Azacytidine induces necrosis of multiple myeloma cells through oxidative stress
© Tian et al.; licensee BioMed Central Ltd. 2013
Received: 13 November 2012
Accepted: 7 June 2013
Published: 13 June 2013
Azacytidine is an inhibitor of DNA methyltransferase and is known to be an anti-leukemic agent to induce cancer cell apoptosis. In the present study, multiple myeloma cells were treated with azacytidine at clinically relevant concentrations to induce necrosis through oxidative stress. Necrotic myeloma cells exhibit unique characteristics, including enrichment of the cell-bound albumin and overexpression of endoplasmic reticulum (ER)- and mitochondrial-specific chaperones, which were not observed in other necrotic cells, including HUH-7, A2780, A549, and Hoc1a. Proteomic analysis shows that HSP60 is the most abundant up-regulated mitochondrial specific chaperone, and azacytidine-induced overexpression of HSP60 is confirmed by western blot analysis. In contrast, expression levels of cytosolic chaperones such as HSP90 and HSP71 were down-regulated in azacytidine-treated myeloma cells, concomitant with an increase of these chaperones in the cell culture medium, suggesting that mitochondrial chaperones and cytosolic chaperones behave differently in necrotic myeloma cells; ER- and mitochondrial-chaperones being retained, and cytosolic chaperones being released into the cell culture medium through the ruptured cell membrane. Our data suggest that HSP60 is potentially a new target for multiple myeloma chemotherapy.
Multiple myeloma (MM) is a clonal B-cell disorder in which malignant plasma cells (PC) accumulate in the bone marrow, resulting in lytic bone lesions and excessive amounts of monoclonal proteins. It accounts for 10% of hematologic malignancies. Although therapeutic interventions have been developed and the overall survival has been improved over the last decade , myeloma is still incurable and most multiple myeloma patients who survive initial treatment will develop drug resistance, and eventually relapse. Development of new therapeutic interventions is strikingly needed for increasing patient survival rate. It has been shown that abnormal methylation of tumor suppressor genes is a common event in malignant plasma cell disorders [2–4] and aberrant global methylation patterns also affect the molecular pathogenesis of myeloma . Azacytidine, a ring analog of the naturally occurring pyrimidine nucleoside cytidine, is an inhibitor of DNA methyltransferase. Following incorporation into DNA, azacytidine is capable of covalently binding to DNA methyltransferase, resulting in hypomethylation and transcriptional reactivation of some silenced genes. This has led to development of azacytidine as a therapeutic anti-cancer agent. Azacytidine has been used to treat patients with myelodysplastic syndrome and acute myeloid leukemia at the dose of 75-100 mg/day [5–7]. In vitro studies have shown that azacytidine alone or in combination with other antitumor agents induces tumor cell apoptosis. It has been reported that azacytidine induced ATR-mediated DNA double-strand break responses, apoptosis, and synergistic cytotoxicity in multiple myeloma cells . It has also been demonstrated that a combination of azacytidine and arsenic trioxide generates a synergistic anti-tumor activity in myeloma . Moreover, azacytidine has been shown to activate the interleukin-6 and nuclear factor-kB signaling pathways , and to induce overexpression of semenogelin I in myeloma cells . To the best of our knowledge, azacytidine induced necrosis in myeloma cells has not yet been reported.
Necrosis is one type of cell death that lacks characteristics of apoptosis and autophagy [12–16]. Over the last several years, the occurrence and course of necrosis was found to be programmed and tightly regulated. Extensive studies show that death ligands (e.g., CD95L, TNF and TNF-related apoptosis-inducing ligand) induce caspase-independent necrotic-like cell death that relies on the activity of the death domain (DD)-containing kinase RIP1. Although the induction mechanisms of necrosis are becoming increasingly clear, the execution of this process remains somewhat elusive. Necrosis is accompanied by a complex sequence of cellular processes including mitochondrial dysfunction with enhanced generation of reactive oxygen species (ROS) and ATP depletion, proteolysis by calpains and cathepsins, and early plasma membrane rupture. One important consequence of necrosis is the induction of immunogenic responses pursuant to the release of immunogens from necrotic cells [17–20]. Basu and colleagues reported that heat shock proteins (HSPs) including gp96, calreticulin, HSP90 and HSP72 were released into the culture supernatant from necrotic cells in response to freeze thaw, but not from apoptotic cells [21, 22]. It was further shown that the released HSPs activated the NF-κB pathway, stimulated macrophages to secrete cytokines, induced the expression of co-stimulatory molecules, and enhanced antigen presentation in dendritic cells [23–28].
A few studies have been reported on necrosis of myeloma cells. Kigamicin, a compound derived from actinomycetes, induces necrosis in human myeloma cells by inhibition of cyclin D1, p21, p-AKT, and p-ERK . A D-amino acid-containing peptide HYD1 increases the reactive oxygen species production, leading to necrotic cell death in multiple myeloma cells . When cells are treated with azacytidine, not only DNA methyltransferase are inhibited, but ROS generation is also observed . For example, ROS generation is used as an indicator for the synergistic and cytotoxic effects of azacytidine in AML and acute lymphoblastic leukemia cells [32, 33]. Within cells, mitochondria are susceptible targets for oxidant stress. ROS can modify mitochondrial lipids, proteins, and DNA. The lack of histones in mtDNA also makes mitochondria more vulnerable to oxidative stress [34, 35]. Oxidative stress also may lead to modifications and alterations of endoplasmic reticulum (ER) chaperone proteins , causing the accumulation of unfolded or misfolded proteins, and decreases in protein synthesis. In the present work, we show that azacytidine-treatment induces necrosis of myeloma cells through oxidative stress, and that necrotic myeloma cells exhibit unique characteristics, including enrichment of cell-bound albumin and overexpression of the ER- and mitochondrial-specific chaperones. Expression of HSP60 has been shown to exhibit the largest increase upon azacytidine treatment and HSP60 is a potential binding partner of cell-bound albumin.
Chemicals and reagents
RPMI1640 medium, phosphate-buffered saline (PBS) and fetal bovine serum were purchased from Wisent (Montreal, QC) and used without further purification. Dithiothreitol (DTT) was purchased from Merck (Whitehouse Station, NJ). Sequencing grade modified trypsin was purchased from Promega (Fitchburg, WI). 5-azacytidine, iodoacetamide (IAA) and RNase A were purchased from Sigma (St Louis, MO). Dimethyl sulfoxide was purchased from Applichem (St Louis, MO). A BCA protein assay kit was purchased from Solarbio (Tongzhou District, Beijing). TMT® Mass Tagging Kits and Reagents were purchased from Thermo Scientific (Rockford, IL).
Cell Culture and Sample Preparation
Human MM cell line U266 was purchased from the Tumor Cell Bank of Chinese Academy of Medical Sciences (Beijing, China), and NCI-H929 and RPMI-8226 cells were kindly provided by Dr. Wenming Chen (Beijing Chao-Yang Hospital Affiliated to the Capital University of Medical Science). All three cell lines were cultured in RPMI 1640 (Wisent) containing 10% or 15% fetal bovine serum with 100 units/mL penicillin and 100 μg/mL streptomycin at 37°C in a humidified incubator with 5% CO2. Prior to treatment, cells were cultured for at least 12 h to reach exponential growth phase. Cells were treated with azacytidine dissolved in dimethyl sulfoxide and the control cells were treated with the same amount of DMSO for the same time periods. After treatments, cells were washed twice with ice-cold PBS and lysed with RIPA lysis buffer (25 mmol/L Tris-HCl pH 7.6, 150 mmol/L NaCl, 0.1% SDS, 1% NP-40, 1% sodium deoxycholate, 1 mmol/L PMSF, and Roche Complete Protease Inhibitor Cocktail) for 30 min on ice. Cell lysates were clarified by centrifugation at 14, 000 ×g for 20 min at 4°C. The protein concentration in the supernatant of each sample was determined using a BCA protein assay kit.
Protein Separation by 1D SDS-PAGE and Proteomics Analysis
Equal amount of proteins from untreated- and azacytidine-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. Peptides were extracted twice with 0.1% trifluoroacetic acid in 50% acetonitrile aqueous solution for 30 min. The extracts were then centrifuged in a speedvac to reduce the volume. To analyze the proteins in the cell culture medium of untreated and azacytidine-treated U266 cells, cells were cultured in RPMI 1640 medium containing 0.5% fetal bovine serum for 48 h and the culture mediums were collected. Proteins from the same volume of cell culture mediums were separated by 1D SDS-PAGE, and the gel bands were excised and digested with trypsin. For protein quantitation, peptides from different samples were labeled with TMT reagents purchased from Thermo-Pierce Biotechnology (Rockford, IL) according to the manufacturer’s instructions. Briefly, TMT reagents were dissolved in anhydrous acetonitrile. Labeling reaction was carried out by incubation of tryptic peptides with the TMT reagents for 1 h at room temperature, and the reaction was quenched with hydroxylamine. TMT-labeled peptides were desalted using the stage tips.
For LC-MS/MS analysis, the digestion product was separated by a 65 min gradient elution at a flow rate 0.250 μL/min with an EASY-nLCII™ integrated nano-HPLC system (Proxeon, Denmark) which was directly interfaced with a Thermo LTQ-Orbitrap 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 LTQ-Orbitrap mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 2.0.7 software and there was a single full-scan mass spectrum in the Orbitrap (m/z 400 to m/z 1800, 30,000 resolution) followed by 20 data-dependent MS/MS scans in the ion trap at 35% normalized collision energy (CID). The MS/MS spectra from each LC-MS/MS run were searched against the selected database using an in-house Proteome Discovery searching algorithm. For quantitation by TMT labeling, TMT-labeled peptides were analyzed by nano-LC-MS/MS with a Q Exactive mass spectrometer that was also operated in the data-dependent acquisition mode using the Xcalibur 2.1.2 software and there was a single full-scan mass spectrum in an Orbitrap (m/z 350 to m/z 1600 Da) followed by 10 MS/MS scans. The MS/MS spectra from each LC-MS/MS run were analyzed using Proteome Discoverer (Version 1.3) for protein identification and quantitation. Intensity ratios of the TMT reporter ions were used to determine relative concentrations of labeled proteins.
The search criteria were as follows: full tryptic specificity was required; one missed cleavages was allowed; carbamidomethylation was set as fixed modification; oxidation (M) was set as variable modification; precursor ion mass tolerances were set at 10 ppm for all MS acquired in the Orbitrap mass analyzer; and the fragment ion mass tolerance was set at 0.8 Da for all MS/MS spectra acquired in the linear ion trap. A proteins was designated as a “hit” only when 2 or more unique peptides with high confidence scores (FDR < 1%) were identified and their corresponding MS/MS spectra were manually inspected. When several proteins matched the same sets of peptides, only the proteins with the greater percentage of coverage was selected. Significance was regarded only when the ratio of spectral counts between two groups were more than 2 or less than 0.5.
DNA Fragment Assay
DNA fragment assay was performed following the procedure described by Mazars et al . Briefly, cells were washed with PBS twice and collected by centrifugation. Cells were suspended in 250 μl lysis buffer (1% NP-40, 20 mM EDTA, 50 mM Tris-HCl, pH 7.5). The supernatants were collected by centrifugation for 5 min at 1,600 × g. The supernatant was incubated with 0.71 mg/ml RNase A for 2 h at 56°C. Then 100 μg/ml pronase E was added and incubated with the supernatants overnight at 37°C. DNA fragments were precipitated with 0.5 volumes of 10 M ammonium acetate and 2 volumes of ethanol at -20°C for 12 h and centrifugated for 15 min at 15,000 ×g. The precipitate was washed with 70% ethanol and resuspended in loading buffer. Electrophoresis was performed in 0.5 × Tris-borate-EDTA buffer for 30 min.
Cells were spun down at 1000 ×g for 5 min. The medium was discarded and washed with PBS twice. Cells were resuspended in 75% ethanol, vortexed to mix briefly, and fixed at 4°C overnight. After vortex, the fixed cells were re-suspended with PBS solution containing Propidium Iodide (PI) (50 μg/ml), followed by RNaseA treatment (1 mg/ml) for 30 min at 37°C and analyzed with a BD FACSCalibur™ Flow Cytometer using 488 nm excitation and a 515 nm bandpass filter for fluorescein detection and a filter >560 nm for PI detection. Dot plots and histograms were analyzed by CellQuest Pro software (BD Biosciences, Heidelberg, Germany).
Western Blot Analysis
Untreated- and azacytidine treated-cells were collected and lysed on ice with Biyuntian cell lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, and sodium pyrophosphate, ß-glycerophosphate, EDTA, and Na3VO4 for Western and IP supplied with the protease inhibitor cocktail. 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 PVDF 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 PBST buffer for 3 times, then incubated with 1000× diluted anti-mouse or anti-rabbit secondary antibody labeled with HRP at room temperature for 2 h. The membrane was further washed with PBST buffer 3 times and developed using the Enlight Kit (Engreen, China). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected with anti- GAPDH antibody as an internal control.
Quantitative real-time PCR (qPCR)
Cells were harvested after being treated with azacytidine for different periods of time. Total RNA was extracted by the SV Total RNA Isolation System. cDNA was synthesized from 4 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.edu/primerbank/). 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 Additional file 1: Table S1.
Cells were lysed with Biyuntian cell lysis buffer for Western and IP supplied with the protease inhibitor cocktail. The protein concentrations of the cell lysates were determined using a BCA assay. The protein A/G agarose beads were washed three times with cell lysis buffer. 2 mg of the cell lysate was incubated with 6 μg anti-bovine albumin antibody and 20 μl protein A/G agarose beads overnight at 4°C. The beads were centrifuged at 1000 ×g for 1 min and washed three times with the cell lysis buffer, and proteins bound to the Protein A/G agarose beads were then eluted by boiling the beads for 5 min in 1 × SDS loading buffer. Each eluent was separated by 1D SDS-PAGE followed by western blot analysis.
Detection of Reactive Oxygen Species (ROS) in Untreated and Azacytidine-treated Cells
ROS in untreated and azacytidine-treated cells was detected using an Image-iT™ LIVE Green Reactive Oxygen Species Detection Kit (Molecular Probes, Inc. Eugene, OR) following the manufacturer’s instructions. Briefly, the cells were collected by centrifugation and washed once with warm HBSS/Ca/Mg. Cells were re-suspended 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 a final concentration of 1.0 μM and incubation for 5 min. The final products were washed gently with 1 ml HBSS/Ca/Mg immediately followed by imaging with Zeiss 710 Confocal Microscopy.
Results and discussion
Azacytidine induces myeloma cell necrosis through oxidative stress
Proteomic analysis and identification of cell-bound albumin in myeloma cell necrosis
Using western blot analysis, we confirmed that enrichment of cell-bound albumin was a concentration-dependent event Additional file 3: Figure S2 (a)). The intensity of the BSA band became more intense when cells were treated with higher concentrations of azacytidine for 24 h. Moreover, western blotting also shows the up-regulation of HSP60 when cells were treated with higher concentrations of azacytidine. A recent study has shown that HSP60 localizes in the tumor cell plasma membrane associated with lipid rafts . In order to determine whether BSA binds to HSP60 in necrotic myeloma cells, we used an anti-BSA antibody to immune-precipitate BSA and associated proteins from untreated and azacytidine-treated myeloma cells. Immunoprecipitated proteins were separated and probed with anti-HSP60 antibodies, and western blot analysis showed that HSP60 was co-immunoprecipitated by an anti-BSA antibody (Additional file 3: Figure S2 (b)). An early study shows that the cell-bound albumin binds to peptidoglycan-, lipopolysacchride-, and lipoteichoic acid in lymphocytes and macrophages . Our data indicate that HSP60 is an additional binding partner of the cell-bound albumin.
A similar protein band pattern by 1D SDS-PAGE was exhibited in Additional file: 4: Figure S3 for U266 cells treated with hydrogen peroxide, showing the characteristic band of enriched cell-bound albumin at about 70 kDa. Moreover, enrichment of BSA was observed in other myeloma cell lines RPMI8226 and NCI-H929 when they were treated with azacytidine (Additional file 5: Figure S4). It is worth mentioning that enrichment of albumin is not observed in azacytidine-treated non-hematopoietic cell lines including A549, A2780, and HUH-7 from lung, ovarian, and liver cancer, respectively. Taken together, out data suggest that enrichment of cell-bound albumin was unique to oxidative stress induced-necrosis in myeloma cell lines.
Oxidative stress-induced over-expression of ER- and mitochondrial-specific chaperones in necrotic myeloma cells
Up-regulated proteins after treatment with 100 μM azacytidine
Up regulated proteins
60 kDa heat shock protein, mitochondrial
Protein disulfide-isomerase A4
Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1
Aspartate aminotransferase, mitochondrial
ATP synthase subunit alpha, mitochondrial
Malate dehydrogenase, mitochondrial
Histone H2A type 2-C
Histone H2B type 1-L
Isoform 1 of Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial
Isoform Long of Sodium/potassium-transporting ATPase subunit alpha-1
Acetyl-CoA acetyltransferase, mitochondrial
ATP synthase subunit beta, mitochondrial
Voltage-dependent anion-selective channel protein 1
3-ketoacyl-CoA thiolase, mitochondrial
60S acidic ribosomal protein P2
DNA topoisomerase 1
Isoform Cytoplasmic of Fumaratehydratase, mitochondrial
Peptidyl-prolylcis-trans isomerase B
Down-regulated proteins after treatment with 100 μM azacytidine
Down regulated proteins
Isoform 1 of Heat shock cognate 71 kDa protein
Heat shock protein HSP 90-beta
Isoform 1 of Heat shock protein HSP 90-alpha
Isoform 2 of Nucleophosmin
T-complex protein 1 subunit eta
Ubiquitin-40S ribosomal protein S27a
ATP-dependent RNA helicase DDX3X isoform 3
DNA replication licensing factor MCM7 isoform 2
DNA-dependent protein kinase catalytic subunit-like
Heat shock 70 kDa protein 4
elongation factor Tu, mitochondrial precursor
Fatty acid synthase
Insulin-like growth factor 2 mRNA-binding protein 1
Isoform 1 of 14-3-3 protein epsilon
Isoform 1 of Insulin-like growth factor 2 mRNA-binding protein 3
Isoform 2 of Clathrin heavy chain 1
Isoform 2 of Cytosolic acyl coenzyme A thioester hydrolase
Isoform Long of Trifunctional purine biosynthetic protein adenosine-3
Lamina-associated polypeptide 2, isoform alpha
Leucine-rich repeat-containing protein 47
Poly(rC)-binding protein 1
Probable ATP-dependent RNA helicase DDX5
Serine/threonine-protein phosphatase PP1-alpha catalytic subunit
suprabasin isoform 1 precursor
Isoform 1 of Heterogeneous nuclear ribonucleoprotein K
Isoform 1 of Polypyrimidine tract-binding protein 1
Leucine-rich PPR motif-containing protein, mitochondrial
Elongation factor 1-alpha 1
High mobility group protein B1
Nuclease-sensitive element-binding protein 1
Multifunctional protein ADE2
116 kDa U5 small nuclear ribonucleoprotein component isoform b
Isoform 1 of Cullin-associated NEDD8-dissociated protein 1
Isoform 1 of Importin-5
Poly [ADP-ribose] polymerase 1
Fatty acid-binding protein, epidermal
Isoform alpha-enolase of Alpha-enolase
Ubiquitin-like modifier-activating enzyme 1
PDI catalyzes formation and breakage of disulfide bonds for proteins to achieve their fully folded state and aids wrongly folded proteins to reach a correctly folded state as a chaperone [48, 49]. Endoplasmin chaperone functions in the processing and transport of secreted proteins in ER and possesses the ATPase activity and calcium-binding property. Calreticulin is a lectin-like, calcium binding ER-specific chaperone that binds to misfolded proteins and prevents them from being exported from the ER to the Golgi apparatus. Overexpression of calreticulin in many cancer cells promotes macrophages to engulf hazardous cancerous cells . Azacytidine also induces up-regulation of calnexin (CNX), whose main function is to assist protein folding and quality control. Calreticulin, calnexin, and ERp57 constitute the calreticulin/calnexin cycle functioning in the quality control of transmembrane and secreted glycoproteins in ER. Up-regulation of ER-specific chaperones indicates that oxidative stress activates intracellular signal transduction pathways and induces the transcriptional upregulation of genes to enhance the ER protein-folding capacity and quality control. Azacytidine induced up-regulation of mitochondrial and ER-chaperones were also observed in other treated myeloma cells as quantified by TMT labeling (Additional file 7: Table S3).
Necrosis-induced release of HSP71 and HSP90 into the cell medium
Taken together, our results show that azacytidine and hydrogen peroxide induce necrosis in myeloma cells through oxidative stress, resulting in enrichment of cell-bound albumin and up-regulation of ER- and mitochondrial-specific chaperones and suggesting that mitochondria and ER are major targets of ROS. Expression levels of HSP90 and HSP71 are down-regulated in azacytidine-treated cells, concomitant with enhanced release of these cytosolic chaperones into the cell culture medium. Inhibition of HSP60 may be a new therapeutic approach for myeloma treatment.
We thank the Cell Biology facility and the Protein Chemistry Facility at the Center for Biomedical Analysis of Tsinghua University for sample analysis. We thank Dr Xiaoyong Jiang for helpful discussions. This work was supported in part by the Center for Life Sciences (Tsinghua University), the National Natural Science Foundation of China (No. 30872391 and 31270871), and MOEC (No. 2012Z02293).
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