The purpose of this study was to identify the roles of non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways in repairing DNA double-strand breaks (DSBs) induced by exposure to high-energy protons and carbon ions (C ions) versus gamma rays in Chinese hamster cells. Two Chinese hamster cell lines, ovary AA8 and lung fibroblast V79, as well as various mutant sublines lacking DNA-PKcs (V3), X-ray repair cross-complementing protein-4 [XRCC4 (XR1), XRCC3 (irs1SF) and XRCC2 (irs1)] were exposed to gamma rays (137Cs), protons (200 MeV; 2.2 keV/μm) and C ions (290 MeV; 50 keV/μm). V3 and XR1 cells lack the NHEJ pathway, whereas irs1 and irs1SF cells lack the HR pathway. After each exposure, survival was measured using a clonogenic survival assay, in situ DSB induction was evaluated by immunocytochemical analysis of histone H2AX phosphorylation at serine 139 (γ-H2AX foci) and chromosome aberrations were examined using solid staining. The findings from this study showed that clonogenic survival clearly depended on the NHEJ and HR pathway statuses, and that the DNA-PKcs−/− cells (V3) were the most sensitive to all radiation types. While protons and γ rays yielded almost the same biological effects, C-ion exposure greatly enhanced the sensitivity of wild-type and HR-deficient cells. However, no significant enhancement of sensitivity in cell killing was seen after C-ion irradiation of NHEJ deficient cells. Decreases in the number of γ-H2AX foci after irradiation occurred more slowly in the NHEJ deficient cells. In particular, V3 cells had the highest number of residual γ-H2AX foci at 24 h after C-ion irradiation. Chromosomal aberrations were significantly higher in both the NHEJ- and HR-deficient cell lines than in wild-type cell lines in response to all radiation types. Protons and gamma rays induced the same aberration levels in each cell line, whereas C ions introduced higher but not significantly different aberration levels. Our results suggest that the NHEJ pathway plays an important role in repairing DSBs induced by both clinical proton and C-ion beams. Furthermore, in C ions the HR pathway appears to be involved in the repair of DSBs to a greater extent compared to gamma rays and protons.

Background. Refractoriness of glioblastoma multiforme (GBM) largely depends on its radioresistance. We investigated the radiosensitizing effects of celecoxib on GBM cell lines under both normoxic and hypoxic conditions. Methods. Two human GBM cell lines, U87MG and U251MG, and a mouse GBM cell line, GL261, were treated with celecoxib or g-irradiation either alone or in combination under normoxic and hypoxic conditions. Radiosensitizing effects were analyzed by clonogenic survival assays and cell growth assays and by assessing apoptosis and autophagy. Expression of apoptosis-, autophagy-, and endoplasmic reticulum (ER) stressrelated genes was analyzed by immunoblotting. Results. Celecoxib significantly enhanced the radiosensitivity of GBM cells under both normoxic and hypoxic conditions. In addition, combined treatment with celecoxib and g-irradiation induced marked autophagy, particularly in hypoxic cells. The mechanism underlying the radiosensitizing effect of celecoxib was determined to be ER stress loading on GBM cells. Conclusion. Celecoxib enhances the radiosensitivity of GBM cells by a mechanism that is different from cyclooxygenase-2 inhibition. Our results indicate that celecoxib may be a promising radiosensitizing drug for clinical use in patients with GBM. Keywords:autophagy, celecoxib, ER stress, glioblastoma, hypoxia, radiosensitivity. G lioblastoma multiforme (GBM) is the most frequently occurring malignant tumor of the central nervous system. 1 Despite standard carecomprising maximum surgical resection, 60 Gy of conventional radiotherapy, and chemotherapy with temozolomide (TMZ), the median survival time among patients with GBM is 1 year. 2The molecular mechanisms underlying the initiation and progression of GBM reported to date fall into 2 categories: (i) primary GBM with increased epidermal growth factor receptor (EGFR) expression and mutation of the phosphatase and tensin homolog and (ii) secondary GBM that is transformed from benign glioma types with stepwise mutations in the p53 and retinoblastoma genes. It is known that these genetic alterations in GBM are significantly associated with clinical prognosis.1 It has also been reported that methylation of O6-methylguanine-DNA methyltransferase increases the susceptibility of GBM tumor cells to alkylating agents, such as TMZ, 3 and mutation in the isocitrate dehydrogenase 1 gene was recently reported to significantly affect the prognosis in patients with GBM. 4 On the basis of these findings, therapy that targets these specific molecules could be a promising approach for controlling GBM. One possible target is cyclooxygenase-2 (COX-2) because a positive association has been reported among the levels of COX-2, EGFR/EGFRvIII, and activated signal transducer and activator of transcription 3 (STAT3).5 COX-2 expression was reportedly increased in human GBM and was negatively correlated with clinical outcomes in patients. 6 Celecoxib, a nonsteroidal anti-inflammatory drug (NSAID), selectively inhibits This drug is used clinically both for patients with ...

Cell inactivation may differ according to different timings and/or endpoints. Proton beams demonstrated higher cell inactivation than X-rays in the early phases. These data may facilitate the understanding of the biological properties of clinical proton beams.

One-third of patients with medulloblastoma die due to recurrence after various treatments including radiotherapy. Although it has been postulated that cancer stem-like cells are radio-resistant and play an important role in tumor recurrence, the “stemness” of medulloblastoma cells surviving irradiation has not yet been elucidated. Using a medulloblastoma cell line ONS-76, cells that survived gamma irradiation were investigated on their “stemness” in vitro. From 10 500 cells, 20 radio-resistant clones were selected after gamma ray irradiation (5 Gy × two fractions) using the replica micro-well technique. These 20 resistant clones were screened for CD133 positivity by flow cytometry followed by side population assay, tumor sphere formation assay and clonogenic survival assay. Results revealed CD133 fractions were significantly elevated in three clones, which also exhibited significantly increased levels of tumor sphere formation ability and side population fraction. Clonogenic survival assay demonstrated that their radio-resistance was significantly higher than the parental ONS-76. This may support the hypothesis that a small number of cancer stem-like cells (CSCs) are the main culprits in local recurrence after radiotherapy, and disruption of the resistance mechanism of these CSCs is a critical future issue in improving the outcome of patients with medulloblastoma.

Many reports have demonstrated that radiation stimulates reactive oxygen species (ROS) production by mitochondria for a few hours to a few days after irradiation. However, these studies were performed using cell lines, and there is a lack of information about redox homeostasis in irradiated animals and humans. Blood redox homeostasis reflects the body condition well and can be used as a diagnostic marker. However, most redox homeostasis studies have focused on plasma or serum, and the anti-oxidant capacity of whole blood has scarcely been investigated. Here, we report changes in the anti-oxidant capacity of whole blood after X-ray irradiation using C57BL/6 J mice. Whole-blood anti-oxidant capacity was measured by electron spin resonance (ESR) spin trapping using a novel spin-trapping agent, 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (DPhPMPO). We found that whole-blood anti-oxidant capacity decreased in a dose-dependent manner (correlation factor, r > 0.9; P < 0.05) from 2 to 24 days after irradiation with 0.5–3 Gy. We further found that the red blood cell (RBC) glutathione level decreased and lipid peroxidation level increased in a dose-dependent manner from 2 to 6 days after irradiation. These findings suggest that blood redox state may be a useful biomarker for estimating exposure doses during nuclear and/or radiation accidents.