The role of intracellular Ca²⁺ in apoptosis induced by hyperthermia and its enhancement by verapamil in U937 cells

Purpose: The relationship between apoptosis induced by 42°C and 44°C hyperthermia alone or in combination with verapamil and changes in intracellular Ca²⁺ concentration ([Ca²⁺]i) was investigated in U937 cells. Methods: Apoptosis induced by hyperthermia was assessed according to DNA fragmentation, nuclear morphologic changes, and expression of phosphatidylserine on the outside plasma cell membrane. These changes were measured by flow cytometry. The [Ca²⁺]i of individual cells after hyperthermia was monitored by a digital image-analyzing technique using Fura-2. Results: Hyperthermia-induced apoptosis reached a plateau after 6 h and was found to be both time and temperature-dependent. DNA fragmentation was maximum at 44°C after 30 min. Verapamil enhanced the apoptosis induced by 42°C and 44°C hyperthermia in normal cells and by 44°C hyperthermia in thermotolerant cells. The number of cells containing higher [Ca²⁺]i (more than 200 nM) was significantly increased by hyperthermia and further elevated by the addition of verapamil in both normal and thermotolerant cells. Apoptosis induced by hyperthermia was markedly decreased by an intracellular Ca²⁺ chelator, BAPTA-AM, in a dose-dependent manner. Conclusion: These results indicate that [Ca²⁺]i increase plays a crucial role in apoptosis induced by hyperthermia and the combined treatment with verapamil in normal and thermotolerant U937 cells. Furthermore, hyperthermia-combined drug therapy has potential significance in cancer therapy.