ESTRO 38 Abstract book

S1198 ESTRO 38

Conclusion Ro90-7501 was identified as a radiosensitizer, which represses ATM phosphorylation, inhibits DNA repair after irradiation, and then increases cell apoptosis. EP-2167 Radiosensitizing effect of eribulin mesylate in human cervical carcinoma cells in vitro R. Benlloch Rodríguez 1 , J. Romero Fernández 1 , R. Castejón Díaz 2 , S. Rosado García 3 , P. Sánchez Rubio 4 , S. Sánchez García 1 , I. Martínez Montesinos 1 , I. Zapata Paz 1 , M. López Valcárcel 1 1 Hospital Universitario Puerta de Hierro, Radiation Oncology, Majadahonda - Madrid, Spain; 2 IDIPHIM, Internal Medicine, Majadahonda - Madrid, Spain; 3 IDIPHIM, Cellular Culture Unit, Majadahonda - Madrid, Spain; 4 Hospital Universitario Puerta de Hierro, Radiophysics, Majadahonda - Madrid, Spain Purpose or Objective To determine the radiosensitizing properties of eribulin mesylate, a synthetic analogue of the marine natural compound halichondrin B, in human cervical carcinoma cells; and study the potential mechanisms of radiosensitization Material and Methods Cell line: HeLa cells, derived from a human cervical carcinoma, were maintained in DMEM medium, supplemented with FCS and antibiotics. In vitro chemo- and radiosensitivity were assessed by the crystal violet method. To determine chemosensitivity, cells were treated with a 24 h. exposure time of eribulin at different concentrations and, after 10 days of incubation, a dose- response curve was constructed from which the IC10 and IC50 were calculated. Radiosensitivity was evaluated by irradiating the cells at different doses (1,2,4,6 and 8 Gy) in absence or presence of the drug at different concentrations. Survival data were adjusted to the LQ model by a least squares algorithm. Surviving fraction at 2 Gy (SF2) and 4 Gy (SF4) from each survival curve were calculated from the fitted data. Cell cycle changes for control and treated cells were assessed by propidium iodide (PI) staining and flow cytometry analysis. Apoptotic cells were detected by FTIC- conjugated Annexyn V labelling method using flow cytometry. The percentage of apoptotic cells was obtained from a bivariate histogram of Annexyn V labelled-cells versu PI labeled cells for control, 1.5 nM and 3 nM eribulin exposure, irradiation (6 Gy) and combined eribulin-irradiation groups. Results Hela cell are sensitive to eribulin at nanomolar range. The IC10 and IC50 for 24 h eribulin exposure were 0.75 nM and 2.1 nM respectively . A dose-dependent radiosensitization was observed. The FS2 for control, eribulin 0.3 nM, 0.75 nM and 1.5 nM were 0.94; 0.96; 0.84; and 0.71, respectively. The corresponding figures to FS4 were 0.79; 0.85; 0.67; and 0.47, respectively. The dose enhancement factor (DEF) calculated at 2 and 4 Gy for 1.5 nM eribulin were 1.25 and 1.68, respectively. A G2M cell cycle arrest was induced by eribulin. In the time-course experiment, the percentage of cells in G2M phase for control and 8, 24 and 48 h after 24 h exposure to 1.5 nM eribulin were 11.6%, 20%, 26.3% and 74.7%. The cultures treated with 1.5 nM and 3 nM eribulin showed a percentage of apoptotic cells of 22.5 and 28.1%, respectively. The combined treatment of irradiation at 6 Gy with 3 nM eribulin produced a significant increase in the percentage of apoptosis up to 45% of cells. Conclusion Eribulin mesylate confers radiosensitizing effect in Hela cells in vitro. The potential mechanisms involved in this effect are an arrest in the most radiosensitive G2M cell cycle phase and an increase in apoptosis in combined radiation-drug treatment.

EP-2168 Analysis of Chromosomal Aberrations by FISH in FaDu tumor cells after in vivo X-ray MRT irradiation A. Porth 1 , A. Hunger 1,2 , T. Setzkorn 2 , N. Mehrabi 1 , K. Burger 1,3,4 , S.E. Combs 1,2 , T.E. Schmid 1,2 1 Technische Universität München, Klinik und Poliklinik für RadioOnkologie und Strahlentherapie, München, Germany; 2 Helmholtz Zentrum München, Institut für innovative Radiotherapie, Neuherberg, Germany; 3 Technische Universität München, Lehrstuhl für Biomedizinische Physik- Physik-Department, Garching, Germany; 4 Technische Universität München, Munich School of Bio Engineering, Garching, Germany Purpose or Objective X-ray microbeam radiation therapy (MRT) is a new irradiation technique in which the dose is deposited spatially separated with high peak doses. Studies comparing this technique to conventional X-ray irradiation have shown reduced toxicity in normal tissue and a better tumor control. Requiring high dose rates and precise beam collimation, MRT studies are mainly performed at synchrotron radiation facilities. In contrast to these, more cost effective and compact novel X-ray sources like the Munich Compact Light Source, used in this study, overcome the main limitations for the transfer of MRT to a clinical setting. In this study fluorescence in situ hybridization (FISH) was performed to detect chromosomal aberrations in order to assess the cellular response to MRT. Material and Methods The human head and neck cancer cell line (FaDu) was injected subcutaneously into the ear of immunocompromised NMRI nu/nu mice. Tumors were irradiated conventionally or with MRT at mean doses of 3 Gy or 5 Gy using 25 keV X-rays. Sham irradiated and unirradiated mice served as control group. When a tumor reached the 15-fold of its initial volume, the tumor cells were isolated and cultivated for further in vitro analysis. Applying standard protocols, metaphase chromosome spreads were prepared and then hybridized with probes staining chromosomes 2 and 4. For each cell line, 500 metaphases were counted manually under a fluorescence microscope and chromosome aberrations were evaluated, focusing on translocations and aneuploidy. Results 24 cell lines were successfully isolated and analyzed via FISH. Three to seven replicates constituted the control and experimental groups respectively. In the control group, 5 ±1% of the cells showed structural aberrations. In irradiated cells this portion was higher: 11 ±1% for cells treated conventionally with 3 Gy, 10 ±1% for cells treated conventionally with 5 Gy, 15 ±2% of the cells irradiated with 3 Gy MRT and 11 ±2% of those irradiated with 5 Gy MRT. Numerical aberrations were found in 4 ±3% of the cells in the control group, 1 ±2% in the group treated with 3 Gy conventional X-rays, 2 ±1% in the group treated conventionally with 5 Gy and 2 ±1% for both MRT irradiated groups. Conclusion Our study demonstrated an overall higher percentage of stable chromosome aberrations in all irradiated groups, corresponding to the clastogenic effect of ionizing radiation. In respect to this effect, no difference was visible between conventional and microbeam X-ray irradiation. Aneuploidy rates were high, both in irradiated and non-irradiated cells, which is a typical finding for tumor cells. Interestingly, this rate was lower in conventionally irradiated groups but not in the MRT groups. This might indicate a different influence of conventional and MRT irradiation on genomic instability.