Amiloride modulates urokinase gene expression at both transcription and post-transcription levels in human colon cancer cells.
Activity of receptor-bound urokinase plasminogen activator (uPA) on the surface of colon cancer cells appears to be a function of the number of uPA receptors. The regulation of uPA therefore may determine the invasive phenotype. The effects of amiloride on the modulation of uPA mRNA and protein induced by phorbol ester (PMA) and cycloheximide (CHX) were studied in four colon cancer cell lines, HCT116, KM12SM, LIM1215 and LS123. Northern blot analyses showed that PMA induced uPA mRNA that peaked at 2-48 h in HCT116 cells. In all colon cancer cell lines tested, the expression of uPA mRNA by PMA was super-induced after the addition of the protein synthesis inhibitor CHX, suggesting that stimulation of uPA gene expression does not require de novo protein synthesis. uPA mRNA was also induced by CHX alone, indicating that there may be a labile protein which inhibits uPA mRNA processing. Amiloride profoundly inhibited uPA mRNA production at concentrations between 0.1-1 mM in the presence or absence of PMA or CHX. uPA protein levels on the colon cancer cell surface reflected PMA induction and amiloride inhibition of uPA mRNA levels. Transcriptional elongation experiments using isolated nuclei indicated that while the induction effects of PMA or CHX on uPA gene expression were mediated at the post-transcriptional level, amiloride acted at both transcription and post-transcription levels. The inhibitory effects of amiloride on uPA gene expression reported in this paper may offer the prospect of developing new therapeutic approaches to the prevention of invasion and metastasis by adenocarcinomas.
Wang
Y
Y
Division of Clinical Sciences, John Curtin School of Medical Research, Australian National University, Canberra.
Dang
J
J
Liang
X
X
Doe
W F
WF
D000584:Amiloride / Q000494:pharmacology*; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000235:genetics*; D003513:Cycloheximide / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects*; D014407:Tumor Cells, Cultured / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects*; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics* / Q000378:metabolism
D012333:RNA, Messenger; D000584:Amiloride; D003513:Cycloheximide; D014568:Urokinase-Type Plasminogen Activator; D013755:Tetradecanoylphorbol Acetate