艾黴素是一種幾十年用來作為治療不同癌症的抗生素化療藥物，例如:乳癌、膀胱癌、卵巢癌以及子宮癌。然而艾黴素會產生reactive oxygen species (ROS)，造成細胞功能不正常導致細胞走向凋亡以及心肌損傷，其副作用限制了艾黴素在臨床醫學上的應用。在此研究中，為了探究艾黴素產生的ROS所造成的心臟病，利用螢光對比二維凝膠蛋白質電泳系統搭配半胱氨酸標定技術，來監控在艾黴素處理之下所誘發之氧化還原調節相關蛋白上巰基修飾的變化；此外，最近的研究指出癌細胞藉由降低細胞內抗氧化物而具有對藥物的抗性。然而尚未有關於探討抗藥性相關的細胞標的蛋白中氧化還原的調節變化相關研究發表；於是，我們使用半胱氨酸標定技術搭配基質輔助雷射脫附游離飛行時間式質譜儀，來探究具抗藥性癌細胞是如何透過與藥物敏感癌細胞不同的氧化還原的調節來保護自己以避免艾黴素所產生的ROS傷害。藥物抗藥性是造成艾黴素在醫學上使用受限的另一原因。為了抗藥性研究，我們使用了一對子宮癌細胞株：MES-SA和對艾黴素有抗性的MES-SA/Dx5來作為研究模式細胞以觀測抗藥性所誘發的細胞反應；再者，為了進一步在藥物抗性更高的細胞中發掘更多與抗藥性相關的標的蛋白，我們培養出一系列對艾黴素具有不同抗藥性程度的子宮癌細胞株: MES-SA, MES-SA/DxR-2μM 和 MES-SA/DxR-8μM。首先，我們利用此組細胞株進行螢光對比二維凝膠蛋白質電泳搭配基質輔助雷射脫附游離飛行時間式質譜儀，來觀測對艾黴素具抗性的癌細胞與對藥物敏感的癌細胞相較下所有細胞內蛋白的表現差異變化。接著我們將探討在不同抗藥性細胞中的特定胞器對於艾黴素所誘發的抗藥性機制，我們使用定量蛋白質體學策略來監測次細胞胞器中蛋白質含量的變化，包含粒線體、細胞核以及分泌胞外蛋白。從這些鑑定出的蛋白中，我們使用核糖核酸干擾技術來探討Asparagine synthetase (ASNS)和progesterone receptor membrane component 1 (PGRMC1)和Reticulocalbin-1 (RCN1)蛋白和粒線體蛋白acetyl-CoA acetyltransferase (ACAT1)以及malate dehydrogenase (MDH2)以及細胞核蛋白XRCC3 在藥物抗藥性上可能扮演的角色。重要的是，我們進一步使用高度表現以及抑制蛋白技術來雙重驗證PGRMC1在子宮癌增生、抗凋亡以及癌細胞侵入能力中扮演重要的角色。 總結來說，我們使用半胱氨酸標定策略來闡述艾黴素所誘發的心肌毒殺相關的細胞反應；另外，我們比較藥物敏感的癌細胞與具抗藥性的癌細胞兩者氧化還原調節蛋白反應程度之差異，來發掘可能造成抗藥性的相關蛋白。更甚者，這個研究提供許多藥物抗藥性的標的蛋白作為未來藥物目標治療來克服化學治療癌症上的困境。

Doxorubicin is one of chemotherapeutic drug in treatment with a board range of cancers (such as breast, bladder, ovaries and uterine cancer) for decades. However, the side effect of doxorubicin in producing ROS to cause cell dysfunction leading to cell apoptosis and myocyte damage to limit its usage in clinics. In current study, in order to monitor the doxorubicin-mediated cardiopathy-induced by ROS, a cysteine-labeling version of 2D-DIGE was employed to monitor the thiol modifications of redox-modulated proteins associated with doxorubicin treatment. In addition, recent studies indicated that reduction of intracellular anti-oxidants could result in drug resistance. However, the redox-modifications of resistance-associated cellular targets have not been reported. For this, we conduct ICy dyes-based labeling and MALDI-TOF MS to investigate how resistant cell protect itself from doxorubicin induced ROS in comparison with sensitive cell according to their differences in redox regulation. The other limited application of doxorubicin is drug resistance. For this, we used a pair of uterine sarcoma cancer lines, MES-SA, and the doxorubicin resistant MES-SA/Dx5 as a model system to examine resistance-induced cellular responses. Furthermore, in order to explore more drug resistance-associated markers in higher resistance cell model with resistance-dependent manner, we developed a panel of differential doxorubicin resistant levels of uterine sarcoma cell lines: MES-SA, MES-SA/DxR-2μM and MES-SA/DxR-8μM. First, we applied 2D-DIGE combined with MALDI-TOF/TOF MS to examine the global protein expression changes in doxorubicin resistant cells. Second, subcellular quantitative proteomic analysis has been performed to enrich low-abundant resistance-associated proteins from subcellular organelles, such as mitochondrial, nuclear and secreted fractions. From these identified proteins, we investigated the possible role of Asparagine synthetase (ASNS), progesterone receptor membrane component 1 (PGRMC1), Reticulocalbin-1 (RCN1), mitochondrial acetyl-CoA acetyltransferase (ACAT1) and malate dehydrogenase (MDH2) and nuclear XRCC3 in drug resistance by RNA interference technique. Importantly, we further focus on PGRMC1 by overexpression and knockdown methods to double confirm its important role in uterine cancer proliferation, anti-apoptosis and invasion ability. To sum up, we utilized ICy dye labeling strategy to elucidate the cell responses to doxorubicin-induced cardiotoxicity; additionally, we compared the differentially redox-modulated proteins between sensitive and resistant cell lines to explore potential cellular proteins in the formation of doxorubicin resistance. Moreover, our proteomic approaches allowed us to identify numerous low abundant proteins involved in drug-resistance-forming mechanism. This study have been provided numerous potential resistance-associated proteins as therapeutic targets to overcome chemotherapy-induced drug resistance.

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