1. Volume 131, Issue 4, Pages 1142-1152 (October 2006)
Nitric Oxide-Induced Down-Regulation of β-Catenin in Colon Cancer Cells by a Proteasome-Independent Specific Pathway 
Laurent Prévotat, Rodolphe Filomenko, Eric Solary, Jean-François Jeannin, Ali Bettaieb 
Gastroenterology 
Volume 131, Issue 4, Pages (October 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

2. Figure 1
GTN-inhibited β-catenin/TCF-mediated transcription. (A) SW480 cells were treated for 48 hours with indicated concentrations of GTN in the presence or absence of the NO scavenger PTIO (100 μmol/L) before measuring accumulation of nitrites and nitrates in the medium. (B) Indicated cells (SW480 or RKO) were transiently transfected with pGL3-OT (TCF4-binding sites upstream of a luciferase gene) or with the control construct pGL3-OF (TCF4 mutated sites). The luciferase activity was measured after a 48-hour exposure to 500 μmol/L GTN in combination or not with PTIO (100 μmol/L). A β-galactosidase expressing vector was transfected simultaneously for normalization. (B, insert:) Western blot analysis of β-catenin expression in the 2 cell lines. (C and D) 293T cells were cotransfected with a plasmid encoding murine iNOS (or the empty vector), pGL3-OT, and the β-galactosidase encoding vector then treated or not with 0.5 mmol/L aminoguanidine (AG) before measuring (C) nitrite and nitrate production (C, insert: Western blot analysis of iNOS protein in cells expressing iNOS) and (D) luciferase activity (normalized against β-galactosidase activity). (E and F) SW480 cells expressing pGL3-OT were treated with 500 μmol/L GTN for indicated times before (E) measuring luciferase activity (internal control: β-galactosidase expressing vector) and (F) immunopreciptation of cell lysates with an anti-NO-tyrosine mAb. Immunoprecipitates and whole cell extracts were resolved in SDS-PAGE and blotted with an anti-β-catenin mAb. Results are mean ± SD of 3 independent experiments or 1 representative experiment of at least 3 independent experiments. MW are in kilodaltons.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

3. Figure 2
GTN-induced β-catenin cleavage in colon cancer cells. (A) SW480 and (B) HCT116 cells were treated with indicated doses of GTN for 48 hours before assessing the percentage of cells with nuclear chromatin condensation and fragmentation after Hoechst staining (upper panel) and analyzing β-catenin expression by immunoblotting (lower panel). Mean ± SD of 3 independent experiments and 1 representative blot of 3 is shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

4. Figure 3
Proteasome-independent GTN-induced β-catenin degradation. (A) SW480 cells were left untreated or treated with 500 μmol/L GTN for 48 hours in the absence or presence of 25 μmol/L MG132 added 12 hours before the measurement of the ability of cell lysates to cleave the substrate Suc-LLVY-AMC. Mean ± SD of 3 independent experiments. (B) 293T cells, in which a plasmid encoding Flag-tagged ubiquitin (Flag-Ub) was transiently expressed, were treated with indicated doses of GTN for 48 hours, in the absence or presence of 25 μmol/L MG132 during the last 12 hours of treatment, before immunoblot analysis of ubiquitinated proteins in cell lysates using an anti-Flag mAb (upper panel) or β-catenin using an anti-β-catenin mAb (lower panel). (C) SW480 cells were treated as in A. Cell lysates were then analyzed by Western blot with Abs that recognized β-catenin, either phosphorylated on serine 33 or serine 37 or theronine 41 (pβc S33/S37/T41) or phosphorylated on serine 45 or threonine 41 (pβc S45/T41) or nonphosphorylated (βc). (D) Western blot analysis of casein kinase Iα (CKI α) in SW480 cell lysates obtained after a 48-hour treatment with indicated concentrations of GTN. An anti-Hsc 70 mAb was used for loading control. (E) SW480, HCT116, and HT29 cells were treated with indicated concentrations of GTN for 48 hours before analysis of the expression of nonphosphorylated (GSK-3β) and Ser9-phosphorylated GSK-3β (S9p-GSK-3β) in cell lysates. (F) SW480 cells were treated with 500 μmol/L for indicated times before immunoprecipitation of β-catenin followed by immunoblot analysis of β-catenin, GSK-3β, and S9p-GSK-3β. Whole cell extracts were used as controls. (G) Fluorescence microscopy analysis of GSK-3β in untreated (NT) and GTN-treated (GTN; 500 μmol/L for 24 hours) SW480 cells. Nuclei were simultaneously labelled with Hoeschst In each panel except A, 1 representative experiment of at least 3 experiments is shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

5. Figure 4
Caspase-mediated cleavage of β-catenin in GTN-treated cells. (A) SW480 cells were left untreated (NT) or treated with 500 μmol/L GTN (GTN) for 48 hours; in the absence or presence of MG132 (25 μmol/L, added 12 hours before the end of treatment); ALLM (10 μmol/L); or calpastatine (10 μmol/L) before immunoblot analysis of β-catenin. *Indicates cleavage fragments. Hsc70 was used as loading control. One representative experiment of 3 experiments is shown. (B) SW480 cells were transiently transfected with pGL3-OT and treated as in A before measuring luciferase activity in whole cell extracts. Results were normalized with renilla expression. Mean ± SD of 3 independent experiments. (C) SW480 cells were left untreated or treated with 500 μmol/L GTN for 48 hours, in the absence or presence of indicated concentrations of z-VAD-fmk, before immunoblot analysis of β-catenin expression. Actin was used as loading control. (D) SW480 cells were transiently transfected with pGL3-OT and treated with 500 μmol/L GTN for 48 hours in the absence or presence of 75 and 200 μmol/L of z-VAD-fmk before measuring luciferase activity. Mean ± SD of 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

6. Figure 5
GTN-induced β-catenin degradation can be prevented by the serine protease inhibitors TPCK and AEBSF. (A) SW480 cells were left untreated (NT) or treated with 500 μmol/L GTN for 48 hours (GTN) in the absence or presence of TPCK (3 or 5 μmol/L), AEBSF (50 μmol/L), and/or indicated concentrations of z-VAD-fmk before immunoblot analyis of β-catenin expression. Hsc 70, loading control. The percentage of apoptotic cells, as identified by Hoechst staining of nuclear chromatin, is indicated below. One representative experiment of 3 experiments is shown. (B) SW480 cells were transiently transfected with pGL3-OT and left untreated (Co) or treated with 500 μmol/L GTN for 48 hours (GTN) in the absence or presence of TPCK (5 μmol/L), z-VAD-fmk (75 or 200 μmol/L), chymostatin (10 μmol/L), AEBSF (50 μmol/L), PTIO (100 μmol/L), or a combination of TPCK (5 μmol/L) and z-VAD-fmk (75 μmol/L) before measuring luciferase activity in whole cell extracts. Results were normalized with renilla expression. Mean ± SD of 3 independent experiments. (C) SW480 cells were treated as in B before RT-PCR analysis of matrilysin (MMP7) and GAPDH (used as internal control) mRNA. One representative experiment of 3 experiments is shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

7. Figure 6
TPCK prevents GTN-induced dissociation of TCF4 from β-catenin. SW480 cells were left untreated (NT) or treated with 500 μmol/L GTN for 24 hours (GTN) in the absence or presence of 5 μmol/L TPCK (GTN + TPCK) or 75 μmol/L z-VAD-fmk (GTN + zVAD) or both (GTN + z-VAD + TPCK). At this time point, cell death never exceeded 15%. (A) Confocal fluorescence microscopy analysis of β-catenin (green) or TCF4 (red). One of 3 independent experiments is shown. (B) EMSA analysis of β-catenin interaction with DNA in cells treated as above. One of 3 independent experiments is shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

8. Figure 7
GTN induces complete regression of subcutaneous murine CT26 colon tumors. Tumors were induced in Balb/c mice by subcutaneous injection of 5 × 105 CT26 colon cancer cells. When tumors reached an average volume of 50–100 mm3 (10 days), animals were assigned randomly to 1 of 2 groups (10 animals per group). Those from the first group were treated with GTN (0.2 mg/kg per day, open circles). Those from the other group received a similar volume of vehicle (solid circles). An intratumor injection was performed every day, and tumor volume was measured at indicated time points.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions