臺灣博碩士論文加值系統

在台灣，大腸癌為發生率第一高與死亡率第三高之癌症，因此尋找有效之治療大腸癌之藥物乃當務之急。轉錄因子STAT3在大腸癌中普遍具有持續性活化的現象，並與不良預後有重要關係，因此可作為一個有效之治療標的。Tanshione I為中藥丹參中之生物活性成分，並在許多癌症中顯示其抗癌能力，是一個極具抗癌潛力之天然物，然而，對於其抗大腸癌效應之研究仍非常缺乏。在我們研究中發現 Tanshinone I能有效抑制人類大腸癌細胞株之存活與群落生成能力，並誘發細胞凋亡。Tanshinone I 能抑制人類大腸癌細胞中持續性之STAT3活化，當我們在HCT116、DLD-1細胞株大量表達持續性活化STAT3能夠顯著拮抗Tanshinone I抑制之細胞凋亡，證實抑制STAT3活化是Tanshinone I誘導大腸癌細胞株細胞凋亡之必要機制。研究中發現Tanshinone I對JAK2活性沒有影響；但我們證實Tanshinone I能抑制大腸癌細胞株SRC的活性，並在大量表現SRC活性之穩定細胞株中，Tanshinone I抑制STAT3活性能力受到拮抗，證實Tanshinone I乃透過抑制SRC活性以壓抑STAT3活性進而促進細胞凋亡。西方墨點法結果顯示Tanshinone I能抑制STAT3下游之抗凋亡蛋白BCL-2及BCL-xL蛋白表現量；此外，在大量表達持續性活化STAT3之HCT116、DLD-1穩定細胞株中， BCL-2及BCL-xL無法被Tanshinone I所抑制，顯示Tanshinone I乃透過抑制STAT3活性以抑制BCL-2、BCL-xL之轉錄。我們將BCL-2、BCL-xL在HCT116、DLD-1中大量表現，西方墨點法及Annexin V assay結果顯示大量表達BCL-2、BCL-xL能拮抗Tanshinone I誘導之大腸癌細胞之凋亡，證明Tanshinone I抑制STAT3以調降BCL-2與BCL-xL，進而誘發細胞凋亡。在我們的研究中發現HT-29為唯一對Tanshinone I具有抗藥性之細胞株，並且Tanshinone I無法抑制其SRC與STAT3活性，此結果暗示抑制STAT3活化對Tanshinone I抗大腸癌效應之重要性。我們對HT-29給予Dasatinib-一種pan-SRC inhibitor，能有效誘發HT-29細胞凋亡以抑制HT-29生存；同時，我們共同給予Tanshinone I與Dasatinib後也發現，Dasatinib能明顯提昇HT-29對Tanshinone I之敏感性。綜上，我們證實Tanshinone I之抗大腸癌效應之機制為透過抑制SRC-STAT3-BCL-2/BCL-xL生存路徑以誘發細胞凋亡。

Colorectal cancer is the most commonly diagnosed cancer and the third cause of cancer mortality in Taiwan. Tanshinone I, one of the bioactive constituent of Salvia miltiorrhiza (Danshen), has been shown to have various pharmacological effects, including anti-inflammatory, anticancer, and cardio-cerebrovascular protection effects. However, the underlying mechanisms of anti-colorectal cancer effect of Tanshinone I are still poorly understood. Here, we reported that Tanshinone I induce apoptosis and markedly suppresse colony formation of colorectal cancer cell lines HCT116, DLD-1, and LoVo. Constitutive activation of signal transducer and activator of transcription 3 (STAT3) is highly correlated with colorectal cancer tumorigenesis and poor prognosis, implicating STAT3 as a promising drug target for colorectal cancer therapy. We found that Tanshinone I suppresse constitutive activation of STAT3, as evidenced by reduced levels of phosphorylated tyrosine 705 of STAT3 (activated STAT3). Notably, overexpression of constitutively active STAT3 mutant (STAT3-CA) reversed apoptosis of Tanshinone I-treated cells. Furthermore, Tanshinone I was found to reduce tyrosine 416 phosphorylation of SRC, suggesting that Tanshinone I inhibits STAT3 activation by repressing SRC activity. Additionally, we found that Tanshinone I downregulate BCL-2 and BCL-xL, two known transcriptional targets of STAT3. Immunobloting revealed that the protein levels of BCL-2 and BCL-xL reduced by Tanshinone I were reversed after STAT3-CA overexpression. Importantly, overexpression of BCL-2 or BCL-xL inhibited apoptosis and restored colony formation of Tanshinone I-treated cells. In this study, we found that HT-29 is the only colorectal cancer cell which has resistance to Tanshinone I. In addition, Tanshinone I doesn’t inhibit activation of SRC kinase and transcription factor STAT3.We demonstrated that the SRC inhibitor, Dasatinib, shown the capability to repress the viability of HT-29 cell, and to induce apoptosis in HT-29 cell.Last but not least, combination of Tanshinone I and Dasatinib synergistically suppress the viability of HT-29. In conclusion, we herein provide the first evidence supporting that suppression of SRC- STAT3-BCL-2/BCL-xL-mediated survival pathway accounts for the anti-colorectal cancer effect of Tanshinone I.

中文摘要 i
Abstract iii
前言（Introduction） 1
一、 大腸癌 1
二、 丹參酮I (Tanshinone I) 4
三、 STAT3 (Signaling transducer and activator of transcription 3) 5
四、 STAT3與大腸癌 6
五、 Src（Proto-oncogene tyrosine-protein kinase Src） 7
六、 細胞凋亡（Apoptosis） 8
七、 Bcl-2家族 9
研究目的（Aim） 11
實驗材料及試劑配方 (Materials) 12
一、 藥物 12
二、 試劑 (Buffer) 12
三、 質體建構（Plasmid construction） 14
四、 抗體配製 16
實驗方法 （Methods） 18
一、 細胞培養 （Cell lines and cell culture） 18
二、 細胞冷凍保存與解凍 19
三、 細胞存活率測試（Cell viability assay） 19
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、 Tanshinone I為抗大腸癌效應最佳之丹參酮成員 26
二、 Tanshinone I能有效抑制人類大腸癌細胞株群落生成能力 26
三、 Tanshinone I誘發大腸癌細胞株細胞凋亡以毒殺細胞 26
四、 Tanshinone I抑制大腸癌細胞株內持續性之STAT3活化 27
五、 Tanshinone I可抑制IL-6誘導之STAT3活化 28
六、 抑制STAT3活化為Tanshinone I誘發人類大腸癌細胞凋亡之必要機制 28
七、 Tanshinone I透過抑制SRC以抑制STAT3活化 29
八、 Tanshinone I透過SRC-STAT3路徑促使大腸癌細胞之細胞凋亡 29
九、 Tanshinone I抑制STAT3轉錄其下游基因BCL-2及BCL-xL 30
十、 Tanshinone I抑制STAT3以調降BCL-2及BCL-xL以誘發大腸癌細胞之凋亡 30
十一、 SRC抑制劑Dasatinib能有效抑制HT-29細胞株存活率 31
十二、 Tanshinone I可協同SRC抑制劑Dasatinib抑制HT-29存活 31
討論（Discussion） 32
一、 本論文首度發現之結果 32
二、 探討Tahshinone I之抗大腸癌潛力 32
三、 探討Tanshinone I對大腸癌細胞存活率之影響 34
四、 抑制轉錄因子STAT3活性在Tanshinone I誘導之人類大腸癌細胞凋亡之意義 35
五、 Tanshinone I透過抑制SRC以壓抑STAT3活化促使細胞凋亡 36
六、 Tanshinone I抑制STAT3轉錄下游標的基因BCL-2、BCL-xL，誘發大腸癌細胞凋亡 37
七、 SRC抑制劑Dasatinib能對HT-29細胞株造成細胞毒性，並能提高其對Tanshinone I之敏感性 38
八、 其餘可能機制之探討 38
結論 (Conclusion) 39
實驗結果圖表 (Results and Figures) 40
圖一 (A)、Tanshinone I有效抑制大腸癌細胞株之存活率 40
圖一 (B)、Tanshinone I無法抑制大腸癌細胞株HT-29之存活率 41
圖二、Tanshinone I有效抑制人類大腸癌細胞群落生成能力 42
圖三 (A)、Tanshinone I促使PARP截切活化影響細胞生存 43
圖三 (B)、在HT-29中Tanshinone I無法使PARP截切活化 44
圖四、Tanshinone I 處理促進人類大腸癌細胞株細胞凋亡 45
圖五(A)、Tanshinone I抑制多株人類大腸癌細胞STAT3持續性活化 46
圖五 (B)、Tanshinone I無法抑制HT-29細胞株中STAT3活性 47
圖六、Tanshinone I抑制人類大腸癌細胞株持續性STAT3活化 48
圖七、Tanshinone I能有效抑制誘導性STAT3活化 49
圖八、抑制STAT3之持續性活化為Tanshinone I誘導大腸癌細胞細胞凋亡之必要機制 50
圖九、抑制STAT3之持續性活化為Tanshinone I誘導大腸癌細胞細胞凋亡之必要機制 51
圖十、Tanshinone I並非透過JAK2路徑影響人類大腸癌細胞株中STAT3活性 52
圖十一 (A)、Tanshinone I抑制STAT3上游蛋白SRC磷酸化使人類大腸癌細胞株STAT3活性下降 53
圖十一 (B)、Tanshinone I無法抑制HT-29細胞株中SRC活性 54
圖十二、Tanshinone I透過SRC-STAT3路徑促使大腸癌細胞細胞凋亡 55
圖十三、Tanshinone I抑制STAT3下游人類大腸癌細胞株之抗凋亡蛋白 BCL-2、BCL-xL蛋白表現量 56
圖十四、在STAT3持續性活化之人類大腸癌細胞株HCT116、DLD-1中 Tanshinone I抗凋亡蛋白BCL-2、BCL-xL蛋白表現量 57
圖十五、在大量表現constitutively active BCL-2之人類大腸癌細胞中， Tanshinone I所誘導之細胞凋亡受到抑制 58
圖十六、在大量表現constitutively active BCL-2之人類大腸癌細胞中， Tanshinone I所誘導之細胞凋亡受到抑制 59
圖十七、在大量表現BCL-xL之人類大腸癌細胞株中，Tanshinone I所誘導之細胞凋亡受到抑制 60
圖十八、在大量表現BCL-xL之人類大腸癌細胞株中，Tanshinone I所誘導之細胞凋亡受到抑制 61
圖十九、SRC抑制劑Dasatinib能有效抑制HT-29細胞株存活率 62
圖二十、SRC抑制劑Dasatinib促使HT-29細胞PARP截切活化影響細胞生存 63
圖二十一、Tanshinone I協同SRC抑制劑Dasatinib抑制HT-29細胞生存 64
圖二十二、大腸癌細胞中內生性SRC活性 65
附錄（Appendix） 66
附錄一 Tanshinones 之化學結構與與抗癌機致 66
附錄二 STAT3路徑示意圖 67
附錄三 Apoptosis 路徑示意圖 68
參考文獻（References） 69

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