乳癌是現代婦女最常見的癌症，也是癌症死亡的第二大原因，過去十年，雖然乳癌的死亡率逐漸下降，但發病率正逐漸增加，目前能透過外科手術、放射治療、藥物治療、免疫治療等方式治療乳癌，但臨床上的一些化療藥物常伴隨著副作用的產生，像是噁心、體重下降、掉髮、白血球及血小板數過低等，另外也需解決三重陰性乳癌對傳統藥物治療效果較差的情形，因此我們仍需要新穎且更有效的治療策略，使病人在較低的負擔下達到更好的療效，其中合併治療能透過化療藥物間不同的分子機制，增加細胞毒殺效果、降低抗藥性、並使重疊的毒性最小化。Etoposide是typeⅡ的拓撲異構酶抑制劑，會使癌細胞產生DNA錯誤，造成DNA雙股斷裂並且誘發細胞凋亡，另外組織蛋白去乙醯酶抑制劑(HDACi)則被發現會抑制DNA修復並誘發細胞凋亡，但HDACi抑制DNA修復的分子機制目前尚有待釐清。因此我們利用體外試驗以及原部位乳癌動物模式，探討新的HDACi YCW-3合併處理etoposide是否對乳癌細胞具有協同毒殺效果，並了解是否與HDACi抑制DNA的修復有關，並進一步探討HDACi抑制DNA的修復是否為透過調控蛋白質泛素化的結果。在體外試驗方面，分別以etoposide和YCW-3單獨與合併處理小鼠三重陰性乳腺癌細胞株4T1，以MTT assay分析細胞毒殺效果；利用流式細胞儀測定細胞死亡模式；透過彗星試驗及西方墨點法分析DNA之損傷與修復；另外使用免疫沉澱觀察蛋白的泛素化及蛋白之間的交互作用；mRNA的表現程度則透過即時聚合酶鏈式反應分析。在動物試驗方面，建立原部位乳癌動物模式，並利用活體影像系統監測腫瘤的生長，犧牲後取出腫瘤利用免疫組織化學染色及西方墨點法分析相關蛋白的表現量變化。本研究首先比較了YCW-3與傳統組織蛋白去乙醯酶抑制劑SAHA，發現YCW-3在藥物動力學、組織蛋白去乙醯酶抑制能力及細胞毒殺效果方面皆優於傳統藥物，表示YCW-3具有發展的潛力。於細胞及動物實驗中，相較於單獨處理組別，合併處理YCW-3與etoposide能協同增強細胞毒殺效果並有效抑制腫瘤生長。為釐清協同作用的機制，首先利用彗星試驗發現藥物合併處理組別比單獨處理產生更嚴重的DNA損傷，且DNA損傷指標蛋白γH2AX的表現量於合併處理的組別也有明顯的增加。而在處理YCW-3的組別中發現DNA關鍵修復蛋白DNA-PK的表現量會受到抑制；透過即時聚合酶鏈鎖反應分析，得知處理YCW-3細胞中DNA-PK mRNA表現程度並無顯著差異。而透過處理MG132能夠恢復YCW-3抑制的DN-PK表現量，表示DNA-PK蛋白的抑制是透過泛素-蛋白酶體路徑所降解；進一步利用免疫沉澱法發現YCW-3會促進DNA-PK與其E3連接酶RNF144A之間的交互作用，並誘發DNA-PK蛋白的泛素化，最後使DNA-PK受到蛋白酶體降解。於細胞死亡模式的研究結果中也發現合併處理的組別會顯著增加細胞凋亡和細胞自噬的百分比，並且細胞自噬是扮演促進死亡的角色。綜合以上結果得知，合併處理YCW-3可經由調控修復蛋白泛素化以抑制DNA修復，增加etoposide對於乳癌細胞之抗癌效果。

Breast cancer is the most common cancer in modern women. Although there are currently a variety of therapeutic methods for the treatment of breast cancer, severe problems still exist due to serious side effects triggered by clinical drugs and less anti-cancer activity in triple negative breast cancer. In this study, triple negative breast cancer cell line was applied to investigate the anti-cancer effects of new histone deacetylase inhibitor, YCW-3 alone or in combination with topoisomerase inhibitor, etoposide and to determine the mechanisms of these effects in vitro and in vivo. Firstly, we confirm that YCW-3 is a potent pan-HDAC inhibitor in which expressed a higher capability to inhibit HDACs and superior pharmacokinetics profile than traditional HDACi. Combination treatment with YCW-3 and etoposide resulted a synergistic cytotoxicity effect and induced remarkable programmed cell death in breast cancer cell line. The combined treatment enhanced significantly DNA damage, quantified by comment assay, when compared to YCW-3 or etoposide treatment alone. From a mechanistic insight, YCW-3 inhibited the expression level of the crucial DNA repair protein, DNA-PK. The effect of YCW-3 on the degradation of DNA-PK protein could be through the ubiquitin proteasome pathway, and the proteasomal degradation of DNA-PK protein was suggested by enhancing the association of DNA-PK with the ubiquitin E3 ligase RNF144A.Thus, our results demonstrated that the combination of YCW-3 and etoposide exerts strong cytotoxic effects in breast cancer and deserve further clinical-related investigation.

第一章、序論 1
第二章、文獻回顧 2
第一節、乳癌(Breast cancer) 2
第二節、合併治療(combination therapy) 3
第三節、拓撲異構酶抑制劑(topoisomerase inhibitor, TOPi) 4
第四節、組織蛋白去乙醯酶抑制劑(histone deacetylase inhibitor, HDACi) 6
第五節、DNA雙股斷裂與DNA修復(DNA double strand break and DNA repair) 7
第六節、泛素化 (ubiquitination) 9
第七節、細胞凋亡與細胞自噬(Apoptosis and autophagy) 10
第三章、研究目的 13
第四章、研究材料與方法 14
第一節、研究材料 14
第二節、研究方法與實驗步驟 20
第五章、研究架構 27
In vitro study 27
In vivo study 28
第六章、實驗結果 29
第一節、YCW-3與傳統的組織蛋白去乙醯酶抑制劑SAHA之比較 29
第二節、拓撲異構酶抑制劑Etoposide合併組織蛋白去乙醯酶抑制劑YCW-3對於4T1細胞之毒性的劑量效應 29
第三節、探討合併處理YCW-3與Etoposide所誘發之DNA損壞及DNA修復 30
第四節、探討組織蛋白去乙醯酶抑制劑YCW-3抑制DNA修復蛋白DNA-PK之作用機制 31
第五節、分析合併處理YCW-3與Etoposide影響4T1細胞之細胞凋亡與細胞自噬的變化 32
第六節、探討合併處理YCW-3與Etoposide對4T1細胞誘發自體吞噬的現象 33
第七節、乳癌原部位腫瘤動物模式 34
第七章、討論 36
第八章、結論及建議 40
第九章、參考文獻 41
圖表 50

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