LCE為從葉形軟珊瑚Lobophytum crassum之粗萃物，其中主要活性成分13-Acetoxysarcocrassolide (13-AC)，在先前研究指出有抗腫瘤之功效。前列腺癌是男性中第二大最常見的癌症診斷，也是全球癌症相關第五大死亡原因，雖前列腺癌為早期治療痊癒率高之疾病，一旦癌細胞發展至後期轉移至其他骨盆淋巴或其他器官使癌症五年存活率降至30%。因此，本研究探討LCE對於前列腺癌之抗癌活性測試與抗腫瘤機制作用，且針對LCE前列腺癌轉移和上皮間質轉化 （EMT）之影響。使用MTT測定及細胞群落形成試驗測定LCE對三種前列腺癌細胞LNcap 、PC3和Du145之細胞毒殺活性並發現其對三種癌細胞皆有劑量依賴性地抑制細胞增生之作用。在體內異種移植腫瘤之動物實驗分別顯示LCE對前列腺癌細胞PC3和Du145具有有效的抗腫瘤作用，並顯著減少腫瘤體積43.9% （PC3）、49.2%（Du145）和抑制腫瘤重量48.8%（PC3）、7.8%（Du145）。接著通過流式細胞儀分析、DAPI螢光染色結果顯示LCE處理可促使增加細胞自噬及細胞凋亡之發生，並且增加自噬標記蛋白LC3α/β與細胞凋亡活化蛋白cleaved caspase-3的表現，代表LCE透過細胞自噬及凋亡以誘導癌細胞死亡。另外在癌細胞轉移的部分，EMT是上皮細胞轉化為間質細胞的生物過程，過去研究指出EMT除了調控胚胎發育也在癌症中促進腫瘤侵襲和癌細胞之轉移。利用傷口癒合分析、transwell細胞遷移及侵襲試驗之結果證實，LCE非常顯著地減少前列腺癌細胞轉移及侵襲的能力，且增加上皮表達蛋白E-cadherin的表達並抑制EMT相關蛋白，進而影響癌細胞EMT。接著使用TGF-β生長因子處理誘發PC3和Du145細胞高度表現EMT，並使用LCE粗萃物觀察其抑制效果，結果表示在LCE處理下依然能有效抑制TGF-β所誘導之細胞轉移作用。本研究在未來可作為抗腫瘤與抗癌轉移的藥物開發之應用。

LCE (Lobophytum crassum extract) is a crude extract obtained from the soft coral Lobophytum crassum. 13-acetoxysarcocrassolide (13-AC) is the major active component and potent anti-tumor effects in LCE. Prostate cancer is the second most common diagnosed cancers in men and the fifth leading cause of cancer-related deaths worldwide. Prostate cancer shows a high cure rate following early detection. However, if the cancer develops undetected, the cancer cells metastasize to pelvic lymph nodes or other organs dropping the five-year survival rate to 30%. The huge health burden of this disease encouraged us to investigate cytotoxic activity and antitumor mechanism of LCE in prostate cancer cells. The metastasis and epithelial-mesenchymal transition (EMT) of prostate cancer cells. The cytotoxic activity and growth inhibition of LCE on three prostate cancer cells lines (LNcap, PC3 and Du145 cells) were dose-dependently suppressed in all three cancer cells using MTT assay, cell colony formation assay. The in vivo animal model with tumor xenografts also showed the potent antitumor activity of LCE on prostate tumor from PC3. LCE administration significantly diminished the tumor volume 43.9% (PC3), 49.2% (Du145) and tumor weight 48.8% (PC3), 7.8% (Du145) in in vivo PC3 and Du145-xenograft animal model, respectively. The results of the flow cytometry analysis, DAPI fluorescence staining analysis suggested that LCE treatment induced cancer cell death through autophagy and apoptosis. The result of western blotting showed LCE treatment could significantly increase the expression of autophagy macker, LC3α/β and apoptosis-related protein, cleaved caspase-3. In addition, EMT is a biological process in which epithelial cells transform into mesenchymal cells. Previous studies indicated that EMT could be the regulatory factor in embryo development and promots tumor invasion and metastasis in the malignant progression. The results of wound healing assay, transwell cell migration and invasion assay confirmed that LCE significantly allevated metastasis and invasion of prostate cancer cells. Western blotting analysis showed that LCE increased the expression of epithelial marker protein, E-cadherin and inhibited the expression of EMT-related proteins in the cells. The co-treatment with TGF-β growth factor induced excessive EMT of PC3 and Du145 that was inhibited by the treatment of LCE. The results of this study indicated the potential applications of LCE as a source for drug leads to tackle prostate cancer.

摘要 I
Abstract III
目錄 V
圖目錄 IX
表目錄 XI
第一章、緒論 1
第二章、文獻回顧 3
2-1　海洋天然物 (Marine natural product) 3
2-1-1　海洋珊瑚 5
2-1-2　Lobophytum 屬所含抗癌活性成分之文獻回顧 5
2-2　癌症-前列腺癌 8
2-2-1　癌症 (cancer) 8
2-2-2　前列腺癌 (Prostate Cancer) 9
2-3　細胞凋亡 (Apoptosis) 11
2-4　細胞自噬 (Autophagy) 12
2-5　上皮間質轉化( EMT) 14
2-6　生長因子TGF-β 17
2-7　自噬對EMT的影響 18
第三章、材料方法 21
3-1　實驗材料 21
3-1-1　珊瑚 21
3-1-2　細胞株 (Cell lines) 22
3-2　實驗方法 22
3-2-1　細胞培養 (Cell culture) 22
3-2-2　細胞冷凍保存 (Cell cryopreservation) 22
3-2-3　冷凍細胞活化 (Cell thawing) 23
3-2-4　細胞存活率試驗（MTT assay） 23
3-2-5　細胞群落形成分析 (Colony formation assay) 23
3-2-6　傷口癒合分析 (Wound healing assay) 23
3-2-7　DAPI染色 (4, 6-diamidino-2-phenylindole stain) 24
3-2-8　檢測細胞內粒線體膜電位之變化 24
3-2-9　Annexin V/PI 雙重染色檢測細胞凋亡之程度 24
3-2-10　西方墨點法 24
3-2-11　transwell之細胞遷移及侵襲分析試驗 (migration and invasion assay) 25
3-2-12　二維電泳 26
3-2-13　人類前列腺癌細胞異植動物之抗癌試驗 (Human prostate cancer cells xenograft animal model) 26
3-2-14　數據統計 26
第四章、結果 27
4-1　LCE萃取物對人類前列腺癌細胞生長影響 27
4-2　LCE萃取物對人類前列腺癌細胞增生群落之影響 28
4-3　LCE萃取物處理是否誘導人類前列腺癌細胞自噬之發生 30
4-4　LCE萃取物處理是否誘導人類前列腺癌細胞凋亡之發生 32
4-5　LCE萃取物處理誘導癌細胞之自噬作用對於細胞凋亡和細胞存活之影響 36
4-6　LCE萃取物對人類前列腺癌細胞上皮間質轉化和細胞轉移侵襲之影響 39
4-7　LCE萃取物抑制TGF-β誘導癌細胞之轉移、侵襲活性之作用 44
4-8　利用蛋白質體學尋找LCE抗癌作用之標靶蛋白 47
4-9　利用動物異植試驗檢測LCE對PC3及Du145腫瘤生長之影響 50
第五章、結論與討論 55
5-1　由Lobophytum crassum萃取之粗萃物LCE具有抗人類前列腺癌活性與抑制腫瘤增長之效果 55
5-2　粗萃物LCE對人類前列腺癌作用機制探討 55
5-3　LCE萃取物抑制人類前列腺癌細胞上皮間質轉化和細胞轉移侵襲 56
5-4　LCE萃取物抑制TGF-β誘導之癌細胞轉移、侵襲活性和EMT 58
5-5　篩選LCE可能作用之標靶蛋白 59
參考文獻 61

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