The anti-diabetes drug metformin has been shown to have anti-neoplastic effects in several tumor models through its effects on energy metabolism and protein synthesis. signaling seems to be promising from a therapeutic point of view in vitro, more research is needed when implementing this combination strategy in vivo. < 0.05 vs. control; (C) Protein expression ... In line with this, cyclin D1 protein expression was drastically decreased after treatment with 5 mM metformin, especially in the rapidly proliferating PC3 Bay 65-1942 and DU145 cell lines (Figure 1C). Additionally, metformin activated its downstream signaling components AMPK and Acetyl-CoA carboxylase (ACC) in a dose-dependent manner in all PCa cell lines (Figure 1C). 2.2. Metformin Increases Radiosensitivity of PCa Cells Independent of Adenosine Monophosphate (AMP)-Activated Protein Kinase (AMPK) Activation Metformin (5 mM) increased radiosensitivity of DU145 and 22Rv1 cells with a dose-enhancement factor (DEF) of 1.6 0.15 (< 0.05) and 1.36 0.08 (< 0.05) respectively. In contrast, the radiosensitivity of PC3 cells was not affected by metformin (Figure 2A). To evaluate the role of AMPK in the metformin-induced radiosensitization effect in the DU145 and 22Rv1 cells, AMPK was silenced by means of silencing RNA (siRNA). Downregulation of (phospho)AMPK did not affect Rabbit polyclonal to GnT V the intrinsic radiosensitivity of either cell line nor did it change the metformin-induced radiosensitization (Figure 2B). Figure 2 Effect of metformin (MF) on radiosensitivity of PCa cells. (A) Clonogenic survival after 72-h treatment with Bay 65-1942 metformin (5 mM) prior to/during ionizing radiation (IR); (B) Clonogenic survival of DU145 and 22Rv1 cells transfected with AMPK Bay 65-1942 silencing RNA … 2.3. Metformin Regulates Hedgehog Signaling in an AMPK-Dependent Manner Next, we investigated if there was a link between metformin and Hh signaling in PCa cells. Indeed, metformin (5 mM) significantly decreased glioma-associated oncogene homolog 1 (and gene expression after 72-h metformin treatment. Means SEM of two independent experiments. * < 0.05 vs. control; (B) PTCH1, GLI1 and GLI2 protein expression after ... 2.4. Combination of Metformin and GANT61 (GLI-ANTagonist 61) Synergistically Decreases PCa Cell Growth The link between AMPK and GLI1 led to the question as to whether the combination of metformin with Hh inhibitors could enhance the cytotoxic effect of the individual drugs. We have previously shown that the GLI1/2 inhibitor GANT61 significantly decreased cell survival of PC3 and Bay 65-1942 22Rv1 cells . Indeed, combining metformin and GANT61 significantly decreased cell Bay 65-1942 growth of all PCa cell lines, resulting in an almost complete blockage of cell growth in PC3 and 22Rv1 cells (Figure 4A). Additionally, we confirmed decreased gene expression in all cells treated with the drug combination (Figure S2). Cell cycle analyses revealed that the drug combination in the PC3 cells led to a G2/M-arrest after only 24 h, which persisted until 72 h of treatment (Figure 4B). This corresponds to the dramatic decrease in cell growth already observed after 24 h of treatment. The drug combination also significantly increased the sub-G1 population which peaked at 48 h (Figure 4C). In the DU145 cells, no significant cell cycle effects were observed after 24C72 h of either treatment (Figure 4B), whereas the combination treatment did significantly increase apoptosis after 72 h compared to either single agent (Figure 4C). In the 22Rv1 cells, GANT61 induced a G1-arrest after only 24 h. Metformin alone did not have a significant effect.