Proteomic Analysis of Pancreatic Ductal Carcinoma Cells after Combined Treatment with Gemcitabine and Trichostatin A Daniela Cecconi,#,† Massimo Donadelli,#,‡ Aldo Scarpa,§ Alberto Milli,† Marta Palmieri,‡ Mahmoud Hamdan,| Liliana B. Areces,⊥ Juri Rappsilber,⊥ and Pier Giorgio Righetti†,* University of Verona, Department of Agricultural and Industrial Biotechnologies, Verona, Italy, University of Verona, Department of Neurological and Visual Sciences, Section of Biochemistry, Verona, Italy, University of Verona, Department of Pathology, Section of Anatomical Pathology, Verona, Italy, Computational, Analytical & Structural Sciences, GlaxoSmithKline, Verona, Italy, and FIRC Institute for Molecular Oncology Foundation (IFOM) and European Institute of Oncology (IEO), Via Adamello 16, Milano, Italy Received May 27, 2005
The human pancreatic adenocarcinoma cell line T3M4 has been treated with two agents, gemcitabine (2′,2′-difluorodeoxycytidine, a drug interfering with DNA synthesis) and trichostatin A (a drug interfering with histone acetylation), both separately and in association. The association of the two drugs showed a marked cooperative effect in inhibiting proliferation and inducing apoptosis of the cells. In an effort to identify differentially expressed proteins in the different drug treatments, the proteomic expression has been studied by two-dimensional gel electrophoresis comparing untreated cells with cells treated with trichostatin A and/or gemcitabine. A total of 81 differentially expressed polypeptide chains have been visualized by setting a 2.5-fold threshold value. Of these, 56 were identified via MALDI-TOF and Q-TOF MS analyses. Most of the regulated proteins are involved in two major biological processes, namely apoptotic cell death and proliferation. Our results demonstrate that the level of activation/ repression of the proteins involved in these processes correlates with the growth inhibition and the apoptotic response of the cells subjected to single or combined drug treatment. Keywords: proteomics • pancreatic tumors • ductal carcinomas • gemcitabine (2′,2′-difluorodeoxycytidine) • trichostatin A
Introduction Proteomic tools have been extensively used in the past decade for unravelling the early mechanisms of cancer onset,1 studying chemoresistance2 as well as for discovery of biomarkers3,4 toward preventive, predictive, and personalized medicine.5 Pancreatic adenocarcinoma is one of the most lethal human cancers. The extremely poor prognosis of this cancer type6 is mainly due to the delayed diagnosis, early metastasis, and resistance to almost all classes of cytotoxic drugs.6 Currently, the most active agent against pancreatic adenocarcinoma is 2′,2′-difluorodeoxycytidine (dFdC), commonly named gemcitabine (GEM),7,8 but even with this drug the objective response rate is less than 20% in clinical studies.9 GEM is a nucleoside * To whom correspondence should be addressed. Prof. Pier Giorgio Righetti, Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, Via Mancinelli 7, Milano 20131, Italy. Tel: +39-02-23993082. Fax: +39-02-23993080. E-mail: piergiorgio.righetti@ polimi.it. † University of Verona, Department of Agricultural and Industrial Biotechnologies. ‡ University of Verona, Department of Neurological and Visual Sciences, Section of Biochemistry. § University of Verona, Department of Pathology, Section of Anatomical Pathology. | Computational, Analytical & Structural Sciences, GlaxoSmithKline. ⊥ FIRC Institute for Molecular Oncology Foundation (IFOM) and European Institute of Oncology (IEO). # D.C. and M.D. contributed equally and share the first authorship. 10.1021/pr050154j CCC: $30.25
2005 American Chemical Society
analogue that is phosphorylated inside the cell into dFd-CDP and dFd-CTP. The first inhibits ribonucleotide reductase (RNR), and dFd-CTP competes with dCTP for incorporation into DNA determining DNA synthesis arrest.10 A peculiar feature of GEM is its ability, once integrated into DNA, to allow the incorporation of an additional natural nucleoside.11 This prevents DNA repair11 and determines apoptotic cell death, which is generally mediated by p53 activation. The presence of inactivation of the p53 gene in a high proportion of pancreatic cancers12 is one of the main causes of resistance to GEM treatment.13 Indeed, it has been shown that reintroduction of the wild type p53 gene in pancreatic cancer cell lines strongly increases GEM sensitivity.14 In human cancer, the importance of epigenetic events, including DNA methylation and histone acetylation, has become increasingly apparent.15 Several drugs that suppress tumor growth by inhibiting DNA methylation or histone deacetylation have entered into clinical trials during the past decade.16,17 In particular, histone deacetylase (HDAC) inhibitors have been shown to act selectively against cancer cells18,19 by altering gene expression of a small number of genes.20,21 We have recently reported that the HDAC inhibitor trichostatin A (TSA) induces apoptosis and G2-phase cell cycle arrest in p53inactivated pancreatic cancer cell lines.22 We have also shown, using a proteomic profiling with 2-D maps, that regulation of specific proteins correlates with TSA induced cell growth inhibition.23 Journal of Proteome Research 2005, 4, 1909-1916
1909
Published on Web 10/05/2005
research articles The aim of this study was to verify whether TSA may sensitize pancreatic cancer cells to GEM treatment and to investigate this effect at the molecular level by proteomic analysis. We report here that TSA enhances growth inhibition and apoptosis of the pancreatic adenocarcinoma cell line T3M4 by GEM. The differential protein expression profiles of T3M4 cells untreated or treated with TSA and/or GEM by using two-dimensional gel electrophoresis and mass spectrometry analysis have identified 56 out of 81 differentially expressed polypeptide chains.
Materials and Methods Chemicals. Trichostatin A (TSA) was obtained from SigmaAldrich Company Ltd., solubilized in DMSO and stored at -80 °C until use. Gemcitabine (GEM) (Gemzar, Lilly) was freshly prepared in sterile water. Cell Culture. Human pancreatic adenocarcinoma cell line T3M4 (see ref 12 for genetic characterization and primary tissue source) was grown in RPMI 1640 supplemented with 20mM glutamine, 10% FBS and 50 µg/mL gentamicin sulfate (BioWhittaker, Italy) and were incubated at 37 °C with 5% CO2. Cell Proliferation Assay. Cells were plated in 96-well cell culture plates (4 × 103 cells/well) and were treated with TSA and/or GEM for 48 h at the indicated concentrations, and then stained with Crystal Violet (Sigma). The dye was solubilized in 1% SDS in PBS and measured photometrically (A595 nm) to determine cell growth. Four independent experiments were performed for each assay condition. Apoptosis. Apoptosis was analyzed by using the Cell-Death Detection ELISAPlus assay (Roche Molecular Biochemicals). The assay is based on a quantitative sandwich-enzyme-immunoassay principle using monoclonal antibodies anti-histone-biotin and anti-DNA-peroxidase (POD). POD was determined photometrically (A405 nm - A492 nm) with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic) acid (ABTS) as a substrate. Apoptosis was assessed by detection of histone-associated DNA fragments after 0.2 µM TSA and/or 2 µM GEM treatment at the time points described. This assay allows the apoptotic determination of the enrichment of nucleosomes in the cytoplasmic fraction of cell lysates from treated cells relative to control cells. Four independent experiments were performed for each assay condition. Two-Dimensional Gel Electrophoresis. Protein extraction from cells untreated, treated with 0.2 µM TSA for 18 h, and/or 2 µM GEM for 18 h was performed with lysis buffer (40 mM Tris, 1% NP40, 1 mM Na3VO4, 1 mM NaF, 1 mM PMSF, protease inhibitor cocktail). Cells were left in lysis buffer for 30 min in ice. After centrifugation at 14.000 × g at 4 °C (for removal of particulate material) the protein solution was collected and stored at -80 °C until used. Seventeen cm long, pH 3-10 immobilized pH gradient strips (IPG; Bio-Rad Labs., Hercules, CA) were rehydrated for 8 h with 450 µL of 2-D solubilizing solution (7 M urea, 2 M thiourea, 3% w/v CHAPS, 5 mM tributylphosphine, 40 mM Tris and 20 mM iodoacetamide) containing 2 mg/mL of total protein from T3M4 cells. Isoelectric focusing (IEF) was carried out with a Protean IEF Cell (Biorad, Hercules, CA), with a low initial voltage and then by applying a voltage gradient up to 10 000 V with a limiting current of 50 µA/strip. The total product time x voltage applied was 70 000 Vh for each strip and the temperature was set at 20 °C. For the second dimension, the IPGs strips were equilibrated for 26 min by rocking in a solution of 6 M urea, 2% SDS, 20% glycerol, 375 mM Tris-HCl, pH 8.8. The IPG strips were then laid on an 8-18%T gradient SDS-PAGE with 0.5% agarose in the cathode buffer (192 mM glycine, 0.1% SDS and Tris to pH 1910
Journal of Proteome Research • Vol. 4, No. 6, 2005
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8.3).24 The anodic buffer was a solution of 375 mM Tris HCl, pH 8.8. The electrophoretic run was performed by setting a current of 2 mA for each gel for 2 h, then 5 mA/gel for 1 h, 10 mA/gel for 20 h and 20 mA/gel until the end of the run. During the whole run the temperature was set at 11 °C. The protein zones were revealed with Sypro Ruby stain.25 Protein Pattern Differential Analysis. The 2-DE gels were scanned with a Versa Doc (Bio-Rad), and analyzed with the software PDQuest Version 7.1 (Bio-Rad, Labs, Hercules, CA). A match set was created from the protein patterns of six replicate gels for each independent cellular extract (control, TSA, GEM, TSA+GEM). A standard gel was generated out of the image with the highest spot number. Spot quantities of all gels were normalized to remove non expression-related variations in spot intensity, so the raw quantity of each spot in a gel was divided by the total quantity of all the spots in that gel that have been included in the standard. The results were evaluated in terms of spot OD (optical density). Statistical analysis of PDQuest by t-test allowed the study of proteins that were significantly increased or decreased after the treatments of the cell line. A p-value
