CellROX Green Research Articles

EXTH-39. A BENCH-TOP MODEL FOR THE OPTIMIZATION OF ULTRASOUND PARAMETERS FOR SONODYNAMIC THERAPY OF GLIOBLASTOMA

Abstract BACKGROUND In-vitro studies have demonstrated that low frequency sound waves can interact with hemo-porphyrin molecule to induce the production of reactive oxygen species. This biophysical phenomenon can induce tumour cell death in glioblastoma cells in-vitro. The purpose of this study was to evaluate the production of reactive oxygen species and expression of apoptotic markers in glioblastoma cells in response to varying total treatment time and ultrasound power using a bench-top focused ultrasound (FUS) device. METHODS U251 cells were exposed to 5-Aminolevulenic Acid (5-ALA) at varying concentrations for up to 24 hours. The time point with peak proto-porphyrin IX (PPIX) fluorescence was established using fluorescence activated cell sorting (FACS). A transducer utilized at 0.91MHz was placed on an inverted stage with the focus targeted to the bottom of a standard 96-well cell culture plate. Pressure wave delivery was confirmed using a hydrophone. GBM cells were treated with 5-ALA alone, FUS alone, or 5-ALA + FUS and the cells were incubated for 24 hours prior to measuring ROS induction and cell death. ROS was measured using FACS with the CellROX Green Kit for Oxidative Stress Detection and Annexin-V was measured using the Dead Cell Stain Kit. RESULTS Peak fluorescence intensity for PPIX in the U251 cell line was found to be induced through incubation with 5-ALA for 24 hours with a concentration 200mg/mL. The fluorescence intensity of PPIX was found to be 313% greater in the treated group compared to the control group. Peak ROS was observed using a 0.91MHz transducer frequency for a total time of 120s with 6W average power. ROS was 61% and 66% greater in the SDT group compared to the FUS and 5-ALA group respectively. CONCLUSION Using this bench-top set up we successfully induced ROS and apoptosis in U251 cells. Establishing a reliable in-vitro model will allow us to further investigate the effects of other sonication parameters such as burst length.

Comparison of CellRox green fluorescence upon thawing on in vitro Bos taurus and Bos indicus embryos cryopreserved by slow freezing or vitrification.

The aim of this study was to compare the levels of reactive oxygen species (ROS) in Bos taurus and Bos indicus in vitro embryos cryopreserved using either slow freezing or vitrification. Embryos were divided into four groups based on subspecies and freezing method: Bos indicus slow freezing (BiSF; n = 8), Bos indicus vitrification (BiVT; n = 10), Bos taurus slow freezing (BtSF; n = 9), and Bos taurus vitrification (BtVT; n = 6). After thawing, the embryos were incubated with CellRox Green and images were obtained using a confocal microscope. The fluorescence intensity of each cell was measured and expressed as arbitrary units of fluorescence (auf) and compared using a multiple regression and unpaired t-test with α = 0.05. Results showed that subspecies and the freezing method significantly affected auf (P < 0.001; R2 = 0.1213). Bos indicus embryos had higher auf than Bos taurus embryos, whether frozen by slow freezing (67.05 ± 23.18 vs 51.30 ± 16.84, P < 0.001) or vitrification (64.44 ± 23.32 vs 47.86 ± 17.53, P < 0.001). Slow freezing induced higher auf than vitrification in both Bos taurus (51.30 ± 16.84 vs 47.86 ± 17.53, P < 0.001) and Bos indicus (67.05 ± 23.18 vs 64.44 ± 23.32, P < 0.014). In conclusion, Bos taurus embryos had lower ROS levels when frozen using vitrification, while Bos indicus embryos had consistent ROS patterns regardless of the freezing method. However, Bos indicus embryos frozen by slow freezing tended to have a higher number of cells with elevated ROS levels.

Activation of the Nrf-2 pathway improves the mitochondrial phenotype in skeletal muscle cells

Aging muscle displays a reduction in mitochondrial content, along with elevated reactive oxygen species (ROS) emission and reduced levels of antioxidant enzymes. These characteristics enhance tissue susceptibility to oxidative damage, facilitating sub-optimal cellular metabolism and subsequent muscle atrophy. While these perturbations to metabolic homeostasis are a natural course of the aging process, there has been ongoing research investigating how to implement interventions to reduce this decline in muscle health. Regular exercise is one possible treatment modality, however this may be unsuitable for some populations who are unable to participate due to their health status. The objective of this study was to examine the activation of the Nrf-2 pathway using the nutraceutical Sulforaphane (SFN) and evaluate its role as a potential exercise mimetic. We hypothesized that SFN would act through similar pathways activated with chronic exercise to improve mitochondrial content, respiration, and ROS emission. To address our objective, C2C12 myotubes were treated with SFN for various timepoints basally, and in the presence of the Complex-I inhibitor rotenone to induce oxidative stress. Western blotting revealed an 8-fold increase in Nrf-2 nuclear localization following 24hrs of SFN treatment, which subsequently led to the upregulation of antioxidant enzymes NQO1, Heme Oxygenase-1, catalase, glutathione reductase and Glucose-6-Phosphate Dehydrogenase by 2-to 3-fold. These protein changes, were accompanied by dramatic reductions in both total cellular and mitochondrial ROS emission with SFN, detected with CellROX Green and MitoSOX Red respectively. Interestingly, this led to an increase in mitochondrial membrane potential, detected via MitoTracker Red (MTR), despite rotenone-induced oxidative stress. Western blotting revealed TFAM upregulation at 24hrs, suggesting an increase in mitochondrial DNA (mtDNA) replication and transcription. This was corroborated by an increase in COX subunit I protein, a mitochondrially-encoded subunit of cytochrome c oxidase, as well as MitoTracker Green and MTR staining by 48hrs. Mitochondrial respiration was evaluated using Seahorse analysis, demonstrating a marked increase in ATP production as well as basal and maximal respiration. Together these data suggest that activation of the Nrf-2 pathway via SFN may lead to adaptations similar to chronic exercise. Additionally, SFN may have applications in disorders characterized by mitochondrial loss and high ROS emission. To fully elucidate the precise mechanisms underlying this agent, SFN will be combined with chronic contractile activity to determine if these two interventions operate through independent pathways. This work was supported by funds from the Natural Science and Engineering Research Council (NSERC). Sabrina Champsi is the recipient of the NSERC Alexander Graham Bell Canada Graduate Scholarship-Master’s (CGS-M). David A. Hood is the holder of a Canadian Research Chair in Cell Physiology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition of the HMGB1/RAGE axis protects against cisplatin-induced ototoxicity via suppression of inflammation and oxidative stress.

As an anti-tumor drug widely used in the clinic, cisplatin is limited by its ototoxic side effects associated with various factors, including inflammatory responses. Receptor for Advanced Glycation Endproducts (RAGE) recognizes damage-associated molecular patterns (DAMPs) and promotes stress and inflammation. This study intended to determine the potential behavior of the HMGB1/RAGE axis after cisplatin injury and whether it has a protective effect after inhibiting this pathway. We used FPS-ZM1, a RAGE inhibitor, to modulate the axis of HMGB1/RAGE in neonatal mouse cochlear explants and C57BL/6 mice in vivo. Apoptosis was identified by Annexin V-FITC/PI assay, Cleaved Caspase-3, and TUNEL staining. Reactive oxygen species (ROS) level was assessed by MitoSOX Red and CellROX Green assay. The expression of proteins associated with the HMGB1/RAGE axis and apoptosis was observed by western blotting. The expression of inflammatory cytokines was evaluated by qPCR. The protective effect of HMGB1/RAGE knockdown was also assessed on cisplatin-induced ototoxicity. These results demonstrated that cisplatin could activate the HMGB1/RAGE pathway in cochlear hair cells and release inflammatory factors. Pretreatment with FPS-ZM1 alleviated cisplatin-induced ototoxicity in vivo and in vitro. Knocking down HMGB1 and RAGE achieved specific protective effects. Altogether, inhibiting HMGB1/RAGE axis can reverse the increase of ROS accumulation, the activation of apoptosis, and the production of inflammatory reactions after cisplatin injury. FPS-ZM1 could resist the ototoxicity of cisplatin by suppressing the HMGB1/RAGE signal pathway, and it may be considered the new otoprotective potential strategy for hearing loss.

Heterozygous Variants in the Clpbgene Associated with Neutropenia Affect Neutrophil Function

Introduction The CLPB gene encodes a caseinolytic peptidase belonging to the ATPase family and acts as a mitochondrial chaperone. While biallelic mutations in CLPB are associated with the 3-methylglutaconic aciduria and severe neutropenia, (MIM number: 616271 3), some heterozygous variants seem to induce neutropenia exclusively. In the current study, we evaluated for the first time a function of neutrophils among neutropenic carriers of CLPB mutation. Methods Among 282 individuals with neutropenia studied with targeted Next-Generation Sequencing (panel of 725 genes related to hematological disorders), three neutropenic probands with heterozygous CLPB amino acid substitutions were identified. Neutrophils from patients and control healthy donors were isolated from peripheral blood with a Pancoll density gradient. The percentages of freshly isolated neutrophils undergoing apoptosis or necrosis were determined by flow cytometry using Annexin V - FITC and Propidium Iodide (PI) staining. Phagocytic capacity was evaluated using pHrodo Green S. aureus BioParticles Conjugates. A Boyden's chamber model was used to determine the level of the neutrophil's mobility and chemotaxis response to 10% human serum. Formation of Neutrophils Extracellular Traps (NETs) and induction of the oxidative burst were studied upon phorbol 12-myristate 13-acetate (PMA) stimulation (25 nM) with SYTOX™ Green Nucleic Acid and flow cytometry with CellROX Green assay, respectively. The intracellular and circulating concentrations of anti-microbial compounds (Myeloperoxidase MPO, Lactotransferrin LTF, Cathelicidin-LL37, and defensins) were measured using commercially available ELISA assays. The results were evaluated with an unpaired t-test using GraphPad Prism 9. Quantitative data presented as medians (25%-75%). Results Genetic analysis identified three heterozygous variants of CLPB gene p.Arg327Trp, p.Arg628Cys, p .Arg629Cys in three different families. Two of these mutations were novel p.Arg327Trp and p .Arg629Cys. Interestingly, the p.Arg327Trp variant was located outside the ATPase domain. In total 7 family members who suffered from neutropenia and were carriers of the CLPB variants were available for neutrophil functional assays (Figure 1). Neutrophils collected from CLPB-deficient individuals have a higher rate of apoptosis as compared to healthy neutrophils: 57(52-66)% vs 23(20-27)%, P&amp;lt;0.0001, and necrosis: 0.56(0.31-1.07)% vs 0.13(0.10-0.21)%, P=0.02. CLPB-deficient neutrophils display reduced mobility in the absence of stimulation: 298(191-413) vs 1011(936-1201) migrating neutrophils, P&amp;lt;0.0001, and after 10% human serum treatment: 2090(1746-2458) vs 3736(3071-4828) migrating neutrophils, P=0.0014. Similarly, reduced phagocytosis of S. aureus bioparticles was observed in CLPB-deficient neutrophils: 1890(1679-2063) vs 3352(2472-4440) Fluorescence Units, respectively, P=0.0029. Upon PMA stimulation, neutrophils from CLPB patients produced less reactive oxygen species compared to controls: 311(175-542)FMI vs 775(766-1150)FMI, P=0.08, and their ability to produce NETs is almost entirely abolished: 0.5(0.21-0.79)ng/µl vs 1.3(1.08-3.05)ng/µl, P=0.0075. Moreover, CLPB-deficient neutrophils have reduced intracellular concentration of myeloperoxidase: 35(31-39)ng/mg vs 55(41-63)ng/mg, P=0.0043 and cathelicidin LL-37: 5.5(5-6.3)ng/µg vs 19.22(14.21-23.28)ng/µg, P=0.0003. The concertation of the LL-37 is also reduced in patients' plasma 1.34(0.84-1.78)ng/ml vs 7.17(5.38-8.03)ng/ml, P&amp;lt;0.0001. Both intracellular and circulating concentrations of LTF and defensins did not differ between study groups. Conclusion Neutrophils from neutropenic carriers of CLPB mutations are functionally impaired, especially concerning their ability for NETs formation and cathelicidin LL-37 production.

Gut-microbiota-derived phenylacetylglutamine induces oxidative stress in endothelial cells via a NOX2-dependent pathway

Abstract Introduction The gut microbiota-derived metabolite phenylacetylglutamine (PAG) is clinically and mechanistically linked to cardiovascular disease (CVD)(1). Here, we aim to identify the effect of PAG on endothelial cells and elucidate the underlying molecular pathways. Methods We treated human aortic endothelial cells (HAEC) at a dose of 100 µM PAG (a concentration which can be observed in vivo) for 72h. As a positive control, cells were treated with H2O2 for 1h. For the determination of reactive oxygen species (ROS) formation, we used the CellROX Green assay with subsequent fluorescence microscopy. For signaling studies, protein expression levels of the NADPH oxidases NOX2 and NOX4 as well as the transcription factor NF-kB were determined by western blot analyses. Results Our studies showed that PAG treatment for 72h significantly increased the fluorescence intensity after CellROX Green staining (p&amp;lt;0.0001). Concomitantly, our immunoblotting results revealed that NOX2 (p&amp;lt;0.01) and NF-kB (p&amp;lt;0.001) protein levels were upregulated, whereas NOX4 remained unchanged in response to PAG. Conclusion We conclude that PAG-induced oxidative stress in endothelial cells is mediated by a NOX2 and NF-kB but not by a NOX4 dependent pathway. This study is a first step towards better understanding the molecular mechanisms of cellular responses to the gut microbiota-derived metabolite PAG.

Ferroptosis Inhibition Generates TCF-1 + CAR-T Cells with Enhanced Persistence and Cytotoxicity

[Introduction] Long-term in vivo persistence of chimeric antigen receptor (CAR) T-cells is associated with sustained response after CAR-T cell therapy against hematological malignancies. Ferroptosis is a novel form of programmed cell death that refers to iron-dependent regulated cell death resulting from lipid peroxidation and consequent cytomembrane rupture. The iron, lipid, amino acid, and glutathione metabolisms strictly govern the beginning and execution of ferroptosis. Recent studies demonstrated that ferroptosis limited longevity of virus-specific memory T cells (Wang Y: Nat Immunol 2022). However, the role of ferroptosis in CAR-T cells remains to be clarified. We herein explored the role of ferroptosis in CAR-T cells both in vitro and in vivo. [Methods] For CAR-T cell generation, purified T cells from naïve mice were stimulated with anti-CD3/CD28 antibodies for 48 hrs and then incubated with retroviral vector encoding anti-CD19 CAR (1D3-28Z.1-3; Addgene) for another 48 hrs, in the presence or absence of 32 µM of ferrostatin-1 (Fer-1), an inhibitor of the iron-dependent production of lipid hydroperoxide, or 10 µM of deferoxamine (DFO), an iron chelator. Flow cytometric analyses of phenotype of CAR-T cells, accumulation of reactive oxygen species (ROS), lipid peroxidation, and cytokine production were performed 96 hrs after T-cell stimulation. ROS were quantified using CellROX Green dye, and lipid peroxidation was evaluated using Lipid Peroxidation Assay Kit (abcam). [Results] First, we tested whether CAR-T manufacturing process could induce ferroptosis. We found a substantial accumulation of ROS in CAR-T cells compared to their pre-culture levels, along with a significant increase in lipid peroxidation, indicating that manufacturing process induces ferroptosis of CAR-T cells. Next, we tested whether ferroptosis inhibition by the lipid peroxidation inhibitor Fer-1, could impact the quality of CAR-T cells. When added to the culture, Fer-1 significantly reduced lipid peroxidation in CAR-T cells and significantly increased the proportion of CAR-T cells with a progenitor phenotype, TCF-1 +Tim-3 -, which is known to be associated with longevity of CAR-T cells. Importantly, ferroptosis inhibition significantly enhanced proliferative capacity and cytotoxicity against syngeneic A20 B-cell leukemia in vitro. Next, we tested whether ferroptosis inhibition by the iron chelator DFO could improve CAR-T cell functions. Similar to the effects of Fer-1, DFO mitigated lipid peroxidation of CAR-T cells in association with increased TCF-1 +Tim-3 - population, while it did not affect ROS accumulation. DFO-treated CAR-T cells demonstrated enhanced cytotoxicity against A20 cells compared with control CAR-T cells, confirming that ferroptosis inhibition improves anti-tumor effects of CAR-T cells. Finally, we examined whether ferroptosis inhibition could enhance antitumor effects of CAR-T cells in vivo. Naïve BALB/c mice were intravenously (i.v.) injected with 1.0 x 10 6 luciferase-expressing A20 cells on day 0, followed by i.v. injection of 1.0 x 10 5 Fer-1- or DFO-treated CAR-T cells or control CAR-T cells 7 days later. In mice injected with Fer-1-treated CAR-T cells, the numbers of CAR-T cells in peripheral blood on day 16 was significantly higher than those in mice injected with control CAR-T cells, suggesting that ferroptosis inhibition improved the persistence of CAR-T cells in vivo ( Fig. 1). In vivo bioluminescence imaging showed that both Fer-1-treated and DFO-treated CAR-T cells demonstrated significantly improved anti-tumor effects compared to control CAR-T cells ( Fig. 2). [Conclusions] We for the first time showed that ferroptosis limits the persistence and anti-tumor effects of CAR-T cells, while ferroptosis inhibition induces TCF-1 +Tim-3 - CAR-T cells and improves the therapeutic effect of CAR-T cell therapy. Figure 1. The number of CAR-T cells in the peripheral blood on day 16 after tumor inoculation. Fer-1, ferrostatin-1; *, P &amp;lt; 0.05; P &amp;lt; 0.01. Figure 2. The fold change of the total flux at each timepoint relative to the total flux of the baseline in the mice injected with DFO-treated CAR-T cells, Fer-1-treated CAR-T cells, and control CAR-T cells on day 7 after tumor inoculation. Fer-1, ferrostatin-1; DFO, deferoxamine; *, P &amp;lt; 0.05.

Electrical stimulation facilitates NADPH production in pentose phosphate pathway and exerts an anti-inflammatory effect in macrophages

Macrophages play an important role as effector cells in innate immune system. Meanwhile, macrophages activated in a pro-inflammatory direction alter intracellular metabolism and damage intact tissues by increasing reactive oxygen species (ROS). Electrical stimulation (ES), a predominant physical agent to control metabolism in cells and tissues, has been reported to exert anti-inflammatory effect on immune cells. However, the mechanism underlying the anti-inflammatory effects by ES is unknown. This study aimed to investigate the effect of ES on metabolism in glycolytic-tricarboxylic acid cycle (TCA) cycle and inflammatory responses in macrophages. ES was performed on bone marrow-derived macrophages and followed by a stimulation with LPS. The inflammatory cytokine expression levels were analyzed by real-time polymerase chain reaction and ELISA. ROS production was analyzed by CellRox Green Reagent and metabolites by capillary electrophoresis-mass spectrometry. As a result, ES significantly reduced proinflammatory cytokine expression levels and ROS generation compared to the LPS group and increased glucose-1-phosphate, a metabolite of glycogen. ES also increased intermediate metabolites of the pentose phosphate pathway (PPP); ribulose-5-phosphate, rebose-5 phosphate, and nicotinamide adenine dinucleotide phosphate, a key factor of cellular antioxidation systems, as well as α-Ketoglutarate, an anti-oxidative metabolite in the TCA cycle. Our findings imply that ES enhanced NADPH production with enhancement of PPP, and also decreased oxidative stress and inflammatory responses in macrophages.

Human hair proteins as natural reactive oxygen species scavengers for in vitro applications.

Human hair proteins are recognized for their intrinsically high cysteine content. They can be solubilized while preserving their highly reductive thiol groups for free radical scavenging applications. The presence of aromatic and nucleophilic amino acids such as methionine, serine, phenylalanine, and threonine further contribute to the antioxidative potential of this material. Herein, utilizing the DPPH (2,2-diphenyl-1-picrylhydrazyl) and acellular 2',7'-dichlorodihydrofluorescein diacetate (H2 DCFDA) assays, keratins are demonstrated to possess the highest radical scavenging activity among the studied hair proteins. Consequently, protection against hydrogen peroxide-induced oxidative stress in human dermal fibroblasts (HDFs) cultured in human hair keratin supplemented media is demonstrated. Quenching of reactive oxygen species in the HDF is observed using the CellROX Green dye and the expression levels of antioxidant (HMOX1, SOD2, GPX1) and tumor suppressor (TP53) genes is analyzed using qPCR. Collectively, this study presents further evidence and demonstrates the in vitro application potential of hair proteins, especially keratins, as an antioxidizing supplement.

Flavor Classification/Categorization and Differential Toxicity of Oral Nicotine Pouches (ONPs) in Oral Gingival Epithelial Cells and Bronchial Epithelial Cells.

Oral nicotine pouches (ONPs) are a modern form of smokeless tobacco products sold by several brands in the U.S., which comprise a significant portion of non-combustible nicotine-containing product (NCNP) sales to date. ONPs are available in various flavors and may contain either tobacco-derived nicotine (TDN) or tobacco-free nicotine (TFN). The growth in popularity of these products has raised concerns that flavored ONPs may cause adverse oral health effects and promote systemic toxic effects due to nicotine and other ONP by-products being absorbed into the circulatory system through oral mucosa. We hypothesized that flavored ONPs are unsafe and likely to cause oral and pulmonary inflammation in oral and respiratory epithelial cells. Before analyzing the effects of ONPs, we first classified ONPs sold in the U.S. based on their flavor and the flavor category to which they belonged using a wheel diagram. Human gingival epithelial cells (HGEP) were treated with flavored ONP extracts of tobacco (original, smooth), menthol (wintergreen and cool cider), and fruit flavor (americana and citrus), each from the TDN and TFN groups. The levels of ONP-induced inflammatory cytokine release (TNF-α, IL-6, and IL-8) by ELISA, cellular reactive oxygen species (ROS) production by CellRox Green, and cytotoxicity by lactate dehydrogenase (LDH) release assay in HGEP cells were assessed. Flavored ONP extracts elicited differential toxicities in a dose- and extract-dependent manner in HGEP cells 24 h post-treatment. Both fruit TDN and TFN extracts resulted in the greatest cytotoxicity. Tobacco- and fruit-flavored, but not menthol-flavored, ONPs resulted in increased ROS production 4 h post-treatment. Flavored ONPs led to differential cytokine release (TNF-α, IL-6, and IL-8) which varied by flavor (menthol, tobacco, or fruit) and nicotine (TDN vs. TFN) 24 h post-treatment. Menthol-flavored ONPs led to the most significant TNF-α release; fruit TFN resulted in the most significant IL-6 release; and fruit TDN and tobacco TFN led to the highest release of IL-8. Subsequently, human bronchial epithelial cells (16-HBE and BEAS-2B) were also treated with flavored ONP extracts, and similar assays were evaluated. Here, the lowest concentration treatments displayed increased cytotoxicity. The most striking response was observed among cells treated with spearmint and tobacco flavored ONPs. Our data suggest that flavored ONPs are unsafe and likely to cause systemic and local toxicological responses during chronic usage.

Effects of fetal bovine serum on trophectoderm and primitive endoderm cell allocation of in vitro-produced bovine embryos.

Supplementing embryonic culture medium with fetal bovine serum (FBS) renders this medium undefined. Glucose and growth factors present in FBS may affect the results of cell differentiation studies. This study tested the hypothesis that FBS supplementation during in vitro culture (IVC) alters cell differentiation in early bovine embryo development. Bovine embryos were produced in vitro and randomly distributed into three experimental groups at 90 h post insemination (90 hpi): the KSOM-FBS group, which consisted of a 5% (v/v) FBS supplementation; the KSOM33 group, with the renewal of 33% of medium volume; and the KSOM-Zero group, without FBS supplementation nor renewal of the culture medium. The results showed that the blastocyst rate (blastocyst/oocytes) at 210 hpi in the KSOM-FBS group was higher than in the KSOM-Zero group but not different from the KSOM33 group. There were no significant changes in metabolism-related aspects, such as fluorescence intensities of CellROX Green and MitoTracker Red or reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD+). Immunofluorescence analysis of CDX2 revealed that the lack of FBS or medium supplementation reduced the number of trophectoderm (TE) cells and total cells. Immunofluorescence analysis revealed a reduction of SOX17-positive cell numbers after FBS supplementation compared with the KSOM33 group. Therefore, we concluded that FBS absence reduced blastocyst rates; however, no reduction occurred when there was a 33% volume renewal of the medium at 90 hpi. We also concluded that FBS supplementation altered TE and primitive endoderm cell allocation during early bovine embryo development.

ALKBH5 inhibits thyroid cancer progression by promoting ferroptosis through TIAM1-Nrf2/HO-1 axis.

As a critical catalytic subunit of N6-methyladenosine (m6A) modification in messenger RNA, ALKBH5 has been reported to affect the progression of numerous tumors. However, the functions and mechanisms of ALKBH5 in thyroid cancer remain largely unknown. Relative mRNA and protein levels in thyroid cancer tissues and cells were detected by qRT-PCR and western blot, respectively. The proliferation and viability were evaluated using colony formation and CCK-8 assays. Intracellular ironlevel was measured by an iron colorimetric assay kit. ROS level was determined using CellRox Green reagent. TIAM1 mRNA m6A level was detected by MeRIP. Xenograft tumor growth was performed to examine the role of ALKBH5 in thyroid tumor growth in vivo. ALKBH5 was decreased in thyroid cancer tissues and cells. ALKBH5 overexpression inhibited thyroid cancer cell proliferation and increased the levels of Fe2+ and ROS and reduced the proteins expression of GPX4 and SLC7A11. Furthermore, overexpression of ALKBH5 inhibited TIAM1 expression by m6A modification, and overexpression of TIAM1 reversed the regulatory of oe-ALKBH5 on cell proliferation and ferroptosis in thyroid cancer. In addition, TIAM1 was elevated in thyroid cancer, and TIAM1 knockdown repressed thyroid cancer cell proliferation and promoted ferroptosis through regulating Nrf2/HO-1 axis. In addition, in vivo evidences also showed that ALKBH5 suppressed thyroid cancer progression by decreasing the m6A level of TIAM1. Our findings suggested that ALKBH5 inhibited thyroid cancer progression by inducing ferroptosis through m6A-TIAM1-Nrf2/HO-1 axis, suggesting ALKBH5 might be a potential target molecule for the treatment and diagnosis of thyroid cancer.

Photoactivation of Chemotherapeutic Agents with Cerenkov Radiation for Chemo-Photodynamic Therapy.

Cerenkov radiation (CR) can be used as an internal light source in photodynamic therapy (PDT). Methotrexate (MTX) and paclitaxel (PTX), chemotherapeutic agents with wide clinical use, have characteristics of photosensitizers (PS). This work evaluates the possibility of photoexciting MTX and PTX with CR from 18F-FDG to produce reactive oxygen species (ROS) capable of inducing cytotoxicity. PTX did not produce ROS when excited by CR from 18F-FDG, so it is not useful for PDT. In contrast, MTX produces 1O2 (detected by ABMA) in amounts sufficient to significantly decrease the viability of the T47D cells. MTX solutions of 100 nM combined with 18F-FDG activities of 50 (1.85 MBq) and 100 μCi (3.7 MBq) produced a significant decrease in cell viability to (50.09 ± 4.95) and (47.96 ± 11.19)%, respectively, compared to MTX (66.29 ± 5.92)% and 18F-FDG (91.35 ± 7.00% for 50 μCi and 99.43 ± 11.03% for 100 μCi) alone. Using the CellRox Green reagent, the intracellular production of ROS was confirmed as the main mechanism of cytotoxicity. The results confirm the therapeutic potential of photoactivation with CR and the synergy of the combined treatment with chemotherapy + photodynamic therapy (CMT + PDT). The combination of chemotherapeutic agents with PS properties and β-emitting radiopharmaceuticals, previously approved for clinical use, will make it possible to shorten the evaluation stages of new CMT + PDT systems.

Puerarin attenuates cisplatin-induced apoptosis of hair cells through the mitochondrial apoptotic pathway

Puerarin, one of the main components of Pueraria lobata, has been reported to possess a wide range of pharmacological activities, including anti-inflammatory, antioxidative and anti-apoptotic effects. However, the role of puerarin in ototoxic drug-induced hair cell injury has not been well characterized. This study explored whether puerarin protects against cisplatin-induced hair cell damage and its potential mechanisms. The viability of puerarin-treated HEI-OC1 cells was assessed by CCK8 assay. Reactive oxygen species (ROS) was estimated with flow cytometric analysis using Cellrox Green fluorescent probe. Apoptosis-related protein levels were detected by western blot analysis. Immunostaining of the organ of Corti was performed to determine mice cochlear hair cell survival. Our results showed that puerarin improved cell viability and suppressed apoptosis in the cisplatin-damaged HEI-OC1 cells and cochlear hair cells. Mechanistic studies revealed that puerarin attenuated mitochondrial apoptosis pathway by regulating apoptotic related proteins, such as Bax and cleaved caspase-3, and attenuated ROS accumulation after cisplatin damage. Moreover, puerarin was involved in regulating the Akt pathway in HEI-OC1 cells in response to cisplatin. Our results demonstrated that puerarin administration decreased the sensitivity to apoptosis dependent on the mitochondrial apoptotic pathway by reducing ROS generation, which could be used as a new protective agent against cisplatin-induced ototoxicity.

Ninjinyoeito ameliorated cigarette smoke extract-induced apoptosis and inflammation through JNK signaling inhibition in human lung fibroblasts

BackgroundCigarette smoke is a major risk factor for various lung diseases, such as chronic obstructive pulmonary disease (COPD). Ninjinyoeito (NYT), a traditional Chinese medicine, has been prescribed for patients with post-illness or post-operative weakness, fatigue, loss of appetite, rash, cold limbs, and anemia. In addition to its traditional use, NYT has been prescribed for treating frailty in gastrointestinal, respiratory, and urinary functions. Further, NYT treatment can ameliorate cigarette smoke-induced lung injury, which is a destructive index in mice; however, the detailed underlying mechanism remains unknown. The purpose of this study was to investigate whether NYT ameliorates cigarette smoke-induced cell injury and inflammation in human lung fibroblasts and determine its mechanism of action.MethodsWe prepared a cigarette smoke extract (CSE) from commercially available cigarettes to induce cell injury and inflammation in the human lung fibroblast cell line HFL1. The cells were pretreated with NYT for 24 h prior to CSE exposure. Cytotoxicity and cell viability were measured by lactate dehydrogenase (LDH) cytotoxicity assay and cell counting kit (CCK)-8. IL-8 level in the cell culture medium was measured by performing Enzyme-Linked Immuno Sorbent Assay (ELISA). To clarify the mechanisms of NYT, we used CellROX Green Reagent for reactive oxygen species (ROS) production and western blotting analysis for cell signaling.ResultsExposure of HFL1 cells to CSE for 24 h induced apoptosis and interleukin (IL)-8 release. Pretreatment with NYT inhibited apoptosis and IL-8 release. Furthermore, CSE exposure for 24 h increased the production of ROS and phosphorylation levels of p38 and JNK. Pretreatment with NYT only inhibited CSE-induced JNK phosphorylation, and not ROS production and p38 phosphorylation. These results suggest that NYT acts as a JNK-specific inhibitor.ConclusionNYT treatment ameliorated CSE-induced apoptosis and inflammation by inhibiting the JNK signaling pathway. Finally, these results suggest that NYT may be a promising therapeutic agent for patients with COPD.

Abstract P5-08-18: Disruption of redox balance in glutaminolytic triple negative breast cancer by inhibition of glutamate transport and glutaminase

Abstract OBJECTIVES: Upregulated glutamine metabolism is an important metabolic signature for aggressive and treatment-resistant cancers, including many triple negative breast cancers (TNBCs). Glutamate (Glu) produced from glutamine (Gln) via mitochondrial glutaminase (GLS) feeds into the TCA cycle to provide energy and biosynthetic precursors for tumor growth. Recent studies from our lab and others have revealed that Glu also plays an important role in maintaining redox homeostasis which is essential for survival of cancer cells. Glu is a component amino acid of glutathione, the most abundant cellular antioxidant. Furthermore, via the xCT antiporter (SLC7A11), Glu is exported for import of cystine, a rate-limiting step for glutathione synthesis. We assessed the effect of metabolic inhibition of GLS and/or xCT pathways on cellular reactive oxygen species (ROS) level and induction of apoptosis. To track cytosolic Glu transport cross plasma membrane in vivo, we tested (4S)-4-(3-[18F]fluoropropyl)-L-glutamic acid ([18F]FSPG), a non-metabolized structural analog of Glu. We hypothesize that mitochondrial Glu supply is necessary for maintaining cellular redox homeostasis and [18F]FSPG PET can serve as a tool to track Glu transport in glutaminolytic TNBC and guide treatment targeting GLS and redox balance leading to unmitigated oxidative stress and cell death. METHODS: A human glutaminolytic TNBC cell line (HCC1806) and a non-glutaminolytic estrogen-receptor positive cell line (MCF-7) were used to generate xenografts in athymic mice. Cells were exposed to culture media containing GLS inhibitor CB839 (1 µM), xCT inhibitor Erastin (ERA, 3 µM) and/or chemotherapy doxorubicin (DOX, 0.2 µM) for 24 h. ROS were measured by DHE and CellROX Green assays; early apoptosis was estimated by flow cytometry analysis of cells double-stained with Annexin V-FITC and TOR-PR-3. In vivo dynamic [18F]FSPG PET imaging (1 h) was performed on HCC1806 and MCF-7 xenograft models. Lipid peroxidation induced by treatments was examined using C11-Bodipy, a fluorescent sensor. RESULTS: ROS level in HCC1806 cells increased robustly with CB839 (20% over untreated cells), ERA (30%), or ERA plus CB839 (50%), whereas ROS responses in MCF-7 cells were more blunted. Compared to untreated cells, DOX treatment increased ROS level in HCC1806 cells by 40% whereas DOX combined with CB839 plus ERA led to a remarkable increase of cellular ROS by more than 200%. Combined treatment also led to 31% of cells undergoing early apoptosis, compared to 3% in DOX only and 2% in untreated cells. Time-activity curves (TACs) from [18F]FSPG PET of these xenograft models were fit by a reversible one-compartment model in keeping with the known biology of the tracer. The tumor-to-blood ratio (T/B) averaged from the last 6 points (30 min) of the TACs reveal a large difference between HCC1806 vs. MCF-7 by a factor &amp;gt; 2. C11-Bodipy signal in HCC1806 cells was not changed after CB839 but increased substantially after ERA only (45%) or DOX only (68%) treatment and after combined treatment of CB839, ERA and DOX (111%). DISCUSSIONS &amp; CONCLUSION: Our preliminary data support the efficacy of dual targeting of glutaminolysis and Glu transport for sensitizing glutaminolytic TNBC to cytotoxic chemotherapy. T/B of [18F]FSPG PET appears to be distinct for glutaminolytic versus non-glutaminolytic breast cancers. Future work will focus on developing [18F]FSPG PET, complementing our prior work with a Gln analog as a measure of Gln metabolism. The two PET tracers may guide treatment targeting both Gln and Glu usage to overcome chemoresistant TNBC. SUPPORT: R01CA211337 and Komen SAC130060. Reference: 1. Gross MI, et al. Mol Cancer Ther 2014;13(4):890-901. 2. Viswanath V, et al. J Nucl Med 2020. 3. Zhou R, et al. Cancer Res 2017;77(6):1476-84. Citation Format: Hoon Choi, Hsiaoju Lee, Austin Pantel, Christopher Hensley, David Mankoff, Rong Zhou. Disruption of redox balance in glutaminolytic triple negative breast cancer by inhibition of glutamate transport and glutaminase [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-18.

The role of synthetic coolants, WS-3 and WS-23, in modulating E-cigarette-induced reactive oxygen species (ROS) in lung epithelial cells

There has been a substantial rise in e-cigarette (e-cig) use or vaping in the past decade, prompting growing concerns about their adverse health effects. Recently, e-cig manufacturers have been using synthetic cooling agents, like WS-23 and WS-3, to provide a cooling sensation without the “menthol taste”. Studies have shown that aerosols/vapes generated by e-cigs can contain significant levels of reactive oxygen species (ROS). However, studies investigating the role of synthetic coolants in modulating ROS levels generated by e-cigs are lacking. This study seeks to understand how synthetic coolants, e-cig additives that have become increasingly prevalent in e-liquids sold in the United States (US), impact acellular ROS production from e-liquid aerosols as well as cellular ROS levels from pulmonary epithelial cells exposed to these e-liquids. To further explain, our study aims to understand whether the addition of WS-3 and WS-23 to e-liquid base and e-liquid base with nicotine significantly modifies generated acellular ROS levels within aerosolized e-liquids, as well as cellular ROS within BEAS-2B cells treated with these same e-liquids. Aerosols were generated from e-liquids with and without synthetic coolants through a single-puff aerosol generator; subsequently, acellular ROS was semi-quantified in H2O2 equivalents via fluorescence spectroscopy. Our acellular ROS data suggest that adding WS-3 to e-liquid base (PG:VG), regardless of nicotine content, has a minimal impact on modifying e-cig generated acellular ROS levels. Additionally, we also measured cellular ROS in lung epithelial cells using both e-liquids containing and not containing synthetic coolants via the CellROX Green fluorescent sensor. Similar comparable results were found in BEAS2B cells though ROS was increased by WS-3 and WS-23 treated in e-cig nicotine groups. Altogether, our data suggest that neither the addition of WS-23 nor WS-3 to e-liquid base solution, with and without nicotine, significantly modifies e-cig generated acellular ROS levels within aerosolized e-liquids and cellular ROS levels within treated BEAS-2B cells. Together, our data provide insight into whether synthetic coolants added to e-liquids could impact vaping-induced oxidative stress in the lungs.

Novel methods to detect ROS in viable spermatozoa of native semen samples

A crucial issue in male infertility work-up is to have reliable methods to detect oxidative stress in native semen samples. Here, we explored flow cytometric detection of Reactive Oxygen Species (ROS) in viable spermatozoa using native semen samples. To this aim, we challenged three fluorescent probes: CM-H2DCFDA, CellROX Green and MitoSOX Red. After excluding all non-sperm cells, each probe was coupled to a suitable stain to eliminate also semen apoptotic bodies and non-viable spermatozoa: Merocyanine 540 (M540) for CM-H2DCFDA and CellROX Green, and LIVE/DEAD Fixable Green Dead Cell Stain (LD-G) for MitoSOX Red. We found that CM-H2DCFDA was confined in the sperm midpiece, whereas CellROX Green and MitoSOX Red were localized in the head of spermatozoa. Treatment with H2O2 highly increased MitoSOX Red fluorescence (36.20 ± 5.24 vs 18.02 ± 2.25, %, p < 0.01), but not, or only slightly, the labelling with CMH2DCFDA (2.57 ± 1.70 vs 2.77 ± 1.43, p > 0.05) and CellROX Green (5.34 ± 3.18 vs 3.76 ± 2.04, p < 0.05), respectively. Menadione treatment highly increased CellROX Green (10.13 ± 5.85 vs 3.82 ± 2.70, p < 0.01) and MitoSOX Red (69.20 ± 27.14 vs 21.18 ± 7.96, %, p < 0.05), but not CM-H2DCFDA fluorescence (8.30 ± 11.56 vs 7.30 ± 9.19, p > 0.05). Further, only MitoSOX Red was able to detect spontaneous ROS generation during in vitro sperm incubation. We also detected DNA fragmentation by Comet and SCD Assay after sorting MitoSOX Red positive and negative sperm viable fractions. Results indicated that MitoSOX labelling in viable spermatozoa was strictly associated to sperm DNA fragmentation. In conclusion, MitoSOX Red/LD-G appears to be a promising method to detect oxidative stress in human semen for male infertility work-up.

Детекция окислительного стресса в артериальных эндотелиальных клетках человека при воздействии кальций-фосфатных бионов

Введение. Кальций-фосфатные бионы (КФБ) формируются в организме человека при перенасыщении сыворотки ионами кальция и фосфора и вызывают дисфункцию эндотелия, однако молекулярные механизмы нарушения функционирования эндотелия при воздействии КФБ не ясны. Цель исследования - выяснение роли кальций-фосфатных бионов различной формы в развитии окислительного стресса в артериальных эндотелиальных клетках (ЭК) человека. Методика. Для детекции окислительного стресса к конфлюэнтным культурам первичных ЭК коронарной и внутренней грудной артерии человека добавляли равные концентрации КФБ сферической или игольчатой формы (СКФБ и ИКФБ соответственно) с последующим культивированием в течение 1 и 4 ч, добавлением флюоресцентных индикаторов окислительного стресса MitoSOX Red и CellROX Green и конфокальной микроскопией. Измеряли концентрацию продуктов перекисного окисления липидов в культуральной жидкости через 24 ч экспозиции эндотелиальных клеток КФБ. Анализ нейтрализации цитотоксических эффектов перекисного окисления липидов проводили путем добавления к ЭК супероксиддисмутазы и каталазы на 4 или 24 ч (одновременно с КФБ). Для сравнения механизмов клеточной гибели при воздействии СКФБ и ИКФБ анализировали цитотоксичность обоих типов бионов при одновременном воздействии лизосомального ингибитора бафиломицина А1. Результаты. Значимого увеличения генерации активных форм кислорода (АФК) в результате экспозиции СКФБ (независимо от линии ЭК и продолжительности экспозиции) не было выявлено. В то же время наблюдалось повышение генерации супероксида через 4 ч, а иных свободных радикалов через 1 ч после добавления ИКФБ к ЭК. Предварительная нейтрализация АФК супероксиддисмутазой и каталазой частично защищала ЭК от индуцируемой ИКФБ гибели. При этом добавление бафиломицина А1 к ЭК частично защищало их от гибели только при воздействии СКФБ, но не ИКФБ. Заключение. Гибель ЭК при воздействии СКФБ происходит в результате первичного повреждения лизосом, а при воздействии ИКФБ - в первую очередь вследствие окислительного стресса. Background. Calcium phosphate bions (CPB) form in the human blood upon its supersaturation with calcium and phosphate and provoke endothelial dysfunction; however, the molecular mechanisms of these pathological processes remain unclear. Aim. To elucidate the role of differently shaped CPBs in induction of oxidative stress in human arterial endothelial cells (Ecs). Methods. For detection of oxidative stress, equal concentrations of spherical CPB (CPB-S) or needle-shaped CPB (CPB-N) were added to confluent cultures of primary human coronary artery and internal thoracic artery ECs for 1 and 4 h; this was followed by MitoSOX Red and CellROX Green staining and subsequent confocal microscopy. Concentration of thiobarbituric acid-reactive substances was measured in the EC culture supernatant at 24 h of the CPB exposure. The lipid peroxidation cytotoxicity was neutralized by adding superoxide dismutase and catalase to ECs for 4 or 24 h. To compare cell death subroutines induced by CPB-S and CPB-N, the effect of bafilomycin A1, a lysosomal inhibitor, on CRB cytotoxicity was studied. Results. No increase in reactive oxygen species generation was observed in the CPB-S exposure, regardless of the EC line and exposure duration. However, addition of CPB-N to ECs increased the production of superoxide and other free radicals after four- and one-hour exposure, respectively. Prior neutralization of reactive oxygen species with superoxide dismutase and catalase partially protected ECs from CPB-N- but not CPB-S-induced death while bafilomycin A1, vice versa, protected ECs from CPB-S- but not CPB-N-induced death. Conclusion. CPB-S cause cell death due to primary damage of lysosomes whereas CPB-N induce apoptosis due to oxidative stress.

Ram semen quality can be assessed by flow cytometry several hours after post-fixation.

Ram spermatozoa are very sensitive to any cold shock or oxidative damage, therefore making them unsuitable for prolonged storage or distant transport to specialized laboratories for flow-cytometric analysis. The aim of this study was to stain ram semen samples with several fluorescent markers and analyse their stability during formaldehyde fixation. Briefly, freshly collected semen samples were stained for apoptosis (annexin V-FITC, YO-PRO™-1 and FLICA), acrosomal damage (PNA-AF488 and FITC-conjugated antibody against GAPDHS), mitochondrial activity (Mitotracker probes), oxidative damage [dihydroethidium (DHE) and CellROX™ Green] and cell viability (live/dead fixable viability dyes). Next, samples were fixed in buffer containing formaldehyde and then washed. Stained sample were analyzed using flow cytometer before fixation, immediately after fixation, and at 5 h and 20 h post-fixation. Fluorescent signals and the proportion of positively stained spermatozoa were compared statistically in fresh and post-fixed samples. All examined markers, except YO-PRO-1 (decreased significantly, P < 0.05), retained their fluorescence intensities after fixation. In conclusion, several tested markers were able to withstand formaldehyde fixation of ram semen samples as follows: annexin V and FLICA for apoptosis; PNA for acrosomal status; MitoTracker Red CMXRos for mitochondrial activity; and CellROX Green for oxidative status in combination with a suitable live/dead fixable viability dye. This optimized methodology could help to comprehensively analyse the quality of ram semen from local farms countrywide.