Okadaic acid stimulates expression of Fas receptor and Fas ligand by activation of nuclear factor kappa-B in human oral squamous carcinoma cells.
In the present study, we used western blot and RT-PCR analysis to examine the expression of proteins and mRNAs of Fas receptor and Fas ligand in human oral squamous carcinoma SCC-25 cells treated with okadaic acid. Treatment with okadaic acid enhanced the expression of proteins and mRNAs of both Fas receptor and Fas ligand in SCC-25 cells. The amount of IkappaB-alpha in whole cell lysates decreased, while the level of NF-kappaB in nucleus increased, in the okadaic acid-treated cells. Okadaic acid-treatment also alters the cellular localization of NF-kappaB, from cytoplasm to nuclei. To investigate the activation of NF-kappaB in okadaic acid-treated SCC-25 cells, we performed electrophoretic mobility gel shift assay using nuclear extracts and the consensus oligonucleotide for NF-kappaB DNA binding site. The binding of nuclear proteins to the oligonucleotide of NF-kappaB increased when the cells had been treated with 20 nM okadaic acid for 4 h. We transfected the cells with pFLF1, which has the promoter region of Fas receptor gene containing NF-kappaB binding site. A luciferase reporter gene assay demonstrated that the activity in the cells transfected with pFLF1 and treated with 20 nM okadaic acid increased in a time-dependent manner and that the activity was more than three-fold over that in the control cells. Our results suggest that NF-kappaB activated at early stages in the okadaic acid-treated SCC-25 cells stimulated the promoter activity of Fas receptor in the cells leading to the apoptotic death of these cells.
Fujita
M
M
Department of Histology and Oral Histology, The University of Tokushima 3-18-15, Tokushima 770-8504, Japan.
Goto
K
K
Yoshida
K
K
Okamura
H
H
Morimoto
H
H
Kito
S
S
Fukuda
J
J
Haneji
T
T
D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053222:Fas Ligand Protein; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D008024:Ligands; D008562:Membrane Glycoproteins / Q000187:drug effects* / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000187:drug effects / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014062:Tongue Neoplasms / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism
D004791:Enzyme Inhibitors; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D020672:I-kappa B Proteins; D008024:Ligands; D008562:Membrane Glycoproteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D009363:Neoplasm Proteins; D019014:fas Receptor; D000072000:NF-KappaB Inhibitor alpha; D019319:Okadaic Acid