Biochemical and microarray analyses of bupivacaine-induced apoptosis.
The mechanism by which apoptosis is induced by local anesthetic bupivacaine, a potent uncoupler of mitochondrial oxidative phosphorylation, was investigated. In promyelocytic leukemia cells HL-60, bupivacaine induced formation of apoptotic bodies and DNA fragmentation in a time- and dose-dependent manner similar to typical apoptosis inducers. Caspase-3, -8 and -9, which play a pivotal role in the initiation and execution of receptor- or mitochondria-mediated apoptosis, were all clearly activated by bupivacaine in good correlation with the degree of DNA fragmentation. However, bupivacaine did not induce either mitochondrial permeability transition (PT) or release of cytochrome c in experiments with isolated mitochondria. These results suggest that an indirect action of bupivacaine on mitochondria occurs and that other mechanisms may be involved in bupivacaine-induced apoptosis. To obtain additional information concerning the mechanism of action involved in bupivacaine-induced apoptosis, a microarray analysis of gene expression in bupivacaine-treated HL-60 cells was carried out. Several apoptosis-related genes were found to be transcriptionally regulated by bupivacaine using a high-density cDNA microarray. The expression levels of heat shock protein 70 (HSP70), c-jun and c-fos genes were remarkably up-regulated and those of c-myc and poly (ADP ribose) polymerase (PARP) were down-regulated in bupivacaine-treated cells. These results are of value in developing a better understanding of the molecular mechanism of bupivacaine-induced apoptosis leading to neuro- or myotoxicity.
Unami
Akira
A
Faculty of Pharmaceutical Sciences, University of Tokushima, 1 Shomachi, Tokushima 770-8505, Japan.
Shinohara
Yasuo
Y
Ichikawa
Tomokazu
T
Baba
Yoshinobu
Y
D000779:Anesthetics, Local / Q000633:toxicity*; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002045:Bupivacaine / Q000633:toxicity*; D002166:Camptothecin / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D003574:Cytochrome c Group / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D053938:DNA Fragmentation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D017403:In Situ Hybridization; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism; D008933:Mitochondrial Swelling / Q000187:drug effects; D009474:Neurons / Q000187:drug effects; D020411:Oligonucleotide Array Sequence Analysis; D010101:Oxygen Consumption / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000779:Anesthetics, Local; D000972:Antineoplastic Agents, Phytogenic; D003574:Cytochrome c Group; D012333:RNA, Messenger; D004247:DNA; D020169:Caspases; D002166:Camptothecin; D002045:Bupivacaine