Background & objective: Recent studies have revealed that some transcription factors may be translationally regulated by eukaryotic initiation factor-4E (eIF-4E) in human cancer. These modifications in the expression levels of the key transcription factors will alter the expression of some malignancy-related gene products at the transcriptional levels. The current study was designed to investigate the effect of eIF-4E on the expression and activity of NF-kappaB and observe how the activity level of NF-kappaB contributes, as a secondary effect, to the transcription of heparanase in human colon adenocarcinoma LS-174T cells.
Methods: In order to repress the expression of eIF-4E, a 20-mer antisense oligodeoxynucleotide (ASODN) targeted against the translation start site of eIF-4E mRNA was transfected into human colorectal cancer cell line LS-174T via liposome reagent, followed by assessment of the activity and the protein expression of NF-kappaB by an electrophoretic gel mobility shift assay (EMSA) and Western blot analysis respectively. The alterations of heparanase expression were examined by RT-PCR and Western blot analysis, and heparanase activity in LS-174T cells was measured by specific enzymatic activity test using radiolabeled heparan sulfate as the substrate and gel filtration chromatography for the analysis of the degradation product.
Results: The 20-mer ASODN against eIF-4E specifically and significantly inhibited eIF-4E expression at both transcriptional and translational levels, and the repression of eIF-4E gene expression was correlated with decreased expression levels and activity of NF-kappaB protein. Furthermore, this down regulation of the ubiquitous transcription factor NF-kappaB led to reduced transcription of the heparanase gene, and the transfected cells also showed a considerable decrease in heparanase protein and activity.
Conclusion: These results suggest that eIF-4E play an important role in translational regulation of NF-kappaB expression in LS-174T cells.NF-kappaB is an essential factor in the regulatory mechanisms of heparanase gene transcription, and the suppressed NF-kappaB activity in LS-174T cells may significantly reduce the transcriptional expression of heparanase that consequently leads to decreased heparanase protein expression and enzymatic activity.