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

Shin BS, Kim HG, Choi OH - J Menopausal Med (2014)

Bottom Line: To examine the effect of diazoxide on hypoxia-induced soluble fms-like tyrosin kinase-1 (sFlt-1) release in JEG-3 choriocarcinoma cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Pusan National University School of Medicine, Busan, Korea.

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-α) 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 (KATP) 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.

No MeSH data available.


Related in: MedlinePlus

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)
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Figure 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)

Mentions: Small interfering RNA approaches were used to confirm further the role of HO-1 in the diazoxide-induced inhibition of sFlt-1 release and ROS production. qRT-PCR and Western blot analysis showed that the basal expression of the HO-1 mRNA and protein was significantly diminished by transfection with the HO-1 siRNA. In these cells, CoPP and diazoxide failed to increase the expression of HO-1 mRNA and protein (Fig. 8). In analogy, in cells transfected with the HO-1 siRNA, diazoxide did not exert any effect on sFlt release and ROS production under hypoxia (Fig. 9).


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

Shin BS, Kim HG, Choi OH - J Menopausal Med (2014)

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)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4217563&req=5

Figure 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)
Mentions: Small interfering RNA approaches were used to confirm further the role of HO-1 in the diazoxide-induced inhibition of sFlt-1 release and ROS production. qRT-PCR and Western blot analysis showed that the basal expression of the HO-1 mRNA and protein was significantly diminished by transfection with the HO-1 siRNA. In these cells, CoPP and diazoxide failed to increase the expression of HO-1 mRNA and protein (Fig. 8). In analogy, in cells transfected with the HO-1 siRNA, diazoxide did not exert any effect on sFlt release and ROS production under hypoxia (Fig. 9).

Bottom Line: To examine the effect of diazoxide on hypoxia-induced soluble fms-like tyrosin kinase-1 (sFlt-1) release in JEG-3 choriocarcinoma cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Pusan National University School of Medicine, Busan, Korea.

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-α) 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 (KATP) 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.

No MeSH data available.


Related in: MedlinePlus