Title page for etd-0728107-113601


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URN etd-0728107-113601
Author Mei-jen Wu
Author's Email Address No Public.
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Department Biological Sciences
Year 2006
Semester 2
Degree Master
Type of Document
Language zh-TW.Big5 Chinese
Title The mechanisms of isoobtusilactone A-induced apoptosis in
human hepatoblastoma cell line (Hep G2)
Date of Defense 2006-07-23
Page Count 85
Keyword
  • Isoobtusilactone A
  • Abstract Chemoprevention using naturally occurring substances has now been
    considered a promising strategy for the prevention of cancer. In this study, the
    effects of isoobtusilactone A, a novel constituent isolated from the leaves of
    Cinnamomum kotoense, on the proliferation of human hepatoma Hep G2 cells
    and the underlying mechanisms in isoobtusilactone A-induced apoptosis are
    thoroughly evaluated. Under the experimental conditions adapted by this
    study, isoobtusilactone A was found to exhibit a concentration-dependent
    growth impediment (IC50 = 37.5 μM). The demise of the cells induced by
    isoobtusilactone A was apoptotic in nature, showing progressive sub-G1
    fraction and DNA fragmentation when the concentration of the substrate was
    increased. Subcellular fractionation analysis further revealed that Bax
    translocation to mitochondria resulted in a rapid release of cytochrome c,
    followed by the activation of caspase 3 and PARP cleavage, and finally cell
    death. Isoobtusilactone A-treated cells also displayed transient increase of
    ROS during the earlier stage of the experiment, followed by the disruption of
    mitochondrial transmembrane potential ( m). The presence of a ROS
    scavenger (N-acetyl-L-cysteine,NAC) and an inhibitor of NADPH oxidase
    (diphenyleneiodonium chloride,DPI) blocked ROS production and the
    subsequent apoptotic cell death. Taken together, our data suggest that ROS
    generated through the activation of NADPH oxidase plays an essential role in
    apoptosis induced by isoobtusilactone A.
    To clarify whether caspases were the sole mediators for eliciting the
    observed apoptotic process, the effects of a broad caspases inhibitor,
    Z-VAD.fmk, was studied. Interestingly, Z-VAD.fmk was found to completely
    inhibit the isoobtusilactone A-induced oligonucleosomal DNA fragmentation,
    yet it could only prevent limited amount of cells from becoming
    apoptosis-prone. These data implied that other mechanism(s) might also be
    important factors and led us to study the possible involvement of
    apoptosis-inducing factor (AIF), a mediator arbitrating caspase-independent
    apoptosis, in isoobtusilactone A-induced apoptotic process. Our data indicated
    that isoobtusilactone A could elicit the nuclear translocation of AIF observed
    along with the occurrence of large-scale DNA fragmentation. Reduction of
    AIF expression by AIF-siRNA transfection suppressed large-scale DNA
    fragmentation. Interestingly, inhibition of AIF expression by AIF-siRNA did
    not prevent isoobtusilactone A-induced oligonucleosomal DNA fragmentation.
    When the cells were simultaneously treated with AIF-siRNA and Z-VAD.fmk,
    both large-scale DNA and oligonucleosomal DNA fragmentations were
    almost completely prevented. In conclusion, our data suggest that
    isoobtusilactone A induced apoptotic cell death was caused by the increase of
    ROS, followed by the disruption of mitochondrial transmembrane potential
    ( m), further mediated by both caspase-dependent and caspase-independent
    pathways.
    Advisory Committee
  • Liu, Tsan-Zon - chair
  • Liu, Ray-H. - co-chair
  • Chern, Chi-Liang - advisor
  • Cheng, Jiin-Tsuey - advisor
  • Files
  • etd-0728107-113601.pdf
  • indicate accessible in a year
    Date of Submission 2007-07-28

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