To explore combined antiglioma effect of
nitric oxide (NO) and
hyperthermia, the rat C6 and human U251
glioma cells were exposed to NO-releasing agents
sodium nitroprusside(SNP),
S-nitrosoglutathione or
PAPA-NONOate, followed by
hyperthermia (1 h, 43 degrees C). While each treatment alone showed only moderate efficiency, a synergistic cytotoxicity of NO donors and
hyperthermia was clearly demonstrated by
crystal violet and MTT cytotoxicity assays. The flow cytometric analysis with the appropriate reporter
fluorochromes confirmed that
hyperthermia and SNP cooperated in inducing oxidative stress, mitochondrial depolarization,
caspase activation and DNA fragmentation, leading to both necrotic and
caspase-dependent apoptotic cell death. The
acridine orange staining of intracellular acidic compartments revealed that SNP completely blocked
hyperthermia-induced autophagy, while the inhibition of autophagy by 3-methyl
adenine mimicked SNP-triggered oxidative stress,
caspase activation and cell death in
hyperthermia-exposed cells. Therefore, the synergistic cytotoxicity of SNP and
hyperthermia could result from NO-mediated suppression of protective autophagic response in
glioma cells.