The dual inhibitor of lipoxygenase and cyclooxygenase ML3000 decreases the expression of CXCR3 ligands.
OBJECTIVE
To find previously unknown properties of ML3000, a competitive inhibitor of the cyclooxygenase and the lipoxygenase (LO) pathway.


METHODS
Gene expression of ML3000 treated and untreated rheumatoid arthritis synovial fibroblasts were measured with Affymetrix gene arrays. Downregulation of chemokine (C-X-C motif) ligands CXCL9, CXCL10 and CXCL11 was verified with Real-time polymerase chain reaction, CXCL10 protein levels were determined with ELISA. Rheumatoid arthritis synovial fibroblasts were treated with the cyclooxygenase inhibitor naproxen, the 5-LO inhibitor BWA4C and the 5-lipoxygenase-activating protein (FLAP) inhibitor MK886, and consecutive changes in CXCL10 protein levels measured. 5-LO expression was determined by polymerase chain reaction and Western blot.


RESULTS
In synovial fibroblasts and monocyte-derived macrophages ML3000 inhibited the tumour necrosis factor induced expression of CXCL9, CXCL10 and CXCL11, which are all ligands of the chemokine receptor CXCR3. No effect was observed in monocytes. Whereas inhibition of the cyclooxygenase pathway or the FLAP protein showed no effect, blockade of 5-LO significantly downregulated CXCL10 protein levels. 5-LO mRNA was detected in monocytes and in monocyte-derived macrophages. All tested cell types expressed 5-LO protein.


CONCLUSIONS
ML3000 effectively downregulates CXCR3 ligands. This study confirms that a thorough analysis of the impact of a drug on its target cells cannot only reveal unexpected properties of a substance, but also helps to understand the underlying molecular mechanisms. Accordingly, our data provide the basis for further clinical studies testing the application of ML3000 in diseases such as rheumatoid arthritis or multiple sclerosis.
Ospelt
C
C
Center of Experimental Rheumatology, University Hospital Zurich, Switzerland. Caroline.Ospelt@usz.ch
Kurowska-Stolarska
M
M
Neidhart
M
M
Michel
B A
BA
Gay
R E
RE
Laufer
S
S
Gay
S
S
D000085:Acetates / Q000494:pharmacology*; D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D019743:Chemokines, CXC / Q000096:biosynthesis / Q000235:genetics; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D015536:Down-Regulation / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008024:Ligands; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods; D011758:Pyrroles / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D054367:Receptors, CXCR3 / Q000235:genetics / Q000378:metabolism*; D013583:Synovial Membrane / Q000187:drug effects / Q000378:metabolism
D000085:Acetates; C515187:CXCR3 protein, human; D019743:Chemokines, CXC; D016861:Cyclooxygenase Inhibitors; D008024:Ligands; D016859:Lipoxygenase Inhibitors; D011758:Pyrroles; D012333:RNA, Messenger; D054367:Receptors, CXCR3; C088092:licofelone