抑制轉錄因子NF-κB活化之微生物代謝物篩選研究

Screening of Microbial Metabolites with Inhibitory Activity against Transcription Factor NF-κB Activation

陳延順(Yen-Shun Chen)

指導教授 ： 許元勳

臺北醫學大學/醫學院/醫學科學研究所/碩士(2001年)

摘要

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-3T3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。

關鍵字

微生物 ； 抗氧化劑 ； 轉錄因子 ； 癌細胞 ； 細胞週期

並列摘要

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 have function in promoting cell proliferation, in addition to resulting cell carcinogenesis by expressing some particular anti-apoptosis genes in some abnormal cells. In this study, we have focused on the establishment of a rapid and precise screening system in search of natural products 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-EGFP). The completed expression gene was afterwards transfected into Huh-7 human cancer cell line to produce the IκB-EGFP fusion protein constantly in the cytoplasm. The IκB-EGFP was used as an indirect signal for diagnosing the level of NF-κB activation when IκB degradation 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 in 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 affect 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 ICR 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 cells with anticancer drug, mitomycin C or 5-fluorouracil combined with HQ, in vitro revealed that HQ could not enhance the antitumor effect, 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 reporter gene of another option for drug screening. Due to the superior performance of luciferase, the drug screening system may be more sensitive to detect the inhibitors of NF-κB from natural resources.

並列關鍵字

NF-κB ； Mitomycin C ； Hydroquinone ； IκB ； Cancer ； 5-FU ； microbial metabolites ； antioxidant

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