探討奈酚所影響之生理機能與拓樸異構酶之參與

Abstract

在人體中，拓樸異構酶(topoisomerase I/II/III, TOP1/2/3)在解決細胞進行複製、轉錄等過程時所伴隨產生之結構性立體空間障礙扮演了重要的角色，拓樸酶催化的反應主要要有三個步驟：和DNA結合，造成可逆的單股(TOP1/3)或雙股(TOP2)斷裂，再接回斷裂的DNA，完成反應循環。透過改變DNA的正負螺旋數目，拓樸異構酶可以幫助細胞進行與完成正常的生理功能。另外，其中TOP1及TOP2是許多抗癌藥物的標的物，這些藥物統稱為拓樸異構酶活性抑制劑(catalytic inhibitors);其中有部分化合物能造成拓墣異構酶可切割復合物(topoisomerase cleavable complex, TOPcc)而產生DNA斷裂，又特別稱為抗拓樸酶毒素(poisons)。奈酚(naphthoquinone, NQ)是一種普遍存在於植物、防腐劑、空氣汙染物當中的化學物質，有許多研究指出奈酚在細胞內具有兩種機制可以影響胞內蛋白：透過和蛋白質上的特定胺基酸形成共價鍵結或是藉由形成自由基而造成氧化壓力進而影響胞內蛋白。我們的研究發現1,2-奈酚(1,2-NQ)以及1,4-奈酚(1,4-NQ)在細胞內可以造成明顯的DNA傷害, 而這個傷害包含了活性含氧物種(reactive oxygen species, ROS)以及第二型拓樸酶β型(TOP2β)的參予。而在試管內以及細胞內我們也發現1,2奈酚及1,4-奈酚可以抑制第二型拓僕異構酶解決連環體DNA (catenane DNA)的能力,並且以奈酚之抗體證實哺乳類第二型拓樸異構酶是可以直接被奈酚修飾的。綜合以上，我們的結果證實1,2-奈酚及1,4-奈酚不管是在調控第二型拓樸酶所造成之DNA傷害或是抑制第二型拓樸異構酶在胞內之活性都有其參與角色，說明第二型拓樸酶參與奈酚影響的的生物機能與與傷害。

Topoisomerase I/II/III (TOP1/2/3) plays an important roles in solving topological problems during cellular DNA replication and transcription. There are three main reactions catalyzed by topoisomerase: binding to DNA, formation of reversible single strand DNA breakage (TOP1/3) or double strand DNA breakage (TOP2) and religation of the DNA breakage to complete the catalytic cycle. Topoisomerases help cells to operate their normal functions through changing the numbers of positive or negative supercoilings in DNA. Moreover, TOP1 and TOP2 are important targets for many anticancer drugs, these compounds are called “topoisomerase catalytic“ inhibitors. Some of these inhibitors can cause the formation of topoisomerase cleavable complex (TOPcc) and subsequently DNA damage, which are termed ”topoisomerase poisons”. Naphthoquinones (NQs) are chemical compounds that widely existed in plants, preservatives and air pollutant. Many researches indicated that NQs can affect cellular proteins and corresponding functions by two proposed mechanisms: by forming covalent linkage to proteins or by going through redox cycling to generate ROS then leading to protein damages. In my study, we found that 1,2-NQ and 1,4-NQ can cause DNA damage in cells, and the damage is possibly mediated through both ROS and the TOP2β isozyme. Importantly, we have also found that 1,2-NQ and 1,4-NQ inhibit the segregation process of chromosome DNA through inhibiting the decatenation activity of TOP2 both in vitro and in cells. The immuno blotting analyzes also showed that NQs can directly modify TOP2. Combining the above results, we suggested that 1,2-NQ and 1,4-NQ participate in the TOP2-mediated DNA damage and cause inhibition of TOP2 activity, thus contributing to the NQ-mediated cellular and biological responses.