Lower ROS Levels Research Articles

Initial biological evaluations of 18F-KS1, a novel ascorbate derivative to image oxidative stress in cancer

BackgroundReactive oxygen species (ROS)-induced oxidative stress damages many cellular components such as fatty acids, DNA, and proteins. This damage is implicated in many disease pathologies including cancer and neurodegenerative and cardiovascular diseases. Antioxidants like ascorbate (vitamin C, ascorbic acid) have been shown to protect against the deleterious effects of oxidative stress in patients with cancer. In contrast, other data indicate potential tumor-promoting activity of antioxidants, demonstrating a potential temporal benefit of ROS. However, quantifying real-time tumor ROS is currently not feasible, since there is no way to directly probe global tumor ROS. In order to study this ROS-induced damage and design novel therapeutics to prevent its sequelae, the quantitative nature of positron emission tomography (PET) can be harnessed to measure in vivo concentrations of ROS. Therefore, our goal is to develop a novel translational ascorbate-based probe to image ROS in cancer in vivo using noninvasive PET imaging of tumor tissue. The real-time evaluations of ROS state can prove critical in developing new therapies and stratifying patients to therapies that are affected by tumor ROS.MethodsWe designed, synthesized, and characterized a novel ascorbate derivative (E)-5-(2-chloroethylidene)-3-((4-(2-fluoroethoxy)benzyl)oxy)-4-hydroxyfuran-2(5H)-one (KS1). We used KS1 in an in vitro ROS MitoSOX-based assay in two different head and neck squamous cancer cells (HNSCC) that express different ROS levels, with ascorbate as reference standard. We radiolabeled 18F-KS1 following 18F-based nucleophilic substitution reactions and determined in vitro reactivity and specificity of 18F-KS1 in HNSCC and prostate cancer (PCa) cells. MicroPET imaging and standard biodistribution studies of 18F-KS1 were performed in mice bearing PCa cells. To further demonstrate specificity, we performed microPET blocking experiments using nonradioactive KS1 as a blocker.ResultsKS1 was synthesized and characterized using 1H NMR spectra. MitoSOX assay demonstrated good correlations between increasing concentrations of KS1 and ascorbate and increased reactivity in SCC-61 cells (with high ROS levels) versus rSCC-61cells (with low ROS levels). 18F-KS1 was radiolabeled with high radiochemical purity (> 94%) and specific activity (~ 100 GBq/μmol) at end of synthesis (EOS). Cell uptake of 18F-KS1 was high in both types of cancer cells, and the uptake was significantly blocked by nonradioactive KS1, and the ROS blocker, superoxide dismutase (SOD) demonstrating specificity. Furthermore, 18F-KS1 uptake was increased in PCa cells under hypoxic conditions, which have been shown to generate high ROS. Initial in vivo tumor uptake studies in PCa tumor-bearing mice demonstrated that 18F-KS1 specifically bound to tumor, which was significantly blocked (threefold) by pre-injecting unlabeled KS1. Furthermore, biodistribution studies in the same tumor-bearing mice showed high tumor to muscle (target to nontarget) ratios.ConclusionThis work demonstrates the strong preliminary support of 18F-KS1, both in vitro and in vivo for imaging ROS in cancer. If successful, this work will provide a new paradigm to directly probe real-time oxidative stress levels in vivo. Our work could enhance precision medicine approaches to treat cancer, as well as neurodegenerative and cardiovascular diseases affected by ROS.

Antioxidative effect of melatonin on cryopreserved chicken semen

This is a unique study because is the first time we are adding melatonin into an extender in order to determine its influence on cryopreserved chicken semen. The primary focus of our present study was to evaluate the influence of different concentrations of Melatonin on cryopreserved chicken semen. Semen samples were allocated into four treatments, being one control and three different combinations of antioxidants and after the freeze-thaw operation, the sperm motility, plasma membrane integrity, acrosome integrity, endogenous enzymes (GSH-Px, CAT, SOD), MDA and ROS of chicken spermatozoa were all evaluated. The collection of the semen samples was from 40 Arbor Acre roosters and this procedure was repeated twice a week and then mixed in an extender that contained different MEL treatments as follows: a diluent without MEL (control, M 0), a diluent comprising 0.125 mg/mL (M 0.125) 0.25 mg/mL, (M 0.25) and 0.5 mg/mL (M 0.5). It was revealed that the supplementation of the base extender with an optimal 0.25 mg/mL MEL led to a higher significant difference in the motility of chicken sperm (P < 0.01), higher acrosome integrity (P < 0.05) and a higher plasma membrane integrity (P < 0.01) when compared to the control group at post-thaw. Furthermore, when compared to the control group, 0.25 mg/mL MEL addition into the extender significantly enhanced the activity of endogenous enzymes (GSH-Px, CAT, and SOD) in the chicken spermatozoa at post-thaw (P < 0.05). Moreover, 0.5 mg/mL MEL supplementation into the extender enhanced the GSH-Px activity in the chicken spermatozoa when compared with the control group (P < 0.05) at post-thaw. In contrast, the addition of 0.25 mg/mL MEL into the extender resulted in a significantly lower MDA in comparison to the 0.125 mg/mL, 0.5 mg/mL MEL treatment group and the control group (P < 0.05). Also, compared to the control group, MEL concentration of 0.125 mg/mL and 0.5 mg/mL MEL into the extender resulted in a significantly low ROS concentration (P < 0.05) but the addition of 0.25 mg/mL MEL concentration resulted in a significantly lower ROS level when compared to the control group (P < 0.01). In summary, MEL improved the quality of cryopreserved chicken sperm quality by decreasing oxidative stress level and the most optimal concentration was 0.25 mg/mL.

Mitochondrial Topoisomerase Dysfunction Contributes to Myocardial Mitochondrial Dysfunction and Mitochondrial DNA (mtDNA) Degradation

Diabetes promotes mtDNA degradation as an antecedent event to cardiomyocyte cell death and we have reported that mitochondrial topoisomerase dysfunction participates in this pathway. In post mitotic cells, the mitochondrial genome continues to replicate about once a month and resolving the topology of mtDNA replication would be almost insurmountable in the absence of the mitochondrial topoisomerases. It remains unclear how chronic hyperglycemia induces this shift in mitochondrial topoisomerase function from physiologic to pathologic. To that end, we hypothesize that low levels of ROS alter mitochondrial topoisomerase function to accelerate mtDNA damage. DNA relaxation of supercoiled DNA is a hallmark measurement of topoisomerase function. Supercoiled DNA incubated with isolated myocardial mitochondria or purified topoisomerase 1 generated the same isopane pattern of relaxation. In the absence of mitochondrial extracts, H2O2 (125–1000 μM) did not alter DNA relaxation of supercoiled DNA. In contrast, in the presence of myocardial mitochondrial extracts, H2O2 (125–1000 μM) significantly increased DNA relaxation in a dose dependent manner. Similar results were obtained following the DNA cleavage protocol. Using the iron chelator EDTA, we determined that DNA relaxation was not due to a Fenton effect. Following immunoprecipitation using mtTop1 or Topo‐2beta antibodies, mitochondrial extracts retained DNA relaxation function indicating the overlapping presence of functional mitochondrial topoisomerases. Using isolated intact mitochondria, incubation with H2O2 significantly increased mitochondrial superoxide levels; an effect that could be blocked with MitoTempo (mitochondrial specific antioxidant). NOX4 is a member of the NADPH oxidase family, but is unique in its preferential generation of H2O2 over superoxide (O2¾). Others have reported that NOX4 is localized to the outer mitochondrial membrane. Overexpression of NOX4 in H9c2 cells exacerbated intracellular and mitochondrial superoxide levels when incubated in the present of high glucose. Diabetes increases pentose shunt pathway activity, which in turn may fuel mitochondrial localized NOX4 to promote the generation of H2O2 a diffusible ROS species. Collectively our results suggest that increased mitochondrial ROS presentation alters mitochondrial topoisomerase function. Separate from a direct impact of oxidative stress, our studies find that alteration of mitochondrial topoisomerase function accelerates and propagates an increase in mtDNA damage. These findings indicate that the functional state of the mitochondrial topoisomerases participates in the pathophysiology leading to the development of diabetic cardiomyopathy.Support or Funding InformationSupported in part by NIH RO3HD065555101 and TCUS Biomedical Education FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparative physiological and metabolomic analyses reveal natural variations of tulip in response to storage temperatures

Three tulip cultivars were screened out with successful bloom after a short-term cold treatment, and the differential responses to postharvest cold treatment were analyzed between two contrasting tulip cultivars. Tulip is one of the most important ornamental bulbous plants in the world. A precious precooling treatment during bulb postharvest is required for optimal floral stalk elongation and flower development in tulip. In this study, the naturally growing and flowering variations of tulip to storage temperatures were analyzed after long-term cold (LTC) and short-term cold (STC) treatments. Three cultivars were screened out with successful blooming after STC, which included 'Dow Jones' (DJ), 'Van Eijk' (VE) and 'World's Favourite' (WF) (5°C for 2weeks). Comparative analysis revealed that DJ cultivar maintained normal and intact reproductive organs under STC condition, while the 'Orange Emperor' (OE) cultivar, which failed blooming after STC treatment, showed gradually destroyed reproductive organs under STC condition. In addition, the DJ cultivar accumulated lower ROS levels and higher antioxidant enzyme activities, as well as significantly higher contents of total primary metabolites than OE to maintain normal shoot growth and floral organ development under STC condition. The relative expression levels of genes involved in vernalization and/or flower time regulation in DJ were significantly higher than those in OE after STC treatment. This study provides new insights into understanding the underlying mechanism of natural variation of tulip cultivars during postharvest storage treatment.

Promoted Proliferation of Hematopoietic Stem Cells Enabled by a Hyaluronic Acid/Carbon Nanotubes Antioxidant Hydrogel

AbstractReactive oxygen species (ROS) level is closely associated with the physiological function of hematopoietic stem cells (HSCs) in ex vivo culture systems. Previously developed hydrogels in use of HSCs culture have generally failed in considering their antioxidant property. In the present work, antioxidant hydrogels are constructed to evaluate the biological effect of ROS on HSCs proliferation by suppressing oxidative stress damage. Functionalized carbon nanotubes (CNTs), which act as potential antioxidants, are physically encapsulated in biocompatible hyaluronic acid (HA) hydrogels to achieve long‐term antioxidant activity. Consequently, these hybrid hydrogels exhibit enhanced physical properties and superior scavenging capability on oxides and peroxides compared to the pure HA hydrogel. The results indicate that ROS significantly inhibit the biological characteristics of HSCs. Nevertheless, the proliferation ability and pluripotency are dramatically improved when the culture system is supplied with the antioxidant hydrogel, revealing that the CNT‐incorporated hydrogel can effectively relieve oxidative stress response in HSCs and reduce apoptosis from ROS. Therefore, the HA/CNT hydrogel can provide a novel strategy to establish an artificial microenvironment with a low ROS level for HSCs proliferation.

Hepatotoxicity of perfluorooctanoic acid and two emerging alternatives based on a 3D spheroid model

Perfluorooctanoic acid (PFOA) toxicity is of considerable concern due to its wide application, environmental persistence, and bioaccumulation. In the current study, we used a scaffold-free three-dimensional (3D) spheroid model of mouse liver cells (AML12) to explore the toxicity of PFOA and emerging alternatives (HFPO-DA and PFO4DA). Comparing the short-term (24 and 72 h treatment) toxicity of PFOA between conventional 2D monolayer cells and 3D spheroids, we found that spheroids had higher EC50 values and lower ROS levels after treatment, indicating their greater resistance to PFOA. Cell viability (i.e., adenosine triphosphate (ATP) content and lactate dehydrogenase (LDH) leakage) and liver-specific function (i.e., albumin secretion) were stable in spheroids through 28 day of culture. However, under 100 and 200 μM-PFOA treatment for 28 day, ROS levels, LDH leakage, and caspase3/7 activity all increased significantly. As a sensitive parameter, ROS showed a significant increase at 21 day, even in the 50 μM-PFOA group. Consistent with the elevation of ROS and caspase3/7, the expressions of oxidative stress- and apoptosis-related genes, including Gsta2, Nqo1, Ho-1, caspase3, p53, and p21, were induced in dose- and time-dependent manners after PFOA exposure. The peroxisome proliferator-activated receptor alpha (PPARα) pathway was also activated after treatment, with significant induction of its target genes, Fabp4 and Scd1. Similar to PFOA, both HFPO-DA and PFO4DA activated the PPARα pathway, induced ROS levels, and initiated cell damage, though at a relatively lower extent than that of PFOA. Our results imply that the 3D spheroid model is a valuable tool in chronic toxicological studies.

Anti-inflammatory and immune-modulating effects of rice callus suspension culture (RCSC) and bioactive fractions in an in vitro inflammatory bowel disease model

BackgroundRice callus suspension culture (RCSC) has been shown to exhibit potent antiproliferative activity in multiple cancer cell lines. RCSC and its bioactive compounds can fill the need for drugs with no side effects. Hypothesis/PurposeThe anti-inflammatory potential of RCSC and its bioactive fractions on normal colon epithelial cell lines, was investigated. Study designThree cell lines, InEpC, NCM356 and CCD841-CoN were treated with proinflammatory cytokines followed by RCSC. Cytoplasmic and nuclear ROS were assayed with fluorescent microscopy and flow cytometer. Expression analysis of immune-related genes was performed in RCSC-treated cell lines. RCSC was fractionated using column chromatography and HPLC. Pooled fractions 10–18 was used to test for antiproliferative activity using colon adenocarcinoma cell line, SW620 and anti-inflammatory activity using CCD841-CoN. Mass spectrometric analysis was performed to identify candidate compounds in four fractions. ResultsRCSC treatment showed differential effects with higher cytoplasmic ROS levels in NCM356 and CCD841-CoN and lower ROS levels in InEpC. Nuclear generated ROS levels increased in all three treated cell lines. Flow cytometry analysis of propidium iodide stained cells indicated mitigation of cell death caused by inflammation in RCSC treated groups in both NCM356 and CCD841-CoN. Genes encoding transcription factors and cytokines were differentially regulated in NCM356 and CCD841-CoN cell lines treated with RCSC which provided insights into possible pathways. Analysis of pooled fractions 10–18 by HPLC identified 8 peaks. Cell viability assay with fractions 10–18 using SW620 showed that the number of viable cells were greatly reduced which was similar to 6X and 33X RCSC with very little effect on normal cells which similar to 1X RCSC. RCSC fractions increased nuclear and cytoplasmic ROS vs. both untreated and inflammatory control. Analysis of four fractions by mass spectrometry identified 4-deoxyphloridzin, 5′-methoxycurcumin, piceid and lupeol as candidate compounds which are likely to be responsible for the antiproliferative, anti-inflammatory and immune-regulating properties of RCSC. ConclusionRCSC and its fractions showed anti-inflammatory activity on inflamed colon epithelial cells. Downstream target candidate genes which are likely to mediate RCSC effects were identified. Candidate compounds responsible for the antiproliferative and anti-inflammatory activity of RCSC and its fractions provide possible drug targets.

Blood-Brain Barrier Cellular Responses Toward Organophosphates: Natural Compensatory Processes and Exogenous Interventions to Rescue Barrier Properties.

Organophosphorus compounds (OPs) are highly toxic chemicals widely used as pesticides (e.g., paraoxon (PX)- the active metabolite of the insecticide parathion) and as chemical warfare nerve agents. Blood-brain barrier (BBB) leakage has been shown in rodents exposed to PX, which is an organophosphate oxon. In this study, we investigated the cellular mechanisms involved in BBB reaction after acute exposure to PX in an established in vitro BBB system made of stem-cell derived, human brain-like endothelial cells (BLECs) together with brain pericytes that closely mimic the in vivo BBB. Our results show that PX directly affects the BBB in vitro both at toxic and non-toxic concentrations by attenuating tight junctional (TJ) protein expression and that only above a certain threshold the paracellular barrier integrity is compromised. Below this threshold, BLECs exhibit a morphological coping mechanism in which they enlarge their cell area thus preventing the formation of meaningful intercellular gaps and maintaining barrier integrity. Importantly, we demonstrate that reversal of the apoptotic cell death induced by PX, by a pan-caspase-inhibitor ZVAD-FMK (ZVAD) can reduce PX-induced cell death and elevate cell area but do not prevent the induced BBB permeability, implying that TJ complex functionality is hindered. This is corroborated by formation of ROS at all toxic concentrations of PX and which are even higher with ZVAD. We suggest that while lower levels of ROS can induce compensating mechanisms, higher PX-induced oxidative stress levels interfere with barrier integrity.

Abstract 281: The Calcineurin/Rcan1 Axis Influences Mitochondrial Dynamics, Metabolism, and Biogenesis

Rationale and Objective: The Regulator of Calcineurin 1 (RCAN1) inhibits calcineurin (CN), a Ca 2+ -activated protein phosphatase important in cardiac remodeling. The hearts and brains of Rcan1 KO mice are more susceptible to damage from ischemia/reperfusion (I/R). As mitochondria are known to be key mediators of I/R damage, the goal of our studies was to determine the impact of the CN/RCAN1 signaling axis on the mitochondrial network and function. Methods and Results: Using both neonatal and isolated adult cardiomyocytes, we found that, the mitochondrial network was more fragmented in the absence of RCAN1, due to increased CN-dependent activation of the fission protein DRP1. RCAN1-deficient cardiomyocytes had reduced mitochondrial membrane potential, consumed less O 2 , and generated lower levels of ROS. Importantly, they also displayed a reduced capacity for mitochondrial Ca 2+ uptake. RCAN1-depleted cardiomyocytes were more sensitive to I/R, however, pharmacological inhibition of CN, DRP1, or calpains (Ca 2+ -activated proteases) restored protection, suggesting that, in the absence of RCAN1, calpain-mediated damage following I/R is greater because of a decrease in the ability of mitochondria to buffer cytoplasmic Ca 2+ . Over-expression of RCAN1 was sufficient to enhance fusion. In addition to its impact on mitochondrial dynamics, CN activation also promoted mitochondrial biogenesis. We demonstrate that expression of the 1b isoform of PGC1α (a master regulator of mitochondrial biogenesis) is directly under CN control . In vivo, a cardiomyocyte-specific CN transgene caused a more rapid increase in: transcript levels for P gc 1α-1b and PolG (the mitochondrial DNA polymerase), mtDNA content, and the abundance of electron transport complex proteins. Despite the increase in mitochondrial content, changes in metabolic enzymes were consistent with a shift toward glycolysis rather than fatty acid oxidation. Conclusions: The CN/RCAN1 signaling axis helps determine both the number of mitochondria (DRP1-dependent fission) and the total mitochondrial mass ( Pgc1α -dependent biogenesis). Maintaining the proper level of these two processes is essential to cardiovascular health and metabolic flexibility.

PO-125 Role of NRF2 in breast cancer stem cells resistance and tumourrecurrence

IntroductionAdvancements in radio/chemo therapies have significantly decreased the mortality rate in breast cancer patients; however, resistance and recurrence of the tumour are major concerns with these therapies. Recurrence after radiation therapy is initiated by a subpopulation that is highly radio resistant, known as cancer stem cells (CSCs). One of the main ways of radio therapy to kill cancer cells is by increasing the cellular oxidative stress. Due to higher metabolic and proliferative rate, cancer cells become well-adapted to intrinsic oxidative stress. CSCs are quiescent and work under low levels of oxidative stress It is assumed that CSCs escape radio therapy by modulating redox homeostasis. Nrf2 pathway is a central cellular pathway that protects against oxidative stress. Nrf2 plays a dual role in cancer protection as well as cancer progression by up-regulating the expression of cytoprotective genes and reducing oxidative stress. In our study, we hypothesised that Nrf2 signalling pathway might be involved in radio resistance and low ROS levels in Breast CSCs (BCSCs).Material and methodsMCF-7 and MDA-MB-231 cell lines were used for CSC isolation and CSCs were characterised by CD44+/CD24-/ESA+, ALDH+ activity. BCSCs were irradiated with fractionated dose of γ-radiation. Protein and mRNA levels were analysed by western blotting and real time-PCR respectively. Radio resistance was checked by colony formation assays.Results and discussionsWe observed an increase in the CD44+/CD24-/ESA+, ALDH+ population and expression of embryonic stem cell markers after fractionated irradiation. BCSCs and non-BCSCs were isolated as CD44+CD24-ESA+ and CD44-CD24+ESA-. We examined the ROS levels in BCSCs and non-BCSCs after fractionated irradiation. ROS levels were found to be decreased in BCSCs as compare to non-BCSCs after fractionated dose irradiation. We checked the radio-resistance in irradiated cells via radio-resistance assay. More numbers of radio-resistant colonies were formed in the fractionated dose irradiated BCSCs than non-BCSCs. Expression levels of Nrf2 and its downstream regulators were checked in fractionated dose irradiation and were found to be elevated in BCSCs irradiated with fractionated dose than non-BCSCs.ConclusionBCSCs have low levels of ROS and high levels of antioxidants which may contribute to the radio-resistance of BCSCs. Elucidation of role of Nrf2 in BCSCs resistance and tumour recurrence may provide an effective strategy to overcome radio-resistance.

Suppression of ROS generation mediated by higher InsP3 level is critical for the delay of seed germination in lpa rice

The breeding of low phytic acid (lpa) crop is considered as an effective strategy to improve crop nutrition, but these crops have an inferior performance in seed germination. In this paper, two rice genotypes, cv. 9311 and its lpa mutant (9311-lpa), were used to clarify the relationship of InsP3 and ROS concentration in sprouting seeds with the occurrence of retarded seed germination for lpa crop. Results showed that the inferior performance of seed germination for 9311-lpa was mostly characterized by the delay of seed germination progression, with insignificant difference in seed germination rate between the two genotypes after a longer imbibition duration. The retarded seed germination of 9311-lpa was closely associated with the genotypic difference in InsP3 and ROS concentrations in germinating seeds, with the considerably higher InsP3 level and lower ROS concentration for 9311-lpa relative to the wild type. Under an appropriate range of concentration, exogenous H2O2 treatment promoted seed germination, while the activator of InsP3 (m-3M3FBS) had an inhibitory effect on seed germination. The delay of seed germination occurred mainly as the results of the higher InsP3 level and the lower ROS concentration in the germinating seeds of 9311-lpa. Furthermore, the germinating seeds of 9311-lpa had relatively lower activities of ROS scavenging enzymes (SOD, CAT, POD, and APX) and ROS generating enzyme (NOX) than those of the wild type. m-3M3FBS significantly suppressed NOX activity and NOX transcripts in sprouting seeds, which then resulted in the lowered ROS generation in sprouting seeds and the decreased seed germination rate. Thus, the retarded seed germination of 9311-lpa, with lower ROS level in germinating seeds, was mostly caused by the weaker ability of ROS generation in the presence of higher InsP3 level, rather than the effective detoxification of ROS burst during seed germination.

ROS-independent ER stress-mediated NRF2 activation promotes warburg effect to maintain stemness-associated properties of cancer-initiating cells

Cancer-initiating cells (CICs) are responsible for tumor initiation, progression, and therapeutic resistance; moreover, redox homeostasis is important in regulating cancer stemness. Previously, we have identified that cancer cells containing low intracellular reactive oxygen species levels (ROSLow cells) display enhanced features of CICs. However, the specific metabolic signatures of CICs remain unclear and are required for further characterization by systemic screenings. Herein, we first showed CICs mainly relying on glycolysis that was important for the maintenance of stemness properties. Next, we revealed that NRF2, a master regulator of antioxidants, was able to maintain low intracellular ROS levels of CICs, even though in the absence of oxidative stress. We further characterized that NRF2 activation was required for the maintenance of CICs properties. Of ROSLow cells, NRF2 activation not only directly activates the transcription of genes encoding glycolytic enzymes but also inhibited the conversion of pyruvate to acetyl-CoA by directly activating pyruvate dehydrogenase kinase 1 (PDK1) to lead to inhibition of tricarboxylic acid (TCA) cycle; therefore, to promote Warburg effect. A positive regulatory ROS-independent ER stress pathway (GRP78/p-PERK/NRF2 signaling) was identified to mediate the metabolic shift (Warburg effect) and stemness of CICs. Lastly, co-expression of p-PERK and p-NRF2 was significantly associated with the clinical outcome. Our data show that NRF2 acting as a central node in the maintenance of low ROS levels and stemness associated properties of the CICs, which is significantly associated with the clinical outcome, but independent from ROS stress. Future treatments by inhibiting NRF2 activation may exhibit great potential in targeting CICs.

Murine Bone Marrow Mesenchymal Stromal Cells Respond Efficiently to Oxidative Stress Despite the Low Level of Heme Oxygenases 1 and 2.

Mesenchymal stromal cells (MSCs) are heterogeneous cells from adult tissues that are able to differentiate in vitro into adipocytes, osteoblasts, or chondrocytes. Such cells are widely studied in regenerative medicine. However, the success of cellular therapy depends on the cell survival. Heme oxygenase-1 (HO-1, encoded by the Hmox1 gene), an enzyme converting heme to biliverdin, carbon monoxide, and Fe2+, is cytoprotective and can affect stem cell performance. Therefore, our study aimed at assessing whether Hmox1 is critical for survival and functions of murine bone marrow MSCs. Both MSC Hmox1+/+ and Hmox1-/- showed similar phenotype, differentiation capacities, and production of cytokines or growth factors. Hmox1+/+ and Hmox1-/- cells showed similar survival in response to 50 μmol/L hemin even in increased glucose concentration, conditions that were unfavorable for Hmox1-/- bone marrow-derived proangiogenic cells (BDMC). Hmox1+/+ MSCs but not fibroblasts retained low ROS levels even after prolonged incubation with 50 μmol/L hemin, although both cell types have a comparable Hmox1 expression and similarly increase its levels in response to hemin. MSCs Hmox1-/- treated with hemin efficiently induced expression of a vast panel of antioxidant genes, especially enzymes of the glutathione pathway. Innovation and Conclusion: Hmox1 overexpression is a popular strategy to enhance viability and performance of MSCs after the transplantation. However, murine MSCs Hmox1-/- do not differ from wild-type MSCs in phenotype and functions. MSC Hmox1-/- show better resistance to hemin than fibroblasts and BDMCs and rapidly react to the stress by upregulation of quintessential genes in antioxidant response. Antioxid. Redox Signal. 00, 000-000.

Oxidative stress and aging: Learning from yeast lessons

The yeast Saccharomyces cerevisiae has played a vital role in the understanding of the molecular basis of aging and the relationship of aging process with oxidative stress (non-homeostatic accumulation of Reactive Oxygen Species, ROS). The mammalian and yeast antioxidant responses are similar and over 25 % of human-degenerative disease related genes have close homologues in yeast. The reduced genetic redundancy of yeast facilitates visualization of the effect of a deleted or mutated gene. By manipulating growth conditions, yeast cells can survive only fermenting (low ROS levels) or respiring (increased ROS levels), which facilitates the elucidation of the mechanisms involved with acquisition of tolerance to oxidative stress. Furthermore, the yeast databases are the most complete of all eukaryotic models. In this work, we highlight the value of S. cerevisiae as a model to investigate the oxidative stress response and its potential impact on aging and age-related diseases.

MiRNA alteration is an important mechanism in sugarcane response to low-temperature environment

BackgroundCold is a major abiotic stress limiting the production of tropical and subtropical crops in new production areas. Sugarcane (Saccharum spp.) originates from the tropics but is cultivated primarily in the sub-tropics where it frequently encounters cold stress. Besides regulating plant growth, miRNAs play an important role in environmental adaption.ResultsIn this study, a total of 412 sugarcane miRNAs, including 261 known and 151 novel miRNAs, were obtained from 4 small RNA libraries through the Illumina sequencing method. Among them, 62 exhibited significant differential expression under cold stress, with 34 being upregulated and 28 being downregulated. The expression of 13 miRNAs and 12 corresponding targets was validated by RT-qPCR, with the majority being consistent with the sequencing data. GO and KEGG analysis indicated that these miRNAs were involved in stress-related biological pathways. To further investigate the involvement of these miRNAs in tolerance to abiotic stresses, sugarcane miR156 was selected for functional analysis. RT-qPCR revealed that miR156 levels increased in sugarcane during cold, salt and drought stress treatments. Nicotiana benthamiana plants transiently overexpressing miR156 exhibited better growth status, lower ROS levels, higher anthocyanin contents as well as the induction of some cold-responsive genes, suggesting its positive role in the plant cold stress response.ConclusionsThis study provides a global view of the association of miRNA expression with the sugarcane response to cold stress. The findings have enriched the present miRNA resource and have made an attempt to verify the involvement of miR156 in plant response to cold stress.

PDK1 plays a vital role on hematopoietic stem cell function

3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a pivotal regulator in the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway that have been shown to play key roles in the functional development of B and T cells via activation of AGC protein kinases during hematopoiesis. However, the role of PDK1 in HSCs has not been fully defined. Here we specifically deleted the PDK1 gene in the hematopoietic system and found that PDK1-deficient HSCs exhibited impaired function and defective lineage commitment abilities. Lack of PDK1 caused HSCs to be less quiescent and to produce a higher number of phenotypic HSCs and fewer progenitors. PDK1-deficient HSCs were also unable to reconstitute the hematopoietic system. Notably, HSC function was more dependent on PDK1 than on mTORC2, which indicates that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function. PDK1-deficient HSCs also exhibited reduced ROS levels, and treatment of PDK1-deficient HSCs with L-butathioninesulfoximine in vitro elevated the low ROS level and promoted colony formation. Therefore, PDK1 appears to contribute to HSC function partially via regulating ROS levels.

Ectopic expression of CSD1 and CSD2 targeting genes of miR398 in grapevine is associated with oxidative stress tolerance.

MicroRNAs (miRNAs) are endogenous small RNAs of -21 nucleotides that play an important role in diverse plant physiological processes at the post-transcriptional level by directing mRNA cleavage or translational inhibition. Previous studies have indicated that down-regulation of miR398 in response to oxidative stress allows up-regulation of the two target genes, cytosolic CSD1 and chloroplastic CSD2 (copper/zinc superoxide dismutase), resulting in protecting the plants to tolerate oxidative stress. In this study, we provide evidence that grapevine miR398 (Vv-miR398), by regulating the expression of its target genes, VvCSD1 and VvCSD2, mediates responses of grapevine to copper (Cu) stress which have been magnified due to increase in Cu-containing pesticide application. The expression of Vv-miR398 was inhibited by different concentrations of Cu stress; on the other hand, there was a steady increase in the activity of VvCSD1 and VvCSD2 genes. The function of VvCSD1 and VvCSD2 under Cu stress was thoroughly examined by overexpressing the use of the VvCSD1 and VvCSD2 in transgenic tobacco (Nicotiana tabacum). We found that both the overexpressed transgenic lines had lower Cu sensitivity and higher Cu tolerance compared with the wild type. In addition, lower levels of ROS and higher levels of SOD activities were accumulated in the transgenic lines in comparison with the wild type under the higher Cu conditions. Furthermore, these transgenic tobacco lines also recorded a higher UV and salt tolerance than the WT plants. These results suggested that overexpressing the VvCSDs will enhance the ROS-scavenging systems and protect the plant against more oxidative damage. Also, more investigations in this line are needed that would provide significant improvements in our understanding the resistance of fruit crops to environmental stress.

Abstract LB-212: Antioxidant therapy reprograms macrophages from protumor M2 to antitumor M1

Abstract Macrophages (Mϕ) have high plasticity and can polarize to two extremes, pro-inflammatory M1 Mϕ and immuno-suppressive M2 Mϕ. In triple negative breast cancer, tumor-associated Mϕ (TAM) have a pro-tumor M2-like phenotype and are associated with decreased survival. Thus, it is vital to develop treatments to reprogram TAM away from protumor M2 Mϕ toward antitumor M1 Mϕ. A recent study reported a lipid peroxide scavenger selectively inhibits M2 Mϕ markers and cytokines. However, little is known about the differences in ROS metabolism between M1 Mϕ and M2 Mϕ. Our hypothesis is pro-tumorigenic M2 Mϕ possess a distinct profile of pro- and anti-oxidant enzymes, as well as a different sensitivity to ROS modulation versus the anti-tumor M1 Mϕ. Our results show M2 Mϕ, derived from primary human monocytes, have a significant 20-30% reduction in intracellular ROS levels versus M1 Mϕ by DCFH and DHE staining. Extracellular ROS synthesis was dramatically reduced by ~70% in M2 Mϕ, in part due to reduced mRNA expression of Nox2, Nox5, and their accessory protein CYBA in M2 Mϕ. Another contributing factor to lower ROS levels is that M2 Mϕ have significantly increased Catalase and Gpx4 mRNA expression, as well as increased Cu/ZnSOD and Gpx1 protein and activity. These data suggest M2 Mϕ have increased ROS metabolism versus M1 Mϕ, which may provide an advantage in the oxidative tumor microenvironment. Furthermore, EcSOD re-expression in MB231, a triple negative breast cancer cell line, inhibited the MB231-mediated increase in M2 Mϕ markers and decrease in M1 Mϕ markers. We moved forward utilizing Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE), a small molecule SOD mimetic, shown to have anti-tumor effects. MnTE treatment increased M1 Mϕ markers, IL-12b and CD86, while concurrently decreasing M2 Mϕ markers, IL-10, CD163, and CD206. MnTE also inhibited the ability of M2 Mϕ to inhibit T cell activation suggesting ROS is required for M2 Mϕ polarization and function. Increasing ROS levels by addition of exogenous H2O2, lead to increased M2 markers and decreased M1 markers, which implicates the oxidative tumor microenvironment in promoting a M2-like TAM phenotype. These studies provide the rationale for antioxidant treatment of breast cancer to reprogram TAM from pro-tumorigenic M2-like TAM phenotype to a more tumoricidal M1-like phenotype. Citation Format: Brandon Griess, Kaustubh Datta, Melissa Teoh-Fitzgerald. Antioxidant therapy reprograms macrophages from protumor M2 to antitumor M1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-212. doi:10.1158/1538-7445.AM2017-LB-212

Adenylate kinase hCINAP determines self-renewal of colorectal cancer stem cells by facilitating LDHA phosphorylation

Targeting the specific metabolic phenotypes of colorectal cancer stem cells (CRCSCs) is an innovative therapeutic strategy for colorectal cancer (CRC) patients with poor prognosis and relapse. However, the context-dependent metabolic traits of CRCSCs remain poorly elucidated. Here we report that adenylate kinase hCINAP is overexpressed in CRC tissues. Depletion of hCINAP inhibits invasion, self-renewal, tumorigenesis and chemoresistance of CRCSCs with a loss of mesenchymal signature. Mechanistically, hCINAP binds to the C-terminal domain of LDHA, the key regulator of glycolysis, and depends on its adenylate kinase activity to promote LDHA phosphorylation at tyrosine 10, resulting in the hyperactive Warburg effect and the lower cellular ROS level and conferring metabolic advantage to CRCSC invasion. Moreover, hCINAP expression is positively correlated with the level of Y10-phosphorylated LDHA in CRC patients. This study identifies hCINAP as a potent modulator of metabolic reprogramming in CRCSCs and a promising drug target for CRC invasion and metastasis.

Increased lignocellulosic inhibitor tolerance of Saccharomyces cerevisiae cell populations in early stationary phase

BackgroundProduction of second-generation bioethanol and other bulk chemicals by yeast fermentation requires cells that tolerate inhibitory lignocellulosic compounds at low pH. Saccharomyces cerevisiae displays high plasticity with regard to inhibitor tolerance, and adaptation of cell populations to process conditions is essential for reaching efficient and robust fermentations.ResultsIn this study, we assessed responses of isogenic yeast cell populations in different physiological states to combinations of acetic acid, vanillin and furfural at low pH. We found that cells in early stationary phase (ESP) exhibited significantly increased tolerance compared to cells in logarithmic phase, and had a similar ability to initiate growth in the presence of inhibitors as pre-adapted cells. The ESP cultures consisted of subpopulations with different buoyant cell densities which were isolated with flotation and analysed separately. These so-called quiescent (Q) and non-quiescent (NQ) cells were found to possess similar abilities to initiate growth in the presence of lignocellulosic inhibitors at pH 3.7, and had similar viabilities under static conditions. Therefore, differentiation into Q-cells was not the cause for increased tolerance of ESP cultures. Flow cytometry analysis of cell viability, intracellular pH and reactive oxygen species levels revealed that tolerant cell populations had a characteristic response upon inhibitor perturbations. Growth in the presence of a combination of inhibitors at low pH correlated with pre-cultures having a high frequency of cells with low pHi and low ROS levels. Furthermore, only a subpopulation of ESP cultures was able to tolerate lignocellulosic inhibitors at low pH, while pre-adapted cell populations displayed an almost uniform high tolerance to the adverse condition. This was in stark contrast to cell populations growing exponentially in non-inhibitory medium that were uniformly sensitive to the inhibitors at low pH.ConclusionsESP cultures of S. cerevisiae were found to have high tolerance to lignocellulosic inhibitors at low pH, and were able to initiate growth to the same degree as cells that were pre-adapted to inhibitors at a slightly acidic pH. Carbon starvation may thus be a potential strategy to prepare cell populations for adjacent stressful environments which may be beneficial from a process perspective for fermentation of non-detoxified lignocellulosic substrates at low pH. Furthermore, flow cytometry analysis of pHi and ROS level distributions in ESP cultures revealed responses that were characteristic for populations with high tolerance to lignocellulosic inhibitors. Measurement of population distribution responses as described herein may be applied to predict the outcome of environmental perturbations and thus can function as feedback for process control of yeast fitness during lignocellulosic fermentation.