Knockdown of checkpoint kinase 1 is associated with the increased radiosensitivity of glioblastoma stem-like cells.
ABSTRACT Glioblastoma multiforme is an aggressive brain tumor with a poor prognosis. The glioblastoma stem-like cells (GSCs) represent a rare fraction of human glioblastoma cells with the capacity for multi-lineage differentiation, self-renewal and exact recapitulation of the original tumor. Interestingly, GSCs are more radioresistant compared with other tumor cells. In addition, the remarkable radioresistance of GSCs has been known to promote radiotherapy failure and therefore is associated with a significantly higher risk of a local tumor recurrence. Moreover, the hyperactive cell cycle checkpoint kinase (Chk) 1 and 2 play a pivotal role in the DNA damage response including radiation and chemical therapy. Based on aforementioned, we hypothesized that knockdown of Chk1 or Chk2 might confer radiosensitivity on GSCs and thereby increases the efficiency of radiotherapy. In this study, we knocked down the expression of Chk1 or Chk2 in human GSCs using lentivirus-delivered short hairpin RNA (shRNA) to examine its effect on the radiosensitivity. After radiation, the apoptosis rate and the cell cycle of GSCs were measured with Flow Cytometry. Compared with control GSCs (apoptosis, 7.82 ± 0.38%; G2/M arrest, 60.20 ± 1.28%), Chk1 knockdown in GSCs increased the apoptosis rate (37.87 ± 0.32%) and decreased the degree of the G2/M arrest (22.37 ± 2.01%). In contrast, the radiosensitivity was not enhanced by Chk2 knockdown in GSCs. These results suggest that depletion of Chk1 may improve the radio-sensitivity of GSCs via inducing cell apoptosis. In summary, the therapy targeting Chk1 gene in the GSCs may be a novel way to treat glioblastoma.
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ABSTRACT: Aim:NVP-BEZ235 is a novel dual PI3K/mTOR inhibitor and shows dramatic effects on gliomas. The aim of this study was to investigate the effects of NVP-BEZ235 on the radiosensitivity and autophagy of glioma stem cells (GSCs) in vitro.Methods:Human GSCs (SU-2) were tested. The cell viability and survival from ionizing radiation (IR) were evaluated using MTT and clonogenic survival assay, respectively. Immunofluorescence assays were used to identify the formation of autophagosomes. The apoptotic cells were quantified with annexin V-FITC/PI staining and flow cytometry, and observed using Hoechst 33258 staining and fluorescence microscope. Western blot analysis was used to analyze the expression levels of proteins. Cell cycle status was determined by measuring DNA content after staining with PI. DNA repair in the cells was assessed using a comet assay.Results:Treatment of SU-2 cells with NVP-BEZ235 (10-320 nmol/L) alone suppressed the cell growth in a concentration-dependent manner. A low concentration of NVP-BEZ235 (10 nmol/L) significantly increased the radiation sensitivity of SU-2 cells, which could be blocked by co-treatment with 3-MA (50 μmol/L). In NVP-BEZ235-treated SU-2 cells, more punctate patterns of microtubule-associated protein LC3 immunoreactivity was observed, and the level of membrane-bound LC3-II was significantly increased. A combination of IR with NVP-BEZ235 significantly increased the apoptosis of SU-2 cells, as shown in the increased levels of BID, Bax, and active caspase-3, and decreased level of Bcl-2. Furthermore, the combination of IR with NVP-BEZ235 led to G1 cell cycle arrest. Moreover, NVP-BEZ235 significantly attenuated the repair of IR-induced DNA damage as reflected by the tail length of the comet.Conclusion:NVP-BEZ235 increases the radiosensitivity of GSCs in vitro by activating autophagy that is associated with synergistic increase of apoptosis and cell-cycle arrest and decrease of DNA repair capacity.Acta Pharmacologica Sinica 04/2013; · 2.35 Impact Factor
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ABSTRACT: Abstract Purpose: The comparison of cell lines with differing radiosensitivities and their molecular response to radiation exposure has been used in a number of human cancer models to study the molecular response to radiation. This review proposes to analyse and compare the protocols used by investigators for the development and validation of these isogenic models of radioresistance. Conclusion: There is large variability in the strategies used to generate and validate isogenic models of radioresistance. Further characterisation of these models is required.International Journal of Radiation Biology 12/2013; · 1.84 Impact Factor