RAD51 Inhibitor and Radiation Toxicity in Vestibular Schwannoma.

OBJECTIVE: To describe the RAD51 response (DNA repair) to radiation-induced DNA damage in patient-derived vestibular schwannoma (VS) cells and investigate the utility of RAD51 inhibitor (RI-1) in enhancing radiation toxicity. STUDY DESIGN: Basic and translational science. SETTING: Tertiary academic facility. METHODS: VS tumors (n = 10) were cultured on 96-well plates and 16-well slides, exposed to radiation (0, 6, 12, or 18 Gy), and treated with RI-1 (0, 5, or 10 µM). Immunofluorescence was performed at 6 hours for γ-H2AX (DNA damage marker), RAD51 (DNA repair protein), and p21 (cell cycle arrest protein). Viability assays were performed at 96 hours, and capillary Western blotting was utilized to determine RAD51 expression in naïve VS tumors (n = 5). RESULTS: VS tumors expressed RAD51. In cultured VS cells, radiation initiated dose-dependent increases in γ-H2AX and p21 expression. VS cells upregulated RAD51 to repair DNA damage following radiation. Addition of RI-1 reduced RAD51 expression in a dose-dependent manner and was associated with increased γ-H2AX levels and decreased viability in a majority of cultured VS tumors. CONCLUSION: VS may evade radiation injury by entering cell cycle arrest and upregulating RAD51-dependent repair of radiation-induced double-stranded breaks in DNA. Although there was variability in responses among individual primary VS cells, RAD51 inhibition with RI-1 reduced RAD51-dependent DNA repair to enhance radiation toxicity in VS cells. Further investigations are warranted to understand the mechanisms of radiation resistance in VS and determine whether RI-1 is an effective radiosensitizer in patients with VS.

SELECTION AND TESTING OF EXPERIMENTAL MODELS OF NORMAL AND MALIGNANT HUMAN CELLS IN VITRO AND EVALUATION OF THEIR SENSITIVITY RANGE TO THE NEUTRON/CAPTURE AND PHOTON/CAPTURE AGENTS AND PHOTOSENSITIZERS. / VIDBIR TA APROBUVANNIa EKSPERYMENTAL'NYKh MODELEI NORMAL'NYKh TA ZLOIaKISNYKh KLITYN LIuDYNY IN VITRO TA DOSLIDZhENIa MEZh ÏKhN'OÏ ChUTLYVOSTI DLIa NEITRONOZAKhVATNYKh, FOTON/ZAKhVATNYKh AGENTIV TA FOTOSENSYBILIZATORIV.

OBJECTIVE: to investigate the structural and morpho-functional changes in test systems of malignant (A-549 cellline) and normal (fibroblasts of the 6th passage) human cells during incubation with gadolinium-containing pho-ton-capture agent «Dotavist¼ and photosensitizer «Fotolon¼. METHODS: The passaged (continuously interweaved) cell culture technique on normal human fibroblasts and malig-nant human cells; cytological, biophysical, statistical methods. RESULTS: The cytotoxic properties of «Dotavist¼ gadolinium-containing photon-capturing agent and «Photolon¼photosensitizer in a wide range of concentrations (5, 10, 25, 50, 100 and 200 µl/ml) were studied by the morpho-functional characteristics (growth kinetics, proliferative and mitotic activity, presence of atypical cells) in the invitro test systems of malignant (non-small cell lung cancer cell line A-549) and normal (6th passage fibroblasts)human cells. It was found that the cytotoxic properties of «Dotavist¼ in test systems of malignant and normal cellsare expressed under its administration in high concentrations (100 and 200 µl/ml). During incubation with«Photolon¼ photosensitizer the cytotoxic effect on malignant cells was determined at the lowest concentrations (5and 10 µl/ml). Photosensitizer administration in the increasing concentrations has lead to genotoxic effects.Cytotoxic effect of photosensitizer on the normal human fibroblasts was evident in the 5-200 µl/ml concentrationrange. There was a moderate decrease in mitotic activity along with increasing concentration. Genotoxic propertiesof photosensitizer were evident at 25 µl/ml concentration and above. CONCLUSION: Study results of the effectiveness of neutron-capture and photon-capture technologies by the sensi-tivity assay in the in vitro test systems of human malignant cells (non-small cell lung cancer cell line A-549) andnormal cells (transplantable human fibroblast culture, the 6th passage) to the gadolinium-containing photon-cap-ture «Dotavist¼ agent and «Photolon¼ photosensitizer in different concentrations provide the basis for pre-clinicalstage of evaluating the effectiveness of medications used in binary technologies.

MODELLING γ-H2AX FOCI INDUCTION TO MIMIC LIMITATIONS IN THE SCORING TECHNIQUE.

An approach based on track-structure calculations has been developed to take account of artefacts occurring during γ-H2AX foci detection in 2D images of samples analyzed through immunocytochemistry. The need of this works stems from the observed saturation in foci yields measured after X-ray doses higher than few grays, hindering an unambiguous quantification of DNA damage and of radiation effectiveness. The proposed modelling approach allows to simulate the observer's point of view for foci scoring, mimicking the selection of a slice Δz of the cell nucleus due to the microscope depth of field, and applying a clustering algorithm to group together damages within a resolution parameter r. Calculation results were benchmarked with experimental measurements at an early time-point for mouse breast cancer cells, irradiated with X-ray doses in the range 0-5 Gy. The model is able to reproduce the saturation in experimental data.

INVESTIGATION INTO THE PROBABILITY FOR MISCOUNTING IN FOCI-BASED ASSAYS.

When early radiation damage to biological systems is studied based on the formation of foci at the location of DNA double-strand breaks, the foci observed in irradiated cells either may be induced by ionizing radiation (IR) interactions or they may be due to other causes that lead to observation of foci also in unirradiated cells. Generally, to take account of the latter, additional samples are taken where the exposure to IR is skipped in the protocol. The data analysis relies on statistical independence of the frequency distributions of background and radiation-induced foci. In microscopy, however, the observed spatial patterns of foci are 2D projections of the spatial distributions of foci in the observed cell nuclei. This may lead to missing foci when scoring their number, particularly if projections of foci overlap or coincide. This paper investigates to what extent the statistical independence of the frequency distribution of the number of foci coming from IR interaction or other causes is compromised by foci overlapping.

BYSTANDER WI-38 CELLS MODULATE DNA DOUBLE-STRAND BREAK REPAIR IN MICROBEAM-TARGETED A549 CELLS THROUGH GAP JUNCTION INTERCELLULAR COMMUNICATION.

Bi-directional signaling involved in radiation-induced bystander effect (RIBE) between irradiated carcinoma cells and their surrounding non-irradiated normal cells is relevant to radiation cancer therapy. Using the SPICE-NIRS microbeam, we delivered 500 protons to A549-GFP lung carcinoma cells, stably expressing H2B-GFP, which were co-cultured with normal WI-38 cells. The level of γ-H2AX, a marker for DNA double-strand breaks (DSB), was subsequently measured up to 24-h post-irradiation in both targeted and bystander cells. As a result, inhibition of gap junction intercellular communication (GJIC) attenuated DSB repair in targeted A549-GFP cells, and suppressed RIBE in bystander WI-38 cells but not in distant A549-GFP cells. This suggests that GJIC plays a two-way role through propagating DNA damage effect between carcinoma to normal cells and reversing the bystander signaling, also called 'rescue effect' from bystander cells to irradiated cells, to enhance the DSB repair in targeted cells.

VISUALIZATION OF THE DNA REPAIR PROCESS IN MAMMALIAN CELLS TRANSFECTED WITH EGFP-EXPRESSING PLASMID DNA AFTER EXPOSURE TO X-RAYS IN VITRO.

To investigate the repair process of DNA damage induced by ionizing radiation in isolation from various types of cytoplasmic damage, we transfected X-irradiated enhanced green fluorescent protein (EGFP)-expressing plasmid DNA into non-irradiated mammalian cells using lipofectamine. The repair kinetics of the irradiated plasmids in the cells were visualized under microscopy as the EGFP fluorescence emitted by transfected cells. Using an agarose gel electrophoresis method, the yields of single- and double-strand breaks of the plasmids were also quantified. As positive control experiments, plasmid DNA with single- or double-strand breaks induced by a nicking or restriction enzyme were also transfected into the cells. The DNA repair rates for X-ray-irradiated plasmids were significantly lower than those of the enzymatically digested positive control samples. These results indicate that X-rays could induce less repairable damage than that induced by enzymes.

Extracellular matrix affects different aspects of cell behaviour potentially involved in response to aminolevulinic acid-based photoinactivation.

Two-dimensional cell cultures do not seem to be reliable models for anticancer drug discovery and validation. Numerous in vitro tumour models of different complexity have been evaluated with the aim to enhance anticancer drug development, but whether all these models could be considered as physiologically relevant is a question. Even type of the extracellular matrix may markedly influence experimental results and supposedly also clinical treatment outcome. By using three human oesophageal cell lines and three-dimensional cultures based on collagen type I, abundant component of stromal tissue, and Matrigel, a surrogate of basement membrane, we tested the impact of extracellular matrix on different aspects of cell behaviour. We applied live cell fluorescence confocal microscopy in combination with image analysis and supplemented it with immunohistochemical analysis of differentiation markers in fixed samples. We found that cell morphogenesis, differentiation, extracellular vesicle formation, protoporphyrin IX production from aminolevulinic acid and response to subsequent photodynamic intervention induced by red light may be affected by the type of extracellular matrix and these modifications occur in a cell-type dependent manner. Our results demonstrate that the choice of the correct extracellular matrix for in vitro tumour models is crucial for gathering clinically relevant information from in vitro experimental studies.

Recovery from sublethal damage and potentially lethal damage : Proton beam irradiation vs. X­ray irradiation.

PURPOSE: In order to clarify the biological response of tumor cells to proton beam irradiation, sublethal damage recovery (SLDR) and potentially lethal damage recovery (PLDR) induced after proton beam irradiation at the center of a 10 cm spread-out Bragg peak (SOBP) were compared with those seen after X­ray irradiation. METHODS: Cell survival was determined by a colony assay using EMT6 and human salivary gland tumor (HSG) cells. First, two doses of 4 Gy/GyE (Gray equivalents, GyE) were given at an interfraction interval of 0-6 h. Second, five fractions of 1.6 Gy/GyE were administered at interfraction intervals of 0-5 min. Third, a delayed-plating assay involving cells in plateau-phase cultures was conducted. The cells were plated in plastic dishes immediately or 2-24 h after being irradiated with 8 Gy/GyE of X­rays or proton beams. Furthermore, we investigated the degree of protection from the effects of X­rays or proton beams afforded by the radical scavenger dimethyl sulfoxide to estimate the contribution of the indirect effect of radiation. RESULTS: In both the first and second experiments, SLDR was more suppressed after proton beam irradiation than after X­ray irradiation. In the third experiment, there was no difference in PLDR between the proton beam and X­ray irradiation conditions. The degree of protection tended to be higher after X­ray irradiation than after proton beam irradiation. CONCLUSION: Compared with that seen after X­ray irradiation, SLDR might take place to a lesser extent after proton beam irradiation at the center of a 10 cm SOBP, while the extent of PLDR does not differ significantly between these two conditions.

A branching process model of heterogeneous DNA damages caused by radiotherapy in in vitro cell cultures.

This paper deals with the dynamic modeling and simulation of cell damage heterogeneity and associated mutant cell phenotypes in the therapeutic responses of cancer cell populations submitted to a radiotherapy session during in vitro assays. Each cell is described by a finite number of phenotypic states with possible transitions between them. The population dynamics is then given by an age-dependent multi-type branching process. From this representation, we obtain formulas for the average size of the global survival population as well as the one of subpopulations associated with 10 mutation phenotypes. The proposed model has been implemented into Matlab© and the numerical results corroborate the ability of the model to reproduce four major types of cell responses: delayed growth, anti-proliferative, cytostatic and cytotoxic.

Nine New Triterpene Glycosides, Magnumosides A1-A4, B1, B2, C1, C2 and C4, from the Vietnamese Sea Cucumber Neothyonidium (=Massinium) magnum: Structures and Activities against Tumor Cells Independently and in Synergy with Radioactive Irradiation.

Nine new sulfated triterpene glycosides, magnumosides A1 (1), A2 (2), A3 (3), A4 (4), B1 (5), B2 (6), C1 (7), C2 (8) and C4 (9) as well as a known colochiroside B2 (10) have been isolated from the tropical Indo-West Pacific sea cucumber Neothynidium (=Massinium) magnum (Phyllophoridae, Dendrochirotida) collected in the Vietnamese shallow waters. The structures of new glycosides were elucidated by 2D NMR spectroscopy and mass-spectrometry. All the isolated new glycosides were characterized by the non-holostane type lanostane aglycones having 18(16)-lactone and 7(8)-double bond and differed from each other by the side chains and carbohydrate moieties structures. Magnumoside A1 (1) has unprecedented 20(24)-epoxy-group in the aglycone side chain. Magnumosides of the group A (1-4) contained disaccharide monosulfated carbohydrate moieties, of the group B (5, 6)-tetrasaccharide monosulfated carbohydrate moieties and, finally, of the group C (7-9)-tetrasaccharide disulfated carbohydrate moieties. The cytotoxic activities of the compounds 1-9 against mouse spleen lymphocytes, the ascites form of mouse Ehrlich carcinoma cells, human colorectal carcinoma DLD-1 cells as well as their hemolytic effects have been studied. Interestingly, the erythrocytes were more sensitive to the glycosides action than spleenocytes and cancer cells tested. The compounds 3 and 7 significantly inhibited the colony formation and decreased the size of colonies of DLD-1 cancer cells at non-cytotoxic concentrations. Moreover, the synergism of effects of radioactive irradiation and compounds 3 and 7-9 at subtoxic doses on proliferation of DLD-1 cells was demonstrated.

The impact of concentration and administration time on the radiomodulating properties of undecylprodigiosin in vitro.

Undecylprodigiosin pigment (UPP) is reported to display cytotoxic activity towards various types of tumours. Nevertheless, its efficacy in modifying the cellular response to ionising radiation is still unknown. In this study, the radiomodulating effects of UPP were investigated. The effects of UPP were assessed in vitro by treating cultures of human peripheral blood with UPP and ionising radiation using two treatment regimens, the UPP pre-irradiation treatment and UPP post-irradiation treatment. The activity of UPP was investigated evaluating its effects on the radiation-induced micronuclei formation, cell proliferation, and induction of apoptosis. The redox modulating effects of UPP were examined measuring the catalase activity and the level of malondialdehyde, as a measure of oxidative stress. The results showed that UPP effects on cellular response to ionising radiation depend on its concentration and the timing of its administration. At low concentration, the UPP displayed radioprotective effects in γ-irradiated human lymphocytes while at higher concentrations, it acted as a radiosensitiser enhancing either mitotic catastrophe or apoptosis depending on the treatment regimen. The UPP modified redox processes in cells, particularly when it was employed prior to γ-irradiation. Our data highlight the importance of further research of the potential of UPP to sensitize tumour cells to radiation therapy by inhibiting pathways that lead to treatment resistance.

Human papillomavirus status and the relative biological effectiveness of proton radiotherapy in head and neck cancer cells.

BACKGROUND: Human papillomavirus (HPV)-positive oropharyngeal carcinomas response better to X-ray therapy (XRT) than HPV-negative disease. Whether HPV status influences the sensitivity of head and neck cancer cells to proton therapy or the relative biological effectiveness (RBE) of protons versus XRT is unknown. METHODS: Clonogenic survival was used to calculate the RBE; immunocytochemical analysis and neutral comet assay were used to evaluate unrepaired DNA double-strand breaks. RESULTS: HPV-positive cells were more sensitive to protons and the unrepaired double-strand breaks were more numerous in HPV-positive cells than in HPV-negative cells (p < .001). Protons killed more cells than did XRT at all fraction sizes (all RBEs > 1.06). Cell line type and radiation fraction size influenced the RBE. CONCLUSION: HPV-positive cells were more sensitive to protons than HPV-negative cells maybe through the effects of HPV on DNA damage and repair. The RBE for protons depends more on cell type and fraction size than on HPV status. © 2016 Wiley Periodicals, Inc. Head Neck 39: 708-715, 2017.

The changing paradigm of tumour response to irradiation.

Tumours contain multiple different cell populations, including cells derived from the bone marrow as well as cancer-associated fibroblasts and various stromal populations including the vasculature. The microenvironment of the tumour cells plays a significant role in the response of the tumour to radiation treatment. Low levels of oxygen (hypoxia) caused by the poorly organized vasculature in tumours have long been known to affect radiation response; however, other aspects of the microenvironment may also play important roles. This article reviews some of the old literature concerning tumour response to irradiation and relates this to current concepts about the role of the tumour microenvironment in tumour response to radiation treatment. Included in the discussion are the role of cancer stem cells, radiation damage to the vasculature and the potential for radiation to enhance immune activity against tumour cells. Radiation treatment can cause a significant influx of bone marrow-derived cell populations into both normal tissues and tumours. Potential roles of such cells may include enhancing vascular recovery as well as modulating immune reactivity.

Modelling responses to spatially fractionated radiation fields using preclinical image-guided radiotherapy.

OBJECTIVE: Radiotherapy is planned to achieve the optimal physical dose distribution to the target tumour volume whilst minimizing dose to the surrounding normal tissue. Recent in vitro experimental evidence has demonstrated an important role for intercellular communication in radiobiological responses following non-uniform exposures. This study aimed to model the impact of these effects in the context of techniques involving highly modulated radiation fields or spatially fractionated treatments such as spatially fractionated radiotherapy (GRID). METHODS: Using the small-animal radiotherapy research platform as a key enabling technology to deliver precision imaged-guided radiotherapy, it is possible to achieve spatially modulated dose distributions that model typical clinical scenarios. In this work, we planned uniform and spatially fractionated dose distributions using multiple isocentres with beam sizes of 0.5-5 mm to obtain 50% volume coverage in a subcutaneous murine tumour model and applied a model of cellular response that incorporates intercellular communication to assess the potential impact of signalling effects with different ranges. RESULTS: Models of GRID treatment plans which incorporate intercellular signalling showed increased cell killing within the low-dose region. This results in an increase in the equivalent uniform dose for GRID exposures compared with standard models, with some GRID exposures being predicted to be more effective than uniform delivery of the same physical dose. CONCLUSION: This study demonstrates the potential impact of radiation-induced signalling on tumour cell response for spatially fractionated therapies and identifies key experiments to validate this model and quantify these effects in vivo. Advances in knowledge: This study highlights the unique opportunities now possible using advanced preclinical techniques to develop a foundation for biophysical optimization in radiotherapy treatment planning.

Polychromatic Light (480-3400 nm) Upregulates Sensitivity of Tumor Cells to Lysis by Natural Killers.

OBJECTIVE: This study evaluates the participation of immunological mechanisms of downregulation of murine hepatoma cells MH22a after direct exposure to polychromatic polarized light. BACKGROUND DATA: Previous studies have shown that exposure to a combination of visible (VIS) and infrared (IR) light leads to decreased tumorigenicity of the murine hepatoma cells MH22a, which correlated with an increase in the amount of cells with reorganized cytoskeleton in the submembrane region. The mechanism of tumor inhibition and elimination has not been determined. MATERIALS AND METHODS: Polychromatic light (480-3400 nm) has been used at doses of 4.8 and 9.6 J/cm(2) to determine the sensitivity of murine MH22a cells and human erythroleukemia cells K562 exposed to this light, to lysis by effector cells of innate immunity (NK cells), and enhancement of the glycocalyx of the studied tumor cells. This was determined using flow cytometry, the H(3)-uridine cytotoxic test followed by spectrophotometry. RESULTS: VIS-IR light increases the sensitivity of MH-22a cells at a dose 4.8 J/cm(2) and K562 cells at 9.6 J/cm(2). The enhancement of sensitivity of tumor cells to NK lysis changed their ability to absorb alcian blue, reflecting a change in the expression of the glycocalyx. CONCLUSIONS: Increasing the sensitivity of the murine tumor cells MH22a and human K562 irradiated VIS-IR light correlated with a change in the expression of their glycocalyx. The results of the present study demonstrate that the reduction of tumorigenicity of irradiated tumor cells is due to their sensitivity to lysis by NK cells of the immune system.

Mechanisms of Melanoma Promotion by Ultraviolet Radiation.

The mutagenic properties of ultraviolet radiation drive the initiation of melanoma. Induction of matrix metalloproteinases in melanoma cells by longwave UVA radiation, possibly via a Warburg-like effect, promotes melanoma invasiveness. This is one of several mechanisms by which ultraviolet radiation also promotes further growth of previously established melanomas.

Low-Intensity Laser Irradiation at 636 nm Induces Increased Viability and Proliferation in Isolated Lung Cancer Stem Cells.

OBJECTIVE: The purpose of this in vitro study was to evaluate the effects of low-intensity laser irradiation (LILI) on isolated lung cancer stem cells (CSCs) after several time intervals, using a wavelength of 636 nm and fluences between 5 and 20 J/cm2. BACKGROUND DATA: LILI has been proven to have a biomodulatory effect on various diseased conditions. A number of studies have been conducted on CSCs. METHODS: Lung CSCs were isolated from lung cancer cells (A549), using cell surface marker CD 133. Isolated lung CSCs were divided into four groups: group 1 consisted of control cells receiving no irradiation; groups 2, 3, and 4 were exposed to laser irradiation at fluences of 5, 10, and 20 J/cm2, respectively. LILI was performed using a 636 nm diode laser with a power output of ±85 mW. Cellular responses were evaluated after 24, 48, or 72 h, and included cell morphology, viability, and proliferation. RESULTS: Cellular morphology indicated an increase in cell density caused by cell proliferation over time. Biostimulatory effects were achieved in lung CSCs when examining viability and proliferation. CONCLUSIONS: It should, therefore, be noted that a low wavelength of 636 nm at various fluences induces biostimulation, which may have detrimental effects when using LILI as a form of regeneration.

Wnt/ß-catenin pathway involvement in ionizing radiation-induced invasion of U87 glioblastoma cells.

BACKGROUND: Radiotherapy has been reported to promote the invasion of glioblastoma cells; however, the underlying mechanisms remain unclear. Here, we investigated the role of the Wnt/ß-catenin pathway in radiation-induced invasion of glioblastoma cells. METHODS: U87 cells were irradiated with 3 Gy or sham irradiated in the presence or absence of the Wnt/ß-catenin pathway inhibitor XAV 939. Cell invasion was determined by an xCELLigence real-time cell analyser and matrigel invasion assays. The intracellular distribution of ß-catenin in U87 cells with or without irradiation was examined by immunofluorescence and Western blotting of nuclear fractions. We next investigated the effect of irradiation on Wnt/ß-catenin pathway activity using TOP/FOP flash luciferase assays and quantitative polymerase chain reaction analysis of ß-catenin target genes. The expression levels and activities of two target genes, matrix metalloproteinase (MMP)-2 and MMP-9, were examined further by Western blotting and zymography. RESULTS: U87 cell invasiveness was increased significantly by ionizing radiation. Interestingly, ionizing radiation induced nuclear translocation and accumulation of ß-catenin. Moreover, we found increased ß-catenin/TCF transcriptional activities, followed by up-regulation of downstream genes in the Wnt/ß-catenin pathway in irradiated U87 cells. Importantly, inhibition of the Wnt/ß-catenin pathway by XAV 939, which promotes degradation of ß-catenin, significantly abrogated the pro-invasion effects of irradiation. Mechanistically, XAV 939 suppressed ionizing radiation-triggered up-regulation of MMP-2 and MMP-9, and inhibited the activities of these gelatinases. CONCLUSION: Our data demonstrate a pivotal role of the Wnt/ß-catenin pathway in ionizing radiation-induced invasion of glioblastoma cells, and suggest that targeting ß-catenin is a promising therapeutic approach to overcoming glioma radioresistance.

Impact of flattening-filter-free radiation on the clonogenic survival of astrocytic cell lines.

BACKGROUND AND PURPOSE: Flattening-filter-free (FFF) beams are increasingly used in radiotherapy as delivery times can be substantially reduced. However, the relative biologic effectiveness (RBE) of FFF may be increased relative to conventional flattened (FLAT) beams due to differences in energy spectra. Therefore, we investigated the effects of FFF and FLAT beams on the clonogenic survival of astrocytoma cells. MATERIAL AND METHODS: Three cell lines (U251, U251-MGMT, and U87) were irradiated with 6-MV and 10-MV X-rays from a linear accelerator in FFF- or FLAT-beam modes at dose rates in the range of 0.5-24 Gy/min. The surviving fraction (SF) as function of dose (2-12 Gy) was determined by the colony formation assay and fitted by the linear-quadratic model. For both beams (FFF or FLAT), the cells were pelleted in conical 15-ml centrifuge tubes and irradiated at 2-cm depth in a 1 × 1-cm(2) area on the central axis of a 30 × 30-cm(2) field. Dosimetry was performed with a 0.3-cm(3) rigid ionization chamber. RBE was determined for FFF versus FLAT irradiation. RESULTS: The RBE of FFF at 7.3-11.3 Gy was 1.027 ± 0.013 and 1.063 ± 0.018 relative to FLAT beams for 6- and 10-MV beams, respectively, and was only significantly higher than 1 for 10 MV. Significantly increased survival rates were seen for lower dose rates (0.5 Gy/min FLAT vs. 5 Gy/min FLAT) at higher doses (11.9 Gy), while no differences were seen at dose rates ≥ 1.4 Gy/min (1.4 Gy/min FFF vs. 14 Gy/min FFF and 2.4 Gy/min FFF vs. 24 Gy/min FFF). CONCLUSIONS: FFF beams showed only a slightly increased RBE relative to FLAT beams in this experimental set-up, which is unlikely to result in clinically relevant differences in outcome.

Targeting of EGFR and HER2 with therapeutic antibodies and siRNA: a comparative study in glioblastoma cells.

BACKGROUND: The epidermal growth factor receptors, EGFR (HER1) and HER2, have proven prognostic relevance in a variety of human malignancies and both are functionally involved in the molecular pathogenesis of malignant gliomas. MATERIAL AND METHODS: We selectively inhibited EGFR and HER2 in glioblastoma cell lines via EGFR- and HER2-specific siRNAs and through the binding of the therapeutic antibodies cetuximab and trastuzumab. The expression of EGFR and HER2 was verified by real-time PCR and western blot analyses. We examined the growth rate, cell cycle distribution, cell migration, clonogenic survival, and radiosensitivity of U251MG and LN-229 glioblastoma cell lines to determine the physiological and cell biological effects of EGFR and HER2 targeting. RESULTS: EGFR and HER2 targeting using the therapeutic antibodies cetuximab and trastuzumab had no effect on cellular growth rate, cell cycle distribution, cell migration, clonogenic survival, and radiosensitivity in the cell lines U251 and LN-229. In contrast, siRNA knock-down of EGFR and HER2, reduced the growth rate by 40-65 %. The knock-down of EGFR did not change the cell migration rate in the cell lines U251 and LN-229. However, knock-down of HER2 reduced the cell migration rate by 50 %. Radiobiological analysis revealed that EGFR knock-down induced no radiosensitization in U251MG and LN-229 cells. However, the knock-down of HER2 induced radiosensitization in U251MG cells. CONCLUSION: The epidermal growth factor receptor HER2 is a promising anti-tumor target for the therapy of glioblastoma. HER2 targeting may represent a promising strategy to induce cell physiological and radiobiological anti-tumor effects in glioblastoma.