Novel Pan-Pim Kinase Inhibitors With Imidazopyridazine and Thiazolidinedione Structure Exert Potent Antitumor Activities.

Pim kinases are overexpressed in various types of hematological malignancies and solid carcinomas, and promote cell proliferation and survival. Here in this study, we investigated the preclinical profile of novel pan-Pim kinase inhibitors with imidazopyridazine and thiazolidinedione structure. Imidazopyridazine-thiazolidinediones inhibited activities of Pim kinases with IC50 values of tens to hundreds nanomolar. With YPC-21440 and/or YPC-21817, which exhibited especially high inhibitory activities against Pim kinases, we investigated in vitro and in vivo activities of imidazopyridazine-thiazolidinediones. In silico analysis of binding mode of YPC-21440 and Pim kinases revealed that it directly bound to ATP-binding pockets of Pim kinases. In the kinase panel tested, YPC-21440 and YPC-21817 were highly specific to Pim kinases. These compounds exerted antiproliferative activities against various cancer cell lines derived from hematological malignancies and solid carcinomas. Furthermore, they suppressed phosphorylation of Pim kinase substrates, arrested cell cycle at the G1 phase, and induced apoptosis in cultured cancer cells. In tumor xenograft models, YPC-21440 methanesulfonate and YPC-21817 methanesulfonate exerted antitumor activities. Furthermore, pharmacodynamic analysis with a xenograft model suggested that YPC-21817 methanesulfonate inhibited Pim kinases in tumors. In conclusion, our data revealed that imidazopyridazine-thiazolidinediones are novel Pim kinases inhibitors, effective on various types of cancer cell lines both in vitro and in vivo.

Discovery of a Potent Anticancer Agent PVHD303 with in Vivo Activity.

As a part of our continuous structure-activity relationship (SAR) studies on 1-(quinazolin-4-yl)-1-(4-methoxyphenyl)ethan-1-ols, the synthesis of derivatives and their cytotoxicity against the human lung cancer cell line A549 were explored. This led to the discovery of 1-(2-(furan-3-yl)quinazolin-4-yl)-1-(4-methoxyphenyl)ethan-1-ol (PVHD303) with potent antiproliferative activity. PVHD303 disturbed microtubule formation at the centrosomes and inhibited the growth of tumors dose-dependently in the HCT116 human colon cancer xenograft model in vivo.

Rational Design of a Potent Pan-Pim Kinases Inhibitor with a Rhodanine-Benzoimidazole Structure.

BACKGROUND/AIM: The serine/threonine Pim kinases are overexpressed in various types of solid carcinomas and hematological malignancies, and contribute to regulating cell-cycle progression and cell survival. The aim of this study was to discover a novel pan-Pim kinases inhibitor with potent anti-proliferative activities against cancer cell lines. MATERIALS AND METHODS: We screened a panel of small molecule compounds for their ability to inhibit Pim-1 kinase activity, and the hit compound was optimized using the docking analysis to Pim-1. We evaluated kinase-inhibition activities of the rationally-designed compound against Pim-1, 2, 3 and another five kinases. Furthermore, in order to characterize the cellular activities, both solid and hematological cancer cell lines treated with the compound were subjected to anti-proliferative assay, western blotting, FACS and apoptosis assays. RESULTS: We discovered a pan-Pim kinases inhibitor, compound 1, with a rhodanine-benzylidene structure via Pim-1 inhibitor screening. Using docking analysis of compound 1 and Pim-1, we optimized it and found a potent- and selective-Pim kinases inhibitor, compound 2, with a rhodanine-benzoimidazole structure. Compound 2 inhibited Pim-1, 2, 3 with IC50 values of 16, 13, and 6.4 nM, respectively, and suppressed proliferation of solid and hematological cancer cell lines at submicromolar concentrations. In both types of cell lines, compound 2 inhibited phosphorylation of Pim signaling substrates and cell-cycle progression and induced apoptosis. CONCLUSION: We identified a pan-Pim kinases inhibitor, compound 2, with a rhodanine-benzoimidazole structure. Our data suggest that compound 2 can serve as a lead to novel anticancer agents, effective in the treatment of both solid carcinomas and hematological malignancies.

YPC-21661 and YPC-22026, novel small molecules, inhibit ZNF143 activity in vitro and in vivo.

Zinc-finger protein 143 (ZNF143) is a transcription factor that is involved in anticancer drug resistance and cancer cell survival. In the present study, we identified a novel small molecule N-(5-bromo-2-methoxyphenyl)-3-(pyridine-3-yl) propiolamide (YPC-21661) that inhibited ZNF143 promoter activity and down-regulated the expression of the ZNF143-regulated genes, RAD51, PLK1, and Survivin, by inhibiting the binding of ZNF143 to DNA. In addition, YPC-21661 was cytotoxic and induced apoptosis in the human colon cancer cell line, HCT116 and human prostate cancer cell line, PC-3. 2-(pyridine-3-ylethynyl)-5-(2-(trifluoromethoxy)phenyl)-1,3,4-oxadiazole (YPC-22026), a metabolically stable derivative of YPC-21661, induced tumor regression accompanied by the suppression of ZNF143-regulated genes in a mouse xenograft model. The present study revealed that the inhibition of ZNF143 activity by small molecules induced tumor regression in vitro and in vivo; therefore, ZNF143 is a promising target of cancer therapeutics.

Establishment of a Novel In Vitro Model for Predicting Incidence and Severity of Microtubule-targeting Agent-induced Peripheral Neuropathy.

Peripheral neuropathy (PN) is a major dose-limiting side-effect of microtubule-targeting agents (MTAs), considered to be induced by inhibition of axonal microtubules. Therefore, it was thought that a useful method for predicting the frequencies of severe sensory-PN (FPN) would be to evaluate the neurite-disrupting effects of MTAs. Using neurite outgrowth from neuron-like cell lines, we comprehensively evaluated the neurite-disrupting effects of several anti-cancer drugs including MTAs, and the reversibility of the effects of MTAs. MTAs that induce PN showed neurite-disrupting effects more strongly than MTAs and anticancer drugs that do not induce PN, but the effects were not related to the FPN. On the other hand, MTAs with high FPN exhibited lower reversibility than those with low FPN. These findings suggest that neurite-disrupting effects are associated with the incidence of PN, and the reversibility of the effects is associated with FPN.

LdrP, a cAMP receptor protein/FNR family transcriptional regulator, serves as a positive regulator for the light-inducible gene cluster in the megaplasmid of Thermus thermophilus.

LdrP (TT_P0055) (LitR-dependent regulatory protein) is one of the four cAMP receptor protein (CRP)/FNR family transcriptional regulators retained by the extremely thermophilic bacterium Thermus thermophilus. Previously, we reported that LdrP served as a positive regulator for the light-induced transcription of crtB, a carotenoid biosynthesis gene encoded on the megaplasmid of this organism. Here, we showed that LdrP also functions as an activator of the expression of genes clustered around the crtB gene under the control of LitR, an adenosyl B12-bound light-sensitive regulator. Transcriptome analysis revealed the existence of 19 LitR-dependent genes on the megaplasmid. S1 nuclease protection assay confirmed that the promoters preceding TT_P0044 (P44), TT_P0049 (P49) and TT_P0070 (P70) were activated upon illumination in the WT strain. An ldrP mutant lost the ability to activate P44, P49 and P70, whilst disruption of litR resulted in constitutive transcription from these promoters irrespective of illumination, indicating that these genes were photo-dependently regulated by LdrP and LitR. An in vitro transcription experiment demonstrated that LdrP directly activated mRNA synthesis from P44 and P70 by the Thermus RNA polymerase holocomplex. The present evidence indicated that LdrP was the positive regulator essential for the transcription of the T. thermophilus light-inducible cluster encoded on the megaplasmid.

Antitumor effect of a novel phenanthroindolizidine alkaloid derivative through inhibition of protein synthesis.

AIM: The present study aimed to determine the antitumor efficacy of a new Phenanthroindolizidine alkaloid (PA) derivative, YPC-10157, and to elucidate its mechanism of action. MATERIALS AND METHODS: The in vitro and in vivo antitumor activity of YPC-10157 was evaluated against several human cancer cell lines and mouse xenograft models, respectively. Cell apoptosis was determined by measuring caspase-3/7 activity. The effect on protein synthesis was assessed using an in vitro cell-free translation assay system. RESULTS: In vitro, YPC-10157 exhibited marked cell growth inhibition and induced apoptosis. In vivo, YPC-10157 had a strong antitumor effect on xenograft models of human colon cancer and leukemia. Moreover, YPC-10157 and its derivatives inhibited protein synthesis and their inhibitory activity on protein synthesis significantly correlated regarding cell growth. CONCLUSION: YPC-10157 has promising antitumor effects and suggest that its cytotoxic mechanism might involve the inhibition of protein synthesis.

ABCG2 inhibitor YHO-13351 sensitizes cancer stem/initiating-like side population cells to irinotecan.

BACKGROUND/AIM: The aim of this study was to determine the efficacy of the combination of irinotecan and newly-synthesized ABCG2 (breast cancer-resistant protein) inhibitor YHO-13351 in cancer chemotherapy. MATERIALS AND METHODS: Side population (SP) and non-SP cells from the human cervical carcinoma cell line HeLa were isolated by fluorescence-activated cell sorting. The antitumor activity of combination therapy with irinotecan and YHO-13351 was evaluated in xenograft studies in athymic BALB/c nude mice. RESULTS: While SP cells exhibited cancer stem/initiating cell-like properties and low sensitivity to irinotecan-alone, YHO-13351 sensitized them to irinotecan in both in vitro and in vivo studies. YHO-13351 in conjunction with irinotecan reduced the increase of the SP cell ratio in the tumors compared to those observed with treatment with irinotecan-alone. CONCLUSION: Combination therapy with irinotecan and YHO-13351 would not only accelerate the antitumor effect of this regimen, but also play a crucial role in preventing resistance or relapse.

Synthesis of phenanthroindolizidine alkaloids and evaluation of their antitumor activities and toxicities.

We previously reported that phenanthroindolizidine alkaloid 3 and its derivatives had markedly potent in vitro cytotoxicity. However, they had low in vivo antitumor activities and high in vivo toxicities, which was a serious problem. To address this problem, new phenanthroindolizidine derivatives were synthesized and their antitumor activities and toxicities were evaluated. This study describes the relationship between the chemical structures, antitumor activities, and toxicities of these phenanthroindolizidine derivatives. Based on its properties, compound 8 was found to be the most suitable potential antitumor agent.

Novel acrylonitrile derivatives, YHO-13177 and YHO-13351, reverse BCRP/ABCG2-mediated drug resistance in vitro and in vivo.

Breast cancer resistance protein (BCRP/ABCG2) confers resistance to anticancer drugs such as 7-ethyl-10-hydroxycamptothecin (SN-38, an active metabolite of irinotecan), mitoxantrone, and topotecan. In this study, we examined the reversing effects of YHO-13177, a novel acrylonitrile derivative, and its water-soluble diethylaminoacetate prodrug YHO-13351 on the BCRP-mediated drug resistance. YHO-13177 potentiated the cytotoxicity of SN-38, mitoxantrone, and topotecan in both BCRP-transduced human colon cancer HCT116 (HCT116/BCRP) cells and SN-38-resistant human lung cancer A549 (A549/SN4) cells that express BCRP, but had little effect in the parental cells. In addition, YHO-13177 potentiated the cytotoxicity of SN-38 in human lung cancer NCI-H460 and NCI-H23, myeloma RPMI-8226, and pancreatic cancer AsPC-1 cells that intrinsically expressed BCRP. In contrast, it had no effect on P-glycoprotein-mediated paclitaxel resistance in MDR1-transduced human leukemia K562 cells and multidrug resistance-related protein 1-mediated doxorubicin resistance in MRP1-transfected human epidermoid cancer KB-3-1 cells. YHO-13177 increased the intracellular accumulation of Hoechst 33342, a substrate of BCRP, at 30 minutes and partially suppressed the expression of BCRP protein at more than 24 hours after its treatment in both HCT116/BCRP and A549/SN4 cells. In mice, YHO-13351 was rapidly converted into YHO-13177 after its oral or intravenous administration. Coadministration of irinotecan with YHO-13351 significantly increased the survival time of mice inoculated with BCRP-transduced murine leukemia P388 cells and suppressed the tumor growth in an HCT116/BCRP xenograft model, whereas irinotecan alone had little effect in these tumor models. These findings suggest that YHO-13351, a prodrug of YHO-13177, could be clinically useful for reversing BCRP-mediated drug resistance in cancer chemotherapy.

Involvement of CarA/LitR and CRP/FNR family transcriptional regulators in light-induced carotenoid production in Thermus thermophilus.

Members of the CarA/LitR family are MerR-type transcriptional regulators that contain a C-terminal cobalamin-binding domain. They are thought to be involved in light-induced transcriptional regulation in a wide variety of nonphototrophic bacteria. Based on the distribution of this kind of regulator, the current study examined carotenoid production in Thermus thermophilus, and it was found to occur in a light-induced manner. litR and carotenoid and cobalamin biosynthesis genes were all located on the large plasmid of this organism. litR or cobalamin biosynthesis gene knockout mutants were unable to switch off carotenoid production under dark conditions, while a mutant with a mutation in the downstream gene adjacent to litR (TT_P0055), which encodes a CRP/FNR family transcriptional regulator, was unable to produce carotenoids, irrespective of light conditions. Overall, genetic and biochemical evidence indicates that LitR is bound by cobalamin and associates with the intergenic promoter region between litR and crtB (phytoene synthase gene), repressing the bidirectional transcription of litR and crtB. It is probable that derepression of LitR caused by some photodependent mechanism induces the expression of TT_P0055 protein, which serves as a transcriptional activator for the crtB operon and hence causes the expression of carotenoid biosynthesis and the DNA repair system under light condition.

Role of ZNF143 in tumor growth through transcriptional regulation of DNA replication and cell-cycle-associated genes.

The cell cycle is strictly regulated by numerous mechanisms to ensure cell division. The transcriptional regulation of cell-cycle-related genes is poorly understood, with the exception of the E2F family that governs the cell cycle. Here, we show that a transcription factor, zinc finger protein 143 (ZNF143), positively regulates many cell-cycle-associated genes and is highly expressed in multiple solid tumors. RNA-interference (RNAi)-mediated knockdown of ZNF143 showed that expression of 152 genes was downregulated in human prostate cancer PC3 cells. Among these ZNF143 targets, 41 genes (27%) were associated with cell cycle and DNA replication including cell division cycle 6 homolog (CDC6), polo-like kinase 1 (PLK1) and minichromosome maintenance complex component (MCM) DNA replication proteins. Furthermore, RNAi of ZNF143 induced apoptosis following G2/M cell cycle arrest. Cell growth of 10 lung cancer cell lines was significantly correlated with cellular expression of ZNF143. Our data suggest that ZNF143 might be a master regulator of the cell cycle. Our findings also indicate that ZNF143 is a member of the growing list of non-oncogenes that are promising cancer drug targets.

Pro-apoptotic effect of nonsteroidal anti-inflammatory drugs on synovial fibroblasts.

Rheumatoid arthritis (RA) is a systemic inflammatory disease that mainly affects the articular synovial tissues. Although the etiology of RA has not yet been elucidated, physical and biochemical inhibition of synovial hyperplasia, which is the origin of articular destruction, may be an effective treatment for RA. Nonsteroidal anti-inflammatory drugs (NSAIDs) have long been used for the treatment of RA. The mechanism of action of NSAIDs generally involves the inhibition of cyclooxygenase (COX) at sites of inflammation. Thus, NSAIDs were not generally considered to have a so-called anti-rheumatic effect, including inhibition of progressive joint destruction and induction of remission. However, certain conventional NSAIDs and celecoxib, a selective COX-2 inhibitor, have been reported to inhibit synovial hyperplasia by inducing the apoptosis of human synovial fibroblasts. Therefore, it has been suggested that such NSAIDs may not only have an anti-inflammatory effect but also an anti-rheumatic effect. In this review, we summarize findings about the pro-apoptotic effect, in other words, anti-proliferative effect of NSAIDs on synovial fibroblasts from patients with RA.

G1P3, an interferon inducible gene 6-16, is expressed in gastric cancers and inhibits mitochondrial-mediated apoptosis in gastric cancer cell line TMK-1 cell.

Expression of an interferon inducible gene 6-16, G1P3, increases not only in type I interferon-treated cells but also in human senescent fibroblasts. However, the function of 6-16 protein is unknown. Here we report that 6-16 is 34 kDa glycosylated protein and localized at mitochondria. Interestingly, 6-16 is expressed at high levels in gastric cancer cell lines and tissues. One of exceptional gastric cancer cell line, TMK-1, which do not express detectable 6-16, is sensitive to apoptosis induced by cycloheximide (CHX), 5-fluorouracil (5-FU) and serum-deprivation. Ectopic expression of 6-16 gene restored the induction of apoptosis and inhibited caspase-3 activity in TMK-1 cells. Thus 6-16 protein has anti-apoptotic function through inhibiting caspas-3. This anti-apoptotic function is expressed through inhibition of the depolarization of mitochondrial membrane potential and release of cytochrome c. By two-hybrid screening, we found that 6-16 protein interacts with calcium and integrin binding protein, CIB/KIP/Calmyrin (CIB), which interacts with presenilin 2, a protein involved in Alzheimer's disease. These protein interactions possibly play a pivotal role in the regulation of apoptosis, for which further detailed analyses are need. These results overall indicate that 6-16 protein may have function as a cell survival protein by inhibiting mitochondrial-mediated apoptosis.

Triptolide, an active compound identified in a traditional Chinese herb, induces apoptosis of rheumatoid synovial fibroblasts.

BACKGROUND: Extracts of Tripterygium wilfordii Hook F (TWHF), a traditional Chinese herb, have been reported to show efficacy in patients with rheumatoid arthritis (RA). Since RA is not only characterized by inflammation but also by synovial proliferation in the joints, we examined whether triptolide (a constituent of TWHF) could influence the proliferation of rheumatoid synovial fibroblasts (RSF) by induction of apoptosis. RESULTS: RSF were obtained from RA patients during surgery and were treated with triptolide under various conditions. The viability and proliferation of RSF were measured by the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay and by 5-bromo-2'-deoxyuridine incorporation, respectively. Apoptosis was identified by detection of DNA fragmentation using an enzyme-linked immunosorbent assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). The role of caspases in apoptosis of RSF was analyzed by measuring caspase-3 activity. Activation of the peroxisome proliferator-activated receptor (PPAR) gamma was assessed by a luciferase reporter gene assay using RSF transfected with a plasmid containing the peroxisome proliferator response element. Triptolide decreased viability, inhibited proliferation, and induced apoptosis of RSF in a concentration-dependent manner at very low (nM) concentrations. Caspase-3 activity was increased by treatment with triptolide and was suppressed by caspase inhibitors. Although PPARgamma activation was induced by 15-deoxy-Delta12,14-prostaglandin J2, triptolide did not induce it under the same experimental conditions. An extract of TWHF also induced DNA fragmentation in RSF. CONCLUSION: The mechanism of action remains to be studied; however, triptolide may possibly have a disease-modifying effect in patients with RA.

Induction of apoptosis in rheumatoid synovial fibroblasts by celecoxib, but not by other selective cyclooxygenase 2 inhibitors.

OBJECTIVE: Selective cyclooxygenase 2 (COX-2) inhibitors are now being used as antiinflammatory agents that cause fewer gastrointestinal complications, compared with other antiinflammatory drugs, in patients with rheumatoid arthritis (RA). This study was undertaken to investigate whether selective COX-2 inhibitors could induce apoptosis of RA synovial fibroblasts (RASFs). METHODS: RASFs were exposed to selective COX-2 inhibitors, i.e., celecoxib, etodolac, meloxicam, nimesulide, N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methanesulfonamide, and rofecoxib, under various conditions. Cell proliferation and cell viability were assessed by 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 identifying DNA fragmentation. Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) was measured by the luciferase reporter gene assay with a PPAR response element-driven luciferase reporter plasmid and a PPARgamma expression plasmid. RESULTS: Celecoxib strongly inhibited the proliferation of RASFs, whereas other selective COX-2 inhibitors had little or no effect. In addition, celecoxib reduced the viability of RASFs by induction of apoptosis, in a concentration-dependent manner. This action was abolished by addition of caspase inhibitors. Interleukin-1beta had a weak enhancing effect on celecoxib-induced apoptosis in RASFs. In contrast, other selective COX-2 inhibitors at concentrations up to 100 microM did not induce apoptosis of RASFs. Indomethacin, a nonselective COX inhibitor, activated PPARgamma transcription, while celecoxib did not. CONCLUSION: Celecoxib suppressed the proliferation of RASFs by COX-2-independent and PPARgamma-independent induction of apoptosis. Although the mechanism involved remains unclear, celecoxib may have not only antiinflammatory activity, but also a disease-modifying effect on rheumatoid synovial proliferation.

Selective cyclooxygenase-2 inhibitors show a differential ability to inhibit proliferation and induce apoptosis of colon adenocarcinoma cells.

Although the influence of selective cyclooxygenase (COX)-2 inhibitors on the proliferation of colon adenocarcinoma cells have been the subject of much investigation, relatively little research has compared the effects of different COX-2 inhibitors. Celecoxib strongly suppressed the proliferation of COX-2 expressing HT-29 cells at 10-40 microM. NS-398 and nimesulide also inhibited cell proliferation, whereas rofecoxib, meloxicam, and etodolac did not. Only celecoxib induced apoptosis of HT-29 cells, as detected on the basis of DNA fragmentation, TUNEL positivity, and caspase-3/7 activation. DNA fragmentation was also increasd in COX-2 non-expressing cell lines (SW-480 and HCT-116) by exposure to celecoxib for 6-24 h. All six COX-2 inhibitors suppressed the production of prostaglandin E(2) by HT-29 cells, suggesting that the pro-apoptotic effect of celecoxib was unrelated to inhibition of COX-2. Inactivation of Akt might explain the differential pro-apoptotic effect of these selective COX-2 inhibitors on colon adenocarcinoma cells.

SN-38 induces cell cycle arrest and apoptosis in human testicular cancer.

OBJECTIVE: CPT-11 is one of the most widely used camptothecin analogues and is converted to form the active metabolite SN-38. Clinical trials are ongoing to better characterize its spectra of clinical activity, to determine the optimal schedules of administration, and to define the usage in combination with other chemotherapeutic compounds. MATERIALS AND METHODS: KU-MT, an AFP-producing testicular carcinoma cell line, was exposed to SN-38, etoposide, or cisplatin for 24 h, and the resulting cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay. This paper describes the effects of SN-38 on the cell proliferation and cell cycle of testicular tumor cells in culture. RESULTS: SN-38 was shown to inhibit KU-MT cell growth more potently than either etoposide or cisplatin. A marked decrease in the percentage of S phase cells was accompanied by the enhancement of cyclin E levels. In concentrations of >30 nmol/l, SN-38 arrested the cell cycle in G2 and induced cell death via apoptosis. The apoptosis was promoted by Bax and p53 protein, which were both shown to be present by flow cytometric and Western blot analyses. CONCLUSION: These results suggest that CPT-11, a pro-drug of SN-38, may be clinically useful for the treatment of testicular cancer, and that the mechanism of this agent's cytotoxicity consists of cell cycle arrest and concomitant apoptosis.

Nonsteroidal anti-inflammatory drugs induce apoptosis in association with activation of peroxisome proliferator-activated receptor gamma in rheumatoid synovial cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been reported to induce apoptosis in a variety of cell lines. In this study, we examined the effect of NSAIDs on the growth and apoptosis of synovial cells from patients with rheumatoid arthritis and analyzed the activation of peroxisome proliferator-activated receptor gamma (PPARgamma) as a possible mechanism of action of NSAIDs. Cell proliferation and viability were assessed from 5-bromo-2'-deoxyuridine incorporation and by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay, respectively. The apoptosis of synovial cells was identified by DNA fragmentation assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Indometacin, diclofenac, oxaprozin, and zaltoprofen reduced cell proliferation and induced apoptotic cell death in synovial cells, whereas ketoprofen and acetaminophen did not. N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methanesulfonamide (NS-398), a selective cyclooxygenase-2 inhibitor, also inhibited cell proliferation, whereas it did not cause apoptosis. Rheumatoid synovial cells expressed PPARgamma mRNA, and the PPARgamma ligands 15-deoxy-Delta(12,14)-prostaglandin J(2) and troglitazone reduced the proliferation and induced apoptosis in synovial cells. Luciferase reporter assay demonstrated that not only PPARgamma ligands but also NSAIDs, which could induce apoptosis, increased the activation of PPARgamma in synovial cells. Furthermore, the ability of NSAIDs and PPARgamma ligands to stimulate the activation of PPARgamma correlated with their ability to decrease cell viability(r = 0.92, p < 0.01) and ability to induce DNA fragmentation (r = 0.97, p < 0.001) in synovial cells. These results suggest that PPARgamma is an attractive target for induction of apoptosis in rheumatoid synovial cells and that the activation of the PPARgamma pathway is associated with the apoptotic action of NSAIDs.

Aspirin and sodium salicylate inhibit proliferation and induce apoptosis in rheumatoid synovial cells.

Aspirin has been reported to induce apoptosis in a variety of cell lines. In this study, we examined whether aspirin and sodium salicylate inhibit cell growth and induce apoptosis in rheumatoid synovial cells. Synovial cells were obtained from patients with rheumatoid arthritis, and the cells were treated with aspirin or sodium salicylate (0.1-10 mM) for 24 h. Cell proliferation and viability were assessed by 5-bromo-2'-deoxyuridine incorporation and by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay, respectively. The apoptosis of synovial cells was identified by DNA fragmentation assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay. Aspirin and sodium salicylate suppressed the proliferation (IC50 (concentration causing 50% inhibition of cell proliferation): 2.1 and 1.2 mM, respectively) and reduced the viability (IC50: 2.0 and 1.4 mM, respectively) of synovial cells in a concentration-dependent manner at 0.3-10 mM. Furthermore, they induced DNA fragmentation and increased the number of TUNEL-positive synovial cells. These results suggest that aspirin and sodium salicylate can inhibit the proliferation of rheumatoid synovial cells through induction of apoptosis.