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On the Anti-STAT3 Decoy Oligonucleotides for Cancer Cell Theranostics
decoy oligonucleotide,cancer cell,theranostics,transcription factor,
|Publication Year :||2015|
oligodeoxynucleotide, dODN) 對於轉錄訊息傳遞活化子3 (signal transducer and activator of transcription 3, STAT3) 的親和力，並將此系統性方法之產物運用於癌細胞抑制，以及延伸發展成活化之轉錄因子的感測方法與靶向性診療系統。過度活化的STAT3 可導致癌細胞的抗凋亡表現與抗藥性，其過度表現的下游蛋白質在此過程扮演了重要的角色。以Bcl-xL 蛋白質為例，其已被證實會影響細胞凋亡機制並降低多柔比星 (doxorubicin, DOX) 抑制癌細胞的功效。為了阻礙過度活化的STAT3 之作用，目前廣泛使用的圈套寡核苷酸模仿了核酸中與轉錄因子結合的啟動子 (promoter) 序列，可抑制轉錄因子並進而促進癌細胞進入凋亡程序。事實上圈套寡核苷酸可以被視為一種細胞內核酸抗體，其透過結構（雙股螺旋）以及特定鹼基序列達到選擇性蛋白質結合。借鏡適體 (aptamer) 的發展概念，本研究的目標為藉由系統性方法重新設計STAT3 圈套寡核苷酸的鹼基序列，讓其擁有更高的轉錄因子親合力，進而更有效地抑制異常活化的轉錄因子。本研究所提出的系統性方法結合了網路伺服器運算 (PiDNA)、寡核苷酸親合力試驗以及寡核苷酸細胞抑制試驗。該系統性方法的產物 (命名為hpdODN-Pi) 較目前廣泛使用的序列具備 (1) 10%~29%的STAT3 親合力提升，(2) 1.7~2.3 倍的選擇性提升 (STAT3-to-STAT1)，(3) 約20% 的抗凋亡蛋白質負調控能力提升，以及(4) 約20% 的癌細胞抑制效力提升。相較於hpdODN-Pi 成功抑制癌細胞，其對纖維母細胞（對照組，沒有STAT3 過度活化）則不具顯著作用，顯示hpdODN-Pi可選擇性抑制STAT3 過度活化之癌細胞。
基於hpdODN-Pi 的親合力特性，本研究提出一個運用hpdODN-Pi 與螢光分子形成之複合物 (complex of hpdODN-Pi and dye, 簡稱PiD) 感測活化之轉錄因子的方法(in situ)。當此複合物遭遇活化之轉錄因子時，嵌入於hpdODN-Pi 雙股序列中的螢光分子 (SYBR green, 或 Quinacrine) 便會因轉錄因子的結合競爭（轉錄因子對序列的親合力高於螢光分子）而自圈套寡核苷酸脫離，此導致的螢光訊號
變化便可用以定量感測活化的轉錄因子，感測線性區間為5 nM 到100 nM。直接給予活細胞PiD 時，因活化的轉錄因子觸發之螢光訊號變化亦可由螢光顯微鏡直接觀察，顯示PiD 具有類似細胞內蛋白質顯影的功能。
根據hpdODN-Pi 的高STAT3 親合力與PiD 的成果，本研究提出以
hpdODN-Pi 和DOX 形成之複合物 (DOX-intercalated hpdODN-Pi, 簡稱 DOXON)對STAT3 異常活化之癌細胞進行靶向性診療的概念與方法。靶向性診療系統需具備三個必要機制，包含辨識目標、發出訊號、以及觸發藥物作用。DOXON 以hpdODN-Pi 辨識活化之STAT3 蛋白質並與其進行結合，嵌入的DOX 因為結合競爭而被釋放至細胞質中，在488 nm 光激發下可發出590 nm 螢光訊號。因STAT3的核酸結合位被hpdODN-Pi 佔據，導致細胞的抗凋亡基因表現下降，而且被釋放
的DOX 進入細胞核並嵌入核酸中，促使細胞啟動凋亡機制。其中，由於hpdODN-Pi 的基因調節與DOX 的機轉可產生協同作用，所以複合物的癌細胞抑制效果大於各別的單獨抑制效果總合。此外，DOXON 只於STAT3 過度活化之細胞內產生作用（如MCF-7 和HepG2 細胞），反之則沒有效果(如心肌細胞和纖維母細胞），證實了DOXON 為一有效的靶向性診療系統。本研究的貢獻在於提出並驗證了圈套寡核苷酸可經由系統性方法重新設計以提高其親合力與選擇性，進而提升圈套寡核苷酸抑制癌細胞的功效。此外，本研究根據其親合力特性發展出的轉錄因子感測方法與靶向性診療系統，發掘出了圈套寡核苷酸的多功能性，讓其不限縮於疾病治療研究而可更進一步運用於感測和診療。上述的研究成果亦可望推廣至其它種類的轉錄因子，促進圈套寡核苷酸在治療、診斷以及診療的研究發展。
This study designed and proved a process that enhances the potency of the decoy oligodeoxynucleotide (dODN) against the signal transducer and activator 3 (STAT3), which is kind of oncogenic transcription factor (TF) in adult human cell. Furthermore, we discovered the undisclosed potential of the potency-enhanced STAT3-dODN in the diagnostics and theranostics. The results point out that the dODN is not only a therapeutic agent, but also is worthwhile to be researched in the applications relative to the diagnostic, and theranostic use.
Over-activated STAT3 plays an important role in a variety of malignant tumour. It not only facilitates the cancer proliferation and metastasis, but also decrease the susceptibility of the cell to chemotherapeutic agents through over-expressed anti-apoptotic proteins (e.g. cyclin D1 and Bcl-xL). To inhibit the abnormal STAT3 protein, small molecule inhibitors have achieved considerable outcome. However, they are usually involved in many other pathway and hence cause the undesired side effect. Searching for a drug with high specificity, effectiveness, and low cytotoxicity become an important issue. To this end, the double-stranded oligodeoxynucleotide that mimics the c-fos promoter sequence binding to the transcription factor is generated to block the activity of STAT3. Although the STAT3-dODN has much higher specificity, its binding affinity is not high enough to be a potent drug. Referring to the research idea of aptamer, we propose the first hypothesis in this study: the c-fos derived STAT3-dODN can be mutated and selected based on the binding affinity to discover a new dODN with higher cancer cell inhibitory potency.
In the first part of the study, we report an in silico and in vitro combined selection to quickly evolve a potent anti-STAT3 hairpin dODN (named hpdODN-Pi). On the basis of a protein-DNA interaction prediction web server (PiDNA) and the concept of aptamer selection, hpdODN-Pi is selected from a 109 dODN library within few days by performing (I) in silico library construction, (II) in silico selection, (III) in silico counter selection, (IV) in vitro affinity-based selection, and (V) in vitro function-based selection sequentially. Binding assays proved that hpdODN-Pi featured the best STAT3-binding affinity and STAT3-to-STAT1 selectivity among the dODNs reported to date. In the MCF-7 cell model, RT-qPCR confirmed that hpdODN-Pi effectively suppressed the downstream mRNAs of STAT3 without affecting STAT1-related expression after transfection. Accordingly, hpdODN-Pi showed twice higher cancer cell killing efficacy than the c-fos consensus dODN, and its enhanced apoptotic efficacy was confirmed by the Annexin V-PI (flow cytometry) and TUNEL (confocal microscopy) assays. In summary, STEP is a useful approach for rapidly identifying a potent TF-inhibitory dODN, and hpdODN-Pi can be further investigated for STAT3-related therapeutics. The overall result proved the proposed hypothesis.
The dODN and aptamer are the only two nucleotides that able to bind to the protein. On the basis of the feature, the aptamer have been used in the diagnostic sensor in many researches. However, the sensor-relative approach of dODN has not been discussed yet. Therefore, we propose the second hypothesis in this study: the dODN can be used in sensing transcription factor protein based on its high binding affinity.
In the second part of the study, we propose a dye-intercalated dODN system for detecting the oncogenic STAT3. This system is constructed with the hpdODN (the product from the first part of study) and the intercalated fluorescent dye. For in vitro STAT3 detection and intracellular STAT3 imaging, the signal comes from the release of intercalated dye due to the binding competition from STAT3. The fluorescence signal increases while the “signal-off” type of dye is used (e.g. SYBR green) and decrease while the “signal-on” type of molecule is used (e.g. Quinacrine). Either system show a detection range between 10 nM to 100 nM of protein. And also show the potential in cell imaging. Overall, the utility of hpdODN in protein sensing is proved.
One inspiring result is observed in the hpdODN-Quinacrine complex treated MCF-7 cell. The complex treated cell shows a much lower viability compared to the cells that treated with only hpdODN or Quinacrine. This result suggests the possible synergistic effect of the drug-intercalated hpdODN complex. Hence, we propose the third hypothesis: the dODN can be used in developing a selective complex for targeted co-delivery, STAT3 probing and synergistic anti-cancer effect.
In the third part of the study, we prove the idea by tackling STAT3-associated cancer cell lines with our DOXON complex, which is composed of a hpdODN and a broad-spectrum anti-cancer drug DOX that is also a structure-sensitive fluorescent reporter. Before internalisation, DOXON remains non-dissociated and does not emit fluorescence. After internalisation mainly through the pinocytosis and specific recognition of over-activated STAT3, DOXON dissociates into therapeutic dODN for STAT3 binding and releases DOX for taking anti-cancer effect. In addition, free DOX emits fluorescence indicating the presence of over-activated STAT3. Since the disassembly of DOXON is caused by STAT3 binding competition, the complex is selective and will not affect the normal cells without STAT3 over-activation. Moreover, the disassembled dODN and DOX exert dual pathway intervention and yield a synergistic anti-cancer effect.
|Appears in Collections:||生物機電工程學系|
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