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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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Quantitative PCR analysis of mitochondrial fission/fusion actors and relevant differentially-expressed miRNA-mRNA complexes in human T98G glioblastoma cells.5A: Quantitative PCR analysis of mitochondrial fission/fusion actors (FIS1 and MFN2) in T98G cells. The data are expressed in units (mRNA expression of a specific gene normalized to β-globin mRNA expression) that are relative to the control, which was assigned a unit value. 5B: Quantitative PCR analysis of relevant differentially expressed miRNA in T98G cells. The data are expressed in units (miRNA expression relative to U5 snRNA) that are relative to the control, which was assigned a unit value. 5C: Quantitative PCR analysis of PTEN and NAIP mRNA, directly targeted by miR-21 and miR-221, respectively. FGFR3 expression was used as negative control of miR-100, which expression level was unchanged by peptide treatment. The data are expressed in units (mRNA expression of a specific gene normalized to β-globin mRNA expression) that are relative to the control, which was assigned a unit value. The values are the average ± SD for three separate determinations (N = 3). *: P<0.05 versus control.
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pone-0098473-g005: Quantitative PCR analysis of mitochondrial fission/fusion actors and relevant differentially-expressed miRNA-mRNA complexes in human T98G glioblastoma cells.5A: Quantitative PCR analysis of mitochondrial fission/fusion actors (FIS1 and MFN2) in T98G cells. The data are expressed in units (mRNA expression of a specific gene normalized to β-globin mRNA expression) that are relative to the control, which was assigned a unit value. 5B: Quantitative PCR analysis of relevant differentially expressed miRNA in T98G cells. The data are expressed in units (miRNA expression relative to U5 snRNA) that are relative to the control, which was assigned a unit value. 5C: Quantitative PCR analysis of PTEN and NAIP mRNA, directly targeted by miR-21 and miR-221, respectively. FGFR3 expression was used as negative control of miR-100, which expression level was unchanged by peptide treatment. The data are expressed in units (mRNA expression of a specific gene normalized to β-globin mRNA expression) that are relative to the control, which was assigned a unit value. The values are the average ± SD for three separate determinations (N = 3). *: P<0.05 versus control.

Mentions: We explored the impact of NFL-TBS.40-63 on the mitochondrial fission–fusion balance using the master regulator of mitochondrial dynamics, MFN2, which is responsible for mitochondrial multiplication and FIS1, which is involved in mitochondrial fission (Figure 5A). Here, we showed that, even if both factors presented a significant decrease in expression level, the FIS1/MFN2 ratio, which refers to the balance between dynamic events, was conserved. Rather, this conserved modeling balance involved differences mainly in mitochondrial motility resulting from abnormal cytoskeletal anchorage. Thus, we have observed a decrease in mitochondrial motility (mean speed motility 7-times slower) using mitochondrial network imaging as well as decrease in cell motility (53% mean decrease in cell migration) using the transwell assay, in peptide-treated cells compared to scramble-treated (Figures S2 and S3 in File S1, respectively).


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)

Quantitative PCR analysis of mitochondrial fission/fusion actors and relevant differentially-expressed miRNA-mRNA complexes in human T98G glioblastoma cells.5A: Quantitative PCR analysis of mitochondrial fission/fusion actors (FIS1 and MFN2) in T98G cells. The data are expressed in units (mRNA expression of a specific gene normalized to β-globin mRNA expression) that are relative to the control, which was assigned a unit value. 5B: Quantitative PCR analysis of relevant differentially expressed miRNA in T98G cells. The data are expressed in units (miRNA expression relative to U5 snRNA) that are relative to the control, which was assigned a unit value. 5C: Quantitative PCR analysis of PTEN and NAIP mRNA, directly targeted by miR-21 and miR-221, respectively. FGFR3 expression was used as negative control of miR-100, which expression level was unchanged by peptide treatment. The data are expressed in units (mRNA expression of a specific gene normalized to β-globin mRNA expression) that are relative to the control, which was assigned a unit value. The values are the average ± SD for three separate determinations (N = 3). *: P<0.05 versus control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4045719&req=5

pone-0098473-g005: Quantitative PCR analysis of mitochondrial fission/fusion actors and relevant differentially-expressed miRNA-mRNA complexes in human T98G glioblastoma cells.5A: Quantitative PCR analysis of mitochondrial fission/fusion actors (FIS1 and MFN2) in T98G cells. The data are expressed in units (mRNA expression of a specific gene normalized to β-globin mRNA expression) that are relative to the control, which was assigned a unit value. 5B: Quantitative PCR analysis of relevant differentially expressed miRNA in T98G cells. The data are expressed in units (miRNA expression relative to U5 snRNA) that are relative to the control, which was assigned a unit value. 5C: Quantitative PCR analysis of PTEN and NAIP mRNA, directly targeted by miR-21 and miR-221, respectively. FGFR3 expression was used as negative control of miR-100, which expression level was unchanged by peptide treatment. The data are expressed in units (mRNA expression of a specific gene normalized to β-globin mRNA expression) that are relative to the control, which was assigned a unit value. The values are the average ± SD for three separate determinations (N = 3). *: P<0.05 versus control.
Mentions: We explored the impact of NFL-TBS.40-63 on the mitochondrial fission–fusion balance using the master regulator of mitochondrial dynamics, MFN2, which is responsible for mitochondrial multiplication and FIS1, which is involved in mitochondrial fission (Figure 5A). Here, we showed that, even if both factors presented a significant decrease in expression level, the FIS1/MFN2 ratio, which refers to the balance between dynamic events, was conserved. Rather, this conserved modeling balance involved differences mainly in mitochondrial motility resulting from abnormal cytoskeletal anchorage. Thus, we have observed a decrease in mitochondrial motility (mean speed motility 7-times slower) using mitochondrial network imaging as well as decrease in cell motility (53% mean decrease in cell migration) using the transwell assay, in peptide-treated cells compared to scramble-treated (Figures S2 and S3 in File S1, respectively).

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