Article pubs.acs.org/jpr
PTRF/Cavin‑1 is Essential for Multidrug Resistance in Cancer Cells Jae-Sung Yi,†,∥ Dong-Gi Mun,‡,∥ Hyun Lee,† Jun-sub Park,† Jung-Woo Lee,† Jae-Seon Lee,§ Su-Jin Kim,‡ Bong-Rae Cho,‡ Sang-Won Lee,‡,* and Young-Gyu Ko†,* †
Division of Life Sciences, College of Life Sciences and Biotechnology, and ‡Department of Chemistry, Research Institute for Natural Sciences, Korea University, Seoul, 136-701, South Korea, and § Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul, 139-706, South Korea S Supporting Information *
ABSTRACT: Since detergent-resistant lipid rafts play important roles in multidrug resistance (MDR), their comprehensive proteomics could provide new insights to understand the underlying molecular mechanism of MDR in cancer cells. In the present work, lipid rafts were isolated from MCF-7 and adriamycin-resistant MCF-7/ADR cells and their proteomes were analyzed by label-free quantitative proteomics. Polymerase I and transcript release factor (PTRF)/cavin-1 was measured to be upregulated along with multidrugresistant P-glycoprotein, caveolin-1, and serum deprivation protein response/cavin-2 in the lipid rafts of MCF-7/ADR cells. PTRF knockdown led to reduction in the amount of lipid rafts on the surface of MCF7/ADR cells as determined by cellular staining with lipid raft-specific dyes such as S-laurdan2 and FITC-conjugated cholera toxin B. PTRF knockdown also reduced MDR in MCF-7/ADR cells. These data indicate that PTRF is necessary for MDR in cancer cells via the fortification of lipid rafts. KEYWORDS: lipid rafts, label-free quantitative proteomics, PTRF, cavin-1, multidrug resistance
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INTRODUCTION In the presence of anticancer drugs, cancer cells have been known to preserve their survival and tumorigenicity by developing multidrug resistance (MDR) and leading to a significant obstacle to chemotherapeutic approach in many cancers.1,2 The MDR mechanisms have been investigated in various MDR cancer cell lines selected by the prolonged exposure to different anticancer drugs. P-glycoprotein (P-gp), an ATP-dependent drug efflux pump, is upregulated in the plasma membrane of all MDR cancer cells.3,4 Knockdown and knockout experiments have revealed that P-gp is required for exporting anticancer drugs into the extracellular space.5−7 In addition to upregulation of P-gp, MDR cancer cells survive and highly proliferate by upregulating receptor tyrosine kinases (RTKs) and antiapoptotic proteins (Bcl-2 family proteins and inhibitor of apoptosis family proteins).8−12 In order to enrich and organize cell-signaling molecules, there are specific compartments called as lipid rafts in the plasma membrane. Lipid rafts are less fluid due to the presence of a rigid ring of cholesterol and long and saturated fatty acids of glycosphingolipids, when compared to unsaturated fatty acids-containing phospholipid membrane.13 Lipid rafts are invaginated to form omegatyped caveolae, which are involved in various cellular events such as endocytosis, lipid trafficking, tumorigenesis, and MDR.14−18 Caveolae are assembled by caveolin family proteins (caveolin-1, -2, and -3) and cavin family proteins (PTRF/cavin-1, SDPR/cavin-2, SRBC/cavin-3 and MURC/cavin-4).19 Global loss of caveolae exhibits dyslipidemia, type 2 diabetes and muscular dystrophy in caveolin-1- or PTRF-deficient mice.20−24 © 2012 American Chemical Society
There are accumulating data that caveolae are abundant in various MDR cancer cells including adriamycin-resistant breast cancer MCF-7/ADR, etoposide-resistant lung cancer A549-R2, and colchicine-resistant colon carcinoma HT29-col. These MDR cancer cells exhibit the enrichment of P-gp in lipid rafts or caveolae with higher level of caveolin-1, cholesterol and glycosphingolipids, when compared to their parental cancer cells.25,26 In addition, MDR activity is reduced when MDR cancer cells are exposed to methylβ-cyclodextrin (MβCD), which is a lipid raft disruptor.27−29 These data imply that MDR might be dependent on the fortification of lipid rafts or the abundance of caveolae. Since detergent-resistant lipid rafts concentrate various signaling molecules and have low protein complexity,13 comparative proteomic analysis of lipid rafts might provide novel insights on different cellular events such as cell signaling, cancer metastasis, tumorigenesis and MDR. For example, many novel signaling molecules including TRIM72, gC1qR, ezrin, and PTRF have been identified from comparative proteomic analysis by using two-dimensional electrophoresis (2DE), isotope-coded affinity tags (ICAT), and isobaric tagging for relative and absolute quantification (iTRAQ) with quantitative mass spectrometry.30−33 However, 2DE is not suitable for high throughput quantification because of labor-intensive and time-consuming procedure and ICAT and iTRAQ possess the risk of technical variation due to multiple and complicated steps for sample preparation.34 Received: July 16, 2012 Published: December 6, 2012 605
dx.doi.org/10.1021/pr300651m | J. Proteome Res. 2013, 12, 605−614
Journal of Proteome Research
Article
1 mM PMSF, and protease inhibitor cocktail), and subjected to discontinuous sucrose gradient ultracentrifugation (40%, 30%, 5%) using a SW41 Ti rotor (39,000 rpm) for 18 h at 4 °C. After centrifugation, the sucrose gradients were fractionated into 13 fractions including the pellet fraction. An opaque buoyant band corresponding to the lipid rafts was collected at the interface between the 30% and 5% sucrose gradients. The raft proteins were separated on 8% or 12% SDS-PAGE gels, and visulalized using a PlusOneTM Silver-staining kit (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer’s instruction. Cholesterol and protein concentrations were determined using a cholesterol test kit and the bicinchoninic acid method (Pierce, Rockford, IL), respectively, according to the manufacturer’s protocols.
Recently, label-free method has been developed for high throughput protein quantification by multiple LC−MS/MS analyses without using any labeling techniques. Since the label-free approach is nonbiased, reliable, versatile, and cost-effective,34 we applied this method on differential lipid raft proteomics. To attain novel insights into the underlying mechanisms of MDR, we examined the expression pattern of detergent-resistant lipid rafts proteins isolated from MCF-7 breast cancer cells and adriamycin-resistant MCF-7/ADR cells by label-free quantitative proteomics because both cells have been widely used to understand the mechanisms of MDR.35−39 Among 936 lipid rafts proteins, 62 proteins such as caveolin-1, PTRF, SDRP, CD44, EGFR, and P-gp were found to be upregulated in MCF-7/ADR cells. Herein, we show that PTRF was necessary for the regulation of membrane fluidity and MDR in MCF-7/ADR cells.
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Immunoblotting
Cells were lysed with RIPA buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM PMSF and protease cocktail and phosphatase inhibitors) at 4 °C. After microcentrifugation (14 000 rpm) for 10 min at 4 °C, the whole cell lysates (supernatants) were separated by SDS-PAGE and transferred to a polyvinyl difluoride membrane. The membranes were blocked with 5% (w/v) dry milk in TTBS buffer for 1 h at 37 °C. Subsequent incubations with primary and secondary antibodies were conducted for 1 h at room temperature. The signal was detected using enhanced chemiluminescence and recorded on an X-ray film.
MATERIALS AND METHODS
Cell Culture
Both, MCF-7 and MCF-7/ADR cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C and 5% CO2. To maintain the drug resistance phenotype of MCF-7/ADR, 10 ng/mL of adriamycin was added into the growth medium. Materials
Anti-Flotillin-1 and -actin antibodies were purchased from Santa Cruz, anti-Caveolin-1 and -2 and -Clathrin heavy chain antibodies from BD Transduction Laboratories, anti-P-gp antibody from Enzo Life Sciences and E-cadherin from Cellular Signaling. Anti-PTRF, -SDPR, and -CD44 antibodies were obtained from Abcam.
Protein Lysis and Digestion for Mass Spectrometric Analysis
A modified FASP (Filter-Aided Sample Preparation) method was used for protein digestion.53 Briefly, lipid rafts of MCF-7 and MCF-7/ADR cell were dissolved in SDT-lysis buffer (4% SDS and 0.1 M DTT in 0.1 M Tris-HCl, pH 7.6) and were subsequently reduced for 45 min at 37 °C and boiled for 10 min. After sonication, the samples were centrifuged for 5 min at 14 000g to remove the debris. The proteins in SDT buffer were mixed with 200 μL of 8 M urea (in 0.1 M Tris-HCl, pH 8.5) on a 10 k membrane filter (Micron device) and filters were centrifuged for 50−60 min at 14 000g at 20 °C to remove SDS. Typically, 100 μL of 50 mM iodoacetamide in 8 M urea was subsequently added to the concentrates for alkylation. The concentrate was washed with 200 μL of 8 M urea again and the urea was replaced with 100 μL of 50 mM NH4HCO3. The resultant protein concentrate within the filter was subjected to proteolytic digestion using trypsin (1:50 of enzyme to protein ratio in 50 mM NH4HCO3, Promega, WI, U.S.) with gentle shaking in the Thermomixer (Eppendorf) at 600 rpm for 1 min before subsequent overnight digestion without shaking at 37 °C. After the first digestion, trypsin (in 1:200 of enzyme to protein ratio) was again added for additional digestion. The resultant tryptic peptides after digestion were eluted from the filter by centrifugation for 20−30 min at 14 000g, and the filter was rinsed with 50 μL of 50 mM NH4HCO3 and the flow-through was mixed with the first eluent. The combined flow-through was vacuum-dried using a SpeedVac concentrator (Thermo) and the dried peptides were stored at −70 °C for LC−MS/MS analysis.
Two- −Photon Microscopy
Two-photon fluorescence microscopy images of SL2-labeled cells were obtained with spectral confocal and multiphoton microscopes (Leica TCS SP2) with a ×10−100 objective, numerical aperture (NA) = 1.30. The two-photon fluorescence microscopy images were obtained with a DM IRE2Microscope (Leica) by exciting the probes with a mode-locked titanium-sapphire laser source (Coherent Chameleon, 90 MHz, 200 fs) set at wavelength 800 nm and output power 1488 mW, which corresponded to approximately 10 mW average power in the focal plane. To obtain images at 410−530 nm (channel 1) range, internal PMTs were used to collect the signals in an 8 bit unsigned 512 × 512 pixels at 400 Hz scan speed. Fluorescence Recovery after Photobleaching (FRAP) Technique
Cells on a coverslip were stained with BODIPY-GM1 (5 μM) in DMEM for 30 min at 4 °C. After staining, live cell imaging was performed on a confocal laser scanning microscope (Zeiss CLSM 510 META) by using the 488-nm line of an Ar laser with a ×40 objective. Images were taken both before and immediately after bleaching. Photobleaching was done with 100% intensity of the 488-nm laser for 200 iterations. Fluorescence recovery was monitored at low laser intensity at 2 s intervals for 180 s. FRAP experiments were performed on at least 7 independent cells, and data were averaged to generate a single FRAP curve.
LC−MS/MS experiments
Each of lipid rafts tryptic peptides from MCF-7 and MCF-7/ ADR cells were subjected to triplicate LC−MS/MS experiments, resulting in a total of six LC−MS/MS data sets. The lipid raft tryptic peptides were separated using a modified nanoACQUITY UPLC (Waters) system that has been described before.54 The analytical column (75 μm i.d. × 360 μm o.d., 70 cm) was
Isolation of Detergent Resistant Lipid Rafts
Lipid rafts were isolated as previously described.52 Briefly, MCF-7 and MCF-7/ADR cells, grown to 70−80% confluence in five 150-mm dishes, were lysed with 1 mL of lysis buffer (1% Triton X-100, 25 mM HEPES, pH 6.5, 150 mM NaCl, 1 mM EDTA, 606
dx.doi.org/10.1021/pr300651m | J. Proteome Res. 2013, 12, 605−614
Journal of Proteome Research
Article
Figure 1. MCF-7/ADR cells possess more lipid rafts than MCF-7 cells. (A and B) MCF-7 and MCF-7/ADR cells were stained with SL-2 (10 μM) for 10 min at room temperature. After staining, SL2 fluorescence image was observed under a two-photon microscope (bar = 10 μm) (A). The SL2 fluorescence intensities were quantified from three different fields. p < 0.05. (B). (c and d) FRAP analysis of BODIPY-GM1-stained MCF-7 and MCF-7/ ADR cells. The cells were stained with BODIPY-GM1 (5 μM) for 30 min at 4 °C and then imaged both before and during recovery after photobleaching in an area indicated by a circle. Images were taken at the indicated times after the bleach pulse (bar = 10 μm) (C). Quantitative analysis of FRAP. Curves represent mean values (±SEM) from measurements of 7 different fields (D).
Database Search for Peptide Identification
manufactured in-house by acetonitrile slurry packing of a fusedsilica capillary (Polymicro Technologies) with C18 material of 3 μm diameter, 300 Å pore size (Jupiter, Phenomenex). The solid phase extraction (SPE) column was prepared by packing the same C18 materials into the 1-cm long liner (250 μm i.d.) of an internal reducer (1/16 in. to 1/32 in, VICI) The temperature of analytical column was set to 50 °C using a semirigid gasline heater (1/4” i.d, 60-cm long, WATLOW). The RPLC was performed at a flow rate of 400 nL/min using the following gradient: 98% solvent A (0.1% formic acid in H2O) in 5 min, 5% to 50% of solvent B (0.1% formic acid in 99.9% acetonitrile) in 115 min, 50% to 80% of solvent B in 10 min, and 80% of solvent B in 10 min. A 7-T Fourier transform ion cyclotron resonance mass spectrometer (FTICR, LTQ-FT, Thermo) was used for acquisition of tandem mass spectrometric data. The eluted peptides from the LC were ionized by a home-built nanoelectrospray interface at an electric potential of 2.0 kV. The spray emitter was manufactured by chemical etching of fused-silica capillary emitter (20 μm i.d. × 150 μm o.d.).55 The temperature of the desolvation capillary was set to 200 °C. MS precursor ion scans (m/z 400−2000) were acquired in full-profile mode with an AGC target value of 1 × 106, a mass resolution of 1 × 105, and a maximum ion accumulation time of 500 ms. The mass spectrometer was operated in datadependent MS/MS mode. The seven most abundant ions detected in a precursor MS scan were dynamically selected for MS/MS experiments simultaneously incorporating a dynamic exclusion option (exclusion mass width low: 1.10 Th; exclusion mass width high: 2.10 Th; exclusion list size: 120; exclusion duration: 30 s) to prevent reacquisition of MS/MS spectra of the same peptides. Collision-induced dissociations of the precursor ions were performed in an ion trap (LTQ) with the collisional energy and isolation width set to 35% and 3 Th, respectively.
LC−MS/MS data was first processed by iPE-MMR analysis, which was previously demonstrated to assign accurate precursor masses to the tandem mass spectrometric data before the subsequent protein database search.56 The resultant MS/MS data were searched against a composite databaseIPI (International Protein Index) ver. 3.63 database (84 140 protein entries) and 180 common contaminants as well as their reversed complementsusing SORCERER-SEQUEST (Version 3.5) search algorithm (Sage-N Research, Milpitas, CA, U.S.). The search was performed allowing semitryptic peptides and the maximum number of missed cleavage sites was set to 3. The mass tolerance was 10 ppm for precursor ions and 1 Da for fragment ions. The carbamidomethylation of cysteine (57.02 146 0 Da) was set to static modification. Oxidation of methionine (15.99 492 0 Da) and the carbamylation of N-terminal site (43.00 581 0 Da) were used as variable modification options. The FP rate of peptide assignment was estimated through a composite target-decoy database search. The values of Xcorr and the ΔCn threshold for the 1% FP rate were used to obtain list of peptide IDs.57 Estimating Peptide Abundance
Label-free quantitative analysis was performed to MCF-7 and MCF-7/ADR LC−/MS/MS data (6 LC−MS/MS data) based on MS intensities of of peptide ions. In LC−MS/MS experiments, a peptide MS peak emerged over a period of LC elution time. With all MS data deisotoped by THRASH algorithm,58 a peptide’s MS peaks that have similar monoisotopic masses (
