Cancer Research Grants Metabolism
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 67, 6502, July 1, 2007. doi: 10.1158/0008-5472.CAN-06-4438
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kassie, F.
Right arrow Articles by Hecht, S. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kassie, F.
Right arrow Articles by Hecht, S. S.
Related Collections
Right arrow Preclinical Intervention
Right arrow Preclinical Intervention: In Vivo (Animals): Drugs, Nutritional Interventions, Mechanisms

Epidemiology and Prevention

Indole-3-carbinol Inhibits 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Plus Benzo(a)pyrene–Induced Lung Tumorigenesis in A/J Mice and Modulates Carcinogen-Induced Alterations in Protein Levels

Fekadu Kassie1, Lorraine B. Anderson2, Robyn Scherber1, Nanxiong Yu1, David Lahti1, Pramod Upadhyaya1 and Stephen S. Hecht1

1 The Cancer Center and 2 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota

Requests for reprints: Stephen S. Hecht, The Cancer Center, University of Minnesota, MMC 806, 420 Delaware Street Southeast, Minneapolis, MN 55455. Phone: 612-626-7604; Fax: 612-626-5135; E-mail: hecht002{at}umn.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We tested the chemopreventive efficacy of indole-3-carbinol (I3C), a constituent of Brassica vegetables, and its major condensation product, 3,3'-diindolylmethane (DIM), against lung tumorigenesis induced by a mixture of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP) in A/J mice. The mixture of NNK plus BaP (2 µmol each) was administered by gavage as eight weekly doses, whereas I3C (112 µmol/g diet) and DIM (2 and 30 µmol/g diet in experiments 1 and 2, respectively) were given in the diet for 23 weeks beginning at 50% of carcinogen treatment. I3C reduced NNK plus BaP–induced tumor multiplicity by 78% in experiment 1 and 86% in experiment 2; the respective reductions in tumor multiplicity by DIM were 5% and 66%. Using a quantitative proteomics method, isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry, we identified and quantified at least 250 proteins in lung tissues. Of these proteins, nine showed differences in relative abundance in lung tissues of carcinogen-treated versus untreated mice: fatty acid synthase, transketolase, pulmonary surfactant-associated protein C (SP-C), L-plastin, annexin A1, and haptoglobin increased, whereas transferrin, {alpha}-1-antitrypsin, and apolipoprotein A-1 decreased. Supplementation of the diet of carcinogen-treated mice with I3C reduced the level of SP-C, L-plastin, annexin A1, and haptoglobin to that of untreated controls. These results were verified using immunoblotting. We show here that tumor-associated signature proteins are increased during NNK plus BaP–induced lung carcinogenesis, and I3C inhibits this effect, suggesting that the lung tumor chemopreventive activity of I3C might be related to modulation of carcinogen-induced alterations in protein levels. [Cancer Res 2007;67(13):6502–11]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lung cancer is the most common cause of cancer death worldwide (1). In the United States, an estimated 174,500 new cases and 162,500 deaths are expected in 2006 (2). Because the proportion of lung cancer cases attributable to cigarette smoking has reached 90%, the best approaches to curb lung cancer mortality are prevention of smoking initiation and improved methods for smoking cessation. However, the prevalence of tobacco smoking has not changed much during the last three decades (3). Moreover, former smokers are at high risk for lung cancer. Therefore, there is an urgent need to develop alternate approaches to reduce lung cancer mortality. Two promising approaches are development of effective chemopreventive agents for both current and former smokers and identification of biomarkers that may help in the early detection of lung cancer.

3-Indolylmethyl glucosinolate (also known as glucobrassicin), the parent compound of indole-3-carbinol (I3C), occurs in a wide variety of cruciferous vegetables. Hydrolysis of this glucosinolate to I3C is catalyzed by myrosinase when plant tissues are disrupted, as during chewing or cutting (4). In the stomach, I3C undergoes condensation reactions to produce various products, the major one being 3,3'-diindolylmethane (DIM; ref. 5). Dietary intake of glucobrassicin and its breakdown products varies widely. In the United States and the United Kingdom, estimated per capita intakes of glucobrassicin are 8.1 and 19.4 mg/d, respectively (6, 7), whereas in some populations in Asia, the level reaches 46 mg/d (8). Considerable evidence shows that I3C inhibits carcinogenesis induced by different classes of carcinogens at many sites through induction of phase I and II enzymes, inhibition of proliferation of tumor cells, induction of apoptosis in tumor cells, and modulation of estrogen metabolism (4). However, I3C also promotes tumorigenesis depending on the experimental design (9). In a recent phase I trial in women with a high risk of breast cancer, daily administration of I3C at doses of 400 and 800 mg was well tolerated and significantly increased activities of glutathione S-transferase (GST) and cytochrome P450 1A2. I3C also markedly increased the 2-hydroxyestrone/16{alpha}-hydroxyestrone ratio in a manner consistent with chemoprevention (10).

In this study, we assessed the chemopreventive efficacy of I3C and DIM against lung carcinogenesis induced by a mixture of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) plus benzo[a]pyrene (BaP) in A/J mice. NNK and BaP are two of the most important lung carcinogens in tobacco smoke. We used a quantitative proteomics method, isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry (MS; ref. 11), to evaluate changes in relative protein abundance in lung tissues of carcinogen-treated versus untreated mice and to determine if co-administration of I3C modifies these alterations. In this method, a set of four isobaric reagents is used to label, in parallel, tryptic peptides from up to four samples by forming an amide linkage to any peptide amine (NH2-terminal or {varepsilon}-amino group of the lysine side chain). This multiplex strategy may simultaneously quantify changes in protein levels under four biological conditions. Due to the isobaric mass design of the iTRAQ reagents, differentially labeled peptides appear as single peaks in MS scans. When iTRAQ-tagged peptides are subjected to tandem MS (MS/MS) analysis, the mass-balancing carbonyl moiety is released, thereby liberating isotope-encoded reporter ions that provide relative quantitative information on proteins.

To our knowledge, this is the first study showing changes in levels of tumor-related proteins during NNK plus BaP–induced lung carcinogenesis in the absence or presence of a chemopreventive agent.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals, reagents, and diets. HEPES, MgCl2, NaCl, protease inhibitor cocktail, BaP, and I3C were from Sigma. DIM was from BioResponse. Urea was purchased from Amersham Pharmacia Biotech. NNK was synthesized as described previously (12). iTRAQ reagent kits were obtained from Applied Biosystems (ABI). Mouse diets (AIN-93G and AIN-93M) were purchased from Harlan Teklad. AIN-93G diet, high in protein and fat, is used to support rapid growth of the mice during early age, whereas AIN-93M diet, low in protein and fat, is recommended for adult maintenance.

A/J mouse tumorigenicity experiments. Two experiments were carried out using female A/J mice, 5 to 6 weeks of age, obtained from The Jackson Laboratory. Although both female and male A/J mice are sensitive to develop lung tumors (13), female mice are easier to handle. The experimental design is shown in Fig. 1A . Upon arrival, the mice were housed in the specific pathogen-free animal quarters of the Research Animal Resources, at the University of Minnesota Academic Health Center. The mice were randomized into four groups (group 1, carcinogen treated; group 2, I3C plus carcinogen treated; group 3, DIM plus carcinogen treated; and group 4, untreated control) and maintained on AIN-93G pelleted diet. One week after arrival, the mice were switched to AIN-93G powdered diet and treated by gavage with either a mixture of BaP plus NNK (2 µmol of each: groups 1–3) in 0.1 mL cottonseed oil or cottonseed oil alone (group 4) once weekly for eight treatments. Mice in groups 2 and 3 were given a diet supplemented with I3C or DIM, respectively, beginning 1 day after the fourth treatment with the carcinogens until sacrifice at 19 weeks after the last carcinogen treatment. We used the 50% carcinogen treatment time point because smokers would not normally use chemopreventive agents in their early years of smoking. The dose of I3C in experiment 1 was 150 µmol/g diet but was reduced, due to a lower food intake and body weight, to 112 µmol/g diet beginning at week 6 of the experiment. The dose of I3C in experiment 2 was 112 µmol/g diet. DIM was administered at 2 µmol/g diet in experiment 1 and 30 µmol/g diet in experiment 2. The dose level of I3C was determined on the basis of the relative abundance in the human diet of I3C precursors relative to those of 2-phenethyl isothiocyanate, which has been previously shown to be an effective chemopreventive agent in the same experimental model (4). Although this dose level is about 10 times higher (4,938 versus 422 mg/m2) than that used in phase I clinical studies (10), administration of drugs in the diet is known to result in lower peak plasma and tissue concentrations and AUC (≥10 times) than gavage dosing (14), which is similar to oral administration in humans. The lower dose of DIM was chosen on the basis of previous studies (4), whereas the higher dose of DIM was determined by calculating the approximate amount of DIM expected to be formed upon consumption of 112 µmol/g diet of I3C (5). One week after the last carcinogen treatment, the mouse diet was changed from AIN-93G to AIN-93M. I3C- or DIM-supplemented diets were prepared every 4 weeks and stored in airtight plastic bags at 4°C. High-performance liquid chromatography (HPLC) analysis showed that both I3C and DIM were stable in the diet under these conditions. The powdered diet was administered using metal boxfeeders (Lab Products, Inc.), which allowed monitoring of food consumption and minimized diet waste. Fresh diet was provided every 3 to 4 days. Food consumption was monitored twice a week. Body weight and water consumption were recorded every week. The mice were sacrificed 19 weeks after the final carcinogen administration. Immediately upon sacrifice, lungs were perfused with cold PBS and harvested, and tumors were scored using a dissecting microscope. During the tumor count, the lungs were protected from drying by moistening them with PBS. Subsequent to the tumor count, the lungs were frozen in liquid nitrogen.


Figure 1
View larger version (10K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. A, study design for evaluating the chemopreventive efficacy of I3C and DIM against BaP plus NNK–induced lung tumorigenesis in A/J mice. B, workflow for iTRAQ proteomics with mouse lung tissues.

 
Protein extraction and iTRAQ labeling. Frozen mouse lungs chosen randomly (eight per group) were pulverized using a mortar and pestle on dry ice. The pulverized lung tissue (weighing 1.2 g) was suspended in 800 µL of lysis buffer [15 mmol/L MgCl2, 50 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 8 mol/L urea, 0.1% Triton] and a cocktail of protease inhibitors [10 µL/g tissue, leupeptin, pepstatin A, aprotinin, Bestatin hydrochloride, N-(trans-epoxysuccinyl)-L-leucine 4-guanidinbutylamide, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride], sonicated for 1 min and centrifuged at 15,000 rpm for 20 min. Recovered supernatants were frozen at –80°C. The protein content of the samples was determined according to the Lowry method (15). The scheme for iTRAQ analysis is shown in Fig. 1B. For iTRAQ labeling, 90 µg of protein from each group was mixed with 100 µL of 0.5 mol/L triethylammonium bicarbonate buffer (pH 8.5) and denatured and reduced [final concentration, 0.05% SDS and 0.5 mmol/L tris-(2-carboxyethyl)phosphine, respectively] for 1 h at 37°C, and cysteine residues were then blocked [2 mmol/L methyl methane thiosulfonate (MMTS)] at room temperature for 10 min. Subsequently, protein was digested by addition of 10 µL of trypsin (1.0 µg/10 µg protein sample) and incubated at 37°C overnight. The final concentrations of protease inhibitor cocktail and urea were 0.4 µL/mL and 0.6 mol/L, respectively. Protein digests from the different groups were differentially labeled with iTRAQ reagent solutions [untreated control (114), carcinogen plus I3C–treated mice (115), and carcinogen-treated mice (116)] and incubated at room temperature for 1 h. Because I3C and DIM have been reported to act similarly at the transcriptional level (16), we did proteomics studies with I3C only. Three iTRAQ runs (technical replicates) were carried out on different days, each time using aliquots of homogenates from pooled mouse lungs (eight per group) for each group. All steps of sample processing, including iTRAQ labeling, were done similarly thrice. Additional iTRAQ experiments were done to compare changes in protein levels in internal duplicates (untreated/untreated and carcinogen treated/carcinogen treated) and untreated/carcinogen-treated aliquots. Labeled samples were pooled, dried, resuspended in 0.1% trifluoroacetic acid, and applied to a Sep-Pak C18 cartridge (Waters Corp.) to remove buffer, trypsin, and salts. The samples were then analyzed by two-dimensional liquid chromatography MS/MS (2D LC-MS/MS; ref. 17).

Strong cation exchange liquid chromatography. To increase the detection of peptides from low-abundance proteins, labeled peptide mixtures were fractionated using strong cation exchange (SCX) liquid chromatography in the first dimension. Off-line SCX chromatography was carried out on a Magic 2002 HPLC system (Michrom Bioresources, Inc.) with a polysulfoethyl A (Poly LC, Inc.) column (150 mm x 1.0 mm inner diameter, 5-µm particle size, and 300 Å pore size). The peptide mixture was rehydrated in 350 µL of buffer A [20% (v/v) acetonitrile, 5 mmol/L KH2PO4 (pH 3.2)] and loaded onto the SCX column. Peptides were eluted with a gradient of 0% to 20% LC solvent B (solvent A with additional 500 mmol/L KCl) over 40 min and from 20% to 100% for 20 min. The column flow rate for loading and elution was 37 µL/min. Absorbance was monitored at 280 nm, and fractions were collected at 3-min intervals and dried in vacuo. Fractions with a mAU280 > 2, (fractions 13–23) were analyzed in the second dimension by reversed-phase LC-MS/MS.

Reversed-phase LC-MS/MS analysis. Each dried SCX fraction was reconstituted with reversed-phase load buffer (98:2, water/acetonitrile, 0.1% formic acid) and injected onto a Dionex/LC packings capillary LC system, online with a QSTAR pulsar i mass spectrometer (ABI) as described previously (18). Briefly, peptides were loaded onto a 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 eluted with a linear gradient from 0% to 35% B over 40 min where solvent B was 5:95 water/acetonitrile, 0.1% formic acid and solvent A was 95:5 water/acetonitrile, 0.1% formic acid, and 35% to 80% B over 5 min, and 80% to 100% B over 2 min. Product ion spectra were collected in an information-dependent acquisition (IDA) mode. IDA mode settings included continuous cycles of one full-scan time-of-flight (TOF) MS from m/z 400 to 1,100 (1.5 s) plus four product ion scans from m/z 50 to 2,000 (3 s each). Precursor m/z values were selected from a peak list automatically generated by Analyst QS software (ABI) from the TOF MS scan during acquisition, starting with the most intense ion.

Data processing. ProQUANT 1.1 software (ABI) with the Interrogator Algorithm (19) was used for the identification and relative quantification of proteins. MS/MS data were searched against the mouse protein database from 3-2-2005 (Celera Discovery System, ABI). The search variables were minimum peptide confidence levels of 90%; peptide mass and fragment ion mass tolerances of 0.15 and 0.1 Da, respectively; one missed cleavage of trypsin; and MMTS-labeled cysteines and oxidized methionine as fixed modifications. The peak areas of the "reporter" ions (m/z 114, 115, 116, and 117) were used by ProQUANT for relative protein quantification. Peptides found in more than one protein were not used in quantitation. The threshold for protein identification was set at >95% confidence. ProGroup Viewer (ABI) was used to compile the results from the database searches into protein groups, to remove protein redundancy, to provide protein-based ratios of relative abundance, and to export data and statistical variables for further analysis and calculations. ProQUANT calculates average iTRAQ ratios and estimates the P value and error factor (EF) for each protein hit. EF expresses the 95% confidence interval (95% CI) of the average iTRAQ ratio [EF = 1095% CI, where 95% CI = (ratio x EF) – (ratio ÷ EF)] for each protein. To normalize differences in protein loading across the samples, all observed protein ratios were divided by the average iTRAQ ratio for that experiment. The rationale for this is that the relative abundance of the majority of proteins is close to 1. Only proteins identified with a minimum of two peptides, quantitation results with P < 0.05 and SD of the quantitation below 20%, which is indicated by EF < 2, were considered as changed in level.

Western analysis. Protein extraction and protein content determination were carried out as for the iTRAQ study. Forty micrograms of protein obtained from homogenates of pooled lungs (eight per group) were loaded onto a 4% to 12% Novex Tris-glycine gel (Invitrogen) and run for 100 min at 125 V. The proteins were then transferred onto a nitrocellulose membrane (Bio-Rad) for 2 h at 25 V. Adequate transfer of proteins was confirmed by staining membranes with BLOT-FastStain (Chemicon). Subsequently, membranes were blocked in 5% Blotto nonfat dry milk in Tris buffer containing 1% Tween 20 for 1 h and probed overnight with the following primary antibodies (Santa Cruz Biotechnology): anti–pulmonary surfactant-associated protein C (SP-C), 1:100; anti-L-plastin, 1:200; and anti–annexin A1, 1:200. After incubating with secondary antibody (Santa Cruz Biotechnology) for 1 h (1:20,000 donkey anti-goat IgG for SP-C and L-plastin and 1:20,000 goat anti-rabbit IgG for annexin A1), 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-ß-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) and normalized relative to total protein to compensate for experimental variation.

Statistical analysis. Lung tumor multiplicity and body weight comparisons were made using one-way ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
I3C and DIM reduced NNK plus BaP–induced lung tumor multiplicity. We carried out two experiments to assess the chemopreventive efficacy of I3C and DIM against NNK plus BaP–induced lung tumorigenesis (Table 1 ). In experiment 1, mice treated with carcinogens and fed conventional AIN-93 diet had 13.8 ± 4.3 lung tumors per mouse. Carcinogen-treated mice given I3C-supplemented diet beginning at 50% carcinogen treatment had statistically significantly lower tumor multiplicities than carcinogen-treated mice fed the control diet (3.1 ± 1.5 tumors per mouse, corresponding to a 78% reduction). Dietary administration of DIM at a dose level of 2 µmol/g diet did not reduce lung tumor multiplicity (13.1 ± 4.8 tumors per mouse). In experiment 2, we examined the reproducibility of lung tumor chemopreventive activity of I3C following the same experimental protocol and tested the effect of a higher dose of DIM (30 µmol/g diet). Mice treated with the carcinogens and fed conventional AIN-93 diet developed 19.3 ± 9.1 tumors per mouse. Upon supplementing the diet with I3C or DIM beginning at 50% of carcinogen treatment, tumor multiplicity was significantly reduced to 2.6 ± 1.7 and 6.5 ± 3.7 tumors per mouse, respectively, corresponding to 86% and 66% reductions compared with the group treated with carcinogens and maintained on conventional AIN-93 diet. Tumor incidence was not significantly reduced either by I3C or DIM. This was not unexpected because the most sensitive indicator in the A/J mouse lung tumorigenicity model is tumor multiplicity.


View this table:
[in this window]
[in a new window]

  		Table 1. Effects of I3C and DIM on lung tumor induction by a mixture of NNK plus BaP in A/J mice

 
Observation of the mice once a week for signs of toxicity, such as changes in fur color or texture, motor and behavioral abnormalities, and palpable masses, did not reveal any of these effects. However, at termination, the mean body weight of the mice given the I3C-supplemented diet was significantly lower (P < 0.05) than the group given the conventional AIN-93 diet. The lower body weight gain was not due to reduced food consumption because the mean food consumption in the group maintained on the I3C-supplemented diet was 9% higher than that of the group given the conventional AIN-93 diet (2.76 g diet per mouse per day for mice fed on the I3C-supplemented diet versus 2.52 g diet per mouse per day for mice fed on the untreated diet), whereas food consumption of the group given DIM-supplemented diet was similar to that of the group given conventional AIN-93 diet. Histopathologic analysis of liver tissues from I3C-treated mice did not reveal any abnormalities.

Two-dimensional LC-MS/MS of iTRAQ-labeled lung tissue samples led to identification of proteins. iTRAQ experiments 1, 2, and 3 led to the identification of 662, 496, and 476 unique proteins, respectively, with >95% confidence, using one or more peptides. Following exclusion of proteins identified with a single unique peptide, the number of proteins identified in experiments 1, 2, and 3 decreased to 348, 287, and 256 proteins, respectively. These proteins were used for further analysis. An example of a MS/MS spectrum used for the identification and quantitation of L-plastin is shown in Fig. 2A . The b- and y-ion series indicate the sequence ISFDEFIK, which is one of the three peptide fragments used to identify L-plastin (20). The iTRAQ reporter ion peak areas at m/z 114, 115, and 116 were used to measure the relative amount of L-plastin in lung tissues from untreated, carcinogen plus I3C–treated, and carcinogen only–treated mice, respectively. Biological process terms (Celera database) indicated that the identified proteins are involved in a variety of biological processes including cell cycle, oncogenesis, and signal transduction. The largest percentage of proteins are involved in protein metabolism (15.5%) followed by those responsible for signal transduction (14.2%; Fig. 2B).


Figure 2
View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. A, example of L-plastin identification and quantitation using the MS/MS spectrum of iTRAQ 114-, 115-, and 116-labeled doubly protonated peptide fragment (ISFDEFIK, m/z 643.85) from the protein. The mass of the peptide reflects an iTRAQ label on the NH2 terminus and the lysine. The reporter ions with m/z 114, 115, and 116 appear in the low mass region of the spectrum and are used to determine the relative amount of L-plastin in lung tissues of carcinogen-treated (iTRAQ 116) or carcinogen plus I3C–treated (iTRAQ 115) versus untreated (iTRAQ 114) mice. B, classification, using Celera Discovery System panther bioprocess terms, of proteins identified with two or more peptides and >95% confidence.

 
NNK plus BaP treatment altered the expression profile of proteins in lung tissues, but this effect was inhibited by dietary administration of I3C. To assess proteomic changes in lung tissues of carcinogen-treated (iTRAQ reagent 116 labeled) or carcinogen plus I3C–treated mice (iTRAQ reagent 115 labeled) versus untreated (iTRAQ reagent 114 labeled) mice, relative quantification of proteins was done by pairwise comparison (116:114 or 115:114) of the areas under the peaks at m/z 114, 115, and 116 Da, which are the masses of the reporter ions that are derived from the iTRAQ reagents. Only proteins quantitated with ≥2 peptides that had ratios with an error factor <2 and P < 0.05 were considered for the determination of changes in relative abundance. The spread of iTRAQ ratios for 100 proteins obtained from internal duplicate lung tissue samples (untreated/untreated or carcinogen treated/carcinogen treated) and lung tissue samples from carcinogen-treated versus untreated mice were compared (Fig. 3C ). The ratios of the replicates differed by less than 20% (Fig. 3A and B), whereas that from carcinogen-treated mice versus untreated mice gave a greater spread of data (Fig. 3C). Hence, the relative abundance was considered changed only when the difference in the iTRAQ ratio was more than 20%. Figure 3D shows the iTRAQ ratios of SP-C, L-plastin, annexin A1, and haptoglobin in replicate samples from untreated and carcinogen-treated mice or carcinogen-treated/untreated mice. There was no difference in protein levels among replicate samples (untreated/untreated and carcinogen treated/carcinogen treated) for all categories. The average ratio for all four proteins was 0.90–1.02. On the other hand, the level of SP-C, L-plastin, annexin A1, and haptoglobin increased by 42%, 66%, 52%, and 58%, respectively, in lung tissues from carcinogen-treated compared with untreated mice.


Figure 3
View larger version (19K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Scatter plot showing iTRAQ ratios of 100 representative proteins from either internal duplicate samples from (A) untreated mice, (B) carcinogen-treated mice, or (C) samples from carcinogen-treated versus untreated mice. X-axis, number of peptides used in the identification of each representative protein. D, iTRAQ ratios of SP-C, L-plastin, annexin A1, and haptoglobin from either internal duplicate lung tissue samples (untreated/untreated or carcinogen treated/carcinogen treated) or lung tissue samples from carcinogen-treated versus untreated mice.

 
Table 2 lists nine proteins that showed changes in relative abundance in lung tissues from carcinogen-treated versus untreated mice. Transferrin (TF), {alpha}-antitrypsin precursor (AAT), and apolipoprotein A-1 (Apo A1) decreased by 56%, 69%, and 68%, respectively, whereas fatty acid synthase, transketolase, SP-C, L-plastin, annexin A1, and haptoglobin increased by 34%, 33%, 42%, 66%, 52%, and 58%, respectively. Interestingly, dietary administration of I3C to carcinogen-treated mice reduced the level of SP-C (1.42 ± 0.06 to 1.09 ± 0.04), L-plastin (1.66 ± 0.06 to 1.16 ± 0.06), annexin A1 (1.52 ± 0.05 to 1.07 ± 0.01), and haptoglobin (1.58 ± 0.03 to 0.71 ± 0.06) to the level measured in the lungs of untreated mice. Compared with the amount measured in lung tissues of untreated mice (114 labeled), cytochrome P450 2F2 and GST mu 1 enzymes were increased (33% and 42%, respectively) in lung tissues of mice treated with I3C plus carcinogen (115 labeled) but not in tissues of mice treated with the carcinogens alone (116 labeled).


View this table:
[in this window]
[in a new window]

  		Table 2. Proteins identified as differentially expressed in lung tissues of A/J mice in response to carcinogen or carcinogen plus I3C treatment

 
Immunoblotting verified changes in protein levels found by iTRAQ technique. To confirm changes in protein levels in lungs of carcinogen-treated mice and reversal of the effect by I3C administration, we carried out immunoblotting analysis on three selected proteins. Consistent with the observations in iTRAQ experiments, levels of SP-C, L-plastin, and annexin A1 increased in lung tissues of carcinogen-treated mice versus untreated mice (Fig. 4A , lanes 5 and 6). In addition, the percentage change for these proteins was similar to that observed in iTRAQ experiments (Fig. 4B). In lung tissues from mice treated with carcinogens and I3C, the relative abundance of all three proteins was close to the levels found in tissues from untreated mice (Fig. 4A, lanes 3 and 4).


Figure 4
View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. A, Western blot analysis of expression of SP-C, L-plastin, and annexin A1 in lung tissues from untreated (lanes 1 and 2), carcinogen plus I3C–treated (lanes 3 and 4), and carcinogen-treated (lanes 5 and 6) mice. Equal amounts of protein from each group were loaded onto a 4% to 12% SDS-PAGE and processed for Western immunoblotting with the respective antibodies. B, percentage expression of SP-C, L-plastin, and annexin A1. Quantification of protein expression differences using U-scan-It software was carried out after normalizing of total protein loading by ß-actin expression. *, P < 0.02 by Student's t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study provides interesting data on the chemopreventive efficacy of I3C against lung tumorigenesis induced by a mixture of two of the most important tobacco smoke carcinogens (i.e., NNK and BaP) in A/J mice. First, highly significant inhibition of lung tumor multiplicity (78% and 86% reduction in experiments 1 and 2, respectively) was observed when I3C was administered in the diet beginning at 50% into the carcinogen administration phase. Earlier studies of lung tumor chemopreventive activity of I3C have been carried out by giving this chemopreventive agent either before (21) or before, during, and after carcinogen treatment (22), and the maximum reduction in lung tumor multiplicity was about 40%. Second, DIM, one of the condensation products of I3C, also significantly inhibited NNK plus BaP–induced lung tumorigenesis when given in the diet at a dose level that would be expected to be similar to the plasma level of DIM achieved upon administering 112 µmol/g diet of I3C (5). These studies suggest that I3C and DIM should be further investigated for lung cancer chemoprevention in smokers who are in transition to quitting. Third, we identified, using the iTRAQ proteomics technique, nine proteins that showed changes in relative abundance in lung tissues of mice treated with a mixture of NNK plus BaP when compared with untreated mice. The level of four of these proteins was modulated upon dietary administration of I3C to that of untreated mice. Moreover, we found increased levels of cytochrome P450 2F2 and GST mu 1 in lung tissues of mice treated with I3C when compared with lung tissues from untreated mice. Collectively, these findings suggest mechanisms that may partially explain the lung tumor chemopreventive activity of I3C and DIM.

The mean body weight of the mice maintained on the I3C-supplemented diet was about 18% lower than the group given conventional AIN-93 diet. Lower body weight gain was not due to reduced food consumption because mice given I3C-supplemented diet consumed about 9% more food than mice maintained on AIN-93 conventional diet. A similar trend has been observed in one previous chemoprevention study with I3C in which the food consumption of F344 rats given an I3C-supplemented diet (0.5%) was about 13% higher than rats maintained on a control diet, but their mean body weight was about 7% lower (23). Lower body weight gain in I3C-treated mice and rats may result from decreased nutrient absorption or increased rate of metabolism. It is not known whether lower body weights in the present study contributed to the chemopreventive activity of I3C. Although calorie restriction is the most potent cancer prevention regimen (24), effects of calorie restriction and reduced body weight gain on lung tumor development have not been extensively studied. In one study, restriction of food intake by 40% and 60% reduced body weight by about 23% and 34%, respectively, but lung tumor multiplicity was reduced only by 25% in both groups (25).

I3C inhibits carcinogenesis through modulation of expression of various proteins involved in carcinogen detoxification, proliferation and apoptosis of tumor cells, and estrogen metabolism (4). Induction of cytochrome P450 enzymes, in particular the P450 1A family, in the liver has been shown to be a major mechanism through which I3C inhibits NNK-induced pulmonary carcinogenesis in mice (21). Similar findings have been reported in smokers who consumed cruciferous vegetables rich in I3C (8) or took I3C (26). A higher induction of P450 1A enzymes in mouse liver by I3C resulted in increased clearance of the carcinogen and reduced bioavailability to the lung. Unlike in mouse liver, in mouse lung, the constitutive expression of P450 1A enzymes is poor (27). In the present study, we found a higher level of P450 2F2, an enzyme with a high catalytic activity for naphthalene and related compounds (28), in lung tissues of I3C-treated mice. As a bifunctional enzyme inducer, I3C induces not only phase I enzymes but also phase II enzymes such as GST. GST inhibits BaP-induced carcinogenesis by catalyzing the conjugation of glutathione with various metabolites of BaP (29).

The level of SP-C, L-plastin, annexin A1, and haptoglobin increased in lung tissues of mice treated with the carcinogens alone. However, upon dietary administration of I3C to carcinogen-treated mice, the expression level of these proteins was decreased to the level measured in untreated mice. These findings suggest the relevance of SP-C, L-plastin, annexin A1, and haptoglobin for the development of NNK plus BaP–induced lung carcinogenesis, and that these proteins may be targets for the lung tumor chemopreventive activity of I3C. Indeed, earlier reports indicate the involvement of SP-C, L-plastin, annexin A1, and haptoglobin in the development of tumors in the lung and other organs.

SP-C is a member of pulmonary surfactant proteins, a complex mixture of lipids and proteins that reduce the surface tension at the air-liquid interface and prevent alveolar collapse during respiration. Surfactant proteins are overexpressed during lung carcinogenesis induced by different carcinogens (30–32). In the present study, pulmonary surfactant proteins A, B, and C were identified, but only SP-C levels increased in carcinogen-treated versus untreated mice. Surfactant proteins are overexpressed not only during lung carcinogenesis but also during lung inflammation and, therefore, cannot be used as specific markers of lung tumors. However, because surfactant proteins are expressed only in the lung, modulation of their expression could help to differentiate between tumors that originate in the lung and those that metastasize from other organs. Moreover, modulation of carcinogen-induced increases in the level of SP-C by I3C indicates that surfactant proteins could be included in a multi-protein tumor marker index to evaluate the efficacy of lung cancer chemopreventive agents.

L-plastin, a protein usually expressed only in cells of hematopoietic origin, is constitutively expressed in many types of malignant cells of solid cancers (33). Overexpression of L-plastin leads to increased cell proliferation and invasion of tumor cells (34), probably as a result of suppression of E-cadherin (34), an epithelium-specific tumor suppressor gene whose loss of expression is directly associated with increased cell proliferation. Up-regulation of E-cadherin expression in I3C-treated prostate cancer cells decreased invasion and proliferation of tumor cells (35). In addition, antisense constructs of L-plastin suppressed prostate carcinoma cell proliferation and invasion (36).

Annexin A1 is a substrate for tyrosine kinases such as epidermal growth factor receptor (EGFR) and protein kinases (37). Although EGFR overexpression and mutations are quite heterogeneous in human lung cancer (38), annexin A1 level may increase in some types of lung cancer. Radioimmunotherapy to annexin A1 in rats bearing lung tumors destroyed the tumors and increased survival (39). Attenuation by I3C of NNK and BaP–induced increases in the level of annexin A1 in this study might be related to I3C-induced down-regulation of EGFR expression observed in PC-3 prostate cancer cells (40).

Haptoglobin, a major hemoglobin binding protein, is a member of acute-phase proteins, which are synthesized by the liver in response to disturbances of the organism homeostasis due to infection, tissue injury, neoplastic growth, or immunologic disorders. Haptoglobin was elevated in the serum of lung cancer patients (41, 42). However, the biological implication of this is not clear. Yang et al. (43) reported severalfold increases in the expression of the haptoglobin gene in mouse and baboon alveolar type II cells, progenitors of lung adenoma, during inflammation and some disease states, suggesting protective roles of haptoglobin in lung tissues. Although we attempted to flush the blood out of the lungs, it was not possible to completely remove the blood, especially in lungs of mice with the highest tumor burden (group treated with carcinogens alone). Therefore, it is possible that serum contributes to the higher level of haptoglobin in this group. Expression of the other acute-phase proteins AAT, TF, and Apo A1, decreased in lung tissues of NNK plus BaP–treated mice, but dietary administration of I3C did not modulate this effect. Decreased levels of AAT, TF, and Apo A-1 were reported in murine or human lung tumors (41, 44, 45). These proteins and haptoglobin originate not from tumorous lung tissues but from the liver; therefore, their usefulness as lung cancer biomarkers is questioned.

Metabolic alteration is a common phenomenon during carcinogenesis. We observed overexpression of transketolase and fatty acid synthase in lung tissues of carcinogen-treated mice (Table 2). Transketolase catalyzes, through a thiamine-dependent mechanism, the non-oxidative conversion of glucose into ribose phosphate, a building block for nucleic acids and nucleotides. Therefore, transketolase plays an important role in proliferating tissues, especially in tumors (46). Using microarray analysis, Hackett et al. (46) reported increased expression of transketolase in airway epithelium of cigarette smokers versus nonsmokers. This suggests a possible contribution of transketolase in early-stage airway carcinogenesis in smokers. Elevated levels of fatty acid synthase, a protein capable of the reductive de novo synthesis of long-chain fatty acids, have been identified in the blood of patients with breast, prostate, colon, and ovarian cancers compared with normal subjects (47). Fatty acid synthase and a related protein (fatty-acid-binding protein) were detected in 60% and 67% of lung carcinoma cases, respectively (48). Inhibition of fatty acid synthase expression reduces tumor development (49). I3C did not modulate NNK plus BaP–induced increases in the level of either transketolase or fatty acid synthase.

Overall, we showed, using the iTRAQ proteomics technique, that the level of some tumor-related proteins increases during lung tumorigenesis, and that some of these proteins are modulated in tandem with the chemopreventive activity of I3C. These proteins can be further investigated as biomarkers for early detection of lung tumorigenesis and as leads to a better understanding of the molecular alterations that take place during the development of lung cancer. Modulation of protein levels by chemopreventive agents could potentially be used as a measure of chemopreventive efficacy. A major limitation of this study is that less abundant but important proteins involved in the development of lung carcinogenesis, such as cyclooxygenase-2, EGFR, and mitogen-activated protein kinase, were not identified, and that the total number of identified proteins (500–600) was low compared with what might be expected on the basis of the ~40,000 protein coding genes in the mouse genome (50). This might be attributed to instrumental limitations and suppression of low-abundance proteins by the high-abundance proteins commonly identified in crude organ homogenate samples. The relative levels of less-abundance proteins compared with the dynamic range of the entire sample may have resulted in peptide signal intensities for proteins to fall below the selection intensity threshold for MS/MS precursor ion detection after the automated sampling of the four most intense peaks. This problem with sample preparation might be surmounted using subcellular fractions, which allow identification of proteins specific to subcellular compartments, such as membrane proteins. We will address this issue in future studies.

In spite of the considerable evidence on the tumor inhibitory effects of I3C, the compound also promotes chemically induced carcinogenesis, depending upon the initiator, exposure protocol, and species of test animal (4). This effect may be attributed to indolo[3,2-b]carbazole, another condensation product of I3C that resembles dioxin in structure and enzyme-inducing ability and is a very strong agonist of the aryl hydrocarbon receptor (51). DIM is a weak agonist of the aryl hydrocarbon receptor (51) and, therefore, could be a relatively safe alternative to I3C as a chemopreventive agent.

In summary, our results show that I3C and DIM inhibit NNK plus BaP–induced lung tumorigenesis, and that this effect might be related in part to modulation of carcinogen-induced alterations in protein levels.


   Acknowledgments
 
Grant support: NIH/National Cancer Institute grant CA-102502 (S.S. Hecht).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank David Jewison for his help in the preparation of the diets, Dr. LeeAnn Higgins of the Center for Mass Spectrometry and Proteomics, University of Minnesota, for her assistance in the analysis of the samples, and Dr. Michael Zeligs (BioResponse LLC, Boulder, CO) for providing DIM in a bioavailable formulation.

Received 12/10/06. Revised 3/27/07. Accepted 4/23/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74–108.[Abstract/Free Full Text]
  2. Jemal A, Siegel R, Ward E, et al. Cancer statistics 2006. CA Cancer J Clin 2006;56:106–30.[Abstract/Free Full Text]
  3. Centers for Disease Control and Prevention (CDC). Cigarette smoking among adults: United States 2004. MMWR 2005;54:1121–24.[Medline]
  4. IARC. Cruciferous vegetables, isothiocyanates and indoles. IARC handbooks of cancer prevention. Vol. 9. Lyon: IARC Press; 2004.
  5. Anderton MJ, Manson MM, Vershoyle RD, et al. Pharmacokinetics and tissue disposition of indole-3-carbinol and its acid condensation products after oral administration to mice. Clin Cancer Res 2204;10:5233–41.[CrossRef]
  6. Broadbent TA, Broadbent HS. The chemistry and pharmacology of indole-3-carbinol (indole-3-methanol) and 3-(methoxymethyl)indole. Curr Med Chem 1988;5:337–52.
  7. Sones K, Heaney RK, Fenwick GR. An estimate of the mean daily intake of glucosinolates from cruciferous vegetables in the UK. J Sci Food Agric 1984;35:712–20.[CrossRef]
  8. Hecht SS, Carmella SG, Kenny PMJ, Low SH, Arakawa K, Yu MC. Effects of cruciferous vegetable consumption on urinary metabolites of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in Singapore Chinese. Cancer Epidemiol Biomarkers Prev 2004;13:997–1004.[Abstract/Free Full Text]
  9. Dashwood RH. Indole-3-carbinol: anticarcinogen or tumor promoter in Brassica vegetables? Chem Biol Interact 1998;110:1–5.[CrossRef][Medline]
  10. Reed GA, Peterson KS, Smith HJ et al. A phase I study of indole-3-carbinol in women: tolerability and effects. Cancer Epidemiol Biomarkers Prev 2005;14:1953–60.[Abstract/Free Full Text]
  11. Ross PL, Huang YN, Marchese JN et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 2004;12:1154–69.
  12. Hecht SS, Lin D, Castonguay A. Effects of {alpha}-deuterium substitution on the mutagenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Carcinogenesis 1983;4:305–10.[Abstract/Free Full Text]
  13. Bogen KT, Witschi H. Lung tumors in A/J mice exposed to environmental tobacco smoke: estimated potency and implied human risk. Carcinogenesis 2002;23:511–9.[Abstract/Free Full Text]
  14. Kapetanovic IM, Krishnaraj R, Martin-Jimenez T, Yuan L, van Breemen RB, Lybimov A. Effects of oral dosing paradigms (gavage versus diet) on pharmacokinetics and pharmacodynamics. Chem Biol Interact 2006;164:68–75.[CrossRef][Medline]
  15. Lowry OH, Rosebrough NJ, Farr AL, Randall NJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75.[Free Full Text]
  16. Tilton SC, Givan SA, Pereira CB, Bailey GS, Williams DE. Toxicogenomic profiling of the hepatic tumor promoters indole-3-carbinol, 17-beta-estradiol and beta-naphthoflavone in rainbow trout. Toxicol Sci 2006;90:61–72.[Abstract/Free Full Text]
  17. Radia M, Schulz-Knappe P, Heine G, Forssmann WG. Liquid chromatography and electrospray mass spectrometric mapping of peptides from human plasma filtrate. J Am Soc Mass Spectrom 1999;10:45–54.[CrossRef][Medline]
  18. Nelsestuen GL, Zhang Y, Martinez MB, et al. Plasma protein profiling: unique and stable features of individuals. Proteomics 2005;5:4012–24.[CrossRef][Medline]
  19. Tang WH, Halpern BR, Shilov IV, Seymour SL, Keating SP, Loboda A. Discovering known and unanticipated protein modifications using MS/MS database searching. Anal Chem 2005;77:3931–46.[Medline]
  20. Biemann K. Contributions of mass spectrometry to peptide and protein structure. Biomed Mass Spectrom 1988;16:99–111.[CrossRef]
  21. Morse MA, LaGreca SD, Amin SG, Chung, FL. Effects of indole-3-carbinol on lung tumorigenesis and DNA methylation induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and on the metabolism and disposition of NNK in A/J mice. Cancer Res 1990;50:2613–7.[Abstract/Free Full Text]
  22. El Bayoumy K, Upadhyaya P, Desai DH, et al. Effects of 1,4-phenylenebis (methylene)selenocyanate, phenethyl isothiocyanate, indole-3-carbinol, and D-limonene individually and in combination on the tumorigenicity of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mouse lung. Anticancer Res 1996;16:2709–12.[Medline]
  23. Jang JJ, Cho KJ, LeeYS, Bae JH. Modifiying responses of allyl sulfide, indole-3-carbinol and germanium in a rat multi-organ carcinogenesis model. Carcinogenesis 1991;12:691–5.[Abstract/Free Full Text]
  24. Hursting SD, Lavigne JA, Berrigan D, Perkins SN, Barrett JC. Calorie restriction, aging, and cancer prevention: mechanisms of action and applicability to humans. Annu Rev Med 2003;54:131–52.[CrossRef][Medline]
  25. Stinn W, Teredesai A, Kuhl P, Knoerr-Wittmann C, Kindt R. Mechanisms involved in A/J mouse lung tumorigenesis induced by inhalation of an environmental tobacco smoke surrogate. Inhal Toxicol 2005;17:263–76.[CrossRef][Medline]
  26. Taioli E, Garbers S, Bradlow HL, Carmella SG, Akerkar S, Hecht SS. Effects of indole-3-carbinol on the metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in smokers. Cancer Epidemiol Biomarkers Prev 1997;6:517–22.[Abstract]
  27. Chichester CH, Philpot RM, Weir AJ, Buckpitt AR, Plopper CG. Characterization of the cytochrome P-450 monooxygenase system in non-ciliated bronchial epithelial (clara) cells isolated from mouse lung. Am J Respir Cell Mol Biol 1991;4:179–86.[Medline]
  28. Shultz MA, Morin D, Chang A, Buckpitt A. Metabolic capabilities of CYP 2F2 with various pulmonary toxicants and its relative abundance in mouse lung subcompartments. J Pharmacol Exp Ther 2001;296:510–9.[Abstract/Free Full Text]
  29. Sparnins VL, Mott AW, Barany G, Wattenberg LKW. Effects of allyl methyl trisulfide on glutathione S-transferase activity and BP-induced neoplasia in the mouse. Nutr Cancer 1986;8:211–5.[Medline]
  30. Mason RJ, Kalina M, Nielsen LD, Malkinson AM, Shannon JM. Surfactant protein C expression in urethane-induced murine pulmonary tumors. Am J Pathol 2000;156:175–82.[Abstract/Free Full Text]
  31. Zhang F, Pao W, Umphress SM, et al. Serum levels of surfactant protein D are increased in mice with lung tumors. Cancer Res 2003;63:5889–94.[Abstract/Free Full Text]
  32. Okubo C, Morishita Y, Minami Y, et al. Phenotypic characteristics of mouse lung adenoma induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Mol Carcinog 2005;42:121–6.[CrossRef][Medline]
  33. Park T, Chen ZP, Leavitt Z. Activation of the leukocyte plastin gene occurs in most human cancer cells. Cancer Res 1994;54:1775–81.[Abstract/Free Full Text]
  34. Foran E, McWilliam P, Kelleher D, Croke DT, Long A. The leukocyte protein L-plastin induces proliferation, invasion and loss of E-cadherin expression in colon cancer cells. Int J Cancer 2006;118:2098–104.[CrossRef][Medline]
  35. Nwanko JO. Anti-metastatic activities of all-transretinoic acid, indole-3-carbinol and catechin in Dunning rat invasive prostate adenocarcinoma cells. Anticancer Res 2002;22:4129–35.[Medline]
  36. Zheng J, Rudra-Ganguly N, Powell WC, Roy-Burman P. Suppression of prostate carcinoma cell invasion by expression of antisense L-plastin gene. Am J Pathol 1999;155:115–22.[Abstract/Free Full Text]
  37. De BK, Misono KS, Lukas TJ, Mroczkowski B, Cohen S. A calcium-dependent 35-kilodalton substrate for epidermal growth factor receptor/kinase from normal tissue. J Biol Chem 1986;261:13784–92.[Abstract/Free Full Text]
  38. Sharma SV, Bell DW, Settleman J, Haber DA. Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer 2007;7:169–81.[CrossRef][Medline]
  39. Oh P, Li Y, Yu J et al. Subtractive proteomics mapping of the endothelial surface in lung and solid tumors for tissue-specific therapy. Nature 2004;429:629–35.[CrossRef][Medline]
  40. Chinni SR, Sarkan FH. Akt inactivation is a key event in indole-3-carbinol- induced apoptosis in PC-3 cells. Clin Cancer Res 2002;8:1228–36.[Abstract/Free Full Text]
  41. Maciel CM, Junqueira M, Paschoal ME, et al. Differential proteomic serum pattern of low molecular weight proteins expressed by adenocarcinoma patients. J Exp Ther Oncol 2005;5:31–8.[Medline]
  42. Bharti A, Ma PC, Maulik G, et al. Haptoglobin-alpha subunit and hepatocyte growth factor can potentially serve as serum tumor biomarkers in small cell lung cancer. Anticancer Res 2004;24:1031–8.[Abstract/Free Full Text]
  43. Yang F, Firedrichs WE, Navarijo-Ashbaugh AL, deGraffenried LA, Bowmann BH, Coalson JJ. Inflammatory induction and cell type specific expression of haptoglobin gene in baboon and mouse lung. Lab Invest 1995;73:433–40.[Medline]
  44. Lin L, Wang, Y, Bergman G, Kelloff GJ, Lubet RA, You M. Detection of differentially expressed genes in mouse lung adenocarcinomas. Exp Lung Res 2001;27:217–29.[Medline]
  45. Sun Z, Yang P. Role of imbalance between neutrophil elastase and alpha-1-antitrypsin in cancer development and progression. Lancet Oncol 2005;5:182–90.
  46. Boros LG, Puigjaner J, Cascante M. Role of thiamine (Vitamin B1) and transketolase in tumor cell proliferation. Nutr Cancer 2000;36:150–4.[CrossRef][Medline]
  47. Hackett NR, Heguy A, Harvey BG, et al. Variability of antioxidant-related gene expression in the airway epithelium of cigarette smokers. Am J Respir Cell Mol Biol 2003;29:331–43.[Abstract/Free Full Text]
  48. Kawamura T, Kanno R, Fujii H, Suzuki T. Expression of liver type fatty-acid-binding protein, fatty acid synthase and vascular endothelial growth factor in human lung carcinoma. Pathology 2005;72:233–40.
  49. Lu S, Archer MC. Fatty acid synthase is a potential molecular target for the chemoprevention of breast cancer. Carcinogenesis 2005;26:153–7.[Abstract/Free Full Text]
  50. Nekrutenko A. Reconciling the numbers: ESTS versus protein coding genes. Mol Biol Evol 2004;21:1278–82.[Abstract/Free Full Text]
  51. Bjeldanes LF, Kim JY, Grose KR, Bartholomew JC, Bradfield CA. Aromatic hydrocarbon receptor agonist generated from indole-3-carbinol in vitro and in vivo: comparisons with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Proc Natl Acad Sci U S A 1991;88:9543–7.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Prevention ResearchHome page
F. Kassie, I. Matise, M. Negia, P. Upadhyaya, and S. S. Hecht
Dose-Dependent Inhibition of Tobacco Smoke Carcinogen-Induced Lung Tumorigenesis in A/J Mice by Indole-3-Carbinol
Cancer Prevention Research, December 1, 2008; 1(7): 568 - 576.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Prevention ResearchHome page
T. E. Johnson, F. Kassie, M. G. O'Sullivan, M. Negia, T. E. Hanson, P. Upadhyaya, P. P. Ruvolo, S. S. Hecht, and C. Xing
Chemopreventive Effect of Kava on 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone plus Benzo[a]pyrene-Induced Lung Tumorigenesis in A/J Mice
Cancer Prevention Research, November 1, 2008; 1(6): 430 - 438.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
G. D. Stoner, A. A. Dombkowski, R. K. Reen, D. Cukovic, S. Salagrama, L.-S. Wang, and J. F. Lechner
Carcinogen-Altered Genes in Rat Esophagus Positively Modulated to Normal Levels of Expression by Both Black Raspberries and Phenylethyl Isothiocyanate
Cancer Res., August 1, 2008; 68(15): 6460 - 6467.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
H. Orita, J. Coulter, E. Tully, F. P. Kuhajda, and E. Gabrielson
Inhibiting Fatty Acid Synthase for Chemoprevention of Chemically Induced Lung Tumors
Clin. Cancer Res., April 15, 2008; 14(8): 2458 - 2464.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
F. Kassie, L. B. Anderson, L. Higgins, Y. Pan, I. Matise, M. Negia, P. Upadhyaya, M. Wang, and S. S. Hecht
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
Carcinogenesis, March 1, 2008; 29(3): 610 - 619.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kassie, F.
Right arrow Articles by Hecht, S. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kassie, F.
Right arrow Articles by Hecht, S. S.
Related Collections
Right arrow Preclinical Intervention
Right arrow Preclinical Intervention: In Vivo (Animals): Drugs, Nutritional Interventions, Mechanisms

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online