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

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.

Fig. 1

Schematic representation of the mitochondrial respiratory chain. After permeabilization of the cells with digitonin, the electron flow through the different complexes was measured. Complex I NADH reductase. Complex II succinate dehydrogenase. Complex III cytochrome c reductase. Complex IV cytochrome c oxidase. Complex V ATP synthase. MDH malate dehydrogenase. GOT glutamate-oxaloacetate transaminase

Fig. 2

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

Fig. 3

Scavenging of mitochondrial ROS by mitoQ. a SJNB10 and SY5Y cells were incubated for 2 h with MitoQ followed by co-incubation for 2 h with 20 μM 4HPR. Control black bars; 4HPR dark grey bars; 4HPR and MitoQ (4 μM) light grey bars; 4HPR and MitoQ (8 μM) white bars. b SJNB10 and SY5Y cells were pre-incubated for 2 h with MitoQ followed by culturing in mitoQ-free medium for 1 h and subsequently incubation with 20 μM 4HPR for 2 h. Control black bars; 4HPR dark grey bars; 4HPR and MitoQ (4 μM) light grey bars; 4HPR and MitoQ (8 μM) white bars. Each bar represents the mean ± SD of three experiments

Fig. 4

ROS production in Rho zero cells (open square) and control cells (black diamond) measured after 4 h incubation with 0–20 μM 4HPR. ROS was measured using the DFCDA probe. The amount of ROS was expressed relative to that in untreated cells. Each figure represents the mean ± SD of three experiments

Fig. 5

The effect of 4HPR on ATP synthesis and aspartate formation (complex I) and ATP synthesis and malate formation (complex II) in digitonin-permeabilized neuroblastoma cells. All cell lines were treated with 0–30 μM 4HPR for 4 h. To measure the flux through complex I–V and II–V, malate (plus glutamate) and succinate (plus rotenone) were used as substrates, respectively, and the products a Aspartate (black diamond)-ATP (open square) and b malate (black square)-ATP (open square) were measured. Each figure represents the mean ± SD of three experiments

Fig. 6

The effect of 4HPR on ATP and aspartate, and ATP and malate synthesis in digitonin-permeabilized SY5Y and SJNB10 cells incubated for 4 h with the uncoupling agent CCCP (5 μM for malate–ATP, 15 μM for aspartate–ATP in SJNB10, 20 μM for aspartate–ATP in SY5Y) and 0–30 μM 4HPR. a Aspartate (black triangle) –ATP(open square) and b malate (black triangle)–ATP (open square). Each figure represents the mean ± SD of three experiments

Fig. 7

Effect of Trolox on loss of viability in 4HPR-treated cells. SJNB10 cells were incubated with 0–40 μM 4HPR with (grey bars) or without (black bars) 500 μM Trolox for 24 h. Viability was measured using the MTS method and was depicted as percentage of control. Each figure represents the mean ± SD of four experiments

Fig. 8

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