Abstract
Abstract Colon cancer is the fourth most common form of cancer in the United States and the third leading cause of cancer-related death in the Western world. Progression to colon cancer is distinguished with a loss of the colonic calcium sensing receptor (CaSR). It has been observed that calcium-dependent proteolytic enzyme, Calpain (Capn) overexpressed in colon cancer may contribute to its metastasis. We attempted to identify role of calcium sensing receptor CaSR on regulation of capn activity and its influence on calmodulin (CaM) homeostasis. We have also investigated the role of curcumin in activation of CaSR. Curcumin (1μM and 20μM) increases expression (qPCR) and protein (2-3 fold) of the CaSR in wild type intestinal cells (RKO; wild type APC, wild type β-catenin) and cells with truncated APC (HT-29, SW480). Transient transfection of siRNA (200 nM) duplex against CaSR reduced CaSR protein expression and prevented reduction of Capn activity after 3 mM Ca2+ challenge (340+/-24 Units, p<0.05 n=4). Western blotting of HT-29 cell lysates after 3 mM Ca2+ challenge demonstrated no change in amount of Calpain-4 (Capn4, small subunit of Capn essential for its activity) but a 6 fold increase in CaM. Bioinformatic analysis of Capn4 revealed a presence of putative CaM binding site. Using circular dichroism, isothermal titration calorimetry and nuclear magnetic resonance it was confirmed that Capn4 binds to CaM. This is a first report of its kind demonstrating interaction of two EF-hand proteins. Capn after 30 min incubation with CaM demonstrated a 3 fold increase in autolysed Capn and 5 fold reduction in 75 kDa large-subunit of Capn. CaM antagonists W7 or W13 (100 μM) prevented CaSR-mediated decrease in Calpain activity (p<0.05, n=4). We hereby conclude that curcumin will increase CaSR expression and that calcium will stimulate the CaSR to increase calmodulin in colonic adenocarcinoma to produce Capn autolysis which will reduce Capn activity. We speculate that CaSR-mediated reduction in Capn activity may be an important determinant of calcium chemoprevention of colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4226. doi:10.1158/1538-7445.AM2011-4226
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