Synergistic effect of heat shock protein 90 inhibitor, 17-allylamino-17-demethoxygeldanamycin and X-rays, but not carbon-ion beams, on lethality in human oral squamous cell carcinoma cells

Abstract

The purpose of this study is to clarify the effect of a heat shock protein 90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in combination with X-rays or carbon-ion beams on cell killing in human oral squamous cell carcinoma LMF4 cells. Cell survival was measured by colony formation assay. Cell-cycle distribution was analyzed by flow cytometry. Expression of DNA repair-related proteins was investigated by western blotting. The results showed 17-AAG to have synergistic effects on cell lethality with X-rays, but not with carbon-ion beams. The 17-AAG decreased G(2)/M arrest induced by X-rays, but not by carbon-ion beams. Both X-ray and carbon-ion irradiation up-regulated expression of non-homologous end-joining-associated proteins, Ku70 and Ku80, but 17-AAG inhibited only X-ray-induced up-regulation of these proteins. These results show that 17-AAG with X-rays releases G(2)/M phase arrest; cells carrying misrepaired DNA damage then move on to the G(1) phase. We demonstrate, for the first time, that the radiosensitization effect of 17-AAG is not seen with carbon-ion beams because 17-AAG does not affect these changes.

Fig. 1.

Effect of 17-AAG on radiosensitivity in human oral SCC LMF4 cells

Fig. 2.

Effect of 17-AAG or irradiation on percentage of cells in the G₂/M phase. The percentage of cells in the G₂/M phase was obtained by flow cytometry. Cells were either treated with 17-AAG (a), irradiated with X-rays (b) or irradiated with carbon-ion beams (c). (A) white circles, untreated; black circles, 17-AAG alone. (B) White circle4s: untreated; black circles: 5-Gy; white triangles: 7-Gy; black triangles: 10-Gy. (C) White circles: untreated; black circles: 1-Gy; white triangles: 2-Gy. Note that a lower-dose range of carbon-ion beams (1–2 Gy) was used than forX-rays (5–10 Gy). (D) Population size of G₂/M phase cells at 12 h after treatment with 100 nM 17-AAG for 24 h and/or exposure to 10-Gy X-rays or 2-Gy carbon-ion beams. (Da) is untreated cells. Cells were either treated with 17AAG (Db), 10-Gy X-rays (Dc), 10-Gy X-rays combined with 17-AAG (Dd), 2-Gy carbon-ion beams (De) or 2-Gy carbon-ion beams in combination with 17-AAG (Df). Data are presented as the mean ± SD. *P < 0.05; **P < 0.01 (Student's t test, compared with several treated cells).

Fig. 3.

Accumulation of NHEJ-related proteins, 24 h after treatment with 100 nM 17-AAG for 24 h, and/or exposure to 10-Gy X-rays, or 2-Gy carbon-ion beams.