB), which in turn promoted the nuclear translocation of NF-kappaB and its DNA binding activity. In contrast, we found that glucocorticoids could antagonize paclitaxel-mediated NF-kappaB nuclear translocation and activation through induction of IkappaB-alpha protein synthesis. Northern blotting analyses demonstrated that the steady-state level of IkappaB-alpha mRNA was not affected by paclitaxel, which suggests that the down-regulation of IkappaB-alpha 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 IkappaB-alpha expression vectors remarkably increased their sensitivity to paclitaxel-induced apoptosis. Finally, we found that a key subunit of IkappaB kinase (IKK) complex, IKKbeta, was up-regulated by paclitaxel, which implies that paclitaxel might down-regulate IkappaB-alpha through modulation of IKKbeta activity. All of these results suggest that the NF-kappaB/IkappaB-alpha signaling pathway may contribute to the mediation of paclitaxel-induced cell death in solid tumor cells.
- Journal of the American Chemical Society 06/1971; 93(9):2325-7. · 10.68 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Taxol, an antimicrotubule agent, has shown promise for efficacy in treatment of breast cancer, but severe hypersensitivity reactions led to cessation of many phase I clinical trials. Consequently, investigators and the National Cancer Institute recommended that phase I and II studies of this agent use 24-hour infusions and antiallergic medications. Using a premedication regimen effective in preventing hypersensitivity reactions, we have performed a phase II trial of taxol in patients with metastatic breast cancer. Taxol was administered to 25 patients at a dose of 250 mg/m2 by 24-hour infusion every 21 days. These patients had received only one prior chemotherapy regimen, either adjuvant to surgery or for metastatic disease; all but two had received doxorubicin. In 60% of the patients, the dominant site of disease was the viscera. All patients were assessable. In April 1991, at a median time on study of 9 months (range, 5–13+months), the objective response rate was 56% (12% complete and 44% partial; 95% confidence interval, 35%–76%). Disease progressed in only 8% of the patients. The median number of courses of therapy was 11. Granulocytopenia was the dose-limiting toxic effect, but neutropenia with fever occurred in only 5% of 232 courses. A chronic glove-and-stocking neuropathy developed in most patients, but no altergic reactions occurred. We conclude that taxol is an active agent in the treatment of metastatic breast cancer and that it warrants continued study. Currently, we are conducting a phase I trial of taxol plus doxorubicin. Future trials should address the optimal effective dose, the optimal sequencing of combinations, mechanisms of drug resistance in tumors, and dose-limiting toxic effects (particularly cardiac toxic effects of taxol given as a single agent or in drug combinations).JNCI Journal of the National Cancer Institute 01/1992; · 14.34 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Taxol is a chemotherapeutic drug which acts by stabilizing microtubules, preventing normal mitosis and resulting in a block of the cell cycle at G2 and M. The drug is isolated from the yew, Taxus sp. L., and is currently being evaluated in a series of Phase II and Phase III clinical trials. Taxol blocks cells in the most radiosensitive phases of the cell cycle and thus could act as a cell cycle-specific radiosensitizer. We report the results of combined taxol-radiation exposures in the human Grade III astrocytoma cell line, G18. Taxol is a potent inhibitor of G18 cell division; a concentration of 10 nM is cytostatic for a cell population observed for at least two doubling times. Cell survival curves for G18 cells showed a significant concentration-dependent interaction between taxol and radiation. Treatment of G18 cells with a fixed taxol concentration and radiation dose showed the interaction to be dependent on the duration of taxol exposure and consequently the fraction of cells in the G2 or M phase of the cell cycle. The sensitizer enhancement ratio for 10 nM taxol at 10% survival is 1.8 and, for 1 nM taxol, it is 1.2. These results suggest that appropriate combinations of taxol have a more than additive interaction in human tissue culture and may have a role in clinical protocols.Cancer Research 07/1992; 52(12):3495-7. · 8.65 Impact Factor
[CANCER RESEARCH 60, 4426–4432, August 15, 2000]
Nuclear Factor-?B/I?B Signaling Pathway May Contribute to the Mediation of
Paclitaxel-induced Apoptosis in Solid Tumor Cells1
Yi Huang, Korey R. Johnson, James S. Norris, and Weimin Fan2
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
Paclitaxel (Taxol®), a naturally occurring antimitotic agent, has shown
significant cell-killing activity in a variety of tumor cells through induction of
apoptosis. The mechanism by which paclitaxel induces cell death is not
entirely clear. Recent studies in our laboratory demonstrated that glucocor-
ticoids selectively inhibited paclitaxel-induced apoptosis without affecting the
ability of paclitaxel to induce microtubule bundling and mitotic arrest. This
finding suggests that apoptotic cell death induced by paclitaxel may occur via
a pathway independent of mitotic arrest. In the current study, through
analyses of a number of apoptosis-associated genes or regulatory proteins, we
discovered that paclitaxel significantly down-regulated I?B-?, the cytoplas-
mic 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 pacli-
taxel-mediated NF-?B nuclear translocation and activation through induc-
the steady-state level of I?B-? mRNA was not affected by paclitaxel, which
suggests that the down-regulation of I?B-? by paclitaxel is attributable to
protein degradation rather than suppression of transcription. Furthermore,
through transfection assays, we demonstrated that tumor cells stably trans-
fected with antisense I?B-? expression vectors remarkably increased their
sensitivity to paclitaxel-induced apoptosis. Finally, we found that a key sub-
unit of I?B kinase (IKK) complex, IKK?, was up-regulated by paclitaxel,
of IKK? activity. All of these results suggest that the NF-?B/I?B-? signaling
pathway may contribute to the mediation of paclitaxel-induced cell death in
solid tumor cells.
Paclitaxel (Taxol®), a naturally occurring antineoplastic agent, has
shown great promise in the therapeutic treatment of certain human
solid tumors, particularly in metastatic breast cancer and drug-refrac-
tory ovarian cancer (1–4). However, the exact mechanism by which
paclitaxel exerts its cytotoxic action remains unclear. Previous studies
demonstrated that paclitaxel is a unique antimicrotubule agent (5).
Unlike other classical antimicrotubule agents (e.g., colchicine, vin-
cristine, and vinblastine) that induce microtubule disassembly and/or
paracrystal formation, paclitaxel acts by inhibiting microtubule depo-
lymerization and promoting the formation of unusually stable micro-
tubules, thereby disrupting normal dynamic reorganization of the
microtubule network required for mitosis and cell proliferation (6).
Thus, it has been generally believed that the antitumor effects of
paclitaxel result mainly from interference with the normal function of
microtubule and blockage of cell cycle progression in late G2-M
phases via prevention of mitotic spindle formation (7).
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 ap-
optosis in a number of solid tumor cells (8–12). These results clearly
indicated that paclitaxel, in addition to its classical activity against
microtubule, also possesses cell-killing activity by induction of apo-
ptosis. 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 apopto-
sis without affecting mitotic arrest has suggested two possibilities: (a)
arrest; or (b) paclitaxel-induced apoptosis may occur via a separate
pathway that can be blocked by glucocorticoids. No matter which path-
way is correct, glucocorticoids are hypothesized to interfere with the
those genes or proteins whose activation or altered expression is poten-
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-1? con-
verting 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
NF-?B,3a member of Rel transcription factor family, and its specific
intracellular inhibitor I?B-? participate in the mediation of many biolog-
ical activities including inflammation, immune response, cell prolifera-
tion, and apoptotic cell death (21). NF-?B normally resides in the cyto-
plasm as an inactivated form by forming a complex with I?B-?. Upon
certain stimulations, I?B-? is rapidly phosphorylated and degraded, al-
lowing NF-?B to translocate to the nucleus, where it participates in
transcriptional regulation of numerous genes (21, 22). In recent years,
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
Received 1/18/00; accepted 6/16/00.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1This 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.
2To 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@
3The abbreviations used are: NF-?B, nuclear factor-?B; I?B-?, inhibitor ?B ?; TA,
triamcinolone acetonide; TNF, tumor necrosis factor; PI, propidium iodide; EMSA,
electrophoretic mobility shift assay; PVDF, polyvinylidene difluoride.
profoundly down-regulated I?B-?, which in turn promoted the nuclear
translocation and DNA binding activities of NF-?B. In contrast, pacli-
taxel-induced I?B-? degradation and the activation of NF-?B were
blocked if tumor cells were coadministered with glucocorticoids. Trans-
fection assays demonstrated that induction of antisense I?B-? cDNA into
BCap37 cells remarkably increased the sensitivity of tumor cells to
paclitaxel-induced apoptosis. Moreover, one subunit of I?B kinase com-
plex IKK ? was revealed to be up-regulated by paclitaxel. These findings
suggest the possible involvement of NF-?B/I?B-? in the mediation of
paclitaxel-induced apoptotic cell death.
MATERIALS AND METHODS
Drugs and Cell Culture. Paclitaxel was purchased from Calbiochem (La
Jolla, CA) and dissolved in 100% DMSO to make a stock solution of 1.0 mM,
which was then diluted in culture medium to obtain the desired concentrations. TA
was dissolved in 100% ethanol as 10?2to 10?5M stock solutions. Human breast
tumor BCap37 cells, human ovarian tumor OV2008 cells, and human epidermoid
tumor KB cells were cultured in RPMI 1640 (Mediatech) supplemented with 10%
fetal bovine serum and 1% penicillin/streptomycin (Hyclone, Logan, UT).
Western Blotting. After exposure to paclitaxel with or without pretreat-
ment of glucocorticoids (10-7M 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 ?g/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, I?B-?, IKK ?, and IKK ?
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).
?-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?7M TA, 24 h prior to paclitaxel
treatment) for 12, 24, and 48 h. Total RNA was isolated, and 20 ?g were
fractionated in 1% agarose-formaldehyde gel, transferred to nitrocellulose
membrane, and UV cross-linked. The membrane was probed with [32P]UTP-
labeled antisense I?B-? RNA probes generated from the subcloned I?B-?
cDNA fragments in pCDNA3 vectors. The membrane was then washed and
autoradiographed. The same membrane was stripped and reprobed with human
antisense ?-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?7M 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?-CAGTGGGGTCTGTGAATTCCCGGGGGTGAT-
TTCA-3?) (24, 25) were prepared by PCR and purified on a 5% polyacrylamide
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?7M glucocorticoids for 12, 24, and 48 h. Cells were harvested
and resuspended in 800 ?l 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 phenylmethyl-
sulfonyl fluoride] and incubated on ice for 15 min. Then, 50 ?l 10% NP-40 were
added, and cells were vigorously mixed and centrifuged. The nuclei pellets were
suspended in 50 ?l of buffer containing 50 mM KCl, 300 mM NaCl, 0.1 mM
and mixed for 20 min and centrifuged to produce supernatant containing nuclear
proteins. EMSA binding reaction mixture contained 1 ?g of protein of nuclear
extract, 2 ?g 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 I?B-? 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
I?B-5? (5?-CTCGTCCGCGCCATGTTC-3?) and I?B-3? (5?-CTTTGCACT-
CATAACGTCAGA-3?) designed according to the published I?B-? cDNA
sequence (26). The PCR products were inserted into pCR II vectors (Invitro-
gen) and sequenced. Sense and antisense I?B-? 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 ?g per 6-cm plate) containing either sense or antisense I?B-?
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 ?g/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 I?B-? expression by Western blotting assays. Positive
colonies were maintained in culture medium with G418 for additional exper-
iments. 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 previ-
ously (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 ?g/ml ethidium bromide and visualized under UV illumination.
Flow Cytometry Analysis. Cell sample preparation and PI staining for flow
et al. (27). BCap37 cells transfected with empty expression vector pcDNA3
(control), I?B-? sense cDNA (I?B-?-SEN8), and I?B-? antisense cDNA (I?B-
?-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
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).
Paclitaxel and Glucocorticoids Have Opposite Regulatory Ac-
tions on I?B-?. Several apoptosis-associated genes or regulatory
proteins were reported previously to be activated or regulated by
paclitaxel in various normal or tumor cells. These genes include p53,
bcl-2, bax, I?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 expres-
sions in paclitaxel-sensitive BCap37 cells under treatment of pacli-
taxel at the clinically relevant concentration of 100 nM with or without
NF-?B/I?B MEDIATES PACLITAXEL-INDUCED APOPTOSIS
pretreatment of glucocorticoids (10?7M 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 pacli-
taxel 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 I?B-?.
Conversely, pretreatment of cells with glucocorticoids remarkably
enhanced the protein levels of I?B-? in BCap37 cells (Fig. 1A). To
confirm the down-regulation of I?B-? 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 I?B-? also oc-
curred in these two tumor cell lines (Fig. 1B). To further characterize
whether paclitaxel-induced down-regulation of I?B-? occurs at lower
concentrations of paclitaxel, we examined the expression of I?B-? 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
I?B-? was observed at concentrations as low as 1 nM and greater. This
result implied that paclitaxel-induced down-regulation of I?B-? might
be independent of microtubule stabilization because previous studies
from this laboratory and others have revealed that microtubule stabi-
lization was not detected at such low concentrations of paclitaxel
treatment (1–10 nM; Refs. 9, 11, 13, and 28–30).
Down-Regulation of I?B-? by Paclitaxel Is Caused by Protein
Degradation. To elucidate the possible mechanism by which pacli-
taxel down-regulates I?B-? and how glucocorticoids block this pro-
cedure, Northern blotting was performed to determine the I?B-?
mRNA level in BCap37 cells under the treatment of paclitaxel with or
without pretreatment of glucocorticoids. As depicted in Fig. 2, the
transcription of I?B-? (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 I?B-? may be
caused by protein degradation rather than the repression of transcrip-
tion. However, we found that pretreatment of cells with glucocorti-
coids (10?7M TA) significantly induced I?B-? transcription. This
result indicates that paclitaxel and glucocorticoids possess opposite
regulatory effects on I?B-? at different levels.
Paclitaxel Promotes the Nuclear Translocation and DNA Bind-
ing Activity of NF-?B. I?B-? is the specific cytoplasmic inhibitor of
NF-?B. To address whether the opposite regulatory effects on I?B-?
by paclitaxel and glucocorticoids were manifested by alternations in
the nuclear translocation of NF-?B, we performed immunofluores-
cence 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 non-
stimulated (Fig. 3A) or glucocorticoid-treated (10?7M 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?7M
TA) 24 h prior to paclitaxel treatment, paclitaxel-promoted nuclear
translocation of NF-?B was clearly inhibited (Fig. 3D).
Furthermore, we examined the possible effects that paclitaxel and
glucocorticoids may have on DNA-binding activities of NF-?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). Con-
versely, when glucocorticoids (10?7M 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.
Fig. 1. Influence of paclitaxel and glucocorticoids on apoptosis-related gene expres-
sions. A, BCap37 cells with or without pretreatment of glucocorticoids (10?7M TA) were
incubated with 100 nM paclitaxel for 12, 24, and 48 h. B, OV2008 cells and KB cells were
treated with 100 nM paclitaxel for 12, 24, and 48 h. C, BCap37 cells were treated with 1,
10, and 50 nM paclitaxel for 24 h. Equal amounts (50 ?g/lane) of cellular protein were
fractionated on 12.5% SDS-PAGE gels and transferred to PVDF membranes, followed by
immunoblotting with anti-p53, Bcl-2, Bax, c-raf-1, c-myc, and I?B-? monoclonal or
polyclonal antibodies and analyzed as described in “Materials and Methods.” ?-actin
protein was blotted as a control.
Fig. 2. Northern blotting analyses of the effect of paclitaxel and glucocorticoids on the
steady-state mRNA level of I?B-?. Total RNA was isolated from BCap37 cells treated
with 100 nM paclitaxel in the presence or absence of glucocorticoids for 12, 24, and 48 h.
Twenty ?g/lane of RNA were sized fractionated by formaldehyde/agarose gel electro-
phoresis. After transfer to the nitrocellulose membrane and UV cross-linking, RNA was
hybridized at 42°C in 50% formamide with [32P]UTP-labeled antisense riboprobes syn-
thesized from I?B-? pCDNA3 vectors using T7 RNA polymerase. ?-actin probes were
used to confirm comparable RNA loading.
NF-?B/I?B MEDIATES PACLITAXEL-INDUCED APOPTOSIS
Suppression of I?B-? Sensitizes Paclitaxel-induced Apoptosis.
To determine whether the NF-?B/I?B signaling cascade is involved in
the mediation of paclitaxel-induced apoptosis, I?B-? 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 I?B-?
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 I?B-?-SEN8
(sense I?B-? transfection) and I?B-?-ANT5 (antisense I?B-? trans-
fection) is presented from Figs. 5–7. Fig. 5 indicates that the protein
level of I?B-? was significantly increased in I?B-?-SEN8 cells.
However, I?B-? was still down-regulated in the presence of pacli-
taxel, suggesting that paclitaxel-induced down-regulation also oc-
curred for exogenous I?B-?. For I?B-?-ANT5 cells, the endogenous
expression of I?B-? was markedly blocked by antisense I?B-?
mRNA. Interestingly, we noticed that glucocorticoids could still sig-
nificantly induce I?B-? expression, even in cells transfected with
antisense I?B-?. We suspected that the induction of glucocorticoids
on I?B-? expression might be dominant so that the exogenous anti-
sense I?B-? mRNA was unable to significantly alter the protein level
of I?B-? 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 I?B-? cDNA exhibited a marked increase in their sensitivity
to paclitaxel-induced apoptosis as compared with the empty vector or
sense I?B-? 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 I?B-? (Fig.
6, Lane 7). However, DNA fragments were observed in antisense
I?B-? 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 I?B-? cDNA were induced to undergo apo-
ptosis by 50 nM paclitaxel for 24 h treatment (pre-G1peak AP
represents the apoptotic cells). However, the cells transfected with
antisense I?B-? were found to significantly increase their sensitivity
Fig. 3. Paclitaxel induces NF-?B nuclear translocation. BCap37 cells were incubated
with 100 nM paclitaxel in the presence or absence of glucocorticoids (10?7M TA) for 24 h.
Cells were fixed with formaldehyde, and the intracellular locations of NF-?B were
determined by indirect immunofluorescence using anti-NF-?B (p65) antibody and de-
tected as described in “Materials and Methods.” Shown are typical fields of stained
BCap37 cells indicating the exclusive cytoplasmic location of NF-?B in untreated control
(A), glucocorticoid-treated only (B), paclitaxel with glucocorticoid-pretreated (D) cells,
and its nuclear translocation in paclitaxel-treated cells (C).
Fig. 4. Effect of glucocorticoids on paclitaxel-enhanced DNA-binding activity of
NF-?B in BCap37 cells. Equal amounts of nuclear extracts were subjected to EMSAs with
[32P]UTP-labeled oligonucleotide encompassing the NF-?B binding site of TNF-? pro-
moter region. Lane 1, free probe; Lanes 2–5, extracts of control and 100 nM paclitaxel-
treated cells in a time course of 12, 24, and 48 h, respectively; Lanes 6–9, extracts of cells
pretreated with glucocorticoids (10?7M TA) with or without paclitaxel. The NF-?B-
specific complex and the free probe are indicated (arrows).
Fig. 5. Effect of paclitaxel and glucocorticoids on I?B-? in transfected Bcap37 cells.
Control vector (pCDNA3), sense I?B-? (I?B-?-SEN8), and antisense I?B-? (I?B-?-
ANT5) transfected cells were incubated with 100 nM paclitaxel with or without pretreat-
ment of glucocorticoids (10?7M TA) for 24 h. Equal amounts (50 ?g/lane) of cellular
proteins were fractionated on 12.5% SDS-PAGE gel and transferred to PVDF membranes,
followed by immunoblotting with anti-I?B-? polyclonal antibodies and analyzed as
described in “Materials and Methods.” ?-actin proteins were used as a control.
Fig. 6. Paclitaxel-induced internucleosomal DNA fragmentation in I?B-?-transfected
BCap37 cells. Control vector (pCDNA3, Lanes 1–4), sense I?B-? (I?B-?-SEN8, Lanes
5–8), and antisense I?B-? (I?B-?-ANT5, Lanes 9–12) transfected cells were incubated
with 1, 10, and 50 nM paclitaxel for 48 h. Then, cells were harvested, and fragmented DNA
was extracted as described in “Materials and Methods.” Fragmented DNA was analyzed
by electrophoresis in 1.2% agarose gel containing 0.1% ethidium bromide.
NF-?B/I?B MEDIATES PACLITAXEL-INDUCED APOPTOSIS
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 I?B-? transfectants. However, at this time
point, 10 nM or greater concentrations of paclitaxel were required to
cause apoptotic cell death in sense I?B-? or empty vector transfec-
tants (Fig. 7B). These results indicate that BCap37 cells transfected
with antisense I?B-? are more sensitive to paclitaxel-induced apop-
Paclitaxel Up-Regulates IKK?. Recent studies have revealed that
I?B kinases (consisting of IKK ? and ? subunits) are responsible for
I?B protein degradation and NF-?B activation (31–33). To determine
the possible involvement of the I?B kinase complex in paclitaxel-
mediated down-regulation of I?B-?, we have examined whether the
expressions of IKK? or IKK? 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, pacli-
taxel markedly increased the protein level of IKK? but glucocorti-
coids did not affect the up-regulation of IKK? by paclitaxel. This
finding raised the possibility that the primary target of paclitaxel in the
regulation of the I?B/NF-?B pathway might be IKK?.
Our previous studies demonstrated that glucocorticoids could selec-
tively inhibit paclitaxel-induced apoptosis but do not affect the ability of
paclitaxel to induce mitotic arrest (13, 14). This finding suggests that
glucocorticoids might specifically interfere with the signaling pathway
leading to paclitaxel-induced apoptotic cell death (14). Although there is
no solid evidence that paclitaxel-induced apoptosis occurs through a
Fig. 7. Effect of paclitaxel on cell cycle distribution of I?B-? transfectants. Control vector (pCDNA3), sense I?B-? (I?B-?-SEN8), and antisense I?B-? (I?B-?-ANT5) transfected
cells were incubated with 1, 10, and 50 nM paclitaxel. After 24 and 48 h incubation, cells were harvested and stained for DNA with PI, and flow cytometry was performed as described
in “Materials and Methods.” The peaks corresponding to G1and G2-M phases of the cell cycle are indicated. The peaks labeled AP represent apoptotic cells.
NF-?B/I?B MEDIATES PACLITAXEL-INDUCED APOPTOSIS
gene-directed process, the possible existence of this pathway has been
proposed by many investigators (17–19). In this study, by using the
unique inhibitory effect of glucocorticoids on paclitaxel-induced apopto-
cell lines (Fig. 1, A and 1B). Conversely, glucocorticoids (10?7M TA)
were found to possess an inverse regulatory effect on I?B-? through
inducing I?B-? expression (Fig. 1A). The inverse regulatory effect of
paclitaxel and glucocorticoids on I?B-? implies that I?B-? might be an
paclitaxel-induced apoptosis. Further studies revealed that down-regula-
tion of I?B-? was also observed when BCap37 cells were treated with
lower concentrations of paclitaxel (1 or 10 nM; Fig. 1C). Because micro-
tubule stabilization is usually not detectable at such low concentrations of
paclitaxel treatment (28–30), this result implies that paclitaxel-induced
down-regulation of I?B-? may occur independently of microtubule sta-
I?B-?, the specific cytoplasmic inhibitory protein of transcription
factor NF-?B, normally forms a complex with NF-?B in the cyto-
plasm of nonstimulated cells. In various cell lines, the endogenous
I?B-? is rapidly degraded as a consequence of stimulation by proin-
flammatory 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 I?B-?
degradation is the critical step for activation of NF-?B (38–40).
Northern blotting assay in this study indicated that a steady-state level
of I?B-? mRNA was not affected by paclitaxel treatment (Fig. 2).
This result suggests that decreased levels of I?B-? 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 glucocor-
ticoids induced a significant increase in I?B-? mRNA levels. Further
analyses revealed that the degradation of I?B-?, 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 and 4). These
results suggest that paclitaxel and glucocorticoids regulate the NF-
?B/I?B signaling pathway at different levels.
The phenomenon of paclitaxel-induced I?B-? degradation and NF-?B
activation raised a question as to the possible role of activation of
I?B/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 apo-
ptosis. Qin et at (43) found that NF-?B activation contributed to the
excitotoxin-induced death of striatal neurons. However, somewhat incon-
sistent 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/I?B 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 I?B-?
significantly increased their sensitivity to paclitaxel-induced apoptosis
(Figs. 6 and 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/I?B-? 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 deg-
radation of I?B-? 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/I?B
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 I?B-?
and the activation of the NF-?B signaling cascade? Because I?B-?
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 I?B-?. Recent
studies have identified a high molecular weight complex of I?B
kinases (IKK? and IKK?) that play a key role in I?B protein phos-
phorylation 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 I?B-? degradation. By Western blot-
ting, we examined the possible influence of paclitaxel and glucocor-
ticoids on protein expression of both IKK? and IKK? in BCap37
cells. Our results indicated that the protein level of IKK? was remark-
ably increased by paclitaxel, whereas IKK? was essentially not af-
fected (Fig. 8). This result is in agreement with the recent reports by
other laboratories that IKK?, and not IKK?, was responsible for
Fig. 8. Effect of paclitaxel on I?B kinases. BCap37 cells were exposed to 100 nM
paclitaxel with or without the pretreatment of glucocorticoids (10?7M TA) for 12, 24, and
48 h. Equal amounts (50 ?g/lane) of cellular protein were fractionated on 12.5%
SDS-PAGE gel and transferred to PVDF membranes, followed by immunoblotting with
anti-IKK-? and anti-IKK-? polyclonal antibodies and analyzed as described in “Materials
and Methods.” ?-actin protein was used as control.
Fig. 9. Proposed model for NF-?B/I?B-? signaling pathway in the mediation of
paclitaxel-induced apoptosis in solid tumor cells and the potential inhibitory action of
glucocorticoids. MT, microtubule; REC, glucocorticoid receptor.
NF-?B/I?B MEDIATES PACLITAXEL-INDUCED APOPTOSIS
cytokine-induced activation of NF-?B (34). From the same experi-
ment, we also found that glucocorticoids did not interfere with the
action of paclitaxel on IKK? (Fig. 8), which suggests that glucocor-
ticoids might mediate the NF-?B/I?B cascade by stimulating I?B-?
gene transcription, not by modulating its upstream regulatory fac-
tor(s). Therefore, it is possible that IKK? is the primary target of
paclitaxel and may play a critical role in the mediation of the activa-
tion of I?B/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/I?B-? 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/I?B-? mediates paclitaxel-induced apoptosis and the in-
hibitory action of glucocorticoids (Fig. 9). Briefly, exposure of tumor
cells to paclitaxel leads to the enhanced expression of IKK?, which
causes the degradation of I?B-? and the disassociation of NF-?B/I?B-?
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 I?B-? protein
synthesis, which antagonizes paclitaxel-mediated NF-?B nuclear trans-
location and activation. These results suggest that the NF-?B/I?B-?
signaling pathway may contribute to the mediation of paclitaxel-induced
cell death in solid tumor cells.
We thank Drs. David Priest and Debra Hazen-Martin for their critical
review of the manuscript and helpful advice.
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