利用高通量平台篩選天然藥物藥庫尋找肝癌特異性之NRF2抑制劑

抗藥性是癌症復發的主要原因之一，也是癌症治療成功與否的主要障礙。正常情況下，NRF2是一個主導抗氧化及解毒功能的轉錄因子，但從先前的研究中已證實NRF2與癌症抗藥性的發生有所關連，抑制NRF2可以使癌細胞對於抗癌藥物更為敏感。為了找到肝癌專一性NRF2抑制劑，我們選用肝癌細胞(Huh7)及正常的皮膚細胞(HaCaT)，並透過luciferase reporter assay的方式建立了高通量藥物篩選的平台，尋找只抑制肝癌細胞中NRF2活性的抑制劑。我們總共篩選了兩個植物藥庫，其中一個藥庫ISC，是由高醫藥學系陳益昇教授所提供，共計3,000個植物粗萃物，另一個藥庫KBCC，是由辜嚴倬雲植物保種中心所提供的，共計1,440個植物粗萃物。最後篩選結果發現，ISC藥庫中抑制活性最強的前三名，有一植物為華河瓊楠，其化學成分先前已由陳益昇教授進行研究過。華河瓊楠是一種樟科、常綠的喬木植物，先前的研究已經發現華河瓊楠的成份具有抗發炎的活性，我們再針對由華河瓊楠所分離出的23種化合物，進行NRF2的活性測試。木犀草素(Luteolin, Lut)為一個已知的NRF2抑制劑，會抑制Huh7和HaCaT兩種細胞的NRF2的活性。相較於木犀草素，華河瓊楠所分離出之化合物rel-(7R,8R,7′R,8′R)-3,4,3′,4′-dimethylene-dioxy-5,5′-dimethoxy-7,7′-epoxylignan (BT04) 可以顯著的抑制肝癌細胞(Huh7)中NRF2的活性，其IC50 為17μM。相對的，在正常的皮膚細胞(HaCaT)中，BT04能促進NRF2的活性，並有明顯的劑量依存關係。BT04能專一性的抑制Huh7的NRF2下游基因表現，而HaCaT則不受BT04的影響，其NRF2的下游基因表現維持不變或增加。從高內含影像分析的結果來看， Lut會造成細胞核及細胞質內的NRF2蛋白顯著減少，但BT04僅會造成細胞核內的蛋白質些微降低。在抗癌藥物與BT04合併使用的試驗中，結果顯示BT04能明顯增加cisplatin及sorafenib對肝癌細胞的毒殺作用。因此，我們的研究結果證實了從天然物中找尋癌症專一性NRF2抑制劑的可行性，如BT04。而這類的藥物有著能應用於改善癌症抗藥性的潛力。

關鍵字

高通量平台；肝癌

並列摘要

Drug resistance is the main cause of cancer recurrence and a major obstacle to the success of anticancer therapy. NRF2, a pivotal transcription factor regulates antioxidant response and detoxification, has been shown to participate in the development of cancer drug resistance. Functional suppression of NRF2 rendered cancer cell more susceptible to anticancer treatments. In order to find the specific NRF2 inhibitor in liver cancer, we established luciferase reporter assay-based high-throughput drug screening platforms in a liver cancer cell line-Huh7 and a normal skin cell line-HaCaT. Two natural product libraries-the ISC and KBCC libraries were used in this study. The ISC library which was provided by professor Ih-Sheng Chen contained 3,000 crude extracts from Taiwan indigenous plants. The plant materials of KBCC library was provided by Dr. Cecilia Koo Botanic Conservation Center. There are 1,440 crude extracts have been used in this study. Among the top three hits list, the chemical composition of Beilschmiedia tsangii Merr., a medium-sized evergreen tree, has been studied and there were 23 compounds isolated from the B. tsangii still accessible. We then identified rel-(7R,8R,7′R,8′R)-3,4,3′,4′-dimethylene-dioxy-5,5′-dimethoxy-7,7′-epoxylignan (BT04) significantly inhibited NRF2 activity in a liver cancer cell (Huh7) with an IC50 value of 17 μM, while it increased NRF2 activity dose-dependently in keratinocyte (HaCaT cell). By contrast, luteolin (Lut), a known NRF2 inhibitor, suppressed NRF2 activity in both Huh7 cell and HaCaT cell. Moreover, the mRNA level of NRF2 target genes were significantly decreased in Huh7 cells upon BT04 treatment, while those NRF2 target genes remained unchanged or even increased (HO-1) in BT04-treated HaCaT cells. High content image analysis revealed that, unlike Lut which reduced NRF2 expression both in the nuclei and cytoplasm, BT04 only caused a slight reduction in nuclear NRF2 expression. Furthermore, notable combinatory effects of BT04 and cisplatin or sorafenib were observed. Accordingly, our result suggested that BT04 can specifically inhibit NRF2 activity in liver cancer and could be a potential adjuvant to improve drug resistance.

並列關鍵字

high-throughput ； liver cancer ； NRF2

參考文獻

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