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Carcinogenesis Advance Access originally published online on January 12, 2008
Carcinogenesis 2008 29(3):610-619; doi:10.1093/carcin/bgn014
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Chemopreventive agents modulate the protein expression profile of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone plus benzo[a]pyrene-induced lung tumors in A/J mice

Fekadu Kassie*, Lorraine B. Anderson1, LeeAnn Higgins1, Yunqian Pan, Ilze Matise, Mesfin Negia, Pramod Upadhyaya, Mingyao Wang and Stephen S. Hecht

University of Minnesota Cancer Center, Mayo Mail Code 806, 420 Delaware Street SE, MN 55455, USA
1 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, MN 55455, USA

* To whom correspondence should be addressed. Tel: +1 612 626 5143; Fax: +1 612 626 5135; Email: kassi012{at}umn.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
We used isobaric tag labeling coupled with mass spectrometry to compare the relative abundance of proteins in lung tumors from A/J mice treated with a mixture of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene versus normal mouse lung tissues. Levels of 59 proteins changed—30 increased and 29 decreased—in tumor tissues versus normal tissues. Among proteins that showed increased levels in tumor tissues versus normal tissues were glycolytic enzymes, ribosomal proteins, fatty acid synthase, cathepsins D and H and carbonic anhydrase 2. On the other hand, the levels of cytochrome P450 enzymes 2B10 and 2F2, glutathione S-transferases mu-1, procollagen VI, Clara cell 10 kDA (CC10) protein, histones, receptor advanced glycation end product, and lung carbonyl reductase were lower in tumor tissues versus normal lung tissues. Upon dietary administration of a combination of N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine plus myo-inositol or indole-3-carbinol to carcinogen-treated mice, the relative abundance of 60S ribosomal protein L4 and carbonic anhydrase in tumor tissues decreased whereas that of histones, glutathione S-transferases mu, receptor advanced glycation end product, transglutaminase, and procollagen VI increased. Western assays with lung tissue homogenates not only verified the proteomics results for selected proteins but also showed differential expression of hypoxia inducible factor-1{alpha}, a transcription factor for most of the proteins that showed changes in relative abundance. This is the first report on the application of quantitative proteomics to study the relative abundance of proteins in a mouse model of lung carcinogenesis. These proteins may have utility for development of candidate lung cancer biomarkers and as targets of chemopreventive/chemotherapeutic agents.

Abbreviations: ACN, acetonitrile; BaP, benzo[a]pyrene; CC10, Clara cell 10 kDA; CI, confidence interval; EF, error factor; FAS, fatty acid synthase; HIF-1{alpha}, hypoxia inducible factor-1{alpha}; iTRAQ, isobaric tags for relative and absolute quantitation; I3C, indole-3-carbinol; MI, myo-inositol; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; PEITC-NAC, N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine; PSPB, pulmonary surfactant-associated protein B; SCX, strong cation exchange


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Lung cancer is the most common cause of death from cancer worldwide (1). In the USA, an estimated 213 380 new cases and 160 390 deaths are expected in 2007, accounting for 15% of cancer diagnoses and 29% of all cancer deaths, respectively (2). Treatment of lung cancer has generally been unsuccessful as the majority of lung cancers are diagnosed at an advanced stage when prognosis is poor. Since ~90% of lung cancer deaths are attributed to tobacco smoking, prevention of smoking initiation and smoking cessation are the best approaches to decrease lung cancer mortality. However, there are still 1.3 billion smokers in the world (3). Moreover, former smokers have a higher lung cancer risk than non-smokers. The heavy burden of lung cancer and the failure of standard approaches to reduce mortality have motivated an interest in identification of lung cancer signature proteins and chemopreventive agents. The signature proteins could be used as biomarkers for early detection of the disease and targets of the chemopreventive agents.

The human diet contains a variety of promising cancer preventive compounds. Among these are isothiocyanates, indole-3-carbinol (I3C) and myo-inositol (MI). Isothiocyanates and I3C are derived from glucosinolate precursors in cruciferous vegetables. MI occurs in a wide variety of food plants or is formed from inositol hexaphosphate in the gastrointestinal tract by the action of phytase (4). Considerable evidence indicates that isothiocyanates and I3C inhibit carcinogenesis induced by various classes of carcinogens at many sites (5). MI inhibits tumorigenesis in the mammary gland, colon and lung (6).

Until recently, the approach to the identification of carcinogenesis-related proteins has been a reductionist biochemical analysis method in which the effects of single or a few proteins are examined at a time. However, cancer is not a disease of changes in one protein but dozens of proteins acting in concert. With the advent of proteomics, analysis of large-scale expression of proteins became possible. The proteomics approach, at the very least, could guide the traditional biochemical approach towards proteins most worthy of attention among thousands of newly identified proteins. In earlier studies, we used a proteomics approach—isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry—to compare protein expression patterns in whole lung tissues of mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) plus benzo[a]pyrene (BaP), the carcinogens and I3C or vehicle (7). The principle behind the iTRAQ approach is that a set of four isobaric reagents is used to differentially isotopically label, in parallel, tryptic peptides from up to four samples by forming an amide linkage to any peptide amino groups thereby enabling simultaneous quantitation of changes in protein levels under four biological conditions (8). Due to the isobaric mass design of the iTRAQ reagents, differentially labeled peptides appear as single peaks in mass spectrometry scans. When iTRAQ-tagged peptides are subjected to tandem mass spectrometry analysis, the mass balancing carbonyl moiety is released, thereby liberating isotope-encoded reporter ions that provide relative quantitative information on proteins.

Here, we extended our study and examined, using the same approach, changes in the relative abundance of proteins in lung tumors versus normal lung tissues, and determined whether protein levels in tumor tissues were modulated by the chemopreventive agents N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine (PEITC-NAC) plus MI or I3C. We also attempted to identify potential lung tumor biomarker proteins in the serum, which might have been released from the tumor tissue into the blood. We showed that (i) the relative abundance of several lung tumorigenesis-related proteins changed in lung tumor tissues versus normal lung tissues, (ii) potential surrogate biomarker proteins were released from the tumor tissue into the blood and (iii) PEITC-NAC plus MI or I3C modulated the levels of tumor proteins in lung tissues or sera. To our knowledge, there are no other published studies that have used proteomics techniques to investigate protein levels in lung tumors and sera of laboratory animals treated with lung carcinogens and chemopreventive agents.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Chemicals, reagents and diets
BaP, MI, I3C, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, MgCl2, NaCl and protease inhibitor cocktail were obtained from Sigma (St Louis, MO). Urea was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). PEITC-NAC and NNK were synthesized (9,10). iTRAQTM reagent kits were obtained from Applied Biosystems (ABI, Foster City, CA). All reagents and primary and secondary antibodies for western assays were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse diets (AIN-93G and AIN-93M) were purchased from Harlan Teklad (Madison, WI). AIN-93G diet, high in protein and fat, was used to support rapid growth of the mice from the time they arrived until they were 15 weeks old, whereas AIN-93M diet, low in protein and fat, was given to maintain growth of the animals beginning from the time the mice were 15 weeks old until the termination of the experiment (11). Chemiluminescent immunodetection reagents and autoradiography films were purchased from Pierce (Rockford, IL) and Denville Scientific (Metuchen, NJ), respectively.

Animals and treatments
Lung tissues (normal and tumor) were from earlier chemoprevention studies with PEITC-NAC plus MI or I3C (7,12). Briefly, female A/J mice, 5–6 weeks of age, obtained from The Jackson Laboratory (Bar Harbor, ME) were housed in the specific pathogen-free animal quarters of Research Animal Resources, University of Minnesota Academic Health Center. Eighty mice were randomly allocated into four groups (20 mice per group) as follows: group 1, vehicle treated; group 2, PEITC-NAC plus MI and NNK plus BaP treated; group 3, I3C and NNK plus BaP treated and group 4, NNK plus BaP treated. The mice were maintained for 1 week on AIN-93G pelleted diet and then switched to AIN-93G powdered diet and treated by gavage with either a mixture of BaP plus NNK (2 µmol of each, groups 2, 3 and 4) in 0.1 ml cottonseed oil or cottonseed oil alone (group 1), once weekly for eight treatments. Mice in groups 2 and 3 were given the powdered diet supplemented with PEITC-NAC (15 µmol/g diet) plus MI (56 µmol/g diet) or I3C (112 µmol/g diet) as described elsewhere (7,12), respectively, beginning 1 day after the fourth treatment with the carcinogens (50% carcinogen treatment) until sacrifice at 19 weeks after the last dose of carcinogen. The experimental design is depicted in Figure 1A. The selection of the 50% point of carcinogen treatment to begin administration of the chemopreventive agents was intended to model the fact that smokers normally would not begin using chemopreventive agents in their early years of smoking. Immediately upon sacrifice by CO2 asphyxiation, blood was collected by cardiac puncture and lungs were perfused with cold phosphate-buffered saline and harvested. The tumors were counted and excised. Normal lungs from vehicle-treated mice and excised lung tumors from carcinogen-treated mice were frozen in liquid nitrogen and stored at –80°C.


Figure 1
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  		Fig. 1. Study design for effects of PEITC-NAC plus MI on lung tumor induction by NNK plus BaP in A/J mice (A) and workflow of iTRAQ experiment (B).

 
Protein extraction and iTRAQ labeling
Aliquots of normal lungs from eight mice (30 mg/mouse) or excised tumors from mice in groups 2, 3 and 4 were pooled (a total of 240 mg, from eight mice per group) and pulverized using a mortar and pestle. The pulverized tissues from each group of mice were suspended in 100 µl lysis buffer [15 mM MgCl2, 50 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (pH 7.4), 150 mM NaCl, 8 M urea, 0.1% Triton] and a cocktail of protease inhibitors [1 µM leupeptin, 0.1 µM pepstatin A, 0.1 µg/ml aprotinin and 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride], sonicated for 1 min and centrifuged at 22 000 g for 20 min. Recovered supernatants were frozen at –80°C. The protein content of the samples was determined (13) and iTRAQ labeling carried out as described elsewhere (7). Briefly, 100 µg of protein (contained in ~5 µl of sample) from each group of mice was mixed with 90 µl 0.5 M triethylammonium bicarbonate buffer (pH 8.5), denatured with 0.05% sodium dodecyl sulfate, reduced with 0.5 mM tris-(2-carboxyethyl)phosphine for 1 h at 37°C and cysteine residues were then blocked (2 mM methylmethanethiosulfonate at room temperature for 10 min). Subsequently, the samples were digested with trypsin with a protein to enzyme ratio of 20:1 (wt/wt) at 37°C overnight. The samples were dried and protein digests from vehicle-treated, carcinogen and PEITC-NAC plus MI-treated, carcinogen and I3C-treated and carcinogen-only-treated mice were labeled with iTRAQ reagents 114, 115, 116 and 117, respectively, by incubating at room temperature for 1 h. Labeled samples were pooled, dried, resuspended in 0.1% trifluoroacetic acid and applied to a Sep-Pak C18 cartridge (Waters Corporation, Milford, MA) to remove buffer, trypsin and salts. The iTRAQ workflow is shown in Figure 1B.

Strong cation exchange chromatography
The combined peptide mixture was fractionated by strong cation exchange (SCX) chromatography on a Magic 2002 high-performance liquid chromatography system (Michrom Bioresources, Auburn, CA) using a polysulfoethyl ATM (Poly LC, Columbia, MD) column (150 x 1.0 mm ID, 5 µm particle size and 300 Å pore size) as described elsewhere (7). The iTRAQ-labeled peptides were next rehydrated in solvent A [(20% vol/vol acetonitrile (ACN), 5 mM KH2PO4 (pH 3.2)] and loaded onto the SCX column. Peptides were eluted with a linear gradient of 0–20% solvent B (solvent A plus 500 mM KCl) >40 min and from 20 to 100% solvent B for 20 min. The column flow rate was 37 µl/min. Absorbance was monitored at 214 and 280 nm. Fractions were collected at 3 min intervals and dried in vacuo. Fractions with mAU280 > 2, fractions 13 through 23, were analyzed in the second dimension by reversed phase liquid chromatography/mass spectrometry/mass spectrometry.

Reversed phase liquid chromatography/mass spectrometry/mass spectrometry analysis
Each dried SCX fraction was reconstituted with reversed phase load buffer (98:2, water:ACN, 0.1% formic acid) and injected onto a Dionex/LC Packings (LCP, Sunnyvale, CA) capillary liquid chromatography system, online with a QSTAR Pulsar i mass spectrometer (Applied Biosystems) as described previously (14). Briefly, peptides were loaded onto an LCP C18 nano-precolumn (0.3 mm internal diameter x 5 mm length) and desalted with load buffer for 17 min at 35 µl/min. Peptides were then eluted onto a 75 µm ID capillary C18 analytical column with a linear gradient from 0 to 35% solvent B where solvent B was 5:95 water/ACN and 0.1% formic acid and solvent A was 95:5 water/ACN, 0.1% formic acid for 40 min, 35–80% solvent B over 5 min and 80–100% solvent B over 2 min. Product ion spectra were collected in an information dependent acquisition mode. Information dependent acquisition mode settings included continuous cycles of one full scan from m/z 400–1100 (1.5 s) plus four product ion scans from m/z 50–2000 (3 s each). Precursor m/z values were selected from a peak list automatically generated by Analyst QS software (ABI) from the time-of-flight mass spectrometry scan during acquisition, starting with the most intense ion.

The iTRAQ experiment was carried out three times (three technical replicates) using aliquots from the same pooled lung tissue homogenates on different days. All steps of sample processing, including iTRAQ labeling, were done three times.

Data processing
ProteinPilotTM 2.0 software (Applied Biosystems) with the Paragon Algorithm (15) was used for the identification and relative abundance quantification of proteins. Tandem mass spectrometry data were searched against the mouse protein database from 3-2-2005 containing 115 658 protein sequences (Celera Discovery System, ABI) and separately against the NCBI mouse protein database plus common contaminants (179 proteins) from 12-12-2006, for a total of 107 806 protein sequences. The search parameters were 95% confidence for protein identification threshold, trypsin as enzyme, methylmethanethiosulfonate-labeled cysteines as fixed modification and rapid ID as search method. Common modifications such as oxidation of methionine and deamidation of asparagine were included automatically as variable modifications in every search. Using ProteinPilot, the relative abundance of each peptide in samples from carcinogen-only-treated versus that of vehicle-control mice was determined by dividing signature-ion peak areas at m/z 117 by signature-ion peak areas at m/z 114. Similarly, the level of peptides in samples from carcinogen and PEITC-NAC plus MI-treated or carcinogen and I3C-treated mice relative to that of samples from mice treated with carcinogens only was calculated by dividing signature-ion peak areas at m/z 115 and m/z 116, respectively, by signature-ion peak areas at m/z 117. Peptides shared among related, but distinct, proteins were not used in quantitation. ProGroup Algorithm (within ProteinPilot) was used to compile the results from the database searches into protein groups and to report protein-based ratios of relative abundance. ProteinPilot calculates average iTRAQ ratios and estimates the P-value and error factor (EF) for each protein hit. The EF defines the quantitative 95% confidence interval (CI) of a ratio, which is the range within which the true protein ratio is 95% probably to fall. The 95% CI for quantitation is calculated as follows: lower border of 95% CI = protein ratio/EF; upper border of 95% CI = protein ratio x EF. ProteinPilot determined protein ratios only if all four iTRAQ reporter ions could be detected. To correct for experimental errors, the median protein ratio was calculated for all proteins reported, adjusted to unity and then the same bias factor was applied to all ratios (within ProteinPilot). This normalizing factor is based on the assumption that most of the protein levels in the tumor tissues should be similar to those from normal lung tissues with the exception of those that are related to carcinogenesis. Only proteins identified with a minimum of two peptides, quantitation results with P-value <0.05 and EF <2, which implies that the certainty of change varies by a factor <2, were considered to be statistically different from unity.

Western analysis
Western immunoblotting studies were carried out using lung tissue homogenates prepared from pooled lungs or tumors as described for the proteomics assay or from pooled sera (eight mice per group). Serum was diluted 3-fold with phosphate-buffered saline before being used in the assay. After determining the protein content of the samples, 40 µg of protein obtained from tissue homogenates or sera were loaded onto a 4–12% Novex Tris-glycine gel (Invitrogen, Carlsbad, CA) and run for 100 min at 125 V. The proteins were then transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA) for 2 h at 30 V. Subsequently, membranes were blocked in 5% Blotto non-fat dry milk in Tris buffer containing 1% Tween-20 for 1 h and probed overnight with the following primary antibodies at a 1:200 ratio of antibody to milk: anti hypoxia inducible factor-1{alpha} (HIF-1{alpha}), anti-fatty acid synthase (FAS), anti-L-plastin, anti-cathepsin D, anti-pulmonary surfactant-associated protein B (PSPB) and anti-caveolin-1. After incubating with an appropriate secondary antibody for 1 h (1:5000 donkey anti-goat IgG for FAS, L-plastin, cathepsin-D and PSPB; 1:5000 goat anti-rabbit IgG for HIF-1{alpha} and caveolin-1), chemiluminescent immunodetection was employed using a kit from Pierce. Signal was visualized by exposure of membranes to HyBolt CL autoradiography film from Denville Scientific. Membranes were stripped and probed with anti-β-actin to check for differences in the amount of protein loaded in each lane. Relative band densities were quantified using the U-Scan-It software (Silk Scientific, Orem, UT) and normalized relative to total protein to compensate for experimental variation.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
2D liquid chromatography/mass spectrometry/mass spectrometry analyses of iTRAQ-labeled lung tissue samples led to identification of carcinogenesis-related proteins
Three iTRAQ experiments were carried out on different days, each time using aliquots (technical replicates) from homogenates of pooled (eight mice per group) normal lungs or lung tumors. We identified 488, 579 and 799 proteins at >95% confidence in iTRAQ experiments 1, 2 and 3, respectively, based on one or more peptide hits per protein. In experiment 3, we were able to identify a higher number of proteins, and new proteins not identified in experiments 1 and 2, by injecting SCX fractions in two portions (50% each time). Following exclusion of proteins identified with a single peptide, the number of proteins identified in iTRAQ experiments 1, 2 and 3 decreased to 248, 298 and 484 proteins, respectively.

The original protein lists generated by ProteinPilot were further filtered as follows: minimum of two peptides for the calculation of iTRAQ ratios; ratios with EF <2 (SD < 20%) and P-values <0.05; exclusion of highly abundant acute phase liver proteins. Moreover, on the basis of our previous studies (7) and reports of others (16–19), only changes beyond 20% (>1.2 or <0.8) from unity, the median ratio of all reported proteins, were considered real changes. Figure 2 depicts an example—carbonic anhydrase 2—of protein identification and quantitation. The b- and y-ion series in the tandem mass spectrometry spectrum (Figure 2A) of the peptide fragment DFPIANGDR were used to identify carbonic anhydrase 2. The iTRAQ reporter ion peak areas at m/z 114, 115, 116 and 117 of the four peptide fragments (DFPIANGDR, TLNFNEEGDAEEAMVDNWRPAQPLK, AVQQPDGLAVLGIFLK and QSPVDIDTATAHHDPALQPLLI-SYDK, Figure 2A and D) were used to measure the relative amount of carbonic anhydrase 2 in normal lung tissues from vehicle controls and tumor tissues from mice treated with carcinogen and PEITC-NAC plus MI, carcinogen and I3C or carcinogen only, respectively.


Figure 2
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  		Fig. 2. (A) An example of tandem mass spectrometry spectra that exhibited significant differences in iTRAQ ratios (inset). The reporter ion signals of DFPIANGDR (bold faced and underlined), a peptide fragment from carbonic anhydrase 2, appear in the low-mass region of the spectrum and are used to determine the relative amount of carbonic anhydrase 2 in lung tissues of mice treated with vehicle control (iTRAQ 114), carcinogen and PEITC-NAC plus MI (iTRAQ 115), carcinogen and I3C (iTRAQ 116) or carcinogen only (iTRAQ 117). (B–D) Reporter ion signals for other peptide fragments (bold faced in Figure 2A) of carbonic anhydrase 2.

 
The relative abundance of tumor-related proteins changed in lung tumors
Using the above criteria, we found reproducible changes in the relative abundance of 59 proteins in lung tumor tissues from mice treated with the carcinogens only compared with normal lungs from vehicle controls (Table I). Of these proteins, 30 (51%) increased and 29 (49%) decreased. The levels of 16 proteins were changed in only one out of three iTRAQ experiments and therefore were not included in Table I. Proteins that increased in abundance in lung tumors from carcinogen-treated mice versus normal lungs from vehicle controls (Table IA and C) were those involved in glyolysis (L-lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, glucose phosphate isomerase 1, triose phosphate isomerase and transketolase), protein synthesis (40S ribosomal proteins S16 and S7 and 60S ribosomal protein L4), lipid synthesis (FAS and ATP citrate synthase), extracellular matrix metabolism (cathepsins D and H), pH regulation (carbonic anhydrase 2), proteasome pathway (ubiquitin conjugation enzyme), cell structure and motility (L-plastin, keratin, cytokeratin endo A, coronin and guanosine triphosphatase-activating-like protein) and stress-mediated chaperonage (heat shock protein 90, 170 kDa glucose-regulated protein and protein disulfide isomerase). Levels of several proteins were reduced in lung tumors versus normal lungs (Table IA and C) including cytoskeletal proteins (alpha actin, myosin heavy chain protein, vinculin, alpha catenin and moesin), xenobiotic metabolism enzymes (cytochrome P450 2B10, cytochrome P450 2F2 and glutathione S-transferases mu 1), extracellular matrix protein (procollagen VI), calcium-binding proteins (calmodulin, transglutaminase 2, spectrin, cytochrome C oxidase and annexin A6), histones, lung carbonyl reductase, Clara cell 10 kDA (CC10) protein, receptor advanced glycation end product, angiotensin converting enzyme and platelet glycoprotein IV.


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  		Table I. Changes in the relative abundance of proteins in lung tissues

 
PEITC-NAC plus MI or I3C modulated the levels of carcinogenesis-related proteins
Dietary administration of PEITC-NAC plus MI- or I3C- to carcinogen-treated mice led to modulation of the levels of 19 proteins, including three proteins that did not change in abundance in tumor tissues versus normal tissues (Table IC). Among these 19 proteins, the levels of 60S ribosomal protein L4, somatic cytochrome C, carbonic anhydrase 2 and pzp protein decreased, whereas the relative abundances of lamin A, histones, lysozyme C, procollagen VI, transglutaminase 2, calmodulin, receptor advanced glycation end product, lung carbonyl reductase and glutathione S-transferases mu 1 increased in tumors from carcinogen and PEITC-NAC plus MI- or I3C-treated mice relative to the levels in tumor tissues from mice treated with carcinogens only (Table IC).

Immunoblotting verified changes in protein levels found by iTRAQ and revealed other tumorigenesis-related proteins
Selected proteins that showed changes in relative abundance in lung tumors versus normal lungs upon iTRAQ analyses (FAS, L-plastin, cathepsin D, PSPB and caveolin-1) were validated by western immunoblots. The rationale for selection of these proteins was that the levels of L-plastin (20), FAS (21) and cathepsin D (22) are elevated in various human cancer tissues and have been used as biomarkers and molecular targets for chemotherapeutic agents. Although PSPB and caveolin-1 were identified in one iTRAQ run only and therefore not included in Table I, these proteins play an important role in lung carcinogenesis (23,24). Thus, we analyzed, using western assay, the levels of PSPB and caveolin-1 in lung tissues of the different groups of mice.

As shown in Figure 3A and C, the levels of FAS, L-plastin, cathepsin D and PSPB increased 3.6-, 1.9-, 3.3-, and 3.1-fold, respectively, whereas caveolin-1 decreased by 40% in lung tumors from carcinogen-only-treated mice compared with normal lungs from vehicle controls. Generally, these data are in agreement with the iTRAQ data. The relative abundance of FAS, cathepsin D and PSPB was reduced (by 63, 36 and 38%, respectively) in tumors from carcinogen and PEITC-NAC plus MI-treated mice and from carcinogen and I3C-treated mice (by 71, 62 and 77%, respectively) relative to that found in tumors from the corresponding controls (treated with carcinogens only) (Figure 3D). The effect of I3C was consistently stronger than that of PEITC-NAC plus MI. The levels of L-plastin and caveolin-1 were modulated neither by PEITC-NAC plus MI nor I3C. The western results for L-plastin were in agreement with the iTRAQ data but not that of FAS and cathepsin D, presumably due to differences in protein isoforms. Although PSPB and caveolin-1 were identified in only one out of three iTRAQ experiments, the changes in iTRAQ and western assays were of similar magnitude.


Figure 3
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  		Fig. 3. Verification of changes determined in iTRAQ experiment by western immunoblotting. (A) Representative immunoblot, out of three experiments, showing treatment-related changes in the levels of HIF-1{alpha}, FAS, L-plastin, PSPB, cathepsin D and caveolin-1 in mouse lung tissues; 1, normal lung tissues from vehicle controls; 2, tumors from carcinogen and PEITC-NAC plus MI-treated mice; 3, tumors from carcinogen and I3C-treated mice; 4, tumors from mice treated with carcinogens only. (B) Representative immunoblot, out of three experiments, showing treatment-related changes in the levels of PSPB and cathepsin D in mouse sera. (C) Mean levels of HIF-1{alpha}, FAS, L-plastin, PSPB, cathepsin D and caveolin-1 in lung tumor tissues relative to those in normal lung tissues. (D) Mean levels of HIF-1{alpha}, FAS, L-plastin, PSPB, cathepsin D and caveolin-1 in lung tumor tissues from carcinogen and PEITC-NAC plus MI-treated mice (white columns) or carcinogen and I3C-treated mice (dark columns) relative to those in tumors from mice treated with the carcinogens alone. (E) Average levels of PSPB and cathepsin D in the sera of mice treated with the carcinogens relative to those in vehicle controls. (F) Average levels of PSPB and cathepsin D in the sera of mice treated with carcinogens and PEITC-NAC plus MI (white columns) or carcinogen and I3C (dark columns) relative to those treated with carcinogens only. *P<0.05; **P<0.005.

 
It was noteworthy that many of the differentially expressed proteins in lung tumors such as glycolytic proteins, carbonic anhydrase, cathepsins, cytoskeletal proteins and transglutaminase are regulated by hypoxia inducible factor-1{alpha} (HIF-1{alpha}), a transcription factor that responds to hypoxia (25). Therefore, we assayed levels of HIF-1{alpha} in lung tissues from the different groups of mice using western assays. Interestingly, the level of HIF-1{alpha} was 4.2-fold higher in tumors from carcinogen-only-treated mice compared with normal lung tissues from vehicle controls (Figure 3A and C) and was reduced by 55 and 61% in tumors from mice treated with carcinogens and PEITC-NAC plus MI and carcinogens and I3C, respectively, relative to the levels in tumors from mice treated with carcinogens only (Figure 3D). HIF-1{alpha} was presumably not detected in iTRAQ studies due to a low-expression level.

Tumor-associated proteins are believed to be shed from tumor cells into the blood. However, the study of the blood proteome is challenging due to the relatively low abundance of potential biomarker proteins in the presence of several high-abundance proteins such as serum albumin. Therefore, we used western immunoblotting to examine the presence and level in the serum of L-plastin, FAS, PSPB and cathepsin D. As shown in Figure 3B and E, we identified PSPB and cathepsin D in the serum and their levels were 5- and 13-fold higher, respectively, in the sera of mice treated with carcinogens alone compared with that from vehicle controls. Serum levels of PSPB and cathepsin D were reduced in mice treated with the carcinogens and given a diet supplemented with PEITC-NAC plus MI (72 and 66%, respectively) or I3C (62 and 60%, respectively, Figure 3F) relative to the level in the serum of mice treated with the carcinogens alone.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
We have used a quantitative proteomics approach, iTRAQ labeling coupled with mass spectrometry, to compare the relative abundance of proteins in lung tumors from mice treated with NNK plus BaP versus normal mouse lungs from vehicle controls and determine whether protein levels in tumor tissues are modulated by the chemopreventive agents PEITC-NAC plus MI or I3C. The tissues used in this study were obtained from a lung carcinogenesis chemoprevention study in which dietary administration of PEITC-NAC plus MI or I3C reduced NNK plus BaP-induced lung tumor multiplicity by 77 and 86%, respectively (7,12).

Our data indicate changes in the relative abundance of 59 proteins in tumor tissues from carcinogen-treated mice versus normal lung tissue. Of these proteins, 37 (63%) were identified in two out of three iTRAQ experiments, whereas 22 (37%) were identified in all three experiments. Sixteen differentially expressed proteins were identified in only one of the three iTRAQ runs (data not shown). Of these proteins, the expression of PSPB and caveolin-1 was further verified in western assays. The problem with run-to-run reproducibility of the results in iTRAQ experiments may be explained by differences in the abundance of the proteins such that peptide ions from more abundant proteins would have a higher probability of being identified than those from less abundant proteins. For example, in one study, 74 out of 80 (93%) proteins at expression levels >105 copies per cell were identified in all of nine analyses, but only 12 out of 80 (15 %) proteins expressed <103 copies per cell were identified in the nine combined LC/LC/MS/MS runs (26). However, frequent identification of a protein does not necessarily lend credence to the accuracy of the data, which are solely determined by the quality of the tandem mass spectra.

Of the 59 differentially expressed proteins, the levels of 19 proteins, including three proteins with unaltered levels in tumor tissues versus normal tissues, were modulated upon administration of PEITC-NAC plus MI or I3C to carcinogen-treated mice. The lung tumor chemopreventive effect of PEITC-NAC plus MI or I3C may be explained, at least partly, by the modulation of these proteins. To our knowledge, this is the first report on differential modulation by chemopreventive agents of carcinogenesis-related proteins in a mouse model of lung carcinogenesis and chemoprevention. In earlier studies, genomics (27) as well as genomics and proteomics (28) approaches have been used to identify differentially expressed genes or genes and their products, respectively, in lung tissues of mice treated with carcinogens alone or carcinogens and chemopreventive agents. However, it is difficult to compare our results with those from gene expression studies since the correlation between gene expression levels and protein abundance is known to be good for a limited number of highly abundant proteins but poor for proteins with lower expression levels (29). The proteomics study mentioned above (28) compared the proteome of lung tissues from rats treated with environmental tobacco smoke alone or with chemopreventive agents using a glass microarray coated with antibodies. These antibodies were developed for the detection of low-abundance proteins that are rarely, if at all, detected with proteomics techniques involving mass spectrometry. Therefore, comparison of their results with ours is not possible.

Our discussion of proteins whose expression patterns were found to be altered upon treatment of mice with lung carcinogens alone or with carcinogens and PEITC-NAC plus MI or I3C focuses on selected proteins: HIF-1{alpha}, a transcriptional activator of many genes that code for proteins involved in several pathways intimately related to cancer (25), PSPB and CC10, proteins expressed exclusively in the lung, and histones, whose levels were highly altered upon administration of PEITC-NAC plus MI or I3C to carcinogen-treated mice.

HIF-1{alpha} is a transcription factor that is present at very low levels during normoxic conditions, owing to hydroxylation by prolyl hydroxylase enzymes, and degradation by proteasomes but stabilized during hypoxic conditions, activation by oncogenes or mutation of certain tumor suppressor proteins (30). Stabilization of HIF-1{alpha} leads to activation of genes involved in glucose, amino acid, extracellular matrix and energy metabolism, pH regulation, cytoskeletal structure, cell proliferation and angiogenesis (25). Although we failed to identify HIF-1{alpha} in iTRAQ experiments, probably because it is a low-abundance protein, western assays showed an increase in the level of HIF-1{alpha} in lung tumors versus normal lungs but a decrease in lung tumors from carcinogen and PEITC-NAC plus MI- or carcinogen and I3C-treated mice compared with that in lung tumors from mice treated with the carcinogens only. This is the first study that indicates an increase in the level of HIF-1{alpha} in lung tumor tissues from animal models of lung carcinogenesis and inhibition of the level of the protein by chemopreventive agents in the same model. Several reports have shown an increased level of HIF-1{alpha} in resected human lung tumors (31–34) and a close association with that of EGFR and HIF-1{alpha} target proteins (31). Therefore, HIF-1{alpha} is considered as an important target for a number of chemotherapeutic drugs (35) as well as naturally occurring agents (36,37).

Among proteins regulated by HIF-1{alpha} and found to have increased levels in tumor tissues in the present study are glycolytic enzymes, carbonic anhydrase 2, cathepsins D and H and prolyl 4-hydroxylase. A high rate of glycolysis in tumor cells results in increased production of lactate which contributes to the acidosis in the extracellular space of tumors through excretion via the H+/lactate co-transporter protein (38). A parallel increase in the expression of HIF-1{alpha}-induced carbonic anhydrase, a membrane-bound enzyme that catalyzes the production of H+ and Formula ions from H2O and CO2, contributes to a further fall in the pH of the extracellular milieu. The accumulation of intracellular Formula in cancer cells and active secretion of H+ ions are mediated by the Na+-dependent Cl–/Formula cation antiport protein (band 3), which has a binding site for carbonic anhydrase 2 (39) and Na+/H+ exchanger, respectively. Increased levels of carbonic anhydrase 2 have been reported in brain, gastric and pancreatic tumors (40) and inhibition of the growth of cancer cells was possible by targeting carbonic anhydrase 2 (41). Acidification of the extracellular milieu of cancer cells results in upregulation of cathepsins and lysosomal proteolytic enzymes, among other metastasis-promoting proteins (42), and leads to damage to the extracellular matrix thereby promoting invasion and metastasis. Elevated levels of cathepsins D (22) and H (43) were reported in tumors and sera of lung cancer patients. We are intrigued by the increased level of prolyl 4-hydroxylase in lung tumor tissues relative to normal lung tissues. Similar results were found in head and neck squamous cell carcinoma in which prolyl 4-hydroxylase expression was strongly elevated both at the mRNA and protein level (44).

Administration of PEITC-NAC plus MI or I3C reduced the level of carbonic anhydrase 2 and band 3 anion transport protein in tumor tissues, suggesting that these proteins may be novel targets for the lung tumor chemopreventive activity of PEITC-NAC plus MI or I3C. In western assays, we observed an increased level of cathepsin D in both lung tumor tissues and sera from tumor-bearing mice, indicating the release of this protein from the tumor cells into the circulation and its potential as a biomarker for early detection of lung cancer. Western assays also showed modulation by PEITC-NAC plus MI or I3C of carcinogen-induced changes in the level of cathepsin D in tumor tissues and in the serum, but this was not observed in iTRAQ experiments. This discrepancy may be related to differences in cathepsin D protein isoforms found during bioinformatic assignment in the iTRAQ assay versus those detected by western immunoblotting.

Among proteins, the levels of which were changed in lung tumors versus normal lungs from vehicle-treated mice, PSPB and CC10 are the only lung-specific proteins. Both iTRAQ experiments and western assays indicated an increased level of PSPB in tumor tissues versus normal tissues, suggesting that the tumors are predominantly derived from type II pneuomocytes from which surfactant proteins are secreted. Expression of PSPB precursor and PSPB increased in pulmonary adenocarcinoma but not in squamous cell and large cell carcinomas of the lung and non-pulmonary adenocarcinomas (23), suggesting the potential use of this protein not only as a marker of lung cancer but also in the differential diagnosis of different forms of lung cancer. We found the level of PSPB to be decreased in tumor tissues and serum of tumor-bearing mice upon supplementation of the diet with PEITC-NAC plus MI or I3C. In contrast to that of PSPB, the relative abundance of CC10 decreased in lung tumor tissues versus normal tissues. This finding is in line with the significant reduction of CC10 in NNK-exposed hamsters (45) and N-methyl-N-nitrosourea-induced mouse lung tumors (46). A reduction in the level of CC10 may indicate an increased susceptibility to lung tumors as demonstrated with NNK-treated CC10-knock-out mice (47). On the other hand, overexpression of CC10 modified neoplastic potential of lung cancer cells (48).

Although histones were originally considered as simple protein spools around which DNA in the nucleus is wound (H2A, H2B, H3 and H4) or mere binders of the nucleosome (H1), they are now recognized to have a role in multiple functions including regulating gene transcription (49), apoptosis (50), cell proliferation (51), DNA repair (52) and chromosomal segregation (53). In the present study, we found a reduction in the relative abundance of Histone2h2aa-613, Histone2h2aa1, Histone1h2a, Histone1h2b-616, Histoneh2a(B)-613 and Histoneh2az in tumor tissues versus normal lungs. More interestingly, the level of the aforementioned histones as well as other histones whose level did not change in tumor tissues (Histone1h2ak, Histone1h4i and Histone1h2bm) dramatically increased (up to 5-fold) in tumor tissues from carcinogen and PEITC-NAC plus MI- or carcinogen and I3C-treated mice versus tumor tissues from carcinogen-only-treated mice. This is the first report on alteration of the level of histones in tumor tissues by cancer chemopreventive agents and may represent a novel mechanism for the lung tumor inhibitory activity of PEITC-NAC plus MI or I3C. In upcoming studies, we will investigate the functional role of the various histones in chemoprevention by PEITC-NAC plus MI or I3C.

This study has certain limitations. First, the number of identified proteins (488, 579 and 799 in iTRAQ experiments 1, 2 and 3, respectively) in lung tissues represents only a small fraction of the proteins expected to be coded by the ~40 000 genes in the mouse genome (54). This is mainly due to signal suppression from co-eluting peptides as well as the fact that the number of co-eluting peptides often exceeds the necessary time scale for tandem mass spectrometry detection of each individual species (limited analytical power of the mass spectrometry instrument). Second, some of the changes in protein levels we observed here may result from comparison of different cell types in the normal lung with more limited cell types in the tumor tissues. Nevertheless, iTRAQ proteomics enabled identification and measurement of the relative abundance of several proteins involved in various crucial aspects of cancer biology such as glucose metabolism, cell proliferation, cell survival and invasion and modulation of their levels by PEITC-NAC plus MI or I3C.

In summary, comparison of the protein profile of lung tumors and normal lung tissues obtained from a mouse model could contribute significantly to the identification of candidate biomarker proteins and potential targets for chemopreventive/therapeutic agents. Currently, we are performing functional studies to confirm the role of the differentially expressed proteins in carcinogenesis and as targets for PEITC-NAC plus MI or I3C.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
The National Institutes of Health/National Cancer Institute (CA-102502) to S.S.H.


   Acknowledgments
 
We thank David Lahti for the synthesis of PEITC-NAC and David Jewison for the preparation of the diets. Mass spectral data were generated at the Center for Mass Spectrometry and Proteomics at the University of Minnesota.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received October 16, 2007; revised December 10, 2007; accepted December 17, 2007.


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