RESUMEN
Spironolactone (SPIR) binds to cytoplasmic mineralocorticoid receptors (MR) and functions as an aldosterone (ALDO) antagonist. Recently, however, the drug was shown to have an early MR independent, suppressive effect on immunoactive and inflammatory cytokines as well as an apoptotic effect on blood mononuclear cells (MNC). To elucidate the mechanism behind SPIR's apoptotic effect, we investigated the relation between apoptosis and cytokine suppression for SPIR along with the apoptosis-inducing and antiinflammatory drug sulfasalazine (SFZ). Using human MNC, we found that SPIR and SFZ, at concentrations 10 and 1000 muM, respectively, significantly increased both apoptosis and cell death. Production of inflammatory cytokines was significantly reduced by 3 to 30 muM SPIR and by 300 to 1000 muM SFZ. We also found that 0.4 muM SPIR and 300 muM SFZ significantly reduced the activity of NF-kappaB, a transcription factor involved in both apoptosis and immunoinflammation. ALDO, the MR antagonist, eplerenone, and the SPIR metabolite, 7alpha-thiomethyl-spironolactone, slightly reduced NF-kappaB activity, but they did not interfere with SPIR's effect, showing that MR binding is not involved in SPIR-induced suppression of NF-kappaB activity. Finally, phosphorylation of IkappaBalpha was also significantly reduced by SPIR. These results provide new insight into the apoptotic and anti-inflammatory effects of SPIR.
Asunto(s)
Apoptosis/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Receptores de Mineralocorticoides/metabolismo , Espironolactona/farmacología , Caspasa 3/metabolismo , Citocinas/biosíntesis , Humanos , Proteínas I-kappa B/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Antagonistas de Receptores de Mineralocorticoides/farmacología , Inhibidor NF-kappaB alfa , Fosforilación/efectos de los fármacos , Sulfasalazina/farmacologíaRESUMEN
1 Spironolactone (SPIR) binds to cytoplasmic mineralocorticoid receptors (MR) and functions as an aldosterone antagonist. Recently, the drug was shown to have an early suppressive effect on several immunoactive and proinflammatory cytokines. 2 To elucidate the mechanism behind this, the four MR-binding steroids SPIR, canrenone, 7alpha-thiomethyl-spironolactone and aldosterone (ALDO) were investigated for effects on lipopolysaccharide- and phytohemagglutinin-A-activated human blood mononuclear cells. Gene expression was examined after 4 h using microarrays, and SPIR affected 1018 transcripts of the (=) 22,000 probed. In contrast, the SPIR-related steroids affected 17 or fewer transcripts. Combining SPIR and ALDO resulted in 940 affected transcripts, indicating that SPIR has an early gene-regulatory effect independent of MR. 3 The affected genes encode a large number of signalling proteins and receptors, including immunoinflammatory response genes and apoptosis and antiapoptosis genes. Apoptosis was evident in CD3-, CD14- and CD19-positive cells, but only after 18 h of exposure to SPIR. 4 The transcriptional network involving the differentially regulated genes was examined and the results indicate that SPIR affects genes controlled by the transcription factors NF-kappaB, CEBPbeta and MYC. 5 These observations provide new insight into the non-MR-mediated effects of SPIR.