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903: Single-fraction of gamma radiation induces apoptosis in cultured HeLa cervical cancer cells
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 902 Mesenchymal stem cell therapy antagonizes radiation-induced endothelial cell damage and metastasis D. Klein1 , A. Schmetter1 , V. Kleff2 , H. Jastrow2 , M. Stuschke3 , V. Jendrossek1 . 1 University Hospital Essen, Institute for Cell Biology (Cancer Research), Essen, Germany, 2 University Hospital Essen, Institute for Anatomy, Essen, Germany, 3 University Hospital Essen, Clinic for Radiation Therapy, Essen, Germany Introduction: Radiation-induced normal tissue damage limits the use of curative radiation doses and fosters a therapeutic failure by the incidence of local recurrence and the development of distant metastases. Current research efforts are aimed to protect the stroma of adjacent tissues during radiation therapy for the use of higher radiation doses in order to minimize the risk of tumor recurrence. Material and Methods: GFP-labeled multipotent stem cells (MPSCs) derived either from the bone marrow (BM) or from aorta (Ao) were intravenously injected into C57Bl/6 mice at different time points after whole thorax irradiation (WTI) and their influence was tracked on radiation-induced endothelial cell damage and the formation of lung metastases by intravenously applied metastatic B16F10 cells, respectively. Molecular factors mediating radiation-induced adverse effects were analyzed using qRTPCR, Western blot and IHC. Results: Our data show that whole thorax irradiation (WTI) as well as total body irradiation (TBI) dramatically enhance tumor cell extravasation and lung metastasis. Pro-invasive cellular activities were accompanied by radiation-induced endothelial cell damage and up-regulation of endothelial matrix metalloproteinase 2 (MMP2). The pro-metastatic effect of ionizing radiation was blocked by treating mice with cultured MPSCs within the first weeks after irradiation. Finally we identified radiation-induced changes in the expression of endothelial matrix metalloproteinase 2 (MMP2) as one major factor involved in radiation-induced normal tissue damage and micrometastasis formation. Stem-cell therapy was able to counteract radiation-induced endothelial cell damage, metastasis and MMP2expression in arterial blood vessels. Furthermore, MMP2 inhibitor treatment improved vascular function but was not sufficient to inhibit metastasis formation. Conclusion: Protecting mice from radiation-induced MMP2 production and vascular dysfunction by therapeutic application of MPSC counteracted seeding and growth of circulating tumor cells in previously irradiated lungs. Therapeutic application of MPSCs may be a promising strategy to prevent radiation-induced normal tissue damage and subsequent tumor recurrence. No conflict of interest. 903 Single-fraction of gamma radiation induces apoptosis in cultured HeLa cervical cancer cells W. Khalilia1 , G. Ozcan Arican2 . 1 Institute of Science, Biology, Istanbul, Turkey, 2 Istanbul University-Institute of Science, Biology, Istanbul, Turkey Introduction: Ionizing radiation is commonly used in the treatment of solid tumors, with more than half of all cancer patients receiving radiotherapy during the course of treatment. But it is unclear if radiotherapy exerts its effects via induction of apoptosis. The present study aimed to determine whether a singlefraction of gamma radiation can induce apoptosis. Material and Method: HeLa cells were treated with a single fraction of gamma radiation at various doses (0, 2, 8, 16, 32 and 64 Gy) and investigated after incubation for five time periods (0 h, 24 h, 48 h, 60 h and 72 h). The proliferation of HeLa cells was measured by MTT assay, the apoptotic index and apoptotic morphological features were assessed by fluorescent microscopy using 4’-6Diamidino-2-fenilindol (DAPI). Real-time PCR (qRT-PCR) were used to analyze mRNA level of apoptosis related genes (TNF ligands and their receptors, members of the bcl-2, caspase, IAP, TRAF, CARD, death domain and death effector domain families, as well as genes involved in the p53 and DNA damage pathways). Results and Discussion: Gamma ray irradiation inhibits proliferation of HeLa cells in a dose- and time-dependent manner, and the IC50 dose was 32 Gy. The apoptotic morphological features, such as DNA condensation, shrinkage of the cell, fragmentation into membrane-bound apoptotic bodies and rapid phagocytosis by neighboring cells depends on the value of the dose and duration after exposure to gamma rays. After 60 h of 32 Gy single-dose of gamma radiation the optimum apoptotic index (AI) were determined. Array-based gene expression analysis revealed that gamma radiation regulated the expression of several genes associated with cell survival and apoptosis. BCL2-associated X protein (BAX), caspase-9 precursor (CASP9), tumor necrosis factor receptor super family member 21 precursor (TNFRSF21), TNF receptor-associated factor 2 (TRAF2), nuclear factor NFkappa-B (NF-úB), signal transducer and activator of transcription (STAT), tumor suppressor gene P53 and others which are known apoptosis activators were up regulated by gamma radiation treatment, whereas the anti-apoptosis Bcl-2 family members, inhibitor of apoptosis protein (IAP) members and others, were down regulated.
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Conclusion: These results provide evidence that gamma radiation directly induces anti proliferative effects by altering the expression of genes associated with cell proliferation and apoptosis. No conflict of interest. 904 Investigation on the role of low dose radiation as chemo-potentiator in locally advanced carcinoma cervix: A new treatment paradigm based on radiobiological advantage S. Das1 , T.S. Vijaykumar2 , R.R. Singh1 , A. Chandramohan3 , J. Subhashini1 . 1 Christian Medical College Hospital, Radiation Oncology, Vellore, India, 2 Christian Medical College Hospital, Nephrology, Vellore, India, 3 Christian Medical College Hospital, Radiology, Vellore, India Background: Carcinoma of the uterine cervix is a major public health problem in developing countries in south-east Asia. Majority of the cases are locally advanced (>FIGO stage IIB) with bulky tumors and response to standard chemoradiation is suboptimal due to tumor size and hypoxia. Radiobiologically, chemo-potentiating effect of low dose radiation therapy (LDRT) in the range of 40–100 cGy enhances cell killing by cellular arrest in G2 phase (induced radio-resistance [IRR]) thus augmenting the effect of G2 cycle specific chemotherapy. Objectives: • To examine the response of cervical cancer cell line (HeLa cell line) to low dose radiation using clonogenic assay and mathematical modeling of the low dose response by Joiner’s induced repair model. • Translational study to evaluate the clinical response of ultra-fractionated low dose radiation and chemotherapy in locally advanced carcinoma cervix. Materials and Methods: Survival of HeLa cells following exposure to single and fractionated low doses of g (gamma)-ray was measured by clonogenic assay. The low dose cellular response was modeled by Joiner’s induced repair model (S.F. = exp{−ar [1 + (as /ar − 1)e−D/Dc ]D − bD2 }. In the clinical component of the study (Phase II design) patients with squamous cell carcinoma cervix (FIGO stage IIB–IIIB) were included. Patients received two cycles of paclitaxel (175 mg/m2 ), carboplatin (AUC × 5), and four concurrent 80-cGy fractions of radiotherapy (two each on Days 1 & 2, and 22 & 23). Clinical and radiological response was assessed after 3 weeks. All patients received standard treatment following neo-adjuvant treatment. Results: HeLa cell line demonstrated marked low-dose response consisting of an area of hyper-radiosensitivity (HRS) and IRR in the dose region of <1 Gy. The two gradients of the low-dose region (as and ar ) were distinctly different with a transition dose (Dc ) of 0.30 Gy. In the fractionated radiotherapy experiments, for the schedules 0.5 Gy ×4, 0.8 Gy ×4, 1 Gy ×2 and 2 Gy single dose the survival fractions were 0.36±0.05, 0.26±0.06, 0.13±0.05 and 0.05±0.02 respectively. In the Clinical Phase II design, total 24 patients (80% IIIB and 20% IIB) were recruited. Mean gross tumor volume (GTV), based on T2 high resolution MRI, before and after neoadjuvant treatment were 54.36±9.75 vs. 11.45±2.39, p < 0.0001 (paired t-test). GTV volumes based on CT and MR were highly correlated (R2 = 0.87). There were variations in GTV assessment after neoadjuvant treatment between T2W MR and CT (R2 = 0.49). The incidence of grade 3 and 4 neutropenia was 33% and 8% respectively during the entire period of treatment. There was no grade 3 or 4 acute non-hematological toxicity. Conclusions: HeLa cell line demonstrates HRS and IRR with distinct transition dose. Neo-adjuvant LDRT and chemotherapy prior to definitive chemoradiation is a novel and feasible approach in cervical cancer with a favorable toxicity profile. This treatment paradigm resulted in significant reduction in the GTV, superior response rate and therefore is a good alternative option for bulky, hypoxic cervical tumors. No conflict of interest. 905 A novel role of kaempferol: Enhancing the radiosensitivity on lung cancer cells C. Yao1 , Y. Tsai1 , W. Kuo2 , J. Lian3 , Y. Kuo3 , Y. Chen1 , S. Kuo4 . 1 Department of Biomedical Imaging and Radiological Science, China Medical University, Taiwan, 2 Department of Biomedical Engineering, National Taiwan University, Taiwan, 3 Department of Radiation Oncology, China Medical University Hospital, Taiwan, 4 Department of Biomedical Engineering, I-Shou University, Taiwan Background: Kaempferol reportedly has anti-proliferative and cytotoxic effects on human cancer cells, but no research has demonstrated that it can enhance radiosensitivity. This study provides evidence that kaempferol has a radiosensitization potential for lung cancer in vitro. Materials and Methods: A549 cells was treated with indicated concentrations of kaempferol plus radiation; the cell viability was confirmed by using MTT and clonogenic assays. Annexin V/PI assay for the evaluation of apoptosis and cell cycle phase were determined by flow cytometry, and the expression of apoptosis-related proteins were analyzed by Western blotting.
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Conclusion: These results provide evidence that gamma radiation directly induces anti proliferative effects by altering the expression of genes associated with cell proliferation and apoptosis. No conflict of interest. 904 Investigation on the role of low dose radiation as chemo-potentiator in locally advanced carcinoma cervix: A new treatment paradigm based on radiobiological advantage S. Das1 , T.S. Vijaykumar2 , R.R. Singh1 , A. Chandramohan3 , J. Subhashini1 . 1 Christian Medical College Hospital, Radiation Oncology, Vellore, India, 2 Christian Medical College Hospital, Nephrology, Vellore, India, 3 Christian Medical College Hospital, Radiology, Vellore, India Background: Carcinoma of the uterine cervix is a major public health problem in developing countries in south-east Asia. Majority of the cases are locally advanced (>FIGO stage IIB) with bulky tumors and response to standard chemoradiation is suboptimal due to tumor size and hypoxia. Radiobiologically, chemo-potentiating effect of low dose radiation therapy (LDRT) in the range of 40–100 cGy enhances cell killing by cellular arrest in G2 phase (induced radio-resistance [IRR]) thus augmenting the effect of G2 cycle specific chemotherapy. Objectives: • To examine the response of cervical cancer cell line (HeLa cell line) to low dose radiation using clonogenic assay and mathematical modeling of the low dose response by Joiner’s induced repair model. • Translational study to evaluate the clinical response of ultra-fractionated low dose radiation and chemotherapy in locally advanced carcinoma cervix. Materials and Methods: Survival of HeLa cells following exposure to single and fractionated low doses of g (gamma)-ray was measured by clonogenic assay. The low dose cellular response was modeled by Joiner’s induced repair model (S.F. = exp{−ar [1 + (as /ar − 1)e−D/Dc ]D − bD2 }. In the clinical component of the study (Phase II design) patients with squamous cell carcinoma cervix (FIGO stage IIB–IIIB) were included. Patients received two cycles of paclitaxel (175 mg/m2 ), carboplatin (AUC × 5), and four concurrent 80-cGy fractions of radiotherapy (two each on Days 1 & 2, and 22 & 23). Clinical and radiological response was assessed after 3 weeks. All patients received standard treatment following neo-adjuvant treatment. Results: HeLa cell line demonstrated marked low-dose response consisting of an area of hyper-radiosensitivity (HRS) and IRR in the dose region of <1 Gy. The two gradients of the low-dose region (as and ar ) were distinctly different with a transition dose (Dc ) of 0.30 Gy. In the fractionated radiotherapy experiments, for the schedules 0.5 Gy ×4, 0.8 Gy ×4, 1 Gy ×2 and 2 Gy single dose the survival fractions were 0.36±0.05, 0.26±0.06, 0.13±0.05 and 0.05±0.02 respectively. In the Clinical Phase II design, total 24 patients (80% IIIB and 20% IIB) were recruited. Mean gross tumor volume (GTV), based on T2 high resolution MRI, before and after neoadjuvant treatment were 54.36±9.75 vs. 11.45±2.39, p < 0.0001 (paired t-test). GTV volumes based on CT and MR were highly correlated (R2 = 0.87). There were variations in GTV assessment after neoadjuvant treatment between T2W MR and CT (R2 = 0.49). The incidence of grade 3 and 4 neutropenia was 33% and 8% respectively during the entire period of treatment. There was no grade 3 or 4 acute non-hematological toxicity. Conclusions: HeLa cell line demonstrates HRS and IRR with distinct transition dose. Neo-adjuvant LDRT and chemotherapy prior to definitive chemoradiation is a novel and feasible approach in cervical cancer with a favorable toxicity profile. This treatment paradigm resulted in significant reduction in the GTV, superior response rate and therefore is a good alternative option for bulky, hypoxic cervical tumors. No conflict of interest. 905 A novel role of kaempferol: Enhancing the radiosensitivity on lung cancer cells C. Yao1 , Y. Tsai1 , W. Kuo2 , J. Lian3 , Y. Kuo3 , Y. Chen1 , S. Kuo4 . 1 Department of Biomedical Imaging and Radiological Science, China Medical University, Taiwan, 2 Department of Biomedical Engineering, National Taiwan University, Taiwan, 3 Department of Radiation Oncology, China Medical University Hospital, Taiwan, 4 Department of Biomedical Engineering, I-Shou University, Taiwan Background: Kaempferol reportedly has anti-proliferative and cytotoxic effects on human cancer cells, but no research has demonstrated that it can enhance radiosensitivity. This study provides evidence that kaempferol has a radiosensitization potential for lung cancer in vitro. Materials and Methods: A549 cells was treated with indicated concentrations of kaempferol plus radiation; the cell viability was confirmed by using MTT and clonogenic assays. Annexin V/PI assay for the evaluation of apoptosis and cell cycle phase were determined by flow cytometry, and the expression of apoptosis-related proteins were analyzed by Western blotting.