Attenuation of chronic thermotolerance by KNK437, a benzylidene lactam compound, enhances thermal radiosensitization in mild temperature hyperthermia combined with low dose-rate irradiation

Purpose: We investigated whether the attenuation of chronic thermotolerance by KNK437, a heat shock protein inhibitor, can modify the effect of thermal radiosensitization in mild temperature hyperthermia (MTH) combined with low dose-rate irradiation (LDRI). Materials and methods: The human lung adenocarcinoma cell line A549 was simultaneously exposed to LDRI with MTH at 41 degrees C and KNK437 at a dose of 100 mu M. Cell survival was estimated by a clonogenic assay. Cell cycle change during treatment was analyzed by flow cytometry. Expression levels of the heat shock proteins hsp72, hsp27 and heat shock factor 1 (HSF-1) were measured by Western blotting. Results: KNK437 inhibited the expression of inducible hsp72 and hsp27, but produced no change in the mobility shift of HSF-1. The cytotoxicity of LDRI was enhanced by MTH. The survival curve for LDRI+MTH revealed no development of chronic thermotolerance up to 48 h. Simultaneous LDRI and KNK437 treatment also resulted in enhanced cell killing. The radiosensitizing effect of KNK437 was enhanced by simultaneous exposure of the cells to MTH. Flow cytometry analysis of cell cycle progression demonstrated marked G2 arrest and mild G1 arrest with LDRI alone, but mild G1 arrest with MTH alone, and mild G2-M, S-phase accumulation with KNK437 alone. The marked G2 arrest caused by LDRI was partially suppressed by the addition of MTH, and was also suppressed by KNK437 treatment. Conclusions: Exposure of A549 cells to KNK437 caused inhibition of hsp72 and hsp27 expression. The addition of KNK437 increased not only thermosensitivity to MTH, but also radiosensitivity to LDRI. KNK437 also enhanced the MTH-induced radiosensitization under these experimental conditions.