Quantitative Proteomic Analysis Reveals the Perturbation of Multiple Cellular Pathways in Jurkat-T Cells Induced by Doxorubicin Xiaoli Dong,† Lei Xiong,† Xinning Jiang,‡ and Yinsheng Wang*,† Department of Chemistry, University of California, Riverside, California 92521-0403, and Department of Pathology, School of Medicine, University of California, San Diego, California 92093 Received July 8, 2010
Doxorubicin remains an important part of chemotherapy regimens in the clinic and is considered an effective agent in the treatment of acute lymphoblastic leukemia (ALL). Although the cellular responses induced by doxorubicin treatment have been investigated for years, the precise mechanisms underlying its cytotoxicity and therapeutic activity remain unclear. Here we utilized mass spectrometry, together with stable isotope labeling by amino acids in cell culture (SILAC), to analyze comparatively the protein expression in Jurkat-T cells before and after treatment with a clinically relevant concentration of doxorubicin. We were able to quantify 1066 proteins in Jurkat-T cells with both forward and reverse SILAC measurements, among which 62 were with significantly altered levels of expression induced by doxorubicin treatment. These included the up-regulation of core histones, heterogeneous nuclear ribonucleoproteins, and superoxide dismutase 2 as well as the down-regulation of hydroxymethylglutaryl-CoA synthase and farnesyl diphosphate synthase. The latter two are essential enzymes for cholesterol biosynthesis. We further demonstrated that the doxorubicin-induced growth inhibition of Jurkat-T cells could be rescued by treatment with cholesterol, supporting that doxorubicin exerts its cytotoxic effect, in part, by suppressing the expression of hydroxymethylglutaryl-CoA synthase and farnesyl diphosphate synthase, thereby inhibiting the endogenous production of cholesterol. The results from the present study provide important new knowledge for gaining insights into the molecular mechanisms of action of doxorubicin. Keywords: Doxorubicin • mass spectrometry • quantitative proteomics • SILAC • cholesterol biosynthesis • acute lymphoblastic leukemia
Introduction The antitumor drug doxorubicin (Dox) has been widely used in the clinic for the treatment of a broad spectrum of solid tumors and hematological malignancies since the early 1960s.1 However, the exact mechanism of action of Dox is still somewhat unclear. Doxorubicin is known to intercalate into DNA,2,3 which prohibits the progression of topoisomerase II, an enzyme unwinding DNA for replication and transcription.4 Dox stabilizes the topoisomerase II complex after it has broken the DNA chain for replication, preventing the DNA double helix from being resealed and thereby terminating DNA replication and transcription. Dox can also induce the generation of free oxygen radicals and lipid peroxidation. A major adverse side effect associated with the use of Dox in the clinic is the onset of cardiomyopathy and heart failure.5 Several reports suggest that Dox-induced apoptosis plays an important role in its cardiotoxicity that is linked with the formation of reactive oxygen species (ROS) derived from the redox activation of Dox.6-8 Recent studies have focused on Dox-induced apoptotic signaling mechanisms.9 * To whom correspondence should be addressed: Phone: (951) 827-2700. Fax: (951) 827-4713. E-mail:
[email protected]. † Department of Chemistry. ‡ Department of Pathology. 10.1021/pr1007043
2010 American Chemical Society
Mass spectrometry (MS)-based proteomics allows for the identification and quantification of a large number of proteins in complex samples. Two-dimensional gel electrophoresis (2DE) is a traditional technique for studying the effects of drug treatments on protein expression. In 2-DE, quantification is achieved by recording differences in the stained spot intensities of proteins derived from two states of cell populations or tissues.10 A number of proteomic studies underlying the effect of treatments with different anticancer drugs, such as cisplatin,11,12 etoposide,13 and all-trans retinoic acid,14 have been performed by using MS for protein identification and 2-DE for protein quantification. The combination of 2-DE with mass spectrometry has also been used previously for assessing the doxorubicin-induced alterations in protein expression in human hepatoma cells,15 MCF-7 breast tumor cells,16 and mouse heart.17 Other than 2-DE, several stable isotope labeling-based quantification strategies, such as isotope-coded affinity tag (ICAT),18 isobaric tags for relative and absolute quantitation (iTRAQ),19 and stable isotope labeling by amino acids in cell culture (SILAC),20 have been developed for MS-based analysis of differential protein expression. Among these isotope-labeling strategies, SILAC is a metabolic labeling method, which is simple, efficient, and can facilitate almost complete heavy Journal of Proteome Research 2010, 9, 5943–5951 5943 Published on Web 09/07/2010
research articles isotope incorporation. It is very suitable for the comparative study of protein expression in cells with and without drug treatment.15,21 With the use of SILAC, accurate results could be obtained with minimal bias, facilitating relative quantification of subtle changes in protein abundance.20 In this study, we employed liquid chromatography-tandem mass spectrometry (LC-MS/MS), in conjunction with SILAC, to examine quantitatively the perturbation of protein expression in cultured Jurkat-T human acute lymphoblastic leukemia (ALL) cells upon Dox treatment. A total of 1066 proteins were quantified in both forward and reverse SILAC measurements, among which 62 were significantly altered after Dox treatment. Importantly, we observed, for the first time, the perturbation of cholesterol biosynthesis induced by Dox treatment.
Materials and Methods Materials. Heavy lysine and arginine ([13C6,15N2]-L-lysine and [ C6,15N4]-L-arginine) were purchased from Cambridge Isotope Laboratories (Andover, MA). All reagents unless otherwise stated were from Sigma (St. Louis, MO). Cell Culture. Jurkat-T cells, obtained from ATCC (Manassas, VA), were cultured in Iscove’s modified minimal essential medium (IMEM) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) and penicillin (100 IU/mL). Cells were maintained in a humidified atmosphere with 5% CO2 at 37 °C, with medium renewal at every 2 or 3 days depending on cell density. For SILAC experiments, the IMEM medium without L-lysine or L-arginine was custom-prepared according to the ATCC formulation. The complete light and heavy IMEM media were prepared by the addition of light or heavy lysine and arginine, along with dialyzed FBS, to the above lysine, arginine-depleted medium. The Jurkat-T cells were cultured in heavy IMEM medium for at least five cell doublings to achieve complete isotope incorporation as described by Mann and co-workers.22,23 They demonstrated that culturing cells in heavy medium for five population doublings could result in full heavy isotope incorporation. Doxorubicin Treatment and Cell Lysate Preparation. In forward SILAC experiment, Jurkat-T cells, at a density of approximately 7.5 × 105 cells/mL, cultured in light medium were treated with 1 µM doxorubicin (Sigma) for 24 h, whereas the cells cultured in heavy medium were untreated. Reverse SILAC experiments were also performed in which the cells cultured in the heavy and light medium were treated with Dox and mock-treated, respectively (Figure 1). After 24 h, the light and heavy isotope-labeled cells were collected by centrifugation at 300g and washed three times with ice-cold PBS. The cell pellets were then resuspended in the CelLytic M cell lysis reagent for 30 min with occasional vortexing. Cell lysates were centrifuged at 12000g at 4 °C for 30 min, and the resulting supernatants were collected. To the supernatant was subsequently added a protease inhibitor cocktail, and the protein concentrations of the cell lysates were determined by using Quick Start Bradford Protein Assay kit (Bio-Rad, Hercules, CA). SDS-PAGE Separation and In-Gel Digestion. The light and heavy cell lysates were combined at a 1:1 ratio (w/w), denatured by boiling in Laemmli loading buffer for 5 min and separated by 12% SDS-PAGE with a 4% stacking gel. The gel was stained with Coomassie blue; after destaining, the gel was cut into 20 bands, in-gel reduced with dithiothreitol and alkylated with iodoacetamide. The proteins were digested in-gel with trypsin (Promega, Madison, WI) overnight, after which peptides were 13
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Dong et al. extracted from gels with 5% acetic acid in H2O and then with 5% acetic acid in CH3CN/H2O (1:1, v/v). The resulting peptide mixtures were dried and stored at -20 °C for further analysis. Exogenous Cholesterol Addition and Cell Viability Assay. The cholesterol-BSA complex was prepared following a previously published method.24 A 10-mL aliquot of 1% cholesterol in ethanol was added to the same volume of doubly distilled water under continuous magnetic stirring at room temperature. The milk-like solution was then centrifuged at 2000g for 10 min. The supernatant was discarded, and the pellet was resuspended in a 10-mL solution containing 0.25 M sucrose and 1 mM EDTA (pH 7.3). The resulting white emulsion was stirred gently, to which was slowly added 4 g of BSA at room temperature. Once the BSA was completely dissolved, the pH of the solution was adjusted to 7.3 with Tris, and the solution was then centrifuged at 4 °C at 12000g for 10 min. The supernatant was collected and used for cholesterol incorporation. Jurkat-T cells were resuspended in IMEM medium and seeded in six-well plates at a density of ∼4 × 105 cells/mL. To the cells was added Dox solution until its concentration reached 1 µM. The cholesterol-BSA complex solution was added subsequently to the wells containing the control, or doxorubicin-treated cells until cholesterol concentrations reached 30 or 60 mg/L. After 12 or 24 h of treatment, cells were stained with trypan blue, and counted on a hemocytometer to measure cell viability. Extraction and Determination of the Cellular Level of Cholesterol. Cells that had been washed three times with 0.85% NaCl solution in 10 mM sodium acetate were extracted with chloroform/methanol/water (2:1.1:0.9, v/v/v) as specified by Madden.25 The chloroform (bottom) layer was washed three times with methanol and water (5:4, v/v). The bottom layer was then collected, placed in a test tube, and evaporated to dryness with dry N2 gas. The cholesterol level was determined by HPLC using methanol as mobile phase, and a UV detector, with the wavelength of detection being set at 205 nm, was employed for monitoring the effluent.26 LC-MS/MS for Protein Identification and Quantification. Online LC-MS/MS analysis was performed on an Agilent 6510 Q-TOF system coupled with an Agilent HPLC-Chip Cube MS interface (Agilent Technologies, Santa Clara, CA). The sample injection, enrichment, desalting, and HPLC separation were carried out automatically on the Agilent HPLC Chip with an integrated trapping column (160 nL) and a separation column (Zorbax 300SB-C18, 75 µm × 150 mm, 5 µm in particle size). The peptide mixture was first loaded onto the trapping column with a solvent mixture of 0.1% formic acid in CH3CN/H2O (2: 98, v/v) at a flow rate of 4 µL/min, which was delivered by an Agilent 1200 capillary pump. The peptides were then separated with a 90-min linear gradient of 2-60% acetonitrile in 0.1% formic acid and at a flow rate of 300 nL/min, which was delivered by an Agilent 1200 Nano pump. The Chip spray voltage (VCap) was set as 1950 V and varied depending on the chip conditions. The temperature and flow rate of the drying gas were 325 °C and 4 L/min, respectively. Nitrogen was used as the collision gas; the collision energy followed an equation with a slope of 3 V/100 Da and an offset of 2.5 V. MS/MS experiments were carried out in the datadependent scan mode with a maximum of five MS/MS scans following each MS scan. The m/z ranges for MS and MS/MS were 300-2000 and 60-2000, and the acquisition rates were 6 and 3 spectra/s, respectively.
Doxorubicin-Induced Global Proteome Alteration
research articles
Figure 1. Flowcharts of forward and reverse SILAC combined with LC-MS/MS for the comparative analysis of protein expression in Jurkat-T cells upon Dox treatment (A). Shown in (B) is the distribution of expression ratios (treated/untreated) for the quantified proteins.
Data Processing. Agilent MassHunter workstation software (Version B.01.03) was used to extract the MS and MS/MS data. The data were converted to mzXML files and DTA files using Trapper and Mzxml2Search programs, respectively. They were available at http://sourceforge.net/projects/sashimi/files. Bioworks 3.2 was used for protein identification by searching the m/z Data files against the human IPI protein database (version 3.21) and its reversed complement. The maximum number of misscleavages for trypsin was set as two per peptide. Cysteine carbamidomethylation was set as a fixed modification. Methionine oxidation as well as lysine (+8 Da) and arginine (+10 Da) mass shifts introduced by heavy isotope labeling were considered as variable modifications. The mass tolerances for MS and MS/MS were 100 ppm and 0.6 Da, respectively. The searching results were
then filtered with DTASelect software, developed by Yates and co-workers,27 to achieve a protein false discovery rate
