乳腺癌在全世界是女性死亡的主要原因，並且化學治療法是治療乳腺癌的主要策略之一，但是化療抗藥性常常會引起乳腺癌患者的預後能力差而導致效果不佳。塑化劑衍生物已經被證明會影響基因的表現，且被認為可能影響細胞對於基因毒性的敏感性和DNA修復的能力。近年來已經證實鄰苯二甲酸酯DEHP暴露跟乳腺癌細胞的化療抗藥性有密切相關。由於DEHP使乳腺癌獲得化療抗藥性的機制尚未清楚，因此在本論文中，我們探討DEHP是否會使乳腺癌產生化療抗藥性。初步研究結果顯示DEHP對於乳腺癌細胞MCF-7會干擾喜樹鹼 (CPT) 所誘導的DNA損傷。更進一步分析發現，以DEHP前處理會顯著降低CPT導致MCF-7細胞內DNA雙鏈斷裂（DSB）標誌，包括γH2AX和ATM受磷酸化的程度。同樣地，斑馬魚異種移殖實驗結果也證實DEHP會干擾CPT誘導的抗乳腺癌之效果。因此，我們根據目前結果推測DEHP可能透過抑制CPT所誘導DNA損傷而增加乳腺癌對CPT的化療抗藥性。

Breast cancer is a leading cause of death in women worldwide, and chemotherapy is one of the primary strategies for breast cancer treatment. However, the chemoresistance often causes the poor prognosis of breast cancer patients. Phthalate derivatives have been shown to affect the expression of genes, and could take effects on genotoxic sensitivity and the ability of DNA repair in cells. Recent evidence suggested that phthalate exposure may be closely correlated with the chemoresistance of breast cancer cells. However, the underlying mechanism that phthalate causes chemoresistance is largely unclear. In the study, we investigated whether phthalates affect the chemoresistance by reinforcing the ability of DNA repair. Our results showed that phthalate Bis(2-ethylhexyl) phthalate (DEHP) attenuates the camptothecin (CPT)-induced DNA damage in breast cancer cells MCF-7. Moreover, phthalate DEHP pre-treatment significantly inhibits the two hallmarks of double-strand breaks (DSB) of DNA, including γH2AX and the phosphorylation of ATM, which were significantly reduced in CPT-treated MCF-7 cells. Consistently, the results of zebrafish xenograft assay confirmed the protective effect of DEHP on CPT-induced cytotoxicity of breast cancer. Accordingly, our present work suggests that phthalate DEHP may affect the anti-breast cancer effect of CPT through attenuating the induction of DNA damage.

國立中山大學研究生學位論文審定書 i
國立中山大學博碩士論文公開授權書 ii
誌謝 iii
中文摘要 iv
Abstract v
縮寫表 ix
中英文對照表 x
第一章、 背景介紹 1
壹、乳腺癌分類 (Classification of breast cancer) 1
貳、乳腺癌之治療和瓶頸 2
參、鄰苯二甲酸酯與乳腺癌治療之關係 2
肆、已知抗藥性( Chemoresistance) 產生的途徑 4
伍、喜樹鹼 (Camptothecin) 背景介紹 6
陸、斑馬魚模式 (Zebrafish model) 介紹 7
柒、研究動機 9
第二章、 材料與方法 10
(一) 藥物來源 (Source of Phthalate and CPT) 10
(二) 細胞株與細胞培養 (Cell lines and cell culture) 10
(三) 篩選藥物濃度以細胞計數分析 (Selection of drug concentration in cell proliferation assay) 11
(四) 細胞內生性活性氧測試 (Intracellular reactive oxygen species generation assay) 11
(五) 彗星試驗 (Comet assay) 12
(六) γH2AX之免疫螢光染色 (Immunofluorescence of γH2AX assay) 13
(七) 以流式細胞儀偵測DNA斷裂與修補標誌物之分析 (Flow cytomerer-based detection of DNA damage and repair marker assay) 14
(八) 西方墨點法 (Western blot assay) 15
細胞樣品製備 15
西方墨點法 15
(九) 斑馬魚飼養與相關實驗 17
斑馬魚種飼養 17
斑馬魚胚胎取得 17
人類乳腺癌細胞異體移殖之斑馬魚模式 (Zebrafish human breast cancer xenografts model) 17
斑馬魚藥物處理 18
數據統計分析 18
第三章、 實驗結果 19
DEHP暴露對乳腺癌細胞株之影響 19
(一) 選擇適合的CPT藥物濃度 19
(二) 選擇適合的DEHP藥物濃度 19
(三) 確認暴露DEHP是否干擾CPT對乳腺癌細胞產生之細胞毒性及細胞凋亡相關蛋白質之變化 20
(四) 處理DEHP減少活性氧自由基 20
(五) DEHP干擾CPT誘導DNA損傷 21
(六) DEHP干擾CPT誘導DNA損傷的路徑 22
(七) DEHP干擾CPT所產生DNA損傷相關蛋白質之變化 23
(八) 以斑馬魚異體移殖模型探討DEHP干擾CPT對MCF-7乳腺癌細胞的毒殺能力 23
斑馬魚對於CPT耐受性之毒性測試 23
利用斑馬魚模式評估DEHP是否會干擾CPT藥物測試 23
第四章、 討論 25
第五章、 結論 30
圖 一.決定CPT藥物使用之濃度 32
圖 二. 單獨先暴露DEHP對兩株乳腺癌細胞是否產生細胞毒性以及觀察DEHP是否干擾CPT對於細胞毒殺能力 34
圖 三. DEHP干擾CPT在乳腺癌細胞之內生性活性氧測試 (ROS) 36
圖 四. DEHP干擾CPT誘導DNA損傷 38
圖 五. 利用γH2AX的活化評估DEHP干擾CPT所產生DNA損傷 39
圖 六. DEHP干擾CPT誘導DNA損傷的路徑 42
圖 七. 利用ATM/ATR評估DEHP干擾CPT所產生DNA損傷 45
圖 八. DEHP干擾CPT所產生DNA損傷相關蛋白質改變情形 47
圖 九. 利用斑馬魚模式驗證DEHP對CPT產生的干擾現象 49
圖 十. DEHP透過干擾CPT藥物所產生DNA損傷可能的抗藥性機轉 51
附錄 52
附錄 一. 2014年美國前十大癌症死亡排名 53
附錄 二. Tamoxifen 機轉示意圖 54
附錄 三. 癌細胞產生抗藥性主要路徑之示意圖 55
附錄 四. 鄰苯二甲酸酯基本結構以及鄰苯二甲酸酯衍生物結構式 56
附錄 五. CPT和衍生物結構 57
附錄 六. SDS-PAGE配製表 59
附錄 七. CPT產生ROS導致DNA雙股斷裂損傷(DSB)反應機制圖 60
附錄 八. CPT造成癌細胞產生細胞週期停滯機制 61
附錄 九. 本論文中實驗所使用之抗體列表 62
附錄 十. 國際癌症研究署（International Agency for Research on Cancer）人類致癌因子分類表 63
參考文獻 64

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