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

Open AccessPublished:August 05, 2014DOI:https://doi.org/10.1074/mcp.M113.033217
      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.
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      The abbreviations used are:
      PGP
      phosphoglycerate kinase
      ROS
      reactive oxygen species
      GO
      Gene Ontology
      PKM
      pyruvate kinase.
      1The abbreviations used are:PGP
      phosphoglycerate kinase
      ROS
      reactive oxygen species
      GO
      Gene Ontology
      PKM
      pyruvate kinase.
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      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.

      MATERIALS AND METHODS

      Chemicals and Reagents

      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).

      Cell Culture and Establishment of Cisplatin Resistant Subline

      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.

      Cell Cytotoxicity 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.

      Sample Preparation and Quantitative Proteomic Analysis

      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.

      Real-time Quantitative PCR (qPCR)

      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.

      Western Blotting

      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.

      Determination of Cellular Platinum Accumulation

      The cellular platinum accumulation was determined by the method described by Kayoko Minakata (
      • Minakata K.
      • Nozawa H.
      • Okamoto N.
      • Suzuki O.
      Determination of platinum derived from cisplatin in human tissues using electrospray ionization mass spectrometry.
      ). 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 the solution, in which DDC forms a complex with Pt by replacing other bonded ligands. After 3 min, 30 μl of isoamylalcohol was added and mixed for 30 s, and separated by centrifugation. The isoamylalcohol layer was mixed with 30 μl of 1 m oxalic acid for 10 s and centrifuged. A 1 μl aliquot of the isoamylalcohol layer was subjected to electrospray ionization mass spectrometry. Measurements were done in triplicate to determine standard errors of the mean (shown as error bars).

      Short Hairpin RNA (ShRNA)-mediated Gene Silencing

      The shRNAs against Rab 5C and Rab 11B were designed by the Invitrogen RNAi design tool (http://www.invitrogen.com) and synthesized by Invitrogen, LTD. Nontargeting negative control of shRNA (NCi) was also synthesized. The shRNA sequences are displayed in supplemental Table S2. The oligonucleotides were annealed and inserted into the pll3.7 siRNA expression vector to generate shRNA. The A2780 cells were plated the day before transfection and allowed to grow to 70–80% confluence. The cells were transiently transfected with Rab 5C-shRNA-pll3.7 or Rab 11B-shRNA-pll3.7 respectively with polyethylenimine (PEI) in DMEM. The effectiveness of shRNA in inhibiting Rab 5C and Rab 11B expression was evaluated by real time RT-PCR (48 h after the transfection) and Western blotting analysis (72 h after the transfection). Cells transfected with the plasmid NCi-pll3.7 served as the control.

      Overexpression of Rab 5C in A2780-DR Cells

      The gene of Rab 5C was cloned from the mRNA by RT-PCR from the Raji cell line, which was then subcloned into eukaryotic plasmid pcDNA3.1B (Invitrogen). The primer sequences are displayed in supplemental Table S2. Briefly, A2780-DR cells were plated the day before transfection and allowed to grow to 70–80% confluence. The cells were transiently transfected with pcDNA3.1 and Rab 5C-pcDNA3.1, respectively. The expression of Rab 5C was examined by Western blotting after 72 h transfection.

      Detection of Reactive Oxygen Species (ROS) in A2780 and A2780-DR Cells

      The ROS in untreated and azacytidine-treated cells was detected using the Image-iT™ LIVE Green Reactive Oxygen Species Detection Kit (Molecular Probes, Inc. Eugene, OR) following manufacturer's instructions. Briefly, A2780 and A2780-DR cells (2.5 × 105 each) were plated in triplicates in six-well plate the day before the test. After 48 h growth, the cells were collected by centrifugation and washed once with warm HBSS/Ca/Mg. Cells were resuspended with 500 μl of the 25 μm carboxy-H2DCFDA working solution for 25 min at 37 °C, followed by addition of the Hoechst 33342 reagent to the reaction mixture at the final concentration of 1.0 μm and incubation for 5 min. The final products were gently washed with 1 ml HBSS/Ca/Mg immediately followed by imaging with Zeiss 710 Confocal Microscopy.

      RESULTS

      Characterization of the Drug-resistant A2780 Cell Line

      The drug-resistant cell line A2780-DR was established by the stepwise selection of A2780 cells cultured in growth media with increasing drug concentrations over a period of 6 months. To determine the sensitivity of A2780 and A2780-DR cells to cisplatin, cells were treated with different concentrations of cisplatin for 24 h and cell viability was measured by the Cell Counting Kit-8 (CCK-8) assay that allows sensitive colorimetric determination of cell viability and drug-sensitivity. The dose-dependent effects of cisplatin were represented as the percentage of viable cells as compared with untreated cells (Fig. 1A). When cells were treated with 80 μm cisplatin for 24h, percentages of viable cells were 40 and 100% for A2780 and A2780-DR cells, respectively. The inhibitory concentration 50% (IC50) and resistance index (RI) values of the two cell lines are displayed in Table I, indicating that A2780-DR is cisplatin resistant. The resistant phenotype is stable as the values of IC50 and RI have no significant changes over a period of 4 months in drug-free medium.
      Figure thumbnail gr1
      Fig. 1A, Cell cytotoxicity assays. Percentage of viable A2780-DR and A2780 cells treated with cisplatin at different concentrations for 24 h determined by using CCK-8 assay. Results are expressed as the mean of three experiments with p value <0.001; B, accumulation of cisplatin in 10 μm cisplatin treated A2780 and A2780-DR cells for 24 h as determined by electrospray ionization mass spectrometry. *** p < 0.001.
      Table IIC50 and Resistance Index of A2780-related cells to cisplatin treatment
      IC50 (μm)RI
      A278061.0 ± 2.31
      A2780-DR404.9 ± 15.16.6
      EV-A2780
      Empty vector transfected cells.
      54.6 ± 5.01
      ShRNA(Rab5C)-A2780103.3 ± 6.21.9
      EV-A278045.1 ± 2.71
      ShRNA(Rab11B)-A278062.7 ± 3.21.4
      a Empty vector transfected cells.
      We also analyzed the total cellular Pt accumulation in sensitive and resistant cells following exposure to 10 μm cisplatin treatment for 24 h. The relative cisplatin concentrations were displayed in Fig. 1B as determined by mass spectrometry. After treatment with cisplatin, total intracellular Pt in A2780-DR is about one third of that in the parent cell line A2780, indicating significant reduction of cisplatin accumulation in drug-resistant cells.

      Proteomic Analysis of A2780 and A2780-DR Cells

      Next, proteomic analysis was carried out on A2780 and A2780-DR cells. Equal amounts of proteins from A2780 and A2780-DR cells were loaded and separated by 1D SDS-PAGE (Fig. 2). Differentially expressed proteins were identified and quantified using TMT-labeling. The experiments were repeated three times and ∼1900 proteins were identified for each cell line. The false-positive rate was set to be less than 1%. Based on TMT ratios (>2.0 or <0.6) in proteins that have two or more unique peptides, 340 proteins were found to be differentially expressed between A2780 and A2780-DR cells, of which 268 proteins are down-regulated and 72 up-regulated (Table II and III). The major protein in band 7 that was down-regulated in A2780-DR was identified as vimentin. HSP90α and HSP90β in band 4 were also down-regulated in the drug-resistant cells (Fig. 2). In order to understand the biological relevance of the identified proteins, Gene Ontology (GO) was used to categorize the differentially expressed proteins according to their molecular functions and biological processes. The annotations of gene lists are summarized via a pie plot using the PANTHER bioinformatics platform (http://www.pantherdb.org/) as shown in Fig. 3. Three hundred and thirty nine proteins were classified into several significant groups of biological processes including metabolic processes, cellular processes, cellular compartment organization, and apoptosis.
      Figure thumbnail gr2
      Fig. 21D SDS-PAGE gel image of A2780 and A2780-DR cells. Lane 1, molecular weight markers; Lane 2, proteins from A2780 cells; Lane 3, proteins from A2780-DR cells; and bands excised from the gel with differentially expressed proteins are labeled with numeric numbers.
      Table IIUp-regulated proteins in cisplatin resistant cells
      AccessionDescriptionScoreCoverage (%)Unique peptidesA2780-DR/A2780
      E9PM6926S protease regulatory subunit 6A171553.5
      O0023126S proteasome non-ATPase regulatory subunit 11181665.1
      P3657860S ribosomal protein L47730125.1
      B4DQJ86-phosphogluconate dehydrogenase, decarboxylating232483.7
      G3V4F2Acyl-coenzyme A thioesterase 1141443.6
      P30520Adenylosuccinatesynthetaseisozyme 2161344.4
      P30837Aldehyde dehydrogenase X, mitochondrial221866.3
      F8VS02Alpha-aminoadipicsemialdehyde dehydrogenase171776.2
      P06733Alpha-enolase21753164.8
      B4DT77Annexin231242.4
      P25705ATP synthase subunit alpha, mitochondrial13645196.8
      P06576ATP synthase subunit beta, mitochondrial19952205.4
      R4GMX5Basigin (Fragment)113634.7
      H3BS10Beta-hexosaminidase17832.3
      Q13895Bystin10634.2
      P10644cAMP-dependent protein kinase type I-alpha regulatory subunit131863.4
      C9JP16Cartilage-associated protein121255.3
      P31930Cytochrome b-c1 complex subunit 1, mitochondrial271765.7
      B7Z5W8Dihydrolipoyllysine-residue succinyltransferase component111146.4
      P39656Dolichyl-diphosphooligosaccharide–protein glycosyltransferase 48 kDa subunit462195.5
      Q05639Elongation factor 1-alpha 22562023.1
      P26641Elongation factor 1-gamma6232134.7
      P49411Elongation factor Tu, mitochondrial8827114.5
      F5H0C8Enolase521124.1
      P07099Epoxide hydrolase 14224115.3
      Q96CG1ETF1 protein13622.7
      P38919Eukaryotic initiation factor 4A-III312164.7
      H7BZU1Eukaryotic translation initiation factor 2 subunit 3 (Fragment)101123.9
      O00303Eukaryotic translation initiation factor 3 subunit F171753.6
      J3KNT0Fascin182394.1
      J3QRD1Fatty aldehyde dehydrogenase271458.1
      P39748Flap endonuclease 112834.6
      P00367Glutamate dehydrogenase 1, mitochondrial221885.9
      P52597Heterogeneous nuclear ribonucleoprotein F471423.6
      P31943Heterogeneous nuclear ribonucleoprotein H943354.5
      P55795Heterogeneous nuclear ribonucleoprotein H2511824.3
      B3KWE1Histidine–tRNA ligase, cytoplasmic15633.9
      P0C0S5Histone H2A.Z2833122.1
      H7C3I1Hsc70-interacting protein (Fragment)172336.6
      Q6YN16Hydroxysteroid dehydrogenase-like protein 212838.6
      P43686-2Isoform 2 of 26S protease regulatory subunit 6B151966.0
      P62195-2Isoform 2 of 26S protease regulatory subunit 8151233.5
      Q16401-2Isoform 2 of 26S proteasome non-ATPase regulatory subunit 5352184.1
      P28838-2Isoform 2 of Cytosol aminopeptidase261986.9
      Q9H0S4-2Isoform 2 of Probable ATP-dependent RNA helicase DDX47121037.3
      P35659-2Isoform 2 of Protein DEK171144.3
      P50395-2Isoform 2 of Rab GDP dissociation inhibitor beta332654.9
      Q8NBS9-2Isoform 2 of Thioredoxin domain-containing protein 54739114.3
      O14773-2Isoform 2 of Tripeptidyl-peptidase 110924.9
      P34897-3Isoform 3 of Serine hydroxymethyltransferase, mitochondrial372096.5
      P00390-5Isoform 4 of Glutathione reductase, mitochondrial251146.1
      O60664-4Isoform 4 of Perilipin-3242165.6
      P07954-2Isoform Cytoplasmic of Fumaratehydratase, mitochondrial281365.7
      P05455Lupus La protein191666.3
      Q10713Mitochondrial-processing peptidase subunit alpha131056.3
      E7ERZ4Mitochondrial-processing peptidase subunit beta111235.8
      B4DEH8Polyadenylate-binding protein 2191735.1
      Q9UQ80Proliferation-associated protein 2G4231873.3
      B7Z254Protein disulfide-isomerase A69535127.3
      P49257Protein ERGIC-5317734.8
      P18754Regulator of chromosome condensation221966.2
      F8W914Reticulon121233.6
      P00352Retinal dehydrogenase 14826112.7
      P13489Ribonuclease inhibitor151754.2
      Q9Y265RuvB-like 16731104.6
      Q9Y230RuvB-like 2362183.7
      O15269Serine palmitoyltransferase 1101144.5
      P50454Serpin H117152194.0
      Q13838Spliceosome RNA helicase DDX39B962934.2
      P55084Trifunctional enzyme subunit beta, mitochondrial2124107.0
      P50552Vasodilator-stimulated phosphoprotein101244.6
      P04004Vitronectin11522.7
      Figure thumbnail gr3
      Fig. 3Functional classification of differentially expressed proteins between A2780 and A2780-DR cells with PANTHER (http://www.pantherdb.org).

      Verification of Differentially Expressed Proteins by Western blotting and qPCR

      Among differentially expressed proteins (Table III), Ras-related protein Rab 5C and Rab 11B are down-regulated in the drug-resistant cells. Fig. 4 shows a ms/ms spectrum of a peptide ions that match to fragments of a tryptic peptide GVDLQENNPASR from Rab 5C and the insert shows fragments at the low mass range for the TMT-reporter ions, whose ratio indicates that the expression level of Rab 5C is three times higher in A2780 as compared with A2780-DR cells. Down-regulation of Rab 5C and Rab 11B was confirmed by Western blotting (Fig. 5A) and by qPCR analysis (Fig. 5B). Results from analysis of band intensities of Western blot are displayed in supplemental Table S3. Vimentin was the most abundant with the highest spectra count among the differentially expressed proteins and the down-regulation of vimentin was confirmed by Western blotting (Fig. 5A), showing that two bands at 54 and 56 kDa were barely visible for vimentin in A2780-DR cells. Western blotting also confirms that the expression level of PKM2 is down-regulated in A2780-DR cells (Fig. 5A). Proteomic analysis shows that a redox proteins glutathione reductase (GSR) is up-regulated in A2780-DR cells.
      Table IIIDown-regulated Proteins in cisplatin-resistant cells
      AccessionDescriptionScoreCoverage (%)Unique PeptidesA2780-DR/A2780
      Q0491714-3-3 protein eta422630.3
      P6198114-3-3 protein gamma573850.3
      P2734814-3-3 protein theta552720.3
      P6310414-3-3 protein zeta/delta754360.3
      P6219126S protease regulatory subunit 4362270.6
      Q1500826S proteasome non-ATPase regulatory subunit 6311140.3
      R4GMR526S proteasome non-ATPase regulatory subunit 8191450.3
      P8293028S ribosomal protein S34, mitochondrial122250.5
      P4678340S ribosomal protein S10242330.3
      P6227740S ribosomal protein S13223850.5
      P6226940S ribosomal protein S18212440.6
      P3901940S ribosomal protein S19344890.6
      P6285140S ribosomal protein S25262130.4
      F2Z2S840S ribosomal protein S3153340.6
      P6270140S ribosomal protein S4, X isoform573290.3
      M0R0F040S ribosomal protein S5 (Fragment)192750.5
      Q5JR9540S ribosomal protein S8213860.2
      P4678140S ribosomal protein S9191840.5
      C9J9K340S ribosomal protein SA (Fragment)863660.2
      F8VU6560S acidic ribosomal protein P0 (Fragment)262560.2
      P6290660S ribosomal protein L10a202460.3
      P3005060S ribosomal protein L12132330.5
      P2637360S ribosomal protein L13282760.3
      E7EPB360S ribosomal protein L14293540.2
      P6131360S ribosomal protein L15251850.4
      G3V20360S ribosomal protein L18172540.5
      P6135360S ribosomal protein L27152130.6
      E9PJD960S ribosomal protein L27a153430.5
      P4920760S ribosomal protein L34182130.5
      Q0287860S ribosomal protein L6232570.2
      P1812460S ribosomal protein L7262760.3
      O953366-phosphogluconolactonase111430.4
      P24752Acetyl-CoA acetyltransferase, mitochondrial302280.4
      H0YN26Acidic leucine-rich nuclear phosphoprotein 32 family member A151820.3
      O95433Activator of 90 kDa heat shock protein ATPase homolog 1191850.3
      Q01518Adenylyl cyclase-associated protein 17331140.5
      P12235ADP/ATP translocase 11283220.3
      P05141ADP/ATP translocase 21714040.4
      P61204ADP-ribosylation factor 3122020.4
      H0YN42Annexin (Fragment)283380.4
      P04083Annexin A145766210.2
      P02647Apolipoprotein A-I5253150.2
      B7Z7E9Aspartate aminotransferase171650.3
      P00505Aspartate aminotransferase, mitochondrial481880.5
      P48047ATP synthase subunit112740.5
      O43681ATPase ASNA111730.4
      Q08211ATP-dependent RNA helicase A34634360.5
      Q92499ATP-dependent RNA helicase DDX13114100.5
      O95816BAG family molecular chaperone regulator 2212760.4
      P51572B-cell receptor-associated protein 31171340.4
      E9PK09Bcl-2-associated transcription factor 1 (Fragment)13650.3
      P07686Beta-hexosaminidase subunit beta301360.4
      P07814Bifunctional glutamate/proline–tRNA ligase3912170.4
      P31327Carbamoyl-phosphate synthase [ammonia], mitochondrial22890.4
      E9PFZ2Ceruloplasmin10430.3
      F5GWX5Chromodomain-helicase-DNA-binding protein 423360.4
      B4DJV2Citrate synthase561830.6
      F5H669Cleavage and polyadenylation-specificity factor subunit 7131760.6
      Q9NX63Coiled-coil-helix-coiled-coil-helix domain-containing protein 3151540.4
      Q9P0M6Core histone macro-H2A.2211220.4
      H3BSJ9Cytochrome b-c1 complex subunit 2, mitochondrial313380.4
      P47985Cytochrome b-c1 complex subunit Rieske, mitochondrial121330.4
      Q14204Cytoplasmic dynein 1 heavy chain 1516230.4
      Q9Y295Developmentally-regulated GTP-binding protein 1131440.2
      H0Y8E6DNA replication licensing factor MCM2 (Fragment)361180.3
      P33992DNA replication licensing factor MCM5281280.4
      E9PCY5DNA topoisomerase 2 (Fragment)611690.5
      P11388DNA topoisomerase 2-alpha8916140.4
      E7EUY0DNA-dependent protein kinase catalytic subunit16011420.4
      C9J4M6DNA-directed RNA polymerase22560.5
      B4DX52DnaJ homolog subfamily B member 1111230.3
      P49792E3 SUM11380.5
      Q9HC35Echinoderm microtubule-associated protein-like 413650.4
      P68104Elongation factor 1-alpha 13453460.5
      E9PK01Elongation factor 1-delta (Fragment)152750.2
      Q9Y371Endophilin-B1101450.3
      P30040Endoplasmic reticulum resident protein 29323880.5
      P30084Enoyl-CoA hydratase, mitochondrial121840.6
      P05198Eukaryotic translation initiation factor 2 subunit 1161550.2
      F5H335Eukaryotic translation initiation factor 3 subunit A7815180.3
      Q13347Eukaryotic translation initiation factor 3 subunit I111340.2
      P56537Eukaryotic translation initiation factor 6113050.3
      Q9NPD3Exosome complex component RRP41161530.4
      Q9Y5B9FACT complex subunit SPT1621770.4
      Q08945FACT complex subunit SSRP14516120.6
      P49327Fatty acid synthase9814280.3
      P30043Flavin reductase (NADPH)113140.5
      P04075Fructose-bisphosphatealdolase A17657180.2
      K7EQ48Glucose-6-phosphate isomerase451680.5
      B4DWJ2Glutamine–tRNA ligase171180.5
      O76003Glutaredoxin-3111230.2
      P78417Glutathione S-transferase omega-1281430.4
      P09211Glutathione S-transferase P152540.4
      P04406Glyceraldehyde-3-phosphate dehydrogenase24755160.2
      H3BM42Golgi apparatus protein 110220.4
      Q08379Golgin subfamily A member 216650.4
      Q9UIJ7GTP:AMP phosphotransferase AK3, mitochondrial122660.5
      P62826GTP-binding nuclear protein Ran152550.4
      Q5T3Q7HEAT repeat-containing protein 112350.6
      P07900Heat shock protein HSP 90-alpha71655240.5
      P08238Heat shock protein HSP 90-beta92556240.5
      D6R9P3Heterogeneous nuclear ribonucleoprotein A/B241850.4
      F8W6I7Heterogeneous nuclear ribonucleoprotein A110244100.3
      G3V4W0Heterogeneous nuclear ribonucleoproteins C1/C2 (Fragment)7241130.4
      Q86YZ3Hornerin11420.4
      Q9Y4L1Hypoxia up-regulated protein 118239330.5
      P52292Importin subunit alpha-1171060.6
      Q12905Interleukin enhancer-binding factor 2682270.5
      P50213Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial272680.3
      O75874Isocitrate dehydrogenase [NADP] cytoplasmic151660.2
      B4DFL2Isocitrate dehydrogenase [NADP]191960.4
      Q9P2E9-2Isoform 1 of Ribosome-binding protein 13820120.4
      Q99714-2Isoform 2 of 3-hydroxyacyl-CoA dehydrogenase type-2515480.5
      Q92688-2Isoform 2 of Acidic leucine-rich nuclear phosphoprotein 32111920.3
      P23526-2Isoform 2 of Adenosylhomocysteinase13730.3
      O00571-2Isoform 2 of ATP-dependent RNA helicase DDX3X5424140.6
      Q00610-2Isoform 2 of Clathrin heavy chain 123031400.4
      O15160-2Isoform 2 of DNA-directed RNA polymerases I and III subunit101740.3
      P21333-2Isoform 2 of Filamin-A38529560.4
      P78347-2Isoform 2 of General transcription factor II-I5713120.5
      P51991-2Isoform 2 of Heterogeneous nuclear ribonucleoprotein A3392260.5
      P31942-2Isoform 2 of Heterogeneous nuclear ribonucleoprotein H3251740.3
      Q86UP2-2Isoform 2 of Kinectin6615170.4
      Q9NZM1-2Isoform 2 of Myoferlin357120.5
      P12036-2Isoform 2 of Neurofilament heavy polypeptide94850.4
      Q9Y617-2Isoform 2 of Phosphoserine aminotransferase141750.2
      P11940-2Isoform 2 of Polyadenylate-binding protein 17031150.6
      O75400-2Isoform 2 of Pre-mRNA-processing factor 40 homolog A191070.4
      P28370-2Isoform 2 of Probable global transcription activator SNF2L142920.4
      P25788-2Isoform 2 of Proteasome subunit alpha type-3242460.3
      Q5VT52-2Regulation of nuclear pre-mRNA domain-containing protein 210550.5
      Q92900-2Isoform 2 of Regulator of nonsense transcripts 118760.4
      Q5JTH9-2Isoform 2 of RRP12-like protein24880.5
      P16615-2Sarcoplasmic/endoplasmic reticulum calcium ATPase 24315120.5
      Q9H2G2-2Isoform 2 of STE20-like serine/threonine-protein kinase11450.4
      A6NHR9-2Structural maintenance of CFH domain-containing protein 115240.3
      O14776-2Isoform 2 of Transcription elongation regulator 115460.4
      P60174-1Isoform 2 of Triosephosphateisomerase11262140.3
      P07951-2Isoform 2 of Tropomyosin beta chain212040.2
      O43399-2Isoform 2 of Tumor protein D54193140.4
      Q9UIG0-2Isoform 2 of Tyrosine-protein kinase BAZ1B339120.4
      Q9NYU2-2Isoform 2 of UDP-glucose:glycoproteinglucosyltransferase 127780.6
      P30622-2Isoform 3 of CAP-Gly domain-containing linker protein 116460.4
      Q9Y281-3Isoform 3 of Cofilin-2111820.3
      P33993-3Isoform 3 of DNA replication licensing factor MCM7181770.6
      Q14103-3Isoform 3 of Heterogeneous nuclear ribonucleoprotein D0232770.4
      P06756-3Isoform 3 of Integrin alpha-V11125230.5
      Q7L2E3-3Isoform 3 of Putative ATP-dependent RNA helicase DHX3022880.4
      P08559-3Pyruvate dehydrogenase E1 component subunit alpha,181450.4
      Q13813-3Isoform 3 of Spectrin alpha chain, non-erythrocytic 113516340.4
      Q99832-3Isoform 3 of T-complex protein 1 subunit eta7536140.5
      Q86W42-3Isoform 3 of TH112450.3
      Q14669-4Isoform 4 of E3 ubiquitin-protein ligase TRIP1218450.4
      Q8N766-4Isoform 4 of ER membrane protein complex subunit 114530.5
      P54819-5Isoform 5 of Adenylate kinase 2, mitochondrial163050.3
      O75369-6Isoform 6 of Filamin-B48580.5
      P27816-6Isoform 6 of Microtubule-associated protein 42510100.4
      Q04637-7Isoform 7 of Eukaryotic translation initiation factor 4 gamma 1298110.3
      Q15149-7Isoform 7 of Plectin999340.4
      Q00325-2Isoform B of Phosphate carrier protein, mitochondrial401560.4
      P02788-2Isoform DeltaLf of Lactotransferrin22850.4
      P31946-2Isoform Short of 14–3-3 protein beta/alpha483630.3
      P46013-2Isoform Short of Antigen KI-6720480.5
      O75534-2Isoform Short of Cold shock domain-containing protein E111960.6
      Q15056-2Isoform Short of Eukaryotic translation initiation factor 4H102950.3
      J3KR24Isoleucine–tRNA ligase, cytoplasmic25670.3
      P42704Leucine-rich PPR motif-containing protein, mitochondrial33432430.5
      P00338l-lactate dehydrogenase A chain15920.4
      P07195l-lactate dehydrogenase B chain14920.5
      O00264Membrane-associated progesterone receptor component 1171630.6
      B4E1E9Mitochondrial dicarboxylate carrier121950.5
      Q9BQG0Myb-binding protein 1A6015180.5
      P19105Myosin regulatory light chain 12A131930.5
      P35580Myosin-1036530.4
      P35579Myosin-916320280.4
      O75489NADH dehydrogenase [ubiquinone] iron-sulfur protein 3263370.5
      P48681Nestin409130.4
      Q09666Neuroblast differentiation-associated protein AHNAK17570.4
      P69849Nodal modulator 325990.5
      Q14980Nuclear mitotic apparatus protein 113022390.6
      P49790Nuclear pore complex protein Nup15316670.6
      E9PF10Nuclear pore complex protein Nup15514560.6
      Q92621Nuclear pore complex protein Nup20520490.5
      Q8TEM1Nuclear pore membrane glycoprotein 21021680.5
      Q14978Nucleolar and coiled-body phosphoprotein 12817100.5
      P06748Nucleophosmin11346130.3
      P12270Nucleoprotein TPR8212280.6
      Q02790Peptidyl-prolylcis-trans isomerase FKBP43625100.5
      P32119Peroxiredoxin-2452650.5
      H7C3T4Peroxiredoxin-4 (Fragment)363940.5
      P30041Peroxiredoxin-6492460.3
      O95571Persulfidedioxygenase ETHE1, mitochondrial121630.4
      P00558Phosphoglycerate kinase 13730100.2
      P18669Phosphoglyceratemutase 1714390.3
      E9PBS1Phosphoribosylaminoimidazole carboxylase (Fragment)10930.4
      O15067Phosphoribosylformylglycinamidine synthase10650.3
      Q15102Platelet-activating factor acetylhydrolase IB subunit gamma242250.3
      Q15365Poly(rC)-binding protein 1352840.2
      H3BRU6Poly(rC)-binding protein 2 (Fragment)333040.3
      O75915PRA1 family protein 3131620.4
      Q6P2Q9Pre-mRNA-processing-splicing factor 86111210.4
      Q8IY81pre-rRNA processing protein FTSJ319870.4
      P07737Profilin-1956590.4
      P25789Proteasome subunit alpha type-4141650.3
      P28066Proteasome subunit alpha type-5182340.3
      P60900Proteasome subunit alpha type-6423590.2
      P20618Proteasome subunit beta type-1171740.4
      J3KSM3Proteasome subunit beta type-3152320.4
      P28070Proteasome subunit beta type-4201940.2
      P28074Proteasome subunit beta type-5111740.5
      P28072Proteasome subunit beta type-6191330.4
      Q99436Proteasome subunit beta type-7111840.3
      Q14690Protein RRP5 homolog12350.5
      Q99584Protein S100-A13234850.6
      P05109Protein S100-A8114040.6
      P06702Protein S100-A9223840.4
      P14618Pyruvate kinase PKM37164320.7
      P46940RasGTPase-activating-like protein IQGAP16513180.5
      Q15907Ras-related protein Rab-11B483990.6
      P61106Ras-related protein Rab-14262540.3
      P62820Ras-related protein Rab-1A373660.5
      B4DJA5Ras-related protein Rab-5A132120.4
      P51148Ras-related protein Rab-5C261920.3
      J3QR09Ribosomal protein L19162140.1
      P38159RNA-binding motif protein, X chromosome6144170.6
      P49756RNA-binding protein 2511740.4
      Q5QPM1RNA-binding protein Raly (Fragment)162750.5
      Q14151Scaffold attachment factor B218420.5
      P35270Sepiapterin reductase143360.4
      B5MCX3Septin-2152560.2
      B4E241Serine/arginine-rich-splicing factor 3212530.6
      P62136Serine/threonine-protein phosphatase PP1-alpha catalytic212530.3
      P02768Serum albumin12343250.4
      Q9Y5M8Signal recognition particle receptor subunit beta111430.4
      J3QLE5Small nuclear ribonucleoprotein-associated protein N212640.3
      Q01082Spectrin beta chain, non-erythrocytic 16210210.5
      O75533Splicing factor 3B subunit 19319210.4
      Q13435Splicing factor 3B subunit 2401080.6
      Q15393Splicing factor 3B subunit 35610100.5
      B4E1K7Stomatin-like protein 2, mitochondrial363270.5
      Q14683Structural maintenance of chromosomes protein 1A20570.4
      Q9UQE7Structural maintenance of chromosomes protein 312550.4
      Q6UWP8Suprabasin171520.4
      O60264SWI/SNF-related matrix-associated actin-dependent regulator731690.5
      Q92797Symplekin11440.5
      Q5TCU6Talin-1396120.4
      P17987T-complex protein 1 subunit alpha8540190.6
      P40227T-complex protein 1 subunit zeta9733150.5
      Q9BRA2Thioredoxin domain-containing protein 17173340.5
      E9PH29Thioredoxin-dependent peroxide reductase, mitochondrial732660.6
      Q8NI27TH14580.4
      Q9Y2W1Thyroid hormone receptor-associated protein 33513100.3
      P37837Transaldolase191350.3
      Q01995Transgelin133560.4
      P37802Transgelin-2152340.5
      Q92616Translational activator GCN1315110.4
      P09661U2 small nuclear ribonucleoprotein A'263170.5
      P08579U2 small nuclear ribonucleoprotein B“172450.4
      O75643U5 small nuclear ribonucleoprotein 200 kDa helicase6710180.5
      P09936Ubiquitin carboxyl-terminal hydrolase isozyme L1222660.3
      D6RDM7Ubiquitin-conjugating enzyme E2 K (Fragment)132220.5
      P54727UV excision repair protein RAD23 homolog B181870.5
      P26640Valine–tRNA ligase13440.4
      O75396Vesicle-trafficking protein SEC22b151530.4
      Q00341Vigilin25880.4
      P08670Vimentin198781430.4
      P13010X-ray repair cross-complementing protein 55817110.6
      Figure thumbnail gr4
      Fig. 4MS/MS spectra for identification of Rab 5C: A MS/MS spectrum of a doubly charged TMT-labeled peptide ion at m/z 764. 9025 for MH22+ corresponding to the mass of the peptide GVDLQENNPASR from Rab 5C. Fig. inserts show peaks of TMT reporter ions of two labeled peptides.
      Figure thumbnail gr5
      Fig. 5Confirmation of differentially expressed proteins by Western and qPCR. A, Western blot analysis of selected proteins from A2780 and A2780-DR cells. B, qPCR analysis of selected genes from A2780 and A2780-DR cells. ** for p value <0.001; and *** for p value <0.001.

      Rab 5C Mediated Cisplatin-resistance

      To understand the role of Rab 5C and Rab 11B in drug-resistance, shRNAs were used to silence Rab 5C and Rab 11B in A2780 cells. The plasmid NCi-pll3.7 was also transfected into A2780 as the control to exclude effects of cytotoxicity caused by transfection. The silencing of Rab 5C and Rab 11B was verified by Western blotting and qPCR assays (supplemental Fig. S1). The expression level of Rab 5C mRNA decreased to 30% of that observed for NCi-pll3.7 -transfected cells, whereas the expression level of Rab 11B mRNA decreased to 57% of the control. The sensitivities of shRNA-transfected cells to cisplatin were analyzed by CCK8 assay after cells were treated with different concentrations of cisplatin for 24 h. When cells were treated with 40 μm cisplatin for 24 h, differences in sensitivity to cisplatin were observed among NCi-pll3.7- and Rab 5C shRNA-transfected cells. The change of drug resistance in Rab 11B shRNA-transfected cells is less significant as compared with A2780 cells. Results demonstrate that transfection of A2780 cells with shRNA against Rab 5C increases cell resistance to cisplatin. The IC50 and resistance index values of shRNA-transfected cell lines are 103.3 and 1.89 for Rab 5C, and 62.7 and 1.39 for Rab 11B (Table I).
      To further explore Rab 5C mediated drug resistance, Rab 5C was sub-cloned into eukaryotic plasmid pcDNA3.1 that was transfected into A2780-DR cells. The overexpression of Rab 5C in A2780 cells was examined by Western blotting (supplemental Fig. S1C), showing that the expression level of Rab 5C in A2780-DR cells is three times higher than that in empty vector-transfected cells. The sensitivities of Rab 5C overexpressing cells to cisplatin were analyzed by CCK8 assay after cells were treated with different concentrations of cisplatin for 24 h (Fig. 6C), demonstrating that overexpression of Rab 5C in A2780-DR cells increases cell susceptibility to cisplatin.
      Figure thumbnail gr6
      Fig. 6Cell cytotoxicity assays. Percentage of viable A2780, shRNA-transfected A2780, and Rab 5C-pcDNA3.1-transfected cells treated with cisplatin at different concentrations for 24 h determined by using CCK-8 assay. Results are expressed as the mean of three experiments; A, Rab 5Ci; B, Rab 11Bi; and C, Rab 5C-pcDNA3.1. ***p < 0.001.

      DISCUSSION

      Multidrug resistance is the main reason for the failure of ovarian cancer chemotherapy. The establishment of drug resistant cancer cell lines is an important step in providing an in vitro model for understanding the mechanism of drug resistance and for identifying new therapeutic targets. Cisplatin is a traditional anticancer drug used in clinical settings. In this study, we used human ovarian cell line A2780 as a model system to establish a cisplatin resistant cell line A2780-DR. By the stepwise increase of cisplatin concentration in the growth medium and selection of drug-resistance colonies for six months, we successfully established a cisplatin-resistant cell line with the resistance index 6.76. Using mass spectrometry analysis, it was found that Pt accumulation in A2780-DR cells was only one third of that in A2780 cells, suggesting that a reduction in cellular Pt accumulation is the major cause of drug-resistance in A2780-DR cells.
      To identify factors leading to drug resistance, we used TMT labeling to quantify proteins from two types of cells. TMT-labeling uses an isobaric tag with an amine-reactive NHS-ester group, which enables quantitation of two samples with a single LC-MS/MS run that eliminates experimental variation. It also enhances the ionization efficiency of peptides to make them more amenable for MS analysis. We identified about 1900 proteins in three repeated experiments. Among them, 340 proteins were differentially expressed between A2780 and A2780-DR cells, which participate in a variety of cellular processes including cell metabolism, stress responses, cell cycle, and DNA repair. Based on GO analysis, 135 proteins are associated with the metabolic processes including ferredoxin metabolic process (GO:0006124); nitrogen compound metabolic process (GO:0006807); oxygen and reactive oxygen species metabolic process (GO:0006800); phosphate metabolic process (GO:0006796); and primary metabolic process (GO:0044238).
      Five out of ten glycolytic proteins were down-regulated including Glucose-6-phosphate isomerase (GPI), fructose-bisphosphate aldolase (ALDO), lactate dehydrogenase (LDH), PGK, and pyruvate kinase (PKM), indicating that glycolysis was down-regulated in drug-resistant cells. This is consistent with an earlier report showing that ALDO and PGK are down-regulated in drug-resistant cells (
      • Gong F.
      • Peng X.
      • Zeng Z.
      • Yu M.
      • Zhao Y.
      • Tong A.
      Proteomic analysis of cisplatin resistance in human ovarian cancer using 2-DE method.
      ). The other proteomic study has also linked the decreased pyruvate kinase M2 expression to oxaliplatin resistance in patients with colorectal cancer, showing tumors with the lowest PKM2 levels attain the lowest oxaliplatin response rates and the high PKM2 levels are associated with high p53 levels (
      • Martinez-Balibrea E.
      • Plasencia C.
      • Ginés A.
      • Martinez-Cardús A.
      • Musulén E.
      • Aguilera R.
      • Manzano J.L.
      • Neamati N.
      • Abad A.
      A proteomic approach links decreased pyruvate kinase M2 expression to oxaliplatin resistance in patients with colorectal cancer and in human cell lines.
      ). In the present study, the expression level of PKM in A2780-DR cells was about half of that in A2780 cells (Table III) by quantitative proteomic analysis. This was confirmed by Western blotting (Fig. 5). The mRNA level of PKM2 in A2780-DR cells was also down-regulated compared with A2780 cells by qPCR analysis. PKM2 catalyzes the rate-limiting step of glycolysis, in which phosphoenolpyruvate is converted to pyruvate. As a key enzyme for cancer metabolism and tumor growth, PKM2 and other glycolytic enzymes were found to be up-regulated in most cancer cells (
      • Christofk H.R.
      • Vander Heiden M.G.
      • Harris M.H.
      • Ramanathan A.
      • Gerszten R.E.
      • Wei R.
      • Fleming M.D.
      • Schreiber S.L.
      • Cantley L.C.
      The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth.
      ,
      • Mazurek S.
      Pyruvate kinase M2: a key enzyme of the tumor metabolome and its medical relevance.
      ,
      • Xu R.
      • Pelicano H.
      • Zhou Y.
      • Carew J.S.
      • Feng L.
      • Bhalla K.N.
      • Keating M.J.
      • Huang P.
      Inhibition of glycolysis in cancer cells: a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia.
      ). However, our results and previous studies have shown that down-regulation of glycolytic enzymes are a characteristic of drug-resistant ovarian cancer and colorectal cancer cells (
      • Gong F.
      • Peng X.
      • Zeng Z.
      • Yu M.
      • Zhao Y.
      • Tong A.
      Proteomic analysis of cisplatin resistance in human ovarian cancer using 2-DE method.
      ,
      • Martinez-Balibrea E.
      • Plasencia C.
      • Ginés A.
      • Martinez-Cardús A.
      • Musulén E.
      • Aguilera R.
      • Manzano J.L.
      • Neamati N.
      • Abad A.
      A proteomic approach links decreased pyruvate kinase M2 expression to oxaliplatin resistance in patients with colorectal cancer and in human cell lines.
      ). Although the molecular events leading to down-regulation of glycolytic enzymes are still not clear, we have found that expression levels of c-Myc and HIF1A are down-regulated in drug-resistant cells. Western blots of c-Myc and HIF1A are displayed in supplemental Fig. S2, showing that both c-Myc and HIF1a are down-regulated in A2780-DR cells. c-Myc is an oncogene that regulates transcription of many growth related genes. HIF1A is a master transcriptional regulator of the adaptive response to hypoxia that activates the transcription of glycolytic enzymes. Down-regulation of c-Myc and HIF1A results in decreases in expression levels of glycolytic enzymes that may contribute to drug resistance in ovarian cancer cells.
      Cisplatin is a potent electrophile covalently modifying nucleophilic sites on proteins, lipids, DNA, and RNA to generate reactive oxygen species and to induce cell apoptosis. To explore the difference in endogenous ROS levels between A280 and A2780-DR, the intracellular ROS levels were measured with the Image-iT™ LIVE Green Reactive Oxygen Species Detection Kit in both cells. Results show that the ROS level in A2780-DR cells is lower than that in A2780 cells (supplemental Fig. S3), indicating that A2780_DR cells possess a higher capacity to accommodate cisplatin-induced ROS stress. However, quantitative proteomics showed that some redox proteins such as peroxiredoxin-6 (PRDX6) and thioredoxin reductase 1 (TR1) were down-regulated, whereas glutathione reductase (GSR) is up-regulated in A2780-DR cells (Table II). GSR maintains high levels of reduced glutathione in the cytosol, and the up-regulation of GSR may lead to a decrease of ROS in A2780-DR cells. Studies are underway to understand the complex interactions of ROS and the cellular antioxidant system.
      On 1D SDS-PAGE (Fig. 2), band 7 has the largest change in intensity, in which vimentin was identified as the major protein. Quantitative proteomics showed that expression of vimentin was decreased 2-fold in A2780-DR cells as compared with A2780 cells, which was confirmed by Western blotting (Fig. 5A). Vimentin is a major intermediate filament protein and is ubiquitously expressed to maintain cellular integrity. Overexpression of vimentin has been observed in various epithelial cancers and correlated with accelerated tumor growth, invasion, and poor prognosis (
      • Satelli A.
      • Li S.
      Vimentin in cancer and its potential as a molecular target for cancer therapy.
      ). Furthermore, etoposide resistance in neuroblastoma cell and vinca alkaloid resistance in acute lymphoblastic leukemia have been linked to overexpression of vimentin (
      • Verrills N.M.
      • Walsh B.J.
      • Cobon G.S.
      • Hains P.G.
      • Kavallaris M.
      Proteome analysis of vinca alkaloid response and resistance in acute lymphoblastic leukemia reveals novel cytoskeletal alterations.
      ,
      • Urbani A.
      • Poland J.
      • Bernardini S.
      • Bellincampi L.
      • Biroccio A.
      • Schnölzer M.
      • Sinha P.
      • Federici G.
      A proteomic investigation into etoposide chemo-resistance of neuroblastoma cell lines.
      ). However, down-regulation of vimentin has been found in resistant1A9 cells to the microtubule stabilizing agents, PLA and LAU (
      • Kanakkanthara A.
      • Rawson P.
      • Northcote P.T.
      • Miller J.H.
      Acquired resistance to Peloruside A and laulimalide is associated with down-regulation of vimentin in human ovarian carcinoma cells.
      ), consistent with results from the present study. Therefore, changes in expression levels of vimentin are cancer-type dependent and vimentin is a potential marker for drug-resistance in ovarian cancer.
      Decreased cellular drug accumulation is the most commonly observed phenomenon among drug resistant cells. In the present study, we found that cisplatin accumulation was lower in drug-resistant cells (Fig. 1). It is known that cisplatin was not a substrate of P-glycoprotein the key mediator for drug efflux (
      • Breier A.
      • Gibalova L.
      • Seres M.
      • Barancik M.
      • Sulova Z.
      New Insight into P-glycoprotein as a drug target.
      ,
      • Cocker H.A.
      • Tiffin N.
      • Pritchard-Jones K.
      • Pinkerton C.R.
      • Kelland L.R.
      In vitro prevention of the emergence of multidrug resistance in a pediatric rhabdomyosarcoma cell line.
      ,
      • Yang X.
      • Page M.
      P-glycoprotein expression in ovarian cancer cell line following treatment with cisplatin.
      ,
      • Stordal B.
      • Hamon M.
      • McEneaney V.
      • Roche S.
      • Gillet J.-P.
      • O'Leary J.J.
      • Gottesman M.
      • Clynes M.
      Resistance to paclitaxel in a cisplatin-resistant ovarian cancer cell line is mediated by P-glycoprotein.
      ). Therefore, reduction of cisplatin accumulation in drug-resistant cells may be related to uptake of cisplatin. Uptake of cisplatin can be governed by different mechanisms including passive diffusion, carrier-mediated transporting, and endocytosis (
      • Arnesano F.
      • Natile G.
      Mechanistic insight into the cellular uptake and processing of cisplatin 30 years after its approval by FDA.
      ). An elegant study has shown that several small GTPases (Rab 5, Rac 1, and Rho A) were down-regulated in cisplatin-resistant human hepatoma and epidermal carcinoma cells, demonstrating that GTPase-regulated endocytosis is an important factor in drug-resistance (
      • Shen D.
      • Su A.
      • Liang X.
      • Pai-Panandiker A.
      • Gottesman M.
      Reduced expression of small GTPases and hypermethylation of the folate binding protein gene in cisplatin-resistant cells.
      ). Quantitative proteomic analysis in this study shows that Ras-related proteins Rab 5C and Rab 11B are down-regulated in A2780-DR cells as confirmed by Western blotting and qPCR. Rab 5C is a member of the Rab protein family and a key regulator in endocytosis and early endosome fusion, whereas Rab 11 has been associated with endosome recycling (
      • Schwartz S.L.
      • Cao C.
      • Pylypenko O.
      • Rak A.
      • Wandinger-Ness A.
      Rab GTPases at a glance.
      ). Therefore, down-regulation of Rab 5C and Rab 11B may result in reduced accumulation of cisplatin. To further confirm Rab 5C and Rab 11B mediated drug-resistance, shRNAs against Rab 5C and Rab 11B were used to silence these genes in A2780 cells. As predicted, silencing Rab 5C in A2780 cells lead to increased drug resistance (Fig. 6), but effects of Rab 11B shRNA are less significant (Table I). On the other hand, overexpression of Rab5C in A2780-DR cells increases its sensitivity to cisplatin treatment. These results suggest for the first time that Rab 5C mediated endocytosis regulates drug resistance in ovarian cancer cells.

      CONCLUSIONS

      Taken together, our results show that multiple cellular processes contribute to drug resistance in ovarian cancer cells. Although increased glycolysis is observed in most cancer cells, glycolytic enzymes PKM2, GPI, ALDO, LDH, and PGK are down-regulated in drug-resistant ovarian cancer cells. Drug resistance is also associated with a decrease of the endogenous ROS level, the up-regulation of GSR, as well as down-regulation of vimentin. Furthermore, the down-regulation of Rab 5C-mediated endocytosis contributes to the reduction of cellular cisplatin accumulation and drug-resistance. These results further our understanding of the multifactorial mechanisms in acquisition and development of cisplatin resistance in human cancer cells.

      Acknowledgments

      We thank the Protein Chemistry Facility at the Center for Biomedical Analysis of Tsinghua University for sample analysis. We thank Dr Zhenyu Zhang for valuable discussions.

      Supplementary Material

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