Lung cancer is the leading cause of cancer-related mortalities in men and women, and despite extensive antismoking campaigns, it still accounts for 15% of all new cancers and 29% of all cancer deaths in the U.S. [1
]. Among lung cancers, pulmonary adenocarcinoma is the predominant histological type [1
]. Tobacco smoking is the major risk factor, estimated to cause 87% of lung cancer cases in the U.S. [2
]. Tobacco smoke (TS) contains 4,000 chemical agents, including over 60 carcinogens. Conversion of these compounds to reactive forms (metabolic activation) results in the formation of DNA adducts that cause many of the genetic changes underlying lung cancer. Among the carcinogens found in cigarette smoke, NNK [4-(methylnitrosamino)-1-(3-pyridyl)-butanone] is one of the most abundant [2
], which leads to K-ras
-activating mutations as early events in the pathway leading to lung adenocarcinoma [3
]. Interestingly, NNK has been shown to promote the formation of ROS in the lung [5
] and this ROS is implicated in the formation and proliferation of tumor cells by promoting DNA damage [6
Macrophages are the most abundant immune cells involved in tumor development [7
]. One previous study showed that the pharmacologic depletion of macrophages in different mouse tumor models significantly reduced tumor angiogenesis and progression, suggesting that macrophages are critical components in the tumor microenvironment for tumor progression [8
]. Tumor-associated macrophages (TAMs) are M2-like cells and are responsible for many tumor-promoting activities during tumor initiation, progression and metastasis [9
]. TAMs play a major role in suppressing the anti-tumor responses of dendritic cells (DCs), cytotoxic T lymphocytes (CTLs), and natural killer (NK) cells [10
]. It has been shown that blocking the functions of TAMs inhibits tumorigenesis [12
Our previous study found that ROS plays a critical role in the differentiation of alternatively activated macrophages and the occurrence of TAMs [14
]. In this study, we examined the effect of blocking ROS on NNK-induced tumorigenesis in A/J mice, which is a widely accepted model of lung tumor. Using the ROS inhibitor, BHA, we examined the occurrence of tumors and TAMs in the lungs of mice treated with NNK. We found that the continuous administration of BHA efficiently blocked the occurrence of TAMs and markedly suppressed tumorigenesis in this NNK-induced lung cancer model.
Although substantial epidemiological evidence indicates an association between pulmonary inflammation and tobacco smoke-induced lung cancer, a direct causal interaction between which specific-type of inflammatory infiltrates results in tumor initiation and the molecular events underlying inflammation-related lung tumorigenesis are not well known [16
]. Our previous study found that ROS plays a critical role in the differentiation of alternatively activated macrophages and TAMs [14
]. The continuous administration of the ROS inhibitor, BHA, efficiently blocked the occurrence of TAMs and markedly suppressed tumorigenesis in urethane-induced lung cancer and K-rasLA2
-induced lung cancer [14
]. Since tobacco smoking is the major risk factor of lung cancer (Hecht, 2002), here we explored the role of BHA in tobacco smoke carcinogen-induced lung tumorigenesis.
NNK is the most abundant carcinogen found in cigarette smoke (Hecht, 2002), we therefore used NNK as the chemical to induce lung tumor. Similar to other lung cancer models, the inflammatory microenvironments including macrophages have been reported to play a critical role in promoting lung tumorigenesis in NNK-induced lung cancer model [17
]. In addition, K-ras
mutation in codon 12 of the A/J mice is most commonly detected in preneoplastic hyperplasias or adenocarcinoma induced by NNK treatment [18
], which results in a milieu of downstream changes leading to tumorigenesis. NNK induced overexpression of oncogenic K-ras
leads to the activation of the transcription factor, NF-κB, the enhancement of inflammatory responses and the development of lung adenocarcinomas [19
]. Our previous study showed that blocking TAMs leads to the suppression of tumorigenesis in oncogene-driven K-rasLA2
-induced lung cancer, suggesting that TAMs may play an important role in NNK-induced lung tumorigenesis. Chemicals targeting TAMs could prove to be successful for the treatment of tobacco smoke-induced lung cancer.
As expected, we found that continuous administration of BHA blocked tumor development in NNK-induced lung cancer model. Previous studies have looked at the effect of antioxidants, including BHA, on NNK-induced tumorigenesis [20
] and have found that it affects tumorigenesis. However, the mechanisms of how BHA effect on tumorigenesis is still unclear. Our study examined the continuous administration of BHA and found that it has a protective effect against NNK-induced lung tumorigenesis in A/J mice which is achieved by specifically blocking the occurrence of TAMs in tumors induced by NNK. Our study suggests the possibility of BHA as an inhibitor of tobacco smoke-induced lung cancer development may be achieved by blocking the accumulation of TAMs. Many factors have been shown to affect the accumulation of TAMs, such as macrophage-colony stimulating factor (M-CSF) which induces monocyte to M2 like macrophage or TAM differentiation [15
], IL4 or IL10 which works on the polarization of TAMs, and some chemokines that are specific for M2 macrophages or TAMs accumulations [15
]. Our previous study showed that BHA specifically affects M2 macrophages or TAMs accumulation triggered by these factors. BHA blocks macrophage differentiation and this is overcome when cells are polarized to classically activated (M1), but not M2 macrophages. Since TAMs are M2-like macropahges BHA specifically targets TAMs as seen by the loss of Arg and Ym-1 expressions in mouse macrophages. This study supports our finding that BHA effects tobacco smoke-induced lung tumorigenesis by targeting TAMs [14
Since oxygen free radicals and other oxidative agents produced during the inflammatory response to NNK could lead to DNA damage, some of which could initiate lung tumorigenesis, it is possible that BHA may directly block the oxidative DNA damage that contributes to the K-ras
mutations. However, we did not observe a difference in tumor incidence (data not shown). Our previous data suggested that BHA has no effect on monocyte migration or the proliferation of tumor cells in a NUDE mouse model depleted of macrophages [14
], therefore it is most likely that the BHA suppressed NNK-induced tumor development by specifically affecting the TAMs.
4. Materials and Methods
A/J mice were from the Jackson Laboratory (Bar Harbor, ME, USA). Mice were maintained under pathogen-free conditions, and experimental protocols were approved by NCI, following NIH guidelines.
4.2. Reagents and Antibody
BHA was obtained from Sigma (St. Louis, MO, USA). NNK was obtained from Toronto Research Chemicals (Toronto, ON, Canada). Anti-Ym-1 from STEMCELL Technologies (Vancouver, BC, Canada); anti-Arginase I from Santa Cruz (Dallas, TX, USA); anti-CD206 from Hycult Biotech (Plymouth Meeting, PA, USA); and anti-F4/80 from eBiosciences (San Diego, CA, USA).
4.3. Western Blot Analysis
Cells were collected and lysed in M2 buffer (20 mM Tris at pH 7, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM PMSF, 20 mM β-glycerol phosphate, 1 mM sodium vanadate, and 1 mg/mL leupeptin). Cell lysates were separated by SDS-PAGE and analyzed by immunoblot. The proteins were visualized by enhanced chemiluminescence (ECL, Pierce, Rockford, IL, USA).
4.4. NNK-Induced Lung Tumor Models
Eight-week old female A/J mice that were weight and age matched were used for experiments. Tumors were induced by two i.p. injection of 150 mg/kg NNK. Animals were then divided into normal NIH-31 chow or NIH-31 chow with 7.5 g/Kg BHA. After 6 months lungs were excised and evaluated for tumors.
4.5. Evaluation of Lung Tumors
For determining tumor multiplicity and tumor burden, whole lungs were inflated with and fixed in 4% paraformaldehyde for 24 h. Lungs were paraffin-embedded and serial sections at 400 microns were histologically examined with hematoxylin and eosin (H&E) stain. For quantitation of lung tumor multiplicity, tumor numbers of 5 serial sections per lung were counted and totaled. Tumor burden were shown as the ratio of tumor area to total lung area on 5 sections taken every 400 microns following staining with H&E by using imagescope. 10 lungs were analyzed for each cohort indicated.
4.6. Immunohistochemical Analysis
Paraffin-embedded slides were deparaffinized and antigens were unmasked by autoclaving at 121 °C for 10 min in Sodium Citrate (pH 6.0) buffer. Slides were incubated with primary antibody (anti-F4/80 and anti-CD206) in 4 °C overnight. Signals were detected with VECTASTIN ABC Elite kit (Vector Laboratories, Burlingame, CA, USA) and DAB Substrate Kit (Vector Laboratories). Quantitative analysis of positive cells was performed by counting cells in ten high-power fields (20×) per two tissue sections from 6 to 10 mice per group.
4.7. BALF Leukocyte Counts
BALF was withdrawn after instillation of 800 μL sterile PBS through the trachea, and cytospin preparations of BALF cells were prepared using Shandon Cytospin centrifuge (Thermo Electron Corporation, Waltham, MA, USA). BALF cells were visualized by Wright-Giemsa staining and percentages of leukocyte types were determined by counting 400 leukocytes in a randomly selected portion of the slide under light microscopy. BALF total leukocyte counts were performed using a hemocytometer.
4.8. Statistical Analysis
Statistical analyses were performed using GraphPad Prism and/or Aperio ImageScope Software. Two group comparisons were performed using Student’s t test. All p values less than 0.05 were considered statistically significant.