RESUMO
BACKGROUND: RAS GTPase-activating protein-binding protein (G3BP1) is an RNA-binding protein that is essential for assembling stress granules. Many functions related to the survival and progression of cancer have been reported. The current study aimed to investigate the role of G3BP1 in radio-sensitisation of cancer cells. MATERIALS AND METHODS: Radiation sensitivity and chemosensitivity were analysed in A549 and H460 cells transfected with G3BP1 siRNAs, and N-acetyl-L-cysteine (NAC) was used to elucidate the involvement of reactive oxygen species (ROS). RESULTS: G3BP1 depletion sensitised lung cancer cell lines to radiation, and the effect was related to ROS. G3BP1 depletion impaired the intracellular ROS scavenging system and NAC abolished the radiation-sensitive phenotypes caused by G3BP1 depletion. CONCLUSION: The study suggested G3BP1 as a promising target for radio- and chemosensitisation of lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/radioterapia , Dano ao DNA/efeitos da radiação , DNA Helicases/antagonistas & inibidores , Neoplasias Pulmonares/radioterapia , Estresse Oxidativo/efeitos da radiação , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , RNA Helicases/antagonistas & inibidores , Proteínas com Motivo de Reconhecimento de RNA/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Antibióticos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , DNA Helicases/genética , DNA Helicases/metabolismo , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais CultivadasAssuntos
Desinfecção/métodos , Contaminação de Equipamentos/prevenção & controle , Vírus da Hepatite B , Hepatite B , Temperatura Alta , Hepatite B/prevenção & controle , Hepatite B/virologia , Vírus da Hepatite B/isolamento & purificação , Vírus da Hepatite B/patogenicidade , Vírus da Hepatite B/fisiologia , Humanos , Controle de Infecções/métodosRESUMO
BACKGROUND & AIMS: As hepatitis B virus (HBV) spreads through the infected liver it is simultaneously secreted into the blood. HBV-susceptible in vitro infection models do not efficiently amplify viral progeny or support cell-to-cell spread. We sought to establish a cell culture system for the amplification of infectious HBV from clinical specimens. METHODS: An HBV-susceptible sodium-taurocholate cotransporting polypeptide-overexpressing HepG2 cell clone (HepG2-NTCPsec+) producing high titers of infectious progeny was selected. Secreted HBV progeny were characterized by native gel electrophoresis and electron microscopy. Comparative RNA-seq transcriptomics was performed to quantify the expression of host proviral and restriction factors. Viral spread routes were evaluated using HBV entry- or replication inhibitors, visualization of viral cell-to-cell spread in reporter cells, and nearest neighbor infection determination. Amplification kinetics of HBV genotypes B-D were analyzed. RESULTS: Infected HepG2-NTCPsec+ secreted high levels of large HBV surface protein-enveloped infectious HBV progeny with typical appearance under electron microscopy. RNA-seq transcriptomics revealed that HBV does not induce significant gene expression changes in HepG2-NTCPsec+, however, transcription factors favoring HBV amplification were more strongly expressed than in less permissive HepG2-NTCPsec-. Upon inoculation with HBV-containing patient sera, rates of infected cells increased from 10% initially to 70% by viral spread to adjacent cells, and viral progeny and antigens were efficiently secreted. HepG2-NTCPsec+ supported up to 1,300-fold net amplification of HBV genomes depending on the source of virus. Viral spread and amplification were abolished by entry and replication inhibitors; viral rebound was observed after inhibitor discontinuation. CONCLUSIONS: The novel HepG2-NTCPsec+ cells efficiently support the complete HBV life cycle, long-term viral spread and amplification of HBV derived from patients or cell culture, resembling relevant features of HBV-infected patients. LAY SUMMARY: Currently available laboratory systems are unable to reproduce the dynamics of hepatitis B virus (HBV) spread through the infected liver and release into the blood. We developed a slowly dividing liver-derived cell line which multiplies infectious viral particles upon inoculation with patient- or cell culture-derived HBV. This new infection model can improve therapy by measuring, in advance, the sensitivity of a patient's HBV strain to specific antiviral drugs.
Assuntos
Proliferação de Células , Vírus da Hepatite B/genética , Hepatite B/metabolismo , Hepatócitos/virologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Técnicas de Cultura de Células/métodos , DNA Viral/metabolismo , Regulação Viral da Expressão Gênica , Genótipo , Células Hep G2 , Hepatite B/virologia , Vírus da Hepatite B/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , RNA-Seq , Receptores Virais/metabolismo , Transcriptoma , Internalização do Vírus/efeitos dos fármacosRESUMO
Hepatitis B virus (HBV) infection is considered a major public health problem worldwide, and a significant number of reports on nosocomial and occupational outbreaks have been reported. This systematic investigation of HBV stability and susceptibility to different antiseptics revealed that HBV infectivity was very stable, with a half-life of >22 days at 37°C. At 4°C, infectivity was barely reduced for up to 9 months. Different alcohols and commercially available hand antiseptics had a virucidal effect against HBV. We propose that very strict compliance with established hygienic guidelines should be mandatory to avoid and prevent HBV infections.
Assuntos
Anti-Infecciosos Locais/farmacologia , Infecção Hospitalar/prevenção & controle , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/prevenção & controle , Doenças Profissionais/prevenção & controle , 1-Propanol/farmacologia , 2-Propanol/farmacologia , Linhagem Celular , Infecção Hospitalar/virologia , Meio Ambiente , Etanol/farmacologia , Higiene das Mãos/normas , Higienizadores de Mão/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Doenças Profissionais/virologia , Soro , Temperatura , Fatores de TempoRESUMO
Diacylglycerol acyltransferases (DGATs) play a critical role in the biosynthesis of endogenous triglycerides (TGs) and formation of lipid droplets (LDs) in the liver. In particular, one member of DGATs, DGAT-1 was reported to be an essential host factor for the efficient production of hepatitis C virus (HCV) particles. By utilizing our previously characterized three different groups of twelve DGAT inhibitors, we found that one of the DGAT inhibitors, a 2-((4-adamantylphenoxy) methyl)-N-(furan-2-ylmethyl)-1H-benzo[d]imidazole-5-carboxam (10j) is a potent suppressor of both HCV genome replication and particle production. 10j was able to induce inhibition of these two critical viral functions in a mutually separate manner. Abrogation of the viral genome replication by 10j led to a significant reduction in the viral protein expression as well. Interestingly, we found that its antiviral effect did not depend on the reduction of TG biosynthesis by 10j. This suggests that the inhibitory activity of 10j against DGATs may not be directly related with its antiviral action.