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

1. Fausto N., a. W. E. Liver regeneration (1994).
2. Shafritz, D. A. Regulation of liver gene expression during various physiologic and pathophysiologic states. Prog Liver Dis 9, 39-55 (1990).
3. Bernuau J, F. G.. Fulminat and subfulminant hepatitis: liver regeneration and prognosis. Liver Regeneration, 147 (1992).
4. Baldwin, A. S., Jr. Series introduction: the transcription factor NF-kappaB and human disease. J Clin Invest 107, 3-6 (2001).
5. Fausto, N. & Webber, E. M. Mechanisms of growth regulation in liver regeneration and hepatic carcinogenesis. Prog Liver Dis 11, 115-37 (1993).
6. Michalopoulos, G. K. Liver regeneration: molecular mechanisms of growth control. Faseb J 4, 176-87 (1990).
7. Selden, A. C. & Hodgson, H. J. Growth factors and the liver. Gut 32, 601-3 (1991).
8. Fausto, N. Growth factors in liver development, regeneration and carcinogenesis. Prog Growth Factor Res 3, 219-34 (1991).
9. Fausto, N. & Mead, J. E. Regulation of liver growth: protooncogenes and transforming growth factors. Lab Invest 60, 4-13 (1989).
10. Boyer., Z. Hepatology, A textbook of Liver Disease. 3rd ed 1, 1: 42. (1990).
11. Thorgeirsson, S. S. & Grisham, J. W. Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet 31, 339-46 (2002).
12. Beasley, R. P. Hepatitis B virus. The major etiology of hepatocellular carcinoma. Cancer 61, 1942-56 (1988).
13. Tsukuma, H. et al. Risk factors for hepatocellular carcinoma among patients with chronic liver disease. N Engl J Med 328, 1797-801 (1993).
14. Folkman, J. Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov 6, 273-86 (2007).
15. Aaronson, S. A. Growth factors and cancer. Science 254, 1146-53 (1991).
16. Cross, M. & Dexter, T. M. Growth factors in development, transformation, and tumorigenesis. Cell 64, 271-80 (1991).
17. Folkman, J. & Shing, Y. Angiogenesis. J Biol Chem 267, 10931-4 (1992).
18. Yancopoulos, G. D. et al. Vascular-specific growth factors and blood vessel formation. Nature 407, 242-8 (2000).
19. Kuiper, R. A., Schellens, J. H. et al. Clinical research on antiangiogenic therapy. Pharmacol Res 37, 1-16 (1998).
20. McNamara, D. A., Harmey, J. H. et al. Significance of angiogenesis in cancer therapy. Br J Surg 85, 1044-55 (1998).
21. Pluda, J. M. Tumor-associated angiogenesis: mechanisms, clinical implications, and therapeutic strategies. Semin Oncol 24, 203-18 (1997).
22. Folkman, J. Tumor angiogenesis: therapeutic implications. N Engl J Med 285, 1182-6 (1971).
23. Li, X. M., Tang, Z. Y. et al. Significance of vascular endothelial growth factor mRNA expression in invasion and metastasis of hepatocellular carcinoma. J Exp Clin Cancer Res 17, 13-7 (1998).
24. Torimura, T. et al. Increased expression of vascular endothelial growth factor is associated with tumor progression in hepatocellular carcinoma. Hum Pathol 29, 986-91 (1998).
25. Mise, M. et al. Clinical significance of vascular endothelial growth factor and basic fibroblast growth factor gene expression in liver tumor. Hepatology 23, 455-64 (1996).
26. Jinno, K. et al. Circulating vascular endothelial growth factor (VEGF) is a possible tumor marker for metastasis in human hepatocellular carcinoma. J Gastroenterol 33, 376-82 (1998).
27. Folkman, J. Anti-angiogenesis: new concept for therapy of solid tumors. Ann Surg 175, 409-16 (1972).
28. Shing, Y. et al. Heparin affinity: purification of a tumor-derived capillary endothelial cell growth factor. Science 223, 1296-9 (1984).
29. Sidky Y, A. R. Lymphocyte-induced angiogenesis in tumor-bearing mice. Science 192, 1237-8 (1976).
30. Folkman, J. & Klagsbrun, M. Angiogenic factors. Science 235, 442-7 (1987).
31. Freeman MR., S. F., & Gagnon ML. Peripheral blood T lymphocytes and lymphocytes infiltrating human cancers express vascular endothelial growth factor: a potential role for T cells in angiogenesis. cancer Res 55, 4140-45 (1995).
32. Carmeliet, P. & Jain, R. K. Angiogenesis in cancer and other diseases. Nature 407, 249-57 (2000).
33. Nakamura, H. et al. Partial purification and characterization of human hepatoma-derived growth factor. Clin Chim Acta 183, 273-84 (1989).
34. Hu, T. H. et al. Expression of hepatoma-derived growth factor in hepatocellular carcinoma. Cancer 98, 1444-56 (2003).
35. Nakamura, H. et al. Molecular cloning of complementary DNA for a novel human hepatoma-derived growth factor. Its homology with high mobility group-1 protein. J Biol Chem 269, 25143-9 (1994).
36. Izumoto, Y., Kuroda, T. et al. Hepatoma-derived growth factor belongs to a gene family in mice showing significant homology in the amino terminus. Biochem Biophys Res Commun 238, 26-32 (1997).
37. Everett, A. D. & Bushweller, J. Hepatoma derived growth factor is a nuclear targeted mitogen. Curr Drug Targets 4, 367-71 (2003).
38. Everett, A. D., Stoops, T. et al. Nuclear targeting is required for hepatoma-derived growth factor-stimulated mitogenesis in vascular smooth muscle cells. J Biol Chem 276, 37564-8 (2001).
39. Kishima, Y. et al. Antisense oligonucleotides of hepatoma-derived growth factor (HDGF) suppress the proliferation of hepatoma cells. Hepatogastroenterology 49, 1639-44 (2002).
40. Bustin, M., Lehn, D. A. & Landsman, D. Structural features of the HMG chromosomal proteins and their genes. Biochim Biophys Acta 1049, 231-43 (1990).
41. Javaherian, K., Sadeghi, M. & Liu, L. F. Nonhistone proteins HMG1 and HMG2 unwind DNA double helix. Nucleic Acids Res 6, 3569-80 (1979).
42. Kuehl, L., Rechsteiner, M. & Wu, L. Relationship between the structure of chromosomal protein HMG1 and its accumulation in the cell nucleus. J Biol Chem 260, 10361-8 (1985).
43. Thanos, D. & Maniatis, T. The high mobility group protein HMG I(Y) is required for NF-kappa B-dependent virus induction of the human IFN-beta gene. Cell 71, 777-89 (1992).
44. Reeck, G. R., Isackson, P. J. & Teller, D. C. Domain structure in high molecular weight high mobility group nonhistone chromatin proteins. Nature 300, 76-8 (1982).
45. Einck, L. & Bustin, M. The intracellular distribution and function of the high mobility group chromosomal proteins. Exp Cell Res 156, 295-310 (1985).
46. Mosevitsky, M. I., Novitskaya, V. A. et al. Tissue specificity of nucleo-cytoplasmic distribution of HMG1 and HMG2 proteins and their probable functions. Eur J Biochem 185, 303-10 (1989).
47. Lukasik, S. M. et al. High resolution structure of the HDGF PWWP domain: a potential DNA binding domain. Protein Sci 15, 314-23 (2006).
48. Sue, S. C. et al. PWWP module of human hepatoma-derived growth factor forms a domain-swapped dimer with much higher affinity for heparin. J Mol Biol 367, 456-72 (2007).
49. Yang, J. & Everett, A. D. Hepatoma derived growth factor binds DNA through the N-terminal PWWP domain. BMC Mol Biol 8, 101 (2007).
50. Salvi, A., Arici, B. et al. Small interfering RNA urokinase silencing inhibits invasion and migration of human hepatocellular carcinoma cells. Mol Cancer Ther 3, 671-8 (2004).
51. Clauss, M. et al. A permissive role for tumor necrosis factor in vascular endothelial growth factor-induced vascular permeability. Blood 97, 1321-9 (2001).
52. Kristensen, D. B. et al. Proteome analysis of rat hepatic stellate cells. Hepatology 32, 268-77 (2000).
53. Kawasaki, J., et al. Dissociation between the Ca(2+) signal and tube formation induced by vascular endothelial growth factor in bovine aortic endothelial cells. Eur J Pharmacol 398, 19-29 (2000).
54. Huang, L. et al. HCPTPA, a protein tyrosine phosphatase that regulates vascular endothelial growth factor receptor-mediated signal transduction and biological activity. J Biol Chem 274, 38183-8 (1999).
55. Nicosia, R. F., Lin, Y. J. et al. Endogenous regulation of angiogenesis in the rat aorta model. Role of vascular endothelial growth factor. Am J Pathol 151, 1379-86 (1997).
56. Nicosia, R. F. & Ottinetti, A. Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro. Lab Invest 63, 115-22 (1990).
57. Huang, J. & Kontos, C. D. PTEN modulates vascular endothelial growth factor-mediated signaling and angiogenic effects. J Biol Chem 277, 10760-6 (2002).
58. Enomoto, H., Yoshida, K. et al. Participation of hepatoma-derived growth factor in the regulation of fetal hepatocyte proliferation. J Gastroenterol 37 Suppl 14, 158-61 (2002).
59. Kishima, Y. et al. Hepatoma-derived growth factor stimulates cell growth after translocation to the nucleus by nuclear localization signals. J Biol Chem 277, 10315-22 (2002).
60. Mori, M. et al. Hepatoma-derived growth factor is involved in lung remodeling by stimulating epithelial growth. Am J Respir Cell Mol Biol 30, 459-69 (2004).
61. Everett, A. D., Narron, J. V. et al. Hepatoma-derived growth factor is a pulmonary endothelial cell-expressed angiogenic factor. Am J Physiol Lung Cell Mol Physiol 286, L1194-201 (2004).
62. Everett, A. D., Lobe, D. R., Matsumura, M. E., Nakamura, H. & McNamara, C. A. Hepatoma-derived growth factor stimulates smooth muscle cell growth and is expressed in vascular development. J Clin Invest 105, 567-75 (2000).
63. Culver, K. W. & Blaese, R. M. Gene therapy for cancer. Trends Genet 10, 174-8 (1994).
64. Culver, K. W. Clinical applications of gene therapy for cancer. Clin Chem 40, 510-2 (1994).
65. Mulligan, R. C. The basic science of gene therapy. Science 260, 926-32 (1993).
66. Yoshida, K. et al. Hepatoma-derived growth factor is a novel prognostic factor for hepatocellular carcinoma. Ann Surg Oncol 13, 159-67 (2006).
67. Chang, K. C. et al. Hepatoma-derived growth factor is a novel prognostic factor for gastrointestinal stromal tumors. Int J Cancer 121, 1059-65 (2007).
68. Yamamoto, S. et al. Expression level of hepatoma-derived growth factor correlates with tumor recurrence of esophageal carcinoma. Ann Surg Oncol 14, 2141-9 (2007).
69. Uyama, H. et al. Hepatoma-derived growth factor is a novel prognostic factor for patients with pancreatic cancer. Clin Cancer Res 12, 6043-8 (2006).
70. Iwasaki, T. et al. Hepatoma-derived growth factor as a prognostic marker in completely resected non-small-cell lung cancer. Oncol Rep 13, 1075-80 (2005).
71. Ren, H. et al. Expression of hepatoma-derived growth factor is a strong prognostic predictor for patients with early-stage non-small-cell lung cancer. J Clin Oncol 22, 3230-7 (2004).
72. Yamamoto, S. et al. Expression of hepatoma-derived growth factor is correlated with lymph node metastasis and prognosis of gastric carcinoma. Clin Cancer Res 12, 117-22 (2006).
73. Lauffenburger, D. A. Cell motility. Making connections count. Nature 383, 390-1 (1996).
74. Friedl, P. & Wolf, K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 3, 362-74 (2003).
75. Bernard, K. et al. Functional proteomic analysis of melanoma progression. Cancer Res 63, 6716-25 (2003).
76. Lepourcelet, M. et al. Insights into developmental mechanisms and cancers in the mammalian intestine derived from serial analysis of gene expression and study of the hepatoma-derived growth factor (HDGF). Development 132, 415-27 (2005).
77. Plate, K. H., Breier, G. et al. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 359, 845-848 (1992).
78. Beck, L. a. D., P.A. Vascular development: cellular and molecular regulation. FASEB J. 11, 365-373 (1997 ).
79. Carmeliet, P., Ferreira, V. et al. and Eberhardt, C. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380, 435-439 (1996).
80. Okuda, Y. et al. Hepatoma-derived growth factor induces tumorigenesis in vivo through both direct angiogenic activity and induction of vascular endothelial growth factor. Cancer Sci 94, 1034-41 (2003).
81. Grosjean, J., Kiriakidis, S. et al. Vascular endothelial growth factor signalling in endothelial cell survival: a role for NFkappaB. Biochem Biophys Res Commun 340, 984-94 (2006).
82. Sasaki, H., Ray, P. S. et al. Oxidative stress due to hypoxia/reoxygenation induces angiogenic factor VEGF in adult rat myocardium: possible role of NFkappaB. Toxicology 155, 27-35 (2000).
83. Chiarugi, V., Magnelli, L. et al. Hypoxia induces pivotal tumor angiogenesis control factors including p53, vascular endothelial growth factor and the NFkappaB-dependent inducible nitric oxide synthase and cyclooxygenase-2. J Cancer Res Clin Oncol 125, 525-8 (1999).
84. Graham, F. L. & Prevec, L. Methods for construction of adenovirus vectors. Mol Biotechnol 3, 207-20 (1995).
85. Yeo, M. et al. Novel action of gastric proton pump inhibitor on suppression of Helicobacter pylori induced angiogenesis. Gut 55, 26-33 (2006).
86. McKillop, I. H., Moran, D. M. et al. Molecular pathogenesis of hepatocellular carcinoma. J Surg Res 136, 125-35 (2006).
87. Curran, S. & Murray, G. I. Matrix metalloproteinases: molecular aspects of their roles in tumour invasion and metastasis. Eur J Cancer 36, 1621-30 (2000).
88. Fridman, R., Toth, M. et al. Activation of progelatinase B (MMP-9) by gelatinase A (MMP-2). Cancer Res 55, 2548-55 (1995).
89. Yoshida, K. et al. Expression of hepatoma-derived growth factor in hepatocarcinogenesis. J Gastroenterol Hepatol 18, 1293-301 (2003).
90. Zhang, J. et al. Down-regulation of hepatoma-derived growth factor inhibits anchorage-independent growth and invasion of non-small cell lung cancer cells. Cancer Res 66, 18-23 (2006).
91. Guinebretiere, J. M. [Angiogenesis and breast neoplasms. The pathologist's point of view]. Gynecol Obstet Fertil 33, 140-6 (2005).
92. Folkman, J. Tumor angiogenesis. Adv Cancer Res 43, 175-203 (1985).
93. Ryschich, E. et al. Molecular fingerprinting and autocrine growth regulation of endothelial cells in a murine model of hepatocellular carcinoma. Cancer Res 66, 198-211 (2006).
94. O'Byrne, K. J., Dalgleish, A. G. et al. The relationship between angiogenesis and the immune response in carcinogenesis and the progression of malignant disease. Eur J Cancer 36, 151-69 (2000).
95. Folkman, J. The role of angiogenesis in tumor growth. Semin Cancer Biol 3, 65-71 (1992).
96. Oliver, J. A. & Al-Awqati, Q. An endothelial growth factor involved in rat renal development. J Clin Invest 102, 1208-19 (1998).
97. Shi, C. S. et al. Evidence of human thrombomodulin domain as a novel angiogenic factor. Circulation 111, 1627-36 (2005).
98. O'Reilly, M. S. et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 277-85 (1997).
99. Jimenez, B. et al. Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Nat Med 6, 41-8 (2000).
100. Wu, J. T. & Kral, J. G. The NF-kappaB/IkappaB signaling system: a molecular target in breast cancer therapy. J Surg Res 123, 158-69 (2005).
101. Karihtala, P. & Soini, Y. Reactive oxygen species and antioxidant mechanisms in human tissues and their relation to malignancies. Apmis 115, 81-103 (2007).
102. Culotta, V. C., Yang, M. et al. Activation of superoxide dismutases: putting the metal to the pedal. Biochim Biophys Acta 1763, 747-58 (2006).
103. Tsanou, E. et al. Immunohistochemical expression of superoxide dismutase (MnSOD) anti-oxidant enzyme in invasive breast carcinoma. Histol Histopathol 19, 807-13 (2004).
104. Sikka, S. C. & Hellstrom, W. J. Role of oxidative stress and antioxidants in Peyronie's disease. Int J Impot Res 14, 353-60 (2002).
105. Carter, C. D., Kitchen, L. E. et al. Loss of SOD1 and LYS7 sensitizes Saccharomyces cerevisiae to hydroxyurea and DNA damage agents and downregulates MEC1 pathway effectors. Mol Cell Biol 25, 10273-85 (2005).
106. Ushio-Fukai, M. & Alexander, R. W. Reactive oxygen species as mediators of angiogenesis signaling: role of NAD(P)H oxidase. Mol Cell Biochem 264, 85-97 (2004).
107. Xia, C. et al. Reactive oxygen species regulate angiogenesis and tumor growth through vascular endothelial growth factor. Cancer Res 67, 10823-30 (2007).
108. Guastalla, J. P., Bachelot, T. et al. [Cyclooxygenase 2 and breast cancer. From biological concepts to clinical trials]. Bull Cancer 91 Suppl 2, S99-108 (2004).
109. Jones, D. A., Carlton, D. P. et al. Zimmerman, G. A. & Prescott, S. M. Molecular cloning of human prostaglandin endoperoxide synthase type II and demonstration of expression in response to cytokines. J Biol Chem 268, 9049-54 (1993).
110. Han, C., Michalopoulos, G. K. et al. Prostaglandin E2 receptor EP1 transactivates EGFR/MET receptor tyrosine kinases and enhances invasiveness in human hepatocellular carcinoma cells. J Cell Physiol 207, 261-70 (2006).
111. Sano, H. et al. Expression of cyclooxygenase-1 and -2 in human colorectal cancer. Cancer Res 55, 3785-9 (1995).
112. Molina, M. A., Sitja-Arnau, M. et al.Increased cyclooxygenase-2 expression in human pancreatic carcinomas and cell lines: growth inhibition by nonsteroidal anti-inflammatory drugs. Cancer Res 59, 4356-62 (1999).
113. Koga, H. et al. Expression of cyclooxygenase-2 in human hepatocellular carcinoma: relevance to tumor dedifferentiation. Hepatology 29, 688-96 (1999).
114. Zhao, Q. T. et al. Potential involvement of the cyclooxygenase-2 pathway in hepatocellular carcinoma-associated angiogenesis. Life Sci 80, 484-92 (2007).
115. Lilienbaum, A. & Israel, A. From calcium to NF-kappa B signaling pathways in neurons. Mol Cell Biol 23, 2680-98 (2003).
116. Kane, L. P., Shapiro, V. S. et al. Induction of NF-kappaB by the Akt/PKB kinase. Curr Biol 9, 601-4 (1999).
117. Beraud, C., Henzel, W. J. et al. Involvement of regulatory and catalytic subunits of phosphoinositide 3-kinase in NF-kappaB activation. Proc Natl Acad Sci U S A 96, 429-34 (1999).
118. Jones, P. F., Jakubowicz, T. et al. Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily. Proc Natl Acad Sci U S A 88, 4171-5 (1991).
119. Hayakawa, J. et al. Inhibition of BAD phosphorylation either at serine 112 via extracellular signal-regulated protein kinase cascade or at serine 136 via Akt cascade sensitizes human ovarian cancer cells to cisplatin. Cancer Res 60, 5988-94 (2000).
120. Imrich, A., Ning, Y. Y. et al. Intracellular oxidant production and cytokine responses in lung macrophages: evaluation of fluorescent probes. J Leukoc Biol 65, 499-507 (1999).
121. Guo, Y. S. et al. Synergistic Regulation of COX-2 Expression by Bombesin and Transforming Growth Factor-beta. Dig Dis Sci (2007).
122. Waris, G. & Siddiqui, A. Hepatitis C virus stimulates the expression of cyclooxygenase-2 via oxidative stress: role of prostaglandin E2 in RNA replication. J Virol 79, 9725-34 (2005).
123. Bonavida, B. Rituximab-induced inhibition of antiapoptotic cell survival pathways: implications in chemo/immunoresistance, rituximab unresponsiveness, prognostic and novel therapeutic interventions. Oncogene 26, 3629-36 (2007).
124. DeWire, S. M., Ahn, S. et al. Beta-arrestins and cell signaling. Annu Rev Physiol 69, 483-510 (2007).
125. Hunzelmann, N., Eming, S. et al. [Growth factors]. Z Rheumatol 66, 290, 292-6 (2007).
126. Yano, S. et al. Current status and perspective of angiogenesis and antivascular therapeutic strategy: non-small cell lung cancer. Int J Clin Oncol 11, 73-81 (2006).
127. Gupta, R. A. & Dubois, R. N. Colorectal cancer prevention and treatment by inhibition of cyclooxygenase-2. Nat Rev Cancer 1, 11-21 (2001).
128. Kondo, M. et al. Increased expression of COX-2 in nontumor liver tissue is associated with shorter disease-free survival in patients with hepatocellular carcinoma. Clin Cancer Res 5, 4005-12 (1999).
129. Ikeda, T., Tozuka, S. et al. Prostaglandin-E-producing hepatocellular carcinoma with hypercalcemia. Cancer 61, 1813-4 (1988).
130. Leng, J., Han, C. et al. Cyclooxygenase-2 promotes hepatocellular carcinoma cell growth through Akt activation: evidence for Akt inhibition in celecoxib-induced apoptosis. Hepatology 38, 756-68 (2003).
131. Jiang, B. H. & Liu, L. Z. PI3K/PTEN signaling in tumorigenesis and angiogenesis. Biochim Biophys Acta (2007).
132. Jackson, C. J. & Nguyen, M. Human microvascular endothelial cells differ from macrovascular endothelial cells in their expression of matrix metalloproteinases. Int J Biochem Cell Biol 29, 1167-77 (1997).