Figure 1.
Metformin induces cell growth arrest and apoptosis in ALL cell lines.
Growth inhibition (A) and apoptosis (B) in CCRF-CEM and NALM6 cells treated with metformin (MET, 1, 2, and 5 mM) for 48 h. Apoptosis (C) in T-ALL (CCRF-CEM, Jurkat, and primary T-ALL) and Bp-ALL (NALM6, REH, and primary Bp-ALL) cells treated with metformin (MET, 5 mM) for 48 h. Growth inhibition was expressed relative to control values (mean ±SEM, n = 3). * and # denote p < 0.001 and p < 0.0001 for metformin vs. CTRL, respectively.
Figure 2.
Metformin activates AMPK, Akt, and UPR signaling pathway proteins in ALL primary and cell line models.
A) Western blot analysis of proteins associated with AMPK, and Akt/mTOR signaling pathways in CCRF-CEM and NALM6 cells treated with metformin (MET, 2.5 and 5.0 mM) for 48 h. The density value of each band was normalized to β-actin level and expressed relative to control (shown as fold induction). B) Immunoblotting of UPR signaling factors in CCRF-CEM and NALM6 cells treated with metformin (MET, 2.5 and 5.0 mM) for 48 h. C) Western blots of AMPK, Akt/mTOR and UPR signaling proteins in representative sample of primary T- and Bp-ALL cells treated with metformin (MET, 10 mM) for 24 h. D) Western blot analysis of GRP78 expression in CCRF-CEM and NALM6 cells treated with metformin (MET, 10mM) and tunicamycin (TUN, 2.5 µg/ml for NALM6; 5.0 µg/ml for CCRF-CEM), either alone or in combination for 48 h.
Figure 3.
Inhibition of mTOR-dependent protein synthesis reverses metformin-induced cell death.
A) Apoptosis in CCRF-CEM cells expressing either scramble shRNA (shCTRL) or shRNA against AMPKα1 (shAMPK) treated with metformin (MET, 10 mM) for 48 h. B) Immunoblotting of AMPK, Akt/mTOR, and ER stress/UPR signaling pathway proteins in the cells described in (A). C) Cell death (upper panel) in CCRF-CEM and NALM6 cells treated with metformin (MET, 5 and 10 mM) and rapamycin (RAPA; 0.1 µg/mL), either alone or in combination for 48 h. A statistical value of p < 0.01 was obtained for MET + RAPA vs. either control or each agent alone. The cell death was expressed as a percentage (%) of cells in the population (mean ±SEM, n = 3). Western blot analysis (lower panel) of p-4EBP1 (T70), IRE1α, and CHOP expression in the CCRF-CEM and NALM6 cells treated with metformin plus rapamycin described in the upper panel.
Figure 4.
Metformin induces expression of PIM-2 in ALL cells.
Western blot analysis of PIM-2, p-BAD (S112) and p-AMPK (T172) expression in CCRF-CEM and NALM6 cells treated with metformin (MET, 5 and 10 mM) for 72 h at 37°C.
Figure 5.
Inhibition of PIM-2 and Akt kinases synergistically sensitizes ALL cells to metformin.
A) Cell death in CCRF-CEM and NALM6 cells treated with metformin (MET, 4 mM) and the PIM-1/2 kinase inhibitor V (PKI; 80 µM), either alone or in combination for 72 h at 37°C. The CI values of 0.27 and 0.28 indicate synergism. B) Immunoblotting of p-ACC (S79), GRP78, and PIM-2 expression in the CCRF-CEM and NALM6 cells treated with MET plus PKI described in (A). C) Cell death in NALM6 cells treated with metformin (MET, 5.0 mM) and Akt inhibitor X (AIX; 5 µM) or perifosine (PER; 6 µM), either alone or in combination for 72 h at 37°C. CI values of 0.19 (MET + AIX) and 0.21 (MET + PER) indicate synergism. The cell death values were expressed as a percentage (%) of cells in the population (mean ±SEM, n = 3). D) Immunoblotting of AMPK/ACC, Akt/mTOR, and UPR signaling pathway proteins in the NALM6 cells treated with MET (5.0 mM) plus AIX (5 µM) described in (C).
Figure 6.
Proposed mechanism of action for metformin in ALL cells.
Metformin induces metabolic stress by decreasing the ATP: AMP ratio, which leads to activation of AMPK, and increased level of unfolded/misfolded proteins in the ER lumen. The inability of ALL cells to engage the UPR caused by AMPK-dependent down-regulation of GRP78 leads to ER stress/UPR mediated cell death. The survival PIM and Akt kinases are expressed as compensatory survival mechanisms in response to metformin’s cytotoxicity to down-regulate AMPK allowing cells to effectively engage UPR and process the ER stress.