臺灣博碩士論文加值系統

Heat shock protein 90 (HSP90) 在細胞生長、分化甚至癌症形成過程中，扮演重要的角色，由於它調控許多致癌蛋白以及其下游蛋白(client proteins)，當抑制HSP90之蛋白表現表達即能抑制致癌途徑，因此在癌症治療中HSP90成為有潛力之標的。由隔板葉型軟珊瑚Lobophytum crassum所分離出海洋天然物13-acetoxysarcocrassolide (13-AC)，發現對各種癌細胞具有顯著毒殺效果，並且能抑制HSP90之活性。經由實驗證實，13-AC會透過干擾粒線體膜電位(MMP)之降解以及刺激reactive oxygen species (ROS)產生進而誘導口腔癌細胞Ca9-22凋亡。然而，抗氧化劑N-acetylcysteine (NAC)前處理後會抑制ROS的產生並且避免13-AC造成細胞毒性。雖然13-AC處理後會快速地活化Keap1-Nrf2之表現，但是利用免疫沉澱、免疫細胞螢光及西方轉漬分析發現13-AC處理明顯地抑制Nrf2抗氧化之功能，並增加p62/SQSTM1之表現。以siRNA方式降解p62表現明顯地減少13-AC處理所造成之生長抑制。利用動物異植試驗結果，相較於對照組織組，發現以13-AC治療60天具有抗腫瘤的效果，顯著地抑制腫瘤的體積及重量分別為55.29%與90.33%。經由分子模擬分析，13-AC結合在HSP90分子結構的N端，然而，17-allylaminogeldanamycin (17-AAG)是現今已知HSP90 N端抑制劑，發現13-AC結合親和力較17-AAG強。觀察Ca9-22細胞處理13-AC後，會活化細胞凋亡相關蛋白的表現量，例如caspases-3、caspases-9以及PARP。另外，13-AC會抑制HSP90的下游蛋白(client proteins)，包括p-Akt、CDK4、HIF-1以及MMP-2。總結以上結果，13-AC是一個新穎的HSP90抑制劑，並且藉由促進ROS的產生以及抑制抗氧化蛋白(Nrf2)蛋白活性，進而引發細胞凋亡。因此，13-AC有機會成為HSP90抑制劑，可望未來治療癌症中展現相當之潛力發展為抗癌藥物。

Heat shock protein 90 (HSP90) plays a significant role in cellular proliferation, differentiation, and carcinogenesis. HSP90 has been recognized as a promising therapeutic target in the war on cancer because it affects various oncoproteins, and the inhibition of HSP90 leads to the suppression of multiple oncogenic pathways as well as client proteins. In this study, we found that the marine cytotoxic product 13-acetoxysarcocrassolide (13-AC), recently isolated from the alcyonacean coral Lobophytum crassum, exhibited potent inhibitory activity on HSP90. 13-AC induced apoptosis in oral cancer cells Ca9-22 through the disruption of mitochondrial membrane potential (MMP) and the stimulation of reactive oxygen species (ROS) generation. However, the pretreatment of Ca9-22 cells with N-acetylcysteine (NAC), an antioxidant, inhibited ROS production resulting in the attenuation of the cytotoxic activity of 13-AC. Under stressful conditions, Ca9-22 cells treated with 13-AC showed a rapid induction of Keap1-Nrf2 pathway and an increase in the expression of p62/SQSTM1, but a suppression in antioxidative function of Nrf2 with immunoprecipitation, immunocytofluorecent and western blotting analysis. Inhibition of p62 expression by siRNA considerably attenuated the growth-inhibited by 13-AC treatment. Moreover, 13-AC exerted antitumor effect against oral cancer cells as demonstrated by the in vivo xenograft animal model. It significantly reduced tumor volume (55.29%) and tumor weight (90.33%). The molecular docking analysis demonstrated that 13-AC binds to N-terminal domain of HSP90 protein showing binding affinity more than 17-allylaminogeldanamycin (17-AAG), a HSP90 inhibitor of N-terminal ATP binding site and suppressed HSP90 client proteins including p-Akt, CDK4, HIF-1, and MMP-2. On the proteins level, 13-AC increased the expression of apoptosis related proteins such as cleaved caspases-3 and -9 as well as cleaved PARP in a dose- and time-dependent manner. Moreover, the results suggested that 13-AC exerted its cytotoxic activity through the promotion of ROS generation and the suppression of antioxidant enzyme activities. Altogether, the apoptotic effect of 13-AC was found to be mediated through the inhibition of HSP90 suggesting its potential future application as an anticancer agent.

Abstract .................................................................................... VI
中文摘要 .....................................................................................VII
第一章、 緒論 ............................................................................... 1
第二章、 文獻回顧 ........................................................................... 3
2-1 海洋天然物............................................................................... 3
2-2 海洋珊瑚 ................................................................................ 3
2-3 Lobophytum 屬所含生物活性之天然物.................................................... 4
2-4 13-Acetoxysarcocrassolide之生物活性................................................... 30
2-5 口腔癌 ......................................................................................................... 30
2-6 細胞凋亡 ..................................................................................................... 30
2-7 熱休克蛋白................................................................................................. 31
2-8 活性氧化物(Reactive Oxygen species, ROS)......................................... 33
2-9 Keap1/Nrf2 pathway ................................................................................. 33
第三章、 研究材料與方法.................................................................................... 35
3-1 實驗材料 ..................................................................................................... 35
3-1-1 13-acetoxysarcocrassolide純化及配製.................................................... 35
3-1-2 細胞株 (Cell lines)..................................................................................... 36
3-1-3 藥品試劑 ..................................................................................................... 36
3-2 實驗方法 ..................................................................................................... 37
3-2-1 細胞培養 (Cell culture)............................................................................. 37
3-2-2 細胞冷凍保存............................................................................................. 37
3-2-3 細胞存活率試驗(MTT assay) ................................................................. 37
3-2-4 DAPI (4',6-diamidino-2-phenylindole)染色............................................ 38
3-2-5 Annexin V/PI 雙重染色檢測細胞凋亡之程度........................................ 38
3-2-6 檢測細胞內粒線體膜電位之變化............................................................. 38
3-2-7 檢測細胞內ROS之變化........................................................................... 38
3-2-8 檢測細胞內鈣離子濃度之變化................................................................. 39
3-2-9 蛋白質之定性及定量分析......................................................................... 39
3-2-10 單細胞凝膠電泳試驗(Single cell gel electrophoresis assay)................. 40
3-2-11 人類口腔癌細胞異植動物之抗癌試驗(Human oral cancer cells xenograft animal model) ........................................................................... 40 3-2-12 分子對位分析(Molecular docking analysis).......................................... 40 3-2-13 免疫沉澱法(Immunoprecipitation, IP) .................................................. 41
3-2-14 免疫螢光染色分析(Immunofluorescence analysis) .............................. 41
3-2-15 Inhibition of p62 expression by small RNA interference (siRNA) ........ 41
第四章、結果 ........................................................................................................ 43
4-1 化合物13-AC針對人類癌細胞生長之影響.............................................. 43
4-2 13-AC抑制口腔癌Ca9-22細胞遷徙能力與誘發DNA受損之影響....... 45
4-3 13-AC誘導口腔癌Ca9-22細胞發生細胞凋亡......................................... 48
4-4 13-AC使口腔癌Ca9-22細胞粒線體功能受損......................................... 51
4-5 13-AC誘導口腔癌Ca9-22細胞活性氧化物生成進而影響細胞粒線體功能受損與細胞凋..... 52
4-6 13-AC誘發抗氧化路徑Keap1/Nrf2/p62/SQSTM1之影響..................... 55
4-7 利用動物異植試驗檢測13-AC對Ca9-22腫瘤生長之影響.................... 59
4-8 13-AC影響HSPs表現............................................................ 63
第五章、 結論與討論................................................................ 67
參考文獻 .......................................................................... 70

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