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Fenretinide induces mitochondrial ROS and inhibits the mitochondrial respiratory chain in neuroblastoma.

Cuperus R, Leen R, Tytgat GA, Caron HN, van Kuilenburg AB - Cell. Mol. Life Sci. (2009)

Bottom Line: ROS induction by fenretinide was of mitochondrial origin, demonstrated by detection of superoxide with MitoSOX, the scavenging effect of the mitochondrial antioxidant MitoQ and reduced ROS production in cells without a functional mitochondrial respiratory chain (Rho zero cells).In digitonin-permeabilized cells, a fenretinide concentration-dependent decrease in ATP synthesis and substrate oxidation was observed, reflecting inhibition of the mitochondrial respiratory chain.Co-incubation of fenretinide with inhibitors of different complexes of the respiratory chain suggested that fenretinide-induced ROS production occurred via complex II.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Genetic Metabolic Diseases, Department of Pediatrics/Emma Children's Hospital, Academic Medical Centre, University of Amsterdam, P.O. Box 22700, 1100 DE, Amsterdam, The Netherlands.

ABSTRACT
Fenretinide induces apoptosis in neuroblastoma by induction of reactive oxygen species (ROS). In this study, we investigated the role of mitochondria in fenretinide-induced cytotoxicity and ROS production in six neuroblastoma cell lines. ROS induction by fenretinide was of mitochondrial origin, demonstrated by detection of superoxide with MitoSOX, the scavenging effect of the mitochondrial antioxidant MitoQ and reduced ROS production in cells without a functional mitochondrial respiratory chain (Rho zero cells). In digitonin-permeabilized cells, a fenretinide concentration-dependent decrease in ATP synthesis and substrate oxidation was observed, reflecting inhibition of the mitochondrial respiratory chain. However, inhibition of the mitochondrial respiratory chain was not required for ROS production. Co-incubation of fenretinide with inhibitors of different complexes of the respiratory chain suggested that fenretinide-induced ROS production occurred via complex II. The cytotoxicity of fenretinide was exerted through the generation of mitochondrial ROS and, at higher concentrations, also through inhibition of the mitochondrial respiratory chain.

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The effect of complex II inhibitors on ROS production in SJNB10 cells. Cells were incubated with (grey bars) or without (black bars) 10 μM 4HPR for 4 h. a In combination with 0–2 μM TTFA. b In combination with 0–2 mM Carboxin. After incubation, ROS production was measured using CM-H2DCFDA probe. Percentage of ROS induction compared to control. Each figure represents the mean ± SD of three experiments
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Fig8: The effect of complex II inhibitors on ROS production in SJNB10 cells. Cells were incubated with (grey bars) or without (black bars) 10 μM 4HPR for 4 h. a In combination with 0–2 μM TTFA. b In combination with 0–2 mM Carboxin. After incubation, ROS production was measured using CM-H2DCFDA probe. Percentage of ROS induction compared to control. Each figure represents the mean ± SD of three experiments

Mentions: To investigate the role of each complex in the 4HPR-induced ROS production, the neuroblastoma cells were incubated with specific inhibitors of complex I, II and III. Carboxin and TTFA, two inhibitors of complex II, were the only mitochondrial respiratory chain complex inhibitors that reduced the 4HPR-induced ROS production (Fig. 8). Rotenone and antimycin A, inhibitors of complex I and complex III (cytochrome reductase), respectively, did not reduce the ROS production (data not shown). A similar protective effect of the complex II inhibitor TTFA on 4HPR-induced ROS formation was observed in other neuroblastoma cell lines such as IMR32, SJNB10, NASS, SY5Y and FISK (as well as in the osteosarcoma control cells). This result is in line with the observation that mitochondrial ROS production and inhibition of the electron flux through the mitochondrial respiratory chain are two independent mechanisms of 4HPR.Fig. 8


Fenretinide induces mitochondrial ROS and inhibits the mitochondrial respiratory chain in neuroblastoma.

Cuperus R, Leen R, Tytgat GA, Caron HN, van Kuilenburg AB - Cell. Mol. Life Sci. (2009)

The effect of complex II inhibitors on ROS production in SJNB10 cells. Cells were incubated with (grey bars) or without (black bars) 10 μM 4HPR for 4 h. a In combination with 0–2 μM TTFA. b In combination with 0–2 mM Carboxin. After incubation, ROS production was measured using CM-H2DCFDA probe. Percentage of ROS induction compared to control. Each figure represents the mean ± SD of three experiments
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2824117&req=5

Fig8: The effect of complex II inhibitors on ROS production in SJNB10 cells. Cells were incubated with (grey bars) or without (black bars) 10 μM 4HPR for 4 h. a In combination with 0–2 μM TTFA. b In combination with 0–2 mM Carboxin. After incubation, ROS production was measured using CM-H2DCFDA probe. Percentage of ROS induction compared to control. Each figure represents the mean ± SD of three experiments
Mentions: To investigate the role of each complex in the 4HPR-induced ROS production, the neuroblastoma cells were incubated with specific inhibitors of complex I, II and III. Carboxin and TTFA, two inhibitors of complex II, were the only mitochondrial respiratory chain complex inhibitors that reduced the 4HPR-induced ROS production (Fig. 8). Rotenone and antimycin A, inhibitors of complex I and complex III (cytochrome reductase), respectively, did not reduce the ROS production (data not shown). A similar protective effect of the complex II inhibitor TTFA on 4HPR-induced ROS formation was observed in other neuroblastoma cell lines such as IMR32, SJNB10, NASS, SY5Y and FISK (as well as in the osteosarcoma control cells). This result is in line with the observation that mitochondrial ROS production and inhibition of the electron flux through the mitochondrial respiratory chain are two independent mechanisms of 4HPR.Fig. 8

Bottom Line: ROS induction by fenretinide was of mitochondrial origin, demonstrated by detection of superoxide with MitoSOX, the scavenging effect of the mitochondrial antioxidant MitoQ and reduced ROS production in cells without a functional mitochondrial respiratory chain (Rho zero cells).In digitonin-permeabilized cells, a fenretinide concentration-dependent decrease in ATP synthesis and substrate oxidation was observed, reflecting inhibition of the mitochondrial respiratory chain.Co-incubation of fenretinide with inhibitors of different complexes of the respiratory chain suggested that fenretinide-induced ROS production occurred via complex II.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Genetic Metabolic Diseases, Department of Pediatrics/Emma Children's Hospital, Academic Medical Centre, University of Amsterdam, P.O. Box 22700, 1100 DE, Amsterdam, The Netherlands.

ABSTRACT
Fenretinide induces apoptosis in neuroblastoma by induction of reactive oxygen species (ROS). In this study, we investigated the role of mitochondria in fenretinide-induced cytotoxicity and ROS production in six neuroblastoma cell lines. ROS induction by fenretinide was of mitochondrial origin, demonstrated by detection of superoxide with MitoSOX, the scavenging effect of the mitochondrial antioxidant MitoQ and reduced ROS production in cells without a functional mitochondrial respiratory chain (Rho zero cells). In digitonin-permeabilized cells, a fenretinide concentration-dependent decrease in ATP synthesis and substrate oxidation was observed, reflecting inhibition of the mitochondrial respiratory chain. However, inhibition of the mitochondrial respiratory chain was not required for ROS production. Co-incubation of fenretinide with inhibitors of different complexes of the respiratory chain suggested that fenretinide-induced ROS production occurred via complex II. The cytotoxicity of fenretinide was exerted through the generation of mitochondrial ROS and, at higher concentrations, also through inhibition of the mitochondrial respiratory chain.

Show MeSH
Related in: MedlinePlus