Maspin is a deoxycholate-inducible, anti-apoptotic stress-response protein differentially expressed during colon carcinogenesis

Clinical and Experimental Gastroenterology Dovepress open access to scientific and medical research O r i g i n al Re s ea r c h Clinical and Experimental Gastroenterology downloaded from https://www.dovepress.com/ by 213.32.48.132 on 13-Jul-2018 For personal use only. Open Access Full Text Article Maspin is a deoxycholate-inducible, anti-apoptotic stress-response protein differentially expressed during colon carcinogenesis This article was published in the following Dove Press journal: Clinical and Experimental Gastroenterology 30 September 2011 Number of times this article has been viewed Claire M Payne 1,2 Hana Holubec 1 Cheray Crowley-Skillicorn 1 Huy Nguyen 1 Harris Bernstein 1 George Wilcox 3 Carol Bernstein 1 1 Department of Cellular and Molecular Medicine, College of Medicine, University of Arizona, 2 Biomedical Diagnostics and Research, Inc, 3Tucson Medical Center, Pathology, Tucson, AZ, USA Introduction Correspondence: Claire M Payne Biomedical Diagnostics and Research Inc, 625 South Plumer Ave, Tucson, Arizona 85719, USA Tel +1 520 885 0662 Fax +1 520 296 8223 Email submit your manuscript | www.dovepress.com Dovepress http://dx.doi.org/10.2147/CEG.S24093 Powered by TCPDF (www.tcpdf.org) Abstract: Increased maspin expression in the colon is related to colon cancer risk and patient survival. Maspin is induced by the hydrophobic bile acid, deoxycholate (DOC), which is an endogenous carcinogen and inducer of oxidative stress and DNA damage in the colon. Persistent exposure of colon epithelial cells, in vitro, to high physiologic levels of DOC results in increased constitutive levels of maspin protein expression associated with the development of apoptosis resistance. When an apoptosis-resistant colon epithelial cell line (HCT-116RC) developed in the authors’ laboratory was treated with a maspin-specific siRNA probe, there was a statistically significant increase in apoptosis compared to treatment with an siRNA control probe. These results indicate, for the first time, that maspin is an anti-apoptotic protein in the colon. Immunohistochemical evaluation of maspin expression in human colonic epithelial cells during sporadic colon carcinogenesis (131 human tissues evaluated) indicated a statistically significant increase in maspin protein expression beginning at the polyp stage of carcinogenesis. There was no statistically significant difference in maspin expression between hyperplastic/adenomatous polyps and colonic adenocarcinomas. The absence of “field defects” in the non-neoplastic colonic mucosa of patients with colonic neoplasia indicates that maspin may drive the growth of tumors, in part, through its anti-apoptotic function. Keywords: maspin, anti-apoptotic, bile acid-inducible, immunohistochemistry, colon cancer Colon carcinogenesis is associated with a high-fat diet1 and such a diet results in increased concentrations of endogenous bile acids in the gut.2–4 Although numerous bile acids are released from the gallbladder into the small intestine, only the hydrophobic bile acids are particularly damaging to cells.1 The hydrophobic bile acids, deoxycholic acid, and lithocholic acid, resulting from bacterial metabolism, are the predominant bile acids found in the human colon. Thus, in previous work, the authors explored the effects of exposure of cells of colonic origin to high pathophysiologic concentrations of deoxycholate (DOC). It was shown that repeatedly exposing HCT-116 colon epithelial cells to increasing concentrations of DOC over months resulted in the development of cells with apoptosis resistance,5 a hallmark of cancer.6,7 Apoptosis resistance during colon carcinogenesis is a major process associated with the survival of cells with genomic instability.8–11 2D-gel electrophoresis/matrix-assisted laser desorption/ionization (MALDI)-mass spectroscopy of these apoptosis-resistant cells indicated, unexpectedly, that maspin was one of the proteins that was substantially upregulated.5 Maspin is a 42 kDa protein with high sequence homology to members of the serpin peptidase inhibitor family of proteins.12,13 Early experimental results with Clinical and Experimental Gastroenterology 2011:4 239–253 © 2011 Payne et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited. 239 Dovepress Clinical and Experimental Gastroenterology downloaded from https://www.dovepress.com/ by 213.32.48.132 on 13-Jul-2018 For personal use only. Payne et al maspin in mammary tissue indicated that it acts as a tumor suppressor.14,15 However, maspin does not undergo the S (stressed) to R (relaxed) conformational transition that characterizes active serpins.16,17 Thus, the mechanisms by which it exerts its tumor suppressor activity have been actively sought. One of the mechanisms proposed to explain the tumorsuppressive functions of maspin in noncolon-derived cells is that it sensitizes cells to apoptosis,18–25 thereby preventing the clonal expansion of preneoplastic cells with DNA damage. In contrast to mammary tissue, maspin expression in colon epithelial tissue appears to be related to increased colon cancer risk and reduced patient survival.26–31 High maspin expression is associated with neoplastic transformation and high tumor grade. It was determined, using small interfering RNA (siRNA) technology, that maspin is an anti-apoptotic protein in colon epithelial cells, which can explain the association of increased maspin expression with increased risk of colon carcinogenesis. Human colonic tissues were screened, using immunohistochemical analysis, to determine if maspin was differentially expressed during colon carcinogenesis. Maspin’s first increased expression occurred at the hyperplastic/adenomatous polyp stage of colon tumorigenesis. No evidence of “field defects” with respect to maspin expression was observed in the non-neoplastic mucosa of patients with polyps or adenocarcinomas. The role of maspin as a “double-edged sword” during colon carcinogenesis is discussed in the light of these new findings. Materials and methods SiRNA methodology Potential siRNA probes were identified by Dr Seth Ririe, Senior Technical Service Scientist (QIAGEN, Inc, Valencia, CA) by scanning the mRNA nucleotide sequence for human maspin (GenBank Accession #NM_002639) using QIAGEN Software. Three siRNA probes were selected to have the highest potential for success based on siRNA design (QIAGEN, Inc). A BLAST (Basic Local Alignment Search Tool [designed by scientists at the NIH]) search was performed on these three oligonucleotide sequences, and the 19-mer sequence that was very specific with homology only to maspin mRNA (3′-dTdTGUCACACUUGCUGGUCUGG-5′) was chosen for the siRNA experiments. This sequence started at nucleotide #528 of the human maspin mRNA sequence (GenBank Accession #NM_002639). An siRNA control probe was provided by QIAGEN that does not target any (...truncated)