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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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Mitochondrial ROS generation after 4HPR incubation. a Mitochondrial superoxide production measured by mitoSOX after 4 h incubation with 0 μM (black bars), 10 μM (grey bars) or 20 μM (white bars) 4HPR. b The effect of the specific mitochondrial probe mitoQ against ROS in cell lines incubated for 2 h with (open square) or without (black diamond) 1 μM mitoQ followed by co-incubation for 2 h with 0–20 μM 4HPR. ROS was measured using the DFCDA probe. Each figure represents the mean ± SD of three experiments
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Fig2: Mitochondrial ROS generation after 4HPR incubation. a Mitochondrial superoxide production measured by mitoSOX after 4 h incubation with 0 μM (black bars), 10 μM (grey bars) or 20 μM (white bars) 4HPR. b The effect of the specific mitochondrial probe mitoQ against ROS in cell lines incubated for 2 h with (open square) or without (black diamond) 1 μM mitoQ followed by co-incubation for 2 h with 0–20 μM 4HPR. ROS was measured using the DFCDA probe. Each figure represents the mean ± SD of three experiments

Mentions: Mitochondrial ROS production was measured using the mitoSOX probe, a ROS probe that specifically detects superoxide in mitochondria. A concentration dependent increase of mitochondrial ROS after 4 h 4HPR incubation was observed in all cell lines except FISK and SY5Y (Fig. 2a). MitoQ is a specific mitochondrial antioxidant that attaches to the mitochondrial membrane. To demonstrate that the ROS production induced by 4HPR incubation is indeed generated mitochondrially, cells were pre-incubated with MitoQ followed by co-incubation with 4HPR. 4HPR-induced ROS production, measured using the CM-H2DCFDA probe, was scavenged when cells were pre-incubated for 2 h with 1 μM MitoQ (Fig. 2b). The degree of protection against ROS varied considerably between the various cell lines. A moderate protective effect of MitoQ on ROS scavenging was observed in SJNB10 and SY5Y. However, increasing the concentration of MitoQ up to 8 μM resulted in a complete protection against 4HPR-induced ROS in SJNB10 cells and SY5Y cells (Fig. 3a). The protective effect of mitoQ in SY5Y cells was not observed when cells were incubated with mitoQ and 4HPR separately (Fig. 3b), indicating a low retention of mitoQ in SY5Y cells. The apparent inability of mitoSOX to detect superoxide in mitochondria in SY5Y cells might be caused by low accumulation or retention of mitoSOX in mitochondria in SY5Y cells, as observed for mitoQ. Thus, our data suggest that ROS production induced by 4HPR is formed mitochondrially in our panel of neuroblastoma cells.Fig. 2


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)

Mitochondrial ROS generation after 4HPR incubation. a Mitochondrial superoxide production measured by mitoSOX after 4 h incubation with 0 μM (black bars), 10 μM (grey bars) or 20 μM (white bars) 4HPR. b The effect of the specific mitochondrial probe mitoQ against ROS in cell lines incubated for 2 h with (open square) or without (black diamond) 1 μM mitoQ followed by co-incubation for 2 h with 0–20 μM 4HPR. ROS was measured using the DFCDA probe. Each figure represents the mean ± SD of three experiments
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2824117&req=5

Fig2: Mitochondrial ROS generation after 4HPR incubation. a Mitochondrial superoxide production measured by mitoSOX after 4 h incubation with 0 μM (black bars), 10 μM (grey bars) or 20 μM (white bars) 4HPR. b The effect of the specific mitochondrial probe mitoQ against ROS in cell lines incubated for 2 h with (open square) or without (black diamond) 1 μM mitoQ followed by co-incubation for 2 h with 0–20 μM 4HPR. ROS was measured using the DFCDA probe. Each figure represents the mean ± SD of three experiments
Mentions: Mitochondrial ROS production was measured using the mitoSOX probe, a ROS probe that specifically detects superoxide in mitochondria. A concentration dependent increase of mitochondrial ROS after 4 h 4HPR incubation was observed in all cell lines except FISK and SY5Y (Fig. 2a). MitoQ is a specific mitochondrial antioxidant that attaches to the mitochondrial membrane. To demonstrate that the ROS production induced by 4HPR incubation is indeed generated mitochondrially, cells were pre-incubated with MitoQ followed by co-incubation with 4HPR. 4HPR-induced ROS production, measured using the CM-H2DCFDA probe, was scavenged when cells were pre-incubated for 2 h with 1 μM MitoQ (Fig. 2b). The degree of protection against ROS varied considerably between the various cell lines. A moderate protective effect of MitoQ on ROS scavenging was observed in SJNB10 and SY5Y. However, increasing the concentration of MitoQ up to 8 μM resulted in a complete protection against 4HPR-induced ROS in SJNB10 cells and SY5Y cells (Fig. 3a). The protective effect of mitoQ in SY5Y cells was not observed when cells were incubated with mitoQ and 4HPR separately (Fig. 3b), indicating a low retention of mitoQ in SY5Y cells. The apparent inability of mitoSOX to detect superoxide in mitochondria in SY5Y cells might be caused by low accumulation or retention of mitoSOX in mitochondria in SY5Y cells, as observed for mitoQ. Thus, our data suggest that ROS production induced by 4HPR is formed mitochondrially in our panel of neuroblastoma cells.Fig. 2

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