The molecular chaperone heat shock protein 90 (Hsp90) is involved in the maturation and stabilization of a wide range of oncogenic client proteins for oncogenesis and malignant cell proliferation, which renders this protein a promising target in the development of cancer therapeutics. PU-H71 is a purine-scaffold Hsp90 inhibitor with less toxicity in normal cells than in cancer cells. In this study, we examined the in vitro radiosensitizing activity and molecular mechanisms of action of PU-H71 in human lung cancer cell lines. PU-H71 enhanced the sensitivity of the SQ-5 and A549 cancer cells to radiation. When the cancer cells were pre-treated with PU-H71, the repair of DNA double-strand breaks (DSBs) was markedly inhibited after irradiation compared with the cells that were not pre-treated with PU-H71, as evaluated by counting the foci of phosphorylated histone H2AX (γ-H2AX). We further demonstrated that post-irradiation, PU-H71 inhibited Rad51 foci formation, a critical protein for the homologous recombination pathway of DNA DSB repair. These data indicate that targeting Hsp90 with PU-H71 may be novel therapeutic strategy for radioresistant carcinomas.

Recent studies showed that the stemness of cancer stem cells is maintained under a hypoxic microenvironment. However, the relationship of the hypoxic microenvironment in a three-dimensional cell mass and the induction of cancer stem cell-like phenotype is not well known. We examined the relationship between CD133 expression and the hypoxic microenvironment using glioblastoma spheroids formed with the T98G cell line. CD133(AC133)- and HIF-1α-positive cells were observed in the marginal region of the central hypoxic area positive for HIF-1α 10 days after plating T98G cells. CD133(AC133)-positive cells were positive for nestin. Quantitative PCR analysis showed that the CD133 expression level is not different in spheroids during the tested period after spheroid formation, indicating that post-translational regulation of the CD133 protein mediates positivity to CD133(AC133). When spheroids were trypsinized and the dissociated cells were cultured under the adherent monolayer conditions, the CD133(AC133)-positive cells gradually disappeared. These results show that CD133(AC133)-positive cells, which may incline toward undifferentiated cells because of nestin positivity, are plastically induced under the different culture conditions, spheroid and monolayer. In this plasticity, HIF-1α is involved in the induction and maintenance of CD133(AC133)-positive cells. Spheroids as an in vitro tumor model are useful to study the dynamic changes in the tumor cell phenotype in the different cell microenvironments.

This study supports the hypothesis that syringetin enhances radiosensitivity more effectively in cancer cells than in normal cells through enhancement of the Caspase-3-mediated apoptosis pathway. Syringetin could be useful in the development of novel efficacious radiosensitizers.