| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,§
Departments of
*
Rheumatology and
Orthopedic Surgery, Hospital for Joint Diseases, New York, NY 10003; and Departments of
Pathology and
§
Medicine and Kaplan Cancer Center, New York University Medical Center, New York, NY 10016
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
PGs such as PGE1 and PGE2, which are produced at elevated levels in inflamed tissues like rheumatoid synovium (11, 12), increase local blood flow and potentiate the effects of mediators such as bradykinin that induce vasopermeability (13). PGE2, when overproduced in some cell types (by transfection of cyclooxygenase (COX)-2 cDNA), is known to exert diverse effects on cellular functions, which include activation of MMPs (14), induction and protection of apoptosis depending on the cell type (15, 16), potentiating metastatic growth of tumor (17, 18), inhibiting chondrocyte growth, triggering osteoclastic bone resorption (19), and up-regulating IL-1 transcription and cAMP levels in various cell types (20, 21).
Tetracyclines (doxycycline and minocycline) inhibited NO production and augmented PGE2 production (1- to 2-fold) in human OA-affected cartilage (in the presence or absence of cytokines and endotoxin) in ex vivo conditions and LPS-stimulated murine macrophages (RAW 264.7) in vitro conditions independent of intracellular NO concentrations (22). Thus, the effects of tetracyclines on NO and PGE2 production are independent of each other. Furthermore, tetracycline(s) and L-N-monomethyl arginine (L-NMMA) (NOS inhibitor) showed an additive effect on inhibition of NO and augmentation of PGE2 accumulation. Tetracyclines augment murine COX-2 expression by increasing the accumulation of 1) COX-2 mRNA and 2) cytosolic COX-2 protein (22). These results (unlike L-NMMA) indicated a novel mechanism of action of tetracyclines to augment the expression of COX-2 and PGE2 production independent of endogenous concentration of NO and caution the use of tetracyclines as antiinflammatory drugs in the present ongoing clinical trials for rheumatoid arthritis, OA, leprosy, and scleroderma.
CMTs have recently shown to be potent inhibitors of tissue and matrix break down and attend a longer half-life in serum than tetracyclines (23). CMTs are potent inhibitors of MMPs and iNOS (24). A major advantage of CMTs (unlike tetracyclines) is the lack of antimicrobial activity and development of antibiotic-resistant microbial flora in vivo in long term therapy. Recent studies have shown that CMT-3 and CMT-8 inhibit tumor metastasis and arthritis-affected synoviocyte invasion in animal models (23, 25).
Due to the instigative role of PGE2 in arthritis, its potential proinflammatory effects in joints, its possible effects on metalloproteases, and the "COX-2 stimulating property" of tetracyclines, we evaluated the action of CMTs on COX-2 in LPS-stimulated murine macrophages, bovine chondrocytes, and OA-affected human cartilage, which spontaneously released NO and PGE2 in ex vivo conditions (26).
In the present study, we report the following: 1) like doxycycline, minocycline, and NOS inhibitor (L-NMMA), CMT-3 augments COX-2 protein expression; 2) unlike doxycycline, minocycline and L-NMMA, CMT-3 inhibits net PGE2 production in LPS-stimulated murine macrophages, bovine chondrocytes, or human OA-affected cartilage; 3) doxycycline and minocycline have no significant effect on COX-2 enzyme activity whereas CMT-3 and -8 inhibit COX-2 enzyme activity in vitro; 4) the mechanism of action of the CMT-3 on COX-2 (which, like tetracyclines, is not at the level of COX-2 mRNA accumulation) seems to be at the level of posttranslational modification of COX-2; 5) CMT-3-induced COX-2 modification is distinct from the nonglycosylated COX-2 synthesized in the presence of tunicamycin; 6) CMT-3 does not inhibit COX-1 protein and mRNA accumulation, but significantly inhibits PGE2 production in whole cells but not in cell-free extracts; and 7) the mechanism of action of CMT-3 on COX-1 and -2 seems to be distinct.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
Murine macrophage cells (RAW 264.7) and human lung carcinoma
cells (A549) were obtained from American Type Culture Collection (ATCC,
Manassas, VA). Anti-COX-1 was obtained from Cayman Chemical Company
(Ann Arbor, MI). Anti-NOS and anti-COX-2 Abs were obtained from
Transduction Laboratories (Lexington, KY). Doxycycline, minocycline,
hydrocortisone, swainsonine, deoxymannojirimycin, deoxynojirimycin, and
LPS were obtained from Sigma (St. Louis, MO). NS398 was obtained from
Cayman Chemical Company and celecoxib is a kind gift from G. D.
Searle (Chicago, IL). The CMTs (designated as CMT-1, -2, -3, -5, and
-8) were a generous gift from CollaGenex Pharmaceutical (Newton, PA).
The molecular structures of doxycycline, minocycline, CMT-3, and CMT-8
are depicted in Fig. 1
.
|
Isolation of bovine chondrocytes was conducted by standard methods (27). Briefly, normal bovine cartilage was washed and cut into small pieces and digested with trypsin (30 min), followed by digestion with collagenase P for 12–16 h in RPMI 1640 medium. The chondrocytes were then washed and resuspended in RPMI 1640 medium before they were grown in 24-well plates at a density of 5 x 105 cells per well. The cells were stimulated after 48 h of seeding with LPS ± modulators.
Assay of OA-affected NOS and COX-2 in organ cultures
This assay was basically conducted as described previously
(22). Briefly, OA-affected cartilage was obtained from
tibial plateau and femoral condyle of OA patients undergoing knee
replacement surgery. OA-affected cartilage was cut into 3-mm discs;
four to six discs (
100–200 mg) were placed in organ cultures in 2
ml medium (Ham’s F-12 with 0.1% human albumin) for 24–72 h in the
incubator. The medium was analyzed for nitrite and
PGE2 accumulation by modified Griess reaction
(28) and RIA, respectively (29).
Cultivation of A549 cells
Human lung carcinoma epithelial-like cells were passaged in Ham’s F-12 (Life Technologies, Gaithersburg, MD) containing 10% FBS, 4 mM L-glutamine, streptomysin (50 µg/ml), and penicillin (50 U/L). The cells were adapted in low serum (0.5%) medium 24 h before stimulating with IL-1ß ± modulators.
Preparation of cell-free extracts
RAW 264.7 cells were induced with LPS (100 ng/ml) in the presence or absence of CMTs or hydrocortisone for 14–20 h. Similarly, A549 cells were incubated in the presence and absence of CMTs for 30 min and induced in the presence and absence of IL-1ß (10 ng/ml) for 24–30 h. Following induction, the cells were pelleted at 4°C and resuspended in Tris buffer (10 mM, pH 7.4) containing 10 µg/ml each of chymostatin, antipain, leupeptin, and pepstatin, 1 mM DTT, and 1 mM PMSF. Cells were lysed in a Polytron PA 1200 homogenizer (Kinematica, Switzerland) after 3 cycles of rapid freeze thawing. The lysate was centrifuged at 18,000 x g at 4°C in an Eppendorf centrifuge, and the resulting supernatant was used as cell-free extracts. The protein was measured by BCA assay reagent (Pierce, Rockford, IL) using BSA as standard (30).
Cell-free enzyme assay for COX-1 and COX-2
The cells were lysed by a Polytron homogenizer as shown above in Tris buffer with a mixture of protease inhibitors. The enzyme assay was performed as previously described (31), using total unfractionated cell extracts, and PGE2 was estimated by RIA. The specific enzyme activity was defined as PGE2 released (ng/mg protein) for 20 min at 37°C.
Western blot analysis
Equal amounts of protein (25–50 µg) were loaded onto SDS-PAGE gels and stained to verify the concentrations of various protein fractions by examining the intensities of the protein bands on the gel. The blot was probed with a specific anti-COX-2, anti-NOS, anti-COX-1, anti-actin, and anti-catalase (kindly provided by Dr. Paul Lazarow, NYU-Mt. Sinai Medical System) mAb that cross-reacts with mouse, human, and bovine COX-2, COX-1, iNOS, and catalase, respectively. The blots were developed using the ECL Western blot system (Amersham, Arlington Heights, IL). Quantitation of the bands was performed using a densitometer (Molecular Dynamics, Sunnyvale, CA).
Northern blot analysis
Total RNA was isolated using TRI Reagent (MRC, Cincinnati, OH). Northern blot analysis was conducted as described by Church and Gilbert (32). Briefly, 20 µg of RNA was subjected to electrophoresis in 1% agarose formaldehyde gel. RNA from the gel was then transferred by capillary action onto a nylon membrane (Bio-Rad Laboratories, Melville, NY). The membrane was hybridized with [32P]dCTP-labeled COX-2 and COX-1 cDNA (a kind gift from Dr. Paul Worley, Johns Hopkins University, and T. Hla, University of Connecticut School of Medicine). After hybridization, the blot was exposed to Kodak x-ray film (Kodak, Rochester, NY) for 24–48 h with intensifying screens at -70°C. Quantitation of the intensity of the COX-2 or COX-1/GAPDH bands was performed using a densitometer (Molecular Dynamics).
Statistical analysis
Data are expressed as mean ± SD, and statistical analysis was performed using GraphPad Software (V1.14). The t test or nonparametric (Mann-Whitney or Wilcoxon) test was performed for experiments as described in the figure legends.
|
Results and Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
Effect of CMTs on NO and PGE2 accumulation
We compared the effects of CMTs and minocycline on NO and
PGE2 accumulation in LPS-stimulated murine
macrophages as shown in Table I. CMT-3
(but not CMT-1, -2, -5, or -8) inhibited PGE2
accumulation. CMT-3 inhibited PGE2 accumulation
by more than 50% at 10 µg/ml (27 µM), while that of
hydrocortisone was 10 µM. It should be noted that CMT-8 marginally
inhibited nitrite accumulation but not PGE2
accumulation, whereas CMT-3 inhibited both nitrite and
PGE2 accumulation at the same concentration.
Addition of minocycline (40 µg/ml; 81 µM) augmented
PGE2 production as previously described.
CMT-1,-2, and -5 at concentrations of 10 µg/ml (27 µM) did not
show a significant effect on NO or PGE2
production. Our preliminary studies show that, in contrast to
minocycline and doxycycline (22), CMTs at 40 µg/ml were
found to be toxic to cells since 50% of the cell died within
12 h based on trypan blue exclusion staining (data not shown).
These experiments demonstrate that, in contrast to doxycycline or
minocycline, which augmented PGE2 production
(22), CMT-3 effectively inhibits
PGE2 accumulation in macrophages.
|
In view of the NO-independent COX-2/PGE2-augmenting properties of tetracyclines (doxycycline and minocycline) and NO-dependent COX-2/PGE2-augmentation properties of L-NMMA (22, 31), we next tested the effect of CMTs on PGE2 production in the presence of L-NMMA at a concentration (500 µM) that is known to augment PGE2 production.
RAW 264.7 cells pretreated with L-NMMA, when stimulated
with LPS, showed augmentation of PGE2
accumulation (Fig. 2). This
L-NMMA-mediated augmentation of PGE2
accumulation could be significantly inhibited by CMT-3 at
concentrations as low as 2.5 µg/ml. These experiments suggest that
the action of CMT-3 on PGE2 production (like
doxycycline and minocycline, which have an opposing effect) is
independent of intracellular NO production.
|
A). CMT-3 at a
concentration of 2.5 µg/ml inhibited NO and
PGE2 production by 50% as compared with
LPS-stimulated bovine chondrocytes, similar to that observed in murine
macrophages (8). Addition of L-NMMA
significantly increased PGE2 production in
LPS-stimulated cells in these experiments. Addition of CMT-3 (2.5 to 10
µg/ml) ± L-NMMA significantly inhibited
PGE2 production. Similarly, 2.5 to 10 µg/ml of
CMT-8 also inhibited NO and PGE2 production in
chondrocytes stimulated with LPS in the presence and absence of
L-NMMA in another similar experiment (Fig. 3B). These experiments suggest that CMT-3 and -8 inhibit
PGE2 production independent of intracellular NO
concentration in LPS-stimulated bovine chondrocytes. Furthermore, these
experiments also show that bovine chondrocytes are also relatively more
susceptible to CMT-8 with respect to PGE2
production, a property distinct from murine macrophages.
|
CMT-3 at 5 µg/ml did not significantly inhibit spontaneous NO
production in OA-affected cartilage. However, higher concentrations of
CMT-3 (20 µg/ml) in the presence or absence of IL-1ß inhibited NO
production by 71% (Table II). Similarly,
5 and 20 µg/ml of CMT-8 inhibited NO production in OA-affected
cartilage by 58 and 73% respectively, thus indicating that CMT-8 is
more effective in inhibiting (like bovine chondrocytes) NO production
in OA-affected cartilage. Our results support the previous observation
that the difference in the hydrophorbicity, solubility, and absorption
of CMT-3 and -8 (23) may contribute to the differential
concentration required for inhibition of NO and
PGE2 production. Sasaki et al. (35)
reported that CMT-8 has more tendency to accumulate in bone and teeth,
which might explain this observation.
|
). CMT-3 and -8, (but not CMT-1, -2, and -5)
inhibited PGE2 production significantly in this
ex vivo assay system. Minocycline, as expected, inhibited nitrite
accumulation and augmented PGE2 accumulation
(22). It should be noted that 5 µg/ml of CMT-3 (unlike
in murine macrophages) had no significant effect on NO production in
OA-affected cartilage and therefore it can be inferred that the
inhibitory effect of CMT-3 on PGE2 production in
human OA-affected cartilage in ex vivo conditions is independent of NO
concentrations. These experiments on the whole show that CMT-3 and/or -8 can effectively block PGE2 production (independent of NO) in three different cell systems, indicating a unique property of these compounds, which is distinct from doxycycline and minocycline.
Western blot analysis of COX-2 in presence of CMTs
We selected to examine the mechanism of action of CMTs (with
emphasis on CMT-3) on COX-2 expression in murine macrophages (RAW
264.7) since the regulation of iNOS and COX-2 is well studied in this
cell type (31, 36, 37). In our initial experiments, we
examined the effects of CMT-3 and -8 at various concentrations on COX-2
protein expression. RAW 264.7 cells were stimulated with LPS in the
presence and absence of CMTs for 16 h; cell-free extracts were
prepared and examined for COX-2 expression by Western blot analysis.
Fig. 4 shows that, like minocycline,
CMT-3 (but not CMT-8) augmented the accumulation of the 72-kDa COX-2
protein. CMT-3-treated cells also showed a prominent COX-2 band of
lower molecular mass (68 kDa). The expression of COX-2 in cells
treated with 2.5 µg/ml of CMT-3 was similar to that observed with 20
µg/ml of minocycline, but CMT-3-treated cells showed inhibition of
COX-2-mediated PGE2 accumulation, whereas
minocycline-treated cells showed COX-2-mediated augmentation of
PGE2 accumulation, as previously reported
(22). A dose-dependent inhibition in the accumulation of
PGE2 by increasing concentrations of CMT-3 in the
same experiment was observed although there was an increase in "the
low m.w. modified COX-2 protein" accumulation as seen by Western blot
analysis. It should be noted that there was significant inhibition of
PGE2 production by 10 µg/ml of CMT-8 in RAW
264.7 cells, but there was no COX-2 protein modification with CMT-8 as
observed in CMT-3-treated cells (Fig. 4). Hydrocortisone as expected
inhibited COX-2 expression (38). The blots were also
probed with anti-actin mAb, which showed equal intensity of the
actin band in all lanes. In summary, these results on the action of
CMT-3 on COX-2 and PGE2 production are in
contrast to those observed with minocycline and doxycycline, which
augmented COX-2 expression and PGE2 production,
as reported recently (22).
|
). These experiments suggest that CMT-3,
which augments COX-2 expression, renders the COX-2 protein inactive by
modifying it and thereby decreasing PGE2
production.
|
We tested the above hypothesis by directly examining the effect of
CMTs on COX-2 enzyme activity in vitro. RAW 264.7 cells were stimulated
with LPS (Table III), and cell-free
extracts were prepared as described in Materials and
Methods. Addition of 10 µg/ml of CMT-3 (27 µM) and CMT-8 (27
µM), like aspirin (56 µM) and indomethacin (28 µM), significantly
inhibited the sp. act. of COX-2 whereas the inactive analogue of CMTs
(CMT-5) had no significant effect. These experiments suggest that CMT-3
and CMT-8 interfere with the enzyme activity of COX-2 in cell-free
extracts, and their ability to inhibit COX-2-mediated
PGE2 production in vitro was similar to aspirin
and indomethacin. These experiments show a novel mechanism of action of
CMTs on COX-2 and separates them from tetracyclines such as doxycycline
and minocycline. The possibility of these CMTs binding to COX-2 (like
aspirin) cannot be ruled out. This may also explain the net decrease in
the PGE2 accumulation in CMT-treated cells
although there is a significant increase in the accumulation of the
modified COX-2 protein on the whole.
|
In view of the above observations, we performed experiments to
determine whether CMT-3-mediated decrease in the size of COX-2 on
SDS-PAGE gels was due to changes in glycosylation pattern of COX-2. We
compared the effects of CMTs on COX-2 expression and
PGE2 production with known glycosylation
inhibitors. Inhibition of N-linked glycosylation of COX-2 by
tunicamycin has been reported to inhibit PGE2
production (39). We examined the effect of tunicamycin
(N-linked glycosylation inhibitor) ± CMT-3 on
PGE2 production in RAW 264.7 cells stimulated
with LPS. CMT-3 (5 µg/ml), which inhibited both NO and
PGE2 production (and induced COX-2 modification),
was selected for these experiments. Tunicamycin, more than CMT-3,
inhibited NO and PGE2 production. A combination
of tunicamycin and CMT-3 inhibited PGE2
accumulation, similar to tunicamycin alone (Table IV). PGE2
production was more sensitive to tunicamycin, as compared with nitrite
accumulation.
|
shows that CMT-3, as expected,
increased the accumulation of a COX-2 protein, which exhibited a
decreased m.w. as compared with untreated control on SDS-PAGE gels.
Addition of tunicamycin to these cells in the same experiment resulted
in an appearance of 1) a 70-kDa glycosylated COX-2 and 2) a 60-kDa
nonglycosylated COX-2 with a net decrease in PGE2
production (data not shown). We also tested the effect of other
glycosylation inhibitors on COX-2 expression. Deoxynojirimycin
(
-glucosidase inhibitor), deoxymannojirimycin (mannosidase II),
swainsonine (golgi mannosidase II inhibitor), and CMT-5 (negative
control) had no significant effect on the accumulation of
PGE2 (data not shown) or the size of COX-2
protein as compared with tunicamycin-treated cells (Fig. 6). The
concentrations of deoxymannojirimycin, deoxynomiirimycin, and
swainsonine used in these studies were sufficient to inhibit
glycosylation in activated murine T cells (40, 41). These
experiments demonstrate that tunicamycin (but not other inhibitors of
glycosylation), which inhibits the total N-linked
glycosylation, blocked PGE2 production and also
significantly decreased the size of COX-2 protein. It should be noted
that, although tunicamycin and CMT-3 share the property to block
PGE2 production, the "CMT-3-modified COX-2"
was not similar to the "tunicamycin-modified COX-2" because 1)
there was an increased expression of COX-2 protein by CMT-3 and a
decreased expression of COX-2 protein by tunicamycin; 2) the sizes of
COX-2 obtained from CMT-3-treated cells and tunicamycin-treated cells
were not similar; 3) expression of COX-2 in various expression systems,
which include vaccinia virus, Drosophila 52 cells, baculovirus, and COS
cells, shows heterogeneity in N-linked glycosylation,
especially in the high mannose structures
(Man)n(GlcNAc)2n =
9,8,7,6 (42). Removal of N-glycosylation
consensus sequence by site-directed mutagenesis at
Asn580 or Asn582 resulted
in partial N-glycosylation of COX-2, which retained its
enzyme activity and was also susceptible to diclofenac comparable to
the wild-type (complete N-linked glycosylated) COX-2
(41). Our studies support those of O’Neill et al.
(39), who indicated that total inhibition of
N-linked glycosylation (with tunicamycin) is essential to
inactivate COX-2 activity (39). The appearance of multiple
COX-2 bands (at least four) resolved on a 15% SDS-PAGE (induced by
CMT-3) in bovine COX-2 indicates that the two glycosylation sites on
COX-2 (42) may not be a target of CMT-3. These results
support the hypothesis that CMT-3-induced COX-2 modification may not be
due to changes in glycosylation of COX-2 protein. However, another
posttranslational modification of COX-2 induced by CMTs, which cannot
be ruled out, is degradation of the enzyme due to unfolding of the
protein.
|
Based on our previous studies, which show up-regulation of COX-2
mRNA by tetracyclines doxycycline and minocycline (22), we
examined the effect of CMTs on COX-2 mRNA accumulation in RAW 264.7
cells stimulated with LPS for 16 h. Fig. 7 shows no significant effect (1–11%
inhibition) of CMTs on COX-2 mRNA accumulation in LPS-stimulated RAW
264.7 cells but showed a significant increase in COX-2 mRNA
accumulation by doxycycline, as compared with LPS-stimulated cells as
described previously (22). The values were normalized with
the respective GAPDH signals. Hydrocortisone, as expected, inhibited
the COX-2 mRNA accumulation. In a separate experiment, glycosylation
inhibitors had no significant effect on COX-2 mRNA accumulation (data
not shown). The results suggest that CMTs, unlike doxycycline or
minocycline, had no significant effect on COX-2 mRNA accumulation, and,
therefore, the mechanism of action of CMT-3 on COX-2 expression is
distinct from doxycycline and minocycline.
|
A human lung carcinoma cell line (A549), which under serum-starved
conditions is reported to constitutively express COX-1 and upon
stimulation with IL-1ß expresses COX-2, was selected for these
experiments (43). Serum-starved A549 cells were
preincubated in the presence and absence of CMT-3 and CMT-8 (5.0
µg/ml) for 30 min and stimulated with IL-1ß (10 ng/ml). After
24–30 h, the spontaneous and IL-1ß-induced
PGE2 production was estimated. CMT-3 and -8
significantly inhibited COX-2-mediated PGE2
production more than the spontaneous production of
PGE2 by COX-1 (Table V). To further understand the mechanism
of action of CMTs on COX-1 expression, we studied COX-1 protein
expression by Western blot analysis. Both CMT-3 and -8 had no
significant effect on COX-1 protein expression, whereas, in the
same experiment, "CMT-3 modified COX-2" protein was augmented
(Fig. 8A). These experiments
suggest that CMT-3 has differential effects on COX-1 and COX-2. CMT-3
and CMT-8 had no significant effect on COX-1 mRNA or COX-2 accumulation
(Fig. 8B).
|
|
). CMT-3-, CMT-8-,
and COX-2-specific inhibitors, such as NS398 and celecoxib, have
marginal effect (<10%) on COX-1-specific activity. However,
IL-1-induced extracts in the same experiments showed significant
inhibition (>50%) of PGE2 accumulation by
CMT-3, CMT-8, NS398, and celecoxib. These experiments suggest that
CMT-3 and -8 preferentially inhibit COX-2 enzyme activity but not COX-1
in cell-free extracts. These experiments suggest that the exact
mechanism of action of CMTs on COX-1 remains elusive but distinct from
COX-2. Furthermore, CMT-3 and -8 (like several nonsteroidal
antiinflammatory drugs (NSAIDS)) preferentially inhibit COX-2-mediated
PGE2 production as compared with COX-1. The
observations are summarized in Table VII.
|
|
10%) on the activity of sPLA2;
however, it inhibited sPLA2 activity (by 50%) at
28 µg/ml (CMT-3) and 60 µg/ml (CMT-8), respectively. Our studies
show that CMT-3 can inhibit COX-2-mediated PGE2
production by 30% at concentrations as low as 2.5 µg/ml in whole
cells (Fig. 4). Furthermore, 10 µg/ml of CMT-3 and -8 inhibited
50% COX-2 activity in vitro, similar to that observed with other
NSAIDS such as aspirin and indomethacin. Therefore, the effects of CMTs
analyzed in the study are independent of the effects on
sPLA2 and NO. In summary, this study shows novel
properties of a CMT CMT-3 (metastat) on COX-1 and COX-2 expression and
PGE2 production. CMT-3, like some NSAIDS and
steroids, inhibits both iNOS, COX-1, and COX-2 expression (8, 27), but the mechanism of action seems to be distinct (8, 41) because CMT-3 inhibits 1) iNOS at the mRNA level (8, 39) and 2) COX-2 at the protein and enzyme level and COX-1 at
whole cell level, as observed in this study. Furthermore, metastat (which also harbors anti-MMP activity) is now being used in Phase I clinical trials as an antimetastatic drug for cancer. The action of metastat on PGE2 production is independent of NO concentrations, unlike other NOS inhibitors (22, 46). This added (anti-COX activity) property of metastat may be exploited for the treatment of colon cancer, where COX-2 has been shown to be involved in angiogenesis and tumor growth (17, 47). This broad spectrum anticancer and antiinflammatory activity of CMT-3 (metastat) makes it a favorable candidate for various diseases, including cancer, arthritis, scleroderma, and leprosy, where tetracycline therapy has been recommended.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Ashok R. Amin, Department of Medicine and Rheumatology, Rheumatology Research Laboratories, Room 1600, Hospital for Joint Diseases, 301 East 17th Street, New York, NY 10003. E-mail address:
3 Abbreviations used in this paper: MMP, matrix metalloproteases; NOS, NO synthase; iNOS, inducible NOS; OA, osteoarthritis; OA-NOS, OA-affected NOS; L-NMMA, L-N-monomethyl arginine; COX, cyclooxygenase; CMT, chemically modified tetracyclines; NSAID, nonsteroidal antiinflammatory drug; RIA, Radioimmunoassay.
Received for publication December 21, 1998. Accepted for publication July 7, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
and IL-1ß in murine peritoneal macrophages. J. Immunol. 145:3333.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  R. J. Flockhart, B. L. Diffey, P. M. Farr, J. Lloyd, and N. J. Reynolds NFAT regulates induction of COX-2 and apoptosis of keratinocytes in response to ultraviolet radiation exposure FASEB J, December 1, 2008; 22(12): 4218 - 4227. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Sun, K. E. Duffy, C. T. Ranjith-Kumar, J. Xiong, R. J. Lamb, J. Santos, H. Masarapu, M. Cunningham, A. Holzenburg, R. T. Sarisky, et al. Structural and Functional Analyses of the Human Toll-like Receptor 3: ROLE OF GLYCOSYLATION J. Biol. Chem., April 21, 2006; 281(16): 11144 - 11151. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Kaliski, L. Maggiorella, K. A. Cengel, D. Mathe, V. Rouffiac, P. Opolon, N. Lassau, J. Bourhis, and E. Deutsch Angiogenesis and tumor growth inhibition by a matrix metalloproteinase inhibitor targeting radiation-induced invasion Mol. Cancer Ther., November 1, 2005; 4(11): 1717 - 1728. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. K. Shin, J. S. Park, H. S. Kim, H. J. Jun, G. E. Kim, C. O. Suh, Y. S. Yun, and H. Pyo Radiosensitivity Enhancement by Celecoxib, a Cyclooxygenase (COX)-2 Selective Inhibitor, via COX-2-Dependent Cell Cycle Regulation on Human Cancer Cells Expressing Differential COX-2 Levels Cancer Res., October 15, 2005; 65(20): 9501 - 9509. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G. Attur, M. N. Dave, M. Y. Leung, C. Cipolletta, M. Meseck, S. L. C. Woo, and A. R. Amin Functional Genomic Analysis of Type II IL-1{beta} Decoy Receptor: Potential for Gene Therapy in Human Arthritis and Inflammation J. Immunol., February 15, 2002; 168(4): 2001 - 2010. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Imamura, K. Matsushita, J. Travis, and J. Potempa Inhibition of Trypsin-Like Cysteine Proteinases (Gingipains) from Porphyromonas gingivalis by Tetracycline and Its Analogues Antimicrob. Agents Chemother., October 1, 2001; 45(10): 2871 - 2876. [Abstract] [Full Text]
|
|
|
|
 |
|
 I. I. Kuzin, J. E. Snyder, G. D. Ugine, D. Wu, S. Lee, T. Bushnell Jr, R. A. Insel, F. M. Young, and A. Bottaro Tetracyclines inhibit activated B cell function Int. Immunol., July 1, 2001; 13(7): 921 - 931. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Tikka, B. L. Fiebich, G. Goldsteins, R. Keinanen, and J. Koistinaho Minocycline, a Tetracycline Derivative, Is Neuroprotective against Excitotoxicity by Inhibiting Activation and Proliferation of Microglia J. Neurosci., April 15, 2001; 21(8): 2580 - 2588. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Chang, J. W. Horner, B. R. Conklin, R. A. DePinho, D. Bok, and D. J. Zack Tetracycline-Inducible System for Photoreceptor-Specific Gene Expression Invest. Ophthalmol. Vis. Sci., December 1, 2000; 41(13): 4281 - 4287. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
