tosylphenylalanyl-chloromethyl-ketone and Colonic-Neoplasms

We have previously reported that nitric oxide could induce the death of colon cancer cells. Because an inappropriate activation of beta-catenin has been associated with intestinal cell malignant transformation, we explored whether nitric oxide could affect beta-catenin expression and function.. Human colon cancer cell lines were treated with the nitric oxide donor glyceryl trinitrate (GTN) before analyzing beta-catenin expression by immunofluorescence, immunoblotting, and immunoprecipitation methods and its transcriptional activity using a luciferase reporter gene driven by a T-cell factor-responsive promotor.. GTN induces beta-catenin degradation and down-regulates its transcriptional activity in colon cancer cells. This effect is preceded by GTN-induced tyrosine nitration of beta-catenin, together with its dephosphorylation on serine 33, 37, and 45 and threonine 41. GTN-induced beta-catenin degradation involves proteases that are sensitive to a broad-spectrum caspase inhibitor, z-VAD-fmk, and to serine protease inhibitors N-tosyl-L-phenylalaline chloromethyl ketone (TPCK) and [4-(2-aminoethyl)-benzenesulfonylfluoride] (AEBSF), whereas the ubiquitin/proteasome pathway is not involved. Interestingly, only TPCK and AEBSF restore beta-catenin transcriptional activity and preserve beta-catenin nuclear localization in GTN-treated colon cancer cells.. Exposure of colon cancer cells to nitric oxide unraveled a so-far-unidentified mechanism of beta-catenin regulation. The protein is nitrated and dephosphorylated, and its transcriptional activity is reduced through degradation by a TPCK and AEBSF-sensitive protease.

Topics: Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; beta Catenin; Caspases; Colonic Neoplasms; Down-Regulation; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Nerve Tissue Proteins; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Phosphorylation; Proteasome Endopeptidase Complex; Serine Proteinase Inhibitors; Sulfones; TCF Transcription Factors; Tosylphenylalanyl Chloromethyl Ketone; Transcription Factor 4; Transcription, Genetic; Transfection

Topics: Amino Acid Chloromethyl Ketones; Animals; Carcinoma, Hepatocellular; Colonic Neoplasms; Dimethylhydrazines; DNA Replication; Female; Kinetics; Liver; Liver Neoplasms; Liver Regeneration; Neoplasms, Experimental; Protein Biosynthesis; Rats; Tosylphenylalanyl Chloromethyl Ketone