Hypoxic Radiosensitization By DMAG, an Hsp90 Inhibitor

Abstract

Hypoxia is one of the major targets in radiation biology. Many efforts have been made over a long period to overcome the radioresistance of hypoxic cells. This resistance is caused not only by a reduction of radiation-induced damage to DNA under hypoxic conditions, but also by the effects of hypoxia-related proteins, such as hypoxia-inducible factor (HIF). Heat shock protein (Hsp)90 is constitutively expressed in cells and accounts for about 1% of total protein. It is a molecular chaperone that plays an important role in protein regulation in cells and is overexpressed in a wide range of tumors. Some investigators have already reported the radiosensitizing effect of inhibitors of Hsp90 on normoxic cells. Hsp90 stabilizes HIF-1α by interacting with it. Therefore, there is a possibility that the inhibition of Hsp90 sensitizes hypoxic cells to the effect of radiation more than oxic cells. In this in vitro study, we thus investigated the radiosensitizing activity of 17-[2-(dimethylamino) ethyl]amino-17-desmethoxygeldanamycin (DMAG), an inhibitor of Hsp90, on hypoxic cells. Two human tumor cell lines, HT1080 (fibrosarcoma) and HeLa (uterine cervical carcinoma), were used in this study. The radiosensitivity of cells was assessed by a standard colony-forming assay. The oxygen enhancement ratio (OER) and sensitizer enhancement ratio (SER) were calculated from the ratio of two radiation doses required to reduce the surviving fraction to 0.01. Cells were exposed to the compound for 24 h before and during radiation. We detected changes in the cell cycle using flow cytometry. Protein expression was measured by western blot analysis. The exposure of HeLa cells to 100 nM DMAG for 24 h significantly increased the expression of Hsp70, a marker of Hsp90 inhibition. However, there was no difference in its expression between cases with and without hypoxic treatment for 4 h. Flow cytometry revealed changes in the cell cycle after DMAG exposure, but again there was no difference between cases with and without hypoxic treatment. Exposure to 50–400 nM DMAG for 24 h was slightly cytotoxic (surviving fractions of around 0.8 to 0.9), but no further effect was observed with the additional hypoxic treatment. DMAG at 100 nM showed a weak radiosensitizing effect on both Hela and HT 1080 cells under oxic conditions: SERs were 1.2 (SD: 0.2) and 1.1 (0.06). However, SERs for hypoxic HeLa and HT1080 cells were significantly greater than those for oxic cells: SERs for 50, 100, 200, and 400 nM DMAG for HeLa cells were 1.3 (0.3), 1.5 (0.2), 1.6 (0.3), and 1.6 (0.06), respectively. SER was 1.5 (0.3) for 100 nM DMAG for hypoxic HT1080 cells. The inhibition of Hsp90 by DMAG showed a much greater radiosensitizing effect on hypoxic cells than on normoxic ones. Although there is a need to investigate its detailed mechanism of action, Hsp90 is a potential new target for hypoxic radiosensitization.