Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.

Nuclear factor (erythroid-derived 2)-like 2 antioxidative response mitigates cytoplasmic radiation-induced DNA double-strand breaks.

It has been reported that DNA double-strand breaks (DSB) can be induced by cytoplasm irradiation, and that both reactive free radicals and mitochondria are involved in DSB formation. However, the cellular antioxidative responses that are stimulated and the biological consequences of cytoplasmic irradiation remain unknown. Using the Single Particle Irradiation system to Cell (SPICE) proton microbeam facility at the National Institute of Radiological Sciences ([NIRS] Japan), the response of nuclear factor (erythroid-derived 2)-like 2 (NRF2) antioxidative signaling to cytoplasmic irradiation was studied in normal human lung fibroblast WI-38 cells. Cytoplasmic irradiation stimulated the localization of NRF2 to the nucleus and the expression of its target protein, heme oxygenase 1. Activation of NRF2 by tert-butylhydroquinone mitigated the levels of DSB induced by cytoplasmic irradiation. Mitochondrial fragmentation was also promoted by cytoplasmic irradiation, and treatment with mitochondrial division inhibitor 1 (Mdivi-1) suppressed cytoplasmic irradiation-induced NRF2 activation and aggravated DSB formation. Furthermore, p53 contributed to the induction of mitochondrial fragmentation and activation of NRF2, although the expression of p53 was significantly downregulated by cytoplasmic irradiation. Finally, mitochondrial superoxide (MitoSOX) production was enhanced under cytoplasmic irradiation, and use of the MitoSOX scavenger mitoTEMPOL indicated that MitoSOX caused alterations in p53 expression, mitochondrial dynamics, and NRF2 activation. Overall, NRF2 antioxidative response is suggested to play a key role against genomic DNA damage under cytoplasmic irradiation. Additionally, the upstream regulators of NRF2 provide new clues on cytoplasmic irradiation-induced biological processes and prevention of radiation risks.

Cellular localization of uranium in the renal proximal tubules during acute renal uranium toxicity.

Renal toxicity is a hallmark of uranium exposure, with uranium accumulating specifically in the S3 segment of the proximal tubules causing tubular damage. As the distribution, concentration and dynamics of accumulated uranium at the cellular level is not well understood, here, we report on high-resolution quantitative in situ measurements by high-energy synchrotron radiation X-ray fluorescence analysis in renal sections from a rat model of uranium-induced acute renal toxicity. One day after subcutaneous administration of uranium acetate to male Wistar rats at a dose of 0.5 mg uranium kg(-1) body weight, uranium concentration in the S3 segment of the proximal tubules was 64.9 ± 18.2 µg g(-1) , sevenfold higher than the mean renal uranium concentration (9.7 ± 2.4 µg g(-1) ). Uranium distributed into the epithelium of the S3 segment of the proximal tubules and highly concentrated uranium (50-fold above mean renal concentration) in micro-regions was found near the nuclei. These uranium levels were maintained up to 8 days post-administration, despite more rapid reductions in mean renal concentration. Two weeks after uranium administration, damaged areas were filled with regenerating tubules and morphological signs of tissue recovery, but areas of high uranium concentration (100-fold above mean renal concentration) were still found in the epithelium of regenerating tubules. These data indicate that site-specific accumulation of uranium in micro-regions of the S3 segment of the proximal tubules and retention of uranium in concentrated areas during recovery are characteristics of uranium behavior in the kidney.

Dose Rate Effects on Hydrated Electrons, Hydrogen Peroxide, and a OH Radical Molecular Probe Under Clinical Energy Protons.

We report the dose rate dependence of radiation chemical yields (G value) of water radiolysis products under clinical energy protons (230 MeV) to understand mechanisms of the FLASH radiotherapy performed at ultra-high dose rate (>40 Gy/s). The G value of 7-hydoroxy-coumarin-3-carboxylic acid (7OH-C3CA) produced by reactions of coumarin-3-carboxylic acid (C3CA) with OH radicals and oxygen is evaluated by fluorescence method. Also, those of hydrated electrons and hydrogen peroxide are derived by absorption method using Saltzman and Ghomley techniques, respectively. Both G values of 7OH-C3CA and hydrated electrons decrease with increasing dose rate. The relative evolution of 7OH-C3CA is -39 ± 2% between 0.1 and 50 Gy/s. This value is higher than that of hydrated electrons, measured at -21 ± 4%. The G value of hydrogen peroxide in ultra-pure water also decreases with increasing dose rate. In comparison to these findings, we represent the increase of the G value of hydrogen peroxide with increasing dose rate in the mixture solution of MeOH and NaNO3, which act as scavengers of OH radicals and hydrated electrons, respectively, that decompose hydrogen peroxide. This finding indicates that a complex track structure can be expected with increasing dose rate and the reduction of OH radicals by forming hydrogen peroxide would be related to the sparing effect of healthy tissues.

The threshold number of protons to induce an adaptive response in zebrafish embryos.

In this study, microbeam protons were used to provide the priming dose to induce an in vivo radioadaptive response (RAR) in the embryos of zebrafish, Danio rerio, against subsequent challenging doses provided by x-ray photons. The microbeam irradiation system (Single-Particle Irradiation System to Cell, acronym SPICE) at the National Institute of Radiological Sciences (NIRS), Japan, was employed. The embryos were dechorionated at 4 h post fertilisation (hpf) and irradiated at 5 hpf by microbeam protons. For each embryo, one irradiation point was chosen, to which 5, 10, 20, 30, 40, 50, 100, 200, 300 and 500 protons each with an energy of 3.4 MeV were delivered. The embryos were returned to the incubator until 10 hpf to further receive the challenging exposure, which was achieved using 2 Gy of x-ray irradiation, and then again returned to the incubator until 24 hpf for analyses. The levels of apoptosis in zebrafish embryos at 25 hpf were quantified through terminal dUTP transferase-mediated nick end-labelling (TUNEL) assay. The results revealed that at least 200 protons (with average radiation doses of about 300 and 650 mGy absorbed by an irradiated epithelial and deep cell, respectively) would be required to induce RAR in the zebrafish embryos in vivo. Our previous investigation showed that 5 protons delivered at 10 points on an embryo would already be sufficient to induce RAR in the zebrafish embryos. The difference was explained in terms of the radiation-induced bystander effect as well as the rescue effect.

Stimulation of Nuclear Factor (Erythroid-Derived 2)-like 2 Signaling by Nucleus Targeted Irradiation with Proton Microbeam.

Nuclear factor (erythroid-derived 2)-like 2 (NRF2), well-known as a master antioxidative response regulator in mammalian cells, is considered as a potential target for radiation protection and cancer therapy sensitization. We examined the response of NRF2 signaling in normal human lung fibroblast WI-38 cells to nucleus targeted irradiation by 3.4 MeV proton microbeam. Nucleus targeted irradiation stimulated the nucleus accumulation of NRF2 and the expression of its target gene, heme oxygenase 1 (HO-1). The nucleus accumulation of NRF2 increased from 3 h to 12 h post 500 proton irradiation. In the 500 protons range, higher number of protons resulted in increased NRF2 nucleus accumulation. Activating NRF2 with tert-butylhydroquinone reduced DNA double-strand break (DSB) formation in nucleus targeted irradiation by 15%. Moreover, ATM phosphorylation was found in nucleus targeted irradiation. Inhibiting ATM with ku55933 prevented NRF2 nucleus accumulation. Furthermore, nucleus targeted irradiation activated ERK 1/2, and ROS-ERK 1/2 signaling regulated NRF2 nucleus accumulation. Taken together, NRF2 signaling was activated by nucleus targeted irradiation and mitigated DNA DSB. The discovery of ATM and ERK 1/2 as upstream regulators of NRF2 signaling in nucleus targeted cells revealed new information regarding radiation protection.

Induction of DNA strand breaks and oxidative base damages in plasmid DNA by ultra-high dose rate proton irradiation.

PURPOSE: Radiation cancer therapy with ultra-high dose rate (UHDR) exposure, so-called FLASH radiotherapy, appears to reduce normal tissue damage without compromising tumor response to therapy. The aim of this study was to clarify whether a 59.5 MeV proton beam at an UHDR of 48.6 Gy/s could effectively reduce the DNA damage of pBR322 plasmid DNA in solution compared to the conventional dose rate (CONV) of 0.057 Gy/s. MATERIALS AND METHODS: A simple system, consisting of pBR322 plasmid DNA in 1× Tris-EDTA buffer, was initially employed for proton beam exposure. We then used formamidopyrimidine-DNA glycosylase (Fpg) enzymes. which convert oxidative base damages of oxidized purines to DNA strand breaks, to quantify DNA single strand breaks (SSBs) and double strand breaks (DSBs) by agarose gel electrophoresis. RESULTS: Our findings showed that the SSB induction rate (SSB per plasmid DNA/Gy) at UHDR and the induction of Fpg enzyme sensitive sites (ESS) were significantly reduced in UHDR compared to CONV. However, there was no significant difference in DSB induction and non-DSB cluster damages. CONCLUSIONS: UHDR of a 59.5 MeV proton beam could reduce non-clustered, non-DSB damages, such as SSB and sparsely distributed ESS. However, this effect may not be significant in reducing lethal DNA damage that becomes apparent only in acute radiation effects of mammalian cells and in vivo studies.

The COX-2/PGE2 Response Pathway Upregulates Radioresistance in A549 Human Lung Cancer Cells through Radiation-Induced Bystander Signaling.

This study aimed to determine the mechanism underlying the modulation of radiosensitivity in cancer cells by the radiation-induced bystander effect (RIBE). We hypothesized that the RIBE mediates cyclooxygenase-2 (COX-2) and its metabolite prostaglandin E2 (PGE2) in elevating radioresistance in unirradiated cells. In this study, we used the SPICE-QST microbeam irradiation system to target 0.07-0.7% cells by 3.4-MeV proton microbeam in the cell culture sample, such that most cells in the dish became bystander cells. Twenty-four hours after irradiation, we observed COX-2 protein upregulation in microbeam-irradiated cells compared to that of controls. Additionally, 0.29% of the microbeam-irradiated cells exhibited increased cell survival and a reduced micronucleus rate against X-ray irradiation compared to that of non-microbeam irradiated cells. The radioresistance response was diminished in both cell groups with the hemichannel inhibitor and in COX-2-knockout cells under cell-to-cell contact and sparsely distributed conditions. The results indicate that the RIBE upregulates the cell radioresistance through COX-2/PGE2 intercellular responses, thereby contributing to issues, such as the risk of cancer recurrence.

Primary and Secondary Bystander Effects of Proton Microbeam Irradiation on Human Lung Cancer Cells under Hypoxic Conditions.

Tumor hypoxia is the most common feature of radioresistance to the radiotherapy (RT) of lung cancer and results in poor clinical outcomes. High-linear energy transfer (LET) radiation is a novel RT technique to overcome this problem. However, a limited number of studies have been elucidated on the underlying mechanism(s) of RIBE and RISBE in cancer cells exposed to high-LET radiation under hypoxia. Here, we developed a new method to investigate the RIBE and RISBE under hypoxia using the SPICE-QST proton microbeams and a layered tissue co-culture system. Normal lung fibroblast (WI-38) and lung cancer (A549) cells were exposed in the range of 06 Gy of proton microbeams, wherein only ~0.04-0.15% of the cells were traversed by protons. Subsequently, primary bystander A549 cells were co-cultured with secondary bystander A549 cells in the presence or absence of a GJIC and NO inhibitor using co-culture systems. Studies show that there are differences in RIBE in A549 and WI-38 primary bystander cells under normoxia and hypoxia. Interestingly, treatment with a GJIC inhibitor showed an increase in the toxicity of primary bystander WI-38 cells but a decrease in A549 cells under hypoxia. Our results also show the induction of RISBE in secondary bystander A549 cells under hypoxia, where GJIC and NO inhibitors reduced the stressful effects on secondary bystander A549 cells. Together, these preliminary results, for the first time, represented the involvement of intercellular communications through GJIC in propagation of RIBE and RISBE in hypoxic cancer cells.

DNA strand break induction of aqueous plasmid DNA exposed to 30 MeV protons at ultra-high dose rate.

Radiation cancer therapy with ultra-high dose rate exposure, so called FLASH radiotherapy, appears to reduce normal tissue damage without compromising tumor response. The aim of this study was to clarify whether FLASH exposure of proton beam would be effective in reducing the DNA strand break induction. We applied a simple model system, pBR322 plasmid DNA in aqueous 1 × TE solution, where DNA single strand breaks (SSBs) and double strand breaks (DSBs) can be precisely quantified by gel electrophoresis. Plasmid DNA were exposed to 27.5 MeV protons in the conventional dose rate of 0.05 Gy/s (CONV) and ultra-high dose rate of 40 Gy/s (FLASH). With both dose rate, the kinetics of the SSB and DSB induction were proportional to absorbed dose. The SSB induction of FLASH was significantly less than CONV, which were 8.79 ± 0.14 (10-3 SSB per Gy per molecule) and 10.8 ± 0.68 (10-3 SSB per Gy per molecule), respectively. The DSB induction of FLASH was also slightly less than CONV, but difference was not significant. Altogether, 27.5 MeV proton beam at 40 Gy/s reduced SSB and not DSB, thus its effect may not be significant in reducing lethal DNA damage that become apparent in acute radiation effect.

Radiation Chemical Yields of 7-Hydroxy-Coumarin-3-Carboxylic Acid for Proton- and Carbon-Ion Beams at Ultra-High Dose Rates: Potential Roles in FLASH Effects.

It has been observed that healthy tissues are spared at ultra-high dose rate (UHDR: >40 Gy/s), so called FLASH effect. To elucidate the mechanism of FLASH effect, we evaluate changes in radiation chemical yield (G value) of 7-hydroxy-coumarin-3-carboxylic acid (7OH-C3CA), which is formed by the reaction of hydroxyl radicals with coumarin-3-carboxylic acid (C3CA), under carbon ions (140 MeV/u) and protons (27.5 and 55 MeV) in a wide-dose-rate range up to 100 Gy/s. The relative G value, which is the G value at each dose rate normalized by that at the conventional dose (CONV: 0.1 Gy/s >), 140 MeV/u carbon-ion beam is almost equivalent to 27.5 and 55 MeV proton beams. This finding implies that UHDR irradiations using carbon-ion beams have a potential to spare healthy tissues. Furthermore, we evaluate the G value of 7OH-C3CA under the de-oxygenated condition to investigate roles of oxygen to the generation of 7OH-C3CA effect. The G value of 7OH-C3CA under the de-oxygenated condition is lower than that under the oxygenated condition. The G value of 7OH-C3CA under the de-oxygenated condition is higher than those under UHDR irradiations. By direct measurements of the oxygen concentration during 55 MeV proton irradiations, the oxygen concentration drops by 0.1%/Gy, which is independent of the dose rate. When the oxygen concentration directly affects to yields of 7OH-C3CA, the rate of decrease in the oxygen concentration may be correlated with that of decrease in the G value of 7OH-C3CA. However, the reduction rate of G value under UHDR is significantly higher than the oxygen consumption. This finding implied that the influence of the reaction between water radiolysis species formed by neighborhood tracks could be strongly related to the mechanisms of UHDR effect.

Hypoxia and Proton microbeam: Role of Gap Junction Intercellular Communication in Inducing Bystander Responses on Human Lung Cancer Cells and Normal Cells.

Radiation-induced bystander effect (RIBE) has been identified as an important contributing factor to tumor resistance and normal tissue damage. However, the RIBE in cancer and normal cells under hypoxia remain unclear. In this study, confluent A549 cancer and WI-38 normal cells were subjected to condition of hypoxia or normoxia, before exposure to high-LET protons microbeam. After 6 h incubation, cells were harvested and assayed for colony formation, micronucleus formation, chromosome aberration and western blotting. Our results show that there were differences of RIBE in bystander A549 and WI-38 cells under hypoxia and normoxia. The differences were also observed in the roles of HIF-1α expression in bystander A549 and WI-38 cells under both conditions. Furthermore, inhibition of gap junction intercellular communication (GJIC) showed a decrease in toxicity of hypoxia-treated bystander A549 cells, but increased in bystander WI-38 cells. These findings clearly support that GJIC protection of bystander normal cells from toxicity while enhancing in bystander cancer cells. Together, the data show a promising strategy for high-LET radiation in designing an entire new line of drugs, either increase or restore GJIC in bystander cancer cells which in turn leads to enhancement of radiation accuracy for treatment of hypoxic tumors.

Scaling parameter of the lethal effect of mammalian cells based on radiation-induced OH radicals: effectiveness of direct action in radiation therapy.

We have been studying the effectiveness of direct action, which induces clustered DNA damage leading to cell killing, relative to indirect action. Here a new criterion Direct Ation-Based Biological Effectiveness (DABBLE) is proposed to understand the contribution of direct action for cell killing induced by C ions. DABBLE is defined as the ratio of direct action to indirect action. To derive this ratio, we describe survival curves of mammalian cells as a function of the number of OH radicals produced 1 ps and 100 ns after irradiation, instead of the absorbed dose. By comparing values on the vertical axis of the survival curves at a certain number of OH radicals produced, we successfully discriminate the contribution of direct action induced by C ions from that of indirect action. DABBLE increases monotonically with increasing linear energy transfer (LET) up to 140 keV/µm and then drops, when the survival curves are described by the number of OH radicals 1 ps after irradiation. The trend of DABBLE is in agreement with that of relative biological effectiveness (RBE) of indirect action. In comparison, the value of DABBLE increases monotonically with LET, when the survival curves are described by the number of OH radicals 100 ns after irradiation. This finding implies that the effectiveness of C ion therapy for cancer depends on the contribution of direct action and we can follow the contribution of direct action over time in the chemical phase.

Radiation engenders converse migration and invasion in colorectal cancer cells through opposite modulation of ANXA2/AKT/GSK3ß pathway.

Radiation therapy is an effective non-surgical means to achieve local control for various solid tumors including colorectal cancer (CRC), but metastasis and recurrences after conventional radiotherapy remains a major obstacle in clinical practice, and the knowledge concerning the changes of metastatic potential after heavy ion radiation is still limited. This study investigated how radiation, including γ- and carbon ion radiation, would change the metastatic capacity of two CRC cell lines, HCT116 and DLD-1, and examined the underlying molecular mechanisms. We found that the migration and invasion was enhanced in DLD-1 cells but impaired in HCT116 cells in vitro and in vivo after radiation of γ-rays or carbons, and radiation induced epithelial mesenchymal transition (EMT) in DLD-1 cells but mesenchymal epithelial transition (MET) in HCT116 cells. The expression of snail, a key inducer of EMT, was significantly enhanced by inhibition of glycogen synthase kinase-3ß (GSK3ß) in both cell lines, suggesting the modulation of snail was alike in the two CRC cell lines. However, radiation inactivated GSK3ß through stimulating the phosphorylation of AKT and GSK3ß at Ser473 and Ser9 in DLD-1 cells respectively, but activated GSK3ß by decreasing the expression of pAKTSer473 and pGSK3ßSer9 or increasing the phosphorylation of GSK3ß at Tyr216 in HCT116 cells. Therefore, the above inverted motility changes was due to the opposite modulation of AKT/GSK3ß signaling pathway by radiation, which was further verified in other type of cancer cell lines including MCF-7, U251 and A549 cells. Moreover, it was found that annexin A2 (ANAX2) directly bound with GSK3ß and acted as a negative regulator of GSK3ß upon radiation. Knocking-down ANXA2 gene reversed the enhanced migration of the irradiated DLD-1 cells and strengthened radiation-impaired migration of HCT116 cells. Collectively, this study reveals that the change of cellular motility after radiation is independent of radiation type but is correlated with the inherent of cells.

Cell lines of the same anatomic site and histologic type show large variability in intrinsic radiosensitivity and relative biological effectiveness to protons and carbon ions.

PURPOSE: To show that intrinsic radiosensitivity varies greatly for protons and carbon (C) ions in addition to photons, and that DNA repair capacity remains important in governing this variability. METHODS: We measured or obtained from the literature clonogenic survival data for a number of human cancer cell lines exposed to photons, protons (9.9 keV/µm), and C-ions (13.3-77.1 keV/µm). We characterized their intrinsic radiosensitivity by the dose for 10% or 50% survival (D10% or D50% ), and quantified the variability at each radiation quality by the coefficient of variation (COV) in D10% and D50% . We also treated cells with DNA repair inhibitors prior to irradiation to assess how DNA repair capacity affects their variability. RESULTS: We found no statistically significant differences in the COVs of D10% or D50% between any of the radiation qualities investigated. The same was true regardless of whether the cells were treated with DNA repair inhibitors, or whether they were stratified into histologic subsets. Even within histologic subsets, we found remarkable differences in radiosensitivity for high LET C-ions that were often greater than the variations in RBE, with brain cancer cells varying in D10% (D50% ) up to 100% (131%) for 77.1 keV/µm C-ions, and non-small cell lung cancer and pancreatic cancer cell lines varying up to 55% (76%) and 51% (78%), respectively, for 60.5 keV/µm C-ions. The cell lines with modulated DNA repair capacity had greater variability in intrinsic radiosensitivity across all radiation qualities. CONCLUSIONS: Even for cell lines of the same histologic type, there are remarkable variations in intrinsic radiosensitivity, and these variations do not differ significantly between photon, proton or C-ion radiation. The importance of DNA repair capacity in governing the variability in intrinsic radiosensitivity is not significantly diminished for higher LET radiation.

Biologic Impact of Different Ultra-Low-Fluence Irradiations in Human Fibroblasts.

In this study, we aimed to evaluate the cellular response of healthy human fibroblasts induced by different types of ultra-low-fluence radiations, including gamma rays, neutrons and high linear energy transfer (LET) heavy ions. NB1RGB cells were pretreated with ultra-low-fluence radiations (~0.1 cGy/7-8 h) of 137Cs gamma rays, 241Am-Be neutrons, helium, carbon and iron ions before being exposed to an X-ray-challenging dose (1.5 Gy). Helium (LET = 2.3 keV/µm), carbon (LET = 13.3 keV/µm) and iron (LET = 200 keV/µm) ions were generated with the Heavy Ion Medical Accelerator in Chiba (HIMAC), Japan. No differences in cell death-measured by colony-forming assay-were observed regardless of the radiation type applied. In contrast, mutation frequency, which was detected through cell transformation into 6-thioguanine resistant clones, was 1.9 and 4.0 times higher in cells pretreated with helium and carbon ions, respectively, compared to cells exposed to X-ray-challenging dose alone. Moreover, cells pretreated with iron ions or gamma-rays showed a mutation frequency similar to cells exposed to X-ray-challenging dose alone, while cells pretreated with neutrons had 0.15 times less mutations. These results show that cellular responses triggered by ultra-low-fluence irradiations are radiation-quality dependent. Altogether, this study shows that ultra-low-fluence irradiations with the same level as those reported in the International Space Station are capable of inducing different cellular responses, including radio-adaptive responses triggered by neutrons and genomic instability mediated by high-LET heavy ions, while electromagnetic radiations (gamma rays) seem to have no biologic impact.

Significant changes in yields of 7-hydroxy-coumarin-3-carboxylic acid produced under FLASH radiotherapy conditions.

FLASH radiotherapy appears to kill off tumor cells while sparing healthy tissues, by irradiation at ultra high dose rate (>40 Gy s-1). The present study aims to clarify the mechanism of the sparing effect by proton irradiation under the FLASH conditions from a viewpoint of radiation chemistry. To do so, we evaluate radiation chemical yields (G values) of 7-hydroxy-coumarin-3-carboxylic acid (7OH-C3CA), which is produced by water radiolysis using coumarin-3-carboxylic acid (C3CA) solution as a radical scavenger of hydroxyl radicals. We shoot 27.5 MeV protons in the dose rate ranging from 0.05 to 160 Gy s-1. The recombination process of hydroxyl radicals produced is followed by varying the concentration of C3CA from 0.2 to 20 mM, which corresponds to the scavenging time scale from 7.1 to 714 ns. The G value of 7OH-C3CA produced decreases with increasing dose rate on the same scavenging time scale. Additionally, the trend of the relative G value normalized at a scavenging time scale of 100 ns, where radical-radical reaction subsides, is consistent in the examined dose rate range. This finding implies that G values of 7OH-C3CA produced reduce with increasing dose rate due to the oxygen depletion. We experimentally present that the sparing effect for healthy tissues would be seen even with a proton beam under the FLASH conditions due to the depletion of oxygen.

Cytoplasmic Radiation Induced Radio-Adaptive Response in Human Lung Fibroblast WI-38 Cells.

It has been reported that in cells exposed to low-dose radiation, radio-adaptive responses can be induced which make irradiated cells refractory to subsequent high-dose irradiation. However, whether adaptive responses are possible when only the cytoplasm, not the nucleus, of the cell is exposed to radiation is still unclear. In this study, using the proton microbeam facility at the National Institute of Radiological Sciences (Japan), we found that cytoplasmic irradiation activates radio-adaptive responses in normal human lung fibroblast WI-38 cells. Our results showed that when cells received cytoplasmic irradiation with 500 protons prior to 2 Gy or 6 Gy X-ray broad-beam irradiation, the DNA double-strand break levels were significantly reduced. In contrast, at cytoplasmic irradiation with less than 100 protons, the radio-adaptive response was not detected. Moreover, the time interval between cytoplasmic irradiation and whole-cell X-ray irradiation should be longer than 6 h for the induction of adaptive responses. In addition, cytoplasmic irradiation elevated the level of cellular mitochondrial superoxide, which enhanced the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK 1/2) and its mediated nuclear accumulation of nuclear factor (erythroid-derived 2)-like 2 (NRF2). This signaling pathway contributed to cytoplasmic irradiation-induced adaptive response as supported by the observations that treatment with the mitochondrial superoxide scavenger mito-tempol, ERK 1/2 inhibitor U0126 or NRF2 inhibitor ML385 could repress the adaptive response. Overall, we showed that cytoplasmic irradiation induces radio-adaptive responses and that mitochondrial superoxide/ERK 1/2/NRF2 signaling is a mechanism. Our results provide new information on the biological effects induced by cytoplasm-targeted irradiation.

Enhancement of Radiosensitivity by Eurycomalactone in Human NSCLC Cells Through G2/M Cell Cycle Arrest and Delayed DNA Double-Strand Break Repair.

Radiotherapy (RT) is an important treatment for non-small cell lung cancer (NSCLC). However, the major obstacles to successful RT include the low radiosensitivity of cancer cells and the restricted radiation dose, which is given without damaging normal tissues. Therefore, the sensitizer that increases RT efficacy without dose escalation will be beneficial for NSCLC treatment. Eurycomalactone (ECL), an active quassinoid isolated from Eurycoma longifolia Jack, has been demonstrated to possess anticancer activity. In this study, we aimed to investigate the effect of ECL on sensitizing NSCLC cells to X-radiation (X-ray) as well as the underlying mechanisms. The results showed that ECL exhibited selective cytotoxicity against the NSCLC cells A549 and COR-L23 compared to the normal lung fibroblast. Clonogenic survival results indicated that ECL treatment prior to irradiation synergistically decreased the A549 and COR-L23 colony number. ECL treatment reduced the expression of cyclin B1 and CDK1/2 leading to induce cell cycle arrest at the radiosensitive G2/M phase. Moreover, ECL markedly delayed the repair of radiation-induced DNA double-strand breaks (DSBs). In A549 cells, pretreatment with ECL not only delayed the resolving of radiation-induced γ-H2AX foci but also blocked the formation of 53BP1 foci at the DSB sites. In addition, ECL pretreatment attenuated the expression of DNA repair proteins Ku-80 and KDM4D in both NSCLC cells. Consequently, these effects led to an increase in apoptosis in irradiated cells. Thus, ECL radiosensitized the NSCLC cells to X-ray via G2/M arrest induction and delayed the repair of X-ray-induced DSBs. This study offers a great potential for ECL as an alternative safer radiosensitizer for increasing the RT efficiency against NSCLC.

Role of Endoplasmic Reticulum and Mitochondrion in Proton Microbeam Radiation-Induced Bystander Effect.

It is well known that mitochondria and the endoplasmic reticulum (ER) play important roles in radiation response, but their functions in radiation-induced bystander effect (RIBE) are largely unclear. In this study, we found that when a small portion of cells in a population of human lung fibroblast MRC-5 cells were precisely irradiated through either the nuclei or cytoplasm with counted microbeam protons, the yield of micronuclei (MN) and the levels of intracellular reactive oxygen species (ROS) in nonirradiated cells neighboring irradiated cells were significantly increased. Mito/ER-tracker staining demonstrated that the mitochondria were clearly activated after nuclear irradiation and ER mass approached a higher level after cytoplasmic irradiation. Moreover, the radiation-induced ROS was diminished by rotenone, an inhibitor of mitochondria activation, but it was not influenced by siRNA interference of BiP, an ER regulation protein. While for nuclear irradiation, rotenone-enhanced radiation-induced ER expression, and BiP siRNA eliminated radiation-induced activation of mitochondria, these phenomena were not observed for cytoplasmic irradiation. Bystander MN was reduced by rotenone but enhanced by BiP siRNA. When the cells were treated with both rotenone and BiP siRNA, the MN yield was reduced for nuclear irradiation but was enhanced for cytoplasmic irradiation. Our results suggest that the organelles of mitochondria and ER have different roles in RIBE with respect to nuclear and cytoplasmic irradiation, and the function of ER is a prerequisite for mitochondrial activation.