Sulindac sulfide suppresses 5-lipoxygenase at clinically relevant concentrations

Abstract

Sulindac is a non-selective inhibitor of cyclooxygenases (COX) used to treat inflammation and pain. Additionally, non-COX targets may account for the drug's chemo-preventive efficacy against colorectal cancer and reduced gastrointestinal toxicity. Here, we demonstrate that the pharmacologically active metabolite of sulindac, sulindac sulfide (SSi), targets 5-lipoxygenase (5-LO), the key enzyme in the biosynthesis of proinflammatory leukotrienes (LTs). SSi inhibited 5-LO in ionophore A23187- and LPS/fMLP-stimulated human polymorphonuclear leukocytes (IC(50) approximately 8-10 microM). Importantly, SSi efficiently suppressed 5-LO in human whole blood at clinically relevant plasma levels (IC(50) = 18.7 microM). SSi was 5-LO-selective as no inhibition of related lipoxygenases (12-LO, 15-LO) was observed. The sulindac prodrug and the other metabolite, sulindac sulfone (SSo), failed to inhibit 5-LO. Mechanistic analysis demonstrated that SSi directly suppresses 5-LO with an IC(50) of 20 muM. Together, these findings may provide a novel molecular basis to explain the COX-independent pharmacological effects of sulindac under therapy.

Fig. 1

Effect of SSi and related compounds on LO product formation in intact PMNL and platelets. The experimental procedures were performed as described in “Materials and methods”. All results are mean + SEM comparatively to vehicle control (100%). a Chemical structures of sulindac, SSi, SSo, indomethacin, diclofenac, and MK-886. b Effect of SSi on 5-LO product formation in intact PMNL evoked by 2.5 μM A23187 in absence or presence of AA (2 or 20 μM). 5-LO product formation in untreated control was 152.9 ± 30.2 ng (no AA), 255 ± 12.3 ng (2 μM AA), and 1,181.3 ± 192 ng (20 μM AA); n ≥ 3 c 5-LO-inhibitory effect of the indicated concentrations of SSi, SSo, sulindac, diclofenac, indomethacin, and BWA4C in intact PMNL stimulated with 2.5 μM A23187 + 20 μM AA; n ≥ 3. ***p ≤ 0.001. d Effect of SSi on 5-, 12-, and 15-LO product formation. For analysis of 5-LO and 15-LO, PMNL preparations were incubated with increasing concentrations of SSi and stimulated with 40 μM AA and 2.5 μM A23187. For analysis of 12-LO, human platelets were pre-treated with SSi and incubated with 5 μM AA; n ≥ 3. Product formation in controls (100%) was 979.4 ± 139.4 ng (5-LO products), 136.9 ± 31.4 ng (15-HETE), and 844.6 ± 176.2 ng (12-HETE). BWA BWA4C, Dic diclofenac, Ind indomethacin, SSi sulindac sulfide, SSo sulindac sulfone, Sul sulindac

Fig. 2

Effect of SSi on 5-LO product formation of intact PMNL triggered by different stimuli. The experimental procedures were performed as described in “Materials and methods”. All results are mean + SEM comparatively to vehicle control (100%). a Effect of SSi in intact PMNL stimulated with 1 μg/ml LPS/Ada 0.2 U/ml/1 μM fMLP; n ≥ 3. 5-LO product formation in untreated control was 10.2 ng/10⁷ cells. b Effect of SSi in intact PMNL stimulated with SA, SC and A23187. Cells were preincubated with SSi at the indicated concentrations for 15 min at 37°C. SA (10 μM) and SC (300 mM) were added 3 min prior to the addition of 20 μM AA, ionophore A23187 (2.5 μM) was added together with 20 μM AA; n = 3. 5-LO product formation in controls (100%) was 1,143.5 ± 74.7 ng (A23187), 361.1 ± 13.2 ng (SC), and 313.4 ± 86.8 ng (SA). SA Sodium arsenite, SC sodium chloride

Fig. 3

Effect of SSi and control agents on recombinant 5-LO product formation. The experimental procedures were performed as described in “Materials and methods”. All results are mean + SEM comparatively to vehicle control (100%). a Inhibition of partially purified human recombinant 5-LO by SSi compared to sulindac and SSo; n = 4. b Effect of SSi, SSo, sulindac, indomethacin and BWA4C at the concentrations indicated on recombinant 5-LO activity. 5-LO product formation in controls (100%) was 774.3 ± 118 ng; n ≥ 3. ***p ≤ 0.001. BWA BWA4C, Ind indomethacin, SSi sulindac sulfide, SSo sulindac sulfone, Sul sulindac

Fig. 4

Effect of SSi and other control agents on A23187-induced subcellular redistribution of 5-LO in intact PMNL. Cells were preincubated with the compounds for 15 min at 37°C, A23187 (2.5 μM) was added and nuclear and non-nuclear fractions were prepared by mild detergent (0.1% NP-40) lysis and analyzed for 5-LO expression using western blot as described in “Materials and methods”. A representative out of three independent experiments is shown. BW BWA4C, MK MK-886, non-nuc non-nuclear, nuc nuclear, SSi sulindac sulfide, SSo sulindac sulfone, Sul sulindac

Fig. 5

Effects of SSi and related compounds on A23187-induced 5-LO product formation in PMNL in presence of MK-886. Cells were preincubated with the compounds and 5 μM MK-886 for 10 min and the reaction was started by addition of 40 μM AA and 2.5 μM A23187. 5-LO product formation was assayed as described in “Materials and methods” and expressed relatively to vehicle control (100%). Results are mean + SEM; n = 3–6. Total 5-LO product formation in the control (100%) was 717 + 100.2 ng. ***p ≤ 0.001

Fig. 6

Effect of SSi on 5-LO product formation triggered by different stimuli in human whole blood. The experimental procedures were performed as described in “Materials and methods”. All results are mean + SEM comparatively to vehicle control (100%). a Effect of SSi on 5-LO product formation in human venous whole blood stimulated with A23187 (30 μM) or the combination of LPS 1 μg/ml and 1 μM fMLP; n ≥ 3. 5-LO product formation in untreated plasma was 234.1 ng/ml (A23187) and 59.2 ng/ml (LPS/fMLP). b Inhibitory effect of SSi, SSo, sulindac and BWA4C on 5-LO product formation in human whole blood; n ≥ 3. ***p ≤ 0.001, *p ≤ 0.05. BWA BWA4C, SSi sulindac sulfide, SSo sulindac sulfone, Sul sulindac

Fig. 7

Effect of sulindac, SSo and SSi on eicosanoid formation in human whole blood. The experimental procedures were performed as described in “Materials and methods”. All results are mean + SEM comparatively to vehicle control (100%). a Effect of SSi on the formation of COX-2-derived PGE₂, COX-1-derived 12-HHT and 5-LO products in human venous whole blood; n ≥ 3. Eicosanoid formation in control plasma (100%) was 123.2 ng/ml (12-HHT), 59.2 ng/ml (5-LO products), and 32.76 ng/ml (COX-2 derived PGE₂). b Comparison of inhibition of 12-HHT and PGE₂ synthesis by SSi, SSo, and sulindac in human whole blood; n ≥ 3. ***p ≤ 0.001, *p ≤ 0.05. SSi Sulindac sulfide; SSo sulindac sulfone, Sul sulindac