Purpose: The aim of the study was to investigate the molecular mechanisms involved in apoptosis of human promyelocytic cells (HL60) induced by hyperthermia and to compare this to radiation-induced apoptosis as a reference model.
Materials and methods: Apoptosis of HL60 cells was induced by heat-treatment (43°C during 1h) or by γ-radiation (8Gy) and followed at increasing time periods after treatment with Annexin V binding to phosphatidylserine (PS). The transition of the mitochondrial membrane potential (ΔΨm) was estimated by the extent of mitochondrial JC-1 uptake. Bcl-2 and Bax protein expression levels were monitored using fluorescent-labelled antibodies. Caspase activation was studied using a fluorochrome-labelled pan-caspase inhibitor (FLICA), which also allowed one to study the kinetics of the apoptotic cascade.
Results: After heat-treatment or irradiation of HL60 cells, a decreased ΔΨm as well as PS membrane expression were detectable after 8h. Bcl-2 and Bax protein expression levels were decreased and increased, respectively, 1 h after heat-treatment or irradiation. The apoptotic rate of HL60 cells, as measured by the FLICA binding, was faster with heat-treatment as compared to γ-irradiation. Addition of a pan-caspase inhibitor prevented PS externalization after heat-treatment but not after irradiation. The presence of a pan-caspase inhibitor did not influence the decrease of ΔΨm both after heat-treatment and γ-irradiation. However, the addition of the specific caspase-2 inhibitor zVDVAD-fmk prevented the mitochondrial breakdown after heat-treatment. Inhibition of caspase-2 had no effect on the γ-irradiation induced apoptosis.
Conclusion: These results suggest that the commitment to apoptosis in HL60 cells after heat-treatment is started by mitochondrial membrane transition involving the Bcl-2 family members and is mainly executed in a caspase-dependent pathway. The results suggest that caspase-2 plays a key role in the heat-induced apoptosis.
- Mitochondrial function