RESUMO
BACKGROUND AND PURPOSE: The transcription factor nuclear factor-kappaB (NF-kappaB) has been linked to the cell growth, apoptosis and cell cycle progression. NF-kappaB blockade induces apoptosis of cancer cells. Therefore, NF-kappaB is suggested as a potential therapeutic target for cancer. Here, we have evaluated the anti-cancer potential of a novel NF-kappaB inhibitor, quinoclamine (2-amino-3-chloro-1,4-naphthoquinone). EXPERIMENTAL APPROACH: In a large-scale screening test, we found that quinoclamine was a novel NF-kappaB inhibitor. The global transcriptional profiling of quinoclamine in HepG2 cells was therefore analysed by transcriptomic tools in this study. KEY RESULTS: Quinoclamine suppressed endogenous NF-kappaB activity in HepG2 cells through the inhibition of IkappaB-alpha phosphorylation and p65 translocation. Quinoclamine also inhibited induced NF-kappaB activities in lung and breast cancer cell lines. Quinoclamine-regulated genes interacted with NF-kappaB or its downstream genes by network analysis. Quinoclamine affected the expression levels of genes involved in cell cycle or apoptosis, suggesting that quinoclamine exhibited anti-cancer potential. Furthermore, quinoclamine down-regulated the expressions of UDP glucuronosyltransferase genes involved in phase II drug metabolism, suggesting that quinoclamine might interfere with drug metabolism by slowing down the excretion of drugs. CONCLUSION AND IMPLICATIONS: This study provides a comprehensive evaluation of quinoclamine by transcriptomic analysis. Our findings suggest that quinoclamine is a novel NF-kappaB inhibitor with anti-cancer potential.
Assuntos
Antineoplásicos/farmacologia , Perfilação da Expressão Gênica , NF-kappa B/antagonistas & inibidores , Naftoquinonas/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Glucuronosiltransferase/genética , Humanos , Proteínas I-kappa B/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/genética , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Transporte Proteico , Fator de Transcrição RelA/metabolismo , TransfecçãoRESUMO
The pseudorabies virus (PRV) gene encoding a DNA-binding protein (DBP) was first identified in this study. The DBP gene has an open reading frame of 3531 nucleotides, capable of coding a 1177-amino-acid polypeptide of 125 kDa. The deduced DBP exhibits a conserved zinc-binding motif and a conserved DNA-binding region, suggesting the similar DNA-binding mechanism occurs among alphaherpesviral DBP homologs. To further identify the biochemical properties of PRV DBP, this protein was expressed in Escherichia coli by using a pET expression vector and purified to homogeneity. The PRV DBP binds cooperatively and preferentially to single-stranded DNA with no significant base preference, judged by agarose gel electrophoresis and competitive nitrocellulose filter binding assays. Taken together, these results suggest that PRV DBP may play an important role in PRV DNA replication by binding cooperatively and nonspecifically to single-stranded DNA that is formed during the replication origin unwinding and replication fork movement.
Assuntos
Proteínas de Ligação a DNA/genética , Genes Virais/genética , Herpesvirus Suídeo 1/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Replicação do DNA , DNA Viral/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/isolamento & purificação , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Herpesvirus Suídeo 1/química , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência , SuínosRESUMO
In order to investigate the mechanism of long-term immunity and the effect of protective immunity induced by DNA vaccination, we constructed the expression plasmid containing a pseudorabies virus (PRV) gD gene encoding an envelope glycoprotein. Intramuscular vaccination of mice with the plasmid DNA induced a strong antibody response which lasted for one year after final vaccination. An IgM to IgG class switch occurred, indicating helper T-lymphocyte activity. We further analyzed the persistence and expression of gD gene by polymerase chain reaction and reverse transcriptase polymerase chain reaction. The results showed that gD gene was present and expressed in the muscle cell up to one year after final booster injection. Furthermore, mice vaccinated with the plasmid DNA were protected against a subsequent lethal challenge with PRV. Therefore, the DNA vaccination does induce a protective immunity and long-term antibody response against PRV, which could be maintained by persistent expression of gD gene in muscle cells.