title: Sulindac modulates secreted protein expression from LIM1215 colon carcinoma cells prior to apoptosis.
abstract: Colorectal cancer (CRC) is a major cause of mortality in Western populations. Growing evidence from human and rodent studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) cause regression of existing colon tumors and act as effective chemopreventive agents in sporadic colon tumor formation. Although much is known about the action of the NSAID sulindac, especially its role in inducing apoptosis, mechanisms underlying these effects is poorly understood. In previous secretome-based proteomic studies using 2D-DIGE/MS and cytokine arrays we identified over 150 proteins released from the CRC cell line LIM1215 whose expression levels were dysregulated by treatment with 1mM sulindac over 16h; many of these proteins are implicated in molecular and cellular functions such as cell proliferation, differentiation, adhesion, angiogenesis and apoptosis (Ji et al., Proteomics Clin. Appl. 2009, 3, 433-451). We have extended these studies and describe here an improved protein/peptide separation strategy that facilitated the identification of 987 proteins and peptides released from LIM1215 cells following 1mM sulindac treatment for 8h preceding the onset of apoptosis. This peptidome separation strategy involved fractional centrifugal ultrafiltration of concentrated cell culture media (CM) using nominal molecular weight membrane filters (NMWL 30K, 3K and 1K). Proteins isolated in the >30K and 3-30K fractions were electrophoretically separated by SDS-PAGE and endogenous peptides in the 1-3K membrane filter were fractioned by RP-HPLC; isolated proteins and peptides were identified by nanoLC-MS-MS. Collectively, our data show that LIM1215 cells treated with 1mM sulindac for 8h secrete decreased levels of proteins associated with extracellular matrix remodeling (e.g., collagens, perlecan, syndecans, filamins, dyneins, metalloproteinases and endopeptidases), cell adhesion (e.g., cadherins, integrins, laminins) and mucosal maintenance (e.g., glycoprotein 340 and mucins 5AC, 6, and 13). A salient finding of this study was the increased proteolysis of cell surface proteins following treatment with sulindac for 8h (40% higher than from untreated LIM1215 cells); several of these endogenous peptides contained C-terminal amino acids from transmembrane domains indicative of regulated intramembrane proteolysis (RIP). Taken together these results indicate that during the early-stage onset of sulindac-induced apoptosis (evidenced by increased annexin V binding, dephosphorylation of focal adhesion kinase (FAK), and cleavage of caspase-3), 1mM sulindac treatment of LIM1215 cells results in decreased expression of secreted proteins implicated in ECM remodeling, mucosal maintenance and cell-cell-adhesion. This article is part of a Special Issue entitled: An Updated Secretome.
authors.affiliation: La Trobe Institute for Molecular Science (LIMS), La Trobe University, Bundoora, Victoria, Australia.
authors.forename: David W
authors.initials: DW
authors.lastname: Greening
authors.forename: Hong
authors.initials: H
authors.lastname: Ji
authors.forename: Eugene A
authors.initials: EA
authors.lastname: Kapp
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Simpson
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002277:Carcinoma / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D003106:Colon / Q000187:drug effects / Q000378:metabolism; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D059748:Proteolysis / Q000187:drug effects; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D055571:Secretory Pathway; D013467:Sulindac / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D000970:Antineoplastic Agents; D020543:Proteome; D013467:Sulindac
keywords: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CM; COX; CRC; Chemoprevention; Colon cancer; ECM; FA; FAK; HNPCC; Hereditary nonpolyposis colorectal cancer; MMP; MSI; MSS; MTT; Matrix metalloproteinase; N(SC); NMWC; NSAID; PI; PS; Peptidome; R(SC); RIP; SFM; Secretome; Sulindac; colorectal cancer; culture medium; cyclooxygenase; extracellular matrix; focal adhesion; focal adhesion kinase; microsatellite instable; microsatellite stable; nominal molecular weight cut-off; nonsteroidal anti-inflammatory drug; normalized spectral count; phosphatidylserine; propidium iodide; regulated intramembrane proteolysis; serum-free media; spectral count fold change ratio