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研究生:陳育瑄
研究生(外文):Yu-Hsuan Chen
論文名稱:探討放射線抗敏性的原因及BEZ235做為抗癌藥物治療之放射線增敏劑的機轉
論文名稱(外文):The mechanisms of radioresistance and BEZ235 as a radiosensitizer in cancer treatment
指導教授:鄧哲明鄧哲明引用關係
口試委員:黃德富楊春茂顏茂雄郭頌鑫潘秀玲
口試日期:2015-04-23
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:110
中文關鍵詞:放射治療PI3K/Akt/mTOR訊息傳導路徑VEGF-CBEZ235肺癌大腸直腸癌
外文關鍵詞:RaidotherpayPI3K/Akt/mTOR pathwayVEGF-CBEZ235lung canercolorectal cancer
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放射治療是癌症治療主要治療方式之一,通常會配合手術治療、化學藥物治療或標靶藥物治療。增加放射治療敏感性是改善放射治療效果主要方法之一。放射治殺死腫瘤細胞是經由促進細胞凋亡效應,但相對的放射線也會引起保護機制的訊息傳導路徑,包含PI3K/Akt/mTOR訊息傳導路徑,這也有可能造成是放射線抗敏性的原因之一。
我們的研究顯示放射線會引起PI3K/Akt/mTOR訊息傳導路徑活化並且也會引起肺癌細胞表現VEGF-C蛋白。經由LY294002 (PI3K抑制劑)及 rapamycin (mTOR抑制劑)降低PI3K/Akt/mTOR訊息傳導路徑的活性,進而可抑制放射線引起VEGF-C蛋白表現。
另外利Akt siRNA 可抑制A549肺癌細胞因放射線引起mTOR磷酸化及VEGF-C蛋白表現,因而證明放射線引起VEGF-C蛋白表現是經由PI3K/Akt/mTOR訊息傳導路徑活化。然而將經放射線照射A549肺癌細胞的培養液放置於培養血管內皮細胞及淋巴內皮細胞可促使內皮細胞增生,此現象則不會表現於A549肺癌細胞接受VEGF-¬C siRNA處理,再經放射線照射後的培養液。因此我們的研究顯示放射線抗敏性的原因可能經過活化PI3K/Akt/mTOR訊息傳導路徑,進而引起VEGF-C蛋白表現並促使內皮細胞增生。
BEZ235是PI3K/Akt/mTOR訊息傳導路徑雙重抑制點的抑制劑。進一步使用
BEZ235合併放射治療可增加大腸直腸癌細胞凋亡效應,也可增加γ-H2AX (DNA雙股螺旋斷裂標記)。BEZ235不只抑制PI3K/Akt/mTOR訊息傳導路徑,同時也會抑制ATM and DNA-PKcs活性,進而增加放射治療敏感性。在動物實驗,和單獨BEZ235治療組或放射治療組,在BEZ235合併放射治療組,腫瘤大小是治療效果最好的及腫瘤p-mTOR, p-eIF4E and p-rpS6的表現是被抑制最多的。我們的研究顯示BEZ235合併放射治療在細胞及動物實驗都會增加放射治療效果。
所以造成放射線抗敏性的部分原因之一可能是經由放射線引起PI3K/Akt/mTOR訊息傳導路徑的保護機制,進而促使癌症細胞表現VEGF-C蛋白及內皮細胞增生。而PI3K/Akt/mTOR訊息傳導路徑抑制劑合併放射治療會增加放射治療效果。未來可藉由後續臨床實驗證明PI3K/Akt/mTOR抑制劑合併放射治療在癌症治療上的應用。


Radiotherapy (RT) is one of the major modalities of cancer treatment of patients with solid tumor, often in combination with surgery, chemotherapy or target therapy. Increasing radiosensitivity is the major strategy to improve the efficacy of RT. RT provides anti-tumor effect through its direct pro-apoptotic effect, but it also can induce radio-protective signal pathway including PI3K/Akt/mTOR pathway, one reason of radioresistance.
We demonstrated radiation-induced activation of PI3K/Akt/mTOR pathway and VEGF-C protein with dose- and time-dependent increase in A549 cancer cells. Radiation induced VEGF-C expression was down-regulated by LY294002 and rapamycin through attenuating the AKT/mTOR signaling pathway.
Radiation-induced Akt/mTOR phosphorylation and VEGF-C up-regulation in A549 cells were inhibited by Akt siRNA. It is indicating that PI3K/Akt/mTOR signaling mediates irradiation-induced VEGF-C expression. Furthermore, the irradiated supernatant promoted HUVEC and LEC proliferation, which was inhibited by VEGF-C-siRNA expression in A 549 cancer cells. The data demonstrated the mechanism of radioresistance through radiation-induced activation of PI3K/Akt/mTOR pathway, subsequently increasing VEGF-C expression in cancer cells and promoting endothelial cell proliferation.
Furthermore, the combination of dual PI3K/Akt/mTOR pathway inhibitor, BEZ235, pretreatment and radiation resulted in an increased cell apoptosis, and a significantly increased number of γ-H2AX/cell (DNA double strand breaks). BEZ235 not only inhibited PI3K/Akt/mTOR pathway but also inhibited ATM and DNA-PKcs activity to enhance radiosensitivity. In vivo model, the tumor size and the expression pattern of p-mTOR, p-eIF4E, and p-rpS6 were significantly decreased in combined group than radiation alone or BEZ235 alone. Our findings indicate that the administration of BEZ235 before radiation enhances the radiotherapeutic effect of colorectal cancer cells both in vitro and in vivo.
These encouraging data demonstrated the mechanism of radioresistance through radiation-induced activation of PI3K/Akt/mTOR pathway, subsequently increasing VEGF-C expression and promoting endothelial cell proliferation. The dual PI3K/Akt/mTOR inhibitor could enhance the radiotherapeutic effect. The further clinical study need to approve PI3K/Akt/mTOR inhibitor combination with RT in colorectal cancer treatment.


致謝……………………………………………………………………………….…..I
縮寫表………………………………………………………………………………..II
Abstract…………………………………………………………………………...... IV
中文摘要.…………………………………………………………………………... VI
Chapter1. Introduction
1.1 Radiotherapy in cancer treatment…………………………………………...1
1.2 The Role of PI3K/AKT/mTOR in cancers…………………………………..2
1.3 The Role of PI3K/AKT/mTOR in radioresistance………………………….4
1.4 The microenvironment and radioresistance………………………………...5
1.5 The Role of PI3K/AKT/mTOR in radiation-induced DNA repair………...6
1.6 Dual effect of PI3K/mTOR inhibitor as a radiosensitizer………………….8
Chapter 2. Radiation-induced VEGF-C Expression and Endothelial Cells Proliferation in lung cancer………………………………………………………..21
2.1 Abstract………………………………………………………………………22
2.2 中文摘要……………………………………………………………………...23
2.3 Introduction………………………………………………………………….24
2.4 Methods and Materials……………………………………………………...26
2.5 Results………………………………………………………………………..34
2.6 Discussion…………………………………………………………….………38
Chapter 3. Dual Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Inhibitor is an Effective Radiosensitizer for Colorectal Cancer………………... 49
3.1 Abstract……………………………………………………………………....50
3.2 中文摘要……………………………………………………………………...51
3.3 Introduction………………………………………………………………….52
3.4 Methods and Materials……………………………………………………...54
3.5 Results………………………………………………………………………..61
3.6 Discussion…………………………………………………………………….68
Chapter 4. Conclusion and Future perspective…………………………………...88
Publication…………………………………………………………………………..95
Reference…………………………………………………………………………….99


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