天然物烯烷基對苯二酚[HQ17(3)]與硫醇基反應引發分子及細胞反應之研究

Abstract

先前研究發現烯烷基對苯二酚，10’(Z), 13’(E), 15’(E)-heptadecatrienylhydroquinone [HQ17(3)]為具有抗癌活性 (anti-cancer)的天然抗氧化小分子 (antioxidant)。已知HQ17(3)屬於不可逆的拓樸異構酶II抑制劑 [topoisomerase II (Topo II)]，可於HL-60血癌細胞中造成Topo II poison、引發氧化壓力及細胞毒性。本篇研究發現以高濃度HQ17(3) (10 μM)處理Huh7肝癌細胞時，同樣導致Topo II poison。進一步以質譜儀分析發現HQ17(3)與細胞內Topo IIα的半胱胺酸-427 (Cys-427)形成共價鍵結，HQ17(3)的處理並造成細胞中Topo IIα-DNA複合體增加、DNA雙股斷裂、數個DNA損傷基因的表現量上升及細胞凋亡。HQ17(3)也造成細胞氧化性傷害，然而以N-acetylcysteine抗氧化物前處理細胞，並無法有效拯救細胞的存活率，顯示Topo II poison為HQ17(3)造成Huh7細胞死亡的主要原因。 本論文另以人類皮膚纖維母細胞 (WS1)的研究模式，發現細胞經處理低濃度HQ17(3) (<1 μM)後，可降低過氧化氫所造成的氧化性傷害。HQ17(3)並透過修飾Kelch-like ECH associated protein 1 (Keap1)的半胱胺酸並活化Keap1所調控的nuclear factor-E2 p45-related factor 2 (Nrf2)轉錄因子。以質譜儀分析發現HQ17(3)與細胞內Keap1的Cys-77、-249、-288及-297形成共價鍵結。HQ17(3)的處理進而促進Nrf2活化並誘導Nrf2下游所調控的heme oxygenase-1抗氧化酵素的表現，因此引發Nrf2分子相關的細胞保護機制。 綜合以上結果，HQ17(3)此種結構的小分子可作為Topo II poison發展外，也可當作研發相關預防性化合物的參考。

We previously reported that the botanical compound 10’(Z), 13’(E), 15’(E)-heptadecatrienylhydroquinone [HQ17(3)], an antioxidant, possesses anti-cancer activity. The cytotoxicity of HQ17(3) toward HL-60 leukemia cells is attributed to irreversible topoisomerase II (Topo II) poisoning and induction of oxidative damages. This study further explored that the cytotoxicity of HQ17(3) (10 μM) in Huh7 hepatoma cells was attributed to Topo II poison. Proteomic analyses revealed that HQ17(3) reacted with Cys-427 of cellular Topo IIα. This HQ17(3) modification caused the accumulation of Topo II-DNA complexes, DNA strand breaks, consequently induced the expression of DNA damage-related genes and apoptosis. HQ17(3) also caused oxidative damages in Huh7 cells. However, pretreatment of N-acetylcysteine, the antioxidant, did not diminish HQ17(3)-induced cell death. These results suggest that the cytotoxicity of HQ17(3) is attributed significantly to Topo II poisoning. In the WS1 skin fibroblast cell model, HQ17(3), at low concentration, was demonstrated to diminish H2O2-caused oxidative damages. HQ17(3) was found to modify Kelch-like ECH associated protein 1 (Keap1) and activate nuclear factor-E2 p45-related factor 2 (Nrf2), a transcription factor involved in the cytoprotection pathway. Proteomic analyses indicated that HQ17(3) modified Cys-77、-249、-288 and -297 of cellular Keap1. The nuclear translocation of Nrf2 and increase of HO-1 expression, an anti-oxidation enzyme regulated by Nrf2, in WS1 cells upon HQ17(3) treatment were observed. The activated Nrf2 pathway was found to be involved in HQ17(3)-mediated cytoprotection. In conclusion, the structural feature of HQ17(3) could be used as a model for Topo II poison design as well as preventive agent design.