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

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.

Figure 1. 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.

Figure 2. Expression analysis of the genes involved in the control of mitochondrial biogenesis in T98G cells treated by 10 µM of NFL-TBS.40-63 peptide.

2A: Quantitative PCR analysis: PRC (PPRC1) and PGC-1α (PPARGC1A) coactivators, NRF-1 transcription factor, mitochondrial transcription factor TFAM and a component of the respiratory chain, Cytochrome c (CYCS), were measured. The data are expressed in relative units (mRNA expression of a specific gene normalized to β-globin mRNA expression) and expressed 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. 2B: Western blot analysis: The protein expression of PGC-1α (105KDa, Ab- 54481, Abcam) and NRF-1 (54 KDa, Ab-86516, Abcam) were measured by Western blot analysis after a 6-hours exposure to 10 µM NFL-TBS.40-63 peptide and normalized to the β-actin level (45 KDa; Abcam). Results are expressed relative to protein expression ratio of scramble treated cells which was assigned a unit value. The values are the average ± SD (N = 3). *: P<0.05 versus control.

Figure 3. Effects of 10 µM of NFL-TBS.40-63 peptide on mitochondrial and microtubule networks in human T98G glioblastoma cells.

3A: NFL peptide accumulates within the cell in a polarized manner, limiting the density of the microtubules and mitochondrial networks. 3B: NFL peptide accumulates at the basis of the midbody and excludes the microtubule network. 3C: The peptide surrounds the microtubules' tips and limits filopodia formation. Microtubules were detected using an Alexa647-labeled, anti-α-tubulin antibody (purple); biotinylated NFL-TBS.40-63 peptide was labeled with streptavidin Alexa488 (green), the nuclei with diamidino phénylindole (DAPI; blue) and mitochondria with a mitotracker (RedCMX ROS). The cells were examined with a Nikon A1RSI confocal microscope and the images were analyzed with Nikon NIS-element software. The red bars are the measuring scale.

Figure 4. The NFL-TBS.40-63 peptide reorganizes mitochondrial networks in human T98G glioblastoma cells.

The white arrow indicates a mitochondrial network (bottom left) superposed with a microtubule network (bottom right). The yellow arrow indicates a mitochondrial network (bottom left) superposed with long peptide sequences (top right). The microtubules were detected using an Alexa647-labeled, anti-α-tubulin antibody (purple); biotinylated NFL-TBS.40-63 peptide was labeled with streptavidin Alexa488 (green), the nuclei with diamidino ph?nylindole (DAPI; blue) and the mitochondria with a mitotracker Red CMX ROS). The cells were examined with a Nikon A1RSI confocal microscope and the images were analyzed with Nikon NIS-element software. The red bars are the measuring scale.

Figure 5. 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.