Down-regulation of Ras-related Protein Rab 5C-dependent Endocytosis and Glycolysis in Cisplatin-resistant Ovarian Cancer Cell Lines

• DOI: 10.1074/mcp.m113.033217
• Publication Date: 2014-08-06T02:38:20Z
• Authors: Lixu Jin; Yi Huo; Zhiguo Zheng; Xiaoyong Jiang; Haiyun Deng; Yuling Chen; Qingquan Lian; Renshan Ge; Haiteng Deng
• Keywords: Rab; Anaerobic glycolysis

Drug resistance poses a major challenge to ovarian cancer treatment. Understanding mechanisms of drug is important for finding new therapeutic targets. In the present work, cisplatin-resistant cell line A2780-DR was established with index 6.64. The cellular accumulation cisplatin significantly reduced in cells as compared A2780 consistent general character resistance. Quantitative proteomic analysis identified 340 differentially expressed proteins between and cells, which involve diverse processes, including metabolic process, component biogenesis, stress responses. Expression levels Ras-related Rab 5C 11B were lower than those confirmed by real-time quantitative PCR Western blotting. short hairpin (sh)RNA-mediated knockdown resulted markedly increased whereas overexpression increases sensitivity cisplatin, demonstrating that 5C-dependent endocytosis plays an role Our results also showed expressions glycolytic enzymes pyruvate kinase, glucose-6-phosphate isomerase, fructose-bisphosphate aldolase, lactate dehydrogenase, phosphoglycerate kinase 1 down-regulated resistant indicating directly associated decrease glycolysis. Furthermore, it found glutathione reductase up-regulated A2780-DR, vimentin, HSP90, Annexin A1 A2 down-regulated. Taken together, our suggest caused multifactorial traits, down-regulation enzymes, up-regulation antioxidant proteins, suggesting potential target treatment drug-resistant cancer. 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Dithiothreitol (DTT) Calbiochem (San Diego, obtained Tumor Cell Bank Chinese Academy Medical Sciences (Beijing, China). Sequencing grade modified trypsin Promega (Fitchburg, WI). propidium iodide staining kit Solarbio TMT labeling Thermo-Pierce Biotechnology (Rockford, IL). maintained DMEM media supplemented 10% serum penicillin (100 U/ml)–streptomycin mg/ml) at 37 °C 5% CO2. Cells grown monolayer cultures 10 cm culture plate passaged when they had reached 90% confluence. monoclonal strain separated flow cytometry obtain (A2780-DR) incubation stepwise increasing concentrations. Backups all stored DMSO. Every 20 passages, backup thawed ascertain unchanged during long term cultivation. relative determined viability assay. Effects proliferation analyzed Counting Kit-8 (CCK-8) Dojindo (Japan). (8 × 103 each) seeded wells 96-well microplates incubated 16 h prior then treated different concentrations (0, 20, 40, 80, 160, 320 μm) triplicates 24 h. CCK8 reagent added 2 Optical density (OD) measured 450 nm microplate reader (Bio-Rad, Hercules). calculated percentage variable untreated experiment repeated three times IC50 SPSS13.0 (SPSS Inc., Chicago, IL,). value, higher potency against proliferation. About 6 105 lysed RIPA lysis buffer (Solabio, Beijing, China), BCA method. Equal amount untreated- treated-samples (about μg) 1D SDS-PAGE, respectively. bands interest excised gel, 25 mm dithiotreitol, alkylated 55 iodoacetamide. digestion carried out sequencing 50 ammonium bicarbonate overnight. peptides extracted twice 0.1% trifluoroacetic acid 50% acetonitrile aqueous solution min. Extracts centrifuged speedvac reduce volume. Tryptic redissolved μl 200 Tetraethylammonium Bromide (TEAB), TMTsixplex each sample according manufacture's instruction. reaction room temperature. Then, 0.5 hydroxylamine (pH 9–10) mixture 15 min quench reaction. combined LC-MS/MS. LC-MS/MS analysis, TMT-labeled 65 gradient elution 0.250 μl/min Thermo-Dionex Ultimate 3000 HPLC system, interfaced Thermo Scientific Q Exactive mass spectrometer. analytical column home-made fused silica capillary (75 μm ID, 150 length; Upchurch, Oak Harbor, WA) packed C-18 resin (300 Å, 5 μm, Varian, Lexington, MA). Mobile phase consisted formic acid, mobile B 100% acid. spectrometer operated data-dependent acquisition mode Xcalibur 2.1.2 software there single full-scan spectrum orbitrap (400–1800 m/z, 60,000 resolution) MS/MS scans 27% normalized collision energy. spectra run searched human.fasta UniProt (release date March 19, 2014; 68406 entries) in-house Discoverer (Version PD1.4, Thermo-Fisher Scientific). search criteria follows: full tryptic specificity required; one missed cleavage allowed; carbamidomethylation (C) sixplex (K N-terminal) set fixed modifications; oxidation (M) modification; precursor tolerances ppm MS analyzer; fragment tolerance mmu MS2 acquired. peptide false discovery Percolator provided PD. When q value smaller 1%, match considered correct. False based reverse, decoy database. Peptides only assigned given unique. 0.01 identifications. Relative quantification performed 1.4) manufacturer's instructions reporter intensities per peptide. Quantitation two unique matches. Protein ratios median hits belonging protein. precision ratio variability. Differentially spectrometry deposited ProteomeXchange Consortium via PRIDE partner repository identifier PXD001176. harvested 48 after transfection. Total SV Isolation System. synthesized 0.8 μg total GoScriptTM Reverse Transcription All LightCycler® 480II Detection System SYBR green incorporation instructions. primers either designed Primer Premier (http://pga.mgh.harvard.eduprimerbank/). To amplification DNA, span exon-exon junctions. products melting curve analysis. 2−ΔΔCt sequences listed supplemental Table S1. buffer. transfected 72 supernatants collected centrifugation 14,000 g 4 °C. assay kit. 12% SDS-PAGE transferred onto polyvinyl diflouride transfer electroblotting. After blocking nonfat milk temperature, overnight 1000× diluted antibody, washed Phosphate Buffered Saline Tween (PBST) times, anti-mouse anti-rabbit antibody labeled horseradish peroxidase temperature PBST developed ECL reagents (Engreen, β-actin detected anti-β-actin internal control. BioRad Image Lab analyze images. described Kayoko Minakata (43Minakata Nozawa Okamoto Suzuki O. Determination derived electrospray ionization spectrometry.J. Chromatogr. 832: 286-291Crossref (21) Briefly, equal 106) pellets ice-cold PBS. pellet wet-ashed concentrated HNO3 85 8 pH adjusted 3–7 m NaOH 7 HNO3. Diethyldithiocarbamate (DDC)