Different effects of phosphatidylinositol 3-kinase inhibitor LY294002 and Akt inhibitor SH-5 on cell cycle progression in synchronized HL-60 leukemia cells

Background and Purpose: Pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway have been proposed in treatment of leukemia. Our previous studies demonstrated that PI3K inhibitor LY294002 and Akt inhibitor SH-5 reduced the number of viable HL-60 leukemia cells, but exerted different effects on their differentiation. PI3K inhibitor LY294002 induced an arrest of HL-60 cells in G0/G1 and G2/M phases of the cell cycle, but the effects of Akt inhibitor on cell cycle progression have not been investigated. The present study was undertaken in order to compare the effects of LY294002 and SH-5 on cell cycle progression in a model of aphidicolin and nocodazole-synchronized HL-60 cells. Materials and Methods: HL-60 cells were arrested at G1/S phase by aphidicolin, or G2/M phase by nocodazole, washed and allowed to progress synchronously through the cell cycle. PI3K inhibitor LY294002 (10 μM) and Akt inhibitor SH-5 (20 μM) were added to medium after release from the block. DNA content was determined by propidium iodide staining and flow cytometry. Results: In aphidicolin-synchronizedHL-60 cells, LY294002 induced a delay in progression through S andG2/M phases, while Akt inhibitor had no significant effects. No statistically significant effects were observed in LY294002 or SH-5-treated cells at 1–5 h after release from nocodazole block. Conclusion: PI3K inhibitor LY294002 and Akt inhibitor SH-5, applied at doses that effectively inhibit Akt-activity, have different effects on cell cycle progression in aphidicolin-synchronized HL-60 cells, suggesting that LY294002-induced delay in S and G2/M phase is probably not due to the specific inhibition of Akt-activity

Metabolism and differentiation

Textbook biochemical pathways do not usually apply to intermediary metabolism of highly proliferating, differentiating, or tumor cells. Over 80 years ago, Otto Warburg observed that cancer cells, unlike normal cells, favor glycolysis for energy production, even under aerobic conditions, and proposed that this shift in cancer cell metabolism (termed „aerobic glycolysis”) was due to mitochondrial dysfunction. Recent studies by several groups suggest that aerobic glycolysis in tumor cells is actually caused by oncogene-directed changes in metabolism that are necessary for both continuous proliferation and a block in cellular differentiation. Phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) is one of the principal proliferative and anti-apoptotic signaling pathways, which is known to support glycolysis and anabolism. Our previous studies demonstrated the activation of PI3K and Akt in nuclei of leukemia cells during differentiation, and confirmed that the inhibition of proximal components of the pathway inhibits proliferation, but negatively affects differentiative capacity of the cells. In contrast, use of rapamycin, which inhibits mTOR, a more distal component of the pathway, potentiates differentiation along granulocytic pathway. To further investigate the role of upstream regulators of mTOR in leukemia differentiation, we tested the effects of modulators of AMP-activated protein kinase (AMPK). Our results suggest a strong differentiative property of an AMPK activator, AICAR (5-amino-1-b-D-ribofuranosyl-imidazole-4-carboxamide) in monocytic U937 cells. The mechanism of AICAR-mediated effects will be presented and a possible role of AMPK-modulators in differentiation therapy will be discussed

The mechanism of synergistic effects of arsenic trioxide and rapamycin in acute myeloid leukemia cell lines lacking typical t(15;17) translocation

Arsenic trioxide (ATO) has potent clinical activity in the treatment of patients with acute promyelocytic leukemia (APL), but is much less efficacious in acute myeloid leukemia (AML) lacking t(15;17) translocation. Recent studies have indicated that the addition of mammalian target of rapamycin (mTOR) inhibitors may increase the sensitivity of malignant cells to ATO. The aim of the present study was to test for possible synergistic effects of ATO and rapamycin at therapeutically achievable doses in non-APL AML cells. In HL-60 and U937 cell lines, the inhibitory effects of low concentrations of ATO and rapamycin were synergistic and more pronounced in U937 cells. The combination of drugs increased apoptosis in HL-60 cells and increased the percentage of cells in G(0)/G(1) phase in both cell lines. In U937 cells, rapamycin alone increased the activity of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and the addition of ATO decreased the level of phosphorylated ERK, Ser473 phosphorylated Akt and anti-apoptotic Mcl-1 protein. Primary AML cells show high sensitivity to growth-inhibitory effects of rapamycin alone or in combination with ATO. The results of the present study reveal the mechanism of the synergistic effects of two drugs at therapeutically achievable doses in non-APL AML cells

5-Aminoimidazole-4-carboxamide ribonucleoside-induced autophagy flux during differentiation of monocytic leukemia cells

Pharmacological modulators of AMP-dependent kinase (AMPK) have been suggested in treatment of cancer. The biguanide metformin and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) have been reported to inhibit proliferation of solid tumors and hematological malignancies, but their role in differentiation is less explored. Our previous study demonstrated that AICAR alone induced AMPK-independent expression of differentiation markers in monocytic U937 leukemia cells, and no such effects were observed in response to metformin. The aim of this study was to determine the mechanism of AICAR-mediated effects and to test for the possible role of autophagy in differentiation of leukemia cells. The results showed that AICAR-mediated effects on the expression of differentiation markers were not mimicked by A769662, a more specific direct AMPK activator. Long-term incubation of U937 cells with AICAR and other differentiation agents, all-trans-retinoic acid (ATRA) and phorbol 12-myristate 13-acetate, increased the expression of the autophagy marker LC3B-II, and these effects were not observed in response to metformin. Western blot and immunofluorescence analyses of U937 cells treated with bafilomycin A1 or transfected with mRFP-GFP-LC3 proved that the increase in the expression of LC3B-II was due to an increase in autophagy flux, and not to a decrease in lysosomal degradation. 3-Methyladenine inhibited the expression of differentiation markers in response to all inducers, but had stimulatory effects on autophagy flux at dose that effectively inhibited the production of phosphatidylinositol 3-phosphate. The small inhibitory RNA-mediated down-modulation of Beclin 1 and hVPS34 had no effects on AICAR and ATRA-mediated increase in the expression of differentiation markers. These results show that AICAR and other differentiation agents induce autophagy flux in U937 cells and that the effects of AICAR and ATRA on the expression of differentiation markers do not depend on the normal levels of key proteins of the classical or canonical autophagy pathway

Short-Term and Long-Term-Effects of Phorbol 12-Myristate 13-Acetate and Different Inhibitors on the Ability of Bone Marrow Cells to Form Colonies In Vitro

Peer Reviewe

Metabolism and differentiation

Textbook biochemical pathways do not usually apply to intermediary metabolism of highly proliferating, differentiating, or tumor cells. Over 80 years ago, Otto Warburg observed that cancer cells, unlike normal cells, favor glycolysis for energy production, even under aerobic conditions, and proposed that this shift in cancer cell metabolism (termed „aerobic glycolysis”) was due to mitochondrial dysfunction. Recent studies by several groups suggest that aerobic glycolysis in tumor cells is actually caused by oncogene-directed changes in metabolism that are necessary for both continuous proliferation and a block in cellular differentiation. Phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) is one of the principal proliferative and anti-apoptotic signaling pathways, which is known to support glycolysis and anabolism. Our previous studies demonstrated the activation of PI3K and Akt in nuclei of leukemia cells during differentiation, and confirmed that the inhibition of proximal components of the pathway inhibits proliferation, but negatively affects differentiative capacity of the cells. In contrast, use of rapamycin, which inhibits mTOR, a more distal component of the pathway, potentiates differentiation along granulocytic pathway. To further investigate the role of upstream regulators of mTOR in leukemia differentiation, we tested the effects of modulators of AMP-activated protein kinase (AMPK). Our results suggest a strong differentiative property of an AMPK activator, AICAR (5-amino-1-b-D-ribofuranosyl-imidazole-4-carboxamide) in monocytic U937 cells. The mechanism of AICAR-mediated effects will be presented and a possible role of AMPK-modulators in differentiation therapy will be discussed

AICAr, a Widely Used AMPK Activator with Important AMPK-Independent Effects: A Systematic Review

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr) has been one of the most commonly used pharmacological modulators of AMPK activity. The majority of early studies on the role of AMPK, both in the physiological regulation of metabolism and in cancer pathogenesis, were based solely on the use of AICAr as an AMPK-activator. Even with more complex models of AMPK downregulation and knockout being introduced, AICAr remained a regular starting point for many studies focusing on AMPK biology. However, there is an increasing number of studies showing that numerous AICAr effects, previously attributed to AMPK activation, are in fact AMPK-independent. This review aims to give an overview of the present knowledge on AMPK-dependent and AMPK-independent effects of AICAr on metabolism, hypoxia, exercise, nucleotide synthesis, and cancer, calling for caution in the interpretation of AICAr-based studies in the context of understanding AMPK signaling pathway

Cytarabine-induced differentiation of AML cells depends on Chk1 activation and shares the mechanism with inhibitors of DHODH and pyrimidine synthesis

Abstract Acute myeloid leukemia (AML) is characterized by arrested differentiation making differentiation therapy a promising treatment strategy. Recent success of inhibitors of mutated isocitrate dehydrogenase (IDH) invigorated interest in differentiation therapy of AML so that several new drugs have been proposed, including inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme in pyrimidine synthesis. Cytarabine, a backbone of standard AML therapy, is known to induce differentiation at low doses, but the mechanism is not completely elucidated. We have previously reported that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) and brequinar, a DHODH inhibitor, induced differentiation of myeloid leukemia by activating the ataxia telangiectasia and Rad3-related (ATR)/checkpoint kinase 1 (Chk1) via pyrimidine depletion. In this study, using immunoblotting, flow cytometry analyses, pharmacologic inhibitors and genetic inactivation of Chk1 in myeloid leukemia cell lines, we show that low dose cytarabine induces differentiation by activating Chk1. In addition, cytarabine induces differentiation ex vivo in a subset of primary AML samples that are sensitive to AICAr and DHODH inhibitor. The results of our study suggest that leukemic cell differentiation stimulated by low doses of cytarabine depends on the activation of Chk1 and thus shares the same pathway as pyrimidine synthesis inhibitors