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Anti-tumor B (ATB) is a Chinese herbal mixture of six plants. Previous studies have shown significant chemopreventive efficacy of ATB against human esophageal and lung cancers. We have recently developed a new mouse model for lung squamous cell carcinomas (SCC). In this study, lung SCC mouse model was characterized using small-animal imaging techniques (MRI and CT). ATB decreased lung SCC significantly (3.1 fold, p < 0.05) and increased lung hyperplastic lesions by 2.4 fold (p < 0.05). This observation suggests that ATB can block hyperplasia from progression to SCC. ATB tissue distribution was determined using matrine as a marker chemical. We found that ATB is rapidly absorbed and then distributes to various tissues including the lung. These results indicate that ATB is a potent chemopreventive agent against the development of mouse lung squamous cell carcinomas.
Lung cancer is the leading cause of cancer death in the world and is one of the most preventable diseases (1). Cancer chemoprevention uses natural or synthetic agents to reverse, suppress, or prevent carcinogenic progression. Cancer chemopreventive agents can be classified into two major groups, blocking agents and suppressing agents. As the population of former smokers is markedly growing, chemopreventive agents are urgently needed. Agents such as α-tocopherol, β-carotene (2), aspirin (2), retinol (3, 4, 5), isotretinoin (6), N-acetylcysteine (7), anethole dithiolethione (8) have been tested in clinical trials in smokers. Unfortunately, there are no chemoprevention agents for lung cancer that have clearly shown clinical benefit. There are a variety of agents which have shown promise in animal studies including glucocorticoids, EGFR inhibitors, RXR agonists, tea polyphenols, deguelin, and the herbal mixture Anti tumor B (ATB) (9, 10, 11, 12). Amongst these only ATB has had success in human trials (13). Anti-tumor B (ATB), a Chinese herbal mixture, is a botanical agent composed of six Chinese herbs: Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus, and Dioscorea bulbifera (13). Previous reports have shown that ATB treatment reduced cancer development by 50% in patients with marked esophageal dysplasia (13). In a chemically induced lung adenocarcinoma model, we showed that ATB caused a significant reduction in lung tumor multiplicity and tumor load by 40% and 70%, respectively (10).
Magnetic resonance imaging (MRI) and computed tomography (CT) are powerful imaging modalities for characterizing animal systems and animal models of disease. In-vivo MRI permits a wide variety of non-invasive, non-destructive longitudinal studies not possible with other analytical methods. The lung presents unique challenges for MRI, requiring the development of new and innovative methods (14, 15) Among the complicating factors for the study of lungs by 1H MR methods, are: 1) low tissue density and low water content within the lung, severely limiting signal-to-noise; 2) variations in magnetic susceptibility associated with the many air-tissue interfaces of the alveoli and bronchioles result in short T2* and T2 relaxation times; and 3) respiratory and cardiac motions lead to significant image blurring in the absence of motion-synchronized data acquisition. Using respiratory gated MRI methods, we have recently demonstrated the detection of sub-millimeter lung lesions in mice treated with the carcinogen, benzo[a]pyrene (14). MicroCT is also a non-invasive, non-destructive imaging technique. The small size of mice limits contrast between tissues of similar density without the use of contrast enhancement agents, however, microCT can take advantage of the high contrast between low-density-lung tissue and high-density-cancer tissue without contrast enhancement.
Recently, we reported a chemically induced model for SCC of the lung in mice (16). The objectives of the present study are to further characterize lung SCC mouse model using small-animal imaging techniques (MRI and CT) and to evaluate the effect of ATB on the development of lung SCC in mice. Our results demonstrate that dietary ATB causes a significant inhibition in the development of mouse lung SCC. To our knowledge, this is the first report of significant efficacy against lung tumors in a mouse lung SCC model.
Benzo[a]pyrene (BP), tricaprylin, and acetone were purchased from Sigma Chemical Co. (St. Louis, MO). Vinyl carbamate (VC) and N-nitroso-tris-chloroethylurea (NTCU) were purchased from Toronto Research Chemicals, Inc. (Toronto, Canada). The putative chemopreventive agent, ATB, was purchased from the Cancer Institute, the Chinese Academy of Medical Sciences (Beijing, China). Female A/J mice were received from the Jackson Laboratories (Bar Harbor, ME) at 6 weeks of age. Animals were quarantined for 1 week and housed with wood chip bedding in environmentally controlled, clean-air rooms with a 12-hour light-dark cycle and relative humidity of 50%. Drinking water and diet were supplied ad libitum. The study was approved by the Washington University’s Institutional Animal Care and Use Committee.
Respiratory-gated, spin-echo MR images of mice were collected in an Oxford Instruments (Oxford, United Kingdom) 4.7 tesla, 40-cm bore magnet. The magnet is equipped with Magnex Scientific (Oxford, United Kingdom) actively shielded, high-performance (10 cm inner diameter, 60 G/cm, 100-μs rise-time) gradient coils and is interfaced with a Varian NMR Systems (Palo Alto, CA) INOVA console. All data were collected using a Stark Contrast (Erlangen, Germany) 2.5-cm birdcage radiofrequency coil. Before the imaging experiments, mice were anesthetized with isoflurane and were maintained on isoflurane/O2 (1–1.5% v/v) throughout data collection. Animal core body temperature was maintained at 37 ± 1°C by circulation of warm air through the bore of the magnet. Approximately 500 μl of Omniscan (Gadodiamide, GE Healthcare) contrast agent, diluted 1:10 in saline, was injected intraperitoneally (IP) immediately before placing the animal in the magnet. During the imaging experiments, the respiration rates for all mice were regular and 2 s−1. Synchronization of MR data collection with animal respiration was achieved with a home-built respiratory-gating unit (14), and all images were collected during postexpiratory periods. Imaging parameters are repetition time (TR) = 3 s, echo time (TE) = 20 ms, 2.5 cm FOV (field of view), and slice thickness = 0.5 mm.
Mice were anesthetized in a similar fashion to that of the MR studies. Animal temperature was regulated at ambient room air. Pairs of mice were imaged on a microCAT-II scanner (Seimens-CTI Concorde Inc., Knoxville, TN). Scans were performed at either low (adenocarcinoma) or high (SCC) resolution. Low resolution images used a x-ray source energy of 80 kvP with a 200 ms exposure time and 400 view angles over 360 degrees. High resolution images used x-ray source energy of 60 kvp with a 500 ms exposure time and 600 view angles. Adenocarcinoma images were reconstructed at low resolution (200 μm × 200 μm × 96 μm) and SCC images were reconstructed at 96 μm istotropic voxel size.
A/J mice at 6-8 week of age were randomized into two groups. All mice were treated topically with 0.04 M NTCU in 100-microliter drop, twice a week, with a 3.5-day interval for 22 weeks (16). Two weeks after the start of NTCU treatment, mice in Group 1 were fed AIN-76A Purified Diet # 100 000 (Dyets Inc, Bethlehem, PA) and mice in Group 2 were fed with the same diet plus 250g/kg ATB. Food was changed every other day. Twenty-four weeks after the initial treatment of NTCU, mice were terminated by Co2 asphyxiation. Lungs were fixed in Tellyesniczky’s (90% ethanol (70% v/v), 5% glacial acetic acid, 5% formalin (10% v/v buffered formalin) solution overnight and stored in 70% ethanol for histopathological evaluation. Unlike the mouse lung adenomas/ ACs, mouse SCC dose not form visible solid nodules on the surface of the lung. Serial tissue sections (4-μm each) were made from formalin-fixed lungs, and 1 in every 20 sections (approximately 100 μm apart) was stained with H&E and examined histologically under a light microscope to establish tumor multiplicity and the types of lesions (invasive SCC, SCC in situ, or bronchial hyperplasia/metaplasia).
We hypothesized that chemically induced lung tumors are more likely to occur in the carcinogen control group than in the treatment groups. To test this hypothesis, the Student’s t test was used. The data were obtained from the carcinogen control groups and different treatment groups in each experiment. We applied square-root transformation of tumor numbers since the original data did not follow normal distribution. The transformed data were of normal distribution (data not shown). Accordingly, the Student’s t test was used to test the differences between the control groups and the treatment groups.
Thirty-six A/J female mice weighing about 20 g each were randomly assigned into two groups. The mice were fed with feeds mixed with either 20% or 30% of ATB for 4 weeks. Three mice were removed from each group and sacrificed at pre-determined time points (0, 2, 4, 7, 14, and 28 days) during the experiment. A blood sample was taken from each mouse immediately and was centrifuged at 3000 rpm for 5 min to collect the plasma. The liver, lung, kidney, heart, and spleen also were removed from the carcass; they were snap-frozen in liquid nitrogen. All tissue samples were stored in a -70°C freezer until analysis.
The tissue samples were thawed at room temperature. About 0.3 g of each tissue was weighed and then homogenized with 0.9 ml of distilled water in a Kinemetica GmbH PCU-2-110 tissue homogenizer (Switzerland). The liver and kidney were homogenized individually. The lung, spleen and heart from 3 different mice were combined before being homogenized because of the small organ sizes. The final volumes of the homogenates were recorded. Extraction and measurement of matrine were conducted according to Sit et al., (17) with modification. Briefly, a 1-ml aliquot of each tissue homogenate was mixed with 50 μl of a deuterated matrine solution (5μg/ml) in a 10-ml screw capped glass centrifuged tube. The deuterated matrine was used as an internal standard of the assay. NaOH (0.5 ml, 1 M) was added to make the content of the centrifuge tube alkaline. The mixture was then extracted with 3 ml of toluene:butanol (v/v 7:3) on a mechanical shaker. After the centrifuge tube was centrifuged to separate the layers, the organic layer was removed and put into a new glass centrifuge tube containing 0.5 ml of 0.25 M HCl. The content in the centrifuge tube was mixed, centrifuged and the organic layer was discarded. The remaining aqueous layer was mixed with 0.5 ml of 1M NaOH before being extracted by 300 μl toluene:butanol (v/v 9:1). The organic layer was removed and analysed by a gas chromatograph/mass spectrometer (GC/MS) using the selective ion monitoring mode. The m/z 248 and m/z 250 ions were used to monitor matrine and deuterated matrine, respectively. Matrine concentration in a tissue sample was calculated from the area ratio of m/z 248 : m/z 250, the tissue weight and the homogenate volume (17).
Under light microscopy, normal bronchi are seen as a single layer of bronchial epithelial cells (Figure A-a-1). Upon NTCU treatment, the single layer bronchial epithelial cells are replaced with multiple layers of cells (hyperplasia) with increased production of keratin (Figure A-a-2). Squamous metaplasia is seen locally (Figure A-b). The lung SCC consists of large, flattened, and stratified cells with intra-cytoplasmic keratin/keratin pearls (Figure 1B, arrow-head) and/or intercellular bridges (Figure 1A-d, arrows) which is a less common phenomenon in mouse lung SCC. Anisokaryosis is another dominate feature in SCC tumor cells (Figure 1A-d). Most SCC arises centrally within the bronchi at different levels, including main, lobar, segmental or subsegmental bronchi. The tumor cells can break through the wall of bronchus and invade into lung parenchyma (Figure 1A-c). As shown in Figure 1B, there is a remarkable similarity in the progressive morphological changes during the development of lung SCCs between mice and humans. Multistage development of SCC in human can be seen histologically from normal epithelium, to early stage (hyperplasia, squamous metaplasia), intermediate stage (dysplasia), to late stage (carcinoma in situ, and invasive tumor) (18). Similarly, serial lesions in mouse lung SCC development can easily be identified because of the uniform appearance of the normal bronchial epithelium, with a single layer of columnar cells, is markedly different from the hyperplasia of bronchial epithelium with increased cell number and a multilayered bronchial epithelium (Figure 1). Bronchial epithelium is replaced by a stratified, keratinized, squamous epithelium in squamous metaplasia. Dysplasia is seen by increased epithelial cell layers and increased nucleus/cytoplasm ratio. In the carcinoma in situ, bronchiolar epithelial cells become atypical with irregular shape, increased nucleus/cytoplasm ratio, with mitosis, loss of orderly differentiation through the entire thickened epithelium. The bronchiole basement membrane is intact with no tumor cells in the surrounding stroma. In the invasive SCC, the cancerous cells are not only disordered throughout the entire thickness of the lining, but they invade the tissue underlying the bronchiole basement membrane into the surrounding stroma. The typical SCC can be seen, including keratin pearls, multiple nuclei, and increasing mitotic index. The normal architecture of the lung is disrupted. Cords and nests of tumors can be seen in the subepithelial stroma.
We characterized our mouse lung SCC model using small-animal imaging techniques (MRI and CT) and compared this with the imaging using the well characterized A/J mouse lung adenocarcinoma model. The images were correlated with gross (Figure 2A) and histopathological examinations of the tumors (Figure 1). Both lung ACs and SCCs were successfully detected using both MRI and micro-CT. Figure 2B shows a series of contiguous coronal, respiratory-gated spin-echo images of one mouse with ACs and two mice with SCCs, at early and advanced stages of disease, respectively. As described in the Introduction, lungs present several unique challenges to study by MRI. However, the very factors that make it difficult to image healthy lung parenchyma, including low tissue density, low water content, and variations in magnetic susceptibility within the lung, in fact aid in the detection of tumors by increasing the contrast between healthy and pathologic tissue. Under the selected experimental conditions the MR images of healthy mouse lung parenchyma are completely dark; while signals attributable to the heart and its major blood vessels suppressed because of flow effects and cardiac motion (electrocardiograph gating was not used in this study). The bright spots visible in Figure 2B are attributable to lung tumors compared to the absence of signal in healthy lungs. As seen in Figure 2B, the MR images of adenocarcinomas show well defined nodules that are distributed randomly throughout the lungs. In contrast, the images of early stage SCCs show nodules that are centrally located along the trachea, while diffuse tumor tissue fills the lungs in the later stages of the disease. These results were further confirmed by micro-CT scans (Figure 2C). While tumor number of ACs by both MRI and micro-CT correlated positively with tumor number by necropsy and histopathology, the tumor counts of SCCs from histopathology are difficult to compare. The images of SCCs from both MRI and micro-CT showed lesions that are rather continuous than discreet. To our knowledge, we report for the first time the in vivo detection of primary lung SCCs at a submillimeter level, correlated with histopathology in mice.
In this study, ATB did not cause any symptoms of toxicity or apparent signs of ill health, nor have any significant affect on body weight in mice when given at a dose of 250 g/kg. As shown in Figure 3, in control NTCU - animals, the distributions of lesions are: normal (37.6 ± 8.7%), hyperplasia (18.7 ± 2.8%), metaplasia (9.4 ± 1.9%), carcinoma in situ (8.6 ± 2.0%), and SCC (23.9 ± 8.3%). In animals treated with ATB, the distributions of lesions are: normal (31.0 ± 5.8%), hyperplasia (45.4 ± 4.9%; p < 0.05), metaplasia (6.7 ± 1.6%), carcinoma in situ (9.3 ± 2.8%), and SCC (7.6 ± 3.5%; p < 0.05). ATB decreases lung SCC development significantly (3.1 fold, p < 0.05). At the same time, the percentages of the hyperplastic bronchioles are increased by 2.4 fold (p < 0.05). This observation indicates that ATB can block the progression of hyperplasia to SCC. An important observation in the present study is that ATB inhibits the progression of lung SCC.
Next, we determined the accumulation of ATB in the lung. Since ATB is composed of six Chinese herbs, it would be difficult if not impossible to examine the disposition of each ATB component in the lung tissue. We have chosen matrine as a marker chemical or tracer of ATB because matrine is one of the most abundant ATB components (19). In fact, matrine has been used as an indicator of ATB consistency between different batches by us and other groups (19). The product produced for human consumption is standardized to matrine content with each tablet containing 1.2 to 1.7 mg of matrine (19). Moreover, matrine has been shown to possess anti-cancer activities and appears to be a reasonable substitute for examining the biodistribution of ATB (20, 21). Figure 4 shows that matrine is rapidly absorbed by mice fed with 20% or 30% of ATB in their foods. Thus, matrine could be detected in the mouse tissues at day 2 (the earliest time point of sampling) after initiating the feeding study. The matrine concentration-time profiles were found to peak in mouse tissues at day 7, however differed in the amounts in each tissue. (Figure 4). Little to no matrine could be detected in the heart of mice treated with 20% ATB. Matrine also was not detected in the spleen of mice treated either with 20% or 30% ATB. Since only 3 plasma samples in the 30% ATB treatment group had matrine levels significantly higher than the LOQ of the analytical method (about 13 ng/ml), it was not possible to derive any meaningful pharmacokinetic parameters from the plasma concentration-time curve. All together, these results suggest that only a small fraction of the matrine consumed by the mice is absorbed. Similar results have been observed in the rat and human (19). Although the amount of matrine or ATB absorbed is small, the absorption does occur quickly and then distributes to the various animal tissues. Lung is an important site of ATB disposition.
In this study, we demonstrate that ATB is effective in chemoprevention of lung tumorigenesis in mouse models of lung ACs and SCCs. The results, showing efficacy of ATB against the development of mouse lung SCCs, represent the first successful application of this novel mouse lung cancer model to chemoprevention studies. We also found that feeding ATB diet significantly reduced tumor development of adenomas and ACs in both complete and progression protocols. Similarly, ATB was found to block the progression of bronchial cell hyperplasia and squamous metaplasia to SCC, as a higher percentage of hyperplasia were detected accompanied the decrease in lung SCCs. Thus, the efficacy of ATB on lung tumorigenesis is independent of tumor developmental stages.
Previously, we have demonstrated that ATB displayed a significant reduction in B(a)P – induced lung tumor multiplicity and tumor load in wild type mice, mice harboring a dominant-negative p53, mice with heterozygous deletion of Ink4a/Arf, and mice with compound mutations (10). Taken together, these results provide important scientific evidence in support of clinical chemoprevention trials of ATB in patients with precancerous lesions of non small cell lung cancer. This is because few agents that have proven useful in preventing lung cancer to date. ATB is a promising candidate, since it has been shown to inhibit effectively progression of precancerous lesions of human esophagus (dysplasia) to esophageal SCC (13).
We previously reported a chemically induced model for SCC of the lung in mice (16). The present study further demonstrated that NTCU induction of SCC in mouse lung exhibits significant similarities to human lung SCC when comparing the histological pictures of mouse lung SCC to that of human lung SCC (18). Distinguished histopathological features in early (hyperplasia, squamous metaplasia), intermediate (dysplasia) and late (in situ, SCC) stages human lung SCCs are also seen NTCU-induced lung SCC. This similarity makes this mouse model ideal for testing chemopreventive agents in pre-clinical studies.
Next, we compared lung SCCs and ACs using MRI and micro-CT. We believe that this is the first report of in vivo detection of primary lung SCCs in mice using either MRI or micro-CT imaging. Interestingly, lung ACs were distributed randomly throughout the lungs, while early stage SCCs were found to be more centrally located. At a later stage of disease, SCC lesions were distributed diffusely throughout the lungs. SCCs from both MRI and micro-CT showed continuous lesions while adenomas or ACs are more discreet. Our results indicate that MRI and micro-CT can clearly distinguish between AC and SCC and can be successfully applied for monitoring the effect of chemopreventive agents on lung tumor development and progression in mice. Longitudinal in vivo MRI/microCT are powerful modality that can be of great aid in elucidating the factors that control the onset of lung tumors and can serve as a platform for the development and preclinical testing of novel therapies having a high likelihood of efficacy in human clinical trials.
Using oligonucleotide array analysis, we previously reported that ATB modulated as many as 114 genes belonging to several cellular signaling pathways, including G protein-Ras-MAPK (MAPK3, MAP3K4, rab3A, Rap1, RSG5, PKCh) and apoptosis (BAD, caspase 3) (10). These results suggest that ATB may have a major effect on cell proliferation and cell cycle progression. We investigated the effect of ATB and its fractions on cell growth, cell cycle regulation, and AP-1 activity in mouse cancer epithelial cells. Treatment with ATB and fractions A & B inhibit lung cancer epithelial cell growth, and arrested the cells at G1 phase (Supplementary Data). Treatment of the cells with ATB and fractions A & B result in an inhibition of AP-1 activity (Supplementary Data). These in vitro experiments showed that ATB caused cell growth inhibition, G1 arrest, and AP-1 inhibition.
In conclusion, ATB inhibits the development of lung SCC by blocking the progression of hyperplasia progression to SCC. ATB is rapidly absorbed and then distributes to various tissues including the lung. Mouse lung SCC exhibits significant similarities to human lung SCC. MRI and micro-CT can distinguish between AC and SCC and can be used to monitor tumor progression. Clearly, ATB is a potent chemopreventive agent against the development of mouse lung squamous cell carcinomas in mice.
Grant support: This work was supported by United States Public Health Service Grants CA058554 & N01-CN-25104 (MY). an NIH/NCI Small Animal Imaging Resource Program (SAIRP) grant (R24 CA83060); and the Alvin J. Siteman Cancer Center at Washington University in St. Louis, an NCI Comprehensive Cancer Center (P30 CA91842). We thank Dr. Daolong Wang for statistical analysis on bioassay data. We acknowledge the assistance of members of Chemoprevention Group at The Siteman Cancer Center for careful reading of the manuscript.