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研究生:陸思穎
研究生(外文):Szu-Ying Lu
論文名稱:探討 α腎上腺素受體促進劑對肺癌細胞株的影響
論文名稱(外文):To study the effects and inhibitory mechanisms of an α2A- adrenergic receptor agonist on non-small cell lung cancer cells
指導教授:黃智生黃智生引用關係
指導教授(外文):Jason C Huang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:77
中文關鍵詞:肺癌α腎上腺素受體細胞自噬細胞凋亡
外文關鍵詞:lung cancerα2A- adrenergic receptor agonistAutophagyApoptosis
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肺癌死亡率為 85%,是世界上最常見的癌症,也是癌症死亡的主要原因。在所有肺癌中,非小細胞肺癌 (NSCLC) 佔 80% 至 85%,且大多數患者在初診時患有局部晚期或轉移性疾病。雖然目前已有新療法出現,例如:靶向療法和免疫檢查站阻斷;但是,治療癒後仍然不佳,其五年生存率約為 19.3%。
化合物 A(Compound A) 是一種小分子藥物,可以激活中樞神經系統 α-2A腎上腺素受體,以減少交感神經輸出並降低血管壓力。此藥物目前被用於治療過動症,、高血壓和偏頭痛的注意力不足症。在我們的實驗室中,已經發現化合物 A 可以抑制某些癌細胞中的細胞增殖和遷移。
先前的研究中,化合物 A 在腫瘤細胞中引起內質網壓力 (ER stress) 和細胞自噬 (autophagy)。然而,也有研究發現自噬會抑制細胞凋亡 (apoptosis)。在本篇研究中,我們研究了化合物 A 對人類非小細胞肺癌細胞株的作用。我們將 A549 和 H1299 細胞與化合物 A 作用後,利用細胞活性試驗和 Transwell 測定細胞移動能力分析發現化合物 A 可以抑制細胞遷移和增殖,且經由西方墨點法分析細胞轉移相關蛋白 N-cadherin、vimentin 隨著濃度增加而遞減的趨勢。而且,凋亡相關蛋白表現量 bax、cleaved-caspase3 和自噬相關蛋白 p62、LC3II 都有隨著濃度增加而遞增的趨勢,因此推測化合物 A 會誘導 A549 和 H1299 同時進行細胞自噬與凋亡;另外,化合物 A 還影響 A549 和 H1299中的細胞週期,我們利用流式細胞儀分析發現化合物 A 會使細胞停滯於 G0 期。接著,進一步探討化合物 A 對於 A549 和 H1299 的作用途徑,觀察到內質網壓力相關蛋白會隨著化合物 A 濃度增加而遞增;當我們於 A549 和 H1299 利用 4-苯基丁酸鈉 (Sodium phenylbutyrate; 4-PBA) 或牛磺脫氧膽酸 (Tauroursodeoxycholic acid; TUDCA) 來阻斷內質網壓力,再和化合物 A 作用,則自噬現象會減弱。若利用氯喹 (Chloroquine; CQ) 作為自噬抑制劑預處理後,化合物A 則可以更顯著地提升細胞凋亡的能力。總結而言,化合物 A 除了誘導 A549 和 H1299 使細胞停滯於 G0 期而抑制細胞生長,且會通過內質網壓力來激活細胞自噬;當 A549 和 H1299 先預做阻斷自噬處理後,可以提升化合物 A 的毒殺功能。當了解化合物 A 對於 A549 和 H1299 信號傳導途徑後,我們希望可以藉由阻斷細胞自噬加上化合物 A的處理能夠抑制腫瘤的生長,並能實現更有效的凋亡作用,使化合物 A 在癌症治療中能具有一定的醫療價值。
Lung cancer mortality is 85%, and it is the most commonly diagnosed cancer in the world and the leading cause of cancer death. Among all lung cancers, non-small cell lung cancer (NSCLC) accounts for 80% to 85%, and most patients have locally advanced or metastatic disease at the time of initial diagnosis. The emergence of new therapies, such as targeted therapy and immune checkpoint blockade, has improved patient survival. However, the prognosis still remain poor and its five-year survival rate is about 19.3%.
Compound A is a small molecule drug that can activate the central nervous system alpha-2 adrenoreceptors to reduce sympathetic outflow and reduce vascular tone. It was used to treat Attention Deficit Disorder with Hyperactivity, Gilles de la Tourette's Syndrome, hypertension and migraines. Compound A has been discovered that it can inhibit cell proliferation and migration in various cancer cells in our laboratory.
In our previous studies, because of abnormal proliferation in tumor cell, Compound A caused ER stress-induced autophagy in tumor culture. However, autophagy can antagonize apoptosis in other studies. In this study, we investigated the effects of Compound A in non-small cell lung cancer cells. After treating A549 and H1299 cells with compound A, we found that compound A can inhibit cell migration and proliferation by cell viability and Transwell assay, and the EMT-related protein levels increase in a dose-dependent manner like N-cadherin and vimentin by Western blot. Moreover, the expression levels of apoptosis-related proteins such as bax, cleaved-caspase3 and autophagy-related proteins p62 and LC3II were increased dose-dependently. Therefore, we suggest that compound A induced autophagy and apoptosis in A549 and H1299. In addition, compound A also induce cell arrest at G0 phase in A549 and H1299. Next, we observed that ER stress-related proteins were induced with increasing concentration. When we used 4-PBA or TUDCA to block the ER stress in A549 and H1299, autophagy was weakened. If A549 and H1299 were pretreated with chloroquine (CQ) as an autophagy inhibitor, compound A can significantly enhance apoptosis. In summary, we found that compound A induced cell arrest in A549 and H1299, inhibited cell growth, and activated autophagy through ER stress. After understanding the signal transduction pathway of compound A for A549 and H1299, we expect that treatment with compound A combined with blocking cell autophagy, can effectively inhibit cell growth, and induce high level of apoptosis. Compound A may provide an alternative for cancer therapy.
致謝 i
中文摘要 ii
Abstract iv
目錄 vi
第一章、緒論 1
第一節、肺癌 (Lung cancer) 1
第二節、細胞週期 (Cell Cycle) 2
第三節、上皮細胞間質轉化 (Epithelial- mesenchymal transition, EMT) 3
第四節、細胞死亡 (Cell death) 4
第五節、內質網壓力 (ER stress) 6
第六節、內質網壓力、細胞凋亡與細胞自噬的關係 (the connection of ER stress, apoptosis and autophagy) 6
第七節、α2A 腎上腺素受體促進劑 (Alpha- 2A adrenergic receptor agonist) 7
第八節、順鉑 Cisplatin 8
第九節、研究動機與目的 8
第二章、材料與方法 9
第一節、實驗材料 9
第二節、實驗方法 21
第三章、實驗結果 30
第一節、Compound A 抑制 A549 及 H1299 非小細胞肺癌細胞株增生及改變細胞型態 30
第二節、Compound A 抑制 A549 及 H1299 非小細胞肺癌細胞株的細胞移動能力 30
第三節、Compound A 使 A549 及 H1299 細胞株產生細胞週期停滯於 G0 期且 subG1 比例增加 31
第四節、Compound A 誘導 A549 及 H1299 細胞凋亡 32
第五節、Compound A 誘導 A549 及 H1299 細胞自噬 32
第六節、Compound A 於 A549 及 H1299 藉由內質網壓力來誘發細胞自噬 33
第七節、A549 及 H1299 在抑制細胞自噬後,Compound A 誘發更為顯著的細胞凋亡 34
第八節、不同劑量 Compound A 對種植 LLC1 細胞的 C57BL/6 小鼠腫瘤大小之影響 34
第九節、不同劑量 Compound A 加上 Cisplatin 共同處理 A549 在抑制細胞生長上有加乘的效果 35
第四章、實驗討論 36
第五章、參考文獻 41
第六章、實驗結果圖表 47
Figure 1 47
Figure 2 49
Figure 3 52
Figure 4 55
Figure 5 57
Figure 6 60
Figure 7 64
Figure 8 67
Figure 9 70
第七章、附錄 71
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