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...
| Autores: | ; ; ; ; ; ; ; ; ; ; ; ; ; ; |
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| Tipo de documento: | Artículo |
| Estado: | Published version |
| Fecha de publicación: | 2024 |
| País: | Brasil |
| Institución: | USP |
| Repositorio: | Journal of applied oral science (Online) |
| Lenguaje: | Inglés |
| OAI Identifier: | oai:revistas.usp.br:article/230164 |
| Acceso en línea: | https://www.revistas.usp.br/jaos/article/view/230164 |
| Nivel de acceso: | Acceso abierto |
| Palabra clave: | Bisphosphonate-associated osteonecrosis of the jaw Dynamins Endosomes Fibroblasts Zoledronic acid |
| _version_ | 1878752874060578816 |
|---|---|
| author | Kirby, Jacob |
| 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 |
| author_browse | Barnes, Charles Binkley, Jessica Brown, Evan Cairncross, Tyler Cartwright, Alex Dadisman, Danielle Engelsman, Caleb Haller, Josh Houseman, Theodore Jones, Daniel Kirby, Jacob Mowat, Colten Murphy, Conner Standfest, Makayla Wilmot, Daniel |
| 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 |
| collection | Journal of applied oral science (Online) |
| 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. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | BR_2eaffc607cdce9a585b883b7eb366deb |
| institution | USP |
| instname_str | Universidade de São Paulo (USP) |
| language | eng |
| network_acronym_str | BR |
| network_name_str | Brasil |
| oai_identifier_str | oai:revistas.usp.br:article/230164 |
| publishDate | 2024 |
| publishDateSort | 2024 |
| publisher | Universidade de São Paulo. Faculdade de Odontologia de Bauru |
| publisherStr | Universidade de São Paulo. Faculdade de Odontologia de Bauru |
| reponame_str | Journal of applied oral science (Online) |
| spelling | The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification 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 Bisphosphonate-associated osteonecrosis of the jaw Dynamins Endosomes Fibroblasts Zoledronic acid 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. Universidade de São Paulo. Faculdade de Odontologia de Bauru https://www.revistas.usp.br/jaos/article/view/230164 10.1590/1678-7757-2024-0224 |
| 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 |
| status_str | publishedVersion |
| title | 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_short | 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 |
| topic | Bisphosphonate-associated osteonecrosis of the jaw Dynamins Endosomes Fibroblasts Zoledronic acid |
| topic_facet | Bisphosphonate-associated osteonecrosis of the jaw Dynamins Endosomes Fibroblasts Zoledronic acid |
| url | https://www.revistas.usp.br/jaos/article/view/230164 |