Abstract
Purpose
To determine whether prilocaine, a local anesthetic, induces apoptosis in osteoblastic cells.
Methods
After reaching subconfluence, human osteoblastic Saos-2 and MG63 cells and mouse osteoblastic MC3T3-E1 cells were exposed for 48 hr to varying concentrations of prilocaine up to 10 mM and the cytotoxicity of the cells was analyzed by phase-contrast microscopy and WST-1 assay. Saos-2 cells treated for 48 hr with 5 mM prilocaine were stained with Hoechst 33342 and nuclear fragmentation was examined under a fluorescence microscope. DNA was extracted from the cells treated with 5 mM prilocaine and DNA ladder formation (a hallmark of apoptosis) was analyzed by agarose gel electrophoresis.
Result
Prilocaine induced cell death in Saos-2 cells in a dose- and time-dependent manner up to the concentration of 10 mM. Marked nuclear condensation and fragmentation of chromatin were observed in the prilocainetreated cells. DNA ladder formation also was induced by prilocaine treatment. Prilocaine-induced DNA ladder formation was dose-dependent with maximal effect at a concentration of 5 mM and was time-dependent from 12 to 48 hr. DNA ladder formation was also induced by prilocaine treatment in human osteoblastic MG63 cells and mouse osteoblastic MC3T3-E1 cells. Cycloheximide prevented prilocaine-induced apoptosis in Saos-2 cells in a dose-dependent fashion up to 20 μM as determined by WST-1 assay and DNA ladder formation in agarose gel electrophoresis.
Conclusion
Osteoblastic cells treated with prilocaine exhibit both morphological and biochemical features indicative of apoptosis. The apoptotic mechanisms involve transcriptional regulation of specific proteins or protein synthesis.
Résumé
Objectif
Déterminer si la prilocaïne, un anesthésique local, a induit l’apoptose de cellules ostéoblastiques.
Méthode
Après avoir atteint le stade de sous-confluence, des cellules ostéoblastiques humaines Saos-2 et MG63 et ostéoblastiques de souris MC3T3-E1 ont été exposées pendant 48 h à des concentrations variables de prilocaïne allant jusqu’à 10 mM et la cytotoxicité des cellules a été analysée par microscopie en contraste de phase et par dosage WST-1. Les cellules Saos-2 traitées pendant 48 h avec 5 mM de prilocaïne ont été colorées avec le Hoechst 33342 et la fragmentation nucléaire a été examinée sous microscopie en fluorescence. L’ADN a été extrait et la formation d’échelle d’ADN (signe cardinal de l’apoptose) a été analysée par électrophorèse sur gel d’agarose.
Résultats
La prilocaïne a induit la mort des cellules Saos-2 d’une manière dépendante de la dose et du temps jusqu’à une concentration de 10 mM. En effet, la prilocaïne a induit dans les cellules traitées une condensation nucléaire marquée et la fragmentation de chromatine de même que la formation d’échelle d’ADN. La formation d’ADN, dépendante de la dose, a connu son effet maximal avec une concentration de 5 mM et était dépendante du temps entre 12 et 48 h. La formation d’ADN a été aussi induite par la prilocaïne dans les cellules humaines MG63 et dans les cellules de souris MC3T3-E1. La cycloheximide a empêché l’apoptose induite par la prilocaïne de se produire dans les cellules Saos-2 d’une manière dépendante de la dose pour une concentration jusqu’à 20 μM comme l’ont déterminé le dosage WST-1 et la formation d’échelle d’ADN dans électrophorèse sur gel d’agarose.
Conclusion
Les cellules ostéoblastiques traitées avec de la prilocaïne ont présenté des caractéristiques morphologiques et biochimiques indicatrices d’apoptose. Les mécanismes de l’apoptose impliquent la régulation de la transcription de protéines spécifiques ou de synthèse protéique.
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This study was supported in part by grants from the Grant in Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and the grant-in-aid for cancer research from the Fukuoka Cancer Society, Fukuoka, Japan.
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Nakamura, K., Kido, H., Morimoto, Y. et al. Prilocaine induces apoptosis in osteoblastic cells. Can J Anesth 46, 476–482 (1999). https://doi.org/10.1007/BF03012949
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DOI: https://doi.org/10.1007/BF03012949