Abstract book - ESTRO meets Asia

S127 ESTRO meets Asia 2018

Material and Methods We used gradient dose model to establish radioresistant strains of human nasopharyngeal carcinoma cells 6-10B and CNE2. Plate colony formation assays were used to verify radioresistance of the cells. CCK-8 assay, flow cytometry, scratch test, and transwell were performed to detect cell biological differences in proliferation, apoptosis, cell cycle, migration and invasion of radioresistant strains. We detected the expression of EGFR, LAPTM4B, Beclin1 and autophagy-related proteins p62, LC3I, and LC3II by Western blot and observed GFP- LC3 puncta by confocal microscope. The interaction between proteins was verified by immunofluorescence and co-immunoprecipitation. The effect of autophagy inhibitor, autophagy inducer, and cetuximab on radioresistance were determined by plate formation Radioresistant nasopharyngeal carcinoma cell lines exhibited cell phenotypic differences in cell biology such as reduced proliferation capacity, decreased apoptosis rate after irradiation, increased G2/M phase arrest after irradiation, and increased migration and invasion ability. The EGFR expression and autophagic flux of radioresistant cells were up-regulated, suggesting that EGFR is associated with autophagy level. Plate colony formation assays verified that the autophagy inhibitor chloroquine and autophagy inducer rapamycin can both reduce the radioresistance of radioresistant cells. We observed that EGFR interacts with LAPTM4B and stabilizes each other in endosomes by confocal microscopy. Co- immunoprecipitation experiments demonstrated that LAPTM4B interacts with Beclin1, which in turn promotes the initiation of autophagy. We confirmed that cetuximab can upregulate the expression of EGFR, Beclin1 and autophagy in radioresistant cells. Colony formation assays suggested that cetuximab with autophagy inhibitor chloroquine or knockdown of LAPTM4B gene can significantly reduce radioresistance of radioresistant cells. Conclusion This study has verified the correlation between EGFR and autophagy in radioresistant nasopharyngeal carcinoma cell lines and indicated that upregulated EGFR may induce autophagy through the EGFR-LAPTM4B-Beclin1 pathway, resulting in radioresistance. Cetuximab combined with autophagy inhibitor chloroquine or knockdown of LAPTM4B can achieve significant radiosensitization. This study proposes a possible mechanism of radioresistance of nasopharyngeal carcinoma and provides a new research direction and theoretical basis for solving the radioresistance of nasopharyngeal carcinoma. PO-308 ROS-induced expression of FTS activates Cu-Zn SOD in cervical cancer cells W.Y. Park 1 , S. Muthusami 2 , D. Prabakaran 1 , T. Sivaraman 3 , J. Yu 4 1 Chungbuk National University, Dept of Radiation Oncology, Cheongju, Korea Republic of 2 Karpagam Academy of Higher Education, Department of Biochemistry, Coimbatore, India 3 Karpagam Academy of Higher Education, Department of Biotechnology, Coimbatore, India 4 Konkuk University, Department of Environmental and Tropical Medicine, Chungju, Korea Republic of Purpose or Objective Ionizing radiation (IR) is commonly advocated for the treatment of cervical cancer. The therapeutic efficacy of IR is mainly attributed to reactive oxygen species (ROS)- induced cellular damage. However, the efficacy of IR via ROS is largely reduced by the endogenous antioxidant systems. Superoxide dismutase (SOD) is an important antioxidant enzyme which detoxifies superoxide into assays. Results

hydrogen peroxide (H 2 O 2 ), which is further converted to water and molecular oxygen by catalase and glutathione peroxidase. The present study is aimed to delineate the effects of ROS and radiation-induced alterations in Cu-Zn SOD (SOD1) and Fused toes homolog (FTS). Material and Methods A human cervical carcinoma cell line (ME180) was used. FTS was silenced using siRNA-based approach. Western blotting was done to see protein expression. Immunoprecipitation and molecular modelling, docking and structural analyses were done to see protein-protein interaction. ROS was measured using H 2 DCF-DA. Results H 2 O 2 treatment elicited no obvious change in the viability of cervical cancer ME180 cells at lower concentrations whereas at higher concentrations H 2 O 2 induced cell death. A significant time dependent increase in the expression of SOD1 induction after H 2 O 2 treatment at low concentration demonstrates the scavenging of H 2 O 2 . A significant increase in FTS is also noted after H 2 O 2 treatment at lower concentrations. Immunoprecipitation analysis identified the physical interaction between SOD1 and FTS after H 2 O 2 treatment. Putative residues responsible for the interaction were identified using computational analyses. Silencing of FTS significantly increased the ROS levels in cervical cancer cells exposed to low levels of H 2 O 2 resulting in increased cell death. Silencing of FTS also prevented radiation-induced induction of SOD1 in these cells. Conclusion These results demonstrate a novel role for FTS in the antioxidant defense by SOD1 in ROS-induced cellular death and defense. FTS is an important binding partner of SOD1 and required for the enzymatic activity of SOD1 in cervical cancer cells. PO-309 Evaluation of oxygen level-driven autophagy regulators in radiosensitivity on colorectal cancer cell S.Y. Tam 1 , K.W.H. Law 1 , W.C.V. Wu 1 1 The Hong Kong Polytechnic University, Department of Health Technology and Informatics, Hong Kong, Hong Kong SAR China Purpose or Objective Colorectal cancer is one of the commonest cancers worldwide with metachronous distant metastases still affecting overall survival. Hypoxia has been identified as a major factor that may decrease tumor radiosensitivity. We hypothesize that hypoxia may induce specific autophagy pathways in colorectal cancer cells. The objective of this study is to evaluate the expressions of multiple key regulators in hypoxic conditions. Material and Methods The expressions of autophagy key regulators including AMPK, HIFs and JNK in human colorectal adenocarcinoma cells HT-29 irradiated by 2 Gy at different oxygen concentrations in different time points were evaluated by western blot and compared with control. The radiosensitivity of the cells after being incubated in different oxygen levels was evaluated by clonogenic assay. Results Incubation of cells at 1% or 10% oxygen level induced autophagy. The activation markers were detected at different time points in the order AMPK, HIF-1α, HIF-2α and JNK. Irradiation of the cells at 2Gy down-regulated autophagy. Clonogenic assay showed cells incubated at 1% oxygen level were the least radiosensitive while cells incubated at 10% oxygen level were the most radiosensitive. Conclusion Autophagy plays an important role in hypoxia with multiple regulators involved. The results supported the notion of using autophagy modulation agents for improving treatment efficacy and demonstrated the difference of