抑制轉錄因子 NF-κB 活化之微生物代謝物篩選研究

本實驗室長期從事微生物代謝物的探索研究，而從代謝物中篩選活性抗氧化劑即 是研究的項目之一。近年來有研究報告指出，某些抗氧化劑具有抑制轉錄因子 NF-κB 活性的能力，且此抑制作用將有助於不正常細胞的死亡。因此，本研究目的著重於建立 一個可以快速而且正確地篩選 NF-κB 活性抑制物的系統。我們先將 pIκB-EGFP 質體轉 染到人類肝癌細胞株 Huh-7 細胞內，此質體會持續在細胞質中產生 IκB 和 EGFP 融合蛋 白質，然後再使用抗氧化劑處理轉染細胞，並以流式細胞儀偵測細胞中 IκB-EGFP 蛋白 質的含量。若該物質為 IκB 裂解抑制劑，則原本將裂解消失的 IκB-EGFP 蛋白質，會因 為抗氧化劑之 IκB 降解抑制能力的不同，而偵測到不同的 EGFP 螢光，此螢光量的高低 便可用來反應抗氧化劑對 IκB 裂解抑制能力的強弱。從本實驗室先前篩選所得數種微生 物來源的抗氧化劑中，我們利用此系統選獲其中一具有抑制 IκB 降解能力的天然化合物

： hydroquinone (HQ) ，其抑制細胞內 IκB 降解的活性已進一步使用西方墨點法證實；

此外，我們亦建構含 luciferase 報導基因的質體，以期建立更敏感的篩選系統，達到大 量篩選 NF-κB 活性抑制物的目的。 HQ 經測試發現會影響腫瘤細胞的生長週期並導致 癌細胞死亡，而且我們偵測 HQ 對於動物體內血液細胞的毒性結果顯示， HQ 對於血小 板和白血球細胞並沒有顯著的毒殺活性，而我們比較 HQ 對 K-Balb 癌化細胞和 Balb-3T 3 不死化（ immortalize ）細胞株的細胞毒性，結果發現 HQ 會對癌化細胞有較高的毒殺 效果，這些特性將有利於 HQ 作為治療或預防癌症之應用。此外，我們亦觀察 HQ 搭配 抗癌藥物併用的細胞毒殺效果發現， HQ 結合抗癌藥物 mitomycin C 或 5-FU 並不能夠 增強對於癌細胞的毒殺效果，反而會降低抗癌藥物本身的毒殺能力，因此 HQ 應用在抗 癌藥物的發展，無論是單獨使用或與其他種類的抗癌藥物的併用，在未來均有深入檢討 的必要。

抑制轉錄因子 NF-κB 活化之微生物代謝物篩選研 究

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We have put effort on the discovery of microbial metabolites with biological activity for years, and one of our research interests is in search of antioxidants. It has been reported that certain antioxidants can also show NF-κB inhibitory activity. NF-κB activation was recognized to h ave function in promoting cell proliferation, in addition to resulting cell carcinogenesis by expressing some particular anti-apoptosis genes i n some abnormal cells. In this study, we have focused on the establishment of a rapid and precise screening system in search of natural prod ucts with NF-κB inhibitory activity. Our strategy was firstly focus on the construction of a reporter gene expression system, which contained a plasmid pIκB-EGFP inserted with a chimeric gene that encodes a fusion protein, green fluorescent protein (EGFP) fused with IkB-α(IκB-E GFP). The completed expression gene was afterwards transfected into Huh-7 human cancer cell line to produce the IκB-EGFP fusion protei n constantly in the cytoplasm. The IκB-EGFP was used as an indirect signal for diagnosing the level of NF-κB activation when IκB degradat ion was occurred. The transfected cells were treated with antioxidants of microbial origin, and the cells accumulated with IκB-EGFP fusion protein were then determined by flow cytometer. The expression level of IκB-EGFP fusion protein can reflect the degree of IκB degradation . If antioxidants being tested showed activity against IκB degradation, they would protect the IκB-EGFP from degradation and would result i n the increase of the level of fluorescence in the transfected cells. In the screening program, we obtained hydroquinone (HQ) which showed significant activity against IκB degradation. HQ was previously isolated from fermentation broth of streptomyces sp. in our laboratory as an antioxidant. Immunoblotting assay verified that HQ cause suppression of the degradation of IκB in tumor cell. We also found HQ could affe ct the cell cycle of tumor cell. Because HQ has NF-κB inhibitory activity, its character may provide a therapeutic effect for the treatment or prevention of cancer. Sequentially, we first determined the cytotoxicity activity of HQ to red blood cells, white blood cells and platelets of I CR mice. The results showed that HQ has no or low cytotoxicity activity to these blood cells. We also compared the HQ susceptibility of the immortalized cell line （ Balb-3T3 ） with oncogenic cell lines （ K-Balb ） . We found that HQ has higher cytotoxicity to K-Balb cancer cells than Balb-3T3 cells. The cytotoxic activities of anticancer drugs in combination with HQ were also determined. To treat CT26 cancer c ells with anticancer drug, mitomycin C or 5-fluorouracil combined with HQ, in vitro revealed that HQ could not enhance the antitumor effec t, on the contrary, decrease the cytotocixity of anticancer drugs being tested. Therefor,the issue of HQ for use solely or in combination with other anticancer drugs were remained to be furture discussed. We have also constructed a series of new plasmids that contained luciferase re porter gene of another option for drug screening. Due to the superior performance of luciferase, the drug screening system may be more sens itive to detect the inhibitors of NF-κB from natural resources.

Screening of Microbial Metabolites with Inhibitory

Activity against Transcription Factor NF-κB Activation