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C10ORF10/DEPP, a transcriptional target of FOXO3, regulates ROS-sensitivity in human neuroblastoma.

Salcher S, Hagenbuchner J, Geiger K, Seiter MA, Rainer J, Kofler R, Hermann M, Kiechl-Kohlendorfer U, Ausserlechner MJ, Obexer P - Mol. Cancer (2014)

Bottom Line: The effect of DEPP on cellular ROS was measured by catalase activity assay and live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS.Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells.In parallel, the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARG), a critical regulator of catalase enzyme activity, was strongly upregulated in DEPP-knockdown cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics I, Medical University Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria. michael.j.ausserlechner@i-med.ac.at.

ABSTRACT

Background: FOXO transcription factors control cellular levels of reactive oxygen species (ROS) which critically contribute to cell survival and cell death in neuroblastoma. In the present study we investigated the regulation of C10orf10/DEPP by the transcription factor FOXO3. As a physiological function of C10orf10/DEPP has not been described so far we analyzed its effects on cellular ROS detoxification and death sensitization in human neuroblastoma cells.

Methods: The effect of DEPP on cellular ROS was measured by catalase activity assay and live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS. The cellular localization of DEPP was determined by confocal microscopy of EYFP-tagged DEPP, fluorescent peroxisomal- and mitochondrial probes and co-immunoprecipitation of the PEX7 receptor.

Results: We report for the first time that DEPP regulates ROS detoxification and localizes to peroxisomes and mitochondria in neuroblastoma cells. FOXO3-mediated apoptosis involves a biphasic ROS accumulation. Knockdown of DEPP prevented the primary and secondary ROS wave during FOXO3 activation and attenuated FOXO3- and H2O2-induced apoptosis. Conditional overexpression of DEPP elevates cellular ROS levels and sensitizes to H2O2 and etoposide-induced cell death. In neuronal cells, cellular ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that allows binding to the PEX7 receptor and import into peroxisomes, we analyzed the effect of DEPP on cellular detoxification by measuring enzyme activity of catalase. Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells. DEPP directly interacts with the PEX7 receptor and localizes to the peroxisomal compartment. In parallel, the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARG), a critical regulator of catalase enzyme activity, was strongly upregulated in DEPP-knockdown cells.

Conclusion: The combined data indicate that in neuroblastoma DEPP localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification, which sensitizes tumor cells to ROS-induced cell death.

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DEPP impairs catalase activity and sensitizes to H2O2-induced apoptosis. a + b) A catalase enzyme activity assay was performed in SH-EP/tetEGFP, SH-EP/tetDEPP, SH-EP/tetEYFP-DEPP cells treated with 200 ng/ml doxy (a) as well as of SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT (b) for 24 hours. The catalase enzyme activity was calculated between treated and untreated cells (a). Shown are mean values ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, ***P < 0.01. Cell lysates of SH-EP/tetEGFP, and SH-EP/tetDEPP cells treated with 200 ng/ml doxy for 24 hours as well as of SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT for the indicated time points were subjected to immunoblot analyses using an antibody against catalase. GAPDH served as loading control. Quantification of catalase protein expression normalized to GAPDH (b) was done with the ImageJ 1.48 software. c) Immunoblot analysis of PPARG expression in SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT for 24 hours was performed. GAPDH served as loading control. d) SH-EP/tetEGFP, SH-EP/tetDEPP and SH-EP/tetEYFP-DEPP cells (upper panel) were pre-treated with 200 ng/ml doxy for 24 hours and then incubated with 15 μM H2O2 for one hour. SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP clone-10, -12 and -13 (middle panel) and NB15/FOXO3-shCtr and NB15/FOXO3-shDEPP (bulk, lower panel) were treated with 25 μM H2O2 for 1 hour. PI-FACS analyses were performed to detect apoptotic cells. Shown are mean values ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05, ***P < 0.01.
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Fig5: DEPP impairs catalase activity and sensitizes to H2O2-induced apoptosis. a + b) A catalase enzyme activity assay was performed in SH-EP/tetEGFP, SH-EP/tetDEPP, SH-EP/tetEYFP-DEPP cells treated with 200 ng/ml doxy (a) as well as of SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT (b) for 24 hours. The catalase enzyme activity was calculated between treated and untreated cells (a). Shown are mean values ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, ***P < 0.01. Cell lysates of SH-EP/tetEGFP, and SH-EP/tetDEPP cells treated with 200 ng/ml doxy for 24 hours as well as of SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT for the indicated time points were subjected to immunoblot analyses using an antibody against catalase. GAPDH served as loading control. Quantification of catalase protein expression normalized to GAPDH (b) was done with the ImageJ 1.48 software. c) Immunoblot analysis of PPARG expression in SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT for 24 hours was performed. GAPDH served as loading control. d) SH-EP/tetEGFP, SH-EP/tetDEPP and SH-EP/tetEYFP-DEPP cells (upper panel) were pre-treated with 200 ng/ml doxy for 24 hours and then incubated with 15 μM H2O2 for one hour. SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP clone-10, -12 and -13 (middle panel) and NB15/FOXO3-shCtr and NB15/FOXO3-shDEPP (bulk, lower panel) were treated with 25 μM H2O2 for 1 hour. PI-FACS analyses were performed to detect apoptotic cells. Shown are mean values ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05, ***P < 0.01.

Mentions: As DEPP knockdown efficiently reduced FOXO3-induced ROS accumulation and DEPP overexpression by its own increased steady state cellular ROS we next searched for a possible explanation of this effect. Under normal conditions low amounts of hydrogen peroxide (H2O2) are mainly detoxified in the peroxisomes by the enzyme CAT/catalase[13]. Catalase was also described as a direct transcriptional target of FOXO transcription factors in various cell types[16, 17]. Protein sequence analysis suggests that DEPP contains a peroxisomal-targeting-signal-2 (PTS2) in its N-terminus that allows binding to the PEX7 receptor[27] and might mediate the import of DEPP into peroxisomes[3]. As peroxisomes play an essential role in ROS control especially in cells of neuronal origin[28] we analyzed peroxisomal function by measuring catalase enzyme activity. Catalase activity was significantly reduced in SH-EP/tetDEPP and SH-EP/tetEYFP-DEPP cells after 24 hours of DEPP overexpression compared to SH-EP/tetEGFP cells (Figure 5a). On the other hand, knockdown of DEPP in SH-EP/FOXO3-shCtr cells resulted in significantly increased catalase enzyme activity after 4OHT treatment (Figure 5b). Both, in DEPP-overexpressing and DEPP-knockdown cells, the amount of catalase protein level did not change significantly (Figure 5a,b). We only observed slightly increased catalase expression (1.45 fold compared to untreated controls) after 24 hours of FOXO3 activation in SH-EP/FOXO3-shCtr cells and no regulation in the SH-EP/FOXO3-shDEPP cell clones (Figure 5b). Next, we analyzed protein expression of PPARG, which is described to increase peroxisomal proliferation[29] and to directly regulate catalase enzyme expression and activity[24, 30]. We found protein levels of PPARG strongly upregulated in SH-EP/FOXO3-shDEPP cells compared to SH-EP/FOXO3-shCtr cells (Figure 5c), which is in line with the increased catalase enzyme activity in these cells (Figure 5b). These data suggest that cellular DEPP lowers the ability of neuroblastoma cells to cope with cellular ROS as reflected by reduced PPARG protein levels and catalase enzyme activity.Figure 5


C10ORF10/DEPP, a transcriptional target of FOXO3, regulates ROS-sensitivity in human neuroblastoma.

Salcher S, Hagenbuchner J, Geiger K, Seiter MA, Rainer J, Kofler R, Hermann M, Kiechl-Kohlendorfer U, Ausserlechner MJ, Obexer P - Mol. Cancer (2014)

DEPP impairs catalase activity and sensitizes to H2O2-induced apoptosis. a + b) A catalase enzyme activity assay was performed in SH-EP/tetEGFP, SH-EP/tetDEPP, SH-EP/tetEYFP-DEPP cells treated with 200 ng/ml doxy (a) as well as of SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT (b) for 24 hours. The catalase enzyme activity was calculated between treated and untreated cells (a). Shown are mean values ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, ***P < 0.01. Cell lysates of SH-EP/tetEGFP, and SH-EP/tetDEPP cells treated with 200 ng/ml doxy for 24 hours as well as of SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT for the indicated time points were subjected to immunoblot analyses using an antibody against catalase. GAPDH served as loading control. Quantification of catalase protein expression normalized to GAPDH (b) was done with the ImageJ 1.48 software. c) Immunoblot analysis of PPARG expression in SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT for 24 hours was performed. GAPDH served as loading control. d) SH-EP/tetEGFP, SH-EP/tetDEPP and SH-EP/tetEYFP-DEPP cells (upper panel) were pre-treated with 200 ng/ml doxy for 24 hours and then incubated with 15 μM H2O2 for one hour. SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP clone-10, -12 and -13 (middle panel) and NB15/FOXO3-shCtr and NB15/FOXO3-shDEPP (bulk, lower panel) were treated with 25 μM H2O2 for 1 hour. PI-FACS analyses were performed to detect apoptotic cells. Shown are mean values ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05, ***P < 0.01.
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Fig5: DEPP impairs catalase activity and sensitizes to H2O2-induced apoptosis. a + b) A catalase enzyme activity assay was performed in SH-EP/tetEGFP, SH-EP/tetDEPP, SH-EP/tetEYFP-DEPP cells treated with 200 ng/ml doxy (a) as well as of SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT (b) for 24 hours. The catalase enzyme activity was calculated between treated and untreated cells (a). Shown are mean values ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, ***P < 0.01. Cell lysates of SH-EP/tetEGFP, and SH-EP/tetDEPP cells treated with 200 ng/ml doxy for 24 hours as well as of SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT for the indicated time points were subjected to immunoblot analyses using an antibody against catalase. GAPDH served as loading control. Quantification of catalase protein expression normalized to GAPDH (b) was done with the ImageJ 1.48 software. c) Immunoblot analysis of PPARG expression in SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT for 24 hours was performed. GAPDH served as loading control. d) SH-EP/tetEGFP, SH-EP/tetDEPP and SH-EP/tetEYFP-DEPP cells (upper panel) were pre-treated with 200 ng/ml doxy for 24 hours and then incubated with 15 μM H2O2 for one hour. SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP clone-10, -12 and -13 (middle panel) and NB15/FOXO3-shCtr and NB15/FOXO3-shDEPP (bulk, lower panel) were treated with 25 μM H2O2 for 1 hour. PI-FACS analyses were performed to detect apoptotic cells. Shown are mean values ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05, ***P < 0.01.
Mentions: As DEPP knockdown efficiently reduced FOXO3-induced ROS accumulation and DEPP overexpression by its own increased steady state cellular ROS we next searched for a possible explanation of this effect. Under normal conditions low amounts of hydrogen peroxide (H2O2) are mainly detoxified in the peroxisomes by the enzyme CAT/catalase[13]. Catalase was also described as a direct transcriptional target of FOXO transcription factors in various cell types[16, 17]. Protein sequence analysis suggests that DEPP contains a peroxisomal-targeting-signal-2 (PTS2) in its N-terminus that allows binding to the PEX7 receptor[27] and might mediate the import of DEPP into peroxisomes[3]. As peroxisomes play an essential role in ROS control especially in cells of neuronal origin[28] we analyzed peroxisomal function by measuring catalase enzyme activity. Catalase activity was significantly reduced in SH-EP/tetDEPP and SH-EP/tetEYFP-DEPP cells after 24 hours of DEPP overexpression compared to SH-EP/tetEGFP cells (Figure 5a). On the other hand, knockdown of DEPP in SH-EP/FOXO3-shCtr cells resulted in significantly increased catalase enzyme activity after 4OHT treatment (Figure 5b). Both, in DEPP-overexpressing and DEPP-knockdown cells, the amount of catalase protein level did not change significantly (Figure 5a,b). We only observed slightly increased catalase expression (1.45 fold compared to untreated controls) after 24 hours of FOXO3 activation in SH-EP/FOXO3-shCtr cells and no regulation in the SH-EP/FOXO3-shDEPP cell clones (Figure 5b). Next, we analyzed protein expression of PPARG, which is described to increase peroxisomal proliferation[29] and to directly regulate catalase enzyme expression and activity[24, 30]. We found protein levels of PPARG strongly upregulated in SH-EP/FOXO3-shDEPP cells compared to SH-EP/FOXO3-shCtr cells (Figure 5c), which is in line with the increased catalase enzyme activity in these cells (Figure 5b). These data suggest that cellular DEPP lowers the ability of neuroblastoma cells to cope with cellular ROS as reflected by reduced PPARG protein levels and catalase enzyme activity.Figure 5

Bottom Line: The effect of DEPP on cellular ROS was measured by catalase activity assay and live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS.Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells.In parallel, the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARG), a critical regulator of catalase enzyme activity, was strongly upregulated in DEPP-knockdown cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics I, Medical University Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria. michael.j.ausserlechner@i-med.ac.at.

ABSTRACT

Background: FOXO transcription factors control cellular levels of reactive oxygen species (ROS) which critically contribute to cell survival and cell death in neuroblastoma. In the present study we investigated the regulation of C10orf10/DEPP by the transcription factor FOXO3. As a physiological function of C10orf10/DEPP has not been described so far we analyzed its effects on cellular ROS detoxification and death sensitization in human neuroblastoma cells.

Methods: The effect of DEPP on cellular ROS was measured by catalase activity assay and live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS. The cellular localization of DEPP was determined by confocal microscopy of EYFP-tagged DEPP, fluorescent peroxisomal- and mitochondrial probes and co-immunoprecipitation of the PEX7 receptor.

Results: We report for the first time that DEPP regulates ROS detoxification and localizes to peroxisomes and mitochondria in neuroblastoma cells. FOXO3-mediated apoptosis involves a biphasic ROS accumulation. Knockdown of DEPP prevented the primary and secondary ROS wave during FOXO3 activation and attenuated FOXO3- and H2O2-induced apoptosis. Conditional overexpression of DEPP elevates cellular ROS levels and sensitizes to H2O2 and etoposide-induced cell death. In neuronal cells, cellular ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that allows binding to the PEX7 receptor and import into peroxisomes, we analyzed the effect of DEPP on cellular detoxification by measuring enzyme activity of catalase. Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells. DEPP directly interacts with the PEX7 receptor and localizes to the peroxisomal compartment. In parallel, the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARG), a critical regulator of catalase enzyme activity, was strongly upregulated in DEPP-knockdown cells.

Conclusion: The combined data indicate that in neuroblastoma DEPP localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification, which sensitizes tumor cells to ROS-induced cell death.

Show MeSH
Related in: MedlinePlus