From the Department of Immunochemistry, German Cancer
Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg,
Germany, the § Institute of Pharmaceutical Biology,
Albert-Ludwigs-Universität, Schänzlestrasse 1, 79104 Freiburg, Germany, and the ¶ Medical Clinics, Department of
Internal Medicine I, Otfried-Müller-Str. 10, 72076 Tübingen, Germany
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ABSTRACT |
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Extracts from certain Mexican Indian medicinal
plants used in traditional indigenous medicine for the treatment of
inflammations contain sequiterpene lactones (SLs), which specifically
inhibit the transcription factor NF-B (Bork, P. M., Schmitz,
M. L., Kuhnt, M., Escher, C., and Heinrich, M. (1997) FEBS
Lett. 402, 85-90). Here we show that SLs prevented the
activation of NF-
B by different stimuli such as phorbol esters,
tumor necrosis factor-
, ligation of the T-cell receptor, and
hydrogen peroxide in various cell types. Treatment of cells with SLs
prevented the induced degradation of I
B-
and I
B-
by all
these stimuli, suggesting that they interfere with a rather common step
in the activation of NF-
B. SLs did neither interfere with DNA
binding activity of activated NF-
B nor with the activity of the
protein tyrosine kinases p59fyn and p60src. Micromolar
amounts of SLs prevented the induced expression of the NF-
B target
gene intracellular adhesion molecule 1. Inhibition of NF-
B by
SLs resulted in an enhanced cell killing of murine fibroblast cells by
tumor necrosis factor-
. SLs lacking an exomethylene group in
conjugation with the lactone function displayed no inhibitory activity
on NF-
B. The analysis of the cellular redox state by fluorescence-activated cell sorter showed that the SLs had no direct or
indirect anti-oxidant properties.
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INTRODUCTION |
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The transcription factor NF-B is one of the key regulators of
genes involved in the immune and inflammatory response (for review, see
Ref. 2). In mammalian cells, NF-
B is composed of a homo- or
heterodimer of various DNA-binding subunits. Five different DNA-binding
subunits share a N-terminal homology domain, which confers DNA binding,
dimerization, nuclear translocation, and interaction with the
inhibitory I
B proteins (for review, see Refs. 3 and 4). In most cell
types these proteins sequester NF-
B, which is frequently a
heterodimer of the p50 and p65 (RelA) subunits, in the cytoplasm by
masking their nuclear localization sequence. Constitutive NF-
B
activity in the cell nucleus can only be detected in certain neurons,
some cells of the monocyte/macrophage lineage and B cells (for review,
see Refs. 5 and 6). Stimulation of cells with a variety of pathogenic
agents including inflammatory cytokines, phorbol esters, UV
irradiation, and oxidants finally leads to the intracellular generation
of reactive oxygen intermediates (ROIs)1 as a key event and
results in the activation of NF-
B (for review, see Refs. 7 and 8).
The two major forms of I
B proteins, termed I
B-
and I
B-
,
can be inducibily phosphorylated and ubiquitinylated (9-11). These
post-translational modifications tag the molecule for the subsequent
proteolytical degradation by the ubiquitin-26 S proteasome pathway
(12-14). This induced degradation of I
B proteins unmasks the
nuclear localization sequences of the DNA-binding subunits of the
NF-
B dimer and allows NF-
B to enter the nucleus, to bind to its
DNA sequence, and to induce transcription. The target genes whose
transcription is mainly regulated by NF-
B include many cytokines,
cell adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1, as well as acute-phase
proteins and immunoreceptors (for review, see Ref. 15).
Among its many different biological activities NF-B seems to play an
important role in cell killing. NF-
B has been shown recently to
counteract the induction of apoptosis by the cytokine TNF-
, ionizing
radiation, and the cancer chemotherapeutic agent daunorubicine (for
review, see Ref. 16). However, there is also evidence for
apoptosis-promoting properties of NF-
B. Glutamate was found to
induce NF-
B in neuronal cells and acetylsalicylic acid (aspirin)
protected these cells from NF-
B-induced cell death (17, 18). Along
this line, the overexpression of the cellular and viral anti-apoptotic
proteins Bcl-2 and E1B 19 K both negatively interfered with the
activation of NF-
B under these conditions (19-21).
The role of NF-B in the immune response is also evident from
gene disruption experiments. The targeted deletion of the p50, p65,
RelB, and c-Rel subunits resulted in an impaired immune response and/or
in a reduced viability of the mice (22-26). These findings and the
immunological relevance of most of the NF-
B target genes make this
transcription factor an interesting therapeutical target for the
identification of inhibitors. One group of NF-
B inhibitors exerts
its inhibitory effects by scavenging ROIs. These inhibitors include
N-acetyl-L-cysteine (27, 28), pyrrolidine
dithiocarbamate (29), acetylsalicylic acid (30, 31), or curcumin (32). All these compounds are structurally unrelated, but share the property
of being anti-oxidative. Another group of inhibitors interferes with
the induced degradation of I
B-family members by affecting the
functioning of the 26 S proteasome (14, 33, 34). Further inhibitors of
NF-
B exert their effects only in the cell nucleus by impairing the
transcriptional activity of NF-
B already bound to DNA. Examples are
inhibitors of the p38 MAP kinase (35, 36) and, at least in some cell
types, glucocorticoids. Here, the activated glucocorticoid receptor
directly binds to the NF-
B dimer and thereby prevents
transcriptional activation (37).
We recently identified SLs isolated from extracts of Mexican Indian
medicinal plants as specific inhibitors of NF-B (1). SL-containing
plant extracts are frequently used in the traditional Mexican Indian
medicine for the treatment of infections of the skin and other organs
(for review, see Ref. 38). The SL parthenolide is also contained in
drugs such as Feverfew® (Tanacetum parthenium)
used against migraine, an illness that has been implicated with
neurogenic inflammatory processes (39). The anti-inflammatory activity
of the SL-containing plant extracts was confirmed in the hen egg tests
where they showed a delay in cell culture experiments (40) and the
onset of capillary reactions of the allantois membrane (1).
This study shows that SLs prevent a common step in NF-B activation.
They did not interfere with the generation of oxygen radicals, but
prevented the induced degradation of I
B-
and I
B-
as well as
the induced expression of the NF-
B target gene ICAM-1. Structural
studies identified the exomethylene group in conjugation with the
lactone group as the decisive structural feature for the inhibitory
activity.
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EXPERIMENTAL PROCEDURES |
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Cell Culture--
Jurkat T leukemia cells (subclone JR
"Würzburg") were maintained in RPMI 1640 supplemented with
10% heat-inactivated fetal calf serum, and 1% (w/v)
penicillin/streptomycin (all purchased from Life Technologies, Inc.,
Grand Island, NY). HeLa cells and L929 fibroblasts were grown in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum
and 1% (w/v) penicillin/streptomycin. All cells were grown in an
incubator at 37 °C and 5% CO2. TNF- and poly(dI-dC)
were obtained from Boehringer Mannheim (Mannheim, Germany).
Parthenolide, isohelenin, isophoronoxide, limonenoxide, caryophyllenoxide, sclareolide, and santonin were from Sigma. Antibodies directed against I
B-
and NF-
B were from Santa Cruz Inc. (Santa Cruz, CA),
-CD28 antibodies were obtained from
Pharmingen Inc. (San Diego, CA), and
-CD3 antibodies were isolated
from a hybridoma cell line. The phycoerythrine-conjugated antibody against ICAM-1 was obtained from Dianova (Hamburg, Germany). All other
chemicals were either from Sigma, Aldrich (Steinheim, Germany), or Roth
(Karslruhe, Germany).
Electrophoretic Mobility Shift Assay (EMSA)--
HeLa or L929
cells (5 × 105) were grown overnight on 10-cm dishes,
Jurkat cells (approximately 1 × 106/ml) in cell
culture flasks. One hour prior to stimulation by TNF-,
phorbol-12-myristate 13-acetate (PMA), or hydrogen peroxide, cells were
preincubated with the indicated amounts of the tested substances for 60 min at 37 °C. The tested substances were dissolved in dimethyl
sulfoxide as a solvent. In the following cells were stimulated for 20 min with PMA at a final concentration of 50 ng/ml, TNF-
(2000 units/ml),
-CD3/
-CD28 (1 µg/ml) antibodies or for the indicated
periods with the specified amounts of hydrogen peroxide. Cells were
harvested by centrifugation and washed twice with cold TBS buffer (25 mM Tris/HCl, pH 7.4, 137 mM NaCl, 5 mM KCl, 0.7 mM CaCl2, 0.1 mM MgCl2). The pellet was resuspended in TOTEX
buffer (20 mM Hepes/KOH, pH 7.9, 0.35 M NaCl,
20% (v/v) glycerol, 1% (v/v) Nonidet P-40, 1 mM
MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride) and incubated on ice for
30 min. The samples were carefully vortexed every 10 min. The cell
debris was pelleted upon centrifugation with 14,000 rpm at 4 °C for
10 min. Equal amounts of supernatant were tested for DNA binding
activity as described in detail elsewhere (1). Briefly, the extracts
were incubated with 2 µg of poly(dI-dC), 2 µg of bovine serum
albumin, and 10,000 cpm of a 32P-labeled oligonucleotide on
ice in 5 × binding buffer (20% (w/v) Ficoll 400, 100 mM Hepes/KOH, pH 7.9, 1 mM dithiothreitol, and 300 mM KCl) in a final volume of 20 µl. Subsequently the
free and the oligonucleotide-bound proteins were separated by
electrophoresis on a native 4% polyacrylamide gel. The gel was dried
after electrophoresis and exposed to an x-ray film (Amersham
Hyperfilm). The following oligonucleotides (binding site underlined)
were used: NF-
B, 5
-AGTTGAGGGGACTTTCCCAGGC-3
and
3
-TCAACTCCCCTGAAAGGGTCCG-5
; Oct-1,
5
-TGTCGAATGCAAATCACTAGAA-3
and
3
-ACAGCTTACGTTTAGTGATCTT-5
.
Western Blotting--
Cell extracts were separated on a 12%
reducing SDS-polyacrylamide gel. Subsequently the proteins were
transferred from the SDS gel onto a polyvinylidene difluoride membrane
(Millipore, Bedford, MA) using a semi-dry blotting apparatus (Bio-Rad,
Munich, Germany). Transfer efficiency was monitored by Ponceau S
staining of membranes. Prior to the incubation with the -I
B-
and I
B-
antibodies (Santa Cruz Inc.), the membrane was blocked
with 5% non-fat dry milk powder in TBST buffer (25 mM
Tris/HCl, pH 7.4, 137 mM NaCl, 5 mM KCl, 0.7 mM CaCl2, 0.1 mM MgCl2,
0.05% (v/v) Tween 20). The membrane was then incubated in a small
volume of TBST containing an 1:1000 dilution of the
-I
B
antibodies. After an overnight incubation the membrane was extensively
washed in TBST buffer and incubated for another hour in TBST containing a 1:3000 dilution of
-rabbit antibody coupled to horseradish peroxidase. After extensively washing the membrane, the immunoreactive bands were visualized by enhanced chemiluminescence according to the
instructions of the manufacturer (Amersham Lifescience, Braunschweig,
Germany).
In Vitro Kinase Assays--
The Src family protein tyrosine
kinases p60src and p59fyn were expressed in
baculovirus-infected Sf9 cells and purified by affinity chromatography as described (41). The effect of SLs on protein kinase
activity was determined using enolase as a substrate. Briefly, serial
concentrations of parthenolide and isohelenin were preincubated at
30 °C with p60src or p59fyn protein in 50 µl
kinase buffer (30 mM Hepes, pH 7.2, 5 mM
MgCl2, 5 mM MnCl2, 1 µM ATP, 10 µCi of [-32P]ATP)
containing 5 µg of acid-treated rabbit muscle enolase (Sigma). After
15 min, the reaction was ended by the addition of Laemmli buffer. The
samples were analyzed by SDS-PAGE and subjected to autoradiography.
Cell Killing Assays-- The cytotoxic activity of TNF was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay essentially as described by Mosmann (42). L929 cells were seeded at a density of 1 × 104 cells/well in 96-well microtiter plates (flat bottomed) and incubated for 16 h in 0.2 ml of culture medium. The supernatant was then removed and replaced by fresh medium containing TNF (2000 units/ml) or/and parthenolide (5 µM). At the indicated times 20 µl of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution (5 mg/ml phosphate-buffered saline) was added to all wells. After another 3-h incubation, supernatants were removed followed by addition of 100 µl of a 24:1 (v/v) isopropyl alcohol/HCl solution. After 15 min at room temperature the absorbance of each well was determined with an automated plate reader (Digiscan, Asys Hitech, Austria) at 550 nm.
FACS Analysis--
The intracellular formation of reactive
oxygen intermediates was measured using dichlorofluorescein diacetate
(DFCH) according to Royall and Ischiropoulus (43). Briefly, a 10 mM stock solution of DFCH (Molecular Probes, Eugene, OR)
was prepared in dimethyl sulfoxide under nitrogen and stored at
20 °C. Cells were incubated with 5 µM DFCH in
phosphate-buffered saline for 0.5 h at 37 °C. ICAM-1 expression
on Jurkat cells was determined using a fluorescein isothiocyanate-conjugated anti-CD54 (ICAM-1) antibody (Dianova, Hamburg, Germany). In both cases measurements were performed in duplicates using a FACScan (Becton Dickinson, Heidelberg, Germany) flow
cytometer. Dead cells were excluded by forward/side scatter gating and
staining with propidium iodide.
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RESULTS |
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SLs Inhibit a Common Step in NF-B Activation--
From a
previous study it was evident that SLs prevent the PMA-induced
activation of NF-
B (1). Therefore we studied whether SLs can inhibit
NF-
B activation in response to stimuli different from PMA. As an
example for a receptor-mediated pathway, mouse L929 cells were
stimulated with TNF-
with or without preincubation with various
amounts of isohelenin or parthenolide. After 1 h of preincubation
cells were stimulated for 20 min with murine TNF-
and total extracts
were tested for DNA binding activity by EMSA. The TNF-induced binding
of NF-
B was completely prevented by preincubation of cells with 5 µM parthenolide and 20 µM isohelenin, respectively (data not shown). Since it is known that most inducers of
NF-
B lead to the formation of ROIs, we tested whether SLs would
interfere with H2O2-induced NF-
B binding
activity. Therefore Jurkat JR cells were preincubated for 1 h with
various amounts of parthenolide and isohelenin and treated with various
concentrations of H2O2 for 90 min. The extracts
prepared from these stimulated cells were subsequently analyzed by EMSA
(Fig. 1A). Only
H2O2 concentrations between 100 and 250 µM activated NF-
B, whereas the addition of 1 mM H2O2 failed to efficiently
induce NF-
B, probably due to an oxidative destruction of the
protein. The H2O2-induced DNA binding activity
of NF-
B was completely prevented by preincubation with the two SLs
in low micromolar concentrations. Another stimulus of NF-
B with
special relevance in T-cells is the activation of the CD3/CD 28 pathway, which also leads to an increased concentration of ROIs (44).
T-cell specific activation of NF-
B in Jurkat T-cells by
cross-linking the CD3 and CD28 receptors was prevented by preincubation
with low micromolar concentrations of the two SLs (Fig. 1B).
All these experiments indicate that SLs interfere with one or more
common steps during NF-
B activation in different cell types rather
than with one single event specific for an individual stimulus. They
are therefore likely to display their inhibitory properties after the
point of integration of the different signals.
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SLs Do Not Affect Activity of Src Family Protein Tyrosine
Kinases--
Tyrosine kinases of the Src family have been implicated
in NF-B activation in response to various stimuli including UV
radiation, T-cell receptor ligation, and stimulation with pro-oxidants.
We therefore investigated the effect of parthenolide and isohelenin on
kinase activity of recombinant p60src and p59fyn. After
pretreatment with various concentrations of SLs, kinase activities were
determined by incubation of the protein kinases with the substrate
rabbit muscle enolase and [
-32P]ATP.
Tyrosine-phosphorylated proteins were then separated by SDS-PAGE and
visualized by autoradiography. Neither increasing amounts up to 20 µM isohelenin nor parthenolide affected the ability of
p59fyn to phosphorylate itself or the substrate enolase. Also
the recombinant p60src kinase was completely unchanged in its
phosphorylating activity in the presence of both SLs (data not shown).
These results suggest that SLs do not prevent NF-
B activation by
inhibiting Src tyrosine kinases. Furthermore, these data indicate that
SLs do not unspecifically alter the activity of cytoplasmatic enzymes
by reacting with the sulfhydryl group of cysteine residues, because
both Src family tyrosine kinases possess exposed and redox-reactive
cysteines which were shown to be important for their activity (45).
SLs Do Not Prevent DNA Binding of NF-B--
To investigate the
mechanism of action of SLs we first tested the potential effects of the
SLs parthenolide and isohelenin on the DNA binding activity of NF-
B
in band shift experiments. Therefore HeLa cells were stimulated with
PMA and cell extracts were prepared 20 min after stimulation. These
extracts, which contain the activated nuclear form of NF-
B, were
pooled and incubated either with various concentrations of the two SLs
or with the solvent dimethyl sulfoxide as a control. The preincubated
extracts were analyzed for DNA binding activity of NF-
B in an EMSA.
Increasing concentrations of parthenolide completely abrogating NF-
B
activation in the intact cell (see Fig. 1) did not influence DNA
binding of activated NF-
B in vitro (Fig.
2). The same amounts of isohelenin also
failed to reduce the DNA binding activity of NF-
B (data not shown).
This experiment excludes that SLs directly inhibit the DNA binding
activity of activated NF-
B, e.g. by modifying reactive
amino acids such as cysteine residues in the DNA-binding or
dimerization domains. The induced DNA-binding complex was confirmed to
be a p50/p65 NF-
B dimer by competition assays with unlabeled oligonucleotides and antibody reactivity (data not shown).
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SLs Prevent the Induced Degradation of IB-
and
I
B-
--
The part of the NF-
B activation cascade that is
influenced by SLs was further analyzed by following the fate of I
B
proteins in Western blots. Mouse L929 cells were preincubated for
1 h with 5 µM of either isohelenin or parthenolide.
Subsequently murine TNF-
was added without changing the medium and
kept for 20 min on the cells. Total cell extracts were tested for the
presence of I
B-
in Western blots and simultaneously for DNA
binding of NF-
B and Oct-1 by EMSAs (Fig.
3). The TNF-
-induced DNA binding of
activated NF-
B in the EMSA experiment coincided with the degradation of I
B-
as detected by Western blotting (Fig. 3, A and
B). Preincubation of cells with 5 µM
parthenolide completely prevented the induction of the DNA-binding form
of NF-
B and protected I
B-
from proteolysis by the 26 S
proteasome. Identical results were obtained for HeLa cells preincubated
with 5 µM isohelenin (data not shown). In a control
experiment the DNA binding activity of the Oct-1 protein remained
unchanged (Fig. 3C).
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SLs Irreversibly Inhibit NF-B Activation--
We next tested
whether SLs act as competitive or irreversible inhibitors. HeLa cells
were incubated with 5 µM parthenolide for 1 h.
Subsequently the cells were washed with medium void of SLs and further
grown for various periods as schematically displayed in Fig.
5A. After stimulation with PMA
for 20 min, cells were lyzed and the extracts were assayed for NF-
B
activity by EMSA. The amount of DNA-bound NF-
B dimers was
quantitated with a PhosphorImager and the results are displayed
graphically (Fig. 5B). The total inhibition of NF-
B
binding activity seen after immediate stimulation of cells following
the preincubation with parthenolide was set as 100%. The inhibition
was still almost complete after 2 h of incubation and decreased to
approximately 50% after 18 h of incubation in parthenolide-free
medium (Fig. 5B). This kinetic behavior suggests that the
SLs act by covalently and thus irreversibly modifying their target
molecule(s), presumably by their reactive Michael system in the lactone
ring. The partial restoration of inducible NF-
B activation, which is
occurring 18 h after SL treatment, is most probably due to the
re-synthesis of the inactivated protein(s).
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SLs Promote Killing of Mouse L929 Cells by TNF---
The role
of NF-
B during the TNF-
-induced cell death is still not clear and
seems to depend on the tested cell line (for review, see Refs. 16 and
46). We therefore wanted to address the question whether cell death of
L929 cells in response to TNF-
is influenced by parthenolide. Mouse
L929 cells were incubated either with 5 µM parthenolide
or 2000 units/ml TNF-
alone or by a combination of both. After
various incubation times the cell viability was measured (Fig.
6). Treatment with parthenolide alone did
not influence the cell viability and TNF-
-induced cell death was
enhanced in the presence of parthenolide. This experiment shows that
the NF-
B inhibitor parthenolide is also enhancing the
TNF-
-induced cell killing of mouse L929 cells.
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SLs Prevent the Induced Expression of the NF-B Target Gene
ICAM-1--
The inducible transcription of the ICAM-1 gene in response
to TNF-
, interleukin-1
, and PMA is controlled by a
NF-
B-binding site in its promoter (for review, see Ref. 47). This
feature makes ICAM-1 surface expression a good read-out to test the
effect of SLs on the expression of endogenous NF-
B target genes. The induction of ICAM-1 gene expression and the appearance of the protein
on the cell surface was analyzed by FACS. Treatment of Jurkat T-cells
with TNF-
resulted in a strong induction of ICAM-1 expression, as
displayed in Fig. 7A.
Preincubation with either 5 µM parthenolide almost
completely prevented the induction of ICAM-1 synthesis (Fig.
7B). In a control experiment, parthenolide or isohelenin did
not influence the amount of the T-cell receptor CD3 protein on the
surface of Jurkat cells in the presence or absence of TNF-
(data not
shown). These results indicate that the induced transcription of
NF-
B target genes is specifically inhibited by SLs.
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Structural Determinants for the Inhibitory Activity of SLs--
We
next investigated the structural features of the SLs which confer
inhibitory activity on NF-B activation pathways. Two structural
hallmarks of SLs are an isoprenoide ring system and a lactone ring. In
many cases this lactone ring contains a conjugated exomethylene group.
Both groups together form a reactive Michael system which is a target
for nucleophilic substrates, e.g. for cysteine residues in
proteins. Various isoprenoide substances lacking either the lactone
ring or the exomethylene group were tested for their effects on NF-
B
activation. The structure of these tested compounds is given in Table
I. None of these substances showed
cytotoxic effects on HeLa cells at concentrations of 5 and 10 µM after 10 h of incubation time. One hour after
preincubation with 5 and 10 µM of the respective drugs,
HeLa cells were stimulated for 20 min with PMA. Subsequently total
extracts of these cells were tested by EMSA on NF-
B activation. All
tested isoprenoides that lacked either the lactone or the exomethylene
group in the
-position to the lactone function displayed no
inhibitory effect on the pathway leading to the activation of NF-
B
(see Table I). Another interesting structural element especially of
parthenolide is its epoxide ring which is also a likely site for the
addition of nucleophilic reagents. To investigate the importance of
this epoxide ring we also tested substances with this structural
feature, even in combination with exomethylene groups to provide a
bireactive substrate for (the) target molecule(s). Again none of the
tested substances displayed inhibitory properties on NF-
B as
assessed by EMSA (Table I). This failure of inhibition occurred
irrespective of a synthetic or natural origin of the tested
substances.
|
SLs Inhibit NF-B Activation without Having Anti-oxidative
Properties--
The chemical structure of the SLs suggests that they
do not have anti-oxidative properties such as many other inhibitors of NF-
B. To exclude that SLs display any direct or indirect
anti-oxidative effects we measured the potential influence of
parthenolide preincubation on the TNF-
induced change of the
intracellular redox state in Jurkat T-cells. Quantitation of the
intracellular levels of ROIs by FACS analysis with the dye DFCH
demonstrated that preincubation of Jurkat T-cells for 1 h with 5 µM parthenolide did not reduce the amount of
intracellular ROIs that were generated by TNF-
stimulation (Fig.
8, A and B). This
observation is confirmed by a direct comparison of ROI
concentrations under various conditions (Fig. 8C).
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DISCUSSION |
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Numerous biological activities have been reported for SLs,
including antimicrobial (48), antiviral (49), and antitumor activities
(50). Furthermore, SLs or SL-containing plant extracts were found to
have anti-inflammatory properties (51, 52). Anti-phlogistic activities
were also seen in hen eggs assays, in the reduced production of the
inflammatory cytokine interleukin-6 and in cell culture experiments (1,
40). It was previously reported that the anti-inflammatory effects of
SLs can be assigned, at least to a certain extent, to the inhibition of
transcription factor NF-B, a central mediator of the immune response
(1). Here we show that SLs do not interfere with the generation of ROIs
following the stimulation of cells, but prevent the induced degradation
of I
B-
and I
B-
. The SLs do not directly act on the
DNA-binding subunits of NF-
B. Also the I
B subunits seems not to
be a direct target for the SLs. The incubation of cell extracts from
unstimulated cells with SLs did not change the inducibility of NF-
B
by the dissociating agent
desoxycholate.2 Furthermore,
the resynthesis of the putative target protein(s) after more than
18 h makes the I
B proteins an unlikely candidate for the SLs,
since I
B-
is completely resynthesized within 1 h (53).
Various protein kinases have been implicated in the induced activation
of NF-B, but the kinases participating in these signaling events
remain poorly defined. The signal transducing events leading to NF-
B
include the small GTP-binding proteins Rac1 (54) and Cdc 42 (55) which
then lead to the activation of mitogen-activated protein kinase/ERK
kinase kinase-1 (MEKK-1). The activation of MEKK-1, which has recently
been shown to be necessary for the induction of NF-
B (56), results
in the induced activity of mitogen-activated protein kinase kinase
(MKK4/SEK) and the c-Jun N-terminal kinase (JNK) (57). The kinase
finally phosphorylating I
B-
at serines 32 and 36 is called CHUK
(58) or IKK (59). It is currently not clear which identified or so far
unidentified member(s) of the signaling cascade is the target for SLs.
Furthermore, the number of molecules inhibited by SLs remains
unknown.
The role of NF-B in the induction of cell death is still not
yielding a homogenous picture. The inhibition of NF-
B promotes TNF-
-mediated cell death in HeLa cells, macrophages, fibroblasts, fibrosarcoma cells, and Jurkat cells (60-63), while it is ineffective in other cell lines such as human breast carcinoma cells (64). Part of
the discrepancies may be explained by differences of the cell type
studied and the nature of the apoptosis-inducing stimulus. This study
shows that the incubation with parthenolide facilitates cell killing by
TNF-
in mouse L929 cells, although only additional experiments using
further inhibitors of NF-
B can really prove the protecting role of
this transcription factor for L929 cells. The mechanism of this
cell-protecting effect of NF-
B is still not clear. It might rely on
the induced expression of anti-apoptotic genes such as the zinc finger
protein A20 or manganese superoxide dismutase (for review, see Ref.
16).
The SLs tested in this study display a high degree of specificity for
their inhibitory activity, since they did not influence the activity of
other transcription factors such as AP-1, RBP-J, and Oct-1 (Ref. 1
and this study). Furthermore, the SLs did not impair the activity of
the T-cell kinases p59fyn and p60src. These results
show that the SLs do not interfere in a nonspecific manner with
transcription factors or signaling molecules. A potential target
specificity of SLs may well be explained by considering the fact that
the combination of the reactive Michael-acceptor system together with
the oxygen-substituted isoprenoide rings forms a pattern of potential
noncovalent binding sites (e.g. hydrogen bonds). These
binding sites would allow the SLs to interact with complementary sites
on the surface of the target molecule(s). The relative positions of SLs
and other inhibitors of NF-
B in the signaling cascade are
schematically displayed in Fig. 9. This allows the usage of this drug for the analysis and dissection of the
diverse signal transduction pathways finally resulting in the
activation of NF-
B. Since the various classes of inhibitors interfere with NF-
B activation at different levels in signal transduction it is tempting to speculate that a mixture of these different inhibitors might be highly effective in NF-
B inhibition. Furthermore, the individual doses required for an optimal blocking may
presumably be reduced in such a blend, thereby reducing the respective
side effects.
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The inhibition of NF-B may be of therapeutic use for the treatment
of chronic diseases such as rheumatoid arthritis or for the acute
situations such as septic shock. There is emerging evidence for the
role of NF-
B during rheumatoid arthritis (for review, see Ref. 2)
and the importance of NF-
B during Crohn's disease. Here, the local
administration of antisense phosphororthioate oligonucleotides to the
p65 subunit of NF-
B abrogated established experimental colitis in
mice (65). Also other chronic inflammatory diseases such as
Alzheimer's disease involve the activation of NF-
B. The amyloid
peptide, which is a major component of the plaque, induces an increase
in ROIs and activates the nuclear translocation of the p50 and p65
subunits of NF-
B in the neurons directly surrounding the plaque (5).
Moreover the septic shock syndrome is associated with a massive
activation of NF-
B. Septic shock occurs when microbial products such
as LPS stimulate the expression of inflammatory cytokines. This massive
production of cytokines leads to failure of circulation and general
organ function. Therefore it is of therapeutical interest to develop drugs that are able to interfere with the activity of NF-
B.
The complete inhibition of NF-B is lost only to 50% after 18 h
post-treatment, suggesting an irreversible mechanism such as a covalent
modification of proteins. Also other drugs such as aspirin and
omeprazole (Antra®), lead to to an irreversible
inactivation of their target molecules (for review, see Refs. 66 and
67). The covalent modification of a target protein by a drug reduces
the required frequency of drug application. The determination of the
structural requirements of SLs for NF-
B inhibition presented in this
study provides the basis for a rational development of a new generation
of anti-inflammatory substances interfering with NF-
B.
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ACKNOWLEDGEMENTS |
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We thank Dr. Alexey Ushmorov for help with the FACS analysis and Andreas Dumont for carefully reading the manuscript.
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FOOTNOTES |
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* This work was supported by a European Union Biomed-2 grant (to M. L. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
49-6221-423725; Fax: 49-6221-423746; E-mail:
l.schmitz{at}dkfz-heidelberg.de.
1
The abbreviations used are: ROIs, reactive
oxygen intermediates; CHUK, conserved helix-loop-helix ubiquitous
kinase; DFCH, dichlorofluorescein diacetate; EMSA, electrophoretic
mobility shift assay; ICAM-1, intercellular adhesion molecule-1; IKK,
IB kinase; JNK, c-Jun N-terminal kinase; MEKK-1, mitogen-activated protein kinase/ERK kinase kinase-1; MKK4, mitogen-activated protein kinase kinase 4; PMA, phorbol 12-myristate 13-acetate; PAGE,
polyacrylamide gel electrophoresis; FACS, fluorescence-activated cell
sorter; SLs, sequiterpene lactones; TNF, tumor necrosis factor.
2 M. L. Schmitz, unpublished results.
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REFERENCES |
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