PMC

AICAR treatment inhibits proliferation of human childhood leukemia ALL cells. The ALL cell lines CCRF-CEM (T-lineage), NALM6 (Bp-lineage), REH (Bp-ALL expressing the TEL/AML1 fusion protein), and SupB15 (Bp-ALL expressing the BCR/ABL fusion protein) were treated for 24 h with various concentrations of AICAR (0.25 – 2 mM), and cell growth analyzed by thymidine ribotide ([³H]TdR) incorporation into DNA. The results are expressed as percentage of [³H]thymidine uptake (%) relative to control values (mean ± SEM, n = 3). #, p < 0.001 for AICAR-treated cells vs. control.

The anti-proliferative activity of AICAR is enhanced by the addition of the mTOR inhibitor rapamycin in ALL cells. CCRF-CEM, NALM6, REH, and SupB15 ALL cells were incubated for 24 h with either AICAR alone (0.25 mM for CCRF-CEM, NALM6, REH, and 1.0 mM for SupB15), 1 μg/ml rapamycin alone, or a combination of both drugs (Rapa + AICAR) and cell proliferation determined using the tetrazolium (MTS) reduction assays. The cell proliferation values are expressed as a percentage relative to those obtained with untreated control cells (mean ± SEM, n = 3). #, p < 0.005 for AICAR vs. Rapa + AICAR.

AICAR treatment induces cell cycle arrest in G1-phase in ALL cells. DNA content of CCRF-CEM, NALM6, REH, and SupB15 ALL cells untreated (Control) or treated with AICAR (0.5 and 2.0 mM) for 48 h was measured by fluorescence-activated cell sorting (FACS) using propidium iodide staining. The panels represent distribution of cells (%) in G1-, S-, and G2-phase of the cell cycle obtained from FACS analysis. Data are representative of at least three independent experiments and values are expressed as mean ± SEM.

AICAR treatment on ALL cells results in activation of Akt. Western blot analysis depicting the level of phosphorylated Akt protein (P-Akt, Ser473) in CCRF-CEM, NALM6, REH, and SupB15 cells were treated for 24 h with various concentrations of AICAR (0 – 0.5 mM). β-actin was used as control for the amount of proteins loaded per lane. Density value of each band was normalized to their respective β-actin level and expressed relative to control (untreated). The immunoblots shown are representative of at least 3 independent experiments.

Activation of AMPK is required for phosphorylation of p38-MAPK in AICAR-treated ALL cells. (A) Immunoblot of p38-MAPK phosphorylation (P-p38-MAPK, Thr180/Tyr182) and β-actin (loading control) expressed in CCRF-CEM and NALM6 cells treated with 0.1% DMSO (Control), 0.5 mM AICAR alone, 0.1 mM iodotubericidin alone (Iodo), or both agents together (Iodo + AICAR). (B) Phosphorylation status of AMPK (P-AMPK, Thr172) from CCRF-CEM and NALM6 cells incubated with either 0.5 mM AICAR alone, 10 μM SB 202190 alone (SB) or both inhibitors together (SB + AICAR). Level of β-actin was used as loading controls. Density value of each band was normalized to their respective β-actin level and expressed relative to control. The immunoblots shown are representative of 3 independent experiments, which produced similar results.

AICAR-activation of AMPK mediates apoptosis in ALL cells via the mitochondrial pathway. (A) DNA fragmentation assay. CCRF-CEM, NALM6, REH, and SupB15 cells were treated for 48 h with increased concentrations of AICAR (0 – 2 mM), and nuclear DNA was analyzed by electrophoresis on a 1.6% Tris-Borate-EDTA agarose gel. (B) Western blot analysis of cytochrome C and caspase 9 expression in CCRF-CEM and NALM6 cells treated for 48 h with 0.5 mM AICAR alone, 0.1 μM iodotubericidin alone (Iodo), or both agents together (Iodo + AICAR). Equal amount of loaded protein (50 μg) was confirmed by immunoblotting with anti-β-actin antibody. The data shown are representative of 3 experiments.

AICAR induces upregulation of the p53 and the cyclin dependent kinase inhibitor p27 in ALL cells. Western blot analyses of p53, p27, and p21 were done using cell extracts from CCRF-CEM, NALM6, REH, and SupB15 cells treated with the indicated concentrations of AICAR (0 – 2 mM) for 48 h. Equivalent amount of proteins (50 μg) were separated by SDS-PAGE and immunodetected with antibodies against p53, p27, and p21. Membranes were stripped and reprobed with anti-β-actin antibody to confirm equal amount of proteins loaded in all lanes. Density value of each band was normalized to their respective β-actin level and expressed relative to control (untreated). The data shown are representative of 3 experiments producing similar results.

Anti-proliferative action of AICAR on ALL cells is associated with downstream AMPK-dependent activation of p38-MAPK. (A) CCRF-CEM, NALM6, REH, and SupB15 ALL cells treated with 0.25 mM AICAR for the indicated times (0 – 24 h) were analyzed by Western blot for phosphorylated p38-MAPK protein (P-p38-MAPK, Thr180/Tyr182). β-actin was used as a loading control. Density value of each band was normalized to their respective β-actin level and expressed relative to control (untreated). (B) Cell proliferation assays of CCRF-CEM, NALM6, REH, and SupB15 cells treated with 0.25 mM AICAR alone, 10 μM of the p38-MAPK inhibitor SB 202190 alone (SB), or both agents together (SB + AICAR). The cell proliferation values are expressed as a percentage relative to those obtained with untreated control cells (mean ± SEM, n = 3). Data are representative of at least three independent experiments. *, p < 0.01 for AICAR vs. SB + AICAR; #, p < 0.05 for AICAR vs. SB + AICAR.

Proposed mechanism of action for AICAR in human leukemia ALL cells. After crossing the cell membrane, AICAR is metabolized by the adenosine kinase (AK) to its active mono-phosphorylated form ZMP (AMP analogue) which activates AMPK. The activated AMPK will signal to multiple downstream targets impinging on cell growth, cell proliferation, and cell survival. In ALL cells, induction of AMPK by AICAR increases the levels of p53, the cdk inhibitor p27, and the p38-MAPK leading to inhibition of cell proliferation, cell arrest in G1-phase, and apoptosis. In addition, P-AMPK activates TSC2 reducing the level of mTOR, an important regulatory factor of the PI3K/Akt pathway necessary for cell proliferation [24]. As a cell survival mechanism, the level of Akt is increased to overcome the reduction in mTOR attempting to restore or promote cell growth. AICAR, 5-Aminoimidazole-4-Carboxamide-1-β-4-Ribofuranoside; AK, adenosine kinase; AMPK, AMP activated protein kinase; PI3K, phosphatidylinositol 3-kinase; Akt, proteinase kinase B; TSC, tuberous sclerosis complex; mTOR, mammalian target of rapamycin; MAPK, mitogen-activated protein kinase.

The anti-proliferative effect of AICAR on ALL cells is mediated via activation of AMPK. (A) Western blot analysis of phosphorylated AMPK (P-AMPK, Thr172) expression in CCRF-CEM, NALM6, REH, and SupB15 cells treated with various concentrations of AICAR (0 – 2.0 mM). Total protein was extracted from AICAR-treated cells and AMPK and P-AMPK were immunodetected using specific antibodies. Equal amounts of protein (50 μg) were loaded per lane as confirmed by β-actin level. Density value of P-AMPK bands were normalized to level of AMPK and expressed relative to control. (B) Cell proliferation assays of ALL cells treated for 18 h with AICAR alone (0.25 mM for CCRF-CEM, NALM6, REH, and 1.0 mM for SupB15), the adenosine kinase inhibitor iodotubericidin alone (Iodo, 0.1 μM), or both agents together (Iodo + AICAR). Growth inhibition was determined using the tetrazolium (MTS) reduction assay. Values are expressed as a percentage relative to those obtained with untreated control cells (mean ± SEM). Data are representative of at least three independent experiments. #, p < 0.001 for AICAR vs. Iodo + AICAR.