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Purpose: The use of Monte Carlo (MC) simulations capable of reproducing radiobiological effects of ionising radiation on human cell lines is of great importance, especially for cases involving protons and heavier ion beams. In the latter, huge uncertainties can arise mainly related to the effects of the secondary particles produced in the beam-tissue interaction. This paper reports on a detailed MC study performed using Geant4-based approach on three cancer cell lines, the HTB-177, CRL-5876 and MCF-7, that were previously irradiated with therapeutic proton and carbon ion beams. Methods: A Geant4-based approach used jointly with analytical calculations has been developed to provide a more realistic estimation of the radiobiological damage produced by proton and carbon beams in tissues, reproducing available data obtained from in vitro cell irradiations. The MC “Hadrontherapy” Geant4 application and the Local Effect Model: LEM I, LEM II and LEM III coupled with the different numerical approaches: RapidRusso (RR) and RapidScholz (RS) were used in the study. Results: Experimental survival curves are compared with those evaluated using the highlighted Geant4 MC-based approach via chi-square statistical analysis, for the combinations of radiobiological models and numerical approaches, as outlined above. Conclusion: This study has presented a comparison of the survival data from MC simulations to experimental survival data for three cancer cell lines. An overall best level of agreement was obtained for the HTB-177 cells.

Purpose:ThescientificcommunityshowsgreatinterestinthestudyofDNAdamageinduction,DNAdamagerepair,andthebiologicaleffectsoncellsandcellularsystemsafterexposuretoionizingradiation.Severalin silicomethodshavebeenproposedsofar to study these mechanisms using Monte Carlo simulations. This study outlinesaGeant4-DNAexampleapplication,named“molecularDNA”,publiclyreleasedinthe11.1versionofGeant4(December2022).Methods:ItwasdevelopedfornoviceGeant4usersandrequiresonlyabasicunder-standing of scripting languages to get started. The example includes two differentDNA-scalegeometriesofbiologicaltargets,namely“cylinders”and“humancell”.Thispublicversionisbasedonapreviousprototypeandincludesnewfeatures,suchas:theadoptionofanewapproachforthemodelingofthechemicalstage,theuseofthestan-dardDNAdamageformattodescriberadiation-inducedDNAdamage,andupgradedcomputationaltoolstoestimateDNAdamageresponse.Results:Simulationdataintermsofsingle-strandbreakanddouble-strandbreakyieldswereproducedusingeachoftheavailablegeometries.Theresultswerecomparedwiththeliterature,tovalidatetheexample,producinglessthan5%differenceinallcases.Conclusion:“molecularDNA”isaprototypetoolthatcanbeappliedinawidevarietyofradiobiologystudies,providingthescientificcommunitywithanopen-accessbasefor DNA damage quantification calculations. New DNA and cell geometries for the“molecularDNA”examplewillbeincludedinfutureversionsofGeant4-DNA.

The scientific community has a large interest in the studies of DNA damage and response after exposure to ionizing radiation. Several in-silico methods have been proposed so far to model and study the mechanisms of DNA damage using Monte Carlo simulations. The “molecularDNA” example is one of the most recent applications to simulate the irradiation of human cancer cells and bacteria using Geant4-DNA. This example enables the simulation of the physical, physico-chemical and chemical stages of liquid water irradiation, including radiolytic processes following the particle irradiation of the pre-defined human cell geometries and it can be used to calculate the early direct and non-direct DNA damage such as single (SSB) and double strand breaks (DSB) as well as DNA fragment distribution. The application is user friendly and can be used following simple macro commands. The results of the Monte Carlo simulation are compared to experimental data of DSB yields, as well as with previously published simulation data.

In the present hadrontherapy scenario, there is a growing interest in exploring the capabilities of different ion species other than protons and carbons. The possibility of using different ions paves the way for new radiotherapy approaches, such as the multi-ions treatment, where radiation could vary according to target volume, shape, depth and histologic characteristics of the tumor. For these reasons, in this paper, the study and understanding of biological-relevant quantities was extended for the case of 4He ion. Approach. Geant4 Monte Carlo based algorithms for dose- and track-averaged LET (Linear Energy Transfer) calculations, were validated for 4He ions and for the case of a mixed field characterised by the presence of secondary ions from both target and projectile fragmentation. The simulated dose and track averaged LETs were compared with the corresponding dose and frequency mean values of the lineal energy, and , derived from experimental microdosimetric spectra. Two microdosimetric experimental campaigns were carried out at the Italian eye proton therapy facility of the Laboratori Nazionali del Sud of Istituto Nazionale di Fisica Nucleare (INFN-LNS, Catania, I) using two different microdosimeters: the MicroPlus probe and the nano-TEPC (Tissue Equivalent Proportional Counter). Main results. A good agreement of and with and experimentally measured with both microdosimetric detectors MicroPlus and nano-TEPC in two configurations: full energy and modulated 4He ion beam, was found. Significance. The results of this study certify the use of a very effective tool for the precise calculation of LET, given by a Monte Carlo approach which has the advantage of allowing detailed simulation and tracking of nuclear interactions, even in complex clinical scenarios.

Accurately modeling the radiobiological mechanisms responsible for the induction of DNA damage remains a major scientific challenge, particularly for understanding the effects of low doses of ionizing radiation on living beings, such as the induction of carcinogenesis. A computational approach based on the Monte Carlo technique to simulate track structures in a biological medium is currently the most reliable method for calculating the early effects induced by ionizing radiation on DNA, the primary cellular target of such effects. The Geant4-DNA Monte Carlo toolkit can simulate not only the physical, but also the physico-chemical and chemical stages of water radiolysis. These stages can be combined with simplified geometric models of biological targets, such as DNA, to assess direct and indirect early DNA damage. In this study, DNA damage induced in a human fibroblast cell was evaluated using Geant4-DNA as a function of incident particle type (gammas, protons, and alphas) and energy. The resulting double-strand break yields as a function of linear energy transfer closely reproduced recent experimental data. Other quantities, such as fragment length distribution, scavengeable damage fraction, and time evolution of damage within an analytical repair model also supported the plausibility of predicting DNA damage using Geant4-DNA.The complete simulation chain application “molecularDNA”, an example for users of Geant4-DNA, will soon be distributed through Geant4.

CATANA (Centro di AdroTerapia ed Applicazioni Nucleari Avanzate) was the first Italian protontherapy facility dedicated to the treatment of ocular neoplastic pathologies. It is in operation at the LNS Laboratories of the Italian Institute for Nuclear Physics (INFN-LNS) and to date, 500 patients have been successfully treated. Even though proton therapy has demonstrated success in clinical settings, there is still a need for more accurate models because they are crucial for the estimation of clinically relevant RBE values. Since RBE can vary depending on several physical and biological parameters, there is a clear need for more experimental data to generate predictions. Establishing a database of cell survival experiments is therefore useful to accurately predict the effects of irradiations on both cancerous and normal tissue. The main aim of this work was to compare RBE values obtained from in-vitro experimental data with predictions made by the LEM II (Local Effect Model), Monte Carlo approaches, and semi-empirical models based on LET experimental measurements. For this purpose, the 92.1 uveal melanoma and ARPE-19 cells derived from normal retinal pigmented epithelium were selected and irradiated in the middle of clinical SOBP of the CATANA proton therapy facility. The remarkable results show the potentiality of using microdosimetric spectrum, Monte Carlo simulations and LEM model to predict not only the RBE but also the survival curves.

Even though protons show several benefits for the therapy of ocular tumors, the application of a single RBE value in eye treatment planning is still questionable and requires further investigation. In this work, the effects of protons were experimentally measured on 92.1, a specific uveal melanoma cell line. The chosen irradiation position was the middle of the 62 MeV therapeutic Spread-Out Bragg Peak. The normal, ARPE-19 retinal cells were also used to assess the behaviour of healthy tissue exposed to irradiation. Cells were irradiated with single doses ranging from 1 to 6 Gy, and the clonogenic assay was adopted to evaluate cell survival after irradiation. The radiobiological results suggest that proton irradiation can cause significant lethal damages in both normal and cancerous cells. The obtained experimental data from biological models will serve for comparisons with Monte Carlo simulations and LEM II calculations.

Aim: Newly synthesized platinum(IV) complexes with ethylenediamine-N,N’-diacetate ligands (EDDA-type) (butyl-Pt and pentyl-Pt) were investigated against two cancer (A549 lung, and HTB 140 melanoma) and one non-cancerous (MRC-5 embryonic lung fibroblast) human cell lines. Materials and Methods: The effects of these agents were compared with those of cisplatin after 6-, 24- and 48-h treatment. Sulforhodamine-B (SRB) assay was performed to estimate the cytotoxic effect, while the inhibitory effect on cell proliferation was measured using 5-bromo-2,-deoxyuridine (BrdU) incorporation assay. Cell cycle analysis was performed by flow cytometry. Type of cell death induced by these agents was determined by electrophoretic analysis of DNA, flow cytometry and by western blot analysis of proteins involved in induction of apoptosis. The effects of gamma irradiation, alone and in combination with platinum-based compounds, were examined by clonogenic and SRB assays. Results: All examined platinum-based compounds had inhibitory and antiproliferative effects on A549 cells, but not on HTB140 and MRC-5 cells. Butyl-Pt, pentyl-Pt and cisplatin arrested the cell cycle in the S-phase and induced apoptotic cell death via regulation of expression of B-cell lymphoma 2 (BCL2) and BCL2-associated X (BAX) proteins. Platinum-based compounds increased the sensitivity of A549 cells to gamma irradiation. Butyl-Pt and pentyl-Pt showed better antitumour effects against A549 cells than did cisplatin, by interfering in cell proliferation and the cell cycle, and by triggering apoptosis. Conclusion: The effects of gamma irradiation on tumour cells may be amplified by pre-treatment of cells with platinum-based compounds.

Ionising radiation induced DNA damage and subsequent biological responses to it depend on the radiation's track-structure and its energy loss distribution pattern. To investigate the underlying biological mechanisms involved in such complex system, there is need of predicting biological response by integrated Monte Carlo (MC) simulations across physics, chemistry and biology. Hence, in this work, we have developed an application using the open source Geant4-DNA toolkit to propose a realistic "fully integrated" MC simulation to calculate both early DNA damage and subsequent biological responses with time. We had previously developed an application allowing simulations of radiation induced early DNA damage on a naked cell nucleus model. In the new version presented in this work, we have developed three additional important features: (1) modeling of a realistic cell geometry, (2) inclusion of a biological repair model, (3) refinement of DNA damage parameters for direct damage and indirect damage scoring. The simulation results are validated with experimental data in terms of Single Strand Break (SSB) yields for plasmid and Double Strand Break (DSB) yields for plasmid/human cell. In addition, the yields of indirect DSBs are compatible with the experimental scavengeable damage fraction. The simulation application also demonstrates agreement with experimental data of gamma -H2AX yields for gamma ray irradiation. Using this application, it is now possible to predict biological response along time through track-structure MC simulations.

The specific advantages of ion beams for application in tumor therapy are attributed to their different macroscopic and microscopic energy deposition pattern as compared to conventional photon radiation. On the macroscopic scale, the inverted dose profile with a Bragg peak and small lateral scattering allow a better conformation of the dose to the tumor. On the microscopic scale, the localized energy deposition around the trajectory of the particles leads to an enhanced biological effectiveness, typically expressed in terms of the relative biological effectiveness (RBE). Experimental investigations reveal complex dependencies of RBE on many physical and biological parameters, as e.g., ion species, dose, position in the field and cell or tissue type. In order to complement the experimental work, different approaches are used for the characterization of the specific physical and biological properties of ion beams. In a set of two papers, which are linked by activities within a European HORIZON 2020 project about nuclear science and application (ENSAR2), we describe recent developments in two fields playing a key role in characterizing the increased biological effectiveness. These comprise the biophysical modeling of RBE and the microdosimetric measurements in complex radiation fields. This first paper gives a brief introduction into these fields and then focuses on aspects of biophysical modeling of RBE, specifically on semi-empirical approaches that are currently used in treatment planning for ion beam therapy. It summarizes the status and recent developments of the Local Effect Model (LEM) and its conceptual framework and shows examples of model validation using recent experimental data. The model is compared to other approaches, e.g., to the Microdosimetric-Kinetic Model (MKM), that builds the bridge to the experimental microdosimetric work. © Copyright © 2020 Scholz, Friedrich, Magrin, Colautti, Ristić-Fira and Petrović.

The aim of the present study was to investigate effects of pomegranate peel (PP) extract on different human cancer cell lines. MTT was performed to estimate cytotoxic effects of PP extract against HTB140, HTB177, MCF7, HCT116 human cancer cell lines and MRC-5 normal fibroblasts. Clonogenic assay was used to reveal cell survival after the treatment with PP extract. Cell cycle analysis was done using flow cytometry. Wound healing assay was applied to estimate inhibitory effects of PP extract on migration of cancer cells. The results showed that PP extract expressed selective cytotoxicity for cancer cells compared to normal cell line. Analyzed cancer cell lines displayed individual variations in sensitivity to PP extract reflected through changes in clonogenic survival, cell cycle distribution and migration, which may be due to the specific nature of each tested cell line. In conclusion, PP extract exhibits good inhibitory effects on tested cancer cell lines.

Purpose: Investigation of effects on DNA of γ-irradiated human cancer cells pretreated with free radical scavengers is aimed to create reference data which would enable assessment of the relative efficiency of high linear energy transfer (LET) radiations used in hadron therapy, i.e. protons and carbon ions. Materials and methods: MCF-7 breast and HTB177 lung cancer cells are irradiated with γ-rays. To minimize indirect effects of irradiation, dimethyl sulfoxide (DMSO) or glycerol are applied as free radical scavengers. Biological response to irradiation is evaluated through clonogenic cell survival, immunocytochemical and cell cycle analysis, as well as expression of proteins involved in DNA damage response. Results: Examined cell lines reveal similar level of radioresistance. Application of scavengers leads to the rise of cell survival and decreases the number of DNA double strand breaks in irradiated cells. Differences in cell cycle and protein expression between the two cell lines are probably caused by different DNA damage repair mechanisms that are activated. Conclusion: The obtained results show that DMSO and glycerol have good scavenging capacity, and may be used to minimize DNA damage induced by free radicals. Therefore, they will be used as the reference for comparison with high LET irradiations, as well as good experimental data suitable for validation of numerical simulations. © 2019, © 2019 Taylor & Francis Group, LLC.

Izvod: Chelidonium majus L. (Papaveraceae) u tradicionalnoj medicini ima primenu u lečenju širokog spektra različitih bolesti. Farmakološka svojstva ove biljke predmet su velikog broja in vitro i in vivo istraživanja, kao i kliničkih studija. Međutim, najveći broj istraživanja, naročito antitumorskog dejstva, usmeren je na ispitivanje biološke aktivnosti pojedinačno izolovanih alkaloida i flavonoida. Antitumorsko dejstvo celog ekstrakta nije dovoljno ispitano. U ovoj disertaciji opisano je direktno citotoksično dejstvo ekstrakta na humanim tumorskim ćelijskim linijama dojke (MDA-MB-231, MCF7), kolona (HCT116, SW480) i pluća (A549, H460) i diferencijalna citotoksičnost u odnosu na normalne ćelije, fibroblaste (MRC-5). Opisano je i indirektno antitumorsko dejstvo, odnosno uticaj ekstrakta na sposobnost migracije i radiosenzitivnost malignih ćelija. Ispitivani ekstrakt Chelidonium majus pokazuje vremenski- i dozno-zavisan citotoksični efekat na svim ispitivanim tumorskim ćelijskim linijama, uz dobru selektivnost prema ovim malignim ćelijama u odnosu na zdrave, netransformisane ćelije (fibroblaste). Analiza protočnom citometrijom pokazala je da ekstrakt uzrokuje zastoj u G2/M fazi ćelijskog ciklusa, kao i da indukuje apoptozu, što je potvrđeno određivanjem proteina uključenih u apoptozu i analizom morfoloških promena, karakterističnim za ovaj proces na A549 ćelijama. Utvrđeno je sinergističko dejstvo ekstrakta i doksorubicina, zavisno od tumorske ćelijske linije koja se tretira i od primenjene doze doksorubicina. Ekstrakt je inhibirao migraciju A549, HCT116 i MDA-MB 231 ćelija i povećao osetljivost ove tri ćelijske linije na γ-zračenje, čime je pokazano da ispoljava i indirektna antitumorska dejstva. Dobijeni rezultati pokazuju da ekstrakt ispoljava direktno i indirektno antitumorsko dejstvo u in vitro uslovima, te bi ovaj potencijal trebalo ispitati u in vivo sistemu., Chelidonium majus L. (Papaveraceae) is widely used in alternative medicine for treatment of various disorders. The pharmacological properties of the plant were the subject of numerous in vitro and in vivo investigations as well as clinical studies. However, most research, especially of antitumor activity, were focused on the assessment of biological activities of isolated alkaloids and flavonoids, while there are only a few studies that examine properties of the whole extract In this dissertation ethanol extract of Chelidonium majus was evaluated for its anti-cancer properties on six human cancer cell lines: breast (MDA-MB-231, MCF7), colon (HCT116, SW480) and lung (A549, H460) tumor cell lines and differential cytotoxicity as compared to normal cells, fibroblasts (MRC -5) was estimated. Also, indirect antitumor activity, i.e. effect on migration and radiosensitivity of cancer cells were investigated. Chelidonium majus extract showed selective time- and dose-dependent increase of cytotoxicity in all six cell lines, with individual cell line sensitivities. Extract promoted cell cycle arrest and induced apoptosis, as demonstrated by flow cytometric analysis of apoptosis-related proteins and by assessment of morphological changes typical for apoptosis. Cotreatment with doxorubicin enhanced cytotoxicity of the drug. Inhibitory effect on migration was shown with non-toxic extract concentration on A549, HCT116 and MDA-MB 231 cells. Also, pretreatment with low concentrations of extract increased sensitivity of these cell lines to γ-radiation. These results indicate possible usefulness of Chelidonium majus crude extract in antitumor therapy, whether through its direct cytotoxic effect, by prevention of metastasis, or as adjuvant therapy. Therefore, this potential should be examined in the in vivo system.

The aim of this study was to investigate effects of irradiations with the therapeutic proton and carbon ion beams in two non-small cell lung cancers, CRL5876 adenocarcinoma and HTB177 large cell lung carcinoma. The DNA damage response dynamics, cell cycle regulation, and cell death pathway activation were followed. Viability of both cell lines was lower after carbon ions compared to the therapeutic proton irradiations. HTB177 cells showed higher recovery than CRL5876 cells seven days following the treatments, but the survival rates of both cell lines were lower after exposure to carbon ions with respect to therapeutic protons. When analyzing cell cycle distribution of both CRL5876 and HTB177 cells, it was noticed that therapeutic protons predominantly induced G1 arrest, while the cells after carbon ions were arrested in G2/M phase. The results illustrated that differences in the levels of phosphorylated H2AX, a double-strand break marker, exist after therapeutic proton and carbon ion irradiations. We also observed dose- and time-dependent increase in the p53 and p21 levels after applied irradiations. Carbon ions caused larger increase in the quantity of p53 and p21 compared to therapeutic protons. These results suggested that various repair mechanisms were induced in the treated cells. Considering the fact that we have not observed any distinct change in the Bax/Bcl-2 ratio following irradiations, it seemed that different types of cell death were involved in the response to the two types of irradiations that were applied.

Geant4 is a software toolkit for the simulation of the passage of particles through matter. It is used by a large number of experiments and projects in a variety of application domains, including high energy physics, astrophysics and space science, medical physics and radiation protection. Over the past several years, major changes have been made to the toolkit in order to accommodate the needs of these user communities, and to efficiently exploit the growth of computing power made available by advances in technology. The adaptation of Geant4 to multithreading, advances in physics, detector modeling and visualization, extensions to the toolkit, including biasing and reverse Monte Carlo, and tools for physics and release validation are discussed here.

Background and objectives: The main goal when treating malignancies with radiation is to deprive tumour cells of their reproductive potential. One approach is to induce tumour cell apoptosis. This study was conducted to evaluate the ability of carbon ions (C-12) to induce apoptosis and cell cycle arrest in human HTB140 melanoma cells. Methods: In this in vitro study, human melanoma HTB140 cells were irradiated with the 62 MeV/n carbon (C-12) ion beam, having two different linear energy transfer (LET) values: 197 and 382 keV/mu m. The dose range was 2 to 16 Gy. Cell viability was estimated by the sulforhodamine B assay seven days after irradiation. The cell cycle and apoptosis were evaluated 48 h after irradiation using flow cytometry. At the same time point, protein and gene expression of apoptotic regulators were estimated using the Western blot and q-PCR methods, respectively. Results: Cell viability experiments indicated strong anti-tumour effects of C-12 ions. The analysis of cell cycle showed that C-12 ions blocked HTB140 cells in G2 phase and induced the dose dependent increase of apoptosis. The maximum value of 21.8 per cent was attained after irradiation with LET of 197 keV/mu m at the dose level of 16 Gy. Pro-apoptotic effects of C-12 ions were confirmed by changes of key apoptotic molecules: the p53, Bax, Bcl-2, poly ADP ribose polymerase (PARP) as well as nuclear factor kappa B (NF kappa B). At the level of protein expression, the results indicated significant increases of p53, NF kappa B and Bax/Bcl-2 ratio and PARP cleavage. The Bax/Bcl-2 mRNA ratio was also increased, while no change was detected in the level of NF kappa B mRNA. Interpretation and conclusions: The present results indicated that anti-tumour effects of C-12 ions in human melanoma HTB140 cells were accomplished through induction of the mitochondrial apoptotic pathway as well as G2 arrest.

Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (gamma H2AX). Immunofluorescent staining visualizes formation of gamma H2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of gamma H2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to gamma-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of gamma H2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of gamma H2AX foci.

In most patients with lung cancer radiation treatment is used either as single agent or in combination with radiosensitizing drugs. However, the mechanisms underlying combined therapy and its impact on different modes of cell death have not yet been fully elucidated. We aimed to examine effects of single and combined treatments with gamma-rays and erlotinib on radioresistant CRL-5876 human lung adenocarcinoma cells with particular emphasis on cell death. CRL-5876 cells were treated with gamma-rays and/or erlotinib and changes in cell cycle, DNA repair dynamics, ultrastructure, nuclear morphology and protein expression were monitored at different time points. To reveal the relationship between types of cell death that arise after these treatments, autophagy was blocked with chloroquine. We found that higher dose of gamma-rays causes G2/M arrest while adding of erlotinib to this treatment decreases the number of cells in S phase. Impact of erlotinib on kinetics of disappearance of irradiation-induced DNA double strand breaks is reflected in the increase of residual gamma-H2AX foci after 24 h. gamma-rays provoke cytoprotective autophagy which precedes development of senescence. Erlotinib predominantly induces apoptosis and enlarges the number of apoptotic cells in the irradiated CRL-5876 cells. Chloroquine improved cytotoxicity induced by radiation and erlotinib, increased apoptosis and decreased senescence in the CRL-5876 cells. The results obtained on CRL-5876 cells indicate significant radiosensitizing effect of erlotinib and suggest that chloroquine in the combination with the above treatments may have an additional antitumor effect in lung adenocarcinoma.

Geant4 is a software toolkit for the simulation of the passage of particles through matter. It is used by a large number of experiments and projects in a variety of application domains, including high energy physics, astrophysics and space science, medical physics and radiation protection. Over the past several years, major changes have been made to the toolkit in order to accommodate the needs of these user communities, and to efficiently exploit the growth of computing power made available by advances in technology. The adaptation of Geant4 to multithreading, advances in physics, detector modeling and visualization, extensions to the toolkit, including biasing and reverse Monte Carlo, and tools for physics and release validation are discussed here.