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研究生:林思瑜
研究生(外文):Szu-Yu Lin
論文名稱:利用NF-κB 抑制劑Platonin增加乳癌對化學治療的敏感性
論文名稱(外文):The NF-κB Inhibitor Platonin Markedly Enhances Sensitivity of Breast Cancer Cells to Chemotherapeutic Agents
指導教授:劉俊仁劉俊仁引用關係
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗暨生物技術學系所
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:2014
畢業學年度:102
語文別:中文
論文頁數:87
中文關鍵詞:乳癌化學治療NF-kBPlatonin
外文關鍵詞:Breast cancerChemotherapeutic agentsNF-kBPlatonin
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  • 下載下載:7
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多種臨床抗癌藥物 (如Doxorubicin) 在施打過幾個療程後,往往會產生抗藥性導致治療困難及預後不佳;而先前研究顯示發炎微環境的生成,也是造成癌細胞抗藥性的重要因素之一。因此,本研究的目的在探討(1)抗癌藥物是否會誘導發炎微環境的生成;(2)藉由調節轉錄因子Nuclear factor-κB (NF-κB)是否可以改善發炎微環境形成;(3)具抗藥性的癌細胞是否能透過合併NF-κB抑制劑增加藥物的敏感性。本研究發現,使用抗乳癌藥物Doxorubicin處理乳癌細胞株SKBR3後,與活化發炎微環境形成有關的調節因子NF-κB,會受到活化而有劑量依賴性的影響;進一步分析也發現,Doxorubicin處理也會誘導促發炎細胞激素TNF-?恁BIL-1β及IL-6的產生,尤其是IL-6的分泌會隨Doxorubicin處理時間增長有明顯的上升。而且藉由合併NF-κB抑制劑Platonin及PDTC,能有效抑制Doxorubicin誘發的NF-κB活化及IL-6的表現。進一步實驗顯示,NF-κB 抑制劑也能有效阻斷受到自體分泌活化的IL-6路徑,如STAT3的磷酸化及其轉錄的相關蛋白質,包括抗凋亡蛋白Survivin、Bcl-xL及抗藥性蛋白MDR-1的表現。因抗凋亡蛋白下降的結果,在細胞毒性試驗中(SRB assay 及Trypan blue exclusion assay)可觀察到,合併Doxorubicin與NF-?羠抑制劑能有效增加活化細胞凋亡路徑的Caspase-3及PARP1表現,增強對乳癌細胞株SKBR3的毒殺能力。此外,對照於SKBR3的結果,分析野生型乳癌細胞株MCF-7及抗藥型MCF-7/ADR的IL-6及MDR-1表現狀況,發現MCF-7/ADR較MCF-7細胞株具有更高的IL-6及MDR-1表現。綜合以上結果顯示,透過合併Doxorubicin與NF-?羠抑制劑能有效降低發炎微環境的發生,進而避免癌細胞產生抗藥性問題,提高治療的效果。

Clinical data showed that most of patients receiving several cycles of anticancer drugs, such as doxorubicin, had the symptoms of drug resistance and poor prognosis. Previous studies also revealed that inflammatory microenvironment contributes to the development of drug resistance. Therefore, aims of this study are: explore (1) if anticancer drugs can induce inflammatory microenvironment formation; (2) if regulation of a key inflammatory related transcription factor nuclear factor kappa B (NF-κB) can mediate anticancer drug induced inflammation; and (3) if NF-κB inhibitors sensitize the drug-resistant cancer cell line toward anticancer drugs. In this study, NF-κB was activated in breast cancer cell line SKBR-3 by treatment with doxorubicin. Further study showed that secretion of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6) were be induced by doxorubicin treatment. Especially, the release level of IL-6 was increased while prolonged treatment of doxorubicin was applied. Combined treatment of Doxorubicin with two NF-κB inhibitors (platonin and PDTC) effectively blocked doxorubicin-induced NF-κB activation and IL-6 secretion. Furthermore, NF-κB inhibitors attenuated the stimulation of IL-6 through decreasing phosphorylated STAT3. This attenuation can affect the downstream signal transduction pathway of STAT3, which related to apoptosis and drug resistant. Proteins involved in these pathway including Survivin, Bcl-xL, and MDR-1. Consequently, combined treatment of doxorubicin with NF-κB inhibitors significantly increased the activation of Caspase-9, Caspase-3 and PARP, which enhanced sensitivity of SKBR-3 cells to Doxorubicin. In study of MCF-7/wt and MCF-7/ADR cell lines, RT-qPCR revealed high expression level of IL-6 and MDR-1 gene expression was detected in MCF-7/ADR cells compared to MCF-7/wt. All the data indicated that promotion of inflammatory microenvironment would occur after anticancer drug treatment and it would contribute to drug resistant. Blockage of inflammation tumor-associated microenviroment can enhance the sensitivity of cancer cells to anti cancer drugs.

目 錄 1
致 謝 iv
中文摘要 vi
Abstract vii
縮寫表 ix
第一章、研究背景 11
1.1. 慢性發炎與癌症的關係 11
1.1.1. 發炎微環境的形成 11
1.1.2. 發炎微環境與癌症的發展 12
1.1.3. 化學治療所誘發的發炎現象 12
1.2. NF-κB 調控發炎與癌症間的關係 13
1.2.1. NF-κB 在發炎反應的角色 14
1.2.2. 活化NF-κB的因素與路徑 15
1.2.3. NF-κB 與癌症的關係 16
1.3. NF-κB 抑制劑的抗癌機制 18
1.3.1. 調控細胞週期的分子機轉 19
1.3.2. 細胞凋亡的分子機轉 21
1.3.3. NF-κB抑制劑與癌症治療 22
1.4. Platonin與NF-κB抑制劑 24
1.4.1. NF-κB 抑制劑的侷限 24
1.4.2. Platonin的介紹 24
第二章、研究特定目標 26
第三章、材料與方法 28
3.1. 實驗材料 28
3.2. 實驗方法 36
第四章、結果 45
4.1. 抗癌藥物Doxorubicin誘發乳癌細胞SKBR3發炎微環境的形成 45
4.2. 合併NF-κB 抑制劑具有減緩發炎微環境的效果 45
4.3. 合併處理對促發炎因子基因表現的影響 46
4.4. 癌細胞對合併處理的敏感性 46
4.4.1. 乳癌細胞株SKBR3對Platonin及Doxorubicin的敏感性測試 46
4.4.2. 合併處理對癌細胞的毒殺能力 47
4.5. 合併處理對細胞週期(Cell cycle)的影響 48
4.6. 合併處理對促發炎因子IL-6活化路徑的影響 49
4.7. 合併處理誘發細胞凋亡 50
4.8. Platonin具抑制抗癌機制表現的潛力 50
第五章、討論 52
5.1. 抗癌用藥Doxorubicin所誘發的發炎微環境與NF-κB的關係 52
5.2. 抗癌藥物Doxorubicin與抗發炎藥物Platonin合併處理造成生長抑制 53
5.3. 合併處理對乳癌細胞株的促發炎因子IL-6路徑的影響與細胞週期的關係 54
5.4. IL-6路徑的活化及MDR-1的表現與合併治療增進細胞死亡的關係 57
第六章、結論與展望 58
第七章、參考文獻 59
第八章、圖表 66
圖一、 Doxorubicin處理誘發乳癌細胞株SKBR3中促發炎因子的表現。 66
圖二、 Doxorubicin誘導乳癌細胞株SKBR3之NF-κB的表現情形。 67
圖三、 Doxorubicin合併NF-κB抑制劑Platonin對乳癌細胞株SKBR3的影響。 69
圖四、 Doxorubicin合併NF-κB抑制劑PDTC及Platonin對IL-6基因的影響。 70
圖五、 乳癌細胞株SKBR3對Doxorubicin及Platonin的敏感性。 71
圖六、 Doxorubicin合併(A) PDTC或(B,C) Platonin對乳癌細胞株SKBR3的細胞抑制效果。 73
圖七、 Doxorubicin單一處理與合併Platonin處理對乳癌細胞株SKBR3之細胞週期作用。 75
圖八、 Doxorubicin合併Platonin處理對IL-6訊息傳遞路徑前期相關蛋白表現的影響。 76
圖九、 Doxorubicin合併Platonin處理對IL-6訊息傳遞路徑後期相關蛋白質表現的影響。 77
圖十、 Doxorubicin合併Platonin處理對細胞凋亡相關蛋白質表現的影響。 78
圖十一、 乳癌細胞株SKBR3使用Doxorubicin合併Platonin治療對抗藥性蛋白質MDR-1表現。 79
圖十二、 在高度Doxorubicin抗藥性的乳癌細胞株MCF7/ADR (Anthracycline Drug resistance) 中的發炎因子基因表現。 80
圖十三、 在高度Doxorubicin抗藥性的乳癌細胞株MCF7/ADR 處理Platonin對NF-κB, IL-6 and MDR-1基因的影響。 81
圖十四、 在高度Doxorubicin抗藥性的乳癌細胞株MCF7/ADR 處理Platonin對IL-6訊息傳遞路徑後期相關蛋白質表現的影響。 82
圖十五、 乳癌細胞株SKBR3在Doxorubicin或合併Platonin處理,乳癌細胞株SKBR3細胞型態變化。 84
圖十六、 乳癌細胞株SKBR3在Doxorubicin或合併Platonin處理,乳癌細胞株SKBR3螢光染色細胞型態變化。 85
表一、 Doxorubicin單一處理與合併Platonin處理對乳癌細胞株SKBR3之細胞週期作用 87


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