- Methylation of Notch3 modulates chemoresistance via P-glycoprotein. [Journal Article]
- EJEur J Pharmacol 2016 Oct 22
- The global gene expression and DNA methylation of genes in adriamycin-resistant human breast cancer cells (MCF-7/ADM cells) are similar to those in paclitaxel-resistant MCF-7 cells （MCF-7/PTX） and ar...
The global gene expression and DNA methylation of genes in adriamycin-resistant human breast cancer cells (MCF-7/ADM cells) are similar to those in paclitaxel-resistant MCF-7 cells （MCF-7/PTX） and are significantly different from those in wild-type MCF-7 cells. DNA methylation is associated with chemoresistance in breast cancer and changes the characteristics of chemoresistant and chemosensitive cells. Here, we showed that the tumor-suppressor gene Notch3 was inactivated due to epigenetic silencing DNA hypermethylation in MCF-7/ADM cells. In addition, the drug efflux pump P-glycoprotein was negatively regulated by Notch3 and highly expressed in MCF-7/ADM cells. Taken together, our findings demonstrated that hypermethylation of Notch3 causes activation of P-glycoprotein in adriamycin-resistant cells.
- Insights into the neuroprotective mechanisms of 2-iminobiotin employing an in-vitro model of hypoxic-ischemic cell injury. [Journal Article]
- EJEur J Pharmacol 2016 Oct 22
- Several animal models have been used to simulate cerebral hypoxia-ischemia and suggested neuroprotective effects of the biotin analogue 2-iminobiotin (2-IB). The aims of this study were to employ a h...
Several animal models have been used to simulate cerebral hypoxia-ischemia and suggested neuroprotective effects of the biotin analogue 2-iminobiotin (2-IB). The aims of this study were to employ a human in-vitro hypoxia model to confirm protective effects of 2-IB on neuronal cells, determine the optimal neuroprotective concentrations of 2-IB and scrutinize underlying cellular effects of 2-IB. Neuronal IMR-32 cells were exposed to hypoxia employing an enzymatic hypoxia system and were thereafter incubated with various concentrations of 2-IB (10 to 300ng/ml). Cell damage, metabolic activity and generation of reactive oxygen species were quantified using colorimetric/fluorometric lactate dehydrogenase (LDH), tetrazolium-based (MTS) and reactive oxygen species assays. Proteome profiling arrays were performed to evaluate the regulation of cell stress protein expression by hypoxia and 2-IB. Seven hours of hypoxia led to morphological changes in IMR-32 cultures, increased neuronal cell damage (P<0.001), reduction of metabolic activity (P<0.01) and enhanced reactive oxygen species production (P<0.05). Post-hypoxic application of 2-IB (30ng/ml) attenuated hypoxia-induced LDH release (P<0.05) and increased metabolic activity of IMR-32 cells (P<0.05), while reactive oxygen species production was only by trend decreased. Array-based protein expression profiling revealed that 2-IB attenuated the expression of several hypoxia-induced cell stress-associated proteins by more than 25% (pp38α, HIF2α, ADAMTS1, pHSP27, PON2, PON3 and p27). Hypoxia-induced neuronal cell damage can be simulated using the described in-vitro model. 2-IB inhibits hypoxia-mediated neurotoxicity most efficiently at 30ng/ml and the underlying mechanisms involve a downregulation of stress-associated protein expression. Our results suggest 2-IB as a potential drug for the treatment of perinatal hypoxia-ischemia.
- In vitro functional characterization of novel nociceptin/orphanin FQ receptor agonists in recombinant and native preparations. [Journal Article]
- EJEur J Pharmacol 2016 Oct 22
- Nociceptin/Orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ receptor (NOP). In this study novel nonpeptide NOP ligands were characterized in vitro in r...
Nociceptin/Orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ receptor (NOP). In this study novel nonpeptide NOP ligands were characterized in vitro in receptor binding and [(35)S]GTPγS stimulated binding in membranes of cells expressing human NOP and classical opioid receptors, calcium mobilization assay in cells coexpressing the receptors and chimeric G proteins, bioluminescence resonance energy transfer (BRET) based assay for studying NOP receptor interaction with G protein and arrestin, the electrically stimulated mouse vas deferens and the mouse colon bioassays. The action of the AT compounds were compared with standard NOP agonists (N/OFQ and Ro 65-6570) and the NOP selective antagonist SB-612111. AT compounds displayed high NOP affinity and behaved as NOP agonists in all the functional assays consistently showing the following rank order of potency AT-127 > AT-090 > AT-035 > AT-004 = AT-001. AT compounds behaved as NOP full agonists in the calcium mobilization and mouse colon assays and as partial agonists in the [(35)S]GTPγS and BRET assays. Interestingly AT-090 and AT-127, contrary to standard nonpeptide agonists that display G protein biased agonism, behaved as an unbiased agonists. AT-090 and AT-127 displayed higher NOP selectivity than Ro 65-6570 at native mouse receptors. AT-090 and AT-127 might be useful pharmacological tools for investigating the therapeutic potential of NOP partial agonists.
- Liraglutide attenuates lipopolysaccharide-induced acute lung injury in mice. [Journal Article]
- EJEur J Pharmacol 2016 Oct 15; 791:735-740
- Liraglutide, an effective drug for the treatment of diabetes, has been proven to demonstrate anti-inflammatory and immunomodulatory effects. Hence, this study explored the effects and mechanism of ac...
Liraglutide, an effective drug for the treatment of diabetes, has been proven to demonstrate anti-inflammatory and immunomodulatory effects. Hence, this study explored the effects and mechanism of action of liraglutide on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Male BALB/c mice were pre-conditioned with liraglutide or saline prior to intraperitoneal LPS or saline administration. Histopathological examination of lung, the wet/dry (W/D)weight ratio, protein content, inflammatory cell numbers and pro-inflammatory cytokine levels in broncho-alveolar lavage fluid (BAL fluid) were conducted. The effects of liraglutide on the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome signalling pathway were assessed by Western blot. Pre-treatment with liraglutide decreased the wet-to-dry weight ratio and protein concentrations in BAL fluid and neutrophil infiltration in the lung tissues. Liraglutide also significantly reduced the interleukin-1β and interleukin-18 levels in BAL fluid, as well as effectively inhibited the expression of NLRP3 inflammasome. These results indicated that liraglutide pre-treatment attenuated LPS-induced ALI by inhibiting the NLRP3 inflammasome pathway.
- Synergistic effect of piperine and paclitaxel on cell fate via cyt-c, Bax/Bcl-2-caspase-3 pathway in ovarian adenocarcinomas SKOV-3 cells. [Journal Article]
- EJEur J Pharmacol 2016 Oct 15; 791:751-762
- Ovarian cancer is fourth most common and lethal among all gynecologic malignancies. The chemotherapy usually requires in all stages of ovarian cancer but drugs have several side effects. We hypothesi...
Ovarian cancer is fourth most common and lethal among all gynecologic malignancies. The chemotherapy usually requires in all stages of ovarian cancer but drugs have several side effects. We hypothesized that use of combination therapy of paclitaxel (PTX) and phytochemical piperine (PIP) may reduce the PTX dose as well as toxicity. The human ovarian adenocarcinomas SKOV3 cell treated with PTX-5nM and PIP-10µm after determination of IC50 by MTT assay. Reactive oxygen species generation, mitochondrial membrane potential (MMP), DNA damage, cell death pathway markers as release of cyt-c, Bax/Bcl2-caspase-3 and cell cycle arrest were analyzed. The dose dependent treatment of SKOV-3 cells showed IC50 and synergism at combination of 5nM-PTX and 10µm-PIP in cell viability assay. PTX and PIP increases the accumulation of reactive oxygen species which subsequently leading to increase in JC-1 and fragmented nuclei in mitotracker/DAPI staining. Comet assay showed 4.4-fold increase of tail formation in combined treated cells as compared to control. PTX-PIP arrests the cell cycle in sub-G1 phase. Immunocytochemistry of Bax showed increase in red fluorescence intensity whereas decrease in green fluorescence i.e Bax/Bcl-2 ratio increased. Moreover morphological EB/AO and Hoechst staining confirmed the enhanced apoptosis in combined treatment. Significant upregulation of apoptotic genes, cyt-c (3.4 fold) Bax (2.8 fold), caspase-3 (3.6 fold) whereas no change occurred in Bcl2 mRNA expression and protein expressions. The combination of PTX with PIP produces synergistic effects in SKOV-3 cells via the modulation of pro and anti-apoptotic gene and may compensate the toxicity and side effects of PTX.
- Cytokine production by PBMC and serum from allergic and non-allergic subjects following in vitro histamine stimulation to test fexofenadine and osthole anti-allergic properties. [Journal Article]
- EJEur J Pharmacol 2016 Oct 15; 791:763-772
- FXF is a third-generation antihistamine drug and osthole is assumed a natural antihistamine alternative. This paper compares peripheral blood mononuclear cell (PBMC) incubation with FXF and osthole, ...
FXF is a third-generation antihistamine drug and osthole is assumed a natural antihistamine alternative. This paper compares peripheral blood mononuclear cell (PBMC) incubation with FXF and osthole, by studying FXF, osthole and histamine cytokine secretion in PBMC in vitro cultures. Mabtech kits determined the interleukins IL-1β, IL-4, IL-10, IL-13 and TNF-α. The influence of the above active substances on cytokine secretion in PBMC's and serum was assessed: cytokines were IL-1β, IL-4, IL-10, IL-13 and TNF-α; and cytokine levels secreted by untreated PBMCs in pure culture medium formed the absolute control (ctrl). We determined that osthole affects PBMC cytokine secretion to almost precisely the same extent as FXF (IL-1β, IL-4, IL-10 and TNF). In addition osthole had greater IL-13 blocking ability than FXF. Moreover, we observed significantly decreased IL-4 level in histamine/osthole theatment compared to histamine alone. Meanwhile, FXF not significantly decrease the level of IL-4 increased by histamine. This data indicates osthole's strong role in allergic inflamation. All results confirm our hypothesis that osthole is a natural histamine antagonist and therefore can be beneficially used in antihistamine treatment of conditions such as allergies.
- Esculin attenuates endotoxin shock induced by lipopolysaccharide in mouse and NO production in vitro through inhibition of NF-κB activation. [Journal Article]
- EJEur J Pharmacol 2016 Oct 13; 791:726-734
- Esculin, a coumarin compound derived from the traditional Chinese herbs such as Cortex Fraxini, has long been used for treating inflammatory and vascular diseases. In present study, we analyzed the r...
Esculin, a coumarin compound derived from the traditional Chinese herbs such as Cortex Fraxini, has long been used for treating inflammatory and vascular diseases. In present study, we analyzed the role of esculin against macrophages and endotoxin shock induced by lipopolysaccharide (LPS) in mice. Here, we demonstrated that esculin suppressed inflammatory reactions in macrophages and protected mice from LPS-induced endotoxin shock. We found that esculin significantly inhibited the production of nitric oxide (NO) production via the inhibition of nuclear factor-κB (NF-κB) activation in macrophages. In animal model, esculin pretreatment significantly improved the survival rate of mice. LPS-induced increase of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in serum, lung, liver and kidney were markedly inhibited by esculin. IL-10, an anti-inflammatory cytokine, was up-regulated by esculin. Moreover, the histopathological analyses showed that esculin significantly attenuated the tissues injury of lung, liver, kidney in endotoxic mice. In addition, esculin significantly diminished the protein expression of NF-κB p65 in lung, liver, kidney, which resulted in lower levels of inflammatory mediators. These results suggest that esculin may be a potential drug for treatment of various inflammatory diseases.
- Helium postconditioning regulates expression of caveolin-1 and -3 and induces RISK pathway activation after ischaemia/reperfusion in cardiac tissue of rats. [Journal Article]
- EJEur J Pharmacol 2016 Oct 11; 791:718-725
- Caveolae, lipid enriched invaginations of the plasma membrane, are epicentres of cellular signal transduction. The structural proteins of caveolae, caveolins, regulate effector pathways in anaestheti...
Caveolae, lipid enriched invaginations of the plasma membrane, are epicentres of cellular signal transduction. The structural proteins of caveolae, caveolins, regulate effector pathways in anaesthetic-induced cardioprotection, including the RISK pathway. Helium (He) postconditioning (HePoc) is known to mimic anaesthetic conditioning and to prevent damage from myocardial infarction. We hypothesize that HePoc regulates caveolin-1 and caveolin-3 (Cav-1 and Cav-3) expression in the rat heart and activates the RISK pathway. Male Wistar rats (n=8, each group) were subjected to 25min of cardiac ischaemia followed by reperfusion (I/R) for 5, 15 or 30min (I/R 5/15/30). The HePoc groups underwent I/R with 70% helium ventilation during reperfusion (IR+He 5/15/30min). Sham animals received surgical treatment without I/R. After each protocol blood and hearts were retrieved. Tissue was obtained from the area-at-risk (AAR) and non-area-at-risk (NAAR) and processed for western blot analyses and reverse-transcription-real-time-polymerase-chain-reaction (RT-qPCR). Protein analyses revealed increased amounts of Cav-1 and Cav-3 in the membrane of I/R+He15 (AAR: Cav-1, P<0.05; Cav-3, P<0.05; both vs. I/R15). In serum, Cav-3 was found to be elevated in I/R+He15 (P<0.05 vs. I/R15). RT-qPCR showed increased expression of Cav-1 in IR+He15 in AAR tissue (P<0.05 vs. I/R15). Phosphorylation of RISK pathway proteins pERK1/2 (AAR: P<0.05 vs. I/R15) and pAKT (AAR: P<0.05; NAAR P<0.05; both vs. I/R15) was elevated in the cytosolic fraction of I/R+He15. These results suggest that 15min of HePoc regulates Cav-1 and Cav-3 and activates RISK pathway kinases ERK1/2 and AKT. These processes might be crucially involved in HePoc mediated cardioprotection.
- Galacto-N-biose is neuroprotective against glutamate-induced excitotoxicity in vitro. [Journal Article]
- EJEur J Pharmacol 2016 Oct 8; 791:711-717
- Galacto-N-biose (GNB: Galβ1-3GalNAc) is an O-glycan disaccharide core moiety that is a core component of mucin in the gastrointestinal tract; however, the physiological properties of GNB are not well...
Galacto-N-biose (GNB: Galβ1-3GalNAc) is an O-glycan disaccharide core moiety that is a core component of mucin in the gastrointestinal tract; however, the physiological properties of GNB are not well understood. Glutamate excitotoxicity causes neuronal death in acute neurological disorders including stroke, trauma, and neurodegenerative disease. Therefore the discovery of drugs to treat glutamate excitotoxicity is an important goal. Here, we report that GNB is neuroprotective against glutamate-induced excitotoxicity. We treated 14-15 days in vitro cultured rat cortical neurons with 0.1-1000nM GNB together with 30µm glutamate for various durations. Short-term (3h) GNB treatments showed a modest neuroprotective effect against glutamate neurotoxicity, however, long-term (24h) GNB treatment conferred significant neuroprotective effects, as shown by both MTT and immunocytochemical assays. Prolonged GNB treatment did not alter glutamate-induced calcium influx, but did induce antioxidant-related gene expression. Furthermore, GNB treatment did not induce cell death or alter synaptic connections. These data suggest that GNB is a potential candidate drug that protects against glutamate excitotoxicity without affecting cell viability and synaptic connections.
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- High fructose-mediated attenuation of insulin receptor signaling does not affect PDGF-induced proliferative signaling in vascular smooth muscle cells. [Journal Article]
- EJEur J Pharmacol 2016 Oct 8; 791:703-710
- Insulin resistance is associated with accelerated atherosclerosis. Although high fructose is known to induce insulin resistance, it remains unclear as to how fructose regulates insulin receptor signa...
Insulin resistance is associated with accelerated atherosclerosis. Although high fructose is known to induce insulin resistance, it remains unclear as to how fructose regulates insulin receptor signaling and proliferative phenotype in vascular smooth muscle cells (VSMCs), which play a major role in atherosclerosis. Using human aortic VSMCs, we investigated the effects of high fructose treatment on insulin receptor substrate-1 (IRS-1) serine phosphorylation, insulin versus platelet-derived growth factor (PDGF)-induced phosphorylation of Akt, S6 ribosomal protein, and extracellular signal-regulated kinase (ERK), and cell cycle proteins. In comparison with PDGF (a potent mitogen), neither fructose nor insulin enhanced VSMC proliferation and cyclin D1 expression. d-[(14)C(U)]fructose uptake studies revealed a progressive increase in fructose uptake in a time-dependent manner. Concentration-dependent studies with high fructose (5-25mM) showed marked increases in IRS-1 serine phosphorylation, a key adapter protein in insulin receptor signaling. Accordingly, high fructose treatment led to significant diminutions in insulin-induced phosphorylation of downstream signaling components including Akt and S6. In addition, high fructose significantly diminished insulin-induced ERK phosphorylation. Nevertheless, high fructose did not affect PDGF-induced key proliferative signaling events including phosphorylation of Akt, S6, and ERK and expression of cyclin D1 protein. Together, high fructose dysregulates IRS-1 phosphorylation state and proximal insulin receptor signaling in VSMCs, but does not affect PDGF-induced proliferative signaling. These findings suggest that systemic insulin resistance rather than VSMC-specific dysregulation of insulin receptor signaling by high fructose may play a major role in enhancing atherosclerosis and neointimal hyperplasia.