| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Immunology |
B, Activator Protein-1, and c-Jun NH2-Terminal Kinase1
Cytokine Research Laboratory, Department of Bioimmunotherapy [S. K. M., N. K. S., B. B. A.], and Pharmaceutical Development Center [R. A. N., A. C.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B (NF-B)
and activator protein-1 (AP-1) may be able to block tumorigenesis and
inflammation. Oleandrin, a polyphenolic cardiac glycoside derived from
the leaves of Nerium oleander, is a candidate NF-B
and AP-1 modulator. We investigated the effect of oleandrin on NF-B
activation induced by inflammatory agents. Oleandrin blocked tumor
necrosis factor (TNF)-induced activation of NF-B in a concentration-
and time-dependent manner. This effect was mediated through inhibition
of phosphorylation and degradation of IB
, an inhibitor of
NF-B. A proprietary hot water extract of oleander (Anvirzel) also
blocked TNF-induced NF-B activation; subsequent fractionation of the
extract revealed that this activity was attributable to oleandrin. The
effects of oleandrin were not cell type specific, because it blocked
TNF-induced NF-B activation in a variety of cells. NF-B-dependent
reporter gene transcription activated by TNF was also suppressed by
oleandrin. The TNF-induced NF-B activation cascade involving TNF
receptor 1/TNF receptor-associated death domain/TNF receptor-associated
factor 2/NF-B-inducing kinase/IB kinase was interrupted at the
TNF receptor-associated factor 2 and NF-B-inducing kinase sites by
oleandrin, thus suppressing NF-B reporter gene expression. Oleandrin
blocked NF-B activation induced by phorbol ester and
lipopolysaccharide. Oleandrin also blocked AP-1 activation induced by
TNF and other agents and inhibited the TNF-induced activation of c-Jun
NH2-terminal kinase. Overall, our results indicate that
oleandrin inhibits activation of NF-B and AP-1 and their associated
kinases. This may provide a molecular basis for the ability of
oleandrin to suppress inflammation and perhaps tumorigenesis. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B,4
a nuclear transcription factor, was first identified in 1986 by Sen and
Baltimore, as reported by Baldwin (1)
. Extensive
research during the past few years has indicated that this factor
regulates the expression of various genes that play critical roles in
apoptosis, viral replication, tumorigenesis, various autoimmune
diseases, and inflammation (2, 3)
. Because of its role in
the pathogenesis of various diseases, NF-B is a current target of
interest used by various pharmaceutical companies (4)
. As
part of the stress response, NF-B is activated in response to
various inflammatory stimuli including cytokines, mitogens, bacterial
products, viral proteins, and apoptosis-inducing agents (2, 3)
. Under normal conditions, NF-B is present in the cytoplasm
as an inactive heterotrimer consisting of p50, p65 and IB
subunits. On activation, degradation of IB exposes nuclear
localization signals on the p50-p65 heterodimer, leading to nuclear
translocation and binding to a specific sequence in DNA, which in turn
results in gene transcription.
NF-B is an ideal target for anticancer drug development for several
reasons: (a) activation of NF-B has been shown to block
apoptosis and promote proliferation (5, 6)
; (b)
constitutive expression of NF-B in tumor cells induces proliferation
(7, 8)
; (c) the tumor microenvironment can
induce NF-B activation (9)
; (d) NF-B
activation induces resistance to chemotherapeutic agents (10, 11)
; and (e) several genes involved in tumor
initiation, tumor promotion, and metastasis are regulated by NF-B
(12)
. Thus, because of its role in tumorigenesis, we have
attempted to identify specific inhibitors of NF-B from natural
sources.
Most agents that activate NF-B also activate another transcription
factor, AP-1, which is protein heterodimer composed of the basic region
leucine zipper protein superfamily, specifically, Fos and Jun, and
activating transcription factor proteins (13)
. AP-1
activation requires phosphorylation of JNK (13)
. AP-1
activity has been implicated in various cellular functions including
proliferation, transformation, and differentiation. High AP-1 has been
shown to be involved in tumor promotion and progression of various
types of cancers and induction of resistance to chemotherapeutic
agents. Therefore, inhibitors of AP-1 have been shown to have potential
in blocking carcinogenesis (14)
.
Research within the last few years has shown that certain fruits,
vegetables, herbs, and plants exhibit chemopreventive effects (Ref.
15
and references therein). As many as 70% of the
therapeutic drugs in use today are derived from plants. One potential
candidate for preventive and therapeutic use is oleandrin, a
polyphenolic cardiac glycoside derived from the leaves of Nerium
oleander. Although it has been used to treat congestive heart
failure, oleandrin is known to be toxic to a wide variety of tumor
cells (16–18)
. The mechanism of its cytotoxic effects,
however, is not understood. Because NF-B and AP-1 are known to play
major roles in cell proliferation, tumor promotion, and drug resistance
(11–14)
, it was hypothesized that the effects of
oleandrin were mediated through suppression of NF-B and AP-1.
Therefore, in this report the effect of oleandrin on NF-B and AP-1
activation induced by various tumor promoters, including TNF, phorbol
ester (PMA), LPSs, H2O2,
okadaic acid, and ceramide were investigated. The results demonstrate
that oleandrin is a potent inhibitor of NF-B and AP-1 activated by
these inflammatory agents in a wide variety of different cell types.
The effect of oleandrin on transcription factors was mediated through
inhibition of JNK and MEK activation.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cell Lines.
The cell lines U937 (human histiocytic lymphoma), CaOV3 (human ovarian)
cells, HeLa (human epithelial cells), Jurkat (human T cells), and L-929
(murine fibroblast cells) were obtained from the American Tissue and
Cell Culture Collection (Rockville, MD). Most of the studies were
performed with human histiocytic lymphoma U937 cells because various
cellular responses in these cells have been well characterized in our
laboratory (20)
.
NF-B Activation Assay.
To assay NF-B activation, EMSAs were performed essentially as
described (23)
. Briefly, nuclear extracts prepared from
treated cells (2 x 106/ml) were
incubated with 32P end-labeled 45-mer
double-stranded NF-B oligonucleotide (6 µg of protein with 16 fmol
DNA) from the HIV-LTR,
5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGT
GG- 3' (bold indicates NF-B binding sites) for 15 min at 37°C, and
the DNA-protein complex formed was resolved from free oligonucleotide
on 6.6% native polyacrylamide gels. A double-stranded mutated
oligonucleotide,
5'-TTGTTACAACTCACTTTCCGCTGTCACTTTCCAGGGAGG
CGTGG-3', was used to examine the specificity of binding of
NF-B to the DNA. The specificity of binding was also examined by
competition with the unlabeled oligonucleotide. The dried gels were
visualized, and radioactive bands were quantitated by a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA) using ImageQuant software.
AP-1 Activation Assay.
Activation of AP-1 was determined as described (20)
.
Briefly, 6 µg of nuclear extract, prepared as indicated above, were
incubated with 16 fmol of the 32P-end-labeled
AP-1 consensus oligonucleotide 5'-CGCTTGATGACTCAGCCGGAA-3'
(bold indicates AP-1 binding site) for 15 min at 37°C and analyzed by
using 6% native polyacrylamide gel. The specificity of binding was
examined by competition with unlabeled oligonucleotide. Visualization
and quantitation of radioactive bands were done as indicated above.
Western Blot for IB.
To assay IB, postnuclear (cytoplasmic) extracts were prepared
(24)
from treated cells and resolved on 10%
SDS-polyacrylamide gels. After electrophoresis, the proteins were
electrotransferred to nitrocellulose filters, probed with rabbit
polyclonal antibodies against IB, and detected by
chemiluminescence (ECLl Amersham-Pharmacia Biotechnology, Arlington
Heights, IL).
c-Jun Kinase Assay.
The c-Jun kinase assay was performed by a modified method as described
earlier (20, 25)
.
NF-B-dependent Reporter Gene Transcription.
The effect of oleandrin on TNF-, TRAF-2-, NIK- and p65 (transactivation
subunit of NF-B)-induced NF-B-dependent reporter gene
transcription was measured as described previously (21)
.
Briefly, HeLa cells (0.1 x 106
cells/well) were plated in six-well plates, pretreated with 0.5 µg/ml
oleandrin for 1 h, and then cells were transfected by the calcium
phosphate method with various plasmids. This reporter system was
specific because TNF-induced NF-B SEAP activity was inhibited by
overexpression of either an IB mutant lacking Ser32/36, a kinase
inactive NIK, or a dominant-negative TRAF2 mutant (21)
.
Determination of Lipid Peroxidation.
TNF-induced lipid peroxidation was determined by detection of
thiobarbituric acid-reactive MDA, an end product of the peroxidation of
polyunsaturated fatty acids and related esters, as described
(26)
. U937 (3 x 106/ml) cells pretreated with either media or
different concentrations of oleandrin for 1 h were stimulated with
TNF (1 nM) for 30 min. Then cells were washed with PBS and
subjected to three cycles of freeze-thawing in 200 µl of water. After
protein determination, we added 300 µg of protein (in 0.1 ml) in 800
µl of assay mix containing 0.4% (w/v) thiobarbituric acid, 0.5%
(w/v) SDS, and 9.4% (v/v) acetic acid (pH 3.5). After incubation for
1 h at 95°C, samples were cooled to room temperature and
centrifuged at 14,000 x g for 10 min, and
the absorbance of the supernatants was read at 532 nm. Results were
normalized with the amount of MDA equivalents/mg of protein and
expressed as a percentage of thiobarbituric acid-reactive substances
above control values. Untreated cells showed 0.571 ± 0.126 nmol of MDA equivalents/mg of protein.
Measurement of ROIs.
The production of ROIs after treatment of cells with TNF was determined
by flow cytometry as described (20)
.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. It is an enolide that is soluble in alcohol, chloroform, and DMSO. In
preliminary experiments (data not shown), the concentration and
duration of treatment with oleandrin used in these studies had no
effect on either the TNF receptors or cell viability.
|
B Activation.B
by EMSA. As shown in Fig. 2
A, TNF activated NF-B by almost 12-fold, and oleandrin
inhibited TNF-mediated NF-B activation in a concentration-dependent
manner, with almost 90% inhibition occurring at 1 µg/ml. Oleandrin
(even up to 10 µg/ml) or the DMSO solvent (0.4% v/v) alone did not
activate NF-B.
|
B activation. The cells were incubated with oleandrin
for 240, 120, 60, and 30 min before the addition of TNF, at the same
time as the addition of TNF, or 5, 15, and 30 min after the addition of
TNF. The cells were treated with TNF for 30 min. Only when the cells
were pretreated for 60 min with oleandrin was maximum inhibition of
NF-B activation observed, and the inhibition decreased with shorter
preincubation time (Fig. 2B
). Cotreatment or posttreatment
with oleandrin did not inhibit NF-B activation (Fig. 2B
).
TNF-induced NF-B activation could be seen as early as 5 min, reached
maximum after 15 min, and was maintained until 60 min (Fig. 2C
). Pretreatment of cells with oleandrin suppressed
TNF-induced NF-B activation at all time points. These results
suggest that oleandrin is a very potent inhibitor of NF-B
activation.
Various combinations of Rel/NF-B proteins can constitute an active
NF-B heterodimer that binds to specific sequences in DNA. To show
that the retarded band visualized by EMSA in TNF-treated cells was
indeed NF-B, we incubated nuclear extracts from TNF-activated cells
with antibody to either p50 (NF-BI) or p65 (Rel A) subunits and then
conducted EMSA. Antibodies to either subunit of NF-B shifted the
band to a higher molecular weight (Fig. 2D
), thus suggesting
that the TNF-activated complex consisted of p50 and p65 subunits.
Neither preimmune serum nor such irrelevant antibodies as anti-cRel or
anti-cyclin DI had any effect on the mobility of NF-B. Excess
unlabeled NF-B (100-fold) caused complete disappearance of the band,
indicating the specificity of NF-B. The oligonucleotide with mutated
NF-B site failed to compete with the binding of the NF-B protein.
Inhibition of NF-B Activation by Oleandrin Is Not Cell Type
Specific.
Recent reports indicate that the NF-B activation pathway may differ
in different cell types (27)
. Therefore, we also studied
whether oleandrin affects other cell types. Specifically, we studied
the ability of oleandrin to block TNF-induced NF-B activation in
ovarian (CaCOV3) cells, epithelial (HeLa) cells, and T-cells (Jurkat).
The results of these experiments (Fig. 3
, A–C) indicate that oleandrin inhibited TNF-induced NF-B
activation in all cell types. These results suggest that the effect of
oleandrin is not restricted to myeloid cells but also suppresses
NF-B activation in other cell types.
|
B activation in murine L-929 fibroblast cell line. Results shown
in Fig. 3D
indicate that oleandrin inhibited NF-B
activation in murine cells too and its potency was not significantly
different from that of human cells.
Inhibition of TNF-mediated NF-B Activation by Oleandrin Is Not
through Inhibition of Na/K ATPase.
Most glycosides exert their effects through inhibition of Na/K-ATPase
(28)
. To determine whether oleandrin blocks NF-B
activation by inhibiting this ATPase, we tested the ability of ouabain,
a potent inhibitor of Na/- ATPase (29, 30)
, to block
TNF-induced NF-B activation. Ouabain had no effect on TNF-induced
activation of this transcription factor (Fig. 4
). These results suggest that oleandrin blocks NF-B activation
through some other mechanism.
|
B.B is also activated by a wide variety of other
agents including phorbol ester, LPS, okadaic acid, and ceramide.
However, the signal transduction pathway induced by various activators
may differ (26, 31)
. We therefore examined the effect of
oleandrin on the activation of NF-B by these various agents. The
results shown in Fig. 5
indicate that oleandrin completely blocked the activation of NF-B
induced by PMA and LPS but had no effect on NF-B activation induced
by H2O2, okadaic acid, or
ceramide. These results suggest that oleandrin may act at a step where
TNF, PMA, and LPS converge in the signal transduction pathway leading
to NF-B activation. Because oleandrin had no effect on NF-B
induced by H2O2, OA, or
ceramide, the data suggest that a decrease of TNF-induced
NF-B by oleandrin is specific and not attributable to cell death or
quantitative loss of nuclear protein or degradation.
|
B.B to the nucleus is preceded by the
phosphorylation and proteolytic degradation of IB
(32)
. To determine whether the inhibitory action of
oleandrin was attributable to an effect on IB degradation, the
cytoplasmic levels of IB proteins were examined by Western blot
analysis. IB began to degrade 5 min after TNF treatment of U937
cells and disappeared completely within 15 min. The band reappeared by
30 min. The pretreatment of cells with oleandrin completely abolished
the TNF-induced degradation of IB (Fig. 6A
).
|
B degradation by
oleandrin is attributable to suppression of IB phosphorylation,
cells were treated with the proteosome inhibitor ALLN (33)
for 1 h, and then the hyperphosphorylated form of IB was
assayed by Western blot. The hyperphosphorylated form of IB
appears as a slow-migrating band on SDS-PAGE (Fig. 6B
). The
lack of appearance of a slow-migrating band in oleandrin-treated cells
indicates that oleandrin blocked TNF-induced IB phosphorylation
(see Lane 4 versus Lane 8). These results were further
confirmed by using antibodies that detect only the
serine-phosphorylated form of IB. These results also clearly show
that TNF induces the phosphorylation of IB, and oleandrin
suppresses it (Fig. 6C
).
Oleandrin Represses TNF-induced NF-B-dependent Reporter Gene
Expression.
Thus far, we have shown that oleandrin blocks the DNA binding of
NF-B protein to its consensus sequence. DNA-binding alone does not
always correlate with NF-B-dependent gene transcription, suggesting
the role of additional regulatory steps (34)
. To determine
the effect of oleandrin on TNF-induced NF-B-dependent reporter gene
expression, the oleandrin pretreated or untreated HeLa cells were
transiently transfected with the NF-B SEAP reporter construct and
then stimulated with TNF. An almost 3-fold increase in SEAP activity
over the vector control was noted upon stimulation with TNF (Fig. 7
). However, TNF-induced SEAP activity was almost completely abolished
when the cells were pretreated with oleandrin. These results
demonstrate that oleandrin also represses NF-B-dependent reporter
gene expression induced by TNF.
|
B activation is mediated through sequential
interaction of the TNF receptor with TRADD, TRAF2, NIK, and IKK-ß,
resulting in phosphorylation of IBa (35, 36)
. To
delineate the site of action of oleandrin in the TNF-signaling pathway
leading to NF-B activation, cells were transfected with TRAF2, NIK,
and p65 plasmids, and then NF-B-dependent SEAP expression was
monitored in oleandrin-untreated and -treated cells. As shown in Fig. 7
, TRAF2, NIK, and p65 plasmids induced gene expression, and oleandrin
suppressed TRAF-2 and NIK-induced but had little effect on p65-induced
NF-B reporter expression. RANK, another NF-B-inducing receptor,
was minimally affected by oleandrin, indicating the specificity.
Specificity of the assay results are also indicated by the suppression
of the TNF-induced NF-B reporter activity by the dominant
negative-IB plasmid. Thus, oleandrin must act at a step
downstream from NIK. Because NIK is known to activate IKK-ß, which in
turn phosphorylates IBa, it appears that oleandrin must block the
activity of IKK-ß, a kinase that phosphorylates IB directly.
Oleandrin Inhibits TNF-induced AP-1 Activation.
TNF is also a potent activator of AP-1 (37)
. TNF induced
AP-1 expression by 5-fold in myeloid cells at 1 nM
concentration. The activation of AP-1 was completely inhibited by
oleandrin in a concentration-dependent manner, with maximum suppression
occurring at 0.50 µg/ml (Fig. 8A
). Supershift analysis with specific antibodies against
c-fos and c-jun indicate that TNF-induced AP-1 consists of fos and jun
(data not shown). Lack of supershift by unrelated antibodies and
disappearance of the AP-1 band by competition with cold
oligonucleotide shows the specificity.
|
B also activate AP-1 (37)
.
Therefore, we also investigated the effect of oleandrin on AP-1
activation by PMA, LPS,
H2O2, okadaic acid, and
ceramide. Similar to NF-B, all of the agents activated AP-1, but
only PMA- and LPS-induced AP-1 was blocked by oleandrin (Fig. 8B
). Thus, these results indicate that oleandrin
blocks both NF-B and AP-1 induced by TNF, PMA, and LPS.
Oleandrin Inhibits TNF-induced JNK and MEK Activation.
TNF is a potent activator of MEKK, which activates MEK, which in turn
activates JNK. These kinases are known to be involved in the activation
of both NF-B and AP-1 (37–39)
. Whether these kinases
are modulated by oleandrin was also examined. U937 cells were
pretreated with different concentrations of oleandrin for 1 h and
then stimulated with TNF (1 nM) for 10 min; activation of
JNK was then measured. TNF activated JNK by 
14-fold, an activation
that gradually decreased with increasing concentration of oleandrin. A
1 µg/ml concentration of oleandrin inhibited most of the JNK induced
by TNF (Fig. 9A
). We also found that TNF activated MEK in a
concentration-dependent manner, and oleandrin abolished TNF-induced MEK
activation (Fig. 9B
).
|
B Inhibiting Species in Oleander Extracts Is Oleandrin.B suppression activity and whether this activity is
attributable to oleandrin. Thus, cells were pretreated with different
concentrations of crude oleander extracts and then examined for
TNF-induced NF-B activation. As shown in Fig. 10
A, the extracts inhibited NF-B activation in a
concentration-dependent manner with maximum suppression occurring at
100 µg/ml. TNF activated NF-B in a time-dependent manner, and
oleander extracts inhibited the activation (Fig. 10B
). TNF
also activated AP-1 in a time-dependent manner, and oleander extracts
inhibited this activation (Fig. 10C
). To further investigate
whether oleander extracts also blocked NF-B and AP-1 activated by
other inflammatory agents, cells were treated with these agents as
described for oleandrin and tested for NF-B (Fig. 10D
).
Like oleandrin, only TNF-, PMA-, and LPS-induced NF-B activation was
blocked by oleander extracts, suggesting that the active moiety in the
extracts could be oleandrin. To further confirm that oleandrin is
responsible for suppression of NF-B, the plant extracts were
fractionated by high pressure liquid chromatography, and various
fractions were examined for suppression of NF-B. Only fractions B2
and C blocked TNF-induced NF-B activation. Analysis indicated that
fraction B2 is an oleander extract without oleandrigenin and fraction C
has the complete nonpolar compound content including oleandrin,
oleandrigenenin, and other cardiac glycosides. Lyophilized fractions A
(without oleandrin and oleanderigenin), B1 (without oleandrin), or D
(polysaccharides only) were inactive in suppressing NF-B activation.
Thus, these results clearly indicate that the active principal in the
oleander extracts is oleandrin, which inhibited NF-B and AP-1
activation.
|
B through
generation of ROI (40–42)
. Whether oleandrin suppresses
NF-B activation through suppression of ROI generation was examined
by flow cytometry. As shown in Fig. 11
A, TNF induced ROI generation in a time-dependent manner, and
this was suppressed on pretreatment of cells with oleandrin. Because
lipid peroxidation has also been implicated in TNF-induced NF-B
activation (26)
, we also examined the effect of oleandrin
on TNF-induced lipid peroxidation. Results in Fig. 11B
show
that TNF induced lipid peroxidation in U937 cells, and this was
completely suppressed by oleandrin. Thus, it is quite likely that
oleandrin blocks TNF signaling through suppression of ROI generation
and lipid peroxidation.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B and AP-1 have potential in blocking tumor initiation,
promotion, and metastasis. In the present report, we demonstrate that
oleandrin can block NF-B activation, as determined by consensus DNA
binding, IB degradation, and NF-B-dependent reporter gene
expression. Oleandrin blocked NF-B activation induced by various
inflammatory stimuli including TNF, PMA, and LPS. The effect of
oleandrin was not cell type specific. Besides NF-B activation,
oleandrin also blocked AP-1 activation induced by various inflammatory
stimuli, and it inhibited the TNF-induced activation of JNK and MEK.
The generation of both ROI and lipid peroxidation induced by TNF was
strongly inhibited by oleandrin.
There are several explanations of how oleandrin might inhibit
TNF-induced NF-B activation. We showed that oleandrin does not
interfere with the binding of NF-B to the consensus DNA binding
site. NF-B activation requires sequential phosphorylation,
ubiquitination, and degradation of IB. Because oleandrin blocks
IB degradation, it suggests that the effects of oleandrin on
NF-B is through inhibition of either phosphorylation or IB
proteolysis. The phosphorylated form of IB is known to appear on
the gel as a band with retarded mobility (32, 43)
. The
lack of a retarded IB band after treatment of cells with
oleandrin suggests that oleandrin blocked the phosphorylation of
IB. By using antibodies that specifically detect the
phosphorylated form of IB, it was shown that oleandrin blocks
TNF-induced phosphorylation of IB. The phosphorylation of
IB is regulated by a large number of kinases including IKK-,
IKK-ß, IKK-
, NIK, TGF-ß-activated kinase-1, AKT, and MEKK1
(44–55)
. In addition to MEKK1, MEKK2 and MEKK3 have been
implicated in NF-B activation, whereas MEKK4 activates JNK
(56)
. AKT and NIK are primarily known to activate IKK-,
whereas MEKK1 and atypical protein kinase C activate IKK-ß
(55)
. MEKK is known to induce the phosphorylation of MEK,
which oleandrin inhibited. Thus, it is possible that oleandrin
inhibited IB phosphorylation by inhibiting the activity of MEKK1
or other kinases.
We found that oleandrin blocked NF-B activation induced by a wide
variety of agents including TNF, PMA, and LPS, but had no effect on
okadaic acid, ceramide, and
H2O2-induced NF-B
activation, suggesting a difference in the pathway leading to NF-B
activation by different activators. This is consistent with a recent
report that different inducers activate NF-B by different mechanisms
(26, 31) . Compared with most other agents, NF-B
activation induced by H2O2
does not require IB degradation, again suggesting a difference in
the signaling pathway (31)
.
Our results also indicate that oleandrin is a potent inhibitor of
AP-1. This is not too surprising because most agents that activate
NF-B also activate AP-1. Activation of AP-1 requires JNK, another
kinase of the mitogen-activated protein kinase family. It is possible
that AP-1 is suppressed through inhibition of JNK. Recent studies from
our laboratory showed that overexpression of cells with either
superoxide dismutase (40)
or with -glutamylcysteine
synthetase, a rate-limiting enzyme in the glutathione biosynthesis
pathway (41)
, blocked both NF-B and AP-1 activation
induced by TNF, indicating a similar mechanism of activation of both
transcription factors. These results also suggest that oleandrin may
suppress these factors by regulating the redox status of the cells. We
did indeed find that oleandrin blocks TNF-induced ROI production
and lipid peroxidation. Our results, however, do not rule out
redox-independent mechanism of suppression of NF-B by oleandrin.
Our results indicate that oleandrin blocks TNF-induced NF-B, JNK,
and AP-1 activation. TRAF2, which is known to bind to TNF receptor
through TRADD, is required for NF-B, AP-1, and JNK activation
(35)
. The suppression of TRAF2 activity may explain how
oleandrin inhibits NF-B, AP-1, and JNK. The activity of NIK, which
interacts with TRAF2, was also blocked by oleandrin. This suggests that
oleandrin must inhibit the IKK that phosphorylates IB.
Several recent reports indicate that NF-B activation induces
resistance to apoptosis stimulated by a wide variety of agents
(5–7, 10) . Because oleandrin is known to be cytotoxic to
various tumor cells (17, 18)
, it is possible that this
toxicity is mediated through the suppression of NF-B. There are
several different tumor cell types that express constitutive NF-B
and thus are resistant to apoptosis induced by various agents
(8–10, 40) . Oleandrin should induce apoptosis in these
cells by suppression of NF-B. The mechanism by which oleandrin
induces apoptosis in tumor cells was recently investigated in detail
(57)
. AP-1 is also known to be involved in cell
proliferation (14)
, and thus suppression of AP-1 by
oleandrin may explain its cytotoxic effects. Suppression of AP-1 may
also suggest the role of oleandrin in inhibition of tumor promotion and
progression (12–15)
.
We found that oleandrin blocked NF-B-dependent reporter gene
expression. Several genes are involved in tumor promotion that are
regulated by NF-B. These include cytokines, cyclooxygenase-2,
metalloproteases, urinary plasminogen activator, and cell surface
adhesion molecules (1–3)
. Because NF-B-regulated genes
also play a critical role in carcinogenesis and inflammation, oleandrin
may also exhibit anticarcinogenic and anti-inflammatory effects.
Adenoviral IB, an NF-B inhibitor, and proteosome inhibitors
that block IB degradation are currently being tested to overcome
chemotherapy-induced resistance (10)
. Therefore, NF-B
suppressive ability of oleandrin could be exploited by combination with
chemotherapy. An additional justification for the use of oleandrin may
come from its ability to suppress AP-1, a transcription factor also
known to play a critical role in tumorigenesis. Because replication of
certain viruses such as human immunodeficiency virus-1 is also
dependent on NF-B (1–3)
, oleandrin may also abolish
viral replication.
Similar to oleandrin, the anti-inflammatory drugs sodium salicylate and
aspirin are also known to block the activation of NF-B by preventing
the degradation of IB (58)
. The effects of
salicylate on NF-B activation were observed, however, at
suprapharmacological concentrations (>5 mM). In contrast,
oleandrin was effective at 2000-fold lower concentration, suggesting
that it is a potent inhibitor. Our results suggest that oleandrin may
also have applications for various other diseases including
inflammation and arthritis, where NF-B activation has been shown to
mediate pathogenesis. These possibilities require further investigation
in detail.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This research was conducted by The Clayton
Foundation for Research and with support from Ozelle Pharmaceuticals,
Inc. (to R. A. N.) and a fellowship from the Government of India (to
N. K. S.). 
2 On leave from R. D. College, Sheikhpura
811105, India.
3 To whom requests for reprints should be
addressed, at Department of Bioimmunotherapy, Box 143, The University
of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard,
Houston, TX 77030. Phone: (713) 792-3503/6459; Fax: (713) 794-1613;
E-mail: aggarwal{at}utmdacc.mda.uth.tmc.edu
4 The abbreviations used are: NF-B, nuclear
transcription factor-B; TNF, tumor necrosis factor; AP-1, activator
protein-1; IB, inhibitory subunit of NF-B; EMSA, electrophoretic
mobility shift assay; TRADD, TNF receptor-associated death domain;
TRAF, TNF receptor-associated factor; NIK, NF-B-inducing kinase;
IKK, IB kinase; SA-LPS, serum activated LPS; PMA, phorbol
12-myristate 13-acetate; JNK, c-Jun NH2-terminal kinase;
MEK, mitogen-activated protein kinase kinase; MEKK, MEK kinase; MDA,
malondialdehyde; ROI, reactive oxygen intermediate.
Received 12/ 2/99. Accepted 5/ 8/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B and IB proteins: new discoveries and insights. Annu. Rev. Immunol, 14: 649-683, 1996.[Medline]
B as a frequent target for immuno-suppressive and anti-inflammatory molecules. Adv. Immunol, 65: 111-137, 1997.[Medline]
B or die?. Curr. Biol, 7: R94-R96, 1997.[Medline]
B: important transcription factor and therapeutic target. J. Clin. Pharmacol, 38: 981-993, 1998.B. Science (Washington DC), 274: 784-787, 1996.-induced apoptosis by NF-B. Science (Washington DC), 274: 787-789, 1996.B causes resistance to apoptosis in human cutaneous T cell lymphoma HuT-78 cells: autocrine role of tumor necrosis factor and reactive oxygen intermediates. J. Biol. Chem, 273: 14008-14014, 1998.B during progression of breast cancer to hormone-independent growth. Mol. Cell. Biol, 17: 3629-3639, 1997.[Abstract]
B and proinflammatory cytokine expression during metastatic tumor progression of murine squamous cell carcinoma. Cancer Res, 59: 3495-3504, 1999.B. Nat. Med, 5: 412-417, 1999.[Medline]
B induces expression of the Bcl-2 homologue A1/Bfl-1 to preferentially suppress chemotherapy-induced apoptosis. Mol. Cell. Biol, 19: 5923-5929, 1999.B (NF-B) as a molecular target. Biochem. Pharmacol, 57: 9-17, 1999.[Medline]
-Melanocyte stimulating hormone inhibits the nuclear transcription factor NF-B activation induced by various inflammatory agents. J. Immunol, 161: 2873-2880, 1998.B, c-Jun N terminal kinase, and apoptosis J. Immunol, 162: 6800-6809, 1999.
B by RANK requires TRAF6 and NF-B-inducing kinase (NIK): identification of a novel TRAF6 interaction motif. J. Biol. Chem, 274: 7724-7731, 1999.B induction by TNF, CD95 and IL-1. Nature (Lond.), 385: 540-544, 1997.[Medline]
B activation, IBa phosphorylation, and degradation. J. Biol. Chem, 272: 30129-30134, 1997.B by tumor necrosis factor but not interleukin-1 in the human endothelial cell line ECV304: lack of involvement of H2O2 in NF-B activation by either cytokine in both primary and transformed endothelial cells. J. Biol. Chem, 272: 25941-25950, 1997.B induction by IL-1ß in epithelial and lymphoid cells. J. Immunol, 159: 5264-5272, 1997.[Abstract]
B through two distinct mechanisms. Proc. Natl. Acad. Sci. USA, 95: 13012-13017, 1998. B activity. Trends Genet, 11: 203-205, 1995.[Medline]
B in response to inducers of NF- B activity. Mol. Cell. Biol, 15: 5339-5345, 1995.[Abstract]
B kinase activation and IB degradation by interleukin-1ß and tumor necrosis factor- in human U937 monocytic cells: evidence for additional regulatory steps in B-dependent transcription. J. Biol. Chem, 274: 19965-19972, 1999.B proteins control NF-B activity. Proc. Natl. Acad. Sci. USA, 96: 49-54, 1999.B, which are the targets for MEK kinase 1 action?. Proc. Natl Acad. Sci. USA, 95: 9067-9069, 1998.B kinase complex by MEKK1, a kinase of the JNK pathway. Cell, 88: 213-222, 1997.[Medline]
B and activated protein-1. J. Biol. Chem, 273: 13245-13254, 1998.-glutamylcysteine synthetase abolishes tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor-B and activator protein-1. Oncogene, 18: 4371-4382, 1999.[Medline]
B the sensor of oxidative stress?. FASEB J, 13: 1137-1143, 1999.B is not sufficient for its dissociation from NF-B and is inhibited by protease inhibitors. Proc. Natl. Acad. Sci. USA, 91: 11884-11888, 1994.B phosphorylation sites that signal its ubiquitination and degradation. Mol. Cell. Biol, 16: 1295-1304, 1996.[Abstract]
B kinases. Intern. Immunol, 11: 1357-1362, 1999.B kinase that activates the transcription factor NF-B. Nature (Lond.), 388: 548-554, 1997.[Medline]
B kinase complex (IKK) contains two kinase subunits, IKK and IKKß, necessary for IB phosphorylation and NF-B activation. Cell, 9: 243-252, 1997.
-induced NF-B activation and degradation of IB-. J. Biol. Chem, 271: 13234-13238, 1996.B kinase ß by protein kinase C isoforms. Mol. Cell. Biol, 19: 2180-2188, 1999.B and stimulates its degradation in vitro. J. Biol. Chem, 272: 21281-21288, 1997.B by phosphorylating IB. Proc. Natl Acad. Sci. USA, 91: 6288-6292, 1994.B acting through the NF-B-inducing kinase and IB kinases. Immunity, 10: 271-280, 1999.[Medline]
B p65/RelA subunit. Mol. Cell. Biol, 19: 4798-4805, 1999.B as well as the MAP kinase cascade in the IL-1 signaling pathway. Nature (Lond.), 398: 252-256, 1999.[Medline]
B. Curr. Opin. Cell Biol, 11: 226-232, 1999.[Medline]
B through IB kinase- and IB kinase-ß. J. Biol. Chem, 274: 8355-8358, 1999. B by sodium salicylate and aspirin. Science (Washington DC), 265: 956-959, 1994.This article has been cited by other articles:
|
|
 |
|
  R. A. Newman, P. Yang, A. D. Pawlus, and K. I. Block Cardiac Glycosides as Novel Cancer Therapeutic Agents Mol. Interv., February 1, 2008; 8(1): 36 - 49. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Newman, Y. Kondo, T. Yokoyama, S. Dixon, C. Cartwright, D. Chan, M. Johansen, and Peiying Yang Autophagic Cell Death of Human Pancreatic Tumor Cells Mediated by Oleandrin, a Lipid-Soluble Cardiac Glycoside Integr Cancer Ther, December 1, 2007; 6(4): 354 - 364. [Abstract] [PDF]
|
|
|
|
 |
|
 W. Schoner and G. Scheiner-Bobis Endogenous and exogenous cardiac glycosides: their roles in hypertension, salt metabolism, and cell growth Am J Physiol Cell Physiol, August 1, 2007; 293(2): C509 - C536. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-O. Yoon, S. Shin, H.-J. Lee, H.-K. Chun, and A.-S. Chung Isoginkgetin inhibits tumor cell invasion by regulating phosphatidylinositol 3-kinase/Akt-dependent matrix metalloproteinase-9 expression. Mol. Cancer Ther., November 1, 2006; 5(11): 2666 - 2675. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Frese, M. Frese-Schaper, A.-C. Andres, D. Miescher, B. Zumkehr, and R. A. Schmid Cardiac Glycosides Initiate Apo2L/TRAIL-Induced Apoptosis in Non-Small Cell Lung Cancer Cells by Up-regulation of Death Receptors 4 and 5 Cancer Res., June 1, 2006; 66(11): 5867 - 5874. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. R. Martin Targeting Apoptosis with Dietary Bioactive Agents Experimental Biology and Medicine, February 1, 2006; 231(2): 117 - 129. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Mijatovic, A. Op De Beeck, E. Van Quaquebeke, J. Dewelle, F. Darro, Y. de Launoit, and R. Kiss The cardenolide UNBS1450 is able to deactivate nuclear factor {kappa}B-mediated cytoprotective effects in human non-small cell lung cancer cells. Mol. Cancer Ther., February 1, 2006; 5(2): 391 - 399. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Yang, W. Huang, C. Jozwik, Y. Lin, M. Glasman, H. Caohuy, M. Srivastava, D. Esposito, W. Gillette, J. Hartley, et al. Cardiac glycosides inhibit TNF-{alpha}/NF-{kappa}B signaling by blocking recruitment of TNF receptor-associated death domain to the TNF receptor PNAS, July 5, 2005; 102(27): 9631 - 9636. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Lefranc, J. Brotchi, and R. Kiss Possible Future Issues in the Treatment of Glioblastomas: Special Emphasis on Cell Migration and the Resistance of Migrating Glioblastoma Cells to Apoptosis J. Clin. Oncol., April 1, 2005; 23(10): 2411 - 2422. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Han, J. A. Englert, R. Yang, R. L. Delude, and M. P. Fink Ethyl Pyruvate Inhibits Nuclear Factor-{kappa}B-Dependent Signaling by Directly Targeting p65 J. Pharmacol. Exp. Ther., March 1, 2005; 312(3): 1097 - 1105. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-C. Huang, S.-T. Chan, T.-L. Yang, C.-C. Tzeng, and C.-C. Chen Inhibition of ICAM-1 gene expression, monocyte adhesion and cancer cell invasion by targeting IKK complex: molecular and functional study of novel {alpha}-methylene-{gamma}-butyrolactone derivatives Carcinogenesis, October 1, 2004; 25(10): 1925 - 1934. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Shishodia, D. Koul, and B. B. Aggarwal Cyclooxygenase (COX)-2 Inhibitor Celecoxib Abrogates TNF-Induced NF-{kappa}B Activation through Inhibition of Activation of I{kappa}B{alpha} Kinase and Akt in Human Non-Small Cell Lung Carcinoma: Correlation with Suppression of COX-2 Synthesis J. Immunol., August 1, 2004; 173(3): 2011 - 2022. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Shishodia and B. B. Aggarwal Cyclooxygenase (COX)-2 Inhibitor Celecoxib Abrogates Activation of Cigarette Smoke-Induced Nuclear Factor (NF)-{kappa}B by Suppressing Activation of I-{kappa}B {alpha} Kinase in Human Non-Small Cell Lung Carcinoma: Correlation with Suppression of Cyclin D1, COX-2, and Matrix Metalloproteinase-9 Cancer Res., July 15, 2004; 64(14): 5004 - 5012. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Takada and B. B. Aggarwal Betulinic Acid Suppresses Carcinogen-Induced NF-{kappa}B Activation Through Inhibition of I{kappa}B{alpha} Kinase and p65 Phosphorylation: Abrogation of Cyclooxygenase-2 and Matrix Metalloprotease-9 J. Immunol., September 15, 2003; 171(6): 3278 - 3286. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-S. Chun, Y.-S. Keum, S. S. Han, Y.-S. Song, S.-H. Kim, and Y.-J. Surh Curcumin inhibits phorbol ester-induced expression of cyclooxygenase-2 in mouse skin through suppression of extracellular signal-regulated kinase activity and NF-{kappa}B activation Carcinogenesis, September 1, 2003; 24(9): 1515 - 1524. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Shishodia, S. Majumdar, S. Banerjee, and B. B. Aggarwal Ursolic Acid Inhibits Nuclear Factor-{kappa}B Activation Induced by Carcinogenic Agents through Suppression of I{kappa}B{alpha} Kinase and p65 Phosphorylation: Correlation with Down-Regulation of Cyclooxygenase 2, Matrix Metalloproteinase 9, and Cyclin D1 Cancer Res., August 1, 2003; 63(15): 4375 - 4383. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Shishodia, P. Potdar, C. G. Gairola, and B. B. Aggarwal Curcumin (diferuloylmethane) down-regulates cigarette smoke-induced NF-{kappa}B activation through inhibition of I{kappa}B{alpha} kinase in human lung epithelial cells: correlation with suppression of COX-2, MMP-9 and cyclin D1 Carcinogenesis, July 1, 2003; 24(7): 1269 - 1279. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Wakisaka, S. Murono, T. Yoshizaki, M. Furukawa, and J. S. Pagano Epstein-Barr Virus Latent Membrane Protein 1 Induces and Causes Release of Fibroblast Growth Factor-2 Cancer Res., November 1, 2002; 62(21): 6337 - 6344. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. I. Dmitrieva and P. A. Doris Cardiotonic Steroids: Potential Endogenous Sodium Pump Ligands with Diverse Function Experimental Biology and Medicine, September 1, 2002; 227(8): 561 - 569. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. E. Muscarella and S. E. Bloom Differential Activation of the c-Jun N-Terminal Kinase Pathway in Arsenite-Induced Apoptosis and Sensitization of Chemically Resistant Compared to Susceptible B-Lymphoma Cell Lines Toxicol. Sci., July 1, 2002; 68(1): 82 - 92. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Majumdar and B. B. Aggarwal Methotrexate Suppresses NF-{kappa}B Activation Through Inhibition of I{kappa}B{alpha} Phosphorylation and Degradation J. Immunol., September 1, 2001; 167(5): 2911 - 2920. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. P. Collett, C. N. Robson, J. C. Mathers, and F. C. Campbell Curcumin modifies Apcmin apoptosis resistance and inhibits 2-amino 1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced tumour formation in Apcmin mice Carcinogenesis, May 1, 2001; 22(5): 821 - 825. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Castrillo, B. de las Heras, S. Hortelano, B. Rodriguez, A. Villar, and L. Bosca Inhibition of the Nuclear Factor kappa B (NF-kappa B) Pathway by Tetracyclic Kaurene Diterpenes in Macrophages. SPECIFIC EFFECTS ON NF-kappa B-INDUCING KINASE ACTIVITY AND ON THE COORDINATE ACTIVATION OF ERK AND p38 MAPK J. Biol. Chem., May 4, 2001; 276(19): 15854 - 15860. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-O. Yoon, M.-M. Kim, and A.-S. Chung Inhibitory Effect of Selenite on Invasion of HT1080 Tumor Cells J. Biol. Chem., June 1, 2001; 276(23): 20085 - 20092. [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 |
