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研究生:吳玫貞
研究生(外文):Mei-jen Wu
論文名稱:IsoobtusilactoneA在人類肝癌細胞株(HepG2)所引起細胞凋亡之相關作用機制的探討
論文名稱(外文):The mechanisms of isoobtusilactone A-induced apoptosis inhuman hepatoblastoma cell line (Hep G2)
指導教授:陳志良陳志良引用關係陳錦翠
指導教授(外文):Chern, Chi-LiangCheng, Jiin-Tsuey
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:85
中文關鍵詞:細胞凋亡活性氧Bax蛋白粒線體膜電位caspase 3蛋白AIF蛋白
外文關鍵詞:Isoobtusilactone A
相關次數:
  • 被引用被引用:3
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  • 下載下載:88
  • 收藏至我的研究室書目清單書目收藏:0
使用天然植物所分離之物質進行化學預防(chemoprevention),已成為許多癌症預防的新策略,因此,本研究探討從蘭嶼肉桂(Cinnamomum kotoense)樹葉所分離出來的isoobtusilactone A化合物對Hep G2肝癌細胞株的毒殺作用及其導致細胞凋亡的可能機制。初步實驗結果發現隨著isoobtusilactone A濃度的增加,Hep G2細胞的sub-G1細胞群和小片段DNA斷裂(oligonucleosomal DNA fragmentation)也會跟著上升;進一步,在細胞蛋白的分析也證實了isoobtusilactone A會促使Bax蛋白從細胞質轉移到粒線體中,同時也將cytochrome c釋放至細胞質中,進而活化caspase 3 和PARP cleavage,最後導致Hep G2細胞凋亡;而且也發現isoobtusilactone A 會增加Hep G2細胞活性氧(ROS)的產生,誘導粒線體膜電位下降(mitochondrial transmembrane potential ),而當分別加入活性氧清除劑(NAC)和NADPH oxidase 抑制劑(DPI)時,發現其會抑制因藥物所誘發產生之ROS和細胞凋亡。另外,我們也發現當加入caspases抑制劑Z-VAD.fmk時,並不能完全的保護細胞走向細胞凋亡,意味著還有其它的細胞凋亡機制参與。Apoptosis-inducing factor (AIF) , 為調控caspase-independent apoptosis的蛋白,實驗結果發現,isoobtusilactone A 可促進AIF從粒線體轉移至細胞核內,並伴隨大片段DNA斷裂(large-scale DNA fragmentation)的產生。因此以AIF-siRNA短暫轉染(transient transfection) Hep G2 細胞,發現會抑制isoobtusilactone A誘發之大片段DNA斷裂,但卻不能抑制 isoobtusilactone A誘導小片段DNA斷裂;而同時以AIF-siRNA 和Z-VAD.fmk前處理Hep G2 細胞,則能同時抑制isoobtusilactone A誘導小片段和大片段DNA 斷裂。綜合上述,isoobtusilactone A 誘導Hep G2細胞凋亡路徑是經由ROS產生來破壞粒線體膜電位,進而同時誘發caspase-dependent and caspase-independent
pathway來執行完成。
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.
中文摘要 ----------------------------------------------------------------------------- 1
英文摘要 ----------------------------------------------------------------------------- 3
第一章 緒論 ------------------------------------------------------------- 5
第二章 文獻探討 ------------------------------------------------------------- 6
第三章 材料與方法 -------------------------------------------------------------- 16
第四章 實驗結果 -------------------------------------------------------------- 31
第五章 討論與結論 -------------------------------------------------------------- 38
參考文獻 -------------------------------------------------------------------------------- 41
圖----------------------------------------------------------------------------- 49
表----------------------------------------------------------------------------- 76
附錄一 -------------------------------------------------------------------------------- 77
附錄二 -------------------------------------------------------------------------------- 78
Anderson, J.E., Wenwen M., Smith, D.L., Chang, C.J., McLaughlin, J.L.,
1992. Biologically active -lactones and methylketoalkenes from
Lindera benzoin. J. Nat. Prod. 55, 71–83.
Arends, M.J., Wyllie, A.H., 1991. Apoptosis: mechanisms and roles in
pathology. Int. Rev. Exp. Pathol. 32, 223–254.
Arends, M.J., Morris, R.G., Wyllie, A.H., 1990. Apoptosis: the role of the
endonuclease. Am. J. Pathol. 136, 593–598.
Bras, M., Queenan, B., Susin, S.A., 2005. Programmed cell death via
mitochondria: different modes of dying. Biochemistry (Mosc.) 70,
231–239.
Babior, B.M., 2000. The NADPH oxidase of endothelial cells. IUBMB
Life 50, 267–269.
Babior, B.M., 1999. NADPH oxidase: an update. Blood 93, 1464–1476.
Chen, C.H., Lo, W.L., Liu, Y.C., Chen, C.Y., 2006. Chemical and
cytotoxic constituents from the leaves of Cinnamomum kotoense. J.
Nat. Prod. 69, 927–933.
Chen, F.C., Peng, C.F., Tsai, I.L., Chen, I.S., 2005. Antitubercular
constituents from the stem wood of Cinnamomum kotoense. J. Nat.
Prod. 68, 1318–1323.
Cande, C., Cohen, I., Daugas, E., Ravagnan, L., Larochette, N., Zamzami,
N., Kroemer, G., 2002. Apoptosis-inducing factor(AIF): a novel
caspase-independent death effector released from mitochondria.
Biochimie 84, 215–222.
Cregan, S.P., Fortin, A., MacLaurin, J.G., Callaghan, S.M., Cecconi, F.,
Yu, S.W., Dawson, T.M., Dawson, V.L., Park, D.S., Kroemer, G.,
Slack, R.S., 2002. Apoptosis-inducing factor is involved in the
regulation of caspase-independent neuronal cell death. J. Cell Biol.
158, 507–517.
Curtin, J.F., Donovan, M., Cotter, T.G., 2002. Regulation and
measurement of oxidative stress in apoptosis. J. Immunol. Methods.
265, 49–72.
Cohen, J.J., Duke, R.C., 1984. Glucocorticoid activation of a
calcium-dependent endonuclease in thymocyte nuclei leads to cell
death. J. Immunol. 132, 38–42.
Demaurex, N., Distelhorst, C., 2003. Apoptosis—the calcium connection.
Science 300, 65–67.
Debatin, K.M., Poncet, D., Kroemer, G., 2002. Chemotherapy: targeting
the mitochondrial cell death pathway. Oncogene 21, 8786–8803.
Evans, J.L., Goldfine, I.D., Maddux, B.A., Grodsky, G.M., 2002.
Oxidative stress and stress-activated signaling pathways: a unifying
hypothesis of type 2 diabetes. Endocr. 23, 599–622.
Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., Nagata,
S., 1998. A caspase-activated DNase that degrades DNA during
apoptosis, and its inhibitor ICAD. Nature 391, 43–50.
Fadeel, B., Orrenius, S., Zhivotovsky, B., 1999. Apoptosis in human
disease: a new skin for the old ceremony? Biochem. Biophys. Res.
Commun. 266, 699–717.
Gupta, S., Agrawal, A., Agrawal, S., Su, H., Gollapudi, S., 2006. A
paradox of immunodeficiency and inflammation in human
aging: lessons learned from apoptosis. Immunity & Ageing 19;3:5.
doi:10.1186/1742-4933-3-5
Garcez, F.R., Garcez, W.S., Martins, M., Matos, M.F., Guterres, Z.R.,
Mantovani, M.S., Misu, C.K., Nakashita, S.T., 2005. Cytotoxic and
genotoxic butanolides and lignans from Aiouea trinervis. Planta. Med.
71, 923–927.
Groenendyk, J., Michalak, M., 2005. Endoplasmic reticulum quality
control and apoptosis. Acta. Biochim. Pol. 52, 381–395.
Joza, N., Susin, S.A., Daugas, E., Stanford, W.L., Cho, S.K., Li, C.Y.,
Sasaki, T., Elia, A.J., Cheng, H.Y., Ravagnan, L., Ferri, K.F., Zamzami,
N., Wakeham, A., Hakem, R., Yoshida, H., Kong, Y.Y., Mak, T.W.,
Zuniga-Pflucker, J.C., Kroemer, G., Penninger, J.M., 2001. Essential
role of the mitochondrial apoptosis-inducing factor in programmed cell
death. Nature 410, 549–554
Jacobson, M.D., Weil, M., Raff, M.C., 1997. Programmed cell death in
animal development. Cell 88, 347–354.
Kohler, C., Orrenius, S., Zhivotovsky, B., 2002. Evaluation of caspase
activity in apoptotic cells. J. Immunol. Methods 265, 97–110.
Kerr, J.F.R., Winterford, C.M., Harmon, B.V., 1994. Apoptosis, it’s
significance in cancer and cancer therapy. Cancer 73, 2013–2025.
Kerr, J.F.R., Wvllie, A.H., Currie, A.R., 1972. Apoptosis: a basic
biological phenomenon with wide ranging implic ations in tissue
kinetics. British. Br. J. Cancer 26, 239–257.
Larner, S. F., Hayes, R. L., Wang, K. K., 2006. Unfolded protein
response after neurotrauma. J. Neurotrauma 23, 807–829.
Liu, R.H., 2004. Potential synergy of phytochemicals in cancer
prevention: mechanism of action. J. Nutr. 134, 3479–3485.
Liu, X., Li, P., Widlak, P., 1998. The 40-kDa subunit of DNA
fragmentation factor induces DNA fragmentation and chromatin
condensation during apoptosis. Proc. Natl. Acad. Sci. 95,
8461–8466.
Masud, A., Mohapatra, A., Lakhani, S.A., Ferrandino, A., Hakem, R.,
Flavell, R.A., 2007. ER stress-induced death of mouse embryonic
fibroblasts requires the intrinsic pathway of apoptosis. J. Biol. Chem.
19, 14132–14139.
Mohapatra, S., Chu, B., Zhao, X., Pledger, E.J., 2005. Accumulation of
p53 and reductions in XIAP abundance promote the apoptosis of
prostate cancer cells. Cancer Research 65, 7717–7723.
Martin, L.J., Price, A.C., KaiSeries, A., Shaikh, A.Y., Liu, Z., 2000.
Mechanisms for neuronal degeneration in amyotrophic lateral sclerosis
and in models of motor neuron death. Int. J. Mol. Med. 5,
3–13.
Michael, O.H., 2000. The biochemistry of apoptosis. Nature
407, 770–776.
McGuire, W.P., Hoskins, W.J., Brady M.F., 1996. A randomized trial of
cyclophosphamide/cisplatin versus Paclitaxel/cisplatin in suboptimal
stage Ⅲ and Ⅳ ovarian cancer: a gynecologic oncology group study.
N. Engl. J. Med. 334, 1–6.
Nakagawa, T., 1999. Caspase-12 mediates endoplasmic reticulum
specific apoptosis and cytotoxicity by amyloid- . Nature 403, 98–103.
Orrenius, S., Zhivotovsky, B., Nicotera, P., 2003. Regulation of cell
death: the calcium-apoptosis link. Nat. Rev. Mol. Cell
Bio. 7, 552–565.
Ravagnan, L., Gurbuxani, S., Susin, S.A., Maisse, C., Daugas, E.,
Zamzami, N., Mak, T., Jaattela, M., Penninger, J.M., Garrido, C.,
Kroemer, G., 2001. Heat-shock protein 70 antagonizes
apoptosis-inducing factor. Nat. Cell Biol. 3, 839–843.
Rao, R.V., Hermel, E., Castro-Obregon, S., Del Rio, G., Ellerby, L.M.,
Ellerby, H.M., Bredesen, D.E., 2001. Coupling endoplasmic reticulum
stress to the cell death program:mechanism of caspase activation. J.
Biol.Chem. 276, 33869–33874.
Scorrano, L., Oakes, S.A., Opferman, J. T., Cheng, E.H., Sorcinelli, M.
D., Pozzan, T.,Korsmeyer, S.J., 2003. BAX and BAK regulation of
endoplasmic reticulum Ca2+: a control point for apoptosis. Science
300, 135–139.
Saikumar, P., Dong, Z., Mikhailov, V., Denton, M., Weinberg, J.M.,
Venkatachalam, M.A., 1999. Apoptosis: Definition, mechanisms and
relevance to disease. Am. J. Med. 107, 489–506.
Susin, S.A., Lorenzo, H.K., Zamzami, N. 1999. Molecular
characterization of mitochondrial apoptosis-inducing factor. Nature
397, 441–446.
Susin, S.A., Lorenzo, H.K., Zamzami, N., Marzo, I., Snow, B.E.,
Brothers, G.M., Mangion, J., Jacotot, E., Costantini, P., Loeffler, M.,
Larochette, N., Goodlett, D.R., Aebersold, R., Siderovski, D.P.,
Penninger, J.M., Kroemer, G., 1999. Molecular characterization of
mitochondrial apoptosis-inducing factor. Nature 397, 441–446.
Talior, I., Tennenbaum, T., Kuroki, T., Finkelman1, H.E., 2005. PKC-δ
-dependent activation of oxidative stress in adipocytes of obese and
insulin-resistant mice: role for NADPH oxidase. Am. J. Physiol.
Endocrinol. Metab. 288, 405–411.
Tsai, I.L., Hung, C.H., Duh, C.Y., Chen, I.S., 2002. Cytotoxic butanolides
and secobutanolides from the stem wood of Formosan Lindera
communis. Planta Med. 68, 142–145.
Tan, S., Sagara, Y., Liu, Y., Mather, P., Schubert, D., 1998. The
regulation of reactive oxygen species production during
programmed cell death. J. Cell Biol. 141, 1423–1432.
Van H.L., Meischl, C., Stooker, W., Meijer, C.J., Niessen, H.W., Roos, D.,
2002. NADPH oxidase(s): new source(s) of reactive oxygen species in
the vascular system? J. Clin. Pathol. 55, 561–568.
Wyllie, A.H., 1980. Glucocorticoid-induced thymocyte apoptosis is
associated with endomuclese activation. Nature 284, 555–557.
Yu, S.W., Wang, H., Poitras, M.F., Coombs, C., Bowers, W.J., Federoff,
H.J., Poirier, G.G., Dawson, T.M., Dawson, V.L., 2002. Mediation of
Poly(ADP-ribose) polymerase-1-dependent cell death by
apoptosis-inducing factor. Science 297, 259–263.
Yoneda, T., Imaizumi, K., Oono, K., Yui, D., Gomi, F., Katayama, T.,
and Tohyama, M., 2001. Activation of caspase-12, an endoplastic
reticulum (ER) resident caspase, through tumor necrosis factor
receptor-associated factor 2-dependent mechanism in response to the ER stress. J. Biol. Chem. 276, 13935–13940.
Yeh, W.C., Pompa, J.L., McCurrach, M.E., 1998. FADD essential for
embryo development and signaling from some, but not all, inducers of
apoptosis. Science 279, 1954–1958.
Zhang, J., Cado, D., Chen, A., Kabra, N.H., Winoto, A., 1998.
Fas-mediated apoptosis and activation-induced T-cell proliferation are
defective in mice lacking FADD/Mort1. Nature 392, 296–300.
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