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研究生:陳薇至
研究生(外文):Wei-Chih Chen
論文名稱:探討天然物薑黃素、紫檀芪及新穎化合物CSC-3436在三陰性乳癌之抗癌活性
論文名稱(外文):Investigation of the anti-tumor efficiency of natural products (curcumin and pterostilbene) and novel compound CSC-3436 in triple-negative breast cancer cells
指導教授:郭盛助郭盛助引用關係
指導教授(外文):Sheng-Chu Kuo
學位類別:博士
校院名稱:中國醫藥大學
系所名稱:癌症生物與藥物研發博士學位學程
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:2014
畢業學年度:102
語文別:英文
論文頁數:107
中文關鍵詞:三陰性乳癌上皮間質轉化doxorubicin薑黃素三陰性乳癌紫檀氏vimentinFas訊息細胞自噬三陰性乳癌tamoxifenCSC-3436細胞自噬現象轉換成細胞凋亡現象雌激素受體回復
外文關鍵詞:Triple negative breast cancerDoxorubicinEpithelial and mesenchymal transitionCurcuminTriple-negative breast cancerPterostilbeneVimentinFas signalingAutophagyTriple negative breast cancerTamoxifenCSC-3436Autophagy switch to apoptosisERalpha re-expression
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第一部份:
三陰性乳癌主要特徵為缺乏雌激素受體、黃體素受體與第二型人類表皮生長因子受體表現,因此無法適用於標靶治療,僅侷限於化學療法。使用化療藥物doxorubicin治療的癌細胞經常伴隨上皮間質轉化與抗藥性發生。本研究首先想探討doxorubicin是否會造成三陰性乳癌細胞發生上皮間質轉化。由結果可知,doxorubicin可藉由活化TGF-β及PI3K/AKT 訊息傳遞路徑改變細胞型態、降低E-cadherin表現與增加vimentin表現進而誘發三陰性乳癌細胞發生上皮間質轉化現象。為了抑制doxorubicin誘發上皮間質轉化現象。我們選用化學預防劑-薑黃素,探討薑黃素是否有效抑制doxorubicin誘發上皮間質轉化現象。結果顯示,薑黃素除了可以有效的阻斷TGF-β及PI3K/AKT 訊息傳遞路徑達成抑制doxorubicin誘發上皮間質轉化。薑黃素亦能增強doxorubicin抑制細胞生長能力。綜觀上述,合併處理薑黃素與doxorubicin可望成為有效治療三陰性乳癌細胞方式。
第二部份:
據臨床統計,高度表現vimentin (間質細胞標誌)與三陰性乳癌病人預後不佳有關,故vimentin被當成具潛力的生物標誌,應用於提昇三陰性乳癌治療。紫檀芪為白藜蘆醇的結構類似物,具有抑制細胞轉移入侵能力。因此,本研究欲探討紫檀芪是否具有抑制三陰性乳癌細胞發生上皮間質轉化之能力。結果顯示,紫檀芪僅能短暫地抑制三陰性乳癌細胞發生上皮間質轉化。探其原因為紫檀芪可剌激Fas訊息活化導致ERK1/2及GSK3β/β-catenin訊息路徑活化,並進一步驅使三陰性乳癌細胞發生上皮間質轉化。處理Fas抑制劑可有效維持紫檀芪抑制上皮間質轉移能力,亦可顯著增加細胞存活抑制率,由上述可知,由紫檀芪活化之Fas訊息為調控三陰性乳癌細胞上皮間質轉化因子之一。紫檀芪亦能誘發三陰性乳癌發生細胞自噬。處理細胞自噬抑制劑3-MA不僅能維持紫檀芪抑制上皮間質轉化的能力且能顯著性的增加細胞存活抑制率,由此可知由紫檀芪所誘發的細胞自噬在此扮演一個細胞保護的角色,同時也參與上皮間質轉化調控。綜觀上述,活化Fas訊息與誘導細胞自噬能增強三陰性乳癌惡性侵犯能力。故阻斷Fas訊息或抑制細胞自噬可望成為一個新的治療三陰性乳癌策略。
第三部份:
臨床上三陰性乳癌因缺乏特定療法,故找出有效療法為本研究之目的。研究指出,先誘發細胞產生保護性的細胞自噬,再抑制細胞自噬現象可以增強腫瘤細胞走向細胞凋亡路徑。本研究結果發現,Tamoxifen會使三陰性乳(MDA-MB-231)產生內質網壓力並活化AMPK/mTOR訊息傳導,進而誘發細胞自噬。CSC-3436為一具抗腫瘤活性的新穎化合物。CSC-3436可誘導ATG5-truncated,使癌細胞從細胞自噬轉換成細胞凋亡。本研究亦發現,CSC-3436可有效的回復雌激素受體表現,由螢火蟲冷光活性試驗及細胞存活率試驗可知,回復的雌激素受體可以有效的與雌激素及tamoxifen作用。綜觀上述,CSC-3436可有效的提高tamoxifen在三陰性乳癌細胞中抗腫瘤活性,其機制為 (i) CSC-3436可將tamoxifen所以發的細胞自噬轉換成細胞凋亡; (ii) CSC-3436可回復雌激素受體表現,增加三陰性乳癌細胞對tamoxifen之敏感性。CSC-3436值得更進一步研究,提供另一可行治療三陰性乳癌病患方法。

Part1:
Triple negative breast cancer (TNBC) is defined by a lack of expression of the estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER 2). Therefore, targeted therapy agents may not be used, and therapy is largely limited to chemotherapy. Doxorubicin-treatment consequently acquires undesired malignance characteristics (i.e., epithelial-mesenchymal transition (EMT) and multidrug resistance). Our results illustrated that doxorubicin triggered EMT and resulted in the acquisition of a mesenchymal phenotype in TNBC cells. Moreover, we found that TGF-β and PI3K/AKT signaling pathways were acquired for doxorubicin-induced EMT. Interestingly, we found that curcumin suppressed doxorubicin-induced EMT. Curcumin reversed doxorubicin-induced morphological changes, inhibited doxorubicin-induced downregulation of E-cadherin expressions and inhibited doxorubicin-induced up-regulation of vimentin expression. We also found that curcumin inhibited doxorubicin-induced EMT by inhibiting the TGF-β and PI3K/AKT signaling pathways. Moreover, curcumin enhanced the anti-proliferative effects of doxorubicin in TNBC cells. In summary, our results suggest that doxorubicin in combination with curcumin may be a potential therapy for TNBC.
Part2:
High expression of vimentin (canonical mesenchymal marker) links with poor prognosis in triple negative breast cancer (TNBC), implying that vimentin may be a potential biomarker in the application of TNBC therapy. Pterostilbene (PTE) shows anti-invasion activity, hence, we investigated whether PTE inhibited epithelial-mesenchymal transition (EMT) in TNBC. Here, we showed that PTE decreased vimentin level, but the effect was transient. PTE stimulated Fas signaling which drove EMT by ERK1/2 and GSK3β/β-catenin pathways, supporting Fas signaling induction involved in EMT regulation. PTE also triggered autophagy in TNBC. The treatment of TNBC with 3-MA (autophagy inhibitor) not only sustained PTE-inhibited EMT but also significantly promoted anti-proliferation, supporting that the autophagy played a cyto-protective role and was associated with EMT. Taken together, these data showed that Fas signaling and autophagy accelerated the aggressiveness of TNBC. Inhibition of autophagy or Fas signaling may provide novel targets for TNBC therapy.
Part3:
TNBC in breast cancer correlate with high aggressive characteristics, poor prognosis and lack of targeted use of therapies. Autophagy maintains energy homeostasis and plays a critical role in survival. Inhibition of autophagy sensitizes tumor cells undergo apoptosis. Hence, investigating the autophagy witch to apoptosis may prove a potential approach to improve efficacy of anticancer therapeutics. We assumed that novel compound, CSC-3436, improved tamoxifen effect in TNBC. Our findings demonstrated that tamoxifen induced autophagy via endoplasmic reticulum stress and AMPK/mTOR signaling. CSC-3436 could inhibit the effect of tamoxifen on AMPK/mTRO signaling and caused the cleavage of ATG5. Additionally, CSC-3436 could induce ER?? mRNA and protein re-expressions. Luciferase reporter gene assay and MTT assay showed that, upon exposure to CSC-3436, estrogen receptor-?? (ER??) re-expression became responsive to ER antagonist, tamoxifen. Taken together, CSC-3436 sensitized ER??-negative MDA-MB-231 breast cancer cells to tamoxifen may through two ways. One was that CSC-3436 contributes apoptosis switch to tamoxifen-induced. The other was that CSC-3436 restored functional ER?? re-expression which could be targeted by tamoxifen. As such, CSC-3436 combined with tamoxifen may be a potential approach for treatment ER??-negative breast cancer.


Acknowledge ⅰ
Abbreviations ⅱ
Brief Contents ⅲ
第一部份:
中文摘要 4
Abstract 5
1. Introduction 6
2. Materials and methods 8
2. 1. Cell lines and culture conditions 8
2. 2. Reagents and antibodies 8
2. 3. Western blot analysis 8
2. 4. Morphology observation 9
2. 5. Cellular Fractionation Analysis 9
2. 6. Growth inhibition assay 9
2. 7. Statistical analysis 10
3. Results 11
3. 1. EMT characteristics induction by doxorubicin in TNBC cells 11
3. 2. TGF-β signaling activation involved in doxorubicin-induced EMT 12
3. 3. Doxorubicin induced activation of β-catenin in TNBC cells 13
3. 4. Curcumin inhibited doxorubicin-induced EMT properties 14
3. 5. Curcumin inhibited doxorubicin-induced EMT by TGF-β and PI3K/AKT signaling pathways 14
3. 6. Curcumin sensitized the anti-proliferative effect of doxorubicin in BT-20 cells 15
4. Discussion 16
References 28
第二部份:
中文摘要 35
Abstract 36
1. Introduction 37
2. Materials and methods 40
2. 1. Cell culture 40
2. 2. Reagents and Antibodies 40
2. 3. Cell viability assay 41
2. 4. Morphology observation 41
2. 5. Real-time PCR 41
2. 6. Western blot analysis 42
2. 7. Statistical analysis 42
3. Results 43
3. 1. Anti-proliferation effect of PTE on TNBC cells 43
3. 2. PTE regulated EMT transiently in TNBC cells 43
3. 3. Fas signaling contributed to PTE transiently regulated EMT 44
3. 4. Activation of ERK1/2 related with PTE transiently regulated EMT 45
3. 5. Glycogen synthase kinase-3 beta (GSK3β) and ??-catenin involved in PTE transiently regulated EMT 46
3. 6. Autophagy induction involved in PTE transiently regulated EMT 46
4. Discussion 48
References 61
第三部份:
中文摘要 72
Abstract 73
1. Introduction 74
2. Materials and methods 76
2. 1. Cell culture 76
2. 2. Reagents and Antibodies 76
2. 3. Supravital cell staining with acridine orange (AO) detection 76
2. 4. Monodansylcadaverine staining 77
2. 5. Cell viability assay 77
2. 6. Western blot analysis 77
2. 7. Cell cycle analysis 78
2. 8. Real-time PCR 78
2. 9. RNA interference 79
2. 10. Luciferase activity assay 79
2. 11. Animal experiments 79
2. 12. Statistical analysis 80
3. Results 81
3. 1. Tamoxifen induced autophagy in TNBC cells 81
3. 2. Tamoxifen-induced AV formation and MDC-stained autophagosomes/ autophagic bodies 81
3. 3. Tamoxifen-induced autophagy related with ER stress and AMPK/mTOR pathway 82
3. 4. CSC-3436 plus tamoxifen enhanced cell death in MDA-MB-231 cells 82
3. 5. ATG-5 and AMPK/mTOR pathway involved in CSC-3436 switched tamoxifen- induced autophagy to apoptosis 83
3. 6. In vivo efficacy of the combination of tamoxifen and CSC-3436 in MDA-MB-231 tumor xenograft model 84
3. 7. CSC-3436 reactivated ER?? expression in MDA-MB-231 cells 84
4. Discussion 86
References 100


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