methylnitronitrosoguanidine and Nasopharyngeal-Neoplasms

Seroepidemiological studies imply a correlation between Epstein-Barr virus (EBV) reactivation and the development of nasopharyngeal carcinoma (NPC). N-nitroso compounds, phorbols, and butyrates are chemicals found in food and herb samples collected from NPC high-risk areas. These chemicals have been reported to be risk factors contributing to the development of NPC, however, the underlying mechanism is not fully understood. We have demonstrated previously that low dose N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.1 µg/ml) had a synergistic effect with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate (SB) in enhancing EBV reactivation and genome instability in NPC cells harboring EBV. Considering that residents in NPC high-risk areas may contact regularly with these chemical carcinogens, it is vital to elucidate the relation between chemicals and EBV and their contributions to the carcinogenesis of NPC. In this study, we constructed a cell culture model to show that genome instability, alterations of cancer hallmark gene expression, and tumorigenicity were increased after recurrent EBV reactivation in NPC cells following combined treatment of TPA/SB and MNNG. NPC cells latently infected with EBV, NA, and the corresponding EBV-negative cell, NPC-TW01, were periodically treated with MNNG, TPA/SB, or TPA/SB combined with MNNG. With chemically-induced recurrent reactivation of EBV, the degree of genome instability was significantly enhanced in NA cells treated with a combination of TPA/SB and MNNG than those treated individually. The Matrigel invasiveness, as well as the tumorigenicity in mouse, was also enhanced in NA cells after recurrent EBV reactivation. Expression profile analysis by microarray indicates that many carcinogenesis-related genes were altered after recurrent EBV reactivation, and several aberrations observed in cell lines correspond to alterations in NPC lesions. These results indicate that cooperation between chemical carcinogens can enhance the reactivation of EBV and, over recurrent reactivations, lead to alteration of cancer hallmark gene expression with resultant enhancement of tumorigenesis in NPC.

Topics: Animals; Butyrates; Carcinogens; Carcinoma; Cell Line, Tumor; Cell Transformation, Viral; Disease Progression; Drug Synergism; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genome, Human; Genomic Instability; Herpesvirus 4, Human; Humans; Methylnitronitrosoguanidine; Mice; Mice, SCID; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasm Invasiveness; Tetradecanoylphorbol Acetate; Virus Activation

Seroepidemiological studies implicate a correlation between Epstein-Barr virus (EBV) reactivation and the development of nasopharyngeal carcinoma (NPC). Moreover, N-nitroso compounds are known chemical carcinogens in preserved foodstuffs and cigarettes and have been implicated as risk factors contributing to the development of NPC. Here, NPC cell lines latently infected with EBV, NA and HA, and the corresponding EBV-negative NPC cell lines, NPC-TW01 and HONE-1, were used as the model system in this study. We demonstrate that the reactivation of EBV increases with increasing concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MNNG at a single non-toxic concentration (0.1μg/ml) did not induce discernible reactivation of EBV, but repeated treatment with this concentration of MNNG significantly induced viral reactivation. Furthermore, low dose MNNG (0.1μg/ml) had a synergistic effect with 12-O-tetradecanoylphorbol-1,3-acetate (TPA)/sodium butyrate (SB) (10ng/ml and 0.75mM, respectively) on EBV reactivation. Through promoter activity assay, MNNG was found to enhance the transcriptional activity of Rta on Rta and Zta promoters. Using siZta to block EBV reactivation, the concomitant induction of genome instability was diminished indicating that reactivation is critical for enhancing genome instability. Co-treatment with TPA/SB and MNNG markedly increased the levels of γH2AX and ROS formation in NPC cells, which may be responsible for the increase of genome instability. Our findings offer a possible mechanism by which N-nitroso compounds induce reactivation of EBV and contribute to malignant progression by enhancing genome instability in NPC cells.

Topics: Butyric Acid; Cell Line, Tumor; DNA Breaks, Double-Stranded; Dose-Response Relationship, Drug; Drug Synergism; Genomic Instability; Herpesvirus 4, Human; Humans; Immediate-Early Proteins; Methylnitronitrosoguanidine; Micronucleus Tests; Nasopharyngeal Neoplasms; Phorbol Esters; Promoter Regions, Genetic; Reactive Oxygen Species; RNA, Small Interfering; Tetradecanoylphorbol Acetate; Trans-Activators; Transcriptional Activation; Virus Activation

Sister chromatid exchange (SCE) is a genetic indicator of DNA damage in mammalian cells and may afford a sensitive monitor to follow genomic instability of some individuals with fragile chromosomal diseases or malignancies. In studies on the effect of dinitrosopiperazine (DNP), aflatoxin B1 (AFB1), methyl-nitro-nitroso-guanidine (MNNG) and Epstein-Barr virus (EBV) infection on SCE in lymphocytes from nasopharyngeal carcinoma (NPC) patients, we found that: (1) the spontaneous SCEs in peripheral blood lymphocytes (PBLs) from 75 NPC patients were significantly higher than those of PBLs from 44 normal adults, 24 cord blood (CBL) specimens, and PBLs from 20 patients with chronic inflammation of the nasopharynx; (2) PBLs from NPC patients who were positive for EBV virus capsid antigen (VCA) IgA antibody had a higher SCE frequency as compared with PBLs from VCA IgA-negative NPC patients; (3) the chemical carcinogens used induced significantly higher SCEs in lymphocytes from NPC patients than in PBLs from normal adults and CBLs; (4) the mean SCEs of EBV growth-transformed CBLs increased from 5.17 to 14.12 after infection and was similar to the level of SCEs found in PBLs from the VCA IgA-positive NPC patients. The data suggest that lymphocytes of NPC patients might be more fragile than the lymphocytes of the control groups studied.

Topics: Adult; Aflatoxin B1; Aflatoxins; Carcinogens; Carcinoma; Cell Line; DNA Damage; DNA, Neoplasm; Genes, Viral; Herpesvirus 4, Human; Humans; Lymphocytes; Methylnitronitrosoguanidine; Nasopharyngeal Neoplasms; Nitrosamines; Sister Chromatid Exchange; Tumor Cells, Cultured