Immune Netw.  2014 Oct;14(5):241-248. 10.4110/in.2014.14.5.241.

5-aminoimidazole-4-carboxamide Riboside Induces Apoptosis Through AMP-activated Protein Kinase-independent and NADPH Oxidase-dependent Pathways

• Wi SM
• Lee KY

• KMID: 2391720
• DOI: http://doi.org/10.4110/in.2014.14.5.241

Abstract

It is debatable whether AMP-activated protein kinase (AMPK) activation is involved in anti-apoptotic or pro-apoptotic signaling. AICAR treatment increases AMPK-alpha1 phosphorylation, decreases intracellular reactive oxygen species (ROS) levels, and significantly increases Annexin V-positive cells, DNA laddering, and caspase activity in human myeloid cell. AMPK activation is therefore implicated in apoptosis. However, AMPK-alpha1-knockdown THP-1 cells are more sensitive to apoptosis than control THP-1 cells are, suggesting that the apoptosis is AMPK-independent. Low doses of AICAR induce cell proliferation, whereas high doses of AICAR suppress cell proliferation. Moreover, these effects are significantly correlated with the downregulation of intracellular ROS, strongly suggesting that AICAR-induced apoptosis is critically associated with the inhibition of NADPH oxidase by AICAR. Collectively, our results demonstrate that in AICAR-induced apoptosis, intracellular ROS levels are far more relevant than AMPK activation.

Keyword

5-aminoimidazole-4-carboxamide (AICA) riboside; AMP-activated protein kinase; Apoptosis; NADPH oxidase; Reactive oxygen species; Lymphoid cells

MeSH Terms

AMP-Activated Protein Kinases
Apoptosis*
Cell Proliferation
DNA
Down-Regulation
Humans
Lymphocytes
Myeloid Cells
NADP*
NADPH Oxidase
Phosphorylation
Reactive Oxygen Species
AMP-Activated Protein Kinases
DNA
NADP
NADPH Oxidase
Reactive Oxygen Species

Figure

• 

  Figure 1 The bifunctional role of AICAR as an AMPK activator and NADPH oxidase inhibitor induces cytotoxicity in human lymphoid cells. (A and B) Jurkat (A) and THP-1 cells (B) were treated with or without 5 mM AICAR for the times indicated. Cell lysates were analyzed by immunoblot with antibodies specific for phospho-AMPK. The immunoblot was reprobed with an anti-GAPDH antibody. Data are representative of three independent experiments. (C and D) Jurkat (C) and THP-1 cells (D) were treated with or without 5 mM AICAR for the times indicated. Intracellular ROS concentrations were measured by DCF-DA staining. Data are the averages±SD of mean fluorescence intensity (MFI) obtained from three independent experiments. (E and F) Jurkat (closed circles) and THP-1 cells (open circles) were treated with different concentrations of AICAR for 24 h. A 3H-thymidine-incorporation assay was performed as described in Materials and Methods. The inhibitory effects of AICAR are expressed as relative cell viabilities, as calculated by cpm (AICAR-treated)/cpm (untreated) ×100. Data are the means±SD of the triplicate samples.

• 

  Figure 2 AICAR induces apoptosis in human lymphoid cells. (A and B) Jurkat (A) and THP-1 cells (B) were treated with 10 mM AICAR for the times indicated. Cells were stained with Annexin V-FITC and PI and analyzed by flow cytometry. Data are representative of three independent experiments. Early apoptotic cells (Annexin V-positive cells) and late apoptotic cells (Annexin V-positive and PI-positive cells) were analyzed by flow cytometry. Data are the means±SD of a percentage of cells obtained from three independent experiments. (C) Jurkat cells were treated with or without 10 mM AICAR for the times indicated. Cell lysates were analyzed by immunoblot with antibodies specific for caspase-3, caspase-8, and caspase-9. The immunoblot was reprobed with an anti-GAPDH antibody. Data are representative of three independent experiments. (D) Jurkat cells were treated with or without 10 mM AICAR for the times indicated and a DNA laddering assay was performed as described in Materials and Methods. (E and F) Jurkat cells were treated with or without 5 mM AICAR for the times indicated. Caspase-3 (E) and caspase-9 (F) activities were analyzed as described in Materials and Methods. Data are the means±SD of three independent experiments.

• 

  Figure 3 AMPK-α1-knockdown THP-1 cells are highly sensitive to treatment with AICAR. (A~D) Wild type (A and C) and AMPK-α1-knockdown THP-1 cells (B and D) were treated with 10 mM AICAR for the times indicated. Cells were stained with Annexin V-FITC and PI (A and B) or a cell cycle analysis kit (C and D), and then analyzed by flow cytometry. Data are representative of three independent experiments. Data are the means±SD of a percentage of cells obtained from three independent experiments. (E) Wild type and AMPK-α1-knockdown THP-1 cells were treated with or without 10 mM AICAR for the times indicated. Cell lysates were analyzed by immunoblot with antibodies specific for caspase-3, caspase-8, and caspase-9. The immunoblot was reprobed with an anti-GAPDH antibody. Data are representative of three independent experiments. (F~H) Wild type and AMPK-α1-knockdown THP-1 cells were treated with or without 10 mM AICAR for the times indicated. Caspase-3 (F), caspase-9 (G), and caspase-8 (H) activities were analyzed as described in Materials and Methods. Data are the means±SD obtained from three independent experiments.

• 

  Figure 4 AICAR functions through the activation of AMPK or inhibition of NADPH oxidase in human lymphoid cells. (A and B) Jurkat (A) and THP-1 (B) cells were treated with or without different concentrations of AICAR for different times as indicated. A 3H-thymidine incorporation assay was performed as described in Materials and Methods. Data are the means±SD of the triplicate samples. (C and D) Jurkat (C) and THP-1 (D) cells were treated with or without different concentrations of AICAR for different times as indicated. DCF staining was performed as described in Materials and Methods, and flow cytometry analysis was performed. Data are the averages±SD of the mean fluorescence intensity in triplicate samples. (E) A model for the dual functions of AICAR: AMPK activation and NADPH oxidase inhibition. The activation of AMPK induced by AICAR may initially promote cellular proliferation at early time points (left). However, the inhibition of NADPH oxidase by AICAR induces the downregulation of intracellular ROS levels, which induces cytotoxicity and ultimately apoptosis (right).

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