Korean Circ J.  2016 Jan;46(1):93-98. 10.4070/kcj.2016.46.1.93.

Isoproterenol Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Human Embryonic Kidney Cells through Death Receptor 5 up-Regulation

• Eom YW
• Jung HY
• Oh JE
• Lee JW
• Ahn MS
• Youn YJ
• Ahn SG
• Kim JY
• Lee SH
• Yoon J
• Yoo BS

• KMID: 2223774
• DOI: http://doi.org/10.4070/kcj.2016.46.1.93

Abstract

BACKGROUND AND OBJECTIVES
Chronic impairment of beta-adrenergic receptor signaling increases cardiac apoptosis, hypertrophy and fibrosis. The aim of this study was to investigate whether isoproterenol (ISO), an agonist of the adrenergic receptor, can enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human embryonic kidney (HEK) 293 cells.
MATERIALS AND METHODS
HEK 293 cells were treated with ISO and/or TRAIL for 24 hours. Cell viability was evaluated by microscopy and an established viability assay, and apoptotic cell death was analyzed by staining with fluorescein isothiocynate-annexin-V/propidium iodide (PI) and caspase activation. To confirm the mechanism of cell death induced by co-treatment with ISO and TRAIL, expression of TRAIL receptor 2 (death receptor 5, DR5) was evaluated by immunoblotting.
RESULTS
Although ISO or TRAIL treatment decreased HEK 293 cell viability by 13% and 17%, respectively, co-treatment with ISO and TRAIL resulted in a markedly higher death rate of 35% after 24 hours. Increases were evident in early apoptotic cells (i.e., annexin-V positive/PI negative; 19.4%), late apoptotic cells (i.e., annexin-V positive/PI positive; 6.3%) and dead cells (i.e., annexin-V negative/PI positive; 1.1%) when cells were co-treated with ISO and TRAIL, compared to cells treated with either ISO or TRAIL. In addition, marked increases of cleaved cas-3, cleaved poly (adenosine diphosphate-ribose) polymerase and DR5 were observed in HEK 293 cells co-treated with ISO and TRAIL.
CONCLUSION
Treatments combining ISO with TRAIL may be responsible for death of HEK 293 cells through DR5 up-regulation. Activation of adrenergic receptors is responsible for the synergistic cell death observed with TRAIL.

Keyword

Isoproterenol; TNF-related apoptosis-inducing ligand; Apoptosis; Death receptor 5

MeSH Terms

Apoptosis*
Cell Death
Cell Survival
Fibrosis
Fluorescein
HEK293 Cells
Humans*
Hypertrophy
Immunoblotting
Isoproterenol*
Kidney*
Microscopy
Mortality
Necrosis*
Receptors, Adrenergic
Receptors, TNF-Related Apoptosis-Inducing Ligand*
TNF-Related Apoptosis-Inducing Ligand
Up-Regulation*
Fluorescein
Isoproterenol
Receptors, Adrenergic
Receptors, TNF-Related Apoptosis-Inducing Ligand
TNF-Related Apoptosis-Inducing Ligand

Figure

• 

  Fig. 1 Cell viability of HEK 293 cells treated with ISO and/or TRAIL. (A) Cell death induced by treatment with ISO and/or TRAIL after 24 hours. Cells were photographed (100X magnification) using a microscope. (B) Cytotoxicity caused by treatment with ISO and/or TRAIL after 24 hours. Cytotoxicity was measured by the MTT assay. Standard error was determined on the basis of three independent experiments. HEK: human embryonic kidney, ISO: isoproterenol, TRAIL: tumor necrosis factor-related apoptosis-inducing ligand, MTT: methylthiazolyldiphenyl-tetrazolium bromide.

• 

  Fig. 2 Synergistic apoptotic cell death caused by treatment with ISO and/or TRAIL. HEK 293 cells were treated with ISO (100 µM) and/or TRAIL (100 ng/mL) for 24 hours. (A and B) Apoptotic cell death of HEK 293 cells treated with ISO and/or TRAIL. The percentage of apoptotic cells was determined by FITC-annexin-V/PI staining. (C) Caspase-3 activation by ISO and TRAIL treatment. Caspase-3 activation and PARP cleavage were observed in HEK 293 cells treated with ISO and TRAIL. (D) The band intensities of cleaved cas-3 and PARP were analyzed by densitometry. GAPDH was used as the housekeeping gene. Standard error was determined on the basis of three independent experiments; *p<0.05. ISO: isoproterenol, TRAIL: tumor necrosis factor-related apoptosis-inducing ligand, HEK: human embryonic kidney, FITC: fluorescein isothiocynate, PI: propidium iodide, PARP: polymerase, GAPDH: glyceraldehydes-3-phosphate dehydrogenase.

• 

  Fig. 3 Up-regulation of DR5 in HEK 293 cells treated with ISO and TRAIL. HEK 293 cells were treated with ISO (100 µM) and/or TRAIL (100 ng/mL) for 24 hours. DR5 expression was evaluated by immunoblotting, and the band intensity of DR5 was analyzed by densitometry. GAPDH was used as the housekeeping gene. Standard error was determined on the basis of three independent experiments; *p<0.05. DR: death receptor, HEK: human embryonic kidney, ISO: isoproterenol, TRAIL: tumor necrosis factor-related apoptosis-inducing ligand, GAPDH: glyceraldehydes-3-phosphate dehydrogenase.

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