Figure 4.

Metformin activates AMPK, but does not affect PMA-induced MMP-9 activation. (A) After HT-1080 cells were treated with metformin or AICAR at the indicated concentrations, AMPK phosphorylation was examined by Western blotting. Each blot is representative of at least three others. (B) The suppressive effects of metformin and AICAR were independent of AMPK activation. Cells were pretreated with compound C (AMPKi; 1 µM) for 1 h prior to metformin or AICAR treatment, and the phosphorylation levels of AMPK was measured by Western blotting. Cells were pretreated for 1 h with compound C (AMPKi, 1 µM) followed by metformin or AICAR treatment for 24 h. Each blot is representative of at least three others. (C) The MMP-9 mRNA expression in the cells was analysed by semi-quantitative RT–PCR or real-time PCR. (D and E) Conditioned media were collected after 24 h, and then Western blotting or gelatin zymography was performed. Each blot is representative of at least three others. (F) Cells were transfected with pGL-AP-1 Luc reporter plasmids. The transfected cells were treated with compound C (AMPKi, 1 µM) for 1 h, and then metformin or AICAR for 24 h. The luciferase activity in the cell extract was determined. Data are expressed as the means ± SD of triplicate experiments. *Significantly different from control (P < 0.01). (G) Cells were transfected with DN-AMPK or pcDNA control for 24 h, and then cells were treated with metformin (5 mM) or AICAR (2 mM) for 24 h. Conditioned media were collected after 24 h, and then gelatin zymography was performed. AMPK expression in cell lysates was analysed by Western blotting. β-Actin in the cell lysates is shown as a control.