The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification

Objective: For treatment of medication-related osteonecrosis of the jaw, one proposed approach is the use of a topical agent to block entry of these medications in oral soft tissues. We tested the ability of phosphonoformic acid (PFA), an inhibitor of bisphosphonate entry through certain sodium-depe...

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Autores: Kirby, Jacob, Standfest, Makayla, Binkley, Jessica, Barnes, Charles, Brown, Evan, Cairncross, Tyler, Cartwright, Alex, Dadisman, Danielle, Mowat, Colten, Wilmot, Daniel, Houseman, Theodore, Murphy, Conner, Engelsman, Caleb, Haller, Josh, Jones, Daniel
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2024
País:Brasil
Institución:Universidade de São Paulo (USP)
Repositorio:Journal of applied oral science (Online)
Idioma:inglés
OAI Identifier:oai:revistas.usp.br:article/230164
Acceso en línea:https://www.revistas.usp.br/jaos/article/view/230164
Access Level:acceso abierto
Palabra clave:Bisphosphonate-associated osteonecrosis of the jaw
Dynamins
Endosomes
Fibroblasts
Zoledronic acid
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spelling The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidificationBisphosphonate-associated osteonecrosis of the jawDynaminsEndosomesFibroblastsZoledronic acidObjective: For treatment of medication-related osteonecrosis of the jaw, one proposed approach is the use of a topical agent to block entry of these medications in oral soft tissues. We tested the ability of phosphonoformic acid (PFA), an inhibitor of bisphosphonate entry through certain sodium-dependent phosphate contransporters (SLC20A1, 20A2, 34A1-3) as well as Dynasore, a macropinocytosis inhibitor, for their abilities to prevent zoledronate-induced (ZOL) death in human gingival fibroblasts (HGFs). Methodology: MTT assay dose-response curves were performed to determine non-cytotoxic levels of both PFA and Dynasore. In the presence of 50 μM ZOL, optimized PFA and Dynasore doses were tested for their ability to restore HGF viability. To determine SLC expression in HGFs, total HGF RNA was subjected to quantitative real-time RT-PCR. Confocal fluorescence microscopy was employed to see if Dynasore inhibited macropinocytotic HGF entry of AF647-ZOL. Endosomal acidification in the presence of Dynasore was measured by live cell imaging utilizing LysoSensor Green DND-189. As a further test of Dynasore’s ability to interfere with ZOL-containing endosomal maturation, perinuclear localization of mature endosomes containing AF647-ZOL or TRITC-dextran as a control were assessed via confocal fluorescence microscopy with CellProfiler™ software analysis of the resulting photomicrographs. Results: 0.5 mM PFA did not rescue  HGFs from ZOL-induced viability loss at 72 hours while 10 and 30 μM geranylgeraniol did partially rescue. HGFs did not express the SLC transporters as compared to the expression in positive control tissues. 10 μM Dynasore completely prevented ZOL-induced viability loss. In the presence of Dynasore, AF647-ZOL and FITC-dextran co-localized in endosomes. Endosomal acidification was inhibited by Dynasore and perinuclear localization of both TRITC-dextran- and AF647-ZOL-containing endosomes was inhibited by 30 μM Dynasore. Conclusion: Dynasore prevents ZOL-induced viability loss in HGFs by partially interfering with macropinocytosis and by inhibiting the endosomal maturation pathway thought to be needed for ZOL delivery to the cytoplasm.Universidade de São Paulo. Faculdade de Odontologia de Bauru2024-10-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://www.revistas.usp.br/jaos/article/view/23016410.1590/1678-7757-2024-0224Journal of Applied Oral Science; Vol. 32 (2024); e20240224Journal of Applied Oral Science; v. 32 (2024); e20240224Journal of Applied Oral Science; Vol. 32 (2024); e202402241678-77651678-7757reponame:Journal of applied oral science (Online)instname:Universidade de São Paulo (USP)instacron:USPenghttps://www.revistas.usp.br/jaos/article/view/230164/208834Copyright (c) 2024 Jacob Kirby, Makayla Standfest, Jessica Binkley, Charles Barnes, Evan Brown, Tyler Cairncross, Alex Cartwright, Danielle Dadisman, Colten Mowat, Daniel Wilmot, Theodore Houseman, Conner Murphy, Caleb Engelsman, Josh Haller, Daniel Joneshttps://creativecommons.org/licenses/by/4.0info:eu-repo/semantics/openAccessKirby, JacobStandfest, MakaylaBinkley, JessicaBarnes, CharlesBrown, EvanCairncross, TylerCartwright, AlexDadisman, DanielleMowat, ColtenWilmot, DanielHouseman, TheodoreMurphy, ConnerEngelsman, CalebHaller, JoshJones, Daniel2025-01-14T14:27:38Zoai:revistas.usp.br:article/230164Revistahttp://www.scielo.br/jaosPUBhttps://www.revistas.usp.br/jaos/oai||jaos@usp.br1678-77651678-7757opendoar:2025-01-14T14:27:38Journal of applied oral science (Online) - Universidade de São Paulo (USP)false
dc.title.none.fl_str_mv The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification
title The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification
spellingShingle The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification
Kirby, Jacob
Bisphosphonate-associated osteonecrosis of the jaw
Dynamins
Endosomes
Fibroblasts
Zoledronic acid
title_short The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification
title_full The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification
title_fullStr The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification
title_full_unstemmed The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification
title_sort The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification
dc.creator.none.fl_str_mv Kirby, Jacob
Standfest, Makayla
Binkley, Jessica
Barnes, Charles
Brown, Evan
Cairncross, Tyler
Cartwright, Alex
Dadisman, Danielle
Mowat, Colten
Wilmot, Daniel
Houseman, Theodore
Murphy, Conner
Engelsman, Caleb
Haller, Josh
Jones, Daniel
author Kirby, Jacob
author_facet Kirby, Jacob
Standfest, Makayla
Binkley, Jessica
Barnes, Charles
Brown, Evan
Cairncross, Tyler
Cartwright, Alex
Dadisman, Danielle
Mowat, Colten
Wilmot, Daniel
Houseman, Theodore
Murphy, Conner
Engelsman, Caleb
Haller, Josh
Jones, Daniel
author_role author
author2 Standfest, Makayla
Binkley, Jessica
Barnes, Charles
Brown, Evan
Cairncross, Tyler
Cartwright, Alex
Dadisman, Danielle
Mowat, Colten
Wilmot, Daniel
Houseman, Theodore
Murphy, Conner
Engelsman, Caleb
Haller, Josh
Jones, Daniel
author2_role author
author
author
author
author
author
author
author
author
author
author
author
author
author
dc.subject.por.fl_str_mv Bisphosphonate-associated osteonecrosis of the jaw
Dynamins
Endosomes
Fibroblasts
Zoledronic acid
topic Bisphosphonate-associated osteonecrosis of the jaw
Dynamins
Endosomes
Fibroblasts
Zoledronic acid
description Objective: For treatment of medication-related osteonecrosis of the jaw, one proposed approach is the use of a topical agent to block entry of these medications in oral soft tissues. We tested the ability of phosphonoformic acid (PFA), an inhibitor of bisphosphonate entry through certain sodium-dependent phosphate contransporters (SLC20A1, 20A2, 34A1-3) as well as Dynasore, a macropinocytosis inhibitor, for their abilities to prevent zoledronate-induced (ZOL) death in human gingival fibroblasts (HGFs). Methodology: MTT assay dose-response curves were performed to determine non-cytotoxic levels of both PFA and Dynasore. In the presence of 50 μM ZOL, optimized PFA and Dynasore doses were tested for their ability to restore HGF viability. To determine SLC expression in HGFs, total HGF RNA was subjected to quantitative real-time RT-PCR. Confocal fluorescence microscopy was employed to see if Dynasore inhibited macropinocytotic HGF entry of AF647-ZOL. Endosomal acidification in the presence of Dynasore was measured by live cell imaging utilizing LysoSensor Green DND-189. As a further test of Dynasore’s ability to interfere with ZOL-containing endosomal maturation, perinuclear localization of mature endosomes containing AF647-ZOL or TRITC-dextran as a control were assessed via confocal fluorescence microscopy with CellProfiler™ software analysis of the resulting photomicrographs. Results: 0.5 mM PFA did not rescue  HGFs from ZOL-induced viability loss at 72 hours while 10 and 30 μM geranylgeraniol did partially rescue. HGFs did not express the SLC transporters as compared to the expression in positive control tissues. 10 μM Dynasore completely prevented ZOL-induced viability loss. In the presence of Dynasore, AF647-ZOL and FITC-dextran co-localized in endosomes. Endosomal acidification was inhibited by Dynasore and perinuclear localization of both TRITC-dextran- and AF647-ZOL-containing endosomes was inhibited by 30 μM Dynasore. Conclusion: Dynasore prevents ZOL-induced viability loss in HGFs by partially interfering with macropinocytosis and by inhibiting the endosomal maturation pathway thought to be needed for ZOL delivery to the cytoplasm.
publishDate 2024
dc.date.none.fl_str_mv 2024-10-01
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv https://www.revistas.usp.br/jaos/article/view/230164
10.1590/1678-7757-2024-0224
url https://www.revistas.usp.br/jaos/article/view/230164
identifier_str_mv 10.1590/1678-7757-2024-0224
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv https://www.revistas.usp.br/jaos/article/view/230164/208834
dc.rights.driver.fl_str_mv https://creativecommons.org/licenses/by/4.0
info:eu-repo/semantics/openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/4.0
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Universidade de São Paulo. Faculdade de Odontologia de Bauru
publisher.none.fl_str_mv Universidade de São Paulo. Faculdade de Odontologia de Bauru
dc.source.none.fl_str_mv Journal of Applied Oral Science; Vol. 32 (2024); e20240224
Journal of Applied Oral Science; v. 32 (2024); e20240224
Journal of Applied Oral Science; Vol. 32 (2024); e20240224
1678-7765
1678-7757
reponame:Journal of applied oral science (Online)
instname:Universidade de São Paulo (USP)
instacron:USP
instname_str Universidade de São Paulo (USP)
instacron_str USP
institution USP
reponame_str Journal of applied oral science (Online)
collection Journal of applied oral science (Online)
repository.name.fl_str_mv Journal of applied oral science (Online) - Universidade de São Paulo (USP)
repository.mail.fl_str_mv ||jaos@usp.br
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