Deoxyelephantopin及其衍生物DETD-35影響三陰性乳癌細胞中粒線體之蛋白質體並誘導氧化壓力介導的粒線體功能障礙

Abstract

乳癌是女性癌症死亡的主要原因之一。在所有乳癌亞型中，三陰性乳癌（ER− / PR−/ HER2−，TNBC）具有高度轉移性且沒有合適的標靶治療。我們先前的研究顯示，植物倍半萜類化合物deoxyelephantopin（DET）及其新穎的半有機合成衍生物（DETD-35）的處理對於人類TNBC細胞MDA-MB-231在體外和異種移植腫瘤小鼠中均具有抑制作用。值得注意的是，DET及DETD-35的處理都會顯著誘導活性氧化物（ROS）的產生，進而導致MDA-MB-231細胞中粒線體結構的損傷和功能障礙。我們假設DET和DETD-35在TNBC細胞中會誘導氧化壓力相關的粒線體功能障礙。因此，本研究旨在解密DET及DETD-35誘導的氧化壓力和粒線體功能障礙對TNBC細胞活性影響之機制。我們首先使用粒線體專一性超氧陰離子（O2−）指示劑—MitoSOX，觀察到DET及DETD-35在1小時內分別誘導增加1.52及1.35倍之粒線體超氧化物產生；以粒線體ETC複合物III（位點IIIQo）特異性超氧化物抑制劑S3QEL-2預先處理後，可部分降低DET誘導之超氧化物的產生，但對於DETD-35的處理並無影響。使用膜通透性染劑calcein AM與螢光淬滅劑CoCl2結合螢光顯微鏡分析顯示，DET及DETD-35在四小時處理下影響粒線體通透性轉換孔洞（mPTP）之開啟。此外，DET及DETD-35處理4小時之後，TNBC細胞內的ATP水平也分別降低0.53及0.66倍。這些結果支持這兩個化合物在TNBC細胞中均會導致粒線體功能障礙。我們進一步使用以iTRAQ為基礎的定量蛋白質體學技術，對控制組及兩種化合物處理1和4小時後之TNBC細胞內的粒線體進行比較性蛋白質體分析。根據三批次生物性重複之蛋白質體數據，我們發現DET及DETD-35處理後所共同誘導的粒線體蛋白質，其參與共同的粒線體相關生物過程/網絡，包括氧化磷酸化、粒線體功能障礙及ATP生合成等。粒線體內與轉譯延長和終止、減少粒線體去極化及活性氧化物生合成相關蛋白質是專一地在DET處理的組別發現，而與粒線體自噬及穀胱甘肽之結合有關的蛋白質則是專一地在DETD-35處理的組別發現。將DET及DETD-35處理而誘導表現量上升的粒線體跨膜電位及酮分解相關蛋白質HSP27及ACAT-2，被選擇來進一步驗證其表現量與化合物誘導的氧化壓力間之關聯。特別的是，預先處理活性氧化物清除劑（NAC）可以逆轉此兩個蛋白質因受到DET及DETD-35處理而誘導的表現。綜合言之，我們目前的實驗結果顯示，DET及DETD-35處理後對抗TNBC細胞的活性至少部分是透過粒線體功能和粒線體相關蛋白質表達的失調，而這些失調可能與化合物誘導之氧化壓力相關。針對DET及DETD-35處理具反應性的粒線體蛋白質的功能與機轉仍有待進一步研究。

Breast cancer is one of the major causes of cancer death in women. Among all breast cancer subtypes, triple negative breast cancer (ER−/PR−/HER2−, TNBC) is highly metastatic and currently there is no suitable targeted therapy available. Our previous study showed that phyto-sesquiterpene lactone deoxyelephantopin (DET) and its derivative (DETD-35), a novel semi-organically synthesized compound, have potent effects against human TNBC cell MDA-MB-231 activities in vitro and in xenograft tumor mouse model. Of note, treatment of either compound can significantly induce reactive oxygen species (ROS) production that subsequently caused structural damage and dysfunction of mitochondria in MDA-MB-231 cells. We hypothesized that DET and DETD-35 induce oxidative stress-associated mitochondria dysfunction in the TNBC cells. This study thus aimed to decipher the mechanistic insight of DET– and DETD-35–induced oxidative stress and mitochondria dysfunction against TNBC cell activity. We first observed that DET and DETD-35 induced 1.52- and 1.35-fold increase of mitochondrial superoxide production, respectively, within 1 h treatment examined using mitochondrial specific superoxide anion (O2−) indicator MitoSOX, while pretreatment with a mitochondrial ETC complex III (site IIIQo) specific superoxide inhibitor S3QEL-2 that can partially decrease DET–induced superoxide production, but had no effect on DETD-35 treatment. Fluorescence microscopy in couple with the staining of membrane-permeable dye calcein AM and fluorescence quencher CoCl2 showed that DET and DETD-35 affected mitochondrial permeability transition pore (mPTP) opening at 4 h treatment. Further, the intracellular ATP level in DET– and DETD-35–treated TNBC cells also decreased 0.53- and 0.66-fold, respectively, within 4 h treatment. These results support in part that both compounds cause mitochondrial dysfunction in TNBC cells. We further carried out comparative mitochondria proteome analysis of TNBC cells treated with vehicle and either compound for 1 and 4 h using iTRAQ-based quantitative proteomics approach. On the basis of the proteomics data from three biological replicates, we observed that DET– and DETD-35–responsive mitochondrial proteins were commonly involved in mitochondria-related biological processes/networks, including oxidative phosphorylation, mitochondrial dysfunction, synthesis of ATP, among others. Meanwhile, mitochondrial translational elongation and termination, decrease depolarization of mitochondria, and synthesis of ROS were unique in DET treatment, and autophagy of mitochondria, conjugation of glutathione were specifically observed in DETD-35 treatment. The observed up-regulated mitochondrial transmembrane potential-related protein HSP27 and ketolysis-related protein ACAT-2, upon DET and DETD-35 treatment were selected for further validation of their expression and correlation with compound-induced oxidative stress in MDA-MB-231 cells. Notably, pretreatment with ROS scavenger N-acetylcysteine (NAC) can reverse DET– and DETD-35–induced both protein expression levels in the TNBC cells. Together, our current results indicate that the anti-TNBC cell activity of DET and DETD-35 are at least in part through deregulation of mitochondrial functions and mitochondria-associated protein expression that are likely associated with compound induced oxidative stress. More in-depth molecular functions and mechanisms of DET– and DETD-35–responsive mitochondrial protein are warranted for further investigation.