Molecular insights of phytoagent deoxyelephantopin and its derivative against human MDA-MB-231 triple negative breast cancer cell activities

• Main Authors: Jeng-Yuan Shiau, 蕭証元
• Other Authors: 徐麗芬
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/5xxawe

博士 === 國立臺灣大學 === 生物科技研究所 === 105 === 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.