Synergistic effect of Gemcitabin-loaded metal organic frameworks nanoparticles with particle therapy.

Combination of nanoagents with radiations has opened up new perspectives in cancer treatment, improving both tumor diagnosis and therapeutic index. This work presents the first investigation of an innovative strategy that combines porous metal-organic frameworks (nanoMOFs) loaded with the anti-cancer drug Gemcitabine monophosphate (GemMP) and particle therapy-a globally emerging technique that offers more precise radiation targeting and enhanced biological efficacy compared to conventional radiotherapy. This radiochemotherapy has been confronted with two major obstacles limiting the efficacy of therapeutics when tested in vivo: (i) the presence of hypoxia, one of the most important causes for radiotherapy failure and (ii) the presence of a microenvironment, main biological barrier to the direct penetration of nanoparticles into cancer cells. On the one hand, this study explore the effects of hypoxia on drug delivery systems in combination with radiation, demonstrating that GemMP-loaded nanoMOFs significantly enhance the anticancer efficacy of particle therapy under both normoxic (pO2 = 20 %) and hypoxic (pO2 = 0.5 %) conditions. Notably, the presence of GemMP-loaded nanoMOFs allows the irradiation dose to be reduced by 1.4-fold in normoxia and at least 1.6-fold in hypoxia, achieving the same cytotoxic effect (SF=10 %) as carbon or helium ions alone. Synergistic effects between GemMP-loaded nanoMOFs and radiations have been observed and quantified. On the other hand, we also highlighted the ability of the nanoMOFs to diffuse through an extracellular matrix and accumulate in cells. An higher effect of the encapsulated GemMP than the free drug was observed, confirming the key role of the nanoMOFs in transporting the active substance to the cancer cells as a Trojan horse. This paves the way to the design of "all-in-one" nanodrugs where each component plays a role in the optimization of cancer therapy to maximize cytotoxic effects on hypoxic tumor cells while minimizing toxicity on healthy tissue.

Dimethyl Sulfoxide Attenuates Ionizing Radiation-induced Centrosome Overduplication and Multipolar Cell Division in Human Induced Pluripotent Stem Cells.

Centrosomes are important organelles for cell division and genome stability. Ionizing radiation exposure efficiently induces centrosome overduplication via the disconnection of the cell and centrosome duplication cycles. Over duplicated centrosomes cause mitotic catastrophe or chromosome aberrations, leading to cell death or tumorigenesis. Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can differentiate into all organs. To maintain pluripotency, PSCs show specific cellular dynamics, such as a short G1 phase and silenced cell-cycle checkpoints for high cellular proliferation. However, how exogenous DNA damage affects cell cycle-dependent centrosome number regulation in PSCs remains unknown. This study used human iPSCs (hiPSCs) derived from primary skin fibroblasts as a PSC model to address this question. hiPSCs derived from somatic cells could be a useful tool for addressing the radiation response in cell lineage differentiation. After radiation exposure, the hiPSCs showed a higher frequency of centrosome overduplication and multipolar cell division than the differentiated cells. To suppress the indirect effect of radiation exposure, we used the radical scavenger dimethyl sulfoxide (DMSO). Combined treatment with radiation and DMSO efficiently suppressed DNA damage and centrosome overduplication in hiPSCs. Our results will contribute to the understanding of the dynamics of stem cells and the assessment of the risk of genome instability for regenerative medicine.

Recent Trends and Potential of Radiotherapy in the Treatment of Anaplastic Thyroid Cancer.

Anaplastic thyroid cancer (ATC) is a rare but highly aggressive malignancy characterized by advanced disease at diagnosis and a poor prognosis. Despite multimodal therapeutic approaches that include surgery, radiotherapy, and chemotherapy, an optimal treatment strategy remains elusive. Current developments in targeted therapies and immunotherapy offer promising avenues for improved outcomes, particularly for BRAF-mutant patients. However, challenges remain regarding overcoming drug resistance and developing effective treatments for BRAF-wild-type tumors. This comprehensive review examines the clinical and biological features of ATC, outlines the current standards of care, and discusses recent developments with a focus on the evolving role of radiotherapy. Moreover, it emphasizes the necessity of a multidisciplinary approach and highlights the urgent need for further research to better understand ATC pathogenesis and identify new therapeutic targets. Collaborative efforts, including large-scale clinical trials, are essential for translating these findings into improved patient outcomes.

Experimental evidence that carbon-ion radiotherapy utilizes cytotoxic T lymphocyte-mediated anti-tumor immunity for shrinking tumors compared to X-ray therapy.

The therapeutic efficacy of radiotherapy (RT) is primarily driven by two factors: biophysical DNA damage in cancer cells and radiation-induced anti-tumor immunity. However, Anti-tumor immune responses between X-ray RT (XRT) and carbon-ion RT (CIRT) remain unclear. In this study, we, employed mouse models to assess the immunological contribution, especially cytotoxic T-lymphocyte (CTL)-mediated immunity, to the therapeutic effectiveness of XRT and CIRT in shrinking tumors. We irradiated mouse intradermal tumors of B16F10-ovalbumin (OVA) mouse melanoma cells and 3LL-OVA mouse lung cancer cells with carbon-ion beams or X-rays in the presence or absence of CTLs. CTL removal was performed by administration of anti-CD8 monoclonal antibody (mAb) in mice. Based on tumor growth delay, we determined the tumor growth and regression curves. The enhancement ratio (ER) of the slope of regression lines in the presence of CTLs, relative to the absence of CTLs, indicates the dependency of RT on CTLs for shrinking mouse tumors, and the biological effectiveness (RBE) of CIRT relative to XRT were calculated. Tumor growth curves revealed that the elimination of CD8+ CTLs by administrating anti-CD8 mAb accelerated tumor growth compared to the presence of CTLs in both RTs. The ERs were larger in CIRT compared to XRT in the B16F10-OVA tumor models, but not in the 3LL-OVA models, suggesting a greater contribution of CTL-mediated anti-tumor immunity to tumor reduction in CIRT compared to XRT in the B16F10-OVA tumor model. In addition, the RBE values for both models were larger in the presence of CTLs compared to models without CTLs, suggesting that CIRT may utilize CTL-mediated anti-tumor immunity more than X-ray. The findings from this study suggest that although immunological contribution to therapeutic efficacy may vary depending on the type of tumor cell, CIRT utilizes CTL-mediated immunity to a greater extent compared to XRT.

Increase of OH radical yields due to the decomposition of hydrogen peroxide by gold nanoparticles under X-ray irradiation.

We elucidate the decomposition mechanism of hydrogen peroxide, which is formed by water radiolysis, by gold nanoparticles (GNPs) under X-ray irradiation. The variations in yields of hydrogen peroxide generated in the presence of GNPs are evaluated using the Ghormley technique. The increase of yields of OH radicals has been quantified using Ampliflu® Red solutions. Almost all hydrogen peroxide generated by irradiation of <25 Gy is decomposed by GNPs, while the yield of OH radicals increases by 1.6 times. The amount of OH radicals thus obtained is almost equivalent to that of the decomposed hydrogen peroxide. The decomposition of hydrogen peroxide is an essential reaction to produce additional OH radicals efficiently in the vicinity of GNPs.

Neutron Capture Enhances Dose and Reduces Cancer Cell Viability in and out of Beam During Helium and Carbon Ion Therapy.

PURPOSE: Neutron capture enhanced particle therapy (NCEPT) is a proposed augmentation of charged particle therapy that exploits thermal neutrons generated internally, within the treatment volume via nuclear fragmentation, to deliver a biochemically targeted radiation dose to cancer cells. This work is the first experimental demonstration of NCEPT, performed using both carbon and helium ion beams with 2 different targeted neutron capture agents (NCAs). METHODS AND MATERIALS: Human glioblastoma cells (T98G) were irradiated by carbon and helium ion beams in the presence of NCAs [10B]-BPA and [157Gd]-DOTA-TPP. Cells were positioned within a polymethyl methacrylate phantom either laterally adjacent to or within a 100 × 100 × 60 mm spread out Bragg peak (SOBP). The effect of NCAs and location relative to the SOBP on the cells was measured by cell growth and survival assays in 6 independent experiments. Neutron fluence within the phantom was characterized by quantifying the neutron activation of gold foil. RESULTS: Cells placed inside the treatment volume reached 10% survival by 2 Gy of carbon or 2 to 3 Gy of helium in the presence of NCAs compared with 5 Gy of carbon and 7 Gy of helium with no NCA. Cells placed adjacent to the treatment volume showed a dose-dependent decrease in cell growth when treated with NCAs, reaching 10% survival by 6 Gy of carbon or helium (to the treatment volume), compared with no detectable effect on cells without NCA. The mean thermal neutron fluence at the center of the SOBP was approximately 2.2 × 109 n/cm2/Gy (relative biological effectiveness) for the carbon beam and 5.8 × 109 n/cm2/Gy (relative biological effectiveness) for the helium beam and gradually decreased in all directions. CONCLUSIONS: The addition of NCAs to cancer cells during carbon and helium beam irradiation has a measurable effect on cell survival and growth in vitro. Through the capture of internally generated neutrons, NCEPT introduces the concept of a biochemically targeted radiation dose to charged particle therapy. NCEPT enables the established pharmaceuticals and concepts of neutron capture therapy to be applied to a wider range of deeply situated and diffuse tumors, by targeting this dose to microinfiltrates and cells outside of defined treatment regions. These results also demonstrate the potential for NCEPT to provide an increased dose to tumor tissue within the treatment volume, with a reduction in radiation doses to off-target tissue.

Neon ion (20 Ne10 + ) charged particle beams manipulate rapid tumor reoxygenation in syngeneic mouse models.

Charged particle beams induce various biological effects by creating high-density ionization through the deposition of energy along the beam's trajectory. Charged particle beams composed of neon ions (20 Ne10+ ) hold great potential for biomedical applications, but their physiological effects on living organs remain uncertain. In this study, we demonstrate that neon-ion beams expedite the process of reoxygenation in tumor models. We simulated mouse SCCVII syngeneic tumors and exposed them to either X-ray or neon-ion beams. Through an in vivo radiobiological assay, we observed a reduction in the hypoxic fraction in tumors irradiated with 8.2 Gy of neon-ion beams 30 h after irradiation compared to 6 h post-irradiation. Conversely, no significant changes in hypoxia were observed in tumors irradiated with 8.2 Gy of X-rays. To directly quantify hypoxia in the irradiated living tumors, we utilized dynamic contrast-enhanced magnetic resonance imaging (MRI) and diffusion-weighted imaging. These combined MRI techniques revealed that the non-hypoxic fraction in neon-irradiated tumors was significantly higher than that in X-irradiated tumors (69.53% vs. 47.67%). Simultaneously, the hypoxic fraction in neon-ion-irradiated tumors (2.77%) was lower than that in X-irradiated tumors (4.27%) and non-irradiated tumors (32.44%). These results support the notion that accelerated reoxygenation occurs more effectively with neon-ion beam irradiation compared to X-rays. These findings shed light on the physiological effects of neon-ion beams on tumors and their microenvironment, emphasizing the therapeutic advantage of using neon-ion charged particle beams to manipulate tumor reoxygenation.

Prediction of DNA rejoining kinetics and cell survival after proton irradiation for V79 cells using Geant4-DNA.

PURPOSE: Track structure Monte Carlo (MC) codes have achieved successful outcomes in the quantitative investigation of radiation-induced initial DNA damage. The aim of the present study is to extend a Geant4-DNA radiobiological application by incorporating a feature allowing for the prediction of DNA rejoining kinetics and corresponding cell surviving fraction along time after irradiation, for a Chinese hamster V79 cell line, which is one of the most popular and widely investigated cell lines in radiobiology. METHODS: We implemented the Two-Lesion Kinetics (TLK) model, originally proposed by Stewart, which allows for simulations to calculate residual DNA damage and surviving fraction along time via the number of initial DNA damage and its complexity as inputs. RESULTS: By optimizing the model parameters of the TLK model in accordance to the experimental data on V79, we were able to predict both DNA rejoining kinetics at low linear energy transfers (LET) and cell surviving fraction. CONCLUSION: This is the first study to demonstrate the implementation of both the cell surviving fraction and the DNA rejoining kinetics with the estimated initial DNA damage, in a realistic cell geometrical model simulated by full track structure MC simulations at DNA level and for various LET. These simulation and model make the link between mechanistic physical/chemical damage processes and these two specific biological endpoints.

Evaluation of X-ray and carbon-ion beam irradiation with chemotherapy for the treatment of cervical adenocarcinoma cells in 2D and 3D cultures.

BACKGROUND: Cervical cancer is the second most common cancer in women and causes more than 250,000 deaths worldwide. Among these, the incidence of cervical adenocarcinomas is increasing. Cervical adenocarcinoma is not only difficult to detect and prevent in the early stages with screening, but it is also resistant to chemotherapy and radiotherapy, and its prognosis worsens significantly as the disease progresses. Furthermore, when recurrence or metastasis is observed, treatment options are limited and there is no curative treatment. Recently, heavy-particle radiotherapy has attracted attention owing to its high tumor control and minimal damage to normal tissues. In addition, heavy particle irradiation is effective for cancer stem cells and hypoxic regions, which are difficult to treat. METHODS: In this study, we cultured cervical adenocarcinoma cell lines (HeLa and HCA-1) in two-dimensional (2D) or three-dimensional (3D) spheroid cultures and evaluated the effects of X-ray and carbon-ion (C-ion) beams. RESULTS: X-ray irradiation decreased the cell viability in a dose-dependent manner in 2D cultures, whereas this effect was attenuated in 3D spheroid cultures. In contrast, C-ion irradiation demonstrated the same antitumor effect in 3D spheroid cultures as in 2D cultures. In 3D spheroid cultures, X-rays and anticancer drugs are attenuated because of hypoxia inside the spheroids. However, the impact of the C-ion beam was almost the same as that of the 2D culture, because heavy-particle irradiation was not affected by hypoxia. CONCLUSION: These results suggest that heavy-particle radiotherapy may be a new therapeutic strategy for overcoming the resistance of cervical adenocarcinoma to treatment.

Formation of clustered DNA damage in vivo upon irradiation with ionizing radiation: Visualization and analysis with atomic force microscopy.

SignificanceDNA damage causes loss of or alterations in genetic information, resulting in cell death or mutations. Ionizing radiations produce local, multiple DNA damage sites called clustered DNA damage. In this study, a complete protocol was established to analyze the damage complexity of clustered DNA damage, wherein damage-containing genomic DNA fragments were selectively concentrated via pulldown, and clustered DNA damage was visualized by atomic force microscopy. It was found that X-rays and Fe ion beams caused clustered DNA damage. Fe ion beams also produced clustered DNA damage with high complexity. Fe ion beam-induced complex DNA double-strand breaks (DSBs) containing one or more base lesion(s) near the DSB end were refractory to repair, implying their lethal effects.

Radiosensitization Effect of Gold Nanoparticles on Plasmid DNA Damage Induced by Therapeutic MV X-rays.

Gold nanoparticles (AuNPs) can be used with megavolt (MV) X-rays to exert radiosensitization effects, as demonstrated in cell survival assays and mouse experiments. However, the detailed mechanisms are not clear; besides physical dose enhancement, several chemical and biological processes have been proposed. Reducing the AuNP concentration while achieving sufficient enhancement is necessary for the clinical application of AuNPs. Here, we used positively charged (+) AuNPs to determine the radiosensitization effects of AuNPs combined with MV X-rays on DNA damage in vitro. We examined the effect of low concentrations of AuNPs on DNA damage and reactive oxygen species (ROS) generation. DNA damage was promoted by 1.4 nm +AuNP with dose enhancement factors of 1.4 ± 0.2 for single-strand breaks and 1.2 ± 0.1 for double-strand breaks. +AuNPs combined with MV X-rays induced radiosensitization at the DNA level, indicating that the effects were physical and/or chemical. Although -AuNPs induced similar ROS levels, they did not cause considerable DNA damage. Thus, dose enhancement by low concentrations of +AuNPs may have occurred with the increase in the local +AuNP concentration around DNA or via DNA binding. +AuNPs showed stronger radiosensitization effects than -AuNPs. Combining +AuNPs with MV X-rays in radiation therapy may improve clinical outcomes.

Dose- and LET-dependent changes in mouse skin contracture up to a year after either single dose or fractionated doses of carbon ion or gamma rays.

Time dependence of relative biological effectiveness (RBE) of carbon ions for skin damage was investigated to answer the question of whether the flat distribution of biological doses within a Spread-Out Bragg peak (SOBP) which is designed based on in vitro cell kill could also be flat for in vivo late responding tissue. Two spots of Indian ink intracutaneously injected into the legs of C3H mice were measured by calipers. An equieffective dose to produce 30% skin contraction was calculated from a dose-response curve and used to calculate the RBE of carbon ion beams. We discovered skin contraction progressed after irradiation and then reached a stable/slow progression phase. Equieffective doses decreased with time and the decrease was most prominent for gamma rays and least prominent for 100 keV/µm carbon ions. Survival parameter of alpha but not beta in the linear-quadratic model is closely related to the RBE of carbon ions. Biological doses within the SOBP increased with time but their distribution was still flat up to 1 year after irradiation. The outcomes of skin contraction studies suggest that (i) despite the higher RBE for skin contracture after carbon ions compared to gamma rays, gamma rays can result in a more severe late effect of skin contracture. This is due to the carbon effect saturating at a lower dose than gamma rays, and (ii) the biological dose distribution throughout the SOBP remains approximately the same even one year after exposure.

Performance Evaluation for Repair of HSGc-C5 Carcinoma Cell Using Geant4-DNA.

Track-structure Monte Carlo simulations are useful tools to evaluate initial DNA damage induced by irradiation. In the previous study, we have developed a Gean4-DNA-based application to estimate the cell surviving fraction of V79 cells after irradiation, bridging the gap between the initial DNA damage and the DNA rejoining kinetics by means of the two-lesion kinetics (TLK) model. However, since the DNA repair performance depends on cell line, the same model parameters cannot be used for different cell lines. Thus, we extended the Geant4-DNA application with a TLK model for the evaluation of DNA damage repair performance in HSGc-C5 carcinoma cells which are typically used for evaluating proton/carbon radiation treatment effects. For this evaluation, we also performed experimental measurements for cell surviving fractions and DNA rejoining kinetics of the HSGc-C5 cells irradiated by 70 MeV protons at the cyclotron facility at the National Institutes for Quantum and Radiological Science and Technology (QST). Concerning fast- and slow-DNA rejoining, the TLK model parameters were adequately optimized with the simulated initial DNA damage. The optimized DNA rejoining speeds were reasonably agreed with the experimental DNA rejoining speeds. Using the optimized TLK model, the Geant4-DNA simulation is now able to predict cell survival and DNA-rejoining kinetics for HSGc-C5 cells.

Comparative analysis of the immune responses in cancer cells irradiated with X-ray, proton and carbon-ion beams.

Radiotherapy (RT) is an effective treatment option for cancer; however, its efficacy remains less than optimal in locally advanced cancer. Immune checkpoint inhibitor-based therapy, including the administration of anti-PD-L1 antibodies, is a promising approach that works synergistically with RT. Proton beam therapy and carbon-ion therapy are common options for patients with cancer. Proton and carbon ions are reported to induce an immune reaction in cancer cells; however, the underlying mechanisms remain unclear. Here, we aimed to compare the immune responses after irradiation (IR) with X-ray, protons, and carbon ions in an oesophageal cancer cell line and the underlying mechanisms. An oesophageal cancer cell line, KYSE450, was irradiated with 1 fraction/15 GyE (Gy equivalent) of X-ray, proton, or carbon-ion beams, and then, the cells were harvested for RNA sequencing and gene enrichment analysis. We also knocked out STING and STAT1 in the quest for mechanistic insights. RNA sequencing data revealed that gene expression signatures and biological processes were different in KYSE450 irradiated with X-ray, proton, and carbon-ion beams 6-24 h after IR. However, after 3 days, a common gene expression signature was detected, associated with biological pathways involved in innate immune responses. Gene knock-out experiments revealed that the STING-STAT1 axis underlies the immune reactions after IR. X-Ray, proton, and carbon-ion IRs induced similar immune responses, regulated by the STING-STAT1 axis.

Comparable radiation sensitivity in p53 wild-type and p53 deficient tumor cells associated with different cell death modalities.

Studies of radiation interaction with tumor cells often take apoptosis as the desired results. However, mitotic catastrophe and senescence are also promoted by clinically relevant doses of radiation. Furthermore, p53 is a well-known transcription factor that is closely associated with radiosensitivity and radiation-induced cell death. Therefore, we aimed to investigate the involvement of radiosensitivity, cell death modalities and p53 status in response to carbon-ion radiation (CIR) here. Isogenic human colorectal cancer cell lines HCT116 (p53+/+ and p53-/-) were irradiated with high-LET carbon ions. Cell survival was determined by the standard colony-forming assay. 53BP1 foci were visualized to identify the repair kinetics of DNA double-strand breaks (DSBs). Cellular senescence was measured by SA-ß-Gal and Ki67 staining. Mitotic catastrophe was determined with DAPI staining. Comparable radiosensitivities of p53+/+ and p53-/- HCT116 colorectal cells induced by CIR were demonstrated, as well as persistent 53BP1 foci indicated DNA repair deficiency in both cell lines. Different degree of premature senescence in isogenic HCT116 colorectal cancer cells suggested that CIR-induced premature senescence was more dependent on p21 but not p53. Sustained upregulation of p21 played multifunctional roles in senescence enhancement and apoptosis inhibition in p53+/+ cells. p21 inhibition further increased radiosensitivity of p53+/+ cells. Complex cell death modalities rather than single cell death were induced in both p53+/+ and p53-/- cells after 5 Gy CIR. Mitotic catastrophe was predominant in p53-/- cells due to inefficient activation of Chk1 and Chk2 phosphorylation in combination with p53 null. Senescence was the major cell death mechanism in p53+/+ cells via p21-dependent pathway. Taken together, p21-mediated premature senescence might be used by tumor cells to escape from CIR-induced cytotoxicity, at least for a time.

Protective Effects of Amino Acids on Plasmid DNA Damage Induced by Therapeutic Carbon Ions.

Radioprotectors with few side effects are useful for carbon-ion therapy, which directly induces clustering damage in DNA. With the aim of finding the most effective radioprotector, we investigated the effects of selected amino acids which might have chemical DNA-repair functions against therapeutic carbon ions. In the current study, we employed five amino acids: tryptophan (Trp), cysteine (Cys), methionine (Met), valine (Val) and alanine (Ala). Samples of supercoiled pBR322 plasmid DNA with a 17 mM amino acid were prepared in TE buffer (10 mM Tris, 1 mM ethylenediaminetetraacetic acid, pH 7.5). Phosphate buffered saline (PBS) was also used in assays of the 0.17 mM amino acid. The samples were irradiated with carbon-ion beams (290 MeV/u) on 6 cm spread-out Bragg peak at the National Institute of Radiological Sciences and Heavy Ion Medical Accelerator in Chiba, Japan. Breaks in the DNA were detected as changes in the plasmids and quantified by subsequent electrophoresis on agarose gels. DNA damage yields and protection factors for each amino acid were calculated as ratios relative to reagent-free controls. Trp and Cys showed radioprotective effects against plasmid DNA damage induced by carbon-ion beam, both in PBS and TE buffer, comparable to those of Met. The double-strand break (DSB) yields and protective effects of Trp were comparable to those of Cys. The yields of both single-strand breaks and DSBs correlated with the scavenging capacity of hydroxyl radicals (rate constant for scavenging hydroxyl radicals multiplied by the amino acid concentration) in bulk solution. These data indicate that the radioprotective effects of amino acids against plasmid DNA damage induced by carbon ions could be explained primarily by the scavenging capacity of hydroxyl radicals. These findings suggest that some amino acids, such as Trp, Cys and Met, have good potential as radioprotectors for preventing DNA damage in normal tissues in carbon-ion therapy.

Health effects triggered by tritium: how do we get public understanding based on scientifically supported evidence?

The Commission for 'Corresponding to Radiation Disaster of the Japanese Radiation Research Society' formulated a description of potential health effects triggered by tritium. This was in response to the issue of discharging water containing tritium filtered by the Advanced Liquid Processing System (ALPS), generated and stored in Fukushima Daiichi Nuclear Power Station after the accident. In this review article, the contents of the description, originally provided in Japanese, which gives clear and detailed explanation about potential health effects triggered by tritium based on reliable scientific evidence in an understandable way for the public, were summarized. Then, additional information about biochemical or environmental behavior of organically bound tritium (OBT) were summarized in order to help scientists who communicate with general public.

Lethal DNA Lesions Caused by Direct and Indirect Actions of X rays are Repaired via Different DSB Repair Pathways under Aerobic and Anoxic Conditions.

We examined lethal damages of X rays induced by direct and indirect actions, in terms of double-strand break (DSB) repair susceptibility using two kinds of repair-deficient Chinese hamster ovary (CHO) cell lines. These CHO mutants (51D1 and xrs6) are genetically deficient in one of the two important DNA repair pathways after genotoxic injury [homologous recombination (HR) and non-homologous end binding (NHEJ) pathways, respectively]. The contribution of indirect action on cell killing can be estimated by applying the maximum level of dimethylsulfoxide (DMSO) to get rid of OH radicals. To control the proportion of direct and indirect actions in lethal damage, we irradiated CHO mutant cells under aerobic and anoxic conditions. The contributions of indirect action on HR-defective 51D1 cells were 76% and 57% under aerobic and anoxic conditions, respectively. Interestingly, these percentages were similar to those of the wild-type cells even if the radiosensitivity was different. However, the contributions of indirect action to cell killing on NHEJ-defective xrs6 cells were 52% and 33% under aerobic and anoxic conditions, respectively. Cell killing by indirect action was significantly affected by the oxygen concentration and the DSB repair pathways but was not correlated with radiosensitivity. These results suggest that the lethal damage induced by direct action is mostly repaired by NHEJ repair pathway since killing of NHEJ-defective cells has significantly higher contribution by the direct action. In other words, the HR repair pathway may not effectively repair the DSB by direct action in place of the NHEJ repair pathway. We conclude that the type of DSB produced by direct action is different from that of DSB induced by indirect action.

Effect of Gold Nanoparticle Radiosensitization on Plasmid DNA Damage Induced by High-Dose-Rate Brachytherapy.

PURPOSE: Gold nanoparticles (AuNPs) are candidate radiosensitizers for medium-energy photon treatment, such as γ-ray radiation in high-dose-rate (HDR) brachytherapy. However, high AuNP concentrations are required for sufficient dose enhancement for clinical applications. Here, we investigated the effect of positively (+) charged AuNP radiosensitization of plasmid DNA damage induced by 192Ir γ-rays, and compared it with that of negatively (-) charged AuNPs. METHODS: We observed DNA breaks and reactive oxygen species (ROS) generation in the presence of AuNPs at low concentrations. pBR322 plasmid DNA exposed to 64 ng/mL AuNPs was irradiated with 192Ir γ-rays via HDR brachytherapy. DNA breaks were detected by observing the changes in the form of the plasmid and quantified by agarose gel electrophoresis. The ROS generated by the AuNPs were measured with the fluorescent probe sensitive to ROS. The effects of positively (+) and negatively (-) charged AuNPs were compared to study the effect of surface charge on dose enhancement. RESULTS: +AuNPs at lower concentrations promoted a comparable level of radiosensitization by producing both single-stranded breaks (SSBs) and double-stranded breaks (DSBs) than those used in cell assays and Monte Carlo simulation experiments. The dose enhancement factor (DEF) for +AuNPs was 1.3 ± 0.2 for SSBs and 1.5 ± 0.4 for DSBs. The ability of +AuNPs to augment plasmid DNA damage is due to enhanced ROS generation. While -AuNPs generated similar ROS levels, they did not cause significant DNA damage. Thus, dose enhancement using low concentrations of +AuNPs presumably occurred via DNA binding or increasing local +AuNP concentration around the DNA. CONCLUSION: +AuNPs at low concentrations displayed stronger radiosensitization compared to -AuNPs. Combining +AuNPs with 192Ir γ-rays in HDR brachytherapy is a candidate method for improving clinical outcomes. Future development of cancer cell-specific +AuNPs would allow their wider application for HDR brachytherapy.