Sulindac modulates secreted protein expression from LIM1215 colon carcinoma cells prior to apoptosis

https://doi.org/10.1016/j.bbapap.2013.07.007Get rights and content

Highlights

  • Understand events associated with early-onset stage of apoptosis.

  • Develop protein (bottom-up proteomic) separation strategy to characterise secretome.

  • Develop top-down approach to characterise low-Mr secreted polypeptides.

  • Demonstrate increased proteolysis of cell surface (ectodomain) proteins.

  • Secreted proteins implicated in mucosal maintenance and cell-cell-adhesion.

  • Contribute to extracellular modulators of apoptosis and chemoprevention.

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 1 mM sulindac over 16 h; 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 1 mM sulindac treatment for 8 h 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 30 K, 3 K and 1 K). Proteins isolated in the > 30 K and 3–30 K fractions were electrophoretically separated by SDS-PAGE and endogenous peptides in the 1–3 K 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 1 mM sulindac for 8 h 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 5 AC, 6, and 13). A salient finding of this study was the increased proteolysis of cell surface proteins following treatment with sulindac for 8 h (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), 1 mM 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.

Introduction

Colorectal cancer (CRC) is the second leading cause of cancer death in Western populations, with more than a million new cases and half a million deaths worldwide annually [1]. One of the most widely studied and promising chemopreventive agents for sporadic CRC [2] and therapeutic agent for the treatment of adenomas in patients with familial adenomatous polyposis (FAP) [3] is the non-steroidal anti-inflammatory drug (NSAID) sulindac; for reviews see [4], [5]. While epidemiological evidence indicates that continued use of NSAIDs such as aspirin is associated with reduced risk of CRC in the general population [6], animal (rodent) data for aspirin is less conclusive [7]. However, the potential harms associated with the chronic use of NSAIDs such as gastrointestinal and cardiovascular complications are well documented [8], [9], [10] and warrant careful consideration before adoption for general clinical use. Investigation of mechanisms associated with antineoplastic effects of NSAIDs may provide insight that could lead to new drug candidates that are potentially safer and more efficacious for CRC chemoprevention.

The chemopreventive effects of NSAIDs occur primarily through inhibition of the cyclooxygenase-(COX)-1 and COX-2 enzymes [11], [12], which are involved in the synthesis of prostaglandins leading to a decreased activation of the inflammatory response [13], inhibition of crypt cell proliferation [14], angiogenesis [15] and increased ap-optosis [16]. At the molecular level NSAIDs are also reported to target a number of COX-independent cellular processes, including peroxisome proliferator-activated receptor (PPAR) subtypes γ and δ, nuclear factor-κB (NF-κB) pathway, and the multidrug-resistance protein 4 (MRP4) [17]. The mechanism(s) by which sulindac prevents CRC are complex and poorly understood, especially in the early-onset stage of apoptosis; for a review of the role of NSAIDs in CRC progression and underlying mechanisms of action, see Antonakopoulos and Karamanolis [18].

Although our understanding of cancer cell biology has advanced considerably [19], there is, however, a growing awareness that targeting the cancer cell in isolation is not sufficient because of the intricate reciprocal interplay between cancer cells and the tumor microenvironment [20], [21]. In addition to epithelial tumor cells, the microenvironment consists of several non-malignant, albeit genetically altered, heterotypic cell types (e.g., fibroblasts, endothelial cells and leukocytes) which crosstalk either physically or via secretion (collectively referred to as the secretome) of paracrine signaling molecules [22]. As well as cytokines, chemokines, growth factors, proteases and protease inhibitors, the secretome also contains extracellular vesicles [23]. It is now recognized that pathophysiology of the tumor microenvironment is critical for neoplastic induction and progression and is now a primary target for cancer chemoprevention [24], [25]. While most proteomics-based studies on NSAID action have focused on cellular proteins, systematic analyses of proteins released from CRC cells into the tumor microenvironment in response to NSAID treatment are limited [26].

As a first step towards understanding the molecular events associated with the chemopreventive action of the NSAID sulindac in the context of the tumour environment, we have employed the human colon carcinoma cell line LIM1215 to assess the early effects of sulindac (i.e., prior to onset of apoptosis) on secretome protein expression levels. This study focuses on identifying secreted modulators involved in sulindac-mediated apoptosis in CRC adenomas. Specifically, we have employed two different proteomic analyses; a bottom-up approach (focused on characterising secreted proteins) and a top-down approach (focused on characterising low-Mr secreted polypeptides). It is anticipated that this approach will improve our understanding of the extracellular contribution to the chemopreventive effects of sulindac on CRC and provide the foundation for improved chemopreventive target design.

Section snippets

Experimental procedures

Cell culture and reagents—The human colon carcinoma cell line LIM1215 [27] was routinely cultured on 150-mm diameter cell culture dishes (2 × 106 cells/dish) in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 5% FCS (CSL, Melbourne), 100 U penicillin and 100 mg/mL streptomycin (Sigma-Aldrich), and incubated at 37 °C in a humidified atmosphere containing 10% CO2 until sub-confluent (80–90%). RPMI-1640 medium containing with sulindac (0.2–1 mM), was prepared fresh by adding appropriate

Results and discussion

The use of the NSAID sulindac has been attributed to inhibitory effects on tumour growth in gastric, breast, lung, and CRC in nude mice, resulting in a decrease in cell growth and increased apoptosis [50], [51], [52], [53]. Although studies have been performed on the chemopreventive actions of sulindac and its metabolites in these systems, the underlying mechanisms of action, especially events associated with apoptotic-onset, are still poorly understood. As a first step toward understanding

Concluding remarks

In summary, we have developed a protein/peptide separation strategy using differential centrifugal ultrafiltration in combination with 1D-SDS-PAGE/RP-HPLC and nanoLC-MS–MS to study for the first time the profile of proteins released into the culture (secretome) of LIM1215 cells following treatment with 1 mM sulindac for 8 h. Collectively, our data shows that under these conditions LIM1215 cells secrete decreased levels of proteins associated with extracellular matrix remodeling (e.g., collagens,

Acknowledgements

Funding was provided by the Australian National Health and Medical Research Council under Program Grant #487922 (RJS). We acknowledge the NHMRC-funded Australian Proteomics Computational Facility (APCF) under Enabling Grant #381413. This work was supported, in part, by funds from the Operational Infrastructure Support Program provided by the Victorian Government, Australia. We acknowledge the Australian Cancer Research Foundation for providing funds to purchase the Orbitrap™ mass spectrometer.

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