- Open Access
Cytotoxic effect of 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy
© Sengupta et al; licensee BioMed Central Ltd. 2007
- Received: 17 April 2007
- Accepted: 10 July 2007
- Published: 10 July 2007
Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting children. Despite significant progress and success in the treatment of ALL, a significant number of children continue to relapse and for them, outcome remains poor. Therefore, the search for novel therapeutic approaches is warranted. The aim of this study was to investigate the AMP activated protein kinase (AMPK) as a potential target in childhood acute lymphoblastic leukemia (ALL) subtypes characterized by non-random translocation signature profiles. We evaluated the effects of the AMPK activator AICAR on cell growth, cell cycle regulators and apoptosis of various childhood ALL cells.
We found that treatment with AICAR inhibited cell proliferation, induced cell cycle arrest in G1-phase, and apoptosis in CCRF-CEM (T-ALL), NALM6 (Bp-ALL), REH (Bp-ALL, TEL/AML1) and SupB15 (Bp-ALL, BCR/ABL) cells. These effects were abolished by treatment with the adenosine kinase inhibitor 5'-iodotubericidin prior to addition of AICAR indicating that AICAR's cytotoxicity is mediated through AMPK activation. Moreover, we determined that growth inhibition exerted by AICAR was associated with activation of p38-MAPK and increased expression of the cell cycle regulators p27 and p53. We also demonstrated that AICAR mediated apoptosis through the mitochondrial pathway as revealed by the release of cytochrome C and cleavage of caspase 9. Additionally, AICAR treatment resulted in phosphorylation of Akt suggesting that activation of the PI3K/Akt pathway may represent a compensatory survival mechanism in response to apoptosis and/or cell cycle arrest. Combined treatment with AICAR and the mTOR inhibitor rapamycin resulted in additive anti-proliferative activity ALL cells.
AICAR-mediated AMPK activation was found to be a proficient cytotoxic agent in ALL cells and the mechanism of its anti-proliferative and apoptotic effect appear to be mediated via activation of p38-MAPK pathway, increased expression of cell cycle inhibitory proteins p27 and p53, and downstream effects on the mTOR pathway, hence exhibiting therapeutic potential as a molecular target for the treatment of childhood ALL. Therefore, activation of AMPK by AICAR represents a novel approach to targeted therapy, and suggests a role for AICAR in combination therapy with inhibitors of the PI3K/Akt/mTOR pathways for the treatment of childhood in ALL.
- Acute Lymphoblastic Leukemia
- mTOR Pathway
- AMPK Activation
- Childhood Acute Lymphoblastic Leukemia
AMP activated protein kinase (AMPK) is a highly conserved heterotrimeric serine/threonine protein kinase that regulates the intracellular ratio of AMP to ATP, and it is activated under conditions that deplete cellular ATP and hence increase AMP levels [1–3]. Therefore, the AMPK cascade is a sensor of cellular energy status that is activated by multiple stimuli such as metabolic stresses including ischemia, hypoxia and glucose deprivation, environmental stresses like heat shock, oxidative and osmotic stress [4, 5]. It is also activated by various pharmacological agents including respiratory chain inhibitors (actinomycin D, nitric oxide), ATP synthase inhibitors (oligomycin), mitochondrial uncouplers (dinitrophenol), TCA cycle inhibitors (arsenite), biguanides (metformin) and nucleosides (adenosine analogue AICAR) [6–9]. The AMPK pathway is also implicated in the regulation of cell cycle and cell proliferation and it has recently been determined that its activation by AICAR results in pro-apoptotic effect [10–12].
Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting children and adolescents . Significant advances in our understanding of the biology and molecular genetics of ALL have led to the identification of molecularly defined subgroups important for therapy stratification and prognosis . Despite significant progress and success in the treatment of ALL, a significant number of children continue to relapse and for them, outcome remains poor . Likewise, the outcome for others who are diagnosed with chemotherapy resistant phenotypes continues to be poor. In this context, childhood ALL continues to be a major cause of cancer related mortality in children and adolescents and therefore, novel treatment strategies are needed. During recent years, novel targeted and molecular agents have been introduced in the treatment of hematological malignancies in adults , but the experience with these agents in pediatric leukemia remains minimal. Our data presented herein, supports the role of AMPK and its downstream pathways as a suitable target for molecular therapies in childhood ALL. The recognition of this pathway's physiological importance in terms of cell cycle regulation, cell proliferation, survival and apoptosis is highlighted by recent reports in prostatic and breast carcinomas, as well as gliomas, among others [16, 17].
The anti-proliferative and pro-apoptotic activity of AMPK have been linked to the tumor suppressor genes LKB1 (a serine/threonine protein kinase formerly identified as STK11) and TSC2 t uberous s clerosis c omplex 2) [6, 18, 19]. LKB1 mutations result in Peutz-Jeghers syndrome, which leads to predisposition to cancers of the colon, pancreas, breast, and other sites [20–22]. Mutations of LKB1 typically occur in the catalytic domain, leading to loss of its kinase activity . TSC2 forms a complex with TSC1 and inhibits mTOR m ammalian t arget o f r apamycin), leading to inhibition in protein synthesis and negative regulation of cell size and growth . Mutations of TSC1·TSC2 cause tuberous sclerosis, a condition associated with hamartomatous polyps in multiple tissues and an increased risk of cancers .
Structurally, AMPK consists of a catalytic (α) and two regulatory subunits (β and γ), each subunit having at least two isoforms [1, 26]. AMPK activation requires a conformational change induced by AMP binding to the α and γ subunits, which in turn allows its phosphorylation/activation by the upstream protein kinase LBK1 [6, 27, 28]. The conformational change required for AMPK activation can also be induced by compounds that act as AMP analogs and therefore under conditions that do not involve changes in the ratio of AMP/ATP. AICAR, a nucleoside widely used as AMPK activator, is converted inside the cell to its mono-phosphorylated form ZMP (5-amino-4-imidazolecarboxamide ribotide), and as such behaves as an AMP analogue capable of activating AMPK upstream of LKB1 . AICAR mediated AMPK activation has been reported to inhibit cell proliferation and cell cycle progression via inhibition of the PI3K/Akt pathway and the cell cycle regulatory proteins p21, p27 and p53 .
In the present study we have investigated the effect of AMPK activation by AICAR on the proliferation, cell cycle progression and apoptosis of various childhood ALL cell models characterized by non-random translocation signature profiles, and representing chemotherapy sensitive and resistant phenotypes [29–32]. AICAR-mediated AMPK activation was found to be a proficient cytotoxic agent in ALL cells and the mechanism of its anti-proliferative and apoptotic effect appear mediated via activation of p38-MAPK pathway, increased expression of cell cycle inhibitory proteins p27 and p53, and downstream effects on the mTOR pathway, hence exhibiting therapeutic potential as a molecular target for the treatment of childhood ALL.
RPMI 1640 medium was obtained from Mediatech, Inc. (Herndon, VA). Iscove's modified Dulbecco medium (IMDM) and fetal bovine serum (FBS) were obtained from GIBCO/Invitrogen (Carlsbad, CA). AICAR was purchased from Toronto Research Chemicals (Ontario, Canada). Iodotubericidin and SB 202190 were obtained from Calbiochem (San Diego, CA). CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit was purchased from Promega (Madison, WI). [3H]Thymidine ribotide ([3H]TdR) was purchased from Amersham Biosciences (GE Healthcare, Piscataway, NJ). The Propidium Iodide (PI)-RNase Staining kit was obtained from BD Pharmigen (Franklin, NJ). The enhanced chemiluminescence (ECL) detecting reagent was from Amersham Biosciences. Primary antibodies against p21, p27, and p53 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phosphospecific as well as pan-Akt, AMPK and p38-MAPK were obtained from Cell Signaling (Beverly, MA).
Childhood ALL Cell Lines
The following childhood ALL cell lines were used in this study: CCRF-CEM (T-lineage ALL), NALM6 (B-lineage precursor Bp-ALL), REH (Bp-ALL expressing the TEL/AML1 fusion protein) as representative of a chemotherapy sensitive phenotype, and SupB15 (Bp-ALL expressing the BCR/ABL fusion protein) as a model of a chemotherapy resistant phenotype. CCRF-CEM, REH and SupB15 cells were obtained from ATCC (Rockville, MD), NALM6 cells were purchased from DSMZ (Braunschweig, Germany).
The childhood ALL cell lines CCRF-CEM, NALM6 and REH were maintained in RPMI 1640 medium supplemented with 10% FBS and antibiotics as described elsewhere . SupB15 cells were maintained in IMDM medium with 20% FBS. All cells were grown at 37°C and 5% CO2 atmosphere, and all drug treatments were done in the presence of serum.
Cell Proliferation Assay
Cell growth and viability were assessed using the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt] (MTS) (Promega). Briefly, 0.5 × 106 cells/well of each cell lines were plated and incubated for 18 – 24 h with or without AICAR at various concentrations. Next, 20 μl of MTS solution was added to each well and cells were incubated for an additional 2 to 4 h, after which, absorbance at 490 nm was determined using a microplate reader as a reflection of MTS reduction by viable cells. Values were expressed as a percentage relative to those obtained in untreated controls.
Thymidine Incorporation Assay
Proliferation of cells was also determined by [3H]thymidine ribotide ([3H]TdR) incorporation into DNA. Each cell line was plated at a density of 0.25 × 106 cells/well, and cells were incubated for 18–24 h with or without AICAR at various concentrations and then exposed to 37 kBq/ml [methyl-3H]thymidine for 6 h. Suspension cell cultures were harvested using a cell harvester (Packard instrument Co., Meriden, CT), and radioactivity was measured using a 1450 microbeta liquid scintillation counter (PerkinElmer Life Sciences).
Flow Cytometry Assessment of Cell Cycle
Cells were cultured in 6-well plates and treated with AICAR prior to assessment of cellular DNA content by flow cytometry. Following treatment, cells were washed with PBS, and 1.0 × 106 cells were resuspended in 100 μl of PBS, and 5 ml of 70% ethanol were added slowly, while under continuous vortexing, for fixation overnight. Subsequently, cells were washed, and suspended in 500 μl of PI/RNase solution and cell cycle progression was determined by flow cytometry (BD Biosciences FACSCalibur flow cytometer) using the Modfit LT software.
Apoptosis/DNA Ladder Gel Assay
Ten million purified ALL cells were obtained by centrifugation after exposure to 0.1 to 1.0 mM AICAR for 48 h. Cells were lysed in 0.5 ml of 20 mM Tris (pH 7.4), 0.4 mM EDTA, 0.25% Triton X-100 (American Bioanalytical, Natick, MA). After 15 min of incubation at room temperature, nuclei were removed by centrifugation at 14,000 rpm (RCF = 16,000). The supernatant was transferred to a new tube and nuclear DNA was precipitated overnight at -20°C using 55 μl of 5 M NaCl and 550 μl of isopropanol. After centrifugation at 14,000 rpm for 10 minutes, the pellet was washed with 70% ethanol and resuspended in 20 μl of 10 mM Tris (pH 8.0), 1 mM EDTA, and 0.1 mg/ml RNase. The DNA preparations were separated by 1.6% Tris borate EDTA agarose gel electrophoresis and visualized by ethidium bromide staining.
After stipulated incubation times in the presence or absence of AICAR, cells were harvested, washed with PBS, and sonicated in 50 mM Tris-HCl (pH 7.4) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml antipain, 5 μg/ml pepstatin A, and 5 μg/ml leupeptin). Proteins (50 μg/lane) were resolved by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked for 1 h in 5% nonfat dry milk in TTBS (20 mM Tris, 500 mM NaCl, and 0.1% Tween 20, pH 7.5) and incubated overnight in primary antibody (p21, p27, p53, AMPK, p38-MAPK, Akt, β-actin, at a 1:2000 dilution) containing 5% nonfat dry milk for non-phospho antibodies and containing 5% albumin for phospho-antibodies (P-Akt, P-AMPK, P-p38-MAPK, at a 1:1000 dilution). The blots were washed four times (5 min) with TTBS and incubated for 45 min at room temperature with the respective horseradish peroxidase-conjugated secondary antibody (1:5000). The blots were washed three times in TTBS and once in 0.1 M PBS (pH 7.4) at room temperature, and protein expression levels determined using the ECL detection kit (Amersham Biosciences). The relative integrated density value (IDV) of each immunodetected band was determined using the ChemiDoc XRS digital imaging system with the Quantity One 1-D Analysis Software Version 4.6.3 (Bio-Rad Laboratories, Inc., Hercules, CA). The IDV data were normalized to β-actin levels and expressed relative to control.
Multiple comparisons of cell proliferation, and number of viable cells (%) were assessed by one-way ANOVA followed by the Newman-Keuls multiple comparison test. Individual comparisons were achieved using one-tailed, unpaired t test according to the Graph Pad PRISM software version 2 (GraphPad Software, Inc., San Diego, CA). The data were expressed as mean ± SEM.
AICAR induces growth inhibition in ALL cells via AMP-activated protein kinase (AMPK)
AICAR-induced regulation of cyclin dependent kinase inhibitors leads to cell cycle arrest and AMPK activation dependent apoptosis in ALL cells
Anti-proliferative action of AICAR on ALL cells is associated with downstream AMPK-dependent activation of p38-MAPK
AICAR treatment in ALL cells results in activation of Akt
The anti-proliferative activity of AICAR is enhanced by the addition of the mTOR inhibitor rapamycin in ALL cells
Multiple studies have demonstrated that a key function of AMPK is regulation of the intracellular energy balance. This is supported by AMPK's activation in response to low levels of ATP [1, 2]. AMPK activation in turn results in phosphorylation of multiple downstream targets to switch off ATP-consuming (fatty acid and cholesterol synthetic pathways) pathways and to turn on ATP-generating pathways (glycolysis and fatty acid oxidation) . The end result is protein synthesis, cell growth, proliferation and survival. Several observations support this role of AMPK in cancer cells [44, 45]. Indeed, many cancers have increased expression of enzymes that are inhibited by AMPK, such as FAS and mTOR [17, 46]. Paradoxically, when activated pharmacologically, AMPK is able to induce apoptosis in various tumor cell types [10, 16]. Two tumor suppressors gene products have been identified as the upstream activator and downstream effector of AMPK, namely LKB1 and TSC2, respectively [6, 18, 19]. A small, but emerging, body of literature demonstrates that AMPK activation is capable of inhibiting growth in cancer cell in vitro [16, 17, 47]. The mechanisms responsible for these opposing effects of AMPK activation are yet to be fully understood but the anti-proliferative and pro-apoptotic effect of AMPK has been shown to be mediated via the negative regulation of mTOR by LKB1 . Others have also reported that AMPK's growth inhibitory properties are mediated by various other mechanisms, including inhibition of de novo fatty acid synthesis and p70S6K mediated inhibition of protein synthesis, inhibition of cell cycle progression by p21, and attenuation of PI3K and Akt pathways .
Although advances in the treatment of children with ALL have resulted in survival rates approaching 90% for all subtypes combined, outcome for patients diagnosed with resistant phenotypes and for those who relapse continues to be dismal [29–32]. Consequently, ALL continues to be a leading cause of cancer related death in children and adolescents, underscoring the need to discover new targets and develop novel treatment strategies for patients with this disease . Our results suggest that AMPK is one such target, and that its activation by the nucleoside AICAR is an efficient strategy to induce apoptosis in childhood ALL cells. AICAR induced growth inhibition as determined by [3H]thymidine incorporation assays in a dose dependent manner in all ALL cell lines studied. Further, AICAR was also capable of inducing growth inhibition in those ALL cell lines representative of more resistant phenotypes, such as CCRF-CEM (T-ALL) and SupB15 (BCR/ABL positive Bp-ALL), even though higher concentrations of AICAR were required to inhibit the latter cell type. Although AMPK-independent effects have been reported for AICAR , our data demonstrate that its growth inhibitory effects were mediated via phosphorylation of AMPK as confirmed by the ability of the adenosine kinase inhibitor iodotubericidin to reverse the cytotoxic effects of AICAR.
AMPK activation by AICAR has also been reported to induce cell cycle arrest in cancer cell culture models . It has been shown that AMPK activation induces the expression of wild-type p53 and p21 in rat hepatoma cells . Most reports indicate AMPK activation induces apoptosis by increasing the phosphorylation of p53 at Ser15 [49–51]. The upstream activator of AMPK has also been implicated in increasing the concentration of p21 when transfected in LKB1-deficient A549 lung adenocarcinoma cells . Others have reported cell cycle arrest in S-phase after AMPK activation by AICAR using C6 glioma and U87MG astrocytic cell lines . Our data are consistent with these reports, as AICAR induced cell cycle arrest in G1-phase in all lymphoid leukemia cell lines we examined, including the BCR/ABL positive cell line SupB15. Although we also observed an increase in p53 expression leading to cell cycle arrest in these cell lines, no consistent change in the level of p21 expression was detected. In contrast, our data demonstrate increased expression of another cell cycle regulatory protein, p27. The TSC proteins tuberin and hamartin are known to be positive regulators of the cyclin dependent kinase p27 and tuberin (TSC2) has been reported to protect the ubiquitin-dependent degradation of p27 [53, 54]. In addition, it has been shown that tuberin can affect p27 localization as p27 is translocated to the nucleus [55, 56]. Our data also demonstrate increase phosphorylation of the mitogen-activated protein kinase p38-MAPK. Several reports indicate that activation of p38-MAPK results in inhibition of the cell cycle at the G1/S boundary, such as it was seen in ALL cells after AICAR mediated activation of AMPK (and increased P-p38-MAPK). The exact mechanism leading to p53 and p27 mediated cell cycle arrest in ALL cell line following activation of AMPK is not fully understood and it is currently under investigation in our laboratory.
AICAR induced phosphorylation of AMPK triggered apoptosis in all cell lines studied, although higher concentrations of AICAR were required for SupB15 cells. Both cytochrome C release and caspase 9 cleavage were observed in NALM6 and CCRF-CEM cells treated with AICAR, and co-incubation with the inhibitor of AICAR metabolism to its activated form ZMP, iodotubericidin, was able to block these apoptotic effects. While others have reported concomitant inhibition of the PI3K/Akt pathway after exposure to AICAR , we consistently observed increase phosphorylation of Akt in ALL cell lines treated with AICAR. It is known that AMPK and Akt have opposite regulatory effects on the mTOR pathway . Activated AMPK via its downstream effector TSC2 negatively regulates mTOR, leading to apoptosis, while Akt promotes activation of the mTOR pathway [24, 43]. We interpreted the consistent activation of Akt in ALL cell lines after AICAR induced AMPK activation as a potentially compensatory pro-survival mechanism. Therefore, we hypothesized that targeting the mTOR pathway while simultaneously activating AMPK should result in increased cytotoxicity. Indeed, our data demonstrate that the combination of AICAR and rapamycin resulted in additive growth inhibitory effects in all ALL cell lines studied. These results suggest simultaneous activation of AMPK and inhibition of the PI3K/Akt/mTOR pathway is an attractive combination targeted therapy for the treatment of childhood ALL leukemia, and may be active in the treatment of resistant phenotypes.
The authors would like to thank Drs. Sumita Bandyopadhyay, Shailendra Giri, and Narender Nath for helpful discussions and technical assistance. This investigation was supported by the National Cancer Institute (NCI) research grant CA098152-02 (to J.C.B.) and the Monica Kreber Golf Classic, Charleston, SC.
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