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Background: Our previous data demonstrated that targeting non-homologous end-joining repair (NHEJR) yields a higher radiosensitivity than targeting homologous recombination repair (HRR) to heavy ions using DNA repair gene knockouts (KO) in mouse embryonic fibroblast (MEF). In this study, we determined if combining the use of an NHEJR inhibitor with carbon (C) ion irradiation was more efficient in killing human cancer cells compared with only targeting a HRR inhibitor.Methods: The TP53-null human non-small cell lung cancer cell line H1299 was used for testing the radiosensitizing effect of NHEJR-related DNA-dependent protein kinase (DNA-PK) inhibitor NU7026, HRR-related Rad51 inhibitor B02, or both to C ion irradiation using colony forming assays. The mechanism underlying the inhibitor radiosensitization was determined by flow cytometry after H2AX phosphorylation staining. HRR-related Rad54-KO, NHEJR-related Lig4-KO, and wild-type TP53-KO MEF were also included to confirm the suppressing effect specificity of these inhibitors.Results: NU7026 showed significant sensitizing effect to C ion irradiation in a concentration-dependent manner. In contrast, B02 showed a slight sensitizing effect to C ion irradiation. The addition of NU7026 significantly increased H2AX phosphorylation after C ion and x-ray irradiations in H1299 cells, but not B02. NU7026 had no effect on radiosensitivity to Lig4-KO MEF and B02 had no effect on radiosensitivity to Rad54-KO MEF in both irradiations.Conclusion: These results suggest that inhibitors targeting the NHEJR pathway could significantly enhance radiosensitivity of human cancer cells to C ion irradiation, rather than targeting the HRR pathway. [ABSTRACT FROM AUTHOR]

Purpose: Recently carbon-ion beams have been reported to be remarkably effective for controlling various cancers with less toxicity and are thought to be a promising modality for cancer treatment. However, the biological effect of carbon-ion beams arising on normal neuron remains unknown. Therefore, this study was undertaken to investigate the effect of carbon-ion beams on neurons by using both morphological and functional assays. Materials and methods: Dorsal root ganglia (DRG) and sympathetic ganglion chains (SYMP) were isolated from day-8 and day-16 chick embryos and cultured for 20 h. Cultured neurons were exposed to carbon-ion beams and X-rays. Morphological changes, apoptosis and cell viability were evaluated with the Growth Cone Collapse (GCC), Terminal deoxynucleotidyl Transferase (TdT)-mediated deoxyUridine TriPhosphate (dUTP) nick End Labeling [TUNEL] assay and 4-[3-(4-iodophenyl)- 2-(4-nitrophenyl)- 2H-5-tetrazolio]- 1,3-benzenedisulfonate [WST-1] assays, respectively. Results: Irradiation caused GCC and neurite destruction on a time- and irradiation dose-dependent manner. Changes in morphological characteristics were similar following either irradiation. Morphological and functional assays showed that day-8 neurons were more radiosensitive than day-16 neurons, whereas, radiosensitivity of DRG was comparable to that of SYMP. The dose-response fitting curve utilising both GCC and TUNEL labeling index showed higher relative biological effectiveness (RBE) values were associated with lower lethal dose (LD) values, while lower RBE was associated with higher LD values. Conclusion: Exposure to high-linear energy transfer (LET) irradiation is up to 3.2 more efficient to induce GCC and apoptosis, in early developed neuronal cells, than low-LET irradiation. GCC is a reliable method to assess the radiobiological response of neurons. [ABSTRACT FROM AUTHOR]

Simple Summary: Carbon ion radiotherapy is an emerging cancer treatment modality that has a greater therapeutic window than conventional photon radiotherapy. To maximize the efficacy of this extremely scarce medical resource, it is important to identify predictive biomarkers of higher carbon ion relative biological effectiveness (RBE) over photons. Here we show that the carbon ion RBE in human cancer cells correlates with the cellular uptake of 64Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM), a potential radioligand that reflects an over-reduced intracellular environment. High RBE/64Cu-ATSM cells show greater steady-state levels of antioxidant proteins and increased capacity to scavenge reactive oxygen species in response to X-rays than low RBE/64Cu-ATSM counterparts. These data suggest that the cellular antioxidant activity is a possible determinant of carbon ion RBE predictable by 64Cu-ATSM uptake. Carbon ion radiotherapy is an emerging cancer treatment modality that has a greater therapeutic window than conventional photon radiotherapy. To maximize the efficacy of this extremely scarce medical resource, it is important to identify predictive biomarkers of higher carbon ion relative biological effectiveness (RBE) over photons. We addressed this issue by focusing on cellular antioxidant capacity and investigated 64Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM), a potential radioligand that reflects an over-reduced intracellular environment. We found that the carbon ion RBE correlated with 64Cu-ATSM uptake both in vitro and in vivo. High RBE/64Cu-ATSM cells showed greater steady-state levels of antioxidant proteins and increased capacity to scavenge reactive oxygen species in response to X-rays than low RBE/64Cu-ATSM counterparts; this upregulation of antioxidant systems was associated with downregulation of TCA cycle intermediates. Furthermore, inhibition of nuclear factor erythroid 2-related factor 2 (Nrf2) sensitized high RBE/64Cu-ATSM cells to X-rays, thereby reducing RBE values to levels comparable to those in low RBE/64Cu-ATSM cells. These data suggest that the cellular activity of Nrf2-driven antioxidant systems is a possible determinant of carbon ion RBE predictable by 64Cu-ATSM uptake. These new findings highlight the potential clinical utility of 64Cu-ATSM imaging to identify high RBE tumors that will benefit from carbon ion radiotherapy. [ABSTRACT FROM AUTHOR]

Carbon-ion radiotherapy (CIRT) holds promise to treat inoperable locally-advanced non-small cell lung carcinoma (NSCLC), a disease poorly controlled by standard chemoradiotherapy using X-rays. Since CIRT is an extremely limited medical resource, selection of NSCLC patients likely to benefit from it is important; however, biological predictors of response to CIRT are ill-defined. The present study investigated the association between the mutational status of EGFR and KRAS, driver genes frequently mutated in NSCLC, and the relative biological effectiveness (RBE) of carbon-ion beams over X-rays. The assessment of 15 NSCLC lines of different EGFR/KRAS mutational status and that of isogenic NSCLC lines expressing wild-type or mutant EGFR revealed that EGFR-mutant NSCLC cells, but not KRAS-mutant cells, show low RBE. This was attributable to (i) the high X-ray sensitivity of EGFR-mutant cells, since EGFR mutation is associated with a defect in non-homologous end joining, a major pathway for DNA double-strand break (DSB) repair, and (ii) the strong cell-killing effect of carbon-ion beams due to poor repair of carbon-ion beam-induced DSBs regardless of EGFR mutation status. These data highlight the potential of EGFR mutation status as a predictor of response to CIRT, i.e., CIRT may show a high therapeutic index in EGFR mutation-negative NSCLC. [ABSTRACT FROM AUTHOR]