Enhancement of hyperthermia-induced apoptosis by a free radical initiator,2,2 '-azobis (2-amidinopropane) dihydrochloride, in human histiocytic lymphoma U937 cells

To elucidate the mechanism how a free radical initiator, 2,2'-azobis (2-ami dinopropane) dihydrochloride (AAPH), induces cell death at hyperthermic tem peratures, apoptosis in a human histiocytic lymphoma cell line, U937, was i nvestigated. Free radical formation deriving from the thermal decomposition of AAPH was examined by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxid e (DMPO). An assay for DNA fragmentation, observation of nuclear morphologi cal changes, and flow cytometry for phosphatidylserine (PS) externalization were used to detect apoptosis and revealed enhancement of 44.0 degreesC hy perthermia-induced apoptosis by free radicals due to AAPH. However, free ra dicals alone derived from AAPH did not induce apoptosis. Hyperthermia induc ed the production of lipid peroxidation (LPO), an increase in intracellular Ca2+ concentration ([Ca2+](i)) and enhanced expression. of the type 1 inos itol 1,4,5-trisphosphate receptor (IP(3)R1). The effects of hyperthermia on LPO and [Ca2+](i) were enhanced markedly by the combination with AAPH. A s ignificant decrease in Bcl-2 expression, increase in Bax expression, a loss of mitochondrial membrane potential (Delta Psim) and a marked increase in cytochrome c expression were found only in cells treated with hyperthermia and AAPH. Although an intracellular Ca2+ ion chelator, BAPTA-AM, completely inhibited DNA fragmentation, water-soluble vitamine E, Trolox, only partia lly suppressed DNA fragmentation and the increase in [Ca2+](i). In contrast , LPO was inhibited completely by Trolox, but no inhibition by BAPTA-AM was found. These results suggest that apoptosis induced by hyperthermia alone is due to the increase in [Ca2+](i) arising from increased expression Of IP (3)R1 and LPO. Additional increase in [Ca2+](i) due to increased LPO and th e activation of mitochondria-caspase dependent pathway play a major role in the enhancement of apoptosis by the combination with hyperthermia and AAPH .