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標題: 檸檬苦素抑制雌激素受體-α過度表現之乳癌細胞中雌激素受體-α訊息傳遞路徑與抗癌活性之研究
Nomilin exerts anticancer activity through the inhibition of ER-α signaling pathway in ER-α positive breast cancer cells
作者: 吳柏慧
Po-Huei Wu
關鍵字: 雌激素受體;檸檬苦素;細胞轉移;Estrogen receptor-α;Nomilin;Metastasis
引用: 1 Rundhaug, J. E. & Fischer, S. M. Molecular mechanisms of mouse skin tumor promotion. Cancers 2, 436-482 (2010). 2 Zhang, L. et al. [Expression of VEGF in endothelial cells and the effects of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside]. Yao xue xue bao = Acta pharmaceutica Sinica 39, 406-409 (2004). 3 Frasor, J. et al. Profiling of Estrogen Up- and Down-Regulated Gene Expression in Human Breast Cancer Cells: Insights into Gene Networks and Pathways Underlying Estrogenic Control of Proliferation and Cell Phenotype. Endocrinology 144, 4562-4574 (2003). 4 Ciruelos Gil, E. M. Targeting the PI3K/AKT/mTOR pathway in estrogen receptor-positive breast cancer. Cancer Treatment Reviews 40, 862-871 (2014). 5 Meera, S. & Gerhard, C. Rebuilding cancer metastasis in the mouse. Molecular Oncology 7, 283-296 (2013). 6 Vergara, D. et al. Translating epithelial mesenchymal transition markers into the clinic: Novel insights from proteomics. EuPA Open Proteomics 10, 31-41(2016). 7 Kalra, J. et al. Suppression of Her2/neu expression through ILK inhibition is regulated by a pathway involving TWIST and YB-1. Oncogene 29 (2010). 8 Vikram, A., Jesudhasan, P. R., Jayaprakasha, G. K., Pillai, B. S. & Patil, B. S. Grapefruit bioactive limonoids modulate E. coli O157:H7 TTSS and biofilm. International journal of food microbiology 140, 109-116 (2010). 9 Xiang, Y. et al. Simultaneous purification of limonin, nomilin and isoobacunoic acid from pomelo fruit (Citrus grandis) segment membrane. Journal of food science 79, C1956-1963 (2014). 10 Kouam, A. F. et al. Induction of Mkp-1 and Nuclear Translocation of Nrf2 by Limonoids from Khaya grandifoliola C.DC Protect L-02 Hepatocytes against Acetaminophen-Induced Hepatotoxicity. Frontiers in pharmacology 8, 653 (2017). 11 Subramani, R. et al. Nimbolide inhibits pancreatic cancer growth and metastasis through ROS-mediated apoptosis and inhibition of epithelial-to-mesenchymal transition. Scientific reports 6, 19819 (2016). 12 Tian, Q., Miller, E. G., Ahmad, H., Tang, L. & Patil, B. S. Differential inhibition of human cancer cell proliferation by citrus limonoids. Nutrition and cancer 40, 180-184 (2001). 13 Zhang, J. et al. Nimbolide-Induced Oxidative Stress Abrogates STAT3 Signaling Cascade and Inhibits Tumor Growth in Transgenic Adenocarcinoma of Mouse Prostate Model. Antioxidants & redox signaling 24, 575-589 (2016). 14 Kim, J., Jayaprakasha, G. K. & Patil, B. S. Limonoids and their anti-proliferative and anti-aromatase properties in human breast cancer cells. Food & function 4, 258-265 (2013). 15 An, F. L. et al. Trijugin- and mexicanolide-type limonoids from the fruits of Heynea trijuga that reverse multidrug resistance in MCF-7/DOX cells. Phytochemistry 151, 42-49 (2018). 16 Elumalai, P. et al. Induction of apoptosis in human breast cancer cells by nimbolide through extrinsic and intrinsic pathway. Toxicology letters 215, 131-142 (2012).
雌激素受體(Estrogen Receptor-α)是依賴雌激素配體(Estrogen)活化的核轉錄因子,存在於正常乳腺細胞中,當細胞質內的Estrogen Receptor-α與Estrogen結合後,會進入細胞核內形成轉錄因子進行轉錄作用,促使細胞增生及分化。檸檬苦素廣泛存在柑橘、檸檬類植物的皮跟籽之中,目前已知的檸檬苦素多達300多種,先前文獻指出檸檬苦素具有抗癌活性,可以促進乳癌細胞凋亡,在ER(+)乳癌和ER(-)乳癌中又以對ER(+)乳癌的效果較為明顯。本篇研究主要探討檸檬苦素(Nomilin)是否會調控ER(+)乳癌中的ER-α訊息傳遞路徑。首先利用細胞存活率分析(MTT assay)來看Nomilin抑制ER(+)乳癌和ER(-)乳癌增生的情形,證實Nomilin抑制ER(+)乳癌增生效果確實比ER(-)乳癌好,並且確認半數抑制濃度不會對正常乳腺細胞造成傷害。再來使用西方墨點法與免疫螢光染色證實Nomilin可以抑制Estrogen Receptor-α蛋白表現與其下游調控目標HIF-1α也會受到影響,此外受到Estrogen Receptor-α磷酸化的蛋白ρ-AKT也會受到Nomilin的抑制,使ρ-AKT的蛋白表現下降,進而調控AKT/mTOR訊息傳遞路徑,抑制癌細胞增生,另外HIF-1α會藉由調控Twist、Snail等轉錄因子進而降低E-cadherin蛋白表現而使癌細胞易轉移,而本篇研究證實Nomilin藉由抑制Twist、Snail,使得E-cadherin蛋白表現增加最終可抑制乳癌細胞的轉移。過去研究已知Y-box binding protein (YB-1)會受到Twist轉錄因子的調控,本篇研究證實Nomilin藉由調控Estrogen Receptor-α進而抑制YB-1。最後利用反轉錄即時定量聚合連鎖反應證實Nomilin不會影響Estrogen Receptor-α之mRNA表現。本篇研究對於日後研究檸檬苦素與乳癌中雌激素受體之關係極具重要性。

Estrogen receptor-α is a ligand-activated transcription factor implicated in breast cell and plays an important role in cancer cell proliferation and metastasis. The binding of estrogen and the estrogen receptor-α in the cytoplasm causes two estrogen receptors to dimerize spontaneously, and translocated into the nucleus. Citrus fruits and its related genera contain about 300 limonoids have been known nowadays. In the present study, we found that limonoids inhibit breast cancer cell proliferation. Interestingly, the effect of limonoids inhibited the growth of ER-α positive breast cancer cells was better than the ER-α negative breast cancer cells. Therefore, we aimed at the relationship between limonoids and ER-α signaling pathway. Our data indicated that among the abundant of the limonoids, the cytotoxic effect of Nomilin was stronger than Limonin in breast cancer cells. Moreover, we demonstrated that Nomilin reduced the expression of ER-α protein levels in ER-α positive breast cancer cells. Nomilin repressed cell proliferation through the inhibition of AKT/mTOR signaling pathway in MCF7 cells. We also found that Nomilin inhibited ER-α positive breast cancer cell metastasis through the regulation of E-cadherin. According to previous studies, Twist was transcription factor that can induced transcription of Y-box binding protein-1. We found that Nomilin reduced the expression of Y-box binding protein-1 through the inhibition of ER-α. This work sheds light on the pathways by which Nomilin inhibits ER-α positive breast cancer cells proliferation and metastasis.
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