Scholarly article on topic 'Labeling of Oxidizable Proteins with a Photoactivatable Analog of the Antitumor Agent DMXAA: Evidence for Redox Signaling in Its Mode of Action'

Labeling of Oxidizable Proteins with a Photoactivatable Analog of the Antitumor Agent DMXAA: Evidence for Redox Signaling in Its Mode of Action Academic research paper on "Biological sciences"

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Abstract of research paper on Biological sciences, author of scientific article — Romy Brauer, Liang-Chuan S. Wang, See-Tarn Woon, David J.A. Bridewell, Kimiora Henare, et al.

Abstract The signaling pathway(s) and molecular target(s) for 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a tumor vascular disrupting agent in late stages of clinical development, are still undefined. As an approach toward identifying potential targets for DMXAA, a tritiated azido-analog of DMXAA was used to probe for cellular binding proteins. More than 20 cytosolic proteins from murine splenocytes, RAW 264.7 cells, and the HECPP immortalized endothelial cells were photoaffinity-labeled. Although no protein domain, fold, or binding site for a specific ligand was found to be shared by all the candidate proteins, essentially all were noted to be oxidizable proteins, implicating a role for redox signaling in the action of DMXAA. Consistent with this hypothesis, DMXAA caused an increase in concentrations of reactive oxygen species (ROS) in RAW264.7 cells during the first 2 hours. This increase in ROS was suppressed in the presence of the antioxidant, N-acetyl-L-cysteine, which also suppressed DMXAA-induced cytokine production in the RAW 264.7 cells with no effects on cell viability. Short interfering RNA (siRNA)-mediated knockdown of one of the photoaffinity-labeled proteins, superoxide dismutase 1, an ROS scavenger, resulted in an increase in tumor necrosis factor-α production by RAW 264.7 cells in response to DMXAA compared with negative or positive controls transfected with nontargeting or lamin A/C-targeting siRNA molecules, respectively. The results from these lines of study all suggest that redox signaling plays a central role in cytokine induction by DMXAA.

Academic research paper on topic "Labeling of Oxidizable Proteins with a Photoactivatable Analog of the Antitumor Agent DMXAA: Evidence for Redox Signaling in Its Mode of Action"

NEOPLASIA

www.neoplasia.com

Volume 12 Number 9 September 2010 pp. 755-765 755

Abstract

The signaling pathway(s) and molecular target(s) for 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a tumor vascular-disrupting agent in late stages of clinical development, are still undefined. As an approach toward identifying potential targets for DMXAA, a tritiated azido-analog of DMXAA was used to probe for cellular binding proteins. More than 20 cytosolic proteins from murine splenocytes, RAW 264.7 cells, and the HECPP immortalized endothelial cells were photoaffinity-labeled. Although no protein domain, fold, or binding site for a specific ligand was found to be shared by all the candidate proteins, essentially all were noted to be oxidizable proteins, implicating a role for redox signaling in the action of DMXAA. Consistent with this hypothesis, DMXAA caused an increase in concentrations of reactive oxygen species (ROS) in RAW 264.7 cells during the first 2 hours. This increase in ROS was suppressed in the presence of the antioxidant, N-acetyl-L-cysteine, which also suppressed DMXAA-induced cytokine production in the RAW 264.7 cells with no effects on cell viability. Short interfering RNA (siRNA)-mediated knockdown of one of the photoaffinity-labeled proteins, superoxide dismutase 1, an ROS scavenger, resulted in an increase in tumor necrosis factor-a production by RAW 264.7 cells in response to DMXAA compared with negative or positive controls transfected with nontargeting or lamin A/C-targeting siRNA molecules, respectively. The results from these lines of study all suggest that redox signaling plays a central role in cytokine induction by DMXAA.

Neoplasia (2010) 12, 755-765

Labeling of Oxidizable Proteins with a Photoactivatable Analog of the Antitumor Agent DMXAA: Evidence for Redox Signaling in Its Mode of Action1,2

Romy Brauer* See-Tarn Woon*

-*'3, Liang-Chuan S. Wang*'3, * David J.A. Bridewell*, Kimiora Henare*, Dieter Malinger*, Brian D. Palmer*, Stefanie N. Vogef, Claudine Kieda*, Sofian M. Tijono* and Lai-Ming Ching*

^Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand; "University of Maryland, Baltimore, Baltimore, MD, USA; ^Centre de Biophysique Moleculaire, CNRS UPR4301, Orleans, France

Introduction

A number of low-molecular tumor vascular-disrupting agents are in late clinical evaluation. Most of these agents, including the combretastatins, taxanes, and vinca alkaloids, have disruption ofnormal tubulin polymerization in endothelial cells as their primary mode of action [1]. Tubulin does not seem to be a primary target for 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a small molecule that has both vascular-disrupting activity and cytokine modulatory activity. DMXAA was synthesized at the Auckland Cancer Society Research Centre [2] as a derivative of flavone acetic acid (FAA), a flavonoid initially synthesized by Lyonnaise Industrielle Pharmaceutique (LIPHA, Lyon, France) as part of their antiinflammatory program [3]. When FAA was tested by the National Cancer Institute, Bethesda, MD, it showed curative properties against a number of transplantable murine tumors that were resistant to current chemotherapies [4]. A hallmark activity of DMXAA and of FAA is the

rapid onset of hemorrhagic necrosis of the implanted tumors [5], resulting from vascular collapse [6], caused by the induction of apoptosis

Abbreviations: DMXAA, 5,6-dimethylxanthenone-4-acetic acid; FCS, fetal calf serum; LPS, lipopolysaccharide; NAC, N-acetyl-L-cysteine; 5-AzXAA, 5-azidoxanthenone-4-acetic acid; DTT, dithiothreitol; ROS, reactive oxygen species; IEF, isoelectric focusing Address all correspondence to: Associate Professor Lai-Ming Ching, Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, New Zealand. E-mail: l.ching@auckland.ac.nz

1The work was funded by a Health Research Council of New Zealand grant to L.-M.C.

and a National Institutes of Health grant (AI-18797) to S.N.V.

2This article refers to a supplementary material, which is designated by Figure W1 and

is available online at www.neoplasia.com.

3R.B. and L.-C.S.W. contributed equally to this article.

Received 5 May 2010; Revised 14 June 2010; Accepted 15 June 2010

Copyright © 2010 Neoplasia Press, Inc. All rights reserved 1522-8002/10/$25.00 DOI 10.1593/neo.10636

selectively in tumor vascular endothelial cells [7]. After the initial direct antivascular effects, a large panel of cytokines are produced [8,9], leading to a cascade of secondary host antitumor responses. Tumor necrosis factor-a (TNF-a), itself a potent vascular-disrupting agent [10], is suggested to amplify and prolong the direct antivascular effects of DMXAA and FAA [11,12], whereas the production of type 1 interferons (IFNs) [13] has been attributed to systemic increases in tumor-specific CD8+ T lymphocytes [14]. More recently, the major influx of neutrophils into tumors after DMXAA treatment was suggested to be linked to the production of chemokines that included IFN-Y-inducible protein 10, RANTES, macrophage inflammatory protein-1(MIP-1), and monocyte chemoattractant protein-1 [8,15].

The molecular mechanism of cytokine induction by DMXAA is not fully understood, although there is strong evidence for the involvement of the nuclear factor (NF) kB pathway [16-18], as well as the TANK-binding kinase 1 (TBK1)-interferon (IFN) regulatory factor 3 (IRF-3) signaling axis [13]. Previous studies from our laboratory using tritiated-DMXAA indicated that the compound diffused rapidly into cells [18], but specific binding to any cellular proteins could not be determined because of the low affinity of binding of the compound. To overcome this problem, photoactivatable azido-analogs of DMXAA were synthesized in an approach to photoaffinity label potential target proteins [19]. Azido substitution at the 5- or 6- position of the xanthenone ring produced analogs capable of inducing NF-kB activation and cytokine production in cultured splenocytes and inducing hemorrhagic necrosis of tumors in mice [19]. Those studies indicated that the azido-analogs had the same profile of activities as DMXAA and were therefore likely to have the same target(s). Covalent bonds formed between the azido-compound and the interacting proteins after photoactivation were predicted to overcome the problems of the reversible low-affinity binding that occur with DMXAA and its target(s). The receptors for a number of drugs including verapamil [20] and paclitaxel [21] were successfully located using a photoaffinity labeling approach. We report here studies using a tritiated azido-XAA analog to photoaffinity label potential DMXAA-binding proteins. More than 20 oxidizable proteins were labeled, leading to the hypothesis that DMXAA may be acting through modulation of redox signaling. Subsequent studies measuring concentrations of reactive oxygen species (ROS) in cells and the effect of the antioxidant N-acetyl-L-cysteine (NAC) on DMXAA-induced cytokine production support this hypothesis.

Materials and Methods

Drugs and Reagents

DMXAA was synthesized as the sodium salt at the Auckland Cancer Society Research Centre [2] and dissolved in a-minimal essential medium (a-MEM). 5-Azidoxanthenone-4-acetic acid (5-AzXAA) was also synthesized at the center [19] and was dissolved in acetonitrile. For photoaffinity labeling experiments, 5-AzXAA was custom radiolabeled with tritium by AmBios Labs, Inc (Newington, CT) to display a specific activity of 0.1 Ci/mmol. NAC (Sigma-Aldrich, Inc, St Louis, MO) was dissolved in a-MEM.

Preparation of Cell Lysates

Murine RAW 264.7 macrophage-like cell line was maintained in a-MEM supplemented with 10% (vol./vol.) fetal calf serum (FCS),

100 U/ml penicillin-G, and 100 p.g/ml streptomycin sulfate at 37°C in a humidified atmosphere of 5% CO2/air. The murine HECPP endothelial cell line [22] was maintained in M199 medium supplemented with FCS (10%) and antibiotics. Murine splenocytes were obtained from C57BL/6 mice after cervical dislocation. Spleen cells were collected, and red blood cells were removed by osmotic lysis.

All cells were lysed with potassium phosphate buffer (8.02 mM K2HPO4 and 1.98 mM KH2PO4, pH 7.4) in the presence of 0.5% Nonidet-P40 (Roche Product Ltd, Auckland, New Zealand) and protease inhibitor cocktail from Sigma-Aldrich. Protein concentrations in the lysates were determined by the Bradford assay [23]. Aliquots were stored at -80°C until use.

Photoaffinity Labeling and Gel Electrophoresis

Cell lysates (150 Mg) were incubated with 1.5 Mg of [3H]-5-AzXAA for 30 minutes on ice and UV-irradiated for 10 minutes (Stratlinker 1800; Stratagene, La Jolla, CA). The samples were then precipitated using 2D Clean-up Kit (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's instructions. The resulting protein pellets were resuspended in 125 Ml of rehydration buffer (9.8 M urea, 2% wt./vol. CHAPS, 0.5% vol./vol. ampholyte, pH 3-10 NL, 0.002% wt./vol. bromophenol blue, 2.8 mg/ml dithiothreitol [DTT] in H2O) and subjected to two-dimensional PAGE using 7-cm isoelec-tric focusing (IEF) strips containing an immobilized nonlinear pH gradient ranging from pH 3 to 10 (Immobiline DryStrips pH 3-10 NL;

Figure 1. (A) SDS-PAGE gel of RAW 264.7 proteins incubated with [3H]-5-AzXAA without UV treatment (lane 2), UV-treated (lane 3), pre-incubated for 30 minutes with 10, 100, and 500-fold excess cold DMXAA (lanes 4, 5 and 6 respectively), and preincubated with 10-and 100-fold excess cold 5-AzXAA before UV treatment (lanes 7 and 8). (B) Autoradiograph of the gel showing radioactive protein bands (0.45 jg of [3H]-5-AzXAA was used for 45 jg of proteins and 30 jg of protein was loaded per lane).

Figure 2. The autoradiograph (left) and the Coomassie Blue-stained two-dimensional gel (right) of lysates from murine splenocytes (A), RAW264.7 cells (B), or HECPP endothelial cells (C), photoreacted with [3H]-5-AzXAA with protein spots that matched the radiolabeled spots from the autoradiograph encircled.

GE Healthcare Bio-Sciences, Uppsala, Sweden). After overnight gel re-hydration, IEF was carried out at 20°C with a current limit of 50 pA per strip using the Ettan IPGphor IEF System (Amersham Biosciences, Uppsala, Sweden). The focused IEF strips were equilibrated in 2.5 ml of equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% wt./vol. glycerol, 2% wt./vol. SDS, and 0.002% wt./vol. bromophenol blue) containing 10 mg/ml DTT, followed by alkylation in 2.5 ml of equilibration buffer containing 25 mg/ml iodoacetamide for 15 minutes each. Equilibrated IEF strips were loaded onto 12% SDS-polyacrylamide gels, and electrophoresis was performed in a Mini PROTEAN 3 cell (Bio-Rad Laboratories, Hercules, CA) for 1.5 hours at 120 V. Two-dimensional gels were stained with Coomassie Blue and soaked in Amplify Fluorographic Solution (Amersham Biosciences, Buckinghamshire, England) for 30 minutes before transferring onto 3-mm filter paper, and vacuum-dried. The dried amplify-treated gels were exposed to autoradiographic film (Kodak BioMax XAR Scientific Imaging Film; Carestream Health, Paris, France) at -80°C for 8 weeks. After autoradi-

ography, films were developed and overlaid on the dried Coomassie Blue-stained gels to locate radiolabeled protein spots.

In-gel Digestion and Mass Spectrometry for Protein Identification

Protein spots that had been radiolabeled were excised from fresh two-dimensional gels. Gel pieces were destained in 0.1 M ammonium bicarbonate/50% acetonitrile (45 minutes at 37°C), dehydrated in 100% acetonitrile, dried in a vacuum centrifuge for 5 minutes, and rehydrated in 50 pi of 20 mM DTT/0.1 M ammonium bicarbonate for 30 minutes at 56°C. After another dehydration step in 100% acetonitrile, gel pieces were incubated with 50 pl of 55 mM iodoacetamide/ 0.1 M ammonium bicarbonate for 15 minutes at room temperature in the dark. Subsequently, the gel pieces were washed with 0.1 M ammonium bicarbonate, followed by a dehydration step (100% acetonitrile), and another wash with milli-Q water. After a final dehydration step

Table 1. Proteins in Murine Cellular Lysates Photoaffinity Labeled with 5-AzXAA.

MW (kDa)

Mascot Score

SwissProt Accession Number

(A) Spleen cell lysates

Proteins with known role in cytokine production S100A9/Calgranulin B/MRP14 Cyclophilin A Superoxide dismutase Protein disulfide isomerase A3 precursor Cytoskeletal proteins ß-Actin ß-Tubulin

Tropomyosin a chain Proteins that alter cytoskeleton dynamics Rho GDP-dissociation inhibitor 1 Rho GDP-dissociation inhibitor 2 Cofilin-1 Profilin-1 Coronin-1A Miscellaneous proteins Hemoglobin a-chain Hemoglobin ß-chain Albumin

SH3 domain-binding glutamic acid-rich-like protein 3

Chloride intracellular channel protein 1

(B) RAW 264.7 cell lysates

Proteins with known role in cytokine production Macrophage migration inhibitory factor Thioredoxin 1 Peroxiredoxin 1

60 kDa heat shock protein, mitochondrial Protein disulfide isomerase A6 precursor Superoxide dismutase [Mn], mitochondrial Chaperones/stress proteins Stress-70 protein, mitochondrial Prostaglandin E synthase 3, cytosolic Glycolytic enzymes Glyceraldehyde-3-phosphate dehydrogenase Aldose reductase Triosephosphate isomerase L-Lactate dehydrogenase A chain Cytoskeletal proteins ß-Actin a-Tubulin ß-Tubulin Vimentin Miscellaneous proteins Inositol monophosphatase 26S protease regulatory subunit 6B Chloride intracellular channel protein 1

(C) HECPP cell lysates

Proteins with known role in cytokine production Cyclophilin A Thioredoxin 1 Peroxiredoxin 1

60 kDa heat shock protein, mitochondrial Protein disulfide isomerase A1 precursor Macrophage migration inhibitory factor Chaperones/stress proteins Prostaglandin E synthase 3, cytosolic Calreticulin Glycolytic enzymes Glyceraldehyde-3-phosphate dehydrogenase Triosephosphate isomerase Cytoskeletal proteins a-Tubulin ß-Tubulin ß-Actin Vimentin

Proteins that alter cytoskeleton dynamics

Rho GDP-dissociation inhibitor 1 Miscellaneous proteins Albumin

ATP synthase ß chain Nucleophosmin

22 18 16

110,11,12

13,14 12

1 6 11

13.0 18.0 16.0

50.1 32.9

23.3 22.8 18.6 15.0 51.5

15.0 15.7

71.2 10.5

12.7 12.0

22.4 61.1

36.1 36.1

30.8 47.4

18.0 12.0

22.4 61.1

57.5 12.7

50.1 42.0

71.2 56.2 32.7

6.73 7.88 6.03 5.98

5.29 4.78 4.68

4.97 8.26 8.50 6.05

8.08 7.26 5.82 5.02

6.90 4.80 8.26

5.91 5.00 8.8

5.91 4.36

8.44 6.71 6.90 8.44

5.29 4.96 4.79 5.06

5.08 5.18

7.88 4.80 8.26 5.91 4.79 6.9

4.36 4.33

8.44 6.90

4.96 4.78 5.29 5.06

5.82 5.15 4.62

80 187

38 193

155 273 206

76 47 142 123

95 817

94 207 237

338 89

60 120 55 98

87 224 287 234

163 82 94 297 356 70

359 527 198 69

64 134 138

P31725 P17742 P08228 P11598

Q6ZWM3

P68372

P58771

P31725

P17742

Q99PT1

P18760

P62962

P01942 P02088 P07724 Q91VW3

Q921Q5

P34884 P10639 P35700 P63038 Q922R8 P09671

P38647 Q9R0Q7

P16858 P45376 P17751 P06151

P60710 P68373 P68372 P20151

055023 P54775 Q9Z1Q5

P17742 P10639 P35700 P63038 P09103 P34884

Q9R0Q7 P14211

P16858 P17751

P68373 P68372 Q6ZWM3 P20151

P31725

P07724 P56480 Q61937

Table 1. (continued)

Protein Spot* MW (kDa) pi Mascot Score ^ SwissProt Accession Number

Proteasome subunit a6 10 27.8 6.34 234 Q9QUM9

SH3 domain-binding glutamic acid-rich-like 13 10.5 5.02 45 Q91VW3 protein 3

*Spots as in Figure 2A (splenocytes), Figure 2B (RAW 264.7 cells), or Figure 2C (HECPP cells); spots 15, 19, 20, and 21 (Figure 2B; RAW 264.7 cells) and spots 8, 18, 20, 22, 23, and 24 (Figure 2C; HECPP cells) could not be identified.

^Mascot score of 30 or greater indicates identity or extensive homology (P < .05).

with 100% acetonitrile, the gel pieces were vacuum-dried for 5 minutes. The dried gel pieces were left to absorb 15 |l of trypsin solution (50 ng/|l trypsin [sequencing grade; Promega, Madison, WI], 0.1 M ammonium bicarbonate, 10% acetonitrile) for 10 minutes, after which 30 |il of 0.1 M Tris-HCl (pH 9.2)/10% acetonitrile was added, and left overnight at 37°C. The supernatants were collected the following day, and the peptides were extracted by two incubations in 150 |l of 0.1% trifluoroacetic acid/60% acetonitrile at 37°C for 30 minutes each. The peptide extracts were reduced in volume to 1 to 2 |l by vacuum centrifugation. Fifteen microliters of solvent A (0.1% acetic acid/0.005% heptafluorobutyric acid) was added, and samples were processed using a high-performance liquid chromatography system coupled to an ion trap mass spectrometer (Agilent 1100 Series LC/ MSD Trap System; Agilent Technologies, Boeblingen, Germany). A 0.5 x 150-mm Zorbax SB C18 column (Agilent Technologies) was pre-equilibrated with solvent A and kept at a constant temperature of 2°C, onto which 8 |l of peptide samples was injected. Peptides were eluted off the column at a flow rate of 12 | l/min using a linear gradient from 90% solvent A (0.1% acetic acid, 0.005% heptafluorobutyric acid in water) and 10% solvent B (0.1% acetic acid, 0.005% heptafluorobutyric acid, 80% acetonitrile in water) 70% solvent B for 45 minutes. The eluted peptides were directly fed into the electrospray ionize of the mass spectrometer, with a spray voltage of 3.5 kV. The electrospray interface was set in positive mode, the nebulizer gas was set at 12 psi, and the drying gas (nitrogen) was delivered at a flow rate of 4.4 L/min at a temperature of325°C. Ion mass spectra were collected in the range of 200 to 2000 m/z with a threshold of 15,000. The LC/ MSD Trap 5.2 software (DataAnalysis version 3.2; Bruker Daltonik, Agilent Technologies) was used to identify compounds for each ion mass spectrum. The resulting data were entered into the Mascot MS/ MS Ion Search Engine [24] and compared with spectra in the SwissProt database (www.expasy.ch/sprot).

Measurement of Intracellular ROS

Intracellular ROS concentrations were determined by oxidation of 2',7'-dichlorodihydrofluorescein. RAW 264.7 cells (106 cells/well) cultured in 24-well plates were incubated for different periods with DMXAA (10 |g/ml). The cells were washed and incubated in the dark for 20 minutes in PBS containing 0.5% FCS (vol./vol.) and H2DCF diacetate (Sigma-Aldrich). After another wash, the cells were resuspended in saline. The mean fluorescence intensity (excitation, 488 nm; emission, 515-540 nm) was measured using flow cytometry (FACScan Flow Cytometer; BD Bioscience, San Diego, CA).

Effects of NAC on DMXAA Activity

RAW 264.7 cells were seeded in triplicate at in flat-

bottomed 96-well plates and preincubated with NAC (1-20 mM) for

1 hour. DMXAA (10 Mg/ml final concentration) was then added, and ROS was measured after 2 hours of incubation at 37°C. Culture supernatants were collected 8 hours after the addition of DMXAA and assayed using ELISA cytokine kits (OptEIA murine IL-6 and murine TNF-a; BD Biosciences) or with a multiplex cytokine kit (catalog no. MCYT0-70K; Linco Research, St Charles, MO) and a Luminex 100 instrument (Luminex Corporation, Austin, TX). Viability of the cells was determined using the sulforhodamine assay [25]. Each treatment was assayed in triplicate, and results were expressed as mean ± SEM. Data between two groups were compared using unpaired Student t test or ANOVA if multiple comparisons were made and were considered significant when the P value was <.05.

RNA Interference of SOD1

A pool of four predesigned small interfering RNA (siRNA) molecules ("SmartPool") targeting murine SOD1 were purchased from Dharmacon, Inc (Thermo Fisher Scientific, Inc, Lafayette, CT), together with the positive control siRNA molecules targeting lamin A/C, and the negative control nontargeting siRNA molecule no. 2. SiRNA molecules were introduced into cells at 40 nM using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). RAW 264.7 cells (5 x 105/well) were seeded onto the preformed transfection complexes in six-well plates in OPTIMEM medium (Invitrogen) without serum. At 4 hours after transfection, a-MEM supplemented with 20% FCS was added to each well, and the cells were allowed to grow. At 48 hours after transfection, the cells were treated with DMXAA (10 Mg/ml) for 4 hours, after which the supernatant was harvested for determination of TNF-a concentrations using ELISA, whereas the cells were washed in ice-cold PBS and their proteins were extracted using RIPA buffer containing 1 x Halt protease cocktail inhibitor (Thermo Scientific, Rockford, IL). The lysates were used for immunoblot analysis to assess the degree of knockdown of the target protein. Samples (25 Mg protein/well) were electrophoresed using precast NuPAGE Novex Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane that was blocked in PBS containing 0.5% Tween 20 (PBS-T) and 5% nonfat dried milk powder. Membranes were incubated overnight at 4°C with rabbit anti-SOD1 primary antibodies (Santa Cruz, San Diego, CA) diluted at 1:2500 and then for 1 hour at room temperature with HRS-conjugated secondary antibodies (Santa Cruz) diluted at 1:10,000 in PBS-T containing 5% milk powder. Signals were detected using SuperSignal West Pico Chemiluminescent substrate (Pierce Thermo Scientific, Rockford, IL), and images were captured on a Fujifilm LAS 3000 imaging system (Fujifilm, Tokyo, Japan). The blots were stripped in Restore Western Blot Stripping Buffer (Pierce Thermo Scientific) before reblocking in PBS-T containing 5% nonfat dried milk powder for determination of loading using a mouse monoclonal antibody to actin (Milli-pore, Billerica, MA).

Results

Specificity of Labeling with 5-AzXAA

The specificity of the photoaffinity labeling with [3H]-5-AzXAA was examined using competitive binding studies with cold DMXAA or cold 5-AzXAA (Figure 1). Cytosolic protein extracts from RAW 264.7 cells were preincubated with up to 500-fold excess concentrations of cold 5-AzXAA or cold DMXAA before the addition of [3H]-5-AzXAA. The extracts were then exposed to UV irradiation and then analyzed by SDS-PAGE and autoradiography. The intensity of the radiolabeling was markedly decreased in the presence of 100- and 500-fold excess cold DMXAA (Figure 1B, lanes 5 and 6) when compared with the cytosolic protein extracts that were irradiated without competitor (lane 3) and was completely blocked by 10-and 100-fold excess cold 5-AzXAA (lanes 7 and 8).

Photoaffinity Labeling of Cytosolic Proteins

Cytosolic proteins from murine splenocytes and RAW 264.7 macrophage-like cells, used previously for studying cytokine induction [8,13], and the HECPP endothelial cells, used previously for studying apoptosis induction by DMXAA [7], were photoaffinity-labeled with

[3H]-5-AzXAA and resolved by two-dimensional PAGE. The two-dimensional gels were exposed to radiographic film, after which the radiolabeled protein spots were located by overlaying the autoradiograph onto the respective two-dimensional gel. The autoradiograph and the corresponding Coomassie Blue-stained gel from a representative experiment with murine splenocytes, RAW 264.7 cells, or HECPP cells are shown in Figure 2, A, B, and C, respectively. Each autoradiograph (Figure 2, left) shows a number of darkened spots that could be matched with protein spots on the Coomassie Blue-stained two-dimensional gel (Figure 2, right). Protein spots that were radiolabeled were excised for identification using mass spectrometry and Mascot Search against spectra in SwissProt database. The proteins identified corresponding to each experiment in Figure 2 are listed in Table 1.

The separation of proteins was not influenced by incubation with [3H]-5-AzXAA or by UV irradiation. Coomassie Blue-stained control two-dimensional gels of protein samples that had not been exposed to [3H]-5-AzXAA or irradiation (Figure W1A), treated with UV-light only (Figure W1B), or incubated with [3H]-5-AzXAA without photoactivation (Figure W1C) all showed a similar pattern of distribution of protein spots.

Table 2. Oxidizable Proteins Photoaffinity-Labeled with 5-AzXAA.

Protein Murine RAW 264.7 Murine Splenocytes Murine HECPP Oxidative Thiol Modifications*

a-Tubulin • • D

ß-Tubulin • G, D

ß-Actin • • G, D

40S Ribosomal protein 5A -

60 kDa heat shock protein, mitochondrial • G

Heat shock cognate 71 kDa protein G

Albumin • • G, D

Alcohol dehydrogenase [NADP+] -

Aldose reductase • G

Annexin A1/2 G

ATP synthase a chain -

Calreticulin D

Cathepsin B • G

Chloride intracellular channel protein 1 • • D

Cofilin-1A • G

Coronin-1A • G or D

Cyclophilin A • • G

Elongation factor-1 • G

Glyceraldehyde-3-phosphate dehydrogenase • G, D

Hsc70-interaction protein • • -

Inorganic pyrophosphatase • G

Inositol monophosphatase -

L-Lactate dehydrogenase A chain • G, D

Macrophage migration inhibitory factor G

Nucleophosmin G

Peroxiredoxin-1 • G, D

Phosphoglycerate mutase 1 G or D

Profilin-1A • G

Prostaglandin E synthase 3 • -

Proteasome subunit a • -

Protein disulfide isomerase A6 • • G

Rho GDP-dissociation inhibitor 1/2 • G

S100A9 • G

SH3 domain-binding glutamic acid-rich-like protein 3 • • -

Stress-70 protein, mitochondrial • • G

Superoxide dismutase [Cu/Zn] • D

Superoxide dismutase [Mn] • D

Thioredoxin 1 • • • G

Triosephosphate isomerase • • G, D

Tropomyosin a chain • • G, D

Vimentin • • G

*Denotes sensitivity to oxidation as determined by oxidative modification by glutathionylation (G) or protein disulfide bond formation (D) of cysteine residues.

Figure 3. (A) Accumulation of ROS after the addition of DMXAA (10 ug/ml) in cultures of RAW 264.7 cells in three independent experiments. (B) Effect of 1 hour of preincubation with NAC (20 mM) on ROS concentrations in RAW 264.7 cells untreated or after treatment with DMXAA (10 ug/ml) for 2 hours. Mean ± SEM of triplicate cultures. (C) Effect of 1 hour of pretreatment with various concentrations of NAC on cell viability (•), TNF-a (o), and IL-6 production (▼) in cultures of RAW 264.7 cells stimulated 8 hours with DMXAA (10 ug/ml). Mean ± SEM of triplicate cultures. ^Statistical significance compared with cultures treated with DMXAA only and no NAC (P < .05, one way ANOVA). (D) Cytokine levels in RAW 264.7 cell cultures without treatment (black bar), preincubated with NAC (1 hour, 20 mM) (open bars), or cultured 8 hours with DMXAA (10 ug/ml) without preincubation with NAC (hatched bars) or with preincubation with NAC (striped bars). Mean ± SEM of triplicate cultures. ^Statistical significance compared with untreated cultures. #Statistical difference compared with cultures treated with DMXAA but without NAC preincubation. 'Value above maximum detection limit.

A minimum of three independent experiments was carried out with each cell type. A list of the identified proteins compiled from all the experiments for each cell type is presented in Table 2. Spots 12 and 13 from Figure 2A, identified as hemoglobin a and hemoglobin P, respectively, were not included in the final list because they most likely represent contaminants from red blood cells in the original spleen suspension and were not consistently detected in repeat experiments. A total of 24, 18, and 30 labeled proteins were identified for RAW 264.7 cells, splenocytes, and HECPP cells, respectively (Table 2). Of these, eight proteins (thioredoxin 1, stress-70 protein, SH3 domain-binding glutamic acid-rich-like protein-3, protein di-sulfide isomerase A3/6, Hsc70-interaction protein, albumin, P-actin, and a-tubulin) were detected from lysates from all three cell types (Table 2), although albumin is likely a contaminant from tissue culture. Almost all of the photoaffinity-labeled proteins have been reported to be oxidizable, either by glutathionylation and/or by forming disulfide bonds at one of their cysteine residues in response to oxidative stress [26-36].

of ROS in RAW 264.7 cells. Intracellular concentrations of ROS increased during the first 2 hours after the addition of DMXAA in three independent experiments (Figure 3A). Preincubation with the antioxidant NAC decreased background concentrations of ROS and reduced DMXAA-induced ROS concentrations (Figure 3B). We next tested the ability of NAC to modulate DMXAA-induced TNF-a and IL-6 production in RAW 264.7 cells. At the concentrations tested, NAC had no effects on cell viability but reduced the production of both TNF-a and IL-6 induced with DMXAA in a dose-dependent manner (Figure 3C ). Using a 32-plex cytokine assay, 10 cytokines (G-CSF, IL-6, IFN-Y-inducible protein 10, keratinocyte chemoattrac-tant, monocyte chemoattractant protein-1, MIP-1a, MIP-1P, MIP-2, RANTES, and TNF-a) from the panel were found to be induced by DMXAA in the RAW 264.7 cells. Supernatants from cultures pre-incubated with NAC before the addition of DMXAA had lower concentrations of all 10 cytokines (Figure 3D). NAC alone did not induce cytokines (Figure 3D). Concentration of cytokines in the entire panel assayed is presented in Table 3.

Modulation of Cellular ROS Concentrations by DMXAA and Its Effects on Cytokine Production

The observation that oxidizable proteins were preferentially labeled using 5-AzXAA led us to investigate whether modulation of redox signaling was involved in DMXAA-mediated cytokine production. We measured DMXAA-induced changes in intracellular concentrations

SiRNA Knockdown of SOD1

RNA interference was used to knock down the expression of SOD1, a protein with antioxidant functions that was photoaffinity-labeled in both RAW 264.7 cell and spleen cell extracts (Table 2), to examine the effect of reducing its expression on TNF-a induction by DMXAA. Because SOD1 is a scavenger of ROS, we hypothesized

that knockdown of SOD1 would attenuate ROS scavenging activity in the cells, resulting in higher ROS concentrations and increased TNF-a production. Consistent with the hypothesis, in four independent experiments, DMXAA-induced TNF-a production in cultures of SOD1 knockdown cells was significantly higher than that of the control cultures of cells transfected with the nontargeting negative control siRNA molecules (P = .003, P < .001, P < .001, and P = .003) or cells transfected with the lamin A/C-positive control molecules (P = .007, P = .007, P = .002, and P = .003). In addition, in all experiments, RAW 264.7 cells transfected with the negative nontargeting control siRNA molecule or the positive control siRNA molecule targeting lamin A/C showed similar levels of TNF-a production as those treated with Lipofectamine 2000 alone, and each was lower than that of untransfected cells. TNF-a levels from a representative experiment are shown in Figure 4A, together with the Western blot of SOD1 in the protein extracts from the various treatment groups (Figure 4B).

Discussion

The present study sought to identify the cellular target protein(s) of DMXAA, a vascular-disrupting agent that is currently undergoing phase 3 clinical evaluation, but whose mode of action is still only partly understood. To this end, a photoaffinity labeling approach was taken using tritiated 5-AzXAA as the photoactive ligand. The specificity of this method was confirmed in competitive binding ex-

Table 3. Cytokine Production in RAW264.7 Cells Untreated, NAC Only, or DMXAA-Treated with or without Previous Incubation with NAC.

Cytokine Concentration (pg/ml)*

Untreated NAC DMXAA DMXAA + NAC

Eotaxin 6 ± 0 6 ± 0 9 ± 1 6 ± 0

G-CSF 7 ± 1 6 ± 0 392 ± 79 40 ± 24

GM-CSF 6 ± 0 6 ±0 8 ± 1 8 ± 1

IFN-y 6 ± 0 6 ± 0 9 ± 0 6 ± 0

IL-10 6 ± 0 6 ±0 6 ± 0 6 ± 0

IL-12(p40) 6 ± 0 6 ±0 6 ± 0 6 ± 0

IL-12(p70) 6 ± 0 6 ±0 19 ± 1 10 ± 1

IL-13 6 ± 0 6 ±0 6 ± 0 6 ± 0

IL-15 6 ± 0 6 ±0 6 ± 0 6 ± 0

IL-17 6 ± 0 6 ±0 26 ± 3 14 ± 3

IL-1a 6 ± 0 6 ± 0 27 ± 8 7 ± 0

IL-1 ß 19 ± 2 44 ±4 27 ± 5 51 ± 6

IL-2 7 ± 1 7 ±1 12 ± 0 8 ± 1

IL-3 6 ± 0 6 ±0 6 ± 0 6 ± 0

IL-4 6 ± 0 6 ±0 6 ± 0 6 ± 0

IL-5 6 ± 0 6 ±0 27 ± 3 11 ± 1

IL-6 6 ± 0 6 ±0 1002 ± 129 309 ± 117

IL-7 6 ± 0 6 ±0 6 ± 0 6 ± 0

IL-9 126 ± 19 70 ± 16 146 ± 2 92 ± 11

IP-10 17 ± 1 6 ±0 7609 ± 904 2636 ± 402

KC 6 ± 0 6 ± 0 311 ± 23 20 ± 4

LIF 6 ± 0 6 ±0 11 ± 1 8 ± 1

LIX 6 ± 0 6 ±0 287 ± 43 121 ± 44

M-CSF 6 ± 0 6 ±0 10 ± 1 8 ± 2

MCP-1 546 ± 42 141 ± 11 3781 ± 145 1149 ± 152

MIG 6 ± 0 6 ±0 11 ± 4 6 ± 0

MIP-1a 161 ± 7 113 ±3 25,171 ± 0 8766 ± 1897

MIP-1ß 75 ± 5 29 ±7 19,834 ± 5337 8183 ± 418

MIP-2 300 ± 16 315 ± 26 4078 ± 347 2779 ± 651

RANTES 8 ± 2 6 ±0 698 ± 13 39 ± 3

TNF-a 8 ± 1 6 ±0 3052 ± 1494 431 ± 98

VEGF 313 ± 19 6 ±0 355 ± 25 6 ± 0

*Mean ± SEM of three cultures per treatment group.

GM-CSF indicates granulocyte/macrophage colony-stimulating factor; MIG, monokine induced by IFN-y; VEGF, vascular endothelial growth factor.

periments with splenocyte extracts [19] and with RAW 264.7 cellular lysates (Figure 1).

A total of 24, 18, and 30 proteins were successfully identified in this study as being photoaffinity-labeled with [3H]-5-AzXAA in cytosolic extracts from RAW 264.7 cells, murine splenocytes, and HECPP cells, respectively. In terms of their broad physiological function, the labeled cytosolic proteins included those with a known role in cytokine production, cytoskeletal proteins, proteins that alter cytoskeleton dynamics, chaperones, glycolytic enzymes, and proteins with miscellaneous functions (Table 1). This large number of potential DMXAA target proteins was unexpected, particularly because the two-dimensional gel system used was capable of resolving only the more abundant cellular proteins. Essentially all the labeled proteins shared a common feature, namely, oxidizable thiols. This conclusion was derived from literature reports showing that those proteins could undergo thiol-specific oxidative modification through glutathionylation and/or di-sulfide bridge formation, on exposure of the cell to oxidative stress [26-36], and led us to consider that DMXAA may interact with target proteins through their accessible and oxidizable thiol groups, for example, cysteine residues. Determination of whether the photoaffinity label is indeed linked to peptide fragments containing cysteine residues is planned. Of note, the cytoskeletal proteins actin and tubulin were among the eight proteins that were photoaffinity-labeled in all cell types, and treatment of endothelial cells with DMXAA has been shown to cause partial dissolution of the actin cytoskeleton, which may be part of its antivascular action [37].

Although the results here suggest that 5-AzXAA with UV irradiation, covalently binds to cellular proteins in vitro, it is not yet known whether such adducts are formed with this class of compounds under physiological conditions in vivo. Adduct formation between proteins and xenobiotics, including taxol [21], 1,4-benzoquinone, and 1,4-naphthoquinone [38] or endogenous compounds, such as dopamine or its metabolite dihydroxyphenylacetic acid [39], is widely reported in the literature. Covalent binding of DMXAA to target proteins conceivably may also occur. In this regard, DMXAA and its predecessor FAA have been proposed to undergo intramolecular protonation to form pyrylium-type cationic salts, which would be expected to display a high affinity for electrons and be capable of electron transfer [40]. Furthermore, oxidation of FAA generates carbon radical species after decarboxylation [41], which could also lead to covalent bond formation with proteins. DMXAA also decarboxylates in solution when exposed to sunlight [42]. Agents capable of electron transfer are able to transmit an electron to oxygen and generate a wide range of ROS. The formation of ROS in RAW 264.7 cells in response to DMXAA (Figure 3B) supports the idea that DMXAA might indeed be capable of electron transfer. The acetic acid group at positions 4 and 8, respectively, is essential for the formation of DMXAA- and FAA-derived pyrylium salts [40] and for the generation of radicals after a decarboxylation pathway [41]. Interestingly, structure-activity studies of xanthone and flavone analogs showed an absolute requirement for the acetic acid group at those positions for analogs with antitumor activity [2,3]. It is not known at this point whether oxidation of DMXAA can occur spontaneously under physiological conditions or is an enzymatically catalyzed process.

The discovery that oxidizable proteins were preferentially labeled in all three cell types (Table 2) suggests that DMXAA might act through the modulation of redox signaling. Many key effects of DMXAA are inducible by redox signaling: reorganization of the actin cytoskeleton and stimulation of apoptosis in endothelial cells [43], activation of

Figure 4. (A) TNF-a in culture supernatants untreated and DMXAA-treated (300 ug/ml) in RAW 264.7 cells that had been transfected using Lipofectamine 2000 with siRNA molecules to SOD1, negative control nontargeting molecules, positive control lamin A/C targeting molecules, or cells treated with Lipofectamine 2000 only or untransfected cells. (B) Immunoblots showing SOD1 protein levels in the various siRNA treatment groups.

NF-kB [44], and TNF-a production [45]. DMXAA induced an increase in the generation of ROS in RAW 264.7 cells, and preincubation with the antioxidant NAC reversed both ROS generation and induction of a number of cytokines, including TNF-a by DMXAA (Figure 3), consistent with the hypothesis that ROS play a central role in its molecular action. SiRNA knockdown of SOD1, a direct scavenger of ROS, increased TNF-a production in response to DMXAA (Figure 4), again consistent with the involvement of ROS in DMXAA-induced cytokine production.

The suggestion that DMXAA induces cytokine production through modulation of redox signaling does not rule out that activation of classic signaling pathway(s) after binding to yet unidentified receptor(s) also occurs. In many inflammatory responses, cytokine production can occur through both ROS-dependent and ROS-independent pathways, and lipopolysaccharide (LPS)-mediated TNF-a production would be a prime example [45]. The ROS-dependent pathway of NF-KB-mediated transcriptional activation of the TNF gene after exposure to LPS in RAW 264.7 cells was shown to be responsible for approximately 50% of the TNF-a produced [45].

Synergistic TNF-a production by human peripheral blood leukocytes and murine splenocytes in culture in response to DMXAA has been shown with the addition of a costimulator [46,47]. Agents that were effective as costimulators with DMXAA for TNF-a production included LPS, IL-1, phorbol myristate acetate, and okadaic acid at suboptimal concentrations that did not, by themselves, induce the cy-

tokine [46,47]. Interestingly, all these compounds have been reported to promote the production of ROS [44,48,49]. Approaches that increase cellular ROS concentrations might provide beneficial strategies for improving the activity of DMXAA. Of note, the phase 3 evaluation of DMXAA is in combination with carboplatin and paclitaxel (ATTRACT-1 and ATTRACT-2; Novartis, Basel, Switzerland). Both carboplatin [50] and paclitaxel have been demonstrated to induce the generation of ROS, with the accumulation of hydrogen peroxide being crucial for paclitaxel-induced cancer cell death in vivo [51].

In summary, the studies here support a role of redox signaling in the action of DMXAA. Whether the observed increase in ROS is a direct or indirect effect of the compound and the identification of the enzymes and mechanisms involved in generating a radical species from DMXAA under physiological conditions require further investigation.

Acknowledgments

The authors thank Aron B. Fisher and Steven M. Albelda, University of Pennsylvania, and Tobias P. Dick, German Cancer Research Centre, for their valuable discussion and critical assessment of the article.

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* • *

# « 1 . mm

Figure W1. Coomassie Blue-stained two-dimensional gels of HECPP cellular lysates without exposure to radiation or [3H]-5-AzXAA (A), treated with UV radiation but no or [3H]-5-AzXAA (B), or incubated with or [3H]-5-AzXAA but without radiation (C).