ESTRO 38 Abstract book

S1194 ESTRO 38

Real-time fluorescent quantitative PCR assay the level of Nrf2 and Western blot to analyze the Nrf2, GCLC, HO-1, NQO1, Bax, Bcl-2. Cells were treated with siRNA-Nrf2 before X-irradiation (4 Gy) and incubated for 24 h. After staining with anti-Nrf2 antibody and Alexa Fluor 647- conjugated secondary antibody and were visualized under a fluorescence microscope. Immunofluorescence staining for Notch1 in A549 cells, Alexa Fluor 647-conjugated secondary antibody and nuclei were counterstained with DAPI.

Pavia, Italy ; 8 Fondazione CNAO- Istituto Europeo di Oncologia, Radiation Oncology, Pavia- Miano, Italy

Purpose or Objective Recently, a role of L-Dopa for the diagnosis and treatment of glioblastoma has been hypothesized. Four hours preload with L-Dopa has been previously reported to increase incorporation of BPA (boron-phenyl-alanine) in tumors both in vivo and in vitro, likely activating the LAT system and thus increasing the efficacy of BNCT (Boron Neutron Capture Therapy). This approach looks very appealing because in theory it could be employed to increase the tumor accumulation of 18-FDOPA in PET diagnostics or of the 11 B substrate in BPCT (Boron Proton Capture Therapy). In light of these experimental applications, the aim of this study was to assess the effects of pretreatment with L-Dopa on the biological behavior of human T98G cells, a chemo/radioresistant glioblastoma cell line. Material and Methods Briefly, 4-hour pretreatment with 50 μg/mL or 100 μg/mL of L-Dopa can influence the morphology, the growth rate, the ability to form colonies, and the migratory capacity of T98G cell in exponential growth, in basal conditions and after carbon ion irradiation (2 and 4 GyRBE). Statistical analysis has been performed with ANOVA and paired t test. Results After L-dopa pretreatment cells show a lower tendency to aggregate and a round-like pattern. Recovery of the scratched area was significantly faster and higher by T98G cells pretreated cells. Boyden Chamber test confirmed enhanced migration of the treated cells. Carbon Ions irradiation increases the migratory efficiency either at 15 and 24 hours. Conclusion The results show that L-Dopa induced significant changes in T98G cells behavior. These aspects have been never previously evaluated and open further questions about real utility of L-Dopa preload in BNCT and indicate a lack of experimental data for a possible application in PET with FDOPA and BPCT. EP-2160 Downregulation of Nrf2 promotes radiation- induced apoptosis in non-small cell lung cancer cells H. Zhang 1 1 Institute of Modern Physics- Chinese Academy of Sciences, Department of Radiation Medicine, Lanzhou, China Purpose or Objective The nuclear factor erythroid-2-related factor 2 (Nrf2) is a crucial regulator of the cellular antioxidant system. Nrf2 is often constitutively activated in non-small cell lung cancer (NSCLC) cell lines, which promotes cytoprotection against oxidative stress and xenobiotics. Notch1 signaling is critically implicated in cell fate determination. It has been reported that Nf2 strongly regulates Notch1 activity. However, the role of Nrf2 mediated Notch1 signaling in response to ionizing radiation (IR) remains elusive. We report that knockdown of Nrf2 promotes radiation-induced apoptosis through Nrf2 mediated Notch1 signaling in NSCLC cells. Material and Methods The human lung cancer cells (A549, NCI-H1299 (H1299), NCI-H460 (H460) were exposed to ionizing radiation at room temperature using Faxitron RX-650 X-rays (Faxitron Bioptics, LLC, USA). The dose rates were 0.765 Gy/min. siRNA against Nrf2, Notch1 and non-targeting negative control siRNA were purchased from Invitrogen (Invitrogen Life Technologies, Carlsbad, CA, USA). Electronic Poster: Radiobiology track: Radiation- induced signalling pathways

Results

Figure 1. IR-induced nuclear translocation of Nrf2 is suppressed by siRNA-Nrf2 in A549 cells. (A) mRNA levels of Nrf2 at 24 h after 4 Gy of X-ray irradiation. (B) Cells were treated with siRNA-Nrf2 before X-irradiation (4 Gy) and incubated for 24 h. After staining with anti-Nrf2 antibody and Alexa Fluor 647-conjugated secondary antibody (red) and DAPI for nuclear staining (blue), cells were visualized under a fluorescence microscope. *p<0.05 versus NC+IR group.

Figure 2. Expression of Notch1 and related gene in the knockdown of Nrf2 cells. (A) Quantification of protein expression. (B) Hes1 mRNA levels were measured by quantitative RT-PCR. (C) Immunofluorescence staining for Notch1 in A549 cells. *p<0.05 and **p<0.01 versus NC+IR group. Conclusion 1) Nrf2 is induced by ionizing radiation in A549 cells 2) Knockdown of Nrf2 decreases radiation-upregulated Nrf2 in A549 cells. 3) Knockdown of Nrf2 decreased Notch1 expression after IR EP-2161 miR-454-3p regulates cellular radio-sensitivity by targeting to BTG1 in renal carcinoma cells J. Wang 1 1 Institute of Modern Physics- Chinese Academy of Sciences, Biophysics, Lanzhou, China