J Menopausal Med.  2014 Apr;20(1):21-31.

Mitochondrial Channel Opener Diazoxide Attenuates Hypoxia-Induced sFlt-1 Release in Human Choriocarcinoma Cells

• Shin BS
• Kim HG
• Choi OH

Abstract

OBJECTIVES
To examine the effect of diazoxide on hypoxia-induced soluble fms-like tyrosin kinase-1 (sFlt-1) release in JEG-3 choriocarcinoma cells.
METHODS
Cells were cultured under normoxia (20% O2) or hypoxia (1% O2), and expression of sFlt-1 mRNA and protein release was determined by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) assays and enzyme-linked immunosorbent assay (ELISA).
RESULTS
Tumor necrosis factor-alpha (TNF-alpha) as well as hypoxia stimulated sFlt-1 release and diazoxide inhibited both of them. The selective inhibitor of mitochondrial adenosine triphosphat (ATP)-sensitive K+ channel opener (K(ATP)) 5-hydroxydecanoate (5-HD) completely reversed the diazoxide-induced inhibition of hypoxia-stimulated sFlt-1 release. qRT-PCR and Western blot analyses showed that diazoxide up-regulated the heme oxygenase-1 (HO-1) expression. In addition, the HO-1 inducer cobalt protoporphyrin (CoPP) and the metabolic product of HO-1 bilirubin mimicked diazoxide to inhibit sFlt-1 release and reactive oxygen species (ROS) production under hypoxia, whereas the HO-1 inhibitor zinc protoporphyrin IX (ZnPP IX) antagonized the effect of diazoxide. In cells transfected with the HO-1 siRNA, diazoxide did not exert any effect on sFlt-1 release and ROS production under hypoxia.
CONCLUSION
These results, taken together, strongly suggest that up-regulation of the HO-1 expression is the crucial mechanism responsible for the diazoxide-induced inhibition of the sFlt-1 release and ROS production under hypoxia.

Keyword

Diazoxide; Heme oxygenase-1; Reactive oxygen speciese; Vascular endothelial growth factor receptor-1

MeSH Terms

Adenosine
Anoxia
Bilirubin
Blotting, Western
Choriocarcinoma*
Cobalt
Diazoxide*
Enzyme-Linked Immunosorbent Assay
Female
Heme Oxygenase-1
Humans
Polymerase Chain Reaction
Pregnancy
Reactive Oxygen Species
RNA, Messenger
RNA, Small Interfering
Tumor Necrosis Factor-alpha
Up-Regulation
Vascular Endothelial Growth Factor Receptor-1
Zinc
Adenosine
Bilirubin
Cobalt
Diazoxide
Heme Oxygenase-1
RNA, Messenger
RNA, Small Interfering
Reactive Oxygen Species
Tumor Necrosis Factor-alpha
Vascular Endothelial Growth Factor Receptor-1
Zinc

Figure

• 

  Fig. 1 Upregulated expression of soluble fms-like tyrosine kinase 1 (sFlt-1) in response to hypoxia and tumor necrosis factor alpha (TNF-α) in JEG-3 cells. Cells were cultured under normoxia (20% O2), under hypoxia (1% O2), or under normoxia with TNF-α (50 ng/mL) for 24 h. (A) sFlt-1 mRNA expression was determined by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR). (B) sFlt-1 protein release was determined by enzyme-linked immunosorbent assay (ELISA) analysis of the culture supernatants. Mean ± SE of six independent duplicate experiments. *P < 0.01 vs. normoxic control. (sFlt-1: soluble fms-like tyrosine kinase 1, TNF-α: tumor necrosis factor alpha)

• 

  Fig. 2 Deterioration of functional integrity of human umbilical vein endothelial cell (HUVEC) by conditioned media from hypoxic JEG-3 cells. HUVECs were grown as confluent monolayers on permeable polycarbonate membrane filters and then exposed to conditioned media from JEG-3 cells cultured for 24 h under normoxia (20% O2, open circle in A, normoxia control [NC] in B) or hypoxia (1% O2, solid circle in A, hyperoxia control [HC] group in B). After indicated time periods (in A) or 24 h further incubation (in B), changes in transendothelial resistance (TER) (A) and nitrite production (B) were determined. Mean ± SE of four independent duplicate experiments. *P < 0.01 vs. normoxic conditioned media. (TER: transendothelial resistance, NC: normoxia control, HC: hyperoxia control)

• 

  Fig. 3 Diazoxide-induced inhibition of hyoxia- and tumor necrosis factor alpha (TNF-α)-stimulated soluble fms-like tyrosine kinase 1 (sFlt-1) release. Cells were cultured under normoxia (20% O2), hypoxia (1% O2), or under normoxia with TNF-α (50 ng/mL) in the presence and absence of diazoxide (10 µM) for 24 h. (A) sFlt-1 mRNA expression was determined by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR). (B) sFlt-1 protein release was determined by enzyme-linked immunosorbent assay (ELISA) analysis of the culture supernatants. Mean ± SE of four independent duplicate experiments. *P < 0.01 vs. normoxic control. #P < 0.05 vs without diazoxide. (TER: transendothelial resistance, NC: normoxia control, HC: hyperoxia control)

• 

  Fig. 4 Effects of mitochondrial K+ channel openers and their inhibitors on soluble fms-like tyrosine kinase 1 (sFlt-1) release. Cells were cultured under normoxia (20% O2) or hypoxia (1% O2) with each 10 µM of diazoxide or NS 1619 in the presence and absence of their selective inhibitors, 5-hydroxydecanoate (5-HD) and paxilline (each 10 µM). sFlt-1 protein release was determined by enzyme-linked immunosorbent assay (ELISA) analysis of the culture supernatants and presented as % of normoxic control. Mean ± SE of four independent duplicate experiments. *P < 0.01 vs. the respective control. #P < 0.05 vs. vehicle (without inhibitors). (sFlt-1: soluble fms-like tyrosine kinase 1, 5-HD: 5-hydroxydecanoate)

• 

  Fig. 5 Up-regulation of heme oxygenase-1 (HO-1) expression by diazoxide. Cells were cultured under normoxia (20% O2) or hypoxia (1% O2) with or without diazoxide (10 µM) and cobalt protoporphyrin (CoPP; 10 µM) as a positive control for 24 h. (A) HO-1 mRNA expression was determined by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR). (B) HO-1 protein expression was determined by Western blot analysis of the cell extracts. Mean ± SE of four independent duplicate experiments. *P < 0.01 vs. normoxia. #P < 0.01 vs. the respective control. (HO-1: heme oxygenase-1, NO : nitric oxide, CoPP: cobalt protoporphyrin)

• 

  Fig. 6 Effects of heme oxygenase-1 (HO-1) related agents on soluble fms-like tyrosine kinase 1 (sFlt-1) release. Cells were cultured under normoxia (20% O2) or hypoxia (1% O2) with or without 10 µM of each agent. sFlt-1 protein release was determined by enzyme-linked immunosorbent assay (ELISA) analysis of the culture supernatants and presented as % of normoxic control. Mean ± SE of three independent duplicate experiments. *P < 0.01 vs. control. #P < 0.05 vs. diazoxide alone. (sFlt-1: soluble fmslike tyrosine kinase 1, CoPP: cobalt protopor phyrin, ZnPP: zinc protoporphyrin)

• 

  Fig. 7 Effects of heme oxygenase-1 (HO-1) related agents on reactive oxygen species (ROS) production. Cells were cultured under normoxia (20% O2) or hypoxia (1% O2) with or without 10 µM of each agents. ROS formation was determined by fluorospectrometric analysis of dichlorofluorescein (DCF) fluorescence and presented as % of normoxic control. Mean ± SE of three independent duplicate experiments. *P < 0.01 vs. control. #P < 0.05 vs diazoxide alone. (ROS: reactive oxygen species, CoPP: cobalt protoporphyrin, ZnPP: zinc protoporphyrin)

• 

  Fig. 8 Down-regulation of heme oxygenase-1 (HO-1) mRNA and protein expression by siRNA transfection. Cells were transfected with HO-1 siRNA or scrambled siRNA (siRNA control). (A) HO-1 mRNA expression was determined by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR). (B) HO-a protein expression was determined by Western blot analysis of the cell extracts. *P < 0.01 vs. siRNA control. #P < 0.05 vs. the respective control (without drug). (HO-1: heme oxygenase-1, CoPP: cobalt protoporphyrin, Cont: Control, Diaz: Diazoxide)

• 

  Fig. 9 Suppression of the diazoxide- and cobalt protoporphyrin (CoPP)-induced effects on soluble fms-like tyrosine kinase 1 (sFlt) release and reactive oxygen species (ROS) formation in heme oxygenase-1 (HO-1) siRNA-transfected cells. Cells were transfected with 30 pi comoles of HO-1 siRNA and incubated for 24 h normoxia (20% O2) or hypoxia (1% O2) with or without each 10 µM of with each 10 µM of diazoxide or CoPP. (A) sFlt-1 protein release was determined by enzyme-linked immunosorbent assay (ELISA) analysis of the culture supernatants. (B) ROS formation was determined by a fluorospectrometric analysis of dichlorofluorescein (DCF) fluorescence. Data were presented as % of normoxic control. Mean ± SE of four independent duplicate experiments. (sFlt-1: soluble fms-like tyrosine kinase 1, CoPP: cobalt protoporphyrin, ROS: reactive oxygen species)

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