JOP. J Pancreas (Online) 2014 Sep 28; 15(5): 465-474 ORIGINAL ARTICLE Nicotine Alters the Proteome of Two Human Pancreatic Duct Cell Lines Joao A Paulo Department of Cell Biology, Harvard Medical School, Boston, USA ABSTRACT Context Cigarette smoking is a known risk factor of pancreatic disease. Nicotine - a major cigarette tobacco component - can adenocarcinoma, respectively. However, at the biomolecular level, particularly in pancreatic research, the effects of nicotine remaintraffic through unresolved. the circulatory Methods system The effects and may of nicotine induce fibrosis on the andproteomes metastasis, of two hallmarks pancreatic of chronic duct cellpancreatitis lines–an and immortalized pancreatic normal cell line (HPNE) and a cancer cell line (PanC1)- were investigated using mass spectrometry-based proteomics. For each cell line, the global proteomesof cells exposed to nicotine for 24 hrswere compared with untreated cells in triplicate using 6-plex tandem mass tag-based isobaric labeling techniques. Results Over 5,000 proteins were detectedper cell line. Of treatment, 57 of which were so in both cell lines. Amyloid precursor protein, previously observed to increase expression in pancreaticthese, over stellate 900 proteins cells when were exposed differentially to nicotine, abundant was alsowith up-regulated statistical significance in both cell (corrected lines.In general, p-value the <0.01) two cell upon lines nicotine varied in the classes of proteins altered by nicotine treatment, supporting published evidence that nicotine may play different roles in the initiation and progression of pancreatic disease. Conclusions Understanding the underlying mechanisms associating nicotine with pancreatic function is paramount to intervention aiming to retard, arrest, or ameliorate pancreatic disease. INTRODUCTION promote the development of chronic pancreatitis by In the United States, pancreatic cancer is the fourth most common cause of death due to cancer and has theincreasing underlying disease cellular severity events and promoted pancreatic by calcifications smoking in thewhich development can further of damage pancreatic the pancreasdisease is [6]. vital Elucidating to timely intervention aimed at counteracting the disease. thean averagemajor form one ofand pancreatic five year cancersurvival - tumorrate of cells25% arise and from5%, respectivelypancreatic ductal[1]. In epithelium.pancreas adenocarcinomaPancreatic ductal - Many of the toxic compounds found in tobacco smoke are absorbed into the blood stream and several have adenocarcinoma has a mortality rate of nearly 100% an admixture of over 4,000 compounds, for which over linewithin the 2 branched years and tubes comprises of the organ,over 90% secrete of all bicarbonate, pancreatic been detected in pancreatic fluid [7]. Cigarette smoke is andcancers thus [2]. conduct Pancreatic enzymes duct produced cells are byepithelial acinar cells thatinto implicated in cancer, nicotine is a major toxic component of tobacco.50 are suspected In addition, carcinogens cigarette smoking [8]. Although cessation not programs directly regulating pancreatic duct cell function, in addition to often rely on nicotine replacement therapy (NRT), alterationsthe duodenum produced [3]. Delineating by cellular the stresses, molecular such mechanisms as nicotine, such as transdermal patches, nasal sprays or emerging will add to our general understanding of the disease. e-Cigarette technology. Nicotine is readily absorbed by the lungs as well as other, more distal organs via systemic One of the most prominent risk factors correlated with the nicotinic acetylcholine receptors (nAChR), with varying circulation [9]. Nicotine interacts with a population of todevelopment investigate of this pancreatic association. cancer Cigarette is cigarette smoking smoking may [4, 5]. However, only limited research has been performed affinities and downstream effectors [10] and as such is as Received May 31st, 2014 – Accepted July 10th, 2014 bindinga contributing of nicotine factor to to cell myocardial surface receptors[11, 12] , transducesatrial [13], Key words Amyloid; Nicotine; Pancreatic Ducts; Pancreatic extracellularpulmonary [14, signals 15] and to biliarythe intracellular fibrosis [16] space diseases. resulting The Neoplasms; Receptors, Nicotinic in ion transport and/or initiation of phosphorylation Abbreviations APP: amyloid precursor protein; FBS: fetal bovine serum; PaDC: pancreatic duct cells; TMT: tandem mass tag Correspondence Joao A Paulo cascadesVarious cancers and other and signaling chronic pathwaysdiseases have [17-19]. been linked to Department of Cell Biology, Harvard Medical School, Boston, nicotine, yet the mechanisms of such are poorly understood MA 02115, USA Phone + 617/432-6767; Fax: + 617/264-5277 E-mail [email protected] pancreatitis[20, 21]. Concerning and pancreatic pancreatic adenocarcinoma, disease, cigarette respectively smoking may induce fibrosis and metastasis, hallmarks of chronic JOP. Journal of the Pancreas–http://www.serena.unina.it/index.php/jop–Vol. 15 No. 5 – Sep 2014. [ISSN 1590-8577] 4 65 JOP. J Pancreas (Online) 2014 Sep 28; 15(5): 465-474 Experimental Strategy pancreatic duct cells (PaDC), and such cellular changes are The experimental strategy was outlined in Figure 1. This implicated[22] . Evidence in pancreatic suggests that cancer protein and expression chronic pancreatitis is altered in procedure was performed in parallel for both HPNE and PanC1 cell lines. Cells were lysed and proteins were effects of nicotine on PaDC in an in vitro environment has extracted via methanol-chloroform precipitation and then been[23]. However,published. to In date,various no cellcomprehensive types, nicotine analysis is known of the to digested with LysC and trypsin. Each sample was labeled induce alterations in protein expression that affect cellular proliferation, chemotaxis, and attachment to various was fractionated by basic pH reversed-phase (BpRP) chromatographywith a specific andTMT subjected isobaric totag. LC-MS3 The analysis.pooled sample Here, I focus on changes in the global proteome of cultured surfaces [24, 25]. Cell Growth and Harvesting of Pancreatic Duct Cells pancreatic cells when subjected to nicotine treatment. I Methods of cell growth and propagation followed cell line, HPNE (human pancreatic Nestin-expressing), and secondinvestigate a well-studied two PaDC cellpancreatic lines, first cancer a normal cell line, epithelial PanC1 (pancreatic cancer 1). Identifying differences in proteins previously utilized techniques [28, 29]. In brief, cells were that are altered in abundance under nicotine stress is an propagated in Dulbecco's modified Eagle's-F12 medium initial step to gain a comprehensive understanding of aspirated(DMEM) supplemented and the cells werewith 10%washed fetal 3 bovine times withserum ice-cold (FBS). the cellular physiology regulating the development and phosphate-bufferedUpon achieving 85-90% saline confluency, (PBS). This the growthcell density media was progression of pancreatic disease. Research such as that chosen so as to maximize the amount of cells for harvesting presented herein may broaden our knowledge of the while ensuring that the cells are proliferating. Assigned cell effects of nicotine on the pancreas and further validation culture dishes were supplemented with 1 µM nicotine and via targeted studies may provide targets for drug therapies control cell culture dishes were mock treated with an equal aiming to retard or reverse the clinical manifestations of volume of sterile deionized water. Twenty-four hours after pancreatic disease. the addition of fresh media, the cells were dislodged with a non-enzymatic reagent, harvested by trituration following MATERIALS AND METHODS the addition of 10 mL PBS, pelleted by centrifugation Materials at 3,000 x g for 5 min at 4°C, and the supernatant was removed. One milliliter of buffer containing TBSp (50 mM Tris, 150 mM NaCl, pH 7.4 supplemented with 1X Roche 11330) was purchased from Gibco (Carlsbad, CA). Fetal Dulbecco's modified Eagle's-F12 medium (DMEM/F12; bovine serum (FBS; F0392) was purchased from Sigma (St. SDS was added to each 10 cm cell culture dish. Louis, MO). CellStripper (25-056-CL) for non-enzymatic Complete protease inhibitors), 1% Triton X-100 and 0.5% cell dislodgement was purchased from Mediatech Cell Lysis and Protein Digestion (Manassas, VA). Tandem mass tag (TMT) isobaric reagents Cells were homogenized by 12 passes through a 27 gauge (1.25 inches long) needle and incubated at 4°C with gentle organic solvents were purchased from J.T. Baker (Center agitation for 1 hr. The homogenate was sedimented by were from Thermo Scientific (Rockford, IL). Water and ultracentrifugation at 100,000 x g for 60 min at 4°C. Protein concentrations were determined using the trypsinValley, PA).(V5111) (-)-Nicotine was obtained (≥99%) from (N3876) Promega was purchased(Madison, from Sigma (St. Louis, MO). Sequencing-grade modified were from Sigma-Aldrich and Burdick & Jackson, 5bicinchoninic mM dithiotreitol acid (BCA)(37°C, assay 25 min) (ThermoFisher and alkylation Scientific). with 10 respectively.WI). Unless otherwise Primary antibody noted, other against reagents amyloid and precursor solvents mMProteins iodo wereacetamide subjected (room to disulfidetemperature, bond 30reduction min in with the protein (ab15272) was from Abcam (Cambridge, MA), dark). Excess iodoacetamide was quenched with 15 mM while secondary horseradish peroxidase anti-rabbit dithiotreitol (room temperature,