臺灣博碩士論文加值系統

在台灣因癌症而死亡的案例，居於十大死因之首。其中大腸癌更是占所有癌症的第三順位。目前臨床上治療大腸癌，利用化學療法來治療，但是許多的化療藥物對人體都有一定的負擔以及副作用，甚至出現了抗性。許多癌症研究中，漸漸趨向使用天然萃取物來治療與預防。並且於近期研究中有許多使用兩藥劑結合的方式來增加治療效果，也解決了部分臨床用藥的抗性問題。Liriodenine (Li) 是一個生物鹼，從蘭嶼烏心石葉部萃取出來的，具有良好的抗癌活性，可抑制大腸癌以及肝癌細胞的生長。但於癌症研究中，除了抑制癌細胞的生長外，更希望癌細胞能走向細胞凋亡。Valproic acid ( VPA ) 是一個已經用為臨床之抗癲癇藥物，對於病患的耐受性佳且安全性高。VPA 具有良好的抗癌活性，可以增強許多抗癌藥物作用。故本篇的目的，觀察 Li 以及 VPA 的共同作用下，VPA 是否能更加強 Li 的抑癌作用，並探討其機制路徑。本篇論文發現，共同處理 Li 與 VPA 可以影響到癌細胞，會經由增加凋亡蛋白 PARP-1 的裂解活性以及抑制抗凋亡蛋白 Bcl-2 的表現量而細胞凋亡。此機轉會透過影響粒線體膜電位、粒線體的電子傳遞鏈 Complex III 以及 NADPH Oxidase 增加細胞內 ROS 的含量，進一步的抑制磷酸化 Akt 的表現量。共同處理 Li 與 VPA 比之兩藥劑單獨處理，更能誘導大腸癌細胞走向凋亡。故 Li 以及 VPA 的共同處理，推測其具有潛力可作為治療大腸癌的治療方針。

Human colon cancer is one of the leading causes of cancer death in Taiwan. A large number of preclinical studies had examined various agents for the prevention and treatment of colon cancer. Recently, combination with two agents is the strategy of the cancer therapy. Liriodenine (Li), a 7-oxoaporphine alkaloid was isolated from the leaves of M. compressa. It had reported that Li showed prominent antiarrhythmic activity, antimicrobial, antibacterial, and antifungal activity, mutagenicity and antiplatelet actions. The effect of the Li responsed to cell cycle arrest in human hepatoma cells and colon cancer cells but not apoptosis. It was necessary to find the synergistic drug that could enhance the apoptotic effect of Li. Valproic acid (VPA), which was well publicly known as a first line and widely used antiepileptic agent, was well tolerated in most patients and had safety profile. In some studies, VPA could enhance effect of apoptosis in many drug of anti-cancer. In this study, we wanted to know whether the VPA could enhance the apoptotic effect of Li response in colon cancer DLD-1 cells, and evaluate the possible synergistic anticancer efficacy of Li/VPA combination treatment.This study found that the VPA can enhance effect of Li-induced apoptosis. It was also increased the expression of apoptotic protein (PARP cleavage) and decreased anti-apoptotic protein (Bcl-2). Li/VPA combination treatment enhanced the intracellular reactive oxygen species (ROS) via affected mitochondrial membrane potential, complex III of mitochondrial respiration chain, and NAPDH Oxidase. Furthermore, increase of ROS could inhibit the expression of phosphorylated Akt. In conclusion, VPA can enhance the effect of Li-induced apoptosis. This pathway induced apoptosis might via increase the ROS and decrease expression of P-Akt protein. Therefore, in this study suggested that Li/VPA combination treatment had the potential to be a strategy of colon cancer therapy.

中文摘要 I
英文摘要 II
第一節 前言 1
第二節 細胞凋亡 (Apoptosis) 2
一、細胞壞死 2
二、細胞自噬 2
三、細胞凋亡 2
第三節Liriodenine (Li) 6
第四節Valproic acid (VPA) 7
第五節 Reactive oxygen species (ROS) 8
第二章 研究目的 10
第三章 材料與方法 11
第一節 材料 11
一、使用的細胞株 11
二、其他藥品 11
三、軟體分析 14
第二節 方法 14
一、細胞培養─貼壁細胞 14
二、細胞活性測試(MTT assay) 15
三、蛋白質的萃取(總蛋白質) 15
四、蛋白質定量分析 16
五、西方點墨法 16
六、流式細胞儀 18
七、穩定表現 Akt phosphorylation overexpression 的細胞之建立 20
八、統計分析 21
第三節 配方 22
一、細胞培養液 22
二、蛋白質定量 22
三、蛋白質轉印配方 22
四、穩定表現 Akt phosphorylation overexpression 細胞之建立 23
第四章 結果 24
第一節 觀察 Li + VPA 對於大腸癌細胞 DLD-1 細胞存活率之影響 24
第二節Li + VPA 對於大腸癌細胞之細胞凋亡作用的影響 24
一、VPA 會加強 Li 誘導細胞凋亡的能力 24
二、Li + VPA 會影響凋亡蛋白 PARP-1 以及抗凋亡蛋白 Bcl-2 的表現量 26
三、Li + VPA 對於三株大腸癌細胞 DLD-1、SW480 以及 HT-29 之細胞凋亡的影響 26
第三節Li + VPA 對大腸癌細胞內 ROS 的影響 27
一、細胞內 ROS 含量的變化 27
二、細胞內 GSH 含量以及 GSH 合成酶的影響 28
三、Li + VPA 誘導細胞內的 ROS 增加是造成細胞凋亡的原因 29
四、Li + VPA 誘導的 ROS 是源自於粒線體 30
五、Li + VPA 所誘導的 ROS 是源自於電子傳遞練 Complex III 以及 NADPH Oxidase 31
第四節 處理 Li + VPA 影響 Akt 以及其下游 GSK 3/ 磷酸化的表現量 32
一、Li + VPA 會抑制 Akt 與 GSK 3/ 的磷酸化 32
二、Li + VPA 會藉由抑制細胞內 Akt 的磷酸化誘導細胞凋亡 33
第五節 Li + VPA 藉由增加細胞內 ROS，進一步抑制 Akt 的磷酸化而誘導細胞凋亡 34
第五章 討論 35
第七章 參考文獻 44
第八章 圖 50
圖一Liriodenine(Li) 的化學結構圖 50
圖二 Li + VPA 處理下對於大腸癌細胞 DLD-1的影響。 51
圖三 Li + VPA 處理下對於大腸癌細胞 DLD-1 細胞凋亡作用的影響。 52
圖四 Li + VPA 處理下對於大腸癌細胞 DLD-1 細胞凋亡的影響。 53
圖五 Li + VPA 處理下對於凋亡相關蛋白 PARP-1 以及 Bcl-2 表現量的影響。 54
圖六 Li + VPA 處理下對於三株大腸癌細胞 DLD-1、SW480 以及 HT-29 之細胞凋亡影響。 55
圖七 Li + VPA 處理下對大腸癌細胞內 ROS 的影響。 56
圖八 Li + VPA 處理下對大腸癌細胞內 GSH 耗空的影響。 57
圖九 Li + VPA處理下對大腸癌細胞內 GSH 合成酶的影響。 58
圖十 細胞內的 ROS 是 Li + VPA 誘導細胞凋亡的原因。 59
圖十一 Li + VPA 處理下對粒線體膜電位的影響。 60
圖十二 Li 與 VPA 的共同處理產生的 ROS 是來自於粒線體膜電位的增加。 61
圖十三 Li 以及 VPA 所誘導的 ROS 是源自於電子傳遞鍊 Complex III 以及 NADPH Oxidase。 62
圖十四 Li 與 VPA 的共同處理抑制 Akt 以及其下游 GSK 3/ 磷酸化的表現量。 63
圖十五 穩定表現 Akt 磷酸化的建立。 64
圖十六 Li 以及 VPA 經由抑制細胞內 Akt 的磷酸化誘導細胞凋亡。 65
圖十七 Li 以及 VPA 經由增加細胞內 ROS 含量，進一步抑制細胞內 Akt 的磷酸化而誘導細胞凋亡。 66
圖十八 Li + VPA 抗大腸癌之機轉圖 67

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