臺灣博碩士論文加值系統

多形膠質母細胞瘤（GBM）是目前最具有侵略性的原發性腦腫瘤，其預後較差，在很大程度上是由於腫瘤對目前的放射療法和替莫唑胺化學療法有較大的抵抗性。而信號轉導子和轉錄激活子3（STAT3）的大量表現被證明是GBM發病機理和治療耐藥性的關鍵驅動力，因此是有希望成為GBM藥物標靶。 5-乙酰氧基-6,7,8,4’-四甲氧基黃酮（5-Acetyloxy-6,7,8,4’–Tetramethoxyflavone；5-AcTMF）是橘皮苷的乙酰化衍生物，已被在乳癌，結腸癌，肺癌和多發性骨髓瘤被發現具有抗癌作用；但是，它對GBM的影響仍然不清楚。本研究中我們發現了5-AcTMF可以抑制了多種人類GBM細胞株中的存活和群落形成能力，並誘導了其凋亡。進一步的作用機制分析，顯示5-AcTMF可以抑制STAT3（p-STAT3）酪氨酸705磷酸化的（STAT3激活的典型標誌物），並且也抑制了白介素6引起的p-STAT3上調。值得注意的是，顯性活性STAT3的異位表達阻礙了5-AcTMF誘導的GBM細胞活力和群落形成性抑制以及凋亡誘導，這證實了阻斷STAT3 是5-AcTMF對GBM細胞存活和生長的抑製作用的必要條件。此外，5-AcTMF也阻斷了STAT3上游JAK2激酶的活化，但也以STAT3依賴性方式下調了抗凋亡BCL-2和BCL-xL。此外，BCL-2或BCL-xL的過表達會弱化了GBM細胞中5-AcTMF介導的生存力降低和凋亡誘導。總體而言，我們首次發現了5-AcTMF對GBM細胞的抗癌作用，這是通過阻止JAK2-STAT3-BCL-2/BCL-xL信號傳遞路徑來實現的。本研究顯示5-AcTMF對GBM具有治療的潛力。

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with poor prognosis, largely due to resistance to current radiotherapy and Temozolomide chemotherapy. The constitutive activation of Signal Transducer and Activator of Transcription 3 (STAT3) is evidenced as a pivotal driver of GBM pathogenesis and therapy resistance, and hence, is a promising GBM drug target. 5-Acetyloxy-6,7,8,4’-tetramethoxyflavone (5-AcTMF) is an acetylated derivative of Tangeretin which is known to exert anticancer effects on breast, colon, lung, and multiple myeloma; however, its effect on GBM remains elusive. Herein, we reported that 5-AcTMF suppressed the viability and clonogenicity along with inducing apoptosis in multiple human GBM cell lines. Mechanistic analyses further revealed that 5-AcTMF lowered the levels of Tyrosine 705-phosphorylated STAT3 (p-STAT3), a canonical marker of STAT3 activation, but also dampened p-STAT3 upregulation elicited by Interleukin-6. Notably, ectopic expression of dominant-active STAT3 impeded 5-AcTMF-induced suppression of viability and clonogenicity plus apoptosis induction in GBM cells, confirming the prerequisite of STAT3 blockage for the inhibitory action of 5-AcTMF on GBM cell survival and growth. Additionally, 5-AcTMF impaired the activation of STAT3 upstream kinase JAK2 but also downregulated antiapoptotic BCL-2 and BCL-xL in a STAT3-dependent manner. Moreover, the overexpression of either BCL-2 or BCL-xL abrogated 5-AcTMF-mediated viability reduction and apoptosis induction in GBM cells. Collectively, we, for the first time, revealed the anticancer effect of 5-AcTMF on GBM cells, which was executed via thwarting the JAK2-STAT3-BCL-2/BCL-xL signaling axis. Our findings further implicate the therapeutic potential of 5-AcTMF for GBM treatment.

目次
中文摘要………………………………………………………………………………i
Abstract………………………………………………………………………………ii
前言（Introduction）………………………………………………………………..1
一、多形性膠質母細胞瘤……………………………………………………………1
二、5-Acetyloxy-6,7,8,4’–Tetramethoxyflavone (5-AcTMF)……….…………….….3
三、STAT3 (Signaling transducer and activator of transcription 3)……………4
四、STAT3與多形性膠質母細胞瘤……………………………………………...6
五、JAK(Janus kinase)……………………………………………………………7
六、細胞凋亡（Apoptosis）……………………………………………………….9
七、Bcl-2家族……………………………………………………………………11
研究目的（Aim）…………………………………………………………………..13
實驗材料及試劑配方 (Materials)………………………………………………….14
一、藥物……………………………………………………………………………14
二、試劑 (Buffer) …………………………………………………………14
三、質體建構（Plasmid construction）…………………………………………..16
四、抗體配製………………………………………………………………………18
實驗方法（Methods）……………………………………………….……………….19
一、細胞培養 （Cell lines and cell culture）……………………..……………….19
二、細胞冷凍保存與解凍……………………………………..……………………19
三、細胞存活率測試（Cell viability assay）………...………………………….…20
四、細胞群落形成能力檢測（Colony formation assay）…………….…..……….20
五、細胞凋亡試驗 （Apoptosis assay）………………………………..…………20
六、細胞總量蛋白萃取（Whole protein extraction）……………….….…………21
七、蛋白濃度定量分析（Protein quantification）………………...………………21
八、西方墨點法（Western blotting）……………………..……………….………22
九、大腸桿菌質體轉型作用（Transformation）………..…………………….......24
十、病毒製備與病毒感染（Retrovirus and Lentivirus production and infection） ………………………………………..…………………………………….…...…24
結果 (Results)…………………………………………………………………….. 26
一、5-AcTMF有效抑制多形膠質母細胞瘤細胞株之存活率.......................26
二、5-AcTMF能有效抑制人類多形膠質母細胞瘤細胞株群落生成能
力……………………………………………………….………………26
三、5-AcTMF誘發多形膠質母細胞瘤細胞株細胞凋亡以毒殺細
胞…………………………………………………..………………………...26
四、5-AcTMF抑制多形膠質母細胞瘤細胞株內持續性之STAT3活
化…………………………………..…………………………………………27
五、5-AcTMF可抑制IL-6誘導之STAT3活化………..…………27
六、抑制STAT3活化為5-AcTMF誘發人類多形膠質母細胞瘤細胞凋亡之必要機
制………………………………………………………….…………………27
七、5-AcTMF透過抑制JAK2以抑制STAT3活化……………………28
八、5-AcTMF抑制STAT3轉錄其下游基因BCL-2及BCL-xL………………28
九、5-AcTMF抑制STAT3以調降BCL-2及BCL-xL以誘發人類多形膠質母細胞
瘤之凋亡…………………………………………………………29
討論（Discussion）…………………………………………………………….…..30
一、本論文首度發現之結果………………………………………………………..30
二、探討STAT3對人類多形膠質母細胞瘤的影響……………………………30
三、探討5-AcTMF對血腦屏障（BBB）的作用…………………………………….31
四、探討5-AcTMF抑制STAT3是否引發其他保護機制……….……..31
五、其餘可能機制之探討………………………………………………….31
結論 (Conclusion) ………………………………………………………………33
實驗結果圖表 (Results and Figures) ……………………………………………..34
圖一、5-AcTMF有效抑制多形膠質母細胞瘤細胞株之存活率…………………34
圖二、5-AcTMF有效抑制人類多形膠質母細胞瘤細胞群落生成能力…………35
圖三、5-AcTMF促使PARP截切活化影響細胞生存…….………………….36
圖四、5-AcTMF抑制多株人類多形膠質母細胞瘤細胞STAT3持續性活化….….37
圖五、5-AcTMF有效抑制誘導性STAT3活化…………….………………..39
圖六、抑制STAT3之持續性活化為5-AcTMF誘導人類多形膠質母細胞瘤
細胞凋亡之必要機制……………………………………………………….40
圖七、抑制STAT3之持續性活化為 5-AcTMF誘導多形膠質母細
胞瘤細胞凋亡之必要機制………………………………………….41
圖八、抑制STAT3之持續性活化為5-AcTMF抑制多形膠質母細胞瘤細胞株
細胞存活率之必要機制…………………………………………………42
圖九、5-AcTMF抑制STAT3上游蛋白JAK2磷酸化使人類多形膠質母細胞瘤細胞
株STAT3活性下降…………………...…………………………………43
圖十、5-AcTMF抑制STAT3下游人類多形膠質母細胞瘤細胞株之抗凋亡蛋白
BCL-2、BCL-xL蛋白表現量…………….……….………………………..44
圖十一、在STAT3持續性活化之人類多形膠質母細胞瘤細胞株T98G中5-AcTMF
抗凋亡蛋白BCL-2、 BCL-xL蛋白表現量……………………….45
圖十二、在大量表現constitutively active BCL-2之人類多形膠質母細胞瘤細
胞中，5-AcTMF所誘導之細胞凋亡受到抑制……………………46
圖十三 、在大量表現constitutively active BCL-2之人類多形膠質母細胞瘤細
胞中，BCL-2之持續性活化為5-AcTMF抑制人類多形膠質母細胞瘤細胞
存活率之必要機制……………………………………………………….47
圖十四、在大量表現constitutively active BCL-2之人類多形膠質母細胞瘤細
胞中，5-AcTMF所誘導之細胞凋亡受到抑制…………………………48
圖十五、在大量表現constitutively active BCL-xL之人類多形膠質母細胞瘤細
胞中，5-AcTMF所誘導之細胞凋亡受到抑制…………………………49
圖十六、在大量表現constitutively active BCL-xL之人類多形膠質母細胞瘤細
胞中，BCL-xL之持續性活化為抑制人類多形膠質母細胞瘤細胞存活率之
必要機制……………………………………………………………….50
圖十七、在大量表現constitutively active BCL-xL之人類多形膠質母細胞瘤細
胞中，5-AcTMF所誘導之細胞凋亡受到抑制……………………………51
附錄…………………………………………………………………………………52
參考文獻………………………………………………………………. ………………55

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