Radiosensitizing Effects of Irinotecan versus Oxaliplatin Alone and in Combination with 5-Fluorouracil on Human Colorectal Cancer Cells.

To date, oxaliplatin and irinotecan are used in combination with 5-flourouracil (5-FU) for metastatic colorectal cancer. In this study it was tested whether oxaliplatin and irinotecan and their combinations with 5-FU have an enhanced effect when treated simultaneously with ionizing radiation. In addition, it should be compared whether one combination therapy is more effective than the other. Colorectal cancer cells (HT-29) were treated with irinotecan or oxaliplatin, both alone and in combination with 5-FU, and subsequently irradiated. The cell growth, metabolic activity and proliferation of cells were investigated, and the clonogenic survival was determined. Furthermore, the assessment of radiation-induced DNA damage and the influence of the drugs and their combinations on DNA damage repair was investigated. Treatment with irinotecan or oxaliplatin in combination with 5-FU inhibited proliferation and metabolic activity as well as clonogenic survival and the DNA damage repair capacity of the tumor cells. The comparison of oxaliplatin and irinotecan with simultaneous irradiation showed the same effect of both drugs. When oxaliplatin or irinotecan was combined with 5-FU, tumor cell survival was significantly lower than with monotherapy; however, there was no superiority of either combination regimen. Our results have shown that the combination of 5-FU and irinotecan is as effective as the combination of 5-FU with oxaliplatin. Therefore, our data support the use of FOLFIRI as a radiosensitizer.

Induction of Radiodermatitis in Nude Mouse Model Using Gamma Irradiator IBL 637.

INTRODUCTION: Acute radiodermatitis is a common, though severe, side effect of radiotherapy against cancer that may lead to an interruption or even abortion of the radiotherapy. Mouse models provide an excellent tool to study pathomechanisms of a radiation-induced dermatitis as well as to test and develop novel innovative treatment strategies. OBJECTIVE: The aim of this study was to provide an overview of different mouse models and irradiation devices that have been used so far and to describe the process of the induction of a radiation dermatitis in an immune proficient nude mouse model (SKH1-Hrhr) using a IBL 637 cesium-137γ-ray machine. METHODS: This process includes the construction of a radiation shielding chamber, restricting the radiation to the right hind leg of the mouse, a dosimetry, and a dose finding study to identify the appropriate irradiation dose to induce a moderate radiation dermatitis. RESULTS: A radiation shielding chamber was successfully constructed allowing selective irradiation of the right hind leg. A moderate radiodermatitis is induced with irradiation doses in the range of 60-70 Gy under the here described conditions. Symptoms peak about 8 days after irradiation and decrease relatively quickly thereafter. Histological analyses confirmed typical signs of inflammation. CONCLUSION: This study describes for the first time a protocol to induce a moderate radiodermatitis in the nude mouse model SKH1-Hrhr using a IBL 637 gamma irradiator. This protocol will allow researchers to study novel treatment strategies to alleviate the burden of a radiodermatitis as a side effect of cancer treatment.

Perspectives for microbeam irradiation at the SYRMEP beamline.

It has been shown previously both in vitro and in vivo that microbeam irradiation (MBI) can control malignant tumour cells more effectively than the clinically established concepts of broad beam irradiation. With the aim to extend the international capacity for microbeam research, the first MBI experiment at the biomedical beamline SYRMEP of the Italian synchrotron facility ELETTRA has been conducted. Using a multislit collimator produced by the company TECOMET, arrays of quasi-parallel microbeams were successfully generated with a beam width of 50 µm and a centre-to-centre distance of 400 µm. Murine melanoma cell cultures were irradiated with a target dose of approximately 65 Gy at a mean photon energy of ∼30 keV with a dose rate of 70 Gy s-1 and a peak-to-valley dose of ∼123. This work demonstrated a melanoma cell reduction of approximately 80% after MBI. It is suggested that, while a high energy is essential to achieve high dose rates in order to deposit high treatment doses in a short time in a deep-seated target, for in vitro studies and for the treatment of superficial tumours a spectrum in the lower energy range might be equally suitable or even advantageous.

Perampanel Add-on to Standard Radiochemotherapy in vivo Promotes Neuroprotection in a Rodent F98 Glioma Model.

An abnormal glutamate signaling of glioblastoma may contribute to both tumor progression and the generation of glioma-associated epileptic seizures. We hypothesized that the AMPA receptor antagonist perampanel (PER) could attenuate tumor growth and epileptic events. F98 glioma cells, grown orthotopically in Fischer rats, were employed as a model of glioma to investigate the therapeutic efficiency of PER (15 mg/kg) as adjuvant to standard radiochemotherapy (RCT). The epileptiform phenotype was investigated by video-EEG analysis and field potential recordings. Effects on glioma progression were estimated by tumor size quantification, survival analysis and immunohistological staining. Our data revealed that orthotopically-growing F98 glioma promote an epileptiform phenotype in rats. RCT reduced the tumor size and prolonged the survival of the animals. The adjuvant administration of PER had no effect on tumor progression. The tumor-associated epileptic events were abolished by PER application or RCT respectively, to initial baseline levels. Remarkably, PER preserved the glutamatergic network activity on healthy peritumoral tissue in RCT-treated animals. F98 tumors are not only a robust model to investigate glioma progression, but also a viable model to simulate a glioma-associated epileptiform phenotype. Furthermore, our data indicate that PER acts as a potent anticonvulsant and may protect the tumor-surrounding tissue as adjuvant to RCT, but failed to attenuate tumor growth or promote animal survival.

The solvent and treatment regimen of sodium selenite cause its effects to vary on the radiation response of human bronchial cells from tumour and normal tissues.

Sodium selenite is often given to moderate the side effects of cancer therapy to enhance the cellular defence of non-cancerous cells. To determine whether sodium selenite during radiotherapy protects not only normal cells but also cancer cells, which would imply a reduction of the desired effect of irradiation on tumour during radiotherapy, the effect of the combined treatment of irradiation and sodium selenite was investigated. Human bronchial cells from carcinoma (A549) and normal tissue (BEAS-2B) were treated with sodium selenite and effects on growth and in combination with radiation on metabolic activity and cell cycle distribution were studied. The influence on radiosensitivity was determined via colony forming assays using different solvents of sodium selenite and treatment schedules. It was shown that sodium selenite inhibits growth and influences cell cycle distribution of both normal and tumour cells. Metabolic activity of normal cells decreased more rapidly compared to that of cancer cells. The influence of sodium selenite on radiation response depended on the different treatment schedules and was strongly affected by the solvent of the agent. It could be shown that the effect of sodium selenite on radiation response is strongly dependent on the respective experimental in vitro conditions and ranges from lead to an initially suspected but ultimately no real radioprotection to radiosensitizing up to no effect in one and the same cell line. This might be a reason for controversially described cell responses to radiation under the influence of sodium selenite in studies so far.

Radiosensitizing effects of trabectedin on human A549 lung cancer cells and HT-29 colon cancer cells.

Background and Purpose Trabectedin is a unique alkylating agent with promising effects against a range of solid tumors. In this study, we aimed to examine the cytotoxic and radiosensitizing effects of trabectedin on two human epithelial tumor cell lines in vitro, and its effects on DNA repair capacity. Methods Cancer cells (A549: human lung cancer cells, HT-29: colon cancer cells) were treated with either trabectedin alone for the determination of their growth, or in combination with radiation for the determination of their metabolic activity, proliferation, and clonogenic survival. Besides, the γH2AX foci assay was performed for the assessment of ionizing radiation-induced DNA damage and to evaluate the influence of trabectedin on DNA damage repair. Results Treatment with trabectedin resulted in a growth-inhibiting effect on both cell lines, with the IC50 values remaining within a low nanomolar range. Analyses of metabolic activity confirmed a cytotoxic influence of trabectedin and a BrdU assay demonstrated an antiproliferative effect. When combined with radiation, incubation with trabectedin was found to enhance the radiosensitivity of the tumor cells. The γH2AX foci assay resulted in an increased number of DNA double-strand breaks (DSBs) in cells treated with trabectedin. Conclusion The results of this study underline the antitumor activity of trabectedin at low nanomolar concentrations. We demonstrated that trabectedin enhanced radiation response in human lung (A549) cancer cells and colon (HT-29) cancer cells. Further studies are necessary to examine trabectedin as a potential candidate for future applications in radiotherapy.

First Insights into the Effect of Low-Dose X-Ray Irradiation in Adipose-Derived Stem Cells.

(1) Background: Emerging interest of physicians to use adipose-derived stem cells (ADSCs) for regenerative therapies and the fact that low-dose irradiation (LD-IR ≤ 0.1 Gy) has been reported to enhance the proliferation of several human normal and bone-marrow stem cells, but not that of tumor cells, lead to the idea of improving stem cell therapies via low-dose radiation. Therefore, the aim of this study was to investigate unwanted side effects, as well as proliferation-stimulating mechanisms of LD-IR on ADSCs. (2) Methods: To avoid donor specific effects, ADSCs isolated from mamma reductions of 10 donors were pooled and used for the radiobiological analysis. The clonogenic survival assay was used to classify the long-term effects of low-dose radiation in ADSCs. Afterwards, cytotoxicity and genotoxicity, as well as the effect of irradiation on proliferation of ADSCs were investigated. (3) Results: LD (≤ 0.1 Gy) of ionizing radiation promoted the proliferation and survival of ADSCs. Within this dose range neither geno- nor cytotoxic effects were detectable. In contrast, greater doses within the dose range of >0.1-2.0 Gy induced residual double-strand breaks and reduced the long-term survival, as well as the proliferation rate of ADSCs. (4) Conclusions: Our data suggest that ADSCs are resistant to LD-IR. Furthermore, LD-IR could be a possible mediator to improve approaches of stem cells in the field of regenerative medicine.

Effect of Ionizing Radiation on Human EA.hy926 Endothelial Cells under Inflammatory Conditions and Their Interactions with A549 Tumour Cells.

PURPOSE: Most tumours are characterized by an inflammatory microenvironment, and correlations between inflammation and cancer progression have been shown. Endothelial cells (ECs), as part of the tumour microenvironment, play a crucial role in inflammatory processes as well as in angiogenesis and could be critical targets of cancer therapy like irradiation. Therefore, in the present study we investigated the effect of ionizing radiation on endothelial cells under inflammatory conditions and their interactions with tumour cells. METHODS: Nonactivated and TNF-α treatment-activated human EC EA.hy926 were irradiated with doses between 0.1 Gy and 6 Gy with a linear accelerator. Using a multiplex assay, the accumulation of various chemokines (IL-8, MCP-1, E-selectin, and P-selectin) and soluble adhesion molecules (sICAM-1 and VCAM-1) as well as protein values of the vascular endothelial growth factor (VEGF) was measured in the supernatant at different time points. The adhesion capability of irradiated and nonirradiated A549 tumour cells to EA.hy926 cells was measured using flow cytometry, and the migration of tumour cells was investigated with a scratch motility assay. RESULTS: In contrast to unirradiated cells, IR of ECs resulted in a modified release of chemokines IL-8 and MCP-1 as well as the adhesion molecules sICAM-1 and VCAM-1 in the EC, whereas concentrations of E-selectin and P-selectin as well as VEGF were not influenced. IR always affected the adhesion capability of tumour cells to ECs with the effect dependent on the IR-treated cell type. TNF-α treatment generally increased adhesion ability of the tumour cells. Tumour cell migration was clearly inhibited after IR. This inhibitory effect was eliminated for radiation doses from 0.5 to 2 Gy when, additionally, an inflammatory environment was predominant. CONCLUSIONS: Our results support past findings suggesting that ECs, as part of the inflammatory microenvironment of tumours, are important regulators of the actual tumour response to radiation therapy.

Immunomodulatory properties of low-dose ionizing radiation on human endothelial cells.

PURPOSE: The application of radiation therapy (RT) is not only used to treat cancer, in Germany, it is also an accepted and empirically established treatment of patients with benign diseases at low doses. The immune modulatory response generated by low-dose RT has a supporting anti-inflammatory effect within the treatment of inflammation-related diseases. The aim of this study was to investigate the effect of ionizing radiation (IR) on the expression and secretion of inflammatory mediators by endothelial cells (ECs) exposed to low and moderate doses. METHODS: Non-activated and activated EC were irradiated with doses between 0.01 Gy and 2 Gy with X-rays. Using a multiplex-assay, protein values of interleukin-8 (IL-8), granulocyte macrophage colony-stimulating factor (G-CSF) and platelet-derived growth factor (PDGF-BB) were measured in the supernatant at different time-points. To investigate possible differences between mRNA expression and protein secretion after IR, the mRNA expression of IL-8, G-CSF and PDGF-BB was determined by real-time quantitative PCR. RESULTS: Radiation treatment caused non-linear dose dependent effects on pro-inflammatory cytokine secretion of IL-8; G-CSF and PDGF-BB. The mRNA-expression levels of those cytokines were non-linear dose-dependent and differed from protein level in the culture supernatant. CONCLUSIONS: This study provides deeper insights into the radiobiological effects of radiation doses below 0.3 Gy, in particular 0.05 Gy, and their significant immunomodulatory properties on EC, which is very important in order to assess the effect of LD-IR on EC.

Modulation of Inflammatory Reactions by Low-Dose Ionizing Radiation: Cytokine Release of Murine Endothelial Cells Is Dependent on Culture Conditions.

BACKGROUND: In many European countries, patients with a variety of chronical inflammatory diseases are treated with low-dose radiotherapy (LD-RT). In contrast to high-dose irradiation given to tumor patients, little is known about radiobiological mechanisms underlying this clinical successful LD-RT application. The objective of this study was to gain a better insight into the modulation of inflammatory reactions after LD-RT on the basis of endothelial cells (EC) as major participants and regulators of inflammation. METHODS: Three murine EC lines were cultivated under 2D and 3D culture conditions and irradiated with doses from 0.01 Gy to 2 Gy. To simulate an inflammatory situation, cells were activated with TNF-α. After LD-RT, a screening of numerous inflammatory markers was determined by multiplex assay, followed by detailed analyses of four cytokines (KC, MCP-1, RANTES, and G-CSF). Additionally, the monocyte binding to EC was analyzed. RESULTS: Cytokine concentrations were dependent on culture condition, IR dose, time point after IR, and EC origin. IR caused nonlinear dose-dependent effects on secretion of the proinflammatory cytokines KC, MCP-1, and RANTES. The monocyte adhesion was significantly enhanced after IR as well as activation. CONCLUSIONS: The study shows that LD-RT, also using very low radiation doses, has a clear immunomodulatory effect on EC as major participants and regulators of inflammation.

Comparative study of the effects of different radiation qualities on normal human breast cells.

BACKGROUND: As there is a growing number of long-term cancer survivors, the incidence of carcinogenesis as a late effect of radiotherapy is getting more and more into the focus. The risk for the development of secondary malignant neoplasms might be significantly increased due to exposure of healthy tissue outside of the target field to secondary neutrons, in particular in proton therapy. Thus far, the radiobiological effects of these neutrons and a comparison with photons on normal breast cells have not been sufficiently characterised. METHODS: MCF10A cells were irradiated with doses of up to 2 Gy with neutrons of different energy spectra and X-rays for comparison. The biological effects of neutrons with a broad energy distribution ( = 5.8 MeV), monoenergetic neutrons (1.2 MeV, 0.56 MeV) and of the mixed field of gamma's and secondary neutrons ( = 70.5 MeV) produced by 190 MeV protons impinging on a water phantom, were analysed. The clonogenic survival and the DNA repair capacity were determined and values of relative biological effectiveness were compared. Furthermore, the influence of radiation on the sphere formation was observed to examine the radiation response of the potential fraction of stem like cells within the MCF10A cell population. RESULTS: X-rays and neutrons caused dose-dependent decreases of survival fractions after irradiations with up to 2 Gy. Monoenergetic neutrons with an energy of 0.56 MeV had a higher effectiveness on the survival fraction with respect to neutrons with higher energies and to the mixed gamma - secondary neutron field induced by proton interactions in water. Similar effects were observed for the DNA repair capacity after exposure to ionising radiation (IR). Both experimental endpoints provided comparable values of the relative biological effectiveness. Significant changes in the sphere formation were notable following the various radiation qualities. CONCLUSION: The present study compared the radiation response of MCF10A cells after IR with neutrons and photons. For the first time it was shown that monoenergetic neutrons with energies around 1 MeV have stronger radiobiological effects on normal human breast cells with respect to X rays, to neutrons with a broad energy distribution ( = 5.8 MeV), and to the mixed gamma - secondary neutron field given by interactions of 190 MeV protons in water. The results of the present study are highly relevant for further investigations of radiation-induced carcinogenesis and are very important in perspective for a better risk assessment after secondary neutron exposure in the field of conventional and proton radiotherapy.

Investigation of epothilone B-induced cell death mechanisms in human epithelial cancer cells -in consideration of combined treatment with ionizing radiation.

Epothilone B was shown to have promising chemo- and radiosensitizing effects on cells, but the mechanisms underlying cell death remain ambiguous. The aim of the study was to examine selected cell death pathways on the basis of FaDu and A549 cells. Western blot analyses were used for investigation of specific apoptotic markers. Immunofluorescence imaging and flow cytometry were utilized for examination of cell death mechanisms. DNA-staining was used for studying influence of epothilone B on micronucleus rate. We showed that epothilone B can initiate cell death via apoptosis and mitotic catastrophe, but induction of cell death was cell type specific.

Effect of epothilone B on cell cycle, metabolic activity, and apoptosis induction on human epithelial cancer cells-under special attention of combined treatment with ionizing radiation.

In recent studies, epothilone B was shown to have a cytotoxic and radiosensitizing effect on cells. The aim of our investigation was to explain this impact by examining the mode of action of epothilone B on FaDu and A549 tumor cells. Flow cytometry was used for cell cycle distribution and for the evaluation of apoptosis. Metabolic activity was studied by proliferation assay. Influence on nuclei morphology was investigated by DNA-staining. We showed that epothilone B-induced G2/M accumulation is the main rationale for drug-induced radiosensitivity. The cytotoxic effect resulted in apoptotic cell death, decreased metabolic activity, and formation of multinucleated cells.

Omega-3 fatty acid supplementation in cancer therapy : does eicosapentanoic acid influence the radiosensitivity of tumor cells?

PURPOSE: The aim of this study was to evaluate whether the omega-3 polyunsaturated fatty acid cis-5,8,11,14,17-eicosapentanoic acid (EPA) can enhance the radiosensitivity of different human tumor cell lines. MATERIALS AND METHODS: Colon adenocarcinoma cells HT-29, and two glioblastoma multiforme tumor cells T98G and U251 were cultured under standard conditions. Cell growth was observed during administration with different concentrations of EPA, using it as the free fatty acid dissolved in ethanol or bound to bovine serum albumin. To investigate the influence of EPA (free and bound) on radiosensitivity, tumor cells were pretreated 30 minutes or 24 hours prior to irradiation with the fatty acid. Cell survival was measured by colony-forming assays. RESULTS: When combined with irradiation, incubation with EPA was found to result in enhanced radiosensitivity with substantial variation: while there was strong radiosensitization for HT-29 and U251 cells, almost no effect for T98G cells was observed. A marked radiosensitization was clearly dependent on the treatment schedule. CONCLUSION: The observations suggest that EPA is not only a nutritional adjuvant but also may be a potential candidate to enhance the efficacy of irradiation on human cancer cells.

[Verification of the VEF photon beam model for dose calculations by the Voxel-Monte-Carlo-Algorithm]. / Verifikation des VEF-Strahlerkopfmodells für Photonen zur Dosisberechnung mit dem Voxel-Monte-Carlo-Algorithmus.

The VEF linac head model (VEF, virtual energy fluence) was developed at the University of Tübingen to determine the primary fluence for calculations of dose distributions in patients by the Voxel-Monte-Carlo-Algorithm (XVMC). This analytical model can be fitted to any therapy accelerator head by measuring only a few basic dose data; therefore, time-consuming Monte-Carlo simulations of the linac head become unnecessary. The aim of the present study was the verification of the VEF model by means of water-phantom measurements, as well as the comparison of this system with a common analytical linac head model of a commercial planning system (TMS, formerly HELAX or MDS Nordion, respectively). The results show that both the VEF and the TMS models can very well simulate the primary fluence. However, the VEF model proved superior in the simulations of scattered radiation and in the calculations of strongly irregular MLC fields. Thus, an accurate and clinically practicable tool for the determination of the primary fluence for Monte-Carlo-Simulations with photons was established, especially for the use in IMRT planning.

A virtual photon energy fluence model for Monte Carlo dose calculation.

The presented virtual energy fluence (VEF) model of the patient-independent part of the medical linear accelerator heads, consists of two Gaussian-shaped photon sources and one uniform electron source. The planar photon sources are located close to the bremsstrahlung target (primary source) and to the flattening filter (secondary source), respectively. The electron contamination source is located in the plane defining the lower end of the filter. The standard deviations or widths and the relative weights of each source are free parameters. Five other parameters correct for fluence variations, i.e., the horn or central depression effect. If these parameters and the field widths in the X and Y directions are given, the corresponding energy fluence distribution can be calculated analytically and compared to measured dose distributions in air. This provides a method of fitting the free parameters using the measurements for various square and rectangular fields and a fixed number of monitor units. The next step in generating the whole set of base data is to calculate monoenergetic central axis depth dose distributions in water which are used to derive the energy spectrum by deconvolving the measured depth dose curves. This spectrum is also corrected to take the off-axis softening into account. The VEF model is implemented together with geometry modules for the patient specific part of the treatment head (jaws, multileaf collimator) into the XVMC dose calculation engine. The implementation into other Monte Carlo codes is possible based on the information in this paper. Experiments are performed to verify the model by comparing measured and calculated dose distributions and output factors in water. It is demonstrated that open photon beams of linear accelerators from two different vendors are accurately simulated using the VEF model. The commissioning procedure of the VEF model is clinically feasible because it is based on standard measurements in air and water. It is also useful for IMRT applications because a full Monte Carlo simulation of the treatment head would be too time-consuming for many small fields.