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

Interaction of OGG1 with NKX3.1 due to oxidative DNA damage

WOS:0006294512000038-Oxoguanine DNA glycosylase 1 (OGG1) is a member of base excision repair, responsible for the repair of 8-oxoG base damage induced by reactive oxygen species. As oxidative DNA damage contributes to prostate carcinogenesis, investigating the interaction of OGG1 with prostate-specific proteins, which function in DNA damage and repair mechanisms in prostate cells, is important to determine appropriate therapeutic targets and ultimately support the cancer treatment strategies. Therefore, in this study we investigated the protein-protein interaction of OGG1 with androgen receptor (AR), which is critical for prostate cell proliferation as well as NKX3.1, which is a tumor suppressor protein specific to prostate cells. in addition, S326C, a polymorphic variant of OGG1 formed by a single amino acid change, has been reported in literature to cause a deficiency in repair activity leading OGG1 to be a predisposition factor for prostate cancer. in our immunoprecipitation results, OGG1 was detected to physically interact with NKX3.1 and AR upon increased oxidative DNA damage by menadione treatment. Further, immunofluorescence microscopy results showed that OGG1 localizes in the nuclear speckles at basal and induced level of DNA damage. Although NKX3.1 co-localize with OGG1 in the nucleus, localization of OGG1 was not observed in nuclear speckles in the presence of NKX3.1 possibly due to reduced oxidative DNA damage in NKX3.1 expressing cells. However, reduced physical association of OGG1-S326C variant form in comparison to wild type and further no co-localization of variant form with NKX3.1 was detected supporting the idea that OGG1-S326C variant form contributes to the prostate carcinogenesis

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

Use of Non-steroidal Anti-inflammatory Drugs for Chemoprevention of Inflammation-induced Prostate Cancer

WOS: 000417379400009Objectives: Chronic inflammation has been known as one of the major causes of cancer progression and 25% of cancer cases initiate due to chronic inflammation according to epidemiologic data. It has been determined that chronic inflammation induces carcinogenesis through the abrogation of cell proliferation, apoptosis, and angiogenesis mechanisms. Therefore, it is believed that inhibition of inflammation-induced carcinogenic mechanisms is an efficient therapeutic strategy in drug development studies of cancer chemoprevention. It has also been observed that use of anti-inflammatory drugs reduces the incidence of cancer, and the risk of developing prostate cancer decreases 15-20% with regular use of aspirin and non-steroidal anti-inflammatory drugs (NSAID). Materials and Methods: In this study, we investigated the effects of some clinically used NSAIDs on cellular mechanisms that play a role in inflammation-induced prostate carcinogenesis. Inhibition activities on the nuclear factor kappa-B signaling pathway, which activates tumorigenic mechanisms, as well as alterations on androgen receptor signaling, which regulates the proliferation of prostate cells, were investigated. In addition, protein kinase B (Akt) activation, which is stimulated a the inflammatory microenvironment, was examined. Results: The results showed that anti-inflammatory agents alter the protein levels of androgen receptors as well as tumor suppressor NKX3.1, and might trigger an unexpected increase in Akt((S473)) level, which induces tumorigenesis. Conclusion: It is suggested that inflammatory pathways and prostate carcinogenesis-specific mechanisms should be taken into account for the use of anti-inflammatory drugs for chemoprevention of inflammation-induced prostate cancer.TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [113Z078]; Ege UniversityEge University [2014/BIL/002]This study was supported by TUBITAK with the grant number 113Z078 to BDB and Ege University with the grant number 2014/BIL/002 to BDB

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

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

SIRT1 siRNA-loaded lipid nanoparticles enhanced doxorubicin-induced cell death in prostate cancer cell lines

Most chemotherapeutics induce apoptosis by creating DNA damage in tumor cells. However, high expression of Sirtuin 1 (SIRT1), which is a histone deacetylase activates DNA repair mechanisms leading to the repair of chemotherapeutic-induced DNA damage and subsequently prevention of apoptosis. Therefore, inhibition of SIRT1 is a useful strategy in cancer therapy to achieve a more efficient eradication of tumor cells. In this study, novel lipoplexes composed of 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) containing lipid nanoparticles and SIRT1 siRNA have been formulated by the freeze-drying method. SIRT1 silencing effect has been demonstrated by a 2.5-to 4-fold decrease in mRNA and protein expression levels. The efficiency of the lipoplexes to enhance the activity of doxorubicin, a DNA damage-inducing chemotherapeutic agent, has been studied on prostate cancer cell lines with different p53 expression patterns, namely LNCaP, DU145, and PC3. SIRT1 silencing by lipoplexes enhanced DNA damage recognition and increased the activity of doxorubicin on cancer cell death. These results have shown that SIRT1 siRNA-loaded lipid nanoparticles can enhance the efficiency of doxorubicin on prostate cancer cell lines.Scientific and Technological Research Council of Turkey (TUBITAK) [216S734]This research was supported by Scientific and Technological Research Council of Turkey (TUBITAK) [Grant number: 216S734]

Nutlin3a-Loaded Nanoparticles Show Enhanced Apoptotic Activity on Prostate Cancer Cells

###EgeUn###Escape from apoptosis, one of the characteristic features of cancer cells, is a case that reduces the therapeutic efficacy of apoptosis-inducing molecules used in the cancer treatment. Stabilization of the P53 protein, which is responsible for the regulation of apoptosis mechanism in the cell, is therefore an important therapeutic goal. Nutlin3a inhibits the degradation of the P53 protein, triggers P53-mediated apoptosis in cancer cells and enhances the effectiveness of chemotherapeutics. However, its low aqueous solubility is the major disadvantage when it comes to in vivo administration. In order to facilitate an aqueous formulation of Nutlin3a and to enhance its apoptotic activity on cancer cells, Nutlin3a was encapsulated in solid lipid nanoparticles (SLNs) prepared by Ouzo method. Physicochemical characterization was performed and activity of apoptosis induction on wild-type P53 expressing LNCaP prostate cancer cell line was evaluated. Nutlin3a-loaded cationic solid lipid nanoparticles were found to stabilize functional P53 at protein level. In addition, induction rate of apoptosis by nanoparticles was higher than Nutlin3a solution in DMSO, proving this nanoparticle formulation is a promising candidate for increasing the efficiency of Nutlin3a for P53(+) cancer cases. Thus, it is anticipated that the results will contribute to evaluate the use of lipid-based nanocarriers to enhance the therapeutic potential of small molecules that are important in cancer cure.Ege UniversityEge University [16ECZ031]This research is supported by a grant (16ECZ031) from Ege University Scientific Research Projects Funding to BDB

The redox biology network in cancer pathophysiology and therapeutics

The review pinpoints operational concepts related to the redox biology network applied to the pathophysiology and therapeutics of solid tumors. A sophisticated network of intrinsic and extrinsic cues, integrated in the tumor niche, drives tumorigenesis and tumor progression. Critical mutations and distorted redox signaling pathways orchestrate pathologic events inside cancer cells, resulting in resistance to stress and death signals, aberrant proliferation and efficient repair mechanisms. Additionally, the complex inter-cellular crosstalk within the tumor niche, mediated by cytokines, redox-sensitive danger signals (HMGB1) and exosomes, under the pressure of multiple stresses (oxidative, inflammatory, metabolic), greatly contributes to the malignant phenotype. The tumor-associated inflammatory stress and its suppressive action on the anti-tumor immune response are highlighted. We further emphasize that ROS may act either as supporter or enemy of cancer cells, depending on the context. Oxidative stress-based therapies, such as radiotherapy and photodynamic therapy, take advantage of the cytotoxic face of ROS for killing tumor cells by a non-physiologically sudden, localized and intense oxidative burst. The type of tumor cell death elicited by these therapies is discussed. Therapy outcome depends on the differential sensitivity to oxidative stress of particular tumor cells, such as cancer stem cells, and therefore co-therapies that transiently down-regulate their intrinsic antioxidant system hold great promise. We draw attention on the consequences of the damage signals delivered by oxidative stress-injured cells to neighboring and distant cells, and emphasize the benefits of therapeutically triggered immunologic cell death in metastatic cancer. An integrative approach should be applied when designing therapeutic strategies in cancer, taking into consideration the mutational, metabolic, inflammatory and oxidative status of tumor cells, cellular heterogeneity and the hypoxia map in the tumor niche, along with the adjoining and systemic effects of oxidative stress-based therapies. Keywords: Cancer, Tumor niche, Redox signaling, Radiotherapy, Photodynamic therapy, Bystander and abscopal effect