Colon cancer is characterized by the progressive loss of E-cadherin, a Ca2+-dependent adherens junction component and epithelial marker. Furthermore, inhibition of the phosphatase SHP2, essential in the cell survival signaling via the epidermal growth factor receptor pathway, has been shown to upregulate E-cadherin in breast cancer cell lines. This suggests that unregulated increases in SHP2 may promote a cancer cell phenotype. The aim of this study was to define molecular mechanisms by which dietary Ca2+ is chemoprotective against colon cancer. The extracellular Ca2+-sensing receptor (CaSR) has been implicated in this process and we speculated that there was a relationship between CaSR activation and E-cadherin and SHP2 expression levels on colonic epithelia. A colonic adenocarcinoma cell line which lacks endogenous E-cadherin expression, SW480, was used as a model cell. CaSR levels were manipulated by transient transfection and E-cadherin and SHP2 expression levels were determined by Western blotting. The E-cadherin expression pattern was also assessed with RT-PCR and immunocytochemistry. We found that after CaSR activation via Ca2+ or other known CaSR agonists (neomycin sulphate, spermine), E-cadherin expression was increased and SHP2 expression was decreased. However, the E-cadherin expression pattern was altered in the presence of a dominant-negative CaSR (R185Q). Furthermore, pharmacological inhibition of p38 MAPK inhibited CaSR-mediated increases of E-cadherin protein in SW480 cells. Inhibition of p38 MAPK had no effect on CaSR-stimulation of E-cadherin transcript or promoter activity. SHP2 decreases usually seen after Ca2+-treatment were reduced after pharmacological inhibition of JNK in the presence of high Ca2+. CaSR activation also increased cell-cell adherence as assessed by electronic cell sizing and this adherence was lost when both CaSR-mediated changes in E-cadherin and SHP2 expression were inhibited. We conclude that CaSR activation in colonic epithelial cancer cells stimulated E-cadherin increases, through a mechanism involving p38 MAPK, as well as inhibited SHP2 expression, through a JNK-mediated mechanism. Further, these protein changes post-CaSR activation cause an increase in cell-cell adherence. Our results suggest that the chemoprotective nature of dietary Ca2+ supplements may involve reciprocal regulation of E-cadherin and SHP2 via the CaSR. / Thesis (Master, Physiology) -- Queen's University, 2009-06-09 17:56:44.843
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/1943 |
| Date | 10 June 2009 |
| Creators | VANDERLEE, AMANDA |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | 918850 bytes, application/pdf |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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