Figure 3

AMPK activation is not required for metformin inhibition of prostate cancer cells. Prostate cancer cells were treated with increasing concentrations of AICAR, an AMPK activator, Compound C, an AMPK inhibitor or a combination of AMPK inhibitor and metformin, respectively. LNCaP cells were treated with the AMPK activator AICAR. After 96 h AR levels were quantified by western blot and normalized to GAPDH. Activation of AMPK was verified by detection of phosphorylated AMPK by western blot (P-AMPK/AMPK ratio) (A). Prostate cancer cells were treated with increasing concentrations of the AMPK inhibitor compound-C (0–10 μM) and cell numbers (B) and AR protein levels (C) in LNCaP and DuCaP cells were determined after 96 h. Inhibition of AMPK was verified by detection of phosporylated AMPK (P-AMPK/AMPK ratio) (C)). Representative fluoroscan images of LNCaP and DuCaP cell western blots are shown in (D). Addition of 1 mM of metformin to the AMPK inhibitor compound-C resulted in enhanced inhibition of cell proliferation in LNCaP and PC3 cells (E) and further reduction of AR protein levels in LNCaP cells (F). AR levels and P-AMPK/AMPK ratios in Figure 3 were quantified by western blot densitometry. The histograms show the data of at least three independent experiments, the fluoroscan image shows representative western blots. Statistical significant differences are indicated as *, p < 0.05; **, p <0.01 and ***, p < 0.001.