Specific COX-2 Inhibitor Research Articles

Cyclooxygenase-2-Derived Prostacyclin Protective Role on Endotoxin-Induced Mouse Cardiomyocyte Mortality

Cardiovascular dysfunction characterizes septic shock, inducing multiple organ failure and a high mortality rate. In the heart, it has been shown an up-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions with subsequent overproduction of nitric oxide (NO) and eicosanoids. This study is focused on the links between these products of inflammation and cell loss of mouse cardiomyocytes during treatment by the Salmonella typhimurium lipopolysaccharide (LPS) in presence or in absence of NOS or COX inhibitors. LPS induced RelA/NF-κB p65 activation, iNOS and COX-2 up-regulations, resulting in NO and prostacyclin releases. These effects were reversed by the NO-synthase inhibitor and increased by the specific COX-2 inhibitor. Immunostainings with FITC-conjugated anti-Annexin-V and propidium iodide and caspase 3/7 activity assay showed that cardiomyocyte necrosis was inhibited by L-NA during LPS treatment challenge, while apoptosis was induced in presence of both LPS and NS-398. No effect on LPS cellular injury was observed using the specific cyclooxygenase-1 (COX-1) inhibitor, SC-560. These findings strongly support the hypothesis of a link between iNOS-dependent NO overproduction and LPS-induced cell loss with a selective protective role allotted to COX-2 and deriving prostacyclins.

Pharmacological profile of FK881(ASP6537), a novel potent and selective cyclooxygenase-1 inhibitor

Nonsteroidal anti-inflammatory drugs (NSAIDs) are now understood to fall into one of two agent classes in clinical use. Traditional NSAIDs inhibit both cyclooxygenases-1 and 2 (COX-1, 2), which act as key enzymes catalyzing the same reaction in the production of prostaglandins (PGs), while the second class of NSAIDs selectively inhibit COX-2. Inhibition of the inducible COX-2 isoform is believed to be responsible for the therapeutic effects of NSAIDs, such as anti-inflammatory, analgesic, and antipyretic effects, while COX-1 inhibition results in side-effects on the gastrointestinal (GI) system. In the present study, however, we changed this notion that inhibiting only COX-1 causes adverse effects. We discovered FK881, a specific COX-1 inhibitor which exhibits a 650-fold ratio for human whole blood COX-1/COX-2 and rats in vivo. In rats, FK881 dose dependently inhibited carrageenan-induced paw edema (ED30: 22 mg/kg; diclofenac ED30: 3.6 mg/kg, rofecoxib ED30: 26 mg/kg) and paw swelling associated with adjuvant arthritis (ED50: 17 mg/kg; diclofenac ED50: 1.4 mg/kg, rofecoxib ED50: 1.8 mg/kg). Further, FK881 dose dependently inhibited acetic acid-induced writhing in mice (ED50: 19 mg/kg; diclofenac ED50: 14 mg/kg, rofecoxib ED50: >100 mg/kg) and adjuvant arthritis hyperalgesia in rats (ED50: 1.8 mg/kg; diclofenac ED50: 1.0 mg/kg, rofecoxib ED50: 0.8 mg/kg). However, unlike traditional NSAIDs, GI tolerability was improved, although the antipyretic effect of FK881 was weak (NOEL: >320 mg/kg; diclofenac NOEL: <1 mg/kg, rofecoxib NOEL: 100 mg/kg). These results suggest that FK881 may be useful in treating symptoms of rheumatoid arthritis and osteoarthritis.

Selective cyclooxygenase-2 inhibitor cross-reactivity in aspirin-exacerbated respiratory disease

Aspirin-induced asthma (AIA) is a severe and difficult-to-treat allergic disease in which acute asthma attacks are induced by nonsteroidal anti-inflammatory drugs. Patients with AIA rarely experience asthma attacks when taking celecoxib, a specific inhibitor of cyclooxygenase (COX) 2. A 33-year-old woman had a severe asthma attack with hypoxia and lost consciousness after oral provocation testing with 15 mg of aspirin and also with 50 mg of celecoxib. After 2 months of treatment with 10 mg/day of oral prednisolone, 1600 μg/day of inhaled fluticasone propionate, montelukast as a leukotriene receptor antagonist (LTRA), and long-term beta-agonist, we again challenged her with a provocation test with up to 200 mg of celecoxib; this time there were neither allergic symptoms nor decrease in forced expiratory volume in 1 second. Patients with severe or poorly controlled asthma may experience asthma attacks even if using selective COX-2 inhibitors. However, treatment with steroids and an LTRAs may inhibit asthma attacks induced by celecoxib.

Inflammatory Nitrergic Dysfunction Evokes Intestinal Dysmotility

COX-2 expressing vector, and HCA-7, a COX-2 expressing human colon cancer cells were used. Stemness was estimated by ability to form spheroid known to enrich in stem cells and oct-4 expression. DNA methyltransferase activity, demethylase activity, and DNA global hypomethylation were measured. Promoter methylation of tumor suppressor genes (p16, E-cadherin, and hMLH1), and an oncogene, S100A, was determined by MSP or COBRA. Expressions of DNMT-1, -3A, and -3B and demethylase genes (AID and glycosylase) were determined by RT-PCR. [Results] RIE-COX showed significantly elevated potential for spheroid formation, oct-4 expression, DNA demethylase activity and expression, promoter hypermethylation of tumor suppressors and hypomethylation of S100A, and DNMT-3a expression and activity, and it also showed significantly downregulated global DNA methylation, compared with RIE. siRNA for COX-2 or celecoxib, a COX-2 specific inhibitor, significantly inhibited spheroid formation, global hypomethylation, hypermethylation of tumor suppressor genes, and DNA demethylase activity/expression in HCA-7. siRNA for DNMT3a or AID partly suppressed spheroid formation of HCA-7 and oct-4 expression. [Conclusion] These results indicate that COX-2 is involved in intricate epigenetic regulation; simultaneous global hypomethylation and gene-specific hypermethylation, through discrete modulation of DNMT/demethylase expression, and it is also suggested that COX-2 inhibitors have a potential to prevent epigenetic dysregulation and aberrant biology of cancer stem cell in carcinogenesis.

Novel Role of Macrophage in the Induction of TH2 Immunity

COX-2 expressing vector, and HCA-7, a COX-2 expressing human colon cancer cells were used. Stemness was estimated by ability to form spheroid known to enrich in stem cells and oct-4 expression. DNA methyltransferase activity, demethylase activity, and DNA global hypomethylation were measured. Promoter methylation of tumor suppressor genes (p16, E-cadherin, and hMLH1), and an oncogene, S100A, was determined by MSP or COBRA. Expressions of DNMT-1, -3A, and -3B and demethylase genes (AID and glycosylase) were determined by RT-PCR. [Results] RIE-COX showed significantly elevated potential for spheroid formation, oct-4 expression, DNA demethylase activity and expression, promoter hypermethylation of tumor suppressors and hypomethylation of S100A, and DNMT-3a expression and activity, and it also showed significantly downregulated global DNA methylation, compared with RIE. siRNA for COX-2 or celecoxib, a COX-2 specific inhibitor, significantly inhibited spheroid formation, global hypomethylation, hypermethylation of tumor suppressor genes, and DNA demethylase activity/expression in HCA-7. siRNA for DNMT3a or AID partly suppressed spheroid formation of HCA-7 and oct-4 expression. [Conclusion] These results indicate that COX-2 is involved in intricate epigenetic regulation; simultaneous global hypomethylation and gene-specific hypermethylation, through discrete modulation of DNMT/demethylase expression, and it is also suggested that COX-2 inhibitors have a potential to prevent epigenetic dysregulation and aberrant biology of cancer stem cell in carcinogenesis.

A Novel Potential of Cyclooxygenase-2 Specific Inhibitors to Prevent Intricate Epigenetic Dysregulation and Aberrant Cancer Stem Cell Biology

A Novel Potential of Cyclooxygenase-2 Specific Inhibitors to Prevent Intricate Epigenetic Dysregulation and Aberrant Cancer Stem Cell Biology

Abstract 2900: COX-2 modulates murine lung adenocarcinoma cell survival by activating Akt and NF-κB pathways and inhibiting p38MAPK

Abstract Cyclooxygenase-2 (COX-2) expression is increased in a number of tumors along with elevated prostaglandin (PG) production. The treatment with COX-2 inhibitors was found to have antitumor properties in different types of cancers, although the dependency of COX-2 inhibition remains controversial. We aimed to study the role of COX-2 in the progression of a murine lung adenocarcinoma. COX-2-specific inhibitor celecoxib delayed tumor growth (p &amp;lt; 0.001 by day 30 after subcutaneous tumor inoculation) and the metastatic outcome of LP07 cells. The treatment with celecoxib reduced LP07 cell motility, invasiveness and the activity of secreted matrix metalloproteinase-2 (MMP-2). Celecoxib (25 μM) also increased the susceptibility of LP07 cells to serum deprivation (p &amp;lt; 0.001) and exogenously added PGE2 (10 μM) counteracted this effect. The reduced viability of tumor cells by specific COX-2 inhibition correlated with decreased levels (2.25 fold) of phosphorylated Akt in serum-stimulated LP07 cells and a reduced activity of NF-κB (p &amp;lt; 0.001), a transcription factor with survival/anti-apoptotic properties. We also found increased levels of phosphorylated p38 MAPK in serum-starved LP07 cells treated with celecoxib. The effect of COX-2 inhibition on cell viability was partially abolished by infecting LP07 cells with a retroviral vector coding for a constitutively active form of Akt or by transfection with a dominant negative p38. The activation of the Akt pathway partially reverted the inhibition of NF-κB activity. To further study the role of COX-2 in LP07 tumor biology, we inhibited COX-2 expression by infection with a retroviral vector with specific shRNAs. Clones with the lowest COX-2 expression were selected. Controls expressed a shRNA for GFP or carried an empty vector. The inhibition of COX-2 expression resulted in reduced tumor growth in mice (p &amp;lt; 0.001 in all cases compared to the shGFP control). Homogenates from these tumors showed a low level of phosphorylated Akt, in line with in vitro experiments where LP07 cells were less responsive to serum pulses in phosphorylating Akt if COX-2 expression was blocked. The inhibition of COX-2 expression reduced the viability of tumor cells after serum depletion (p &amp;lt; 0.01), and this effect was completely abolished by PGE2 and partially by the expression of a constitutively active form of Akt and PI3K, or a dominant negative p38. NF-κB activity was also reduced by the inhibition of COX-2 expression (p &amp;lt; 0.01). Taken together, we found that COX-2 is a key player for the malignant behavior of the LP07 lung tumor. COX-2 and PGE2 had an important role for LP07 cell survival by modulating the Akt and p38 MAPK and NF-κB pathways Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2900. doi:10.1158/1538-7445.AM2011-2900

C6-ceramide facilitates IL-12-mediated T helper-1 cell differentiation through a COX-2/PGE2-dependent pathway (152.17)

Abstract Ceramides, lipid molecules that are found predominantly in the plasma membrane of a cell, can serve as a second messenger and have been known to perform a number of cellular functions. Our study was designed to investigate the effect of ceramides on T helper cell responses and their underlying mechanisms. We found that a cell-permeable C6-ceramide (C6) together with IL-12 synergistically increased the populations of IFN-γ+ T cells and up-regulated IFN-γ production from CD4+ T cells, whereas C2-ceramide and long chain ceramides (C16 and C24) did not affect. C6 treatment increased the expression of T-bet, a master transcription factor of Th1 responses, in a dose-dependent manner. Moreover, C6 induced expression of cyclooxygenase-2 (COX-2) accompanied by prostaglandin E2 (PGE2) production in CD4+ T cells. The IFN-γ+ T cells populations and IFN-γ production induced by C6 were significantly suppressed by both a COX-2 specific inhibitor (NS398) and an anti-PGE2 neutralizing antibody in a dose-dependent manner. T-bet expression was also decreased by NS398 treatment. Taken together, C6 promotes the production of PGE2 in CD4+ T cells through a COX-2 dependent mechanism, thereby facilitating Th1 cell differentiation. These results suggest that C6 can be utilized in the therapeutics of diseases such as cancer and allergic by enhancing the Th1 activity.

Hansch analysis of novel pyrimidine derivatives as highly potent and specific COX-2 inhibitors

A QSAR study on novel Pyrimidine derivatives as specific COX-2 inhibitory agents was performed with 69 (59 training + 10 test) compounds. Molecular modeling studies were performed using chemoffice 6.0 supplied by cambridgesoft. The sketched structures were subjected to energy minimization and the lowest energy structure was used to calculate the physiochemical properties. The regression analysis was carried out using a computer program called SYSTAT 10.2. The best models were selected from the various statistically significant equations. The study revealed that the hydrogen bond donar groups at position-4 enhances the activity, electron with-drawing groups at position-2 reduces the activity, electron donating groups at position-6 enhances the activity. The analysis resulted in QSAR equation, which suggests that, n = 59, r = 0.957, r2 = 0.915, adjusted squared multiple R = 0.901, Standard error of estimate(s) = 0.294 & validated r2(q2) = 0.642. This study can help in rational drug design and synthesis of new selective cyclooxygenase-2 inhibitor with predetermined affinity.

Effects of chelerythrine, a specific inhibitor of cyclooxygenase-2, on acute inflammation in mice

Chelerythrine (CHE), a quaternary benzo[c]phenanthridine alkaloid, which is an agent in traditional Chinese medicine exhibits a wide spectrum of pharmacological effects. In this study, we examined the anti-inflammatory activities and mechanism of CHE in vivo and in vitro, respectively. Further, in the analgesic test, CHE also showed pronounced inhibition of the acetic acid-induced writhing response. These results clearly suggested that CHE is a bioactive agent which has a significant anti-inflammatory action, which may be relevant to the inhibition of the release/production of exudates and prostaglandin E 2 mediated through cyclooxygenase-2 regulation.

Interleukin-6 and soluble interleukin-6 receptor suppress osteoclastic differentiation by inducing PGE2 production in chondrocytes

This study examined how interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6r) influence osteoclastic differentiation through the function of chondrocytes. Chondrocytes were cultured with or without IL-6 and/or sIL-6r in the presence or absence of NS398, a specific inhibitor of cyclooxygenase (COX)-2, for up to 28 days. Chondrocytes were also cultured with or without IL-6 and sIL-6r for 28 days, and the conditioned medium from cells cultured without IL-6 and sIL-6r was used to induce differentiation of RAW264.7 cells into osteoclast precursors. Osteoclastic differentiation was assessed by tartrate-resistant acid phosphatase (TRAP) staining. Expression of osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), COX-2, and prostaglandin E(2) (PGE(2)) increased in cells exposed to IL-6 and sIL-6r, whereas expression of macrophage colony-stimulating factor (M-CSF) and bone resorption-related enzymes decreased. NS398 blocked the stimulatory/suppressive effects of IL-6 and sIL-6r on the expression of OPG, RANKL, and M-CSF. Fewer TRAP-positive multinucleated cells were detected after treatment with conditioned medium from IL-6- and sIL-6r-treated chondrocytes than after treatment with conditioned medium from untreated chondrocytes. These results suggest that IL-6 and sIL-6r interfere with osteoclast function through the involvement of chondrocytes. Specifically, they appear to suppress the differentiation of osteoclast precursors into osteoclasts by inducing chondrocytic PGE(2) production, which, in turn, increases OPG secretion and decreases M-CSF secretion by chondrocytes.

Systematic Analyses of Genes Associated with Radiosensitizing Effect by Celecoxib, a Specific Cyclooxygenase-2 Inhibitor

To investigate genes regulated by COX-2 or a COX-2 specific inhibitor, celecoxib, in irradiated cancer cells, we analyzed changes in gene expression using complementary DNA microarray following celecoxib or combined celecoxib and ionizing radiation (IR) treatment in a stable COX-2 knockdown A549 (AS) and a mock cell line (AN). Thirty-six genes were differentially expressed by COX-2 knockdown. Celecoxib changed the expressions of 40 and 69 genes in AN and AS cells, respectively. Twenty-seven genes were synchronously regulated by COX-2 and celecoxib. Among these, celecoxib regulated ras homolog gene family B and mitosin protein expression in a COX-2 dependent manner, especially in irradiated cells. In addition, we identified 11 genes that changed by more than 1.5 times the expected additive values after celecoxib and IR treatment. The current study may provide evidence that COX-2 or celecoxib regulates various intracellular functions in addition to their enzymatic activity regulation. We also identified candidate molecules that may be responsible for COX-2-dependent radiosensitization by celecoxib.

Ibuprofen Inhibits Activation of Nuclear β-Catenin in Human Colon Adenomas and Induces the Phosphorylation of GSK-3β

Nonselective cyclooxygenase (COX) inhibitors target many of the same cancer-associated molecular pathways as COX-2-specific inhibitors. Although these nonsteroidal anti-inflammatory drugs (NSAIDs) are often associated with gastrointestinal toxicity, there is renewed interest in their use as colorectal cancer (CRC) chemopreventive agents due to the adverse side effects associated with long-term use of selective COX-2 inhibitors. In this study, we investigated the effects of long-term use (up to 25 years) of NSAIDs (ibuprofen or aspirin) on adenoma pathology and β-catenin-mediated signaling in sporadic human colon adenomas. Although NSAID use did not impact overall adenoma size or degree of dysplasia, it did cause a significant inhibition of nuclear β-catenin localization, which correlated with suppression of cyclin D1 expression. To further elucidate the effect of these agents in regulating β-catenin, we treated SW480 colon cancer cells with a panel of NSAIDs and determined their effects on β-catenin levels and cellular localization. In agreement with our in vivo results, both S-ibuprofen and aspirin were found to decrease total levels of β-catenin while increasing its phosphorylation. In addition, S-ibuprofen induced both degradation of IκBα and nuclear localization of NF-κB. Despite its nuclear localization, however, the activation of the NF-κB target genes, Bcl-2, survivin, and cyclin D1, was suppressed. This reduction in NF-κB transcriptional activity may be due to increased phosphorylation of GSK-3β following S-ibuprofen treatment. These data suggest that ibuprofen can effectively target both the Wnt/β-catenin and NF-κB pathways, and potentially uncovers a novel mechanism through which NSAIDS may exert their chemopreventive efficacy.

Etodolac Attenuates Hippocampal Blood Flow Responses Induced by Tooth Pulp Stimulation in Rats

In this study, we investigated whether increases in hippocampal blood flow (HBF) by tooth pulp stimulation (TPS) are mediated via the activation of cyclooxygenase-2 (COX-2). We measured mean arterial blood pressure (MABP) and HBF in urethane-anesthetized rats using laser Doppler flowmetry and examined the effects of etodolac, a specific COX-2 inhibitor, on TPS-induced MABP and HBF responses. The MABP response was not attenuated by the intraperitoneal (i.p.) administration of etodolac (10 mg/kg); however, the HBF response was significantly attenuated by etodolac administration. These results suggest that COX-2 may be involved in the mechanisms regulating TPS-induced HBF responses.

Tumor-secreted PGE2 Inhibits CCL5 Production in Activated Macrophages through cAMP/PKA Signaling Pathway

One of the major characteristics of tumors is their ability to evade immunosurveillance through altering the properties and functions of host stromal and/or immune cells. CCL5 has been shown to play important roles in T cell proliferation, IFN-γ, and IL-2 production, which promotes the differentiation and proliferation of Th1 cells important for immune defense against intracellular infection. In this study we found that tumor-bearing mice were more susceptible to bacterial infection and showed reduced CCL5 levels in serum during endotoxic shock. Our data further demonstrated that the soluble factors secreted by mammary gland tumor cells but not normal mammary gland epithelial cells inhibited CCL5 expression in macrophages in response to LPS, but not to TNF-α stimulation. The inhibitory effect of tumor-secreted molecules on LPS-induced CCL5 expression was regulated at the post-transcriptional level. Blocking PGE(2) synthesis by NS398 or through the use of PGE(2) receptor antagonists AH-6809 (EP2 antagonist) and AH-23848 (EP4 antagonist) completely reversed the inhibitory effect of tumor-conditioned medium (TCM) on LPS-induced CCL5 expression. Moreover, PGE(2) and the cAMP analog forskolin could mimic tumor-mediated CCL5 inhibition, and the inhibitory effects of TCM, PGE(2), and cAMP analog on LPS-induced CCL5 expression could be completely reversed by the PKA inhibitor H89. Furthermore, blocking PGE(2) synthesis in vivo led to partial recovery of CCL5 production during endotoxic shock. Taken together, our data indicate that PGE(2) secreted from breast cancer cells suppresses CCL5 secretion in LPS-activated macrophages through a cAMP/PKA signaling pathway, which may result in suppression of host immune responses against subsequent bacterial infection.

Resveratrol and apoptosis

Resveratrol is a naturally occurring stilbene with desirable cardioprotective and anti-cancer properties. We have demonstrated the existence of a plasma membrane receptor for resveratrol near the arginine-glycine-aspartate (RGD) recognition site on integrin α(v)β₃ that is involved in stilbene-induced apoptosis of cancer cells. Resveratrol treatment in vitro causes activation and nuclear translocation of mitogen-activated protein kinase (ERK1/2), consequent phosphorylation of Ser-15 of p53, and apoptosis. An RGD peptide blocks these actions of resveratrol. By a PD98059-inhibitable process, resveratrol causes inducible COX-2 to accumulate in the nucleus where it complexes with pERK1/2 and p53. Chromatin immunoprecipitation reveals binding of nuclear COX-2 to promoters of certain p53-responsive genes, including PIG3 and Bax. NS-398, a specific pharmacologic inhibitor of COX-2, prevents resveratrol-induced complexing of nuclear ERK1/2 with COX-2 and with pSer-15-p53 and subsequent apoptosis; cyclooxygenase enzyme activity is not involved. Molecular steps in the pro-apoptotic action of resveratrol in cancer cells include induction of intranuclear COX-2 accumulation relevant to activation of p53. Epidermal growth factor, estrogen, and thyroid hormone act downstream of ERK1/2 to prevent resveratrol-induced apoptosis.

The effect of Cox-2 specific inhibition on direct fracture healing in the rabbit tibia

ObjectiveThe purpose of this study was to evaluate the effect of Cox-2 administration on direct (primary) fracture healing. MethodsA transverse tibial osteotomy was created in adult male rabbits and rigidly fixed in compression using a 2.7-mm dynamic compression plate. Animals were randomized to receive either rofecoxib (12.5mg orally per day) or placebo. Animals were killed at 4 weeks and fracture healing assessed by mechanical testing. ResultsThere were no significant differences between the control and Cox-2 treated animals in terms of mechanical strength at 4 weeks. There was a high complication rate of peri-implant fractures during the daily medication administration. ConclusionThe immediate administration of a Cox-2 specific inhibitor did not impair primary (direct) bone healing at the dose administered in this rabbit tibial osteotomy model.

Coxibs strike back

Coxibs strike back

Houttuynia cordata, a novel and selective COX-2 inhibitor with anti-inflammatory activity

Houttuynia cordata Thunb. (Saururaceae; HC) has been long used in traditional oriental medicine for the treatment of inflammation diseases. Modern research has implicated inducible cyclooxygenase-2 (COX-2) as a key regulator of the inflammatory process. In the present study, we aimed to investigate the effect of HC on COX-2. We examined the effects of HC on lipopolysaccharide (LPS)-induced prostaglandin (PG) E(2) production, an indirect indicator of COX-2 activity, and COX-2 gene and protein expression in mouse peritoneal macrophages. LPS-induced mouse peritoneal macrophages were employed as an in vitro model system. LPS-induced PGE(2) production was assessed by enzyme-linked immunosorbant assay and COX-2 protein expression was assessed by Western blot assay. The results showed that HC was able to inhibit the release of LPS-induced PGE(2) from mouse peritoneal macrophages (IC50 value: 44.8 μg/mL). Moreover, the inhibitory activity of HC essential oil elicited a dose-dependent inhibition of COX-2 enzyme activity (IC50 value: 30.9 μg/mL). HC was also found to cause reduction in LPS-induced COX-2 mRNA and protein expression, but did not affect COX-1 expression. The non-steroidal anti-inflammatory drug (NSAID) and specific COX-2 inhibitor NS398 functioned similarly in LPS-induced mouse peritoneal macrophages. Taken together, our data suggest HC mediates inhibition of COX-2 enzyme activity and can affect related gene and protein expression. HC works by a mechanism of action similar to that of NSAIDs. These results add a novel aspect to the biological profile of HC.

Optimization of cell-based assays to quantify the anti-inflammatory/allergic potential of test substances in 96-well format

There is an insistent need for robust, reliable, and optimized assays for screening novel drugs targeting the inflammatory/allergic markers. The present study describes about the optimization of eight cell-based assays utilizing mammalian cell lines in 96-well format for quantifying anti-inflammatory/allergic drug candidates. We estimated the inhibitory response of reference compounds: 1400 W dihydrochloride on LPS-induced NO release, celecoxib on LPS-induced PGE(2) production and dexamethasone on LPS-induced pro-inflammatory cytokines IL-1 beta, IL-6, and TNF-alpha production by J774A.1 murine macrophages. Response of acetylsalicylic acid and celecoxib was studied on A23187-induced TXB(2) production; captopril on A23187-stimulated LTB(4) production by HL-60 cells. Effect of ketotifen fumarate was evaluated on A23187-elicited histamine release by RBL-2H3 cells. Each experiment was repeated twice to assess the reproducibility and suitability of the assays by determining appropriate statistical tools viz. %CV, S/B and Z' factor. 1400 W dihydrochloride was capable of inhibiting LPS-induced NO levels (IC(50) = 10.7 μM). Dexamethasone attenuated LPS-induced IL-1 beta (IC(50) = 70 nM), IL-6 (IC(50) = 58 nM) and TNF-alpha (IC(50) = 44 nM) release, whereas celecoxib, a specific COX-2 inhibitor showed marked reduction in LPS-induced PGE(2) (IC(50) = 23 nM) production. Captopril (IC(50) = 48 μM) and ketotifen fumarate (IC(50) = 36.4 μM) demonstrated potent inhibitory effect against A23187-stimulated LTB(4) and histamine levels, respectively. Both acetylsalicylic acid (IC(50) = 5.5 μM) and celecoxib (IC(50) = 7.9 nM) exhibited concentration-dependent decrease in TXB(2) production. Results for all the cell assays from two experiments showed a Z' factor varying from 0.30 to 0.99; the S/B ratio ranged from 2.39 to 24.92; %CV ranged between 1.52 and 20.14. The results proclaim that these cell-based assays can act as ideal tools for screening new anti-inflammatory/anti-allergic compounds.