研究目的
口腔癌是一種常見的惡性疾病，口腔癌之死亡率並沒有因為治療方法改變而降低，因此找到安全且有效的藥物對這些病人非常重要。Arginine是腫瘤細胞生存非常重要的胺基酸，去除arginine會抑制腫瘤細胞的生長，Arginine deiminase (ADI)可以將arginine代謝成citrulline，而Argininosuccinate synthetase (ASS)是citrulline合成arginine最重要的酵素，因此本研究主要是要探討頭頸部腫瘤細胞株及口腔癌病理切片之ASS表現量以及ADI在口腔癌之治療及應用。
研究方法
藉由腫瘤細胞培養，來分析頭頸部腫瘤細胞中ASS蛋白表現之情形，並藉由免疫組織化學染色，來研究口腔癌病理組織中ASS之表現量，再藉由ADI來探討細胞存活和ASS表現量之相關性，最後我們會收集病人臨床資料，比較ASS表現量和臨床特徵及存活率間之相關性。
主要發現
由Western blot可知，頭頸部腫瘤細胞之ASS表現量並不相同，且口腔癌檢體免疫組織化學染色顯示有56%(41/73)的病人組織ASS表現量相對較低，但ASS的表現量和年紀、性別、腫瘤分期、抽菸、喝酒及嚼檳榔之相關性並沒有達到統計學上顯著差別。ASS表現量較低的病人其overall survival 及disease free survival較ASS表現量比較高的病人好。
結論
頭頸部腫瘤細胞之ASS表現量並不相同，且腫瘤檢體免疫組織染色顯示有些病人ASS表現量較低，ASS的表現量可以當作口腔癌治療效果的生物標記。

Purpose
The prevalence of oral squamous cell carcinoma (OSCC) patients is increasing in Taiwan. Overall survival of these patients did not improved significantly in recent years. It is urgent to identified novel drugs for OSCC. Amino acid deprivation therapy has been proved to be an effective method to treat various kinds of cancers. Argininosuccinate synthetase (ASS) is the rate-limiting enzyme for production of arginine. Arginine deiminase (ADI) can degrade arginine into citrulline. Some murine tumor cells were found to be unable to synthesize arginine due to ASS deficiency. The aim of this study is to investigate the ASS expression level in OSCC specimens and to explore the antitumor potential of ADI for OSCC patients.
Experimental Design
Head and neck cancer cell lines were be cultured and ASS expression level in head and neck cancer cells lines were be confirmed by western blot. OSCC specimens ASS expression levels were be examined by immunohistochemistry (IHC). The ADI antitumor efficacy of head and neck cancer cell lines were be tested.
Results
Head and neck cancer cell lines have different ASS expression level. Forty-one (56%) patients show low ASS expression. The specimens ASS expression level was not significantly different from age, gender, TNM staging, smoking, alcohol consummation and betel nut chewing. The overall survival and disease free survival of low ASS expression group was better than high ASS expression group
Conclusions
Head and neck cancer cell lines and OSCC specimens have different ASS expression levels. Forty-one (56%) patients show low ASS expression level. The increasing expression of argininosuccinate synthetase may be an independent predictive factor for oral squamous cell carcinoma patients with poor prognosis.

American Joint Committee on Cancer staging manual for staging of cancer. 6th ed. Philadelphia: J. B. Lippincott 17-32; 2002

Ascierto PA, Scala S, Castello G, Daponte A, Simeone E, Ottaiano A et al. Pegylated arginine deiminase treatment of patients with metastatic melanoma: results from phase I and II studies. J Clin Oncol 23:7660-7668, 2005.

Agra IMG , Carvalho AL,Ulbrich FS, Campos OD, Martins EP, Magrin J et al. Prognostic factors in salvage surgery for recurrent oral and oropharyngeal cancer. Head Neck 28: 107-113,2006.

Boyle P, Macfarlane G, Maisonneuve P, Zeng T, Scully C,Tedesco B. Epidemiology of mouth cancer in 1989. A review.J Royal Soc Med 83:724–730, 1990.

Bornstein S, White R, Malkoski S, Oka M, Han G, Cleaver T et al. Smad4 loss in mice causes spontaneous head and neck cancer with increased genomic instability and inflammation. J Clin Invest 119:3408–3419, 2009.

Cheng PNM, Lam TL, Lam WM, Tsui SM, Cheng AWM, Lo WH et al. Pegylated recombinant human arginase (rhArg-peg5,000mw) inhibits the in vitro and in vivo proliferation of human hepatocellular carcinoma through arginine depletion. Cancer Res 67:309-317, 2007.

Dillon BJ, Prieto VG, Curley SA, Ensor CM, Holtsberg FW, Bomalaski JS, Clark MA Incidence and distribution of argininosuccinate synthetase deficiency in human cancers Cancer 100:826–833,2004.

Delage B, Fennell DA, Nicholson L, McNeish I, Lemoine NR,Crook T et al. Arginine deprivation and argininosuccinate synthetase expression in the treatment of cancer Int J Cancer 126: 2762–2772, 2010.

Ensor CM, Holtsberg FW, Bomalaski JS, Clark MA. Pegylated arginine deiminase (ADI-SS PEG20,000 mw) inhibits human melanoma and hepatocellular carcinomas in vitro and in vivo. Cancer Res 62:5443–5450, 2002.

Feun L, Savaraj N. Pegylated arginine deiminase: a novel anticancer enzyme agent. Expert Opin Investig Drugs 15:815-22,2006.

Gonzalez GG, Byus CV. Effect of dietary arginine restriction upon ornithine and polyamine metabolism during two-stage epidermal carcinogenesis in the mouse. Cancer Res 51:2932–2939, 1991.

Harvey JM, Clark GM, Osborne CK, Allred DC. Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol 17: 1474–1481, 1999.

Holtsberg FW , Ensor CM, Steiner MR, Bomalaski JS, Clark MA. Poly(ethylene glycol)(PEG) conjugated arginine deiminase:effects of PEG formulations on its pharmacological properties. J Control Release 80:259–271,2002.

Husson A, Brasse-Lagnel C, Fairand A, Renouf S, Lavoinne A. Argininosuccinate synthetase rom the urea cycle to the citrulline-NO cycle. Eur J Biochem 270:1887–1899,2003.

Hunter KD, Parkinson EK, Harrison PR. Profiling early head and neck cancer. Nat Rev Cancer 5:127–135,2005.

Izzo F, Marra P, Beneduce G, Castello G, Vallone P, Rosa VD et al. Pegylated arginine deiminase treatment of patients with unresectable hepatocellular carcinoma: results from phase I/II studies. J Clin Oncol 22:1815-1822,2004.

Johnson NW. Orofacial neoplasm: global epidemiology, risk factors and recommendations for research. Int Dent J 41:365–375,1991.

Ko YC, Huang YL, Lee CH, Chen MJ, Lin LM, Tsai CC.Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan. J Oral Pathol Med 24:450–453,1993.

Kao SY, Chu YW, Chen YW, Chang KW, Liu TY. Detection and screening of oral cancer and pre-cancerous lesions. J Chin Med Assoc 72:227-233,2009.

Kobayashi E, Masuda M, Nakayama R, Ichikawa H, Satow R, Shitashige M et al.Reduced argininosuccinate synthetase is a predictive biomarker for the development of pulmonary metastasis in patients with psteosarcoma. Mol Cancer Ther 9: 535–544,2010.

Lagarde SM, Ver Loren van Themaat PE, Moerland PD, Gilhuijs-Pederson LA, ten Kate FJW, Reitsma PH et al. Analysis of gene expression identifies differentially expressed genes and pathways associated with lymphatic dissemination in patients with adenocarcinoma of the esophagus. Ann Surg Oncol 15:3459–3470,2008.

Lind DS. Arginine metabolism: enzymology, nutrition,and clinical significance. J Nutr 134:2837S–2841S, 2004.

Luiking YC, Hallemeesch MM, van de Poll MC, Dejong CH, de Jonge WJ, Lamers WH et al. Reduced citrulline availability by OTC deficiency in mice is related to reduced nitric oxide production. Am J Physiol Endocrinol Metab 295:E1315-1322, 2008.

Miyazaki K, Takaku H, Umeda M, Fujita T, Huang W, Kimura T, et al. Potent growth inhibition of human tumor cells in culture by arginine deiminase purified from a culture medium of a Mycoplasma-infected cell line. Cancer Res 50: 4522–4527, 1990.

Mehvar R. Modulation of the pharmacokinetics and pharmacodynamics of proteins by polyethylene glycol conjugation. J Pharm Pharm Sci 3:125–136, 2000.

Park IS, Kang SW, Shin YJ, Chae KY, Park MO, Kim MY et al. Arginine deiminase: a potential inhibitor of angiogenesis and tumour growth. Br J Cancer 89:907-914,2003.

Shen LJ, LinWC, Beloussow K, ShenWC. Resistance to the anti-proliferative activity of recombinant arginine deiminase in cell culture correlates with the endogenous enzyme, argininosuccinate synthetase. Cancer Lett 191:165-170,2003.

Szlosarek PW, Klabatsa A, Pallaska A, Sheaff M, Smith P, Crook T et al. In vivo loss of expression of argininosuccinate synthetase in malignant pleural mesothelioma is a biomarker for susceptibility to arginine depletion. Clin Cancer Res 12:7126-7131, 2006.

Takaku H, Takase M, Abe SI. Hayashi H, Miyazaki K. In vivo anti-tumor activity of arginine deiminase purified from Mycoplasma arginini. Int J Cancer 51:244–249, 1992.

Takaku H., Misawa S, Hayashi H, Miyazaki K. Chemical modification by polyethylene glycol of the anti-tumor enzyme arginine deiminase from Mycoplasma arginini. Jpn J Cancer Res 84: 1195–1200, 1993.

Takaku H, Matsumoto M, Misawa S, Miyazaki M. Anti-tumor activity of arginine deiminase from Mycoplasma arginini and its growth-inhibitory mechanism. Jpn J Cancer Res 86: 840–846, 1995.

Tsai WB, Aiba I, Lee SY, Feun L, Savaraj N, Kuo MT. Resistance to arginine deiminase treatment in melanoma cells is associated with induced argininosuccinate synthetase expression involving c-Myc/HIF-1α/Sp4. Mol Cancer Ther 8:3223–3233, 2009.

Wu G, Morris SM, Jr. Arginine metabolism: nitric oxide and beyond. Biochem J 336:1–17,1998.

Xia Y, Zweier JL. Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc Natl Acad Sci 94:6954-6958, 1998.