臺灣博碩士論文加值系統

三陰性乳癌(TNBC)是一種具高度轉移性且在臨床治療出現棘手挑戰的乳癌，其因缺乏有效的標靶藥物的運用。本研究的目的是探索藥用植物衍生之植化物倍半萜類化合物deoxyelephantopin (DET)及其新型衍生物DETD-35抑制TNBC細胞活性的作用機轉。DETD-35的處理對於抑制人類三陰性MDA-MB-231細胞增長的表現優於DET，其半致死率的用藥劑量降低3.5倍 (3.62 vs. 11.24 uM)。DET和DETD-35的短時間處理(2小時)會明顯地刺激活性氧化物(ROS)的產生，而造成泛素化蛋白質和自噬體標誌物LC3的積累以及MDA-MB-231細胞中大量胞質液泡的形成。透射電子顯微鏡(TEM)和共聚焦顯微鏡分析顯示DET或DETD-35的處理並不會影響TNBC細胞的質膜完整性，而DET與DETD-35所引發的胞質空泡化起因於內質網(ER)或粒線體的腫脹。同時，DET和DETD-35處理也會誘發ER壓力相關蛋白的表達及線粒體膜電位的喪失(27％和23.5％)相對於對照組(100％)。值得注意的是兩種化合物處理所引起的抑制作用會因ROS清除劑(N-乙酰半胱氨酸與穀胱甘肽)及蛋白質合成抑製劑的預處理而被阻斷，而上述現象完全吻合了paraptotic程序性細胞死亡（PCD）的表型和生物化學特徵。再者，藉由剔除MEK上游調節者RAF1與自噬體蛋白LC3的表現及JNK與ERK1/2抑製劑的共同處理下會顯著減弱DETD-35所誘發的細胞毒性作用，而DET引起的抑制作用卻不受MAPK抑製劑的影響。另外，DET和DETD-35處理明顯地刺激TNBC細胞衍生的外泌體(exosomes)釋放到培養基中起因為細胞質中游離鈣離子的增加，而鈣螯合劑BAPTA-AM預處理會明顯消除DET或DETD-35處理所引發的外泌體釋放。有趣的是，DET和DETD-35誘發的外泌體擁有非典型的功能，其對於MDA-MB-231細胞具有抗增殖的活性，此表明DET和DETD-35處理會影響外泌體的活性與功能。質譜（MS）為基礎的定量蛋白質組結合IPA資料庫分析顯示DET和DETD-35處理會減弱TNBC細胞外泌體中與細胞遷移，細胞粘附和血管生成等生物過程相關蛋白質的表達，並且發現這些因DET和DETD-35處理而失調的蛋白質也會涉及氧化壓力和粒線體跨膜電位的降低等生物機轉。在本研究中也進行了比較蛋白質組學分析，以了解人類TNBC腫瘤以及DET或化療藥物卡培他濱(CAP)處理之老鼠三陰性4T1乳腺腫瘤中蛋白質的變化和信號網絡。因DET和CAP處理而有所反應的蛋白質群會參與氧化壓力和粒線體功能障礙或損傷相關的生物機制，例如粒線體腫脹和粒線體跨膜電位的變化，而人類TNBC腫瘤的蛋白質組學研究了解到TNBC腫瘤於不同階段會有其差異性的蛋白質表達模式。在2個IIB期的TNBC腫瘤蛋白質體中發現了21個蛋白質(如annexin A1 and A5, SLC2A1, protein Z-dependent protease inhibitor, alpha-1-acid glycoprotein 1, hemopexin等)表達與DET處理4T1腫瘤蛋白質體出現相似的表達水平。總體而言，我們的研究結果表明了DET和DETD-35抑制TNBC細胞的活性是通過氧化壓力所誘發的paraptosis似的細胞死亡及改變癌細胞釋放的外泌體內蛋白質的組成及其功能。本研究提供了藥用植物來源的倍半萜類化合物DET及其新型衍生物DETD-35抑制TNBC細胞活性之新穎的機轉見解，並用以支持於未來在開發干預TNBC活性的潛力。

The triple negative breast cancer (ER-/PR-/HER2-, TNBC) is a highly metastatic breast cancer subgroup and a thorny issue of this cancer disease for clinical therapy is the absent of efficient targeted therapeutic approaches. The objectives of this thesis study were to investigate the mechanisms of action of a phyto-sesquiterpene lactone deoxyelephantopin (DET) and its novel derivative DETD-35 for anti-TNBC cell activities. DETD-35 displayed superior activity than DET on inhibition of human TNBC MDA-MB-231 cell proliferation, with a 3.5-fold decrease of IC50 value (3.62 vs. 11.24 uM). Both DET and DETD-35 compounds can stimulate the reactive oxygen species (ROS) production within an early stage (2 h) of treatment that resulted in significant ubiquitinated protein and autophagosomal marker LC3 accumulation along with induction of massive cytoplasmic vacuole formation in MDA-MB-231 cells. Transmission electron microscopy (TEM) and confocal microscopy analysis showed that the TNBC cell plasma membrane integrity was not affected by DET or DETD-35, and both compound-induced cytoplasmic vacuolation were derived from swelling of endoplasmic reticulum (ER) or mitochondria. Meanwhile, both DET and DETD-35 treatments also evoked the expression of ER stress-related proteins and the loss of mitochondrial membrane potential in TNBC cells (27% and 23.5%) relative to vehicle control (100%). Of note, the observed DET and DETD-35 effects were blockaded by pretreatment with ROS scavengers N-acetylcysteine and glutathione, and protein synthesis inhibitor, cycloheximide, that perfectly matched the phenotypic and biochemical characteristic features of paraptotic programmed cell death (PCD) of cancer cells. Further, knockdown of MEK upstream regulator RAF1 and autophagosomal protein LC3, or co-treatment with JNK or ERK1/2 inhibitor can significantly attenuated DETD-35-triggered cytotoxic effects in TNBC cells, while the anti-cancer cell effect of DET was not influenced by MAPK inhibitor treatment. Moreover, both DET and DETD-35 treatment significantly stimulated the release of TNBC cell-derived exosomes into culture media due to increase of cytosolic free calcium ion level, as calcium chelator BAPTA-AM pretreatment significantly abolished exosome release in DET or DETD-35 treated cancer cells. Interestingly, both DET- and DETD-35-induced exosomes had an atypical function that showed anti-proliferative activity against MDA-MB-231 cells, suggesting that both DET and DETD-35 treatments affected exosome activities and functions. Comparative LC-mass spectrometry (MS)-based quantitative proteomics coupled with Ingenuity Pathway Analysis (IPA) database analysis of exosome from DET- and DETD-35-treated TNBC cells showed that DET and DETD-35 attenuated the expression of exosomal proteins related to several biological processes, such as cell migration, cell adhesion, and angiogenesis. Furthermore, several exosomal proteins participating in biological mechanisms such as oxidative stress and decrease of transmembrane potential of mitochondria were found deregulated by treatment with either compound. Comparative proteomic analysis was also conducted in this study to decipher the proteins and signaling networks in human TNBC specimens and murine triple negative 4T1 mammary tumors with or without DET or chemotherapeutic drug capecitabine (CAP) treatment. DET- and CAP-responsive proteomes could involve in a few biological mechanisms related to oxidative stress and mitochondria dysfunction or damage, such as mitochondria swelling and change of transmembrane potential of mitochondria. A parallel proteomic study on the clinical human TNBC specimens obtained the differential protein expression patterns in different stages of TNBC tumors. Among 2 TNBC tumors at stage IIB, 21 proteins, such as annexin A1 and A5, SLC2A1, protein Z-dependent protease inhibitor, alpha-1-acid glycoprotein 1, hemopexin, etc., displayed similarly expressed levels with those observed in murine 4T1 tumors and that were responsive and deregulated by DET treatment. Overall, this study demonstrates that the DET and DETD-35 inhibitory TNBC cell activities were through promoting exosome release from the cancer cells and oxidative stress-induced paraptosis-like programmed cell death by calcium dependent mechanism. The DET and DETD-35 induced cancer cell releasing exosomes was in tandem with alteration of exosomal protein composition and functions. Overall, this study provides novel mechanistic insights of medicinal plant derived sesquiterpene lactose DET and its novel derivative DETD-35 against TNBC cell actives in support of the potential for future developing these agents in TNBC intervention.

口試委員審定書...........................................i
誌謝...................................................ii
摘要..................................................iii
Abstract................................................v
Table of Contents....................................viii
List of Figures......................................xiii
List of Tables.........................................xv
Abbreviations.........................................xvi
Chapter 1 General Introduction..........................1
1.1 Triple negative breast cancer (TNBC)................1
1.2 Highlights of current proteomic research of TNBC....3
1.3 Cytoplasmic vacuolation-related cell death..........5
1.4 Cancer cell-derived exosomes........................6
1.5 Anti-cancer plant sesquiterpene lactones (SLs) and deoxyelephantopin from Elephantopus scaber L. and its novel derivatives.......................................9
1.6 Novel deoxyelephantopin derivatives (DETDs)........12
1.7 General objective and specific aims................13
Chapter 2 Materials and Methods........................16
2.1 Isolation and structure elucidation of DET.........16
2.2 Chemicals and antibodies...........................17
2.3 Cell culture.......................................17
2.4 Cell viability assay...............................18
2.5 Animals............................................18
2.6 Inhibition of triple negative tumor growth in NOD/SCID mice..........................................18
2.7 Western blot analysis..............................19
2.8 Immunofluorescence cell staining...................20
2.9 Immunohistochemistry analysis......................21
2.10 Measurement of reactive oxygen species (ROS)......21
2.11 Measurement of mitochondrial membrane potential (MMP)..................................................22
2.12 Transmission electron microscopy (TEM)............22
2.13 Transfection of small hairpin RNA.................22
2.14 Protein digestion and iTRAQ labeling..............23
2.15 Strong cation exchange fractionation and LC-MS/MS analysis...............................................24
2.16 Protein identification and quantitation...........26
2.17 Bioinformatics analysis...........................27
2.18 Statistical analysis..............................28
Chapter 3 Results......................................29
3.1 To elucidate the mechanisms of action of DET and its derivative DETD-35 against TNBC cell activities........29
3.1.1 DET and DETD-35 inhibited cell proliferation and growth and induced cytoplasmic vacuolation formation in MDA-MB-231 cells.......................................29
3.1.2 DETD-35 promotes non-autophagic cytoplasmic vacuolation death in TNBC cells........................31
3.1.3 DET- and DETD-35-caused cytoplasmic vacuolation death is associated with endoplasmic reticulum (ER) stress and ubiquitinated protein accumulation..........34
3.1.4 DET and DETD-35 induce the loss of mitochondrial membrane potential (MMP) and the formation of mitochondria-derived vacuoles in MDA-MB-231 cells......36
3.1.5 DET- and DETD-35-induced cytoplasmic vacuolation require protein synthesis..............................38
3.1.6 Role of MAP kinases in DET- and DETD-35-induced cytoplasmic vacuolation death in TNCB cells............39
3.1.7 DET- or DETD-35-induced ROS mediates cytoplasmic vacuolation-associated cell death......................41
3.2 To investigate the DET and DETD-35 effects on TNBC cell release of exosomes and exosomal proteins.........43
3.2.1 DET and DETD-35 promoted Ca2+-dependent release of exosomes from TNBC cells...............................44
3.2.2 Comparative exosomal proteomes analysis..........46
3.2.3 Analysis of differentially expressed proteins in exosomes...............................................48
3.3 To investigate the comparative proteome of human triple negative breast tumors and murine 4T1 mammary tumors with DET or anti-cancer drug treatment..........51
3.3.1 Comparative proteomics study of human clinical triple negative breast tumors and adjacent normal mammary tissues................................................51
3.3.2 Analysis of differentially expressed proteins in TNBC 4T1 tumor with or without DET treatment...........54
Chapter 4 Discussion...................................60
4.1 DET and DETD-35 inhibited TNBC cell activity by induction of ROS-dependent, cytoplasmic vacuole-mediated paraptosis-like programmed cell death (PCD)............60
4.1.1 DET and DETD-35 induced the formation of ER or mitochondria-originated cytoplasmic vacuoles in MDA-MB-231 cells..............................................60
4.1.2 DET and DETD-35 augmented autophagosome accumulation leading to non-autophagic PCD in TNBC cells..................................................60
4.1.3 DET- and DETD-35-caused cytoplasmic vacuolation death was associated with ER stress and ubiquitinated protein accumulation that was reversed by cycloheximide..........................................63
4.1.4 DET- or DETD-35-induced ROS plays as a role as upstream initiator to initiate paraptotic PCD in TNBC cells..................................................64
4.1.5 Cytotoxic effects of PTX in TNBC cells...........66
4.2 DET and DETD-35 significantly induced exosome release from TNBC cells and DET/DETD-35 changed exosomal protein profile and content was associated with their anti-TNBC cell activity..........................................67
4.2.1 DET and DETD-35 induced deteriorate effect on structural integrity and function of ER and mitochondria, and promoted calcium-dependent exosome release from TNBC cells..................................................67
4.2.2 DET- or DETD-35-induced oxidative stress altered the protein composition and activity of TNBC cell-secreted exosomes......................................69
4.2.3 DET or DETD-35 deregulated the expression of oxidative stress- and transmembrane potential of mitochondria-related proteins in TNBC cell-secreted exososmes..............................................70
4.3 Comparative proteomes of human triple negative breast tumors and the mouse triple negative 4T1 mammary tumors with DET or chemotherapeutic drug CAP treatment........73
4.3.1 Significantly high or low expressed proteins in human triple negative breast tumors....................73
4.3.2 Comparative protein profiling in the 4T1 tumors with or without DET treatment..........................77
Chapter 5 Conclusions..................................82
References............................................148
Appendix I Published article 1......................162
Appendix II Published article 2......................163
Appendix III Published article 3......................164

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