Canonical Wnt Signaling Pathway 小分子化學調節物細胞篩選平臺之建立

Wnt signaling pathway在多細胞動物中具有高度保留性。此訊息傳導途徑可於發育時期調節身體結構（body patterning），並且終身都具有決定幹細胞命運的能力。根據其對於特定組織幹細胞的影響力，在這些特定組織中癌症也隨著Wnt 訊息傳導途徑的異常活化而發生。此一關係顯示了尋找幹細胞及癌細胞之外來Wnt 訊息傳導途徑調節物的重要性及進一步運用於再生醫學及癌症治療的潛力。此研究的目的在於建立一個以細胞為平台的報導系統，此系統可做為一個強而有力並具對於Wnt 訊息傳導途徑專一性的指標，之後並依此系統篩選新的小分子調節物。在比較過兩種小鼠的纖維母細胞株後，3T3-L1細胞被選為建立Wnt 訊息傳導途徑報導系統的模式細胞株。我們所建立帶有穩定結合Wnt 訊息傳導途徑報導質體Super(8X)TopFlash的3T3-L1 B2細胞株，可以表現對於外來Wnt-3a分子在細胞數、作用時間、劑量上相關的的專一反應。在利用九個GSK3抑制劑及六個與Wnt 訊息傳導途徑不相關的已知化學小分子的預先篩選上，有四個GSK3抑制劑可以誘導冷光酶（luciferase）活性的產生，與Wnt 訊息傳導途徑不相關的化學小分子則皆不具有此能力，此結果確認了此系統在篩選Wnt/beta-catenin 訊息傳導途徑小分子活化物的應用性。當處理以連續稀釋的已知Wnt 訊息傳導途徑抑制物，包括槲黃素（Quercetin）及兒茶素益多酚（EGCG）後，由50％ L Wnt-3a條件培養液(conditioned medium)所誘導產生的冷光酶（luciferase）活性出現了具劑量相關的被抑制現象，說明了B2細胞株可應用在篩選Wnt/beta-catenin 訊息傳導途徑小分子抑制物的能力。在篩選過兩個系列的小分子，一個系列為合成的gamma-secretase 可能抑制物，另一為植物萃取物及其衍生物後，我們找到了兩個Wnt/beta-catenin 訊息傳導途徑的活化物及十五個抑制物。這兩個活化物名為SY202及SY203，十五個抑制物包括了九個合成的化學物及六個植物萃取物，九個合成的化學物分別名為Jia138、SY15、SY57、SY85、SY173、SY186、SY188、SY202、SY203，六個植物萃取物則包含CCL GS-SE、CCL IC-F、CCL kts、CCL ktf、CCL CD-X、CCL BB。在進一步的半定量（titration）實驗中，四個合成化學物包括SY186、SY188、SY202、SY203同時顯示了Wnt/beta-catenin 訊息傳導途徑活化物及抑制物的活性。根據這些合成化合物在之前的實驗中所表現的gamma-secretase抑制物活性，這些化合物也許可以做為一個探針，幫助我們進一步了解Wnt與Notch兩個訊息傳導途徑間的聯繫情形。經由後續對於這些新Wnt/beta-catenin 訊息傳導途徑調節物的確認及功能性分析，我們期待在不久的將來，這些新調節物能在幹細胞及癌症研究上有所貢獻。

關鍵字

Wnt 訊息傳導途徑；細胞篩選平台:小分子調節物

並列摘要

Wnt signaling pathway is highly conserved in metazoan. This pathway regulates body patterning during development and determinates stem cell fate throughout lifetime. With its influence in the stem cell from specific tissue, cancer ensues upon dysregulated activation of the Wnt pathway. This connection reveals the importance of searching extrinsic modulators of the Wnt signaling pathway in stem cells and cancer cells with potential applications in regenerative medicine and cancer therapy. The aim of this research is to establish a cell-based reporter system which can be used as a robust specific indicator for the canonical Wnt signaling pathway and to screen for small novel modulators. After comparing two mouse fibroblastoid cell lines, 3T3-L1 was chosen as a model cell line to establish the Wnt/beta-catenin signaling reporter system. The established 3T3-L1 B2 cell clone, which harbored a stably integrated Super (8X) TopFlash Wnt signaling reporter plasmid, exhibits a specific response to exogenous Wnt3A molecule in a cell number-, time- and dose-dependent manner. With the prescreening of known modulators, including 9 GSK3 inhibitors and 6 irrelevant small chemicals, 4 GSK3 inhibitors but no irrelevant small chemicals showed positive response, confirmed the practicable ability of this system in screening novel Wnt/beta-catenin signaling activators. When treated with serious diluted well-documented Wnt signaling inhibitors, Quercetin and EGCG, a dose-dependent inhibitory curve was revealed in the present of 50% L Wnt3a CM, illustrating the B2 cell clone can be applied in screening novel Wnt/beta-catenin signaling inhibitors. There are two activators and fourteen inhibitors of Wnt/beta-catenin signaling pathway found by the established system from the screening of two libraries including one synthesized compounds and another plant extracts. Four compounds showed a biphasic activity of Wnt signaling in the further titration. With the gamma-secretase inhibitor activity revealed in previous data, these compounds might be probes for investigation the crosstalk of Wnt/Notch signaling in the future. By further identification and functional assay of the new candidate modulators, we expect these new modulators could be used in stem cell and cancer research in the near future.

並列關鍵字

Wnt signaling pathway ； cell-based screening system ； small novel modulator(s)

參考文獻

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國際替代計量

Canonical Wnt Signaling Pathway 小分子化學調節物細胞篩選平臺之建立

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