Comprehensive evaluation of a novel nuclear factor-kappaB inhibitor, quinoclamine, by transcriptomic analysis.

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.

Identification, expression, and characterization of the pseudorabies virus DNA-binding protein gene and gene product.

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.

DNA vaccination induces a long-term antibody response and protective immunity against pseudorabies virus in mice.

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.

Increased gene expression in human promyeloid leukemia cells exposed to trans,trans-muconaldehyde, a hematotoxic benzene metabolite.

The hematotoxicity of benzene, a human leukemogen, has been postulated to be mediated by reactive metabolites and involve cell damage caused by reactive oxygen species. Because expression of the transcription factors AP-1 and NF-kappaB is sensitive to the redox state in eukaryotic cells, the DNA binding activity of AP-1 and NF-kappaB was examined in HL-60 promyeloid leukemia cells exposed to trans,trans-muconaldehyde, a microsomal hematotoxic metabolite of benzene. There was little AP-1 binding activity in nuclear extracts from control HL-60 cells based on electrophoretic mobility shift assays. Exposure to 0.1 microM MUC for 4 h resulted in significantly increased levels of nuclear protein with high sequence specificity for the consensus AP-1 sequence. In addition, electrophoretic mobility shift assays showed a strong increase in the binding of a factor to the NF-kappaB site. The latter was highest in nuclear extracts from HL-60 cells treated with 1.0 microM muconaldehyde and cultured for 4 h. Exposure of HL-60 cells to muconaldehyde resulted in an increase in c-fos and c-jun mRNA levels. Western blot analysis showed that the protein levels of c-jun increased in HL-60 cells treated with 1 microM muconaldehyde and cultured for 4-6 h and subsequently decreased gradually. Increased AP-1 binding was observed in bone marrow cells from B6C3F1 mice 2 h after administration of 440 mg/kg benzene. We suggest that increased gene expression of NF-kappaB and AP-1 binding activity and up-regulation of c-fos and c-jun may play a role in the mechanism of benzene leukemogenesis.

Polypeptide pattern of human breast epithelial cells following human chorionic gonadotropin (hCG) treatment.

Numerous attempts have made to describe the particular protein pattern of malignant cells by using high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The placental hormone human chorionic gonadotropin (hCG) inhibits tumor initiation and progression in experimental animals and has an inhibitory effect on the proliferation of human breast epithelial cells (HBEC) in vitro. The inhibitory effect on the immortalized HBEC MCF-10F is accompanied by the immunocytochemical expression of inhibin alpha and beta subunits by treated cells. With the purpose of clarifying the molecular mechanisms involved in this effect, the pattern of protein synthesis and mRNA were studied by 2-D PAGE in the immortalized HBEC MCF-10F cells treated in vitro 1001U for 24 h. The effect of hCG treatment on the synthesis of MCF-10F cells was monitored by labeling both control and treated cells with [S35]methionine and separation by 2-D PAGE. At least 11 proteins were preferentially synthesized and five specific polypeptides were decreased in hCG treated cells in comparison with controls. The hCG induced at least four new mRNAs which encoded protein in the molecular mass range of 24-72 kDa. It also increased the expression of at least six mRNAs and reduced the expression of least four mRNAs in comparison with control cells. The hCG-treated cells actively synthesized a 33-kDa polypeptide which was not present in control cells. The nature of this hCG-inducible 33 kDa protein elucidated by immunoprecipating [S35]methionine-labeled proteins with antisera directed against rat inhibin subunit alpha and beta b.(ABSTRACT TRUNCATED AT 250 WORDS)