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The NFL-TBS.40-63 anti-glioblastoma peptide disrupts microtubule and mitochondrial networks in the T98G glioma cell line.

Rivalin R, Lepinoux-Chambaud C, Eyer J, Savagner F - PLoS ONE (2014)

Bottom Line: Despite aggressive therapies, including combinations of surgery, radiotherapy and chemotherapy, glioblastoma remains a highly aggressive brain cancer with the worst prognosis of any central nervous system disease.We show that the internalized peptide disturbs mitochondrial and microtubule networks, interferes with mitochondrial dynamics and induces a rapid depletion of global cell respiration.This effect may be related to reduced expression of the NRF-1 transcription factor and of specific miRNAs, which may impact mitochondrial biogenesis, in regard to default mitochondrial mobility.

View Article: PubMed Central - PubMed

Affiliation: Université d'Angers, Angers, France; Laboratoire Neurobiologie & Transgenese, LNBT, UPRES EA-3143, Université d'Angers, Bâtiment IBS-IRIS, Angers, France.

ABSTRACT
Despite aggressive therapies, including combinations of surgery, radiotherapy and chemotherapy, glioblastoma remains a highly aggressive brain cancer with the worst prognosis of any central nervous system disease. We have previously identified a neurofilament-derived cell-penetrating peptide, NFL-TBS.40-63, that specifically enters by endocytosis in glioblastoma cells, where it induces microtubule destruction and inhibits cell proliferation. Here, we explore the impact of NFL-TBS.40-63 peptide on the mitochondrial network and its functions by using global cell respiration, quantitative PCR analysis of the main actors directing mitochondrial biogenesis, western blot analysis of the oxidative phosphorylation (OXPHOS) subunits and confocal microscopy. We show that the internalized peptide disturbs mitochondrial and microtubule networks, interferes with mitochondrial dynamics and induces a rapid depletion of global cell respiration. This effect may be related to reduced expression of the NRF-1 transcription factor and of specific miRNAs, which may impact mitochondrial biogenesis, in regard to default mitochondrial mobility.

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Related in: MedlinePlus

Action of the NFL-TBS.40-63 peptide on mitochondrial number and functions in T98G cells and NIH 3T3 control cells.1A: The relative oxygen consumption was measured using a mitostress kit and a Seahorse XF-24 apparatus (from Seahorse Bioscience, North Billerica, MA, USA). The oligomycin-insensitive fraction represents non-phosphorylating respiration, which was recorded after the inhibition of ATP synthase with oligomycin. The oligomycin-sensitive fraction represents the phosphorylating respiration, i.e., the fraction used for ATP synthesis. Results are expressed relative to oxygen consumption of scramble treated cells used as control (pmol/min/mg protein). 1B: The protein expression of mitochondrial subunit IV of complex IV (COX4, MS408, Mitosciences) and subunit Ip of complex II (SDHB, MS203, Mitosciences) were measured by Western blot analysis after a 6-hour exposure to 10 µM NFL-TBS.40-63 peptide and normalized to the α-tubulin level (65 KDa; Abcam, Cambridge, UK). The protein expression for the peptide-treated samples was expressed relative to that of the scramble-treated samples. S: Scramble; P: NFL-TBS.40-63 peptide. Results are expressed relative to protein expression ratio of scramble treated cells used as control. The values represent the average ± SD for three separate determinations (N = 3). *: P<0.05 versus control.
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pone-0098473-g001: Action of the NFL-TBS.40-63 peptide on mitochondrial number and functions in T98G cells and NIH 3T3 control cells.1A: The relative oxygen consumption was measured using a mitostress kit and a Seahorse XF-24 apparatus (from Seahorse Bioscience, North Billerica, MA, USA). The oligomycin-insensitive fraction represents non-phosphorylating respiration, which was recorded after the inhibition of ATP synthase with oligomycin. The oligomycin-sensitive fraction represents the phosphorylating respiration, i.e., the fraction used for ATP synthesis. Results are expressed relative to oxygen consumption of scramble treated cells used as control (pmol/min/mg protein). 1B: The protein expression of mitochondrial subunit IV of complex IV (COX4, MS408, Mitosciences) and subunit Ip of complex II (SDHB, MS203, Mitosciences) were measured by Western blot analysis after a 6-hour exposure to 10 µM NFL-TBS.40-63 peptide and normalized to the α-tubulin level (65 KDa; Abcam, Cambridge, UK). The protein expression for the peptide-treated samples was expressed relative to that of the scramble-treated samples. S: Scramble; P: NFL-TBS.40-63 peptide. Results are expressed relative to protein expression ratio of scramble treated cells used as control. The values represent the average ± SD for three separate determinations (N = 3). *: P<0.05 versus control.

Mentions: We investigated the impact of a 6 hours-incubation in various NFL-TBS.40-63 peptide concentrations (2 to 10 µM) on mitochondrial respiratory function and biogenesis from T98G cells and NIH3T3 control cells [8], [9]. A mitostress test was performed to investigate the main parameters of the OXPHOS process, including basal respiration rate, ATP turnover, proton leak and maximal oxygen consumption rate. Mitochondrial respiration is divided into two fractions. The oligomycin insensitive fraction corresponds to non-phosphorylating respiration and is recorded after the inhibition of ATP synthase with 1 µM oligomycin. The oligomycin-sensitive fraction represents the phosphorylating respiration and is the fraction used for ATP synthesis, which is calculated by subtracting the nonphosphorylating respiration rate from the basal respiration rate. The NFL-TBS.40-63 peptide significantly affected both oligomycin-sensitive and insensitive fractions at 10 µM (Figure 1A). The global oxygen consumption rate was reduced at all concentrations, with specific and significant decreases in the oligomycin-insensitive fractions of approximately 20% at the 2 µM and 5 µM concentrations. However, no defect in the fraction used for ATP synthesis (oligomycin-sensitive fraction) could be noticed at these lowest concentrations, contrary to that observed for 10 µM peptide. Conversely, for the NIH-3T3 cells, none of the two oligomycin fractions seemed to be significantly affected by the peptide even if a tendency to decrease could be noticed at 10 µM peptide for the oligomycin-sensitive fraction. To explore the impact of 10 µM NFL-TBS.40-63 treatment on mitochondrial biogenesis, we have investigated the protein expression level of mitochondrial complex subunits using Western blot analysis (Figure 1B). Our results show a significant reduction in SDHB (complex II subunit) by 30% and of COX4 (complex IV subunit) by 20%, respectively, in T98G cells, when compared to the scrambled peptide. In NIH-3T3 cells, no such reduction was observed, in accordance with our previous results for non-permeant NIH-3T3 cells for this peptide [8]. These results indicate a primary impact of the peptide on mitochondrial functions at low concentrations (2 and 5 µM), which had no detectable effect on the microtubule network. However, at 10 µM of peptide, both networks were affected [8].


The NFL-TBS.40-63 anti-glioblastoma peptide disrupts microtubule and mitochondrial networks in the T98G glioma cell line.

Rivalin R, Lepinoux-Chambaud C, Eyer J, Savagner F - PLoS ONE (2014)

Action of the NFL-TBS.40-63 peptide on mitochondrial number and functions in T98G cells and NIH 3T3 control cells.1A: The relative oxygen consumption was measured using a mitostress kit and a Seahorse XF-24 apparatus (from Seahorse Bioscience, North Billerica, MA, USA). The oligomycin-insensitive fraction represents non-phosphorylating respiration, which was recorded after the inhibition of ATP synthase with oligomycin. The oligomycin-sensitive fraction represents the phosphorylating respiration, i.e., the fraction used for ATP synthesis. Results are expressed relative to oxygen consumption of scramble treated cells used as control (pmol/min/mg protein). 1B: The protein expression of mitochondrial subunit IV of complex IV (COX4, MS408, Mitosciences) and subunit Ip of complex II (SDHB, MS203, Mitosciences) were measured by Western blot analysis after a 6-hour exposure to 10 µM NFL-TBS.40-63 peptide and normalized to the α-tubulin level (65 KDa; Abcam, Cambridge, UK). The protein expression for the peptide-treated samples was expressed relative to that of the scramble-treated samples. S: Scramble; P: NFL-TBS.40-63 peptide. Results are expressed relative to protein expression ratio of scramble treated cells used as control. The values represent the average ± SD for three separate determinations (N = 3). *: P<0.05 versus control.
© Copyright Policy
Related In: Results  -  Collection

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pone-0098473-g001: Action of the NFL-TBS.40-63 peptide on mitochondrial number and functions in T98G cells and NIH 3T3 control cells.1A: The relative oxygen consumption was measured using a mitostress kit and a Seahorse XF-24 apparatus (from Seahorse Bioscience, North Billerica, MA, USA). The oligomycin-insensitive fraction represents non-phosphorylating respiration, which was recorded after the inhibition of ATP synthase with oligomycin. The oligomycin-sensitive fraction represents the phosphorylating respiration, i.e., the fraction used for ATP synthesis. Results are expressed relative to oxygen consumption of scramble treated cells used as control (pmol/min/mg protein). 1B: The protein expression of mitochondrial subunit IV of complex IV (COX4, MS408, Mitosciences) and subunit Ip of complex II (SDHB, MS203, Mitosciences) were measured by Western blot analysis after a 6-hour exposure to 10 µM NFL-TBS.40-63 peptide and normalized to the α-tubulin level (65 KDa; Abcam, Cambridge, UK). The protein expression for the peptide-treated samples was expressed relative to that of the scramble-treated samples. S: Scramble; P: NFL-TBS.40-63 peptide. Results are expressed relative to protein expression ratio of scramble treated cells used as control. The values represent the average ± SD for three separate determinations (N = 3). *: P<0.05 versus control.
Mentions: We investigated the impact of a 6 hours-incubation in various NFL-TBS.40-63 peptide concentrations (2 to 10 µM) on mitochondrial respiratory function and biogenesis from T98G cells and NIH3T3 control cells [8], [9]. A mitostress test was performed to investigate the main parameters of the OXPHOS process, including basal respiration rate, ATP turnover, proton leak and maximal oxygen consumption rate. Mitochondrial respiration is divided into two fractions. The oligomycin insensitive fraction corresponds to non-phosphorylating respiration and is recorded after the inhibition of ATP synthase with 1 µM oligomycin. The oligomycin-sensitive fraction represents the phosphorylating respiration and is the fraction used for ATP synthesis, which is calculated by subtracting the nonphosphorylating respiration rate from the basal respiration rate. The NFL-TBS.40-63 peptide significantly affected both oligomycin-sensitive and insensitive fractions at 10 µM (Figure 1A). The global oxygen consumption rate was reduced at all concentrations, with specific and significant decreases in the oligomycin-insensitive fractions of approximately 20% at the 2 µM and 5 µM concentrations. However, no defect in the fraction used for ATP synthesis (oligomycin-sensitive fraction) could be noticed at these lowest concentrations, contrary to that observed for 10 µM peptide. Conversely, for the NIH-3T3 cells, none of the two oligomycin fractions seemed to be significantly affected by the peptide even if a tendency to decrease could be noticed at 10 µM peptide for the oligomycin-sensitive fraction. To explore the impact of 10 µM NFL-TBS.40-63 treatment on mitochondrial biogenesis, we have investigated the protein expression level of mitochondrial complex subunits using Western blot analysis (Figure 1B). Our results show a significant reduction in SDHB (complex II subunit) by 30% and of COX4 (complex IV subunit) by 20%, respectively, in T98G cells, when compared to the scrambled peptide. In NIH-3T3 cells, no such reduction was observed, in accordance with our previous results for non-permeant NIH-3T3 cells for this peptide [8]. These results indicate a primary impact of the peptide on mitochondrial functions at low concentrations (2 and 5 µM), which had no detectable effect on the microtubule network. However, at 10 µM of peptide, both networks were affected [8].

Bottom Line: Despite aggressive therapies, including combinations of surgery, radiotherapy and chemotherapy, glioblastoma remains a highly aggressive brain cancer with the worst prognosis of any central nervous system disease.We show that the internalized peptide disturbs mitochondrial and microtubule networks, interferes with mitochondrial dynamics and induces a rapid depletion of global cell respiration.This effect may be related to reduced expression of the NRF-1 transcription factor and of specific miRNAs, which may impact mitochondrial biogenesis, in regard to default mitochondrial mobility.

View Article: PubMed Central - PubMed

Affiliation: Université d'Angers, Angers, France; Laboratoire Neurobiologie & Transgenese, LNBT, UPRES EA-3143, Université d'Angers, Bâtiment IBS-IRIS, Angers, France.

ABSTRACT
Despite aggressive therapies, including combinations of surgery, radiotherapy and chemotherapy, glioblastoma remains a highly aggressive brain cancer with the worst prognosis of any central nervous system disease. We have previously identified a neurofilament-derived cell-penetrating peptide, NFL-TBS.40-63, that specifically enters by endocytosis in glioblastoma cells, where it induces microtubule destruction and inhibits cell proliferation. Here, we explore the impact of NFL-TBS.40-63 peptide on the mitochondrial network and its functions by using global cell respiration, quantitative PCR analysis of the main actors directing mitochondrial biogenesis, western blot analysis of the oxidative phosphorylation (OXPHOS) subunits and confocal microscopy. We show that the internalized peptide disturbs mitochondrial and microtubule networks, interferes with mitochondrial dynamics and induces a rapid depletion of global cell respiration. This effect may be related to reduced expression of the NRF-1 transcription factor and of specific miRNAs, which may impact mitochondrial biogenesis, in regard to default mitochondrial mobility.

Show MeSH
Related in: MedlinePlus