天然海洋化合物構成的豐富資源在現今人類疾病藥物發展中扮演重要的角色。 L是海洋軟珊瑚Cladiella australis所萃取出具抗發炎的衍生物。在受到內毒素刺激的巨噬細胞中，L可抑制誘導型一氧化氮合成酶（iNOS）和環氧合酶-2（COX-2），此外，在大鼠頸動脈模式中內膜增生的動脈粥樣硬化動物模型亦有治療效果。由於在內膜生成時，血管新生扮演極為重要的角色。因此本研究著重於在利用L評估血管新生上之功能進而應用於其他相關之疾病。結果發現，L抑制內皮細胞增殖、遷移、管柱形成和基質金屬蛋白酶的活性。此外L對大鼠主動脈環微血管及斑馬魚的血管生長也有明顯的抑制生長。機制研究也發現，在血管內皮細胞中，L抑制血管內皮生長因子（VEGF）和血管內皮生長因子受體（VEGFR2）。另一方面結果表示L也可以抑制肝癌及黑色素癌細胞的生長。因此，本研究主要探討L發展成新的血管內皮生長因子和抗腫瘤血管生成抑製劑。希望進而能抑制腫瘤及腫瘤幹細胞，發展該藥物能成為低副作用的癌症治療用藥。
肝癌在台灣是常見的癌症之一，肝癌目前的治療方式，主要有手術切除、放射線療法、化學治療等。無論如何，整體的肝癌預後效果是很差的。在此研究中，我們也探討了血管新生在肝癌是否有實質性之作用。研究發現 L有效抑制肝癌細胞癌化的功能，包括細胞增殖、集落形成、細胞遷移；並且發現L也同時誘發細胞凋亡。另外也發現L有效的抑制癌症幹細胞的功能例如幹細胞的自我更新以及側群效應。並且有效的抑制了癌症幹細胞的標記CD133及CD44。相同的結果由大鼠的原位肝癌動物模式中也可以觀察到相同情形。
血管新生的過程對絕大多數固體腫瘤的發育與轉移具有關鍵性之影響，包括惡性黑色素瘤。在此研究中，我們也探討了血管新生在黑色素瘤是否有實質性之 作用。我們利用小鼠黑色素瘤細胞（B16F10）來研究L對黑色素癌的作用及其相關機轉。結果也顯示L可抑制黑色素瘤細胞的轉移，主要透過抑制細胞增殖、集落形成、細胞遷移，並且有效的抑制黑色素腫瘤的生長。由以上結果總結我們證實L是一種新的血管新生抑制劑且可用於治療黑色素瘤、肝癌及其他與血管新生有關之疾病。由以上總結L是一種新的血管新生抑制劑具有用於癌症的治療包括黑色素瘤、肝癌及其他與血管新生有關之疾病的潛力。

Naturally occurring marine compounds constitute a bountiful drugs resource for human diseases. L is an anti-inflammatory, synthetic intermediate of marine compound E-2 derived from soft coral Cladiella australis. L suppresses the expression of inducible nitric oxide syntheses (iNOS) and cyclooxygenase-II (COX-2) in endotoxin-stimulated macrophage cells, thereby reducing the neointima formation in rat atherosclerosis model. Angiogenesis plays an important role in neointima genesis. Thus, the present study investigated the angiogenic function and mechanism of L. Application of L perturbed the development of intersegmental vessels in transgenic zebrafish. Moreover, L potently suppressed microvessel sprouting in organotypic rat aortic rings. Among cultured endothelial cells, L significantly inhibited MMP-2/MMP-9 expression, cell proliferation, migration and tube formation in human umbilical vein endothelial cells (HUVECs). Mechanistic studies revealed that L significantly reduced the VEGF released by reducing VEGF expression at the mRNA and protein levels. In addition, L reduced surface VEGF receptor 2 (VEGFR2/Flk-1) expressions by repressing the VEGFR2 mRNA level. Finally, an exogenous VEGF supply partially rescued the L-induced angiogenesis blockage in vitro and in vivo.
Hepatocellular carcinoma (HCC) is one of the most common malignancies in Taiwan. Current HCC therapies include surgery, chemotherapy, radiofrequency ablation and target therapy. However, the overall prognosis of the patients with HCC remains poor, underscoring the demand of novel therapeutic agents. In this study, we explored the therapeutic potential and mechanism of L in a pre-clinical HCC model. The therapeutic efficacy of L regimen (10 mg/kg/d) was investigated in rats bearing established Novikoff hepatoma by serial ultrasound (US) and histological studies. In addition, the effect of L on distinct tumor growth processes, including cell proliferation and invasion, as well as colony formation, was studied using N1-S1 and Huh7 cells line. L effectively suppressed the self-renewal and drug-pumping functions by sphere and flow cytometry assay in HCC cells. L also depleted the abundance of CD133/CD44 cancer stem cell markers in HCC cells. A 7-day. L therapy significantly perturbed the progression of rat Novikoff hepatoma. Histological analysis revealed that L therapy result is the same as in vitro.
The process of angiogenesis is crucial for progression and metastasis of the majority of solid tumors including melanomas. In this study, we try to investigate whether angiogenesis is a relevant process in melanoma. We studied this effect and relevant signaling pathways in mouse melanoma cells (B16F10). These findings suggest that L suppressed the metastatic properties, including proliferation, anchorage-independent via colony formation, invasion. L also inhibits the melanoma tumor growth. Above of all, L is an anti-angiogenesis drug which also can inhibit the tumor and tumor stem cells, including melanoma, HCC and other angiogenesis related diseases.

論文審定書 i
論文公開授權書 ii
誌謝 iii
Abstract vi
Chapter 1 1
INTRODUCTION 1
1-1 Marine natural products 1
1-2 Angiogenesis 2
1-3 L (dihydroaustrasulfone alcohol) 4
1-4 Hepatocellular carcinoma 5
1-5 Cancer stem cells and HCC 6
1-6 Drug-resistance in HCC 7
1-7 Melanoma tumor and Metastasis 10
MATERIALS AND METHODS 12
2-1 Coral compounds and antibodies 12
2-2 Aortic Ring Assay 12
2-3 Zebrafish angiogenesis model 13
2-4 Cell culture 15
2-5 Gelatin zymography 16
2-6 Proliferation assay 16
2-7 Migration assay 17
2-8 Tube formation assay 18
2-9 Immunofluorescence assay 18
2-10 Flow cytometric analysis 19
2-11 Colony formation assay 19
2-12 Invasion Assay 20
2-13 Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 20
2-14 Western blot analysis 21
2-15 Enzyme-linked immunosorbent assay (ELISA) 22
2-16 Nitric oxide measurement 23
2-17 Sphere formation assay 23
2-18 Side population cells (SPCs) analysis 23
2-19 Animal experiments 24
2-20 Retrovirus production and infection 25
2-21 Primary melanoma models 25
2-22 Metastasis melanoma models 26
2-23 Scratch Migration Assay 26
Chapter 3 28
Coral-derived Compound L Inhibits Angiogenesis by At tenuating VEGF/VEGFR2 Signaling Pathway 28
3-1 Results 28
3-2 DISCUSSION 37
3-3 Figure and Legends 41
Chapter 4 60
Coral-derived Compound L suppresses hepatoma stemness and progression 60
4-1 Reuslts 60
4-4 DISCUSSION 68
4-5 FIGURES AND LEGENDS 70
Chapter 5 84
Coral-derived Compound L suppresses melanoma and melanoma metastasis 84
5-1 Results 84
5-2 DISCUSSION 86
5-3 FIGURES AND LEGENDS 89
APPENDIX 95
REFERENCES 99
Shih-Wei Lin CV 105

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