Gy Carbon Ion Research Articles

Synergistic Effects of Melatonin on Radiosensitization in Carbon-ion Radiotherapy.

Despite the established antitumor effectiveness and synergistic interactions of melatonin with photon irradiation, its role in carbon-ion radiotherapy remains uncertain. This study aimed to elucidate the mechanisms and potential clinical advantages of combining exogenous melatonin therapy with carbon-ion radiotherapy. The investigation assessed the impact of combining exogenous melatonin with photon or carbon-ion irradiation on cell-cycle modulation and DNA-repair capability using the melanoma cell line B16F10. RNA sequencing and bioinformatics analysis were conducted to explore mechanisms and evaluate potential clinical benefits, with validation performed on the osteosarcoma cell line LM8. Pre-treatment with melatonin reduced the survival fraction of B16F10 and LM8 cells upon exposure to photon and carbon-ion radiation. Mechanistically, melatonin was found to inhibit G2/M arrest, preserve DNA damage, and suppress key genes involved in DNA double-strand break repair after 8 Gy carbon-ion radiation. Furthermore, RNA sequencing and bioinformatics analysis revealed favorable changes in genes associated with survival and metastasis, highlighting potential clinical significance. LM8 cells treated with melatonin exhibited increased radiosensitivity and suppression of DNA-repair proteins. The combination of exogenous melatonin not only heightened radiosensitivity and modulated hallmark tumor gene sets in vitro but also markedly suppressed the efficiency of DNA double-strand break-repair pathway, thus enhancing the cytotoxicity of carbon-ion radiotherapy.

Lost in Space? Unmasking the T Cell Reaction to Simulated Space Stressors

The space environment will expose astronauts to stressors like ionizing radiation, altered gravity fields and elevated cortisol levels, which pose a health risk. Understanding how the interplay between these stressors changes T cells’ response is important to better characterize space-related immune dysfunction. We have exposed stimulated Jurkat cells to simulated space stressors (1 Gy, carbon ions/1 Gy photons, 1 µM hydrocortisone (HC), Mars, moon, and microgravity) in a single or combined manner. Pro-inflammatory cytokine IL-2 was measured in the supernatant of Jurkat cells and at the mRNA level. Results show that alone, HC, Mars gravity and microgravity significantly decrease IL-2 presence in the supernatant. 1 Gy carbon ion irradiation showed a smaller impact on IL-2 levels than photon irradiation. Combining exposure to different simulated space stressors seems to have less immunosuppressive effects. Gene expression was less impacted at the time-point collected. These findings showcase a complex T cell response to different conditions and suggest the importance of elevated cortisol levels in the context of space flight, also highlighting the need to use simulated partial gravity technologies to better understand the immune system’s response to the space environment.

Multi-omics analyses of the effect of carbon ion beam irradiation on cannabis fructus (Cannabis sativa L.) composition

Cannabis fructus (Cannabis sativa L.) belongs to the mulberry family, which is one of the earliest cultivated crops by humans and has industrial, agricultural, and medicinal values. In this study, the effect of carbon ion beam irradiation on the physiological and biochemical pathways of cannabis fructus was analyzed by multi-omics. Compared to the unirradiated cannabis fructus stem, 2,891 differentially-expressed genes, 1,145 differentially-expressed proteins, and 954 altered ubiquitination modification sites were identified in cannabis fructus irradiated by carbon ion beam irradiation (60 Gy dosage). Correlation analyses between proteomic, transcriptomic, and ubiquitin modification groups were carried out, and we found that the carbon fixation pathway, fatty acid synthesis pathway, JA synthesis, and glutathione metabolism pathway were enriched in relevant protein levels, and activates the MAPK signaling pathway regulated by gene expression. Further analysis found that the content of some proteins (LOX, AOC, and MDR) was correlated with their respective transcriptional levels, and the content of some proteins (APX, G-6-PD, GST, GAPHD, FBA, AOS, PR1, and ChiB) was correlated with their ubiquitination levels. In addition, M2 generation cannabis fructus seeds exhibited a larger volume, higher thousand-grain weight, and increased linolenic acid content compared to the control group, which was consistent with the trends of our multi-omics analyses. In summary, we demonstrated that 60 Gy carbon ion beam irradiation can enhance the carbon fixation pathway in cannabis fructus and enhance linolenic acid synthesis. Taken together, our multi-omics analysis of the response of cannabis fructus to carbon ion beam irradiation provides a theoretical foundation for readily producing cannabis fructus varieties with advantageous traits in the future.

Analysis of the relationship between LET, γH2AX foci volume and cell killing effect of carbon ions using high-resolution imaging technology.

The strong cell killing effect of high linear energy transfer (LET) carbon ions is dependent on lethal DNA damage. Our recent studies suggest that induction of clusters of double-strand breaks (DSBs) in close proximity is one of the potential mechanisms. However, the relationship between LET, the degree of DSB clustering and the cell killing effect of carbon ions remains unclear. Here, we used high-resolution imaging technology to analyze the volume of γH2AX foci induced by monoenergetic carbon ions with a clinically-relevant range of LET (13-100keV/μm). We obtained data from 3317 γH2AX foci and used a gaussian function to approximate the probability (p) that 1Gy-carbon ions induce γH2AX foci of a given volume (vth) or greater per nucleus. Cell killing effects were assessed in clonogenic assays. The cell killing effect showed high concordance with p at vth = 0.7μm3 across various LET values; the difference between the two was 4.7% ± 2.2%. This relationship was also true for clinical carbon ion beams harboring a mixed LET profile throughout a spread-out Bragg peak width (30-120mm), with the difference at vth = 0.7μm3 being 1.6% ± 1.2% when a Monte Carlo simulation-derived dose-averaged LET was used to calculate p. These data indicate that the cell killing effect of carbon ions is predictable by the ability of carbon ions to induce γH2AX foci containing clustered DSBs, which is linked to LET, providing the biological basis for LET modulation in the planning of carbon ion radiotherapy.

LIF Inhibits Proliferation of Esophageal Squamous Carcinoma Cells by Radiation Mediated Through JAK-STAT Signaling Pathway.

Background: Esophagus cancer is a malignant tumor with a high incidence rate, and radiation is an important modality for esophageal cancer therapy. However, therapeutic failure in the treatment of ESCC is often attributed to an inherent radio-resistance of the tumor cells. This study discusses effect and mechanism of carbon ion exerts tumor-inhibiting proliferation via down-regulation of LIF in esophageal squamous cell carcinoma. Methods: Colony formation, CCK8 and EdU assays were used to detect cell survival and proliferation after 0 and 2Gy carbon ion irradiation of ECA109 cells. Proteomics changes were probed in response to carbon ion irradiation using quantitative proteomics approach incorporating TMT isotope tags. Then, candidate genes were identified via bioinformatics analysis methods and microarray results were verified by real-time qPCR. Paired ESCC tumor tissues and adjacent non-tumor samples from 17 patients were collected and used for detecting expression by immunohistochemistry. Furthermore, small interfering RNA (siRNA) was transfected into ECA109 and KYSE150 cells and cell proliferation was analyzed by EdU assay. Flow cytometry and Western blot were performed to measure the and apoptosis and JAK-STAT3 protein expression level of ECA109 and KYSE150 cells combined drugs after siLIF transfection. Results: When compared with the control (0Gy), Inhibition of ECA109 cell proliferation and clonogenic survival by 2 Gy carbon ions, radiation group screened 360 differentially expressed proteins, 156 of which were up-regulated and 144 were down-regulated. Downregulation of LIF expression by siRNA enhances apoptotic in the ECA109 and KYSE150 cells, significantly inhibited esophageal squamous cell carcinoma cells proliferation. In ESCC cells, the JAK/STAT3 signaling pathway is inhibited in a LIF-dependent manner, resulting in the expression of STAT3 downstream target genes. Carbon ions combined with siLIF inhibited cell proliferation more significantly. The inhibitory cell proliferation effect was more pronounced by the combined intervention of carbon ion irradiation with siLIF. LIF expression was 18.51±9.84 and 5.82±4.50 in 17 paired ESCC tissues and adjacent non-cancerous tissues, respectively. LIF protein expression was lower in ESCC than in the adjacent normal tissue. Conclusion: The findings of this study reveal that Carbon ion knockdown was shown to downregulate LIF in ESCC cells. LIF is involved in ESCC proliferation and inhibited the ESCC cell proliferation by activating the STAT3 signaling pathways.

Abstract 9722: P53-phlda3 Signaling Induced by Carbon-Ion Irradiation is a Novel Therapeutic Target to Suppress Cardiac Hypertrophy in Mice

Background: Irradiation to the heart increases the risk of several cardiac disease including coronary artery disease, heart failure, pericardial disease and cardiac arrythmia, therefore, the heart has been considered one of the organs at risk in thoracic irradiation. On the other hand, recent experimental studies suggested irradiation may ameliorate cardiac injury and arrhythmia after myocardial infarction. To test irradiation effects on pathological cardiac remodeling, we examine the effects from irradiation in a mouse model of heart failure. Methods and Results: We used cardiac hypertrophy and heart failure mice model induced by transverse aortic constriction (TAC), irradiated by X-ray (20, 30 Gy) or carbon-ion beam (10, 30 Gy) at 2 weeks after pressure overload, and examined the changes in cardiac function by echocardiography. Concerning to the X-ray, 1 week after irradiation, ventricular systolic dysfunction induced by TAC was attenuated compared with control group. Among the X-irradiated groups, the higher dose improved cardiac function better. However, at 2 weeks post-irradiation, the cardiac function decreased again. On the other hand, carbon-ion irradiation improved left ventricular function and cardiac hypertrophy even at 2 weeks post-irradiation. The BNP expression was increased in TAC-treated mice and markedly decreased in irradiated mice, suggesting improvement of cardiac hypertrophy and cardiac remodeling. Cardiac p53 phosphorylation is markedly induced by cardiac irradiation. RNA sequencing using irradiated heart revealed pleckstrin homology-like domain family A, member 3 (PHILDA3), a downstream of p53 was significantly upregulated after X-ray or carbon-ion irradiation. PHLDA3 was decreased by TAC treatment, but improved especially in 30 Gy carbon-ion irradiation group. Akt phosphorylation, a hypertrophic signal and downstream target of PHLDA3, was suppressed by irradiation after TAC. Conclusion: Irradiation, especially with carbon-ion beam, brought a beneficial effect on pathological cardiac remodeling. Irradiation induced p53-PHLDA3-Akt signaling might be novel therapeutic target in the pathogenesis of heart failure.

Time Course Analysis of Genome-Wide Identification of Mutations Induced by and Genes Expressed in Response to Carbon Ion Beam Irradiation in Rice (Oryza sativa L.).

Heavy-ion irradiation is a powerful mutagen and is widely used for mutation breeding. In this study, using whole-genome sequencing (WGS) and RNA sequencing (RNA-seq) techniques, we comprehensively characterized these dynamic changes caused by mutations at three time points (48, 96, and 144 h after irradiation) and the expression profiles of rice seeds irradiated with C ions at two doses. Subsequent WGS analysis revealed that more mutations were detected in response to 40 Gy carbon ion beam (CIB) irradiation than 80 Gy of CIB irradiation at the initial stage (48 h post-irradiation). In the mutants generated from both irradiation doses, single-base substitutions (SBSs) were the most frequent type of mutation induced by CIB irradiation. Among the mutations, the predominant ones were C:T and A:G transitions. CIB irradiation also induced many short InDel mutations. RNA-seq analysis at the three time points showed that the number of differentially expressed genes (DEGs) was highest at 48 h post-irradiation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the DEGs showed that the “replication and repair” pathway was enriched specifically 48 h post-irradiation. These results indicate that the DNA damage response (DDR) and the mechanism of DNA repair tend to quickly start within the initial stage (48 h) after irradiation.

Effect of Carbon Ion Radiation Induces Bystander Effect on Metastasis of A549 Cells and Metabonomic Correlation Analysis.

ObjectiveTo analyze the effect of carbon ion (12C6+) radiation may induce bystander effect on A549 cell metastasis and metabonomics.MethodsA549 cell was irradiated with carbon ion to establish the clone survival model and the transwell matrix assay was applied to measure the effect of carbon ion on cell viability, migration, and invasion, respectively. Normal human embryonic lung fibroblasts (WI-38) were irradiated with carbon ions of 0 and 2 Gy and then transferred to A549 cell co-culture medium for 24 h. The migration and invasion of A549 cells were detected by the Transwell chamber. The analysis of metabonomic information in transfer medium by liquid phase mass spectrometry (LC-MS), The differential molecules were obtained by principal pomponent analysis (PCA) and the target proteins of significant differences (p = 1.7 × 10−3) obtained by combining with the STICH database. KEGG pathway was used to analyze the enrichment of the target protein pathway.ResultsCompared with 0 Gy, the colony formation, migration, and invasion of A549 cells were significantly inhibited by carbon ion 2 and 4 Gy irradiation, while the inhibitory effect was not significant after 1 Gy irradiation. Compared with 0 Gy, the culture medium 24 h after carbon ion 2 Gy irradiation significantly inhibited the metastasis of tumor cells (p = 0.03). LC-MS analysis showed that 23 differential metabolites were obtained in the cell culture medium 24 h after carbon ion 0 and 2 Gy irradiation (9 up-regulated and 14 down-regulated). Among them, two were up-regulated and two down-regulated (p = 2.9 × 10−3). 41 target proteins were corresponding to these four differential molecules. Through the analysis of the KEGG signal pathway, it was found that these target molecules were mainly enriched in purine metabolism, tyrosine metabolism, cysteine and methionine metabolism, peroxisome, and carbon metabolism. Neuroactive ligand-receptor interaction, calcium signaling pathway, arachidonic acid metabolism, and Fc epsilon RI signaling pathway.ConclusionThe bystander effect induced by 2 Gy carbon ion radiation inhibits the metastasis of tumor cells, which indicates that carbon ions may change the metabolites of irradiated cells, so that it may indirectly affect the metabolism of tumor cell growth microenvironment, thus inhibiting the metastasis of malignant tumor cells.

Carbon ion dosimetry on a fluorescent nuclear track detector using widefield microscopy

Fluorescent nuclear track detectors (FNTDs) are solid-state dosimeters used in a wide range of dosimetric and biomedical applications in research worldwide. FNTDs are a core but currently underutilized dosimetry tool in the field of radiation biology which are inherently capable of visualizing the tracks of ions used in hadron therapy. The ions that traverse the FNTD deposit their energy according to their linear energy transfer and transform colour centres to form trackspots around their trajectory. These trackspots have fluorescent properties which can be visualized by fluorescence microscopy enabling a well-defined dosimetric readout with a spatial component indicating the trajectory of individual ions. The current method used to analyse the FNTDs is laser scanning confocal microscopy (LSM). LSM enables a precise localization of track spots in x, y and z however due to the scanning of the laser spot across the sample, requires a long time for large samples. This body of work conclusively shows for the first time that the readout of the trackspots present after 0.5 Gy carbon ion irradiation in the FNTD can be captured with a widefield microscope (WF). The WF readout of the FNTD is a factor ∼10 faster, for an area 2.97 times the size making the method nearly a factor 19 faster in track acquisition than LSM. The dramatic decrease in image acquisition time in WF presents an alternative to LSM in FNTD workflows which are limited by time, such as biomedical sensors which combine FNTDs with live cell imaging.

Regulation of DNA Damage Signal-dependent PD-L1 Upregulation In Cancer Cells After Ionizing Irradiation

Studies have suggested that the combination of radiotherapy and anti-PD-1/PD-L1 antibody is promising strategy for cancer treatment. PD-L1 upregulation induced by radiotherapy has been considered as one of the favorable backgrounds. We demonstrated that DNA double-strand break (DSB)-related DNA damage signaling induced by X-ray irradiation upregulates PD-L1 expression. However, the mechanisms underlying PD-L1 upregulation mediated by DNA damage signals after ionizing irradiation have not been completely elucidated. Therefore, we examined the mechanisms underlying PD-L1 upregulation after carbon-ion irradiation with high linear energy transfer (LET) or oxidative DNA stress. Human cancer cells (U2OS, MCF7, HCT116, and H1299) were irradiated by X-ray or carbon-ion beam with high LET (60 keV/μm) or applied hydrogen peroxide (H2O2). PD-L1 expression was analyzed by immunoblotting, real-time PCR, and flow cytometry. We investigated the involvement of ataxia telangiectasia and Rad3-related protein (ATR) or Chk1 in PD-L1 upregulation after DNA damage using their specific inhibitors. Then, siRNA-depleted cells were analyzed to determine if base excision repair (BER) or single-strand break repair (SSBR) genes are involved in PD-L1 upregulation after DNA damage. PD-L1 distribution on cell surfaces was visualized by super-resolution microscopy. TCGA dataset analysis was used to examine PD-L1 levels in neoplastic samples to confirm reproducibility in clinical samples. PD-L1 upregulation was observed 48 h after DNA damage induced by 10 Gy carbon-ion irradiation or H2O2 treatment. PD-L1 upregulation was higher after carbon-ion irradiation than that with the same dose of X-rays. PD-L1 upregulations were significantly suppressed after ATR/Chk1 pathway inhibition. Furthermore, PD-L1 upregulation after DNA damage required STAT1 phosphorylation and IRF1 expression. siRNA screening revealed that the majority of BER/SSBR siRNAs augmented PD-L1 upregulation compared with that in control cells after X-ray irradiation. TCGA dataset analysis showed that BER-mutated cancer with high neoantigen production exhibited further increase in PD-L1 expression compared with the BER wild-type cancer with high neoantigen production. Thus, high PD-L1 expression in BER-mutated cancer is not only dependent on the neoantigen pathway but also dependent on the DNA damage signals. Our study demonstrates that DNA damage signaling from DSB repair, SSBR, and BER after ionizing radiation or oxidative DNA stress regulates the expression of PD-L1 in cancer cells. These findings suggest that the dataset of the expression status of DNA repair genes prior to the treatment would be informative for considering the combination of radiotherapy and immune checkpoint therapy.

Hadron Therapy, Magnetic Nanoparticles and Hyperthermia: A Promising Combined Tool for Pancreatic Cancer Treatment.

A combination of carbon ions/photons irradiation and hyperthermia as a novel therapeutic approach for the in-vitro treatment of pancreatic cancer BxPC3 cells is presented. The radiation doses used are 0–2 Gy for carbon ions and 0–7 Gy for 6 MV photons. Hyperthermia is realized via a standard heating bath, assisted by magnetic fluid hyperthermia (MFH) that utilizes magnetic nanoparticles (MNPs) exposed to an alternating magnetic field of amplitude 19.5 mTesla and frequency 109.8 kHz. Starting from 37 °C, the temperature is gradually increased and the sample is kept at 42 °C for 30 min. For MFH, MNPs with a mean diameter of 19 nm and specific absorption rate of 110 ± 30 W/gFe3o4 coated with a biocompatible ligand to ensure stability in physiological media are used. Irradiation diminishes the clonogenic survival at an extent that depends on the radiation type, and its decrease is amplified both by the MNPs cellular uptake and the hyperthermia protocol. Significant increases in DNA double-strand breaks at 6 h are observed in samples exposed to MNP uptake, treated with 0.75 Gy carbon-ion irradiation and hyperthermia. The proposed experimental protocol, based on the combination of hadron irradiation and hyperthermia, represents a first step towards an innovative clinical option for pancreatic cancer.

Biological effects of three types of ionizing radiation on creeping bentgrass

Purpose: Gamma-rays and carbon ions are frequently used for mutation breeding in diverse plant species, whereas proton ions have been rarely used for this purpose. This study assessed the potential of proton ions for plant mutation breeding.Materials and methods: We compared the effects of radiation on creeping bentgrass seeds with γ-rays, proton ions, and carbon ions on seed germination, plant growth parameters, and DNA fragmentation.Results and conclusions: The lethal dose 50 (LD50) doses based on seed germinability were 115.9 Gy (γ-rays), 225.1 Gy (proton ions), and 57.7 Gy (carbon ions). Threshold doses for survival were 150 Gy (γ-rays), 150 Gy (proton ions), and 25 Gy (carbon ions). Suppression of plant growth was displayed at 100 Gy (γ-rays), 25 Gy (proton ions), and 25 Gy (carbon ions). Similar patterns of decreasing head DNA percentage were observed for γ-rays and proton ions. Carbon ions induced the lowest frequency of DNA fragmentation. The biological effects of the ionizing radiation types on creeping bentgrass are summarizable as follows: germination, carbon ions (C)>γ-rays (G)>proton ions (P); survival, C > P = G; growth, C ≥ P > G; DNA fragmentation, G ≥ P > C. These results indicate that proton ions are useful as a physical mutagen in plant mutation breeding.

HMGB1 mediated autophagy protects glioblastoma cells from carbon-ion beam irradiation injury

The present study investigated autophagy changes and the expression of HMGB1 in human glioblastoma cells, responding to carbon-ion beam irradiation (35 keV/μm, 80.55 MeV/u). U251 cells were irradiated with carbon-ion beams and cell proliferation was measured by counting the number of living cells. The expression of Light Chain 3 beta (LC3B), Beclin 1, high-mobility-group box 1 (HMGB1), pro-form caspase-3 and Cellular FLICE-like inhibitory protein (c-FLIP) was analyzed by western blotting. Caspase enzyme activity was determined via a caspase cleavage based florescent substrate commercial Kit. Living cell counting demonstrated a time- and dose-dependent cell death in U251 cells. The expression of LC3B and Beclin 1 revealed that, a high level of autophagy was induced 24 h after irradiation with 1 Gy carbon ions and then decreased in a time- and dose-dependent manner. The expression of the whole HMGB1 showed a well correlation with the dynamic autophagic level. Cytoplasmic HMGB1 maintained autophagy was concluded. Enzyme-Linked Immuno Sorbent Assay (ELISA) measurement found that, HMGB1 was released into the extracellular space in a time- and dose-dependent manner. Lower intracellular HMGB1 levels correlated with decreased autophagy as measured by the expression of LC3B. Decreased expression of pro-form caspase-3 and c-FLIP as well as the increased caspase enzyme activity indicated that apoptosis was induced by carbon-ion beam irradiation. Inhibition of HMGB1 release from the area of intracellular to that of extracellular significantly increased cell survival. In summary, carbon-ion beam irradiation could elevate autophagy and HMGB1 expression efficiently, which would protect the cells from programmed cell death via inducing autophagy. Apoptosis as measured by expression of caspase activities increased as the dose increased, which was accompanied with decreased levels of LC3B and HMGB1.

Comparative Analysis of Mitochondrial Proteome Reveals the Mechanism of Enhanced Ram Sperm Motility Induced by Carbon Ion Radiation After In Vitro Liquid Storage.

The aim of this study was to reveal the mechanism of enhanced ram sperm motility induced by heavy ion radiation (HIR) after in vitro liquid storage. Ram semen was stored for 24 hours at 5°C and then irradiated with 0.1 Gy carbon ion radiation (CIR). In comparison to nonirradiated (NIR) sperm, the motility, viability, and adenosine triphosphate content were all higher in CIR sperm, and the reactive oxygen species levels were lower. Moreover, 87 differential mitochondrial protein spots were detected in 2-dimensional gels between CIR and NIR sperm and were identified as 52 corresponding proteins. In addition, 33 differential proteins were involved in a main pathway network, including COX5B, ERAB/HSD17B10, ETFA, SDHB, and SOD2, which are known to be involved in cell communication, energy production, and antioxidant responses. We used immunoblotting and immunofluorescence to analyze the content and localization of these proteins, respectively, and the levels of these proteins in CIR sperm were lower than those in NIR sperm. An understanding of the molecular function of these proteins could provide further insight into the mechanisms underlying high sperm motility induced by HIR in rams.

Ameliorating mitochondrial dysfunction restores carbon ion-induced cognitive deficits via co-activation of NRF2 and PINK1 signaling pathway

Carbon ion therapy is a promising modality in radiotherapy to treat tumors, however, a potential risk of induction of late normal tissue damage should still be investigated and protected. The aim of the present study was to explore the long-term cognitive deficits provoked by a high-linear energy transfer (high-LET) carbon ions in mice by targeting to hippocampus which plays a crucial role in memory and learning. Our data showed that, one month after 4 Gy carbon ion exposure, carbon ion irradiation conspicuously resulted in the impaired cognitive performance, neurodegeneration and neuronal cell death, as well as the reduced mitochondrial integrity, the disrupted activities of tricarboxylic acid cycle flux and electron transport chain, and the depressed antioxidant defense system, consequently leading to a decline of ATP production and persistent oxidative damage in the hippocampus region. Mechanistically, we demonstrated the disruptions of mitochondrial homeostasis and redox balance typically characterized by the disordered mitochondrial dynamics, mitophagy and glutathione redox couple, which is closely associated with the inhibitions of PINK1 and NRF2 signaling pathway as the key regulators of molecular responses in the context of neurotoxicity and neurodegenerative disorders. Most importantly, we found that administration with melatonin as a mitochondria-targeted antioxidant promoted the PINK1 accumulation on the mitochondrial membrane, and augmented the NRF2 accumulation and translocation. Moreover, melatonin pronouncedly enhanced the molecular interplay between NRF2 and PINK1. Furthermore, in the mouse hippocampal neuronal cells, overexpression of NRF2/PINK1 strikingly protected the hippocampal neurons from carbon ion-elicited toxic insults. Thus, these data suggest that alleviation of the sustained mitochondrial dysfunction and oxidative stress through co-modulation of NRF2 and PINK1 may be in charge of restoration of the cognitive impairments in a mouse model of high-LET carbon ion irradiation.

Comparative Profiling of MicroRNAs Reveals the Underlying Toxicological Mechanism in Mice Testis Following Carbon Ion Radiation.

This study investigated the toxicity of heavy ion radiation to mice testis by microRNA (miRNA) sequencing and bioinformatics analyses. Testicular indices and histology were measured following enterocoelia irradiation with a 2 Gy carbon ion beam, with the testes exhibiting the most serious injuries at 4 weeks after carbon ion radiation (CIR) exposure. Illumina sequencing technology was used to sequence small RNA libraries of the control and irradiated groups at 4 weeks after CIR. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses implicated differential miRNAs in the regulation of target genes involved in metabolism, development, and reproduction. Here, 8 miRNAs, including miR-34c-5p, miR-138, and 6 let-7 miRNA family members previously reported in testis after radiation, were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to validate miRNA sequencing data. The differentially expressed miRNAs described here provided a novel perspective for the role of miRNAs in testis toxicity following CIR.

Characterizing the Potency and Impact of Carbon Ion Therapy in a Primary Mouse Model of Soft Tissue Sarcoma.

Carbon ion therapy (CIT) offers several potential advantages for treating cancers compared with X-ray and proton radiotherapy, including increased biological efficacy and more conformal dosimetry. However, CIT potency has not been characterized in primary tumor animal models. Here, we calculate the relative biological effectiveness (RBE) of carbon ions compared with X-rays in an autochthonous mouse model of soft tissue sarcoma. We used Cre/loxP technology to generate primary sarcomas in KrasLSL-G12D/+; p53fl/fl mice. Primary tumors were irradiated with a single fraction of carbon ions (10 Gy), X-rays (20 Gy, 25 Gy, or 30 Gy), or observed as controls. The RBE was calculated by determining the dose of X-rays that resulted in similar time to posttreatment tumor volume quintupling and exponential growth rate as 10 Gy carbon ions. The median tumor volume quintupling time and exponential growth rate of sarcomas treated with 10 Gy carbon ions and 30 Gy X-rays were similar: 27.3 and 28.1 days and 0.060 and 0.059 mm3/day, respectively. Tumors treated with lower doses of X-rays had faster regrowth. Thus, the RBE of carbon ions in this primary tumor model is 3. When isoeffective treatments of carbon ions and X-rays were compared, we observed significant differences in tumor growth kinetics, proliferative indices, and immune infiltrates. We found that carbon ions were three times as potent as X-rays in this aggressive tumor model and identified unanticipated differences in radiation response that may have clinical implications. Mol Cancer Ther; 17(4); 858-68. ©2018 AACR.

Impact of Co-Culturing with Fractionated Carbon-Ion-Irradiated Cancer Cells on Bystander Normal Cells and Their Progeny.

The purpose of this study was to compare the biological effects of fractionated doses versus a single dose of high-LET carbon ions in bystander normal cells, and determine the effect on their progeny using the layered tissue co-culture system. Briefly, confluent human glioblastoma (T98G) cells received a single dose of 6 Gy or three daily doses of 2 Gy carbon ions, which were then seeded on top of an insert with bystander normal skin fibroblasts (NB1RGB) growing underneath. Cells were co-cultured for 6 h or allowed to grow for 20 population doublings, then harvested and assayed for different end points. A single dose of carbon ions resulted in less damage in bystander normal NB1RGB cells than the fractionated doses. In contrast, the progeny of bystander NB1RGB cells co-cultured with T98G cells exposed to fractionated doses showed less damage than progeny from bystander cells co-cultured with single dose glioblastoma cells. Furthermore, inhibition of gap junction communication demonstrated its involvement in the stressful effects in bystander cells and their progeny. These results indicate that dose fractionation reduced the late effect of carbon-ion exposure in the progeny of bystander cells compared to the effect in the initial bystander cells.

The bystander cell-killing effect mediated by nitric oxide in normal human fibroblasts varies with irradiation dose but not with radiation quality

Purpose: To investigate the dependence of the bystander cell-killing effect on radiation dose and quality, and to elucidate related molecular mechanisms.Materials and methods: Normal human fibroblast WI-38 cells were irradiated with 0.125 - 2 Gy of γ-rays or carbon ions and were co-cultured with non-irradiated cells. Survival rates of bystander cells were investigated using the colony formation assays, and nitrite concentrations in the medium were measured using the modified Saltzman method.Results: Survival rates of bystander cells decreased with doses of γ-rays and carbon ions of ≤ 0.5 Gy. Treatment of the specific nitric oxide (NO) radical scavenger prevented reductions in survival rates of bystander cells. Moreover, nitrite concentrations increased with doses of less than 0.25 Gy (γ-rays) and 1 Gy (carbon ions). The dose responses of increased nitrite concentrations as well as survival reduction were similar between γ-rays and carbon ions. In addition, negative relationships were observed between survival rates and nitrite concentrations.Conclusion: The bystander cell-killing effect mediated by NO radicals in normal human fibroblasts depends on irradiation doses of up to 0.5 Gy, but not on radiation quality. NO radical production appears to be an important determinant of γ-ray- and carbon-ion-induced bystander effects.

Combination of carbon ion beam and gemcitabine causes irreparable DNA damage and death of radioresistant pancreatic cancer stem-like cells in vitro and in vivo.

We try to elucidate whether a carbon ion beam alone or in combination with gemcitabine has advantages over X-ray in targeting putative pancreatic cancer stem-like cells (CSCs) in vitro and in vivo. Colony, spheroid formation and tumorigenicity assays confirmed that CD44+/ESA+ cells sorted from PANC1 and PK45 cells have more CSC properties than CD44-/ESA- cells. The number of colonies and spheroids formed from CSCs after carbon ion beam irradiation was significantly reduced compared to after X-ray irradiation, and they were extremely highly suppressed when carbon ion beam combined with gemcitabine. The relative biological effectiveness (RBE) values for the carbon ion beam relative to X-ray at the D10 levels for CSCs were 2.23-2.66. Expressions of multiple cell death-related genes were remarkably highly induced, and large numbers of γH2AX foci in CSCs were formed after carbon ion beam combined with gemcitabine. The highly expressed CSC markers were significantly inhibited after 30 Gy of carbon ion beam and almost lost after 25 Gy carbon ion beam combined with 50 mg/kg gemcitabine. In conclusion, a carbon ion beam combined with gemcitabine has superior potential to kill pancreatic CSCs via irreparable clustered DSB compared to a carbon ion alone or X-rays combined with gemcitabine.