Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on May 3, 2005; DOI: 10.1124/jpet.105.086116
0022-3565/05/3142-796-803$20.00
JPET 314:796-803, 2005
INFLAMMATION AND IMMUNOPHARMACOLOGY
A Novel Celecoxib Derivative Potently Induces Apoptosis of Human Synovial Fibroblasts
Natsuko Kusunoki,
Takumi Ito,
Nobuyuki Sakurai,
Toru Suguro,
Hiroshi Handa, and
Shinichi Kawai
Division of Rheumatology, Department of Internal Medicine, Toho University Omori Medical Center, Tokyo, Japan (N.K., S.K.); Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan (T.I., N.S., H.H.); and Department of Orthopaedic Surgery, Toho University Omori Medical Center, Tokyo, Japan (T.S.)
Received for publication
March 9, 2005
Accepted
April 29, 2005.
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Abstract
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We have already demonstrated that celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, has a proapoptotic effect on synovial fibroblasts obtained from patients with rheumatoid arthritis (RA). Here we report on the development of two novel derivatives of celecoxib, N-(2-aminoethyl)-4-[5-(4-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (TT101) and 4-[5-(4-aminophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (TT201), including whether these compounds have a proapoptotic effect on synovial fibroblasts. Synovial fibroblasts were harvested from the synovial tissues of patients with RA or osteoarthritis (OA). Cell proliferation and cell viability were assessed by the incorporation of 5-bromo-2'-deoxyuridine and by the 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay, respectively. Apoptosis was detected by the identification of DNA fragmentation, and activation of caspase-3 was detected by the addition of a caspase-3 substrate to cell lysates. Production of prostaglandin E2 by RA synovial fibroblasts was analyzed by enzyme-linked immunosorbent assay. TT101 inhibited the proliferation of RA and OA synovial fibroblasts in a concentration-dependent manner. It caused a marked decrease of cell viability and induced DNA fragmentation more potently than either celecoxib or SC-236 (4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide). TT101 also increased caspase-3 activity. The order of potency of the COX-2 inhibitory activity of these drugs in RA synovial fibroblasts was celecoxib = SC-236 > rofecoxib > TT201 > TT101. In conclusion, we developed TT101 with about a 5- to 10-fold stronger proapoptotic effect on RA and OA synovial fibroblasts compared with that of celecoxib. Although the mechanism of action of TT101 remains unclear, it may have potential as a novel antirheumatic agent.
Celecoxib is one of the nonsteroidal anti-inflammatory drugs (NSAIDs) that selectively inhibit cyclooxygenase (COX)-2. It is widely used for treatment of rheumatoid arthritis (RA) with the expectation that an anti-inflammatory effect will result from inhibiting the production of prostanoids, like prostaglandin (PG) E2, through the suppression of COX-2 activity. On the other hand, we previously demonstrated that celecoxib induces the apoptosis of synovial fibroblasts obtained from patients with RA (Kusunoki et al., 2002
).
Apoptosis is considered to be one of the mechanisms regulating autoimmune diseases such as RA (Thompson, 1995). In the pathogenesis of RA, it is thought that the normal balance between proliferation and apoptosis of synovial fibroblasts is lost, leading to hyperplasia of these fibroblasts (Perlman et al., 2001). Activated synovial cells cause growth of synovium in the articular cavity along with angiogenesis, invade the adjacent bone, promote production of inflammatory cytokines by inflammatory cells, and cause cartilage and bone destruction (Ospelt et al., 2004). Therefore, it has been shown that stimulation of the apoptosis of synovial fibroblasts might be useful for the treatment of RA (Nishioka et al., 1998; Baier et al., 2003).
Recently, celecoxib was reported to cause a significant reduction in the number of colorectal polyps in patients with familial adenomatous polyposis (Steinbach et al., 2000), and it has attracted attention as an antiproliferative agent in animal and cell culture studies. We have previously shown that celecoxib induces the apoptosis of colorectal carcinoma cells (Yamazaki et al., 2002). In addition, several studies conducted by other investigators have demonstrated that celecoxib suppresses the proliferation of various cells by inducing apoptosis (Myers et al., 2001; reviewed by Arun and Goss, 2004; Kismet et al., 2004; Sandler and Dubinett, 2004; Sinicrope and Gill, 2004; Xiong, 2004), suggesting that the proapoptotic action of celecoxib may be useful for the chemoprevention of tumorigenesis (Kismet et al., 2004; Sinicrope et al., 2004). These effects may represent an action that is unique to the drug celecoxib rather than being a class effect of COX-2 inhibitors.
In our in vitro study (Kusunoki et al., 2002; Yamazaki et al., 2002), the concentration of celecoxib required to induce apoptosis of synovial fibroblasts obtained from patients with RA and colon adenocarcinoma cells was slightly higher than the blood level of the drug achieved in healthy individuals (McAdam et al., 1999). Therefore, for adequately proapoptotic activity to achieve an antirheumatic effect in clinical use, celecoxib may need to be used administered at higher dose levels than those used clinically. Accordingly, we have attempted to develop a drug with more potent apoptosis-inducing activity for this purpose.
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Materials and Methods
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Materials. We synthesized TT101 and TT201. The purity of TT101 was >99%, as assessed by high-performance liquid chromatography under the following conditions: MeOH solution (1 mg/ml) was injected into the L-column octadecylsilane (4.6 x 150 mm), and the mobile phase was MeCN/20 mM phosphate buffer (pH 6.5) (35: 65) at a flow rate of 1 ml/min. TT101 was detected with a UV detector at a wavelength of 254 nm and a retention time of 21.2 min. 1H NMR [dimethyl sulfoxide (DMSO)-d6], 
2.31 (s, 3H), 2.50 (br, 5H), 2.76 (t, J = 7.0 Hz, 2H), 7.20 (br, 5H), 7.55 (dd, J = 2.0, 6.0 Hz, 2H), and 7.84 (dd, J = 2.0, 6.0 Hz, 2H). The purity of TT201 was >99%, as assessed by high-performance liquid chromatography under the following conditions: MeOH solution (1 mg/ml) was injected into the L-column octadecylsilane (4.6 x 150 mm), and the mobile phase was MeCN/20 mM phosphate buffer (pH 6.5) (1:1) at a flow rate of 1 ml/min. TT201, as previously reported by Penning et al. (1997), was detected with a UV detector at a wavelength of 254 nm and a retention time of 8.6 min (m.p., 202–203°C). Celecoxib and SC-236 were kindly supplied by Pfizer Japan Inc. (Tokyo, Japan). Rofecoxib was synthesized as reported elsewhere (Ducharme et al., 1995; Kato et al., 2001).
Z-DEVD-FMK (a caspase-3 inhibitor), Z-IETD-FMK (a caspase-8 inhibitor), and Z-LEHD-FMK (a caspase-9 inhibitor) were purchased from R&D Systems, Inc. (Minneapolis, MN). Interleukin (IL)-1
was purchased from Techne Corporation (Minneapolis, MN). The test drugs were dissolved in DMSO [the final concentration of DMSO in all cultures was 0.1% (v/v)]. RPMI 1640 medium, penicillin-streptomycin solution, fetal bovine serum (FBS), and trypsin-EDTA were obtained from Invitrogen (Frederick, MD). All other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan).
Culture of Human Synovial Fibroblasts. Synovial fibroblasts were prepared from harvested synovial tissues, as described previously (Kawai et al., 1998). The synovial tissues were obtained during the performance of total knee or hip replacement in patients with RA or osteoarthritis (OA) who fulfilled the respective criteria of the American College of Rheumatology (Altman et al., 1986, 1991; Arnett et al., 1988). The protocol for the study was approved by the Ethics Committees of St. Marianna University (Kawasaki, Japan) and Toho University (Tokyo, Japan). All patients gave written informed consent for their tissues to be used in this research. The synovial tissues were digested for 2 h in 0.2% (w/v) bacterial collagenase (Immunobiological Laboratories Co., Ltd., Gunma, Japan), and the synovial cells thus obtained were suspended in RPMI 1640 medium with 10% (v/v) FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Incubation was performed at 37°C in 5% CO2 for several days, after which nonadherent cells were removed. The fibroblast-like adherent cells were cultured in RPMI 1640 medium containing 1% (v/v) FBS under a 5% CO2 atmosphere and were used as synovial fibroblasts within two passages. These adherent cells contained no T cells (CD3+) or macrophages/monocytes (CD14+) on two-color immunofluorescence and flow cytometry (Kusunoki et al., 2002).
Cell Proliferation. The proliferative activity of cultured human synovial fibroblasts was estimated from the uptake of 5-bromo-2'-deoxyuridine (BrdU). Cells (1 x 104 /well) were exposed to the test drugs during culture in 96-well plates under the conditions described above. After 24 h, 10 µM BrdU was added to the medium, and the cells were incubated for another 18 h. Then the cells were fixed, and nuclear incorporation of BrdU was measured by using a cell proliferation ELISA kit (Roche Diagnostics, Mannheim, Germany) in accordance with the manufacturer's instructions. Each measurement was performed in triplicate, and the results are presented as percentages relative to the value for untreated control cultures.
Cell Viability. Human synovial fibroblasts in 96-well culture plates (2 x 104 cells/well) were exposed to the test drugs under the conditions described above. After 24 h, cell viability was determined by measuring mitochondrial NADH-dependent dehydrogenase activity with a Cell Counting kit (Dojindo Laboratories, Kumamoto, Japan) that used a sulfonated tetrazolium salt and WST-1, in accordance with the manufacturer's instructions. Each measurement was performed in triplicate, and the results are presented as percentages relative to the value for untreated control cultures.
DNA Fragmentation. Human synovial fibroblasts in 96-well culture plates (2 x 104 cells/well) were exposed to the test drugs under the conditions described above. After 24 h, cytoplasmic DNA fragmentation (an indicator of apoptosis) was detected by using a DNA cell death detection ELISAPLUS kit (Roche Diagnostics) in accordance with the manufacturer's instructions. Each measurement was performed in triplicate, and the results are presented relative to that for untreated control cultures. Cell morphology was also examined under a BX51 light microscope (Olympus Optical Co., Ltd., Nagano, Japan) at a magnification of 100x.
Caspase Activity. RA synovial fibroblasts were incubated in tissue culture flasks in the presence or absence of TT101 and/or Z-IETD-FMK and Z-LEHD-FMK under the conditions described above. After 24 h, cellular caspase-3 activity was measured by using the CaspACE assay system (Promega Corporation, Madison, WI) in accordance with the manufacturer's instructions. Then the cells were washed in ice-cold phosphate-buffered saline (PBS; 9.57 mM, pH 7.35–7.65; Takara Shuzo Co., Ltd., Shiga, Japan) and resuspended in cell lysis buffer. After lysis of the cells by freezing and thawing, the lysates were centrifuged, and the supernatants were used as cell extracts. The protein content of each extract was determined by the bicinchoninic acid protein assay method (Pierce Biotechnology, Rockford, IL) with bovine serum albumin as the standard, and the protein content of was adjusted to 1 mg/ml.
Effect of Caspase Inhibitors on DNA Fragmentation Induced by TT101. RA synovial fibroblasts in 96-well culture plates (2 x 104 cells/well) were exposed to Z-DEVD-FMK, Z-IETD-FMK, or Z-LEHD-FMK in the presence or absence of TT101 under the conditions described above. After 24 h, cytoplasmic DNA fragmentation was detected by using a DNA cell death detection ELISAPLUS kit (Roche Diagnostics) in accordance with the manufacturer's instructions.
Effect of Rofecoxib on DNA Fragmentation Induced by TT101. Human synovial fibroblasts in 96-well culture plates (2 x 104 cells/well) were pretreated or not pretreated with 1 µM rofecoxib and then were incubated with TT101 or celecoxib under the conditions described above. After 24 h, cytoplasmic DNA fragmentation was assessed by using a DNA cell death detection ELISAPLUS kit (Roche Diagnostics) in accordance with the manufacturer's instructions.
Western Blotting. RA synovial fibroblasts were lysed in Chaps cell extract buffer (50 mM Pipes/HCl, pH 6.5, 0.1% (w/v) Chaps, 5 mM dithiothreitol, 2 mM EDTA, 10 µg/ml pepstatin, 20 µg/ml leupeptin, and 10 µg/ml aprotinin) and then centrifuged at 14,000 rpm for 30 min to remove debris. Subsequently, the protein content of the supernatant was determined by the bicinchoninic acid protein assay (Pierce Biotechnology) with bovine serum albumin as the standard, and the protein content of the extracts was adjusted to 1 mg/ml. Then the extracts were subjected to SDS-polyacrylamide gel electrophoresis using 15% (w/v) acrylamide slab gels under reducing conditions. The proteins thus separated were electroblotted onto Immobilon-P poly (vinylidene difluoride) membranes (Millipore Corporation, Billerica, MA) with a semidry blotter (Atto Technology, Inc., Tokyo, Japan). After the membranes had been blocked in 10 mM TBS containing 0.1% Tween 20 (TBS-T) and 5% skim milk for 1 h at room temperature, rabbit anti-Bcl-2 polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or rabbit anti-BID polyclonal antibody (Cell Signaling Technology, Inc., Beverly, MA) was applied for 18 h at 4°C. Then the membranes were washed with TBS-T, and incubation with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit antibody at a dilution of 1:10,000 in TBS-T) was performed for 1 h. After further washing with TBS-T, the protein bands were detected by using an enhanced chemiluminescence Western blot analysis system.
Assay of PGE2 Production. RA synovial fibroblasts were incubated in 24-well plates (5 x 105 cells/well) with IL-1 (1 ng/ml) for 18 h washed with PBS and then exposed to the test drugs for 1 h under the conditions described above. Then 3 µM arachidonic acid (Cayman Chemical Company, Ann Arbor, MI) was added to the medium. After incubation for 30 min, the culture medium was harvested using a syringe and filtered through a 0.22-µm filter (Millipore Corporation). The PGE2 concentration in the medium was measured using an ELISA kit (Cayman Chemical Company) in accordance with the manufacturer's instructions. Each measurement was performed in triplicate.
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Results
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Effect on Cell Proliferation. To determine whether TT101 and TT201 had an inhibitory effect on the proliferation of RA or OA synovial fibroblasts, we first examined the influence of these drugs on cell proliferation (DNA synthesis) by measuring the nuclear incorporation of BrdU (Fig. 1). TT101 inhibited the proliferation of both RA synovial fibroblasts (Fig. 1A) and OA synovial fibroblasts (Fig. 1B) in a concentration-dependent manner. TT201, celecoxib, and SC-236 also inhibited cell proliferation, but their effects were weaker than that of TT101. Rofecoxib had no effect on cell proliferation up to a concentration of 100 µM.
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Fig. 1. Effect of the each drug on the proliferation of synovial fibroblasts obtained from patients with rheumatoid arthritis (RA; A) and osteoarthritis (OA; B). Cells were incubated with celecoxib (closed diamonds), TT101 (closed triangles), TT201 (open squares), SC-236 (crosses), or rofecoxib (open circles) for 24 h. Then proliferative activity was estimated from the nuclear incorporation of BrdU and was expressed as a percentage of the control value (untreated cells). Data are the mean ± S.D. for triplicate cultures, and representative results from three independent experiments are shown.
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Effect on Cell Viability. To determine whether TT101 and TT201 had an influence on the viability of RA synovial fibroblasts and OA synovial fibroblasts, we evaluated cell viability using the WST-1 assay (Fig. 2). TT101 caused a marked decrease of the cell viability of both RA synovial fibroblasts (Fig. 2A) and OA synovial fibroblasts (Fig. 2B) in a concentration-dependent manner. The order of potency of the effect of each drug on cell viability was as follows: TT101 > celecoxib = SC-236 > TT201 (for both types of cells). Rofecoxib had no effect at all on cell viability.
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Fig. 2. Effect of each test drug on the viability of synovial fibroblasts obtained from patients with RA (A) and OA (B). Cells were incubated with celecoxib (closed diamonds), TT101 (closed triangles), TT201 (open squares), SC-236 (crosses), or rofecoxib (open circles) for 24 h. Then cell viability was determined by the WST-1 assay and expressed as a percentage of the control value (untreated cells). Data are the mean ± S.D. for triplicate cultures, and representative results from three independent experiments are shown.
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Effect on DNA Fragmentation. TT101 induced DNA fragmentation, the hallmark of apoptosis, in both RA synovial fibroblasts (Fig. 3A) and OA synovial fibroblasts (Fig. 3B), and its effect was more potent than that of celecoxib or SC-236. TT201 also induced DNA fragmentation but was weaker than celecoxib. In contrast, rofecoxib did not induce any DNA fragmentation.
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Fig. 3. DNA fragmentation in synovial fibroblasts from RA patients (A) and OA patients (B). Cells were incubated with celecoxib (closed diamonds), TT101 (closed triangles), TT201 (open squares), SC-236 (crosses), or rofecoxib (open circles) for 24 h, after which cytoplasmic DNA fragmentation was measured by enzyme immunoassay and expressed relative to the control value (untreated cells). Data are the mean ± S.D. for triplicate cultures, and representative results from three independent experiments are shown.
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The cell morphology was examined under a light microscope after exposure to TT101 (Fig. 4). When the cells were incubated with 7 µM TT101 for 24 h (Fig. 4, C and D), distinctive morphological changes were observed, such as rounding and shrinkage, and the cells became detached from their neighbors.
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Fig. 4. Morphological changes of the synovial fibroblasts from RA patients (A and C) or OA patients (B and D) as observed by light microscopy. Cells were incubated for 24 h without (A and B) or with (C and D) TT101 at a concentration of 7 µM. Bar = 60 µm.
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Effects of TT101 and Caspase Inhibitors. Caspases are responsible for many of the biochemical and morphological changes that occur during apoptosis, so we investigated whether TT101 induced the activation of caspase-3 (a terminal enzyme in the apoptotic pathway). As shown in Fig. 5A, incubation of RA synovial fibroblasts with 7 µM TT101 for 24 h induced the activation of caspase-3, whereas this activation was completely blocked by incubation with Z-IETD-FMK or Z-LEHD-FMK (inhibitors of caspase-8 and -9, respectively). Next, we examined the effects of these caspase inhibitors on the TT101-induced apoptosis (Fig. 5B). Induction of DNA fragmentation in RA synovial fibroblasts by TT101 was suppressed by Z-DEVD-FMK, Z-IETD-FMK, and Z-LEHD-FMK (inhibitors of caspase-3, -8, and -9, respectively) in a concentration-dependent manner.
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Fig. 5. Effect of TT101 on caspase activity in RA synovial fibroblasts (A). Cells were cultured for 24 h in the absence of any of agents or with 7 µM TT101 alone, 7 µM TT101 plus Z-IETD-FMK (a caspase-8 inhibitor), or 7 µM TT101 plus Z-LEHD-FMK (a caspase-9 inhibitor), after which caspase activity was detected by using the CaspACE assay system (the absorbance was measured at 405 nm). Data are the mean ± S.D. from three independent experiments. *, p < 0.01 versus cells treated with TT101 alone. Significance was evaluated by Tukey's multiple comparison test. Effects of TT101 plus a caspase inhibitor on DNA fragmentation in RA synovial fibroblasts (B). Cells were incubated with TT101 with/without a caspase inhibitor for 24 h, after which the cytoplasmic DNA fragmentation was measured by enzyme immunoassay and expressed relative to the control value (untreated cells). Data are the mean ± S.D. for triplicate cultures, and representative results from three independent experiments are shown. *, p < 0.01 versus cells treated with TT101 alone. Significance was evaluated by Tukey's multiple comparison test.
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Western Blot Analysis of Bcl-2 and BID. We examined the effects of the test drugs on the expression of Bcl-2, which is linked to inhibition of apoptosis, by RA synovial fibroblasts. Western blotting demonstrated expression of Bcl-2 by RA synovial fibroblasts, and little change in the level of expression was seen after treatment with any of the COX-2 inhibitors (Fig. 6, top). We also studied the effects of the each drug on cleavage of BID (a substrate of caspase-8) in RA synovial fibroblasts, since truncated BID (tBID; 15 kDa) activates the mitochondrial proapoptotic pathway. Full-length BID was expressed in RA synovial fibroblasts, but none of the test drugs caused cleavage of BID under the conditions tested (Fig. 6, bottom).
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Fig. 6. Effects of TT101 and celecoxib on expression of Bcl-2 and cleavage of BID. RA synovial fibroblasts were cultured for 6 h without any agents (lane 1) or were incubated with 7 µM TT101 (lane 2), 100 µM TT201 (lane 3), or 40 µM celecoxib (lane 4). Protein extracts were prepared from the cells and subjected to Western blotting using an antibody that detected Bcl-2 (top) or BID (bottom), as described in Materials and Methods.
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Effect of Rofecoxib Pretreatment on DNA Fragmentation Induced by TT101. To investigate whether the interaction between TT101 and the COX-2 was related to the induction of apoptosis, we studied the effect of pretreatment with another COX-2 inhibitor (rofecoxib) on TT101-induced apoptosis. One hour before the addition of TT101, rofecoxib was added to cultures of human synovial fibroblasts at a concentration that was sufficient to inhibit COX-2, and then DNA fragmentation was assessed as described above. As shown in Fig. 7, TT101 induced the same extent of apoptosis after pretreatment with rofecoxib as that seen in the absence of COX-2 inhibition by rofecoxib; this was true for both in RA synovial fibroblasts (Fig. 7A) and OA synovial fibroblasts (Fig. 7B).
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Fig. 7. Effect of rofecoxib on TT101-induced DNA fragmentation. RA synovial fibroblasts (A) and OA synovial fibroblasts (B) were incubated with (continuous line)/without (broken line) 1 µM rofecoxib for 1 h and then were incubated in the presence/absence of TT101 (triangles) or celecoxib (diamonds) for 24 h. Cytoplasmic DNA fragmentation was measured by enzyme immunoassay and expressed relative to the control value (untreated cells). Data are means ± S.D. from triplicate cultures, and representative results from three independent experiments are shown.
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Effect on Production of PGE2. We investigated the effect of each test drug on the production of PGE2 by RA synovial fibroblasts. As shown in Fig. 8, all of the drugs suppressed the production of PGE2 (stimulated by the addition 3 µM of arachidonic acid) in a concentration-dependent manner, but TT101 had the weakest suppressive effect on PGE2 production.
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Fig. 8. Effect of the test drugs on the production of PGE2 by RA synovial fibroblasts. Cells were stimulated with IL-1 (1 µg/ml) for 24 h, washed twice with PBS, and incubated for 1 h at 37°C with various concentrations of celecoxib (closed diamonds), TT101 (closed triangles), TT201 (open squares), SC-236 (crosses), or rofecoxib (open circles). Then 3 µM arachidonic acid was added, and incubation was continued for another 30 min, after which the PGE2 level in the culture medium was measured by enzyme immunoassay. Representative results from three independent experiments are shown, and values are the mean ± S.D. from triplicate cultures. The PGE2 level in control cultures was 38.0 ± 5.9 ng/ml.
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Discussion
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TT101, a novel celecoxib derivative, was shown to be a powerful inducer of apoptosis in both RA synovial fibroblasts and OA synovial fibroblasts. The potency of the inhibitory effect of TT101 on cell proliferation, evaluated in terms of the 50% effective concentration, was about 5-fold stronger than that of celecoxib for RA synovial fibroblasts and 15-fold stronger for OA synovial fibroblasts. On the other hand, the potency of TT101 for inhibiting COX-2 (50% inhibitory concentration) was about 70-fold weaker than that of celecoxib. Thus, the proapoptotic activity of TT101 was far stronger than that of celecoxib, whereas its COX-2 inhibitory activity was much weaker. The proapoptotic activity of TT101 was not altered by the binding of rofecoxib with COX-2 after pretreatment of cultured cells with adequate concentrations of rofecoxib (Fig. 7). This allows us to rule out the possibility that binding of TT101 to COX-2 alters the structure of the enzyme and thus triggers apoptosis, suggesting that the mechanism of apoptosis induction by TT101 (as well as celecoxib) involves neither COX-2 inhibition nor structural changes to the COX-2 molecule.
We examined the proapoptotic effect of TT101 on U937, a human monocyte cell line. TT101 as well as celecoxib induced cell death in U937 at almost the same potency as those in synovial cells (date not shown). A strong proapoptotic activity of TT101 on human synovial cells might be one of the therapeutic approaches for rheumatoid arthritis (Nishioka et al., 1998); however, a major improvement of TT101 itself and/or a proper targeting technique for the drug will be necessary for the clinical application.
In our previous study of adenocarcinoma cells, phosphorylation of Akt was only induced by celecoxib among the selective COX-2 inhibitors tested (Yamazaki et al., 2002). Accordingly, the results of the present study using synovial fibroblast and OA synovial fibroblast differ from those of previous studies conducted using tumor cell lines. Zhu et al. (2004) synthesized various derivatives of celecoxib and examined each derivative for induction of the apoptosis of PC-3 human prostate cancer cells. They demonstrated that stronger inhibition of 3-phosphoinositide-dependent kinase-1, upstream kinase of Akt activation, was associated with stronger induction of apoptosis by celecoxib analogs. In the present study, however, phosphorylated Akt was detected in RA synovial fibroblasts, but it was unaffected by treatment with TT101 or celecoxib (data not shown). The different types of cells used in the two studies may explain the differences in the proapoptotic effects of celecoxib.
Apoptosis can be induced by internal (mitochondria-dependent) and external (death receptor-dependent) pathways (Baier et al., 2003). In the mitochondria-dependent pathway, cytochrome c and apoptotic protease activating factor (Apaf)-1 are released from the mitochondria and then bind to pro-caspase-9 to produce active caspase-9. In the death receptor-dependent pathway, extracellular death ligands bind to receptors and cause activation of caspase-8. The present study showed that TT101 activated caspase-3, which is at the end of the caspase cascade. Also, the induction of DNA fragmentation induced by TT101 was suppressed by all of the caspase inhibitors tested (inhibitors of caspases-3, -8, and -9). These findings suggest that the proapoptotic activity of TT101 may involve two signal transduction pathways, i.e., both the internal (mitochondria-dependent) pathway and the external (death receptor-dependent) pathway.
The Bcl-2 family is thought to be involved in the regulation of these pathways (Tsujimoto and Shimizu, 2000). Bcl-2 prevents various apoptotic mitochondrial changes, including cytochrome c release and loss of the membrane potential (Zamzami et al., 1996). Similar to the observations obtained in our previous study using HT-29 cells (Yamazaki et al., 2002), in which celecoxib did not affect Bcl-2 expression, TT101 did not alter the expression of this anti-apoptotic protein. BID is a specific substrate of caspase-8 involved in the external death receptor-dependent pathway (Li et al., 1998; Luo et al., 1998). Death receptor-dependent signals cause cleavage of cytosolic BID to release tBID, which tBID translocates to mitochondria and thus transduces apoptotic signals from the cytoplasmic membrane. Although TT101-induced DNA fragmentation was suppressed by the addition of a caspase-8 inhibitor, we found that BID was not degraded by TT101. It seems likely that the mechanisms of mitochondria-mediated apoptosis are not only regulated by Bcl-2 and BID but also by various other factors, including Bad, Bim, and Bcl-XL (Tsujimoto and Shimizu, 2000). We cannot rule out the involvement of such mechanisms in the induction of apoptosis by TT101. Further studies including in vitro experiments to investigate the mechanisms of TT101-induced apoptosis and in vivo experiments to examine the antirheumatic activity using animal arthritis models remain to be conducted.
We modified the sulfonamide group of celecoxib to an N-(2-aminoethyl)-sulfonamide group when developing TT101, whereas the tolyl group in the terminal aromatic ring of celecoxib was modified to an aminophenyl group to create TT201. In the case of SC-236, this region was changed to a chlorophenyl group. The structures of these celecoxib derivatives and rofecoxib are shown in Fig. 9. Whereas TT101 had a strong proapoptotic effect, the proapoptotic activity of TT201 for RA and OA synovial fibroblasts was much weaker than that of celecoxib. This result suggests that the methyl group of celecoxib may be essential for a proapoptotic effect on synovial fibroblasts, when the inhibition of PGE2 production via COX-2 inhibition was evaluated, TT201 was less potent than celecoxib, but the 50% inhibitory concentration of TT201 was comparable with that of rofecoxib. As suggested by Zhu et al. (2004), the basic frame and electron density of TT101 and TT201 may influence their apoptosis-inducing activity and COX-2 inhibitory activity.
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Acknowledgements
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We thank Drs. Moroe Beppu and Haruhito Aoki (St. Marianna University School of Medicine) for providing clinical samples from patients and for valuable advice. We also thank Drs. Fumiaki Kojima and Ryuta Yamazaki for suggestions about the methodology of the experiments, as well as Reiko Oura and Sonoko Sakurai for secretarial assistance.
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Footnotes
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This work was supported in part by research grants from The Japanese Ministry of Education, Culture, Sports, Science and Technology (no. 14572167 to S.K.) and a Research Promotion grant from Toho University Graduate School of Medicine (no. 04-01 to S.K.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.105.086116.
ABBREVIATIONS: NSAIDs, nonsteroidal anti-inflammatory drugs; COX, cyclooxygenase; RA, rheumatoid arthritis; PG, prostaglandin; TT101, N-(2-aminoethyl)-4-[5-(4-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide; TT201, 4-[5-(4-aminophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide; DMSO, dimethyl sulfoxide; SC-236, 4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzene sulfonamide; IL-1, interleukin-1; FBS, fetal bovine serum; OA, osteoarthritis; BrdU, 5-bromo-2'-deoxyuridine; WST-1,4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; Pipes, 1,4-piperazinediethanesulfonic acid; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TBS, Tris-buffered saline; TSB-T, TBS containing 0.1% Tween 20; tBID, truncated BID; Z-LEHD-FMK, N-benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethyl ketone; Z-DEVD-FMK, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone; Z-IETD-FMK, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethyl ketone.
Address correspondence to: Natsuko Kusunoki, Division of Rheumatology, Department of Internal Medicine, Toho University Omori Medical Center, 6-11-1 Omori-Nishi, Ota-ku, Tokyo 143-8541, Japan. E-mail: kusunoki{at}med.toho-u.ac.jp
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References
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Altman R, Alarcon G, Appelrouth D, Bloch D, Borenstein D, Brandt K, Brown C, Cooke TD, Daniel W, Feldman D, et al. (1991) The American College of Rheumatology criteria for the classification and reporting of osteoarthritis of the hip. Arthritis Rheum 34: 505-514.[Medline]
Altman R, Asch E, Bloch D, Bole G, Borenstein D, Brandt K, Christy W, Cooke TD, Greenwald R, Hochberg M, et al. (1986) Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association. Arthritis Rheum 29: 1039-1049.[Medline]
Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS, et al. (1988) The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31: 315-324.[Medline]
Arun B and Goss P (2004) The role of COX-2 inhibition in breast cancer treatment and prevention (Review). Semin Oncol 31 (Suppl 7): 22-29.
Baier A, Meineckel I, Gay S, and Pap T (2003) Apoptosis in rheumatoid arthritis. Curr Opin Rheumatol 15: 274-279.[CrossRef][Medline]
Ducharme Y, Gauthier JY, Prasit P, Leblanc Y, Wang Z, Leger S, and Therien M (1995) inventors, Merck Frosst Canada Inc., assignee. Phenyl heterocycles as cyclooxygenase-2 inhibitors. International Patent Publication WO95/00501.
Kato M, Nishida S, Kitasato H, Sakata N, and Kawai S (2001) Cyclooxygenase-1 and cyclooxygenase-2 selectivity of non-steroidal anti-inflammatory drugs: investigation using human peripheral monocytes. J Pharm Pharmacol 53: 1679-1685.[CrossRef][Medline]
Kawai S, Nishida S, Kato M, Furumaya Y, Okamoto R, Koshino T, and Mizushima Y (1998) Comparison of cyclooxygenase-1 and -2 inhibitory activities of various nonsteroidal anti-inflammatory drugs using human platelets and synovial cells. Eur J Pharmacol 347: 87-94.[CrossRef][Medline]
Kismet K, Akay MT, Abbasoglu O, and Ercan A (2004) Celecoxib: a potent cyclooxygenase-2 inhibitor in cancer prevention. Cancer Detect Prev 28: 127-142.[CrossRef][Medline]
Kusunoki N, Yamazaki R, and Kawai S (2002) Induction of apoptosis in rheumatoid synovial fibroblasts by celecoxib, but not by other selective cyclooxygenase 2 inhibitors. Arthritis Rheum 46: 3159-3167.[CrossRef][Medline]
Li H, Zhu H, Xu CJ, and Yuan J (1998) Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94: 491-501.[CrossRef][Medline]
Luo X, Budihardjo I, Zou H, Slaughter C, and Wang X (1998) Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94: 481-490.[CrossRef][Medline]
McAdam BF, Catella-Lawson F, Mardini IA, Kapoor S, Lawson JA, and FitzGerald G (1999) Systemic biosynthesis of prostacyclin by cyclooxygenase (COX)-2: the human pharmacology of a selective inhibitor of COX-2. Proc Natl Acad Sci USA 96: 272-277.[Abstract/Free Full Text]
Myers C, Koki A, Pamukcu R, Wechter W, and Padley RJ (2001) Proapoptotic anti-inflammatory drugs. Urology 57: 73-76.[CrossRef][Medline]
Nishioka K, Hasunuma T, Kato T, Sumida T, and Kobata T (1998) Apoptosis in rheumatoid arthritis: a novel pathway in the regulation of synovial tissue. Arthritis Rheum 41: 1-9.[CrossRef][Medline]
Ospelt C, Neidhart M, Gay RE, and Gay S (2004) Synovial activation in rheumatoid arthritis. Front Biosci 9: 2323-2334.[Medline]
Penning TD, Talley JJ, Bertenshaw SR, Carter JS, Collins PW, Docter S, Graneto MJ, Lee LF, Malecha JW, Miyashiro JM, et al. (1997) Synthesis and biological evaluation of the 1,5-diarylpyrazole class of cyclooxygenase-2 inhibitors: identification of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (SC-58635, celecoxib). J Med Chem 40: 1347-1365.[CrossRef][Medline]
Perlman H, Pagliari LJ, and Volin MV (2001) Regulation of apoptosis and cell cycle activity in rheumatoid arthritis. Curr Mol Med 1: 597-608.[CrossRef][Medline]
Sandler AB and Dubinett SM (2004) COX-2 inhibition and lung cancer (Review). Semin Oncol 31 (Suppl 7): 45-52.
Sinicrope FA and Gill S (2004) Role of cyclooxygenase-2 in colorectal cancer (Review). Cancer Metastasis Rev 23: 63-75.[CrossRef][Medline]
Sinicrope FA, Half E, Morris JS, Lynch PM, Morrow JD, Levin B, Hawk ET, Cohen DS, Ayers GD, and Stephens LC (2004) Familial Adenomatous Polyposis Study Group. Cell proliferation and apoptotic indices predict adenoma regression in a placebo-controlled trial of celecoxib in familial adenomatous polyposis patients. Cancer Epidemiol Biomarkers Prev 13: 920-927.[Abstract/Free Full Text]
Steinbach G, Lynch PM, Phillips RK, Wallace MH, Hawk E, Gordon GB, Wakabayashi N, Saunders B, Shen Y, Fujimura T, et al. (2000) The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis. N Engl J Med 342: 1946-1952.[Abstract/Free Full Text]
Thompson CB (1995) Apoptosis in the pathogenesis and treatment of disease. Science (Wash DC) 267: 1456-1462.[Abstract/Free Full Text]
Tsujimoto Y and Shimizu S (2000) Bcl-2 family: life-or-death switch. FEBS Lett 466: 6-10.[CrossRef][Medline]
Xiong HQ (2004) Molecular targeting therapy for pancreatic cancer (Review). Cancer Chemother Pharmacol 54 (Suppl 1): S69-S77.
Yamazaki R, Kusunoki N, Matsuzaki T, Hashimoto S, and Kawai S (2002) Selective cyclooxygenase-2 inhibitors show a differential ability to inhibit proliferation and induce apoptosis of colon adenocarcinoma cells. FEBS Lett 531: 278-284.[CrossRef][Medline]
Zamzami N, Susin SA, Marchetti P, Hirsch T, Gomez-Monterrey I, Castedo M, and Kroemer G (1996) Mitochondrial control of nuclear apoptosis. J Exp Med 183: 1533-1544.[Abstract/Free Full Text]
Zhu J, Huang JW, Tseng PH, Yang YT, Fowble J, Shiau CW, Shaw YJ, Kulp SK, and Chen CS (2004) From the cyclooxygenase-2 inhibitor celecoxib to a novel class of 3-phosphoinositide-dependent protein kinase-1 inhibitors. Cancer Res 64: 4309-4318.[Abstract/Free Full Text]
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Abstract
Materials and Methods
