肝癌為世界上最盛行的癌症之一，經統計指出全世界每年有超過一佰萬人罹患肝癌。現行針對肝癌的治療策略不外乎手術切除惡性腫瘤組織，以及施行經導管動脈栓塞術(transcatheter arterial embolization, TAE) 與抗癌藥物化學療法。然而，因為肝癌早期病兆
不易查覺，所以肝癌患者發病時接受手術及拴塞治療的效果不甚理想。因此，發展適於肝癌特性的新治療方式更是刻不容緩。PTEN基因為一腫瘤抑制基因，主要是對抗PI3K調控路徑，在人類很多的癌症上都可以發現PTEN基因的突變或缺失。先前的研究結果顯示，在人類肝癌檢體中高達40-50%有PTEN基因缺損的現象，而PTEN基因可能在肝癌的疾病發展過程中扮演著關鍵的角色。因此，若能恢復PTEN基因的功能，可能在治療癌症上可以成為另一種治療方法。最近的研究證實，當肝細胞暴露在酒精中會增加腫瘤壞死因子引起細胞毒殺的敏感性，並且也會增加PTEN基因啟動區的活性。本研究重點在於評估以PTEN基因傳送及酒精毒殺作用於治療肝癌的效率和可行性。本實驗發展以腺病毒(Adenovirus)攜帶重組PTEN基因(Ad-PTEN)以及綠螢光蛋白基因(Ad-GFP)作為基因傳送的研究。研究結果發現，在培養的N1-S1肝癌細胞株中，理想的腺病毒感染濃度約在MOI 100-200。而藉由腺病毒攜帶PTEN基因傳送的N1-S1細胞中，Ad-PTEN 可造成N1-S1細胞株的細胞周期停止，進而抑制N1-S1的細胞增生達40-50%。此外，理想的酒精抑制濃度為6%。在細胞實驗方面證實，合併腺病毒攜帶PTEN基因傳送及酒精治療下，N1-S1細胞明顯的增生抑制高達約70%。而動物實驗結果，在以N1-S1細胞肝內注射誘導肝癌的實驗中，先經 Ad-PTEN感染的實驗動物組別中有較低的發生率，其中六隻實驗大白鼠只有一隻有誘導長出肝腫瘤，但在控制組及先經Ad-GFP感染的組別中均有高達80%的肝腫瘤發生率。這種先經 Ad-PTEN感染的動物實驗模式，顯示其具有效抑制腫瘤形成的可能性。經由本實驗結果顯示，PTEN基因傳送對於治療免疫完全老鼠的原位肝癌具有發展成新興肝癌治療方式的潛力。

Hepatocellular carcinoma (HCC) is one of the most common cancerous diseases worldwide. The annual occurrences exceed one million peoples affected. Currently, the treatment modalities for HCC include surgical resection, trans-arterial embolization (TAE) and chemotherapy. However, these modalities are not completely effective, underscoring the need for development of novel therapeutic approaches.
PTEN, a tumor suppressor that antagonizes the PI3K pathway, is frequently mutated or deleted in various human cancers. Loss of PTEN occurs in 40-50% of surgical resected HCC samples and predicts poor prognosis for HCC patients, suggesting PTEN restoration may constitute
a treatment alternative for HCC. Since PTEN increased ethanol-induced cytotoxicity in hepatoma cells, PTEN gene delivery may serve as an adjuvant therapy in conjunction with ethanol TAE for HCC. In the present study, we evaluated the efficacy of PTEN gene therapy and its combination with ethanol in a syngenic Novikoff hepatoma model by implantation of N1-S1 cells into livers of Sprague Dawley rats. Adenovirus encoding PTEN (Ad-PTEN) or green fluorescent protein (Ad-GFP) was generated for gene delivery studies. The optimal condition for adenovirus vectors to infect N1-S1 cells was determined at multiplicity of infection (MOI) of 100-200. Infection of N1-S1 cells with Ad-PTEN, but not Ad-GFP, increased PTEN levels and led to 40-50% inhibition of cell proliferation via cell cycle arrest. Besides, the half maximal -inhibitory concentration (IC50) for ethanol in N1-S1 cells was determined at 6%. Combination with PTEN gene delivery further augmented the cytotoxicity of ethanol in N1-S1 cells from 40% to 70% inhibition. To evaluate the prevention efficacy of PTEN gene delivery, N1-S1 cells were infected with adenovirus vectors then implanted into livers of Sprague-Dawley rats to induce Novikoff hepatoma. Injection of PBS- or Ad-GFP-treated N1-S1 cells led to large hepatoma (with an average size of 3-4 cm) with tumor incidence of 80-90%. In contrast, injection of Ad-PTEN-infected N1-S1 cells only induced one hepatoma (with size of 0.1 cm) in six rats, suggesting that pretreatment with PTEN gene delivery effectively abolished the tumorigenic potential of N1-S1 hepatoma cells in vivo. In summary, these results validate the feasibility of PTEN gene delivery as a new promising therapeutic strategy for the treatment of orthotopic hepatoma in immune-competent rats.

Contents II
Abstract in Chinese VI
Abstract in English VIII
Abbreviations X












Contents
Page
Introduction
Hpatocellularcarcinoma 1
HCC and Tumor Suppressor Gene PTEN 3
Specific aims 6
Materials and Methods
Cell culture 7
Treatment with methylase inhibitors 7
Determination of optimal MOI for adenovirus to
infect hepatoma cells 7
Determination of optimal cytotoxic concentration
for ethanol tohepatoma cells 8
Generation of Ad-PTEN and Ad-p53 8
Amplification and purification of adenovirus vectors 9
RT-PCR 10
Cell proliferation assay 11
Western blot analysis 11
Animal model 12
Immunohistochemistry 13
Flow cytometry Assay 14
Statistical analysis 14
Results
Reduced PTEN level, but elevated p53 and PCNA expression,
in Novikoff hepatoma N1-S1 cells 15
Mutation of p53 gene in Novikoff hepatoma N1-S1 cells 15
Addition of methylase inhibitor, 5-Aza-dC, did not alter the
PTEN level in N1-S1 cells 15
PTEN downregulation, p53 overexpression and increased
PCNA level in orthotpic Novikoff hepatoma 16

Gene delivery of PTEN or p53 effectively inhibited the
proliferation of N1-S1 cells 16
Gene delivery of PTEN and p53 induced apoptosis and cell
cycle arrest in N1-S1 cells 17
Implantation of PTEN- or p53-transduced N1-S1 cells
led to retarded hepatoma growth in rats 17
Cytotoxicity of ethanol in Clone 9 normal epithelial cells
and N1-S1 hepatoma cells 18
Ethanol induced cellular apoptosis in Clone 9 normal epithelial
cells and N1-S1 hepatoma cells 18
Gene delivery of PTEN or p53 enhanced the sensitivity of
N1-S1 cells to ethanol 19
Discussion 20
References 25
Figures and legends 34
Tables 57
Index 58

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