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系統識別號 U0026-0308201320140300
論文名稱(中文) ARNT在抗癌藥物cisplatin誘導癌細胞死亡中所扮演的角色
論文名稱(英文) The potential role of ARNT in the regulation of cisplatin-induced cancer cell death
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 101
學期 2
出版年 102
研究生(中文) 詹雅衣
研究生(英文) Ya-Yi Chan
學號 S26001119
學位類別 碩士
語文別 英文
論文頁數 67頁
口試委員 指導教授-陳炳焜
口試委員-張文昌
口試委員-張雋曦
中文關鍵字 ARNT  MDR1  抗藥性 
英文關鍵字 ARNT  MDR1  drug resistance 
學科別分類
中文摘要 Aryl hydrocarbon receptor nuclear translocator (ARNT)屬於bHLH-PAS (basic helix-loop-helix-Per-ARNT-Sim)的成員,也叫做hypoxia-inducible factor-1β (HIF-1β),可和HIF-1α一起調控許多在缺氧環境中活化的基因,當細胞感受外來物質(xenobiotic compounds)時,ARNT也可和其他bHLH-PAS的成員形成異源雙體(heterodimer),並且ARNT 也是胚胎發育的重要因子,且可能與腫瘤的生長有關。在目前癌症的臨床治療中,對抗癌藥物產生的抗藥性是癌症治療失敗的主因之一,而其中主要用於許多癌症治療的抗癌藥物cisplatin,藉由造成癌細胞的DNA受損,使癌細胞凋亡,來達到治療效果,但在臨床上也已經有許多對cisplatin產生抗藥性的案例。本篇研究主要是探討ARNT是否會影響到癌細胞的抗藥性,並且又是藉由何種機制。由實驗結果可知,在抗藥性的細胞中給予cisplatin,ARNT仍可穩定表現,但對於cisplatin敏感的細胞,在給予cisplatin下,其ARNT表現會受到抑制。而我們也發現,在ARNT缺乏的細胞中,會造成藥物抗藥性的蛋白,例如multidrug resistance protein 1 (MDR1)會減少,並且ARNT是藉由調控MDR1的啟動子來影響MDR1的轉錄,ARNT可與Sp1形成複合物作用到MDR1啟動子上的Sp1 site來調控MDR1的轉錄,若利用ARNT small hairpin RNA (shRNA) 導致癌細胞ARNT減少,MDR1的啟動也會受到抑制,反之若ARNT大量表現,MDR1的啟動則會增加,而我們也證實ARNT的入核能力和活化轉錄能力會影響到MDR1的轉錄啟動。更進一步在ARNT缺乏的癌細胞中加入cisplatin,其細胞死亡有明顯增加的情形,若是將ARNT大量表現,細胞受到cisplatin毒殺死亡的情形則減少。本篇研究證實,ARNT可藉由影響到MDR1的表現而影響到對抗癌藥物的抗藥性,對於癌症治療的抗藥性提供了一個新的治療方向。
英文摘要 Aryl hydrocarbon receptor nuclear translocator (ARNT), which is known as hypoxia-inducible factor-1β (HIF-1β), belongs to bHLH-PAS (basic helix-loop-helix-Per-ARNT-Sim) family. The complex of ARNT/HIF-1α regulates hypoxic response gene. ARNT also forms heterodimer with family members in response to xenobiotic stimulation. In addition, ARNT is essential for embryonic development and is related to tumor growth. In clinical cancer therapy, drug resistance is one of the main obstacles. Cisplatin, which is a common anti-cancer drug, induces cancer cells DNA damage, resulting in induction of cell apoptosis. However, tumor resistance to cisplatin still occurs. The aim of this study is to investigate whether ARNT alters the sensitivity of cancer cells to anti-cancer drugs and mechanisms involved in this process. In our study, we found that the expression of ARNT was reduced in cisplatin-sensitive tumor cells, but not cisplatin-resistant cells under cisplatin treatment. We also found that multidrug resistance protein 1 (MDR1), which had been reported to produce P-glycoprotein (P-gp) and cause multidrug resistance in chemotherapy, was decreased in ARNT-knockdown cancer cells. Our data showed that ARNT regulated the MDR1 promoter activity through the interaction with Sp1 and the binding of ARNT/Sp1 complex to the promoter. MDR1 promoter activity was decreased in stable ARNT-knockdown cells (shARNT cells). On the contrary, overexpression of ARNT induced MDR1 promoter activity. In addition, the nuclear localization and transcription activity of ARNT were essential for MDR1 promoter activity. Moreover, Sp1 was essential for the binding of ARNT/Sp1 to Sp1 sites of MDR1 promoter to regulate promoter activity. Furthermore, cisplatin-induced cell death was increased in ARNT-silenced cancer cells and overexpression of ARNT reversed the effect of cisplatin on cell death. These results suggested that ARNT reduced the effect of anti-cancer drugs on tumor cell death by the regulation of MDR1 expression.
論文目次 Absract in Chinese I
Abstract II
Acknowledgement IV
Contents V
Index VI
Introduction 1
The regulation of ARNT in angiogenesis and embryonic development 1
ARNT is involved in cancer 2
The difference between ARNT and HIF-1α 2
Growth factor regulates ARNT in cancer cells 3
The mechanism of cisplatin resistance 4
Drug resistance and drug efflux transporters 4
Materials and Methods 6
Results 15
ARNT regulates MDR1 expression 16
ARNT regulates the promoter activity of MDR1 16
Sp1 sites of MDR1 promoter are essential for ARNT to regulate MDR1 promoter activity 17
ARNT-knockdown cancer cells are more sensitive to cisplatin 18
ROS is induced in ARNT-knockdown cells 20
Effect of nucleolin on EGF- and PMA-induced A549 cells migration 20
Cisplatin-induced cacner cell death was increased in nucleolin-knockdown cells 21
Discussion 22
The mechanisms that stimulate MDR1 expression causing drug resistance 22
The activity of ARNT to regulate MDR1 promoter in HONE-1 cells and HONE-1-C15 cells 24
Other drug efflux transporters and multidrug resistance 24
ROS and drug resistance in cancer 25
ARNT stability and drug resistance 26
Reference 28
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