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Anti-migratory effect of vinflunine in endothelial and glioblastoma cells is associated with changes in EB1 C-terminal detyrosinated/tyrosinated status.

Rovini A, Gauthier G, Bergès R, Kruczynski A, Braguer D, Honoré S - PLoS ONE (2013)

Bottom Line: Such effect was associated with a reduction of EB1 comet length at microtubule (+) ends.Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration.Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin.

View Article: PubMed Central - PubMed

Affiliation: Aix Marseille Université, Institut National de la Santé et de la Recherche Médicale UMR_S 911, Marseille, France.

ABSTRACT
We previously showed that vinflunine, a microtubule-targeting drug of the Vinca-alkaloid family exerted its anti-angiogenic/anti-migratory activities through an increase in microtubule dynamics and an inhibition of microtubule targeting to adhesion sites. Such effect was associated with a reduction of EB1 comet length at microtubule (+) ends. In this work we first showed that the pro-angiogenic vascular endothelial growth factor VEGF suppressed microtubule dynamics in living Human Umbilical Vein Endothelial Cells (HUVECs), increased EB1 comet length by 40%, and induced EB1 to bind all along the microtubules, without modifying its expression level. Such microtubule (+) end stabilization occurred close to the plasma membrane in the vicinity of focal adhesion as shown by TIRF microscopy experiments. Vinflunine completely abolished the effect of VEGF on EB1 comets. Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration. By using 2D gel electrophoresis we demonstrated for the first time that EB1 underwent several post-translational modifications in endothelial and tumor cells. Particularly, the C-terminal EEY sequence was poorly detectable in control and VEGF-treated HUVECs suggesting the existence of a non-tyrosinated form of EB1. By using specific antibodies that specifically recognized and discriminated the native tyrosinated form of EB1 and a putative C-terminal detyrosinated form, we showed that a detyrosinated form of EB1 exists in HUVECs and tumor cells. Interestingly, vinflunine decreased the level of the detyrosinated form and increased the native tyrosinated form of EB1. Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin. Altogether, our results show that vinflunine inhibits endothelial cell migration through an alteration of EB1 comet length and EB1 detyrosination/retyrosination cycle.

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Anti-migratory vinflunine altered EB1 detyrosination/retyrosination cycle in both endothelial and glioblastoma cells.Western blotting detection of detyrosinated or tyrosinated EB1 in HUVECs (A) and U87 cells (B) under VFL treatment. Relative ratios detyrosinated EB1 (EB1ΔY)/total EB1 or tyrosinated EB1 (EB1-EEY)/total EB1, from at least five independent experiments, are presented under the blot. (C) Transwell migration assay with control sh0 U87cells treated or not with VFL (10 nM). Data show the average number of cells that migrated through the filter. At least three independent experiments were performed for each condition. Bar ± SEM (**) indicates significant differences from control (p<0.05).
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pone-0065694-g006: Anti-migratory vinflunine altered EB1 detyrosination/retyrosination cycle in both endothelial and glioblastoma cells.Western blotting detection of detyrosinated or tyrosinated EB1 in HUVECs (A) and U87 cells (B) under VFL treatment. Relative ratios detyrosinated EB1 (EB1ΔY)/total EB1 or tyrosinated EB1 (EB1-EEY)/total EB1, from at least five independent experiments, are presented under the blot. (C) Transwell migration assay with control sh0 U87cells treated or not with VFL (10 nM). Data show the average number of cells that migrated through the filter. At least three independent experiments were performed for each condition. Bar ± SEM (**) indicates significant differences from control (p<0.05).

Mentions: We thus determined the effect of VFL on the equilibrium between tyrosinated and detyrosinated EB1 in both endothelial (Figure 6A) and glioblastoma cells (Figure 6B) by using specific antibodies [22]. As expected, according to the results obtained with YL½ in HUVECs, VFL increased the amount of tyrosinated EB1 while decreasing the amount of detyrosinated EB1 in a concentration-dependant manner, in both HUVECs and U87. Moreover, similarly to the results obtained in HUVECs at the concentration that induced the highest changes on EB1 tyrosination and detyrosination status, VFL inhibited glioblastoma cell migration (Figure 6C). Altogether, our results suggest that, in link with its anti-migratory potential, VFL interfered with EB1 tyrosination/detyrosination cycle.


Anti-migratory effect of vinflunine in endothelial and glioblastoma cells is associated with changes in EB1 C-terminal detyrosinated/tyrosinated status.

Rovini A, Gauthier G, Bergès R, Kruczynski A, Braguer D, Honoré S - PLoS ONE (2013)

Anti-migratory vinflunine altered EB1 detyrosination/retyrosination cycle in both endothelial and glioblastoma cells.Western blotting detection of detyrosinated or tyrosinated EB1 in HUVECs (A) and U87 cells (B) under VFL treatment. Relative ratios detyrosinated EB1 (EB1ΔY)/total EB1 or tyrosinated EB1 (EB1-EEY)/total EB1, from at least five independent experiments, are presented under the blot. (C) Transwell migration assay with control sh0 U87cells treated or not with VFL (10 nM). Data show the average number of cells that migrated through the filter. At least three independent experiments were performed for each condition. Bar ± SEM (**) indicates significant differences from control (p<0.05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672205&req=5

pone-0065694-g006: Anti-migratory vinflunine altered EB1 detyrosination/retyrosination cycle in both endothelial and glioblastoma cells.Western blotting detection of detyrosinated or tyrosinated EB1 in HUVECs (A) and U87 cells (B) under VFL treatment. Relative ratios detyrosinated EB1 (EB1ΔY)/total EB1 or tyrosinated EB1 (EB1-EEY)/total EB1, from at least five independent experiments, are presented under the blot. (C) Transwell migration assay with control sh0 U87cells treated or not with VFL (10 nM). Data show the average number of cells that migrated through the filter. At least three independent experiments were performed for each condition. Bar ± SEM (**) indicates significant differences from control (p<0.05).
Mentions: We thus determined the effect of VFL on the equilibrium between tyrosinated and detyrosinated EB1 in both endothelial (Figure 6A) and glioblastoma cells (Figure 6B) by using specific antibodies [22]. As expected, according to the results obtained with YL½ in HUVECs, VFL increased the amount of tyrosinated EB1 while decreasing the amount of detyrosinated EB1 in a concentration-dependant manner, in both HUVECs and U87. Moreover, similarly to the results obtained in HUVECs at the concentration that induced the highest changes on EB1 tyrosination and detyrosination status, VFL inhibited glioblastoma cell migration (Figure 6C). Altogether, our results suggest that, in link with its anti-migratory potential, VFL interfered with EB1 tyrosination/detyrosination cycle.

Bottom Line: Such effect was associated with a reduction of EB1 comet length at microtubule (+) ends.Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration.Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin.

View Article: PubMed Central - PubMed

Affiliation: Aix Marseille Université, Institut National de la Santé et de la Recherche Médicale UMR_S 911, Marseille, France.

ABSTRACT
We previously showed that vinflunine, a microtubule-targeting drug of the Vinca-alkaloid family exerted its anti-angiogenic/anti-migratory activities through an increase in microtubule dynamics and an inhibition of microtubule targeting to adhesion sites. Such effect was associated with a reduction of EB1 comet length at microtubule (+) ends. In this work we first showed that the pro-angiogenic vascular endothelial growth factor VEGF suppressed microtubule dynamics in living Human Umbilical Vein Endothelial Cells (HUVECs), increased EB1 comet length by 40%, and induced EB1 to bind all along the microtubules, without modifying its expression level. Such microtubule (+) end stabilization occurred close to the plasma membrane in the vicinity of focal adhesion as shown by TIRF microscopy experiments. Vinflunine completely abolished the effect of VEGF on EB1 comets. Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration. By using 2D gel electrophoresis we demonstrated for the first time that EB1 underwent several post-translational modifications in endothelial and tumor cells. Particularly, the C-terminal EEY sequence was poorly detectable in control and VEGF-treated HUVECs suggesting the existence of a non-tyrosinated form of EB1. By using specific antibodies that specifically recognized and discriminated the native tyrosinated form of EB1 and a putative C-terminal detyrosinated form, we showed that a detyrosinated form of EB1 exists in HUVECs and tumor cells. Interestingly, vinflunine decreased the level of the detyrosinated form and increased the native tyrosinated form of EB1. Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin. Altogether, our results show that vinflunine inhibits endothelial cell migration through an alteration of EB1 comet length and EB1 detyrosination/retyrosination cycle.

Show MeSH
Related in: MedlinePlus