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 (NF£eB) 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. NF£eB is a pivotal transcription factor for regulation of pro-inflammatory cytokines and genes such as VEGF and cycloxygenase¡V2 (COX-2). Application of HDGF stimulated NF£eB-driven luciferase activities. This was correlated with a dose- and time-depedent increment of NF£eB (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 NF£eB/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.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0830110-170618 |
| Date | 30 August 2010 |
| Creators | Kuo, Hsiao-Mei |
| Contributors | Tai, Ming-Hong, Cheng, Jiin-Tsuey, Hsu, Ching-Mei, Cho, Chung-Lung, Tsung-Hui Hu |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0830110-170618 |
| Rights | not_available, Copyright information available at source archive |
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