Regulation of Apoptosis in Eicosapentaenoic Acid-Treated HL-60 Cells

doi:10.1016/j.jss.2006.08.012

Journal of Surgical Research

Volume 137, Issue 1, January 2007, Pages 141-150

https://doi.org/10.1016/j.jss.2006.08.012 Get rights and content

Background

Neutrophil apoptosis is an important physiological process in the resolution of pulmonary inflammation. Previous studies have shown that eicosapentaenoic acid (EPA; 20:5n-3) increases the rate of apoptosis in a concentration- and time-dependent manner in HL-60 cells. However, it is not known if the EPA-induced apoptosis involves the lipoxygenase (LO) and cyclooxygenase (COX) enzymes or the downstream metabolic products of these enzymes. Thus, the objective of this study was to determine the effects of inhibitors LO and COX enzymes on apoptosis, viability, and necrosis in EPA-treated HL-60 cells.

Materials and methods

Cells were incubated with 50 μm EPA in the presence of an enzyme inhibitor (1–10 μm) for 12 h. Compounds were used to inhibit COX 1 and 2 (ibuprofen), 5-, 12-, 15-LO (NDGA), 12-LO (baicalein), 5-LO (AA-861), and 5-LO activating protein (MK-886). Eicosanoid (0.001–1.0 μm) add-back experiments were also conducted; LTB₄ and 5-HETE with 5-LO inhibition and 12-HETE with 12-LO inhibition. Flow cytometry was used to assess apoptosis.

Results

Inhibition of COX 1 and 2 had no effect on apoptosis. Inhibition of 5-LO and 12-LO significantly increased apoptosis in EPA-treated HL-60 cells. Addition of LTB₄ reduced apoptosis to levels significantly lower than in HL-60 cells treated with EPA alone; 5-HETE and 12-HETE also lowered apoptosis to control levels.

Conclusions

These data indicate that inhibition of LO, particularly 5-LO, increased apoptosis in EPA-treated HL-60 cells. Furthermore, this study demonstrated that the products of the LO enzymes, particularly LTB₄, are critical in the regulation of apoptosis in EPA-treated HL-60 cells.

Introduction

The acute respiratory distress syndrome (ARDS) is characterized by an increase in neutrophil accumulation in the airways and alveoli [1]. Pulmonary injury is caused by the release of oxygen free radicals by activated neutrophils in ARDS [2]. The clearance of apoptotic neutrophils by alveolar macrophages limits tissue damage [3] and may play a critical physiological role in the resolution of pulmonary inflammation in acute lung injury and ARDS [4]. Serial bronchoalveolar lavage data demonstrate a progressive decrease in lung neutrophils as ARDS patients improve and are weaned from mechanical ventilation [1]. These findings demonstrate a central role for neutrophils in the pathogenesis of ARDS and suggest that interventions should be aimed at regulating the rate of neutrophil apoptosis to resolve the inflammatory response in the lung.

Apoptosis can be induced in many cell types, including neutrophils, by in vitro exposure to various n-3 and n-6 polyunsaturated fatty acids (PUFAs) including eicosapentaenoic acid (EPA; 20:5n-3), γ-linolenic acid (GLA; 18:4n-6), and arachidonic acid (AA; 20:4n-6) [5, 6, 7, 8, 9, 10]. Enteral nutrition with EPA and GLA attenuate some of the pro-inflammatory effects of AA-derived eicosanoids [3, 11, 12, 13, 14]. Several enteral nutrition studies have demonstrated that EPA and GLA decreased the number of neutrophils and LTB₄ levels in the bronchoalveolar lavage fluid of both patients and animals with ARDS [11, 13]. EPA and GLA also reduced pulmonary inflammation and improved clinical outcomes for patients with ARDS [11]. These improvements may be because of fewer neutrophils in the lung, which may be a direct result of neutrophil apoptosis induced by changes in the leukotriene profile in the lung.

Human promyelocytic HL-60 cells share many functional characteristics with peripheral blood neutrophils, such as containing azurophilic granules, and are therefore considered an excellent model for human neutrophil responses in vitro [15, 16]. Several recent studies have demonstrated that fatty acids induce apoptosis in HL-60 cells [17, 18]. In addition, we previously showed that the rate of apoptosis increased in a time- and concentration-dependent manner in EPA-treated HL-60 cells [5]. Furthermore, studies have demonstrated that the rate of apoptosis is significantly increased with inhibition of lipoxygenase (LO) enzymes in AA-treated and EPA/GLA-treated HL-60 cells [6, 10]. However, it is not known whether EPA-induced apoptosis involves the LO and cyclooxygenase (COX) enzymes or the downstream metabolic products of these enzymes. Thus, the objective of this study was to determine the effect of inhibitors of LO and COX enzymes on apoptosis, viability, and necrosis in EPA-treated HL-60 cells. In addition, eicosanoid add-back experiments were conducted to investigate products of the lipoxygenase enzymes involved in the regulation of apoptosis in EPA-treated HL-60 cells.

Section snippets

Cells

The human promyelocytic leukemia cell line HL-60 was obtained from the American Type Tissue Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 medium with phenol red supplemented with HEPES (25 mmol/L; BioWhittaker, Walkersville, MD), 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin solution (Sigma Chemical, St. Louis, MO), and 1% l-glutamine (BioWhittaker). Cells were maintained at 37°C in an atmosphere of 5% CO₂ and 95% room air in a NAPCO model 5410

Results

PUFAs can be metabolized by a variety of pathways that can be blocked with enzyme inhibitors (Fig. 1.) Oleic acid, methanol, and DMSO did not induce cell death (apoptosis and necrosis), and viability was maintained after 12-h incubation. Ibuprofen, NDGA, and baicalein did not induce cell death, and viability was maintained after a 12-h incubation. AA-861 and MK-886 did not induce cell death and did not affect viability at the lower concentrations at 12 h. However, AA-861 (20 μm) and MK-886 (10 μ

Discussion

We have previously shown that treatment of HL-60 cells with EPA (20:5n-3) increased apoptosis and secondary necrosis and reduced viability in a concentration and time-dependent manner. However, it was not known if the induction of cell death by EPA involved the eicosanoid products of 5- or 12-LO or COX 1 and 2. This study demonstrates that the increase in apoptosis and secondary necrosis because of EPA was increased further with inhibitors of either 5- or 12-LO in HL-60 cells. Furthermore,

Acknowledgment

The authors gratefully thank R. B. Andrews for his technical assistance with measurements of flow cytometry.

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Metabolism/nutritionRegulation of Apoptosis in Eicosapentaenoic Acid-Treated HL-60 Cells

Background

Materials and methods

Results

Conclusions

Introduction

Section snippets

Cells

Results

Discussion

Acknowledgment

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Regulation of Apoptosis in Eicosapentaenoic Acid-Treated HL-60 Cells