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749: Targeting cell-surface nucleolin in metastatic breast cancer
S180
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
749 Targeting cell-surface nucleolin in metastatic breast cancer A. Gregorio ´ 1 , N. Fonseca2 , V. Moura3 , G. Domingues4 , M. Lacerda5 , P. Figueireido6 , S. Simoes ˜ 7 , S. Dias4 , J.N. Moreira7 . 1 Center for Neuroscience and Cell Biology and IIIUC − Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal, 2 Center for Neuroscience and Cell Biology and FFUC − Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal, 3 Center for Neuroscience and Cell Biology and TREAT U SA, University of Coimbra, Coimbra, Portugal, 4 IMM − Molecular Medicine Institute, University of Lisbon, Lisbon, Portugal, 5 IPATIMUP − Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal, 6 Portuguese Institute of Oncology Francisco Gentil, Coimbra, Coimbra, Portugal, 7 CNC − Center for Neurosciences and Cell Biology and FFUC − Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal Introduction: Breast cancer (BC) is the most common malignant disease among women. Despite the developments and great progress achieved in BC treatment, metastatic disease remains an incurable condition and is responsible for cancer-associated mortality. Therefore, new rationally designed therapeutic approaches, targeting both primary and metastatic disease, are required. Herein, we aim to validate cell-surface nucleolin as therapeutic target for previously developed F3 peptide-targeted nanoparticle. Material and Method: 4T1 and E0771 mouse cell lines were used as Metastatic breast cancer (MBC) models. To investigate the relevance of nucleolin in this cell lines, cellular association studies were performed by flow cytometry; cells were incubated with fluorescently-labeled liposomes, either non-targeted (SLpH), or targeted by a non-specific (SLpHNS) or F3 (SLpHF3) peptide, for 1, 4 and 7 h at 37ºC, and analyzed for cell-associated fluorescence. Aiming at assessing cytotoxicity, cells were incubated, as previously described, with targeted or non-targeted formulations encapsulating doxorubicin (DXR), for 1 h at 37ºC. Cell proliferation was evaluated by resazurin assay, and IC50 values were determined from dose–response curves. To access clinical potential of the nucleolin-targeting strategy, immunohistochemistry of human breast tumors were performed in collaboration with Portuguese Institute of Oncology FG, EPE, Coimbra. Results and Discussion: A 31 to 48-fold increase in cellular association of F3targeted liposomes by 4T1 and E0771 cells relative to non-targeted or targeted by a non-specific peptide, suggests a ligand-specific interaction mediated by surface nucleolin. Superior association led to improved intracellular delivery of encapsulated DXR by SLpHF3 liposomes, resulting in 11 to 20-fold increase of drug cytotoxicity relative to the other tested formulations. Clinical relevance of targeting nucleolin as a therapeutical approach for MBC treatment is emphasized by its overexpression on primary tumor human samples. Conclusion: The results show that liposomes targeting cell-surface nucleolin are a therapeutical strategy with great potential outcome for MBC treatment. Further preclinical evaluation will be conducted using the 4T1 and E0771 as mouse models of metastatic disease. Ana Gregorio ´ is the recipient of a fellowship from FCT (ref.: SFRH/BD/51190/ 2010). The work was supported by the grants PTDC/SAU-BMA/121028/2010 (FEDER, COMPETE, FCT) and PEst-C/SAU/LA0001/2011. No conflict of interest. 750 Human amniotic membrane secreted factors plus chemotherapy: A mishmash of effects? S. Guerra1 , A.C. Mamede2 , M. Laranjo3 , A.S. Pires4 , M.J. Carvalho5 , A.F. Brito3 , P. Moura6 , A.M. Abrantes3 , C.J. Maia7 , M.F. Botelho3 . 1 Biophysics Unit/FCTUC, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 2 Biophysics Unit/CICS-UBI − Health Sciences Research Centre/IBILI/CIMAGO, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 3 Biophysics Unit/IBILI/CIMAGO, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 4 Biophysics Unit/FCTUC/IBILI/CIMAGO, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 5 Obstetrics Service/Biophysics Unit/IBILI/CIMAGO, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 6 Obstetrics Service, Coimbra Hospital and University Centre, Coimbra, Portugal, 7 CICS-UBI − Health Sciences ˜ Portugal Research Centre, University of Beira Interior, Covilha, Introduction: Hepatocellular carcinoma (HCC) is a primary liver cancer and is one of the leading causes of death worldwide, in part due to its ability to resist to chemotherapy and other conventional therapies. Even though, doxorubicin, 5-fluorouracil, cisplatin and sorafenib are used in HCC management. However, side effects depend on drug dose and administration period. Studies showed that human amniotic membrane (hAM) secretes several cytotoxic cytokines, such as M-CSF, TNF-a, TNF-b, IFN-g and TGF-b, as well as various interleukins such as IL-1, IL-2, IL-3, IL-4, IL-6 and IL-8. Thus, the aim of this study is to prove that factors secreted by hAM (F-hAM) are able to potentiate the effect of conventional chemotherapy in the treatment of HCC. Material and Methods: hAM was obtained from caesarean sections with informed consent according to the guidelines of Ethical Committee of Coimbra Hospital and University Centre (Coimbra, Portugal). hAM was washed with phosphate buffered solution, cut in pieces with 3 cm of diameter and placed
in a petri dish with culture medium. After 72 h, 100 mL of culture medium were collected and incubated with three human cancer cell lines: Hep3B2.1−7, HuH7 and HepG2. Cells were also treated with 5-fluoracil (5-FU), doxorubicin, cisplatin or sorafenib in several concentrations. After 72 h, MTT assay was performed. Results: Our preliminary results indicate that F-hAM can potentiate the effect of 5-FU, doxorubicin and sorafenib in Hep3B2.1−7 cell line. On the other hand, F-hAM inhibits the effect of cisplatin in this cell line. This profile was also observed in HepG2 and Huh7 cell lines. It should be noted, however, that in these cell lines the effect of 5-FU, doxorubicin and sorafenib are more enhanced than in Hep3B2.1−7 cell line. Interestingly, the effect of sorafenib seems to be highly potentiated in HuH7 cell line. Conclusions: Our results suggest that the combination of F-hAM with some chemotherapeutic agents can potentiate their effect. The fact that F-hAM inhibit the effect of cisplatin, contrary to what happens with other drugs, is worthy of investigation thereof. Further studies are necessary to confirm which cytokines and/or interleukins may be involved in observed effects. No conflict of interest. 751 Butyrate and irinotecan combination as a new therapeutic approach for colon cancer J.C. Encarna¸cao ˜ 1 , A.S. Pires2 , T.J. Gon¸calves1 , J.E. Casalta-Lopes3 , A.C. Gon¸calves4 , A.M. Abrantes5 , A.B. Sarmento-Ribeiro4 , M.F. Botelho5 . 1 Biophysics Unit/FFUC, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 2 Biophysics Unit/FCTUC/IBILI/CIMAGO, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 3 Biophysics Unit, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 4 Applied Molecular Biology and University Clinic of Hematology/CIMAGO, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 5 Biophysics Unit/IBILI/CIMAGO, Faculty of Medicine University of Coimbra, Coimbra, Portugal Background: An important diet based on high levels of dietary fiber is related with a lower risk for developing colon cancer (CC). Microbial fermentation of dietary fiber by the gut results in production of short chain fatty acid (SCFA) such as butyrate. Butyrate is an important energy source for colonocytes and it plays an important role in maintenance of the colon homeostasis. It was also reported that butyrate may be a chemopreventive agent. Irinotecan is used as second-line treatment, however, there is uncertainty about the balance between benefits and risks, namely, the large interindividual variability in pharmacogenetic behavior. The use of natural compounds to turn the resistant cells more sensitive to the chemotherapy seems to be a probably solution. The aim of this study is to evaluate the effect of the combination of butyrate and irinotecan on three colon cancer cell lines. Methods: C2BBe1, WiDr and LS1034 cells were separately incubated with different sodium butyrate (1−50 mM) and irinotecan (0.1–100 mM) concentrations. In order to determine the IC50 (half maximal inhibitory concentration) after 48, 72 and 96 hours, cell proliferation was evaluated through MTT assay. Flow cytometry was also performed to study the butyrate effect on cell viability and types of death, apoptosis (evaluating the BAX/BCL2 ratio) and expression of reduced glutathione (GSH). Results: It was observed that when cells are incubated for a longer time (48 h, 72 h and 96 h) cell proliferation decreases, being obtained lower IC50 values. The data obtained also showed that the combination of butyrate and irinotecan significantly decrease cell proliferation compared to monotherapy. These results are similar in all cell lines, being the multi-drug resistant cell line (LS1034) the most sensitive to the combination at longer incubation times. Regarding cell viability, preliminary results showed that as the butyrate concentration increases, cell viability decreases in all cell lines. When C2BBe1 and LS1034 cells are incubated with higher butyrate concentrations, there is an increase in BAX/BCL2 ratio. There is also a slight increase in GSH expression, comparing to control. Conclusions: Our study suggests that butyrate and irinotecan combination has a significant anti-proliferative effect on the three CC cell lines despite of the different genetic background and organ localization. The use of natural compounds as butyrate in combination with chemotherapeutic agents can be a new solution for CC treatment. No conflict of interest.
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
749 Targeting cell-surface nucleolin in metastatic breast cancer A. Gregorio ´ 1 , N. Fonseca2 , V. Moura3 , G. Domingues4 , M. Lacerda5 , P. Figueireido6 , S. Simoes ˜ 7 , S. Dias4 , J.N. Moreira7 . 1 Center for Neuroscience and Cell Biology and IIIUC − Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal, 2 Center for Neuroscience and Cell Biology and FFUC − Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal, 3 Center for Neuroscience and Cell Biology and TREAT U SA, University of Coimbra, Coimbra, Portugal, 4 IMM − Molecular Medicine Institute, University of Lisbon, Lisbon, Portugal, 5 IPATIMUP − Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal, 6 Portuguese Institute of Oncology Francisco Gentil, Coimbra, Coimbra, Portugal, 7 CNC − Center for Neurosciences and Cell Biology and FFUC − Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal Introduction: Breast cancer (BC) is the most common malignant disease among women. Despite the developments and great progress achieved in BC treatment, metastatic disease remains an incurable condition and is responsible for cancer-associated mortality. Therefore, new rationally designed therapeutic approaches, targeting both primary and metastatic disease, are required. Herein, we aim to validate cell-surface nucleolin as therapeutic target for previously developed F3 peptide-targeted nanoparticle. Material and Method: 4T1 and E0771 mouse cell lines were used as Metastatic breast cancer (MBC) models. To investigate the relevance of nucleolin in this cell lines, cellular association studies were performed by flow cytometry; cells were incubated with fluorescently-labeled liposomes, either non-targeted (SLpH), or targeted by a non-specific (SLpHNS) or F3 (SLpHF3) peptide, for 1, 4 and 7 h at 37ºC, and analyzed for cell-associated fluorescence. Aiming at assessing cytotoxicity, cells were incubated, as previously described, with targeted or non-targeted formulations encapsulating doxorubicin (DXR), for 1 h at 37ºC. Cell proliferation was evaluated by resazurin assay, and IC50 values were determined from dose–response curves. To access clinical potential of the nucleolin-targeting strategy, immunohistochemistry of human breast tumors were performed in collaboration with Portuguese Institute of Oncology FG, EPE, Coimbra. Results and Discussion: A 31 to 48-fold increase in cellular association of F3targeted liposomes by 4T1 and E0771 cells relative to non-targeted or targeted by a non-specific peptide, suggests a ligand-specific interaction mediated by surface nucleolin. Superior association led to improved intracellular delivery of encapsulated DXR by SLpHF3 liposomes, resulting in 11 to 20-fold increase of drug cytotoxicity relative to the other tested formulations. Clinical relevance of targeting nucleolin as a therapeutical approach for MBC treatment is emphasized by its overexpression on primary tumor human samples. Conclusion: The results show that liposomes targeting cell-surface nucleolin are a therapeutical strategy with great potential outcome for MBC treatment. Further preclinical evaluation will be conducted using the 4T1 and E0771 as mouse models of metastatic disease. Ana Gregorio ´ is the recipient of a fellowship from FCT (ref.: SFRH/BD/51190/ 2010). The work was supported by the grants PTDC/SAU-BMA/121028/2010 (FEDER, COMPETE, FCT) and PEst-C/SAU/LA0001/2011. No conflict of interest. 750 Human amniotic membrane secreted factors plus chemotherapy: A mishmash of effects? S. Guerra1 , A.C. Mamede2 , M. Laranjo3 , A.S. Pires4 , M.J. Carvalho5 , A.F. Brito3 , P. Moura6 , A.M. Abrantes3 , C.J. Maia7 , M.F. Botelho3 . 1 Biophysics Unit/FCTUC, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 2 Biophysics Unit/CICS-UBI − Health Sciences Research Centre/IBILI/CIMAGO, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 3 Biophysics Unit/IBILI/CIMAGO, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 4 Biophysics Unit/FCTUC/IBILI/CIMAGO, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 5 Obstetrics Service/Biophysics Unit/IBILI/CIMAGO, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 6 Obstetrics Service, Coimbra Hospital and University Centre, Coimbra, Portugal, 7 CICS-UBI − Health Sciences ˜ Portugal Research Centre, University of Beira Interior, Covilha, Introduction: Hepatocellular carcinoma (HCC) is a primary liver cancer and is one of the leading causes of death worldwide, in part due to its ability to resist to chemotherapy and other conventional therapies. Even though, doxorubicin, 5-fluorouracil, cisplatin and sorafenib are used in HCC management. However, side effects depend on drug dose and administration period. Studies showed that human amniotic membrane (hAM) secretes several cytotoxic cytokines, such as M-CSF, TNF-a, TNF-b, IFN-g and TGF-b, as well as various interleukins such as IL-1, IL-2, IL-3, IL-4, IL-6 and IL-8. Thus, the aim of this study is to prove that factors secreted by hAM (F-hAM) are able to potentiate the effect of conventional chemotherapy in the treatment of HCC. Material and Methods: hAM was obtained from caesarean sections with informed consent according to the guidelines of Ethical Committee of Coimbra Hospital and University Centre (Coimbra, Portugal). hAM was washed with phosphate buffered solution, cut in pieces with 3 cm of diameter and placed
in a petri dish with culture medium. After 72 h, 100 mL of culture medium were collected and incubated with three human cancer cell lines: Hep3B2.1−7, HuH7 and HepG2. Cells were also treated with 5-fluoracil (5-FU), doxorubicin, cisplatin or sorafenib in several concentrations. After 72 h, MTT assay was performed. Results: Our preliminary results indicate that F-hAM can potentiate the effect of 5-FU, doxorubicin and sorafenib in Hep3B2.1−7 cell line. On the other hand, F-hAM inhibits the effect of cisplatin in this cell line. This profile was also observed in HepG2 and Huh7 cell lines. It should be noted, however, that in these cell lines the effect of 5-FU, doxorubicin and sorafenib are more enhanced than in Hep3B2.1−7 cell line. Interestingly, the effect of sorafenib seems to be highly potentiated in HuH7 cell line. Conclusions: Our results suggest that the combination of F-hAM with some chemotherapeutic agents can potentiate their effect. The fact that F-hAM inhibit the effect of cisplatin, contrary to what happens with other drugs, is worthy of investigation thereof. Further studies are necessary to confirm which cytokines and/or interleukins may be involved in observed effects. No conflict of interest. 751 Butyrate and irinotecan combination as a new therapeutic approach for colon cancer J.C. Encarna¸cao ˜ 1 , A.S. Pires2 , T.J. Gon¸calves1 , J.E. Casalta-Lopes3 , A.C. Gon¸calves4 , A.M. Abrantes5 , A.B. Sarmento-Ribeiro4 , M.F. Botelho5 . 1 Biophysics Unit/FFUC, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 2 Biophysics Unit/FCTUC/IBILI/CIMAGO, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 3 Biophysics Unit, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 4 Applied Molecular Biology and University Clinic of Hematology/CIMAGO, Faculty of Medicine University of Coimbra, Coimbra, Portugal, 5 Biophysics Unit/IBILI/CIMAGO, Faculty of Medicine University of Coimbra, Coimbra, Portugal Background: An important diet based on high levels of dietary fiber is related with a lower risk for developing colon cancer (CC). Microbial fermentation of dietary fiber by the gut results in production of short chain fatty acid (SCFA) such as butyrate. Butyrate is an important energy source for colonocytes and it plays an important role in maintenance of the colon homeostasis. It was also reported that butyrate may be a chemopreventive agent. Irinotecan is used as second-line treatment, however, there is uncertainty about the balance between benefits and risks, namely, the large interindividual variability in pharmacogenetic behavior. The use of natural compounds to turn the resistant cells more sensitive to the chemotherapy seems to be a probably solution. The aim of this study is to evaluate the effect of the combination of butyrate and irinotecan on three colon cancer cell lines. Methods: C2BBe1, WiDr and LS1034 cells were separately incubated with different sodium butyrate (1−50 mM) and irinotecan (0.1–100 mM) concentrations. In order to determine the IC50 (half maximal inhibitory concentration) after 48, 72 and 96 hours, cell proliferation was evaluated through MTT assay. Flow cytometry was also performed to study the butyrate effect on cell viability and types of death, apoptosis (evaluating the BAX/BCL2 ratio) and expression of reduced glutathione (GSH). Results: It was observed that when cells are incubated for a longer time (48 h, 72 h and 96 h) cell proliferation decreases, being obtained lower IC50 values. The data obtained also showed that the combination of butyrate and irinotecan significantly decrease cell proliferation compared to monotherapy. These results are similar in all cell lines, being the multi-drug resistant cell line (LS1034) the most sensitive to the combination at longer incubation times. Regarding cell viability, preliminary results showed that as the butyrate concentration increases, cell viability decreases in all cell lines. When C2BBe1 and LS1034 cells are incubated with higher butyrate concentrations, there is an increase in BAX/BCL2 ratio. There is also a slight increase in GSH expression, comparing to control. Conclusions: Our study suggests that butyrate and irinotecan combination has a significant anti-proliferative effect on the three CC cell lines despite of the different genetic background and organ localization. The use of natural compounds as butyrate in combination with chemotherapeutic agents can be a new solution for CC treatment. No conflict of interest.