bims-noxint 2019-03-31 papers

bims-noxint

Biomed News

on NADPH oxidases in tumorigenesis

Issue of 2019–03–31
ten papers selected by
Laia Caja Puigsubira, Uppsala University

Monitoring structural modulation of redox-sensitive proteins in cells with MS-CETSA.
Apoptosis signal-regulating kinase 1 activation by Nox1-derived oxidants is required for TNFα receptor endocytosis.
Reactive Oxygen Species and Cancer: A complex interaction.
The M1 muscarinic acetylcholine receptor subtype is important for retinal neuron survival in aging mice.
Antioxidant Defenses Confer Resistance to High Dose Melphalan in Multiple Myeloma Cells.
The impact of exenatide (a GLP-1 agonist) on markers of inflammation and oxidative stress in normal human astrocytes subjected to various glycemic conditions.
Engagement with tNOX (ENOX2) to Inhibit SIRT1 and Activate p53-Dependent and -Independent Apoptotic Pathways by Novel 4,11-Diaminoanthra[2,3-b]furan-5,10-diones in Hepatocellular Carcinoma Cells.
Palmitic acid-induced autophagy increases reactive oxygen species via the Ca2+/PKCα/NOX4 pathway and impairs endothelial function in human umbilical vein endothelial cells.
MCUR1 facilitates epithelial-mesenchymal transition and metastasis via the mitochondrial calcium dependent ROS/Nrf2/Notch pathway in hepatocellular carcinoma.
Mitochondria and oxidative stress in ovarian endometriosis.

Redox Biol. 2019 Mar 14. pii: S2213-2317(19)30096-5. [Epub ahead of print]24 101168

Monitoring structural modulation of redox-sensitive proteins in cells with MS-CETSA.

Wendi Sun, Lingyun Dai, Han Yu, Brenda Puspita, Tianyun Zhao, Feng Li, Justin L Tan, Yan Ting Lim, Ming Wei Chen, Radoslaw M Sobota, Daniel G Tenen, Nayana Prabhu, Pär Nordlund.

  Reactive oxygen species (ROS) induce different cellular stress responses but can also mediate cellular signaling. Augmented levels of ROS are associated with aging, cancer as well as various metabolic and neurological disorders. ROS can also affect the efficacy and adverse effects of drugs. Although proteins are key mediators of most ROS effects, direct studies of ROS-modulated-protein function in the cellular context are very challenging. Therefore the understanding of specific roles of different proteins in cellular ROS responses is still relatively rudimentary. In the present work we show that Mass Spectrometry-Cellular Thermal Shift Assay (MS-CETSA) can directly monitor ROS and redox modulations of protein structure at the proteome level. By altering ROS levels in cultured human hepatocellular carcinoma cell lysates and intact cells, we detected CETSA responses in many proteins known to be redox sensitive, and also revealed novel candidate ROS sensitive proteins. Studies in intact cells treated with hydrogen peroxide and sulfasalazine, a ROS modulating drug, identified not only proteins that are directly modified, but also proteins reporting on downstream cellular effects. Comprehensive changes are seen on rate-limiting proteins regulating key cellular processes, including known redox control systems, protein degradation, epigenetic control and protein translational processes. Interestingly, concerted shifts on ATP-binding proteins revealed redox-induced modulation of ATP levels, which likely control many cellular processes. Collectively, these studies establish CETSA as a novel method for cellular studies of redox modulations of proteins, which implicated in a wide range of processes and for the discovery of CETSA-based biomarkers reporting on the efficacy as well as adverse effects of drugs.

Keywords:  ATP levels; Cellular thermal shift assay; Glutathione; Quantitative proteomics; Reactive cysteines; Reactive oxygen species; Sulfasalazine

DOI:  https://doi.org/10.1016/j.redox.2019.101168
Am J Physiol Heart Circ Physiol. 2019 Mar 29.

Apoptosis signal-regulating kinase 1 activation by Nox1-derived oxidants is required for TNFα receptor endocytosis.

Hyehun Choi, Ryan J Stark, Benjamin S Raja, Anna Dikalova, Fred S Lamb.

  Tumor necrosis factor-α (TNFα) is pro-inflammatory cytokine that is closely linked to the development of cardiovascular disease. TNFα activates NADPH oxidase 1 (Nox1), and reactive oxygen species (ROS) including superoxide (O2•-) production extracellularly are required for subsequent signaling in vascular smooth muscle cells (VSMC). Apoptosis signal-regulating kinase 1 (ASK1) is a MAPKKK that is activated by oxidation of associated thioredoxin. The role of ASK1 in Nox1-mediated signaling by TNFα is poorly defined. We hypothesized that ASK1 is required for TNFα receptor endocytosis and subsequent inflammatory TNFα signaling. We employed a knockdown strategy to explore the role of ASK1 in TNFα signaling in VSMCs. Small interfering RNA (siRNA) targeting ASK1 had no effect on TNFα-induced extracellular O2•- production. However, siASK1 inhibited receptor endocytosis, as well as phosphorylation of two endocytosis-related proteins, dynamin1 and caveolin1. Intracellular O2•- production was subsequently reduced, as were other inflammatory signaling steps including; NF-κB activation, IL-6 production, iNOS and VCAM expression and VSMC proliferation. Prolonged exposure to TNFα (24 hours) increased TNFR subtype 1 and 2 expression and these effects were also attenuated by siASK1. ASK1 co-immunoprecipitated with both Nox1 and the LRRC8A anion channel, two essential components of the TNFR1 signaling complex. Activation of ASK1 by autophosphorylation at Thr845 occurs following thioredoxin dissociation and this requires the presence of Nox1. Thus, Nox1 is part of the multi-protein ASK1 signaling complex. In response to TNFα, ASK1 is activated by Nox1-derived oxidants and this plays a critical role in translating these ROS into a physiologic response in VSMCs.

Keywords:  Apoptosis signal-regulating kinase 1; NADPH oxidase 1; Tumor Necrosis Factor-α; endocytosis; vascular smooth muscle cells

DOI:  https://doi.org/10.1152/ajpheart.00741.2018
Cancer Lett. 2019 Mar 21. pii: S0304-3835(19)30176-4. [Epub ahead of print]

Reactive Oxygen Species and Cancer: A complex interaction.

Saikolappan Sankaralingam, Binod Kumar, Gauri Shishodia, Sweaty Koul, Hari Koul.

  Elevated levels of Reactive Oxygen Species (ROS), increased antioxidant ability and the maintenance of redox homeostasis can cumulatively contribute to tumor progression and metastasis. The sources and the role of ROS in a heterogeneous tumor microenvironment can vary at different stages of tumor: initiation, development, and progression, thus making it a complex subject. In this review, we have summarized the sources of ROS generation in cancer cells, its role in the tumor microenvironment, the possible functions of ROS and its important scavenger systems in tumor progression with special emphasis on solid tumors.

Keywords:  Cancer; Hypoxia Re-oxygenation; ROS

DOI:  https://doi.org/10.1016/j.canlet.2019.03.020
Sci Rep. 2019 Mar 26. 9(1): 5222

The M1 muscarinic acetylcholine receptor subtype is important for retinal neuron survival in aging mice.

Panagiotis Laspas, Mayagozel B Zhutdieva, Christoph Brochhausen, Aytan Musayeva, Jenia Kouchek Zadeh, Norbert Pfeiffer, Ning Xia, Huige Li, Juergen Wess, Adrian Gericke.

Muscarinic acetylcholine receptors have been implicated as potential neuroprotective targets for glaucoma. We tested the hypothesis that the lack of a single muscarinic receptor subtype leads to age-dependent neuron reduction in the retinal ganglion cell layer. Mice with targeted disruption of single muscarinic acetylcholine receptor subtype genes (M1 to M5) and wild-type controls were examined at two age categories, 5 and 15 months, respectively. We found no differences in intraocular pressure between individual mouse groups. Remarkably, in 15-month-old mice devoid of the M1 receptor, neuron number in the retinal ganglion cell layer and axon number in the optic nerve were markedly reduced. Moreover, mRNA expression for the prooxidative enzyme, NOX2, was increased, while mRNA expression for the antioxidative enzymes, SOD1, GPx1 and HO-1, was reduced in aged M1 receptor-deficient mice compared to age-matched wild-type mice. In line with these findings, the reactive oxygen species level was also elevated in the retinal ganglion cell layer of aged M1 receptor-deficient mice. In conclusion, M1 receptor deficiency results in retinal ganglion cell loss in aged mice via involvement of oxidative stress. Based on these findings, activation of M1 receptor signaling may become therapeutically useful to promote retinal ganglion cell survival.

DOI: https://doi.org/10.1038/s41598-019-41425-5
Cancers (Basel). 2019 Mar 28. pii: E439. [Epub ahead of print]11(4):

Antioxidant Defenses Confer Resistance to High Dose Melphalan in Multiple Myeloma Cells.

Claire Gourzones, Céline Bellanger, Sylvain Lamure, Ouissem Karmous Gadacha, Elvira Garcia De Paco, Laure Vincent, Guillaume Cartron, Bernard Klein, Jérôme Moreaux.

   BACKGROUND: Multiple myeloma (MM) is the second most common hematological cancer after lymphoma. It is characterized by the accumulation of clonal malignant plasma cells within the bone marrow. The development of drug resistance remains a major problem for effective treatment of MM. Understand the mechanisms underlying drug resistance in MM is a focal point to improve MM treatment.
METHODS: In the current study, we analyzed further the role of redox imbalance induction in melphalan-induced toxicity both in human myeloma cell lines (HMCLs) and primary myeloma cells from patients.
RESULTS: We developed an in-vitro model of short-term resistance to high-dose melphalan and identified that pretreatment with physiological concentration of GSH protects HMCLs from melphalan-induced cell cycle arrest and cytotoxicity. We validated these results using primary MM cells from patients co-cultured with their bone marrow microenvironment. GSH did not affect the ability of melphalan to induce DNA damages in MM cells. Interestingly, melphalan induced reactive oxygen species, a significant decrease in GSH concentration, protein and lipd oxydation together with NRF2 (NF-E2-related factor 2) pathway activation.
CONCLUSIONS: Our data demonstrate that antioxidant defenses confers resistance to high dose melphalan in MM cells, supporting that redox status in MM cells could be determinant for patients' response to melphalan.

Keywords:  GSH; NRF2; drug resistance; melphalan; multiple myeloma; reactive oxygen species

DOI:  https://doi.org/10.3390/cancers11040439
Exp Ther Med. 2019 Apr;17(4): 2861-2869

The impact of exenatide (a GLP-1 agonist) on markers of inflammation and oxidative stress in normal human astrocytes subjected to various glycemic conditions.

Łukasz Bułdak, Grzegorz Machnik, Estera Skudrzyk, Aleksandra Bołdys, Bogusław Okopień.

  GLP-1 agonists such as exenatide and liraglutide are novel drugs for the treatment of diabetes and obesity. While improvements in glycemic control can rely on an incretin effect, the mechanisms behind the loss of weight following therapy have yet to be completely elucidated, and seem to be associated with alterations in eating habits, resulting from changes in cytokines e.g. interleukin 1β (IL-1β) and oxidative signaling in the central nervous system (CNS). Increased levels of IL-1β and reactive oxygen species have been demonstrated to exert anorexigenic properties, and astrocytes appear to actively participate in maintaining the integrity of the CNS, which includes the paracrine secretion of inflammatory cytokines and involvement in the redox status. Therefore, the present study decided to explore the influence of exenatide [a glucagon-like peptide 1 (GLP-1 agonist)] on inflammatory and oxidative stress markers in cultured human astrocytes as a potential target for weight reduction therapies. In an experimental setting, normal human astrocytes were subjected to various glycemic conditions, including 40 mg/dl-hypoglycemic, 100 mg/dl-normoglycemic and 400 mg/dl-hyperglycemic, and exenatide, which is a GLP-1 agonist. The involvement of intracellular signaling by a protein kinase A (PKA) in the action of exenatide was estimated using a specific PKA inhibitor-PKI (14-22). The expression levels of IL-1β, nuclear factor kappa κB (NFκB), glial-fibrillary acidic protein (GFAP), p22 NADPH oxidase, glutathione peroxidase, catalase, superoxide dismutase 1, and reactive oxidative species were measured. The present study demonstrated that varying glucose concentrations in the culture media did not affect the protein expression or the level of reactive oxygen species. Conversely, exenatide led to an increase in IL-1β in normoglycemic culture conditions, which was accompanied by the increased expression of p22, glutathione peroxidase and the reduced expression of GFAP. Changes in the expression of IL-1β and p22 were dependent on the activation of PKA. The present study concluded that exenatide predominantly affected astrocytes in normoglycemic conditions, and hypothesize that this impact demonstrated one of novel mechanisms associated with astrocyte signaling that may contribute to weight loss.

Keywords:  Glucagon-like peptide-1; astrocytes; exenatide; interleukin-1β; oxidative stress

DOI:  https://doi.org/10.3892/etm.2019.7245
Cancers (Basel). 2019 Mar 24. pii: E420. [Epub ahead of print]11(3):

Engagement with tNOX (ENOX2) to Inhibit SIRT1 and Activate p53-Dependent and -Independent Apoptotic Pathways by Novel 4,11-Diaminoanthra[2,3-b]furan-5,10-diones in Hepatocellular Carcinoma Cells.

Chia-Yang Lin, Atikul Islam, Claire J Su, Alexander S Tikhomirov, Andrey E Shchekotikhin, Show-Mei Chuang, Pin Ju Chueh, Yao Li Chen.

  Hepatocellular carcinoma (HCC) is the most frequent primary malignancy of the liver and is among the top three causes of cancer-associated death worldwide. However, the clinical use of chemotherapy for HCC has been limited by various challenges, emphasizing the urgent need for novel agents with improved anticancer properties. We recently synthesized and characterized a series of 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives that exhibit potent apoptotic activity against an array of cancer cell lines, including variants with multidrug resistance. Their effect on liver cancer cells, however, was unknown. Here, we investigated three selected 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives (compounds 1⁻3) for their cytotoxicity and the underlying molecular mechanisms in wild-type or p53-deficient HCC cells. Cytotoxicity was determined by WST-1 assays and cell impedance measurements and apoptosis was analyzed by flow cytometry. The interaction between compounds and tumor-associated NADH oxidase (tNOX, ENOX2) was studied by cellular thermal shift assay (CETSA). We found that compound 1 and 2 induced significant cytotoxicity in both HepG2 and Hep3B lines. CETSA revealed that compounds 1 and 2 directly engaged with tNOX, leading to a decrease in the cellular NAD⁺/NADH ratio. This decreased the NAD⁺-dependent activity of Sirtuin 1 (SIRT1) deacetylase. In p53-wild-type HepG2 cells, p53 acetylation/activation was enhanced, possibly due to the reduction in SIRT1 activity, and apoptosis was observed. In p53-deficient Hep3B cells, the reduction in SIRT1 activity increased the acetylation of c-Myc, thereby reactivating the TRAIL pathway and, ultimately leading to apoptosis. These compounds thus trigger apoptosis in both cell types, but via different pathways. Taken together, our data show that derivatives 1 and 2 of 4,11-diaminoanthra[2,3-b]furan-5,10-diones engage with tNOX and inhibit its oxidase activity. This results in cytotoxicity via apoptosis through tNOX-SIRT1 axis to enhance the acetylation of p53 or c-Myc in HCC cells, depending on their p53 status.

Keywords:  anthraquinone derivatives; apoptosis; c-Myc; hepatocellular carcinoma; p53; protein acetylation; sirtuin 1 (SIRT1); tumor-associated NADH oxidase (tNOX or ENOX2)

DOI:  https://doi.org/10.3390/cancers11030420
Exp Ther Med. 2019 Apr;17(4): 2425-2432

Palmitic acid-induced autophagy increases reactive oxygen species via the Ca2+/PKCα/NOX4 pathway and impairs endothelial function in human umbilical vein endothelial cells.

Pan Chen, Hengdao Liu, Hong Xiang, Jianda Zhou, Zhengpeng Zeng, Ruifang Chen, Shaoli Zhao, Jie Xiao, Zhihao Shu, Shuhua Chen, Hongwei Lu.

  It is well known that the lipotoxic mechanism of palmitic acid (PA), a main constituent of triglyceride, is dependent on reactive oxygen species (ROS). Recently, it has also been reported that PA is an autophagy inducer. However, the causal association and underlying mechanism of induced autophagy and ROS in PA toxicity remain unclear. The present study demonstrates for the first time that PA-induced autophagy enhances ROS generation via activating the calcium ion/protein kinase Cα/nicotinamide adenine dinucleotide phosphate oxidase 4 (Ca2+/PKCα/NOX4) pathway in human umbilical vein endothelial cells (HUVECs). It was revealed that PA treatment resulted in a significant increase in ROS generation and autophagic activity, leading to endothelial dysfunction as indicated by downregulated nitric oxide synthesis, decreased capillary-like structure formation and damaged cell repair capability. Furthermore, PA effectively activated the Ca2+/PKCα/NOX4 pathway, which is indicative of upregulated cytosolic Ca2+ levels, activated PKCα and increased NOX4 protein expression. 3-Methyladenine was then used to inhibit autophagy, which significantly reduced PA-induced ROS generation and blocked the Ca2+/PKCα/NOX4 pathway. The endothelial dysfunction caused by PA was ameliorated by downregulating ROS generation using a NOX4 inhibitor. In conclusion, PA-induced autophagy contributes to endothelial dysfunction by increasing oxidative stress via the Ca2+/PKCα/NOX4 pathway in HUVECs.

Keywords:  Endothelial cell dysfunction; autophagy; lipotoxicy; palmitic acid; reactive oxygen species

DOI:  https://doi.org/10.3892/etm.2019.7269
J Exp Clin Cancer Res. 2019 Mar 25. 38(1): 136

MCUR1 facilitates epithelial-mesenchymal transition and metastasis via the mitochondrial calcium dependent ROS/Nrf2/Notch pathway in hepatocellular carcinoma.

Mingpeng Jin, Jiaojiao Wang, Xiaoying Ji, Haiyan Cao, Jianjun Zhu, Yibing Chen, Jin Yang, Zheng Zhao, Tingting Ren, Jinliang Xing.

   BACKGROUND: Mitochondrial Ca2+ plays a critical role in tumorigenesis, including cell proliferation and metastasis. Mitochondrial calcium uniporter regulator 1 (MCUR1) has been shown to be frequently upregulated in HCC and promote cancer cell survival. However, whether MCUR1 is involved in the metastasis of HCC and its underlying mechanisms remain unknown.
METHODS: The effect of MCUR1 expression on epithelial-mesenchymal transition (EMT) in HCC cells was first evaluated by immunofluorescent staining and Western blot. Then, in vitro invasion and in vivo metastasis assays were used to evaluate the function of MCUR1 in HCC metastasis. The underlying mechanism has also been explored by investigating the effect of MCUR1 on ROS/Nrf2/Notch1 pathway.
RESULTS: MCUR1 expression was significantly higher in HCC with metastasis and associated with tumor progression. MCUR1 promoted in vitro invasion and in vivo metastasis of HCC cells by promoting EMT via Snail. Mechanistically, MCUR1-mediated mitochondrial Ca2+ signaling promoted the EMT of HCC cells by activating ROS/Nrf2/Notch1 pathway. Inhibition of ROS production, mitochondrial Ca2+ uptake, Nrf2 expression or Notch1 activity significantly suppressed MCUR1-induced EMT of HCC cells. In addition, treatment with the mitochondrial Ca2+-buffering protein parvalbumin significantly inhibited ROS/Nrf2/Notch pathway and MCUR1-induced EMT and HCC metastasis.
CONCLUSIONS: Our study provides evidence supporting a metastasis-promoting role for MCUR1-dependent mitochondrial Ca2+ uptake in HCC. Our findings suggest that MCUR1 may be a potential therapeutic target for HCC treatment.

Keywords:  EMT; Hepatocellular carcinoma; Metastasis; Mitochondrial calcium uniporter regulator 1; Notch 1

DOI:  https://doi.org/10.1186/s13046-019-1135-x
Free Radic Biol Med. 2019 Mar 26. pii: S0891-5849(19)30264-3. [Epub ahead of print]

Mitochondria and oxidative stress in ovarian endometriosis.

Chaolu Chen, Yong Zhou, Changchang Hu, Yinfeng Wang, Zhuqing Yan, Zhi Li, Ruijin Wu.

  Endometriosis is associated with inflammatory reaction, and reactive oxidative species (ROS) are highly pro-inflammatory factors. Mitochondria are responsible for the production of ROS and energy. However, little is known about how mitochondria regulate ROS generation and energy metabolism in endometriosis. In our study, we investigated mitochondrial structure and function of ectopic endometrial stromal cells (ESCs) in ovarian endometriosis. We found mitochondria in ectopic ESCs generated more ROS and energy than controlled groups. Mitochondrial superoxide dismutase (SOD2), as an antioxidant enzyme, was found highly expressed in ectopic endometrium compared with normal endometrium. Due to its antioxidant role, SOD2 promoted the development of endometriosis by maintaining functional mitochondria to support high energetic metabolism of ectopic ESCs. We also showed that SOD2 promoted cell proliferation and migration in ovarian endometriosis. Inhibiting SOD2 expression reduced proliferation and migration of ectopic ESCS, and increased cell apoptosis. Therefore, understanding the role of mitochondrial dysfunction and SOD2 in ovarian endometriosis may provide new strategies to treat this disease.

Keywords:  Mitochondria; Mitochondrial superoxide dismutase; Ovarian endometriosis; Oxidative stress; Reactive oxidative species

DOI:  https://doi.org/10.1016/j.freeradbiomed.2019.03.027