The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification
DOI:
https://doi.org/10.1590/1678-7757-2024-0224Keywords:
Bisphosphonate-associated osteonecrosis of the jaw, Dynamins, Endosomes, Fibroblasts, Zoledronic acidAbstract
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.
Downloads
References
Boquete-Castro A, Gómez-Mereno G, Calvo-Guirado JL, Aguilar-Salvatierra A, Delgado-Ruiz RA. Denosumab and osteonecrosis of the jaw: a systematic analysis of events reported in clinical trials. Clin Oral Implants Res. 2016;27(3):367-75. doi: 10.1111/clr.12556
Otto S, Pautke C, Van den Wyngaert T, Niepel D, Schiødt M. Medication-related osteonecrosis of the jaw: Prevention, diagnosis and management in patients with cancer and bone metastases. Cancer Treat Rev. 2018;69:177-87. doi: 10.1016/j.ctrv.2018.06.007
Bamias A, Kastritis E, Bamia C, Moulopoulos LA, Melakopoulos I, Bozas G, et al. Osteonecrosis of the jaw in cancer after treatment with bisphosphonates: incidence and risk factors. J Clin Oncol. 2005;23(34):8580-7. doi: 10.1200/JCO.2005.02.8670
Dimopoulos MA, Kastritis E, Anagnostopoulos A, Melakopoulos I, Gika D, Moulopoulos LA. Osteonecrosis of the jaw in patients with multiple myeloma treated with bisphosphonates: evidence of increased risk after treatment with zoledronic acid. Haematologica. 2006;91(7):968-71.
Cafro AM, Barbarano L, Nosari AM, D'Avanzo G, Nichelatti M, Bibas M, et al. Osteonecrosis of the jaw in patients with multiple myeloma treated with bisphosphonates: definition and management of the risk related to zoledronic acid. Clin Lymphoma Myeloma. 2008;8(2):111-6. doi: 10.3816/clm.2008.n.013
Smith A, Kressley A, Saif MW. Oral osteonecrosis associated with the use of zoledronic acid: First case of a patient with advanced pancreatic cancer and bone metastases. JOP. 2009;10(2):212-4.
Woo SB, Hellstein JW, Kalmar JR. Systematic review: bisphosphonates and osteonecrosis of the jaws. Ann Intern Med. 2006;144(10):753-61. doi: 10.7326/0003-4819-144-10-200605160-00009
Acil Y, Moller B, Niehoff P, Rachko K, Gassling V, Wiltfang J, et al. The cytotoxic effects of three different bisphosphonates in-vitro on human gingival fibroblasts, osteoblasts and osteogenic sarcoma cells. J Craniomaxillofac Surg. 2012;40(8):e229-e235. doi: 10.1016/j.jcms.2011.10.024
Scheper MA, Badros A, Chaisuparat R, Cullen KJ, Meiller TF. Effect of zoledronic acid on oral fibroblasts and epithelial cells: a potential mechanism of bisphosphonate-associated osteonecrosis. Br J Haematol. 2009;144(5):667-76. doi: 10.1111/j.1365-2141.2008.07504.x
Yuan A, Munz A, Reinert S, Hoefert S. Gingival fibroblasts and medication-related osteonecrosis of the jaw: results by real-time and wound healing in vitro assays. J Craniomaxillofac Surg. 2019;47(9):1464-74. doi: 10.1016/j.jcms.2019.06.004
Kim RH, Lee RS, Williams D, Bae S, Woo J, Lieberman M, et al. Bisphosphonates induce senescence in normal human oral keratinocytes. J Dent Res. 2011;90(6):810-6. doi: 10.1177/0022034511402995
Pabst AM, Krüger M, Ziebart T, Jacobs C, Sagheb K, Walter C. The influence of geranylgeraniol on human oral keratinocytes after bisphosphonate treatment: an in vitro study. J Craniomaxillofac Surg. 2015;43(5):688-95. doi: 10.1016/j.jcms.2015.03.014
Rattanawonsakul K, Bullock G, Bolt R, Claeyssens F, Atkins S, Hearnden S. In vitro effect of geranylgeraniol (GGOH) on bisphosphonate-induced cytotoxicity of oral mucosa cells. Front Oral Health. 2022;3:892615. doi:10.3389/froh.2022.892615
Ziebart T, Koch F, Klein MO, Guth J, Adler J, Pabst A, et al. Geranylgeraniol: a new potential therapeutic approach to bisphosphonate associated osteonecrosis of the jaw. Oral Oncol. 2011;47(3):195-201. doi: 10.1016.j.oraloncology.2010.12.003
Zafar S, Coates DE, Cullinan MP, Drummond BK, Milne T, Seymour GJ. Zoledronic acid and geranylgeraniol regulate cellular behaviour and angiogenic gene expression in human gingival fibroblasts. J Oral Pathol Med. 2014;43(9):711-21. doi: 10.1111/jop.12181
Kiyama T, Tsuchiya M, Okada S, Oizuma T, Yamaguchi K, Sasaki K, et al. Phosphonocarboxylates can protect mice against the inflammatory and necrotic side effects of nitrogen-containing bisphosphonates by inhibiting their entry into cells via phosphate transporters. Biol Pharm Bull. 2016;39(5):712-20. doi: 10.1248/bpb.b15-00770
Baba TT, Miyazaki T, Ohara-Nemoto Y, Nemoto TK. Suppressive effects of N-bisphosphonate in osteoblastic cells mitigated by non-N-bisphosphonate but not by sodium-dependent phosphate cotransporter inhibitor. Cell Biochem Funct. 2019;37(6):400-7. doi: 10.1002/cbf.3418
Ip YK, Boo MV, Poo JS, Wong WP, Chew SF. Sodium-dependent phosphate transporter protein 1 is involved in the active uptake of inorganic phosphate in nephrocytes of the kidney and the translocation of P i into the tubular epithelial cells in the outer mantle of the giant clam, Tridacna squamosa. Front Mar Sci. 2021;8:655714. doi: 10.3389/fmars.2021.655714
Bottino P, Pastrone L, Curtoni A, Bondi A, Sidoti F, Zanotto E, et al. Antiviral approach to cytomegalovirus infection: an overview of conventional and novel strategies. Microorganisms. 2023;11(10):2372. doi: 10.3390/microorganisms11102372
Zlatev HP, Auriola S, Mönkkönen J, M??tt? J. Uptake of free, calcium-bound and liposomal encapsulated nitrogen containing bisphosphonates by breast cancer cells. Eur J Pharm Sci. 2016;86:58-66. doi: 10.1016/j.ejps.2016.02.016
Glogauer M, Lee W, McCulloch CA. Induced endocytosis in human fibroblasts by electrical fields. Exp Cell Res. 1993;208(1):232-40. doi: 10.1006/excr.1993.1242
Verhulst A, Sun S, McKenna CE, D'Haese PC. Endocytotic uptake of zoledronic acid by tubular cells may explain its renal effects in cancer patients receiving high doses of the compound. PLoS One. 2015;10(3): e0121861. doi: 10.1371/journal.pone.0121861
Lin HP, Singla B, Ghoshal P, Faulkner JL, Cherian-Shaw M, O'Conner PM, et al. Identification of novel macropinocytosis inhibitors using a rational screen of Food and Drug Administration-approved drugs. Br J Pharmacol. 2018;175(18):3640-55. doi: 10.1111/bph.14429
Preta G, Cronin JG, Sheldon IM. Dynasore: not just a dynamin inhibitor. Cell Commun Signal. 2015;13:24. doi: 10.1186/s12964-015-0102-1
Chaumet A, Wright GD, Seet SH, Tham KM, Gounko NV, Bard F. Nuclear envelope-associated endosomes deliver surface proteins to the nucleus. Nat Commun. 2015;6:8218. doi: 10.1038/ncomms9218
Mesaki K, Tenabe K, Obayashi M, Oe N, Takei K. Fission of tubular endosomes triggers endosomal acidification and movement. PLoS One 2011;6(5):e19764. doi: 10.1371/journal.prone.0019764
Thompson K, Rogers MJ, Coxon FP, Crockett JC. Cytosolic entry of bisphosphonate drugs require acidification of vesicles after fluid-phase endocytosis. Mol Pharmacol. 2006;69(5):1624-32. doi: 10.1124/mol.105.020776
Leemans B, Stout TA, Van Soom A, Gadella BM. pH-dependent effects of procaine on equine gamete activation. Biol Reprod. 2019;101(5):1056-74. doi: 10.1093/biolre/ioz131
Das S, Edwards PA, Crockett JC, Rogers MJ. Upregulation of endogenous farnesyl diphosphate synthase overcomes the inhibitory effect of bisphosphonate on protein prenylation in Hela cells. Biochim Biophys Acta. 2014;1841(4):569-73. doi: 10.1016/j.bbalip.2013.12.010
Thul PJ, Åkesson L, Wiking M, Mahdessian D, Geladaki A, Blal HA, et al. A subcellular map of the human proteome. Science. 2017;356(6340):eaaI3321. doi: 10.1126/science.aaI3321
Brock DJ, Kondow-McConaghy HM, Hager EC, Pellois JP. Endosomal escape and cytosolic penetration of macromolecules mediated by synthetic delivery agents. Bioconjug Chem. 2019;30(2):293-304. doi: 10.1021/acs.bioconjugchem.8b00799
Au KM, Satterlee A, Min Y, Tian X, Kim YS, Caster JM, et al. Folate-targeted pH-responsive calcium zoledronate nanoscale metal-organic frameworks: turning a bone antiresorptive agent into an anticancer therapeutic. Biomaterials. 2016;82:178-93. doi: 10/1016/j.biomaterials.2015.12.018
Barriere H, Apaja P, Okiyonida T, Lukacs GL. Endocytic sorting of CFTR variants monitored by single cell fluorescence ratio image analysis (FRIA) in living cells. Methods Mol Biol. 2011;741:301-17. doi: 10.1007/978-1-61779-117-8_20
Okada S, Kiyama T, Sato E, Yukinori T, Oizumi T, Kuroishi T, et al. Inhibition of phosphate transporters ameliorates the inflammatory and necrotic side effects of the nitrogen-containing bisphosphonate zoledronate in mice. Tohoku J Exp Med. 2013;231(2):145-58. doi: 10.1620/tjem.231.145
Virkki LV, Biber J, Murer H, Forster IC. Phosphate transporters: a tale of two solute carrier families. Am J Physiol Renal Physiol. 2007;293(3):F643-54. doi: 10.1152/ajprenal.00228.2007
Crumpacker CS. Mechanism of action of foscarnet against viral polymerases. Am J Med. 1992;92(2A):3S-7S. doi: 10.1016/0002-9343(92)90329-a
Sapkota D, Wang D, Schreurs O, Vallenari EM, Dhakal SP, Küntziger T, et al. Investigation of roles of SLC38A1 in proliferation and differentiation of mouse tongue epithelium and expression in human oral tongue squamous cell carcinoma. Cancers (Basel). 2024;16(2):405. doi: 10.3390/cancers16020405
Kirchhausen T, Macia E, Pelish HE. Use of dynasore, the small molecule inhibitor of dynamin, in the regulation of endocytosis. Methods Enzymol. 2008;438:77-93. doi: 10.1016/S0076-6879(07)38006-3
McCluskey A, Daniel JA, Hadzic G, Chau N, Clayton EL, Mariana A, et al. Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis. Traffic. 2013;14(12):1272-89. doi: 10.1111/tra.12119
Prabhakara C, Godbole R, Sil P, Jahnavi S, Gulzar SE, van Zanten TS, et al. Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors. PLoS Pathog. 2021;17(7):e1009706. doi: 10.1371/journal.ppat.1009706
Downloads
Published
Versions
- 2024-10-01 (2)
- 2024-09-30 (1)
Issue
Section
License
Copyright (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 Jones
This work is licensed under a Creative Commons Attribution 4.0 International License.
Todo o conteúdo do periódico, exceto onde está identificado, está licenciado sob uma Licença Creative Commons do tipo atribuição CC-BY.
