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PWE-001 Bile acids modulate Golgi structure in gastrointestinal cancer via a PKC\η\PKD-dependent pathway
  1. A Byrne1,
  2. E Foran1,
  3. R Shamra1,
  4. J O Sullivan2,
  5. D O Donoghue2,
  6. D Kelleher1,
  7. A Long1
  1. 1Department of Clinical Medicine, Institute of Molecular Medicine, Trinity Centre for Health Science, Dublin, Ireland
  2. 2Centre for Colorectal Disease, St Vincent's University Hospital, Dublin, Ireland

Abstract

Introduction Deoxycholic acid (DCA) is a bile acid implicated in pathogenesis of colon cancer as (i) it activates pathways associated with tumorigenesis, particularly the protein kinase C (PKC) pathway (ii) it is pro-tumorigenic in rodent models of colon cancer and (iii) elevated serum and faecal levels are found in patients with colorectal cancer. Ursodeoxycholic acid (UDCA) is currently used clinically to treat biliary disease. Studies have demonstrated a chemopreventative effect in rat models of colon cancer and retrospective analyses of clinical trials have demonstrated chemopreventative effects in development of colon cancer.

Bile acids are known to modulate the activation of a number of PKC isoforms (α, β, δ and ε). In this study we investigated the effects of bile acids on subcellular localisation and activity of Protein Kinase Cη (PKCη) and its downstream effects on Golgi structure in gastrointestinal cell lines and colorectal tissue.

Methods Subcellular localisation and activity of PKCη and PKD was assessed by immunofluorescence and Western Blot analysis, respectively. Golgi were visualised by immunofluorescence and Golgi fragmentation was quantified by high content screening using the Incell-1000 and GE-software analysis. The effect of DCA on secretory mechanisms in the cell was assessed using a Gaussian luciferase reporter assay. Since UDCA has anti-inflammatory properties, the involvement of the glucocorticoid receptor (GR) in UDCA signalling was assessed using pharmacological inhibition and an siRNA approach. Tissue microarrays were utilised to assess Golgi structure in normal, ulcerative colitis (UC) and colon cancer tissue.

Results Analysis of Golgi architecture showed significant increases in Golgi fragmentation in UC and colorectal cancer tissue (p<0.0005 vs normal) suggesting that Golgi fragmentation may be an important mechanism in disease. We hypothesised that this could be mediated through bile acid exposure. Exposure of HCT116 colon cancer cells to DCA caused phosphorylation of PKCη and PKD and fragmentation of the Golgi. Furthermore DCA exposure decreased secretion of Gaussian luciferase by HCT116 cells (p<0.001 vs control). Pre-treatment of cells with UDCA or dexamethasone, inhibited DCA-induced PKCη/ PKD phosphorylation and Golgi fragmentation. Inhibition of the GR abrogated UDCA effects on DCA-induced Golgi fragmentation.

Conclusion DCA disrupted the structure and function of the Golgi, an organelle critical for normal cell homeostasis whereas UDCA could attenuate these effects. This study identified novel mechanisms of bile acid signalling which may play a role in the pathogenesis of benign and malignant gastrointestinal disease.

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