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Proteomics was used to analyze the arsenic trioxide-induced protein alterations in multiple myeloma cell line U266. Significantly regulated 14-3-3ζ a...
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Proteomic and Functional Analyses Reveal a Dual Molecular Mechanism Underlying Arsenic-Induced Apoptosis in Human Multiple Myeloma Cells Feng Ge,†,‡,# Xin-Peng Lu,†,‡,# Hui-Lan Zeng,§ Quan-Yuan He,| Sheng Xiong,†,‡ Lin Jin,†,‡ and Qing-Yu He*,†,‡ Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, China, Guangdong Provincial Key Laboratory of Bioengineering Pharmaceutics/National Research Center of Genetic Medicine, Jinan University, Guangzhou 510632, China, Department of Hematology, The First Affiliated Hospital, Jinan University, Guangzhou 510632, China, and The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha 410081, China Received February 7, 2009

Multiple myeloma (MM) is an incurable plasma cell malignancy with a terminal phase marked by increased proliferation and resistance to therapy. Arsenic trioxide (ATO), an antitumor agent with a multifaceted mechanism of action, displayed clinical activity in patients with late-stage multiple myeloma. However, the precise mechanism(s) of action of ATO has not been completely elucidated. In the present study, we used proteomics to analyze the ATO-induced protein alterations in MM cell line U266 and then investigated the molecular pathways responsible for the anticancer actions of ATO. Several clusters of proteins altered in expression in U266 cells upon ATO treatment were identified, including down-regulated signal transduction proteins and ubiquitin/proteasome members, and upregulated immunity and defense proteins. Significantly regulated 14-3-3ζ and heat shock proteins (HSPs) were selected for further functional studies. Overexpression of 14-3-3ζ in MM cells attenuated ATOinduced cell death, whereas RNAi-based 14-3-3ζ knock-down or the inhibition of HSP90 enhanced tumor cell sensitivity to the ATO induction. These observations implicate 14-3-3ζ and HSP90 as potential molecular targets for drug intervention of multiple myeloma and thus improve our understanding on the mechanisms of antitumor activity of ATO. Keywords: Multiple myeloma • Arsenic trioxide • Proteomics • 14-3-3ζ • Heat shock proteins

Introduction Multiple myeloma (MM) is a hematologic malignancy characterized by the clonal proliferation of plasma cells in the bone marrow and, usually, the presence of a monoclonal immunoglobulin in the blood and/or urine.1 MM is the second most commonly diagnosed hematologic malignancy in the Western world, and the most common cancer with skeleton as its primary site. It has an incidence of 19 900 new cases per year in the U.S.A., and accounts for 10% of hematologic malignancies and 1% of all cancer deaths.2 In China, the annual incidence of multiple myeloma is approximately one case per 100 000 persons.3 At present, the disease is incurable; and although survival has improved, there remains a need for new therapeutic options for patients with this B-cell malignancy.4 * To whom correspondence should be addressed. Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, China. Phone/ Fax: +86-20-85227039. E-mail: [email protected]. † Institute of Life and Health Engineering, Jinan University. ‡ Guangdong Provincial Key Laboratory of Bioengineering Pharmaceutics/ National Research Center of Genetic Medicine, Jinan University. # Equally contributed to this work. § Department of Hematology, The First Affiliated Hospital, Jinan University. | Hunan Normal University.

3006 Journal of Proteome Research 2009, 8, 3006–3019 Published on Web 04/13/2009

Arsenic trioxide (ATO), an ancient drug used in traditional Chinese medicine and, in the last century, in Western medicine, is very effective in the treatment of acute promyelocytic leukemia (APL) patients, with very little toxicity.5 ATO is a potent inducer of apoptosis in a number of other cell types such as acute myeloid leukemia (AML),6 gastric cancer7 and neuroblastoma.8 ATO is also a promising agent for progressive and refractory MM by inducing growth inhibition and apoptosis in MM cells.9-11 The ATO activity was observed as a single agent or in combination with ascorbic acid in several Phase 1 and 2 trials.12-14 A number of reports have addressed various molecular targets of ATO action including promyelocytic leukemia (PML) receptors and other nuclear body proteins,15,16 nuclear factorκB (NF-κB),17 glucocorticoid nuclear receptors,18 as well as components of the mitogen-activated protein kinase (MAPK) signaling cascade.19 However, the precise mechanism(s) of ATO action in myeloma is unclear. Advances in the use of proteomic technologies provide a robust approach to study multiple signaling pathways simultaneously. The altered proteins identified by proteomic approach can be further characterized as potential drug targets and the global analysis of the protein alterations can result in 10.1021/pr9001004 CCC: $40.75

 2009 American Chemical Society

Arsenic-Induced Apoptosis in Multiple Myeloma valuable information to understand the drug action mechanisms.20-24 In this study, we used a global proteomic-based approach to look at ATO-induced apoptosis in U266 myeloma cells. When the protein profiles of U266 cells treated by ATO were compared to these untreated control, we identified 76 differentially expressed proteins. Further functional studies showed that ATO induces a dual apoptotic pathway with mitochondria- and ubiquitination-proteasome (UPS)-dependence. Overexpression of 14-3-3ζ in MM cells modestly attenuated ATO-induced cell death, whereas suppression of 143-3ζ or HSP90 activity enhanced tumor cell sensitivity to ATO inhibition. When the proteomic data is correlated with these functional studies, the current results suggest potential therapeutic targets for the MM drug intervention and provide direct implications for the development of novel anti-MM therapeutic strategies.

Experimental Procedures Reagents and Cell Line. A 1 mg/mL ATO preparation for intravenous (iv) administration was preserved and protected from air at 4 °C in aliquots and diluted to a working concentration in RPMI 1640 before use. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) was purchased from ALEXIS Biochemicals (San Diego, CA), dissolved in DMSO at a concentration of 1 mmol/L, and stored at -20 °C until use. All other chemicals, except otherwise noted, were purchased from GE Healthcare (Uppsala, Sweden). The human myeloma cell line U266 was purchased from American type Culture Collections (Rockville, MD). U266 cells were cultured in RPMI 1640 supplemented with 1% penicillin/ streptomycin, 1 mmol/L L-glutamine, and 10% fetal bovine serum at 37 °C, 5% CO2 in air. Two-Dimensional Gel Electrophoresis (2-DE). U266 cells were treated with 2 µM ATO for 24 h, according to the halfmaximal inhibitory concentration (IC50) measured by Karasavvas et al.25 After treatment, cells were washed three times with ice-cold washing buffer (10 µM Tris-HCl, 250 µM sucrose, pH 7.0) and transferred to a clean 1.5 mL Eppendorf tube. Cells were lysed with a buffer containing 7 M urea, 2 M thiourea, 4% CHAPS and 1% DTT, 2% (v/v) IPG buffer 3-10 NL or 4-7 linear, 0.2 mg/mL PMSF and protease-inhibitors (Complete kit, Roche Diagnostics, Germany). Cellular debris was removed by centrifugation for 30 min at 13 200g and at 4 °C. The lysis supernatant was used for 2-DE. Protein concentrations were determined using Bradford assay. All the samples were stored at -80 °C prior to electrophoresis. Two-dimensional gel electrophoresis was carried out with Amersham Biosciences IPGphor IEF System and Hoefer SE 600 (GE healthcare, Uppsala, Sweden) electrophoresis (13 cm) units, in accordance with a previously described protocol.21 Proteins were detected by a silver nitrate staining protocol adapted from Wang et al.21 Image Analysis and Mass Spectrometry Peptide Sequencing. Analytical gels were scanned on an Image Scanner (GE Healthcare, Uppsala, Sweden), and images were analyzed using the ImageMaster 2D Platinum (GE Healthcare, Uppsala, Sweden). Only protein spots that were reproducibly different in all three experiments by at least a factor of 2 were considered to be significant and were excised from gels for analysis by MS. Tryptic in-gel digestion was performed in accordance with a previously described protocol.21 Peptide mass spectra were obtained on an ABI 4800 plus MALDI TOF/TOF mass spectrometer (Applied Biosystems, Foster City, CA). Mass spectra were obtained using the settings presented in the Supporting

research articles Information Data 1. Both the MS and MS/MS data were interpreted and processed by using the GPS Explorer software (V3.6, Applied Biosystems), then the obtained MS and MS/MS spectra per spot were combined and submitted to MASCOT search engine (V2.1, Matrix Science, London, U.K.) by GPS Explorer software. MASCOT protein scores (based on combined MS and MS/MS spectra) of greater than 65 were considered statistically significant (p e 0.05). The individual MS/MS spectrum with statistically significant (p e 0.05) best ion score (based on MS/MS spectra) was also accepted. The MS spectra of all identified proteins were presented in the Supporting Information Data 2 and Tables 1-3. Protein Categorization and Network Construction. Differentially expressed proteins were classified based on the PANTHER (Protein ANalysis THrough Evolutionary Relationships) system (http://www.pantherdb.org), which is a unique resource that classifies genes and proteins by their functions.26,27 Some proteins were annotated manually based on literature searches and closely related homologues. The differentially expressed protein interaction network was build automatically by the STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) system with default setting except that organism, confidence(score), and interactors shown were set to “human”, “0.20”, and “no more than 10 interactors”, respectively.28,29 The gene name list of these proteins was input to search against the database which contains known and predicted protein-protein interactions. The retrieve included a detailed network which highlights several hub proteins. Detection of Apoptosis. Apoptosis was detected by using Annexin V/ PI staining. In brief, cells (1 × 106) from 24- or 48-h cultures were washed once in phosphate-buffered saline (PBS) and were stained with Annexin V-FITC and PI (2 mg/mL) according to manufacturer’s instructions. Samples were acquired on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and analyzed with the WinMDI 2.8 software program. Annexin V-positive, PI-negative cells reflect those cells in the early stages of apoptosis, whereas Annexin V-positive, PIpositive cells reflect dead cells or cells at the late stages of apoptosis. Both subpopulations were considered as the overall apoptotic cells. Western Blot Analysis. Protein extracts (30 µg) prepared with RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF, 100 mM leupeptin, and 2 mg/mL aprotinin, pH 8.0) were resolved by a 10% SDS-PAGE gel, and transferred onto a Immobilon-P PVDF transfer membrane (Millipore, Bedford, MA) by electroblotting. After blocking with 5% nonfat milk, the membranes were probed with anti-Bax, anti-Bad, anti-Bcl-2, anti-HSP27, antiHSP70, anti-14-3-3ζ (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bid, anti-PARP, anti-Mcl-1 (Cell signaling, Danvers, MA), anti-HSP90, anti-Bcl-x (Epitomics, Burlingame, CA), anticaspase 3 and anti-tubulin antibody (LabVision, Fremont, California). Blots were then incubated with peroxidaseconjugated anti-mouse, anti-rabbit or anti-goat IgG (KPL, Gaithersburg, Maryland) for 1 h at room temperature at a 1:3000 dilution and then developed by using the SuperSignal West Pico kit (Pierce Biotechnology, Rockford, IL). Proteasome Activity Assay. Untreated or ATO-treated U266 cells were harvested after 24 h exposure, washed twice with PBS and homogenized in a lysis buffer (50 mM Tris (pH 8.0), 5 mM ethylenediamine tetraacetic acid, 150 mM NaCl, 0.5% NP40). After 30 min of rocking at 4 °C, the mixtures were Journal of Proteome Research • Vol. 8, No. 6, 2009 3007

research articles centrifuged at 12 000g for 15 min and the supernatants were collected as whole-cell extracts. Proteasome activity was measured using the 20S proteasome activity assay kit (Chemicon, Temecula, CA) according to the manufacturer’s protocol. Fluorescence was measured on a Perkin-Elmer fluorometer using an excitation wavelength of 380 nm and an emission wavelength of 460 nm. siRNA for 14-3-3ζ and Transfection of siRNA. 14-3-3ζ RNA interference mediated by duplexes of 21-nucleotide RNA was performed in U266 cells. The siRNA duplex targeted against 14-3-3ζ and the nonsilencing negative control siRNA duplex were purchased from Shanghai GenePharma Co. (Shanghai, China). For initial screening of siRNAs, U266 cells were transfected and the knockdown was estimated by using Western blotting. From this screen, YWHAZ siRNA #694 (sense, 5′CCAAGGAGACGAAGCUGAAT T-3′; antisense, 5′-UUCAGCUUCGUCUCCUUGGGT-3′) was chosen as the main siRNA with the most efficient knockdown. The RNA sequence used as a negative control for siRNA activity was sense, 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense, 5′-ACGUGACACGUUCGG AGAATT-3′. All duplexes were dissolved in RNase-free water to a final concentration of 20 µM. Dissolved siRNA duplexes were aliquoted and stored at-20 °C. In preliminary experiments, we optimized conditions for the efficient transfection using FAM-labeled negative control siRNA duplex. Streptolysin-O (SLO) (Sigma Chemical Co, Poole, England) was used to reversibly permeabilize cells toward siRNA30,31 according to a recently revised protocol.32 In all experiments, more than 95% of cells were permeabilized toward siRNA without loss of viability (data not shown). Transfection efficiency and 14-3-3ζ protein knockdown was assessed 24 and 48 h postpermeabilization by flow cytometric analysis and Western blotting analysis. Plasmid Constructs and Transient Transfection. The human 14-3-3ζ expression plasmid (Catalog No.: EX-C0032-M03) and blank plasmid were purchased from GeneCopoeia, Inc. (Germantown, MD). The detailed information of the plasmids is available upon request. The 14-3-3ζ and control constructs were expressed in U266 cells by transient transfection using SLO permeabilization method as described above. Cells were collected 48 h later and overexpression of 14-3-3ζ was confirmed by Western blotting assays. Cell survival was assessed by using Annexin V/ PI staining, as described above. Statistical Analysis. Statistical analysis was performed using a two-tailed Student’s t-test. Results were considered significant for p < 0.05. Data were expressed as the mean ( SD of triplicate samples. All experiments were done at least thrice to ensure reproducibility of the results.

Results Proteome Profiles of Control and ATO-Treated Myeloma Cells. Protein profiles of control and ATO-treated U266 cells were studied by comparative proteomic analysis. Figure 1 shows a pair of representative 2-D gel images for whole cell proteins extracted from U266 cells with and without ATO treatments for 24 h. To obtain statistically significant results, each protein sample was run in triplicate. To find more differently expressed proteins, two kinds of IPG strips were used. Figure 1A is the gel images using pH 3-10 nonlinear gradient IPG strips, while Figure 1B is the gel images using pH 4-7 linear gradient IPG strips. The proteomic maps of control and ATO-treated cells were compared with ImageMaster 2D Platinum software to identify the protein spot variations. After 3008

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Ge et al. ATO treatment, significantly differentially expressed protein spots (p < 0.05) with 2-fold or more increased or decreased intensity as observed in all three replicate gels were scored. Eighty-four differentially expressed proteins that could be visualized with silver staining were excised from the 2-D gels and identified by MALDI-TOF/TOF followed by database interpretation. Altogether, 76 proteins including 29 upregulated and 47 down-regulated proteins were identified successfully. Table 1 lists the proteins identities, together with their spot numbers, molecular weights and pI’s, reported functions and fold differences of alterations corresponding to the ATO treatments. The MS and MS/MS spectra were presented in the Supporting Information Data 2. As was the case for the 2-DE gels, more than one spot could often be identified as the same protein (Table 1). For example, four spots of 76B, 78B, 79B and 80B were identified as the same protein, ubiquilin-1, and two spots of 109B and 110B were identified as the same protein, 14-3-3 protein zeta. Functional Categories of Identified Proteins. To understand the biological relevance of the changes in protein expression in response to ATO treatment, PANTHER classification system was used to classify the 76 identified proteins according to their functions. The PANTHER classification system revealed that the proteins can be classified into eight groups according to their functional properties (Table 1): (i) carbohydrate metabolism; (ii) nucleoside, nucleotide and nucleic acid metabolism; (iii) protein metabolism and modification; (iv) signal transduction; (v) ubiquitination-proteasome pathway; (vi) cell cycle; (vii) cell structure and motility; and (viii) immunity and defense. Among these dys-regulated proteins, of particular interest are upregulated HSPs and down-regulated 14-3-3 and proteasome proteins. We expect that these proteins play important roles in ATO-induced cytotoxicity in multiple myeloma cells. Protein Association Network among the Identified Proteins. Among the 76 identified proteins, 65 of them can be linked through direct interaction into a protein-protein interaction network based on the prediction results of STRING system (Figure 2). Notably, several 14-3-3 proteins were hubs in this network. By comparing with 427 known interacting proteins of 14-3-3ζ (http://www.genecards.org/cgi-bin/ carddisp.pl?gene)YWHAZ&search)YWHAZ& interactions)427# pathways), 33 out of the 76 proteins are known 14-3-3 ζ interactors. The list of these 14-3-3ζ interacting proteins was presented in the Supporting Information Table 4. These results suggested that 14-3-3ζ may play a vital role in mediating ATOinduced apoptosis in multiple myeloma cells. 14-3-3ζ as the Major Target of ATO Cytotoxicity. To investigate whether 14-3-3ζ has a role in ATO-induced apoptosis and to explore the molecular sequelae of 14-3-3ζ inhibition, we used RNAi to reduce cellular 14-3-3ζ levels and examine the effect of 14-3-3ζ knock-down. The effect of the 14-3-3ζ siRNA in silencing the 14-3-3ζ gene in human U266 cells was examined by directly measuring changes in 14-3-3ζ protein levels. As shown in Figure 3, transfection of cells with the 14-3-3ζ siRNA resulted in a significant decrease in the level of 14-3-3ζ protein, similar to the effect of ATO treatment, whereas mock transfection with a random siRNA had no such effect. This effect was specific because the 14-3-3ζ siRNA did not change the levels of tubulin protein (Figure 3). To analyze whether 14-3-3ζ knock-down has an effect on apoptosis of multiple myeloma cells, we did Annexin V/PI flow cytometry on U266 cell lines. As shown in Figure 4A, knockdown of 14-3-3ζ resulted in a marked increase in the number

Arsenic-Induced Apoptosis in Multiple Myeloma

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Figure 1. 2-D gel maps of protein expression in control and ATO-treated human myeloma U266 cells. Arrows indicate the changed proteins that were successfully identified. Proteins from the U266 cells treated with ATO for 24 h were extracted and separated on a pH 3-10 NL (A) or pH 4-7 linear (B) IPG strip, followed by SDS-PAGE. After staining and image analysis, the spots were analyzed by MALDI-TOF/TOF. The identified proteins, as labeled by numbers on the gel, are listed in Table 1. Results were from one representative experiment out of three.

of apoptotic cells. Quantitative analysis indicated that the percentage of apoptotic cells in 14-3-3ζ siRNA-transfected cells (14.9 ( 1.9%) was significantly higher than that of a random siRNA-transfected group, which is 6.7 ( 0.9% (p < 0.03; Figure 4B). In cells transfected with 14-3-3ζ siRNA, ATO-induced inhibition (54.6 ( 4.1%) was also significantly higher than that of ATO-treated cells with a random siRNA transfection, which

is 37.2 ( 4.9% (p < 0.02; Figure 4B). These observations suggest that 14-3-3ζ plays a role in maintaining U266 cell survival and thus is a target of ATO cytotoxicity. Because caspases are critical intermediates in apoptosis pathways, we next investigated the impact of the 14-3-3ζ knockdown on caspase-3 activity. Executioner caspase activities were monitored by their cleavage of polyADP ribose polymerase Journal of Proteome Research • Vol. 8, No. 6, 2009 3009

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Ge et al. a

Table 1. Summary of the Identified Proteins with Different Expressions in ATO-Treated U266 Cells spot no.b 89B 98B 21

36 58B 97B 69 68 45B 66B 62B

67 75 80 38 74 19 72 29 110 20 87

88B 88 82 1B 83B 9B 31B 14 18 15B 28 79 63 84B 95B 42

3010

protein MW/pI

peptide(s) detectede

scoref

F. D. (24 h)g

Carbohydrate Metabolismh Decarboxylase IPI00514944

34789.8/5.46

2

78

-2.2 ( 0.4

Protein phosphatase

IPI00218236

37162.6/5.84

2

163

-5.3 ( 1.1

Decarboxylase

IPI00797038

70684.6/7.57

6

420

-3.8 ( 0.7

Synthetase Dehydratase Dehydrogenase

IPI00147874 IPI00644339 IPI00219217

40281.4/6.29 25014.6/8.2 36615.1/5.71

3 1 2

261 104 158

-2.1 ( 0.8 3.3 ( 0.9 -2.0 ( 0.4

Dehydratase;Hydratase

IPI00017855

85371.9/7.36

5

721

-7.7 ( 1.5

Dehydratase; Hydratase

IPI00008485

98336.6/6.23

2

125

-2.8 ( 0.3

Carbohydrate kinase Synthetase

IPI00019383 IPI00024993

42245.6/6.04 31367.1/8.34

2 1

114 88

2.1 ( 0.2 2.4 ( 0.7

Synthetase

IPI00011416

35793.4/8.16

3

80

3.6 ( 0.6

Nucleoside, Nucleotide and Nucleic Acid Metabolism RNA-binding protein IPI00027442 106743.2/5.34

5

401

-2.4 ( 0.7

mRNA splicing factor

IPI00395775

45542.3/6.19

4

196

-2.8 ( 0.2

Ligase

IPI00218408

46003.8/6.34

5

403

-2.1 ( 0.3

Phosphorylase

IPI00011876

31215.8/6.75

2

94

-2.1 ( 0.5

Hydrolase

IPI00257508

62254.6/5.95

2

92

-2.1 ( 0.2

Synthase Synthase Synthetase

IPI00029079 IPI00029079 IPI00026833

76667/6.42 76667/6.42 50065.8/6.13

4 2 3

204 86 220

-9.8 ( 1.7 -3.0 ( 0.3 -2.8 ( 0.4

Transcription cofactor RNA-binding protein

IPI00021187 IPI00853059

50196.3/6.02 68691.1/6.62

2 3

73 182

3.0 ( 0.5 -7.3 ( 1.9

Glycosyltransferase

IPI00215974

44019.1/6.82

2

70

-2.1 ( 0.3

Protein Metabolism and Modification Hydrolase IPI00009268 Transaminase IPI00219029

45856/5.77 46218.5/6.52

1 4

95 343

-3.8 ( 0.3 -2.3 ( 0.4

Dehydrogenase

IPI00016801

61359.2/7.66

1

103

-2.1 ( 0.4

Glucosidase

IPI00383581

106806.6/5.74

4

142

3.5 ( 0.5

Isomerase

IPI00025252

56746.8/5.98

2

246

-6.8 ( 0.5

Chaperonin

IPI00290770

60424.3/6.1

1

69

3.4 ( 0.2

RNA helicase

IPI00009328

46841.2/6.3

2

143

2.6 ( 0.4

Translation elongation factor Translation elongation factor Chaperonin

IPI00186290 IPI00186290 IPI00027626

95277/6.41 95277/6.41 57987.6/6.23

2 3 2

133 204 130

-15.2 ( 3.1 -2.4 ( 0.1 2.2 ( 0.2

Translation initiation factor

IPI00290460

35588.9/5.87

2

216

-2.3 ( 0.5

Metalloprotease

IPI00789806

52737.9/6.3

5

659

-2.1 ( 0.5

Chaperonin

IPI00297779

57452.1/6.01

2

151

5.6 ( 1.3

Aminoacyl-tRNA synthetase

IPI00412737

48820.4/6.03

4

253

-2.9 ( 0.7

Translation initiation factor

IPI00012795

36478.6/5.38

3

380

-3.0 ( 0.5

Ribosomal protein

IPI00375631

17876.3/6.84

2

92

-2.7 ( 0.6

protein namec Uroporphyrinogen decarboxylase Serine/threonine-protein phosphatase PP1-beta catalytic subunit Mitochondrial phosphoenolpyruvate carboxykinase 2 isoform 1 precursor Sialic acid synthase UDP-galactose-4-epimerase L-lactate dehydrogenase B chain Aconitate hydratase, mitochondrial precursor Iron-responsive element-binding protein 1 Galactokinase Enoyl-CoA hydratase, mitochondrial precursor Delta(3,5)-Delta(2,4)-dienoylCoA isomerase, mitochondrial precursor Alanyl-tRNA synthetase, cytoplasmic Isoform 2 of paraspeckle component Isoform Short of Trifunctional purine biosynthetic protein adenosine-3 S-methyl-5-thioadenosine phosphorylase Dihydropyrimidinase-related protein 2 GMP synthase GMP synthase Adenylosuccinate synthetase isozyme 2 Isoform 1 of RuvB-like 1 Isoform 2 of far upstream element-binding protein 1 Queuine tRNA-ribosyl transferase 1 Aminoacylase-1 Aspartateamino transferase, cytoplasmic Glutamate dehydrogenase 1, mitochondrial precursor Isoform 1 of Neutral alpha-glucosidase AB precursor Protein disulfide-isomerase A3 precursor chaperonin containing TCP1, subunit 3 isoform b Eukaryotic initiation factor 4A-III Elongation factor 2 Elongation factor 2 T-complex protein1 subunit zeta Eukaryotic translation initiation factor 3 subunit 4 Isoform 2 of cytosolamino peptidase T-complex protein 1subunit beta Tryptophanyl-tRNA synthetase isoform b Eukaryotic translation initiation factor 3, subunit 2 Interferon-induced 17 kDa protein precursor

function

Journal of Proteome Research • Vol. 8, No. 6, 2009

accession no.d

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Arsenic-Induced Apoptosis in Multiple Myeloma Table 1. Continued spot no.b

protein namec

function

accession no.d

protein MW/pI

peptide(s) detectede

scoref

F. D. (24 h)g

IPI00106642 IPI00847579 IPI00021828 IPI00873768

66975.2/5.62 14505.5/6.81 11132.6/6.96 8963.9/8.97

3 3 3 3

131 273 169 235

3.6 ( 0.3 -3.6 ( 0.4 -2.1 ( 0.5 -4.4 ( 0.6

121 124 44 46

Dihydropyrimidinase-like 2 Ribosomal protein S12 Cystatin-B NEDD8 precursor

Glycosyltransferase Ribosomal protein Cysteine protease inhibitor Ribosomal protein

111B

Isoform short of 14-3-3 protein beta/alpha Isoform short of 14-3-3 protein beta/alpha 14-3-3 protein zeta/delta 14-3-3 protein zeta/delta 14-3-3 protein gamma 14-3-3 protein gamma Stathmin Stathmin1/oncoprotein 18 Isoform 2 of Acyl-protein thioesterase 1

Chaperones

Signal Transduction IPI00759832

27832.7/4.76

1

91

-6.3 ( 1.2

Chaperones

IPI00759832

27832.7/4.76

3

105

-3.8 ( 0.4

Chaperones Chaperones Chaperones Chaperones Phosphoprotein Phosphoprotein Phospholipase

IPI00021263 IPI00021263 IPI00220642 IPI00220642 IPI00479997 IPI00642012 IPI00398727

27727.7/4.73 27727.7/4.73 28284.9/4.8 28284.9/4.8 17291.9/5.76 9858.3/6.75 22860.6/6.05

1 1 1 2 3 2 3

83 129 192 194 264 277 276

-7.1 ( 0.7 -5.9 ( 0.3 -3.7 ( 0.2 -3.9 ( 0.7 -2.7 ( 0.6 -4.2 ( 0.7 -2.1 ( 0.7

Ubiquitination-Proteasome Pathway Ubiquitin-like protein IPI00071180 Ubiquitin-like protein IPI00071180 Ubiquitin-like protein IPI00099550 Ubiquitin-like protein IPI00071180 Proteases IPI00219445

59182.8/5.01 59182.8/5.01 62479.4/5.02 59182.8/5.01 30867.2/5.79

2 2 4 2 4

185 137 218 76 489

-3.9 ( 0.5 -3.3 ( 0.4 -2.2 ( 0.8 -2.5 ( 0.8 -5.3 ( 1.5

Proteases

IPI00291922

26394.2/4.74

1

79

-3.3 ( 0.7

Hydrolase

IPI00021435

48603.1/5.71

2

235

-5.4 ( 0.8

Proteases

IPI00215824

29750.3/5.53

1

94

-2.5 ( 0.6

IPI00784990

17142.3/7.93

2

157

-2.4 ( 0.1

DNA helicase

IPI00018349

96497.6/6.28

1

137

3.2 ( 0.2

Nonmotor actin binding protein DNA helicase

IPI00010133

50993.8/6.25

1

136

2.3 ( 0.3

IPI00299904

81256.6/6.08

2

166

3.9 ( 0.7

DNA helicase

IPI00031517

92831.2/5.29

5

274

8.4 ( 0.9

Ribonucleoprotein

IPI00216746

50996.4/5.19

2

77

7.2 ( 1.1

Membrane traffic protein

IPI00014053

37869.1/6.79

2

131

-3.0 ( 0.4

Extracellular matrix structural protein Small GTPase

IPI00297646

138826.5/5.6

1

69

5.0 ( 0.5

IPI00167949

75487.2/5.6

6

636

2.8 ( 0.3

Actin binding motor protein Actin binding motor protein

IPI00477649 IPI00218320

28775.8/4.76 28937.8/4.79

1 1

83 153

-4.7 ( 0.4 -3.7 ( 0.3

Nonmotor actin binding protein

IPI00012011

18490.7/8.22

2

168

-5.3 ( 0.8

Immunity and Defense Chaperones IPI00025512 Hsp 70 family chaperone IPI00845339 Hsp 70 family chaperone IPI00845339 Peroxidase IPI00374151 Hsp 70 family chaperone IPI00003865

22768.5/5.98 69995/5.48 69995/5.48 25822.3/7.04 70854.2/5.37

3 5 6 3 2

273 512 786 232 148

6.0 ( 0.8 3.8 ( 0.7 8.9 ( 1.1 3.8 ( 0.9 2.1 ( 0.4

Hsp 90 family chaperone

IPI00796844

49327.3/5.33

1

81

3.3 ( 0.3

Hsp 90 family chaperone

IPI00382470

98099.5/5.07

4

182

6.0 ( 1.5

Hsp 90 family chaperone Hsp 70 family chaperone Hsp 90 family chaperone

IPI00414676 IPI00339269 IPI00027230

83212.1/4.97 70984.2/5.81 92411.3/4.76

6 6 6

656 800 883

12.4 ( 2.3 21.8 ( 3.1 2.7 ( 0.9

112B 109B 110B 116 117 4 124B 3

78B 79B 80B 76B 125 93 86B 96 100

Isoform 2 of ubiquilin-1 Isoform 2 of ubiquilin-1 Isoform 1 of ubiquilin-1 Isoform 2 of ubiquilin-1 Isoform 2 of proteasome activator complex subunit 3 Proteasome subunit alpha type-5 26S protease regulatory subunit 7 Isoform 2 of proteasome subunit beta type-8 precursor Ubiquitin C splice variant

Ribosomal protein Cell Cycle

104 16B

DNA replication licensing factor MCM4 Coronin-1A

6B

Isoform 1 of DNA replication licensing factor MCM7 112 DNA replication licensing factor MCM6 Cell structure and motility 115 Isoform 2 of Heterogeneous nuclear ribonucleo protein K 89 Isoform 1 of probable mitochondrial import receptor subunit 37B Collagenalpha-1(I) chain precursor 49 Interferon-induced GTP-bindin protein Mx1g 104B Tropomyosin 3 isoform 5 105B Isoform 3 of tropomyosin alpha-3 chain 1 Cofilin-1

64 50 51 72B 18B 57 126 60 54 48

Heat shock protein beta-1 Heat shock 70 kDa protein 1A Heat shock 70 kDa protein 1A Peroxiredoxin 3 isoform b Isoform 1 of Heat shock cognate 71 kDa protein Full-length cDNA clone CS0CAP 007YF18 of Thymus of Homo sapiens Heat shock protein 90 kDa alpha Heat shock protein 90-beta Heat shock 70 kDa protein 6 HSP90B1 endoplasmin precursor

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Table 1. Continued spot no.b 64B 118 45

protein namec Peroxiredoxin-4 Translationally controlled tumor protein Macrophage migration inhibitory factor

function

accession no.d

protein MW/pI

peptide(s) detectede

scoref

Peroxidase Nonmotor microtubulebinding protein Cytokine

IPI00011937 IPI00550900

30520.8/5.86 19582.6/4.84

3 1

155 71

2.6 ( 0.8 2.1 ( 0.9

IPI00293276

12468.2/7.74

3

224

-2.5 ( 0.3

F. D. (24 h)g

a Deregulated proteins identified in whole cell lysates of U266 after treatment with ATO are listed, and are classified according to their primary functions. b Spot no. refers to Figure 1. c Protein description in the IPI_HUMAN database. d Accession number in the IPI_HUMAN database. e The number of peptides used for spot identification. f Score is -10 × Log(P), where P is the probability that the observed match is a random event; it is based on NCBInr database using the MASCOT searching program as MALDI-TOF data. g Fold differences between ATO-treated and control U266 cells, which were expressed as positive value or negative value (mean ( SD, n ) 3). Positive value, up-regulation in ATO-treated cells; negative value, down-regulation in ATO-treated cells. h The identified proteins were grouped according to their functions.

Figure 2. The protein-protein interaction network of the identified proteins. The network containing 76 identified proteins was mapped using the STRING system (http://string.embl.de/) based on evidence with different types. In the evidence view, the links between proteins represent possible interactions. Different line colors represent the types of evidence for the associations, which are shown in the legend.

(PARP) and caspase processing. The 32-kDa precursor (procaspase-3) was degraded both in ATO-treated U266 cells and 14-3-3ζ knock-down cells (Figure 3), implying that induction of apoptosis by ATO or 14-3-3ζ knock-down was related to the activation of caspase-3 enzymatic protein. Concerning PARP protein as a major substrate for active caspase-3 and a hallmark of apoptosis, Western blotting showed that the intact 113-kDa moiety of PARP was cleaved to 89-kDa products (Figure 3). Next, we focused on the Bcl-2 family of apoptosis regulators that integrate cellular survival and apoptosis signals through their action on mitochondria.33-35 The Bcl-2 proteins are essential regulators of apoptosis induced by many different stimuli. This family, which contains both pro- and antiapoptotic members, acts in part by controlling the status of the mitochondria. We reasoned that 14-3-3ζ levels may control the status of Bcl-2 family proteins in U266 cells, which, in turn, determine cancer cell susceptibility to ATO treatment. To test 3012

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this model, we monitored the expression levels of some important Bcl-2 family proteins. Among proteins tested, levels of Bax and Bad increased in ATO-treated cells or 14-3-3ζ knockdown cells, while Bcl-2 and Bcl-x showed no change in their expression levels upon ATO treatment or 14-3-3ζ knock down (Figure 3). In contrast, Mcl-1 appeared to decrease with the time upon ATO treatment or 14-3-3ζ knock down. On the other hand, both ATO-treated and 14-3-3ζ knock-down cells exhibited increased levels of cleaved Bid (tBid), whereas total Bid levels remained almost the same (Figure 3). Thus, decreased Mcl-1 and increased Bad and Bax in ATO-treated or 14-3-3ζ knock-down cells along with increased levels of cleaved Bid (tBid) may tip the balance in U266 cells toward enhanced apoptotic potential. Conclusively, these results suggest that the 14-3-3ζ survival signal acts upstream of the Bcl-2 proteins and effector caspases, possibly aiding in the maintenance of mitochondrial integrity.

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Arsenic-Induced Apoptosis in Multiple Myeloma

Figure 3. Western blotting results indicating that both ATO treatment and 14-3-3ζ knock-down of U266 cells were accompanied by the changes of Bcl-2 protein levels and caspase-3 activity. ATO treated, 14-3-3ζ siRNA or random siRNA transfected U266 cells were cultured for 24 and 48 h. Cells were then harvested, lysed, electrophoresed, and immunoblotted as described in Experimental Procedures. Whole cell lysates were immunoblotted with antibodies specific for 14-3-3ζ, caspase 3, PARP, Mcl-1, Bcl-x, Bcl-2, Bax, Bad and Bid. Blots were stripped and reprobed with anti-tubulin antibody to ensure equivalent loading. Typical experiment conducted three times with similar results.

Our results prompted us to further investigate whether overexpression of 14-3-3ζ conferred protection against ATO. We transiently transfected U266 cells with an expression vector containing a cDNA encoding human 14-3-3ζ or a blank vector as a control. The overexpression of 14-3-3ζ was confirmed by Western blotting (Figure 5A). As shown in Figure 5B, transient expression of 14-3-3ζ reduced ATO-sensitivity in U266 cells, compared with the cells transfected with the control vector. Quantitative analysis also revealed that the percentage of apoptotic cells in 14-3-3ζ-transfected and ATO-treated cells (16.6 ( 3.1%) was significantly lower than that of a control vector-transfected group, which is 36.5 ( 4.1% (p < 0.02; Figure 5C). These results support the notion that in principle inhibition of 14-3-3ζ could be a useful therapeutic strategy for the treatment of multiple myeloma. Induction of Stress Proteins and Suppression of the Ubiquitin Proteasome System (UPS) in Myeloma Cells by ATO. Our proteomic analysis also showed that ATO induced a pronounced and global increase in heat shock proteins, including HSP90, HSP70, and HSP27 (Table 1). These changes likely reflect a stress response, which would be consistent with the well-documented role of these molecular chaperones in conferring protection against therapeutic agents. Immunoblotting confirmed the ATO-induced up-regulation of HSP27, HSP70, and HSP90 (Figure 6A). The functional significance of the upregulation of HSPs, in general, and, in particular, HSP90, in conferring a protective effect against therapeutic agents was

validated by the facts that inhibition of the HSP90-chaperoning function by inhibitor 17-AAG sensitized MM cells to ATOmediated apoptosis (Figure 6B) and enhanced the effect of 143-3ζ knock-down in U266 cells (Figure 6C). ATO also induced the decreased expression of proteins in the ubiquitin proteasome system (UPS) (Table 1). Notably, ATO down-regulated expression of a wide range of proteasome subunits, including proteasome activator subunit 3, proteasome subunit 5, 26S protease regulatory subunit 7 and proteasome subunit 8. The 20S proteasome activity, measured with a commercially available kit (see Experimental Procedures), was inhibited by 17%, 29%, and 40% in the cells treated with 1, 2, and 4 µM ATO, respectively (Figure 6D). This coordinated down-regulation of multiple proteasome subunits triggered by ATO suggests that the ability of ATO to suppress the UPS is partially responsible for the induction of apoptosis in U266 myeloma cells.

Discussion To date, studies in multiple myeloma investigating arsenicinduced apoptosis have used pathway-specific approaches; however, advances in technology now enable a more global approach to analyze the protein expression changes. This study is the first to employ proteomic technique to globally search for the dysregulated proteins induced by ATO in multiple myeloma cells. In this experiment, we carried out comparative Journal of Proteome Research • Vol. 8, No. 6, 2009 3013

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Figure 4. Effect of knock-down of 14-3-3ζ on apoptosis in U266 cells as measured by AnnexinV/PI fluorescence-activated cell sorting analysis at 48 h. Results are from one representative experiment of at least three. (A) Knock-down of 14-3-3ζ resulted in a marked increase in the number of apoptotic cells. X-axis, AnnexinV; y-axis, PI staining. Cell percentages are quantified in each quadrant. (I) Untreated cells; (II) control siRNA; (II) 14-3-3ζ siRNA; (IV) 1 µM ATO; (V) the combination of 1 µM ATO and control siRNA; (VI) the combination of 1 µM ATO and 14-3-3ζ siRNA. (B) Quantitative analysis of knock-down of 14-3-3ζ on apoptosis in U266 cell line. Data are expressed as the means ( SD of the fractions of apoptotic cells from at least three experiments. *Means of the 14-3-3ζ siRNA treated cells are significantly higher than those of the control siRNA-treated cells and of control cells (P < 0.03). **Means of the combination of ATO and 14-3-3ζ siRNA treated cells are significantly higher than those of the combination of ATO and control siRNA treated cells or of ATO treated cells (P < 0.02).

proteome analysis of U266 cells with the aim to identify clusters of proteins (and pathways) that showed differential expression 3014

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upon ATO treatment. Our data clearly showed that, in addition to targeting proteins involved in apoptotic pathways, ATO also

Arsenic-Induced Apoptosis in Multiple Myeloma

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Figure 5. The effect of overexpression of 14-3-3ζ on ATO-sensitivity in U266 cells. (A) The overexpression of 14-3-3ζ in ATO-treated U266 cells was confirmed by Western blotting analysis. (B) Overexpression of 14-3-3ζ reduces ATO-sensitivity in U266 cells as measured by AnnexinV/PI fluorescence-activated cell sorting analysis at 48 h. X-axis, AnnexinV; y-axis, PI staining. Cell percentages are quantified in each quadrant. The results are representative of three independent experiments. (I) Untreated cells; (II) cells transfected with blank vector; (III) cells transfected with 14-3-3ζ plasmid; (IV) cells treated with 1 µM ATO; (V) the combination of 1 µM ATO and blank vector; (VI) the combination of 1 µM ATO and 14-3-3ζ plasmid. (C) Quantitative analysis showed that the effect of ATO on U266 cells was modestly attenuated by 14-3-3ζ overexpression in transfectants. Columns are the mean of triplicate samples; bars, ( SD. *Means of the combination of 14-3-3ζ plasmid and ATO-treated cells are significantly lower than those of the combination of blank vector and ATO-treated cells or of ATO-treated cells (P < 0.02). Journal of Proteome Research • Vol. 8, No. 6, 2009 3015

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Figure 6. Stimulation of stress proteins and suppression of the ubiquitin proteasome system (UPS) in myeloma cells by ATO. Columns are the mean of three independent experiments; bars, ( SD; experiments were repeated at least three times with similar results. (A) Immunoblotting confirmed that ATO induced increase in levels of stress-response proteins HSP90, HSP70, and HSP27. (B) The HSP90 inhibitor 17-AAG (0.5 µM, 48 h) sensitized U266 cells to a subtoxic concentration of ATO (0.5 µM, 48 h). (C) Effect of knock-down of 14-3-3ζ in U266 cells was enhanced by 17-AAG treatment (0.5 µM, 48 h). (D) The 20S proteasome activity showed that the proteasomal activity was inhibited by 15%, 20%, and 40% in the cells treated with 1, 2, and 4 µM ATO, respectively. 3016

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Figure 7. Schematic diagram of apoptotic pathways activated by ATO treatment in myeloma cells.

altered the metabolic profile of the cancer cells to induce its anticancer effects. A better understanding of the mechanism whereby ATO mediates its therapeutic actions would certainly aid in the design of better therapeutic intervention. In particular, as suggested by bioinformational analysis, 143-3ζ may play a vital role in mediating ATO-induced apoptosis in multiple myeloma cells. 14-3-3 proteins are a family of multifunctional phosphoserine binding molecules that can serve as effectors of survival signaling.36 They are involved in a variety of important cellular processes that include cell cycle progression, growth, differentiation as well as apoptosis.37 The oncogenic role of 14-3-3ζ has been proposed in recent studies.38,39 By investigating the impact of the 14-3-3ζ knockdown on caspase-3 activity and Bcl-2 proteins, we showed that the apoptosis induced by the 14-3-3ζ knock-down appeared to be mediated in part by up-regulated Bad and Bax, coupled with decreased Mcl-1 and cleavage of Bid. This indicates that 14-3-3ζ can be placed in this network as acting upstream of the Bcl-2 proteins and effector caspases. In contrast to the 143-3ζ knock-down that increased the sensitivity of U266 myeloma cells to ATO, overexpression of 14-3-3ζ reduced ATOsensitivity in U266 cells. Together with the fact that RNAi-based 14-3-3ζ knock-down alone can lead to induction of apoptosis, development of small-molecule inhibitors of 14-3-3ζ may find the therapeutic applications on MM. Our proteomic and functional studies also demonstrated that ATO increased the expression levels of HSPs but suppressed UPS in myeloma cells. Much evidence implicated that HSPs and UPS are participants in keeping proteins folding correctly. They provide an effective protein quality control system essential for cellular functions and survival in many tissues.40 Dysfunction of these systems leads to protein aggregation and induces apoptosis in myeloma cells.41,42 It has been also demonstrated that the inhibition of proteasome can increase the expression of HSPs, implying that HSPs may act as a compensation of UPS or they work coordinatively to regulate the intracellular protein level.43 In this connection, our current finding on the reciprocal regulation of HSPs and UPS may

implicate that ATO-treated tumor cells attempt to compensate for the loss of proteasome activity by synthesizing new chaperones. In addition, we found that 17-AAG, a specific inhibitor of the chaperoning function of HSP90,44 sensitized MM cells to subtoxic concentrations of ATO. This effect can be attributed to the fact that HSP90 inhibitors suppress a wide constellation of antiapoptotic protective cellular pathways, thereby rendering cells more sensitive to various proapoptotic stimuli. The synergistic effect between the HSP90 inhibition and ATO induction confirms the functional significance of the upregulation of HSPs, in general, and, in particular, HSP90, as a protective mechanism against ATO-induced apoptosis and provides a possibility for combination treatments that will include HSP90 inhibitors in an effort to augment clinical efficacy and overcome clinical refractoriness to ATO.

Conclusions The current study characterized the molecular sequelae of ATO treatment in MM and defined apoptotic pathways triggered by this anticancer agent. Our results showed that ATO induced up-regulation of HSPs and down-regulation of 14-33ζ and the members of proteasome/ubiquitin system. Specifically, our in-depth functional studies suggested a dual apoptotic mechanism induced by ATO in MM cells, with a pivotal role for the 14-3-3ζ in one arm and the regulation of UPS in the other. The apoptotic cascades triggered by ATO can be schematically summarized in Figure 7. On the basis of this speculation, combining ATO treatment with 14-3-3ζ inhibition could improve clinical outcome. Similarly, coupling ATO or 143-3ζ inhibitor with agents that block the chaperoning function of HSP90 could also enhance the intervention efficacy of MM. These findings therefore not only shed light into the action mechanisms of ATO against MM cells, but also suggest therapeutic strategies to overcome potential clinical resistance to this antitumor agent. Abbreviations: MM, multiple myeloma; ATO, arsenic trioxide; 17-AAG, 17-allylamino- 17-demethoxygeldanamycin; IAA, Journal of Proteome Research • Vol. 8, No. 6, 2009 3017

research articles iodoacetamide; 2-DE, two-dimensional gel electrophoresis; MS/ MS, tandem MS; MW, molecular weight; PARP, poly (ADPribose) polymerase; SLO, streptolysin O; HSP, heat shock protein; UPS, ubiquitination-proteasome pathway.

Acknowledgment. This work was partially supported by the 2007 Chang-Jiang Scholars Program, “211” Projects, Talents Start-up Foundation of Jinan University (Grant 51207040) and Open Research Fund Program of the State Key Laboratory of Virology of China (Grant 2009003). Supporting Information Available: Supplementary Tables 1-4 and Supplementary Data 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Kyle, R. A.; Rajkumar, S. V. Multiple myeloma. N. Engl. J. Med. 2004, 351, 1860–1873. (2) Jemal, A.; Siegel, R.; Ward, E.; Murray, T.; Xu, J.; Smigal, C.; Thun, M. J. Cancer statistics. CA Cancer J. Clin. 2006, 56, 106–130. (3) Yang, L.; Parkin, D. M.; Ferlay, J.; Li, L.; Chen, Y. Estimates of Cancer Incidence in China for 2000 and Projections for 2005. Cancer Epidemiol. Biomarkers Prev. 2005, 14, 243–250. (4) Kyle, R. A.; Rajkumar, S. V. Multiple myeloma. Blood 2008, 15, 2962–2972. (5) Soignet, S. L.; Maslak, P.; Wang, Z. G.; Jhanwar, S.; Calleja, E.; Dardashti, L. J.; Corso, D.; DeBlasio, A.; Gabrilove, J.; Scheinberg, D. A.; Pandolfi, P. P.; Warrell, R. P. Complete remission after treatment of acute promyelocytic leukemia with arsenic trioxide. N. Engl. J. Med. 1998, 339, 1341–1348. (6) Perkins, C.; Kim, C. N.; Fang, G.; Bhalla, K. N. Arsenic induces apoptosis of multidrug-resistant human myeloid leukemia cells that express Bcr-Abl or overexpress MDR, MRP, Bcl-2, or Bcl-xL. Blood 2000, 95, 1014–1022. (7) Zhang, T. C.; Cao, E. H.; Li, J. F.; Ma, W.; Qin, J. F. Induction of apoptosis and inhibition of human gastric cancer MGC-803 cell growth by arsenic trioxide. Eur. J. Cancer 1999, 35, 1258–1263. (8) Ora, I.; Bondesson, L.; Jo¨nsson, C.; Ljungberg, J.; Po¨rn-Ares, I.; Garwicz, S.; Paˆhlman, S. Arsenic trioxide inhibits neuroblastoma growth in vivo and promotes apoptotic cell death in vitro. Biochem. Biophys. Res. Commun. 2000, 277, 179–185. (9) Rousselot, P.; Labaume, S.; Marolleau, J. P.; Larghero, J.; Noguera, M. H.; Brouet, J. C.; Fermand, J. P. Arsenic trioxide and melarsoprol induce apoptosis in plasma cell lines and in plasma cells from myeloma patients. Cancer Res. 1999, 59, 1041–1048. (10) Park, W. H.; Seol, J. G.; Kim, E. S.; Hyun, J. M.; Jung, C. W.; Lee, C. C.; Kim, B. K.; Lee, Y. Y. Arsenic trioxide-mediated growth inhibition in MC/CAR myeloma cells via cell cycle arrest in association with induction of cyclin-dependent kinase inhibitor, p21, and apoptosis. Cancer Res. 2000, 60, 3065–3071. (11) Kajiguchi, T.; Yamamoto, K.; Iida, S.; Ueda, R.; Emi, N.; Naoe, T. Sustained activation of c-jun-N-terminal kinase plays a critical role in arsenic trioxide-induced cell apoptosis in multiple myeloma cell lines. Cancer Sci. 2006, 97, 540–545. (12) Munshi, N. C.; Tricot, G.; Desikan, R.; Badros, A.; Zangari, M.; Toor, A.; Morris, C.; Anaissie, E.; Barlogie, B. Clinical activity of arsenic trioxide for the treatment of multiple myeloma. Leukemia 2002, 16, 1835–1837. (13) Hussein, M. A.; Saleh, M.; Ravandi, F.; Mason, J.; Rifkin, R. M.; Ellison, R. Phase 2 study of arsenic trioxide in patients with relapsed or refractory multiple myeloma. Br. J. Hamaetol. 2004, 125, 470–476. (14) Bahlis, N. J.; McCafferty-Grad, J.; Jordan-McMurry, I.; Neil, J.; Reis, I.; Kharfan-Dabaja, M.; Eckman, J.; Goodman, M.; Fernandez, H. F.; Boise, L. H.; Lee, K. P. Feasibility and correlates of arsenic trioxide combined with ascorbic acid-mediated depletion of intracellular glutathione for the treatment of relapsed/refractory multiple myeloma. Clin. Cancer Res. 2002, 8, 3658–3668. (15) Kurki, S.; Latonen, L.; Laiho, M. Cellular stress and DNA damage invoke temporally distinct Mdm2, p53 and PML complexes and damage-specific nuclear relocalization. J. Cell Sci. 2003, 116, 3917– 3925. (16) Lallemand-Breitenbach, V.; Zhu, J.; Puvion, F.; Koken, M.; Honore´, N.; Doubeikovsky, A.; Duprez, E.; Pandolfi, P. P.; Puvion, E.; Freemont, P.; de The´, H. Role of promyelocytic leukemia (PML) sumolation in nuclear body formation, 11S proteasome recruitment, and As2O3-induced PML or PML/retinoic acid receptor alpha degradation. J. Exp. Med. 2001, 193, 1361–1371.

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K. C. Aggresome induction by proteasome inhibitor bortezomib and tubulin hyperacetylation by tubulin deacetylase (TDAC) inhibitor LBH589 are synergistic in myeloma cells. Blood 2006, 108, 3441–3449. (43) Kim, D.; Kim, S. H.; Li, G. C. Proteasome inhibitors MG132 and lactacystin hyperphosphorylate HSF1 and induce hsp70 and hsp27 expression. Biochem. Biophys. Res. Commun. 1999, 254, 264–268. (44) Mitsiades, C. S.; Mitsiades, N. S.; McMullan, C. J.; Poulaki, V.; Kung, A. L.; Davies, F. E.; Morgan, G.; Akiyama, M.; Shringarpure, R.; Munshi, N. C.; Richardson, P. G.; Hideshima, T,; Chauhan, D.; Gu, X.; Bailey, C.; Joseph, M.; Libermann, T. A.; Rosen, N. S.; Anderson, K. C. Antimyeloma activity of heat shock protein-90 inhibition. Blood 2006, 107, 1092–1100.

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