總摘要
氧化壓力調控肝癌細胞轉移的實驗證明，迄今仍未被廣泛研究。因此我們想利用一系列已鑑定其分化程度的肝癌細胞株作為研究模式，藉以探討腫瘤細胞內源性氧化壓力調控腫瘤轉移相關基因之表現及其調控之機制。由先前的實驗證明（Liu et al., 2000）分化程度好的肝癌細胞株具有較強的抗氧化能力（GSH + 四種抗氧化酵素之表現程度）[total intracellular antioxidant reserve (TIAR)] 與較高GSH/GSSG比值，依序為HepG2 > Hep3B > J5 > SK-Hep-1，為了再度證實該現象是否屬實，因此我們再利用duplex RT-PCR方法，來分析這些肝癌細胞株各類抗氧化酵素之mRNA的表現，再與蛋白活性做比較。實驗結果證實分化狀態良好的肝癌細胞株有較多的extracellular GPx (eGPx)及catalase mRNA的表現。相反地，分化程度較差之細胞株，如Mahlavu及SK-HEP-I，則該兩種酵素之表現甚少。目前已知 g-GCS蛋白是控制GSH合成最重要的酵素。[ g-GCSh（重鏈催化次單體）與 g-GCSl（輕鏈調控次單體）所組成]。因此我們想進一步證明，是否此酵素的表現也受細胞分化狀態所調控。我們再以HPLC及duplex RT-PCR方法分析這些肝癌細胞內 g -GCSh 蛋白活性及其mRNA表現，結果顯示該酵素之表現強度依序為HepG2 > Hep3B > J5 > SK-Hep-I。由此可知 g-GCSh蛋白之表現，是受細胞分化狀態所調控。相反地， g-GCSl mRNA的表現，則較不受分化狀態所調控。
吾人由文獻獲知NF-kB與AP-1轉錄因子，是受細胞內部氧化還原狀態所調控。先前的實驗證明了分化程度差的肝癌細胞株，其NF-kB與DNA結合活性遠大於分化程度良好的肝癌細胞。同時，我們也以EMSA方法分析AP-1與DNA的結合活性。其結合強度依序為：SK-Hep-I > Mahlavu > J5 > Hep3B > HepG2。目前已知此二轉錄因子調控了許多與腫瘤轉移相關因子的表現，因此我們選取了IL-8 ,Matrix Metalloproteinase (MMP)及E-cadherin三個分子做為評估肝癌細胞轉移傾向的指標，以蛋白活性染色及duplex RT-PCR方法分析肝癌細胞自行分泌的MMP。結果顯示SK-Hep-I細胞主要是可分泌MMP-9；Mahlavu細胞主要是可分泌MMP-2及少量的MMP-9；Hep3B細胞則分泌少量其它型的MMP。HepG2則完全不會分泌，其次我們以ELISA方法測量細胞所分泌的IL-8量，結果依序為SK-Hep-I > HepG2 > Hep3B > J5 > Mahlavu。吾人若再以西方墨點法及duplex RT-PCR方法分析E-cadherin的表現時，發現僅分化程度較良好的HepG2、Hep3B細胞有大量的蛋白及mRNA表現，其餘的肝癌細胞，則完全不表現。
根據上述結果得知：細胞內源性氧化還原狀態與腫瘤轉移相關因子的表現，具某種程度的相關性。為了更進一步證明細胞內氧化還原狀態可調控腫瘤轉移相關基因的表現，我們把 g-GCSh cDNA永久性轉染入SK-Hep-I細胞，得知轉染此DNA序列的細胞株（GCS 30），其 g-GCSh活性，mRNA的表現及GSH含量，均較SK-Hep-I細胞皆有顯著上升，以DCFDA方法分析細胞內部氧化壓力，也證明了GCS 30細胞內部氧化壓力有下降趨勢。但我們發現氧化壓力下降並不影響細胞內GSH/ GSSG比值。以EMSA方法分析NF-kB、AP-1因子與DNA的結合活性，得知GCS 30細胞株有較強的NF-kB結合能力，至於AP-1轉錄因子在兩株細胞內結合能力相當。進一步分析MMP-9的蛋白活性，得知兩株細胞差異甚微，由此可知，肝癌細胞株（SK-Hep-I）分泌MMP-9可能是經其他的訊息傳遞路徑所調控，細胞內氧化壓力並非絕對重要。
綜合上述，肝癌細胞的分化程度可調控TIAR及H2O2的產生,此二因子對於各種腫瘤轉移相關之表現以及其調控機轉，則尚需進一步更深入的研究。

Abstract
The metabolism of reactive oxygen species (ROS) in cancer cells is a research area that has not been intensively pursued. It is generally believed that cancer cells are persistently oxidative stressed. The consequence of this phenomenon will result in changes of the characteristic of a cancer cell. Whether or not the oxidative stress has a role in regulating the metastatic potential of a cancer cell is a research area that has been totally neglected. Using a group of human hepatocellular carcinoma (HCC) cell lines with distinct disparity in their differentiation status, established by their morphological observation and the abilities of synthesizing at least 15 plasma proteins, as the experimental model, we proposed to investigate the possibility that the constitutive oxidative stress status of these HCC cells may be modulated by their differentiation status. As a result, varying degrees of oxidative stress status of these cells will affect the propensity of expression of a few redox-sensitive transcription factors, such as NF-kB and AP-1, which may eventually modulate the metastasis- related gene expression. If these proposed hypotheses turn out to be true, we will then investigate the underlying mechanism(s) associated with the phenomena observed. Firstly, we demonstrated previously (Liu et al., 2000) that the total antioxidative capacity (as expressed by the composite propensities of expressing 4 antioxidant enzymes and the intracellular glutathione contents) as well as GSH/GSSG ratios of the HCC cells we studied were excellently correlated with their differentiation status, with an order of HepG2 > Hep3B > J5 > SK-Hep-I. To further confirm this observed phenomenon, we quantified the steady state mRNA expressions of the four antioxidant enzymes by duplex RT-PCR method. In this study, we further confirmed that well-differentiated HCC cells, such as HepG2 and Hep3B expressed higher levels of extracellular GPx (eGPx) and catalase mRNAs. Conversely, the expression of mRNA for both enzymes in a poorly-differentiated HCC cells, such as SK-Hep-I and Mahlavu, was trace or even negligible. Since GSH biosynthesis is controlled by g-glutamylcysteine synthetase (g-GCS), a rate-limiting enzyme composing of a catalytic heavy subunit ( gGCS h ) and a regulatory light subunit ( gGCSl) ,we wanted to further substantiate that differentiation status-mediated upregulation of GSH is regulated by this enzyme. We demonstrated that the g-GCSh expression was again differentiation status regulated, established by using either a HPLC or a duplex RT-PCR method. The order of ranking for the expression of g-GCS was HepG2 > Hep3B > J5 > SK-Hep-I. In contrast, we found that g-GCSl mRNA seemed not to be influenced by the differentiation status.
It has been documented that the transcription factors NF-kB and AP-1 are redox-sensitive. Thus, we wanted to see if both transcription factors could constitutively be activated. Also, is the expression propensity of these transcription factors modulated by the oxidative stress status of these HCC cells? Using EMSA and supershift techniques, we demonstrated that varying degrees of expressions of both transcription factors can be seen, with an order of expression propensity of SK-Hep-I > Mahlavu > J5 > Hep3B > HepG2. Having known that both NF-kB and AP-1 could modulate a group of metastasis-related gene expression, we then investigate if the constitutive metastatic potential of these HCC cells can be varied depending upon how extensive the expression propensity of both NF-kB and AP-1, we used a panel of markers for evaluating the metastatic potential, namely: Matrix metalloproteinase (MMP), interleukin 8 (IL-8) and an adhesion molecular E-cadherin. Using both activity and duplex RT-PCR methods, we demonstrated that only a poorly differentiated HCC cells were capable of expressing MMPs (SK-Hep-I predominately expressed a large amount of MMP-9 and mRNA; Mahlavu predominately expressed MMP-2 along with a trace amount of MMP-9). HepG2, Hep3B and J5 were completely devoid of MMP expression. Using ELISA assay, we measured the secretion of IL-8 in the culture media by these HCC cells and demonstrated that the propensity of secretion having an order of SK-Hep-I > HepG2 > Hep3B (J5 and Mahlavu expressed only a trace amount). Next, we used western blotting and duplex RT-PCR techniques to demonstrate that the expressions of E-cadherin were predominately existed in only well-differentiated cell lines, HepG2 and Hep3B. J5, Mahlavu and SK-Hep-I were all devoid of expression. Finally, for the purpose of demonstrating that the intracellular oxidative stress status does have a role in regulating the above-mentioned metastasis-related gene expression, we transfected g-GCSh cDNA to SK-Hep-I cell and obtained a cell type, termed GCS 30, in which its g-GCSh activity, mRNA and GSH content has been proved to be higher than its untransfected counterpart, SK-Hep-I. We then measured the oxidative stress status of GCS 30 using DCFDA-flowcytometric method. From our data, we did demonstrate that the oxidative-stress status of GCS 30 was shown to be decreased as compared to its counterpart. However, we were surprised to find out that GSH/GSSG ratio remained unchange in GCS-30 as compared to SK-Hep-I cells. Using EMSA technique, we showed that GCS 30 cells only exhibited relative strong binding activity to NF-kB, but not for AP-1 binding. Surprisingly, we found that the MMPs activities in GCS 30 cells were relatively comparable to SK-Hep-I, indicating that MMP expression might be regulated by a pathway other than AP-1. The mechanism(s) underlying this observed phenomenon await further clarification.

目錄

緒論 1

第一章分化程度不同的HCC其總內源性氧化還原狀態
之比較
前言 7
材料與方法 13
結果 18
討論 30


第二章細胞內源性氧化還原狀態與腫瘤轉移相關因子
表現之探討
前言 31
材料與方法 40
結果 46
討論 57

第三章降低肝癌細胞株氧化壓力，觀察其對AP-1,NF-kB轉
錄因子及type IV collagenase之影響
前言 60
材料與方法 61
結果 69
討論 87
總結論 89
總表 90
參考文獻 92
附錄 100
論文發表 103

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