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746: Metabolic radiotherapy in cholangiocarcinoma: An option in the future?
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 Conclusions: According to these data, AuNP conjugated lignan samples showed strong anti-proliferative effects on MCF-7 cells when compared to free lignan derivatives. The AuNP–lignan conjugation study is promising in development of new drug formulations for prevention or new treatment strategies of malignant breast tumours. This study was supported by The Scientific and Technological Research Council of Turkey, Grant SBAG-112S549. No conflict of interest. 744 Clinical and histopathological characteristics of HER2-positive breast cancer in patients treated with adjuvant trastuzumab P. Jurcic1 . 1 University Hospital for Tumors, Department of Medical Oncology, Zagreb, Croatia Background: Breast cancer is a heterogeneous disease with variable expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor (HER2). Overexpression of HER2, which is found in 15−25% of breast cancers, is generally considered to be a negative prognostic factor. Trastuzumab (Herceptin® ), a monoclonal humanized antibody that binds the extracellular domain in HER2, in combination with cytotoxic chemotherapy improves disease-free survival and overall survival. In adjuvant chemotherapy regimens it is applied during a course of three weeks at loading doses of 8 mg/kg and 6 mg/kg. Materials and Methods: Medical records of breast cancer patients (stages 1−3) treated with adjuvant trastuzumab at the Day-Care Hospital of the University Hospital for Tumors in 2013 were retrospectively analyzed. The patients had previously received chemotherapy in the following order: AC protocol, FAC protocol and, depending on the status of axillas, taxanes − paclitaxel. Patient demographic data was collected, as well as data on tumor characteristics and treatment modalities. HER2 testing was performed by IHC on paraffin-embedded tissue samples and additional CISH testing was performed on all 2+ results. Results: Medical records of 116 patients (age range 25−79) were analyzed, 6 (5%) of whom were in the 25−35 age group, 15 (13%) in the 36−45 age group, 29 (25%) in the 46−55 age-group, 43 (37%) in the 56−65 agegroup and 23 (20%) were in the 66 age group. The results show that 68 patients (59%) had been diagnosed cancer in the left breast, whereas 48 patients (41%) had cancer in the right breast. Tumor size was 10 mm in 10 patients (9%), 11−20 mm in 38 patients (33%), 21−30 mm in 34 patients (29%), and in 27 patients (23%) it exceeded 31 mm in size. Surgical treatments included ablation in 56 patients (48%), segmentectomy in 54 patients (47%), quadrantectomy in 4 patients (3%), re-segmentectomy in one patient (1%) and biopsy in one patient (1%). Axillary dissection was performed on all patients − 50 patients (43%) were node negative. Eighteen patients (16%) had no indications for radiation. Immunotherapy was not followed by hormone therapy in patients with negative hormone receptor status, who accounted for 45% of patients. IHC test was equivocal (2+) in 25 patients, in which case positive results of subsequent CISH tests allowed for the application of trastuzumab. Conclusion: The outcome of trastuzumab therapy cannot be predicted based on the histopathological characteristics of cancer. In adjuvant trastuzumab therapy, outcomes for HER2-positive and for ER-positive, HER2-negative breast cancers are similar. The focus in the future should be on improving the treatment of hormone-receptor negative, HER2 positive tumors. No conflict of interest. 746 Metabolic radiotherapy in cholangiocarcinoma: An option in the future? A.I. Fernandes1 , A.C. Ribeiro2 , A.F. Brito3 , A.M. Abrantes3 , K. Santos4 , A.C. Gon¸calves5 , A.B. Sarmento-Ribeiro5 , J.G. Tralhao ˜ 6 , M.F. Botelho3 . 1 Biophysics Unit − IBILI − CIMAGO Gastroenterology Department, Faculty of Medicine of University of Coimbra and CHUC, Coimbra, Portugal, 2 Biophysics Unit, Faculty of Medicine of University of Coimbra, Coimbra, Portugal, 3 Biophysics Unit − IBILI − CIMAGO, Faculty of Medicine of University of Coimbra, Coimbra, Portugal, 4 Biophysics Unit − CIMAGO, Faculty of Medicine of University of Coimbra, Coimbra, Portugal, 5 CIMAGO − Applied Molecular Biology and Hematology Group, Faculty of Medicine of University of Coimbra, Coimbra, Portugal, 6 Biophysics Unit CIMAGO − Surgical Department Surgery A, Faculty of Medicine of University of Coimbra and CHUC, Coimbra, Portugal Background: Cholangiocarcinoma (CC) has a poor prognosis and limited therapeutic options. Thus, it becomes imperative to investigate new therapeutic options for this highly aggressive type of tumour. Recently, it was shown that CC has increased expression of natrium-iodide symporter (NIS), a molecule that mediates the iodine uptake. It is already known that NIS is a key component in the successful metabolic radiotherapy, using iodine-131 (131 I), in the treatment of thyroid tumours. These data open the possibility of a new therapeutic approach for CC. Thus, the aim of this study was to evaluate
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the therapeutic efficacy of metabolic radiotherapy with 131 I in a human CC cell line. Material and Methods: The human CC cell line used was TFK-1. Influx studies were performed in order to determine the 131 I uptake profile by the cell line. Subsequently, the cells were subjected to 1, 5, 10 and 20 Gy of 131 I, in order to evaluate and characterize the effects of metabolic radiotherapy. The effect on cell survival was assessed by clonogenic assay. Then, it was used flow cytometry in order to evaluate the type of induced cell death, as well as BAX, BCL-2 and cytochrome C expression, mitochondrial membrane potential, cell cycle changes, intracellular peroxides, superoxide anion, and glutathione production. Superoxide dismutase activity was also evaluated. To determine the possible damages in the DNA it was performed the comet assay. Results: Through uptake studies it was observed that the 131 I uptake peak of TFK-1 cells occur in the first minutes, keeping constant for the remaining time. It was observed that treatment with 131 I induced a decrease in cell viability dependent on the dose. The predominant type of cell death was apoptosis, followed by an increase in the BAX/BCL-2 ratio. In agreement with these results, there was also the release of cytochrome C, mitochondrial membrane depolarization and the occurrence of pre-G0 peak during the cell cycle. Interestingly, it wasn’t possible to detect differences in the production of intracellular peroxides, superoxide anion, glutathione, and superoxide dismutase. It was also found that 131 I induce DNA breaks in TFK-1 cells. Conclusions: Metabolic radiotherapy with 131 I causes a decrease in TFK-1 cells survival, inducing cell death mainly by apoptosis, at least in part through mitochondrial pathway. Thus, the 131 I could a promising option for the treatment of CC. No conflict of interest. 747 Novel anthraquinone-thiosemicarbazones with tautomerizable methylene group as anti-metastatic and anti-angiogenic agents B. Kolundzija1 , T. Stanojkovic1 , M.D. Joksovic2 . 1 Institute of Oncology and Radiology of Serbia, Department of Experimental Oncology, Belgrade, Serbia, 2 Faculty of Science University of Kragujevac, Department of Chemistry, Kragujevac, Serbia Background: Anthraquinones, both natural and synthetic, have been widely used in different therapeutic roles, as they poses laxative, anti-inflammatory, antibiotic and anti-tumor effects. Among these, anthracyclines, like doxorubicin and daunorubicin are extremely important as chemotherapeutics, but with many limitations to their use, mainly due to cardiotoxicity. Nine novel anthraquinones with thiosemicarbazone supstituents were synthesized, with additional introduction of tautomerizable group, with aim of increasing biological activity and decreasing toxicity. We have demonstrated previously [1] the good biological activity and satisfactory selectivity of these compounds against malignant cell lines. The aim of this work is to elucidate the potential of these compounds to inhibit cell migration and metastasis. Material and Methods: Anti-metastatic potential of compounds was tested using gelatin zymography and wound-healing assay. Briefly, in both these tests, sub-lethal concentrations of investigated compounds (IC20 , previously determined by MTT test) were used. For gelatin zymography, HeLa cells were treated with the compounds for 24 h. Supernatants were collected, and run on 8% SDS-PAGE gels, with 2 mg/ml final concentration of gelatin. Gels were stained with Coomassie Brilliant Blue, destained and the bands were visualized. Wound-healing assay was performed using EA.hy926 cells. On the first day a scratch was made in a confluent cell monolayer, and the cells were treated during next 48 hours. Every 24 hours, photomicrographs of the same area were taken. Results and Discussion: Matrix metalloproteinases have been shown to play a significant role in angiogenesis and metastatic potential of tumor cells. Our results show that the investigated compounds significantly reduce the activity of MMPs, compared to controls. This reduction is highly significant for five of the tested compounds and significant for other four. Most importantly, the inhibition of MMPs by the investigated compounds was higher in comparison to the action of doxorubicin and cisplatin. In wound-healing assay, all of compounds exhibit between 25 and 70% of inhibition of tumor cell invasion compared to control. Conclusion: Our results show that the investigated novel anthraquinone derivatives are potent inhibitors of MMPs secretion and malignant cells migration and invasion. This could lead toward further in vivo testing and development of these and similar compounds as anti-metastatic and antiangiogenic agents. Reference(s) [1] Markovic V et al. Eur J Med Chem. 2013; 64:228−38. No conflict of interest.
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the therapeutic efficacy of metabolic radiotherapy with 131 I in a human CC cell line. Material and Methods: The human CC cell line used was TFK-1. Influx studies were performed in order to determine the 131 I uptake profile by the cell line. Subsequently, the cells were subjected to 1, 5, 10 and 20 Gy of 131 I, in order to evaluate and characterize the effects of metabolic radiotherapy. The effect on cell survival was assessed by clonogenic assay. Then, it was used flow cytometry in order to evaluate the type of induced cell death, as well as BAX, BCL-2 and cytochrome C expression, mitochondrial membrane potential, cell cycle changes, intracellular peroxides, superoxide anion, and glutathione production. Superoxide dismutase activity was also evaluated. To determine the possible damages in the DNA it was performed the comet assay. Results: Through uptake studies it was observed that the 131 I uptake peak of TFK-1 cells occur in the first minutes, keeping constant for the remaining time. It was observed that treatment with 131 I induced a decrease in cell viability dependent on the dose. The predominant type of cell death was apoptosis, followed by an increase in the BAX/BCL-2 ratio. In agreement with these results, there was also the release of cytochrome C, mitochondrial membrane depolarization and the occurrence of pre-G0 peak during the cell cycle. Interestingly, it wasn’t possible to detect differences in the production of intracellular peroxides, superoxide anion, glutathione, and superoxide dismutase. It was also found that 131 I induce DNA breaks in TFK-1 cells. Conclusions: Metabolic radiotherapy with 131 I causes a decrease in TFK-1 cells survival, inducing cell death mainly by apoptosis, at least in part through mitochondrial pathway. Thus, the 131 I could a promising option for the treatment of CC. No conflict of interest. 747 Novel anthraquinone-thiosemicarbazones with tautomerizable methylene group as anti-metastatic and anti-angiogenic agents B. Kolundzija1 , T. Stanojkovic1 , M.D. Joksovic2 . 1 Institute of Oncology and Radiology of Serbia, Department of Experimental Oncology, Belgrade, Serbia, 2 Faculty of Science University of Kragujevac, Department of Chemistry, Kragujevac, Serbia Background: Anthraquinones, both natural and synthetic, have been widely used in different therapeutic roles, as they poses laxative, anti-inflammatory, antibiotic and anti-tumor effects. Among these, anthracyclines, like doxorubicin and daunorubicin are extremely important as chemotherapeutics, but with many limitations to their use, mainly due to cardiotoxicity. Nine novel anthraquinones with thiosemicarbazone supstituents were synthesized, with additional introduction of tautomerizable group, with aim of increasing biological activity and decreasing toxicity. We have demonstrated previously [1] the good biological activity and satisfactory selectivity of these compounds against malignant cell lines. The aim of this work is to elucidate the potential of these compounds to inhibit cell migration and metastasis. Material and Methods: Anti-metastatic potential of compounds was tested using gelatin zymography and wound-healing assay. Briefly, in both these tests, sub-lethal concentrations of investigated compounds (IC20 , previously determined by MTT test) were used. For gelatin zymography, HeLa cells were treated with the compounds for 24 h. Supernatants were collected, and run on 8% SDS-PAGE gels, with 2 mg/ml final concentration of gelatin. Gels were stained with Coomassie Brilliant Blue, destained and the bands were visualized. Wound-healing assay was performed using EA.hy926 cells. On the first day a scratch was made in a confluent cell monolayer, and the cells were treated during next 48 hours. Every 24 hours, photomicrographs of the same area were taken. Results and Discussion: Matrix metalloproteinases have been shown to play a significant role in angiogenesis and metastatic potential of tumor cells. Our results show that the investigated compounds significantly reduce the activity of MMPs, compared to controls. This reduction is highly significant for five of the tested compounds and significant for other four. Most importantly, the inhibition of MMPs by the investigated compounds was higher in comparison to the action of doxorubicin and cisplatin. In wound-healing assay, all of compounds exhibit between 25 and 70% of inhibition of tumor cell invasion compared to control. Conclusion: Our results show that the investigated novel anthraquinone derivatives are potent inhibitors of MMPs secretion and malignant cells migration and invasion. This could lead toward further in vivo testing and development of these and similar compounds as anti-metastatic and antiangiogenic agents. Reference(s) [1] Markovic V et al. Eur J Med Chem. 2013; 64:228−38. No conflict of interest.