肝癌是全世界為最常見之惡性腫瘤之ㄧ。目前已發現一系列生長因子與其接受器已經被確定在肝癌化的不同的階段作為正和負的調節器。肝癌衍生生長因子(HDGF) 為一由肝癌細胞株Huh-7中發現之生長因子。 HDGF對許多細胞有刺激生長的能力。近年文獻報告中指出HDGF過度表現與人類肝癌，非小型細胞肺癌和黑色素癌之患者術後存活與癌症化進行有密切關聯。然而, HDGF過度表達對細胞癌化的功能和機制並不情楚。在本論文第一部分中，我們首先研究良性HepG2和惡性SK-Hep-1肝癌細胞的HDGF釋放與對外加HDGF的反應。發現無血清培養會刺激SK-Hep-1細胞之HDGF釋放，但HepG2 細胞則無此反應。有趣的是，無血清培養不會增加SK-Hep-1細胞分泌VEGF，一種強力的血管生成因子。而且，SK-Hep-1細胞能對外加的HDGF得到更多的生長和促進遷移效應。我們也確定重組HDGF蛋白質在體外和體內的血管生成功能。在本論文第二部分，我們研究細胞HDGF表現量對肝癌細胞癌化的影響。腺病毒做為載體複製HDGF，Ad-HDGF，和antisense HDGF, Ad-HDGF (-)，使在SK-Hep-1肝臟細胞產生HDGF含量有所變動。Ad-HDGF感染SK-Hep-1會增HDGF表現和釋放，顯著增加細胞的增殖能力, 遷移和無固著依賴性生長。相反的，Ad-HDGF(-)基因傳送至SK-Hep-1細胞會減少HDGF表現和釋放，並且減低致癌的能力。植入過度表現HDGF之SK-Hep-1細胞能促使肝腫瘤於SCID老鼠之成長，當用SK-Hep-1細胞感染HDGF抑制表現植入SCID的老鼠抑制腫瘤成長。組織學分析顯示在過度表現HDGF的腫瘤有增加生長和新生血管的現象。這可歸因於SK-Hep-1細胞會藉由HDGF提升而影響VEGF 表現提高和NFκB活化。在本論文第三部分，我們探索SK-Hep-1細胞在HDGF誘導下VEGF分泌和NFκB活化途徑。以重組HDGF蛋白處理SK-Hep-1細胞會增加VEGF 釋放特別是在血清飢餓期間。這與SK-Hep-1細胞與相伴應增加VEGF蛋白質和mRNA有關。像很多生長因子形成一樣，HDGF會呈劑量效應的增加包括過氧陰離子和過氧化氫等活性氧分子(ROS)的產生。預先用抗氧化劑處理則徹底破壞HDGF 引起VEGF分泌的效應。NFκB是轉錄因子中重要調節炎症性的細胞激素和基因例如VEGF 和COX-2。HDGF的運用刺激NFκB-驅動的luciferase活性。這次藉由HDGF與NFκB (p65)劑量和時間依賴方法增加有關係。SK-Hep-1細胞用HDGF處理也提高COX-2蛋白質和活性。另外，NS-398是COX-2抑制劑會減少HDGF引起的VEGF分泌，提供COX-2牽連。最後，發現HDGF 刺激Akt，Erk1/2 和p38 MAPK的磷酸化。LY294002是Akt的抑制劑也減少HDGF 引起的VEGF分泌。這些研究指出HDGF誘導氧化的壓力的活化NFκB/COX-2/Akt途徑，因此刺激VEGF 表現並與釋放。總結，本論文對HDGF過度表現造成肝細胞癌化之功能和機轉提供更深入之見解。

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. An extensive array of growth factors and their receptors have been identified and may act as positive and negative modulators in different stages of liver carcinogenesis. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from conditioned medium of Huh-7 hepatoma cell line. HDGF has growth stimulating activity for various types of cells. Recent evidence indicates that HDGF upregulation is associated with poor survival outcome and tumor progression in HCC, non-small cell lung carcinoma and melanoma. However, the exact function and molecular mechanism of HDGF overexpression during HCC progression remain largely unknown. In the first project (Chapter 2) of this thesis study, we started with characterizing in HDGF release and response to exogenous HDGF between benign HepG2 and malignant SK-Hep-1 hepatoma cells. It was found that serum deprivation significantly stimulated the HDGF secretion in SK-Hep-1 cells but not HepG2 cells. Interestingly, SK-Hep-1 cells did not increase the secretion of vascular endothelial growth factor (VEGF), a potent angiogenic factor, during serum deprivation. Besides, SK-Hep-1 cells were more responsive to the growth- and migration-promoting effect of exogenous HDGF. We also validated the angiogenic functions of recombinant HDGF protein in vitro and in vivo. In the second project (Chapter 3), we investigated the influence of cellular HDGF level on the neoplastic potential of hepatoma cells. Adenovirus vectors encoding HDGF, Ad-HDGF, and antisense HDGF, Ad-HDGF (-), were generated to modulate the cellular HDGF levels in SK-Hep-1 cells. Adenovirus-mediated HDGF gene delivery increased the HDGF expression and release, and stimulated the proliferation, migration and anchorage-independent growth of SK-Hep-1 cells. In contrast, infection with Ad-HDGF (-) reduced the HDGF expression and secretion, and attenuated the oncogenic behaviors of SK-Hep-1 cells. Implanting HDGF-overexpressing SK-Hep-1 cells led to the accelerated growth of xenografted hepatoma in SCID mice while implantation of HDGF-downregulated SK-Hep-1 cells caused retarded tumor growth. Histological analysis revealed the increased proliferation and neovascularization in HDGF-overexpressing tumors. This could be attributed to elevated VEGF expression and activation of the nuclear factor kappa B (NFκB) activities by HDGF upregulation in SK-Hep-1 cells. In the third project (Chapter 4), we delineated the mechanism underlying HDGF-induced VEGF secretion and activation of NFB pathway in SK-Hep-1 cells. Adding recombinant HDGF protein enhanced the VEGF release by SK-Hep-1 cells particularly during serum starvation. This was associated with a concomitant increment in VEGF protein and mRNA levels in SK-Hep-1 cells. Like many mitogens, HDGF increased the production of reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide in a dose-dependent manner. Pretreatment with antioxidants abolished the HDGF-induced VEGF secretion. NFκB is a pivotal transcription factor for regulation of pro-inflammatory cytokines and genes such as VEGF and cycloxygenase–2 (COX-2). Application of HDGF stimulated NFκB-driven luciferase activities. This was correlated with a dose- and time-depedent increment of NFκB (p65) by HDGF. HDGF treatment also elevated the COX-2 protein levels and activities in SK-Hep-1 cells. In addition, blockade of COX-2 by NS-398 attenuated the HDGF-induced VEGF secretion, suggesting the involvement of COX-2. Finally, it was found that HDGF stimulated the phosphorylation of Akt, Erk1/2, and p38 MAPK. Inhibition of Akt by LY294002 also diminished the HDGF-induced VEGF secretion. These studies suggest that HDGF induces oxidative stress to activate NFκB/COX-2/Akt pathway, thereby stimulating VEGF expression and release. In summary, this thesis study brings functional and mechanistic insights on how aberrant HDGF expression contributes to angiogenesis and tumorigenesis during liver carcinogenesis.

致謝 ----------------------------------------------------------------- 1
摘要 ----------------------------------------------------------------- 3
ABSTRACT ------------------------------------------------------- 5
INDEX -------------------------------------------------------------- 8
LIST OF TABLES AND FIGURES ------------------------- 10
Chapter 1 Background -------------------------------------- 14
1-1 INTRODUCTION ----------------------------------------- 14
1-2 SPEIFIC AIMS --------------------------------------------- 21
1-3 FIGURES AND LEGENDS ---------------------------- 23
Chapter 2 HDGF Secretion and Response to Exogenous HDGF in Hepatoma Cells ------------------ 24
2-1 INTRODUCTION ----------------------------------------- 24
2-2 MATERIALS AND METHODS ------------------------- 26
2-3 RESULTS -------------------------------------------------- 31
2-4 DISCUSSION --------------------------------------------- 34
2-5 FIGURES AND LEGENDS ---------------------------- 35
Chapter 3 Influences of HDGF Expression on Tumorigenesis of Hepatoma Cells ---------------------- 44
3-1 INTRODUCTION ----------------------------------------- 44
3-2 MATERIALS AND METHODS ------------------------- 47
3-3 RESULTS -------------------------------------------------- 57
3-4 DISCUSSION --------------------------------------------- 62
3-5 TABLES ----------------------------------------------------- 67
3-6 FIGURES AND LEGENDS ---------------------------- 69
Chapter 4 Signaling Pathway of HDGF-induced VEGF Expression in Hepatoma Cells --------------------------- 85
4-1 INTRODUCTION ----------------------------------------- 85
4-2 MATERIALS AND METHODS ------------------------- 88
4-3 RESULTS -------------------------------------------------- 93
4-4 DISCUSSION --------------------------------------------- 97
4-5 FIGURES AND LEGENDS --------------------------- 100
Chapter 5 Conclusion ------------------------------------- 117
FURURE PERSPECTIVES ------------------------------- 119
REFERENCES ----------------------------------------------- 120
APPENDIX ----------------------------------------------------- 132
PUBLICATIONS ---------------------------------------------- 135
CONFERENCES -------------------------------------------- 137

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