| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Tumor Biology |
Lineberger Comprehensive Cancer Center [S. M., J. S. P.] and Departments of Medicine [J. S. P.] and Microbiology and Immunology [J. S. P.], University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295, and Department of Otolaryngology, School of Medicine [S. M., T. Y., H. T., M. F.], and Department of Molecular Virology and Oncology, Cancer Research Institute [H. S.], Kanazawa University, Ishikawa 920-8640, Japan
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B and activator protein (AP)-1 (T. Yoshizaki,
et al., Proc. Natl. Acad. Sci. USA, 95:
3621–3626, 1998), we therefore tested whether up-regulation of MMP-9
by LMP1 could be correlated with enhanced invasiveness of tumor cells
in vitro. Whether aspirin and sodium salicylate could
reduce invasiveness and whether LMP1 could enhance MMP-9 expression in
tumors grown in nude mice were also tested. C33A cells stably
expressing LMP1 had increased expression of MMP-9 and showed greater
invasion through reconstituted basement membrane compared with
vector-transfected C33A cells (P < 0.02). Treatment with aspirin or sodium salicylate inhibited
invasiveness of the LMP1-expressing C33A cells
(P < 0.03) and suppressed both the
LMP1-induced MMP-9 expression in zymographic analyses and LMP1-induced
MMP-9 promoter activity in CAT reporter assays
(P < 0.01). Endogenous MMP-2 levels were
unaffected by either drug. Both drugs repressed the CAT activity of the
truncated MMP-9 promoter construct, which only contained a binding site
for AP-1, to the basal level (P < 0.05).
Moreover, EMSA indicated that the effects of the salicylates were
through the inhibition of not only NF-B but also AP-1 binding
activity. Inhibitory effect of salicylates could be reversed by p50/p65
subunits of NF-B or c-Jun overexpression. The inhibitory effect of
aspirin on NF-B activity was attributable to the inhibition of IB
kinase activity. Finally, tumors derived from C33A cells stably
expressing LMP1 grown in nude mice showed enhanced MMP-9 levels
compared with tumors derived from vector-transfected C33A cells. This
enhancement was inhibited by treatment of the mice with aspirin. These
results suggest that aspirin may be able to suppress invasion and
metastasis of EBV-associated tumors that express LMP1 by suppression of
MMP-9. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
EBV is a ubiquitous human herpesvirus and the causative agent of infectious mononucleosis. EBV is also associated with several malignancies such as Burkitt’s lymphoma, Hodgkin’s disease (3) , non-Hodgkin’s lymphoma in immunocompromised hosts (4) , nasal T/natural killer cell lymphoma (5) , and NPC (6 , 7) . In all of these tumors, EBV infection is predominantly latent. EBV latent infection is divided into three types by the pattern of expression of six EBNAs (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNA-LP) and three LMPs (LMP1, LMP2A, and LMP2B). Only EBNA1 is expressed in Burkitt’s lymphoma (type I latency). On the other hand, all six EBNAs and three LMPs are expressed in non-Hodgkin’s lymphoma in immunocompromised hosts (type III latency). LMP1, LMP2A, and LMP2B are expressed in NPC, nasal T/natural killer cell lymphoma and Hodgkin’s disease (type II latency) in addition to EBNA1. Thus, expression of LMP1 is common to type II and type III latency. LMP1 is considered an oncoprotein because it can transform rodent fibroblasts (8) . In addition to its oncogenic properties, LMP1 was shown recently to enhance expression of MMP-9 (9) , which might contribute to the invasion and metastasis of EBV-associated tumors especially in type II and type III latency.
Enhanced expression of MMP-9 by LMP1 is through the activation of
NF-B and AP-1 (9)
. There are two regions that activate
NF-B in the COOH-terminal region of LMP1 (10)
, and both
contribute to the enhanced expression of MMP-9 (11)
.
NF-B remains sequestered in the cytoplasm under unstimulated
conditions by tightly bound inhibitory proteins, the IBs
(12)
. Many of the signals known to activate NF-B result
in phosphorylation of IB
on serine residues 32 and 36 and
subsequent degradation of IB, allowing NF-B to translocate
into the nucleus and activate target genes (13)
.
Acetylsalicylic acid (aspirin) and sodium salicylate have been shown to
suppress the NF-B activation induced by TNF-, lipopolysaccharide,
and interleukin 1 in certain systems (14)
. A recent report
demonstrated that aspirin and sodium salicylate inhibited the activity
of IB kinase ß, which is responsible for phosphorylation of
IB (15)
. Additionally, aspirin was shown recently to
inhibit JNK in several systems (16)
, which resulted in the
inactivation of AP-1. Here we demonstrate that LMP1 increases
invasiveness of tumor cells in vitro and that both aspirin
and sodium salicylate inhibit this invasiveness by suppression of the
induced constitutive MMP-9 expression. In addition, inhibition of
NF-B and AP-1 binding to the MMP-9 promoter is involved in the
suppression of MMP-9 by both drugs. Furthermore, LMP1 induces
expression of MMP-9 in tumors grown in nude mice, which is suppressed
by aspirin. These results indicate that both aspirin and sodium
salicylate may contribute to reduce invasion and metastasis in
EBV-associated tumors.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Plasmids.
LMP1 expression plasmid was described previously (9)
. The
IB(SS32/36AA) expression plasmid, the p50 subunit of NF-B
(referred to as p50) expression plasmid, and the p65 subunit of NF-B
(referred to as p65) expression plasmid were kindly provided by Dr.
Albert Baldwin (University of North Carolina, Chapel Hill, NC).
In the IB(SS32/36AA) expression plasmid, both serines at position
32 and 36 were substituted by alanines, resulting in the prevention of
degradation of IB and subsequent activation of NF-B. A series
of 5' flanking sequences of MMP-9 were inserted upstream of the
CAT reporter gene as described (17)
. A
c-Jun expression plasmid was described previously (18)
.
Transient and Stable Transfection.
C33A cells were transfected with 5 µg of appropriate reporter and/or
effector plasmids using LipofectAMINE reagent (Life Technologies, Inc.,
Grand Island, NY), following the manufacturer’s instructions. To
establish either the vector-transfected C33A cells (referred to as
C33A/pcDNA3 cells) or the C33A cells stably expressing LMP1 (referred
to as C33A/LMP1 cells), 0.5 µg of pcDNA3 (Invitrogen, Carlsbad, CA)
or LMP1 expression plasmid, respectively, was transfected into C33A
cells and cultivated in the presence of geneticin (700 µg/ml
Geneticin; Life Technologies).
Reagents.
Aspirin, sodium salicylate, and MTT were purchased from Sigma Chemical
Co. (St. Louis, MO).
MTT Assay.
MTT assays were performed to evaluate the cytotoxicity of aspirin and
sodium salicylate. One hundred µl of each cell type (1 x 105 cells/ml) were plated and incubated
for 24 h in wells of a 96-well plate. Then various concentrations
of each drug were added to the wells. After another 24 h
incubation, 10 µl of sterile MTT dye were added, and the cells were
incubated for 6 h at 37°C. Then 100 µl of acidic isopropanol
(0.04 M HCl in isopropanol) were added and thoroughly
mixed. Spectrometric absorbance at 595 nm (for formazan dye) was
measured with the absorbance at 655 nm for reference.
Conditioned Media.
Eight x 105 cells were plated and
cultured for 48 h in DMEM with 10% FBS. Then cells were incubated
for 24 h in DMEM with neither FBS nor antibiotics with or without
each drug. Media were harvested and used for gelatin zymography.
Gelatin Zymography.
MMP-2 and MMP-9 enzymatic activity were assayed by gelatinolysis by
means of gelatin zymography as reported previously (9
, 19)
. For mouse tissues, equal amounts of each tissue were
homogenized in SDS sample buffer without reducing agent. The mixture
was then incubated at 37°C for 30 min, and gelatin zymography was
performed. MMP-2 is detected as the clear band appearing at
Mr 72,000 and MMP-9 at
Mr 92,000.
Western Blot Analysis.
C33A/pcDNA3 cells and C33A/LMP1 cells (8 x 105 cells) were plated and cultured for 96 h
in DMEM with 10% FBS with or without either drug for the last 24 h. Protein extracts were obtained, and the concentration was calculated
as described previously (20)
, then mixed with 2x SDS
sample buffer containing reducing agent. For tumors grown in nude mice,
equal amounts of each tumor were homogenized in RIPA buffer [25
mM Tris-Cl (pH 7.5), 150 mM NaCl, 2
mM EDTA, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS,
0.2 mM sodium orthovanadate, 100 mM NaF, 10
µg/ml aprotinin, 5 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride]; then extracts were obtained as
reported (21)
, and the concentration of protein was
calculated. After SDS/PAGE, immunoblotting was performed as described
previously (9)
. Antimouse immunoglobulin chain
antibody (1:2000 dilution; Southern Biotechnology Associates,
Birmingham, AL) was used for the secondary antibody in analyses of
tumors grown in nude mice.
CAT Reporter Assay.
CAT assays were performed as described previously (9)
.
Construction of the MMP-9 promoter reporter has been described (Ref.
17
; Fig. 1C
). Cells were incubated 48 h after transfection with or
without adding either drug 24 h after transfection and then
harvested. Acetylated [14C]chloramphenicol was
quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Transfection efficiency was monitored by cotransfection with
ß-galactosidase reporter construct.
|
B or AP-1 binding sequences
in the promoter region of MMP-9 (9)
. Oligonucleotides were
annealed with the use of Klenow DNA polymerase and then labeled with
[
-32P]ATP with the use of T4 polynucleotide
kinase. The unlabeled oligonucleotides were used for competition. Five
µg of nuclear extracts were incubated with 50,000 cpm labeled probe
and analyzed by 4.8% PAGE; then autoradiography was performed as
reported (9)
.
IB Kinase Assay.
Assays were performed after transient transfection as described
previously (23)
. Briefly, lysates were obtained from C33A
cells transfected with pcDNA3 or with the LMP1 expression construct
with or without treatment with 5 mM aspirin; then
supernatant fluids were collected, and protein concentration was
calculated. Five hundred µg of protein were incubated with 15 µg of
IKK2 CT antibody (kindly provided by Drs. Albert Baldwin and Minnie
Holmes-McNary [University of North Carolina, Chapel Hill,
NC]). Incubation was continued with protein A beads (50 µl).
The immunoprecipitates were then vigorously washed and subjected to an
in vitro kinase assay. Kinase assays were done with 2 µCi
of [-32P]ATP in the presence of 2 µg of
GST-IB or GST-IB(SS32/36TT) (kindly provided by Drs. Albert
Baldwin and Minnie Holmes-McNary) for substrates, followed by SDS-PAGE
analysis and autoradiography.
In Vitro Invasion Assay.
Assays were performed with the use of Biocoat Matrigel Invasion
Chambers (Becton Dickinson Labware, Bedford, MA) as described
elsewhere. Biocoat Cell Culture Inserts were used for uncoated assays.
We established two indices to evaluate in vitro
invasiveness: percent invasion (% invasion) and invasion index. Both
indices were calculated as follows: % invasion = number
of cells invading through Matrigel-coated membrane/number of cells
migrating through uncoated control insert membrane. Invasion
index = % invasion of tested cells/% invasion of
C33A/pcDNA3 cells.
Injection of Cells into Athymic Mice.
Either C33A/pcDNA3 cells or C33A/LMP1 cells (2 x 106 cells) in a volume of 0.1 ml PBS were s.c.
injected into athymic nude mice. Four weeks after injection, PBS alone
or 9 mg/day aspirin were given s.c. at a distance from the tumor for 3
days, and then mice were sacrificed. Equal amounts of each tumor were
homogenized in SDS sample buffer without reducing agent or RIPA buffer
for gelatin zymography or Western blot analysis, respectively.
Statistical Analysis.
Significant differences were determined by the paired t
test. P < 0.05 was considered to be
statistically significant.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), suggesting that LMP1 is a possible
invasiveness-inducing factor.
NF-B and AP-1 Are Involved in Expression of MMP-9 Induced by
LMP1.
We showed previously that transient transfection of LMP1 into C33A
cells enhanced expression of MMP-9 (9)
. To investigate the
contribution of this type IV collagenase to the LMP1-associated
invasion in vitro, we first carried out gelatin zymography
to determine MMP-9 expression. The zymographic analysis showed that
C33A/LMP1 cells constitutively enhanced the expression of MMP-9
compared with C33A/pcDNA3 cells (Fig. 1B)
. As before
(9)
, MMP-2 gelatinolytic activity was not affected by LMP1
expression. This result suggests that MMP-9 associated with LMP1
expression may be involved in cell invasiveness in vitro.
Analysis of the effect of LMP1 expression on MMP-9 promoter constructs
(Fig. 1C)
was then carried out. MMP-9 promoter activity was
increased in C33A/LMP1 cells, as shown by CAT reporter assays with the
-670 CAT construct (P < 0.01; Fig. 1D
, Lanes 1 and 2). C33A/LMP1 cells also
increased activity of the -90 CAT construct, which only contains the
AP-1 binding site and TATA box, compared with the -73 CAT construct in
which the AP-1 binding site was deleted (P < 0.01; Fig. 1D
, Lanes 3–6). These results are
compatible with previous reports that LMP1 activates JNK
(24)
because AP-1 is one of the downstream pathways of
JNK. EMSA showed increases in nuclear factor binding not only to
NF-B but also to AP-1 binding sequence in the MMP-9 promoter in
C33A/LMP1 cells (Fig. 1E)
.
Influence of Aspirin on Cell Viability and LMP1 Expression.
Aspirin and sodium salicylate are cytotoxic in high concentrations;
therefore, we investigated the effect of both drugs on cell viability
by MTT assay. The effects of 1, 2.5, 5, and 10 mM aspirin
or sodium salicylate were tested. Absorbance was not reduced, nor was
there any visible cytotoxic effect by any of the concentrations tested
except with 10 mM aspirin (data not shown). Therefore, 5
mM aspirin and 10 mM sodium salicylate were
used in the experiments. Neither drug affected the expression of LMP1
(Fig. 2
, Lanes 3–5).
|
, Lanes 5 and 6). Neither drug affected
invasiveness of the C33A/pcDNA3 cells (Fig. 3
, Lanes 2 and
3). These results suggest that both drugs can reduce
LMP1-related invasiveness.
|
B and AP-1.
, Lanes 3 and 4). MMP-2 gelatinolytic activity was
affected by neither aspirin nor sodium salicylate. Direct exposure of
collected conditioned medium of C33A/LMP1 cells to aspirin or sodium
salicylate had no effect on MMP-9 and MMP-2 gelatinolytic activity
(Fig. 4
, Lanes 5 and 6), indicating that
inhibition of LMP1-induced MMP-9 was not attributable to a direct
effect of the drugs on secreted MMP-9 itself.
|
, Lanes 3 and 4). Cotransfection with
IB(SS32/36AA) also sharply repressed CAT activity
(P < 0.01; Fig. 5A
, Lane
5). The residual activity might be attributable to the
contribution of AP-1, because at least the AP-1 binding site proximal
to the start site is essential for activation of MMP-9 promoter by LMP1
(9)
. Therefore, we investigated the effect of the drugs on
AP-1 activity with the use of the -90 CAT and -73 CAT constructs. The
activity of the -90 CAT construct was repressed to the basal level by
both aspirin (P < 0.05) and sodium
salicylate (P < 0.05; Fig. 5B
, Lanes
4 and 5), suggesting that LMP1-induced AP-1 activity
was significantly repressed by both drugs. However, residual activity
compared with the -73 CAT construct was still observed. Because
C33A/pcDNA3 cells had more activity with the -90 CAT than with the
-73 CAT construct (Fig. 5B
, Lanes 1 and 6), this
residual activity seems to be independent of LMP1. Finally, binding
activity of nuclear factors not only to NF-B but also to the AP-1
binding sequence in the MMP-9 promoter was reduced by aspirin and
sodium salicylate (Fig. 6A
, Lanes 3, 5, and 7; Fig. 6B
, Lanes 4, 6, and 8). Therefore,
inhibition of LMP1-induced MMP-9 by aspirin and sodium salicylate is
probably through the inactivation of both NF-B and AP-1.
|
|
B p65/p50 and c-Jun Overcomes the
Inhibitory Effect of Aspirin on LMP1-induced MMP-9 Expression.B inhibition by
IB(SS32/36AA), confirming that it is NF-B that is blocked by
IB(SS32/36AA) (Ref. 25
). Therefore, transient
transfection assays were performed to investigate whether the
inhibitory effect of aspirin on LMP1-induced MMP-9 expression could be
reversed by overexpression of NF-B or c-Jun. Zymographic analysis
showed that overexpression of p50 and p65 restored MMP-9 expression
inhibited by aspirin (Fig. 7A
, Lane 4). Overexpression of c-Jun also restored
MMP-9 expression (Fig. 7A
, Lane 5). CAT reporter
assays with the -670 CAT construct also showed that overexpression of
p50 and p65 restored MMP-9 promoter activity inhibited by aspirin (Fig. 7B
, Lane 4). Overexpression of c-Jun also
restored MMP-9 promoter activity (Fig. 7B
, Lane
5). These results suggest that both NF-B and c-Jun are involved
in the inhibitory effect of aspirin on LMP1-induced MMP-9 expression.
|
B on serine residues
32 and 36, which is a critical step in NF-B activation. We tested
whether LMP1 activates NF-B through IKK, then tested whether aspirin
had an effect on phosphorylation of IB by IKK. In
vitro kinase assays showed phosphorylation of IB in
LMP1-transfected cells in contrast to vector-transfected cells (Fig. 8
, Lanes 2 and 3), suggesting that IKK is involved
in LMP1-induced NF-B activation. No phosphorylation of IB by
LMP1 was detected when GST-IB(SS32/36TT) was used as substrate
(Fig. 8
, Lane 7), indicating specificity of this assay.
Furthermore, treatment with aspirin abolished phosphorylation of
IB (Fig. 8
, Lane 4). This result suggests that aspirin
inhibits LMP1-induced NF-B activation through inhibition of IKK
activity.
|
. Gelatin zymography showed that there was enhanced
expression of MMP-9 in C33A/LMP1-derived tumors compared with
C33A/pcDNA3-derived tumor (Fig. 9B
, Lanes 1 and
2), indicating that LMP1 induces MMP-9 expression in
vivo. Next the effect of aspirin on expression of MMP-9 in
vivo was investigated. Treatment with aspirin dramatically
decreased expression of MMP-9 in C33A/LMP1-derived tumor (Fig. 9B
, Lane 3) in contrast to expression of LMP1,
which was not affected (Fig. 9A
, Lane 3). Expression of
MMP-2 was not affected by aspirin. These results suggest that aspirin
may be an inhibitor of LMP1-induced MMP-9 expression in
vivo.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The COOH-terminal region of LMP1 contains two functional domains named
CTAR1 and CTAR2. CTAR1 is needed for binding the TRAF-1, TRAF-2, and
TRAF-3 proteins (30
, 31)
, and CTAR2 has been shown to bind
the TNFR-associated death domain protein (32)
, suggesting
that LMP1 might act as a TNFR family-like molecule. Indeed, downstream
signaling of LMP1 is similar to that of CD40, which is one of the TNFR
family members (33)
. Although LMP1 is not homologous to
any of the known TNFR family members and its activity does not appear
to depend on the binding of a specific ligand, LMP1 activates NF-B
through a pathway including IKK, as shown here and previously
(34)
. Both CTAR1 and CTAR2 contribute to NF-B
activation. In addition, LMP1 also activates the JNK pathway through
CTAR2, which results in the activation of AP-1 (24)
.
Aspirin is used as a nonsteroidal anti-inflammatory drug. The range of
concentrations for the drug is correlated with the amounts in plasma
(1–5 mM) for optimal anti-inflammatory effects in patients
with rheumatoid arthritis. Moreover, both epidemiological and clinical
studies have indicated that aspirin and related compounds have
considerable potential as chemopreventive agents for cancers. It has
been reported that patients with regular aspirin use had a reduced
incidence of colon, lung, and breast cancers (35)
. Aspirin
suppresses NF-B activity or JNK activity induced by certain stimuli
such as TNF-, interleukin 1, and UVB in certain systems (14
, 16
, 36)
. It should be emphasized with respect to activation of
NF-B and JNK that LMP1 is considered to be a constitutively
activated TNFR-like molecule (37)
. Here we show that
aspirin inhibited constitutively active NF-B and AP-1 and that
inhibition could be reversed by p50/p65 or c-Jun overexpression.
Inhibition of LMP1-induced NF-B activation through inhibition of IKK
is direct evidence of the inhibitory effect of aspirin on
constitutively active NF-B. Suppression of LMP1-inducible signaling
pathways may be a potential therapeutic tool for the type II and type
III EBV-associated diseases.
MMP-9 has been shown to have a direct role in tumor metastasis in
certain systems (38)
. Here we demonstrate that
LMP1-induced in vitro invasiveness is correlated with
induction of MMP-9. The possibility that suppression of MMP-9 could
prevent invasion and metastasis has been explored with the use of
anti-MMP-9 ribozyme (39)
or ursolic acid
(40)
. Here we show that aspirin can be a possible
candidate for repression of MMP-9 and inhibition of invasiveness.
Aspirin completely suppressed MMP-9 promoter activity through its
bifunctional effect on NF-B and AP-1. We showed previously that
MMP-9 promoter activity induced by LMP1 was abolished when either the
NF-B site or the proximal AP-1 site was mutated (9)
. On
the basis of this observation, inhibition of either NF-B or AP-1
binding by aspirin might be causal for MMP-9 down-regulation. However,
the data in Fig. 5
suggest that the proximal AP-1 site may be
responsible for mediating down-regulation, although the NF-B site is
essential for full induction, even in the presence of the proximal AP-1
site. A preferential inhibitory effect of aspirin on binding of the
upper NF-B band on EMSA, the one known to contain p65, may be
beneficial because p65 has more transactivation capacity than p50
(41)
. Overexpression of either p50/p65 or c-Jun restored
MMP-9 expression and promoter activity, suggesting that either one of
them may be operative. This possibility may be explained by the
previous reports showing the cross-coupling of NF-B p65 and c-Jun
(41
, 42) . Those reports showed that p65 activates AP-1 and
c-Jun activates NF-B (41)
, and that NF-B inhibitor
blocks AP-1 and AP-1 inhibitor blocks NF-B (42)
.
Accordingly, we showed previously that LMP1 CTAR1 induces AP-1 binding
activity (11)
.
Thus, both aspirin and sodium salicylate significantly suppressed LMP1-induced in vitro invasion. Furthermore, aspirin inhibited LMP1-induced MMP-9 expression in tumors grown in nude mice. The present study is the first report showing that aspirin and sodium salicylate suppress not only the expression of MMP-9 in cell culture but also in vitro invasiveness. It is also the first to show induction of MMP-9 by LMP1 in tumors grown in nude mice and inhibition of that induction by treatment of the tumor-bearing mice with aspirin. Thus, hypothetically, aspirin or an aspirin-like effect may have potential to prevent invasion and metastasis, in addition to its anti-inflammatory and chemopreventive properties. Finally, this study is the first to show suppression of downstream signals of LMP1 by aspirin and sodium salicylate. Aspirin significantly, but not completely, inhibited LMP1-induced tumor cell invasion in vitro. Unfortunately, tumors caused by s.c. injection into nude mice are generally noninvasive because of the ectopic site. However, in vitro invasiveness predicts in vivo metastasis in certain systems (43) . Therefore, we are now establishing an LMP1-induced tumor invasion and metastasis model in nude mice so as to evaluate the effect of aspirin on invasion and metastasis in vivo.
|
ACKNOWLEDGMENTS |
|---|
B(SS32/36AA) and p50 or p65
subunit of NF-B expression plasmids and reviewing the manuscript,
Minnie Holmes-McNary for helping with the IKK assay, and Nancy
Raab-Traub and Shannon Kenney for reviewing the manuscript. |
FOOTNOTES |
|---|
1 This work was supported by Grant P01CA19014 from
the NIH. S. M. is supported in part by the Sumitomo Life
Enterprise Group for Social Welfare, Osaka, Japan. 
2 To whom requests for reprints should be
addressed, at Lineberger Comprehensive Cancer Center, University of
North Carolina, Chapel Hill, NC 27599-7295. Phone: (919) 966-8644;
Fax; (919) 966-9673; E-mail; Joseph_Pagano@med.unc.edu.
3 The abbreviations used are: MMP, matrix
metalloproteinase; NPC, nasopharyngeal carcinoma; EBNA, EBV nuclear
antigen; LMP, latent membrane protein; AP-1, activator protein-1; TNF,
tumor necrosis factor; TNFR, TNF receptor; JNK, c-Jun
NH2-terminal kinase; FBS, fetal bovine serum; CAT,
chloramphenicol acetyltransferase; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-B,
nuclear factor-B; GST, glutathione S-transferase;
CTAR, COOH-terminal activation region; EMSA, electrophoretic mobility
shift assay; IKK, IB kinase; TRAF, TNFR-associated factor.
Received 11/ 1/99. Accepted 3/ 6/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B and cell surface phenotype via two effector regions in its carboxy-terminal cytoplasmic domain. Oncogene, 10: 549-560, 1995.[Medline]
B activation: the IB kinase revealed?. Cell, 91: 299-302, 1997.[Medline]
B kinase: beginning, not the end. Proc. Natl. Acad. Sci. USA, 94: 11758-11760, 1997.B by sodium salicylate and aspirin. Science (Washington DC), 265: 956-959, 1994.B kinase-ß. Nature (Lond.), 396: 77-80, 1998.[Medline]
B during infection. J. Virol., 71: 5051-5059, 1997.[Abstract]
B kinases essential for NF-B activation. Science (Washington DC), 278: 860-866, 1997.B. Science (Washington DC), 274: 784-787, 1996.B activation. Mol. Cell. Biol., 16: 7098-7108, 1996.[Abstract]
B. Proc. Natl. Acad. Sci. USA, 94: 12592-12597, 1997.B through a pathway that includes the NF-B-inducing kinase and the IB kinases IKK and IKKß. Proc. Natl. Acad. Sci. USA, 95: 10106-10111, 1998.B p65 and Fos/Jun transcription factors produces potentiated biological function. EMBO J., 12: 3879-3891, 1993.[Medline]
B and activator protein-1 activation block the neoplastic transformation response. Cancer Res., 57: 3569-3576, 1997.This article has been cited by other articles:
|
|
 |
|
  J.-A. Liang, S.-L. Wu, H.-Y. Lo, C.-Y. Hsiang, and T.-Y. Ho Vanillin Inhibits Matrix Metalloproteinase-9 Expression through Down-Regulation of Nuclear Factor-{kappa}B Signaling Pathway in Human Hepatocellular Carcinoma Cells Mol. Pharmacol., January 1, 2009; 75(1): 151 - 157. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Horikawa, J. Yang, S. Kondo, T. Yoshizaki, I. Joab, M. Furukawa, and J. S. Pagano Twist and Epithelial-Mesenchymal Transition Are Induced by the EBV Oncoprotein Latent Membrane Protein 1 and Are Associated with Metastatic Nasopharyngeal Carcinoma Cancer Res., March 1, 2007; 67(5): 1970 - 1978. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kondo, T. Yoshizaki, N. Wakisaka, T. Horikawa, S. Murono, K. L. Jang, I. Joab, M. Furukawa, and J. S. Pagano MUC1 Induced by Epstein-Barr Virus Latent Membrane Protein 1 Causes Dissociation of the Cell-Matrix Interaction and Cellular Invasiveness via STAT Signaling J. Virol., February 15, 2007; 81(4): 1554 - 1562. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. McCarty and K. I. Block Preadministration of High-Dose Salicylates, Suppressors of NF-{kappa}B Activation, May Increase the Chemosensitivity of Many Cancers: An Example of Proapoptotic Signal Modulation Therapy Integr Cancer Ther, September 1, 2006; 5(3): 252 - 268. [Abstract] [PDF]
|
|
|
|
 |
|
 K. Pal, A. K. Banthia, and D. K. Majumdar Polyvinyl Alcohol--Gelatin Patches of Salicylic Acid: Preparation, Characterization and Drug Release Studies J Biomater Appl, July 1, 2006; 21(1): 75 - 91. [Abstract] [PDF]
|
|
|
|
 |
|
 R. Furst, S. B. Blumenthal, A. K. Kiemer, S. Zahler, and A. M. Vollmar Nuclear Factor-{kappa}B-Independent Anti-Inflammatory Action of Salicylate in Human Endothelial Cells: Induction of Heme Oxygenase-1 by the c-Jun N-Terminal Kinase/Activator Protein-1 Pathway J. Pharmacol. Exp. Ther., July 1, 2006; 318(1): 389 - 394. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. F. McCarty and K. I. Block Toward a Core Nutraceutical Program for Cancer Management Integr Cancer Ther, June 1, 2006; 5(2): 150 - 171. [Abstract] [PDF]
|
|
|
|
 |
|
 W. H. Sun, Y. L. Sun, R. N. Fang, Y. Shao, H. C. Xu, Q. P. Xue, G. X. Ding, and Y. L. Cheng Expression of Cyclooxygenase-2 and Matrix Metalloproteinase-9 in Gastric Carcinoma and its Correlation with Angiogenesis Jpn. J. Clin. Oncol., December 1, 2005; 35(12): 707 - 713. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Kuppers, K. Lan, J. S. Knight, and E. S. Robertson Regulation of Matrix Metalloproteinase 9 Expression by Epstein-Barr Virus Nuclear Antigen 3C and the Suppressor of Metastasis Nm23-H1 J. Virol., August 1, 2005; 79(15): 9714 - 9724. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. S. Apple, A. H.B. Wu, J. Mair, J. Ravkilde, M. Panteghini, J. Tate, F. Pagani, R. H. Christenson, M. Mockel, O. Danne, et al. Future Biomarkers for Detection of Ischemia and Risk Stratification in Acute Coronary Syndrome Clin. Chem., May 1, 2005; 51(5): 810 - 824. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Mayoral, A. Fernandez-Martinez, L. Bosca, and P. Martin-Sanz Prostaglandin E2 promotes migration and adhesion in hepatocellular carcinoma cells Carcinogenesis, April 1, 2005; 26(4): 753 - 761. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Xu, S. Zhao, H. Zhou, H. Ye, and J. Li Atorvastatin Lowers Plasma Matrix Metalloproteinase-9 in Patients with Acute Coronary Syndrome Clin. Chem., April 1, 2004; 50(4): 750 - 753. [Full Text] [PDF]
|
|
|
|
 |
|
 S. Okada, Q. Li, J. C. Whitin, C. Clayberger, and A. M. Krensky Intracellular Mediators of Granulysin-Induced Cell Death J. Immunol., September 1, 2003; 171(5): 2556 - 2562. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Lu, H.-H. Chua, S.-Y. Chen, J.-Y. Chen, and C.-H. Tsai Regulation of Matrix Metalloproteinase-1 by Epstein-Barr Virus Proteins Cancer Res., January 1, 2003; 63(1): 256 - 262. [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]
|
|
|
|
|
|
S.-Y. Chen, J. Lu, Y.-C. Shih, and C.-H. Tsai Epstein-Barr Virus Latent Membrane Protein 2A Regulates c-Jun Protein through Extracellular Signal-Regulated Kinase J. Virol., August 12, 2002; 76(18): 9556 - 9561. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Murono, N. Raab-Traub, and J. S. Pagano Prevention and Inhibition of Nasopharyngeal Carcinoma Growth by Antiviral Phosphonated Nucleoside Analogs Cancer Res., November 1, 2001; 61(21): 7875 - 7877. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. TEGEDER, J. PFEILSCHIFTER, and G. GEISSLINGER Cyclooxygenase-independent actions of cyclooxygenase inhibitors FASEB J, October 1, 2001; 15(12): 2057 - 2072. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Yoshizaki, T. Horikawa, R. Qing-chun, N. Wakisaka, H. Takeshita, T.-S. Sheen, S.-Y. Lee, H. Sato, and M. Furukawa Induction of Interleukin-8 by Epstein-Barr Virus Latent Membrane Protein-1 and Its Correlation to Angiogenesis in Nasopharyngeal Carcinoma Clin. Cancer Res., July 1, 2001; 7(7): 1946 - 1951. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Murono, H. Inoue, T. Tanabe, I. Joab, T. Yoshizaki, M. Furukawa, and J. S. Pagano Induction of cyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells PNAS, May 24, 2001; (2001) 121016998. [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]
|
|
|
|
|
|
S. Murono, H. Inoue, T. Tanabe, I. Joab, T. Yoshizaki, M. Furukawa, and J. S. Pagano Induction of cyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells PNAS, June 5, 2001; 98(12): 6905 - 6910. [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 |
) and
C33A cells stably expressing LMP1 (
). The relative CAT activities
from four experiments are shown; bars, SD.
E, EMSA. Nuclear extracts from C33A cells stably
transfected with pcDNA3 (pcDNA3) or C33A cells stably
expressing LMP1 (LMP1) were mixed with either NF-
