BACKGROUND: Heat-shock protein 90 (Hsp90) has a crucial role in both the stabilisation and regulation of various proteins, including those related to radioresistance. Inhibition of Hsp90 may therefore provide a strategy for enhancing the radiosensitivity of tumour cells. This study explores the responses of four tumour cell lines (A549, GaMG, HT 1080 and SNB19) to combined treatment with ionising radiation (IR) and two novel inhibitors of Hsp90, NVP-AUY922 and NVP-BEP800. The techniques used included cell and colony counts, expression of Hsp90, Hsp70, Akt, survivin, cleaved caspase 3, p53, cell-cycle progression and associated proteins. DNA damage was analysed by histone gH2AX and Comet assays. RESULTS: We found that NVP-AUY922 and NVP-BEP800 enhanced radiosensitivity in all tested cell lines. In contrast, only two cell lines (HT 1080 and GaMG) exhibited an increased rate of apoptosis after drug pretreatment, as revealed by western blot. In all tested cell lines, the expression of histone gH2AX, a marker of DNA double-strand breaks, after combined drug-IR treatment was higher and its decay rate was slower than those after each single treatment modality. Drug-IR treatment also resulted in impaired cell-cycle progression, as indicated by S-phase depletion and G2/M arrest. In addition, the cell cycle-associated proteins, Cdk1 and Cdk4, were downregulated after Hsp90 inhibition. INTERPRETATION: These findings show that the novel inhibitors of Hsp90 can radiosensitise tumour cell lines of different entities through destabilisation and depletion of several Hsp90 client proteins, thus causing the depletion of S phase and G2/M arrest, increased DNA damage and repair protraction and, to some extent, apoptosis. The results might have important implications for the radiotherapy of solid tumours.

Background: Photorhabdus luminescens is a Gram-negative luminescent enterobacterium and a symbiote to soil nematodes belonging to the species Heterorhabditis bacteriophora. P.luminescens is simultaneously highly pathogenic to insects. This bacterium exhibits a complex life cycle, including one symbiotic stage characterized by colonization of the upper nematode gut, and a pathogenic stage, characterized by release from the nematode into the hemocoel of insect larvae, resulting in rapid insect death caused by bacterial toxins. P. luminescens appears to sense and adapt to the novel host environment upon changing hosts, which facilitates the production of factors involved in survival within the host, host-killing, and -exploitation.

Targeting heat shock protein 90 (Hsp90) provides a promising therapeutic approach to enhance the sensitivity of tumor cells to ionizing radiation (IR). To explore the impact of scheduling drug-IR administration, in the present study, we analyzed the response of lung carcinoma A549 and glioblastoma SNB19 cells to simultaneous drug-IR treatment followed by a long-term drug administration. Cellular response was evaluated at different time intervals after IR-alone, drug-alone, or combined drug-IR treatments by colony counts and expression profiles of Hsp90 and its clients, along with several apoptotic markers and cell cycle-related proteins, as well as by IR-drug-induced cell cycle arrest, DNA damage, and repair. A short 30-minute exposure to either Hsp90 inhibitor did not affect the radiosensitivity of both tumor cell lines. Increasing the duration of post-IR-drug treatment progressively enhanced the sensitivity of SNB19 cells to IR. In contrast, the response of A549 cells to drug-IR combination was largely determined by the cytotoxic effects of both drugs without radiosensitization. Combined drug-IR treatment induced more severe DNA damage in both tumor cell lines than each treatment alone and also protracted the kinetics of DNA damage repair in SNB19 cells. In addition to large cell cycle disturbances, drug-IR treatment also caused depletion of the antiapoptotic proteins Akt and Raf-1 in both cell lines, along with a decrease of survivin in A549 cells in case of NVP-AUY922. The data show that simultaneous Hsp90 inhibition and irradiation may induce cell type-specific radiosensitization as well as cytotoxicity against tumor cells.

Pre-silencing of Hsp90α followed by Hsp90 inhibition did not enhance the radiosensitizing effect of NVP-AUY922 in both tested tumour cell lines. Future work will be done on stable transfection with shRNA against Hsp90α or simultaneous silencing of both Hsp90 isoforms, Hsp90α and Hsp90β, in order to optimize tumour cell killing.