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研究生:林奕愷
研究生(外文):Yi-Kai Lin
論文名稱:低氧環境對腸胃道基質瘤中c-Kit和Akt訊息傳遞的影響
論文名稱(外文):The effect of hypoxia on c-Kit and Akt signaling in gastrointestinal stromal tumors
指導教授:賴逸儒賴逸儒引用關係
指導教授(外文):I-Rue Lai
口試委員:龔秀妮李財坤
口試委員(外文):Hsiu-Ni KungTsai-Kun Li
口試日期:2014-07-23
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:解剖學暨細胞生物學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:48
中文關鍵詞:腸胃道基質瘤低氧c-Kit/AKT 訊息傳遞
外文關鍵詞:Gastrointestinal stromal tumorsHypoxiac-Kit/AKT signaling
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前言:腸胃道基質瘤(Gastrointestinal stromal tumors, 簡稱GIST)為腸胃道最常見肉瘤(sarcoma)之一。這種肉瘤起源於腸胃道平滑肌層的神經叢細胞── Interstitial cells of Cajal的不正常增生,而且80%以上的GIST具有c-KIT gene的變異,導致所轉碼的receptor tyrosine kinase出現異常的活化。
腫瘤細胞由小型增長成晚期大型腫瘤的過程中,會面臨低氧(hypoxia)的微環境變化,原因是大型腫瘤內部的細胞較無法接收到外圍血管所供應的氧氣所致。低氧微環境會對腫瘤細胞的生長、凋亡、移動和抗藥性等產生不同程度的影響。目前低氧微環境對GIST影響的研究較少,本研究主要探討在低氧環境對GIST腫瘤細胞株的生長與死亡影響,與相關的因應機制。
材料及方法: 分別用三種GIST細胞株(882、62、48b) 和GIST病人的檢體作為實驗材料。首先,將手術切除的GIST腫瘤組織分為中心部分(central part)和周圍的部分(peripheral part),以西方墨點試驗(western blotanalysis) 和組織免疫染色(IHC)實驗,分析這兩部分所表現生物訊號的差異。再者,將GIST細胞株放在低氧培養箱一至三天,用不同assays觀察其細胞存活率、凋亡率,並以western blot分析其分子訊號表現。
結果: 首先,吾人分析手術切除的腫瘤,發現較大的GIST(7.5cm~15cm)中心部分的HIF-1α以及p-Kit表現比周圍部分多。再以MTT assay分析細胞存活。在低氧箱培養1-3天後, GIST 882比另2種GIST細胞株(62、48b)具有較高的存活率。而且, 西方墨點試驗顯示在低氧培養下的GIST882的p-KIT, p-AKT及p-PKCθ有較明顯的活化。目前已知p-AKT及p-PKCθ分別會對BAD的serine 136、112位置進行磷酸化,使之活性降低,進而減少細胞凋亡。因此使用MTT, western blot, Flow cytometry和TUNEL assay等方法,觀察在低氧環境下,對GIST882細胞株施以AKT的抑制劑(MK2206)或PKCθ的抑制劑(Sotrastaurin)的影響。結果顯示MK2206可以增加細胞凋亡和減少其存活率。
結論: 較大的GIST中心部分可能出現低氧的微環境變化。GIST882細胞株在低氧環境中的生存能力較佳,可能和p-AKT的活化有關。
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Introduction: Gastrointestinal stromal tumors (GIST) are the most common sarcomas of the gastrointestinal(GI) tract. This tumor was reported to spring from the interstitla cells of Cajal, a pace-maker cells of the GI tract. About 80% GISTs harbor c-KIT gene mutation, leading to over-expression and activation on its post-translation product, a receptor tyrosine kinase.
When a tumor grows, a relative low-oxygen (hypoxia) microenvironment develop in the core of the tumor due to insufficient oxygen supply. The hypoxic microenvironment has different impact on growth, apoptosis, migration and drug resistance of the tumors. However, there is limited research about the influence of hypoxia on GISTs. In this study, we investigate the impact of hypoxia on the growth, cell death and molecular signaling of GISTs.
Methods: 3 GIST cell lines (882, 62 and 48b) and GIST tissues were used. The signaling in central and peripheral parts of excised GIST tumors were analyzed by western blot and IHC. The GIST cell lines were incubated in hypoxia incubator for 24~72h, and their viability, apoptosis, proliferation and molecular signals were assessed.
Results: The western blot and IHC results showed that large GISTs (7.5~15cm) expressed more HIF-1α and p-Kit. The MTT assay showed GIST 882 was more resistant to hypoxia than the other 2 cell lines. In addition, western blot also showed p-KIT, p-AKT and p-PKCθ were activated in GIST882. It was reported that p-AKT and p-PKCθ would lead to phosphorylation on serine136, 112 on BAD, reducing the activity of BAD and decreasing apoptosis. So, we treated GIST882 with p-AKT inhibitor (MK2206) and p-PKCθ inhibitor (Sotrastaurin) under hypoxia, and analyze the cell viability, cell death and proliferation by MTT assay, flow cytometry and TUNEL assay. The results showed MK2206 increased the apoptosis and reduced the viability of GIST 882 cells.
Conclusion: The hypoxic microenvironment developed in the central part of large GISTs. The resistance of GIST 882 to hypoxia might be related to the activation of p-AKT.


致謝 i
中文摘要 ii
英文摘要 iv
壹、緒論 1
一、腸胃道基質瘤(Gastrointestinal Stromal Tumors, GISTs) 1
二、GISTs的致癌訊息傳遞路徑(Oncogenic signaling ) 2
三、低氧環境對腫瘤之影響(The hypoxic effect on tumor) 3
四、低氧誘導分子(Hypoxia-inducible factor) 3
五、研究目的 4
貳、實驗材料 5
參、實驗方法 11
肆、實驗結果 17
伍、討論 21
陸、圖表 26
柒、參考文獻 44


Balsara, B. R., J. Pei, Y. Mitsuuchi, R. Page, A. Klein-Szanto, H. Wang, M. Unger and J. R. Testa (2004). "Frequent activation of AKT in non-small cell lung carcinomas and preneoplastic bronchial lesions." Carcinogenesis 25(11): 2053-2059.

Banai, S., D. Shweiki, A. Pinson, M. Chandra, G. Lazarovici and E. Keshet (1994). "Upregulation of vascular endothelial growth factor expression induced by myocardial ischaemia: implications for coronary angiogenesis." Cardiovasc Res 28(8): 1176-1179.

Bauer, B., N. Krumbock, F. Fresser, F. Hochholdinger, M. Spitaler, A. Simm, F. Uberall, B. Schraven and G. Baier (2001). "Complex formation and cooperation of protein kinase C theta and Akt1/protein kinase B alpha in the NF-kappa B transactivation cascade in Jurkat T cells." J Biol Chem 276(34): 31627-31634.

Bauer, S., A. Duensing, G. D. Demetri and J. A. Fletcher (2007). "KIT oncogenic signaling mechanisms in imatinib-resistant gastrointestinal stromal tumor: PI3-kinase/AKT is a crucial survival pathway." Oncogene 26(54): 7560-7568.

Bertolotto, C., L. Maulon, N. Filippa, G. Baier and P. Auberger (2000). "Protein kinase C theta and epsilon promote T-cell survival by a rsk-dependent phosphorylation and inactivation of BAD." J Biol Chem 275(47): 37246-37250.

Blancher, C., J. W. Moore, N. Robertson and A. L. Harris (2001). "Effects of ras and von Hippel-Lindau (VHL) gene mutations on hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, and vascular endothelial growth factor expression and their regulation by the phosphatidylinositol 3''-kinase/Akt signaling pathway." Cancer Res 61(19): 7349-7355.

Bonni, A., A. Brunet, A. E. West, S. R. Datta, M. A. Takasu and M. E. Greenberg (1999). "Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms." Science 286(5443): 1358-1362.

Cardone, M. H. (1998). "Regulation of Cell Death Protease Caspase-9 by Phosphorylation." Science 282(5392): 1318-1321.

Cardone, M. H., N. Roy, H. R. Stennicke, G. S. Salvesen, T. F. Franke, E. Stanbridge, S. Frisch and J. C. Reed (1998). "Regulation of cell death protease caspase-9 by phosphorylation." Science 282(5392): 1318-1321.

Chen, W. T., C. J. Huang, M. T. Wu, S. F. Yang, Y. C. Su and C. Y. Chai (2005). "Hypoxia-inducible factor-1alpha is associated with risk of aggressive behavior and tumor angiogenesis in gastrointestinal stromal tumor." Jpn J Clin Oncol 35(4): 207-213.

Cheng, Y., X. Ren, Y. Zhang, R. Patel, A. Sharma, H. Wu, G. P. Robertson, L. Yan, E. Rubin and J. M. Yang (2011). "eEF-2 kinase dictates cross-talk between autophagy and apoptosis induced by Akt Inhibition, thereby modulating cytotoxicity of novel Akt inhibitor MK-2206." Cancer Res 71(7): 2654-2663.

Datta, S. R., H. Dudek, X. Tao, S. Masters, H. Fu, Y. Gotoh and M. E. Greenberg (1997). "Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery." Cell 91(2): 231-241.

Duensing, A., N. E. Joseph, F. Medeiros, F. Smith, J. L. Hornick, M. C. Heinrich, C. L. Corless, G. D. Demetri, C. D. Fletcher and J. A. Fletcher (2004). "Protein Kinase C theta (PKCtheta) expression and constitutive activation in gastrointestinal stromal tumors (GISTs)." Cancer Res 64(15): 5127-5131.

Duensing, A., F. Medeiros, B. McConarty, N. E. Joseph, D. Panigrahy, S. Singer, C. D. Fletcher, G. D. Demetri and J. A. Fletcher (2004). "Mechanisms of oncogenic KIT signal transduction in primary gastrointestinal stromal tumors (GISTs)." Oncogene 23(22): 3999-4006.

Han, Z. B., H. Ren, H. Zhao, Y. Chi, K. Chen, B. Zhou, Y. J. Liu, L. Zhang, B. Xu, B. Liu, R. Yang and Z. C. Han (2008). "Hypoxia-inducible factor (HIF)-1 alpha directly enhances the transcriptional activity of stem cell factor (SCF) in response to hypoxia and epidermal growth factor (EGF)." Carcinogenesis 29(10): 1853-1861.

Heinrich, M. C., C. L. Corless, C. D. Blanke, G. D. Demetri, H. Joensuu, P. J. Roberts, B. L. Eisenberg, M. von Mehren, C. D. Fletcher, K. Sandau, K. McDougall, W. B. Ou, C. J. Chen and J. A. Fletcher (2006). "Molecular correlates of imatinib resistance in gastrointestinal stromal tumors." J Clin Oncol 24(29): 4764-4774.

Heinrich, M. C., C. L. Corless, A. Duensing, L. McGreevey, C. J. Chen, N. Joseph, S. Singer, D. J. Griffith, A. Haley, A. Town, G. D. Demetri, C. D. Fletcher and J. A. Fletcher (2003). "PDGFRA activating mutations in gastrointestinal stromal tumors." Science 299(5607): 708-710.

Hirano, K., Y. Shishido-Hara, A. Kitazawa, K. Kojima, A. Sumiishi, M. Umino, F. Kikuchi, A. Sakamoto, Y. Fujioka and H. Kamma (2008). "Expression of stem cell factor (SCF), a KIT ligand, in gastrointestinal stromal tumors (GISTs): a potential marker for tumor proliferation." Pathol Res Pract 204(11): 799-807.

Hirota, S. (1998). "Gain-of-Function Mutations of c-kit in Human Gastrointestinal Stromal Tumors." Science 279(5350): 577-580.

Lee, Y., J. Jung, K. J. Cho, S. K. Lee, J. W. Park, I. H. Oh and G. J. Kim (2013). "Increased SCF/c-kit by hypoxia promotes autophagy of human placental chorionic plate-derived mesenchymal stem cells via regulating the phosphorylation of mTOR." J Cell Biochem 114(1): 79-88.

Litz, J. and G. W. Krystal (2006). "Imatinib inhibits c-Kit-induced hypoxia-inducible factor-1alpha activity and vascular endothelial growth factor expression in small cell lung cancer cells." Mol Cancer Ther 5(6): 1415-1422.

Lux, M. L., B. P. Rubin, T. L. Biase, C.-J. Chen, T. Maclure, G. Demetri, S. Xiao, S. Singer, C. D. M. Fletcher and J. A. Fletcher (2000). "KIT Extracellular and Kinase Domain Mutations in Gastrointestinal Stromal Tumors." The American Journal of Pathology 156(3): 791-795.

Mabjeesh, N. J. and S. Amir (2007). "Hypoxia-inducible factor (HIF) in human tumorigenesis." Histol Histopathol 22(5): 559-572.

Monsel, G., N. Ortonne, M. Bagot, A. Bensussan and N. Dumaz (2010). "c-Kit mutants require hypoxia-inducible factor 1alpha to transform melanocytes." Oncogene 29(2): 227-236.

Ou, W. B., M. J. Zhu, G. D. Demetri, C. D. Fletcher and J. A. Fletcher (2008). "Protein kinase C-theta regulates KIT expression and proliferation in gastrointestinal stromal tumors." Oncogene 27(42): 5624-5634.

Poon, E., A. L. Harris and M. Ashcroft (2009). "Targeting the hypoxia-inducible factor (HIF) pathway in cancer." Expert Rev Mol Med 11: e26.

Pore, N., Z. Jiang, H. K. Shu, E. Bernhard, G. D. Kao and A. Maity (2006). "Akt1 activation can augment hypoxia-inducible factor-1alpha expression by increasing protein translation through a mammalian target of rapamycin-independent pathway." Mol Cancer Res 4(7): 471-479.

Rubin, B. P., S. Singer, C. Tsao, A. Duensing, M. L. Lux, R. Ruiz, M. K. Hibbard, C. J. Chen, S. Xiao, D. A. Tuveson, G. D. Demetri, C. D. Fletcher and J. A. Fletcher (2001). "KIT activation is a ubiquitous feature of gastrointestinal stromal tumors." Cancer Res 61(22): 8118-8121.

Scheid, M. P., K. M. Schubert and V. Duronio (1999). "Regulation of bad phosphorylation and association with Bcl-x(L) by the MAPK/Erk kinase." J Biol Chem 274(43): 31108-31113.

Schmid, T., J. Zhou, R. Kohl and B. Brune (2004). "p300 relieves p53-evoked transcriptional repression of hypoxia-inducible factor-1 (HIF-1)." Biochem J 380(Pt 1): 289-295.

Stegeman, H., J. H. Kaanders, D. L. Wheeler, A. J. van der Kogel, M. M. Verheijen, S. J. Waaijer, M. Iida, R. Grenman, P. N. Span and J. Bussink (2012). "Activation of AKT by hypoxia: a potential target for hypoxic tumors of the head and neck." BMC Cancer 12: 463.

Theou-Anton, N., S. Tabone, D. Brouty-Boye, R. Saffroy, L. Ronnstrand, A. Lemoine and J. F. Emile (2006). "Co expression of SCF and KIT in gastrointestinal stromal tumours (GISTs) suggests an autocrine/paracrine mechanism." Br J Cancer 94(8): 1180-1185.

Tsujimoto, Y. and S. Shimizu (2005). "Another way to die: autophagic programmed cell death." Cell Death Differ 12 Suppl 2: 1528-1534.

Turner, K. J., J. W. Moore, A. Jones, C. F. Taylor, D. Cuthbert-Heavens, C. Han, R. D. Leek, K. C. Gatter, P. H. Maxwell, P. J. Ratcliffe, D. Cranston and A. L. Harris (2002). "Expression of hypoxia-inducible factors in human renal cancer: relationship to angiogenesis and to the von Hippel-Lindau gene mutation." Cancer Res 62(10): 2957-2961.

Tuveson, D. A., N. A. Willis, T. Jacks, J. D. Griffin, S. Singer, C. D. Fletcher, J. A. Fletcher and G. D. Demetri (2001). "STI571 inactivation of the gastrointestinal stromal tumor c-KIT oncoprotein: biological and clinical implications." Oncogene 20(36): 5054-5058.

Uchiyama, T., R. M. Engelman, N. Maulik and D. K. Das (2004). "Role of Akt signaling in mitochondrial survival pathway triggered by hypoxic preconditioning." Circulation 109(24): 3042-3049.

Wouters, B. G. and M. Koritzinsky (2008). "Hypoxia signalling through mTOR and the unfolded protein response in cancer." Nat Rev Cancer 8(11): 851-864.

Wu, X., J. Li, M. Zhu, J. A. Fletcher and F. S. Hodi (2012). "Protein kinase C inhibitor AEB071 targets ocular melanoma harboring GNAQ mutations via effects on the PKC/Erk1/2 and PKC/NF-kappaB pathways." Mol Cancer Ther 11(9): 1905-1914.

Yang, Z. J., C. E. Chee, S. Huang and F. A. Sinicrope (2011). "The role of autophagy in cancer: therapeutic implications." Mol Cancer Ther 10(9): 1533-1541.

Zhang, W. and H. T. Liu (2002). "MAPK signal pathways in the regulation of cell proliferation in mammalian cells." Cell Res 12(1): 9-18.

Zhong, H., F. Agani, A. A. Baccala, E. Laughner, N. Rioseco-Camacho, W. B. Isaacs, J. W. Simons and G. L. Semenza (1998). "Increased expression of hypoxia inducible factor-1alpha in rat and human prostate cancer." Cancer Res 58(23): 5280-5284.

Zhu, M. J., W. B. Ou, C. D. Fletcher, P. S. Cohen, G. D. Demetri and J. A. Fletcher (2007). "KIT oncoprotein interactions in gastrointestinal stromal tumors: therapeutic relevance." Oncogene 26(44): 6386-6395.


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