木樨草素抑癌活性與調控細胞組蛋白乙醯化之關聯研究

木樨草素(Luteolin, 3',4',5,7-tetrahydroxyflavone)是一種普遍存在於各種植物的類黃酮，屬類黃酮當中的黃酮，在傳統醫學裡被應用於治療各種疾病，包括高血壓、發炎和癌症。而組蛋白乙醯化可以調控基因的表現，許多癌症產生的原因可能涉及了由於組蛋白去乙醯化作用所產生的染色質濃縮，因而造成許多抑癌基因的轉錄抑制，然而，目前對於木樨草素所具有的抑癌作用與經由組蛋白修飾作用調控基因表現是否有關連性並不清楚；在本實驗中發現木樨草素(35 μM)可以誘導HL-60細胞走向細胞凋亡，為了證實其中的分子機制，在此利用西方點墨法、DNA電泳、聚合酶鏈鎖反應、反轉錄聚合酶鏈鎖反應、染色質免疫沉澱法等技術觀察，結果我們發現，木樨草素可以經由活化JNK MAPK pathway促使轉錄因子c-jun往核內移動，同時增加組蛋白乙醯基轉移酶：p300與FasL基因操縱子區域結合，造成FasL基因操縱子區域高度乙醯化，改變染色質結構而增加FasL轉錄活性，促使FasL新合成而活化Caspase 8、Caspase 3，並進一步造成PARP斷裂導致HL-60細胞走向細胞凋亡；另一方面，我們也發現，木樨草素也能藉由活化Caspase 7造成HDAC 3斷裂而抑制HDAC對組蛋白的去乙醯基酶活性，這可能是造成Fas基因操縱子區域的高度乙醯化而增加其轉錄活性，共同引發外源途徑的細胞凋亡。綜合以上結果，我們知道，木樨草素誘導HL-60血癌細胞細胞凋亡是透過活化訊息傳遞JNK/c-Jun signaling pathway與組蛋白的修飾作用。另外，我們也比較木樨草素對於固體腫瘤是否也具有類似的抗癌活性，發現對MDA-MB-231乳癌細胞處理低劑量(35 μM)木樨草素能夠引起細胞周期停滯，而處理高劑量(75 μM)木樨草素能夠引發細胞凋亡，而兩種劑量處理下都會引起組蛋白H3的乙醯化。綜合以上結果，本研究首度提出木樨草素的抗癌機制與組蛋白修飾作用有密切關係。

關鍵字

木樨草素；組蛋白乙醯化；細胞凋亡

並列摘要

Luteolin, 3',4',5,7-tetrahydroxyflavone, is a common flavonoid that exists in many types of plants including fruits, vegetables, and medicinal herbs. Plants rich in luteolin have been used in Chinese traditional medicine for treating various diseases such as hypertension, inflammatory disorders, and cancer. Histone acetylation can modulate gene expression. Many cancers are involved in the chromatin condensation caused by histone deacetylaton which makes transcriptional repression of several tumor suppressor genes. However, the relationship between histone modification mediating gene regulation and the mechanism of the luteolin-induced apoptosis have not yet been clarified. Here, we found that luteolin was able to induce HL-60 cells apoptosis. For further understanding of this molecular mechanism we applied western blot, DNA electrophoresis, PCR, RT-PCR and ChIP assay. As a result we discovered that luteolin activated JNK MAPK pathway facilitating c-jun transcription factor translocation from cytosol to nuclear which concomitant with integration of histone acetyltransferase, p300 to FasL promoter region. This caused hyperacetylation of FasL promoter and change chromatin structure which increased transcriptional activity of FasL. This de novo FasL induced caspase 8 and caspase 3 activation and PARP cleavage in regulating the luteolin-induced apoptosis. On the other hand, we also discovered, interestingly, that histone decetylase 3 (HDAC 3) was cleaved follow caspase 7 activation. This could be the main reason of the hyperacetylated Fas promoter which induced apoptotsis through extrinsic pathway. Taken together, luteolin induced HL-60 cells apoptosis through JNK/c-Jun signaling pathway and histone modification. Besides cystic tumor, we also evaluated the anti-tumor activity of luteolin on solid tumor. It showed that while 35 μM luteolin led to cell cycle arrest and 75μM luteolin induced apoptosis of MDA-MB-231 cells. No matter low or high dose of luteolin both can cause histone H3 acetylation. Overall, luteolim presents antitumor activity involving mediating histone H3 acetylation.

並列關鍵字

luteolin ； histone acetylation ； apoptosis

參考文獻

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木樨草素抑癌活性與調控細胞組蛋白乙醯化之關聯研究

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