Mild thermotolerance induced at 40 °C protects cells against hyperthermia-induced pro-apoptotic changes in Bcl-2 family proteins

Abstract

Purpose: Despite clinical progress, mechanisms involved in cellular responses to low and high doses of hyperthermia are not entirely clear. This study investigates the role of Bcl-2 family proteins in control of the mitochondrial pathway of apoptosis during hyperthermia at 42–43 °C and the protective effect of a low dose adaptive survival response, mild thermotolerance induced at 40 °C.Materials and methods: Levels of Bcl-2 family proteins were detected in HeLa cells by western blotting, caspase activation by spectrofluorimetry and apoptosis by chromatin condensation.Results: Hyperthermia (42–43 °C) decreased total and mitochondrial expression of anti-apoptotic proteins Bcl-2 and Bcl-xL, while expression of pro-apoptotic proteins Bax, Bak, Puma and Noxa increased. Hyperthermia perturbed the equilibrium between these anti- and pro-apoptotic Bcl-2 family proteins in favour of pro-apoptotic conditions. Hyperthermia also caused activation of caspases-9 and -3, and chromatin condensation. Disruption of the balance between Bcl-2 family proteins was reversed in thermotolerant (40 °C) cells, thus favouring cell survival. Bcl-2/Bcl-xL inhibitor ABT-737 sensitised cells to apoptosis, which indicates that Bcl-2 family proteins play a role in hyperthermia-induced apoptosis. The adaptive response of mild thermotolerance (40 °C) was still able to protect cells against hyperthermia (42–43 °C) when Bcl-2/Bcl-xL were inhibited.Conclusions: These results improve knowledge about the role of Bcl-2 family proteins in cellular apoptotic responses to hyperthermia (42–43 °C), as well as the adaptive survival response induced by exposure to mild stresses, such as a fever temperature (40 °C). This study could provide rationale to explore the manipulation of Bcl-2 family proteins for increasing tumour sensitivity to hyperthermia.