(Ⅰ) γ-麩胺醯轉移酶之特異型作為人類肝細胞癌診斷標記之研究 (Ⅱ) 抑制γ-麩胺醯轉移酶活性影響癌細胞對氧化壓力 和化療感受性之研究

Abstract

γ-麩胺醯轉移酶 (GGT)為一細胞膜結合的醣蛋白，主要功能是分解細胞外的glutathione (GSH)生成半胱胺酸，半胱胺酸可以進入細胞內作為合成GSH的原料。當肝細胞發生損傷時，GGT被釋出造成血液中GGT活性的上升，臨床上是阻塞性黃疸或膽汁鬱積的診斷標記。 本論文的第一部份是有關GGT作為輔助肝癌診斷血清標記的探討。由於肝癌的臨床血清學檢驗是依靠甲型胎兒蛋白的升高來做早期診斷，但是甲型胎兒蛋白診斷的敏感性及特異性都不夠理想，因此本研究擬探討在血液中是否有肝癌相關的GGT特異型可以作為輔助的診斷標記，探討對象包括曾在肝癌組織被發現的GGT mRNA特異型V3，以及血清蛋白電泳中肝癌特有的GGT蛋白質特異型。發現在臨床肝癌血清中沒有偵測到任何mRNA，只有在新鮮抽取的血清能偵測到GAPDH mRNA，可能由於血清中RNA不穩定以及mRNA含量極低。在HepG2肝癌細胞偵測到V3 mRNA，適合作為研究GGT mRNA在肝癌中表現的細胞模式，另外，在蛋白質層次利用非變性膠片電泳將血清蛋白質分離後進行GGT 活性染色，發現肝相關疾病血清相較於正常人，在post-γ和β位置有特異型，本研究認為肝癌特異型應為在β位置的GGT。過去在不同研究由電泳結果所定義出的GGT特異型都不同，本研究中證實GGT蛋白質唾液酸化以及GGT與血清中其他大分子成份，如脂蛋白、脂質…等的結合，都是改變其在電泳中的移動速度而顯現不同特異型的原因，此外本研究發現將唾液酸切除並不會影響GGT酵素的活性。由於過去瞭解fucosyltransferase在肝癌中表現上升，使許多醣蛋白發生fucosylation，因此本研究利用一種能特異性結合fucose的凝集素(AAL)，來探討肝癌中的GGT是否發生fucosylation，結果並未偵測到GGT與AAL結合，推測可能是在肝癌中，GGT 發生fucosylation的程度極低所致。 本篇論文的第二部份是探討細胞GGT活性對癌症治療的影響。由於GGT與細胞內維持GSH恆定有密切關係，而細胞內GSH能協助細胞抵抗氧化壓力及排出毒性物質。過去研究指出癌細胞內常有GGT基因表現量上升的情形，使得癌細胞內GSH含量較正常細胞來得高，利於腫瘤細胞生長和減低藥物毒殺作用，造成治療無效。本研究利用GGT專一性抑制劑GGsTop將GGT 活性抑制，發現抑制GGT能有效減少Huh7細胞內GSH的量，並使之無法抵抗苯醌引起的氧化傷害：添加GSH的組別，苯醌對Huh7 細胞的IC50為113.6 μM，而GGT被抑制的組別IC50降低至77.4 μM，兩者有明顯差異，除此之外，本篇論文中發現Huh7細胞對於Cisplatin有抗性，而當GGT活性被抑制後，會增加Huh7細胞對Cisplatin的敏感性，且在Cisplatin處理下，細胞為了因應藥物刺激而造成GGT基因表現上升。因此我們推測GGT的抑制劑有潛力成為一個癌症治療上藥物的佐劑，藉由抑制GGT活性來增加抗癌藥物毒殺細胞的作用可能成為癌症治療的一種策略。

γ-glutamyltransferase (GGT), a membrane-bound heterodimeric glycoprotein, is abundant in many tissues, including kidney, intestine and liver. GGT plays a key role in glutathione (GSH) homeostasis by breaking down extracellular GSH, which contributes to providing cysteine for intracellular GSH synthesis. Under pathological conditions, when liver cells are damaged, GGT is released to serum. In clinical examination, serum GGT activity is used to detect alcoholism and obstructive jaundice. In the first part of this study, hepatoma cell lines and clinical HCC serum were used to study the differences on GGT between hepatoma and normal, including HCC-specific mRNA variants and protein isoforms, which might be developed as potent diagnostic markers. The results indicated that HepG2 cells express HCC-specific mRNA variant, V3, which might be a valuable cell model for studying the GGT mRNA expression state in HCC. Besides, we use native PAGE and then GGT activity staining to identify the HCC-specific protein isoforms. Different GGT isoforms were found between normal and liver diseases, while β-GGT might be HCC-specific. Owing to the different definitions of HCC-specific isoforms in previous studies, we also demonstrated that sialyation status and interaction with other components in serum contributed to different GGT electrophoresis mobility. However, the sialic acid content on GGT showed no correlation to its activity. On the other hand, in lectin extraction experiment using fucose-specific AAL (Aleuria aurantia), we found that fucosylation on GGT might be minor but needs further study. The second part of this study, we used an effective GGT inhibitor, GGsTop, to investigate the effect of GGT inhibition on chemotherapy efficacy. Previous researches indicated that GSH can maintain intracellular redox potential and participate in the detoxification of xenobiotic compounds. As the result of the important role of GGT in maintaining GSH homeostasis, cancer cells were found to exhibit higher GGT level to assist rapid growth and to resist oxidative stress. We found that the inhibition of GGT activity resulted in the decrease of intracellular GSH content and the failure to defend the oxidative damage induced by benzoquinone (BQ). Moreover, we also demonstrated that GGT mRNA level was elevated after Cisplatin treatment which caused resistance to Cisplatin (IC50 >50 μM ). When GGT was inhibited, Huh7 cells were more sensitive to the cytotoxic action of Cisplatin. Taken together, we concluded that GGT inhibitor might be promising as adjuvant for cancer therapy.