Background Endothelin-1 type A receptor (ETAR) antagonists (e.g. ambrisentan) are currently approved by the U.S Food and Drug administration, representing a well-tolerated treatment of pulmonary arterial hypertension (PAH) for patients with connective tissue diseases such as systemic sclerosis (SSc). Noteworthy, increased numbers of infiltrating neutrophils have been associated with worse clinical outcome in PAH patients. In another context, several studies have reported that endothelin-1 and its receptor ETAR also play a central role in the development of tumour cell invasion and metastasis. However, the effects of ETAR antagonists on migration of neutrophils and tumour cells remain to be determined.
Objectives The objective was to analyse the effects of two ETAR antagonists on migration of neutrophils and tumour cell lines.
Methods The migratory ability of peripheral neutrophils from healthy donors (HD) and different tumour cell lines (myeloid leukaemia HL60 cells and human pancreatic adenocarcinoma COLO357 cells) was analysed in response to N-Formylmethionyl-leucyl-phenylalanine (FMLP) or Protease-activated receptor 2 (Par-2) agonist. Because it has been shown before , IgGs from HD and SSc patients were used as additional stimulus for migration. Neutrophils and HL60 cells were preincubated (1h) with sitaxentan or ambrisentan, respectively, before being tested for migration (1h) using the Transwell assay. COLO357 cells were incubated (48h) in the presence of sitaxentan and migration was tested in the Oris Pro Cell assay. Migration was analysed by automatic cell counting or digital photo analysis and a migration index was calculated.
Results Sitaxentan and/or ambrisentan significantly blocked the migration of neutrophils and tumour cell lines. In more detail, neutrophil migration in response to FMLP, being set to 100%, was completely inhibited by sitaxentan (0,46%). Further, neutrophil migration in response to IgGs from HD and SSc patients was induced equally, again being set to 100%. In the presence of sitaxentan migration was reduced to 60%, respectively. In DMSO-differentiated HL60 cells the migratory capacity in response to FMLP (100%) was reduced to 66% by ambrisentan and to 14% by sitaxentan. Moreover, in the presence of sitaxentan and a Par-2 agonist the migratory ability of COLO357 cells was significantly decreased to 89% compared to Par-2 agonist only, being set to 100%.
Conclusions Our results suggest a pivotal and non-redundant role of ETAR in cell migration, which needs further clarification in order to repurpose the use of ETAR inhibitors. Therapeutic switching of ETAR antagonists from PAH to cancer therapies is a promising adjuvant therapy.
Waxman AB. A review of sitaxsentan sodium in patients with pulmonary arterial hypertension. Vasc Health Risk Manag 2007;3:151–7.
Kill A, Tabelin C, Undeutsch R, et al. Autoantibodies to angiotensin and endothelin receptors in systemic sclerosis induce cellular and systemic events associated with disease pathogenesis. Arthritis R&T, 2014, 16:R29.
Disclosure of Interest None declared