title: Bufalin induces G0/G1 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.
abstract: 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 (ΔΨ(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.
authors.affiliation: Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan.
authors.forename: Wen-Wen
authors.initials: WW
authors.lastname: Huang
authors.forename: Jai-Sing
authors.initials: JS
authors.lastname: Yang
authors.forename: Shu-Jen
authors.initials: SJ
authors.lastname: Pai
authors.forename: Ping-Ping
authors.initials: PP
authors.lastname: Wu
authors.forename: Shu-Jen
authors.initials: SJ
authors.lastname: Chang
authors.forename: Fu-Shin
authors.initials: FS
authors.lastname: Chueh
authors.forename: Ming-Jen
authors.initials: MJ
authors.lastname: Fan
authors.forename: Shang-Ming
authors.initials: SM
authors.lastname: Chiou
authors.forename: Hsiu-Maan
authors.initials: HM
authors.lastname: Kuo
authors.forename: Chin-Chung
authors.initials: CC
authors.lastname: Yeh
authors.forename: Po-Yuan
authors.initials: PY
authors.lastname: Chen
authors.forename: Minoru
authors.initials: M
authors.lastname: Tsuzuki
authors.forename: Jing-Gung
authors.initials: JG
authors.lastname: Chung
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002018:Bufanolides / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D056741:Cyclin D / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism; D004354:Drug Screening Assays, Antitumor; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects*; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D001749:Urinary Bladder Neoplasms
chemical_list: D000970:Antineoplastic Agents; D002018:Bufanolides; D056741:Cyclin D; D019927:Cyclin E; D017382:Reactive Oxygen Species; C495894:CDK2 protein, human; C495900:CDK4 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; C022777:bufalin