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Reactive Oxygen Species Promotes Hsp90-Mediated HBV Capsid Assembly
296 Reactive Oxygen Species Promotes Hsp90-Mediated HBV Capsid Assembly Cheolmin Ha1, Yoon Sik Kim1, and Guhung Jung1 1 Seoul National University, Republic of Korea Hepatitis B virus (HBV) infection increases ROS levels in hepatocyte, and ROS production plays an important role in hepatocarcinogenesis. In high oxidative stress, Hsp90, one of the host factor, and GSH, one of the antioxidant, functions on defense mechanism. Previous report showed that Hsp90 facilitates HBV capsid assembly via interaction with core protein dimer. Capsid assembly is a first important step for HBV production. However, it is not known if molecules regarding oxidative stress are involved in HBV capsid assembly. In this research, we investigated that capsid assembly is associated with molecules, such as ROS, Hsp90, and GSH. First of all, we confirmed that HBV capsid assembly was increased in the presence of Hsp90 while it was decreased in the absence of Hsp90 by ROS treatment. In the comparison to different ROS treatment timing when ROS was treated on Hsp90 before assembly reaction with core protein (pre-treatment of ROS) and ROS was treated during assembly reaction with core protein and Hsp90 (post-treatment of ROS), HBV capsid assembly was increased at low concentration of ROS in pretreatment of ROS while it was increased proportionally following increased concentration of ROS in post-treatment of ROS. Moreover, GSH inhibited function of Hsp90 facilitating HBV capsid assembly, and we found that conformation change of Hsp90 by ROS, GSH induced change of HBV capsid assembly level. Furthermore, In HepG2.2.15 cell which produces HBV virion, HBV DNA and capsid level was increased by ROS treatment, and Buthionine sulphoximine, inhibitor of GSH synthesis, also increased HBV DNA and capsid level. Thus, our results show that ROS influences on HBV production in the liver cell. doi: 10.1016/j.freeradbiomed.2014.10.193
297 Characterization of Redox Homeostasis in Breast Epithelial Cell Lines Fabio Hecht1, Juliana Cazarin1, Rodrigo Soares Fortunato1, and Denise Pires de Carvalho1 1 UFRJ, Brazil Background: Oxidative stress may lead to oxidative damage of cellular components, such as protein and DNA, and activation of carcinogenesis-related signaling pathways, enabling tumor initiation and progression. The in vitro evidences that ROS may promote mammary tumorigenesis create interest in therapeutic approaches involving modulation of the production and degradation of ROS. However, the effectiveness of such approaches rely on deep knowledge of the redox homeostasis of the tumor. Objectives: Characterize the molecular basis of redox homeostasis of normal strains of breast epithelial cells (MCF10A) and tumor cells (MCF7 and MDAMB231), in relation to ROS production and removal by antioxidant enzymes. Results: In our model, the mammary tumor cells have higher extracellular ROS production than the normal lineage. Analysis of the concentration of hydrogen peroxide into the extracellular medium, which reflects the balance between ROS-generating activity and antioxidant activity, showed that the normal line had, paradoxically, higher concentrations of it. The extracellular antioxidant enzyme activity was compared among the three strains and higher activity was observed in MDAMB231 compared to MCF10A and MCF7 cells. Regarding to intracellular antioxidant
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activity, a lower activity of glutathione peroxidase and increased catalase activity in tumor cell lines was observed. The resistance of these strains to hydrogen peroxide was evaluated and it was observed that tumor cell lines are more sensitive to oxidative stress-mediated death than the normal strain, which might be related to their lower antioxidant activity mediated by cytosolic glutathione peroxidase. Conclusions: The differences observed in ROS-generating systems and antioxidants mechanisms of the studied strains are important information for a better understanding of the molecular basis of breast tumors cells, contributing to the background required for the development of new therapeutic approaches targeting the redox homeostasis. doi: 10.1016/j.freeradbiomed.2014.10.194
298 Endogenous Nox4 Limits Fibrosarcoma Development in the Murine 3-Methylcholanthrene Model Valeska Helfinger1, Nina Henke1, and Katrin Schröder1 1 Goethe University Frankfurt, Germany Through the constitutive production of H2O2 the NAPDH Oxidase Nox4 promotes differentiation of cells and contributes to cellular quiescence. Our previous observations in the mesenchymal tissue of the vasculature and the bone suggest that the absence of Nox4 promotes inflammation and de-differentiation. Chronic inflammation and lack of differentiation are pre-carcinogenic states. We therefore hypothesized that lack of Nox4 promotes malignant transformation and tumor development. The process of inflammation-dependent tumor formation was studied in response to the chemical carcinogen 3methylcholanthrene (MCA) which was administered subcutaneously to mice and induces sarcoma formation. Initial fibrosarcoma formation was massively enhanced in Nox4deficient mice and in average their tumors were bigger as compared to those of wildtype mice. Nox1y/- mice, in contrast, developed fewer tumors. Genetic deletion of Nox4 resulted in an increased number of F4/80 positive myeloid cells within the tumor. Simultaneously, the activity of 1)ț% RQH of the most important pro-inflammatory transcription factors, was significantly increased in tumor cells cultured from Nox4-/- mice as compared to WT control mice. As a consequence, pro-inflammatory cytokines and adhesion molecules e.g. IL-1beta, TNF-alpha and VCAM were expressed at higher levels in the tumors of Nox4 knockout mice. Thus, a higher degree of inflammation is present in the absence of Nox4 which may promote tumor development. Mechanistically, the abundance of the tumor suppressor p53 was significantly reduced in the tumor tissue as well as in the cells of Nox4-/-. This was accompanied by an increased phosphorylation of AKT which is presumably responsible for the reduction of p53 through its ability to activate the E3 ubiquitin-protein ligase MDM2 which promotes p53 degradation. Additionally, induction of the AKT pathway in the Nox4-deficient tumor cells may lead to a prosurvival phenotype which contributes to tumor growth. Taken together these results suggest that anti-inflammatory properties of endogenous Nox4 attenuate inflammation-induced tumor formation in the murine 3-methylcholanthrene model. doi: 10.1016/j.freeradbiomed.2014.10.195
SFRBM 2014
297 Characterization of Redox Homeostasis in Breast Epithelial Cell Lines Fabio Hecht1, Juliana Cazarin1, Rodrigo Soares Fortunato1, and Denise Pires de Carvalho1 1 UFRJ, Brazil Background: Oxidative stress may lead to oxidative damage of cellular components, such as protein and DNA, and activation of carcinogenesis-related signaling pathways, enabling tumor initiation and progression. The in vitro evidences that ROS may promote mammary tumorigenesis create interest in therapeutic approaches involving modulation of the production and degradation of ROS. However, the effectiveness of such approaches rely on deep knowledge of the redox homeostasis of the tumor. Objectives: Characterize the molecular basis of redox homeostasis of normal strains of breast epithelial cells (MCF10A) and tumor cells (MCF7 and MDAMB231), in relation to ROS production and removal by antioxidant enzymes. Results: In our model, the mammary tumor cells have higher extracellular ROS production than the normal lineage. Analysis of the concentration of hydrogen peroxide into the extracellular medium, which reflects the balance between ROS-generating activity and antioxidant activity, showed that the normal line had, paradoxically, higher concentrations of it. The extracellular antioxidant enzyme activity was compared among the three strains and higher activity was observed in MDAMB231 compared to MCF10A and MCF7 cells. Regarding to intracellular antioxidant
S126
activity, a lower activity of glutathione peroxidase and increased catalase activity in tumor cell lines was observed. The resistance of these strains to hydrogen peroxide was evaluated and it was observed that tumor cell lines are more sensitive to oxidative stress-mediated death than the normal strain, which might be related to their lower antioxidant activity mediated by cytosolic glutathione peroxidase. Conclusions: The differences observed in ROS-generating systems and antioxidants mechanisms of the studied strains are important information for a better understanding of the molecular basis of breast tumors cells, contributing to the background required for the development of new therapeutic approaches targeting the redox homeostasis. doi: 10.1016/j.freeradbiomed.2014.10.194
298 Endogenous Nox4 Limits Fibrosarcoma Development in the Murine 3-Methylcholanthrene Model Valeska Helfinger1, Nina Henke1, and Katrin Schröder1 1 Goethe University Frankfurt, Germany Through the constitutive production of H2O2 the NAPDH Oxidase Nox4 promotes differentiation of cells and contributes to cellular quiescence. Our previous observations in the mesenchymal tissue of the vasculature and the bone suggest that the absence of Nox4 promotes inflammation and de-differentiation. Chronic inflammation and lack of differentiation are pre-carcinogenic states. We therefore hypothesized that lack of Nox4 promotes malignant transformation and tumor development. The process of inflammation-dependent tumor formation was studied in response to the chemical carcinogen 3methylcholanthrene (MCA) which was administered subcutaneously to mice and induces sarcoma formation. Initial fibrosarcoma formation was massively enhanced in Nox4deficient mice and in average their tumors were bigger as compared to those of wildtype mice. Nox1y/- mice, in contrast, developed fewer tumors. Genetic deletion of Nox4 resulted in an increased number of F4/80 positive myeloid cells within the tumor. Simultaneously, the activity of 1)ț% RQH of the most important pro-inflammatory transcription factors, was significantly increased in tumor cells cultured from Nox4-/- mice as compared to WT control mice. As a consequence, pro-inflammatory cytokines and adhesion molecules e.g. IL-1beta, TNF-alpha and VCAM were expressed at higher levels in the tumors of Nox4 knockout mice. Thus, a higher degree of inflammation is present in the absence of Nox4 which may promote tumor development. Mechanistically, the abundance of the tumor suppressor p53 was significantly reduced in the tumor tissue as well as in the cells of Nox4-/-. This was accompanied by an increased phosphorylation of AKT which is presumably responsible for the reduction of p53 through its ability to activate the E3 ubiquitin-protein ligase MDM2 which promotes p53 degradation. Additionally, induction of the AKT pathway in the Nox4-deficient tumor cells may lead to a prosurvival phenotype which contributes to tumor growth. Taken together these results suggest that anti-inflammatory properties of endogenous Nox4 attenuate inflammation-induced tumor formation in the murine 3-methylcholanthrene model. doi: 10.1016/j.freeradbiomed.2014.10.195
SFRBM 2014