臺灣博碩士論文加值系統

臨床上常用腫瘤標誌 ( Tumor marker ) 的濃度高低來預測病人的預後及追蹤病情。鱗狀上皮細胞癌抗原 ( Squamous Cell Carcinoma Antigen, SCCA ) 是目前較具臨床應用價值的腫瘤標誌之ㄧ。根據臨床的診斷，子宮頸癌患者之 SCCA 指數偏高者，通常較有不良的預後結果。在我們先前研究結果發現 SCCA 的高表達量，對於化學藥物會產生抗性，例如：藜蘆醇 ( Resveratrol ) 及含鉑化合物 ( Cisplatin )。但是SCCA在腫瘤細胞中所扮演的角色及分子機制仍不是很清楚。有研究指出，癌細胞生長速度遠大於正常細胞，其能量來源也偏好利用醣解作用取代正常細胞的有氧循環，此一現象被稱為〝瓦氏效應〞。醣解作用的產物會導致乳酸的堆積，進而造成腫瘤細胞外環境的酸化。也有文獻指出，腫瘤細胞外環境的酸化會促進腫瘤的生成和轉移。在我們過去的研究發現， SCCA會去抑制溶解體蛋白酶酵素 L 的活性 ( Cathepsin L ) 並且可以拮抗癌細胞的死亡。在本次的研究目的發現，癌細胞處在弱酸的環境下，會促使SCCA表現量增加， 對於化學藥物如藜蘆醇 ( Resveratrol ) 及含鉑化合物 ( Cisplatin ) 也會產生抗性。然而酸性環境會引起活性氧化物 ( Reactive oxygen species, ROS ) 的產生，進而引起 MAPK 分子的訊息傳遞以及轉錄因子CREB的磷酸化。我們將癌細胞處理在弱酸的環境下，同時加入活性氧清除劑，可以抑制 SCCA 表現量也可以看到轉錄因子 CREB 的磷酸化會降低。我們利用軟體分析 SCCA1 啟動子結合位區域，發現有轉錄因子CREB 的結合位。我們預測酸可能透過活性氧化物的增加，促進轉錄因子 CREB 的磷酸化進而增加轉錄的能力。由以上結果可知，癌細胞處在弱酸的環境下對於化學藥物產生抗性，可能是透過活性氧化物 ( ROS ) 將轉錄因子CREB 的磷酸化進而促使SCCA表現量增加。

In clinical, tumor markers can be use to help cancer diagnosis , predict the patient’s response to particular therapies and follow-up disease. The squamous cell carcinoma antigen (SCCA) is a tumor marker and has a clinical value. In clinical diagnosis, patients with higher expression of SCCA show poor prognosis. However, SCCA in clinical use was more than 20 years, but the biological function and mechanism still remain unclear. Our previous studies, we found that overexpression of SCCA led to drug resistance, such as RSV (Resveratrol) and Cisplatin. Cancer cells usually grow faster than normal cells. In contrast to normal differentiated cells, which rely primarily on mitochondrial oxidative phosphorylation to generate the energy needed for cellular processes, most cancer cells instead rely on aerobic glycolysis, a phenomenon termed the “Warburg effect”. Many cancer cells consume glucose and produce lactic acid and cause acidification of the tumor extracellular environment. Acidification of the tumor extracellular environment may promote tumor formation and metastasis. In our previous studies showed the SCCA is a lysosomal proteases inhibitor and could inactivate cathepsin L and anti-cell death. This study we show the cancer cells cultured in low pH (Acidosis) media, increase SCCA expression. The cancer cells culture in low pH (Acidosis) media display drug resistance of RSV (Resveratrol) and Cisplatin. The acidosis enhances the formation of reactive oxygen species (ROS) and induces MAPK signaling pathway and transcription factor, CREB phosphorylation. The cancer cells treated with ROS scavenger reduce SCCA expression and CREB phosphorylation in acidosis condition. We found that, there is a CREB binding region in the promoter region of SCCA1 gene. These results suggested that acidosis increased phosphorylation of the transcription factor CREB via ROS, and them increased its transcriptional activity. Taken together, we suggest cancer resist drug-induced cell death in acidosis condition is through ROS-increased CREB transcriptional activity to promote SCCA1 gene expression .

考試合格證明........................................... I
中文摘要 .............................................. II
Abstract ............................................. IV
誌謝 .................................................. VI
總目錄 ................................................ VIII
圖目錄................................................. XI
縮寫 .................................................. XII
第一章 緒論 ............................................ 1
1. 鱗狀上皮細胞癌抗原
1.1 臨床意義 .......................................... 1
1.2 鱗狀上皮細胞癌抗原簡介.............................. 1
1.3 生物功能性 ........................................ 2
2. 腫瘤的微環境
2.1 腫瘤的形成......................................... 3
2.2 瓦氏效應 ( Warburg effect ) 與腫瘤的關係............ 3
2.3 腫瘤的酸性環境..................................... 4
3. 活性氧化物 ( Reactive oxygen species, ROS )
3.1 活性氧化物 ( ROS ) 簡介.............................. 5
3.2 活性氧化物 ( ROS ) 與生物體的代謝關係.................. 6
第二章 研究目的 ......................................... 8
第三章 材料與方法 ........................................ 10
一、 實驗材料
1.1 引子 ( Primer ) ................................. 10
1.2 抗體 ( Antibody ) ................................ 10
1.3 細胞株 ( Cell line ) ............................... 11
1.4 化學藥品及試劑組 ( Chemical reagent and assay kit )... 11
1.5 緩衝液 ( Buffer ) .................................. 14
二、 實驗方法
2.1 細胞株培養 ( Cell culture ) ......................... 19
2.2 細胞數目測定 ( Cell count ) ......................... 19
2.3 細胞冷凍儲存 ( Cell freezing ) ...................... 21
2.4 細胞實驗培養條件 ( Condition medium ) ................ 21
2.5 生物晶片分析 ( Microarray ) ......................... 22
2.6 西方墨點法 ( Western blot analysis ) ................ 22
2.7 互補DNA（Complementary DNA, cDNA ）................. 23
2.8 聚合酶鏈鎖反應 ( Polymerase chain reaction, PCR ) ... 24
2.9 即時半定量聚合酶鏈鎖反應 ( Real time - PCR ) .......... 25
2.10 細胞存活測試 ( Cell Viability ) .................... 25
2.11 細胞免疫染色 ( Cell staining ) ..................... 26
2.12 免疫組織化學染色 ( Immunohistochemistry assay, IHC ) 27
2.13 流式細胞儀分析 ( Flow Cytometry analysis ) ......... 28
第四章 結果 ............................................. 29
一、HeLa cell 在弱酸環境下會誘導 SCCA1 與 SCCA2 基因的表現.. 29 二、HeLa cell 培養在弱酸環境下對於藥物會產生抗性............ 30
三、Lactic acid ( LA ) 和 L(+)-Lactic acid ( L ) 誘導 SCCA1
和SCCA2 蛋白質表現 .................................. 31
四、 HeLa cell 在弱酸環境下會引起活性氧化物 ( ROS ) 的形成...32
五、 弱酸環境下會增加 CREB 的磷酸化 ....................... 33
第五章 結論 ............................................. 34
第六章 討論 ............................................. 36
參考文獻 ................................................ 40
圖表 ................................................... 49

Abe H, Okuno N, Takeda O, Suminami Y, Kato H, Nakamura K. (1994) Analysis on heterogeneity of squamous cell carcinoma antigen by two-dimensional electrophoresis. Electrophoresis. 15, 988-991.

Assaily W, Benchimol S. (2006) Differential utilization of two ATP-generating pathways is regulated by p53. Cancer Cell. 10, 4-6.

Barnes RC, Worrall DM. (1995) Identification of a novel human serpin gene; cloning sequencing and expression of leupin. FEBS Lett. 373, 65-65.

Brookes, P. S., Levonen, A. L., Shiva, S., Sarti, P., and Darley-Usmar, V. M. (2002) Mitochondria: regulators of signal transduction by reactive oxygen and nitrogen species. Free Radic Biol Med. 33, 755-764.

Blázquez-Castro A, Carrasco E, Calvo MI, Jaén P, Stockert JC, Juarranz A, Sánz-Rodríguez F, Espada J. (2012) Protoporphyrin IX-dependent photodynamic production of endogenous ROS stimulates cell proliferation. Eur J Cell Biol. 91, 216-223.

Bokoch, G. M., and B. A. Diebold. (2002) Current molecular models for NADPH oxidase regulation by Rac GTPase. Blood 100, 2692-2706.

Chen CH, Lin H, Chuang SM, Lin SY, Chen JJ. (2010) Acidic stress facilitates tyrosine phosphorylation of HLJ1 to associate with actin cytoskeleton in lung cancer cells. Exp Cell Res. 316, 2910-2921.

Chen Z, Lu W, Garcia-Prieto C, Huang P. (2007) The Warburg effect and its cancer therapeutic implications. J Bioenerg Biomembr. 39, 267-274.

Dhup S, Dadhich RK, Porporato PE, Sonveaux P. (2012) Multiple biological activities of lactic acid in cancer: influences on tumor growth, angiogenesis and metastasis. Curr Pharm Des. 18, 1319-1330.

Davelaar EM, van de Lande J, von Mensdorff-Pouilly S, Blankenstein MA, Verheijen RH, Kenemans P.(2008). A combination of serum tumor markers identifies high-risk patients with early-stage squamous cervical cancer. Tumour Biol. 29, 9-17.

Dooley LM, Potts KL, Wilson LD, Cappello ZJ, Bumpous JM. (2011) Treatment outcome in the residually positive neck after definitive chemotherapy and irradiation. Laryngoscope. 121, 1656-1661.

DeClerck K, Elble RC. (2010) The role of hypoxia and acidosis in promoting metastasis and resistance to chemotherapy. Front Biosci. 15, 213-225.

Ewaschuk JB, Naylor JM, Barabash WA, Zello GA. (2004) High-performance liquid chromatographic assay of lactic, pyruvic and acetic acids and lactic acid stereoisomers in calf feces, rumen fluid and urine. J Chromatogr B Analyt Technol Biomed Life Sci. 805, 347-351.

Elaine M. Sillos, Jerry L. Shenep , George A. Burghen , Ching-Hon Pui , Frederick G. Behm , John T. Sandlund . (2001) Lactic acidosis: A metabolic complication of hematologic malignancies. Case report and review of the literature Cancer Volume 92, Issue 9, pages 2237-2246.

Ferrandina G, Macchia G, Legge F, Deodato F, Forni F, Digesù C, Carone V, Morganti AG, Scambia G. (2008) Squamous cell carcinoma antigen in patients with locally advanced cervical carcinoma undergoing preoperative radiochemotherapy: association with pathological response to treatment and clinical outcome. Oncology. 74, 42-49.

Fan Y, Dickman KG, Zong WX. (2010) Akt and c-Myc differentially activate cellular metabolic programs and prime cells to bioenergetic inhibition. J Biol Chem. 285, 7324-7333.

Federico A, Cardaioli E, Da Pozzo P, Formichi P, Gallus GN, Radi E. (2012) Mitochondria, oxidative stress and neurodegeneration. J Neurol Sci.

Gatenby RA, Gawlinski ET, Gmitro AF, Kaylor B, Gillies RJ. (2006) Acid-mediated tumor invasion: a multidisciplinary study. Cancer Res. 66,5216-5223.

Gatenby RA, Gillies RJ. (2004) Why do cancers have high aerobic glycolysis? Nat Rev Cancer. 4, 891-899.

GERSCHMAN R, GILBERT DL, NYE SW, DWYER P, FENN WO. (1954) Oxygen poisoning and x-irradiation: a mechanism in common. Science. 119, 623-626.

Hordijk, P. L. (2006). Regulation of NADPH oxidases: the role of Rac proteins. Circ Res 98, 453-462.

Hsu KF, Huang SC, Shiau AL, Cheng YM, Shen MR, Chen YF, Lin CY, Lee BH, Chou CY. (2007). Increased expression level of squamous cell carcinoma antigen 2 and 1 ratio is associated with poor prognosis in early-stage uterine cervical cancer. Int J Gynecol Cancer. 17, 174-181.

Hashimoto K, Kiyoshima T, Matsuo K, Ozeki S, Sakai H. (2005) Effect of SCCA1 and SCCA2 on the suppression of TNF-alpha-induced cell death by impeding the release of mitochondrial cytochrome c in an oral squamous cell carcinoma cell line. Tumour Biol. 26, 165-172.

Hsu KF, Wu CL, Huang SC, Wu CM, Hsiao JR, Yo YT, Chen YH, Shiau AL, Chou CY. (2009) Cathepsin L mediates resveratrol-induced autophagy and apoptotic cell death in cervical cancer cells. Autophagy. 5, 451-460.

Hanahan D, Weinberg RA. (2000) The hallmarks of cancer. Cell. 100, 57-70.

Halliwell, B. (1991). Reactive oxygen species in living systems: source, biochemistry, and role in human disease. Am J Med. 91, 14S-22S.

Halliwell B. (1992) Oxygen radicals as key mediators in neurological disease: fact or fiction? Ann Neurol. 32,10-5.

Heimburger O, Mujais S. (2003) Buffer transport in peritoneal dialysis. Kidney Int Suppl. S37- 42. Review.

Ji AR, Ku SY, Cho MS, Kim YY, Kim YJ, Oh SK, Kim SH, Moon SY, Choi YM. (2010) Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage. Exp Mol Med. 42, 175- 186.

Kato H, Torigoe T. (1977) Radioimmunoassay for tumor antigen of human cervical squamous cell carcinoma. Cancer. 40, 1621-1628.

Kato H, Kanematsu M, Tanaka O, Mizuta K, Aoki M, Shibata T, Yamashita T, Hirose Y, Hoshi H. (2009) Head and neck squamous cell carcinoma: usefulness of diffusion-weighted MR imaging in the prediction of a neoadjuvant therapeutic effect. Eur Radiol. 19, 103-109.

Kopfstein L, Christofori G. (2006) Metastasis: cell-autonomous mechanisms versus contributions by the tumor microenvironment. Cell Mol Life Sci. 63, 449-468.

Kang D, Hamasaki N. (2005) Alterations of mitochondrial DNA in common diseases and disease states: aging, neurodegeneration, heart failure, diabetes, and cancer. Curr Med Chem. 12,429-441.

Koh YI, Choi IS. (2002) Lactic acidosis associated with the usual theophylline dose in a patient with asthma. Korean J Intern Med. 17, 147- 149.

Murakami A, Suminami Y, Hirakawa H, Nawata S, Numa F, Kato H. (2001) Squamous cell carcinoma antigen suppresses radiation-induced cell death. Br J Cancer. 84, 851-858.

Matsuzawa, A., and H. Ichijo. (2005) Stress-responsive protein kinases in redox-regulated apoptosis signaling. Antioxid Redox Signal 7, 472-481.

Omole OO, Brocks DR, Nappert G, Naylor JM, Zello GA. (1999) High-performance liquid chromatographic assay of (+/-)-lactic acid and its enantiomers in calf serum. J Chromatogr B Biomed Sci Appl. 727, 23-29.

Omole OO, Nappert G, Naylor JM, Zello GA. (2001) Both L- and D-lactate contribute to metabolic acidosis in diarrheic calves. J Nutr. 131, 2128-2131.

Ohashi K, Pao W. (2011) A new target for therapy in squamous cell carcinoma of the lung. Cancer Discov.1,23-24.

Riemann A, Schneider B, Ihling A, Nowak M, Sauvant C, Thews O, Gekle M. (2011) Acidic environment leads to ROS-induced MAPK signaling in cancer cells. PLoS One. 6, 22445.

Sakaguchi Y, Kishi F, Murakami A, Suminami Y, Kato H. (1999) Structural analysis of human SCC antigen 2 promoter. Biochim Biophys Acta. 1444, 111-116.

Schneider SS, Schick C, Fish KE, Miller E, Pena JC, Treter SD, Hui SM, Silverman GA. (1995) A serine proteinase inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene. Proc Natl Acad Sci U S A. 92, 3147-3151.

Shayan R, Achen MG, Stacker SA. (2006) Lymphatic vessels in cancer metastasis: bridging the gaps. Carcinogenesis. 27, 1729-1738.

Suminami Y, Kishi F, Sekiguchi K, Kato H. (1991) Squamous cell carcinoma antigen is a new member of the serine protease inhibitors. Biochem Biophys Res Commun. 181,51-58 .

Shimada H, Nabeya Y, Okazumi S, Matsubara H, Shiratori T, Gunji Y, Kobayashi S, Hayashi H, Ochiai T. (2003) Prediction of survival with squamous cell carcinoma antigen in patients with resectable esophageal squamous cell carcinoma. Surgery. 133, 486-494.

Stubbs M, McSheehy PM, Griffiths JR, Bashford CL. (2000) Causes and consequences of tumour acidity and implications for treatment. Mol Med Today. 6,15-19.

Schornack PA, Gillies RJ. (2003) Contributions of cell metabolism and H+ diffusion to the acidic pH of tumors. Neoplasia. 5,135-145.

Santos CX, Tanaka LY, Wosniak J, Laurindo FR. (2009) Mechanisms and implications of reactive oxygen species generation during the unfolded protein response: roles of endoplasmic reticulum oxidoreductases, mitochondrial electron transport, and NADPH oxidase. Antioxid Redox Signal. 11, 2409-9427.

Tsuchiya Y, Noguchi T, Matsumoto K, Matsumura T, Hoshi K, Ito H, Osano H, Jinbu Y, Kusama M. (2009) Clinical effects of concurrent chemoradiotherapy (hyperfractionation and cisplatin/5-fluorouracil) for patients with advanced oral squamous cell carcinoma. Gan To Kagaku Ryoho. 36, 101-103 .

Takeda A, Kajiya A, Iwasawa A, Nakamura Y, Hibino T. (2002) Aberrant expression of serpin squamous cell carcinoma antigen 2 in human tumor tissues and cell lines: evidence of protection from tumor necrosis factor-mediated apoptosis. Biol Chem. 383, 1231-1236.

Trédan O, Galmarini CM, Patel K, Tannock IF. (2007) Drug resistance and the solid tumor microenvironment. J Natl Cancer Inst. 99, 1441-1454.

Uribe P, Gonzalez S. (2011) Epidermal growth factor receptor (EGFR) and squamous cell carcinoma of the skin: molecular bases for EGFR-targeted therapy. Pathol Res Pract. 207, 337-342.

Vander Heiden MG, Cantley LC, Thompson CB. (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science.324,1029 - 1033.

Vaupel P, Kallinowski F, Okunieff P. (1989) Blood Flow, Oxygen and Nutrient Supply, and Metabolic Microenvironment of Human Tumors: A Review1 Cancer Res. 49, 6449-6465.

Witz IP, Levy-Nissenbaum O.(2006)The tumor microenvironment in the post-PAGET era. Cancer Lett. 242, 1-10.

WARBURG O. (1956) On the origin of cancer cells. Science. 123, 309- 314.

Yun J, Rago C, Cheong I, Pagliarini R, Angenendt P, Rajagopalan H, Schmidt K, Willson JK, Markowitz S, Zhou S, Diaz LA Jr, Velculescu VE, Lengauer C, Kinzler KW, Vogelstein B, Papadopoulos N. (2009) Glucose deprivation contributes to the development of KRAS pathway mutations in tumor cells. Science. 325, 1555-1559.