臺灣博碩士論文加值系統

靈芝多醣體萃取物 (EORP) 是一種由靈芝水溶性萃取物中分離出來天然具生物活性的多醣體成分，並且已經被發現有免疫調節及抗腫瘤的活性。然而對於靈芝在肺癌細胞增殖的抑制效果是由於哪些分子機制造成目前仍不是很了解。已經有先前的研究指出，當內皮素受體A受到內皮素1的活化後，會引發多重不同的訊息傳遞，其中包括磷脂酰肌醇3激酶 (PI3K)、蛋白激酶B (AKT) 和胞外信號調節激酶 (ERK)，以達到幫助腫瘤的生長。而在肺腺癌的組織中已經被發現可以偵測到內皮素1的存在並且內皮素受體A也會過度表現。這些研究結果都代表著內皮素1相關的蛋白表現及訊息活化在腫瘤的生長是非常重要的。在本篇研究中，我們顯示靈芝多醣體萃取物可以使A549細胞生長受抑制藉由引發細胞週期停留在G0/G1的階段，並且制內皮素受體A的表現以及阻斷下游磷脂酰肌醇3激酶 (PI3K)、蛋白激酶B (AKT) 和胞外信號調節激酶 (ERK) 的活化並且。除此之外，靈芝多醣體萃取物藉由減少Src酪氨酸激酶的活化達到抑制內皮素受體A和表皮生長因子受體 (EGFR) 之間轉活化及訊息交叉的作用。由於抑制內皮素受體A和表皮生長因子受體兩條路徑造成糖原合成酶激酶-3β (GSK3β) 的磷酸化下降，進一步影響乙型連環素 (β-catenin) 轉移進入細胞核的量降低，促使D型週期素 (cyclin D1) 表現量下降，最後抑制A549細胞的增殖。由於不同受體之間的訊息交叉的作用已經在許多癌症中被發現，所以靈芝多醣體萃取物的抑制效果可以為癌症治療上提出一個新的機會。

An extract of Reishi (Ganoderma lucidum or Ling-Zhi) polysaccharides (EORP), a natural bioactive glycan isolated from a water-soluble fraction of Reishi, has been shown to exhibit immuno-modulating and anti-tumor activities. However, the molecular mechanisms responsible for the inhibitory effects of EORP on the proliferation of lung cancer cells remain less understood. It has been reported that endothelin receptor type A (ETAR) activation by endothelin-1 (ET-1) triggers the activation of a network of a multiple signaling including PI3K/AKT and MAPK pathways to contribute to tumor growth. Further more, ET-1 has been detected and ETAR is overexpressed in pulmonary adenocarcinomas. Both show that ET axis plays an important role in tumor proliferation. In this study, we demonstrate that EORP inhibits A549 cells growth via induction of cell cycle arrest at G0/G1 phase. Moreover, EORP down-regulates the expression of ETAR and blocking activation of downstream phosphoinositide 3-kinase (PI3K), Akt and extracellular signal-regulated kinases (ERK). In addition, the regulation of EORP in ETAR causes an inhibition of the transactivation and cross-talk between ETAR and epidermal growth factor receptor (EGFR) by decreasing activation of Src. The inhibition of ETAR and EGFR pathways resulted in reducing the phosphorylation of GSK3β, and leading to down-regulate the translocation of β-catenin to nuclear to repress the expression of cyclin D1, finally suppressing proliferation of A549 cell. Since that cross-talk between different receptors has been implicated in many cancers, the inhibitory effects of EORP indicate a new therapeutic opportunities for cancer.

Contents 1
中文摘要 3
Abstract 4
Introduction 5
Materials and Methods 10
Cell Culture 10
Aantibodies 10
Preparation of EORP 10
Soft Agar Colony Formation Assay 10
MTT assay 11
Cell Cycle Assays 11
Preparation of whole cell extracts 12
Nuclear extracts 12
Western Blotting Analysis 12
Result 14
EORP Inhibits Cell Clonogenic Survival in A549 Cells. 14
Antiproliferative effects of EORP. 14
EORP induces cell cycle arrest at G0-G1 phase in A549 cells. 14
EORP reduces ETAR expression and the activation of ETAR-dependent signaling pathway. 14
EORP disrupts the crosstalk between ETAR and EGFR through Src 15
EORP inhibits ET-1–induced ERK, AKT activation and transactivation of EGFR. 16
EORP disrupts the crosstalk between ETAR and EGFR signaling and inhibits EGF signaling. 16
EORP reduces phosphorylation of GSK3β and cyclin D1 through inhibition of AKT activation. 17
EORP affects β-catenin translocation and down-regulation the expression of cyclin D1. 17
Discussion 18
Reference 22
Figure legend 27

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