The selective Cox-2 inhibitor Celecoxib suppresses angiogenesis and growth of secondary bone tumors: an intravital microscopy study in mice

BACKGROUND: The inhibition of angiogenesis is a promising strategy for the treatment of malignant primary and secondary tumors in addition to established therapies such as surgery, chemotherapy, and radiation. There is strong experimental evidence in primary tumors that Cyclooxygenase-2 (Cox-2) inhibition is a potent mechanism to reduce angiogenesis. For bone metastases which occur in up to 85% of the most frequent malignant primary tumors, the effects of Cox-2 inhibition on angiogenesis and tumor growth remain still unclear. Therefore, the aim of this study was to investigate the effects of Celecoxib, a selective Cox-2 inhibitor, on angiogenesis, microcirculation and growth of secondary bone tumors. METHODS: In 10 male severe combined immunodeficient (SCID) mice, pieces of A549 lung carcinomas were implanted into a newly developed cranial window preparation where the calvaria serves as the site for orthotopic implantation of the tumors. From day 8 after tumor implantation, five animals (Celecoxib) were treated daily with Celecoxib (30 mg/kg body weight, s.c.), and five animals (Control) with the equivalent amount of the CMC-based vehicle. Angiogenesis, microcirculation, and growth of A549 tumors were analyzed by means of intravital microscopy. Apoptosis was quantified using the TUNEL assay. RESULTS: Treatment with Celecoxib reduced both microvessel density and tumor growth. TUNEL reaction showed an increase in apoptotic cell death of tumor cells after treatment with Celecoxib as compared to Controls. CONCLUSION: Celecoxib is a potent inhibitor of tumor growth of secondary bone tumors in vivo which can be explained by its anti-angiogenic and pro-apoptotic effects. The results indicate that a combination of established therapy regimes with Cox-2 inhibition represents a possible application for the treatment of bone metastases.

Cyclooxygenase-2 inhibition selectively attenuates bone morphogenetic protein-6 synthesis and bone formation during guided tissue regeneration in a rat model

OBJECTIVES: Bone formation during guided tissue regeneration is a tightly regulated process involving cells, extracellular matrix and growth factors. The aims of this study were (i) to examine the expression of cyclooxygenase-2 (COX-2) during bone regeneration and (ii) the effects of selective COX-2 inhibition on osseous regeneration and growth factor expression in the rodent femur model. MATERIAL AND METHODS: A standardized transcortical defect of 5 x 1.5 mm was prepared in the femur of 12 male rats and a closed half-cylindrical titanium chamber was placed over the defect. The expression of COX-2 and of platelet-derived growth factor-B (PDGF-B), bone morphogenetic protein-6 (BMP-6) and insulin-like growth factor-I/II (IGF-I/II) was analyzed at Days 3, 7, 21 and 28 semiquantitatively by reverse transcriptase-polymerase chain reaction and immunohistochemistry. The effects of COX-2 inhibition by intraperitoneal injection of NS-398 (3 mg/kg/day) were analyzed in five additional animals sacrificed at Day 14. RESULTS: Histomorphometry revealed that new bone formation occurred in the cortical defect area as well as in the supracortical region, i.e. region within the chamber by Day 7 and increased through Day 28. Immunohistochemical evidence of COX-2 and PDGF-B levels were observed early (i.e. Day 3) and decreased rapidly by Day 7. BMP-6 expression was maximal at Day 3 and slowly declined by Day 28. In contrast, IGF-I/II expression gradually increased during the 28-day period. Systemic administration NS-398 caused a statistically significant reduction (P<0.05) in new bone formation (25-30%) and was associated with a statistically significant reduction in BMP-6 protein and mRNA expression (50% and 65% at P<0.05 and P<0.01, respectively). PDGF-B mRNA or protein expression was not affected by NS-398 treatment. CONCLUSION: COX-2 inhibition resulted in reduced BMP-6 expression and impaired osseous regeneration suggesting an important role for COX-2-induced signaling in BMP synthesis and new bone formation.

Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2

In this study, we investigated the molecular mechanisms underlying the ATP analogue adenosine-5'-O-(3-thio)triphosphate-induced nucleocytoplasmic shuttling of the mRNA stabilizing factor HuR in human (h) mesangial cells (MC). Using synthetic protein kinase C (PKC) inhibitors and small interfering RNA approaches, we demonstrated that knockdown of PKC alpha efficiently blocked the ATP-dependent nuclear HuR export to the cytoplasm. The functional importance of PKC alpha in HuR shuttling is highlighted by the high cytosolic HuR content detected in hMC stably overexpressing PKC alpha compared with mock-transfected cells. The ATP-induced recruitment of HuR to the cytoplasm is preceded by a direct interaction of PKC alpha with nuclear HuR and accompanied by increased Ser phosphorylation as demonstrated by coimmunoprecipitation experiments. Mapping of putative PKC target sites identified serines 158 and 221 as being indispensable for HuR phosphorylation by PKC alpha. RNA pull-down assay and RNA electrophoretic mobility shift assay demonstrated that the HuR shuttling by ATP is accompanied by an increased HuR binding to cyclooxygenase (COX)-2 mRNA. Physiologically, the ATP-dependent increase in RNA binding is linked with an augmentation in COX-2 mRNA stability and subsequent increase in prostaglandin E(2) synthesis. Regulation of HuR via PKC alpha-dependent phosphorylation emphasizes the importance of posttranslational modification for stimulus-dependent HuR shuttling.

Posttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA

The mRNA stabilizing factor HuR is involved in the posttranscriptional regulation of many genes, including that coding for cyclooxygenase 2 (COX-2). Employing RNA interference technology and actinomycin D experiments, we demonstrate that in human mesangial cells (hMC) the amplification of cytokine-induced COX-2 by angiotensin II (AngII) occurs via a HuR-mediated increase of mRNA stability. Using COX-2 promoter constructs with different portions of the 3' untranslated region of COX-2, we found that the increase in COX-2 mRNA stability is attributable to a distal class III type of AU-rich element (ARE). Likewise, the RNA immunoprecipitation assay showed AngII-induced binding of HuR to this ARE. Using the RNA pulldown assay, we demonstrate that the AngII-caused HuR assembly with COX-2 mRNA is found in free and cytoskeleton-bound polysomes indicative of an active RNP complex. Mechanistically, the increased HuR binding to COX-2-ARE by AngII is accompanied by increased nucleocytoplasmic HuR shuttling and depends on protein kinase Cdelta (PKCdelta), which physically interacts with nuclear HuR, thereby promoting its phosphorylation. Mapping of phosphorylation sites identified serines 221 and 318 as critical target sites for PKCdelta-triggered HuR phosphorylation and AngII-induced HuR export to the cytoplasm. Posttranslational modification of HuR by PKCdelta represents an important novel mode of HuR activation implied in renal COX-2 regulation.

Cyclooxygenase-2 and 5-lipoxygenase in dogs with chronic enteropathies.

BACKGROUND Cyclooxygenase-2 (COX-2) is a key enzyme in the synthesis of pro-inflammatory prostaglandins and 5-lipoxygenase (5-LO) is the major source of leukotrienes. Their role in IBD has been demonstrated in humans and animal models, but not in dogs with chronic enteropathies (CCE). HYPOTHESIS COX-2 and 5-LO are upregulated in dogs with CCE. ANIMALS Fifteen healthy control dogs (HCD), 10 dogs with inflammatory bowel disease (IBD), and 15 dogs with food-responsive diarrhea (FRD). METHODS Prospective study. mRNA expression of COX-2, 5-LO, IL-1b, IL-4, IL-6, TNF, IL-10 and TFG-β was evaluated by quantitative real-time RT-PCR in duodenal and colonic biopsies before and after treatment. RESULTS COX-2 expression in the colon was significantly higher in IBD and FRD before and after treatment (all P < .01). IL-1b was higher in FRD in the duodenum after treatment (P = .021). TGF-β expression was significantly higher in the duodenum of HCD compared to FRD/IBD before treatment (both P < .001) and IBD after treatment (P = .012). There were no significant differences among groups and within groups before and after treatment for IL-4, IL-6, TNF, and IL-10. There was a significant correlation between COX-2 and IL-1b in duodenum and colon before treatment in FRD and IBD, whereas 5-LO correlated better with IL-6 and TNF. IL-10 and TGF-β usually were correlated. CONCLUSIONS AND CLINICAL IMPORTANCE COX-2 is upregulated in IBD and FRD, whereas IL-1b and TGF-β seem to be important pro- and anti-inflammatory cytokines, respectively. The use of dual COX/5-LO inhibitors could be an interesting alternative in the treatment of CCE.

Celecoxib enhances radiation response of secondary bone tumors of a human non-small cell lung cancer via antiangiogenesis in vivo

Cyclooxygenase-2 (COX-2) inhibitors mediate a systemic antitumor activity via antiangiogenesis and seem to enhance the response of primary tumors to radiation. Radiosensitizing effects of COX-2 inhibition have not been reported for bone metastases. Therefore, the aim of this study was the investigation of the radiosensitizing effects of the selective COX-2 inhibitor celecoxib in secondary bone tumors of a non-small cell lung carcinoma in vivo.

Pharmacological inhibition of integrin alphavbeta3 aggravates experimental liver fibrosis and suppresses hepatic angiogenesis

The vitronectin receptor integrin alphavbeta3 promotes angiogenesis by mediating migration and proliferation of endothelial cells, but also drives fibrogenic activation of hepatic stellate cells (HSCs) in vitro. Expecting antifibrotic synergism, we studied the effect of alphavbeta3 inhibition in two in vivo models of liver fibrogenesis. Liver fibrosis was induced in rats by way of bile duct ligation (BDL) for 6 weeks or thioacetamide (TAA) injections for 12 weeks. A specific alphavbeta3 (alphavbeta5) inhibitor (Cilengitide) was given intraperitoneally twice daily at 15 mg/kg during BDL or after TAA administration. Liver collagen was determined as hydroxyproline, and gene expression was quantified by way of quantitative polymerase chain reaction. Liver angiogenesis, macrophage infiltration, and hypoxia were assessed by way of CD31, CD68 and hypoxia-inducible factor-1alpha immunostaining. Cilengitide decreased overall vessel formation. This was significant in portal areas of BDL and septal areas of TAA fibrotic rats and was associated with a significant increase of liver collagen by 31% (BDL) and 27% (TAA), and up-regulation of profibrogenic genes and matrix metalloproteinase-13. Treatment increased gamma glutamyl transpeptidase in both models, while other serum markers remained unchanged. alphavbeta3 inhibition resulted in mild liver hypoxia, as evidenced by up-regulation of hypoxia-inducible genes. Liver infiltration by macrophages/Kupffer cells was not affected, although increases in tumor necrosis factor alpha, interleukin-18, and cyclooxygenase-2 messenger RNA indicated modest macrophage activation. CONCLUSION: Specific inhibition of integrin alphavbeta3 (alphavbeta5) in vivo decreased angiogenesis but worsened biliary (BDL) and septal (TAA) fibrosis, despite its antifibrogenic effect on HSCs in vitro. Angiogenesis inhibitors should be used with caution in patients with hepatic fibrosis.

The role of self-monitoring of blood glucose in patients treated with SGLT-2 inhibitors: a European expert recommendation.

The role for the novel treatment approach of sodium-glucose cotransporter-2 (SGLT-2) in type 2 diabetes is increasing. Structured self-monitoring of blood glucose (SMBG), based on a less intensive and a more intensive scheme, may contribute to an optimization of SGLT-2 inhibitor based treatment. The current expert recommendation suggests individualized approaches of SMBG, using simple and clinically applicable schemes. Potential benefits of SMBG in SGLT-2 inhibitor based treatment approaches are early assessment of treatment success or failure, timely modification of treatment, detection of hypoglycemic episodes, assessment of glucose excursions, and support of diabetes management and education. The length and frequency of SMBG should depend on the clinical setting and the quality of metabolic control.

4'-O-methylhonokiol increases levels of 2-arachidonoyl glycerol in mouse brain via selective inhibition of its COX-2-mediated oxygenation

BACKGROUND AND PURPOSE 4'-O-methylhonokiol (MH) is a natural product showing anti-inflammatory, anti-osteoclastogenic, and neuroprotective effects. MH was reported to modulate cannabinoid CB2 receptors as an inverse agonist for cAMP production and an agonist for intracellular [Ca2+]. It was recently shown that MH inhibits cAMP formation via CB2 receptors. In this study, the exact modulation of MH on CB2 receptor activity was elucidated and its endocannabinoid substrate-specific inhibition (SSI) of cyclooxygenase-2 (COX-2) and CNS bioavailability are described for the first time. METHODS CB2 receptor modulation ([35S]GTPγS, cAMP, and β-arrestin) by MH was measured in hCB2-transfected CHO-K1 cells and native conditions (HL60 cells and mouse spleen). The COX-2 SSI was investigated in RAW264.7 cells and in Swiss albino mice by targeted metabolomics using LC-MS/MS. RESULTS MH is a CB2 receptor agonist and a potent COX-2 SSI. It induced partial agonism in both the [35S]GTPγS binding and β-arrestin recruitment assays while being a full agonist in the cAMP pathway. MH selectively inhibited PGE2 glycerol ester formation (over PGE2) in RAW264.7 cells and significantly increased the levels of 2-AG in mouse brain in a dose-dependent manner (3 to 20 mg kg(-1)) without affecting other metabolites. After 7 h from intraperitoneal (i.p.) injection, MH was quantified in significant amounts in the brain (corresponding to 200 to 300 nM). CONCLUSIONS LC-MS/MS quantification shows that MH is bioavailable to the brain and under condition of inflammation exerts significant indirect effects on 2-AG levels. The biphenyl scaffold might serve as valuable source of dual CB2 receptor modulators and COX-2 SSIs as demonstrated by additional MH analogs that show similar effects. The combination of CB2 agonism and COX-2 SSI offers a yet unexplored polypharmacology with expected synergistic effects in neuroinflammatory diseases, thus providing a rationale for the diverse neuroprotective effects reported for MH in animal models.

Leukocyte populations and mRNA expression of inflammatory factors in quarter milk fractions at different somatic cell score levels in dairy cows

The effect of somatic cell count (SCC) and milk fraction on milk composition, distribution of cell populations, and mRNA expression of various inflammatory parameters was studied. Therefore, quarter milk samples were defined as cisternal (C), first 400 g of alveolar (A1), and remaining alveolar milk (A2) during the course of milking. Quarters were assigned to 4 groups according to their total SCC: 1) <12 x 10(3)/mL, 2) 12 to 100 x 10(3)/mL, 3) 100 to 350 x 10(3)/mL, and 4) >350 x 10(3)/mL. Milk constituents of interest were SCC, fat, protein, lactose sodium, and chloride ions as well as electrical conductivity. Cell populations were classified into lymphocytes, macrophages, and neutrophils (PMN). The mRNA expression of the inflammatory factors tumor necrosis factor-alpha, interleukin-1beta, cyclooxygenase-2, lactoferrin, and lysozyme was measured via real-time, quantitative reverse transcription PCR. Somatic cell count decreased from highest levels in C to lowest levels in A1 and increased thereafter to A2 in all groups. Fat content increased from C to A2 and with increasing SCC level. Lactose decreased with increasing SCC level but remained unchanged during milking. Concentrations of sodium and chloride, and electrical conductivity increased with increasing SCC but were higher in C than in A1 and A2. Protein was not affected by milk fraction or SCC level. The distribution of leukocytes was dramatically influenced by milk fraction and SCC. Lymphocytes were the dominating cell population in group 1, but the proportion of lymphocytes was low in groups 2, 3, and 4. Macrophage proportion was highest in group 2 and decreased in groups 3 and 4, whereas that of PMN increased from group 2 to 4. The content of macrophages decreased during milking in all SCC groups whereas that of PMN increased. The proportion of lymphocytes was not affected by milk fraction. The mRNA expression of all inflammatory factors showed an increase with increasing SCC but minor changes occurred during milking. In conclusion, milk fraction and SCC level have a crucial influence on the distribution of leukocyte populations and several milk constituents. The surprisingly high content of lymphocytes and concomitantly low mRNA expression of inflammatory factors in quarters with SCC <12 x 10(3)/mL indicates a different and possibly reduced readiness of the immune system to respond to invading pathogens.

Evidence that N-acetylcysteine inhibits TNF-alpha-induced cerebrovascular endothelin-1 upregulation via inhibition of mitogen- and stress-activated protein kinase

N-acetylcysteine (NAC) is neuroprotective in animal models of acute brain injury such as caused by bacterial meningitis. However, the mechanism(s) by which NAC exerts neuroprotection is unclear. Gene expression of endothelin-1 (ET-1), which contributes to cerebral blood flow decline in acute brain injury, is partially regulated by reactive oxygen species, and thus a potential target of NAC. We therefore examined the effect of NAC on tumor necrosis factor (TNF)-alpha-induced ET-1 production in cerebrovascular endothelial cells. NAC dose dependently inhibited TNF-alpha-induced preproET-1 mRNA upregulation and ET-1 protein secretion, while upregulation of inducible nitric oxide synthase (iNOS) was unaffected. Intriguingly, NAC had no effect on the initial activation (i.e., IkappaB degradation, nuclear p65 translocation, and Ser536 phosphorylation) of NF-kappaB by TNF-alpha. However, transient inhibition of NF-kappaB DNA binding suggested that NAC may inhibit ET-1 upregulation by inhibiting (a) parallel pathway(s) necessary for full transcriptional activation of NF-kappaB-mediated ET-1 gene expression. Similar to NAC, the MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and the protein kinase inhibitor H-89 selectively inhibited ET-1 upregulation without affecting nuclear p65 translocation, suggesting that NAC inhibits ET-1 upregulation via inhibition of mitogen- and stress-activated protein kinase (MSK). Supporting this notion, cotreatment with NAC inhibited the TNF-alpha-induced rise in MSK1 and MSK2 kinase activity, while siRNA knock-down experiments showed that MSK2 is the predominant isoform involved in TNF-alpha-induced ET-1 upregulation.

Modulation of signal transduction by vitamin E

The ability of vitamin E to modulate signal transduction and gene expression has been observed in numerous studies; however, the detailed molecular mechanisms involved are often not clear. The eight natural vitamin E analogues and synthetic derivatives affect signal transduction with different potency, possibly reflecting their different ability to interact with specific proteins. Vitamin E modulates the activity of several enzymes involved in signal transduction, such as protein kinase C, protein kinase B, protein tyrosine kinases, 5-, 12-, and 15-lipoxygenases, cyclooxygenase-2, phospholipase A2, protein phosphatase 2A, protein tyrosine phosphatase, and diacylglycerol kinase. Activation of some these enzymes after stimulation of cell surface receptors with growth factors or cytokines can be normalized by vitamin E. At the molecular level, the translocation of several of these enzymes to the plasma membrane is affected by vitamin E, suggesting that the modulation of protein-membrane interactions may be a common theme for vitamin E action. In this review the main effects of vitamin E on enzymes involved in signal transduction are summarized and the possible mechanisms leading to enzyme modulation evaluated. The elucidation of the molecular and cellular events affected by vitamin E could reveal novel strategies and molecular targets for developing similarly acting compounds.

Effect of risk factors on complicated and uncomplicated ulcers in the TARGET lumiracoxib outcomes study

BACKGROUND ; AIMS: Selective cyclooxygenase-2 inhibitors were developed to reduce the gastrointestinal risk associated with nonsteroidal anti-inflammatory drugs (NSAIDs). The Therapeutic Arthritis Research and Gastrointestinal Event Trial was the largest study to evaluate primarily the gastrointestinal safety outcomes of selective cyclooxygenase-2 inhibitors. Data from the Therapeutic Arthritis Research and Gastrointestinal Event Trial were used to identify risk factors and investigate the safety of lumiracoxib in subgroups. METHODS: Patients with osteoarthritis (age, >or=50 y) were randomized to receive lumiracoxib 400 mg once daily, naproxen 500 mg twice daily, or ibuprofen 800 mg 3 times daily for 12 months. Events were categorized by a blinded adjudication committee. The primary end point was all definite or probable ulcer complications. RESULTS: For patients taking NSAIDs, factors associated with an increased risk of ulcer complications were age 65 years or older (hazard ratio [HR], 2.30; 95% confidence interval [CI], 1.48-3.59), previous history of gastrointestinal bleed or ulcer (HR, 3.61; 95% CI, 1.86-7.00), non-Caucasian racial origin (HR, 2.10; 95% CI, 1.35-3.27), and male sex (HR, 1.70; 95% CI, 1.08-2.68). With lumiracoxib, significant risk factors were age 65 years or older (HR, 3.18; 95% CI, 1.40-7.20), male sex (HR, 2.60; 95% CI, 1.25-5.40), non-Caucasian racial origin (HR, 2.16; 95% CI, 1.02-4.59), and concomitant aspirin use (HR, 2.89; 95% CI, 1.40-5.97). Increased risks in patients age 65 years and older were increased further if other risk factors were present. Lumiracoxib maintained an advantage over NSAIDs across all subgroups except aspirin use. CONCLUSIONS: Lumiracoxib was associated with a reduced risk of ulcer complications compared with NSAIDs in all significant subgroups except aspirin users.

Characterization of a stem cell population in lung cancer A549 cells

We isolated a stem cell subpopulation from human lung cancer A549 cells using FACS/Hoechst 33342. This side population (SP), which comprised 24% of the total cell population, totally disappeared after treatment with the selective ABCG 2 inhibitor fumitremorgin C. In a repopulation study, isolated SP and non-SP cells were each able to generate a heterogeneous population of SP and non-SP cells, but this repopulation occurred more rapidly in SP cells than non-SP. An MTT assay and cell cycle distribution analysis reveal a similar profile between SP and non-SP groups. However, in the presence of doxorubicin (DOX) and methotrexate (MTX), SP cells showed significantly lower Annexin V staining when compared to non-SP cells. Taken together, these results demonstrate that SP cells have an active regeneration capacity and high anti-apoptotic activity compared with non-SP cells. Furthermore, our GeneChip data revealed a heightened mRNA expression of ABCG2 and ABCC2 in SP cells. Overall these data explain why the SP of A549 has a unique ability to resist DOX and MTX treatments. Therefore, we suggest that the expression of the ABCG2 transporter plays an important role in the multidrug resistance phenotype of A549 SP cells.

Cell population, viability, and some key immunomodulatory molecules in different milk somatic cell samples in dairy cows

Immune cells in the milk are most important in combating pathogens that invade the mammary gland. This study investigated the immune competence and viability of somatic milk cells that are already resident in milk and udders free of infection. Cells were studied in freshly removed milk to simulate conditions in the udder. Effects of incubation, cell preparation, and immunological stimulation with 0.5 mug/ml lipopolysaccharide (LPS) from Escherichia coli were analysed. Viability and differential counts of milk cells between high and low somatic cell count (SCC) quarters, and cisternal and alveolar milk with and without LPS stimulation were compared. Incubation and preparation of cells caused a cell loss which further increased with time independently of SCC and milk fraction. The viability of these cells was stable until 3 h post incubation and decreased until 6 h. Cell populations differed between both investigations, but did not change during the course of the experiment. mRNA expression of immune and apoptosis factors of the cells, measured by qPCR, did not change substantially: mRNA expression of caspase 3, Toll like receptor 4, and GM-CSF did not change, whereas the expression of the death receptor Fas/APO-1 (CD95), lactoferrin and lysozyme was decreased at 6 h. Cyclooxygenase-2 and TNF-alpha mRNA expression were decreased after 6 h of LPS treatment. In comparison with other studies in vivo or in vitro (in cell culture), in this study where cells are studied ex vivo (removed from the udder but kept in their natural environment, the milk) resident milk cells seem to be more vulnerable, less viable, less able to respond to stimulation, and thus less immune competent compared with cells that have freshly migrated from blood into milk after pathogen stimulation. The cell viability and differential cell count differed between high- and low-SCC milk and between cisternal and alveolar milk depending on the individual cow. In conclusion, the results support the view that for a most effective defence against invading pathogens the mammary gland is reliant on the recruitment of fresh immune cells from the blood.