br Inhibition of endometrial cancer cell
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
kinase complex from phosphorylating its downstream substrates, in-cluding S6 which mediates AMG 925 progression . As shown in Fig. 6, western immunoblot analysis demonstrated that effects on phos-phorylation of AMPK and S6 following treatment with SIM and MET alone were variable and differed by cell line. Combination MET+SIM treatment, however, increased P-AMPK and decreased P\\S6 by 48 h following treatment in all cell lines (Fig. 6).
3.7. Autophagy is unlikely a significant mechanism of cell death following treatment with MET+SIM in endometrial cancer cells
Macroautophagy (hereafter referred to as autophagy) is a lysosomal-dependent process of degradation of cytoplasmic contents, abnormal protein aggregates, and excess or damaged organelles. This process can promote either survival or cell death and is regulated by mTOR in that mTOR inhibition promotes autophagy signaling. Autoph-agy and apoptosis are interrelated via complex crosstalk between the two mechanisms and this crosstalk is in part caspase-mediated. Given our findings suggestive of apoptosis induction and increased P-AMPK with associated decreased P\\S6 along the mTOR pathway, we next sought to determine if autophagy was also being induced. Beclin-1 is a central regulator of autophagy downstream of mTOR and was thus eval-uated to determine if significant autophagy induction occurred. West-ern blot analysis showed that MET+SIM combination treatment resulted in statistically significant but modest increases in beclin-1 levels at 24 h in all cell lines (Supplementary Fig. S4). By 48 h following treatment, these effects were attenuated: combination-treated HEC-1B cells showed a statistically significant but equally modest increase in beclin-1, and there were no longer significant differences in RL95-2 and Ishikawa cells.
Repurposing of inexpensive, commonly used, FDA-approved medi-cations to exploit their anti-cancer effects may yield the development of cost-effective approaches to cancer therapy. Along these lines, MET and SIM have great potential for repurposing in the endometrial cancer population given the high prevalence of comorbid metabolic syndrome, obesity, and hyperlipidemia . We demonstrate here that the combi-nation of MET+SIM treatment resulted in synergistic growth inhibition in three endometrial cancer cell lines. This was associated with pro-found activation of apoptosis as manifested by markedly increased caspase-3 activity, TUNEL positivity, and Annexin V labeling. Addition-ally, we showed that the combination activates the AMPK pathway.
Interestingly, apoptosis was not consistently induced in our study by MET alone, yet the effect with combination MET+SIM treatment was robust. One prior study found that MET induces apoptosis at 24 h in Ishikawa cells but only at high concentrations . We replicated this finding in Ishikawa cells though we did not find MET-induced apoptosis in HEC-1B or RL95-2 cell lines. Apoptosis induction has also previously been demonstrated in Ishikawa cells following treatment with SIM  alone using similar doses as those used in this study, however the combination of MET+SIM in endometrial cancer has not been previ-ously investigated. To our knowledge, this combination has only been studied in castration-resistant prostate cancer cells in which apoptosis induction with MET+SIM treatment was not observed . This differ-ence may be explained by this prostate cancer cell line's resistance to extrinsic apoptotic cell death due to loss of tumor necrosis factor receptor-associated death domain protein expression.
Silencing Bim in our study reversed both MET+SIM-induced apo-ptosis and synergistic growth inhibition, suggesting that the dramatic apoptotic effects we observed may explain the synergy seen with this treatment combination. Bim may directly interact with BAX to initiate apoptosis . This mechanism of cell death is consistent with prior in-vestigations with statins in human cancer cells, including ovarian cancer cells [22,30]. Of note, Bim can both activate BAX and bind bcl-2,
Fig. 5. Bim is upregulated by combination treatment with metformin and simvastatin and has a primary role in endometrial cancer cell apoptosis. A, Western blot analysis shows that the production of all three isoforms of Bim is increased in RL95-2 cells in response to simvastatin (SIM) and combination metformin and simvastatin (MET+SIM). Metformin (MET) alone significantly increased BimEl and BimL. Mean ± SEM; symbols above bars indicate comparison to control, symbols above lines designate comparisons between indicated treatment groups; *p b 0.05, **p b 0.01, ***p b 0.001, by ANOVA. Ratios of untreated controls are set to 1.0. B, Western blot images and densitometry chart of RL95-2 cells transfected with Bim siRNA or control siRNA show substantial knockdown of Bim production by Bim siRNA (percent knockdown indicated in parentheses). First two lanes show control siRNA, last two lanes show Bim siRNA (each performed in duplicate with composite data shown here). C, RL95-2 cells transfected with Bim siRNA or control siRNA were treated with MET, SIM, or MET+SIM for 48 h. MTS assay demonstrated a significant rescue of cell viability in Bim siRNA-transfected MET+SIM-treated cells compared with control siRNA-transfected MET +SIM-treated cells, as well as in SIM-treated cells. Mean ± SEM; **p b 0.01, ***p = 0.0001, by t-test. D, MET-, SIM-, and MET+SIM-treated RL95-2 endometrial cancer cells were assessed for caspase-3 activity. Bim siRNA transfection resulted in a significant abrogation of caspase-3 activity compared with control siRNA transfection in cells treated with MET+SIM, as well as in cells treated with SIM alone. Mean ± SEM; *p b 0.05, **p b 0.01, by t-test.