- 學位論文
硝基化合物 N-甲基-N'-硝基-N-亞硝基亞胺甲二胺 造成EB病毒再活化及其對鼻咽癌癌化貢獻之研究
Studies on the reactivation of Epstein-Barr virus by N-nitroso compound, N-methyl-N’-nitro-N-nitrosoguanidine, and the contributions on the carcinogenesis of nasopharyngeal carcinoma
摘要
在鼻咽癌高風險地區食物中，已知含有大量致癌物質亞硝基化合物（N-nitroso compounds），且流行病學研究也證實經常接觸亞硝基化合物是罹患鼻咽癌的危險因素。然而，亞硝基化合物如何促進鼻咽癌癌變的機制尚不清楚。此外，許多血清流行病學研究證實，重複且持續性的EB病毒（Epstein-Barr virus）再活化和鼻咽癌（nasopharyngeal carcinoma）癌化發展之間有很強的關聯性。這些研究暗示，亞硝基化合物可能不僅透過本身化學致癌性，還透過引發EB病毒再活化來促進鼻咽癌發展，然而這個重要觀點卻還沒有實際研究證實。因此，本研究最主要目的，著重於探討亞硝基化合物引發EB病毒再活化，以及促進鼻咽癌癌化發展之角色。 本研究首先測試七種亞硝基化合物，並使用潛伏感染EB病毒之NA和HA兩株鼻咽癌細胞株，以及相應無EB病毒感染之TW01和HONE-1鼻咽癌細胞株進行實驗，以探討亞硝基化合物是否引發EB病毒再活化，及病毒再活化對於細胞癌化之影響。此外，利用N-甲基-N'-硝基-N-亞硝基亞胺甲二胺（MNNG），進一步探討亞硝基化合物引發病毒再活化及細胞癌化的機制。本研究率先證明，亞硝基化合物，包括2種亞硝酰胺:N-甲基-N'-硝基-N-亞硝基亞胺甲二胺（MNNG）和N-乙基-N-亞硝基脲（ENU）及4種亞硝胺: N-亞硝基二甲胺（NDMA）、N-亞硝基二乙胺（NDEA）、N-亞硝基吡咯 （NPYR）和N-亞硝基嗎啉（NMOP），能誘導鼻咽癌細胞中EB病毒再活化。而且加入亞硝基化合物處理時，潛伏感染EB病毒的鼻咽癌細胞中微核（MN）形成顯著增加。此外，隨著MNNG 濃度增加，EB病毒再活化程度也隨之增加。雖然單次低濃度（0.1 μg/ml）MNNG處理並無法引發EB病毒明顯再活化，但重複以此一濃度MNNG處理時，卻可以顯著誘導病毒再活化。另外，低劑量MNNG （0.1 μg/ml）具有加強12-O-十四酰佛波-1, 3 -乙酸酯（TPA）和丁酸鈉（SB）引發病毒再活化的協同效應，這兩種化學成分存在於鼻咽癌高風險地區的草藥和食物來源中。而不論經過TPA / SB單獨或以MNNG共同處理，其細胞中的微核（MN）同時隨著EB病毒再活化而明顯增加。使用Zta的干擾RNA (siZta) 阻斷EB病毒再活化，微核顯著增加現象則隨之消失，顯示EB病毒再活化就是造成細胞中微核增加的成因。本研究也觀察到，將EB病毒感染的NA細胞以TPA / SB單獨或合併MNNG連續處理五代，其微核生成會隨著處理次數增加而累積增加。而EB病毒再活化明顯增加鼻咽癌細胞中DNA損傷標記 gamma-H2AX及活性氧自由基 (ROS) 的量，顯示EB病毒活化造成細胞中微核生成增加可能是因為DNA損傷造成。此外，EB病毒再活化也明顯增加NA細胞的遷移和侵襲能力，顯示亞硝基化合物可藉由誘發EB病毒再活化，而增加細胞微核生成、遷移和侵襲能力。 另一方面，本研究首度發現亞硝基化合物觸發EB病毒從潛伏期進行再活化之機制。MNNG啟動病毒再活化過程中，調控EB病毒再活化特早期蛋白Rta，其mRNA率先表現。而透過啟動子活性試驗證實，MNNG可以明顯活化Rta的啟動子（Rp），及增強Rta 蛋白對於Rta及Zta啟動子的轉錄活性。另外，活性氧自由基 (ROS) 拮抗劑，包括N-乙酰基-L-半胱氨酸（NAC）、過氧化氫酶和還原態穀胱甘肽，則能有效抑制MNNG和過氧化氫所誘導的EB病毒再活化，證明活性氧自由基 (ROS)是EB病毒再活化的重要促發者，並且在MNNG誘導EB病毒再活化中擔任中介角色。抑製劑試驗則揭示ATM、p38和JNK蛋白激酶受MNNG產生的活性氧自由基（ROS）所活化，造成 p53蛋白的磷酸化，並且參與MNNG誘導EB病毒再活化。此外，p53蛋白對於MNNG 誘導EB病毒再活化，及活化Rta啟動子（Rp）扮演關鍵角色。MNNG誘發的活性氧自由基（ROS）會造成 p53蛋白的磷酸化，使其在細胞核中累積，而後大量結合至Rta啟動子（Rp）上。這些結果顯示MNNG調控EB病毒從潛伏期進入再活化，其機制是透過刺激活性氧自由基（ROS）生成，而後藉由活化p53啟動Rta啟動子（Rp）。 本研究首度揭露亞硝基化合物引發鼻咽癌發展及惡化的可能機制。亞硝基化合物藉由誘導EB病毒再活化造成細胞基因體不穩定，同時也增加細胞遷移和侵襲的能力，可能因此促進鼻咽癌惡性進展。此外，我們的研究也首度發現亞硝基化合物調控EB病毒再活化的機制。亞硝基化合物透過ROS/p53/Rp的訊號傳遞，進而誘發EB病毒進入再活化，而抗氧化劑能有效抑制此一過程。因此，抗氧化劑的補充，可能是預防及治療EB病毒相關疾病的有效方法。
並列摘要
N-nitroso compounds (NOCs) are carcinogens and known abundant in foodstuffs from NPC high risk areas. Epidemiological studies have implicated that frequently contact with NOCs is a risk factor contributing to the development of nasopharyngeal carcinoma (NPC). However, the underlying mechanism of NOCs for the carcinogenesis of NPC is not fully understood. Moreover, a variety of seroepidemiological studies implicate a strong correlation between recurrent reactivation of Epstein-Barr virus (EBV) and the development of NPC. These studies imply a notion that NOCs may not only through its carcinogenic properties but also through induction of EBV reactivation contribute to the development of NPC, but this theoretical view has not been sufficiently supported by directly researching. The purpose of this study is to examine the effects of NOCs on EBV reactivation and NPC carcinogenesis. In this study, seven kinds of NOCs were examined on EBV reactivation in NPC cells. NPC cell lines latently infected with EBV, NA and HA, and the corresponding EBV-negative NPC cell lines, TW01 and HONE-1, were used as a model system to compare the effects of EBV reactivation by NOCs on NPC cells. N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) was futher employed to explore the mechanisms of EBV reactivation and genomic instability induced by NOCs. Firstly, we demonstrated that NOCs, including two nitrosamides, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N-nitrosourea (ENU), and four nitrosamines, N-nitrosodimethylamine (NDMA), nitrosamines N-nitrosodiethylamine (NDEA), N-nitrosopyrrolidine (NPYR) and N-nitrosomorpholine (NMOP), can induce EBV reactivation in EBV-positive NPC cells. The micronucleus (MN) formation was simultaneously increased in the treated EBV-positive NPC cells. Furthermore, the intensity of EBV reactivation was significantly increased with MNNG concentrations. Although a single treatment of low dose MNNG (0.1 μg/ml) did not induce discernible EBV reactivation, repeated treatments significantly induced viral reactivation. Additionally, low dose MNNG had a synergistic effect with 12-O-tetradecanoylphorbol-1, 3-acetate (TPA) and sodium butyrate (SB), which present in certain herbal medicines and food sources, on EBV reactivation. In EBV-positive NA cell, MN formation was dramatically increased as long as EBV reactivation was induced, no matter after treatment with MNNG alone, TPA/SB alone or in combination with both. Using siZta to block EBV reactivation, the concomitant increase of MN formation was diminished indicating that EBV reactivation is responsible for the increase of MN formation by MNNG. Accumulation of MN formation was observed in NA cells with the treated frequency of TPA/SB alone or in addition with MNNG. EBV reactivation markedly increased the levels of gamma-H2AX and ROS formation in NPC cells, suggesting induction of DNA damage may be responsible for the increase of MN formation by EBV reactivation. In addition, significant elevation in the ability of migration and invasiveness was concomitantly observed only in NA cells with 5 progressive passages, suggesting that MNNG enhanced the MN formation, migration and invasiveness of NPC cells via induction of EBV reactivation. Furthermore, we disclosed the mechanism by MNNG to trigger EBV reactivation from latency. We found that the expression of Rta mRNA was earlier than Zta mRNA on the reactivation by MNNG. Through promoter activity assay, MNNG was found to significantly induce the activation of Rta promoter (Rp) and enhance the Rta transcriptional activity on Rta and Zta promoters (Zp), suggesting MNNG initiates EBV reactivation through induction of Rp activation and ehances Rta transcriptional activity for futher induction of Rp and Zp. Importantly, ROS scavengers N-acetyl-L-cysteine (NAC), catalase and reduced glutathione inhibited EBV reactivation by MNNG and H2O2 treatment, indicating that ROS is an important trigger for EBV reactivation and mediates to MNNG-induced EBV reactivation. In addition, inhibitor experiments revealed ATM, p38 MAPK and JNK were activated by MNNG-induced ROS and involved in EBV reactivation. We also demonstrated that p53 was essential for EBV reactivation and Rp activation by MNNG. The p53 was phosphorylated, translocated into nucleus, and abundantly bound to Rp following MNNG-induced ROS stimulation, further supporting that the ROS-mediated p53-dependent mechanism is critical for regulation of EBV reactivation by MNNG. Our findings firstly provide the evidence that N-nitroso compounds are capable of inducing EBV reactivation and consequently enhancing genomic instability, migration and invasiveness in NPC cells, which may contribute to NPC carcinogenesis. Futhermore, we demonstrated ROS/p53/Rp signaling pathway is critical for MNNG to induce EBV reactivation. Notably, this study indicates that antioxidants are effective for inhibiting N-nitroso compound-induced EBV reactivation and therefore could be promising preventive and therapeutic agents for EBV-associated diseases.