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Selective inhibition of prostacyclin synthase activity by rofecoxib.

Griffoni C, Spisni E, Strillacci A, Toni M, Bachschmid MM, Tomasi V - J. Cell. Mol. Med. (2007 Mar-Apr)

Bottom Line: The inhibitory effect of rofecoxib is associated neither to a decrease of PGIS protein levels nor to an impairment of the enzyme intracellular localization.The results of this study may explain the absence of a clear relationship between COX-2 selectivity and cardiovascular side effects.Moreover, in the light of these results we propose that novel selective COX-2 inhibitors should be tested on PGI2 synthase activity inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Biology, University of Bologna, Via Selmi 3, 40126 Bologna, Italy.

ABSTRACT
The development of cyclooxygenase-2 (COX-2) selective inhibitors prompted studies aimed at treating chronic inflammatory diseases and cancer by using this new generation of drugs.Yet, several recent reports pointed out that long-term treatment of patients with COX-2 selective inhibitors (especially rofecoxib) caused severe cardiovascular complicances. The aim of this study was to ascertain whether, in addition to inhibiting COX-2, rofecoxib may also affect prostacyclin (PGI2) level by inhibiting PGI2 forming enzyme (prostacyclin synthase, PGIS). In order to evaluate if selective (celecoxib, rofecoxib) and non-selective (aspirin, naproxen) anti-inflammatory compounds could decrease PGI2 production in endothelial cells by inhibiting PGIS, we analyzed the effect of anti-inflammatory compounds on the enzyme activity by ELISA assay after addition of exogenous substrate, on PGIS protein levels by Western blotting and on its subcellular distribution by confocal microscopy. We also analyzed the effect of rofecoxib on PGIS activity in bovine aortic microsomal fractions enriched in PGIS. This study demonstrates an inhibitory effect of rofecoxib on PGIS activity in human umbilical vein endothelial (HUVE) cells and in PGIS-enriched bovine aortic microsomal fractions, which is not observed by using other anti-inflammatory compounds. The inhibitory effect of rofecoxib is associated neither to a decrease of PGIS protein levels nor to an impairment of the enzyme intracellular localization. The results of this study may explain the absence of a clear relationship between COX-2 selectivity and cardiovascular side effects. Moreover, in the light of these results we propose that novel selective COX-2 inhibitors should be tested on PGI2 synthase activity inhibition.

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Effect of non-selective NSAIDs and selective COX-2 inhibitors on PGIS intracellular distribution in HUVEC. HUVE cells, seeded on cover slides and treated with acetylsalicylic acid (10−3 M), naproxen (10−3 M), celecoxib (10−4 M) and rofecoxib (10−5 M) for 24 hrs, were stained with PGIS and Cav-1 antibodies and analyzed by confocal microscopy. The figure represents images regarding PGIS and Cav-1 distributions in control (untreated) cells (panels A–D) and in rofecoxib-treated cells (panels E–H), because results were similar for all the treatments tested. Cav-1 distribution (green signal) is represented in panels A and E while PGIS distribution (red signal) is represented in panels B and F. The merging of the two signals (yellow) is reported in panels C and G. The colocalization maps, obtained as described under Materials and Methods, are reported in panels D and H. Bar: 10 μm.
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fig05: Effect of non-selective NSAIDs and selective COX-2 inhibitors on PGIS intracellular distribution in HUVEC. HUVE cells, seeded on cover slides and treated with acetylsalicylic acid (10−3 M), naproxen (10−3 M), celecoxib (10−4 M) and rofecoxib (10−5 M) for 24 hrs, were stained with PGIS and Cav-1 antibodies and analyzed by confocal microscopy. The figure represents images regarding PGIS and Cav-1 distributions in control (untreated) cells (panels A–D) and in rofecoxib-treated cells (panels E–H), because results were similar for all the treatments tested. Cav-1 distribution (green signal) is represented in panels A and E while PGIS distribution (red signal) is represented in panels B and F. The merging of the two signals (yellow) is reported in panels C and G. The colocalization maps, obtained as described under Materials and Methods, are reported in panels D and H. Bar: 10 μm.

Mentions: We also analyzed by confocal microscopy PGIS intracellular distribution in HUVEC treated for 24 hrs with acetylsalicylic acid, naproxen, celecoxib and rofecoxib at the higher doses previously tested by ELISA assay and Western blotting (Fig. 5). All the inhibitors tested, and in particular rofecoxib, did not cause evident changes of PGIS intracellular distribution with respect to control cells (compare panels E–H to panels A–D). In particular, the colocalization of PGIS with caveolin-1 (Cav-1), which is relevant for its activity [18], was not altered following the treatment with rofecoxib. This result indicates that the inhibition of PGIS activity caused by rofecoxib is not related to a misallocation of the enzyme leading to an impairment of its function.


Selective inhibition of prostacyclin synthase activity by rofecoxib.

Griffoni C, Spisni E, Strillacci A, Toni M, Bachschmid MM, Tomasi V - J. Cell. Mol. Med. (2007 Mar-Apr)

Effect of non-selective NSAIDs and selective COX-2 inhibitors on PGIS intracellular distribution in HUVEC. HUVE cells, seeded on cover slides and treated with acetylsalicylic acid (10−3 M), naproxen (10−3 M), celecoxib (10−4 M) and rofecoxib (10−5 M) for 24 hrs, were stained with PGIS and Cav-1 antibodies and analyzed by confocal microscopy. The figure represents images regarding PGIS and Cav-1 distributions in control (untreated) cells (panels A–D) and in rofecoxib-treated cells (panels E–H), because results were similar for all the treatments tested. Cav-1 distribution (green signal) is represented in panels A and E while PGIS distribution (red signal) is represented in panels B and F. The merging of the two signals (yellow) is reported in panels C and G. The colocalization maps, obtained as described under Materials and Methods, are reported in panels D and H. Bar: 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822831&req=5

fig05: Effect of non-selective NSAIDs and selective COX-2 inhibitors on PGIS intracellular distribution in HUVEC. HUVE cells, seeded on cover slides and treated with acetylsalicylic acid (10−3 M), naproxen (10−3 M), celecoxib (10−4 M) and rofecoxib (10−5 M) for 24 hrs, were stained with PGIS and Cav-1 antibodies and analyzed by confocal microscopy. The figure represents images regarding PGIS and Cav-1 distributions in control (untreated) cells (panels A–D) and in rofecoxib-treated cells (panels E–H), because results were similar for all the treatments tested. Cav-1 distribution (green signal) is represented in panels A and E while PGIS distribution (red signal) is represented in panels B and F. The merging of the two signals (yellow) is reported in panels C and G. The colocalization maps, obtained as described under Materials and Methods, are reported in panels D and H. Bar: 10 μm.
Mentions: We also analyzed by confocal microscopy PGIS intracellular distribution in HUVEC treated for 24 hrs with acetylsalicylic acid, naproxen, celecoxib and rofecoxib at the higher doses previously tested by ELISA assay and Western blotting (Fig. 5). All the inhibitors tested, and in particular rofecoxib, did not cause evident changes of PGIS intracellular distribution with respect to control cells (compare panels E–H to panels A–D). In particular, the colocalization of PGIS with caveolin-1 (Cav-1), which is relevant for its activity [18], was not altered following the treatment with rofecoxib. This result indicates that the inhibition of PGIS activity caused by rofecoxib is not related to a misallocation of the enzyme leading to an impairment of its function.

Bottom Line: The inhibitory effect of rofecoxib is associated neither to a decrease of PGIS protein levels nor to an impairment of the enzyme intracellular localization.The results of this study may explain the absence of a clear relationship between COX-2 selectivity and cardiovascular side effects.Moreover, in the light of these results we propose that novel selective COX-2 inhibitors should be tested on PGI2 synthase activity inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Biology, University of Bologna, Via Selmi 3, 40126 Bologna, Italy.

ABSTRACT
The development of cyclooxygenase-2 (COX-2) selective inhibitors prompted studies aimed at treating chronic inflammatory diseases and cancer by using this new generation of drugs.Yet, several recent reports pointed out that long-term treatment of patients with COX-2 selective inhibitors (especially rofecoxib) caused severe cardiovascular complicances. The aim of this study was to ascertain whether, in addition to inhibiting COX-2, rofecoxib may also affect prostacyclin (PGI2) level by inhibiting PGI2 forming enzyme (prostacyclin synthase, PGIS). In order to evaluate if selective (celecoxib, rofecoxib) and non-selective (aspirin, naproxen) anti-inflammatory compounds could decrease PGI2 production in endothelial cells by inhibiting PGIS, we analyzed the effect of anti-inflammatory compounds on the enzyme activity by ELISA assay after addition of exogenous substrate, on PGIS protein levels by Western blotting and on its subcellular distribution by confocal microscopy. We also analyzed the effect of rofecoxib on PGIS activity in bovine aortic microsomal fractions enriched in PGIS. This study demonstrates an inhibitory effect of rofecoxib on PGIS activity in human umbilical vein endothelial (HUVE) cells and in PGIS-enriched bovine aortic microsomal fractions, which is not observed by using other anti-inflammatory compounds. The inhibitory effect of rofecoxib is associated neither to a decrease of PGIS protein levels nor to an impairment of the enzyme intracellular localization. The results of this study may explain the absence of a clear relationship between COX-2 selectivity and cardiovascular side effects. Moreover, in the light of these results we propose that novel selective COX-2 inhibitors should be tested on PGI2 synthase activity inhibition.

Show MeSH
Related in: MedlinePlus