探討腫瘤生成相關的發炎反應中一氧化氮活化第二型拓樸異構酶參與之DNA裂解與突變的機制

Abstract

在慢性發炎反應中與氧自由基或氮自由基相關的分子(ROS & RNOS)已經被證實在癌症產生的過程中扮演重要的角色。而在我們實驗室以往的研究中指出，第二型拓樸異構酶(topoisomerase II, TOP2)會因為氧自由基的反應下，產生DNA與第二型拓樸異構酶複合體(topoisomerase II cleavage complex, TOP2cc)，導致DNA斷裂。因此在本篇的研究中，我們想要解釋在發炎的環境下，氮自由基中的一氧化氮(Nitric oxide, NO)會因為誘導TOP2參與的DNA損傷和染色體基因組的不穩定，而提高細胞癌化的風險。我們利用兩階段小鼠皮膚黑色素瘤形成的動物試驗模型來證明NO提供者，S-亞硝基穀胱甘肽（GSNO）與第二型拓樸異構酶藥物，Etoposide (VP-16)的處理下，可以誘發黑色素瘤的形成。另外，我們也發展細胞共培養系統來證實活化的巨噬細胞因為大量表現誘導型一氧化氮合成酶（iNOS），使得標的細胞染色體DNA斷裂與細胞訊息的活化(γ-H2AX & p53-ser15P)。在有無抑制藥物的條件下，也可以觀察到NO與TOP2的重要性。細胞毒性試驗則對於NO所誘發的反應提供一個有力的證據：在細胞可以忍受的NO毒性下，細胞可以對於NO產生的DNA斷裂進行修補的反應。因此，進一步我們利用DNA重組試驗，觀察到由於NO可以誘發TOP2參與的DNA斷裂，因此提高了DNA重組的頻率。最後我們利用TOP2β基因剔除小鼠來檢查TOP2同工異構酶在NO的處理下，確實可以降低因為NO誘發TOP2引起的DNA損傷和染色體基因組的不穩定，也因此降低小鼠皮膚黑色素瘤產生的風險。在本篇研究中，我們提供了第一個實驗證據證明NO誘導TOP2參與的DNA損傷，會提高DNA突變和細胞癌化的風險。這些研究有助於以分子生物學的角度解釋慢性發炎反應與癌症之間的關係。

Aims: Both cancer-suppressing and cancer-promoting properties of reactive nitrogen and oxygen species (RNOS) have been suggested to play a role in tumor pathology, particularly those activities associated with chronic inflammation. Here, we address the impact of nitric oxide (NO) on the induction of DNA damage and genome instability with a specific focus on the involvement of topoisomerase II (TOP2). We also investigate the contribution of NO to the formation of skin melanoma in mice. Results: Similar to the TOP2-targeting drug, etoposide (VP-16), the NO-donor, S-nitrosoglutathione (GSNO), induces skin melanomas formation in 7,12-dimethyl- benz[a]anthracene (DMBA)-initiated mice. To explore the mechanism(s) underlying this NO-induced tumorigenesis, we use a co-culture model system to demonstrated that inflamed macrophages with inducible NO synthase (iNOS) expression cause γ-H2AX activation, p53 phosphorylation and chromosome DNA breaks in the target cells. Inhibitor experiments revealed that NO and TOP2 isozymes are responsible for the described above cellular phenotypes. Notably, NO unlike VP-16 preferentially induces the formation of TOP2β cleavable complexes (TOP2βcc) in cells. Moreover, GSNO induced TOP2-dependent DNA sequence rearrangements and cytotoxicity. Furthermore, the incidences of GSNO- and VP-16-induced skin melanomas were also observed to be lower in the skin-specific top2β-knockout mice. Our results suggest that TOP2 isozymes contribute to NO-induced mutagenesis and subsequent cancer development during chronic inflammation. Innovation and Conclusions: Here, we provide the first experimental evidence for the functional role of TOP2 in NO-caused DNA damage, mutagenesis and carcinogenesis. Notably, these studies contribute, in part, to our molecular understanding of the cancer-promoting actions of RNOS during chronic inflammation.