European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS).

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.The EU-ROS consortium (COST Action BM1203) was supported by the European Cooperation in Science and Technology (COST). The present overview represents the final Action dissemination summarizing the major achievements of COST Action BM1203 (EU-ROS) as well as research news and personal views of its members. Some authors were also supported by COST Actions BM1005 (ENOG) and BM1307 (PROTEOSTASIS), as well as funding from the European Commission FP7 and H2020 programmes, and several national funding agencies

NKX3.1 binding to GPX2, QSCN6, SOD1, and SOD2 promoters contributes to antioxidant response regulation via transactivation

WOS: 000341523900012NKX3.1 is a prostate-specific transcription factor that is regulated by the androgen receptor in the presence of androgens. It functions as a tumor suppressor against the development of prostatic intraepithelial neoplasia and primary prostate tumors. Here, a recognized approach combining in silico analysis and chromatin immunoprecipitation (ChIP) was used to identify the genes directly regulated by NKX3.1 promoter binding in LNCaP cells. Quantitative PCR using ChIP-captured DNAs as templates verified a subset of NKX3.1 binding motifs. Thus, in the presence of androgens, significant NKX3.1 binding occurs to promoters of GPX2, QSCN6, SOD1, and SOD2 genes that contribute to oxidative stress regulation. Our data demonstrate that NKX3.1 is found in a DNA-bound state transiently at a basal level even in the absence of androgens; an increase in androgens promotes NKX3.1 binding, perhaps temporally rather than spatially, to the specific sites. The overall changes potentiate the transcriptional regulatory activity of NKX3.1, although they are dependent on the androgen receptor for the target promoters. The results suggest that NKX3.1 contributes to an antioxidant response by regulating the transcription of oxidative stress regulators by direct promoter binding.Scientific and Technology Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [106S200]This research was funded by the Scientific and Technology Research Council of Turkey (TUBITAK), Project 106S200 to KSK

OGG1 Does not Interact with NKX3.1 and AR to Repair 8-OHdG Base Damage in LNCaP Cells

OGG1 (8-oxo-G DNA glycosylase 1) is a BER (base excision repair) enzyme responsible for the repair of 8-oxo-G base damage related to oxidative stress through glycosylation. Determination of the interaction of DNA repair enzymes with other proteins that contributes to repairing base damages in the cell, is highly relevant in cancer treatment strategies and therapeutic targeting studies. In a previous study, it is shown that OGG1 associated with the homeobox protein NKX3.1 and androgen receptor (AR) in the 8-OHdG-repair complex in prostate cell line LNCaP, when treated with etoposide. Additionally, it is still uncertain that whether S326C, a polymorphic variant of OGG1 formed by a single amino acid change, might harbor functional loss, which might cause a deficiency in repair activity augmenting the susceptibility to (PCa). Therefore, in our study, we investigated the interaction of OGG1 and its polymorphic variant S326C with prostate specific proteins in LNCaP cells under oxidative conditions. Thus, we confirmed the previously determined interactions, however oxidative induction via menadione treatment impairs these interactions

Inflammation contributes to NKX3.1 loss and augments DNA damage but does not alter the DNA damage response via increased SIRT1 expression

The oxidative stress response is a cellular defense mechanism that protects cells from oxidative damage and cancer development. The exact molecular mechanism by which reactive oxygen species (ROS) contribute to DNA damage and increase genome instability in prostate cancer merits further investigation. Here, we aimed to determine the effects of NKX3.1 loss on antioxidant defense in response to acute and chronic inflammation in an in vitro model. Oxidative stress-induced DNA damage resulted in increased H2AX((S139)) phosphorylation (a hallmark of DNA damage), along with the degradation of the androgen receptor (AR), p53 and NKX3.1, upon treatment with conditioned medium (CM) obtained from activated macrophages or H(2)O(2). Furthermore, the expression and stability of SIRT1 were increased by CM treatment but not by H(2)O(2) treatment, although the level of ATM((S1981)) phosphorylation was not changed compared with controls. Moreover, the deregulated antioxidant response resulted in upregulation of the pro-oxidant QSCN6 and the antioxidant GPX2 and downregulation of the antioxidant GPX3 after CM treatment. Consistently, the intracellular ROS level increased after chronic treatment, leading to a dose-dependent increase in the ability of LNCaP cells to tolerate oxidative damage. These data suggest that the inflammatory microenvironment is a major factor contributing to DNA damage and the deregulation of the oxidative stress response, which may be the underlying cause of the increased genetic heterogeneity during prostate tumor progression

Inflammatory Microenvironment-Mediated E-Cadherin Decrease Induces Migration in LNCaPs

Infection and chronic inflammation contribute to about 1 in 4 of all cancer cases. Emerging evidence suggests that the host factors in the tumor microenvironment may interact with underlying inflammatory prostate cancer cells to make them aggressive. In this study, we hypothesized that soluble factors secreted by immune cells activated by inflammation can induce EMT in prostate cancer and thus promote metastasis. We identify that macrophage-secreted cytokines including TNFα act as mediators for potentiating LNCaP cell proliferation in migration assay. Hence, our data indicate a mechanistic insight of how inflammation may contribute to development of prostatic disease at an early stage through increasing cell proliferation and metastasis of LNCaP cells

3D Cell Culture Model for Prostate Cancer Cells to Mimic Inflammatory Microenvironment

The studies on the relationship between inflammation and cancer progression have been mostly carried out with monolayer cell cultures in vitro, which can be insufficient to mimic tumor tissue. Here, we established a three-dimensional (3D) cell culture model of inflammatory microenvironment for prostate cancer cells to better evaluate the role of inflammation in prostate carcinogenesis. Formation of the cell spheroids has been achieved for LNCaP, Du145, LNCaP-104r2 prostate cancer cell lines but not for RWPE1 normal prostate epithelial cell and PC3 by using 3D Petri Dish®. We also showed that cells in inflammatory conditioned media might have a different response based on the culturing method. Overall, we are suggesting that 3D cell culture model can be a useful tool to study molecular alterations on proliferation and migration/invasion of tumor cells related to inflammation