Systematic analysis of potential targets of the curcumin analog pentagamavunon-1 (PGV-1) in overcoming resistance of glioblastoma cells to bevacizumab.

BACKGROUND: Glioblastoma is one of the most aggressive and deadliest malignant tumors. Acquired resistance decreases the effectiveness of bevacizumab in glioblastoma treatment and thus increases the mortality rate in patients with glioblastoma. In this study, the potential targets of pentagamavunone-1 (PGV-1), a curcumin analog, were explored as a complementary treatment to bevacizumab in glioblastoma therapy. METHODS: Target prediction, data collection, and analysis were conducted using the similarity ensemble approach (SEA), SwissTargetPrediction, STRING DB, and Gene Expression Omnibus (GEO) datasets. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted using Webgestalt and DAVID, respectively. Hub genes were selected based on the highest degree scores using the CytoHubba. Analysis of genetic alterations and gene expression as well as Kaplan-Meier survival analysis of selected genes were conducted with cBioportal and GEPIA. Immune infiltration correlations between selected genes and immune cells were analyzed with database TIMER 2.0. RESULTS: We found 374 targets of PGV-1, 1139 differentially expressed genes (DEGs) from bevacizumab-resistant-glioblastoma cells. A Venn diagram analysis using these two sets of data resulted in 21 genes that were identified as potential targets of PGV-1 against bevacizumab resistance (PBR). PBR regulated the metabolism of xenobiotics by cytochrome P450. Seven potential therapeutic PBR, namely GSTM1, AKR1C3, AKR1C4, PTGS2, ADAM10, AKR1B1, and HSD17B110 were found to have genetic alterations in 1.2%-30% of patients with glioblastoma. Analysis using the GEPIA database showed that the mRNA expression of ADAM10, AKR1B1, and HSD17B10 was significantly upregulated in glioblastoma patients. Kaplan-Meier survival analysis showed that only patients with low mRNA expression of AKR1B1 had significantly better overall survival than the patients in the high mRNA group. We also found a correlation between PBR and immune cells and thus revealed the potential of PGV-1 as an immunotherapeutic agent via targeting of PBR. CONCLUSION: This study highlighted seven PBR, namely, GSTM1, AKR1C3, AKR1C4, PTGS2, ADAM10, AKR1B1, and HSD17B110. This study also emphasized the potential of PBR as a target for immunotherapy with PGV-1. Further validation of the results of this study is required for the development of PGV-1 as an adjunct to immunotherapy for glioblastoma to counteract bevacizumab resistance.

Cucurbita argyrosperma Seed Extracts Attenuate Angiogenesis in a Corneal Chemical Burn Model.

Severe corneal inflammation produces opacity or even perforation, scarring, and angiogenesis, resulting in blindness. In this study, we used the cornea to examine the effect of new anti-angiogenic chemopreventive agents. We researched the anti-angiogenic effect of two extracts, methanol (Met) and hexane (Hex), from the seed of Cucurbita argyrosperma, on inflamed corneas. The corneas of Wistar rats were alkali-injured and treated intragastrically for seven successive days. We evaluated: opacity score, corneal neovascularization (CNV) area, re-epithelialization percentage, and histological changes. Also, we assessed the inflammatory (cyclooxigenase-2, nuclear factor-kappaB, and interleukin-1ß) and angiogenic (vascular endothelial growth factor A, VEGF-A; -receptor 1, VEGFR1; and -receptor 2, VEGFR2) markers. Levels of Cox-2, Il-1ß, and Vegf-a mRNA were also determined. After treatment, we observed a reduction in corneal edema, with lower opacity scores and cell infiltration compared to untreated rats. Treatment also accelerated wound healing and decreased the CNV area. The staining of inflammatory and angiogenic factors was significantly decreased and related to a down-expression of Cox-2, Il-1ß, and Vegf. These results suggest that intake of C. argyrosperma seed has the potential to attenuate the angiogenesis secondary to inflammation in corneal chemical damage.

Anti-inflammatory effects of shikonin in human periodontal ligament cells.

CONTEXT: Shikonin (SHI), an active component extracted from Radix Arnebiae, has been reported to possess anti-inflammatory properties in various cells. However, its effect on lipopolysaccharide (LPS)-stimulated human periodontal ligament cells (hPDLCs) is unknown. OBJECTIVE: To investigate the effects of SHI on the expression of inflammatory related cytokines in LPS-stimulated hPDLCs. MATERIALS AND METHODS: The effects of SHI (0.125, 0.25, 0.5, 1, and 2 µg/mL) on hPDLCs proliferation for 1, 3 and 7 days were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of interleukin-1 (IL-1), IL-6, tumor necrosis factor-α (TNF-α), matrix metalloproteinase-2 (MMP-2), MMP-9 and cyclooxygenase-2 (COX-2) were detected in hPDLCs following SHI treatment (0.25 and 0.5 µg/mL) using Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). The signaling pathways triggered by SHI in hPDLC were evaluated using western blotting. RESULTS: LD50 of SHI is 1.7 µg/mL (day 1) and 1.1 µg/mL (day 3 and 7) in hPDLCs. No morphological changes were observed when hPDLCs were treated with LPS only (1 µg/mL) or LPS with SHI (0.25 and 0.5 µg/mL). Data from qRT-PCR suggests that SHI attenuates LPS-induced increases of IL-1, IL-6, TNF-α, MMP-2, MMP-9 and COX-2 in hPDLCs. Down-regulation of phosphorylated extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB), and up-regulation of I-κB, were observed in LPS-stimulated hPDLCs after exposed to SHI at 0.25 or 0.5 µg/mL. DISCUSSION AND CONCLUSIONS: SHI possesses anti-inflammatory effects in LPS-stimulated hPDLCs via phospho-ERK and NF-κB/I-κB signaling pathways; this suggests that SHI may hold potential as an anti-inflammatory agent against periodontitis.

In vitro comprehensive analysis of VA692 a new chemical entity for the treatment of osteoarthritis.

Selective cyclooxigenase (COX)-2 inhibitors were developed to prevent traditional non-steroidal anti-inflammatory drugs (tNSAIDs) gastro-intestinal adverse effects. VA692, a recently disclosed selective COX-2 inhibitor, structurally related to well-known marketed coxibs, showed anti-inflammatory, and anti-nociceptive properties. The aim of this study was to analyze the anti-inflammatory effect of VA692, in comparison with celecoxib. At this purpose we evaluated the pro-inflammatory cytokines and anti-oxidant enzymes gene expression, apoptosis and ROS production, and PGE2 release in chondrocytes (both primary cultures and immortalized T/C-28a2 cell line) treated with the two drugs. Furthermore, a proteomic analysis has been performed in T/C-28a2 cell line to evaluate modifications in their proteomic profile following drug treatment in presence of IL-1ß. Our results demonstrated the anti-inflammatory effect of the novel synthesized VA692, and confirmed those of celecoxib, in counteracting the stimulus of IL-1ß in both osteoarthritic (OA) chondrocytes and T/C-28a2 cell line. Furthermore, the data underlined the possible anti-apoptotic and anti-oxidant role of VA692, implying its regulation in superoxide anion production as indicated by the modulation of anti-oxidant enzymes. The proteomic analysis provides new information about the effect of VA692 on human T/C-28a2 intracellular proteome, demonstrating the usefulness of this approach in the identification and quantifications of several proteins. Modulation of some proteins such as Hsp90 and SOD by VA692 could explain its role in the therapeutic approach of OA. Based on our results, we can affirm that VA692 has more beneficial effect compared with celecoxib particularly regarding the modulation of oxidant/anti-oxidant system and proteome profile of human articular chondrocytes.

Cyclooxygenase-2 immunoexpression in intestinal epithelium and lamina propria of cats with inflammatory bowel disease and low grade alimentary lymphoma.

BACKGROUND: Cyclooxygenase 2 (COX-2) is an inducible isoform by cellular activation, proinflammatory cytokines and growth factors. The aims of the current study were to evaluate COX-2 immunoexpression in epithelial and lamina propria (LP) of cats with inflammatory bowel disease (IBD) and low grade alimentary lymphoma (LGAL), as well as to correlate them with clinical signs and histopathological scoring. Cats diagnosed with IBD and LGAL (2007-2013) were included in the current study. Feline chronic enteropathy activity index (FCEAI) was calculated for all cases. Control group was composed by 3 healthy indoor cats and 5 sick cats died or were euthanized (non-gastrointestinal illness). Diagnosis and classification of IBD and LGAL was established according to the WSAVA gastrointestinal standardization group template and the National Cancer Institute formulation, respectively. Furthermore, a modified WSAVA template was applied for LGAL evaluation. Immunolabelling for COX-2 (polyclonal rabbit anti-murine antibody) was performed on biopsy samples. Epithelial and LP (inflammatory or neoplastic cells) COX-2 immunolabelling was calculated according to the grade and intensity. The most representative segment scored by the WSAVA and the modified WSAVA were used for statistical analysis. RESULTS: Significant difference was found regarding COX-2 intensity overexpression in the epithelial cells of IBD and LGAL groups when compared to control cats, but not between the groups of sick cats, whereas no differences were found regarding the grade of immunoreactivity between groups. No difference was found for COX-2 immunoexpression at the LP between all groups. However, 3 cats from LGAL group showed COX-2 expression in neoplastic cells at the LP. There were no correlations between epithelial or LP COX-2 expression and FCEAI and histological alterations. CONCLUSIONS: Increased COX-2 intensity at the epithelial cells observed in cats with IBD and LGAL may be secondary to the inflammatory response or a protective function in the intestinal reparation. COX-2 expression at the LP was presented in 33% of LGAL. This result provides a reason for further investigation concerning the role of COX-2 expression in feline alimentary lymphoma.

COX-2 as a determinant of lower disease-free survival for patients affected by ameloblastoma.

Ameloblastoma is a locally aggressive neoplasm with a poorly understood pathogenesis. Therefore, the aim of this study is to investigate whether COX-2 expression is associated with ameloblastoma microvascular density (MVD) and with tumor aggressiveness. Sixty-three cases of primary ameloblastomas arranged in tissue microarray were submitted to immunohistochemistry against cyclooxigenase-2 (COX-2) and CD34. Clinicopathological parameters regarding sex, age, tumour size, tumour duration, tumour location, treatment, recurrences, radiographic features, vestibular/lingual and basal cortical disruption and follow-up data were obtained from patients' medical records and correlated with the proteins expression. The results on BRAF-V600E expression were obtained from our previous study and correlated with COX-2 and CD34 expressions. Log-rank univariate analysis and multivariate Cox regression model were done to investigate the prognostic potential of the molecular markers. Twenty-eight cases (44.4%) exhibited cytoplasmic positivity for COX-2, predominantly in the columnar peripheral cells, with a mean MVD of 2.2 vessels/mm2. COX-2 was significantly associated with recurrences (p < 0.001) and BRAF-V600E expression (p < 0.001), whereas lower MVD was associated with the use of conservative therapy (p = 0.004). Using univariate and multivariate analyses, COX-2 was significantly associated with a lower 5-year disease-free survival (DFS) rate (p < 0.001 and p = 0.012, respectively), but not with a higher MVD (p = 0.68). In conclusion, COX-2 expression in ameloblastomas is not associated with MVD, but it is significantly associated with recurrences and with a lower DFS.

Chondroprotective effect of three different classes of anti-inflammatory agents on human osteoarthritic chondrocytes exposed to IL-1ß.

VA694, a promising cyclooxigenase-2 (COX-2)-inhibiting hybrid drug endowed with nitric oxide (NO) releasing properties (NO-COXIB), showed COX-2-selective inhibitory effects, associated with interesting anti-inflammatory and anti-nociceptive activities. Therefore, we studied the effects of VA694 on cartilage metabolism, in comparison with Naproxcinod, a COX inhibitor and NO donor (CINOD), and Naproxen, a traditional non-steroidal-anti-inflammatory drug (NSAID) on human osteoarthritic chondrocyte cultures. IL-1ß-stimulated chondrocytes showed a significant decrease in cell viability (P<0.001). VA694, Naproxcinod and Naproxen alone didn't significantly affect cell viability, while it restored cell viability in cultures stimulated by IL-1ß. The presence of IL-1ß determined a significant increase (P<0.001) in PGE2 levels measured by an ELISA assay, and in COX-2 and MMP-3, -9, and -13 gene expression analyzed by RT-PCR. VA694, Naproxcinod and Naproxen, at both concentrations analyzed, significantly counteracted the negative effects induced by IL-1ß. VA694, Naproxcinod and Naproxen pre-treatment were able to inhibit IL-1ß-induced NF-κB activation, when measured as its nuclear translocation (p50 and p65 subunits). Naproxcinod and Naproxen pre-treatment didn't affect cytoplasmic NF-κB levels; VA694 decreased the cytoplasmic levels of both subunits. Our data suggest that VA694, Naproxcinod and Naproxen, exert anti-inflammatory and chondroprotective effects on OA chondrocytes.

New 1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides inhibit hepatitis C virus replication via suppression of cyclooxygenase-2.

We report here the synthesis and mechanism of inhibition of pyrazolecarboxamide derivatives as a new class of HCV inhibitors. Compounds 6, 7, 8 and 16 inhibited the subgenomic HCV replicon 1b genotype at EC50 values between 5 and 8 µM and displayed an even higher potency against the infectious Jc1 HCV 2a genotype. Compound 6 exhibited an EC50 of 6.7 µM and selectivity index of 23 against HCV 1b, and reduced the RNA copies of the infectious Jc1 chimeric 2a clone by 82% at 7 µM. Evaluation of the mode of anti-HCV activity of 6 revealed that it suppressed HCV-induced COX-2 mRNA and protein expression, displaying an IC50 of 3.2 µM in COX-2 promoter-linked luciferase reporter assay. Conversely, the anti-HCV activity of 6 was abrogated upon over-expression of COX-2. These findings suggest that 6 as a representative of these pyrazolecarboxamides function as anti-HCV agents via targeting COX-2 at both the transcription and translation levels.

Gut-liver axis improves with meloxicam treatment after cirrhotic liver resection.

AIM: To investigate the effect of meloxicam on the gut-liver axis after cirrhotic liver resection. METHODS: Forty-four male Wistar rats were assigned to three groups: (1) control group (CG); (2) bile duct ligation with meloxicam treatment (BDL + M); and (3) bile duct ligation without meloxicam treatment (BDL). Secondary biliary liver cirrhosis was induced via ligature of the bile duct in the BDL + M and BDL groups. After 2 wk, the animals underwent a 50% hepatectomy. In the BDL + M group 15 min prior to the hepatectomy, one single dose of meloxicam was administered. Parameters measured included: microcirculation of the liver and small bowel; portal venous flow (PVF); gastrointestinal (GI) transit; alanine aminotransferase (ALT); malondialdehyde; interleukin 6 (IL-6), transforming growth factor beta 1 (TGF-ß1) and hypoxia-inducible factor 1 alpha (HIF-1α) levels; mRNA expression of cyclooxigenase-2 (COX-2), IL-6 and TGF-ß1; liver and small bowel histology; immunohistochemical evaluation of hepatocyte and enterocyte proliferation with Ki-67 and COX-2 liver expression. RESULTS: Proliferative activity of hepatocytes after liver resection, liver flow and PVF were significantly higher in CG vs BDL + M and CG vs BDL group (P < 0.05), whereas one single dose of meloxicam ameliorated liver flow and proliferative activity of hepatocytes in BDL + M vs BDL group. COX-2 liver expression at 24 h observation time (OT), IL-6 concentration and mRNA IL-6 expression in the liver especially at 3 h OT, were significantly higher in BDL group when compared with the BDL + M and CG groups (P < 0.01, P < 0.001, P < 0.01, respectively). Liver and small bowel histology, according to a semi quantitative scoring system, showed better integrity in BDL + M and CG as compared to BDL group. ALT release and HIF-1α levels at 1 h OT were significantly higher in BDL + M compared to CG and BDL group (P < 0.001 and P < 0.01, respectively). Moreover, ALT release levels at 3 and 24 h OT were significantly higher in BDL group compared to CG, P < 0.01. GI transit, enterocyte proliferative activity and number of goblet cells were in favor of meloxicam treatment vs BDL group (P < 0.05, P < 0.001, P < 0.01, respectively). Additionally, villus length were higher in BDL + M as compared to BDL group. CONCLUSION: One single dose of meloxicam administered after cirrhotic liver resection was able to cause better function and integrity of the remaining liver and small bowel.

Expresión de ciclo-oxigenasa 2 en carcinoma de laringe / Cyclooxigenase-2 expression in laryngeal carcinoma

Introducción: La sobrevida de carcinoma escamoso de laringe no ha aumentado significativamente en los últimos 20 años. Ciclo-oxigenasa 2 (COX-2) es una molécula que tiene un rol en la progresión tumoral ya que estimula la proliferación y angiogénesis e inhibe apoptosis celular. Objetivo: Evaluar la expresión de COX-2 en muestras de carcinoma escamoso de laringe. Material y método: Pacientes atendidos en los hospitales Barros Luco Trudeau y San Juan de Dios entre 2008 y 2011 con diagnóstico de carcinoma escamoso de laringe sin tratamiento previo. Se recolectaron tacos de biopsia los cuales fueron estudiados mediante inmunohistoquímica para la expresión de COX-2. Protocolo aprobado por Comité de Ética de la Facultad de Medicina de la Universidad de Chile. Resultados: Se incluyeron 32 casos. En 17 de ellos se describe sobreexpresión de COX-2, lo que equivale al 53% de la muestra. No hubo correlación con edad, sexo ni hábito tabáquico. Conclusión: La sobreexpresión de COX-2 es un fenómeno frecuente en carcinoma escamoso de laringe por lo cual es una molécula interesante para considerar como candidata a terapia adyuvante.

Introduction: Survival of laryngeal squamous cell carcinoma has not improved significantly in the last 20 years. Cyclooxigenase 2 (COX-2) has a role in carcinogenesis since it induces proliferation and angiogenesis, and inhibits apoptosis. Aim: To evaluate the expression of COX-2 in samples of laryngeal carcinoma. Material and methods: Patients attending Hospital San Juan de Dios and Barros Luco-Trudeau with diagnose of laryngeal carcinoma between 2008 and 2011 were included. Formalin fixed paraffin embedded samples were collected and COX-2 expression was assayed with immunohistochemistry. The study was approved by the ethics committee of Facultad de Medicina Universidad de Chile, and all patients consented. Results: 32 cases were included. COX-2 was overexpressed in 17, that is 53%. No correlation was identified with age, sex, or smoking habit. Conclusion: COX-2 overexpression is a frequent phenomenon in laryngeal carcinoma.

Paraquat Exposure Up-regulates Cyclooxygenase-2 in the Lungs, Liver and Kidneys in Rats.

Paraquat is a commonly used herbicide in many countries which can lead to systematic poisoning on exposure, In this study, paraquat (PQ)-induced changes in the expression of Cyclooxygenase-2 (COX-2) along with biochemical and histopathological changes in the lungs, liver and kidneys were studied. Twenty four male Wistar rats (180-200 g) were exposed either against saline as control group or various doses of PQ (3.5, 7 and 10 mg/kg, SC) as test groups for 7 consecutive days. The animals in test groups demonstrated a significant increase of malondialdehyde and NO contents, while a remarkable decrease of total thiol molecules was recorded. Histopathological studies revealed a severe alveolar edema and hemorrhages in the lungs, congestion and glycogen degeneration in the liver and multifocal interstitial nephritis along with proximal tubular degeneration in the kidneys. PQ up-regulated the COX-2 expression at mRNA level significantly in the examined organs. This data suggest that the PQ-induced oxidative disturbances and pathological damages can be attributed to the upregulation of COX-2 in examined organs.

Determinación de la cicloxigenasa-2en carcinoma escamocelular de lengua bien diferenciado, moderadamente diferenciado y poco diferenciado / Determination of cyclooxigenase-2 in well-differentiated, moderately differ entiated and P oorly differ entiated squamous cell carcinoma of the tongue

Propósito: Determinar y comparar la expresión de la cicloxigenasa-2 (COX-2) en el carcinomaescamocelular de lengua (CECL) según el grado de diferenciación, con la finalidadde explorar si este puede ser un marcador molecular útil en el diagnóstico y pronóstico delcáncer de la cavidad oral. Métodos: Se utilizaron 45 especímenes con CECL, 15 de ellos biendiferenciados, 12 moderadamente diferenciados, 18 mal diferenciados, un control positivode carcinoma de colon y un control negativo de mucosa oral sana. La identificación de laCOX-2 se obtuvo por medio de inmunohistoquímica. Resultados: La muestra de mucosalingual sana expresó la COX-2 en bajo nivel; el 60 % de las quince muestras de carcinomaescamocelular diferenciado mostraron un bajo nivel de expresión, el 41,3 % de 12 muestrasde carcinoma moderadamente diferenciado mostraron una alta expresión, y el 74 % de 18muestras de carcinoma no diferenciado mostraron una alta expresión de la enzima. Conclusión:La expresión de la COX-2 aumenta si el carcinoma es indiferenciado, lo cual sugiereque esta enzima podría desempeñar un papel importante en el desarrollo histopatológicodel CECL, tanto en las etapas iniciales como en las tardías...

Aim: Determine and compare the expression of cyclooxygenase-2 (COX-2) in squamouscell carcinoma of the tongue (SCCT) in three degrees of differentiation, in order to verify ifthis may be a molecular marker useful in the diagnosis and prognosis of oral cavity cancer.Methods: The sample consisted of 45 specimens with SCCT (15 well-differentiated, 12 moderatelydifferentiated, 18 poorly differentiated), a positive control (colon carcinoma) and anegative control (healthy oral mucosa). The identification of COX-2 was obtained throughimmunohistochemistry. Results: Samples of healthy lingual mucosa showed a low expressionof COX-2, 60 % of the 15 samples of well-differentiated squamous cell carcinoma showeda low expression of COX-2, 41.3 % of 12 samples of moderately differentiated carcinomashowed high expression, and 74 % of the 18 non-differentiated carcinoma samples showeda high expression of the enzyme. Conclusion: The expression of COX-2 increases in lessdifferentiated squamous cell carcinoma, which suggests that identification of COX-2 in thehistologic development of squamous carcinoma of the tongue might be important in thedifferentiation of both, the early and late stag es...

Treatment of mild to moderate Graves' ophthalmopathy with sodium diclofenac: a pilot study / Tratamento da oftalmopatia de Graves leve a moderada com diclofenato de sódio: um estudo piloto

OBJECTIVE: To report the use of sodium diclofenac, an antagonist of PPAR-gamma and cyclooxigenase-2 (COX-2) inhibitor in the treatment of mild to moderate Graves' ophthalmopathy. SUBJECTS AND METHODS: Thirteen patients with clinical activity score (CAS) 2 to 7 were treated during a period ranging from 3 to 12 months (mean 7.8 ± 3.4) with oral sodium diclofenac, 50 mg every 12 hours. RESULTS: Extra-ocular muscle restriction and CAS improved significantly, p = 0.003 and = 0.004, respectively. Ocular pain and diplopia disappeared, except for one patient who reported improvement of these symptoms. No recurrence was found after interruption of treatment. CONCLUSIONS: Treatment of moderate Graves' ophthalmopathy with oral sodium diclofenac is a good, safe and less expensive therapeutic option. Like others new treatment trials, findings must be confirmed in a greater number of patients in a controlled study.

OBJETIVO: Relatar o uso do diclofenato de sódio, um antagonista do PPAR-gama e inibidor da ciclooxigenase-2 (COX-2) no tratamento da leve a moderada oftalmopatia de Graves. SUJEITOS E MÉTODOS: Treze pacientes com CAS (clinical activity score) 2 a 7 foram tratados durante um período de 3 a 12 meses (média 7,6 ± 3,4) com diclofenaco de sódio por via oral na dose de 50 mg a cada 12 horas. RESULTADOS: A restrição da musculatura extraocular e o índice CAS melhoraram de modo significativo, respectivamente p = 0,003 e p = 0,004. A dor ocular e a diplopia desapareceram, com exceção de um paciente que referiu melhora desses sintomas. Não houve recidiva após a interrupção do tratamento. CONCLUSÕES: O tratamento da oftalmopatia de Graves de média gravidade com diclofenaco de sódio por via oral é uma opção boa, segura e de baixo custo. Como outros novos tratamentos, ele deverá ser confirmado em um maior número de pacientes em estudos controlados.