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The peroxisome proliferator-activated receptor {gamma} is an inhibitor of ErbBs activity in human breast cancer cells

Miguel Pignatelli1, Marta Cortés-Canteli1, Cary Lai2, Angel Santos3,* and Ana Perez-Castillo1,*

1 Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas-Universidad Autónoma, 28029-Madrid
2 Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037
3 Departamento de Bioquímica y Biología Molecular, Facultad de.Medicina, Universidad Complutense, Madrid



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  		Fig. 1. Effect of PG-J2 treatment on the phosphorylation state of ErbB2 and ErbB3. (A) Subconfluent MCF-7 cells were cultivated for 3 or 10 hours in the presence or absence of 10 µM PG-J2 as indicated. Thereafter the cells were stimulated with NRG1 or NRG2 for 5 minutes and the lysates were used for immunoprecipitation (IP) with anti-ErbB2 or anti-ErbB3 polyclonal antibodies. The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted with the indicated antibodies. (B) Same experiment as in A; however, the cells were treated with 1 mM ortovanadate prior to PG-J2 treatment.

 


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  		Fig. 2. Effect of PG-J2 treatment on the phosphorylation state of EGF-R and IGF-IR. Subconfluent MCF-7 cells were cultivated for 10 hours in the presence or absence of 10 µM PG-J2 as indicated. Thereafter the cells were stimulated with EGF or IGF-I for 5 minutes and the lysates were used for immunoprecipitation (IP) with anti-EGF-R or anti-IGF-IR polyclonal antibodies. The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted with the indicated antibodies.

 


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  		Fig. 3. Effects of neuregulins and PG-J2 on the growth properties of human MCF-7 cells. (A) Effects of NRG1, NRG2, and PG-J2 treatment on proliferation of MCF-7 cells. MCF-7 cells were treated with 30 nM of NRG1 or NRG2 in the presence or absence of PG-J2, and the incorporation of [3H]thymidine into DNA was determined. Results are the mean of three determinations from three different experiments. **P<=0.01; ***P<=0.001. (B) Effects of PG-J2 and kinase inhibitors on NRG-1 and NRG-2 effects on cell cycle progression. Cells were grown for 24 hours in the presence or absence of NRG1 or NRG2 preincubated or not for 10 hours with PG-J2, and analyzed by PI staining and FACS analysis. Inhibitors of MEK1 and 2 (40 µM PD98059) or PI 3-K (4 µM LY294002) were added 1 hour before treatment with neuregulins. Curves modeling the G0/G1, S, and G2/M compartments, derived by using the ModFit program, are shown. Cell number is plotted on the y axes, and DNA content is plotted on the x axes.

 


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  		Fig. 4. Activation of MAPK and PI 3-K by NRG1 and NRG2. Cells were stimulated with NRG1 or NRG2 and some cultures were preincubated for 1 hour with PD98059 (40 µM) or LY294002 (4 µM), or for 10 hours with PG-J2. (A) PI 3-K activation was determined by measuring the extent of Akt phosphorylation by western blotting using an anti-phospho-Akt-specific goat polyclonal antibody. Total Akt protein was determined by stripping and reprobing the blot with an anti-Akt polyclonal antibody. (B) MAPK activation was determined by measuring the extent of MAPK phosphorylation by western blotting using an antiphospho-MAPK (p42 and p44)-specific mouse monoclonal antibody. Total MAPK protein content was determined by stripping and reprobing the blot using an anti-MAPK-p42 and an anti-MAPK-p44 polyclonal antibody.

 


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  		Fig. 5. Effects of NRG1, NRG2 and/or PG-J2 on the clonogenicity of MCF-7 cells. After 24 hours of seeding, cells were treated as indicated in Materials and Methods. Representative plates and microphotographs (25-fold magnification) of clones are shown.

 


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  		Fig. 6. Fluorescence microphotographs (100-fold magnification) of MCF-7 cells stained with 0.1 µg/ml of Nile red and viewed at yellow-gold fluorescence.

 


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  		Fig. 7. Effects of NRG1, NRG2 and/or PG-J2 on apoptosis of MCF-7 cells. Cells were cultured for 72 hours in serum-free medium containing the indicated factors, and apoptotic cells were detected by measuring annexin V-FITC binding by flow cytometry. Inhibitors of MEK1 and -2 (40 µM PD98059) or PI 3-K (4 µM LY294002) were added 1 hour before treatment with the neuregulins. Results are the mean of three determinations from three different experiments. **P<=0.01; ***P<=0.001.

 


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  		Fig. 8. Effect of PG-J2 on T47D and SKBR3 breast cancer cell lines. (A) Effect of PG-J2 treatment on the phosphorylation state of ErbB2 and ErbB3. Subconfluent cells were cultivated for 10 hours in the presence or absence of 10 µM PG-J2 as indicated. Thereafter the cells were stimulated with NRG1 for 5 minutes and the lysates were used for immunoprecipitation (IP) with anti-ErbB2 or anti-ErbB3 polyclonal antibodies. The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted with the indicated antibodies. (B) Effects of NRG1 and PG-J2 treatment on cell proliferation. Both cell lines were treated with 30 nM of NRG1 in the presence or absence of PG-J2, and the incorporation of [3H]thymidine into DNA was determined. Results are the mean of three determinations from two different experiments. **P<=0.01; ***P<=0.001.

 

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