- 學位論文
氣體性氮氧化物致肺癌惡化及轉移作用之研究
The studies of the effect of gaseous nitrogen oxides (NOx) on tumor promotion and malignant progression
摘要
第一章 氣體性氮氧化物(NOx)是室內外空氣中重要的污染物質之一。許多研究指出，NOx氣體經由其氧化特性及其自由基來造成肺組織的傷害。在我們先前的研究已證實NOx氣體可誘導人類肺纖維細胞MRC-5的增生。在本研究結果發現，NOx氣體誘導人類肺纖維細胞MRC-5的增生是經由活化cyclin-cdk complexes進而磷酸化Rb蛋白。本研究利用西方墨點法與免疫沉澱法證實：NOx氣體處理人類肺纖維細胞MRC-5後9小時可誘使細胞中cyclinA/cdk2、cyclinD1/cdk4、cyclinE/cdk2蛋白增加表現，Rb蛋白磷酸化增加，並且使cdk抑制蛋白p27、p16表現下降。這些結果顯示NOx氣體誘導人類肺纖維細胞MRC-5的增生是經由活化細胞週期蛋白cyclin-cdk complexes，進而磷酸化Rb蛋白，並且抑制cdk抑制蛋白。總而言之，NOx氣體經由刺激細胞週期進行而誘導人類肺纖維細胞的增生可能與空氣污染物質刺激肺部纖維化有關。 第二章 Benzo[a]pyrene (B[a]P) ，是一種多環芳香碳氫化合物(Polycyclic Aromatic Hydrocarbons, PAH)，它廣泛存在於空氣污染與香菸煙霧之中。B[a]P 已有研究指出會誘導人類肝細胞、B細胞、子宮頸細胞、巨噬細胞及大白鼠肺部細胞的凋謝死亡。氣體性氮氧化物(NOx)是另一廣泛存在於室內與室外的空氣污染物質，許多研究指出NOx 經由其氧化特性與自由基而造成肺臟組織的氧化性傷害。我們先前的研究已經證實NOx氣體經由刺激細胞週期進行而誘導人類肺纖維細胞的增生，本部份研究將證明在B[a]P 刺激下造成人類肺纖維細胞凋謝死亡下，氣體性氮氧化物(NOx)將扮演一個抑制的角色。分析細胞週期實驗結果發現，NOx可抑制B[a]P 誘導人類肺纖維細胞的凋謝死亡；此外在西方墨點法實驗結果發現，在B[a]P 誘導凋謝死亡的過程中，NOx可活化抑制凋謝死亡的蛋白(Bcl-2, Mcl-1)表現及抑制凋謝死亡蛋白(Bax, t-Bid, cytochrome c, FasL and caspases)的表現。而更進一步分析發現B[a]P 誘導細胞的凋謝死亡是經由JNK1/FasL與JNK1/p53的訊息路徑。總之，NOx抑制B[a]P 誘導人類肺纖維細胞的凋謝死亡是經由抑制JNK1的路徑以及活化抑制凋謝死亡的蛋白(Bcl-2, Mcl-1)表現。 第三章 空氣中的氮氧化物(NOx)在以前一直被認為僅從燃燒等廢氣產生，是一種體外污染物，最近有許多研究顯示，NOx與生物體中蛋白之相互作用扮演著重要的生理角色(1,2), NO在生體內可與superoxide產生peroxynitrite (3)，在cigarette smoke fracction中也發現peroxynitrite的產生(4)， peroxynitrite已被証實顯示具有神經毒性 (5,6)，上皮細胞傷害(7)及脂質過氧化作用(5)等，peroxynitrite也被證實為一種oxidative stress-inducing compound，能造成人類細胞DNA傷害(4,8),但此是否煙霧或空氣污染物中NOx造成肺癌之原因，仍未被證實，過去我們的研究中除了發現NOx會攻擊DNA產生8-NO2-G(9)外，我們也發現NOx能促進MRC-5細胞生長及S phase分佈增加(10)，本研究過去的工作發現氣體性NOx對人類肺纖維細胞MRC-5之增生作用，其作用係經由加強Cyclin/CDK活化及Rb磷酸化(11) ; 從以上結果及許多相關研究，顯示NOx可能有tumor promotion的作用，因此我們將進一步探討NOx在腫瘤促進作用的角色。由之前的結果顯示NOx有抑制B(a)P (benzo[a]pyrene)致細胞死亡之作用(12)，此作用經由抑制JNK1及增加Bcl-2及Mcl-1之途徑。因此本研究的工作將探討NOx是否有促進肺癌細胞侵入轉移及惡化作用，我們用NOx促進下探討A549肺癌細胞在migration及invasion作用及其在鼠體中之轉移作用及其機轉；緊接之工作在於釐清NOx所調控肺癌惡化增生及血管新生作用之詳細分子機轉，並深入研究Ras及RhoB在肺癌惡化過程中的角色，以確立NOx促進肺癌增生、惡化、轉移、侵入及血管增生之重要性，架構如附圖。 由以上的研究, 我們將首先提出NOx致癌作用之角色可能在於它的腫瘤促進作用及惡化轉移。
關鍵字
氣體性氮氧化物, 惡化, 轉移並列摘要
Part 1 Nitrogen oxides (NOx) are important indoor and outdoor air pollutants. Many studies have indicated that NOx gas causes lung tissue damage by its oxidation properties and its free radicals. In a previous study we demonstrated that NOx gas induced proliferation of human lung .broblast MRC-5 cells. In this study we show that NOx gas stimulates MRC-5 cell proliferation by Rb (retinoblastoma) phosphorylation via activation of cyclin-cell division protein kinase (cdk) complexes. Western blot and immunoprecipitation data showed that NOx gas increased the expressions of cyclinA/cdk2, cyclinD1/cdk4, and cyclinE/cdk2 complexes in the cells at 9 h after treatment. The levels of phospho-Rb were also increased and cdk inhibitors (CKIs) p27 and p16 were apparently decreased. These data suggested that NOx gas stimulates cell-cycle progression by Rb phosphorylation via activation of cyclin-cdk complexes and inhibition of CKIs. In conclusion, the NOx-gas that induced lung .broblast cell proliferation by stimulation of cellcycle progression may contribute to lung .brosis by NOx pollutants. Part 2 Benzo[a]pyrene (B[a]P) present in environmental pollution and cigarette smoke. B[a]P has been shown to induce apoptosis in hepatoma cells, human B cells, human ectocervical cells , macrophages and rat lungs. Nitrogen oxides (NOx) are other important indoor and outdoor air pollutants. Many studies have indicated that NOx gas causes lung tissue damage by its oxidative properties and its free-radicals. In our previous study we demonstrated that NOx gas induced proliferation of human lung fibroblast MRC-5 cells. In this study we showed that NOx gas inhibits B[a]P induced MRC-5 cells apoptosis by cell cycle analysis. Western blot data revealed that NOx gas increased the expressions of antiapoptosis proteins (Bcl-2 and Mcl-1) and decreased the expression of apoptosis proteins (Bax, t-Bid, cytochrome c, FasL and caspases) after B[a]P treatment. We further clarified that B[a]P induced MRC-5 cell apoptosis via JNK1/FasL and JNK1/p53 signals. In conclusion, NOx gas inhibited B[a]P-induced MRC-5 cells apoptosis via inhibition of JNK1 apoptosis pathway and induction of Bcl-2 and Mcl-1 antiapoptosis pathway. Part 3 Nitric oxide (NO), a short-lived radical molecule was considered as an environmental pollutant. Recent studies have demonstrated the biological importance of the interaction of NOx with proteins (1,2). Muller et al. provide evidences that peroxynitrite is formed in aqueous cigarette smoke fractions (3,4). Peroxynitrite, a reaction product of superoxide and nitric oxide, has been implicated in neurotoxicity (5,6), endothelial injury (7) and lipid peroxidative damage (8). Although much work has been performed characterizing the peroxynitrite, there is considerably less data on the cause of lung cancer exposure of NOx. In our previous study, we found that NOx caused DNA damage by the formation of 8-NO2-guanine in MRC-5 cells (9). We also provide evidences to suggest that stimulation of iNOS and accumulation of P53 by NOx gas in MRC-5 cells, we also found that gaseous NOx as a tumor promoter characterized by stimulation of cell proliferation (10) and inflammation via activation of Cyclin/CDK and increase phosphorylation of Rb (11) and gaseous NOx could repressed Benzo[a]pyrene induced MRC-5 cells apoptosis via inhibiting JNK1 signals (12). The present and future work, we designed to study the role of NOx on the tumor promotion via against carcinogens- mediated apoptosis and the role of NOx on the tumor metastasis and angiogenesis. Furthermore, we used A549 cells that exposed to NOx to study the role NOx in lung cancer metastasis and angiogenesis. The results of these studies may be used to firstly demonstrate the tumor promoting effect of NOx from air pollutant or cigarette on lung pathogenesis and tumor metastasis induction, respectively. Moreover, the results also explored that the NOx induced cell proliferation and blocked carcinogens-mediated apoptosis and NOx induced tumor metastasis, invasion and angiogenesis which may contribute to the lung pathogenic process, respectively.