Enhancement of hyperthermia-induced apoptosis by local anesthetics on human histiocytic lymphoma U937 cells

The combined effects of hyperthermia at 44°C and local anesthetics on apoptosis in human histiocytic lymphoma U937 cells were investigated. When the cells were exposed to hyperthermia for 10 min marginal DNA fragmentation and nuclear fragmentation were observed. In the presence of amide-type local anesthetics further enhancement was found depending on concentration. The order of the concentration required for maximum induction was the reverse order of the lipophilicity (prilocaine > lidocaine > bupivacaine). Western blotting revealed that in hyperthermia there was initial release of Ca²⁺ from the intracellular store site as indicated by increased expression of the type 1 inositol-1,4,5-trisphosphate receptor. However, the combination with lidocaine did not induce any further enhancement. Lidocaine enhanced the decrease in ATP content and the increase in intracellular Ca²⁺ concentration in individual cells induced by hyperthermia. In addition, superoxide formation, decrease in the mitochondrial membrane potential, and activation of intracellular caspase-3 were found in the cells treated with hyperthermia and lidocaine. All of these were suppressed in part in the presence of the intracellular Ca²⁺ ion chelator BAPTA-AM (bis-(O-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl). The present results indicate that local anesthetics at optimal concentrations enhance hyperthermia-induced apoptosis via Ca²⁺- and mitochondria-dependent pathways. Initial release of Ca²⁺ from intracellular store sites caused by hyperthermia and followed by the subsequent increase in the intracellular Ca²⁺ concentration and the additional activation of the mitochondrial caspase-dependent path-way (partly regulated by intracellular Ca²⁺ concentration) plays a crucial role in the enhancement of apoptosis induced by the combination of hyperthermia and lidocaine.