Journal of Ethnopharmacology 137 (2011) 503–511 Contents lists available at ScienceDirect Journal of Ethnopharmacology journa l h o me page: www.elsevier.com/locate/jethpharm Subamolide A, a component isolated from Cinnamomum subavenium, induces apoptosis mediated by mitochondria-dependent, p53 and ERK1/2 pathways in human urothelial carcinoma cell line NTUB1 Chiung-Hui Liu a , Chung-Yi Chen b , A-Mei Huang c,∗ , Jih-Heng Li a,∗∗ a School of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan b School of Medical and Health Sciences, Fooyin University, Kaohsiung 831, Taiwan c Department of Medicine, Graduate Institute of Biochemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan a r t i c l e i n f o Article history: Received 31 January 2011 Received in revised form 27 May 2011 Accepted 1 June 2011 Available online 17 June 2011 Keywords: Cinnamomum subavenium Subamolide A Human urothelial carcinoma Mitochondria-mediated apoptosis Chemotherapeutic drugs a b s t r a c t Ethnopharmacological relevance: Cinnamomum subavenium has long been used as a traditional Chinese medicine to treat carcinomatous swelling, abdominal pain and other diseases. Aim of the study: The goal of this work was to study the cytotoxic effect of subamolide A, a constituent isolated from the stems of Cinnamomum subavenium Miq., and to extend its traditional use for clinical applications in treating human urothelial carcinoma. Materials and methods: Cytotoxic effect of subamolide A was determined by the MTT assay in NTUB1, T24, PC3 and SV-HUC-1 cells treated with various concentrations of subamolide A for three days. Apoptosis was detected by the change of cell morphology and flow cytometry analysis. The reactive oxygen species (ROS) level and mitochondria membrane potential (m) were determined by flow cytometry. Western blot analysis was used to quantify the expression of apoptosis-related and stress-induced signaling molecules. Results: Subamolide A selectively induced apoptosis in two cancerous human urothelial carcinoma cell lines (NTUB1 and T24) in comparison with normal immortalized uroepithelial cells (SV-HUC-1). Sub- amolide A reduced mitochondrial membrane potential (m) and caused apoptosis of NTUB1 cells. Subamolide A increased Bax/Bcl-2 ratios, the amount of cytochrome c released from the mitochondria, caspase-3 and PARP cleavage, activated p53 and ERK1/2 and ultimately led to apoptosis in NTUB1 cells. Furthermore, a higher dose (10 M) of subamolide A synergistically enhanced the cytotoxicity of cisplatin and gemcitabine in NTUB1 cells. Conclusions: The current study demonstrated that subamolide A triggered the mitochondria-dependent apoptotic pathways and p53 and ERK1/2 activation in the human urothelial carcinoma cell line NTUB1. In addition, subamolide A synergistically enhanced cytotoxic effect of CDDP and Gem in NTUB1. These data suggested that subamolide A exhibited a potent anti-proliferation activity. This study supports the traditional use of Cinnamomum subavenium stems with a therapeutic potential for the treatment of human urothelial carcinoma. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Cinnamomum subavenium Miq. (Lauraceae), a medium-sized evergreen tree, is distributed in central and southern parts of China, Burma, Cambodia, Taiwan, Malaysia, and Indonesia (Liao, 1996). Cinnamomum subavenium has long been used as a traditional Chinese medicine as recorded in ancient medical lit- eratures for treating a variety of ailments including carcinomatous swelling, stomachache, chest pain, abdominal pain, hernia, diar- ∗ Corresponding author. Tel.: +886 7 312 1101/2138; fax: +886 7 321 8309. ∗∗ Corresponding author. Tel.: +886 7 312 1101/2651; fax: +886 7 321 0683. E-mail addresses: amhuang@kmu.edu.tw (A.-M. Huang), jhlitox@kmu.edu.tw (J.-H. Li). rhea, rheumatism, nausea and vomiting (Hu, 1998; Xie, 1996). Constituents isolated from Cinnamomum subavenium by column chromatography had been shown with potent cytotoxic and geno- toxic effects in several human cancer cell lines (Chen et al., 2007; Kuo et al., 2008; Shen et al., 2010; Wang et al., 2011). Urothelial carcinoma (UC) is the most common malignancy occurred in the human urinary tract. The treatment strategy against UC is mainly surgery followed by chemotherapy or radiotherapy (Kaufman, 2006). Chemotherapeutic agents with various combina- tions have been shown effective against advanced bladder cancer (Shariat et al., 2010). Gemcitabine (2 ′ -deoxy-2 ′ ,2 ′ -difluorocytidine; Gem) plus cisplatin (CDDP) regimen has been shown promis- ing results for treating advanced and metastatic bladder tumors (Roberts et al., 2006; von der Maase et al., 2000). However, drug resistance often leads to treatment failure (Gallagher et al., 2008). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.06.001