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275 SORAFENIB EFFECTIVENESS IS INHIBITED IN PRESENCE OF LAMININ-5 IN HCC CELLS
POSTERS 273 PROPERTIES AND LIMITATIONS OF MONOCYTES AND CANCER CELLS AS CARRIERS FOR REPLICATION-COMPETENT ADENOVIRAL VECTORS IN LIVER METASTASES OF PANCREATIC CANCER E. Garcia-Aragoncillo1 , M. Bunuales1 , R. Casado1 , S. Bortolanza1 , J. Prieto2 , R. Hernandez-Alcoceba1 . 1 Foundation for Applied Medical Research, CIMA, University of Navarra, 2 CIBERehd, Clinica Universitaria, Pamplona, Spain E-mail: [email protected] Oncolytic adenoviruses (OAV) are promising agents for the treatment of cancer, but their clinical efficacy is hampered by a diversity of factors, including the poor biodistribution in solid tumors and the rapid appearance of neutralizing antibodies (NAbs) against adenovirus. The use of carrier cells can overcome some of these limitations. An important feature of such cells is to shield the adenovirus from NAbs in the circulation and allow efficient re-administration over an extended period of time. In the present work we have compared monocytes and cancer cells as carriers for OAV, using a replication-competent adenovirus expressing the luciferase reporter gene (Ad-WTLuc). Experiments performed in athymic nude mice carrying subcutaneous human cancer xenografts indicated that monocytes (from either human or hamster origin) were able to increase the access of Ad-WTLuc to the tumors upon intravenous administration. In vivo bioluminescence detection revealed that up to 15% of the luciferase signal can be detected in the tumors, in contrast with the almost exclusive hepatic localization observed with the free virus. However, when a parallel experiment was performed in immune-competent hamsters carrying liver metastases of pancreatic cancer, systemic administration of infected monocytes caused no significant tumor transduction. In contrast, intratumoral administration of hamster monocytes loaded ex-vivo with Ad-WTLuc allowed repeated cycles of local transgene expression, and achieved partial protection from NAbs. Similar properties were observed when irradiated cancer cells were used as carriers. These results suggest that lethally irradiated cancer cells infected ex-vivo with OAVs expressing therapeutic genes could be efficient tools for immunotherapy, with dual roles as cancer vaccines and carrier cells. 274 TREATMENT OF ANTISENSE INSULIN-LIKE GROWTH FACTOR II CONTROLING THE DEVELOPMENT OF HEPATOCELLULAR CARCINOMA IN RATS B. Mukherjee, M.K. Ghosh, Antisense Therapy in Liver Cancer Research Group. Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India E-mail: [email protected] Aim of the study: Insulin-like Growth Factor-II, a mitogenic polypeptide, has been implicated in the pathogenesis of neoplasm of different tissues including liver of rat and man. The protein is found to express in first few days of neonatal life and also in neoplasia including hepatocellular carcinoma (HCC). Effect of blocking IGF II gene expression using antisense IGF II oligomers during hepatocarcinogenesis (in rats) was studied here. Many important marker enzymes and isoenzymes, along with two very important cellular proliferating genes, IGF II and c-raf.1, were investigated and correlated. Methods: HCC was developed in male Sprague-Dawley rats treated with 2-acetylaminofluorene for 28 weeks. Animals were killed at 36–37 weeks. The modified phosphorothioate antisense IGF II was used to treat the animals from 20–34 weeks, 3 times in a week. Glutathione peroxidase, cytochrome-c-reductase, cytochrome b5 and cytochrome p-450, suproxide dismutase, catalase, reduced glutathione content, glutathione reductase and S114
glucose-6-phosphatase were estimated/detected. Gene expressions were studied by in-situ hybridization procedure. Focal lesions were counted using histopathological staining technique. Results: Decrease in size and numbers of lesions was detected in different carcinogen-treated animals treated with antisense IGF II oligomers. The IGF II expression was found to be predominant in HCC. IGF II and raf expressions in the hepatic tissues were found to vary in the experimental animals. Variable activities/levels of glutathione peroxidase, cytochrome-c-reductase, cytochrome b5 and p-450, the enzymes, super oxide dismutase, catalase, reduced glutathione (GSH) content and GSH-reductase were detected in experimental and control animals. Raf.1 gene was found to express strongly in the glucose-6-phosphatase negative HCC which were basophilic and IGF II expression was observed in the early and then at the late stage lesions as well as in HCC. Inference: Blocking of IGF II gene by antisense IGF II oligomers slowed down the progress of the hepatocellular cancer development in rats. Further, IGF-II induced cellular signaling may be mainly mediated and/or affected by c-raf during HCC or in the late stage of development of cancer. Acknowledgement: The work has been carried out, using the fund from Indian Council of Medical Research (ICMR), grant no. 58/7/2009-BMS. 275 SORAFENIB EFFECTIVENESS IS INHIBITED IN PRESENCE OF LAMININ-5 IN HCC CELLS A. Azzariti1 , L. Porcelli2 , A.E. Quatrale2 , A. Paradiso2 , G. Giannelli3 . 1 Department of Experimental Oncology, 2 Department of Experimental Oncology, Laboratory of Analyses, National Cancer Institute, 3 Department of Emergency and Organ Transplantation, Section of Internal Medicine Allergology and Immunology, University of Bari, Bari, Italy E-mail: [email protected] Sorafenib is currently the gold standard therapy for patients with advanced stage HCC. However, in some patients treatment has to be discontinued because of side effects, while about 30% do not respond and undergo progression. The molecular mechanisms underlying failure to respond to Sorafenib are unknown, making it difficult to select patients most likely to respond to therapy. Aim of this study is to investigate the role of the microenvironment in affecting response to Sorafenib. Methods: Different invasive HCC cell lines (HLE, HLF, Sk-Hep1 and Huh7) were challenged to proliferate in presence of Sorafenib at an IC50 concentration for 72 hours in serum-free conditions. As expected, Sorafenib inhibited HCC proliferation, but in presence of Laminin-5 (Ln-5), an extracellular matrix protein present in the HCC microenvironment, the effectiveness of Sorafenib was strongly (p < 0.01) reduced. Consistently, Sorafenib also stimulated apoptosis of HCC cells, but also in this case the effects of Sorafenib were significantly abrogated in presence of Ln-5. Similar effects were obtained with serum-free conditioned medium of human Hepatic Stellate cells, that we have recently shown to produce and secrete Ln-5. Molecular analysis of different intracellular pathways showed that Akt was dephosphorylated in the presence of Sorafenib, but recovered in drug-treated HCC cells in presence of Ln-5. To further confirm the specificity of the Ln-5 dependent effect, we used Hep3B cells that do not constitutively express the main receptor for Ln-5, integrin a3b1. Hep3B cells were stably transfected to express high levels of integrin a3b1. Sorafenib inhibited cell proliferation and stimulated apoptosis in Hep3B wild type also in the presence of Ln-5, while these effects were completely reversed in Hep3B a3b1 transfected cells in the presence of Ln-5. Conclusion: This is the first evidence of a molecular mechanism that contributes to explain failure to respond to Sorafenib in patients with HCC.
Journal of Hepatology 2012 vol. 56 | S71–S224
glucose-6-phosphatase were estimated/detected. Gene expressions were studied by in-situ hybridization procedure. Focal lesions were counted using histopathological staining technique. Results: Decrease in size and numbers of lesions was detected in different carcinogen-treated animals treated with antisense IGF II oligomers. The IGF II expression was found to be predominant in HCC. IGF II and raf expressions in the hepatic tissues were found to vary in the experimental animals. Variable activities/levels of glutathione peroxidase, cytochrome-c-reductase, cytochrome b5 and p-450, the enzymes, super oxide dismutase, catalase, reduced glutathione (GSH) content and GSH-reductase were detected in experimental and control animals. Raf.1 gene was found to express strongly in the glucose-6-phosphatase negative HCC which were basophilic and IGF II expression was observed in the early and then at the late stage lesions as well as in HCC. Inference: Blocking of IGF II gene by antisense IGF II oligomers slowed down the progress of the hepatocellular cancer development in rats. Further, IGF-II induced cellular signaling may be mainly mediated and/or affected by c-raf during HCC or in the late stage of development of cancer. Acknowledgement: The work has been carried out, using the fund from Indian Council of Medical Research (ICMR), grant no. 58/7/2009-BMS. 275 SORAFENIB EFFECTIVENESS IS INHIBITED IN PRESENCE OF LAMININ-5 IN HCC CELLS A. Azzariti1 , L. Porcelli2 , A.E. Quatrale2 , A. Paradiso2 , G. Giannelli3 . 1 Department of Experimental Oncology, 2 Department of Experimental Oncology, Laboratory of Analyses, National Cancer Institute, 3 Department of Emergency and Organ Transplantation, Section of Internal Medicine Allergology and Immunology, University of Bari, Bari, Italy E-mail: [email protected] Sorafenib is currently the gold standard therapy for patients with advanced stage HCC. However, in some patients treatment has to be discontinued because of side effects, while about 30% do not respond and undergo progression. The molecular mechanisms underlying failure to respond to Sorafenib are unknown, making it difficult to select patients most likely to respond to therapy. Aim of this study is to investigate the role of the microenvironment in affecting response to Sorafenib. Methods: Different invasive HCC cell lines (HLE, HLF, Sk-Hep1 and Huh7) were challenged to proliferate in presence of Sorafenib at an IC50 concentration for 72 hours in serum-free conditions. As expected, Sorafenib inhibited HCC proliferation, but in presence of Laminin-5 (Ln-5), an extracellular matrix protein present in the HCC microenvironment, the effectiveness of Sorafenib was strongly (p < 0.01) reduced. Consistently, Sorafenib also stimulated apoptosis of HCC cells, but also in this case the effects of Sorafenib were significantly abrogated in presence of Ln-5. Similar effects were obtained with serum-free conditioned medium of human Hepatic Stellate cells, that we have recently shown to produce and secrete Ln-5. Molecular analysis of different intracellular pathways showed that Akt was dephosphorylated in the presence of Sorafenib, but recovered in drug-treated HCC cells in presence of Ln-5. To further confirm the specificity of the Ln-5 dependent effect, we used Hep3B cells that do not constitutively express the main receptor for Ln-5, integrin a3b1. Hep3B cells were stably transfected to express high levels of integrin a3b1. Sorafenib inhibited cell proliferation and stimulated apoptosis in Hep3B wild type also in the presence of Ln-5, while these effects were completely reversed in Hep3B a3b1 transfected cells in the presence of Ln-5. Conclusion: This is the first evidence of a molecular mechanism that contributes to explain failure to respond to Sorafenib in patients with HCC.
Journal of Hepatology 2012 vol. 56 | S71–S224