Characterisation of Endothelin-1 binding sites in colorectal cancer and antagonistic action of the Endothelin A receptor antagonist Zibotentan (ZD4054)

Samer-ul Haque1, Marilena Loizidou1, Xu Shiwen3, David Abraham3, Noreen Farooqui1, Hazel Welch1, Olagunju Ogunbiyi1, Michael Dashwood2. 1UCL, Dept. of Surgery, NW3 2QG, United Kingdom, 2UCL, Dept. of Clinical Biochemistry, NW3 2QG, United Kingdom, 3UCL, Centre for Rheumatology and Connective Tissue Disease, NW3 2QG, United Kingdom.

Background: Endothelin-1 (ET-1) acts via two G-protein coupled receptors, ETA and ETB. Overexpressed ET-1 and ETA in colorectal cancer (CRC) promotes tumour growth and progression.

Aim: To investigate the distribution of ETA and ETB in patient tissue sections. ET-1 affinity (Kd) and receptor density (Bmax) was determined in whole tissue homogenates, CRC cell lines and colorectal fibroblasts. In addition the effect of the orally active ETA specific antagonist Zibotentan (ZD4054) on ET-1 receptor binding (IC50) was evaluated against subtype selective laboratory compounds.

Material and Methods: ET-1 receptor distribution and binding characteristics (Kd; Bmax) were determined using in vitro autoradiography on patient sections, tissue homogenates, CRC cell lines and primary fibroblasts. Immunohistochemistry (IHC) identified stromal structures. Study was awarded ethical approval, REC No. 08/H0720/162, University College London Hospitals

Results: ET-1 binding to cancer and normal colon tissue had similar characteristics. There was greater ETA than ETB binding in CRC sections. Both cancer and normal tissues had strongest binding to stromal cells, particularly fibroblasts (IHC). Furthermore, characterising CRC cell lines and primary fibroblasts revealed high density and affinity ET-1 binding (Bmax 1.11 fmol/1x106 cells; Kd 450.5 pmol/L and Bmax 3.03 fmol/1x106 cells; Kd 213.6 pmol/L respectively). Inhibition studies showed ETA antagonists (BQ123; Zibotentan) more effectively reduced ET-1 binding (approximate IC50 values in CRC: 10μM, 0.1μM respectively; fibroblasts: 0.1μM, 10μM respectively) than ETB antagonism BQ788 (approximate IC50; 1mM in both).

Conclusions: ET-1 binds strongly to CRC stromal structures (fibroblasts; endothelial cells), and is consistent with ET-1 signalling contributing to tumourigenesis. We further demonstrated that the orally active ETA antagonist Zibotentan reduces ET-1 binding to CRC tissues. This study provides further evidence for the potential therapeutic use of the specific ETA antagonist Zibotentan as an adjuvant treatment for CRC.

CRC = colorectal cancer; ET-1 = Endothelin-1; IHC = Immunohistochemistry.