臺灣博碩士論文加值系統

頭頸癌是頭頸部以上除了中樞神經之外所源發之癌症總稱，在全球癌症發生率排名第六，而口腔癌(頭頸癌其中之一)在台灣2107年男性癌症十大死因位居第四。患者早期雖然可以經由手術、化學藥物、放射線進行治療，然而可能因為過晚發現、治療後復發或是產生抗藥性，使預後不佳。過去研究指出，具有自我更新能力的癌症幹細胞與癌症復發和轉移有一定關聯性，然而，目前傳統癌症治療大多只針對已分化的癌細胞，因此常引起癌症復發轉移。雖然目前火紅的標靶治療能夠消滅癌症幹細胞，但標靶治療只針對體內表皮生長因子受體大量表現之患者較有效果，因此，若能針對消滅癌症幹細胞之藥物將是癌症治療一大發現。過去實驗室藉由微陣列分析方式篩選出了胺基胜肽酶A，並且發現此蛋白大量表象與大腸直腸癌之轉移與幹細胞有相關性，接著藉由酵素活性試驗在SIGMA LOPAC 1280平台篩選出了一種藥物compound A，能夠抑制胺基胜肽酶A的活性。先前實驗室的研究發現，compound A可以抑制結腸直腸癌細胞以及乳癌細胞之移動、侵襲及聚球能力，是以，本篇論文我們將探討compound A能否抑制口腔癌(頭頸癌)增生及轉移。透過MTS assay及Clonogenic assay，compound A能夠抑制癌細胞增生，再藉由流式細胞儀分析發現compound A會使細胞週期停滯於G0/G1期，抑制細胞增生，我們發現compound A可能是經由抑制細胞週期調控蛋白(cyclin D)的表現來影響細胞生長；透過migration assay及invasion assay得知compound A會抑制細胞移動及侵襲能力。利用sphere formation assay，發現compound A能造成腫瘤微球體的崩解並抑制癌症幹細胞CD44表現。對於細胞增生、轉移訊息傳遞路徑，先前在大腸直腸癌以及乳癌的結果發現compound A可能透過抑制ERK或是AKT達到調控細胞週期的效果，然而在頭頸癌中，compound A並非藉由上述兩路徑抑制細胞增生與轉移。同時，在動物實驗中移植癌細胞到免疫缺陷小鼠並腹腔注射compound A，觀察腫瘤大小與細胞淋巴轉移情形，推測可能因為給予藥物濃度並未達到抑制癌細胞濃度，因此給予藥物最大劑量之組別並未有效抑制腫瘤增生。最後，藉由MTS assay與電腦軟體計算發現compound A和臨床藥物cisplatin具有協同作用，未來可藉由兩者藥物進行合併進一步實驗。綜合以上實驗結果，可得知compound A在細胞實驗能抑制癌症細胞生長、移動，但在體內試驗仍需調高藥物濃度或是與cisplatin進行合併治療。

Head and neck cancer, a general cancer that starts from head and neck, except the central nervous system, is the sixth cancer occurrence all over the world. According to the latest epidemiological statistics from the Ministry of Health and Welfare, the oral cancer, head and neck cancer, is the fourth in the causes of cancer death for males in Taiwan. Most oral cancer patients can be treated by surgery, chemotherapy, or radiation therapy. However, the patients might have poor prognosis due to drug resistance and relapse. Previous studies indicated that cancer metastasis and relapse correlatewith cancer stem cells (CSCs), with self-renewal ability. However, traditional cancer therapies only inhibit differentiated cancer cells, and thus often cause cancer recurrence and metastasis. Although it is able to kill cancer stem cells, the target therapies are more effective in patients with epidermal growth factor receptors (EGFR) overexpression. Therefore, development of a novel drug to eliminate CSCs will be a major task of cancer treatment. Aminopeptidase A (ENPEP), a protein has association with cancer metastasis and stemness, was found overexpressed in colorectal cancerby microarray analysis. By enzyme inhibition assay, SIGMA LOPAC 1280 compound library was screened and compound A was found as a potent inhibitor of aminopeptidase A. According to previous studies, Compound A was shown to inhibit cell migration, invasion and sphere formation ability in colon cancer cells and breast cancer cells. Therefore, we tried to investigate the effects on head and neck cancer cells by compound A. Through MTS assay and clonogenic assay, we found that compound A can inhibit oral cancer proliferation, and compound A can arrest cell cycle at G0/G1 by flow cytometry. Furthermore, cyclin D which controls the cell cycle was downregulated with compound A treatment by Western blot. Our data showed that the migration and invasion ability were inhibited by compound A treatment. In sphere formation assay, the level of CD44 on cancer stem cell was downregulated and sphere break-up by compound A treatment. Next, we tried to identify which signal pathways involved in cancer cell proliferation and metastasis targeted by compound A treatment. Our previous studies with colorectal and breast cancer cells found that PI3K / AKT / mTOR and MAPK / ERK1 / 2 signal pathways were targeted by compound A to inhibit cancer cell proliferation and metastasis. However, our data showed that both pathways may not affect by compound A in head and neck cancer cells. Our animal experiments with cancer xenograft combined with compound A treatment did not see significant therapeutic effects. Finally, we observed that compound A exerted drug synergy with cisplatin. In conclusion, our data evidenced that compound A inhibits cell proliferation, migration in vitro and also inhibits cancer stemness properties. Yet the mechanisms of compound A and effective in vivo treatment dose need further studies.

致謝 i
中文摘要 iii
Abstract v
目錄 vii
圖目錄 ix
第一章 、緒論....1
第一節 、頭頸癌 (Head and neck cancer)....1
第二節 、細胞週期 (Cell cycle)....3
第三節 、癌症幹細胞 (Cancer stem cell, CSC).....4
第四節 、頭頸癌訊息傳遞路徑......5
第五節 、Cisplatin......6
第六節 、Aminopeptidase A inhibitor....6
第七節 、研究動機與目的.7
第二章 、材料與方法.....9
第一節 、實驗材料.......9
第二節 、實驗方法.......21
第三章 、實驗結果.......31
第一節 、Compound A 對 SAS 細胞與 SAS-LN 細胞生長之影響...31
第二節 、Compound A對細胞株細胞週期之影響.......31
第三節 、Compound A 抑制 SAS 細胞與 SAS-LN 細胞移動能力...32
第四節 、Compound A 抑制SAS-LN細胞之侵襲能力....33
第五節 、Compound A 抑制癌症幹細胞標的之表現....34
第六節 、Compound A對細胞訊息傳遞之影響.........35
第七節 、不同劑量compound A對BALB/c小鼠體重與肝臟大小之影響......36
第八節 、不同劑量compound A對種植SAS-LN細胞的裸鼠其腫瘤大小與淋巴轉移之影響.....37
第九節 、Compound A與臨床藥物cisplatin進行藥物協同測試....38
第四章 、實驗討論.......40
第五章 、參考文獻.......47
第六章 、實驗結果圖表...54
第七章 、附錄...........78

圖目錄
FIGURE 1........54
FIGURE 2........56
FIGURE 3........59
FIGURE 4........62
FIGURE 5........63
FIGURE 6........67
FIGURE 7........69
FIGURE 8........70
FIGURE 9........75

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