RESUMO
Epstein-Barr virus (EBV) has a lifelong latency period after initial infection. Rarely, however, when the EBV immediate early gene BZLF1 is expressed by a specific stimulus, the virus switches to the lytic cycle to produce progeny viruses. We found that EBV infection reduced levels of various ceramide species in gastric cancer cells. As ceramide is a bioactive lipid implicated in the infection of various viruses, we assessed the effect of ceramide on the EBV lytic cycle. Treatment with C6-ceramide (C6-Cer) induced an increase in the endogenous ceramide pool and increased production of the viral product as well as BZLF1 expression. Treatment with the ceramidase inhibitor ceranib-2 induced EBV lytic replication with an increase in the endogenous ceramide pool. The glucosylceramide synthase inhibitor Genz-123346 inhibited C6-Cer-induced lytic replication. C6-Cer induced extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB phosphorylation, c-JUN expression, and accumulation of the autophagosome marker LC3B. Treatment with MEK1/2 inhibitor U0126, siERK1&2, or siCREB suppressed C6-Cer-induced EBV lytic replication and autophagy initiation. In contrast, siJUN transfection had no impact on BZLF1 expression. The use of 3-methyladenine (3-MA), an inhibitor targeting class III phosphoinositide 3-kinases (PI3Ks) to inhibit autophagy initiation, resulted in reduced beclin-1 expression, along with suppressed C6-Cer-induced BZLF1 expression and LC3B accumulation. Chloroquine, an inhibitor of autophagosome-lysosome fusion, increased BZLF1 protein intensity and LC3B accumulation. However, siLC3B transfection had minimal effect on BZLF1 expression. The results suggest the significance of ceramide-related sphingolipid metabolism in controlling EBV latency, highlighting the potential use of drugs targeting sphingolipid metabolism for treating EBV-positive gastric cancer.IMPORTANCEEpstein-Barr virus remains dormant in the host cell but occasionally switches to the lytic cycle when stimulated. However, the exact molecular mechanism of this lytic induction is not well understood. In this study, we demonstrate that Epstein-Barr virus infection leads to a reduction in ceramide levels. Additionally, the restoration of ceramide levels triggers lytic replication of Epstein-Barr virus with increase in phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB. Our study suggests that the Epstein-Barr virus can inhibit lytic replication and remain latent through reduction of host cell ceramide levels. This study reports the regulation of lytic replication by ceramide in Epstein-Barr virus-positive gastric cancer.
Assuntos
Carcinoma , Ceramidas , Infecções por Vírus Epstein-Barr , Neoplasias Gástricas , Humanos , Carcinoma/virologia , Linhagem Celular Tumoral , Ceramidas/farmacologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Proteína Quinase 3 Ativada por Mitógeno , Neoplasias Gástricas/virologia , Transativadores/metabolismo , Ativação ViralRESUMO
Sex and gender disparities in biomedical research have been emphasized to improve scientific knowledge applied for the health of both men and women. Despite sex differences in cancer incidence, prognosis, and responses to therapeutic agents, mechanistic explanations at molecular levels are far from enough. Recent studies suggested that cell sex is an important biological variable due to differences in sex chromosome gene expression and differences in events associated with developmental biology. The objective of this study was to analyze the reporting of sex of cells used in cancer research using articles published in Cancer Cell, Molecular Cancer, Journal of Hematology & Oncology, Journal for ImmunoTherapy of Cancer, and Cancer Research in 2020, and to examine whether there exists any sex bias. We found that the percentage of cells with sex notation in the article was 36.5%. Primary cells exhibited higher sex notation compared to cell lines. A higher percentage of female cells were used in cell cultures with sex notation. Also, sex-common cells omitted sex description more often compared to sex-specific cells. None of the cells isolated from embryo and esophagus reported the cell sex in the article. Our results indicate cell sex report in cancer research is limited to a small proportion of cells used in the study. These results call for acknowledging the sex of cells to increase the applicability of biomedical research discoveries.
Assuntos
Pesquisa Biomédica , Células Cultivadas , Neoplasias , Feminino , Humanos , Masculino , Publicações , Fatores Sexuais , SexismoRESUMO
Integrating sex as an important biological variable is imperative to enhance the accuracy and reproducibility of cell-based studies, which provide basic information for subsequent preclinical and clinical study designs. Recently, international funding agencies and renowned journals have been attempting to integrate sex as a variable in every research step. To understand what progress has been made in reporting of cell sex in the articles published in AJP-Cell Physiology since the analysis in 2013, we examined the sex notation of the cells in relevant articles published in the same journal in 2018. Of the 107 articles reporting cell experiments, 53 reported the sex of the cells, 18 used both male and female cells, 23 used male cells only, and 12 used female cells only. Sex omission was more frequent when cell lines were used than when primary cells were used. In the articles describing experiments performed using rodent primary cells, more than half of the studies used only male cells. Our results showed an overall improvement in sex reporting for cells in AJP-Cell Physiology articles from 2013 (25%) to 2018 (50%). However, sex omission and male bias were often found still. Furthermore, the obtained results were rarely analyzed by sex even when both male and female cells were used in the experiments. To boost sex-considerate research implementation in basic biomedical studies, cooperative efforts of the research community, funders, and publishers are urged.
Assuntos
Viés , Publicações Periódicas como Assunto/normas , Projetos de Pesquisa/normas , Caracteres Sexuais , Animais , Linhagem Celular , Lista de Checagem/normas , Políticas Editoriais , Feminino , Guias como Assunto/normas , Humanos , Masculino , Cultura Primária de Células , Fatores SexuaisRESUMO
Epstein-Barr virus (EBV) is associated with several tumors and generates BamHI A rightward transcript (BART) microRNAs (miRNAs) from BART transcript introns. These BART miRNAs are expressed at higher levels in EBV-associated epithelial malignancies than in EBV-infected B lymphomas. To test the effects of EBV miRNA on the cell cycle and cell growth, we transfected miR-BART1-3p, a highly expressed EBV-associated miRNA, into gastric carcinoma cells. We found that miR-BART1-3p induced G0/G1 arrest and suppressed cell growth in gastric carcinoma cells. As our microarray analyses showed that E2F3, a cell cycle regulator, was inhibited by EBV infection, we hypothesized that miR-BART1-3p regulates E2F3. Luciferase assays revealed that miR-BART1-3p directly targeted the 3'-UTR of E2F3 mRNA. Both E2F3 mRNA and encoded protein levels were reduced following miR-BART1-3p transfection. In contrast, E2F3 expression in AGS-EBV cells transfected with a miR-BART1-3p inhibitor was enhanced. As E2F3 has been shown to regulate the expression of highly conserved miR-17-92 clusters in vertebrates, we examined whether this expression is affected by miR-BART1-3p, which can downregulate E2F3. The expression of E2F3, miR-17-92a-1 cluster host gene (MIR17HG), and miR-17-92 cluster miRNAs was significantly reduced in EBV-associated gastric carcinoma (EBVaGC) patients compared with EBV-negative gastric carcinoma (EBVnGC) patients. Further, miR-BART1-3p as well as the siRNA specific to E2F3 inhibited the expression of the miR-17-92 cluster, while inhibition of miR-BART1-3p enhanced the expression of the miR-17-92 cluster in cultured GC cells. Our results suggest a possible role of miR-BART1-3p in cell cycle regulation and in regulation of the miR-17-92 cluster through E2F3 suppression.
Assuntos
Fator de Transcrição E2F3/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Regiões 3' não Traduzidas , Antagomirs/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Fator de Transcrição E2F3/antagonistas & inibidores , Fator de Transcrição E2F3/genética , Pontos de Checagem da Fase G1 do Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Herpesvirus Humano 4/isolamento & purificação , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Interferência de RNA , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/virologiaRESUMO
Hepatocellular carcinoma (HCC), the most common malignant tumor in the liver, grows and metastasizes rapidly. Despite advances in treatment modalities, the five-year survival rate of HCC remains less than 30%. We sought genetic mutations that may affect the oncogenic properties of HCC, using The Cancer Genome Atlas (TCGA) data analysis. We found that the GNAQ T96S mutation (threonine 96 to serine alteration of the Gαq protein) was present in 12 out of 373 HCC patients (3.2%). To examine the effect of the GNAQ T96S mutation on HCC, we transfected the SK-Hep-1 cell line with the wild-type or the mutant GNAQ T96S expression vector. Transfection with the wild-type GNAQ expression vector enhanced anchorage-independent growth, migration, and the MAPK pathways in the SK-Hep-1 cells compared to control vector transfection. Moreover, cell proliferation, anchorage-independent growth, migration, and the MAPK pathways were further enhanced in the SK-Hep-1 cells transfected with the GNAQ T96S expression vector compared to the wild-type GNAQ-transfected cells. In silico structural analysis shows that the substitution of the GNAQ amino acid threonine 96 with a serine may destabilize the interaction between the regulator of G protein signaling (RGS) protein and GNAQ. This may reduce the inhibitory effect of RGS on GNAQ signaling, enhancing the GNAQ signaling pathway. Single nucleotide polymorphism (SNP) genotyping analysis for Korean HCC patients shows that the GNAQ T96S mutation was found in only one of the 456 patients (0.22%). Our data suggest that the GNAQ T96S hotspot mutation may play an oncogenic role in HCC by potentiating the GNAQ signal transduction pathway.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Mutação , Transdução de Sinais , Alelos , Substituição de Aminoácidos , Carcinoma Hepatocelular/patologia , Movimento Celular/genética , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Suscetibilidade a Doenças , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Genótipo , Humanos , Neoplasias Hepáticas/patologia , Sistema de Sinalização das MAP Quinases , Modelos Moleculares , Oncogenes , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Sex has not received enough attention as an important biological variable in basic research, even though the sex of cells often affects cell proliferation, differentiation, apoptosis, and response to stimulation. Knowing and considering the sex of cells used in basic research is essential as preclinical and clinical studies are planned based on basic research results. Cell lines derived from tumor have been widely used for proof-of-concept experiments. However, cell lines may have limitations in testing the effect of sex on cell level, as chromosomal abnormality is the single most characteristic feature of tumor. To examine the status of sex chromosomes in a cell line, 12 commercially available gastric carcinoma (GC) cell lines were analyzed using several different methods. Loss of Y chromosome (LOY) accompanied with X chromosome duplication was found in three (SNU-484, KATO III, and MKN-1) out of the six male-derived cell lines, while one cell line (SNU-638) showed at least partial deletion in the Y chromosome. Two (SNU-5 and MKN-28) out of six female-derived cell lines showed a loss of one X chromosome, while SNU-620 gained one extra copy of the X chromosome, resulting in an XXX karyotype. We found that simple polymerase chain reaction (PCR)-based sex determination gives a clue for LOY for male-derived cells, but it does not provide detailed information for the gain or loss of the X chromosome. Our results suggest that carefully examining the sex chromosome status of cell lines is necessary before using them to test the effect of sex on cell level.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos X/genética , Cromossomos Humanos Y/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Feminino , Humanos , Cariotipagem , Masculino , Células Tumorais CultivadasRESUMO
Several carcinomas including gastric cancer have been reported to contain Epstein-Barr virus (EBV) infection. EBV-associated gastric cancer (EBVaGC) is classified as one of four molecular subtypes of gastric cancer by The Cancer Genome Atlas (TCGA) group with increased immune-related signatures. Identification of EBV-dependent pathways with significant biological roles is needed for EBVaGC. To compare the biological changes between AGS gastric epithelial cells and EBV-infected AGS (AGS-EBV) cells, proliferation assay, CCK-8 assay, invasion assay, cell cycle analysis, RT-PCR, Western blot and ELISA were performed. BI836845, a humanized insulin-like growth factor (IGF) ligand-neutralizing antibody, was used for IGF-related signalling pathway inhibition. AGS-EBV cells showed slower proliferating rate and higher sensitivity to BI836845 compared to AGS cells. Moreover, invasiveness of AGS-EBV was increased than that of AGS, and BI836845 treatment significantly decreased the invasiveness of AGS-EBV. Although no apoptosis was detected, entry into the S phase of the cell cycle was delayed in BI836845-treated AGS-EBV cells. In conclusion, AGS-EBV cells seem to modulate their proliferation and invasion through the IGF signalling pathway. Inhibition of the IGF signalling pathway therefore could be a potential therapeutic strategy for EBVaGC.
Assuntos
Herpesvirus Humano 4/metabolismo , Transdução de Sinais , Somatomedinas/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/virologia , Anticorpos Neutralizantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Biomarcadores Tumorais/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Invasividade Neoplásica , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
UNLABELLED: Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with a variety of tumor types. EBV can establish latency or undergo lytic replication in host cells. In general, EBV remains latent in tumors and expresses a limited repertoire of latent proteins to avoid host immune surveillance. When the lytic cycle is triggered by some as-yet-unknown form of stimulation, lytic gene expression and progeny virus production commence. Thus far, the exact mechanism of EBV latency maintenance and the in vivo triggering signal for lytic induction have yet to be elucidated. Previously, we have shown that the EBV microRNA miR-BART20-5p directly targets the immediate early genes BRLF1 and BZLF1 as well as Bcl-2-associated death promoter (BAD) in EBV-associated gastric carcinoma. In this study, we found that both mRNA and protein levels of BRLF1 and BZLF1 were suppressed in cells following BAD knockdown and increased after BAD overexpression. Progeny virus production was also downregulated by specific knockdown of BAD. Our results demonstrated that caspase-3-dependent apoptosis is a prerequisite for BAD-mediated EBV lytic cycle induction. Therefore, our data suggest that miR-BART20-5p plays an important role in latency maintenance and tumor persistence of EBV-associated gastric carcinoma by inhibiting BAD-mediated caspase-3-dependent apoptosis, which would trigger immediate early gene expression. IMPORTANCE: EBV has an ability to remain latent in host cells, including EBV-associated tumor cells hiding from immune surveillance. However, the exact molecular mechanisms of EBV latency maintenance remain poorly understood. Here, we demonstrated that miR-BART20-5p inhibited the expression of EBV immediate early genes indirectly, by suppressing BAD-induced caspase-3-dependent apoptosis, in addition to directly, as we previously reported. Our study suggests that EBV-associated tumor cells might endure apoptotic stress to some extent and remain latent with the aid of miR-BART20-5p. Blocking the expression or function of BART20-5p may expedite EBV-associated tumor cell death via immune attack and apoptosis.
Assuntos
Apoptose , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , MicroRNAs/metabolismo , Ativação Viral , Proteína de Morte Celular Associada a bcl/metabolismo , Caspase 3/metabolismo , Humanos , Proteínas Imediatamente Precoces/biossíntese , RNA Viral/metabolismo , Transativadores/biossíntese , Latência ViralRESUMO
MicroRNAs (miRNAs) are a class of noncoding RNA molecules approximately 19 to 25 nucleotides in length that downregulate the expression of target genes at the post-transcriptional level by binding to the 3'-untranslated region (3'-UTR). Epstein-Barr virus (EBV) generates at least 44 miRNAs, but the functions of most of these miRNAs have not yet been identified. Previously, we reported BRUCE as a target of miR-BART15-3p, a miRNA produced by EBV, but our data suggested that there might be other apoptosis-associated target genes of miR-BART15-3p. Thus, in this study, we searched for new target genes of miR-BART15-3p using in silico analyses. We found a possible seed match site in the 3'-UTR of Tax1-binding protein 1 (TAX1BP1). The luciferase activity of a reporter vector including the 3'-UTR of TAX1BP1 was decreased by miR-BART15-3p. MiR-BART15-3p downregulated the expression of TAX1BP1 mRNA and protein in AGS cells, while an inhibitor against miR-BART15-3p upregulated the expression of TAX1BP1 mRNA and protein in AGS-EBV cells. Mir-BART15-3p modulated NF-κB activity in gastric cancer cell lines. Moreover, miR-BART15-3p strongly promoted chemosensitivity to 5-fluorouracil (5-FU). Our results suggest that miR-BART15-3p targets the anti-apoptotic TAX1BP1 gene in cancer cells, causing increased apoptosis and chemosensitivity to 5-FU.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Herpesvirus Humano 4/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , MicroRNAs/genética , Proteínas de Neoplasias/genética , RNA Viral/genética , Regiões 3' não Traduzidas , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/patologia , Mucosa Gástrica/virologia , Genes Reporter , Herpesvirus Humano 4/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Luciferases/genética , Luciferases/metabolismo , MicroRNAs/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Transdução de SinaisRESUMO
Extranodal NK/T-cell lymphoma (ENKTCL) is associated with latent Epstein-Barr virus (EBV) infection and frequent relapse even after complete response (CR) to intensive chemotherapy and radiotherapy. The expression of EBV proteins in the tumor provides targets for adoptive immunotherapy with antigen-specific cytotoxic T cells (CTL). To evaluate the efficacy and safety of EBV latent membrane protein (LMP)-1 and LMP-2a-specific CTLs (LMP1/2a CTLs) stimulated with LMP1/2a RNA-transferred dendritic cells, we treated 10 ENKTCL patients who showed complete response to induction therapy. Patients who completed and responded to chemotherapy, radiotherapy, and/or high-dose therapy followed by stem cell transplantation (HDT/SCT) were eligible to receive eight doses of 2 × 10(7) LMP1/2a CTLs/m(2). Following infusion, there were no immediate or delayed toxicities. The 4-year overall survival (OS) and progression-free survival (PFS) were 100%, and 90% (95% CI: 71.4 to 100%) respectively with a median follow-up of 55·5 months. Circulating IFN-γ secreting LMP1 and LMP2a-specific T cells within the peripheral blood corresponded with decline in plasma EBV DNA levels in patients. Adoptive transfer of LMP1/2a CTLs in ENKTCL patients is a safe and effective postremission therapeutic approach. Further randomized studies will be needed to define the role of EBV-CTLs in preventing relapse of ENKTCL.
Assuntos
Imunoterapia Adotiva/métodos , Linfoma Extranodal de Células T-NK/terapia , Linfócitos T Citotóxicos/citologia , Proteínas da Matriz Viral/genética , Adulto , Idoso , Células Dendríticas/citologia , Células Dendríticas/patologia , Intervalo Livre de Doença , Feminino , Terapia Genética , Herpesvirus Humano 4/genética , Humanos , Linfoma Extranodal de Células T-NK/imunologia , Masculino , Recidiva Local de Neoplasia , Recidiva , Indução de Remissão , Transplante de Células-Tronco , Resultado do Tratamento , Adulto JovemRESUMO
UNLABELLED: Epstein-Barr virus (EBV) is a human herpesvirus associated with various tumors. Rather than going through the lytic cycle, EBV maintains latency by limiting the expression of viral genes in tumors. Viral microRNAs (miRNAs) of some herpesviruses have been reported to directly target immediate early genes and suppress lytic induction. In this study, we investigated whether BamHI-A rightward transcript (BART) miRNAs targeted two EBV immediate early genes, BZLF1 and BRLF1. Bioinformatic analysis predicted that 12 different BART miRNAs would target BRLF1. Of these, the results of a luciferase reporter assay indicated that only one interacted with the 3' untranslated region (UTR) of BRLF1: miR-BART20-5p. miR-BART20-5p's effect on gene expression involved two putative seed match sites in the BRLF1 3' UTR, but a mutant version of the miRNA, miR-BART20-5pm, had no effect on expression. As expected from the fact that the entire 3' UTR of BZLF1 resides within the 3' UTR of BRLF1, miR-BART20-5p interacted with the 3' UTR of BZLF1 as well. BZLF1 and BRLF1 mRNA and protein expression was suppressed in cells of an AGS cell line infected with the recombinant Akata strain of EBV (AGS-EBV) transfected with a miR-BART20-5p mimic. The expression of various EBV early proteins was also suppressed by the miR-BART20-5p mimic. In contrast, BZLF1 and BRLF1 expression in AGS-EBV cells transfected with a miR-BART20-5p inhibitor was enhanced. Furthermore, progeny virus production was suppressed by the miR-BART20-5p mimic and enhanced by the miR-BART20-5p inhibitor in AGS-EBV cells induced for the lytic cycle. Our data suggest that miR-BART20-5p plays a key role in latency maintenance in EBV-associated tumors by directly targeting immediate early genes. IMPORTANCE: Herpesviruses maintain latency using various mechanisms and establish lifelong infection in the host. From time to time, herpesviruses are reactivated and express immediate early genes which trigger a lytic cascade, leading to the production of progeny viruses. Recently, some herpesviruses have been shown to use their own microRNAs (miRNAs) to downregulate immediate early genes to inhibit the lytic cycle. This study presents evidence that EBV also downregulates two immediate early genes by miR-BART20-5p to suppress the lytic cycle and progeny virus production. Overall, this is the first study to report the direct regulation of EBV immediate early genes by an EBV miRNA, implying its likely importance in latency maintenance in EBV-associated tumors.
Assuntos
Proteínas de Transporte/metabolismo , Herpesvirus Humano 4/genética , Proteínas Imediatamente Precoces/metabolismo , MicroRNAs/genética , Transativadores/metabolismo , Latência Viral/genética , Regiões 3' não Traduzidas/genética , Proteínas de Transporte/genética , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Expressão Gênica/genética , Genes Precoces/genética , Genes Virais/genética , Células HEK293 , Herpesvirus Humano 4/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Transativadores/genética , Fatores de Transcrição , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ativação Viral/genéticaRESUMO
BACKGROUND: Lymphangiogenesis is one of the major causes of corneal graft rejection. Among the lymphangiogenic factors, vascular endothelial growth factor (VEGF)-C and -D are considered to be the most potent. Both bind to VEGF receptor 3 (VEGFR3) to activate Prospero homeobox 1 (Prox1), a transcription factor essential for the development and maintenance of lymphatic vasculature. MicroRNAs (miRNAs) bind to the 3' untranslated regions (3' UTRs) of target genes in a sequence-specific manner and suppress gene expression. In the current study, we searched for miRNAs that target the pro-lymphangiogenic factor Prox1. RESULTS: Among the miRNAs predicted by the bioinformatic analysis to seed match with the 3' UTR of Prox-1, we chose 3 (miR-466, miR-4305, and miR-4795-5p) for further investigation. Both the miR-466 and miR-4305 mimics, but not the miR-4795-5p mimic, significantly reduced the luciferase activity of the Prox-1 3' UTR reporter vector. In primary lymphatic endothelial cells (HDLEC), miR-466 mimic transfection suppressed Prox1 mRNA and protein expression, while miR-4305 mimic transfection did not. Experiments using mutated reporter constructs of the two possible seed match sites on the 3' UTR of Prox1 suggested that the target site 2 directly bound miR-466. HDLEC transfected with the miR-466 mimic suppressed tube formation as compared to the scrambled control. Furthermore, HDLEC transfected with a miR-466 inhibitor showed enhanced tube formation as compared to control inhibitor transfected cells, and this inhibitory effect was counteracted by Prox1 siRNA. The miR-466 mimic reduced angiogenesis and lymphangiogenesis resulting in clearer corneas in an cornea injury rat model compared to the scrambled control. CONCLUSIONS: Our data suggest that miR-446 may have a protective effect on transplanted corneas by suppressing Prox1 expression at the post-transcriptional level. The results of the current study may provide insights into the mechanisms of lymphangiogenesis resulting from corneal graft rejection and alkali-burn injuries, as well as into the development of new treatments for lymphangiogenic eye diseases.
Assuntos
Queimaduras Químicas/genética , Lesões da Córnea/genética , Queimaduras Oculares/genética , Proteínas de Homeodomínio/genética , Linfangiogênese , MicroRNAs/genética , Proteínas Supressoras de Tumor/genética , Álcalis/toxicidade , Animais , Queimaduras Químicas/etiologia , Lesões da Córnea/induzido quimicamente , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Queimaduras Oculares/induzido quimicamente , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , MicroRNAs/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Supressoras de Tumor/metabolismoRESUMO
Epstein-Barr Virus (EBV) generates a variety of viral microRNAs (miRNAs) by processing the BHRF1 and BamHI A rightward (BART) transcripts. BART miRNAs are expressed in all cells latently infected with EBV, but the functions of most BART miRNAs remain unknown. The results of a cell proliferation assay revealed that miR-BART15-3p inhibited cell proliferation. Fluorescence-activated cell sorting following staining with annexin V or propidium iodide showed that miR-BART15-3p promoted apoptosis. Furthermore, the inhibitor for miR-BART15-3p increased cell growth and reduced apoptosis in EBV-infected cells. Using bioinformatic analyses, we predicted that miR-BART15-3p may target the antiapoptotic B-cell lymphoma 2 (BCL2), BCL2L2, DEAD (Asp-Glu-Ala-Asp) box polypeptide 42 (DDX42), and baculovirus inhibitor of apoptosis repeat-containing ubiquitin-conjugating enzyme (BRUCE) mRNAs. The luciferase reporter assay showed that only the 3' untranslated region (UTR) of BRUCE was affected by miR-BART15-3p. Two putative seed-matched sites for miR-BART15-3p were evident on the BRUCE 3' UTR. The results of a mutation study indicated that miR-BART15-3p hybridized only with the first seed-matched site on the BRUCE 3' UTR. miR-BART15-3p downregulated the BRUCE protein in EBV-negative cells, while the inhibitor for miR-BART15-3p upregulated the BRUCE protein in EBV-infected cells without affecting the BRUCE mRNA level. miR-BART15-3p was secreted from EBV-infected gastric carcinoma cells, and the level of miR-BART15-3p was 2- to 16-fold higher in exosomes than in the corresponding cells. Our data suggest that miR-BART15-3p can induce apoptosis partially by inhibiting the translation of the apoptosis inhibitor BRUCE. Further study is warranted to understand the role of miR-BART15-3p in the EBV life cycle.
Assuntos
Apoptose/fisiologia , Herpesvirus Humano 4/genética , Proteínas Inibidoras de Apoptose/metabolismo , MicroRNAs/metabolismo , Análise de Variância , Anexina A5 , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Biologia Computacional , Primers do DNA/genética , Citometria de Fluxo , Humanos , Luciferases , MicroRNAs/genética , MicroRNAs/farmacologia , Microscopia Eletrônica de Transmissão , Plasmídeos/genética , Propídio , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Epstein-Barr virus (EBV) is a herpesvirus associated with lymphomas and carcinomas. While EBV-associated epithelial cell lines are good model systems to investigate the role of EBV in carcinoma, only a few cell lines are available as they are hard to acquire. A greater variety of naturally EBV-infected cell lines which are derived from tumour patients are needed to represent various features of EBVaGC. We characterized cell line YCCEL1, established from a Korean EBVaGC patient, to ascertain whether it can be used to study the roles of EBV in EBVaGC. The expression of EBV genes and cell surface markers was examined by in situ hybridization, RT-PCR, Western blot analysis, immunofluorescence assay and Northern blot analysis. EBV episomal status was analysed by Southern blotting and real-time PCR. This cell line expressed EBV nuclear antigen 1 (EBNA1) and latent membrane protein 2A (LMP2A), but not EBNA2, LMP2B nor LMP1. The majority of the lytic proteins were not detected in YCCEL1 cells either before or after treatment with 12-O-tetradecanoylphorbol-13-acetate. YCCEL1 cells expressed BART microRNAs (miRNAs) at high level but did not express BHRF1 miRNAs. YCCEL1 cells expressed cytokeratin, but not CD21 and CD19, suggesting CD21-independent EBV infection. The latent EBV gene and EBV miRNA expression pattern of YCCEL1 cells closely resembled that of general EBVaGC cases. Our results support the value of YCCEL1 cells as a good model system to study the role of EBV in gastric carcinogenesis.
Assuntos
Carcinoma/virologia , Herpesvirus Humano 4/fisiologia , Neoplasias Gástricas/virologia , Southern Blotting , Linhagem Celular Tumoral , Regulação da Expressão Gênica/fisiologia , Genoma Viral , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus implicated in lymphomas, such as Burkitt's lymphoma, Hodgkin's lymphoma, and NK/T cell lymphoma. MicroRNAs (miRNAs) are 19-25 nucleotide long single-stranded RNAs involved in post-transcriptional gene regulation. miRNAs are mainly transcribed by RNA polymerase II (pol II) to have stem-loop structures and subsequently processed by Drosha and Dicer. EBV miRNAs are expressed in B cells, nasopharyngeal carcinoma cells, and gastric carcinoma cells infected with EBV. EBV miRNAs can be divided into two groups: BHRF1 miRNAs and BART miRNAs. In this study, we investigated the biogenesis of EBV miRNAs. Treatment of the SNU-719 EBV-positive gastric cancer cell line with α-amanitin at a concentration that selectively inhibits RNA polymerase II activity decreased the expression levels of BART miRNAs. The expression levels of BART miRNAs were also reduced by RNA interference targeting Drosha and Dicer. Two of each C/EBPß and c-Myc binding sites are located upstream of the proposed initiation sites for primary BART miRNA transcripts. Knockdown of C/EBPß but not c-Myc using siRNAs reduced BART miRNA expression by 25-55% compared with the control. These results suggest that BART miRNAs are transcribed by pol II and undergo a similar biogenesis process with cellular miRNAs.
Assuntos
MicroRNAs/biossíntese , RNA Viral/biossíntese , Alfa-Amanitina/farmacologia , Linfócitos B/metabolismo , Sequência de Bases , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Regulação Viral da Expressão Gênica , Técnicas de Silenciamento de Genes , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Humanos , MicroRNAs/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , RNA Viral/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo , Transcrição Gênica , Latência ViralRESUMO
OBJECTIVE: The risk of malignancy after transplantation is regarded to be higher than in the general population. The aim of this study was to evaluate the frequency of gastric cancer in renal transplant recipients. METHODS: A total of 820 renal transplantation recipients were invited for gastric cancer screening. Frequencies of gastric cancer in this cohort and in 10,080 asymptomatic subjects were compared. Cancer specimens were examined for Epstein-Barr virus by in situ hybridization. RESULTS: A total of 509 recipients (mean age, 48.1 ± 10.7 y; men, 56.8%) participated. Fifteen (2.9%) and 10 (0.1%) cases of adenocarcinoma were identified among recipients and controls, respectively (P<0.001; odds ratio, 30.58). Early gastric cancer was detected in 9 of the 15 recipients, and 4 of the 9 were treated by endoscopic resection. Recipient age was found to be a significant factor of gastric cancer development. In cancer tissues, Epstein-Barr virus was detected in 5 (33.3%) renal recipients and in 1 (10%) of the controls, respectively. CONCLUSIONS: The frequency of gastric cancer was found to be higher in renal recipients than in controls. Gastric cancer screening should be considered after transplantation, because it would provide cure by minimally invasive treatment.
Assuntos
Adenocarcinoma/epidemiologia , Imunossupressores/efeitos adversos , Transplante de Rim/imunologia , Neoplasias Gástricas/epidemiologia , Adenocarcinoma/cirurgia , Adenocarcinoma/virologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Intervalos de Confiança , Infecções por Vírus Epstein-Barr/epidemiologia , Feminino , Gastroscopia , Infecções por Helicobacter/epidemiologia , Helicobacter pylori , Herpesvirus Humano 4 , Humanos , Transplante de Rim/efeitos adversos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Vigilância da População , República da Coreia/epidemiologia , Neoplasias Gástricas/cirurgia , Neoplasias Gástricas/virologia , Fatores de Tempo , Adulto JovemRESUMO
The incidence and mortality rates of gastric cancer rank among the highest five of all cancer types worldwide. The chemotherapeutic agent 5-fluorouracil (5-FU) is the gold standard for treating gastric cancer, but its efficacy is limited due to high rates of resistance. To improve the therapeutic efficacy of 5-FU and overcome its resistance, the synergistic effect of chrysin with 5-FU was investigated and its mechanism was elucidated. Chrysin was co-administered with 5-FU in AGS cells and 5-FU-resistant AGS cells (AGS/FR). Cytotoxicity was investigated using MTT assay, followed by calculating the combination index (CI). Several biomarkers were detected using western blotting analysis. Apoptosis and cell cycle distribution were measured by flow cytometry. The combination of chrysin and 5-FU significantly increased cytotoxicity more than chrysin or 5-FU alone. 5-FU induced apoptosis through p53-p21 activity, while chrysin arrested the cell cycle in the G2/M phase. The combination of chrysin and 5-FU showed an anticancer effect via S phase arrest. The results indicated that chrysin and 5-FU exhibited anticancer properties via different pathways. Furthermore, the present study found that chrysin enhanced the chemotherapeutic effect of 5-FU in AGS/FR cells. In the resistant cells, the combination of chrysin and 5-FU improved the anticancer effect via G2/M phase arrest. These findings indicated that chrysin potentiated the chemotherapeutic effect of 5-FU in gastric cancer AGS and AGS/FR cells via cell cycle arrest. Therefore, chrysin may be used to treat gastric cancers that have become resistant to 5-FU.
RESUMO
Gastric cancer is the fifth leading cause of cancer and a global public health problem. 5-Fluorouracil (5-FU) is the primary drug chosen for the treatment of advanced gastric cancer, but acquired cancer drug resistance limits its effectiveness and clinical use. Proliferation assays showed that a gastric carcinoma cell line, AGS and 5-FU-resistant AGS cells (AGS FR) treated with 3-100 µM 5-FU for 48 h or 72 h showed different sensitivities to 5-FU. Immunoblot assay demonstrated that AGS FR cells expressed more COX-2 and PGE2-cognated receptor EP2 than AGS cells. AGS FR cells considerably produced PGE2 than AGS upon stimulation with 5-FU. These results suggest that COX-2 expression is associated with 5-FU resistance. Unlike AGS FR cells, AGS cells showed increased levels of both cleaved caspase-3 and Bax following 5-FU treatment. Treatment of cells with the COX-2 selective inhibitor celecoxib induced cell death of AGS FR cells in a time- and concentration-dependent manner. FACS analysis showed that celecoxib at high doses caused apoptotic cell death, demonstrating a concentration-dependent increase in the cell populations undergoing early apoptosis and late apoptosis. This apoptotic induction was strongly supported by the expression profiles of apoptosis- and survival-associated proteins in response to celecoxib; pro-apoptotic cellular proteins increased while expressions of COX-2 and p-Akt were downregulated in a concentration-dependent manner. An increase in PTEN expression was accompanied with downregulation of p-Akt. Based on the data that downregulation of COX-2 was correlated with the concentrations of celecoxib, COX-2 may play a key role in celecoxib-induced cell death of AGS FR cells. Butaprost, the EP2 agonist, promoted proliferative activity of AGS FR cells in a concentration-dependent manner compared with AGS cells. In cells exposed to butaprost, expressions of COX-2 and p-Akt were increased in a concentration-dependent manner with concomitantly reduced PTEN levels. Taken together, 5-FU-resistance in gastric cancer is correlated with COX-2 expression, and therefore the selective inhibition of COX-2 leads to suppression of cell proliferation of AGS FR cells. Modulation of COX-2 expression and its catalytic activity may be a potential therapeutic strategy to overcome 5-FU-resistant gastric cancer.
RESUMO
BACKGROUND: As EBV-associated gastric cancer has unique features that are different from EBV (-) gastric cancer, EBV is considered to have a key role in gastric carcinogenesis. It has been reported that viral latent membrane protein 2A (LMP2A) in EBV-transformed tumor cells activates the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which provides a survival signal and chemo-resistance to cytotoxic anti-cancer drugs. This study was to evaluate anti-proliferative effect and cell cycle change when 5-FU and LY294002 (LY), a selective inhibitor of PI3K, were treated separately or combined with different schedules in EBV positive gastric cancer cell line, SNU-719. METHODS: After single treatment and sequential combination of 5-FU and LY, cytotoxic activity was measured by MTS assay. When 5-FU and LY were treated in single and sequential combinations, the expression of p-AKT, p-NFkB, p-p53 and bcl-2 was observed on different concentrations by Western blot analysis. We also investigated the effect on apoptosis and cell cycle distribution using flow cytometry. The LMP2A siRNA inhibition was done to confirm the reversal of decreased 5-FU activity and p-AKT. RESULTS: When 5-FU was sequentially combined with LY, the combination index (CI) value indicated synergistic anti-proliferative effect. The expression of p-AKT and p-NF kappaB was upregulated by 5-FU alone but sequential treatment of 5-FU and LY decreased the expression of both p-AKT and p-NF kappaB. When 5-FU was combined with LY, G0/G1 and sub G1 cell population (%) increased. When 5-FU was added to the cells transfected with LMP2A siRNA, its anti-proliferative effect increased and the expression of p-AKT decreased. In sequential combination of 5-FU and LY, the expression of p-p53 was increased and bcl-2 expression was diminished compared to 5-FU alone. CONCLUSION: These data suggest that sequential combination of 5-FU and LY induce synergistic cytotoxicity and overcome intrinsic and acquired resistance of 5-FU via downregulation of activated p-AKT and mitochondria-dependent apoptosis in EBV gastric cancer cell line, SNU-719.