臺灣博碩士論文加值系統

烷化劑至今仍為癌症化學治療的第一線藥物，例如oxaliplatin用於治療大腸癌，cisplatin用於治療肺癌及膀胱癌，mechlorethamine用於治療多發性骨髓瘤。氮芥子屬於烷化劑成員之一，在現今癌症化學治療上不論是單獨使用還是合併其它藥物皆有很重要的角色，例如治療何金杰氏症所使用的合併治療法MOPP（併用mechlorethamine、oncovin、procarbazine以及 prednisone）。氮芥子烷化劑不僅會和癌細胞的DNA作用，也容與其它蛋白質或正常細胞DNA上陰電性強的基團形成共價鍵結，因此造成許多副作用，如嗜中性白血球減少及骨髓抑制，成為氮芥子烷化劑開發成抗癌藥物的一大障礙。為了改善氮芥子烷化劑水溶性不佳及不良作用，中央研究院 蘇燦隆 教授設計合成一系列的芥子氮化合物，將氮芥子以urea作為連結橋，與水溶性之N-phenyl ω-dialkylaminolalkylamide 或 ω-cyclic aminoalkylamide基團連結。本論文利用 alamarBlue®，進一步證明這一系列化合物具有抗人類大腸癌細胞生長之活性。在這些化合物中，BO-2094展現之抗大腸癌細胞效果較強，因此更進一步探討其在異種移植動物模式腫瘤抑制的效果。實驗結果發現BO-2094很顯著地抑制HCT-116及H3347異種移植動物模式的腫瘤生長。此外BO-2094與5-FU合併使用在人類大腸癌細胞HCT-116具有協同細胞毒性（synergistic cytotoxicity）；在HCT-116異種移植動物實驗模式中也證明了HCT-116合併5-FU的治療可以很明顯地抑制腫瘤生長。透過TUNEL assay證實，BO-2094可經由細胞凋亡路徑造成大腸癌細胞死亡而抑制腫瘤生長。透過鹼性膠遷移試驗以及改良型單細胞鹼性膠體電泳法，證明這系列的化合物抑制細胞生長的原因可能為在DNA上形成連結橋（Cross-link）。單一使用此類化合物會造成人類大腸癌細胞HCT-116細胞週期G2/M 期停滯，而將BO-2094合併5-FU會延緩G2/M以及S細胞週期的進行。綜合所有實驗結果可以證明BO-2094極具更進一步往臨床前研究發展的潛力。

Numerous alkylating agents are one of the major classes of frontline chemotherapeutic drugs, such as oxaliplatin against colorectal cancer, cisplatin against lung and bladder cancers and mechlorethamine against multiple myeloma. Nitrogen mustard, the earliest member of alkylating agent family, still plays a key role in treatment of patients with Hodgkin’s disease and multiple myeloma. Alkylating agents may be used alone or in combination with other chemotherapeutic agents, such as MOPP (mechlorethamine combine with oncovin, procarbazine, and prednisone) for Hodgkin’s disease. Since alkylating agents in general non-selectively react and irreversibly bind to DNA as well as protein’s thiols, many unpleasant side effects, such as neutropenia and myelosuppression limit the use of alkylating agents in clinic. To resolve these problems, new series of water-soluble N-mustards link with various hydrophilic N-phenyl ω-dialkylaminolalkylamide or ω-cyclic aminoalkylamide moieties via urea linker were designed and synthesized by Dr. Tsann-Long Su (IBMS of Academia Sinica). DNA-affinic molecule can help compound to be more water soluble and bring compound to target site, and urea linker can reduce the chemical reactivity of N-mustard moiety. Using alamarBlue® cell viability assay, it was found that human colorectal cancer cell lines were relatively more sensitive to these water-soluble N-mustards as compared to other cancer cell lines. Among them, anticancer activity of BO-2094 was evaluated in nude mice bearing HCT-116 or H3347 xenografts. The results showed that BO-2094 given 30 mg/kg for 5 days significantly suppressed the tumor growth of HCT116 xenograft animal model. Furthermore, there is synergistic antitumor activity of BO-2094 in combination with 5-FU in human colon cancer HCT-116 cells. Significant anticancer activity of BO-2094 combined with 5-FU in nude mice bearing HCT-116 xenografts were also observed. Using TUNEL assay improved that BO-2094 could lead to cell apoptosis of human colorectal cancer tissue. Through alkaline agarose gel shift assay and modified comet assay, it was confirmed the potent activity of water-soluble N-mustards on induction of DNA interstrand cross-links. Meanwhile, G2/M arrest in HCT-116 cells treated with these newly synthesized water-soluble N-mustards was observed, whereas HCT-116 cell was arrested at the G2/M and S phase by treatment in combination with BO-2094 and 5-FU. These results implicate that BO-2094 warrants further development and preclinical studies.

目 錄

論文電子檔著作權授權書…………………………………… i
論文審定同意書……………………………………………… ii
誌謝………...…………………………………...……………. iii
英文摘要…………………….………………………………… iv
中文摘要……………………………….………………..……. vi
目錄…………………………………………………………… viii
圖目錄………………………………...……………...………. xi
表目錄……………………………………...…………………. xi
第一章 緒論…………………………………………………………… 1
1. 大腸癌...……………………………………...……..…………….....… 1
(1) 大腸癌之危險因子……………………………………….......…..….. 1
(2) 大腸癌之徵兆…………………………........…………….......…..….. 2
(3) 大腸癌之篩檢…………………………............………….......…..….. 2
(4) 大腸癌之進程分期…………………………........……….......…..….. 3
(5) FDA許可大腸癌使用藥物介紹………………….……..........…..….. 4
(6) 大腸癌之治療……………............................…….…...….......…..….. 5
2. 烷化劑………………….……….......…..…........................................... 7
3. 水溶性phenyl N-mustards benzene conjugates設計概念..................… 8

第二章 研究目的…………………………………………………………….. 10

第三章 材料與方法………………………………………………………….. 11
1. 細胞培養...………………………………………….……………...… 11
(1) 細胞株..........…… ..…..…………………………….…….......…..… 11
(2) 細胞培養液..…….…..…………………………………..........…..… 12
(3) 細胞培養條件…...…..…………....……………………..........…..… 12
(4) 細胞繼代培養..……...…………………………………..........…..… 12
2. 細胞毒性檢測...…………………………………..………...……...… 13
(1) 單一藥物對不同細胞之毒性檢測…………..…………...…....…..… 13
(2) BO-2094合併5-FU物對不同細胞之毒性檢測……...…...........…..… 14
3. 異種移植動物模式探討…………………...…….....……………...… 15
(1) 實驗動物飼養................................…………..…..………….....…..… 15
(2) 大腸癌細胞收集............................…………..…..………….....…..… 15
(3) 大腸癌細胞移植............................…………..…..………….....…..… 16
(4) 藥物給予........................................…………..…..………….....…..… 16
(5) 腫瘤大小測量................................…………..…..………….....…..… 16
4. 腫瘤組織細胞凋亡分析…………………...…….....……………...… 17
5. 鹼性凝膠遷移試驗.........…………………...…….....……………...… 18
6. 改良型單細胞膠體電泳法..………………...…….....……………...… 19
7. 細胞週期分析......................………………...…….....……………...… 21
8. 統計方法..............................………………...…….....……………...… 21

第四章 結果…………………………………………………………………. 22
1. 水溶性phenyl N-mustard benzene conjugates有抑制人類大腸癌細胞生
長之效果...................................…………………….……………...… 22
2. BO-2094與5-FU併用對於人類大腸癌細胞具有協同細胞毒性
（Synergistic effect）.............………………....…….……………...… 22



3. 水溶性phenyl N-mustards benzene conjugates可抑制人類大腸癌細胞
HCT-116及H3347之異種移植裸鼠腫瘤生長，其中以 BO-2094最
佳...........................…….............................…....…….………...…...… 23
4. BO-2094合併5-FU可有效地抑制人類大腸癌細胞HCT-116之異種移
植裸鼠腫瘤生長...............................………....…….……………...… 25
5. BO-2094單獨治療或合併5-FU治療抑制人類大腸癌細胞 HCT-116之
異種移植腫瘤生長的原因可能為造成癌細胞 走向細胞凋亡（
Apoptosis）...................………………....…….........……………...… 26
6. 水溶性phenyl N-mustards benzene conjugates抑制人類大腸 癌細胞生
長的機轉可能為在DNA雙股形成interstrand cross-links..…..…...… 27
7. BO-2094單獨處理會延緩人類大腸癌細胞週期G2/M期的進行，合
併5-FU處理會延緩細胞週期S及G2/M期的進行................……...… 29

第五章 討論………………………………………………………………….. 30

圖…………..………………………………………………………………….. 36

表…………....……………………………………………………………….... 48

參考文獻………......………………………………………………………….. 49

附表………………..………………………………………………………….. 55




圖目錄

圖1. BO-2094合併5-FU處理後協同作用探討 ………………………..…… 36
圖2. BO-2094與BO-2095以及BO-1055對於HCT-116異種移植裸鼠腫瘤之抑
制效果 ……………………………………………………….………… 37
圖3. BO-2094對於HCT-116異種移植裸鼠腫瘤之抑制效果............……… 38
圖4. BO-2094對於H3347異種移植裸鼠腫瘤之抑制效果………………… 39
圖5. BO-2094合併5-FU對於HCT-116異種移植裸鼠腫瘤之治療效果...… 40
圖6. BO-2094合併5-FU對於HCT-116異種移植裸鼠腫瘤之治療效果...… 41
圖7. BO-2094治療後，HCT-116異種移植裸鼠腫瘤細胞凋亡探討........… 42
圖8. BO-2094及BO-2095在DNA上形成連結橋之探討...........................… 43
圖9. BO-2094在HCT-116細胞內DNA上形成連結橋之探討...................… 44
圖10. BO-2094對人類大腸癌細胞週期的影響.........................................… 45
圖11. 5-FU對人類大腸癌細胞週期的影響................................................… 46
圖12. BO-2094合併5-FU對人類大腸癌細胞週期的影響.........................… 47


表目錄

表1. 水溶性phenyl N-mustards benzene conjugates於不同人類大腸癌細胞之
細胞毒性…………………………………….………………………….... 48

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