br BAX Bcl ratio is
3.3. BAX/Bcl-2 ratio is static in SIM- and MET-treated endometrial cancer cells
To characterize the apoptotic mechanism, RL95-2 Cycloheximide treated with 1 μM SIM, 4 mM MET, or the combination for 48 h were tested by west-ern immunoblotting of BAX and Bcl-2 proteins as this pathway has been implicated in SIM-treated cancer cell lines [21,22]. Both BAX and Bcl-2
SIM has also been reported to induce apoptosis via the Bcl-2 like 11 protein (Bim), which upregulates mitochondrial apoptosis . Because the BAX/Bcl-2 ratio did not change, we therefore performed similar ex-periments with Bim as with BAX using western immunoblot analysis. The combination treatment showed significant upregulation of all three isoforms of Bim with the greatest significance being due to BimEl (P b 0.001). SIM showed similarly significant effects with BimL and BimS (p b 0.001) (Fig. 5A).
Fig. 3. Caspase-3 activity increases compared with control following treatment with metformin (MET), (simvastatin) SIM, or the combination (MET+SIM) for 24 and 48 h in all three cell lines. Data are presented as mean ± SEM. Letters above the chart bars indicate pair-wise comparisons: a) compared to control, b) compared to SIM, c) compared to MET, and d) compared to MET+SIM; all are significant at p b 0.001, by ANOVA. Each experiment was run with a minimum of three samples and/or repeated three times.
Fig. 4. BAX and Bcl-2 both increase in RL95-2 endometrial cancer cells treated with simvastatin (SIM), metformin (MET), or the combination (MET+SIM) for 48 h. Western immunoblotting of BAX and Bcl-2 showed similar increases in both proteins and a BAX/ Bcl-2 ratio that was not different between MET+SIM-treated (0.26 ± 0.002) and control (0.27 ± 0.003) cells. Mean ± SEM; *p b 0.01 vs control and MET, p b 0.05 vs SIM; **p b 0.05 versus all other treatments; by ANOVA. Ratios of untreated controls are set to 1.0. Each experiment was run with a minimum of three samples and/or repeated three times.
3.5. Bim silencing reverses MET- and SIM-induced apoptosis and cell growth inhibition
In order to verify Bim involvement in the apoptotic pathway, RL95-2 cells transfected with Bim siRNA or control siRNA were treated with 1 μM SIM, 4 mM MET, or the combination for 48 h and assessed for caspase-3 activity and viability. MTS assay demonstrated a significant rescue of cell viability in Bim siRNA-transfected combination-treated cells compared with control siRNA-transfected combination-treated cells (p = 0.0001) (Fig. 5C). In addition, Bim siRNA transfection resulted in a significant abrogation of caspase-3 activity compared with control siRNA transfection in cells treated with combination MET+SIM (p b 0.01) (Fig. 5D). Taken together, these results indicate that apoptosis in MET+SIM-treated endometrial cells is Bim-mediated and further sug-gest that apoptosis induction is a major mechanism underlying the in-hibitory effect of combination treatment on cell growth.
3.6. Inhibition of endometrial cancer cell growth following MET+SIM treat-ment is partially mediated by AMPK activation and mTOR pathway effects
The mTOR pathway has been a proposed key target in MET- and SIM-associated inhibition of endometrial cancer cells as well as in other cell types including breast, prostate, and ovarian cancer. Combina-tion MET+SIM has demonstrated similar anti-proliferative effects in castrate-resistant prostate cancer cells. We therefore evaluated the ef-fects of treatment with SIM, MET, or combination MET+SIM on targets of this pathway including phosphorylated AMPK (P-AMPK) and phos-phorylated S6 (P\\S6). Phosphorylated AMPK directly phosphorylates the mTOR subunit Raptor which in turn blocks the ability of mTOR