Procyanidin C 1 from Cinnamomi Cortex inhibits TGF-β-induced epithelial-to-mesenchymal transition in the A 549 lung cancer cell line

Cancer metastasis is one of the most critical events in cancer patients, and the median overall survival of stage IIIb or IV patients with metastatic lung cancer in the TNM classification is only 8 or 5 months, respectively. We previously demonstrated that Juzentaihoto, a Japanese traditional medicine, can inhibit cancer metastasis through the activation of macrophages and T cells in mouse cancer metastatic models; however, the mechanism(s) through which Juzentaihoto directly affects tumor cells during the metastasis process and which herbal components from Juzentaihoto inhibit the metastatic potential have not been elucidated. In this study, we focused on the epithelial-to-mesenchymal transition (EMT), which plays an important role in the formation of cancer metastasis. We newly determined that only the Cinnamomi Cortex (CC) extract, one of 10 herbal components of Juzentaihoto, inhibits TGF-β-induced EMT. Moreover, the contents of catechin trimer in CC extracts were significantly correlated with the efficacy of inhibiting TGF-β-induced EMT. Finally, the structure of the catechin trimer from CC extract was chemically identified as procyanidin C1 and the compound showed inhibitory activity against TGF-β-induced EMT. This illustrates that procyanidin C1 is the main active compound in the CC extract responsible for EMT inhibition and that procyanidin C1 could be useful as a lead compound to develop inhibitors of cancer metastasis and other diseases related to EMT. Introduction The essential characteristics of cancer are the ability to invade surrounding tissues and metastasize to distal tissues, known as cancer metastasis, which is the major cause of mortality in cancer patients. In non-small cell lung cancer (NSCLC) patients, the median overall survival of patients with metastatic lung cancer (stage IIIb or IV in the TNM classification) is limited (only 8 or 5 months, respectively) (1); therefore, suppression of cancer metastasis results in the improved survival of lung cancer patients. Cancer metastasis consists of several steps: intravasation, attachment to a vessel, extravasation, angiogenesis and growth in distal tissues (2-5). Among them, epithelial-to-mesenchymal transition (EMT) is involved in an early step of metastasis (6,7). EMT is a phenomenon in which cobblestone-like epithelial cells change into spindle-like mesenchymal cells with downregulation of epithelial markers such as E-cadherin and also upregulation of mesenchymal markers such as N-cadherin (8). In addition to metastasis, this physiological phenomenon is important for resistance to apoptosis, maintenance of cancer stem cells and production of extracellular matrix. Thus, EMT would be an attractive therapeutic target in metastatic cancers. Juzentaihoto, which is a Japanese Kampo medicine, has been widely used for the decline of physical strength, general debility, cold hands and feet, fatigue, night sweats, circulatory problems and anemia (9). Interestingly, our previous studies showed that Juzentaihoto indirectly inhibited cancer metastasis through the activation of macrophages and T cells in mouse models (10-12). Although Juzentaihoto could suppress cancer metastasis, it is not well known how Juzentaihoto can directly affect tumor cells during the metastasis process and which herbal ingredients of Juzentaihoto are involved in the regulation of EMT. Here we firstly report that Cinnamomi Cortex (CC), one of the herbal medicines from Juzentaihoto, inhibits TGF-βinduced EMT phenotypes. After fractionation of CC extract, the content of catechin trimer was well-associated with the inhibitory activities of CC extract. Finally, procyanidin C1 Procyanidin C1 from Cinnamomi Cortex inhibits TGF-β-induced epithelial-to-mesenchymal transition in the A549 lung cancer cell line RYOEI KIN1, SHINICHIRO KATO1, NAOKI KANETO1, HIROAKI SAKURAI1,2, YOSHIHIRO HAYAKAWA1, FENG LI2, KEN TANAKA3, IKUO SAIKI1 and SATORU YOKOYAMA1 1Division of Pathogenic Biochemistry, Institute of Natural Medicine, 2Department of Cancer Cell Biology, Graduate School of Medicine and Pharmaceutical Science, 3Research Promotion Office, 4Division of Pharmacognosy, Institute of Natural Medicine, University of Toyama, Toyama 930-0194, Japan Received July 10, 2013; Accepted September 2, 2013 DOI: 10.3892/ijo.2013. 2139 Correspondence to: Dr Satoru Yokoyama, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194,