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

2014; Elsevier BV; Volume: 13; Issue: 11 Linguagem: Inglês

10.1074/mcp.m113.033217

1535-9484

Lixu Jin, Yi Huo, Zhiguo Zheng, Xiaoyong Jiang, Haiyun Deng, Yuling Chen, Qingquan Lian, Ren‐Shan Ge, Haiteng Deng,

Erythrocyte Function and Pathophysiology

Drug resistance poses a major challenge to ovarian cancer treatment. Understanding mechanisms of drug resistance is important for finding new therapeutic targets. In the present work, a cisplatin-resistant ovarian cancer cell line A2780-DR was established with a resistance index of 6.64. The cellular accumulation of cisplatin was significantly reduced in A2780-DR cells as compared with A2780 cells consistent with the general character of drug resistance. Quantitative proteomic analysis identified 340 differentially expressed proteins between A2780 and A2780-DR cells, which involve in diverse cellular processes, including metabolic process, cellular component biogenesis, cellular processes, and stress responses. Expression levels of Ras-related proteins Rab 5C and Rab 11B in A2780-DR cells were lower than those in A2780 cells as confirmed by real-time quantitative PCR and Western blotting. The short hairpin (sh)RNA-mediated knockdown of Rab 5C in A2780 cells resulted in markedly increased resistance to cisplatin whereas overexpression of Rab 5C in A2780-DR cells increases sensitivity to cisplatin, demonstrating that Rab 5C-dependent endocytosis plays an important role in cisplatin resistance. Our results also showed that expressions of glycolytic enzymes pyruvate kinase, glucose-6-phosphate isomerase, fructose-bisphosphate aldolase, lactate dehydrogenase, and phosphoglycerate kinase 1 were down-regulated in drug resistant cells, indicating drug resistance in ovarian cancer is directly associated with a decrease in glycolysis. Furthermore, it was found that glutathione reductase were up-regulated in A2780-DR, whereas vimentin, HSP90, and Annexin A1 and A2 were down-regulated. Taken together, our results suggest that drug resistance in ovarian cancer cell line A2780 is caused by multifactorial traits, including the down-regulation of Rab 5C-dependent endocytosis of cisplatin, glycolytic enzymes, and vimentin, and up-regulation of antioxidant proteins, suggesting Rab 5C is a potential target for treatment of drug-resistant ovarian cancer. This constitutes a further step toward a comprehensive understanding of drug resistance in ovarian cancer. Drug resistance poses a major challenge to ovarian cancer treatment. Understanding mechanisms of drug resistance is important for finding new therapeutic targets. In the present work, a cisplatin-resistant ovarian cancer cell line A2780-DR was established with a resistance index of 6.64. The cellular accumulation of cisplatin was significantly reduced in A2780-DR cells as compared with A2780 cells consistent with the general character of drug resistance. Quantitative proteomic analysis identified 340 differentially expressed proteins between A2780 and A2780-DR cells, which involve in diverse cellular processes, including metabolic process, cellular component biogenesis, cellular processes, and stress responses. Expression levels of Ras-related proteins Rab 5C and Rab 11B in A2780-DR cells were lower than those in A2780 cells as confirmed by real-time quantitative PCR and Western blotting. The short hairpin (sh)RNA-mediated knockdown of Rab 5C in A2780 cells resulted in markedly increased resistance to cisplatin whereas overexpression of Rab 5C in A2780-DR cells increases sensitivity to cisplatin, demonstrating that Rab 5C-dependent endocytosis plays an important role in cisplatin resistance. Our results also showed that expressions of glycolytic enzymes pyruvate kinase, glucose-6-phosphate isomerase, fructose-bisphosphate aldolase, lactate dehydrogenase, and phosphoglycerate kinase 1 were down-regulated in drug resistant cells, indicating drug resistance in ovarian cancer is directly associated with a decrease in glycolysis. Furthermore, it was found that glutathione reductase were up-regulated in A2780-DR, whereas vimentin, HSP90, and Annexin A1 and A2 were down-regulated. Taken together, our results suggest that drug resistance in ovarian cancer cell line A2780 is caused by multifactorial traits, including the down-regulation of Rab 5C-dependent endocytosis of cisplatin, glycolytic enzymes, and vimentin, and up-regulation of antioxidant proteins, suggesting Rab 5C is a potential target for treatment of drug-resistant ovarian cancer. This constitutes a further step toward a comprehensive understanding of drug resistance in ovarian cancer. Ovarian cancer is the major cause of death in women with gynecological cancer. Early diagnosis of ovarian cancer is difficult, while its progression is fast. The standard treatment is surgical removal followed by platinum-taxane chemotherapy. However, the efficacy of the traditional surgery and chemotherapy is rather compromised and platinum resistant cancer recurs in ∼25% of patients within six months, and the overall five-year survival rate is about 31% (1Bell D. Berchuck A. Birrer M. Chien J. Cramer D. Dao F. Dhir R. Disaia P. Gabra H. Glenn P. Integrated genomic analyses of ovarian carcinoma.Nature. 2011; 474: 609-615Crossref PubMed Scopus (5418) Google Scholar, 2Miller D.S. Blessing J.A. Krasner C.N. Mannel R.S. Hanjani P. Pearl M.L. Waggoner S.E. Boardman C.H. Phase II evaluation of pemetrexed in the treatment of recurrent or persistent platinum-resistant ovarian or primary peritoneal carcinoma: a study of the Gynecologic Oncology Group.J. Clin. 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Identification of platinum-resistance associated proteins through proteomic analysis of human ovarian cancer cells and their platinum-resistant sublines.J. Proteome Res. 2007; 6: 772-780Crossref PubMed Scopus (123) Google Scholar). Employing a similar 2D gel electrophoresis approach, changes in protein expressions of capsid glycoprotein, fructose-bisphosphate aldolase C, heterogeneous nuclear ribonucleoproteins A2/B1, putative RNA-binding protein 3, Ran-specific GTPase-activating protein, ubiquitin carboxyl-terminal hydrolase isozyme L1, stathmin, ATPSH protein, chromobox protein homolog3, and phosphoglycerate kinase 1 (PGK) 1The abbreviations used are:PGPphosphoglycerate kinaseROSreactive oxygen speciesGOGene OntologyPKMpyruvate kinase. 1The abbreviations used are:PGPphosphoglycerate kinaseROSreactive oxygen speciesGOGene OntologyPKMpyruvate kinase. were found in A2780 and drug-resistant A2780 cells (32Gong F. Peng X. Zeng Z. Yu M. Zhao Y. Tong A. 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Mitochondrial proteomic analysis of drug-resistant cells has shown that five mitochondrial proteins (ATP-a, PRDX3, PHB, ETF, and ALDH) that participate in the electron transport respiratory chain are down-regulated in drug-resistant cell lines (41Dai Z. Yin J. He H. Li W. Hou C. Qian X. Mao N. Pan L. Mitochondrial comparative proteomics of human ovarian cancer cells and their platinum-resistant sublines.Proteomics. 2010; 10: 3789-3799Crossref PubMed Scopus (42) Google Scholar). PRDX3 is involved in redox regulation of the cell to protect radical-sensitive enzymes from oxidative damage. However, it is not clear how down-regulation of PRDX3 is associated with drug-resistance. A more recent study showed that activated leukocyte cell adhesion molecule (ALCA) and A kinase anchoring protein 12 (AKAP12) are elevated in drug-resistant A2780-CP20 cells by quantifying the mitochondrial proteins (42Chappell N.P. Teng P.-n. Hood B.L. Wang G. Darcy K.M. Hamilton C.A. Maxwell G.L. Conrads T.P. Mitochondrial proteomic analysis of cisplatin resistance in ovarian cancer.J. Proteome Res. 2012; 11: 4605-4614Crossref PubMed Scopus (40) Google Scholar). Despite these efforts, the drug-resistance mechanisms are not yet well understood. phosphoglycerate kinase reactive oxygen species Gene Ontology pyruvate kinase. phosphoglycerate kinase reactive oxygen species Gene Ontology pyruvate kinase. In this work, we established and characterized a drug-resistant cell line A2780-DR from A2780 cells. We employed a quantitative proteomic method to identify the differentially expressed proteins between A2780 and A2780-DR cells. Expression changes of selected proteins were confirmed by qPCR and Western blotting. We also used shRNA silencing to explore functions of Rab 5C and Rab 11B proteins in drug resistance. Our data indicate that the differentially expressed proteins participate in a variety of cellular processes and enhance our understanding of the mechanisms of drug resistance in ovarian cancer cells. Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum, and penicillin-streptomycin were purchased from Wistent (Saint-Jean-Baptiste, CA). Dithiothreitol (DTT) was purchased from Calbiochem (San Diego, CA). The A2780 cell line was obtained from the Tumor Cell Bank of the Chinese Academy of Medical Sciences (Beijing, China). Sequencing grade modified trypsin was purchased from Promega (Fitchburg, WI). The propidium iodide staining kit was purchased from Solarbio (Beijing, China). The TMT labeling kit was purchased from Thermo-Pierce Biotechnology (Rockford, IL). The human epithelial ovarian cancer cell line A2780 cells were maintained in DMEM media supplemented with 10% fetal bovine serum and penicillin (100 U/ml)–streptomycin (100 mg/ml) at 37 °C with 5% CO2. Cells were grown as monolayer cultures in 10 cm tissue culture plate and passaged when they had reached about 90% confluence. A monoclonal strain was separated by flow cytometry and further cultured to obtain A2780 cisplatin resistant strain (A2780-DR) by incubation with stepwise increasing cisplatin concentrations. Backups of all cells were stored with 10% DMSO. Every 20 passages, a new backup of cells was thawed to ascertain that resistance mechanisms were unchanged during long term cultivation. The relative cisplatin resistance was determined by cell viability assay. Effects of cisplatin on cell proliferation in A2780 and A2780-DR were analyzed with the Cell Counting Kit-8 (CCK-8) from Dojindo (Japan). A2780 and A2780-DR cells (8 × 103 each) were seeded into wells in 96-well cell culture microplates and incubated for 16 h prior to cisplatin treatment. Cells were then treated with cisplatin at different concentrations (0, 20, 40, 80, 160, and 320 μm) in triplicates for 24 h. The CCK8 reagent was added to treated cells and incubated at 37 °C for 2 h. Optical density (OD) was measured at 450 nm with a microplate reader (Bio-Rad, Hercules). Cell viability was calculated as the percentage of variable cells compared with untreated cells. The experiment was repeated three times and the IC50 was calculated by SPSS13.0 (SPSS Inc., Chicago, IL,). The lower the IC50 value, the higher the potency against cell proliferation. About 6 × 105 cells were lysed using RIPA lysis buffer (Solabio, Beijing, China), and protein concentrations were measured using the BCA method. Equal amount of proteins from untreated- and treated-samples (about 30 μg) were separated by 1D SDS-PAGE, respectively. The gel bands of interest were excised from the gel, reduced with 25 mm of dithiotreitol, and alkylated with 55 mm iodoacetamide. In gel digestion was then carried out with sequencing grade modified trypsin in 50 mm ammonium bicarbonate at 37 °C overnight. The peptides were extracted twice with 0.1% trifluoroacetic acid in 50% acetonitrile aqueous solution for 30 min. Extracts were then centrifuged in a speedvac to reduce the volume. Tryptic peptides were redissolved in 50 μl 200 mm Tetraethylammonium Bromide (TEAB), and 2 μl TMTsixplex labeling reagent was added to each sample according to the manufacture's instruction. The reaction was incubated for 1 h at room temperature. Then, 0.5 μl of 5% hydroxylamine (pH 9–10) was added to the reaction mixture and incubated for 15 min to quench the reaction. Equal amount of proteins from A2780 and A2780-DR cells were combined and analyzed by LC-MS/MS. For LC-MS/MS analysis, the TMT-labeled peptides were separated by a 65 min gradient elution at a flow rate 0.250 μl/min with a Thermo-Dionex Ultimate 3000 HPLC system, which was directly interfaced with a Thermo Scientific Q Exactive mass spectrometer. The analytical column was a home-made fused silica capillary column (75 μm ID, 150 mm length; Upchurch, Oak Harbor, WA) packed with C-18 resin (300 Å, 5 μm, Varian, Lexington, MA). Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. The Q Exactive mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 2.1.2 software and there was a single full-scan mass spectrum in the orbitrap (400–1800 m/z, 60,000 resolution) followed by 10 data-dependent MS/MS scans at 27% normalized collision energy. The MS/MS spectra from each LC-MS/MS run were searched against the human.fasta from UniProt (release date of March 19, 2014; 68406 entries) using an in-house Proteome Discoverer (Version PD1.4, Thermo-Fisher Scientific). The search criteria were as follows: full tryptic specificity was required; one missed cleavage was allowed; carbamidomethylation (C) and TMT sixplex (K and N-terminal) were set as the fixed modifications; the oxidation (M) was set as the variable modification; precursor ion mass tolerances were set at 10 ppm for all MS acquired in an orbitrap mass analyzer; and the fragment ion mass tolerance was set at 20 mmu for all MS2 spectra acquired. The peptide false discovery rate was calculated using Percolator provided by PD. When the q value was smaller than 1%, the peptide spectrum match was considered to be correct. False discovery was determined based on peptide spectrum match when searched against the reverse, decoy database. Peptides only assigned to a given protein group were considered as unique. The false discovery rate was also set to 0.01 for protein identifications. Relative protein quantification was performed using Proteome Discoverer software (Version 1.4) according to manufacturer's instructions on the six reporter ion intensities per peptide. Quantitation was carried out only for proteins with two or more unique peptide matches. Protein ratios were calculated as the median of all peptide hits belonging to a protein. Quantitative precision was expressed as protein ratio variability. Differentially expressed proteins were further confirmed by qPCR or Western blotting. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD001176. Cells were harvested 48 h after transfection. Total RNA was extracted by the SV Total RNA Isolation System. cDNA was synthesized from 0.8 μg total RNA using the GoScriptTM Reverse Transcription System. All qPCR was performed using the Roche LightCycler® 480II Detection System with SYBR green incorporation according to the manufacturer's instructions. The primers were either designed by using the Primer Premier 5 software or from Primer Bank (http://pga.mgh.harvard.eduprimerbank/). To prevent amplification of genomic DNA, all target primers span exon-exon junctions. The specific PCR products were confirmed by melting curve analysis. Relative expression was analyzed using the 2−ΔΔCt method. Primer sequences for qPCR are listed in supplemental Table S1. Cells were harvested and lysed in RIPA lysis buffer. For shRNA transfected cells, cells were lysed at 72 h after transfection. The supernatants were collected after centrifugation at 14,000 × g for 10 min at 4 °C. Protein concentrations were determined using the BCA protein assay kit. Proteins were separated on a 12% SDS-PAGE gel and transferred onto a polyvinyl diflouride transfer membrane by electroblotting. After blocking with 5% nonfat milk for 2 h at room temperature, the membrane was incubated overnight at 4 °C with 1000× diluted primary antibody, washed with Phosphate Buffered Saline with Tween 20 (PBST) buffer for three times, then incubated with 1000× diluted anti-mouse or anti-rabbit secondary antibody labeled with horseradish peroxidase at room temperature for 2 h. The membrane was further washed with PBST buffer three times and developed using ECL reagents (Engreen, China). β-actin was detected with anti-β-actin antibody as an internal control. BioRad Image Lab software was used to analyze the images. The cellular platinum accumulation was determined by the method described by Kayoko Minakata (43Minakata K. Nozawa H. Okamoto N. Suzuki O. Determination of platinum derived from cisplatin in human tissues using electrospray ionization mass spectrometry.J. Chromatogr. B. 2006; 832: 286-291Crossref PubMed Scopus (21) Google Scholar). Briefly, equal amount of A2780 and A2780-DR cells (about 4 × 106) were collected after 10 μm cisplatin treatment for 24 h. Cell pellets were washed three times with ice-cold PBS. Cell pellet was wet-ashed in 30 μl concentrated HNO3 at 85 °C for 8 h. The pH of wet-ashed solution was adjusted to 3–7 with either 10 m NaOH or 7 m HNO3. 30 μl of 1 m Diethyldithiocarbamate (DDC) was then added to