Haque, S; (2014) The Contribution of Endothelin-1 in Colorectal Cancer & The Efficacy of the Novel Endothelin Receptor Antagonist ZD4054. Doctoral thesis , UCL (University College London).

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Abstract

The overall aims of this thesis are: to determine the cellular response to ET-1 and effects of receptor antagonism on proliferation, migration and contraction of colonic fibroblasts and cancer cell lines. At the molecular level to identify novel genes that are regulated by ET-1 and whether antagonists including ZD4054 have potentially beneficial effects by blocking expression of these genes. Finally to determine ET-1 binding distribution by autoradiography in patient tumour sections and delineate binding characteristics of ET-1 and its receptor antagonists (Bmax, Kd and IC50). To investigate ET-1 and its antagonistic effects at the cellular level, colorectal cancer cell lines and colonic fibroblasts (isolated from patient colorectal cancer specimens) were studied. They were incubated with ET-1 with/without BQ123, zibotentan (ETAR antagonists) and/or BQ788 (ETBR antagonist). Growth was measured by methylene blue uptake; migration by scratch wound assay and contraction in collagen gels. To identify novel key genes regulated by ET-1, Illumina micro-arrays determined differential gene expression post-ET-1 stimulation of 3 colorectal cancer cell lines and the 4 human colonic fibroblast strains. To confirm expression of genes of interest, we examined time point induction mRNA levels (conventional RT-PCR; quantitative real-time RT-PCR). ETA (Zibotentan, BQ123) and ETB (BQ788) antagonistic effects were measured at the mRNA and protein levels (Immunoblotting). Silencing (SiRNA) was also used to confirm receptor involvement in regulation of these key genes. ET-1 receptor distribution and binding characteristics (Kd; Bmax) were determined using in vitro autoradiography on patient sections, tissue homogenates, CRC cell lines and colonic fibroblasts. Effects of the ETAR specific antagonist zibotentan (ZD4054) on ET-1 receptor binding (IC50) were evaluated against laboratory-standard compounds. Immunohistochemistry (IHC) was used to identify stromal structures and receptor distribution (vascular CD31; Thy-1 fibroblasts; collagen type XI; ETA and ETB). Study was awarded ethical approval, REC No. 08/H0720/162, University College London Hospitals

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