Mutation Research 732 (2012) 26–33 Contents lists available at SciVerse ScienceDirect Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journa l h o me pa g e: www.elsevier.com/locate/molmut Co mm unit y add re ss: www.elsevier.com/locate/mutres Bufalin induces G 0 /G 1 phase arrest through inhibiting the levels of cyclin D, cyclin E, CDK2 and CDK4, and triggers apoptosis via mitochondrial signaling pathway in T24 human bladder cancer cells Wen-Wen Huang a,1 , Jai-Sing Yang b,1 , Shu-Jen Pai a , Ping-Ping Wu c , Shu-Jen Chang c , Fu-Shin Chueh d , Ming-Jen Fan e , Shang-Ming Chiou f,g , Hsiu-Maan Kuo h , Chin-Chung Yeh i , Po-Yuan Chen a , Minoru Tsuzuki j,k , Jing-Gung Chung a,e,∗ a Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan b Department of Pharmacology, China Medical University, Taichung 404, Taiwan c School of Pharmacy, China Medical University, Taichung 404, Taiwan d Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan e Department of Biotechnology, Asia University, Taichung 413, Taiwan f Department of Functional Neurosurgery & Gamma Knife Center, China Medical University Hospital, Taichung 404, Taiwan g School of Medicine, China Medical University, Taichung 404, Taiwan h Department of Parasitology, China Medical University, Taichung 404, Taiwan i Department of Urology, China Medical University Hospital, Taichung 404, Taiwan j Department of Biochemistry, Nihon Pharmaceutical University, Saitama 362-0806, Japan k Tsuzuki Institute for Traditional Medicine, China Medical University, Taichung 404, Taiwan a r t i c l e i n f o Article history: Received 3 February 2011 Received in revised form 25 September 2011 Accepted 27 September 2011 Available online 20 January 2012 Keywords: Bufalin T24 human bladder cancer cells G0/G1 phase arrest Apoptosis Mitochondrial signaling pathway a b s t r a c t Most of the chemotherapy treatments for bladder cancer aim to kill the cancer cells, but a high recurrence rate after medical treatments is still occurred. Bufalin from the skin and parotid venom glands of toad has been shown to induce apoptotic cell death in many types of cancer cell lines. However, there is no report addressing that bufalin induced cell death in human bladder cancer cells. The purpose of this study was investigated the mechanisms of bufalin-induced apoptosis in a human bladder cancer cell line (T24). We demonstrated the effects of bufalin on the cell growth and apoptosis in T24 cells by using DAPI/TUNEL double staining, a PI exclusion and flow cytometric analysis. The effects of bufalin on the production of reactive oxygen species (ROS), the level of mitochondrial membrane potential ( m ), and DNA content including sub-G1 (apoptosis) in T24 cells were also determined by flow cytometry. Western blot analysis was used to examine the expression of G 0 /G 1 phase-regulated and apoptosis-associated protein levels in bufalin-treated T24 cells. The results indicated that bufalin significantly decreased the percentage of viability, induced the G 0 /G 1 phase arrest and triggered apoptosis in T24 cells. The down- regulation of the protein levels for cyclin D, CDK4, cyclin E, CDK2, phospho-Rb, phospho-AKT and Bcl-2 with the simultaneous up-regulation of the cytochrome c, Apaf-1, AIF, caspase-3, -7 and -9 and Bax protein expressions and caspase activities were observed in T24 cells after bufalin treatment. Based on our results, bufalin induces apoptotic cell death in T24 cells through suppressing AKT activity and anti- apoptotic Bcl-2 protein as well as inducing pro-apoptotic Bax protein. The levels of caspase-3, -7 and -9 are also mediated apoptosis in bufalin-treated T24 cells. Therefore, bufalin might be used as a therapeutic agent for the treatment of human bladder cancer in the future. © 2012 Elsevier B.V. All rights reserved. Abbreviations: AIF, apoptosis-inducing factor; CDK, cyclin-dependent kinase; CsA, cyclosporine A; DCFH-DA, 2 ′ -7 ′ -dichlorfluorescein-diacetate; DiOC6, 3,3 ′ - dihexyloxacarbocyanine iodide; DMSO, dimethyl sulfoxide; ECL, enzyme chemiluminescence; FCS, fetal calf serum; HRP, horseradish peroxidase; NAC, N-acetyl-cysteine; PBS, phosphate-buffered saline; PI, propidium iodide; SDS, sodium dodecyl sulfate; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP nick end-labeling; z-VAD-fmk, z-Val-Ala-Asp-fluoromethyl ketone (pan-caspase inhibitor); z-LEHD-fmk, z-Leu-Glu-His-Asp-fluoromethyl ketone (caspase-9 inhibitor). ∗ Corresponding author at: Department of Biological Science and Technology, China Medical University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan. Tel.: +886 4 22053366x2161; fax: +886 4 22053764. E-mail address: jgchung@mail.cmu.edu.tw (J.-G. Chung). 1 These authors contributed equally to this work. 0027-5107/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2011.09.010