Prilocaine induces apoptosis in osteoblastic cells

Purpose: To determine whether prilocaine, a local anesthetic, induces apopt osis in osteoblastic cells. Methods: After reaching subconfluence, human osteoblastic Saos-2 and MG63 c ells and mouse osteoblastic MC3T3-EI cells were exposed for 48 hr to varyin g concentration; of prilocaine up to 10 mM and the cytotoxicity of the cell s was analyzed by phase-contrast microscopy and WST-I assay. Saos-2 cells t reated for 48 hr with 5 mM prilocaine were stained with Hoechst 33342 and n uclear fragmentation was examined under a fluorescence microscope. DNA was extracted from the cells treated with 5 mM prilocaine and DNA ladder format ion (a hallmark of apoptosis) was analyzed by agarose gel electrophoresis. Result: Prilocaine induced-cell death in Saos-2 cells in a dose- and time-d ependent manner up to the concentration of 10 mM. Marked nuclear condensati on and fragmentation of chromatin were observed in the prilocaine treated c ells. DNA ladder formation also was induced by prilocaine treatment. Priloc aine 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 ladde r formation was also induced by prilocaine treatment in human osteoblastic MG63 cells and mouse osteoblastic MC3T3-EI cells. Cycloheximide prevented p rilocaine-induced apoptosis in Saos-2 cells in a dose-dependent fashion up to 20 mu M as determined by WST-I assay and DNA ladder formation in agarose gel electrophoresis. Conclusion: Osteoblastic cells treated with prilocaine exhibit both morphol ogical and biochemical features indicative of apoptosis. The apoptotic mech anisms involve transcriptional regulation of specific proteins or protein s ynthesis.