Thrombosis is reduced by inhibition of COX-1, but unaffected by inhibition of COX-2, in an acute model of platelet activation in the mouse

Background

Clinical use of selective inhibitors of cyclooxygenase (COX)-2 appears associated with increased risk of thrombotic events. This is often hypothesised to reflect reduction in anti-thrombotic prostanoids, notably PGI₂, formed by COX-2 present within endothelial cells. However, whether COX-2 is actually expressed to any significant extent within endothelial cells is controversial. Here we have tested the effects of acute inhibition of COX on platelet reactivity using a functional in vivo approach in mice.

Methodology/Principal Findings

A non-lethal model of platelet-driven thromboembolism in the mouse was used to assess the effects of aspirin (7 days orally as control) diclofenac (1 mg.kg⁻¹, i.v.) and parecoxib (0.5 mg.kg⁻¹, i.v.) on thrombus formation induced by collagen or the thromboxane (TX) A₂-mimetic, U46619. The COX inhibitory profiles of the drugs were confirmed in mouse tissues ex vivo. Collagen and U46619 caused in vivo thrombus formation with the former, but not latter, sensitive to oral dosing with aspirin. Diclofenac inhibited COX-1 and COX-2 ex vivo and reduced thrombus formation in response to collagen, but not U46619. Parecoxib inhibited only COX-2 and had no effect upon thrombus formation caused by either agonist.

Conclusions/Significance

Inhibition of COX-1 by diclofenac or aspirin reduced thrombus formation induced by collagen, which is partly dependent upon platelet-derived TXA₂, but not that induced by U46619, which is independent of platelet TXA₂. These results are consistent with the model demonstrating the effects of COX-1 inhibition in platelets, but provide no support for the hypothesis that acute inhibition of COX-2 in the circulation increases thrombosis.     

Diclofenac Inhibits Tumor Growth in a Murine Model of Pancreatic Cancer by Modulation of VEGF Levels and Arginase Activity

Background

Diclofenac is one of the oldest anti-inflammatory drugs in use. In addition to its inhibition of cyclooxygenases (COX), diclofenac potently inhibits phospholipase A₂ (PLA₂), thus yielding a broad anti-inflammatory effect. Since inflammation is an important factor in the development of pancreatic tumors we explored the potential of diclofenac to inhibit tumor growth in mice inoculated with PANCO2 cells orthotopically.

Methodology/Principal Findings

We found that diclofenac treatment (30 mg/kg/bw for 11 days) of mice inoculated with PANC02 cells, reduced the tumor weight by 60%, correlating with increased apoptosis of tumor cells. Since this effect was not observed in vitro on cultured PANCO2 cells, we theorized that diclofenac beneficial treatment involved other mediators present in vivo. Indeed, diclofenac drastically decreased tumor vascularization by downregulating VEGF in the tumor and in abdominal cavity fluid. Furthermore, diclofenac directly inhibited vascular sprouting ex vivo. Surprisingly, in contrast to other COX-2 inhibitors, diclofenac increased arginase activity/arginase 1 protein content in tumor stroma cells, peritoneal macrophages and white blood cells by 2.4, 4.8 and 2 fold, respectively. We propose that the subsequent arginine depletion and decrease in NO levels, both in serum and peritoneal cavity, adds to tumor growth inhibition by malnourishment and poor vasculature development.

Conclusion/Significance

In conclusion, diclofenac shows pronounced antitumoral properties in pancreatic cancer model that can contribute to further treatment development. The ability of diclofenac to induce arginase activity in tumor stroma, peritoneal macrophages and white blood cells provides a tool to study a controversial issue of pro-and antitumoral effects of arginine depletion.     

Diclofenac Prolongs Repolarization in Ventricular Muscle with Impaired Repolarization Reserve

Background

The aim of the present work was to characterize the electrophysiological effects of the non-steroidal anti-inflammatory drug diclofenac and to study the possible proarrhythmic potency of the drug in ventricular muscle.

Methods

Ion currents were recorded using voltage clamp technique in canine single ventricular cells and action potentials were obtained from canine ventricular preparations using microelectrodes. The proarrhythmic potency of the drug was investigated in an anaesthetized rabbit proarrhythmia model.

Results

Action potentials were slightly lengthened in ventricular muscle but were shortened in Purkinje fibers by diclofenac (20 µM). The maximum upstroke velocity was decreased in both preparations. Larger repolarization prolongation was observed when repolarization reserve was impaired by previous BaCl₂ application. Diclofenac (3 mg/kg) did not prolong while dofetilide (25 µg/kg) significantly lengthened the QT_c interval in anaesthetized rabbits. The addition of diclofenac following reduction of repolarization reserve by dofetilide further prolonged QT_c. Diclofenac alone did not induce Torsades de Pointes ventricular tachycardia (TdP) while TdP incidence following dofetilide was 20%. However, the combination of diclofenac and dofetilide significantly increased TdP incidence (62%). In single ventricular cells diclofenac (30 µM) decreased the amplitude of rapid (I_Kr) and slow (I_Ks) delayed rectifier currents thereby attenuating repolarization reserve. L-type calcium current (I_Ca) was slightly diminished, but the transient outward (I_to) and inward rectifier (I_K1) potassium currents were not influenced.

Conclusions

Diclofenac at therapeutic concentrations and even at high dose does not prolong repolarization markedly and does not increase the risk of arrhythmia in normal heart. However, high dose diclofenac treatment may lengthen repolarization and enhance proarrhythmic risk in hearts with reduced repolarization reserve.     

Atorvastatin reduces lipopolysaccharide-induced expression of cyclooxygenase-2 in human pulmonary epithelial cells

Objective

To explore the effects of atorvastatin on expression of cyclooxygenase-2 (COX-2) in human pulmonary epithelial cells (A549).

Methods

A549 cells were incubated in DMEM medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. After incubation, the medium was collected and the amount of prostaglandin E₂ (PGE₂) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, and COX-2 mRNA and protein were analyzed by RT-PCR and western-blot respectively.

Results

LPS increased the expression of COX-2 mRNA and production of PGE₂ in a dose- and time-dependent manner in A549. Induction of COX-2 mRNA and protein by LPS were inhibited by atorvastatin in a dose-dependent manner. Atorvastatin also significantly decreased LPS-induced production of PGE₂. There was a positive correlation between reduced of COX-2 mRNA and decreased of PGE₂ (r = 0.947, P < 0.05).

Conclusion

Atorvastatin down-regulates LPS-induced expression of the COX-2 and consequently inhibits production of PGE₂ in cultured A549 cells.     

Identification of Novel Piperazinylquinoxaline Derivatives as Potent Phosphoinositide 3-Kinase (PI3K) Inhibitors

Background

Development of small-molecule inhibitors targeting phosphoinositide 3-kinase (PI3K) has been an appealing strategy for the treatment of various types of cancers.

Methodology/Principal Finding

Our approach was to perform structural modification and optimization based on previously identified morpholinoquinoxaline derivative WR1 and piperidinylquinoxaline derivative WR23 with a total of forty-five novel piperazinylquinoxaline derivatives synthesized. Most target compounds showed low micromolar to nanomolar antiproliferative potency against five human cancer cell lines using MTT method. Selected compounds showed potent PI3Kα inhibitory activity in a competitive fluorescent polarization assay, such as compound 22 (IC₅₀ 40 nM) and 41 (IC₅₀: 24 nM), which induced apoptosis in PC3 cells. Molecular docking analysis was performed to explore possible binding modes between target compounds and PI3K.

Conclusions/Significance

The identified novel piperazinylquinoxaline derivatives that showed potent PI3Kα inhibitory activity and cellular antiproliferative potency may be promising agents for potential applications in cancer treatment.     

Cause-Specific Cardiovascular Risk Associated with Nonsteroidal Anti-Inflammatory Drugs among Myocardial Infarction Patients - A Nationwide Study

Background

Non steroidal anti-inflammatory drugs (NSAIDs) increase mortality and morbidity after myocardial infarction (MI). We examined cause-specific mortality and morbidity associated with NSAIDs in a nationwide cohort of MI patients.

Methods and Results

By individual-level linkage of nationwide registries of hospitalization and drug dispensing from pharmacies in Denmark, patients aged >30 years admitted with first-time MI during 1997–2009 and their subsequent NSAID use were identified. The risk of three cardiovascular specific endpoints: cardiovascular death, the composite of coronary death and nonfatal MI, and the composite of fatal and nonfatal stroke, associated with NSAID use was analyzed by Cox proportional hazard analyses. Of 97,698 patients included 44.0% received NSAIDs during follow-up. Overall use of NSAIDs was associated with an increased risk of cardiovascular death (hazard ratio [HR] 1.42, 95% confidence interval [CI] 1.36–1.49). In particular use of the nonselective NSAID diclofenac and the selective cyclooxygenase-2 inhibitor rofecoxib was associated with increased risk of cardiovascular death (HR 1.96 [1.79–2.15] and HR1.66 [1.44–1.91], respectively) with a dose dependent increase in risk. Use of ibuprofen was associated with increased risk of cardiovascular death (HR 1.34[1.26–1.44]), whereas naproxen was associated with the lowest risk of (e.g., HR 1.27[1.01–1.59].

Conclusion

Use of individual NSAIDs is associated with different cause-specific cardiovascular risk and in particular rofecoxib and diclofenac were associated with increased cardiovascular morbidity and mortality. These results support caution with use of all NSAIDs in patients with prior MI.     

Antioxidant biomarkers from Vanda coerulea stems reduce irradiated HaCaT PGE-2 production as a result of COX-2 inhibition

Background

In our investigations towards the isolation of potentially biologically active constituents from Orchidaceae, we carried out phytochemical and biological analyses of Vanda species. A preliminary biological screening revealed that Vanda coerulea (Griff. ex. Lindl) crude hydro-alcoholic stem extract displayed the best DPPH /^•OH radical scavenging activity and in vitro inhibition of type 2 prostaglandin (PGE-2) release from UV_B (60 mJ/cm²) irradiated HaCaT keratinocytes.

Principal Findings

Bio-guided fractionation and phytochemical analysis led to the isolation of five stilbenoids: imbricatin (1) methoxycoelonin (2) gigantol (3) flavidin (4) and coelonin (5). Stilbenoids (1–3) were the most concentrated in crude hydro-alcoholic stem extract and were considered as Vanda coerulea stem biomarkers. Dihydro-phenanthropyran (1) and dihydro-phenanthrene (2) displayed the best DPPH/^•OH radical scavenging activities as well as HaCaT intracellular antioxidant properties (using DCFH-DA probe: IC₅₀ 8.8 µM and 9.4 µM, respectively) compared to bibenzyle (3) (IC₅₀ 20.6 µM). In turn, the latter showed a constant inhibition of PGE-2 production, stronger than stilbenoids (1) and (2) (IC₅₀ 12.2 µM and 19.3 µM, respectively). Western blot analysis revealed that stilbenoids (1–3) inhibited COX-2 expression at 23 µM. Interestingly, stilbenoids (1) and (2) but not (3) were able to inhibit human recombinant COX-2 activity.

Conclusions

Major antioxidant stilbenoids (1–3) from Vanda coerulea stems displayed an inhibition of UV_B-induced COX-2 expression. Imbricatin (1) and methoxycoelonin (2) were also able to inhibit COX-2 activity in a concentration-dependent manner thereby reducing PGE-2 production from irradiated HaCaT cells. Our studies suggest that stilbenoids (1–3) could be potentially used for skin protection against the damage caused by UV_B exposure.     

Raltegravir Cerebrospinal Fluid Concentrations in HIV-1 Infection

Introduction

Raltegravir is an HIV-1 integrase inhibitor currently used in treatment-experienced HIV-1-infected patients resistant to other drug classes. In order to assess its central nervous system penetration, we measured raltegravir concentrations in cerebrospinal fluid (CSF) and plasma in subjects receiving antiretroviral treatment regimens containing this drug.

Methods

Raltegravir concentrations were determined by liquid chromatography tandem mass spectrometry in 25 paired CSF and plasma samples from 16 HIV-1-infected individuals. The lower limit of quantitation was 2.0 ng/ml for CSF and 10 ng/ml for plasma.

Results

Twenty-four of the 25 CSF samples had detectable raltegravir concentrations with a median raltegravir concentration of 18.4 ng/ml (range, <2.0–126.0). The median plasma raltegravir concentration was 448 ng/ml (range, 37–5180). CSF raltegravir concentrations correlated with CSF:plasma albumin ratios and CSF albumin concentrations.

Conclusions

Approximately 50% of the CSF specimens exceeded the IC₉₅ levels reported to inhibit HIV-1 strains without resistance to integrase inhibitors. In addition to contributing to control of systemic HIV-1 infection, raltegravir achieves local inhibitory concentrations in CSF in most, but not all, patients. Blood-brain and blood-CSF barriers likely restrict drug entry, while enhanced permeability of these barriers enhances drug entry.     

Prostaglandin E2 synthesizing enzymes in rheumatoid arthritis B cells and the effects of B cell depleting therapy on enzyme expression

Introduction

B cells may play an important role in promoting immune activation in the rheumatoid synovium and can produce prostaglandin E₂ (PGE₂) when activated. In its turn, PGE₂ formed by cyclooxygenase (COX) and microsomal prostaglandin E₂ synthase 1 (MPGES1) contributes to the rheumatoid arthritis (RA) pathological process. Therapeutic depletion of B cells results in important improvement in controlling disease activity in rheumatoid patients. Therefore we investigated the expression of PGE₂ pathway enzymes in RA B cells and evaluated the effects of B cell depleting therapy on their expression in RA tissue.

Methods

B cells expressing MPGES1 and COX-2 were identified by flow cytometry in in vitro stimulated and control mononuclear cells isolated from synovial fluid and peripheral blood of RA patients. Synovial biopsies were obtained from 24 RA patients before and at two consecutive time points after rituximab therapy. Expression of MPGES1, COX-1 and COX-2, as well as interleukin (IL)-1β and IL-6, known inducers of MPGES1, was quantified in immunostained biopsy sections using computerized image analysis.

Results

Expression of MPGES1 or COX-2 was significantly upregulated upon stimulation of B cells from blood and synovial fluid while control cells displayed no detectable enzymes. In synovial biopsy sections, the expression of MPGES1, COX-1 or COX-2 was resistant to rituximab therapy at 8 or 16 weeks after start of treatment. Furthermore expression of IL-1β in the synovial tissue remained unchanged, while IL-6 tended to decrease after therapy.

Conclusions

Therapy with B cell depleting agents, although efficient in achieving good clinical and radiographic response in RA patients, leaves important inflammatory pathways in the rheumatoid synovium essentially unaffected.     

Pentacycloundecane lactam vs lactone norstatine type protease HIV inhibitors: binding energy calculations and DFT study

Background

Novel pentacycloundecane (PCU)-lactone-CO-EAIS peptide inhibitors were designed, synthesized, and evaluated against wild-type C-South African (C-SA) HIV-1 protease. Three compounds are reported herein, two of which displayed IC₅₀ values of less than 1.00 μM. A comparative MM-PB(GB)SA binding free energy of solvation values of PCU-lactam and lactone models and their enantiomers as well as the PCU-lactam-NH-EAIS and lactone-CO-EAIS peptide inhibitors and their corresponding diastereomers complexed with South African HIV protease (C-SA) was performed. This will enable us to rationalize the considerable difference between inhibitory concentration (IC₅₀) of PCU-lactam-NH-EAIS and PCU-lactone-CO-EAIS peptides.

Results

The PCU-lactam model exhibited more negative calculated binding free energies of solvation than the PCU-lactone model. The same trend was observed for the PCU-peptide inhibitors, which correspond to the experimental activities for the PCU-lactam-NH-EAIS peptide (IC₅₀ = 0.076 μM) and the PCU-lactone-CO-EAIS peptide inhibitors (IC₅₀ = 0.850 μM). Furthermore, a density functional theory (DFT) study on the natural atomic charges of the nitrogen and oxygen atoms of the three PCU-lactam, PCU-lactim and PCU-lactone models were performed using natural bond orbital (NBO) analysis. Electrostatic potential maps were also used to visualize the electron density around electron-rich regions. The asymmetry parameter (η) and quadrupole coupling constant (χ) values of the nitrogen and oxygen nuclei of the model compounds were calculated at the same level of theory. Electronic molecular properties including polarizability and electric dipole moments were also calculated and compared. The Gibbs theoretical free solvation energies of solvation (∆G_solv) were also considered.

Conclusions

A general trend is observed that the lactam species appears to have a larger negative charge distribution around the heteroatoms, larger quadrupole constant, dipole moment and better solvation energy, in comparison to the PCU-lactone model. It can be argued that these characteristics will ensure better eletronic interaction between the lactam and the receptor, corresponding to the observed HIV protease activities in terms of experimental IC₅₀ data.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0115-5) contains supplementary material, which is available to authorized users.     

Evidence for a Pro-Proliferative Feedback Loop in Prostate Cancer: The Role of Epac1 and COX-2-Dependent Pathways

Objective

In human prostate cancer cells, a selective Epac agonist, 8-CPT-2Me-cAMP, upregulates cell proliferation and survival via activation of Ras-MAPK and PI- 3-kinase-Akt-mTOR signaling cascades. Here we examine the role of inflammatory mediators in Epac1-induced cellular proliferation by determining the expression of the pro-inflammatory markers p-cPLA2, COX-2, and PGE₂ in prostate cancer cells treated with 8-CPT-2Me-cAMP.

Methods

We employed inhibitors of COX-2, mTORC1, and mTORC2 to probe cyclic AMP-dependent pathways in human prostate cancer cells. RNAi targeting Epac1, Raptor, and Rictor was also employed in these studies.

Results

8-CPT-2Me-cAMP treatment caused a 2–2.5-fold increase of p-cPLA2^S505, COX-2, and PGE₂ levels in human prostate cancer cell lines. Pretreatment of cells with the COX-2 inhibitor SC-58125 or the EP4 antagonist AH-23848, or with an inhibitor of mTORC1 and mTORC2, Torin1, significantly reduced the Epac1-dependent increase of p-cPLA2 and COX-2, p-S6-kinase^T389, and p-AKT^S473. In addition, Epac1-induced protein and DNA synthesis were greatly reduced upon pretreatment of cells with either COX-2, EP4, or mTOR inhibitors. Transfection of prostate cancer cells with Epac1 dsRNA, Raptor dsRNA, or Rictor dsRNA profoundly reduced Epac1-dependent increases in p-cPLA2 and COX-2.

Conclusion

We show that Epac1, a downstream effector of cAMP, functions as a pro-inflammatory modulator in prostate cancer cells and promotes cell proliferation and survival by upregulating Ras-MAPK, and PI 3-kinase-Akt-mTOR signaling.     

Correlation of MicroRNA-16, MicroRNA-21 and MicroRNA-101 Expression with Cyclooxygenase-2 Expression and Angiogenic Factors in Cirrhotic and Noncirrhotic Human Hepatocellular Carcinoma

Background

Hepatocellular carcinoma (HCC) is a classical example of inflammation-linked cancer and is characterized by hypervascularity suggesting rich angiogenesis. Cycloxygenase-2 (COX-2) is a potent mediator of inflammation and is considered to upregulate angiogenesis. The aims of the study are (1) to analyze expression of Cox-2 mRNA, Cox-2 protein, miR-16, miR-21 and miR-101 in HCC and adjacent liver parenchyma in cirrhotic and noncirrhotic liver, (2) to investigate the relation between COX-2 expression, miR-21 expression and angiogenic factors in these tissues and (3) to investigate the association between miR-16 and miR-101 and COX-2 expression.

Methods

Tissue samples of HCC and adjacent liver parenchyma of 21 noncirrhotic livers and 20 cirrhotic livers were analyzed for COX-2 expression at the mRNA level (qRT-PCR) and at the protein level by Western blot and immunohistochemistry. Gene expression of VEGFA, VEGFR1, VEGFR2, Ang-1, Ang-2 and Tie-2 were correlated with COX-2 levels. miR-16, miR-21 and miR-101 gene expression levels were quantified in HCC tumor tissue.

Results

COX-2 mRNA and protein levels were lower in HCC as compared to adjacent liver parenchyma both in cirrhotic and noncirrhotic liver. COX-2 protein localized mainly in vascular and sinusoidal endothelial cells and in Kupffer cells. At the mRNA level but not at the protein level, COX-2 correlated with mRNA levels of angiogenic factors VEGFR1, Ang-1, and Tie2. miR-21 expression was higher in cirrhotic tissues versus noncirrhotic tissues. MiR-101 expression was lower in cirrhotic versus noncirrhotic adjacent liver parenchyma. None of the miRNAs correlelated with COX-2 expression. miR-21 correlated negatively with Tie-2 receptor in adjacent liver parenchyma.

Conclusions

In human HCC, COX-2 mRNA but not COX-2 protein levels are associated with expression levels of angiogenic factors. MiR-21 levels are not associated with angiogenic molecules. MiR-16 and miR-101 levels do not correlate with COX-2 mRNA and protein levels.     

Drug Synergy of Tenofovir and Nanoparticle-Based Antiretrovirals for HIV Prophylaxis

Background

The use of drug combinations has revolutionized the treatment of HIV but there is no equivalent combination product that exists for prevention, particularly for topical HIV prevention. Strategies to combine chemically incompatible agents may facilitate the discovery of unique drug-drug activities, particularly unexplored combination drug synergy. We fabricated two types of nanoparticles, each loaded with a single antiretroviral (ARV) that acts on a specific step of the viral replication cycle. Here we show unique combination drug activities mediated by our polymeric delivery systems when combined with free tenofovir (TFV).

Methodology/Principal Findings

Biodegradable poly(lactide-co-glycolide) nanoparticles loaded with efavirenz (NP-EFV) or saquinavir (NP-SQV) were individually prepared by emulsion or nanoprecipitation techniques. Nanoparticles had reproducible size (d ∼200 nm) and zeta potential (-25 mV). The drug loading of the nanoparticles was approximately 7% (w/w). NP-EFV and NP-SQV were nontoxic to TZM-bl cells and ectocervical explants. Both NP-EFV and NP-SQV exhibited potent protection against HIV-1 BaL infection in vitro. The HIV inhibitory effect of nanoparticle formulated ARVs showed up to a 50-fold reduction in the 50% inhibitory concentration (IC₅₀) compared to free drug. To quantify the activity arising from delivery of drug combinations, we calculated combination indices (CI) according to the median-effect principle. NP-EFV combined with free TFV demonstrated strong synergistic effects (CI₅₀ = 0.07) at a 1∶50 ratio of IC₅₀ values and additive effects (CI₅₀ = 1.05) at a 1∶1 ratio of IC₅₀ values. TFV combined with NP-SQV at a 1∶1 ratio of IC₅₀ values also showed strong synergy (CI₅₀ = 0.07).

Conclusions

ARVs with different physicochemical properties can be encapsulated individually into nanoparticles to potently inhibit HIV. Our findings demonstrate for the first time that combining TFV with either NP-EFV or NP-SQV results in pronounced combination drug effects, and emphasize the potential of nanoparticles for the realization of unique drug-drug activities.     

Gemcitabine-mediated radiosensitization of human soft tissue sarcoma

Background/Purpose

Local and systemic control of soft tissue sarcoma (STS) remains a clinical challenge, particularly for retroperitoneal, deep truncal, or advanced extremity disease. 2′,2′-Difluoro-2′-deoxycytidine (gemcitabine) is a potent radiosensitizer in many tumor types, but it has not been studied in human STS. The purpose of this study was to determine the radiosensitizing potential of gemcitabine in preclinical models of human STS.

Materials and Methods

The in vitro radiosensitizing activity of gemcitabine was assessed with clonogenic survival assay on three human STS cell lines: SK-LMS-1 (leiomyosarcoma), SW-872 (liposarcoma), and HT-1080 (fibrosarcoma). Cell cycle distribution was determined using dual-channel flow cytometry. The in vivo radiosensitizing activity of gemcitabine was assessed with subcutaneous SK-LMS-1 nude mice xenografts. Tumor-bearing mice were treated with concurrent weekly gemcitabine and fractionated daily radiotherapy (RT) (2 Gy daily) for 3 weeks (a total dose of 30 Gy).

Results

The 50% inhibitory concentration (IC₅₀) of gemcitabine for the human STS cell lines ranged from 10 to 1000 nM. Significant in vitro radiosensitization was demonstrated in all three human STS cell lines using gemcitabine concentrations at and below the IC₅₀. Maximal radiosensitization was associated with accumulation of cells in early S-phase. SK-LMS-1 xenografts displayed significant tumor growth delay with combined gemcitabine and RT compared to either treatment alone. Treatment related toxicity was greatest in the gemcitabine plus RT arm, but remained at an acceptable level.

Conclusions

Gemcitabine is a potent radiosensitizer in preclinical models of human STS. Clinical trials combining gemcitabine and RT in human STS are warranted.     

An intranasal selective antisense oligonucleotide impairs lung cyclooxygenase-2 production and improves inflammation,but worsens airway function,in house dust mite sensitive mice

Background

Despite its reported pro-inflammatory activity, cyclooxygenase (COX)-2 has been proposed to play a protective role in asthma. Accordingly, COX-2 might be down-regulated in the airway cells of asthmatics. This, together with results of experiments to assess the impact of COX-2 blockade in ovalbumin (OVA)-sensitized mice in vivo, led us to propose a novel experimental approach using house dust mite (HDM)-sensitized mice in which we mimicked altered regulation of COX-2.

Methods

Allergic inflammation was induced in BALBc mice by intranasal exposure to HDM for 10 consecutive days. This model reproduces spontaneous exposure to aeroallergens by asthmatic patients. In order to impair, but not fully block, COX-2 production in the airways, some of the animals received an intranasal antisense oligonucleotide. Lung COX-2 expression and activity were measured along with bronchovascular inflammation, airway reactivity, and prostaglandin production.

Results

We observed impaired COX-2 mRNA and protein expression in the lung tissue of selective oligonucleotide-treated sensitized mice. This was accompanied by diminished production of mPGE synthase and PGE₂ in the airways. In sensitized mice, the oligonucleotide induced increased airway hyperreactivity (AHR) to methacholine, but a substantially reduced bronchovascular inflammation. Finally, mRNA levels of hPGD synthase remained unchanged.

Conclusion

Intranasal antisense therapy against COX-2 in vivo mimicked the reported impairment of COX-2 regulation in the airway cells of asthmatic patients. This strategy revealed an unexpected novel dual effect: inflammation was improved but AHR worsened. This approach will provide insights into the differential regulation of inflammation and lung function in asthma, and will help identify pharmacological targets within the COX-2/PG system.     

The Novel Deacetylase Inhibitor AR-42 Demonstrates Pre-Clinical Activity in B-Cell Malignancies In Vitro and In Vivo

Background

While deacetylase (DAC) inhibitors show promise for the treatment of B-cell malignancies, those introduced to date are weak inhibitors of class I and II DACs or potent inhibitors of class I DAC only, and have shown suboptimal activity or unacceptable toxicities. We therefore investigated the novel DAC inhibitor AR-42 to determine its efficacy in B-cell malignancies.

Principal Findings

In mantle cell lymphoma (JeKo-1), Burkitt''s lymphoma (Raji), and acute lymphoblastic leukemia (697) cell lines, the 48-hr IC₅₀ (50% growth inhibitory concentration) of AR-42 is 0.61 µM or less. In chronic lymphocytic leukemia (CLL) patient cells, the 48-hr LC₅₀ (concentration lethal to 50%) of AR-42 is 0.76 µM. AR-42 produces dose- and time-dependent acetylation both of histones and tubulin, and induces caspase-dependent apoptosis that is not reduced in the presence of stromal cells. AR-42 also sensitizes CLL cells to TNF-Related Apoptosis Inducing Ligand (TRAIL), potentially through reduction of c-FLIP. AR-42 significantly reduced leukocyte counts and/or prolonged survival in three separate mouse models of B-cell malignancy without evidence of toxicity.

Conclusions/Significance

Together, these data demonstrate that AR-42 has in vitro and in vivo efficacy at tolerable doses. These results strongly support upcoming phase I testing of AR-42 in B-cell malignancies.     

Inhibition of Human Neutrophil Elastase by Pentacyclic Triterpenes

Scope

Inhibiting human neutrophil elastase (HNE) is a promising strategy for treating inflammatory lung diseases, such as H1N1 and SARS virus infections. The use of sivelestat, the only clinically registered synthesized HNE inhibitor, is largely limited by its risk of organ toxicity because it irreversibly inhibits HNE. Therefore, potent reversible HNE inhibitors are promising alternatives to sivelestat.

Methods and Results

An in vitro HNE inhibition assay was employed to screen a series of triterpenes. Six pentacyclic triterpenes, but not tetracyclic triterpenes, significantly inhibited HNE. Of these pentacyclic triterpenes, ursolic acid exhibited the highest inhibitory potency (IC₅₀ = 5.51 µM). The HNE inhibitory activity of ursolic acid was further verified using a mouse model of acute smoke-induced lung inflammation. The results of nuclear magnetic resonance and HNE inhibition kinetic analysis showed that the pentacyclic triterpenes competitively and reversibly inhibited HNE. Molecular docking experiments indicated that the molecular scaffold, 28-COOH, and a double bond at an appropriate location in the pentacyclic triterpenes are important for their inhibitory activity.

Conclusion

Our results provide insights into the effects of pentacyclic triterpenes on lung inflammatory actions through reversible inhibition of HNE activity.     

Curcumin as a Potent and Selective Inhibitor of 11β-Hydroxysteroid Dehydrogenase 1: Improving Lipid Profiles in High-Fat-Diet-Treated Rats

Background

11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) activates glucocorticoid locally in liver and fat tissues to aggravate metabolic syndrome. 11β-HSD1 selective inhibitor can be used to treat metabolic syndrome. Curcumin and its derivatives as selective inhibitors of 11β-HSD1 have not been reported.

Methodology

Curcumin and its 12 derivatives were tested for their potencies of inhibitory effects on human and rat 11β-HSD1 with selectivity against 11β-HSD2. 200 mg/kg curcumin was gavaged to adult male Sprague-Dawley rats with high-fat-diet-induced metabolic syndrome for 2 months.

Results and Conclusions

Curcumin exhibited inhibitory potency against human and rat 11β-HSD1 in intact cells with IC₅₀ values of 2.29 and 5.79 µM, respectively, with selectivity against 11β-HSD2 (IC₅₀, 14.56 and 11.92 µM). Curcumin was a competitive inhibitor of human and rat 11β-HSD1. Curcumin reduced serum glucose, cholesterol, triglyceride, low density lipoprotein levels in high-fat-diet-induced obese rats. Four curcumin derivatives had much higher potencies for Inhibition of 11β-HSD1. One of them is (1E,4E)-1,5-bis(thiophen-2-yl) penta-1,4-dien-3-one (compound 6), which had IC₅₀ values of 93 and 184 nM for human and rat 11β-HSD1, respectively. Compound 6 did not inhibit human and rat kidney 11β-HSD2 at 100 µM. In conclusion, curcumin is effective for the treatment of metabolic syndrome and four novel curcumin derivatives had high potencies for inhibition of human 11β-HSD1 with selectivity against 11β-HSD2.     

Antitumor activity of Noscapine in combination with Doxorubicin in triple negative breast cancer

Background

The aim of this study was to investigate the anticancer activity and mechanism of action of Noscapine alone and in combination with Doxorubicin against triple negative breast cancer (TNBC).

Methods

TNBC cells were pretreated with Noscapine or Doxorubicin or combination and combination index values were calculated using isobolographic method. Apoptosis was assessed by TUNEL staining. Female athymic Nu/nu mice were xenografted with MDA-MB-231 cells and the efficacy of Noscapine, Doxorubicin and combination was determined. Protein expression, immunohistochemical staining were evaluated in harvested tumor tissues.

Results

Noscapine inhibited growth of MDA-MB-231 and MDA-MB-468 cells with the IC₅₀ values of 36.16±3.76 and 42.7±4.3 µM respectively. The CI values (<0.59) were suggestive of strong synergistic interaction between Noscapine and Doxorubicin and combination treatment showed significant increase in apoptotic cells. Noscapine showed dose dependent reduction in the tumor volumes at a dose of 150–550 mg/kg/day compared to controls. Noscapine (300 mg/kg), Doxorubicin (1.5 mg/kg) and combination treatment reduced tumor volume by 39.4±5.8, 34.2±5.7 and 82.9±4.5 percent respectively and showed decreased expression of NF-KB pathway proteins, VEGF, cell survival, and increased expression of apoptotic and growth inhibitory proteins compared to single-agent treatment and control groups.

Conclusions

Noscapine potentiated the anticancer activity of Doxorubicin in a synergistic manner against TNBC tumors via inactivation of NF-KB and anti-angiogenic pathways while stimulating apoptosis. These findings suggest potential benefit for use of oral Noscapine and Doxorubicin combination therapy for treatment of more aggressive TNBC.     

Activation of interleukin-32 pro-inflammatory pathway in response to influenza A virus infection

Background

Interleukin (IL)-32 is a recently described pro-inflammatory cytokine that has been reported to be induced by bacteria treatment in culture cells. Little is known about IL-32 production by exogenous pathogens infection in human individuals.

Methods and Findings

In this study, we found that IL-32 level was increased by 58.2% in the serum samples from a cohort of 108 patients infected by influenza A virus comparing to that of 115 healthy individuals. Another pro-inflammatory factor cyclooxygenase (COX)-2-associated prostaglandin E2 was also upregulated by 2.7-fold. Expression of IL-32 in influenza A virus infected A549 human lung epithelial cells was blocked by either selective COX-2 inhibitor NS398 or Aspirin, a known anti-inflammatory drug, indicating IL-32 was induced through COX-2 in the inflammatory cascade. Interestingly, we found that COX-2-associate PGE₂ production activated by influenza virus infection was significantly suppressed by over-expression of IL-32 but increased by IL-32-specific siRNA, suggesting there was a feedback mechanism between IL-32 and COX-2.

Conclusions

IL-32 is induced by influenza A virus infection via COX-2 in the inflammatory cascade. Our results provide that IL-32 is a potential target for anti-inflammatory medicine screening.