Selective inhibition of prostacyclin synthase activity by rofecoxib

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.

1

Effect of non-selective NSAIDs and selective COX-2 inhibitors on cyclooxygenase activity in HUVEC. HUVE cells were stimulated with 20 nM and 40 nM TPA, and analyzed for COX-1 and COX-2 protein levels by Western blot (panel A). 40 nM TPA-stimulated HUVE cells (panel B) were treated with acetylsalicylic acid, naproxen, celecoxib and rofecoxib at different doses as described under Materials and Methods. The amount of 6-keto-PGF1 released into the cell medium after 24 hrs was evaluated by ELISA assay and it is reported in the graph as pg/10⁴ cells ±SEM (n = 9).

2

Effect of non-selective NSAIDs and selective COX-2 inhibitors on PGIS activity in HUVEC. HUVE cells were treated with acetylsalicylic acid (panel A), naproxen (panel B), celecoxib (panel C) and rofecoxib (panel D) at different doses as described under Materials and Methods. After 24 hrs, the medium was changed and PGH2 (1 μM) was added for 10 min. The amount of 6-keto-PGF1α released into the cell medium was evaluated by ELISA assay and it is reported in the graphs, normalised for the cell number, as percentage of control (dose 0 of the drug). Values represents the mean ± S.E.M. of three independent experiments performed under the same conditions (n = 12). *= P < 0.05; **= P < 0.01.

3

Enzymatic properties of PGIS in HUVEC, in the absence or in the presence of rofecoxib. HUVE cells were untreated or treated with rofecoxib at 10⁻⁵ M concentration, as described under Materials and Methods. After 24 hrs, the medium was changed and the substrate PGH2 was added at different concentrations (ranging from 0.25 μM to 5 μM). The enzyme reaction was allowed to proceed for 10 min. The amount of 6-keto-PGF1α released into the cell medium was evaluated by ELISA assay. Data are represented by a Lineweaver-Burk plot as mean of three independent experiments ± SEM, (n = 12). The slope of the line, analyzed with SPSS linear regression programme, was 0.08 in the absence of rofecoxib and increased to 0.12 in the presence of rofecoxib at 10⁻⁵ M.

4

Effect of non-selective NSAIDs and selective COX-2 inhibitors on PGIS protein levels and mRNA expression in HUVEC. HUVE cells were treated with acetylsalicylic acid, naproxen, celecoxib and rofecoxib at different doses as described under Materials and Methods (A and B). After 24 hrs, cell lysates were prepared and analyzed by immunoblotting to detect PGIS protein levels. Lane 1: PGIS standard; lanes 2–4: samples treated with acetylsalicylic acid at different doses (10⁻³ M, 10⁻⁴ M, 10⁻⁶ M); lanes 5–7: samples treated with naproxen at different doses (10⁻³ M, 10⁻⁴ M, 10⁻⁶ M); lanes 8–10: samples treated with celecoxib at different doses (10⁻⁴ M, 10⁻⁵ M, 10⁻¹⁰ M); lanes 11–13: samples treated with rofecoxib at different doses (10⁻⁵ M, 10⁻⁶ M, 10⁻¹⁰ M); lane 14: control (untreated sample). For each sample the intensity of the band corresponding to PGIS (panel A) was normalized with respect to that corresponding to β-actin and results are represented in panel B as percentage of control. PGIS and COX-2 mRNA levels were analysed by real-time PCR (panel C) in HUVE cells after 24 hrs of incubation in the absence (lanes 1–2) or in the presence (lanes 3–4) of 10⁻⁵M rofecoxib. No statistically significant differences in PGIS mRNA expression were detectable after rofecoxib treatment. COX-2 mRNA expression, before and after rofecoxib treatment, is also shown. Bars represent values normalized against GUSB mRNA levels. Values are the mean ± S.E.M. of three independent experiments (n = 9) performed under the same experimental conditions.

5

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.

6

Effect of non-selective NSAIDs and selective COX-2 inhibitors on PGIS activity in bovine aortic micro-somes (BAMs). 2 μg of bovine aortic microsomal fractions enriched in PGIS were incubated with acetylsalicylic acid, naproxen, celecoxib and rofecoxib at different doses for 1 h at 37°C as described under Materials and Methods. After addition of the substrate PGH2 (1 μM), the reaction products were extracted, 6-keto-PGF1α production was evaluated by ELISA assay and it is reported in the graph as percentage of control (representing PGIS activity in BAMs in the absence of any treatment). Values are the mean ± S.E.M. of three independent experiments (n = 12) performed under the same experimental conditions. *= P < 0.01.