Project PXD001176

PRIDE Assigned Tags:
Biomedical Dataset

Summary

Title

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

Description

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 to 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 PKM, GPI, Aldolase, LDH, and PGK 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, while 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 towards a comprehensive understanding of drug resistance in ovarian cancer.

Sample Processing Protocol

About 6x105 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 DTT 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 hour at room temperature. Then, 0.5 μl of 5% hydroxylamine (pH 9-10) was added to the reaction mixture and incubated for 15 minutes to quench the reaction. at 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 (HCD).

Data Processing Protocol

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, USA). 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 (FDR) was calculated using Percolator provided by PD. When the q value was smaller than 1%, the peptide spectrum match (PSM) was considered to be correct. FDR was determined based on PSMs when searched against the reverse, decoy database. Peptides only assigned to a given protein group were considered as unique. The false discovery rate (FDR) 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 only carried out for proteins with 2 or more unique peptides. 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.

Contact

Haiteng Deng, Tsinghua University
Haiteng Deng, Tsinghua University ( lab head )

Submission Date

28/07/2014

Publication Date

06/07/2016

Instrument

Q Exactive

Software

Not available

Experiment Type

Shotgun proteomics

Publication

    Jin L, Huo Y, Zheng Z, Jiang X, Deng H, Chen Y, Lian Q, Ge R, Deng H. Down-regulation of Ras-related protein Rab 5C-dependent endocytosis and glycolysis in cisplatin-resistant ovarian cancer cell lines. Mol Cell Proteomics. 2014 Nov;13(11):3138-51 PubMed: 25096996