Honokiol Inhibits Lung Tumorigenesis Through Inhibition of Mitochondrial Function
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Published OnlineFirst September 22, 2014; DOI: 10.1158/1940-6207.CAPR-14-0091 Cancer Prevention Research Article Research Honokiol Inhibits Lung Tumorigenesis through Inhibition of Mitochondrial Function Jing Pan1, Qi Zhang1, Qian Liu1, Steven M. Komas2, Balaraman Kalyanaraman2, Ronald A. Lubet3, Yian Wang1, and Ming You1 Abstract Honokiol is an important bioactive compound found in the bark of Magnolia tree. It is a non- adipogenic PPARg agonist and capable of inhibiting the growth of a variety of tumor types both in vitro and in xenograft models. However, to fully appreciate the potential chemopreventive activity of honokiol, a less artificial model system is required. To that end, this study examined the chemopreven- tive efficacy of honokiol in an initiation model of lung squamous cell carcinoma (SCC). This model system uses the carcinogen N-nitroso-trischloroethylurea (NTCU), which is applied topically, reliably triggering the development of SCC within 24 to 26 weeks. Administration of honokiol significantly reduced the percentage of bronchial that exhibit abnormal lung SCC histology from 24.4% bronchial in control to 11.0% bronchial in honokiol-treated group (P ¼ 0.01) while protecting normal bronchial histology (present in 20.5% of bronchial in control group and 38.5% of bronchial in honokiol-treated group. P ¼ 0.004). P63 staining at the SCC site confirmed the lung SCCs phenotype. In vitro studies revealed that honokiol inhibited lung SCC cells proliferation, arrested cells at the G1–S cell-cycle checkpoint, while also leading to increased apoptosis. Our study showed that interfering with mitochondrial respiration is a novel mechanism by which honokiol changed redox status in the mitochondria, triggered apoptosis, and finally leads to the inhibition of lung SCC. This novel mechanism of targeting mitochondrial suggests honokiol as a potential lung SCC chemopreventive agent. Cancer Prev Res; 7(11); 1149–59. Ó2014 AACR. Introduction it has experienced a number of important milestones over Lung cancer is the leading cause of cancer death world- the past several decades that have validated this approach wide. Though great advances have been made in early (2, 3). Over 80% of lung cancer is non–small cell lung diagnosis, discovery of chemotherapeutic agents, and in carcinoma (NSCLC), with adenocarcinoma and squamous molecular oncology, many common forms of epithelial cell carcinoma (SCC) as the two major subtypes. Although malignancy, especially carcinoma of the lung, remain dif- research suggests that the histopathologic features and ficult to cure. In this context, re-evaluation of basic assump- molecular mechanisms of these two lung cancer subtypes tions about cancer is necessary. Cancer is increasingly are very different, treatment has generally not been tailored recognized as a progressive disease, with frank malignancy to specific tumor subtypes until recently. representing the end stage of a chronic disease process (1). Honokiol is an important bioactive compound present in This end stage is difficult to treat compared with earlier, Magnolia bark extracts, which has been used as a folk premalignant lesions. This raises the potential that focusing remedy for centuries in China, South Korea, and Japan to resources on the targeting of early-stage carcinogenesis may treat gastrointestinal disorders, cough, anxiety, stroke, and provide a way to better control cancer. This approach is allergic diseases (4). Honokiol has been reported to have a defined as chemoprevention, and although a nascent field, variety of broad mechanisms of action, such as antiinflam- matory activity (5, 6), along with antiangiogenic (7) and cardio-beneficiating (8, 9), and neuroprotective activity 1Medical College of Wisconsin Cancer Center and Department of Phar- (10, 11), without appreciable toxicity. Honokiol has also macology and Toxicology, Medical College of Wisconsin, Milwaukee, been identified as a naturally occurred PPARg agonist with- Wisconsin. 2Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin. 3Chemoprevention Branch, National Cancer Insti- out previously known agonist’s side effect, such as adipo- tute, Bethesda, Maryland. genesis (12). It even prevents hyperglycemia and body Corresponding Author: Ming You, Medical College of Wisconsin, 8701 weight gain in diabetic mice. Watertown Plank Road, Milwaukee, WI 53226. Phone: 414-955-2565; Fax: In recent years, honokiol has also been demonstrated as a 414-955-6058; E-mail: [email protected] potential antitumor agent in many cancer cell lines as well doi: 10.1158/1940-6207.CAPR-14-0091 as xenografts, from a variety of tumor types, including Ó2014 American Association for Cancer Research. glioma (13), breast cancer (14), and colorectal carcinoma www.aacrjournals.org 1149 Downloaded from cancerpreventionresearch.aacrjournals.org on September 25, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst September 22, 2014; DOI: 10.1158/1940-6207.CAPR-14-0091 Pan et al. (7). The combination of honokiol with other anticancer Throughout the studies, the health condition of the mice agents such as the mTOR inhibitors and cisplatin had was monitored daily, and body weights were measured synergistic effects (15, 16). To determine the potential of weekly. Twenty-eight weeks after the initial treatment of honokiol as a chemopreventive agent, however, testing its NTCU, mice were euthanized by CO2 asphyxiation. Lungs efficacy in a mouse model with chemically induced precan- were fixed in zinc formalin overnight and stored in 70% cerous lesions is necessary. In this study, we tested honokiol ethanol for histopathology evaluation. Unlike lung ade- in the N-nitroso-trischloroethylurea (NTCU)–induced nocarcinomas, SCC does not form visible solid nodules mouse lung SCC model, which has been shown to be a on the surface of the lung. Serial tissue sections (5-mm successful mouse lung SCC model and faithfully captures each) were made from formalin-fixed lungs, and one in well-defined, typical pathologic development from normal every 20 sections (approximately 100 mmapart)was to bronchiolar hyperplasia, metaplasia, SCC in situ, and stained with hematoxylin and eosin (H&E) and examined finally SCC as seen in humans (17–19). histologically under a light microscope to assess severity Previous studies have shown that honokiol induces apo- of tumor development (invasive SCC, SCC in situ,bron- ptosis in a variety of cancer cell lines, including murine chial metaplasia, and bronchial hyperplasia) as we endothelial SVR cells (6), human leukemia MOLT 4B cells reported previously (17). (7), human colorectal carcinoma RKO cells (8), and human lung SCC CH27 cells (9). However, the precise molecular Histopathology analysis mechanism underlying the induction of apoptosis of cancer The lesions, including invasive SCC, SCC in situ, and the cells by honokiol still needs further investigation. Mito- bronchial hyperplasia/metaplasia, were scored from the chondria are essential cellular organelles that play central H&E-stained sections of each lung by following the guide- roles in energy metabolism and apoptosis (20). During lines as described below. When hyperplasia occurs, a single oxidative phosphorylation, electrons that escape from the layer of bronchiolar epithelial cells becomes multiple mitochondrial electron transport chain react with oxygen to layers. The cells maintain their normal morphology. When & form O2 (21), which is then converted to hydrogen bronchiolar metaplasia occurs, the normal columnar epi- peroxide and other reactive oxygen species (ROS). Unbal- thelium is replaced by flattened squamous epithelium with anced intracellular ROS level can lead to oxidative stress, increased keratin production. When SCC in situ occurs, causing DNA damage, lipid peroxidation, and can ultimate- atypical cells (such as irregular shape, increased nucleus/ ly trigger apoptosis. We therefore hypothesize that honokiol cytoplasm ratio) with visible mitosis and loss of orderly may inhibit lung SCC by affecting mitochondrial function. differentiation replace the entire thickness of the epitheli- In the present study, we used NTCU-induced preclinical um, although the bronchiole basement membrane remains mouse lung SCC model to evaluate the preventive efficacy of intact, with no tumor cell invasion into the surrounding honokiol and the impact of honokiol on mitochondrial stroma. When invasive SCC occurs, general features of SCC respiration and function. such as keratin pearls, multiple nuclei, increasing mitotic index can be seen, and the normal architecture of the lung is Materials and Methods disrupted. The lung SCCs area/lung lobe area ratio was Reagents and animals evaluated using NanoZoomer Digital Pathology Virtual NTCU was purchased from Toronto Research Chemicals, Slide Viewer software (Hamamatsu Photonic Co.). H&E- Inc.. Acetone was purchased from Sigma. Honokiol was stained slides were scanned with the NanoZoomer HT slide purchased from LKT laboratories. scanner (Hamamatsu Photonics), and virtual slides were Lung SCC was induced in mice through NTCU admin- analyzed and quantified. istration as previously reported (18, 22, 23). All studies on animals were approved by the Medical College of Wisconsin Cell lines Institutional Animal Care and Use Committee. Six-to-eight- Human lung SCC cell lines H226 and H520 were week old female NIH Swiss mice were obtained from purchased from the ATCC in 2011, where they are Charles River Laboratories. Animals were housed with regularly authenticated. Both cell lines were