Knockdown of Checkpoint Kinase 1 Is Associated with the Increased Radiosensitivity of Glioblastoma Stem-Like Cells
Department of Neurosurgery, Huazhong University of Science and Technology, Hubei, People's Republic of China. The Tohoku Journal of Experimental Medicine
(Impact Factor: 1.35).
04/2012; 226(4):267-74. DOI: 10.1620/tjem.226.267
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.
Available from: Laure Marignol
- "PKH26 ϩ stem-cell like nasopharyngeal carcinoma cells displayed increased clonogenicity, sphere formation and resistance to radiotherapy through overexpression of c-MYC resulting increased expression of the CHK1 and CHK2 cell cycle checkpoint proteins and activation of the DNA damage response (Wang et al. 2013b). Similarly CHK1 knockdowns increased radiosensitivity of prostate (Wang et al. 2012) and glioma (Wu et al. 2012) cancer stem cells. In gliomainitiating cells, failure of irradiated cells to arrest at the S-cell cycle phase entry checkpoint was associated with increased homologous recombination and increased radioresistance (Lim et al. 2012). "
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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 analyze and compare the protocols used by investigators for the development and validation of these isogenic models of radioresistance.
There is large variability in the strategies used to generate and validate isogenic models of radioresistance. Further characterization of these models is required.
Available from: PubMed Central
- "GSCs can be eliminated in several ways: by employing sonic hedgehog signaling inhibitors in the stemness signature of GBM, by targeting the differentiation pathways, by delivering RNA interference (RNAi) to GBM cells, and by targeting the hyperactive cell cycle checkpoint kinases (Chk1 and Chk2)7,8. The mammalian target of the rapamycin (mTOR) inhibitor, rapamycin, induces autophagy and significantly affects the regulation of self-renewal, differentiation, tumorigenic potential, and radiosensitization of GSCs9. "
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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.
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.
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.
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.
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ABSTRACT: Radioresistance is responsible for treatment failure after radiotherapy in localized prostate cancer, while prostate cancer stem cells promote radioresistance by preferential activation of the DNA damage response. Chk1 inhibition has been shown to sensitize many tumor cells to radiation. However, whether Chk1 inhibition can potentiate the cytotoxic effects of radiation on prostate cancer stem cells remains to be elucidated. In this study, CD133+CD44+ cells were isolated using microbeads and were found to possess cancer stem cell properties. Using shRNA, Chk1 was knocked down in the sorted CD133+CD44+ cells. Our results demonstrated that Chk1 knockdown abrogated the radiation-induced G2/M arrest, inhibited DNA damage repair and promoted premature mitosis, leading to increased apoptosis in the radiated sorted CD133+CD44+ cells. Moreover, these effects were accompanied by caspase-2 activation and the inactivation of phosphorylated Cdc25C and Cdc2. Our results suggest that Chk1 knockdown increases the radiosensitivity of CD133+CD44+ prostate cancer stem cells. Chk1 knockdown in prostate cancer stem cells may be an effective therapeutic strategy against prostate cancer.
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