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The mRNA expression of endocannabinoid-related enzymes in rat prostate AT-1 cells following exposure to lactate and interleukin-6
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.ORCID iD: ORCID.org/0000_0001-8572-5841
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The endocannabinoid system is dysregulated in prostate cancer but the mechanisms responsible for this dysregulation are not known. We hypothesise that the dysregulation is secondary to factors in the tumour microenvironment. In this study we investigated the effects of lactic acid induced low pH and interleukin-6 (IL-6) treatment upon the expression of endocannabinoid related enzymes and the functional effects upon anandamide degradation and cell viability in Dunning R3327 rat prostate AT-1 cancer cells. Cells were exposed for 3 h at 37 °C to Krebs-Ringer-HEPES/bicarbonate buffer at either pH 7.4 or at pH 6.6 (due to the presence of 40 mM lactic acid), and to 0, 25 or 100 ng/ml of IL-6. Neither low pH (pH 6.6) nor IL-6 induced any changes in the mRNA levels of the anandamide metabolic enzymes. However, the expression of the 2- arachidonoylglycerol-synthesizing enzyme DAGLα was increased by low pH and the expression of CB2 receptor mRNA was decreased at the low pH. The DAGL inhibitor orlistat increased extracellular LDH levels after 24 h of incubation of AT-1 cells, suggesting a higher frequency of cell death. It is concluded that under the conditions used, exposure to lactate and IL-6 do not affect the expression of the anandamide metabolic enzymes in AT-1 cells, but do modify the expression of an enzyme involved in the synthesis of 2-arachidonoylglycerol. 

Keywords [en]
Anandamide, 2-arachidonoylglycerol, fatty acid amide hydrolase, interleukin-6, lactic acid, prostate cancer, Dunning R3327 AT-1 cells
National Category
Basic Medicine Pharmacology and Toxicology Cell and Molecular Biology
Research subject
cellforskning; biokemisk farmakologi
Identifiers
URN: urn:nbn:se:umu:diva-147501OAI: oai:DiVA.org:umu-147501DiVA, id: diva2:1203704
Funder
Swedish Research Council, 12158
Note

Forskningsfinansiärer: vetenskapsrådet (12158), Lions cancerforskningsfond (LP16-2129)

Available from: 2018-05-04 Created: 2018-05-04 Last updated: 2018-06-09
In thesis
1. Endocannabinoid metabolism: the impact of inflammatory factors and pharmacological inhibitors
Open this publication in new window or tab >>Endocannabinoid metabolism: the impact of inflammatory factors and pharmacological inhibitors
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Endokannabinoid metabolism : påverkan av inflammatoriska faktorer och farmakologiska inhibitorer
Abstract [en]

The endocannabinoid (eCB) system is an endogenous signaling system consisting of ligands (referred to as endocannabinoids, eCBs), receptors and metabolic enzymes. The eCB system is involved in homeostatic control of a variety of biological functions such as neuronal signaling, mood, appetite and pathological conditions such as pain, inflammation and tumour progression. The main eCBs N- arachidonoylethanolamine (AEA, anandamide) and 2-arachidonoylglycerol (2-AG) are synthesised upon stimuli when and where their action is demanded. The signaling is brief and the eCBs are quickly degraded. The enzyme primarily responsible for eCB degradation is fatty acid amide hydrolase (FAAH) for AEA and monoacylglycerol lipase (MAGL) for 2-AG. In addition, both substances are substrates for cyclooxygenase-2 (COX-2). COX-2 is upregulated in inflammation, pain and in several tumours including prostate cancers, but it is not known whether COX-2 contribute significantly to eCB metabolism under these conditions.

Increasing endogenous levels of eCBs by inhibiting their degradation is exploited as a future therapy for pain conditions. One suggested therapeutic strategy is dual inhibition of enzymes FAAH and COX-2 to raise AEA levels. Paper I and II of this thesis investigates FAAH and COX inhibitory effects of: the major metabolites and enantiomers of derivatives (flu-AM1 and ibu-AM5) of the current clinically used NSAIDs ibuprofen and flurbiprofen. The metabolites 3 ́hydroxyibuprofen and 4 ́hydroxyflurbiprofen retained the FAAH and COX- inhibitory effects seen by the parent compounds although at lower potencies. Both enantiomers of flu-AM1 were equally potent as FAAH inhibitors and displayed a useful substrate selective COX-2 inhibition profile, favoring eCBs as substrates rather than arachidonic acid.

Paper III explores the impact of COX-2 and the effect of (R)-flu-AM1 upon AEA levels and degradation in mouse leukemic macrophage RAW264.7 cells. Despite the high inhibitory potency in enzyme assays, neither (R)-flu-AM1 nor the combination of a FAAH inhibitor with flurbiprofen increased AEA levels in the intact cells to any great extent. This suggests that the eCB turnover in these cells is rather slow. Further, in paper IV, induction of COX-2 did not unmask an ability of this enzyme to “gate” the uptake of AEA analogous to that seen with FAAH.

Paper IV and V focus upon the role of the eCB system in prostate cancer. The eCB system is altered in cancer and is linked to the progression and prognosis of prostate cancer. How and whereby this change occurs is unknown. This thesis explores the impact of the inflammatory factors TNFα, IL-6 and lactic acid induced low pH upon the mRNA levels of eCB related enzymes and the functional impact upon AEA degradation in human DU145 and rat AT-1 prostate cancer cells. TNFα treatment of DU145 and IL-6 and lactic acid induced low pH exposure of AT-1 changed the mRNA levels of 2-AG related enzymes leaving AEA rather unaffected other than for a substantial induction of COX-2 mRNA in DU145 cells. Thus, AEA homeostasis was not shifted in prostate cancer cell lines exposed to inflammatory factors. The results suggest that COX-2 does not gate the uptake of AEA and is a minor contributor to AEA degradation in intact cells. 

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2018. p. 82
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1958
Keywords
endocannabinoid, anandamide, 2-AG, fatty acid amide hydrolase, cyclooxygenase-2, prostate cancer, catabolism, inflammation, inflammatory factors
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-147503 (URN)978-91-7601-870-5 (ISBN)
Public defence
2018-06-01, Major Groove, NUS byggnad 6L, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2018-05-09 Created: 2018-05-04 Last updated: 2018-06-09Bibliographically approved

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