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Blood, 1 June 2002, Vol. 99, No. 11, pp. 4079-4086
NEOPLASIA
Biologic sequelae of nuclear factor- B blockade in
multiple myeloma: therapeutic applications
Nicholas Mitsiades,
Constantine S. Mitsiades,
Vassiliki Poulaki,
Dharminder Chauhan,
Paul G. Richardson,
Teru Hideshima,
Nikhil Munshi,
Steven P. Treon, and
Kenneth C. Anderson
From the Department of Adult Oncology, Dana Farber
Cancer Institute, Harvard Medical School; the Department of Medicine,
Harvard Medical School; and the Retina Research Laboratory,
Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston,
MA.
 |
Abstract |
The transcription factor nuclear factor- B (NF-B)
confers significant survival potential in a variety of tumors. Several established or novel anti-multiple myeloma (anti-MM) agents, such as
dexamethasone, thalidomide, and proteasome inhibitors (PS-341), inhibit
NF-B activity as part of their diverse actions. However, studies to
date have not delineated the effects of specific inhibition of NF-B
activity in MM. We therefore investigated the effect of SN50, a
cell-permeable specific inhibitor of NF-B nuclear translocation and
activity, on MM cells. SN50 induced apoptosis in MM cell lines and
patient cells; down-regulated expression of Bcl-2, A1,
X-chromosome-linked inhibitor-of-apoptosis protein (XIAP),
cellular inhibitor-of-apoptosis protein 1 (cIAP-1), cIAP-2, and
survivin; up-regulated Bax; increased mitochondrial cytochrome c release into the cytoplasm; and activated
caspase-9 and caspase-3, but not caspase-8. We have previously
demonstrated that tumor necrosis factor- (TNF-) is present
locally in the bone marrow microenvironment and induces
NF-B-dependent up-regulation of adhesion molecules on both MM cells
and bone marrow stromal cells, with resultant increased adhesion. In
this study, TNF- alone induced NF-B nuclear translocation, cIAP-1
and cIAP-2 up-regulation, and MM cell proliferation; in contrast, SN50
pretreatment sensitized MM cells to TNF--induced apoptosis
and cleavage of caspase-8 and caspase-3, similar to our previous
finding of SN50-induced sensitization to apoptosis induced by the
TNF- family member TNF-related apoptosis-inducing ligand
(TRAIL)/Apo2L. Moreover, SN50 inhibited TNF--induced
expression of another NF-B target gene, intercellular adhesion
molecule-1. Although the p38 inhibitor PD169316 did not directly kill
MM cells, it potentiated the apoptotic effect of SN50, suggesting an
interaction between the p38 and NF-B pathways. Our results therefore
demonstrate that NF-B activity in MM cells promotes tumor-cell
survival and protects against apoptotic stimuli. These studies provide
the framework for targeting NF-B activity in novel biologically
based therapies for MM.
(Blood. 2002;99:4079-4086)
© 2002 by The American Society of Hematology.
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Introduction |
Multiple myeloma (MM), a presently incurable
B-cell malignancy, affects 14 000 new patients in the United States
annually and is the second most common hematologic
malignancy.1-3 Combination chemotherapy offers initial
response rates of 40% to 70% in MM patients,4 but
refractoriness to these regimens eventually develops. High-dose
chemotherapy with stem cell support has achieved higher response rates
than conventional therapy, but few patients remain in long-term
remission, highlighting the urgent need for novel therapeutic strategies.
The Rel/nuclear factor-B (Rel/NF-B) proteins comprise a family
of transcription factors, which is conserved from Drosophila to humans and regulates a variety of physiological aspects of immune
and inflammatory responses. These proteins bind to 10 base pair DNA
sites (B sites) as dimers and directly regulate gene transcription.
NF-B commonly refers to the p50-RelA heterodimer, which is the major
Rel complex. In most cell types, NF-B is present constitutively in
the cytosol in a latent, inactive form where it is retained
through its interaction with inhibitory IB (inhibitor of NF-B)
proteins, masking its nuclear localization sequence. A
variety of stimuli induce phosphorylation of IB at 2 N-terminal Ser
residues by the IB kinase complex, followed by
ubiquitination and degradation of IB by the
proteasome.5 Rel/NF-B complex then enters the nucleus,
binds to DNA, and activates transcription of target genes whose
products include proteins mediating host immune responses, such as
cytokines, chemokines, major histocompatibility complex molecules;
proteins involved in antigen presentation; and receptors required for
neutrophil adhesion and transmigration across blood vessel
walls.5
NF-B was originally identified as a B-cell nuclear factor, and
NF-B activity is required for proper regulation of B-cell homeostasis.6,7 Constitutive NF-B activity is also
present in some neurons,8 human thymocytes,9
Sertoli cells,10 and photoreceptor cells.11
Members of the NF-B/Rel transcription-factor family are expressed
constitutively during B-cell development, and they are further induced
by mitogen activation.12 Conversely, activated B cells
from mice harboring germline disruptions in individual NF-B subunits
exhibit increased sensitivity to apoptosis.13 There is
also accumulating evidence that many tumor cells have constitutive
NF-B site-binding and transactivation
activity.14-17 Consequently, NF-B has emerged as a
therapeutic target in a variety of neoplasias. For example,
NF-B is constitutively activated in primitive human acute
myelogenous leukemia cells, and the proteasome inhibitor MG-132
inhibits IB degradation and induces tumor cell apoptosis.18,19 Rel/NF-B activity inhibits
apoptosis in WEHI 231 immature B-lymphoma cells, whereas treatment with
inhibitors of NF-B induction, including the proteasome
inhibitor N-tosyl-L-phenylalanine chloromethyl ketone and
microinjection of
glutathione-S-transferase-IB or an antibody
to c-Rel, sensitizes these cells to anti-immunoglobulin M
(anti-IgM)-induced apoptosis.20
The importance of NF-B activity for survival of activated B cells,
coupled with the constitutive activity of NF-B in several malignancies, prompted us to investigate its role in MM
pathophysiology. Moreover, several conventional or emerging anti-MM
agents, such as dexamethasone, proteasome inhibitors, and thalidomide,
possess anti-NF-B activity.21-26 To date, however, the
direct effects of specific NF-B inhibition have not been studied in
MM. We therefore evaluated the effects of SN50, a cell-permeable
peptide that specifically inhibits NF-B nuclear transport and
activity, in MM cell lines and patient cells. As tumor necrosis
factor- (TNF-) is secreted into the bone marrow
microenvironment27 and confers resistance to MM cell
apoptosis, we also investigated the effects of NF-B inhibition on
TNF--induced signaling. Our studies demonstrate that NF-B
activity promotes growth, survival, and drug resistance in MM cells and
that these effects can be inhibited by NF-B blockade. Importantly,
they provide the framework for novel therapies targeting NF-B in MM.
| |
Materials and methods |
MM cell lines and MM patient cells
Dexamethasone-sensitive MM.1S and
dexamethasone-resistant MM.1R human MM cell lines were kindly provided
by Dr Steven Rosen (Northwestern University, Chicago, IL). RPMI-8226/S
cells and its doxorubicin-resistant subline (Dox40) were a kind gift
from Dr William Dalton (Lee Moffit Cancer Center, Tampa, FL).
OCI-My5 cells were kindly provided by Dr H. A. Messner
(Ontario Cancer Institute, Toronto, Ontario, Canada). ARH-77,
HS-Sultan, and IM-9 Epstein-Barr virus (EBV)-transformed B-cell lines
were obtained from the American Type Culture Collection (Manassas, VA).
The MM-AS cell line has been established from an MM patient
sample.28 Malignant plasma cells were freshly isolated
from peripheral blood of a patient with plasma cell leukemia and were
confirmed by flow cytometric analysis to be greater than 95%
CD38+CD45RA .
All cells were cultured in RPMI 1640 medium (GIBCO Laboratories, Grand
Island NY) supplemented with 10% charcoal dextran-treated fetal
bovine serum (Hyclone, Logan, UT) as well as L-glutamine, penicillin,
and streptomycin (GIBCO).
Materials
The peptide SN50, consisting of the nuclear localization
sequence of p50 (residues 360 to 369) fused to the hydrophobic region of the signal sequence of Kaposi fibroblast growth factor to provide cell permeability, specifically inhibits nuclear translocation of
NF-B.29-36 SN50M, a synthetic analog with a mutated
nuclear localization sequence, is inactive and served as a negative
control.29 Both peptides were purchased from BIOMOL
(Plymouth Meeting, PA). TNF-related apoptosis-inducing ligand
(TRAIL)/Apo2L was provided by Genentech (South San Francisco, CA) and
PS-341 by Millennium (Cambridge, MA).
Other reagents were obtained as follows: p38 inhibitor PD169316
(Calbiochem, La Jolla, CA); mouse monoclonal antibodies for Bcl-2,
BclxL, A1, Bax, and tubulin and polyclonal antibody for Mcl-1 (Santa
Cruz Biotechnology, Santa Cruz, CA); human recombinant interleukin-6
(IL-6) and polyclonal antisera against cellular inhibitor-of-apoptosis protein 1 (cIAP-1), cIAP-2, and
X-chromosome-linked IAP (XIAP) (R&D Systems, Minneapolis,
MN); polyclonal antiserum against survivin (Oncogene Research,
Cambridge, MA); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dexamethasone, and doxorubicin (Sigma Chemical, St Louis, MO); a Complete (proprietary name) mixture of
proteinase inhibitors, immunoglobulin-free normal horse serum
and sodium dodecyl sulfate (SDS) (Life Technologies, Gaithersburg, MD);
and the Enhanced Chemiluminescence (ECL) kit, which includes the
peroxidase-labeled antimouse and antirabbit secondary antibodies
(Amersham, Arlington Heights, IL).
Evaluation of NF-B activity
The DNA-binding activity of NF-B in MM.1S cells was
quantified by enzyme linked immunosorbent assay (ELISA) by means of the Trans-AM NF-B p65 Transcription Factor Assay Kit (Active Motif North
America, Carlsbad, CA), according to the manufacturer's instructions.
Briefly, MM.1S cells were cultured with or without SN50 (20 µM) for 1 hour, and then treated with or without TNF- (50 ng/mL) for 4 hours.
Nuclear extracts were prepared as previously described37
and incubated in 96-well plates coated with immobilized oligonucleotide
(5'-AGTTGAGGGGACTTTCCCAGGC-3') containing a consensus (5'-GGGACTTTCC-3') binding site for the p65 subunit of NF-B. NF-B
binding to the target oligonucleotide was detected by incubation with
primary antibody specific for the activated form of p65 (Active Motif
North America), visualized by anti-IgG horseradish peroxidase conjugate
and developing solution, and quantified at 450 nm with a reference
wavelength of 655 nm. Background binding, obtained by incubation with a
2-nucleotide mutant oligonucleotide (5'-AGTTGAGGCCACTTTCCCAGGC-3'), was
subtracted from the value obtained for binding to the consensus DNA sequence.
MTT colorimetric survival assay
The survival of MM cells was examined by means of the MTT
colorimetric assay, as previously described.38 Cells were
plated in 48-well plates at 70% to 80% confluence and then incubated for 18 hours with the indicated concentration of SN50. At the end of
each treatment, cells were incubated with 1 mg/mL MTT for 4 hours at
37°C; a mixture of isopropanol and 1N HCl (23:2, vol/vol) was then
added under vigorous pipetting to dissolve the formazan crystals. Dye
absorbance in viable cells was measured at 570 nm, with 630 nm as a
reference wavelength. Cell survival was estimated as a percentage of
the value of untreated controls. All experiments were repeated
at least 3 times, and each experimental condition was repeated in at
least quadruplicate wells in each experiment.
Annexin V-propidium iodide staining
Detection of early apoptotic cells was performed by means of the
annexin V-propidium iodide (PI) detection kit (Immunotech/Beckman Coulter, Miami, FL). Briefly, 106 MM cells were
exposed for 4 hours to SN50 (20 M), washed with Dulbecco modified Eagle
medium, incubated in the dark at 4°C with annexin V-fluorescein
isothiocyanate (annexin V-FITC) and PI for 15 minutes, and then
analyzed by dual-color flow cytometry. Cells that were annexin
V-FITC-positive (with translocation of phosphatidylserine from the
inner to the outer leaflet of the plasma membrane) and PI-negative
(with intact cellular membrane) were considered early apoptotic cells.
In another experiment, MM cells were preincubated with caspase
inhibitors (pan-caspase inhibitor ZVAD-FMK, caspase-8 inhibitor
IETD-FMK, caspase-3 inhibitor DEVD-FMK, and caspase-9 inhibitor
LEHD-FMK; all used at 20 µM) for 1 hour prior to exposure to SN50
(20 µM).
Immunoblotting analysis
Immunoblotting analysis was performed as previously
described.38 Briefly, cells were lysed for 30 minutes on
ice in lysis buffer (50 mM Tris-HCl, pH 8, with 120 mM NaCl and 1%
NP-40) supplemented with the Complete mixture of proteinase inhibitors.
The samples were cleared by microcentrifugation (14 000 rpm, 30 minutes, 4°C) and assessed for protein concentration. We
electrophoresed 30 µg protein per sample in a 12%
SDS-polyacrylamide gel and electroblotted it onto nitrocellulose
membranes. After 1-hour incubation in blocking solution (20%
IgG-free normal horse serum, in phosphate-buffered saline [PBS]), the
membranes were exposed overnight at 4°C to the primary antibody.
Following washing in PBS, the respective secondary peroxidase-labeled
antibody was applied at 1:10 000 dilution for 1 hour at room
temperature. The proteins were visualized with the ECL technique.
Subcellular fractionation and cytochrome c
detection
MM.1S cells were treated with or without SN50 (20 µM) for 4 hours, washed in cold PBS once, harvested in 100 µL isotonic buffer (210 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 10 mM Hepes, pH 7.5, supplemented with the Complete protease inhibitors cocktail) and homogenized with a Dounce homogenizer. Samples
were centrifuged originally at 1000g to remove the nuclei,
and subsequently at 10 000g for 30 minutes at 4°C to
obtain the heavy membrane, mitochondria-enriched pellet. Both the
mitochondria-enriched and the cytoplasm-enriched supernatant were
assayed for the presence of cytochrome c by means of an
ELISA,39 according to the manufacturer's instructions (R&D Systems).
Cleavage of caspases and poly(ADP-ribose) polymerase
The involvement of caspases in SN50-induced apoptosis in MM
cells was studied by evaluating the levels of procaspase-8,
procaspase-3, and procaspase-9, as well as the emergence of their
cleaved active forms, by immunoblotting in lysates of cells treated
with SN50 (20 µM) for 4, 8, and 16 hours. The cleavage of
poly(ADP-ribose) polymerase (PARP), a well-known target of caspase
activity, was also studied in the same lysates. Treatment with
TRAIL/Apo2L served as a positive control for caspase activation, as
previously reported.40
Statistical analysis
Statistical significance was examined by a 2-way analysis of
variance, followed by a Duncan post hoc test. In all analyses, P < .05 was considered statistically significant.
| |
Results |
SN50 down-regulates constitutive and induced NF-B activity
in MM.1S
We first evaluated the ways in which the DNA-binding activity of
NF-B in MM cells was affected by SN50, a cell-permeable peptide
derived from the nuclear localization sequence of p50, which inhibits
the nuclear translocation of NF-B.9,30 As seen
in Figure 1, constitutive NF-B
DNA-binding activity in MM.1S cells was significantly inhibited by
SN50. Moreover, SN50 also inhibited NF-B activation induced by
TNF.
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| Figure 1.
Effect of SN50 on NF-B activity in MM cells.
SN50 down-regulates constitutive and induced NF-B activity in MM
cells. DNA-binding activity of the transcriptional factor NF-B was
quantified in MM.1S cells pretreated with or without SN50 (20 µM) for
1 hour, and then treated with or without TNF- (50 ng/mL) for 4 hours. The DNA binding activity of NF-B in MM.1S cells was
quantified by ELISA with the use of the Trans-AM NF-B p65
Transcription Factor Assay Kit, according to the manufacturer's
instructions. Values (mean ± SD) were normalized for cellular
protein content. Data are representative of 3 experiments. Constitutive
NF-B DNA-binding activity in MM.1S cells was significantly inhibited
by SN50. Moreover, SN50 inhibited NF-B activation induced by
TNF-.
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SN50 induces apoptosis in MM cells
We next investigated the effect of SN50 on MM cell survival by
means of the MTT assay. As can be seen in Figure
2A-B, SN50 induced
concentration-dependent cell death in 7 of 9 cell lines, including MM
cell lines resistant to dexamethasone or doxorubicin, EBV-transformed
ARH-77 and IM-9 cells, and freshly isolated patient MM cells. In
contrast, normal peripheral B cells were resistant to SN50-induced cell
death. Annexin V-PI labeling revealed early externalization of
phosphatidylserine in MM.1S cells treated with SN50 for 5 hours (Figure
2C-D), confirming that SN50 induced apoptosis. Mutation of 2 amino-acid
residues in SN50, which results in loss of its NF-B inhibitory
activity, also abolished its anti-MM activity (Figure 2E).
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| Figure 2.
Effect of NF-B inhibition on apoptosis in MM cells.
NF-B inhibition induces apoptosis in MM cells. (A) (B) Dose-response
survival curves based on MTT colorimetric assay were generated for MM
cells and healthy donor B cells cultured for 18 hours with SN50 (0 to
25 µM). Data shown (absorbance at 570 nm, mean ± SD) are
representative of 3 experiments. Panel A shows dexamethasone-sensitive
MM.1S ( ) and dexamethasone-resistant MM.1R ( ) cells; RPMI-8226/S
( ) and its cytotoxic drug-resistant subline Dox40 (filled diamond)
cells; and OCI-My5 ( ) and MM-AS ( ) cells. Panel B shows
EBV-transformed ARH-77 () cells, HS Sultan () cells, and IM-9
() cells; freshly isolated patient MM cells (); and healthy donor
peripheral blood B cells (filled diamond). SN50 induced
concentration-dependent cell death in 7 of 9 cell lines, including MM
cell lines resistant to dexamethasone or doxorubicin, as well as in
freshly isolated patient MM cells. In contrast, normal peripheral B
cells were resistant to SN50-induced cell death. (C) (D) Annexin-PI
staining of MM.1S cells cultured without (panel C) or with (panel D)
SN50 (20 µM) for 4 hours revealed early externalization of
phosphatidylserine in MM.1S cells treated with SN50 for 5 hours,
confirming that SN50 induced apoptosis. (E) The proapoptotic activity
of the NF-B inhibitor SN50 () was compared with its mutant (2 amino-acid residue difference), inactive analog SN50M () in MM.1S
cells. Data shown (absorbance at 570 nm, mean ± SD) are
representative of 3 experiments. Loss of NF-B inhibitory activity
abolishes anti-MM activity.
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SN50 decreases expression of apoptosis inhibitors
Having demonstrated that NF-B inhibition with SN50 induces MM
cell apoptosis, we next evaluated its effect on the level of protein
expression of several apoptosis inhibitors. As can be seen in Figure
3, SN50 rapidly decreased protein
expression of Bcl-2, A1, XIAP, cIAP-1, cIAP-2, and survivin. In
contrast, Mcl-1 and BclxL protein levels were not changed. Moreover, we
found that SN50 up-regulated the expression of the proapoptotic
Bax protein.
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| Figure 3.
Effect of NF-B inhibition on expression of apoptosis
inhibitors.
NF-B inhibition decreases expression of apoptosis inhibitors.
Immunoblotting analysis for apoptosis regulators in cell lysates of
MM.1S cells treated with SN50 (20 µM) for 0, 4, 8, or 16 hours. SN50
rapidly decreased protein expression of Bcl-2, A1, XIAP, cIAP-1,
cIAP-2, and survivin. In contrast, Mcl-1 and BclxL protein levels were
not changed. Moreover, SN50 up-regulated the expression of the
proapoptotic Bax protein.
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SN50 induces cytochrome c release from the
mitochondria to the cytoplasm
The above-mentioned effects of NF-B inhibition on Bcl-2 family
member expression (ie, increase of the proapoptotic Bax and decrease of
the antiapoptotic Bcl-2 and A1 proteins) suggested that mitochondrial
events could participate in the resulting induction of apoptosis. A
major mechanism of mitochondrial regulation of apoptosis is via release
of cytochrome c into the cytoplasm. We therefore assayed for
cytochrome c in the cytoplasmic and mitochondrial fractions
in MM.1S cells before and after treatment with SN50. As can be seen in
Figure 4, treatment with SN50 induced a
rapid decrease of cytochrome c in the mitochondrial fraction
of MM.1S cells, associated with an increase of cytochrome c
in the cytosolic fraction. These data suggest that cytochrome
c release is associated with MM cell apoptosis induced by
NF-B inhibition.
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| Figure 4.
Effect of NF-B inhibition on cytochrome
c release from the mitochondria to the cytoplasm.
NF-B inhibition induces cytochrome c release from the
mitochondria to the cytoplasm. Cytochrome c protein levels
(nanograms per microgram of total protein, mean ± SD) were
measured in the cytoplasmic and mitochondrial fractions of MM.1S cells
treated with (white bars) or without (black bars) SN50 (20 µM) for 4 hours. Treatment with SN50 induced a rapid decrease of cytochrome
c in the mitochondrial fraction of MM.1S cells, associated
with an increase of cytochrome c in the cytosolic
fraction.
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Activation of caspases by SN50
We next investigated the potential involvement of downstream
caspases mediating SN50-induced MM cell apoptosis. As seen in Figure
5A, treatment with SN50 induced cleavage
of caspase-9, caspase-3, and PARP. In contrast, no cleavage of
caspase-8 was detected upon SN50 treatment. TRAIL/Apo2L (300 ng/mL for
5 hours) induced caspase-8 cleavage, as in our prior
study,40 and served as a positive control. To evaluate the
functional involvement of caspases in SN50-induced apoptosis, we used
specific inhibitors of caspase-3, caspase-8, and caspase-9. The
caspase-9 inhibitor LEHD-FMK and the caspase-3 inhibitor DEVD-FMK
partially blocked SN50-induced cell death, whereas the caspase-8
inhibitor IETD-FMK had no effect (Figure 5B). These data collectively
suggest that SN50-induced apoptosis is mediated by the mitochondrial
release of cytochrome c, triggering activation of caspase-9
and consequent caspase-3 activation. Caspase-8 is not involved,
suggesting a mechanism distinct from death-receptor-induced cell
death.
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| Figure 5.
Activation of caspases by NF-B inhibition.
(A) Immunoblotting analysis for caspase-9, caspase-3, and
caspase-8, as well as PARP, was performed in lysates of MM.1S cells
treated with SN50 (20 µM) for 0, 4, 8, or 16 hours. Treatment with
SN50 induced cleavage of caspase-9, caspase-3, and PARP. In contrast,
no cleavage of caspase-8 was detected upon SN50 treatment. TRAIL/Apo2L
(300 ng/mL for 5 hours) induced caspase-8 cleavage, as in our prior
studies,40 and served as a positive control. (B) Annexin
V-PI staining was performed to quantify phosphatidylserine
externalization in MM.1S cells treated with or without SN50 (20 µM)
for 4 hours, in the presence or absence of specific inhibitors for
caspase-3, caspase-8, and caspase-9. The caspase-9 inhibitor LEHD-FMK
and the caspase-3 inhibitor DEVD-FMK partially blocked SN50-induced
cell death, whereas the caspase-8 inhibitor IETD-FMK had no
effect.
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SN50 inhibits the stimulatory effect of IL-6 on MM.1S
cells
Since IL-6 is a major growth factor for MM cells, we next
evaluated the impact of SN50 on the stimulatory effect of IL-6 on MM
cells. As seen in Figure 6A, IL-6 (50 ng/mL) increased the number of MM.1S cells (approximately 20% after 16 hours), whereas preincubation with SN50 (20 µM) abolished this
effect. Moreover, our data demonstrate that IL-6 does not protect
against SN50-induced cell death (Figure 6B), suggesting that NF-B
inhibition is not attenuated by IL-6.
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| Figure 6.
Effect of NF-B inhibition on the stimulatory effect
of IL-6 on MM.1S cells.
NF-B inhibition inhibits the stimulatory effect of IL-6 on MM.1S
cells. SN50 overcomes IL-6-induced growth, as evidenced by MTT (panel
A), and protection against apoptosis, as evidenced by PI staining
(panel B), in MM.1S cells. Data shown (panel A, absorbance at 570 nm,
mean ± SD) are representative of 3 experiments.
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Effect of SN50 on TNF-induced sequelae on MM cells
We have previously reported a small, but reproducible, stimulatory
effect of TNF- on MM cell proliferation.27 Here, we investigated the effect of NF-B blockade on this response of MM
cells to TNF-. In MM.1S cells pretreated with a nontoxic
concentration of SN50, TNF- induced decreased, rather than
increased, cell growth (Figure 7A). This
decreased viability was due to MM cell apoptosis, as evidenced by
cleavage and activation of caspase-8 and downstream caspase-3 (Figure
7B). This ability of TNF- to trigger death-receptor-mediated
apoptosis in SN50-pretreated MM.1S cells was associated with
down-regulation of expression of cIAP-1 and cIAP-2 caspase-8 inhibitory
proteins. In contrast, TNF- alone up-regulated cIAP-1 and cIAP-2
expression. These data confirm that TNF- activates a proapoptotic
pathway, via caspase-8 activation, as well as an antiapoptotic pathway,
via NF-B activation and up-regulation of cIAP-1 and cIAP-2, in MM
cells. Our data further suggest that the balance between these 2 pathways is modulated by the level of NF-B activation. Finally, we
have also recently reported that the expression of another known
NF-B target, intercellular adhesion molecule-1 (ICAM-1), is
up-regulated by TNF-.27 As seen in Figure 7B, SN50
strongly inhibits this induction of ICAM-1 on MM.1S cells by TNF-,
further implicating NF-B in the regulation of MM cell adhesion and
interactions of MM cells in the bone marrow microenvironment.
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| Figure 7.
Effect of NF-B activity on TNF--induced
signaling.
TNF--induced signaling is modulated by the activity of NF-B. (A)
Cell viability (mean ± SD) was assayed by MTT in MM.1S cells
treated with or without TNF- (50 ng/mL), in the presence or absence
of SN50 (10 µM) for 18 hours. Data shown (absorbance at 570 nm,
mean ± SD) are representative of 3 experiments. In
MM.1S cells pretreated with SN50, TNF- induced apoptosis, rather
than cell growth. (B) This was evidenced by cleavage and activation of
caspase-8 and downstream caspase-3 and was associated with
down-regulation of expression of the caspase-8 inhibitory proteins
cIAP-1 and cIAP-2 In contrast, TNF- alone up-regulated cIAP-1 and
cIAP-2 expression. Moreover, TNF- up-regulated adhesion molecule
intercellular adhesion molecule-1 (ICAM-1) expression, and SN50
blocked this effect.
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The p38 inhibitor PD169316 sensitizes MM.1S cells to apoptosis
induced by SN50
Since p38 kinase signaling regulates cell survival41
and the activation of NF-B,42 we next investigated the
role of p38 in SN50-induced MM cell apoptosis. The p38 inhibitor
PD169316 did not induce MM cell apoptosis, but did enhance the
apoptotic effect of SN50, suggesting an interaction between the p38 and NF-B pathways (Figure 8).
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| Figure 8.
Effect of PD169316 on MM.1S-cell apoptosis induced by
NF-B inhibition.
The p38 inhibitor PD169316 sensitizes MM.1S cells to apoptosis induced
by NF-B inhibition. Cell death was assayed by MTT in MM.1S cells
treated with a nontoxic concentration of the p38 inhibitor PD169316 (10 µM) and/or the NF-B inhibitor SN50 (10 µM) for 18 hours.
PD169316 did not induce MM cell apoptosis, but did enhance the
apoptotic effect of SN50, suggesting an interaction between the p38 and
NF-B pathways. Data shown (mean ± SD) are representative of 3 experiments.
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SN50 sensitizes MM cells to chemotherapy
The transcription factor NF-B has been implicated in the
regulation of cell sensitivity to chemotherapy in multiple model systems.43,44 We therefore investigated the effect of SN50 on chemotherapy-induced apoptosis in MM.1S cells. As seen in Figure 9A, preincubation with a nontoxic
concentration of the NF-B inhibitor SN50 sensitized MM.1S cells to
low concentrations of doxorubicin, with the percentage of MM cell
survival as follows: 101.6% ± 1.9% with doxorubicin (25 ng/mL)
versus 52.1% ± 0.2% with doxorubicin (25 ng/mL) plus SN50
(P < .016); 55.6% ± 4.0% with doxorubicin (50 ng/mL)
versus 27.0% ± 1.4% with doxorubicin (50 ng/mL) plus SN50
(P < .005). These data suggest that inhibition of NF-B
strongly potentiates the anticancer effects of traditional anti-MM
chemotherapy. We also evaluated the combined effects of SN50 with
dexamethasone and the proteasome inhibitor PS-341 and found a modest
potentiating effect (Figure 9B).
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| Figure 9.
Effects of NF-B inhibition combined with anti-MM
agents.
(A) MM.1S cells were incubated with or without doxorubicin (doxo;
25 and 50 ng/mL). At 24 hours later, the cells were treated with or
without the NF-B inhibitor SN50 (10 µM) for 18 hours. Cell
survival was assayed by MTT. Data shown (mean ± SD) are
representative of 3 experiments. The NF-B inhibitor strongly
sensitized MM.1S cells to doxorubicin. (B) SN50 (10 µM) also
increased, but to a lesser extent, the anti-MM effect of the proteasome
inhibitor PS-341 (5 nM, 18 hours), and dexamethasone (dex; 1 µM,
48 hours).
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| |
Discussion |
In the present study, we investigated the effects of the NF-B
inhibitor SN50 on MM cell lines and patient cells, as well as on normal
B lymphocytes. We found that SN50 inhibited constitutive and induced
NF-B activity and induced apoptosis in MM cell lines and
patient cells, including those resistant to conventional therapies, but
not in normal human B lymphocytes. SN50 down-regulated Bcl-2, A1, XIAP,
cIAP-1, cIAP-2, and survivin protein levels, up-regulated Bax
expression, induced release of mitochondrial cytochrome c into the cytoplasm, and activated caspase-9 and caspase-3 but not
caspase-8. SN50 also sensitized MM cells to TNF- and doxorubicin and
had a synergistic proapoptotic effect with a p38 inhibitor. These
studies provide the framework for targeting NF-B in MM therapies.
Several established or novel anti-MM agents, such as
dexamethasone, thalidomide, proteasome inhibitors, and arsenic
trioxide, have the ability to inhibit NF-B
activation.21-26 As a consequence, NF-B inhibition may
be at least a component of their antitumor activity. However, the exact
contribution of NF-B inhibition to their anti-MM activity has not
been delineated. Moreover, the effect of specific NF-B inhibition on
MM cells is also unknown. We therefore used an inhibitor of the nuclear
translocation of NF-B to characterize the role of this transcription
factor in MM cells.29 We found that the NF-B
inhibitor SN50 induced apoptosis in most MM cell lines, as well as in
primary patient cells, but not in normal B lymphocytes. In previous
studies of our panel of cell lines, the 2 SN50-resistant cell lines
constitutively express the highest levels of various apoptosis
inhibitors, including FLICE/caspase-8 inhibitory protein
(FLIP), cIAP-1, cIAP-2, XIAP, and survivin.40
We then investigated the molecular mechanism of SN50-induced MM
cell apoptosis. We found evidence of mitochondrial involvement, including Bcl-2 and A1 down-regulation, release of
mitochondrial cytochrome c to the cytoplasm, and activation
of caspase-9. Several members of the Bcl-2 family of apoptosis
inhibitors are regulated by Rel/NF-B transcription factors in
various models, including Bcl-2 and A1.44-49 On
the other hand, mitogen-activated B cells from mice lacking c-Rel or
p50 expression are very sensitive to apoptosis and can be rescued by
overexpression of Bcl-213 or A1.48 Moreover,
A1 regulates the survival of macrophages in an NF-B-dependent
fashion by preserving mitochondrial integrity50 and
suppresses apoptosis by inhibiting cytochrome c release and caspase-3 activation.44 In our model, the increase in Bax
and the decrease in Bcl-2 and A1 protein levels shift the balance of
Bcl2-A1/Bax toward apoptosis. Bax induces pore formation in the
mitochondrial membrane and ultimately facilitates the release of
cytochrome c into the cytoplasm,51 as in the
present study. Cytochrome c binds the adaptor molecule
Apaf-1 and forms the "apoptosome" that activates caspase-9, which
cleaves and activates caspase-3.52,53 The role of
caspase-9 and caspase-3 in our model is confirmed, since specific
caspase-3 and caspase-9 inhibitors attenuated SN50-induced apoptosis.
The apoptosis inhibitor XIAP is NF-B dependent in other
models.35,54 Since XIAP inhibits caspase-9 activity, it
appears that NF-B inhibition stimulates caspase-9-dependent
apoptosis in a dual manner: by stimulating the release of mitochondrial cytochrome c, leading to the formation of the apoptosome;
and by down-regulating XIAP expression. Activated caspase-9 then
activates caspase-3. In our study, the expression of survivin, another
member of the IAP family, was also found to be NF-B dependent in MM cells. Since caspase-3 can be inhibited by survivin,55 it
appears that NF-B also regulates MM cell apoptosis at the level of
caspase-3 activity.
We recently demonstrated that TNF- is secreted into the bone
marrow microenvironment by MM cells and induces NF-B-dependent alterations in adhesion molecule expression on both MM cells and bone
marrow stromal cells, with resulting increased cell adhesion. This
binding confers resistance to apoptosis and also triggers NF-B-dependent secretion of IL-6. TNF- also causes a modest, but
reproducible, MM cell proliferation.27 In this study, we investigated the effect of NF-B inhibition on the sequelae of TNF- on MM.1S cells. In contrast to the moderate (approximately 25%
increased) proliferation response to TNF- monotherapy, TNF- also
induced a moderate reduction of survival (approximately 25% decrease)
via activation of caspase-8 and caspase-3 when combined with a
noncytotoxic concentration of SN50. These data suggest that TNF- is
not per se a growth factor for MM cells, but is rather a proapoptotic
factor that activates caspase-8 and caspase-3. Our data further
suggest that constitutive activity of NF-B in MM cells abrogates the
proapoptotic effect of TNF-, owing to the potent up-regulation of
cIAP-1 and cIAP-2, and induces modest proliferation. We have also
recently reported that NF-B inhibition increases the sensitivity of
MM cells to another member of the TNF family,
TRAIL/Apo2L.56 As was observed in this study, this sensitizing effect is associated with down-regulation of cIAP-1 and
cIAP-2, members of the IAP family of antiapoptotic proteins that are
recruited to the signaling complex of TNF receptor I via their
interaction with TNF-receptor-associated factor 1 (TRAF1) and
TRAF2, thereby exerting an inhibitory function on caspase-8 activation.57 The expression of cIAP-2 is also regulated
by NF-B in human leukemic cells.58 Moreover,
overexpression of cIAP-1 and cIAP-2 can protect RelA-deficient cells,
which are highly sensitive to TNF--induced apoptosis, by blocking
the activation of caspase-8.57 Thus, cIAP-1 and/or cIAP-2
mediate, at least in part, the protective effect of NF-B against
apoptosis in MM cells.
These data collectively demonstrate a dual antiapoptotic effect of
NF-B in MM cells: first, at the level of caspase-8 in the
death-receptor pathway and second, at the mitochondrial level. Our work
further suggests that the death-receptor pathway is more sensitive to
NF-B inhibition, since lower concentrations of SN50 are sufficient
to potentiate death-receptor-induced apoptosis in our model, in both
this and prior studies.40 In contrast, mitochondrial
dysfunction apparently requires complete abrogation of constitutive
NF-B activity. It is possible that this dual regulation corresponds
to different conditions in vivo: complete abrogation of NF-B
activity may by itself be sufficient to induce MM cell apoptosis,
whereas a partial block in NF-B activity may require an
extracellular stimulus in the form of a death ligand, such as TNF-
or TRAIL/Apo2L, to efficiently trigger cell death.
NF-B confers protection against apoptosis induced by chemotherapy in
several types of cells,44,57 and numerous tumor cells with
elevated levels of NF-B are resistant to apoptosis induced by
chemotherapy and radiotherapy. Conversely, adenoviral delivery of a
superrepressor form of IB sensitizes chemoresistant tumors to
TNF- and to the chemotherapeutic drug CPT-11, leading to tumor regression.59 In addition, proteasome inhibitors, such as
PS-341, inhibit NF-B by blocking the degradation of IB, thereby
increasing their sensitivity to doxorubicin.60 In this
study, we similarly detected a potent sensitizing effect of SN50 on
doxorubicin-induced apoptosis in MM cells, as well as more modest
increments in apoptosis induced by the proteasome inhibitor PS-341 and
dexamethasone. Finally, SN50 had a synergistic proapoptotic effect with
p38 inhibition, suggesting cross-talk between these 2 signaling
pathways in MM cells.
In summary, we have demonstrated that NF-B protects MM cells from
apoptosis both by maintaining mitochondrial stability via the
expression of Bcl-2 and A1 and by attenuating death-receptor-induced apoptosis via the expression of cIAP-1 and cIAP-2. These studies delineate the role of NF-B as an antiapoptotic factor in MM
cells and suggest the potential utility of combining novel agents
targeting NF-B with standard anti-MM chemotherapeutics.
| |
Footnotes |
Submitted October 22, 2001; accepted December 20, 2001.
Supported by the Multiple Myeloma Research Foundation (N.M.,
C.S.M.); the Laurie Strauss Leukemia Foundation (N.M., C.S.M.); the
Bailey Family Research Fund (N.M., C.S.M.); a National Institutes of
Health Career Development Award (S.P.T.); and the Doris Duke Distinguished Clinical Research Scientist Award (K.C.A.).
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Kenneth C. Anderson, Department of Adult
Oncology, Dana Farber Cancer Institute, 44 Binney St, Boston, MA 02115;
e-mail: kenneth_anderson{at}dfci.harvard.edu.
| |
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Top
Abstract
Introduction