Decreased vascular contraction and changes in oxidative state in middle-aged Wistar rats after exposure to increased levels of dietary zinc.

Both copper and zinc are known to be important for maintaining health, but most research has focused on deficiencies of these elements. Recent studies have shown that high levels of Cu can be toxic, especially to the cardiovascular (CV) system. However, little research has been done on the effects of higher levels of Zn on the CV system. In this study, male Wistar rats aged 12 months were given a diet with twice the recommended daily allowance of zinc (31.8 mg/kg of diet) and compared to a control group (15.9 mg/kg of diet) after 8 weeks. Blood plasma and internal organs of both groups were examined for levels of copper, zinc, selenium and iron, as well as several key enzymes. Aortic rings from both groups were also examined to determine vascular functioning. There were very few changes in the vascular system functioning after chronic exposure to zinc, and only one enzyme, heme oxygenase-1 (HO-1) was elevated, whereas vascular contraction to noradrenaline decreased with no changes in vasodilation to acetylcholine. Of the micronutrients, zinc and selenium were elevated in the blood plasma, while copper decreased. Meanwhile, the total antioxidant status increased. These were not observed in the liver. Therefore, it is proposed that there is a mechanism in place within the vascular system to protect against the overproduction of heme, caused by chronic zinc exposure.

Time-dependent dual mode of action of COX-2 inhibition on mouse serum corticosterone levels.

To elucidate the effect of cyclooxygenase-2 (COX-2) inhibition on corticosterone release, mice were divided into a group receiving NS398, a selective COX-2 inhibitor at a dose of 3 mg/kg for seven days, and a group receiving NS398 for fourteen days. After this time, the mice were sacrificed, and blood serum was collected. An ELISA protocol was used to analyze serum corticosterone levels. Short-term COX-2 inhibition increased corticosterone levels, while long-term inhibition lowered them. The exact schedule of experiments was repeated after the lipopolysaccharide (LPS) Escherichia coli challenge in mice to check the influence of stress stimuli on the tested parameters. In this case, we observed increases in corticosterone levels, significant in a seven-day pattern. These results indicate that corticosterone levels are regulated through a COX-2-dependent mechanism in mice.

The Effect of an Elevated Dietary Copper Level on the Vascular Contractility and Oxidative Stress in Middle-Aged Rats.

Copper (Cu), being an essential mineral, plays a crucial role in maintaining physiological homeostasis across multiple bodily systems, notably the cardiovascular system. However, an increased Cu level in the body may cause blood vessel dysfunction and oxidative stress, which is unfavorable for the cardiovascular system. Middle-aged (7-8 months old) male Wistar rats (n/group = 12) received a diet supplemented with 6.45 mg Cu/kg (100% of the recommended daily dietary quantity of copper) for 8 weeks (Group A). The experimental group received 12.9 mg Cu/kg of diet (200%-Group B). An ex vivo study revealed that supplementation with 200% Cu decreased the contraction of isolated aortic rings to noradrenaline (0.7-fold) through FP receptor modulation. Vasodilation to sodium nitroprusside (1.10-fold) and acetylcholine (1.13-fold) was potentiated due to the increased net effect of prostacyclin derived from cyclooxygenase-1. Nitric oxide (NO, 2.08-fold), superoxide anion (O2•-, 1.5-fold), and hydrogen peroxide (H2O2, 2.33-fold) measured in the aortic rings increased. Blood serum antioxidant status (TAS, 1.6-fold), Cu (1.2-fold), Zn (1.1-fold), and the Cu/Zn ratio (1.4-fold) increased. An increase in Cu (1.12-fold) and the Cu/Zn ratio (1.09-fold) was also seen in the rats' livers. Meanwhile, cyclooxygenase-1 (0.7-fold), cyclooxygenase-2 (0.4-fold) and glyceraldehyde 3-phosphate dehydrogenase (0.5-fold) decreased. Moreover, a negative correlation between Cu and Zn was found (r = -0.80) in rat serum. Supplementation with 200% Cu did not modify the isolated heart functioning. No significant difference was found in the body weight, fat/lean body ratio, and organ weight for either the heart or liver, spleen, kidney, and brain. Neither Fe nor Se, the Cu/Se ratio, the Se/Zn ratio (in serum and liver), heme oxygenase-1 (HO-1), endothelial nitric oxide synthase (eNOS), or intercellular adhesion molecule-1 (iCAM-1) (in serum) were modified. Supplementation with 200% of Cu potentiated pro-oxidant status and modified vascular contractility in middle-aged rats.

The selective cyclooxygenase-2 inhibitor NS398 ameliorates cisplatin-induced impairments in mitochondrial and cognitive function.

Chemobrain is a condition that negatively affects cognition in cancer patients undergoing active chemotherapy, as well as following chemotherapy cessation. Chemobrain is also known as chemotherapy-induced cognitive impairment (CICI) and has emerged as a significant medical contingency. There is no therapy to ameliorate this condition, hence identification of novel therapeutic strategies to prevent CICI is of great interest to cancer survivors. Utilizing the platinum-based chemotherapy cisplatin in an investigative approach for CICI, we identified increased expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in the adult mouse hippocampus, and in human cortical neuron cultures derived from induced pluripotent stem cells (iPSCs). Notably, administration of NS398, a selective COX-2 inhibitor, prevented CICI in vivo without negatively affecting the antitumor efficacy of cisplatin or potentiating tumor growth. Given that dysfunctional mitochondrial bioenergetics plays a prominent role in CICI, we explored the effects of NS398 in cisplatin-induced defects in human cortical mitochondria. We found that cisplatin significantly reduces mitochondrial membrane potential (MMP), increases matrix swelling, causes loss of cristae membrane integrity, impairs ATP production, as well as decreases cell viability and dendrite outgrowth. Pretreatment with NS398 in human cortical neurons attenuated mitochondrial dysfunction caused by cisplatin, while improving cell survival and neurite morphogenesis. These results suggest that aberrant COX-2 inflammatory pathways may contribute in cisplatin-induced mitochondrial damage and cognitive impairments. Therefore, COX-2 signaling may represent a viable therapeutic approach to improve the quality of life for cancer survivors experiencing CICI.

The Interaction of Dietary Pectin, Inulin, and Psyllium with Copper Nanoparticle Induced Changes to the Cardiovascular System.

We aimed to analyze how supplementation with a standard (recommended, 6.5 mg/kg) or enhanced (two-times higher, 13 mg/kg) dose of copper (Cu), in the form of nanoparticles (NPs) along with dietary intervention via the implementation of diverse types of fiber, affects the cardiovascular system in rats. Nine-week-old male Wistar Han rats (n/group = 10) received, for an additional 6 weeks, a controlled diet with cellulose as dietary fiber and ionic Cu (in the form of carbonate salt). The experimental groups received cellulose, pectin, inulin, and psyllium as dietary fiber, together with CuNPs (6.5 or 13 mg/kg diet). After the experimental feeding, samples of blood, hearts, and thoracic arteries were collected for further analysis. Compared to pectin, and under a standard dose of CuNPs, inulin and psyllium beneficially increased the antioxidant capacity of lipid- and water-soluble compounds in the blood, and decreased heart malondialdehyde. Moreover, pectin decreased heart catalase (CAT) and cyclooxygenase (COX)-2 in the aortic rings compared to inulin and psyllium under standard and enhanced doses of copper. When the dose of CuNPs was enhanced, inulin and psyllium potentiated vasodilation to acetylcholine by up-regulation of COX-2-derived vasodilator prostanoids compared to both cellulose and pectin, and this was modulated with selective inducible nitric oxide synthase (iNOS) inhibitor for psyllium only. Moreover, inulin decreased heart CAT compared to psyllium. Our results suggest that supplementation with dietary fiber may protect the vascular system against potentially harmful metal NPs by modulating the antioxidant mechanisms.

Dietary Effects of Chromium Picolinate and Chromium Nanoparticles in Wistar Rats Fed with a High-Fat, Low-Fiber Diet: The Role of Fat Normalization.

We aimed to evaluate how feeding a high-fat-low-fiber (F) diet to rats and dietary intervention with the implementation of a standard-fat-and-fiber (S) diet affects the response of the cardiovascular system to chromium (III) picolinate (Cr-Pic) and, alternatively, chromium nanoparticles (Cr-NPs). Young male Wistar Han rats (n/group = 12) from either the fatty group (18 weeks on F diet) or the intervention group (9 weeks on F diet + 9 weeks on S diet) received a pharmacologically relevant dose of 0.3 mg Cr/kg body weight in the form of Cr-Pic or Cr-NPs for 9 weeks. Our study on rats confirmed the pro-inflammatory effect of an F diet administered for 18 weeks. In the intervention group, both Cr-Pic and Cr-NPs decreased heart glutathione ratio (GSH+GSSG), enhanced participation of nitric oxide (NO) derived from inducible NO synthase (iNOS) in vascular relaxation to acetylcholine (ACh), increased the vasodilator net effect of cyclooxygenase-2 (COX-2)-derived prostanoids, and increased the production of superoxide anion (O2.-) in aortic rings. Meanwhile, in the fatty group, there was increased heart superoxide dismutase (SOD), decreased heart catalase (CAT), and reduced sensitivity in pre-incubated aortic rings to endogenous prostacyclin (PGI2). The factors that significantly differentiated Cr-NPs from Cr-Pic were (i) decreased blood antioxidant capacity of water-soluble compounds (0.75-fold, p = 0.0205), (ii) increased hydrogen peroxide (H2O2) production (1.59-fold, p = 0.0332), and (iii) modified vasodilator response due to PGI2 synthesis inhibition (in the intervention group) vs. modified ACh-induced vasodilator response due to (iv) COX inhibition and v) PGI2 synthesis inhibition with thromboxane receptor blockage after 18 weeks on F diet (in the fatty group). Our results show that supplementation with Cr-Pic rather than with Cr-NPs is more beneficial in rats who regularly consumed an F diet (e.g., for 18 weeks). On the contrary, in the intervention group (9 weeks on F diet + 9 weeks of dietary fat normalization (the S diet)), Cr-Pic and Cr-NPs could function as pro-oxidant agents, initiating free-radical reactions that led to oxidative stress.

Cyclooxygenase-2 Upregulated by Temozolomide in Glioblastoma Cells Is Shuttled In Extracellular Vesicles Modifying Recipient Cell Phenotype.

Temozolomide (TMZ) resistance is frequent in patients with glioblastoma (GBM), a tumor characterized by a marked inflammatory microenvironment. Recently, we reported that cyclooxygenase-2 (COX-2) is upregulated in TMZ-resistant GBM cells treated with high TMZ concentrations. Moreover, COX-2 activity inhibition significantly counteracted TMZ-resistance of GBM cells. Extracellular vesicles (EV) are considered crucial mediators in orchestrating GBM drug resistance by modulating the tumor microenvironment (TME) and affecting the surrounding recipient cell phenotype and behavior. This work aimed to verify whether TMZ, at low and clinically relevant doses (5-20 µM), could induce COX-2 overexpression in GBM cells (T98G and U87MG) and explore if secreted EV shuttled COX-2 to recipient cells. The effect of COX-2 inhibitors (COXIB), Celecoxib (CXB), or NS398, alone or TMZ-combined, was also investigated. Our results indicated that TMZ at clinically relevant doses upregulated COX-2 in GBM cells. COXIB treatment significantly counteracted TMZ-induced COX-2 expression, confirming the crucial role of the COX-2/PGE2 system in TMZ-resistance. The COXIB specificity was verified on U251MG, COX-2 null GBM cells. Western blotting of GBM-EV cells showed the COX-2 presence, with the same intracellular trend, increasing in EV derived from TMZ-treated cells and decreasing in those derived from COXIB+TMZ-treated cells. We then evaluated the effect of EV secreted by TMZ-treated cells on U937 and U251MG, used as recipient cells. In human macrophage cell line U937, the internalization of EV derived by TMZ-T98G cells led to a shift versus a pro-tumor M2-like phenotype. On the other hand, EV from TMZ-T98G induced a significant decrease in TMZ sensitivity in U251MG cells. Overall, our results, in confirming the crucial role played by COX-2 in TMZ-resistance, provide the first evidence of the presence and effective functional transfer of this enzyme through EV derived from GBM cells, with multiple potential consequences at the level of TME.

Inhibition of COX-2 ameliorates murine liver schistosomiasis japonica through splenic cellular immunoregulation.

BACKGROUND: We have reported the positive association of the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) axis with liver fibrosis induced by Schistosoma japonicum (Sj) infection, and TLR4 signaling controlled this axis. However, how COX-2 regulates immune response during Sj infection is still unclear. METHODS: Hematoxylin and eosin staining was used to evaluate the effect of the COX-2-specific inhibitor NS398 on liver granulomatous inflammation and fibrosis. Flow cytometry was used to explore the frequency and amount of different immune cell infiltration in the spleen during Sj infection. RESULTS: NS398 significantly reduced the size of liver granuloma, spleen, and mesenteric lymph node (MLN) and alleviated chronic granulomatous inflammation. Mechanically, this might be by decreasing the number of Sj-induced macrophages and T helper type 1 (Th1), Th2, T follicular helper (Tfh), T follicular regulatory (Tfr), and germinal center B (GC B) cells. There were no differences in the number of neutrophils, myeloid-derived suppressor cells, Th17 cells, regulatory T cells (Treg), or total B cells in the spleen of the mice with or without NS398 treatment. CONCLUSIONS: COX-2/PGE2 inhibition may represent a potential therapeutic approach for schistosomiasis japonica through splenic cellular immunoregulation.

Up-Regulation of Cyclooxygenase-2 (COX-2) Expression by Temozolomide (TMZ) in Human Glioblastoma (GBM) Cell Lines.

TMZ-resistance remains a main limitation in glioblastoma (GBM) treatment. TMZ is an alkylating agent whose cytotoxicity is modulated by O6-methylguanine-DNA methyltransferase (MGMT), whose expression is determined by MGMT gene promoter methylation status. The inflammatory marker COX-2 has been implicated in GBM tumorigenesis, progression, and stemness. COX-2 inhibitors are considered a GBM add-on treatment due to their ability to increase TMZ-sensitivity. We investigated the effect of TMZ on COX-2 expression in GBM cell lines showing different COX-2 levels and TMZ sensitivity (T98G and U251MG). ß-catenin, MGMT, and SOX-2 expression was analyzed. The effects of NS398, COX-2 inhibitor, alone or TMZ-combined, were studied evaluating cell proliferation by the IncuCyte® system, cell cycle/apoptosis, and clonogenic potential. COX-2, ß-catenin, MGMT, and SOX-2 expression was evaluated by RT-PCR, Western blotting, and immunofluorescence and PGE2 by ELISA. Our findings, sustaining the role of COX-2/PGE2 system in TMZ-resistance of GBM, show, for the first time, a relevant, dose-dependent up-regulation of COX-2 expression and activity in TMZ-treated T98G that, in turn, correlated with chemoresistance. Similarly, all the COX-2-dependent signaling pathways involved in TMZ-resistance also resulted in being up-modulated after treatment with TMZ. NS398+TMZ was able to reduce cell proliferation and induce cell cycle arrest and apoptosis. Moreover, NS398+TMZ counteracted the resistance in T98G preventing the TMZ-induced COX-2, ß-catenin, MGMT, and SOX-2 up-regulation.

The Role of 20-HETE, COX, Thromboxane Receptors, and Blood Plasma Antioxidant Status in Vascular Relaxation of Copper-Nanoparticle-Fed WKY Rats.

Recently, the addition of copper nanoparticles (NPs) in a daily diet (6.5 mg/kg) was studied in different animal models as a possible alternative to ionic forms. Male Wistar-Kyoto rats (24-week-old, n = 11) were fed with copper, either in the form of carbonate salt (Cu6.5) or metal-based copper NPs (NP6.5), for 8 weeks. The third group was fed with a half dose of each (NP3.25 + Cu3.25). The thoracic aorta and blood plasma was studied. Supplementation with NP6.5 decreased the Cu (×0.7), Cu/Zn-ratio (×0.6) and catalase (CAT, ×0.7), and increased Zn (×1.2) and superoxide dismutase (SOD, ×1.4). Meanwhile, NP3.25 + Cu3.25 decreased the Cu/Zn-ratio (×0.7), and CAT (×0.7), and increased the daily feed intake (×1.06). Preincubation with either the selective cyclooxygenase (COX)-2 inhibitor, or the non-selective COX-1/2 inhibitor attenuated vasodilation of rat thoracic aorta in the NP6.5 group exclusively. However, an increased vasodilator response was observed in the NP6.5 and NP3.25 + Cu3.25 group of rats after preincubation with an inhibitor of 20-hydroxyeicosatetraenoic acid (20-HETE) formation, and the thromboxane receptor (TP) antagonist. Significant differences were observed between the NP6.5 and NP3.25 + Cu3.25 groups of rats in: dietary intake, acetylcholine-induced vasodilation, and response to COX-inhibitors. Copper NPs in a standard daily dose had more significant effects on the mechanism(s) responsible for the utilization of reactive oxygen species in the blood plasma with the participation of prostanoids derived from COX-2 in the vascular relaxation. Dietary copper NPs in both doses modified vasodilation through the vasoconstrictor 20-HETE and the TP receptors.

Upregulation of the mGlu5 receptor and COX-2 protein in the mouse brain after imipramine and NS398, searching for mechanisms of regulation.

Imipramine belongs to a group of tricyclic antidepressants (TCAs). It has been also documented that its antidepressant activity connects with the modulation of cytosolic phospholipase A2 (cPLA2) and arachidonic acid (AA) turnover. Through this mechanism, imipramine can indirectly modify glutamate (Glu) transmission. Additionally, it has been shown that chronic treatment with imipramine results in the upregulation of the metabotropic glutamate receptor subtype 5 (mGlu5 receptor) in the hippocampus of rats. Our previous study revealed that manipulation of the AA pathway via inhibition of cyclooxygenase-2 (COX-2) by selective COX-2 inhibitor (NS398) could effectively modulate the behavior of mice treated with imipramine. Here, we hypothesized that COX-2 inhibition could similarly to imipramine influence mGlu5 receptor, and thus NS398 can modulate the effect of imipramine on Glu. Moreover, such regulation changes should correspond with alterations in neurotransmission. Increased cPLA activity after imipramine administration may change the activity of the AA pathway and the endocannabinoid metabolism, e.g., 2-Arachidonyl-glycerol (2-AG). To verify the idea, mGlu5 receptor level was investigated in the hippocampus (HC) and prefrontal cortex (PFC) of mice treated for 7 or 14 days with imipramine and/or COX-2 inhibitor: NS398. Western blot and PCR analyses were conducted. Moreover, the excitatory (Glu) and inhibitory (gamma-aminobutyric acid; GABA) neurotransmitters were measured using HPLC and 2-AG using ELISA. A time-dependent change in mGlu5 receptor and COX-2 protein level, COX-2 expression, and 2-AG level in the PFC after imipramine administration was found. Up-regulation of mGlu5 receptor after NS398 was found in HC and PFC. A structure-dependent shift between excitatory vs. inhibitory transmission was detected when NS398 and imipramine were co-administered.

NS398 as a potential drug for autosomal-dominant polycystic kidney disease: Analysis using bioinformatics, and zebrafish and mouse models.

Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by uncontrolled renal cyst formation, and few treatment options are available. There are many parallels between ADPKD and clear-cell renal cell carcinoma (ccRCC); however, few studies have addressed the mechanisms linking them. In this study, we aimed to investigate their convergences and divergences based on bioinformatics and explore the potential of compounds commonly used in cancer research to be repurposed for ADPKD. We analysed gene expression datasets of ADPKD and ccRCC to identify the common and disease-specific differentially expressed genes (DEGs). We then mapped them to the Connectivity Map database to identify small molecular compounds with therapeutic potential. A total of 117 significant DEGs were identified, and enrichment analyses results revealed that they are mainly enriched in arachidonic acid metabolism, p53 signalling pathway and metabolic pathways. In addition, 127 ccRCC-specific up-regulated genes were identified as related to the survival of patients with cancer. We focused on the compound NS398 as it targeted DEGs and found that it inhibited the proliferation of Pkd1-/- and 786-0 cells. Furthermore, its administration curbed cystogenesis in Pkd2 zebrafish and early-onset Pkd1-deficient mouse models. In conclusion, NS398 is a potential therapeutic agent for ADPKD.

Biological effects of selective COX-2 inhibitor NS398 on human glioblastoma cell lines.

BACKGROUND: Cyclooxygenase-2 (COX-2), an inflammation-associated enzyme, has been implicated in tumorigenesis and progression of glioblastoma (GBM). The poor survival of GBM was mainly associated with the presence of glioma stem cells (GSC) and the markedly inflammatory microenvironment. To further explore the involvement of COX-2 in glioma biology, the effects of NS398, a selective COX-2 inhibitor, were evaluated on GSC derived from COX-2 expressing GBM cell lines, i.e., U87MG and T98G, in terms of neurospheres' growth, autophagy, and extracellular vesicle (EV) release. METHODS: Neurospheres' growth and morphology were evaluated by optical and scanning electron microscopy. Autophagy was measured by staining acidic vesicular organelles. Extracellular vesicles (EV), released from neurospheres, were analyzed by transmission electron microscopy. The autophagic proteins Beclin-1 and LC3B, as well as the EV markers CD63 and CD81, were analyzed by western blotting. The scratch assay test was used to evaluate the NS398 influence on GBM cell migration. RESULTS: Both cell lines were strongly influenced by NS398 exposure, as showed by morphological changes, reduced growth rate, and appearance of autophagy. Furthermore, the inhibitor led to a functional change of EV released by neurospheres. Indeed, EV secreted by NS398-treated GSC, but not those from control cells, were able to significantly inhibit adherent U87MG and T98G cell migration and induced autophagy in recipient cells, thus leading to effects quite similar to those directly caused by NS398 in the same cells. CONCLUSION: Despite the intrinsic diversity and individual genetic features of U87MG and T98G, comparable effects were exerted by the COX-2 inhibitor NS398 on both GBM cell lines. Overall, our findings support the crucial role of the inflammatory-associated COX-2/PGE2 system in glioma and glioma stem cell biology.

NS398, a cyclooxygenase-2 inhibitor, reverses memory performance disrupted by imipramine in C57Bl/6J mice.

Imipramine has been widely used as an antidepressant in the clinic over the years. Unfortunately, it produces a detrimental effect on memory. At the same time, COX-2 inhibitors engagement in the mechanisms of memory formation, and synapse plastic changes has been well documented. Our previous studies have demonstrated the contribution of cyclooxygenase-2 (COX-2) inhibition to the parameters of the mGluR5 pathway in memory formation. Because chronic administration of imipramine has been shown to affect mGluR5, the purpose of this study was to verify the hypothesis of COX-2 pathway engagement in disrupting effects of imipramine. Imipramine is currently used as a reference compound, and therefore it seems important to decipher and understand mood-related pathways, as well as cognitive changes activated during its use. This study covers the examination of spatial, and motor parameters. To this end, C57Bl/6J mice received imipramine, and NS398 (a COX-2 inhibitor) alone, or in combination for 7 or 14 days. We performed the modified Barnes maze (MBM), modified rotarod (MR) tests, and electrophysiological studies. The harmful effect of imipramine on MBM learning was improved by NS398 use. The same modulatory role of the COX-2 inhibitor in procedural learning in the MR test was found. In conclusion, our data show the involvement of the COX-2 pathway in changes in the long-term memory, and procedural memory of C57Bl/6J mice after chronic imipramine treatment.

COX-2 inhibitor prevents tumor induced down regulation of classical DC lineage specific transcription factor Zbtb46 resulting in immunocompetent DC and decreased tumor burden.

The interaction between the immune and tumor cells in the microenvironment is an important factor deciding the progression of cancer. Though many of the soluble mediators in the microenvironment that mediate immunosuppression are known, the mechanism by which the tumor affects the distal progenitors is not known. We report that the tumor derived prostanoids down regulated classical dendritic cells DC (cDC) lineage specific transcription factor Zbtb46 in the progenitor cells which affects its differentiation. Prostanoids also induced ERK/CREB/IL-10 signaling pathway in DC that is more important for maturation of DC. This was observed under in vitro as well as in vivo conditions leading to phenotypic and functional impairment of DC. siRNA mediated knockdown of Zbtb46 and not exogenous IL-10 mimicked the effects of tumor conditioned medium (TCM) on suppression of maturation markers. Treatment of tumor cells with COX-2 inhibitor NS-398 averted TCM induced phenotypic impairment of DC in vitro. Treatment of tumor bearing mice with NS-398 prevented tumor induced down regulation of Zbtb46 resulting in immunocompetent DC which in turn led to a decrease in tumor burden. The effects of NS-398 was indeed through immunomodulation was corroborated by no such response in SCID mice. Our study provides novel insight into the distal regulation of progenitor cells by tumor and the importance of Zbtb46 expression in anti-tumor immunity. These results identify Zbtb46 expression as an indicator of immunocompetent DC in tumor and also highlights that COX-2 inhibitors could be useful in cancer immunotherapy.

Co-treatment with Celecoxib or NS398 Strongly Sensitizes Resistant Cancer Cells to Antimitotic Drugs Independent of P-gp Inhibition.

BACKGROUND/AIM: Inhibition of cyclooxygenase-2 (COX-2) has been investigated in clinical trials. Currently, NS398 and celecoxib are the most commonly used COX-2 inhibitors. The purpose of this study was to identify conditions that would increase the sensitivity of resistant cancer cells to antimitotic drugs. MATERIALS AND METHODS: We tested whether COX-2 inhibitors can sensitize drug-resistant KBV20C cancer cells. We also compared the efficacy of NS398 with that of celecoxib. RESULTS: Both NS398 and celecoxib could sensitize KB and KBV20C cells to a similar extent, suggesting that COX-2 inhibitors could be used for sensitive, as well as resistant, cancer cells. We demonstrated that the NS398 and celecoxib sensitization mechanism is independent of the inhibition of p-glycoprotein (P-gp), suggesting that resistant KBV20C cells are sensitized through targeting of signaling pathways by both drugs. Furthermore, through using microscopic observation, assessment of cleaved poly ADP ribose polymerase (C-PARP) and annexin V staining we determined that both COX-2 inhibitors strongly sensitized resistant KBV20C cells to vinblastine (VIB) or paclitaxel (PAC) treatment. These results suggest that antimitotic drug-resistant cancer cells can be strongly sensitized by co-treatment with COX-2 inhibitors, without P-gp inhibitory activity. CONCLUSION: These findings provide important information regarding the sensitization of drug-resistant cells and indicate that COX-2 inhibitors may be used for potentially resistant cancer patients, without the toxic effects of P-gp inhibition.

In vitro and in vivo assessment of meadowsweet (Filipendula ulmaria) as anti-inflammatory agent.

ETHNOPHARMACOLOGICAL RELEVANCE: Meadowsweet (Filipendula ulmaria (L.) Maxim, Rosaceae) has been traditionally used in most European countries for the treatment of inflammatory diseases due to its antipyretic, analgesic, astringent, and anti-rheumatic properties. However, there is little scientific evidence on F. ulmaria anti-inflammatory effects regarding its impact on cyclooxygenases enzymatic activity and in vivo assessment of anti-inflammatory potential. This study aims to reveal the anti-inflammatory activity of methanolic extracts from the aerial parts (FUA) and roots (FUR) of F. ulmaria, both in in vitro and in vivo conditions. MATERIALS AND METHODS: The characteristic phenolic compounds in F. ulmaria extracts were monitored via high performance thin layer chromatography (HPTLC). The in vitro anti-inflammatory activity of F. ulmaria extracts was evaluated using cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzyme assays, and an assay for determining COX-2 gene expression. The in vivo anti-inflammatory effect of F. ulmaria extracts was determined in two doses (100 and 200 mg/kg b.w.) with hot plate test and carrageenan-induced paw edema test in rats. Inflammation was also evaluated by histopathological and immunohistochemical analysis. RESULTS: FUA extract showed the presence of rutoside, spiraeoside, and isoquercitrin. Both F. ulmaria extracts at a concentration of 50µg/mL were able to inhibit COX-1 and -2 enzyme activities, whereby FUA extract (62.84% and 46.43% inhibition, respectively) was double as effective as the root extract (32.11% and 20.20%, respectively). Extracts hardly inhibited the level of COX-2 gene expression in THP-1 cells at a concentration of 25µg/mL (10.19% inhibition by FUA and 8.54% by FUR). In the hot plate test, both extracts in two doses (100 and 200mg/kg b.w.), exhibited an increase in latency time when compared with the control group (p<0.05). In the carrageenan-induced acute inflammation test, FUA at doses of 100 and 200mg/kg b.w., and FUR at 200mg/kg, were able to significantly reduce the mean maximal swelling of rat paw until 6h of treatment. Indomethacin, FUA, and FUR extracts significantly decreased inflammation score and this effect was more pronounced after 24h, compared to the control group (p<0.05). CONCLUSIONS: The observed results of in vitro and, for the first time, in vivo anti-inflammatory activity of meadowsweet extracts, provide support of the traditional use of this plant in the treatment of different inflammatory conditions. Further investigation of the anti-inflammatory compounds could reveal the mechanism of anti-inflammatory action of these extracts.

Novel effects of the cyclooxygenase-2-selective inhibitor NS-398 on IL-1ß-induced cyclooxygenase-2 and IL-8 expression in human ovarian granulosa cells.

Ovulation is a critical inflammation-like event that is central to ovarian physiology. IL-1ß is an immediate early pro-inflammatory cytokine that regulates production of several other inflammatory mediators, such as cyclooxygenase 2 (COX)-2 and IL-8. NS-398 is a selective inhibitor of COX-2 bioactivity and thus this drug is able to mitigate the COX-2-mediated production of downstream prostaglandins and the subsequent inflammatory response. Here we have investigated the action of NS-398 using a human ovarian granulosa cell line, KGN, by exploring IL-1ß-regulated COX-2 and IL-8 expression. First, NS-398, instead of reducing inflammation, appeared to further enhance IL-1ß-mediated COX-2 and IL-8 production. Using selective inhibitors targeting various signaling molecules, MAPK and NF-κB pathways both seemed to be involved in the impact of NS-398 on IL-1ß-induced COX-2 and IL-8 expression. NS-398 also promoted IL-1ß-mediated NF-κB p65 nuclear translocation but had no effect on IL-1ß-activated MAPK phosphorylation. Flow cytometry analysis demonstrated that NS-398, in combination with IL-1ß, significantly enhanced cell cycle progression involving IL-8. Our findings demonstrate a clear pro-inflammatory function for NS-398 in the IL-1ß-mediated inflammatory response of granulosa cells, at least in part, owing to its augmenting effect on the IL-1ß-induced activation of NF-κB.

Inhibition of Kv channel expression by NSAIDs depolarizes membrane potential and inhibits cell migration by disrupting calpain signaling.

Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug-affected signaling pathways that contribute to inhibition of migration by NSAIDs are poorly understood, though previous studies have shown that NSAIDs depolarize membrane potential and suppress expression of calpain proteases and voltage-gated potassium (Kv) channel subunits. Kv channels play significant roles in cell migration and are targets of NSAID activity in white blood cells, but the specific functional effects of NSAID-induced changes in Kv channel expression, particularly on cell migration, are unknown in intestinal epithelial cells. Accordingly, we investigated the effects of NSAIDs on expression of Kv1.3, 1.4, and 1.6 in vitro and/or in vivo and evaluated the functional significance of loss of Kv subunit expression. Indomethacin or NS-398 reduced total and plasma membrane protein expression of Kv1.3 in cultured intestinal epithelial cells (IEC-6). Additionally, depolarization of membrane potential with margatoxin (MgTx), 40mM K(+), or silencing of Kv channel expression with siRNA significantly reduced IEC-6 cell migration and disrupted calpain activity. Furthermore, in rat small intestinal epithelia, indomethacin and NS-398 had significant, yet distinct, effects on gene and protein expression of Kv1.3, 1.4, or 1.6, suggesting that these may be clinically relevant targets. Our results show that inhibition of epithelial cell migration by NSAIDs is associated with decreased expression of Kv channel subunits, and provide a mechanism through which NSAIDs inhibit cell migration and may contribute to NSAID-induced gastrointestinal (GI) toxicity.

Attenuation of renovascular hypertension by cyclooxygenase-2 inhibitor partly through ANP release.

Angiotensin II (Ang II) is an important inflammatory mediator. Ang II induces cyclooxygenase-2 (COX-2) expression and prostaglandin F2α release followed by cardiac hypertrophy. Inhibition of COX-2 may modulate high blood pressure but controversy still exists. The aim of this study was to determine the role of COX-2 in the regulation of blood pressure and to define the mechanisms in two kidney one-clip hypertensive (2K1C) rats. Chronic treatment with nimesulide or NS-398 (5 mg/kg/day) for 3 weeks lowered high blood pressure and cardiac hypertrophy with decreased expression levels of cardiac hypertrophy markers [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP)], Ang type 1 receptor, urotensin II, and urotensin II receptor in 2K1C rats. Plasma level of ANP was markedly increased and plasma levels of Ang II and aldosterone were decreased by treatment with nimesulide or NS-398. In both in vitro and in vivo experiments, nimesulide or NS-398 augmented ANP release in 2K1C rats. The inhibitory effect of NS-398 on blood pressure was attenuated by the pretreatment with natriuretic peptide receptor-A (NPR-A) antagonist (A71915, 30 µg/kg/day). These results suggest that chronic treatment with nimesulide or NS-398 attenuated hypertension and cardiac hypertrophy partly through ANP release in 2K1C rats.