BR Biomedical Reports 2049-9434 2049-9442 D.A. Spandidos 10.3892/br.2016.819 BR-0-0-819 Articles Ascorbic acid does not reduce the anticancer effect of radiotherapy HosokawaYoichiro SagaRyo MonzenSatoru TerashimaShingo TsurugaEichi Department of Radiation Science, Graduate School of Health Sciences, Hirosaki University, Hirosaki, Aomori 036-8564, Japan Correspondence to: Professor Yoichiro Hosokawa, Department of Radiation Science, Graduate School of Health Sciences, Hirosaki University, 66-1 Honcho, Hirosaki, Aomori 036-8564, Japan, E-mail: hosokawa@cc.hirosaki-u.ac.jp 01 2017 29 11 2016 6 1 103 107 17032016 26052016 Copyright © 2016, Spandidos Publications 2016

The present study hypothesized that the therapeutic use of ascorbic acid (AsA) in combination with radiation may reduce therapy-related side effects and increase the antitumor effects. The aim of the study was to examine the association between the scavenged activity of AsA and the biological anticancer effect of hydroxyl (OH) radicals generated by X-ray irradiation. Cell survival, DNA fragmentation of human leukemia HL60 cells and the amount of OH radicals were investigated following X-ray irradiation and AsA treatment. The number of living cells decreased, and DNA fragmentation increased at AsA concentrations >1 mM. Electron spin resonance spectra revealed that X-ray irradiation generated OH radicals, which were scavenged by AsA at concentrations >75 µM. The AsA concentration inside the cell was 75 µM when cells underwent extracellular treatment with 5 mM AsA, which significantly induced HL60 cell death even without irradiation. No increase in the number of viable HL60 cells was observed following AsA treatment with irradiation when compared to irradiation alone. In conclusion, the disappearance of the radiation anticancer effects with AsA treatment in combination with radiotherapy for cancer treatment is not a cause for concern.

 X-ray irradiation ascorbic acid hydroxyl radical cell death radical scavenger Introduction

It is well known that ascorbic acid (AsA) has anticancer effects due to its antioxidant activity, and the results of epidemiological surveys have demonstrated that AsA intake is inversely correlated with carcinogenicity (1). Almost 30 years ago, the study by Cameron and Pauling was the first to demonstrate the antitumor effect of AsA and this finding was applied to a clinical setting (2). A few years earlier, the effectiveness of combining cancer treatment with AsA was reported, but the anticancer activity of AsA was not confirmed in clinical tests at the Mayo Clinic (3).

Recent studies have shown that high plasma concentrations of AsA can only be achieved when AsA is administered intravenously or intraperitoneally as the rate of absorption from the gut is limited with oral administration (4). The plasma concentrations of AsA in humans following high-dose intravenous AsA is ~200 times higher in comparison to oral administration (5). Therefore, we hypothesized that the therapeutic use of AsA in combination with radiation may reduce therapy-related side effects and increase the antitumor effects (6). It has been reported that pharmacological ascorbate sensitized cells to the effects of ionizing radiation (7,8).

Ionizing radiation is known to damage numerous cell organelles through the production of free radicals (9). The hydroxyl (OH) radical (·OH) is considered to have a major role in radiation-induced cell damage and induces apoptotic cell death through activation of a pro-apoptotic pathway (10). However, AsA is a known scavenger of reactive radical species, and it is possible that AsA may reduce the anticancer effect of radiation. The aim of the present study was to examine the association between the scavenged activity of AsA and the biological anticancer effect of OH radicals generated by X-ray irradiation.

 Materials and methods <sec> <title>Cell culture, extracellular AsA treatment and X-ray irradiation

Human promyelocytic leukemia HL60 cells were purchased from the RIKEN Bioresource Center (Tsukuba, Japan). The cells were grown in 100-mm culture dishes containing Roswell Park Memorial Institute (RPMI)-1640 medium (Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 atmosphere. The cells were passaged every third day, and the density in the culture was ≤1×106 cells/ml. The cell concentration was adjusted to 5×105 cells/ml prior to AsA treatment and X-ray irradiation. The AsA solution was titrated with NaOH at pH 7.4. HL60 (5×105 cells/ml) in 50-mm culture dishes were irradiated 1 h after adjusting each experimental AsA dose. The radiation was administered with Sofron BST 1505 CX (Sofron X-ray Industry Corporation, Ltd., Tokyo, Japan) under the following conditions: Tube voltage, 80 kVp (effective 62.3 kV); tube current, 5 mA; focus surface distance, 30 cm; dose rate, 16 Gy/h; filter, 1-mm aluminum; and temperature, room temperature.

 Electron spin resonance (ESR) measurement

OH radicals generated from irradiated phosphate-buffered saline (PBS) were measured by ESR (JES-FR80; JEOL, Tokyo, Japan). The spin-trap technique was used with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (Labotec Corp., Ltd., Tokyo, Japan). The PBS sample (200 µl) contained 150 µl of PBS with or without AsA, 35 µl of diethylenetriaminepentaacetic acid (Labotec Corp. Ltd.) and 15 µl of DMPO. Radicals generated in this sample by X-ray irradiation under the aforementioned condition were measured within 1 min of irradiation. The ESR spectrometer (JES-FR80) was used under the following conditions: Frequency, 9.475 GHz; power, 4 mW; center field, 334.5 mT; sweep width, ±10.0 mT; modulation width, 5×0.1 m; receiver gain, 0.4×100; time constant, 0.1 sec; sweep time, 2.0 min; and temperature, room temperature. The relative signal intensity (RSI) of OH radicals was defined as the ratio of the peak height of OH radicals and a manganese oxide standard marker on the ESR spectra. Each sample was measured three to five times and the results are shown as average values of RSI.

 Cell viability assay and quantitative detection of apoptosis

The cells were stained with 2% trypan blue for 1 min and counted per 1 mm3 in each experiment to estimate the number of unstained cells within 2 min. The number of unstained cells was considered to be the number of viable cells.

Apoptosis was detected using a cellular DNA fragmentation enzyme-linked immunosorbent assay kit (Roche Diagnostics, Basel, Switzerland). The cells were labeled with bromodeoxyuridine (BrdU) (Roche Diagnostics) on the day prior to treatment. Following AsA treatment and/or 4 Gy X-ray irradiation, the cells were cultured for 6 h. After this incubation, 1.5×106 cells were collected and treated with a cell solution (0.05% Triton-X and 650 µM EDTA) (Research Organics, Cleveland, OH, USA) at 4°C for 30 min. The cells were subsequently centrifuged at 4°C and 12,000 × g for 30 min at 4°C, and 160 µl of the supernatant was used as a sample. This sample was plated on a microtiter plate coated with an anti-BrdU antibody (Roche Diagnostics), and color development caused by the tetramethylbenzidine (Roche Diagnostics) substrate reaction was measured using a spectrophotometer (450 nm).

 Analysis of intracellular AsA concentration

The OxiSelect™Ascorbic Acid Assay kit (FRASC) was purchased from Cell Biolabs, Inc. (San Diego, CA, USA). Cells (1.0×106 cells/ml) were lysed by multiple freeze-thaw cycles in four volumes of cold 1X assay buffer. The samples and AsA standard were added to 96-well plate wells. Deionized water (−AO) or diluted 1X ascorbate oxidase (+AO) was added to each well. The reaction reagent was added to all the wells, and the absorbance of each well was measured at a wavelength of 540 nm using a microplate reader (10043; Bio Rad, Tokyo, Japan). The AsA concentration was calculated from a standard curve created from the measured absorbance.

 Statistical analysis

All the data are expressed as the mean ± standard deviation of ≥3 independent experiments. Statistical comparisons between groups were performed by an analysis of variance and Student's t-test, where P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>ESR spectra

The ESR spectra of PBS following irradiating with a 4 Gy X-ray are shown in Fig. 1. The spectra without AsA treatment shows the typical wave type of radicals generated by X-ray irradiation. There were two types, DMPO-OH (αN = αH = 1.49 mT) and DMPO-H (αN = 1.64, αH = 2.25 mT). However, there were unclear waveforms for the OH and H radicals in PBS with 100 µM (0.1 mM) AsA. With 1 mM AsA, the ascorbate radical in the middle of the ESR spectrum of the OH and H radicals disappeared.

 RSIs of OH radicals

Fig. 2 shows the RSIs of the OH radicals generated from the 4 Gy X-ray irradiation with various concentrations of AsA in PBS. The RSIs of the OH radicals were similar for the AsA values <50 µM AsA, suggesting that OH radicals were not scavenged at concentrations <50 µM AsA. The RSIs of the OH radicals decreased with 75 µM AsA with similarly low RSIs at concentrations of 100 µM (0.1 mM) AsA.

 Intracellular AsA concentration

The intracellular AsA concentrations in HL60 cells 1 h after extracellular AsA treatment are shown in Fig. 3. The intracellular concentration of AsA was <20 µM after treatment with 1 mM AsA. However, the intracellular AsA concentration was 75 µM after the 5 mM AsA extracellular treatment.

 HL60 cell viability

Fig. 4A shows the cell viability of HL60 cells 24 h after AsA treatment alone. AsA treatment at concentrations >1 mM AsA decreased the number of living cells, and no effect was observed for concentrations <1 mM. The cell viability of HL60 cells 24 h after 4 Gy X-ray irradiation with/without AsA treatment is shown in Fig. 4B. At concentrations >1 mM AsA, combination AsA treatment and 4 Gy X-ray irradiation decreased the number of living cells compared to X-ray irradiation alone. The number of living cells with AsA treatment combined with 4 Gy X-ray irradiation was not higher than that for cells exposed to X-ray irradiation alone.

 DNA fragmentation

DNA fragmentation following AsA treatment alone and combination AsA treatment with 4 Gy X-ray irradiation is shown in Fig. 5. AsA treatment increased DNA fragmentation at concentrations >1 mM in a concentration-dependent manner. DNA fragmentation with AsA treatment concentrations >5 mM in combination with 4 Gy X-ray irradiation was more than that of 4 Gy X-ray irradiation alone. When AsA was added to 4 Gy X-ray irradiation, a concentration that induced lower DNA fragmentation than 4 Gy irradiation alone was not identified.

 Discussion

When ionizing radiation is delivered to living cells/animals, reactive oxygen species (ROS) such as ·OH, hydrogen radicals (·H) superoxide (O2·−), and hydrogen peroxide (H2O2) are produced. Initial generation of ·OH is via direct radiolysis of water. Highly reactive OH cannot travel long distances, but instead immediately reacts with other nearby molecules (11). For this reason, ·OH generated by ionizing irradiation near DNA in cells have a major role in radiation-induced cell damage. Our previous study reported the significant association between the amount of intracellular ·OH and apoptotic cell death (12).

There is renewed interest in the use of high dose AsA (20 M in plasma) in the treatment of numerous different types of cancer (13–15). AsA has been shown to selectively kill cancer cells via the extracellular formation of H2O2 (16). It was hypothesized that the presence of a catalyst, such as ferric ion, within the extracellular matrix of tumors could oxidize AsA to ascorbate radicals, which could subsequently donate electrons to oxygen to form O2-. O2− can be converted by superoxide dismutase to the tumoricidal peroxide ion (5). In the present study, ascorbate radicals were detected on the ESR spectrum of 1 mM AsA PBS. The number of living cells decreased, and DNA fragmentation increased at concentrations >1 mM AsA. These results indicate that at concentrations >1 mM AsA treatment induces apoptosis and cell death of HL60 cells. Combination treatment with >1 mM AsA and X-ray irradiation decreased the number of living cells more than exposure to cells with X-ray irradiation alone. DNA fragmentation was also higher in cells treated with combination AsA treatment >1 mM and X-ray irradiation compared to that in cells treated with X-ray irradiation alone. The result confirmed that combination AsA treatment with X-ray irradiation increased apoptosis in HL60 cells.

AsA is present in aqueous compartments (such as cytosol, plasma and other body fluids) and has several metabolically important cofactor functions in enzyme reactions, particularly hydroxylation. AsA has been shown to efficiently scavenge O2·−, H2O2, hypochlorite, ·OH and peroxyl radicals (17). The radical scavenging effects of AsA are used to protect normal tissue from radiation injury (18). However, no increase in the number of viable HL60 cells following AsA treatment was observed with irradiation when comparing to irradiation alone. Therefore, the amount of ·OH scavenged by AsA was estimated. ·OH generated with 4 Gy X-ray irradiation were scavenged by AsA at concentrations >75 µM in the ESR analysis. The result suggests that AsA concentrations >75 µM in the fluid inside the cell could scavenge ·OH generated with X-ray irradiation. The actual AsA concentration inside the cell was 75 µM when cells underwent extracellular treatment with 5 mM AsA. However, 5 mM AsA significantly induced HL60 cell death even without irradiation. The result was evidenced in the HL60 cells in the present study. However, our previous study reported that 5 mM AsA treatment suppressed the growth of epithelial cancer cells in vitro (6). It was suggested that extracellular H2O2 generated with >5 mM AsA mainly damaged the cell membrane due to passing more cascades than radiation alone (19,20). The current study revealed that caspase-3, −8 and −9 were activated 2 h after exposure to H2O2, whereas in irradiated cells activation of caspase-3 and −9 occurred 4 h after exposure; caspase-8 activation was not observed (19). These high fluxes of H2O2 appear to have a limited effect on normal cells but can be detrimental to certain tumor cells (13).

In conclusion, treating cancer cells with high extracellular concentrations of AsA (>5 mM) could achieve an intracellular AsA concentration level of 75 µM, leading to scavenging of ·OH generated with X-ray irradiation. However, treatment with the high extracellular concentrations of AsA alone directly induced cancer cell death. As aforementioned, the disappearance of radiation anticancer effects with AsA treatment in combination with radiotherapy for cancer treatment is not a concern. Previously, clinical trials on the effects of high-dose intravenous AsA on patients with cancer have been performed (21,22). We hypothesize that a clinical trial to evaluate the high-dose intravenous AsA combined with radiotherapy in patients with cancer will be launched in the near future.

 Acknowledgements

The present study was supported by the Japan Society for the Promotion of Science (Grants-in-Aid for Scientific Research, project nos. 21591603 and 24591831).

 References LuSHOhshimaHFuHMTianYLiFMBlettnerMWahrendorfJBartschHUrinary excretion of N-nitrosamino acids and nitrate by inhabitants of high- and low-risk areas for esophageal cancer in Northern China: Endogenous formation of nitrosoproline and its inhibition by vitamin CCancer Res461485149119863943105 CameronEPaulingLAscorbic acid and cancer: A reviewProc Natl Acad Sci USA7545383542198710.1073/pnas.75.9.4538 CreaganETMoertelCGO'FallonJRSchuttAJO'ConnellMJRubinJFrytakSFailure of high-dose vitamin C (ascorbic acid) therapy to benefit patients with advanced cancer. A controlled trialN Engl J Med301687690197910.1056/NEJM197909273011303384241 HofferLJLevineMAssoulineSMelnychukDPadayattySJRosadiukKRousseauCRobitailleLMillerWHJrPhase I clinical trial of i.v. ascorbic acid in advanced malignancyAnn Oncol1919691974200810.1093/annonc/mdn37718544557 WilsonMKBaguleyBCWallCJamesonMBFindlayMPReview of high-dose intravenous vitamin C as an anticancer agentAsia Pac J Clin Oncol102237201410.1111/ajco.1217324571058 HosokawaYMonzenSYoshinoHTerashimaSNakanoMToshimaKSagaRKashiwakuraIEffects of X ray irradiation in combination with ascorbic acid on tumor controlMol Med Rep1254495454201526238154 HerstPMBroadleyKWHarperJLMcConnellMJPharmacological concentrations of ascorbate radiosensitize glioblastoma multiforme primary cells by increasing oxidative DNA damage and inhibiting G2/M arrestFree Radic Biol Med5214861493201210.1016/j.freeradbiomed.2012.01.02122342518 ShinozakiKHosokawaYHazawaMKashiwakuraIOkumuraKKakuTNakayamaEAscorbic acid enhances radiation-induced apoptosis in an HL60 human leukemia cell lineJ Radiat Res (Tokyo)52229237201110.1269/jrr.10089 TanejaNTjalkensRPhilbertMARehemtullaAIrradiation of mitochondria initiates apoptosis in a cell free systemOncogene20167177200110.1038/sj.onc.120405411313941 RileyPAFree radicals in biology: Oxidative stress and the effects of ionizing radiationInt J Radiat Biol652733199410.1080/095530094145500417905906 MatsumotoKUenoMNakanishiIAnzaiKDensity of hydroxyl radicals generated in an aqueous solution by irradiating carbon-ion beamChem Pharm Bull (Tokyo)63195199201510.1248/cpb.c14-0073625757490 HosokawaYTanakaLKanekoMSakakuraYTsurugaEIrieKYajimaTApoptosis induced by generated OH radicals inside cells after irradiationArch Histol Cytol65301305200210.1679/aohc.65.30112501887 DuJCullenJJBuettnerGRAscorbic acid: Chemistry, biology and the treatment of cancerBiochim Biophys Acta1826443457201222728050 EspeyMGChenPChalmersBDriskoJSunAYLevineMChenQPharmacologic ascorbate synergizes with gemcitabine in preclinical models of pancreatic cancerFree Radic Biol Med5016101619201110.1016/j.freeradbiomed.2011.03.00721402145 MikirovaNACasciariJJHunninghakeRERiordanNHIntravenous ascorbic acid protocol for cancer patients:scientific rationale, pharmacology, and clinical experienceFunct Foods Health Dis33443662013 TerashimaSHosokawaYYoshinoHYamaguchiMNakamuraTEffect of ascorbic acid and X-irradiation on HL-60 human leukemia cells: The kinetics of reactive oxygen speciesOncol Rep3026532658201324085115 SiesHStahlWVitamins E and C, beta-carotene, and other carotenoids as antioxidantsAm J Clin Nutr621315S1321S19957495226 SatoTKinoshitaMYamamotoTItoMNishidaTTakeuchiMSaitohDSekiSMukaiYTreatment of irradiated mice with high-dose ascorbic acid reduced lethalityPLoS One10e0117020201510.1371/journal.pone.011702025651298 HosokawaYSakakuraYTanakaLOkumuraKYajimaTKanekoMRadiation-induced apoptosis is independent of caspase-8 but dependent on cytochrome c and the caspase-9 cascade in human leukemia HL60 cellsJ Radiat Res (Tokyo)46293303200510.1269/jrr.46.293 MojićMBogdanović PristovJMaksimović-IvanićDJonesDRStanićMMijatovićSSpasojevićIExtracellular iron diminishes anticancer effects of vitamin C: An in vitro studySci Rep45955201410.1038/srep0595525092529 StephensonCMLevinRDSpectorTLisCGPhase I clinical trial to evaluate the safety, tolerability, and pharmacokinetics of high-dose intravenous ascorbic acid in patients with advanced cancerCancer Chemother Pharmacol72139146201310.1007/s00280-013-2179-923670640 MontiDAMitchellEBazzanAJLittmanSZabreckyGYeoCJPillaiMVNewbergABDeshmukhSLevineMPhase I evaluation of intravenous ascorbic acid in combination with gemcitabine and erlotinib in patients with metastatic pancreatic cancerPLoS One7e29794201210.1371/journal.pone.002979422272248

Electron spin resonance spectra in the case of 4 Gy X-ray irradiation with/without ascorbic acid (AsA). On the left, AsA concentrations in phosphate-buffered saline are shown. Arrows show the generation of hydroxyl radicals (OH*) and hydrogen radicals (H*). H* disappeared at 100 µM (0.1 mM) AsA and ascorbate radicals were observed.

Relative signal intensities (RSIs) of hydroxyl (OH) radicals generated with 4 Gy X-ray irradiation in phosphate-buffered saline (PBS). RSIs of OH radicals were similar <50 µM ascorbic acid (AsA). RSI of OH radicals decreased at concentration of AsA >75 µM. *P<0.05 vs. 0 µM AsA.

Intracellular ascorbic acid (AsA) concentration in HL60 cells following extracellular AsA treatment. Control (Cont) was 0 mM AsA. The intracellular AsA concentration was 75 µM after the 5 mM AsA treatment.

(A) Cell viability of HL60 cells following ascorbic acid (AsA) treatment alone and without X-ray irradiation. Control (Cont) was 0 mM AsA. Following AsA treatment alone, the number of living cells decreased >1 mM AsA; *P<0.05 vs. control. (B) Cell viability of HL60 cells 24 h after 4 Gy X-ray irradiation with/without AsA treatment. Following AsA treatment combined with 4 Gy X-ray irradiation, the number of living cells at concentrations >1 mM AsA was lower than those exposed to 4 Gy X-ray irradiation alone; *P<0.05 vs. 0 mM AsA (4 Gy X-ray irradiation alone).

(A) DNA fragmentation 6 h after ascorbic acid (AsA) treatment alone. DNA fragmentation with AsA treatment alone increased in a concentration-dependent manner at concentrations >1 mM; *P<0.05 vs. control. (B) DNA fragmentation 6 h after combination AsA treatment with 4 Gy X-ray irradiation. DNA fragmentation with 4 Gy X-ray irradiation with AsA was not lower than with 4 Gy X-ray irradiation alone; *P<0.05 vs. 0 mM AsA (4 Gy X-ray irradiation alone).