Alterations in the Mitochondrial Proteome of Adriamycin Resistant MCF-7 Breast Cancer Cells Rachael Strong,† Takeo Nakanishi,‡ Douglas Ross,‡ and Catherine Fenselau*,†,‡ Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland, and The Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland Received May 3, 2006
A 2D-gel based comparative proteomic analysis has been conducted of changes in mitochondrial protein abundances in MCF-7 human breast cancer cells selected for resistance to Adriamycin accompanied by verapamil. We identified and compared 156 unique proteins from 184 spots. Eleven mitochondrial proteins were found with abundances altered more than 2-fold. Transcription was evaluated for two of these, using quantitative RT-PCR. Implications of the changes are considered with respect to drug resistance. Keywords: proteomics • comparative proteomics • mitochondria • drug-resistance • breast cancer • mass spectrometry • 2D-gel electrophoresis • qRT-PCR • heme synthesis • ATP synthase • coproporphyrinogen III oxidase • apoptosis
Introduction Chemotherapy is commonly used as part of the treatment against breast cancer and can be advantageous for some time. Chemotherapy is eventually impaired, however, by the development of drug resistance.1 This phenotype emerges when mutations render cancerous cells resistant to the original drug(s) used for treatment. Often resistance is also acquired to a wider array of drugs that are not necessarily similar in chemical structure, mechanism, or cellular target.2 This phenomenon, known as multidrug resistance, represents a most challenging problem in the successful treatment of cancer and is the main reason for the failure of chemotherapy. To date, several mechanisms have been characterized, which contribute to drug resistance. Some have been studied extensively, such as the enhanced efflux of chemotherapeutic drugs out of cancer cells through the ATP-dependent pump Pglycoprotein (PGP/ABCB1). However, not all resistance can be explained by the known mechanisms, and it is generally acknowledged that additional forms of resistance have yet to be determined.2-5 Because resistance is multifactorial and is conferred by alterations in many proteins, it is advantageous to use a proteomic analysis.6,7 In the present study, soluble mitochondrial proteins have been identified, which show changes in abundances in the MCF-7 cell line selected for resistance to Adriamycin in the presence of verapamil (MCF7/ADRVp), as compared to the drug-susceptible parent MCF-7 cell line. Adriamycin is an anthracycline chemotherapeutic agent used to treat solid tumors, including breast cancer. The cytotoxicity of Adriamycin is considered to be two-pronged. The drug is * To whom correspondence should be addressed. E-mail: fenselau@ umd.edu. Telephone: 301-405-8616. Fax: 301-405-8615. † University of Maryland. ‡ University of Maryland School of Medicine. 10.1021/pr060207c CCC: $33.50
2006 American Chemical Society
reduced to its semiquinone form by complex I of the electron transport chain in mitochondria.8 The semiquinone reacts with oxygen, iron, and hydrogen peroxide to produce reactive oxygen species.9,10 Production of these free radicals generates oxidative damage such that apoptosis, or programmed cell death, is initiated.11 In its second mechanism of action, Adriamycin intercalates along the deoxyribonucleic acid (DNA) helix where it interacts with topoisomerase II to cause double strand breaks in the nucleic acid and prevent DNA synthesis.8,12 Verapamil, the second chemical used in the selection of the resistant cell line studied here, is a hydrophobic calcium channel blocker and a competitive inhibitor of PGP. Its presence allowed the selection of Adriamycin-resistant cells in which PGP is not up-regulated.13-15 It has been shown that another ATP-requiring trans-membrane pump, designated as the breast cancer resistance protein (BCRP/ABCG2), is overexpressed in this MCF-7/ADRVp cell line and mediates drug efflux and resistance.16,17 Historically, DNA has been the cellular target for chemotherapeutic drugs. Current understanding, however, is that most chemotherapeutic agents kill cells via apoptosis.18,19 Mitochondria play a central role in mediating apoptosis; many proteins associated with the organelle can promote or inhibit the pathway.20 In addition, mutations within the mitochondrial genome are reported in most cancers, which are believed to promote chemoresistance.21,22 Changes in mitochondrial proteins have been observed in several types of cancers resistant to Adriamycin.23,24 Because of prior evidence that mitochondria are intimately involved in drug resistance, we proposed to study alterations in the abundances of mitochondrial proteins in the drug resistant cell line described above. Two-dimensional gel electrophoresis (2D-GE) and mass spectrometry combined with bioinformatics have been used to characterize a part of the Journal of Proteome Research 2006, 5, 2389-2395
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research articles soluble MCF-7 mitochondrial proteome and to compare protein abundances in the MCF-7 and MCF-7/ADRVp cell lines. Based on the observations of the study, alterations in several mitochondrial pathways are proposed to function to support resistance mechanisms centered elsewhere in the cell, while alterations in other pathways may contribute independently to drug resistance.
Materials and Methods Materials. The two-dimensional quantitation kit, twodimensional clean up kit, Percoll, and immobilized pH gradient (IPG) buffer were obtained from Amersham Biosciences, which is now part of GE Healthcare (Piscataway, NJ). The protein isoelectric focusing cell, immobilized pH gradient (IPG) strips (17 cm, pH 3-10), IPG buffer, Protean II ready gels (8-16% Tris-HCl precast gel 193 × 183 × 1.0 mm IPG comb), and BioSafe Coomassie blue stain were obtained from Bio-Rad (Hercules, CA). ZipTip C 18 columns were obtained from Millipore (Billerica, MA). Sequence grade, modified porcine trypsin was obtained from Promega Corporation (Madison, WI). All additional materials were purchased from Sigma-Aldrich (St. Louis, MO). MCF-7 Cell Culture and Harvest. Dr. K. H. Cowan (The Eppley Institute, University of Nebraska Medical Center, Omaha, NE) generously provided the drug susceptible MCF-7 cell line. The cells were cultured in 150 cm2 flasks in Improved Minimal Essential Media (MEM) supplemented with 10% fetal bovine serum and 1% penicillin streptomycin antibiotic solution. The MCF-7/ADRVp cell line had been earlier selected for resistance to Adriamycin in the presence of verapamil, and was maintained as described previously.16,17 MCF-7/MX cells were kindly obtained from Dr. Erasmus Schneider (Wadsworth Center, New York State Department of Health, Albany, New York) and were maintained as described previously.25 The cells were sustained in a 37 °C incubator with 5% carbon dioxide. The cells were harvested at confluence. Cells were washed with 25 mL of 10 mM phosphate buffered saline solution (PBS). Then, 5 mL of cell culture grade trypsin was added, and the flasks were returned to the incubator for 5 min. Next, 15 mL of MEM was added, and the cells were washed from the flask and pelleted in a centrifuge at 500g for 5 min. The cell pellet was washed twice with PBS. Preparation of Soluble Mitochondrial Proteins. All work was done on ice unless specified otherwise. The cell pellet was suspended in mitochondria isolation buffer [MIB:220 mM mannitol, 70 mM sucrose, 5 mM 4-morpholinepropanesulfonic acid (MOPS), pH 7.4 plus 2 mM ethylene glycol-bis (βaminoethyl ether)-tetraacetic acid (EGTA)] at a ratio of 1 g of cell pellet to 10 mL of MIB.26 The suspension was homogenized and centrifuged at 400g for 10 min to pellet the unbroken cells and nuclei. The supernatant was transferred to a centrifuge tube and kept on ice. The cell pellet was resuspended in MIB at a ratio of 0.5 g of cell pellet to 5 mL of MIB and the procedure was repeated. The supernatants were combined and spun at 7000g for 10 min to pellet the crude mitochondrial fraction. The crude mitochondria were weighed and suspended in mitochondria purification buffer (MPB; 220 mM mannitol, 70 mM sucrose, 5 mM MOPS, pH 7.4) at a ratio of 800 µL of MPB to 0.8 g of crude mitochondria.27 A total of 1 mL of the suspension was layered on top of 14 mL of MPB and 6 mL of Percoll. The sample was centrifuged in a fixed angle rotor (Ti 70, Beckman Coulter, Palo Alto, CA) at 38 900 rotations per minute (rpm). Upon removal from the centrifuge, three 2390
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layers could be distinguished, the bottom two of which contained mitochondria. The bottom, dark yellow layers were collected, diluted 10-fold with MPB buffer and centrifuged at 7000g for 10 min to pellet the mitochondria. The enriched mitochondria were washed twice with MPB buffer. Sample enrichment was monitored based on protein identification. In a 2D gel array of proteins from the sample isolated in the first centrifugation, 52% of the first 93 spots identified, were known to be associated with a cellular compartment other than mitochondria. In the enriched mitochondrial fraction, further processed through the Percoll gradient, 19% of the first 93 spots identified were associated with an organelle other than mitochondria. The mitochondrial pellet was suspended in protein extraction buffer (PEB; 7 M Urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (Chaps), 65 mM dithiothreitol (DTT), and 2% IPG buffer) at a ratio of 0.1 g of mitochondria to 600 µL of PEB.28 To rupture the mitochondria, the suspension was subjected to six 5-s bursts of probe sonication with 1-min rests in between. The sample was centrifuged in a swinging bucket rotor (SW 60, Beckman Coulter) at 28 000 rpm for 1 h to separate the soluble and insoluble proteins. The supernatant or soluble protein fraction was collected and portioned into 1 mL aliquots. Protein concentration was determined using the two-dimensional quantitation kit, and the absorbance was read at 480 nm on a DU 530 life science UV/vis spectrometer (Beckman Coulter). The soluble extract was then flash frozen with liquid nitrogen and stored at -80 °C. Two-Dimensional Gel Electrophoresis. The soluble mitochondrial proteins were separated by 2D-GE. Prior to gel electrophoresis, the mitochondrial proteins were precipitated using the two-dimensional cleanup kit. Following precipitation, the mitochondrial protein pellet was solubilized in 320 µL of rehydration buffer (7 M urea, 2 M thiourea, 2% Chaps, 50 mM DTT, and 1% IPG buffer) and incubated on the benchtop for 1 h at room temperature. Approximately 350 µg of protein was loaded onto the isoelectric focusing cell for first dimension separation by isoelectric point. An electrode wick saturated with 6 µL of distilled water was placed over the anode, while a second wick saturated with 6 µL of 15 mM DTT was placed at the cathode to facilitate focusing of basic proteins.29 A 17 cm IPG strip, pH 3-10 was placed gel side down and covered with 1.5 mL of mineral oil to prevent burning. The strip was rehydrated for 12 h, after which proteins were focused for 60 000 V‚h. Following isoelectric focusing, the IPG strip was placed in 3.5 mL of equilibration buffer (0.375 M tris-HCl, pH 8.8, 6 M urea, 20% glycerol, and 2% sodium dodecyl sulfate (SDS)) containing 2% DTT followed by 2.5% iodoacetamide to reduce and alkylate the proteins, respectively. The strip was then placed on top of an 8-16% Tris-HCl precast gel and covered with agarose solution. The gel was developed at 16 mA for 30 min followed by 24 mA for approximately 5 h. Once the second dimension was developed, the gel was removed from between two glass plates and rocked gently for 1 h in 500 mL of 45% methanol, 5% acetic acid, and 50% distilled water to fix the proteins. After fixation, the gel was washed in water for 15 min and then stained overnight in Bio-safe colloidal Coomassie blue. After 24 h, the gels were destained with gentle rocking in several washes of water. Two-dimensional gel images were visualized using a GS-800 calibrated densitometer (Bio-Rad) and its associated software.
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Alterations in MCF-7 Breast Cancer Cells
Relative Quantitation of Proteins. Densitometric comparative image analysis was performed using Compugen Z3 software (Compugen Limited, Tel Aviv, Israel), following the manufacturer’s instructions. Briefly, two gel images of choice were registered, layered on top of one another, and the spots matched. The difference in abundance between the same spot on two gels was calculated based on the quotient of their relative expression on each gel and was displayed by the computer program. To ensure that significant changes in abundances were not the results of biological or manual variation, each cell line was harvested four separate times, and two gel arrays were developed from each of the harvests for pairwise quantitative comparisons. Mass Spectrometry and Protein Identification. Protein spots were excised from the 2D gels using a plastic pipet. Peptides were analyzed to identify protein spots of interest. An in-gel tryptic digestion was performed on the spots.30,31 Prior to mass spectrometric analysis, peptides were desalted using ZipTip C18 columns, per manufacturers’ instructions. Once desalted, the peptide solution was dried down in a vacuum centrifuge for 10 min and dissolved in 5 µL of electrospray solution (49% water, 49% methanol, and 2% acetic acid). Mass spectrometric analysis was performed on an API QSTAR Pulsar Qq-TOF electrospray ionization instrument (Applied BioSystems, Foster City, CA). To introduce the sample, 1.5 µL of electrospray buffer solution containing the peptide mixture was loaded into a capillary tip (Protana Incorporated, Odense, Denmark) and placed in the nanospray source. A precursor survey (MS) scan was recorded to determine what peptide molecular ions were present in the sample mixture. To do so, the spray voltage was set to 900 V and the MS scan range was set between 350 and 1000 m/z. Doubly charged peptides were manually chosen for fragmentation in the product ion (MS/MS) scan. Nitrogen gas was used for collision induced dissociation (CID) and the collision energy setting ranged from 20 to 45. The MS/MS scan range was set between 50 and 1500 m/z and CID spectra were recorded for one minute. Sequence tags from two to five peptides were used for protein identification. The sequence tags, masses of the peptides and masses on each side of the tag were submitted through the Mascot search engine, available in-house, using the BioExplore software program associated with the mass spectrometer. The protein databases used were SwissProt and NCBI (National Center for Biotechnology Information). Mascot returned a list of candidate proteins from which the sequence tags could have originated. The proteins were ranked and their identity determined to a 95% confidence level. The pI and molecular weight of each candidate protein was checked against the location of that spot on the 2-D gel. Relative Quantification of Coproporphyrinogen III Oxidase (CPO III) and R ATP-Synthase mRNA by Real-Time qRT-PCR. Total RNA was isolated from MCF-7, MCF-7/ADRVp, and MCF7/MX cells using Trizol Reagent (Invitrogen, Carlsbad, CA) and treated with RNase free DNase I (Roche, Indianapolis, IN). The RNase free DNase I-treated total RNA was subjected to qRTPCR by LightCycler (Roche) using SYBR Green Quantitative RTPCR kit (Sigma-Aldrich) to detect mRNA expression of human CPOIII and R ATP-synthase. In general, 250 ng of total RNA was applied for each reaction, and the reaction mixture was prepared according to the manufacturer’s instructions with 0.5 µM of primers specific to either CPOIII, R ATP-synthase, or β-actin cDNA. The sequences of the primers for CPOIII and R
ATP-synthase were as follows: CPOIII (GenBank acs. no. BC023554), either sense: 5′-catcgtgg agaacggc-3′(corresponding to 1033-1328) and antisense: catcgggcagttagagg (1318-1324), or sense:5′-caggt gatagtgcgg-3′ (36-50) and antisense: 5′ccgaggtcttaggcaac-3′ (358-373); R ATP-synthase (ATP5A1, NM_004046), sense: 5′-ttgtggtgtcggctacgg-3′ (908-925) and antisense: 5′-cggcggagcaacagaga-3′ (1063-1079). The sequences of the primers for β-actin were described previously.32 Reverse transcription was done at 48 °C for 25 min, with a denaturation step at 95 °C for 30 s followed by 45 cycles with 95 °C denaturation for 1 s, 58 °C annealing for 15 s, and 72 °C extension for 15 s. Negative controls were run concomitantly to ensure that the samples were not cross-contaminated. Crossing points (Cp) score for these genes were normalized against Cp scores for endogenous β-actin expression. Foldchange relative to MCF-7 cells was determined according 2-∆∆Cp method.33
Results Protein Identification. Figure 1 shows an annotated 2D gel map of the soluble MCF-7 mitochondrial fraction. In this study, proteins in 184 spots have been identified. Typically, two to three sequence tags were used for identification of each protein. For some proteins, however, a positive identification was obtained with only one sequence tag. In each of these cases, a theoretical digest of the protein was obtained, and the experimental mass spectrum was manually interpreted against the appropriate theoretical spectrum. All of the proteins identified in this analysis have Mascot scores that exceed the 95% confidence level set by the software. Also, the pI and molecular weight of each protein were verified, based on the 2D-gel image. A list of the proteins and their ratios is presented as Supporting Information, Table 1. In Figure 1, it can be seen that related isoforms are assigned the same spot number in most cases, and constitute a single entry in the supplementary table. Proteins with Altered Abundances. Comparative densitometry was used to evaluate the protein abundance profiles of the 2D gel arrays obtained from the drug-susceptible MCF-7 cell line relative to the drug-resistant cell line. Each cell line was harvested four separate times and two gels were run from each of the harvests. Thus, eight gels from each cell line were used for comparative analyses. Only changes greater than or equal to 2 are considered significant. Figure 2 shows a pair of gels typical of those compared. Protein spots designated by red arrows were found to be increased in abundance in the resistant cell line, whereas proteins with yellow arrows show decreased abundance. Thirteen proteins isolated in the soluble mitochondrial fraction of the resistant cell line show significantly altered abundances. Eleven are considered to be mitochondrial proteins. Table 1 lists the 13 proteins and their relative abundances, as compared to abundances in the drug susceptible MCF-7 cells. Quantitative RT-PCR Analysis of CPO III and r ATPSynthase. Steady-state levels of these mRNA transcripts are shown in Figure 3. Expression levels of CPO III mRNA are increased (p2 are considered significant based on four cell harvests, two gel pairs each.
superoxide dismutase,41 and heme has been shown to activate transcription of genes that encode both superoxide dismutase and catalase.42 In untreated breast cancer, the capacity for heme biosynthesis is enhanced 20-fold.43 However, rodents treated with Adriamycin show a net decrease in cellular heme content.44,45 Evidence in the present study for an increase in CPO III in Adriamycin-resistant cells suggests a novel contribution to chemoresistance. It is proposed that in parental MCF-7 cancer cells an increase in heme production activates production of the anti-oxidants superoxide dismutase and catalase; when these drug-sensitive MCF-7 cancer cells are treated with Adriamycin, a decrease in heme occurs, concurrent with increased oxidative stress and cell death. In contrast, in the MCF-7/ADRVp cancer cells that have become resistant to Adriamycin, expression of coproporphyrinogen III oxidase is increased, production of heme is enhanced, and the production of superoxide dismutase and catalase extends cell survival.
Fatty Acid Oxidation. Fatty acid oxidation drives the synthesis of ATP and is essential for maximum cellular energy yield. The increased abundance is detected here of two key enzymes involved in fatty acid oxidation, 3,2 trans-enoyl CoA isomerase (spot 23) and the trifunctional enzyme R-subunit (spot 70). Based on their increase, an increase in fatty acid oxidation is proposed in the cell line resistant to Adriamycin, as compared to the drug-susceptible cell line. It should be noted that additional enzymes involved in fatty acid oxidation have been identified, which do not show altered abundances (See supplementary Table 1). Tumor cells have long been known to have altered metabolism, including an increase in glycolysis for the production of ATP, often accompanied by a decrease in fatty acid oxidation.46 Consistent with the present report, a recent study has demonstrated that drug-susceptible cancer cells have increased rates of glycolysis, whereas drug-resistant cancer cells favor fatty acid oxidation to support ATP synthesis.47 It was suggested that this alteration is characteristic of many drug resistant cells. ATP and Oxidative Phosphorylation. In the MCF-7/ADRVp resistant cell line, the up-regulated transmembrane pump BCRP requires ATP to support the major mechanism of drug resistance. The present study finds an increase in abundance of adenylate kinase 2 (spot #87) (Table 1), which catalyzes the phosphorylation of AMP to ADP and thereby helps to regulate the cellular pools of ADP and ATP. Mitochondria are responsible for meeting 80-90% of cellular energy needs through the process of oxidative phosphorylation of ADP. Changes are observed in abundances of two proteins involved in oxidative phosphorylation. Abundance of cytochrome c oxidase, subunit Vb is decreased (spot 7) (Table 1), one of 13 proteins in the cytochrome c oxidase complex in the electron transport chain. An earlier study using Adriamycin resistant leukemia cells has linked alterations in subunits of cytochrome c oxidase, and decreased activity of the complex as a whole, to the drug resistant phenotype.48 Alterations were also detected in the structure and abundance of the R-subunit of ATP synthase. Table 2 summarizes Journal of Proteome Research • Vol. 5, No. 9, 2006 2393
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Figure 3. Results from quantitative real time PCR analyses in drug susceptible and resistant cell lines. (a) mRNA for CPO III; (b) mRNA for R-ATP synthase. RNA was prepared twice on different days and RT-PCR was performed at least twice in replicate. Each bar represents the mean value of individual RT-PCR results with SEM (n ) 6 for CPOIII and n ) 4 for R-ATP synthase). Table 2. Relative Abundances of ATP Synthase Subunits in Adriamycin Resistant and Susceptible MCF-7 Cell Lines
a
ATP synthase subunit (spot number)
relative abundancea
full-length R (127) similar to R (17) truncated R (81) truncated R (82) β (98) ∆ (1) δ (9)
0.75 ( 0.04 8.74 ( 1.17 18.68 ( 3.6 2.31 ( 0.01 1.56 ( 0.10 0.58 ( 0.02 0.82 ( 0.09
n ) 8.
ratios measured for the full-length R-subunit, three truncated isoforms, and three other subunits of ATP synthase. The abundances of the full-length ATP synthase R-subunit, along with those of the ∆, β, and δ-subunits, are not altered significantly (
