Oxygen Species Production Research Articles

Effects of platinum-coexisting dopamine with X-ray irradiation upon human glioblastoma cell proliferation.

In brain tumors, neurotransmitters and platinum drugs may have some interaction, but their role in radiation therapy remains unclear. We investigated the effects of dopamine in combination with platinum on human glioblastoma U-251MG cells upon X-ray irradiation, comparing with L-DOPA, 2-phenylethylamine and temozolomide. Cell proliferation of U-251MG cells was prominently decreased by dopamine in combination with 10μM platinum upon 4Gy of X-ray irradiation, accompanied with intracellular reactive oxygen species generation and mitotic catastrophe. Platinum alone did not increase intracellular reactive oxygen species. On the other hand, L-DOPA in combination with platinum did not decrease cell proliferation regardless of X-ray irradiation. It was clearly shown that 2-phenylethylamine did not suppress cell proliferation as compared to dopamine. Temozolomide decreased cell proliferation in a dose-dependent manner upon X-ray irradiation. However, the combined administration of temozolomide and platinum did not further decrease cell proliferation. The platinum nanoparticles were gradually taken up by cells after administration as determined by ICP analysis. Our results suggest that platinum-coexisting dopamine led cells to mitotic catastrophe due to increased production of intracellular reactive oxygen species which was boosted by X-ray and platinum-catalyzed auto-oxidation of dopamine, and thereby cell proliferation was suppressed. In addition, normal human fibroblast OUMS-36T-1 cells were subjected to experiments. Regarding the effect of the combined administration of dopamine and platinum on each cell which was exposed to X-ray, cell proliferation was decreased in U-251MG cells by the combined administration of platinum, whereas that was not decreased in OUMS-36T-1 cells. This provides one basic insight into the effects of dopamine in combined with platinum on radiation therapy for glioblastoma.

P–004 Effect of varicocelectomy on sperm DNA fragmentation rates in infertile men with clinical varicocele: a systematic review and meta-analysis

Abstract Study question Does varicocelectomy improve sperm DNA quality in men with infertility and clinically detected varicoceles? Summary answer Varicocelectomy reduces sperm DNA fragmentation (SDF) rates in infertile men with clinical varicocele. What is known already Varicocele has been linked to male infertility through various non-mutually exclusive mechanisms, including an increase in reactive oxygen species (ROS) production that may lead to sperm DNA damage. Damage to sperm DNA may result in longer time-to-pregnancy, unexplained infertility, recurrent pregnancy loss, and failed intrauterine insemination or in vitro fertilization/intracytoplasmic sperm injection. Therefore, interventions aimed at decreasing SDF rates, including varicocele repair, have been explored to improve fertility and pregnancy outcomes potentially, either by natural conception or using medically assisted reproduction. Study design, size, duration Systematic review and meta-analysis Participants/materials, setting, methods We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Our systematic search included PubMed/Medline, EMBASE, Scielo, and Google Scholar to identify all relevant studies written in English and published from inception until October 2020. Inclusion criteria were studies comparing SDF rates before and after varicocelectomy in infertile men with clinical varicocele. Articles were included if the following SDF assays were utilized: SCSA, TUNEL, SCD test, or alkaline Comet. Main results and the role of chance Thirteen studies fulfilled the inclusion criteria and were selected for the analysis. The estimated weighted mean difference of SDF rates after varicocelectomy was –6.58% (13 studies, 95% CI –8.33%, –4.84%; I2=90% p < 0.0001). Subgroup analysis revealed a significant decrease in SDF rates using SCSA (eight studies, WMD –6.80%, 95% CI –9.31%, –4.28%; I2=89%, p < 0.0001), and TUNEL (three studies, WMD –4.86%, 95% CI –7.38%, –2.34%; I2=89%, p < 0.0001). The test for subgroup difference revealed that pooled results were conservative using the above SDF assays. Comet and SCD tests were used in only one study each; thus, a meta-analysis was not applicable. The studies were further categorized by the surgical technique (microsurgical versus non-microsurgical). This subgroup analysis showed a significant decrease in SDF rates using microsurgical technique (10 studies, WMD –6.70%, 95% CI –9.04%, –4.37%; I2=91%, p < 0.0001). After varicocelectomy, SDF rates were also decreased when non-microsurgical approaches were used, albeit the effect was not statistically significant (2 studies, WMD –6.84%, 95% CI –10.05%, 1.38%; I2=86%) (Figure 3). The heterogeneity was not materially affected by performing analyses by the above subgroups, suggesting that the SDF assay and surgical technique do not explain the inconsistency in the treatment effect across primary studies. Limitations, reasons for caution There were no randomized controlled trials comparing varicocelectomy to placebo for alleviating SDF levels. Heterogeneity was high, which may be explained by the low number of included studies. Pregnancy data are not available in most studies, thus the impact of reduced SDF after varicocelectomy on pregnancy rates unclear. Wider implications of the findings: Our study indicates a positive association between varicocelectomy and reduced postoperative SDF rates in men with clinical varicocele and infertility, independentetly of the assays used to measure SDF. These findings may help counsel and manage infertile men with varicocele and high SDF levels. Trial registration number Not applicable

Hemosiderin, a possible biomarker for sudep?

Epilepsy is one of the neurological diseases of complex etiology that affects around 50 million people worldwide and is characterized by abnormal electrical activity and recurrent seizures. Uncontrolled generalized repetitive tonic-clonic seizures (GTCS) are the main causes of unexpected sudden death in epilepsy (SUDEP). Hypoxic stress induced by seizure results in neurocardiogenic dysfunctions, including iron overload and cardiomyopathy (IOC) which is related to severe lipid peroxidation caused by the production of reactive oxygen species (ROS). ROS induces recurrent seizure activity, favoring the overexpression of P glycoprotein (P-gp) in the heart. P-gp plays a depolarizing role in cardiomyocyte membranes and potassium (Kir) channels control cellular excitability regarding the repolarization of the cardiac action potential. All these events result in a possible appearance of severe bradycardia and fatal arrhythmia. Several studies have sought evidence for different possible biomarkers for potential prediction of the risk of SUDEP avoiding its fatal outcome.

Regulation of reactive oxygen species in the pathogenesis of matrix vesicles induced calcification of recipient vascular smooth muscle cells.

Increased oxidative stress is associated with vascular calcification in patients with chronic kidney disease (CKD). We have previously demonstrated that cellular-derived matrix vesicles (MV), but not media-derived MV, are endocytosed in the presence of phosphorus by recipient normal rat vascular smooth muscle cells (VSMC) and induce calcification through ERK1/2 and [Ca2+]i signaling. We hypothesized that these changes were mediated by increased reactive oxygen species (ROS) production. MV were co-cultured with recipient VSMC in the presence of high phosphorus and ROS production and cell signaling assessed. The results demonstrated MV endocytosis led to increased ROS production in recipient VSMC with no increase in mitochondrial oxygen consumption or oxidative phosphorylation (OXPHOS), indicating the ROS was not from the mitochondria. The use of inhibitors demonstrated that endocytosis of these MV by VSMC led to a signaling cascade in the cytoplasm beginning with ERK1/2 signaling, then increased [Ca2+]i and stimulation of ROS production, mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)1/4. Media-derived MV did not induce this cascade, indicating endocytosis itself was not a factor. Furthermore, inhibition of either ERK1/2 activation or [Ca2+]i reduced vascular calcification. We conclude that endocytosis of pro-mineralizing MV can induce a series of signaling events in normal VSMC that culminate in generation of ROS via activation of NOX1/4. Understanding these pathways will allow the development of future targeted therapeutics.

Effect of Aerobic Training and High-fat Diet on Enos and Ros in Testicular Tissue of Juvenile Male Rats

Background and Objectives: This study aimed to determine the effect of a course of aerobic exercise with a high-fat diet on eNOS and ROS in testicular tissue of adolescent male rats. Subjects and Methods A total of 40 adolescent male rats (30 days old) were randomized in the following groups: normal diet control, normal diet training, high fat diet control, and high-fat diet training. The high-fat diet rats were under a high-fat regimen (5.817 kcal/g) for 30 days, and then a normal fat diet (3.801 kcal/g) was continued after the 60th day of birth. Aerobic training was conducted for four weeks included three training sessions from the 70th to 98th days of life. Results The results showed that the amount of ROS in the testicular tissue of male mice was higher only in the high-fat diet group. Also, there was no significant difference between the groups regarding eNOS testicular tissue in male mice. Conclusion A high-fat diet increases the production of reactive oxygen species in testicular tissue and is not affected by aerobic exercise. Also, neither exercise nor a high-fat diet had any effect on testicular eNOS. However, due to the limitations of this study and no evidence in this field, further studies are needed on cell phenotype, sperm fate, and identification of pathways involved in the occurrence of oxidative stress and subsequent effects of eNOS activation in testicular tissue in response to exercise and obesity.

Comparative outcomes of exposing human liver and kidney cell lines to tungstate and molybdate

Tungsten has no known function in humans and is a relatively new contaminant, whereas molybdenum, its congener in the periodic table, is a nutritionally essential element. In addition to early studies on molybdosis in ruminants, their toxic effects in the form of tungstate and molybdate have been addressed primarily in rodents and are predominantly mediated by inducing oxidative stress in various tissues. The purpose of this study was to evaluate the differences between tungstate and molybdate in human liver (HepG2) and kidney (HEK293) cell lines in terms of retention in cells, effect on reactive oxygen species, and activities of xanthine oxidase and phosphatases. The cell lines were exposed to tungstate or molybdate (1 µM to 10 mM) for 24 h, lysed and analyzed for the above biochemical parameters. Despite the chemical similarity of the two anions, cell-specific differential effects were observed. At all concentrations, tungstate was retained more in HEK293 cells while molybdate was retained more in HepG2 cells. HepG2 cells were more sensitive to tungstate than molybdate, showing reduced viability at concentrations as low as 10 µM. Exposure to either anion resulted in the inhibition of protein tyrosine phosphatases at 1 mM and an increased production of reactive oxygen species (ROS) at 100 µM despite their inhibition of the ROS-producing molybdenum enzyme xanthine oxidase. In conclusion, the results indicate that excess of nutritionally essential molybdate or non-essential tungstate causes toxicity by affecting ROS- and phosphorylation-dependent signaling pathways and ensuing gene expression.

Cinaciguat Prevents Postnatal Closure of Ductus Arteriosus by Vasodilation and Anti-remodeling in Neonatal Rats.

Closure of the ductus arteriosus (DA) involves vasoconstriction and vascular remodeling. Cinaciguat, a soluble guanylyl cyclase (sGC) activator, was reported with vasodilatory and anti-remodeling effects on pulmonary hypertensive vessels. However, its effects on DA are not understood. Therefore, we investigated whether cinaciguat regulated DA patency and examined its underlying mechanisms. In vivo, we found that cinaciguat (10 mg/kg, i.p. at birth) prevented DA closure at 2 h after birth with luminal patency and attenuated intimal thickening. These anti-remodeling effects were associated with enhanced expression of cyclic guanosine monophosphate (cGMP) in DA. Ex vivo, cinaciguat dilated oxygen-induced DA constriction dose-dependently. Such vasodilatory effect was blunted by KT-5823, a PKG inhibitor. In DA smooth muscle cells (DASMCs), we further showed that cinaciguat inhibited angiotensin II (Ang II)-induced proliferation and migration of DASMCs. In addition, cinaciguat inhibited Ang II-induced mitochondrial reactive oxygen species (ROS) production. Finally, Ang II-activated MAPKs and Akt were also inhibited by cinaciguat. In conclusion, cinaciguat prevents postnatal DA closure by vasodilation and anti-remodeling through the cGMP/PKG pathway. The mechanisms underlying anti-remodeling effects include anti-proliferation and anti-migration, with attenuation of mitochondrial ROS production, MAPKs, and Akt signaling. Thus, this study implicates that sGC activation may be a promising novel strategy to regulate DA patency.

Host–pathogen interaction between macrophage co-cultures with Staphylococcus aureus biofilms

The ability of Staphylococcus aureus to form biofilms is an important virulence factor. During the infectious process, the interaction between biofilms and immune cells is determinant; however, the properties that make biofilms resistant to the immune system are not well characterized. In order to better understand this, we evaluated the in vitro interaction of macrophages during the early stages of S. aureus biofilm formation. Biofilm formation was evaluated by crystal violet staining, light microscopy, and confocal scanning laser microscopy. Furthermore, different activation on L-arginine pathways such as nitric oxide (NO•) release and the arginase, the production of reactive oxygen species (ROS), the total oxidative stress response (OSR), and levels of cytokine liberation, were determined. Our findings show that the interaction between biofilms and macrophages results in stimuli for catabolism of L-arginine via arginase, but not for NO•, an increase of ROS production, and activation of the non-enzymatic OSR. We also observed the production of IL-6, but not of TNFα o IL-10 in these co-cultures. These results contribute to a better understanding of host-pathogen interactions and suggest that biofilms increase resistance against immune cell mechanisms, a phenomenon that could contribute to the ability of S. aureus biofilms to establish mature biofilms.

Protective Effects of Maclurin against Benzo[a]pyrene via Aryl Hydrocarbon Receptor and Nuclear Factor Erythroid 2-Related Factor 2 Targeting.

Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon formed during the incomplete combustion of organic matter, has harmful effects. Therefore, much research is ongoing to develop agents that can mitigate the effects of B[a]P. The aim of this study was to examine the effect of maclurin, one component of the branches of Morus alba L., on the B[a]P-induced effects in HaCaT cells, a human keratinocyte cell line. Maclurin treatment inhibited aryl hydrocarbon receptor (AHR) signaling as evidenced by reduced xenobiotic response element (XRE) reporter activity, decreased expression of cytochrome P450 1A1 (CYP1A1), and reduced nuclear translocation of AHR. The B[a]P-induced dissociation of AHR from AHR-interacting protein (AIP) was suppressed by maclurin. Maclurin also inhibited the production of intracellular reactive oxygen species (ROS) induced by B[a]P. In addition, the antioxidant property of maclurin itself was demonstrated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Furthermore, maclurin activated antioxidant response element (ARE) signaling through enhancement of ARE luciferase reporter activity and the expression of ARE-dependent genes including nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1). Nrf2 activation and its nuclear translocation were promoted by maclurin through p38 MAPK activation. These data indicate that maclurin had antagonistic activity against B[a]P effects through activation of Nrf2-mediated signaling and inhibition of AHR signaling and, suggesting its potential in protecting from harmful B[a]P-containing pollutants.

Distinct Signaling Pathways Distinguish in vivo From in vitro Growth in Murine Ovarian Follicle Activation and Maturation.

Women with cancer and low ovarian reserves face serious challenges in infertility treatment. Ovarian tissue cryopreservation is currently used for such patients to preserve fertility. One major challenge is the activation of dormant ovarian follicles, which is hampered by our limited biological understanding of molecular determinants that activate dormant follicles and help maintain healthy follicles during growth. Here, we investigated the transcriptomes of oocytes isolated from dormant (primordial) and activated (primary) follicles under in vivo and in vitro conditions. We compared the biological relevance of the initial molecular markers of mature metaphase II (MII) oocytes developed in vivo or in vitro. The expression levels of genes involved in the cell cycle, signal transduction, and Wnt signaling were highly enriched in oocytes from primary follicles and MII oocytes. Interestingly, we detected strong downregulation of the expression of genes involved in mitochondrial and reactive oxygen species (ROS) production in oocytes from primordial follicles, in contrast to oocytes from primary follicles and MII oocytes. Our results showed a dynamic pattern in mitochondrial and ROS production-related genes, emphasizing their important role(s) in primordial follicle activation and oocyte maturation. The transcriptome of MII oocytes showed a major divergence from that of oocytes of primordial and primary follicles.

ZBED6 counteracts high-fat diet-induced glucose intolerance by maintaining beta cell area and reducing excess mitochondrial activation

Aims/hypothesisZBED6 (zinc finger, BED-type containing 6) is known to regulate muscle mass by suppression of Igf2 gene transcription. In insulin-producing cell lines, ZBED6 maintains proliferative capacity at the expense of differentiation and beta cell function. The aim was to study the impact of Zbed6 knockout on beta cell function and glucose tolerance in C57BL/6 mice.MethodsBeta cell area and proliferation were determined in Zbed6 knockout mice using immunohistochemical analysis. Muscle and fat distribution were assessed using micro-computed tomography. Islet gene expression was assessed by RNA sequencing. Effects of a high-fat diet were analysed by glucose tolerance and insulin tolerance tests. ZBED6 was overexpressed in EndoC-βH1 cells and human islet cells using an adenoviral vector. Beta cell cell-cycle analysis, insulin release and mitochondrial function were studied in vitro using propidium iodide staining and flow cytometry, ELISA, the Seahorse technique, and the fluorescent probes JC-1 and MitoSox.ResultsIslets from Zbed6 knockout mice showed lowered expression of the cell cycle gene Pttg1, decreased beta cell proliferation and decreased beta cell area, which occurred independently from ZBED6 effects on Igf2 gene expression. Zbed6 knockout mice, but not wild-type mice, developed glucose intolerance when given a high-fat diet. The high-fat diet Zbed6 knockout islets displayed upregulated expression of oxidative phosphorylation genes and genes associated with beta cell differentiation. In vitro, ZBED6 overexpression resulted in increased EndoC-βH1 cell proliferation and a reduced glucose-stimulated insulin release in human islets. ZBED6 also reduced mitochondrial JC-1 J-aggregate formation, mitochondrial oxygen consumption rates (OCR) and mitochondrial reactive oxygen species (ROS) production, both at basal and palmitate + high glucose-stimulated conditions. ZBED6-induced inhibition of OCR was not rescued by IGF2 addition. ZBED6 reduced levels of the mitochondrial regulator PPAR-γ related coactivator 1 protein (PRC) and bound its promoter/enhancer region. Knockdown of PRC resulted in a lowered OCR.Conclusions/interpretationIt is concluded that ZBED6 is required for normal beta cell replication and also limits excessive beta cell mitochondrial activation in response to an increased functional demand. ZBED6 may act, at least in part, by restricting PRC-mediated mitochondrial activation/ROS production, which may lead to protection against beta cell dysfunction and glucose intolerance in vivo.Graphical abstract

Induction of tolerance to cryogenic protocols in Solanum tuberosum by salicylic acid is mediated by enzymatic antioxidant activity and hydrogen peroxide

ABSTRACT Two applications of cryogenic protocols are cryotherapy and cryopreservation for virus cleaning and germplasm storage. They are based on exposing explants to ultra-low temperature (−196°C) in liquid nitrogen. Ultra-low temperature in cryogenics implies abiotic stress, leading to the production of reactive oxygen species (ROS), which are the main reason for the low survival and death of explants. Hydrogen peroxide (H2O2) is an important ROS produced by stress. Enzymatic antioxidants like catalase (CAT) and peroxidase (POX) are essential for avoiding damage due to H2O2, which is a primary signal against stress. Salicylic acid (SA) is involved in tolerance to stress-like cryogeny. Information about the relationship between SA-cryogeny-antioxidant enzymatic activity is scarce. The objective was to evaluate the induction of cryogeny tolerance by SA associated with antioxidant activities like CAT, POX, and H2O2 and proline accumulation in tissue, during cryogenic protocols in two clones of potato (Solanum tuberosum L.). SA-induced cryogenic tolerance in both clones, mediated by reducing CAT enzymatic activity, higher H2O2 content, proline accumulation and higher water potential before cryogeny. Enhanced CAT and POX activities after cryogeny were related to decreased H2O2 content and higher survival. Results suggest the signalling role of H2O2 in the induction of tolerance to cryogenic stress.

An Investigation of the Antiproliferative Effect of Rhododendron luteum Extract on Cervical Cancer (HeLa) Cells via Nrf2 Signaling Pathway

The aim of the present study was to investigate the role of Rhododendron luteum extract (RLE) in the induction of Nrf2‑related oxidative stress and endoplasmic reticulum (ER) stress in human cervical cancer (HeLa) cells. The antiproliferative effect of RLE on HeLa and fibroblast cells was determined using the MTT assay. The effects of RLE on the cell cycle, apoptosis, and production of reactive oxygen species (ROS) in HeLa cells were evaluated using fluorescent probes. The mRNA expression levels of Nrf2 [and its targets glutamate-cysteine ligase catalytic subunit (GCLC), and glucose-6-phosphate dehydrogenase (G6PD)], and C/EBP homologous protein (CHOP, an ER stress marker were determined using reverse transcription‑quantitative polymerase chain reaction (RT-PCR). The results demonstrated that RLE exhibited a selective cytotoxic effect (2.9-fold) on HeLa cells compared to fibroblast cells. RLE arrested the cell cycle at the S phase, and induced apoptosis, ER stress, and ROS formation. In addition, RLE significantly suppressed the expression levels of Nrf2, GCLC and G6PD (0.65, 0.69, and 0.54-fold, respectively) and increased the expression of CHOP (4.48-fold) in HeLa cells at 72 h of treatment (p < 0.05). These results show that the antiproliferative effect of RLE occurs through the Nrf2 and ER stress pathways, and the results should now be supported by further in vivo studies.

Biochanin A Inhibits Glioblastoma Growth via Restricting Glycolysis and Mitochondrial Oxidative Phosphorylation.

Abnormal metabolism serves a critical role in glioblastoma (GBM). Biochanin A (BCA), a flavonoid phenolic compound found in edible and herbal plants, has antioxidative and antitumor activities. However, it remains unclear whether BCA has an effect on energy metabolism. The aim of the present study was to evaluate the anticancer effects and molecular mechanism of the effect of BCA on energy metabolism. We observed that BCA inhibited the growth of U251 cells by the mitochondria-mediated intrinsic apoptotic pathway. BCA treatment reduced metabolic function, repressed mitochondrial membrane potential, and increased the production of reactive oxygen species (ROS) in GBM. In addition, we found that BCA decreased aerobic glycolysis by inactivation of the AKT/mTOR pathway. Taken together, the results demonstrate that treatment with BCA inhibited the proliferation of GBM by regulating metabolic reprogramming.

Walsura robusta Roxb. (Meliaceae), a little-known tree with a rich limonoid profile

The plant Walsura robusta Roxb. (Meliaceae) is a robust tree largely distributed in south-east Asia, including provinces of southern China. A few traditional usages of the plant have been mentioned, notably for the treatment of microbial infections. But experimental studies using different types of plant extracts only revealed modest antibacterial effects, and no major antiparasitic activity. Walsura robusta Roxb. is a rich source of secondary metabolites. Several series of limonoids have been isolated from the leaves or the fruits of the plant, such as walsuronoid A-I, walsurins A-E, walsunoids A-I, walrobsins A-R and other cedrelone- or dihydrocedrelone-type limonoids, in addition to a few other terpenoids. All information about Walsura robusta Roxb. have been collated in this brief review. The analysis underlines the presence of two limonoids endowed with significant anticancer activities, walsuronoid B and cedrelone. They both activate the production of reactive oxygen species in cancer cells, modulate mitochondrial activities and induce apoptosis of cancer cells. Their molecular targets and mechanism of action are discussed. Walsura robusta Roxb. has a potential for the development of anticancer natural products. The use of the plant extracts could be further considered for the treatment of diseases with a cell proliferation component.

The Effect of Pulsed Magnetic Fields on the Production of Reactive Oxygen Species by Neutrophils

The Effect of Pulsed Magnetic Fields on the Production of Reactive Oxygen Species by Neutrophils

Abstract 2017: Dual targeting of glutathione-GPX4 nexus and mitochondrial protease CLPP causes synergistic anti-leukemia effects in acute myeloid leukemia (AML)

Abstract Cancer cells depend on aberrant antioxidant mechanisms to survive increased oxidative stress, which is particularly critical for cancer stem cell maintenance and its therapy resistance phenotype. Mitochondria is the primary site of oxygen consumption and reactive oxygen species (ROS) production, thus is essential for regulating cellular redox homeostasis. We have recently reported that hyperactivation of mitochondrial (mito-) protease CLPP by imipridones (ONC201/212) exerts cancer selective lethality through degradation of mito-proteins including respiratory chain complexes and induction of oxidative stress (Ishizawa et al., Cancer Cell 2019). However, the cell death mechanisms remain to be fully determined. We hypothesized that deregulation of mito-redox homeostasis induces a distinct mode of cell death in CLPP-hyperactivated cells and that concomitant inhibition of both mito- and cytosolic antioxidant mechanisms could enhance the anti-leukemia effects. We first tested if inhibition of glutathione (GSH) synthesis enhances the anti-leukemia effects by CLPP hyperactivation by using buthionine sulfoximine (BSO), a glutamate-cysteine ligase inhibitor. As hypothesized, BSO highly sensitized AML cells to genetic or pharmacologic hyperactivation of CLPP by overexpressing CLPP-Y118A (an activating mutation) or ONC201 treatment. We next investigated if a downstream target of GSH depletion is causing the synergy, and re-analysis of recently published CRISPR-knockout screens using leukemic cells treated with imipridones identified GPX4 as one of the 15 top-hit genes whose knockout sensitized cells to CLPP hyperactivation. GPX4 is a selenoprotein that is known for its GSH-dependent reduction of lipid hydroperoxides to protect cells from oxidative cell death including ferroptosis. We found that CLPP hyperactivation increased GPX4 protein levels in AML cells, prominently in mitochondria. Consistently, the GPX4 inhibitors ML210 and JKE1674 sensitized cells to ONC201 in multiple AML cell lines. Overexpression of the activating CLPP-Y118A was also synthetically lethal to GPX4 inhibition, confirming the on-target effect of ONC201. Mechanistically, GPX4 inhibition induced iron-dependent lipid peroxidation and AML cell killing, thus defining ferroptosis as the operational mechanism of cell death. Dual targeting of CLPP and GPX4 induced enhanced lipid peroxidation and mito-ROS but ferroptosis inhibitors only partially blocked cell death, indicating that other modes of oxidative cell death are also involved in the combinatorial synergism. Collectively, our data indicate therapeutic potential of targeting GPX4 and CLPP in AML and potential identification of a novel mode of cancer cell death. Investigations are ongoing to elucidate the underlying molecular mechanisms and to assess in vivo efficacy of the combinatorial treatment. Citation Format: Hiroki Akiyama, Ran Zhao, Lauren B. Ostermann, Jo Ishizawa, Michael Andreeff. Dual targeting of glutathione-GPX4 nexus and mitochondrial protease CLPP causes synergistic anti-leukemia effects in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2017.

Abstract LB103: Investigating the roles of reactive oxygen species and lysosomes in the cytotoxicity of doxorubicin in breast cancer cells

Abstract Background: Nuclear events play a well-established role in the cytotoxicity of doxorubicin (DOX). Other reported mechanisms include redox cycling for reactive oxygen species (ROS) production, which is getting recognized for a larger role than previously recognized. In addition, ROS production may be implicated in lysosome membrane permeabilization (LMP) and subsequent cell death. We have previously reported DOX-induced LMP in breast cancer cells. The goal of this study is to establish the role of LMP in the cytotoxic process and whether ROS are the initiator of this LMP. Methods: Studies were conducted using DOX concentrations ranging from 0.1 μM to 10 μM for 2, 6, and 24 h exposure periods in MCF-7 and MDA-MB-231 breast cancer cells. Intracellular localization of DOX was examined by fluorescence microscopy using lysosome and nuclear markers. ROS production was determined by the DCFDA assay using a fluorescence plate reader. LMP was demonstrated using fluorescence microscopy as the cytosolic release of a 10 kDa AlexaFluor-488 Dextran conjugate that was preloaded into lysosomes. Mitochondrial membrane potential (MMP) was determined by JC-1 assay. Viability was determined by MTT. Cathepsin B activity will be determined by Z-Arg-Arg-AMC cathepsin B substrate. Experiments will be conducted in the presence of E-64 cathepsin B inhibitor and Tiron superoxide scavenger to establish the importance of LMP and ROS towards DOX cytotoxicity. Results: Nuclear uptake was seen in both cell lines as early as 2 h at a concentration of 10 μM. Furthermore, lysosome accumulation of DOX, although small, was visible by 2 h in MDA cells, increasing in degree with time and concentration. ROS levels were found to be highest at 2 h in MCF-7 cells, increasing by up to 100% of the untreated control, and occurring concurrently with LMP at higher concentrations. Preliminary results suggest much lower ROS levels in MDA-MB-231 cells. In MCF-7 cells, ROS production decreased at 6 h, but increased by 24 h with concurrent increases in LMP. Although less than the MCF-7 cells, MDA cells demonstrated notable LMP for the first time at 5 and 10 μM conditions, occurring concurrently with MMP. By 24 h, viability was substantially reduced for both cell lines, with MCF-7s demonstrating a greater reduction in viability. Significant MMP was not observable until 24 h in MCF-7 cells. Conclusions: The early and strong ROS occurring with LMP suggest a previously unrecognized DOX cytotoxic combination in MCF-7 cells. LMP does not appear to result from lysosomal accumulation of DOX. A reduced role of ROS and LMP may account for the lesser cytotoxicity in MDA-MB-231 cells. Both ROS and LMP appear to be necessary for cytotoxicity in MCF-7 cells. Citation Format: Rachel E. Nicoletto, Clyde M. Ofner. Investigating the roles of reactive oxygen species and lysosomes in the cytotoxicity of doxorubicin in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB103.

Оксидантный стресс и тканевая гипоксия как факторы, способствующие развитию дисфункции предстательной железы и мочевого пузыря при метаболическом синдроме

Introduction. The development of oxidative stress and nonspecific inflammation is one of the leading factors in the development of benign prostatic hyperplasia (BPH) and associated urination disorders in metabolic syndrome (MS). However, the specific mechanisms of these processes are not entirely clear. The purpose of the study. To study the activity of reactive oxygen species production and the functional state of mitochondria in the prostate and bladder and their role in the dysfunction of these organs using an experimental model of MS induction in rats. Material and methods. In 10 adult mongrel male rats MS was induced by keeping them on a high-calorie diet with an increased content of carbohydrates and fats for 3 months. 10 rats kept on a standard vivarium diet served as controls. The development of MS was confirmed by characteristic changes in the biochemical analysis of blood (hyperglycemia, hyperuricemia, dyslipidemia, hyperinsulinemia). In both series of rats, sections of the native prostate and bladder were examined by laser confocal microscopy and stained with fluorescent probes that characterize the activity of the production of reactive oxygen species (dichlorofluorescein-DCF) and the functional state of the mitochondria (tetramethylrodamine ether – TMRE). The activity of a number of intracellular enzymes (AST, ALT. Alkaline phosphatase, LDH) was investigated in the tissues and urine. Results. In rats with MS, the development of BPH and hypertrophy of the bladder were revealed, confirmed histologically. The study of sections of both organs by confocal microscopy revealed a significant increase in the production of reactive oxygen species by their cells and a decrease in the functional activity of mitochondria, which indicated the development of oxidant stress and tissue hypoxia. In the prostate, this was accompanied by a decrease in the secretory activity of the prostate glands, and in the bladder – the release of cytoplasmic enzymes from damaged cells into the urine, indicating cell damage. Conclusion. The causes of the development of a non-specific inflammatory process in the prostate and bladder, leading to dysfunction of these organs in MS, are increased production of reactive oxygen species and the development of tissue hypoxia.

Proteomic Analysis of Liver from Finishing Beef Cattle Supplemented with a Rumen-Protected B-Vitamin Blend and Hydroxy Trace Minerals.

Simple SummaryGreater metabolic needs in high-producing beef cattle might lead to mineral and vitamin deficiency. Previous studies have shown the benefits of B-vitamin and trace mineral supplementation in animal performance of ruminants; however, little is known about the effects of supplementing finishing beef cattle with rumen-protected forms on the liver metabolism. Therefore, the aim of the present study was to determinate the impact of rumen-protected B-vitamin blend and hydroxy trace mineral supplementation on the hepatic proteome of finishing steers. This study reports the first evidence indicating that the supplementation of these micronutrients induces protein changes concerning oxidative metabolism and responses to oxidative stress in the liver tissue.Vitamin B and trace minerals are crucial molecular signals involved in many biological pathways; however, their bioavailability is compromised in high-producing ruminant animals. So far, studies have mainly focused on the effects of these micronutrients on animal performance, but their use in a rumen-protected form and their impact on liver metabolism in finishing beef cattle is poorly known. We used a shotgun proteomic approach combined with biological network analyses to assess the effects of a rumen-protected B-vitamin blend, as well as those of hydroxy trace minerals, on the hepatic proteome. A total of 20 non-castrated Nellore males with 353 ± 43 kg of initial body weight were randomly assigned to one of the following treatments: CTRL—inorganic trace minerals without supplementation of a protected vitamin B blend, or SUP—supplementation of hydroxy trace minerals and a protected vitamin B blend. All animals were fed the same amount of the experimental diet for 106 days, and liver biopsies were performed at the end of the experimental period. Supplemented animals showed 37 up-regulated proteins (p < 0.10), and the enrichment analysis revealed that these proteins were involved in protein folding (p = 0.04), mitochondrial respiratory chain complex I (p = 0.01) and IV (p = 0.01), chaperonin-containing T-complex 2 (p = 0.01), glutathione metabolism (p < 0.01), and other aspects linked to oxidative-stress responses. These results indicate that rumen-protected vitamin B and hydroxy trace mineral supplementation during the finishing phase alters the abundance of proteins associated with the electron transport chain and other oxidation–reduction pathways, boosting the production of reactive oxygen species, which appear to modulate proteins linked to oxidative-damage responses to maintain cellular homeostasis.