Ionomycin downregulates beta-catenin/Tcf signaling in colon cancer cell line.
Functional activation of beta-catenin/Tcf signaling plays an important role in the early events in colorectal carcinogenesis. We examined the effect of ionomycin against beta-catenin/Tcf signaling in colon cancer cells. Reporter gene assay showed that ionomycin inhibited beta-catenin/Tcf signaling efficiently. In addition, the inhibition of beta-catenin/Tcf signaling by ionomycin in HEK293 cells transiently transfected with a constitutively mutant beta-catenin gene, whose product is not phosphorylated by GSK3beta, indicates that its inhibitory mechanism is related to beta-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed immunoprecipitation analysis, western blot and electrophoretic mobility shift assay. As a result, our data reveal that the association of beta-catenin and Tcf-4 is disrupted and the amount of beta-catenin product in the nucleus is decreased by ionomycin in a concentration-dependent manner. Moreover, ionomycin strongly suppressed the binding of the Tcf complexes to its specific DNA-binding sites. The significance of the current work is that ionomycin is a negative regulator of beta-catenin/Tcf signaling in colon cancer cells and its inhibitory mechanism is related to the decreased nuclear beta-catenin products and to the suppressed binding of Tcf complexes to consensus DNA.
Park
Chi Hoon
CH
Division of Chemistry and Molecular Engineering, Seoul National University, Seoul 151-742, Korea.
Hahm
Eun Ryeong
ER
Lee
Ju Hyung
JH
Jung
Kyung Chae
KC
Rhee
Ho Sung
HS
Yang
Chul Hak
CH
D001665:Binding Sites; D015153:Blotting, Western; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D015536:Down-Regulation; D024202:Electrophoretic Mobility Shift Assay; D038362:Glycogen Synthase Kinase 3 / Q000494:pharmacology; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D047468:Immunoprecipitation; D015759:Ionomycin / Q000494:pharmacology*; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D008156:Luciferases; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic; D020218:Response Elements / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects*; D051820:TCF Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection; D051176:beta Catenin / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
D051820:TCF Transcription Factors; D015534:Trans-Activators; D051176:beta Catenin; D015759:Ionomycin; D004247:DNA; D008156:Luciferases; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3