肝細胞癌及腎細胞癌相關基因，精氨基琥珀酸合成酵素，對生長抑制、誘導細胞週期停止及凋亡之影響

Abstract

摘要 肝細胞癌是在肝臟中所產生的惡性腫瘤，其死亡率在所有癌症中居於首位，全球每年被診斷發現的新病例多達五十萬人次，全球發生率約為十萬人口中有5.5-14.9人被診斷為肝細胞癌，其發生以亞洲及非洲地區為主。目前已知肝細胞癌的危險因子包括有B型肝炎病毒及C型肝炎病毒的感染，另外已知在許多食品中，尤其是堅果及穀類製品相當容易受到黃麴黴菌的感染，而此類黴菌的化學產物，黃麴毒素也是相當重要的致癌因子。即使過去在已有相當大量的研究是針對肝細胞癌的致癌途徑，然而真正可供臨床診斷治療使用的結果卻是相當少量。腎細胞癌是由腎小管上皮細胞病變所衍化而成的癌症，佔所有成人腎臟惡性腫瘤中的百分之九十，發生率則約為所有惡性腫瘤的百分之二。腎細胞癌最好根治的方法, 就是利用手術切除，因為大部分的腎細胞癌對目前的化學藥物治療、荷爾蒙治療及放射線治療都具有抗性，整體而言，五年的存活率低於百分之二十。到目前為止，臨床上還沒有可作為預測診斷或治療預後的分子指標。因此，癌症學者們積極探究造成癌症之基因變化，不但可以增進對於癌症分子病理機轉之認識，更可以藉此發現做為診斷、篩檢以及治療之分子指標。而在本實驗室的蔡家櫸先生及唐賽文先生所建構的全長cDNA庫中發現，精氨基琥珀酸合成酵素基因的轉錄調控在肝細胞癌及腎細胞癌組織中都有受到抑制的現象。 精氨基琥珀酸合成酵素是參與在精氨酸生合成過程中的一個重要的酵素，在肝臟中參與尿素生化循環反應，此循環反應在大多數的哺乳類中是一種排出多餘氨的重要機制。除此之外，由於瓜胺酸-一氧化氮循環的發現，使得精氨基琥珀酸合成酵素躍升為一氧化氮生合成反應中速率限制步驟的重要酵素。一氧化氮的生物學作用具有複雜性和多樣性，在過去的研究證據指出，一氧化氮的生物學作用可能是通過誘導抑癌基因p53蛋白的表現量上升或透過促進抑癌基因p53蛋白的轉譯後修飾，進而殺滅腫瘤或抑制腫瘤生長。在最近的研究顯示，精氨基琥珀酸合成酵素的缺失在不同的腫瘤及組織中都有相當高的發生率。然而，精氨基琥珀酸合成酵素的缺失導致癌症生成中値得研究。 在本研究中發現，在多對肝細胞癌組織及腎細胞癌組織與其週邊正常組織配對樣本中，精氨基琥珀酸合成酵素在腎細胞癌中RNA及蛋白層次都有表現量下降的情形。針對精氨基琥珀酸合成酵素在功能性研究上，發現在人類胚胎腎細胞株，HEK293，和人類腎細胞癌細胞株，786-O，中過度表現精氨基琥珀酸合成酵素，會造成抑癌基因p53的蛋白表現量增加以及促進p53後轉譯修飾的產生，而這樣的現象可能是憑藉著一氧化氮訊息路徑而產生的。此外，在持續表現精氨基琥珀酸合成酵素的細胞株有生長速率被抑制的現象，更進一步地，我們亦發現這種生長抑制的現象可能是透過p53調控的G2細胞週期阻滯或細胞凋亡所引發的。綜合以上的實驗結果，顯示出精氨基琥珀酸合成酵素藉由引發癌細胞死亡或是減緩癌細胞生長速率進而抑制癌症的生成及發展。

Abstracts Hepatocellular carcinoma (HCC) arises from hepatocytes, and is the primary malignant disease of the liver. It is the number one cause of cancer death worldwide. It is especially prevalent in parts of Asia and Africa. Although many studies about HCC have been studied, only fragmentary and incomplete data are available about genomic alterations during the development and progression of HCC in humans. Renal cell carcinoma (RCC), the most common tumor of the kidney in adults, accounts for 2% of all cancers and 90% of malignant kidney tumors. RCC is highly resistant to the most therapy and has poor response to chemotherapy, hormonal therapy and radiation therapy, and is associated with a 5-year survival rate of less than 20%. To date there have been no specific tumor markers available for the differential diagnosis of RCC. The identification of new molecular parameters of HCC or RCC will be necessary in cancer research. In pervious studies, the down-regulated expression of arginiosuccinate sythetase (ASS) was identified by comparing the gene-expression profiles of the full-length cDNA libraries both of HCC/normal liver tissues by Mr. C.C. Tsai, and RCC/normal kidney tissues by Mr. S.W. Tang in our laboratory. ASS is an enzyme that participates in the arginine biosynthetic pathway and contributes to ammonia detoxification in the liver. In addition, discovery of the citrulline– nitric oxide (NO) cycle has increased interest in the key enzyme, ASS that was found to represent a potential limiting step in NO synthesis. Then, the evidences for the link between NO and p53 are shown that p53 accumulation and post-translational modifications induced by NO donor treatment in previous studies. Previous studies have demonstrated that the incidence of ASS deficiency varied greatly with the tumor type and tissue of origin. However, the reason of ASS deficiency is unknown. In this study, the down-regulation expression of ASS mRNA and ASS protein was demonstrated in HCC tissues and RCC tissues by quantitative real-time PCR, Western blot analysis and immunohistochemistry analysis. By functional studies, the tumor suppressor p53 protein was up-regulated and phosphorylated by overexpression of ASS in 786-O cells and HEK cells. Furthermore, the accumulation and modification of p53 protein probably mediated by nitric oxide signaling pathway. On the other hand, the cell viability assay showed that stable expression of ASS protein could decrease the cell growth rate. Furthermore, the decrease of the cell growth could be caused by cell cycle arrest or apoptosis which was mediated by p53 protein in 786-O cells and HEK cells, respectively. Taken together, these results suggested that ASS would cause tumor cell death or its growth repression to suppress tumorgenesis.