プリロカインによる培養骨肉腫細胞のアポトーシス誘導

Abstract

Local anesthetics cause cytotoxicity to various types of mammalian cells. To determine whether local anesthetics can induce apoptosis in osteoblastic cells, the effects of prilocaine were examined on cultured human osteoblastic cell lines, Saos-2 and MG63 cells, and mouse MC3T3-E1 cells. The cells in confluence were exposed to varying concentrations of prilocaine. In Saos-2 cells, prilocaine induced cell damage in a dose- and time-dependent manners up to the concentration of 10mM as determined by phase-contrast microscopy and WST-1 assay. Marked nuclear condensation and fragmentation of chromatin were observed in the prilocaine-treated cells by using Hoechst 33342 staining. Prilocaine-induced DNA ladder formation in agarose gel electrophoresis was dose-dependent with maximal effect at a concentration of 5mM and was time-dependent from 12 h to 48 h. Prilocaine induced also DNA ladder formation in both human osteoblastic MG63 cells and mouse osteoblastic MC3T3-E1 cells. Other local anesthetics, including dibcaine, tetracaine, lidocaine, and procaine, also induced DNA ladder formation in Saos-2 cells. Cycloheximide and Actinomycin D prevented prilocaine-induced apoptosis in Saos-2 cells in a dose-dependent fashion up to 10μM and 40nM, respectively, as determined by WST-1 assay and DNA ladder formation. The present results showed that prilocaine and other local anesthetics caused both morphological and/or biochemical features indicative of apoptosis in osteoblastic cells, and that the synthesis of specific proteins would be involved in prilocaine-induced apoptosis.