| 1. |
- Alhouayek, Mireille, et al.
(författare)
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Altered mRNA Expression of Genes Involved in Endocannabinoid Signalling in Squamous Cell Carcinoma of the Oral Tongue
- 2019
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Ingår i: Cancer Investigation. - : Informa UK Limited. - 0735-7907 .- 1532-4192. ; 37:8, s. 327-338
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Tidskriftsartikel (refereegranskat)abstract
- Little is known about the endocannabinoid (eCB) system in squamous cell carcinoma of the oral tongue (SCCOT). Here we have investigated, at the mRNA level, expression of genes coding for the components of the eCB system in tumour and non-malignant samples from SCCOT patients. Expression of NAPEPLD and PLA2G4E, coding for eCB anabolic enzymes, was higher in the tumour tissue than in non-malignant tissue. Among genes coding for eCB catabolic enzymes, expression of MGLL was lower in tumour tissue while PTGS2 was increased. It is concluded that the eCB system may be dysfunctional in SCCOT.
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| 2. |
- Alhouayek, Mireille, et al.
(författare)
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Effects of orthotopic implantation of rat prostate tumour cells upon components of the N-acylethanolamine and monoacylglycerol signalling systems : an mRNA study
- 2020
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- There is good evidence that the N-acylethanolamine (NAE)/monoacylglycerol (MAG) signalling systems are involved in the pathogenesis of cancer. However, it is not known how prostate tumours affect these systems in the surrounding non-malignant tissue and vice versa. In the present study we have investigated at the mRNA level 11 components of these systems (three coding for anabolic enzymes, two for NAE/MAG targets and six coding for catabolic enzymes) in rat prostate tissue following orthotopic injection of low metastatic AT1 cells and high metastatic MLL cells. The MLL tumours expressed higher levels of Napepld, coding for a key enzyme in NAE synthesis, and lower levels of Naaa, coding for the NAE hydrolytic enzyme N-acylethanolamine acid amide hydrolase than the AT1 tumours. mRNA levels of the components of the NAE/MAG signalling systems studied in the tissue surrounding the tumours were not overtly affected by the tumours. AT1 cells in culture expressed Faah, coding for the NAE hydrolytic enzyme fatty acid amide hydrolase, at much lower levels than Naaa. However, the ability of the intact cells to hydrolyse the NAE arachidonoylethanolamide (anandamide) was inhibited by an inhibitor of FAAH, but not of NAAA. Treatment of the AT1 cells with interleukin-6, a cytokine known to be involved in the pathogenesis of prostate cancer, did not affect the expression of the components of the NAE/MAG system studied. It is thus concluded that in the model system studied, the tumours show different expressions of mRNA coding for key the components of the NAE/MAG system compared to the host tissue, but that these changes are not accompanied by alterations in the non-malignant tissue.
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| 3. |
- Alhouayek, Mireille, et al.
(författare)
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Interferon γ treatment increases endocannabinoid and related N-acylethanolamine levels in T84 human colon carcinoma cells
- 2019
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Ingår i: British Journal of Pharmacology. - : Wiley. - 0007-1188 .- 1476-5381. ; 176:10, s. 1470-1480
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Tidskriftsartikel (refereegranskat)abstract
- Background and purpose: Endocannabinoids and related N-acylethanolamines (NAEs) are involved in regulation of gut function, but relatively little is known as to whether inflammatory cytokines such as IFN affect their levels. We have investigated this in vitro using cultures of T84 colon cancer cells.Experimental approach: T84 cells, when cultured in monolayers, differentiate to form adult colonic crypt-like cells with excellent permeability barrier properties. The integrity of the permeability barrier in these monolayers was measured using transepithelial electrical resistance (TEER). NAE levels were determined by ultra-performance liquid chromatography-tandem mass spectrometric analysis. Expression of the enzymes involved in NAE and 2-arachidonoylglycerol (2-AG) turnover were assessed with qPCR.Key results: IFN treatment for 8 or 24h increased levels of both endocannabinoids (anandamide and 2-AG) and the related NAEs. The treatment did not affect the rate of hydrolysis of either anandamide or palmitoylethanolamide by intact cells, and in both cases, fatty acid amide hydrolase (FAAH) rather than NAE-hydrolysing acid amidase (NAAA) was mainly responsible for the hydrolysis of these NAEs. IFN treatment reduced the TEER of the cells in a manner that was not prevented by inhibition of either FAAH or NAAA but was partially reversed by apical administration of the NAE palmitoylethanolamide.Conclusion and implications: IFN treatment mobilized endocannabinoid and related NAE levels in T84 cells. However, blockade of anandamide or NAE hydrolysis was insufficient to negate the deleterious effects of this cytokine upon the permeability barrier of the cell monolayers.
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| 4. |
- Alhouayek, Mireille, et al.
(författare)
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Involvement of CYP1B1 in interferon gamma-induced alterations of epithelial barrier integrity
- 2018
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Ingår i: British Journal of Pharmacology. - : WILEY. - 0007-1188 .- 1476-5381. ; 175:6, s. 877-890
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND AND PURPOSE CYP1B1 and CYP1A1 are important extra-hepatic cytochromes, expressed in the colon and involved in the metabolism of dietary constituents and exogenous compounds. CYP1B1 expression is increased by pro-inflammatory cytokines, and it has been recently implicated in regulation of blood brain barrier function. We investigated its involvement in the increased permeability of the intestinal epithelial barrier observed in inflammatory conditions. EXPERIMENTAL APPROACH Epithelial monolayers formed by human T84 colon carcinoma cells cultured on transwells, were disrupted by incubation with IFN gamma (10 ng.mL(-1)). Monolayer integrity was measured using transepithelial electrical resistance. CYP1A1 and CYP1B1 inhibitors or inducers were applied apically. Potential mechanisms of action were investigated using RT-qPCR. KEY RESULTS IFN gamma disrupts the barrier integrity of the T84 monolayers and increases CYP1B1 and HIF1 alpha mRNA expression. CYP1B1 induction is inhibited by the NF-kappa B inhibitor ammonium pyrrolidinedithiocarbamate (100 mu M) but not by the HIF1 alpha inhibitor 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (50 mu M). Inhibition of CYP1B1 with the selective inhibitor 2,4,3,5-tetramethoxystilbene (100 nM) partly reverses the effects of IFN gamma on epithelial permeability. CONCLUSIONS AND IMPLICATIONS These data suggest that increased expression of CYP1B1 is involved in the effects of IFN gamma on epithelial permeability. Inhibition of CYP1B1 counteracts the alterations of epithelial barrier integrity induced by IFN gamma and could thus have a therapeutic potential in disorders of intestinal permeability associated with inflammation.
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| 5. |
- Alhouayek, Mireille, et al.
(författare)
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Role of pannexin-1 in the cellular uptake, release and hydrolysis of anandamide by T84 colon cancer cells
- 2019
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- The large pore ion channel pannexin-1 (Panx1) has been reported to play a role in the cellular uptake and release of anandamide (AEA) in the hippocampus. It is not known whether this is a general mechanism or limited to the hippocampus. We have investigated this pharmacologically using T84 colon cancer cells. The cells expressed Panx1 at the mRNA level, and released ATP in a manner that could be reduced by treatment with the Panx1 inhibitors carbenoxolone and mefloquine and the Panxl substrate SR101. However, no significant effects of these compounds upon the uptake or hydrolysis of exogenously applied AEA was seen. Uptake by T84 cells of the other main endocannabinoid 2-arachidonoylglycerol and the AEA homologue palmitoylethanolamide was similarly not affected by carbenoxolone or mefloquine. Total release of tritium from [H-3]AEA-prelabelled T84 cells over 10 min was increased, rather than inhibited by carbenoxolone and mefloquine. Finally, AEA uptake by PC3 prostate cancer and SH-SY5Y neuroblastoma cells, which express functional Panx1 channels, was not inhibited by carbenoxolone. Thus, in contrast to the hippocampus, Panx1 does not appear to play a role in AEA uptake and release from the cells studied under the conditions used.
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| 6. |
- Cheng, Ran, et al.
(författare)
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In Vitro and in Vivo Evaluation of C-11-Labeled Azetidinecarboxylates for Imaging Monoacylglycerol Lipase by PET Imaging Studies
- 2018
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 61:6, s. 2278-2291
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Tidskriftsartikel (refereegranskat)abstract
- Monoacylglycerol lipase (MAGL) is the principle enzyme for metabolizing endogenous cannabinoid ligand 2-arachidonoyglycerol (2-AG). Blockade of MAGL increases 2-AG levels, resulting in subsequent activation of the endocannabinoid system, and has emerged as a novel therapeutic strategy to treat drug addiction, inflammation, and neurodegenerative diseases. Herein we report a new series of MAGL inhibitors, which were radiolabeled by site-specific labeling technologies, including C-11-carbonylation and spirocyclic iodonium ylide (SCIDY) radio fluorination. The lead compound [C-11]10 (MAGL-0519) demonstrated high specific binding and selectivity in vitro and in vivo. We also observed unexpected washout kinetics with these irreversible radiotracers, in which in vivo evidence for turnover of the covalent residue was unveiled between MAGL and azetidine carboxylates. This work may lead to new directions for drug discovery and PET tracer development based on azetidine carboxylate inhibitor scaffold.
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| 7. |
- Gabrielsson, Linda, et al.
(författare)
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The anti-inflammatory compound palmitoylethanolamide inhibits prostaglandin and hydroxyeicosatetraenoic acid production by a macrophage cell line
- 2017
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Ingår i: Pharmacology Research & Perspectives. - : John Wiley & Sons. - 2052-1707. ; 5:2
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Tidskriftsartikel (refereegranskat)abstract
- The anti-inflammatory agent palmitoylethanolamide (PEA) reduces cyclooxygenase (COX) activity in vivo in a model of inflammatory pain. It is not known whether the compound reduces prostaglandin production in RAW264.7 cells, whether such an action is affected by compounds preventing the breakdown of endogenous PEA, whether other oxylipins are affected, or whether PEA produces direct effects upon the COX-2 enzyme. RAW264.7 cells were treated with lipopolysaccharide and interferon-c to induce COX-2. At the level of mRNA, COX-2 was induced > 1000-fold following 24 h of the treatment. Coincubation with PEA (10 mu mol/L) did not affect the levels of COX-2, but reduced the levels of prostaglandins D-2 and E-2 as well as 11- and 15-hydroxyeicosatetraenoic acid, which can also be synthesised by a COX-2 pathway in macrophages. These effects were retained when hydrolysis of PEA to palmitic acid was blocked. Linoleic acidderived oxylipin levels were not affected by PEA. No direct effects of PEA upon the oxygenation of either arachidonic acid or 2-arachidonoylglycerol by COX-2 were found. It is concluded that in lipopolysaccharide and interferon-c-stimulated RAW264.7 cells, PEA reduces the production of COX-2-derived oxylipins in a manner that is retained when its metabolism to palmitic acid is inhibited.
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| 8. |
- Hashemian, Sanaz, et al.
(författare)
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TLR4 receptor expression and function in F11 dorsal root ganglion x neuroblastoma hybrid cells
- 2017
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Ingår i: Innate Immunity. - : SAGE Publications. - 1753-4259 .- 1753-4267. ; 23:8, s. 687-696
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Tidskriftsartikel (refereegranskat)abstract
- TLR4 respond to bacterial LPS to produce inflammatory cytokines. TLR4 are expressed in dorsal root ganglia and play a role in pain. F11 dorsal root ganglia x mouse neuroblastoma cells possess many of the properties seen in nociceptive dorsal root ganglia neuronal cells. Here, we investigated the effect of 2h and 6h treatment with LPS upon the expression of inflammatory proteins in undifferentiated and differentiated F11 cells. The cells expressed mRNA for TRL4 (mouse, not rat) and proteins involved in TLR4 signaling. TLR4 expression was confirmed using immunohistochemistry. LPS produced modest increases in mouse and rat IL-6 and in mouse cyclooxygenase-2 levels in undifferentiated cells, but did not significantly affect mouse TNF- expression. This contrasts with the robust effects of LPS upon cyclooxygenase-2 expression in cultured dorsal root ganglia neurons. F11 cells expressed the endocannabinoid metabolizing enzymes fatty acid amide hydrolase and N-acylethanolamine acid amidase (both murine), which were functionally active. These data suggest that F11 cells are not a useful model for the study of LPS-mediated effects but may be useful for the study of endocannabinoid catabolism.
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| 9. |
- Karlsson, Jessica, et al.
(författare)
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Effects of tumour necrosis factor alpha upon the metabolism of the endocannabinoid anandamide in prostate cancer cells
- 2017
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Ingår i: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 12:9
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Tidskriftsartikel (refereegranskat)abstract
- Tumour necrosis factor a (TNF alpha) is involved in the pathogenesis of prostate cancer, a disease where disturbances in the endocannabinoid system are seen. In the present study we have investigated whether treatment of DU145 human prostate cancer cells affects anandamide (AEA) catabolic pathways. Additionally, we have investigated whether cyclooxygenase- 2 (COX-2) can regulate the uptake of AEA into cells. Levels of AEA synthetic and catabolic enzymes were determined by qPCR. AEA uptake and hydrolysis in DU145 and RAW264.7 macrophage cells were assayed using AEA labeled in the arachidonic and ethanolamine portions of the molecule, respectively. Levels of AEA, related N-acylethanolamines (NAEs), prostaglandins (PG) and PG-ethanolamines (PG-EA) in DU145 cells and medium were quantitated by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. TNF alpha treatment of DU145 cells increased mRNA levels of PTSG2 (gene of COX-2) and decreased the mRNA of the AEA synthetic enzyme N-acylphosphatidylethanolamine selective phospholipase D. mRNA levels of the AEA hydrolytic enzymes fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase were not changed. AEA uptake in both DU145 and RAW264.7 cells was inhibited by FAAH inhibition, but not by COX-2 inhibition, even in RAW264.7 cells where the expression of this enzyme had greatly been induced by lipopolysaccharide + interferon. treatment. AEA and related NAEs were detected in DU145 cells, but PGs and PGE(2)-EA were only detected when the cells had been preincubated with 100 nM AEA. The data demonstrate that in DU145 cells, TNFa treatment changes the relative expression of the enzymes involved in the hydrolytic and oxygenation catabolic pathways for AEA. In RAW264.7 cells, COX-2, in contrast to FAAH, does not regulate the cellular accumulation of AEA. Further studies are necessary to determine the extent to which inflammatory mediators are involved in the abnormal endocannabinoid signalling system in prostate cancer.
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| 10. |
- Szeremeta, Janis, et al.
(författare)
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Low mRNA expression and activity of monoacylglycerol lipase in human SH-SY5Y neuroblastoma cells
- 2019
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Ingår i: Prostaglandins & other lipid mediators. - : Elsevier. - 1098-8823 .- 2212-196X. ; 142, s. 59-67
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Tidskriftsartikel (refereegranskat)abstract
- Relatively little is known about the endocannabinoid system in human neuroblastoma cell lines. In the present study, we have investigated the expression of the genes coding for the enzymes involved in the synthesis and catabolism of endocannabinoids in the SH-SY5Y cell line. The expression of MGLL, the gene coding for the 2-arachidonoylglycerol hydrolytic enzyme monoacylglycerol lipase (MAGL), was found to be 85 and 340 fold lower than the expression levels for the genes coding for alpha/beta-hydrolase domain containing 6 and 12 (ABHD6, ABHD12), which are alternative hydrolytic enzymes for this endocannabinoid. In comparison, mRNA levels of MGLL were 1.5 fold higher than ABHD6 and 2 fold lower than the levels of ABHD12 in DU-145 human prostate cells. In functional assays, the hydrolysis of the 2-arachidonoylglycerol homologue 2-oleoylglycerol by intact SH-SY5Y cells was partially inhibited by the ABHD6 inhibitor WWL70, but not by the MAGL inhibitor JZL184, whereas the reverse was true in DU-145 cells. The combination of JZL184 + WWL70 did, however produce a significantly greater inhibition of 2-OG hydrolysis than seen with WWL70 alone in the SH-SY5Y cells. The low MGLL expression in the SH-SY5Y cells was not due to epigenetic silencing, since levels were not affected by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine and/or the histone acetylase inhibitor trichostatin A. The low MGLL expression in SH-SY5Y cells should be taken into account when using these cells in experiments investigating the involvement of the endocannabinoid system in models of physiological and pathological processes.
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