Abstract
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.