Scandium Ion-Promoted Electron-Transfer Disproportionation of 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-Oxide (PTIO•) in Acetonitrile and Its Regeneration Induced by Water.

2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO•), a persistent nitronyl nitroxide radical, has been used for the detection and trapping of nitric oxide, as a redox mediator for batteries, for the activity estimation of antioxidants, and so on. However, there is no report on the reactivity of PTIO• in the presence of redox-inactive metal ions. In this study, it is demonstrated that the addition of scandium triflate, Sc(OTf)3 (OTf = OSO2CF3), to an acetonitrile (MeCN) solution of PTIO• resulted in an electron-transfer disproportionation to generate the corresponding cation (PTIO+) and anion (PTIO-), the latter of which is suggested to be stabilized by Sc3+ to form [(PTIO)Sc]2+. The decay of the absorption band at 361 nm due to PTIO•, monitored using a stopped-flow technique, obeyed second-order kinetics. The second-order rate constant for the disproportionation, thus determined, increased with increasing the Sc(OTf)3 concentration to reach a constant value. A drastic change in the cyclic voltammogram recorded for PTIO• in deaerated MeCN containing 0.10 M Bu4NClO4 was also observed upon addition of Sc(OTf)3, suggesting that the large positive shift of the one-electron reduction potential of PTIO• (equivalent to the one-electron oxidation potential of PTIO-) in the presence of Sc(OTf)3 may result in the disproportionation. When H2O was added to the PTIO•-Sc(OTf)3 system in deaerated MeCN, PTIO• was completely regenerated. It is suggested that the complex formation of Sc3+ with H2O may weaken the interaction between PTIO- and Sc3+, leading to electron-transfer comproportionation to regenerate PTIO•. The reversible disproportionation of PTIO• was also confirmed by electron paramagnetic resonance (EPR) spectroscopy.

Enhanced Inhibition of Cancer Cell Migration by a Planar Catechin Analog.

Cancer cell migration is related to malignancy and patient prognosis. We previously reported that intracellular reactive oxygen species (ROS) promoted cancer cellular migration and invasion and that an antioxidant enzyme could help to attenuate the malignancy. Catechin is known as an antioxidant, and we have developed a catechin analog, planar catechin, which showed an antioxidant activity significantly stronger than that of the parent (+)-catechin. In this study, we examined the effects of the planar catechin on the migration of gastric normal and cancer cells. A scratched assay showed that the planar catechin suppressed the cellular migration rates in both normal and cancer cells, while the prevention levels in cancer cells were remarkable compared to the normal cells. These results suggest that the planar catechin with the enhanced antioxidant activity effectively scavenged the ROS overexpressed in the cancer cells and inhibited cancer cellular activities, including migration.

Photodynamic Therapy for X-ray-Induced Radiation-Resistant Cancer Cells.

Radiotherapy, in which X-rays are commonly used, is one of the most effective procedures for treating cancer. However, some cancer cells become resistant to radiation therapy, leading to poor prognosis. Therefore, a new therapeutic method is required to prevent cancer cells from acquiring radiation resistance. Photodynamic therapy (PDT) is a cancer treatment that uses photosensitizers, such as porphyrin compounds, and low-powered laser irradiation. We previously reported that reactive oxygen species (ROS) derived from mitochondria induce the expression of a porphyrin transporter (HCP1) and that laser irradiation enhances the cytotoxic effect. In addition, X-ray irradiation induces the production of mitochondrial ROS. Therefore, radioresistant cancer cells established with continuous X-ray irradiation would also overexpress ROS, and photodynamic therapy could be an effective therapeutic method. In this study, we established radioresistant cancer cells and examined the therapeutic effects and mechanisms with photodynamic therapy. We confirmed that X-ray-resistant cells showed overgeneration of mitochondrial ROS and elevated expression of HCP1, which led to the active accumulation of porphyrin and an increase in cytotoxicity with laser irradiation. Thus, photodynamic therapy is a promising treatment for X-ray-resistant cancers.

Anti-cancer Effect of a Planar Catechin Analog through the Decrease in Mitochondrial Membrane Potential.

Catechin is one of the best-known antioxidants and is reported to have some favorable physiological activities, including anti-cancer effects. We previously synthesized a catechin analog, planar catechin, which showed a 10-fold larger radical scavenging activity than (+)-catechin. However, the physiological effects of the planar catechin have remained unclear. In this study, we examined cytotoxicity and mitochondrial membrane potential after planar catechin treatment using a rat normal gastric mucosal cell line, RGM1, and its chemically induced cancer-like cell line, RGK1. Interestingly, the planar catechin showed remarkable cytotoxicity compared to (+)-catechin, with cancer cell specificity. Furthermore, the decrease in the mitochondrial membrane potential of cancer cells was observed at specific concentrations of the planar catechin. These results indicate that the planar catechin, possessing higher antioxidant activity, induces its anti-cancer effect through a decrease in the mitochondrial membrane potential and thus can be a promising agent for cancer treatment.

Water-Induced Regeneration of a 2,2-Diphenyl-1-picrylhydrazyl Radical after Its Scandium Ion-Promoted Electron-Transfer Disproportionation in an Aprotic Medium.

A neutral, stable radical, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•), has been frequently used to estimate the activity of antioxidants for more than 60 years. However, the number of reports about the effect of metal ions on the reactivity of DPPH• is quite limited. We have recently reported a unique electron-transfer disproportionation of DPPH• to produce the DPPH cations (DPPH+) and anions (DPPH-) upon the addition of scandium triflate [Sc(OTf)3 (OTf = OSO2CF3)] to an acetonitrile (MeCN) solution of DPPH•. The driving force of this reaction is suggested to be an interaction between DPPH- and Sc3+. In this study, it is demonstrated that the addition of H2O to the DPPH•-Sc(OTf)3 system in MeCN resulted in an increase in the absorption band at 519 nm due to DPPH•. This indicated that an electron-transfer comproportionation occurred to regenerate DPPH•. The regeneration of DPPH• was also confirmed by electron paramagnetic resonance (EPR) spectroscopy. The amount of DPPH• increased with an increasing amount of added H2O to reach a constant value. The detailed mechanism of regeneration of DPPH• was proposed based on the detailed spectroscopic and kinetic analyses, in which the reaction of DPPH+ with [(DPPH)2Sc(H2O)3]+ generated upon the addition of H2O to [(DPPH)2Sc]+ is the rate-determining step.

Importance of Locations of Iron Ions to Elicit Cytotoxicity Induced by a Fenton-Type Reaction.

The impact of the site of the Fenton reaction, i.e., hydroxyl radical (•OH) generation, on cytotoxicity was investigated by estimating cell lethality in rat thymocytes. Cells were incubated with ferrous sulfate (FeSO4) and hydrogen peroxide (H2O2), or pre-incubated with FeSO4 and then H2O2 was added after medium was replaced to remove iron ions or after the medium was not replaced. Cell lethality in rat thymocytes was estimated by measuring cell sizes using flow cytometry. High extracellular concentrations of FeSO4 exerted protective effects against H2O2-induced cell death instead of enhancing cell lethality. The pre-incubation of cells with FeSO4 enhanced cell lethality induced by H2O2, whereas a pre-incubation with a high concentration of FeSO4 exerted protective effects. FeSO4 distributed extracellularly or on the surface of cells neutralized H2O2 outside cells. Cytotoxicity was only enhanced when the Fenton reaction, i.e., the generation of •OH, occurred inside cells. An assessment of plasmid DNA breakage showed that •OH induced by the Fenton reaction system did not break DNA. Therefore, the main target of intracellularly generated •OH does not appear to be DNA.

Simplifying quantitative measurement of free radical species using an X-band EPR spectrometer.

The quantitative measurement of free radicals in liquid using an X-band electron paramagnetic resonance (EPR) was systematized. Quantification of free radicals by EPR requires a standard sample that contains a known spin amount/concentration. When satisfactory reproducibility of the sample material, volume, shape, and positioning in the cavity for EPR measurements can be guaranteed, a sample tested and a standard can be directly compared and the process of quantification can be simplified. The purpose of this study was to simplify manual quantitative EPR measurement. A suitable sample volume for achieving a stable EPR intensity was estimated. The effects of different solvents on the EPR sensitivity were compared. The stability and reproducibility of the EPR intensity of standard nitroxyl radical solutions were compared among different types of sample tubes. When the sample tubes, sample volumes, and/or solvents were the same, the EPR intensity was reproduced with an error of 2% or less for µM samples. The quantified sample and the standard sample in the same solvent and the same volume drawn into the same sample tube was able to be directly compared. The standard sample for quantification should be measured just before or after every daily experiment.

Effect of Magnesium Ion on the Radical-Scavenging Rate of Pterostilbene in an Aprotic Medium: Mechanistic Insight into the Antioxidative Reaction of Pterostilbene.

Pterostilbene (PTS), a methylated analog of resveratrol (RSV), has recently attracted much attention due to its enhanced bioavailability compared to RSV. However, little is known about the radical-scavenging mechanism of PTS. In this study, we investigated the effect of Mg(ClO4)2 on the scavenging reaction of galvinoxyl radical (GO•) by PTS in acetonitrile (MeCN). GO• was used as a model for reactive oxygen radicals. The second-order rate constant (kH) for the GO•-scavenging reaction by PTS was more than threefold larger than that by RSV, although thermodynamic parameters, such as the relative O-H bond dissociation energies of the phenolic OH groups, ionization potentials, and HOMO energies calculated by the density functional theory are about the same between PTS and RSV. The oxidation peak potential of PTS determined by the cyclic voltammetry in MeCN (0.10 M Bu4NClO4) was also virtually the same as that of RSV. On the other hand, no effect of Mg (ClO4)2 on the kH values was observed for PTS, in contrast to the case for RSV. A kinetic isotope effect of 3.4 was observed when PTS was replaced by a deuterated PTS. These results suggest that a one-step hydrogen-atom transfer from PTS to GO• may be the rate-determining step in MeCN.

Estimation of the Local Concentration of the Markedly Dense Hydroxyl Radical Generation Induced by X-rays in Water.

The linear-density (number of molecules on an arbitrary distance) of X-ray-induced markedly dense hydroxyl radicals (•OH) in water was estimated based on EPR spin-trapping measurement. A lower (0.13 mM-2.3 M) concentration series of DMPO water solutions and higher (1.7-6.0 M) concentration series of DMPO water solutions plus neat DMPO liquid (8.8 M as DMPO) were irradiated with 32 Gy of X-rays. Then, the yield of DMPO-OH in DMPO water solutions and the total spin-adduct of DMPO in neat DMPO were quantified. For the higher concentration DMPO series, the EPR peak area was estimated by double integration, and the baseline correction of the integral spectrum is necessary for accurate estimation of the peak area. The preparation of a suitable standard sample corresponding to the electric permittivity according to DMPO concentration was quite important for quantification of DMPO-OH, especially in DMPO concentration beyond 2 M. The linear-density of •OH generation in water by X-ray irradiation was estimated from the inflection point on the plot of the DMPO-OH yield versus DMPO linear-density. The linear-density of X-ray-induced markedly dense •OH was estimated as 1168 µm-1, which was converted to 0.86 nm as the intermolecular distance and 2.6 M as the local concentration.

Tunneling in the Hydrogen-Transfer Reaction from a Vitamin E Analog to an Inclusion Complex of 2,2-Diphenyl-1-picrylhydrazyl Radical with ß-Cyclodextrin in an Aqueous Buffer Solution at Ambient Temperature.

Recently, increasing attention has been paid to quantum mechanical behavior in biology. In this study, we investigated the involvement of quantum mechanical tunneling in the hydrogen-transfer reaction from Trolox, a water-soluble analog of vitamin E (α-tocopherol), to 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) in a phosphate buffer solution (0.05 M, pH 7.0). DPPH• was used as a reactivity model of reactive oxygen species and solubilized in water using ß-cyclodextrin (ß-CD). The second-order rate constants, kH and kD, in 0.05 M phosphate buffer solutions prepared with H2O (pH 7.0) and D2O (pD 7.0), respectively, were determined for the reaction between Trolox and DPPH•, using a stopped-flow technique at various temperatures (283-303 K). Large kinetic isotope effects (KIE, kH/kD) were observed for the hydrogen-transfer reaction from Trolox to the ß-CD-solubilized DPPH• in the whole temperature range. The isotopic ratio of the Arrhenius prefactor (AH/AD = 0.003), as well as the isotopic difference in the activation energies (19 kJ mol-1), indicated that quantum mechanical tunneling plays a role in the reaction.

Effects of LET on oxygen-dependent and-independent generation of hydrogen peroxide in water irradiated by carbon-ion beams.

Linear energy transfer (LET) dependence of yields of O2-dependent and O2-independent hydrogen peroxide (H2O2) in water irradiated by ionizing radiation was investigated. The radiation-induced hydroxyl radical (•OH) generation in an aqueous solution was reported to occur in two different localization densities, the milli-molar (relatively sparse) and/or molar (markedly-dense) levels. In the milli-molar-level •OH generation atmosphere, •OH generated at a molecular distance of ∼7 nm are likely unable to interact. However, in the molar-level •OH generation atmosphere, several •OH were generated with a molecular distance of 1 nm or less, and two •OH can react to directly make H2O2. An aliquot of ultra-pure water was irradiated by 290-MeV/nucleon carbon-ion beams at the Heavy-Ion Medical Accelerator in Chiba (HIMAC, NIRS/QST, Chiba, Japan). Irradiation experiments were performed under aerobic or hypoxic (<0.5% oxygen) conditions, and several LET conditions (13, 20, 40, 60, 80, or >100 keV/µm). H2O2 generation in irradiated samples was estimated by three methods. The amount of H2O2 generated per dose was estimated and compared. O2-independent H2O2 generation, i.e. H2O2 generation under hypoxic conditions, increased with increasing LET. On the other hand, the O2-dependent H2O2 generation, i.e. subtraction of H2O2 generation under hypoxic conditions from H2O2 generation under aerobic conditions, decreased with increasing LET. This suggests that the markedly-dense •OH generation is positively correlated with LET. High-LET beams generate H2O2 in an oxygen-independent manner.

Lipid-soluble polyphenols from sweet potato exert antitumor activity and enhance chemosensitivity in breast cancer.

Polyphenols are abundant in vegetables and fruit. They have been shown to have various antitumor, antioxidant, and anti-inflammatory effects. Here, we extracted the lipid-soluble fraction of polyphenols from fermented sweet potato (Ipomoea batatas). These lipid-soluble polyphenols mainly contained caffeic acid derivatives with strong antioxidant ability, which we hypothesized to affect diseases for which oxidative stress is a factor, such as cancer. We therefore investigated the antitumor and chemo-sensitizing effects of lipid-soluble polyphenols on E0771 murine breast cancer cells. The lipid-soluble polyphenols accumulated in the cells' cytoplasm due to its high lipophilicity, and reduced reactive oxygen species through its strong antioxidant activity. The lipid-soluble polyphenols also arrested the cell cycle at G0/G1 by suppressing Akt activity, and enhanced the cytotoxicity of anticancer agents. In this model, lipid-soluble polyphenols inhibited tumor growth and enhanced the efficacy of chemotherapy drugs. These results suggest the potential of lipid-soluble polyphenols as a functional food to support cancer therapy.

Effects of reaction environments on radical-scavenging mechanisms of ascorbic acid.

The effects of reaction environments on the radical-scavenging mechanisms of ascorbic acid (AscH2) were investigated using 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) as a reactivity model of reactive oxygen species. Water-insoluble DPPH• was solubilized by ß-cyclodextrin (ß-CD) in water. The DPPH•-scavenging rate of AscH2 in methanol (MeOH) was much slower than that in phosphate buffer (0.05 M, pH 7.0). An organic soluble 5,6-isopropylidene-l-ascorbic acid (iAscH2) scavenged DPPH• much slower in acetonitrile (MeCN) than in MeOH. In MeOH, Mg(ClO4)2 significantly decelerated the DPPH•-scavenging reaction by AscH2 and iAscH2, while no effect of Mg(ClO4)2 was observed in MeCN. On the other hand, Mg(ClO4)2 significantly accelerated the reaction between AscH2 and ß-CD-solubilized DPPH• (DPPH•/ß-CD) in phosphate buffer (0.05 M, pH 6.5), although the addition of 0.05 M Mg(ClO4)2 to the AscH2-DPPH•/ß-CD system in phosphate buffer (0.05 M, pH 7.0) resulted in the change in pH of the phosphate buffer to be 6.5. Thus, the DPPH•-scavenging reaction by iAscH2 in MeCN may proceed via a one-step hydrogen-atom transfer, while an electron-transfer pathway is involved in the reaction between AscH2 and DPPH•/ß-CD in phosphate buffer solution. These results demonstrate that the DPPH•-scavenging mechanism of AscH2 are affected by the reaction environments.

Heavy-ion beam-induced reactive oxygen species and redox reactions.

Quantification and local density estimation of radiation-induced reactive oxygen species (ROS) were described focusing on our recent and related studies. Charged particle radiation, i.e. heavy-ion beams, are currently utilized for medical treatment. Differences in ROS generation properties between photon and charged particle radiation may lead to differences in the quality of radiation. Radiation-induced generation of ROS in water was quantified using several different approaches to electron paramagnetic resonance (EPR) techniques. Two different densities of localized hydroxyl radical (•OH) generation, i.e. milli-molar and molar levels, were described. Yields of sparse •OH decreased with increasing linear energy transfer (LET), the yield total •OH was not affected by LET. In the high-density, molar level, •OH environment, •OH can react and directly make hydrogen peroxide (H2O2), and then possible to form a high-density H2O2 cluster. The amount of total oxidation reactions caused by oxidative ROS, such as •OH and hydroperoxyl radial (HO2•), was decreased with increasing LET. Possibilities of the sequential reactions were discussed based on the initial localized density at the generated site. Water-induced ROS have been well investigated. However, little is known about radiation-induced free radical generation in lipidic conditions. Radio-chemistry to understand the sequential radio-biological effects is still under development.

Expression of Ascorbate Peroxidase Derived from Cyanidioschyzon merolae in Mammalian Cells.

BACKGROUND/AIM: Ascorbate peroxidase (APX) derived from Cyanidioschyzon merolae, a primitive red alga living in high temperature and acidic environments, has greater anti-oxidative capacity than similar peroxidases occurring in other plants. In the present study, we examined whether expression of Cyanidioschyzon merolae-derived APX (cAPX) in mammalian cells increases cellular anti-oxidative capacity. MATERIALS AND METHODS: The cAPX gene was introduced into the mouse fibroblast-like cell line C3H10T1/2. Production of reactive oxygen species (ROS) and/or cell viability was assessed after heat, H2O2 and acid stimulation. RESULTS: Heat and H2O2 stimulation resulted in ROS production. cAPX-expressing cells were more tolerant to oxidative stress induced by heat, H2O2 and acid stimulations than control cells lacking cAPX. CONCLUSION: Introduction of cAPX increases the anti-oxidative capacity in mammalian cells.

A large kinetic isotope effect in the reaction of ascorbic acid with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO˙) in aqueous buffer solutions.

A large kinetic isotope effect (KIE, kH/kD) of 12.8 was observed for the hydrogen-transfer reaction from ascorbic acid to 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO˙) in a phosphate buffer solution (0.05 M, pH/pD 7.0) at 298 K. The isotopic difference in the activation energies (6.8 kJ mol-1) determined from the temperature dependence of the KIE suggests that quantum mechanical tunneling may partly play a role in the reaction, although the isotopic ratio of the Arrhenius prefactor (AH/AD = 0.86) is within the semiclassical limits.

Relationship between the radical-scavenging activity of selected flavonols and thermodynamic parameters calculated by density functional theory.

The relationship between radical-scavenging rate constants (k) in an aprotic medium and thermodynamic parameters calculated by density functional theory (DFT) was investigated for 7 flavonols, which are myricetin (Myr), quercetin (Que), morin (Mor), kaempferol (Kae), 2'-methylquercetin (2'-MeQue), 5'-methylquercetin (5'-MeQue), and 2',5'-dimethylquercetin (Me2Que). The k values were determined for the reaction between the flavonols and galvinoxyl radical used as a reactivity model of reactive oxygen species in deaerated acetonitrile at 298 K. The energy difference values (D HT, HT: hydrogen transfer) between the flavonols and the corresponding radicals, which equal to the relative O-H bond dissociation energies of the OH groups in the flavonols and ionisation potentials (IP) were calculated by DFT at the B3LYP/6-31++G(d) level with C-PCM solvation model parameterised for acetonitrile. Among the 7 flavonols used in this study, calculated IP values of 4 flavonols exhibited a linear correlation with log k, suggesting that the radical-scavenging reaction of these flavonols may proceed via an electron transfer as the rate determining step.

Reduction of molecular oxygen by redox active thiols: comparison of glutathione, N-acetylcysteine, cysteine, and homocysteine.

The reaction properties of the thiol compounds, cysteine (Cys), N-acetyl-l-cysteine (NAC), the reduced form glutathione (GSH), and homocysteine (HCS) were compared. The main purpose of this study was to find a thiol-based anti-oxidant suitable for biological experiments and to provide clear reasoning for its selection. The availability of thiol compounds to generate superoxide by reducing molecular oxygen (O2) at a hyperthermal temperature was discussed. An oxidative atmosphere, i.e., superoxide generation by the hypoxanthine-xanthine oxidase reaction, hydroxyl radical generation by X-ray irradiation, or direct one-electron oxidation by ferricyanide, was prepared in a reaction mixture containing 0.1 mM TEMPOL and 1 mM test compound, and the EPR signal decay of TEMPOL was observed. A reaction mixture containing 0.1 mM TEMPOL and 1 mM thiol compound was incubated at 44°C, and the EPR signal decay of TEMPOL was observed. Thiols could function as H-donors to the oxoammonium cation and produce the hydroxylamine form of TEMPOL in an oxidative atmosphere. Thiols could also irreversibly react with the oxoammonium cation. GSH and Cys could reduce O2 to form superoxide/hydroperoxyl radical at hyperthermal temperatures, but HCS and NAC could not reduce O2. GSH and Cys may cause reductive stress, whereas NAC is a simple tractable antioxidant.

Genetic Variants in CD44 and MAT1A Confer Susceptibility to Acute Skin Reaction in Breast Cancer Patients Undergoing Radiation Therapy.

PURPOSE: Heterogeneity in radiation therapy (RT)-induced normal tissue toxicity is observed in 10% of cancer patients, limiting the therapeutic outcomes. In addition to treatment-related factors, normal tissue adverse reactions also manifest from genetic alterations in distinct pathways majorly involving DNA damage-repair genes, inflammatory cytokine genes, cell cycle regulation, and antioxidant response. Therefore, the common sequence variants in these radioresponsive genes might modify the severity of normal tissue toxicity, and the identification of the same could have clinical relevance as a predictive biomarker. METHODS AND MATERIALS: The present study was conducted in a cohort of patients with breast cancer to evaluate the possible associations between genetic variants in radioresponsive genes described previously and the risk of developing RT-induced acute skin adverse reactions. We tested 22 genetic variants reported in 18 genes (ie, NFE2L2, OGG1, NEIL3, RAD17, PTTG1, REV3L, ALAD, CD44, RAD9A, TGFßR3, MAD2L2, MAP3K7, MAT1A, RPS6KB2, ZNF830, SH3GL1, BAX, and XRCC1) using TaqMan assay-based real-time polymerase chain reaction. At the end of RT, the severity of skin damage was scored, and the subjects were dichotomized as nonoverresponders (Radiation Therapy Oncology Group grade <2) and overresponders (Radiation Therapy Oncology Group grade ≥2) for analysis. RESULTS: Of the 22 single nucleotide polymorphisms studied, the rs8193 polymorphism lying in the micro-RNA binding site of 3'-UTR of CD44 was significantly (P=.0270) associated with RT-induced adverse skin reactions. Generalized multifactor dimensionality reduction analysis showed significant (P=.0107) gene-gene interactions between MAT1A and CD44. Furthermore, an increase in the total number of risk alleles was associated with increasing occurrence of overresponses (P=.0302). CONCLUSIONS: The genetic polymorphisms in radioresponsive genes act as genetic modifiers of acute normal tissue toxicity outcomes after RT by acting individually (rs8193), by gene-gene interactions (MAT1A and CD44), and/or by the additive effects of risk alleles.

Effects of carbon ion irradiation and X-ray irradiation on the ubiquitylated protein accumulation.

C-ion radiotherapy is associated with improved local control and survival in several types of tumors. Although C-ion irradiation is widely reported to effectively induce DNA damage in tumor cells, the effects of irradiation on proteins, such as protein stability or degradation in response to radiation stress, remain unknown. We aimed to compare the effects of C-ion and X-ray irradiation focusing on the cellular accumulation of ubiquitylated proteins. Cells from two human colorectal cancer cell lines, SW620 and SW480, were subjected to C-ion or X-ray irradiation and determination of ubiquitylated protein levels. High levels of ubiquitylated protein accumulation were observed in the C-ion-irradiated SW620 with a peak at 3 Gy; the accumulation was significantly lower in the X-ray-irradiated SW620 at all doses. Enhanced levels of ubiquitylated proteins were also detected in C-ion or X-ray-irradiated SW480, however, those levels were significantly lower than the peak detected in the C-ion-irradiated SW620. The levels of irradiation-induced ubiquitylated proteins decreased in a time-dependent manner, suggesting that the proteins were eliminated after irradiation. The treatment of C-ion-irradiated SW620 with a proteasome inhibitor (epoxomicin) enhanced the cell killing activity. The accumulated ubiquitylated proteins were co-localized with γ-H2AX, and with TP53BP1, in C-ion-irradiated SW620, indicating C-ion-induced ubiquitylated proteins may have some functions in the DNA repair system. Overall, we showed C-ion irradiation strongly induces the accumulation of ubiquitylated proteins in SW620. These characteristics may play a role in improving the therapeutic ratio of C-ion beams; blocking the clearance of ubiquitylated proteins may enhance sensitivity to C-ion radiation.