Oxidative Stress In HeLa Cells Research Articles

PLK-1 Interacting Checkpoint Helicase, PICH, Mediates Cellular Oxidative Stress Response.

Cells respond to oxidative stress by elevating the levels of antioxidants, signaling, and transcriptional regulation, often implemented by chromatin remodeling proteins. The study presented here shows that the expression of PICH, a Rad54-like helicase belonging to the ATP-dependent chromatin remodeling protein family, is upregulated during oxidative stress in HeLa cells. We also show that PICH regulates the expression of Nrf2, a transcription factor regulating antioxidant response in both the absence and presence of oxidative stress. The overexpression of PICH in PICH-depleted cells restored Nrf2 as well as antioxidant gene expression. In turn, Nrf2 regulated the expression of PICH in the presence of oxidative stress. ChIP experiments showed that PICH is present on the Nrf2 as well as antioxidant gene promoters, suggesting that the protein might be regulating the expression of these genes directly by binding to the DNA sequences. In addition, Nrf2 and histone acetylation (H3K27ac) also played a role in activating transcription in the presence of oxidative stress. Both Nrf2 and H3K27ac were found to be present on PICH and antioxidant promoters. Their occupancy was dependent on the PICH expression as fold enrichment was found to be decreased in PICH-depleted cells. PICH ablation led to the reduced expression of Nrf2 and impaired antioxidant response, leading to increased ROS content and thus showing PICH is essential for the cell to respond to oxidative stress.

Cerium Oxide Nanoparticles Regulate Oxidative Stress in HeLa Cells by Increasing the Aquaporin-Mediated Hydrogen Peroxide Permeability.

Some aquaporins (AQPs) allow the diffusion of hydrogen peroxide (H2O2), the most abundant ROS, through the cell membranes. Therefore, the possibility of regulating the AQP-mediated permeability to H2O2, and thus ROS scavenging, appears particularly important for controlling the redox state of cells in physiological and pathophysiological conditions. Several compounds have been screened and characterized for this purpose. This study aimed to analyze the effect of cerium oxide nanoparticles (CNPs) presenting antioxidant activity on AQP functioning. HeLa cells express AQP3, 6, 8, and 11, able to facilitate H2O2. AQP3, 6, and 8 are expressed in the plasma membrane and intracellularly, while AQP11 resides only in intracellular structures. CNPs but not cerium ions treatment significantly increased the water and H2O2 permeability by interacting with AQP3, 6, and especially with AQP8. CNPs increased considerably the AQP-mediated water diffusion in cells with oxidative stress. Functional experiments with silenced HeLa cells revealed that CNPs increased the H2O2 diffusion mainly by modulating the AQP8 permeability but also the AQP3 and AQP6, even if to a lesser extent. Current findings suggest that CNPs represent a promising pharmaceutical agent that might potentially be used in numerous pathologies involving oxidative stress as tumors and neurodegenerative diseases.

Hydralazine protects the heart against acute ischaemia/reperfusion injury by inhibiting Drp1-mediated mitochondrial fission.

AimsGenetic and pharmacological inhibition of mitochondrial fission induced by acute myocardial ischaemia/reperfusion injury (IRI) has been shown to reduce myocardial infarct size. The clinically used anti-hypertensive and heart failure medication, hydralazine, is known to have anti-oxidant and anti-apoptotic effects. Here, we investigated whether hydralazine confers acute cardioprotection by inhibiting Drp1-mediated mitochondrial fission.Methods and resultsPre-treatment with hydralazine was shown to inhibit both mitochondrial fission and mitochondrial membrane depolarisation induced by oxidative stress in HeLa cells. In mouse embryonic fibroblasts (MEFs), pre-treatment with hydralazine attenuated mitochondrial fission and cell death induced by oxidative stress, but this effect was absent in MEFs deficient in the mitochondrial fission protein, Drp1. Molecular docking and surface plasmon resonance studies demonstrated binding of hydralazine to the GTPase domain of the mitochondrial fission protein, Drp1 (KD 8.6±1.0 µM), and inhibition of Drp1 GTPase activity in a dose-dependent manner. In isolated adult murine cardiomyocytes subjected to simulated IRI, hydralazine inhibited mitochondrial fission, preserved mitochondrial fusion events, and reduced cardiomyocyte death (hydralazine 24.7±2.5% vs. control 34.1±1.5%, P=0.0012). In ex vivo perfused murine hearts subjected to acute IRI, pre-treatment with hydralazine reduced myocardial infarct size (as % left ventricle: hydralazine 29.6±6.5% vs. vehicle control 54.1±4.9%, P=0.0083), and in the murine heart subjected to in vivo IRI, the administration of hydralazine at reperfusion, decreased myocardial infarct size (as % area-at-risk: hydralazine 28.9±3.0% vs. vehicle control 58.2±3.8%, P<0.001).ConclusionWe show that, in addition to its antioxidant and anti-apoptotic effects, hydralazine, confers acute cardioprotection by inhibiting IRI-induced mitochondrial fission, raising the possibility of repurposing hydralazine as a novel cardioprotective therapy for improving post-infarction outcomes.

Identification, Quantification and &amp;lt;i&amp;gt;In-Vitro&amp;lt;/i&amp;gt; Genotoxicity of Major Polyaromatic Hydrocarbons Produced by Sugarcane Fly Ash Emitted from Sugarmill

Sugarcane burning during harvest and non-harvest season emits various pollutants like volatile organic compounds (VOCs), alkanes, and PAHs (Polyaromatic hydrocarbons) in the surrounding environment. Among these pollutants, PAHs are of uttermost concern due to their high level of toxicity. Burning of sugarcane bagase in sugar mill results in the production of fly ash. Fly ash is produced as a result of sugarcane bagasse burning in sugar mills. In present study, fly ash that comes out from the sugar mill chimney was collected from Western Uttar Pradesh, India and used for further analysis. High temperature and incomplete combustion inside chimney lead to the formation of PAHs. Extraction of PAHs present in fly ash samples was done by ultrasonication method and was identified with GC-FID (gas chromatography-flame ionization detector). Results exhibit the presence of eight PAHs in fly ash samples where the Benzo(a)pyrene and Naphthalene were found to be in high concentration. Furthermore, we have evaluated toxic effects of fly ash and Polyaromatic hydrocarbons (Standard of BaP & Nap) through different methods i.e. MTT, ROS and comet assay. Significant reduction (p < 0.001) in cell viability was noted in cells treated with fly ash as compared to control. Fly ash samples were also found to induce significant oxidative stress in HeLa cells, which ultimately causes DNA damage. Therefore, it may be concluded that the fly ash samples are toxic to the environment due to the presence of PAHs. Hence, the present study plays an important role in determining the harmful effects of PAHs and their source of occurrence.

Interaction of vaginal Lactobacillus strains with HeLa cells plasma membrane.

Vaginal lactobacilli offer protection against recurrent urinary and vaginal infections. The precise mechanisms underlying the interaction between lactobacilli and the host epithelium remain poorly understood at the molecular level. Deciphering such events can provide valuable information on the mode of action of commensal and probiotic bacteria in the vaginal environment. We investigated the effects exerted by five Lactobacillus strains of vaginal origin (Lactobacillus crispatus BC1 and BC2, Lactobacillus gasseri BC9 and BC11 and Lactobacillus vaginalis BC15) on the physical properties of the plasma membrane in a cervical cell line (HeLa). The interaction of the vaginal lactobacilli with the cervical cells determined two kinds of effects on plasma membrane: (1) modification of the membrane polar lipid organisation and the physical properties (L. crispatus BC1 and L. gasseri BC9); (2) modification of α5β1 integrin organisation (L. crispatus BC2, L. gasseri BC11 and L. vaginalis BC15). These two mechanisms can be at the basis of the protective role of lactobacilli against Candida albicans adhesion. Upon stimulation with all Lactobacillus strains, we observed a reduction of the basal oxidative stress in HeLa cells that could be related to modifications in physical properties and organisation of the plasma membrane. These results confirm the strictly strain-specific peculiarities of Lactobacillus and deepen the understanding of the mechanisms underlying the health-promoting role of this genus within the vaginal ecosystem.

Design of chalcogen-containing norepinephrines: efficient GPx mimics and strong cytotoxic agents against HeLa cells.

Numerous chronic diseases exhibit multifactorial etiologies, so focusing on a single therapeutic target is usually an inadequate treatment; instead, multi-target drugs are preferred. Herein, a panel of phenolic thioureas and selenoureas were designed as new prototypes against multifactorial diseases concerning antioxidation and cytotoxicity, as a pro-oxidant environment is usually found in such diseases. Selenoureas were excellent antiradical agents and biomimetic catalysts of glutathione peroxidase for the scavenging of H2O2. They were also potent and selective cytotoxic agents against cancer cells, in particular HeLa (IC50 2.77-6.13 μM), apoptosis being involved. Selenoureas also reduced oxidative stress in HeLa cells (IC50= 3.76 μM). Phenolic selenoureas are promising lead structures for the development of drugs targeting multifactorial diseases like cancer.

Induction of Oxidative Stress in HeLa Cells with Reactive Oxygen Species Generated in Titanium Oxide Nano-tubes

Induction of Oxidative Stress in HeLa Cells with Reactive Oxygen Species Generated in Titanium Oxide Nano-tubes

Diquat-induced cytotoxicity on Vero and HeLa cell lines: effect of melatonin and dihydromelatonin.

Diquat dibromide is a moderately toxic contact herbicide belonging to the bipyridyl group of redox-active compounds that induce a strong oxidative damage. Melatonin (MEL) can protect against oxidative damage under in vivo conditions, probably through its anti-oxidative capacity and ability to induce expression of anti-oxidative enzymes. The objective of this study was to investigate effects of diquat on viability of Vero and HeLa cells and possible protective effects of MEL and its analogue 2,3-dihydromelatonin (DMEL). Cell viability was evaluated with the MTT test. First, we analyzed dose-dependent effects of diquat on cell viability using the concentration range of 0.1–100 μM. Second, we used the diquat dose which reduced cell viability by 50% and treated cells with either MEL or DMEL (both in the concentration range of 1–100 μM) in the presence or absence of diquat. In addition, effects of both diquat and MEL on oxidative stress in HeLa cells were measured by flow cytometry using 2’,7’-dichlorofluorescin diacetate. We confirmed the expected negative effects of diquat on viability of Vero and HeLa cells. Melatonin and DMEL were able to prevent diquat reduced viability of Vero cells in rather low concentrations (1 μM) and DMEL exerted substantially stronger protective effects than MEL. However in HeLa cells, we did not find the same effects and MEL even reduced their viability. Moreover, treatment of HeLa cells with high concentrations of MEL (100 μM) exaggerated the pro-oxidative effects of diquat. The results suggest that in addition to the expected anti-oxidative effects, MEL exerts a pro-oxidative action which is cell type and dose dependent.

Cytological analysis of the response of nucleolar RNA and RNA-binding proteins to oxidative stress in HeLa cells

The organization and functional activity of the nucleoli of mammalian cells can be modified in response to various stress factors. However, data on the reaction of nucleoli to oxidative stress remain limited. In this paper, we have studied the localization of nucleolar RNAs and two RNA-binding proteins--fibrillarin, a pre-rRNA processing factor, and nucleophosmin/B23, a pre-ribosome assembly factor, in HeLa cells exposed to 1 mM H2O2 up to four hours. We have shown that under the conditions used H2O2 does not induce death, but inhibits rDNA transcription, reduces the total RNA content in the cells and the amount of 18S rRNA in the nucleoli, and causes relocalization of fibrillarin and nucleophosmin/B23 to the nucleoplasm. To date, similar changes in the localization of fibrillarin were described in mammalian cells only after HgCl2 treatment. Redistribution of nucleophosmin/B23 observed in H2O2-treated cells occurred in mammalian cells under inhibition of rDNA transcription and early rRNA processing. Overall, this study shows the high sensitivity of the nucleoli of HeLa cells to acute oxidative stress, which is clearly evident on the cytological level.

Cellular Inhibitor of Apoptosis Protein 1 ubiquitinates endonuclease G but does not affect endonuclease G-mediated cell death

Inhibitors of Apoptosis Proteins (IAPs) are evolutionarily well conserved and have been recognized as the key negative regulators of apoptosis. Recently, the role of IAPs as E3 ligases through the Ring domain was revealed. Using proteomic analysis to explore potential target proteins of DIAP1, we identified Drosophila Endonuclease G (dEndoG), which is known as an effector of caspase-independent cell death. In this study, we demonstrate that human EndoG interacts with IAPs, including human cellular Inhibitor of Apoptosis Protein 1 (cIAP1). EndoG was ubiquitinated by IAPs in vitro and in human cell lines. Interestingly, cIAP1 was capable of ubiquitinating EndoG in the presence of wild-type and mutant Ubiquitin, in which all lysines except K63 were mutated to arginine. cIAP1 expression did not change the half-life of EndoG and cIAP1 depletion did not alter its levels. Expression of dEndoG 54310, in which the mitochondrial localization sequence was deleted, led to cell death that could not be suppressed by DIAP1 in S2 cells. Moreover, EndoG-mediated cell death induced by oxidative stress in HeLa cells was not affected by cIAP1. Therefore, these results indicate that IAPs interact and ubiquitinate EndoG via K63-mediated isopeptide linkages without affecting EndoG levels and EndoG-mediated cell death, suggesting that EndoG ubiquitination by IAPs may serve as a regulatory signal independent of proteasomal degradation.

Metabolomics reveals that carnitine palmitoyltransferase‐1 is a novel target for oxidative inactivation in human cells

Oxidative dysfunction in the metabolism has long been implicated in diverse biological disorders. Although a substantial number of metabolic enzymes are targeted for inactivation by oxidative stress, identifying those targets remains difficult due to a lack of comprehensive observations of the metabolism acting through the stress response. We herein developed a metabolomics strategy using integrative liquid chromatography-mass spectrometry (LC-MS) and observing rapid metabolomic changes in response to hydrogen peroxide (H2 O2 )-induced oxidative stress in HeLa cells. Among the many metabolite changes detected, the most characteristic metabolites uniquely indicated carnitine palmitoyltransferase-1 (CPT1), the critical enzyme for mitochondrial β-oxidation of long-chain fatty acids, to be a target for oxidative inactivation. We showed that the enzymatic activity of CPT1 significantly declined by H2 O2 in several human cells. Interestingly, the inactivation was shown to be a direct effect of H2 O2 in vitro, but substantially occurred when cells were cultured with some reagents that generate reactive oxygen species (ROS). Thus, our results suggest the generality of CPT1 inhibition under various stress conditions associated with ROS generation, providing an insight into a mechanism for oxidative dysfunction in mitochondrial metabolism. Our metabolome data additionally suggest that certain methyltransferase(s) may be targets of oxidative stress as well.

Curcumin targeting the thioredoxin system elevates oxidative stress in HeLa cells

The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, is ubiquitous in all cells and involved in many redox-dependent signaling pathways. Curcumin, a naturally occurring pigment that gives a specific yellow color in curry food, is consumed in normal diet up to 100mg per day. This molecule has also been used in traditional medicine for the treatment of a variety of diseases. Curcumin has numerous biological functions, and many of these functions are related to induction of oxidative stress. However, how curcumin elicits oxidative stress in cells is unclear. Our previous work has demonstrated the way by which curcumin interacts with recombinant TrxR1 and alters the antioxidant enzyme into a reactive oxygen species (ROS) generator in vitro. Herein we reported that curcumin can target the cytosolic/nuclear thioredoxin system to eventually elevate oxidative stress in HeLa cells. Curcumin-modified TrxR1 dose-dependently and quantitatively transfers electrons from NADPH to oxygen with the production of ROS. Also, curcumin can drastically down-regulate Trx1 protein level as well as its enzyme activity in HeLa cells, which in turn remarkably decreases intracellular free thiols, shifting the intracellular redox balance to a more oxidative state, and subsequently induces DNA oxidative damage. Furthermore, curcumin-pretreated HeLa cells are more sensitive to oxidative stress. Knockdown of TrxR1 sensitizes HeLa cells to curcumin cytotoxicity, highlighting the physiological significance of targeting TrxR1 by curcumin. Taken together, our data disclose a previously unrecognized prooxidant mechanism of curcumin in cells, and provide a deep insight in understanding how curcumin works in vivo.

Effect of acute vs chronic H2O2-induced oxidative stress on antioxidant enzyme activities

H2O2 can freely crosses membranes and in the presence of Fe2+ (or Cu+) it is prone to participate in Fenton reaction. This study evaluated the concentration and time-dependent effects of H2O2-induced oxidative stress on MnSOD, Se:GPx and catalase and on aconitase. Acute and chronic H2O2 treatments were able to induce oxidative stress in HeLa cells as they significantly decreased aconitase activity and also caused a very significant decrease on antioxidant enzyme activities. The inhibition of enzyme activities was time- and concentration-dependent. Chronic treatment with 5 µM H2O2/h after 24 h was able to decrease all enzyme activities almost at the same level as the acute treatment. Acute and chronic treatments on antioxidant enzyme activities were prevented by cell treatment with ascorbic acid or N-acetylcysteine. These results indicate that antioxidant enzymes can also be affected by the same ROS they produce or neutralize if the time of exposure is long enough.

Induction of DNA strand breaks and oxidative stress in HeLa cells by ethanol is dependent on CYP2E1 expression

Induction of cytochrome P4502E1 (CYP2E1) is considered to be an important mechanism by which ethanol can cause toxicity related to oxidative stress both in vivo and in vitro. In the current study, we used HeLa cells with doxycycline-regulated CYP2E1 expression to test the hypothesis that induction of CYP2E1 could lead to secondary DNA oxidation that could potentially contribute to the carcinogenicity of ethanol in vivo. Overexpression of CYP2E1 protein was not associated with oxidative stress per se as assessed by markers of lipid peroxidation (cis-parinaric acid oxidation), glutathione depletion and elevation of intracellular reactive oxygen species (dichlorofluoroscin oxidation) in the presence or absence of ethanol substrate (10 mM, 24 h). Furthermore, there was no evidence of elevation of frequency of DNA strand breaks as assessed by the comet assay. In contrast, however, after pre-incubation of cells with L-buthionine-(S,R)-sulphoximine (BSO, 10 microM) which caused a 75% reduction in intracellular reduced glutathione (GSH) levels, CYP2E1 expression resulted in oxidative stress as assessed by all of these markers and DNA strand breaks but only in the presence of ethanol (10 mM). No effect was observed under these conditions in control cells not expressing CYP2E1. Furthermore, these effects could be attenuated by co-incubation with 1-aminobenzotriazole (0.5 mM), a suicide inhibitor of P450 activity. In conclusion, in this in vitro model CYP2E1-mediated interaction with ethanol results in the intracellular oxidative stress and the formation of DNA strand breaks which are detectable in cells pre-sensitized by depletion of intracellular levels of GSH.

Dual Phospholipase C/Diacylglycerol Requirement for Protein Kinase D1 Activation in Lymphocytes

The serine/threonine kinase protein kinase D1 (PKD1) is a protein kinase C (PKC) substrate that mediates antigen receptor signal transduction in lymphocytes. PKC phosphorylates serines 744/748 within the PKD1 catalytic domain, and this is proposed to be necessary and sufficient for enzyme activation. Hence, a PKD1 mutant with alanine substituted at positions 744 and 748 (PKD-S744A/S748A) is catalytically inactive. Conversely, a PKD1 mutant with glutamic residues substituted at positions 744 and 748 as phospho-mimics (PKD-S744E/S748E) is constitutively active when expressed in Cos7 or HeLa cells. The present study reveals that Ser-744/Ser-748 phosphorylation is required for PKD1 activation in lymphocytes. However, PKD-S744E/S748E is not constitutively active but, like the wild type enzyme, requires antigen receptor triggering or phorbol ester stimulation. Antigen receptor activation of wild type PKD is dependent on phospholipase C (PLC)/diacylglycerol (DAG) and PKC, whereas PKD-S744E/S748E is only dependent on PLC/DAG but no longer requires PKC. Hence, substitution of serines 744 and 748 with glutamic residues as phospho-mimics bypasses the PKC requirement for PKD1 activation but does not bypass the need for antigen receptors, PLC, or DAG. In lymphocytes, PKD1 is, thus, not regulated by PLC and PKC in a linear pathway; rather, PKD1 activation has more stringent requirements for integration of dual PLC signals, one mediated by PKCs and one that is PKC-independent.

Zinc resistance impairs sensitivity to oxidative stress in hela cells: protection through metallothioneins expression

To analyze the effects of high concentrations of zinc ions on oxidative stress protection, we developed an original model of zinc-resistant HeLa cells (HZR), by using a 200 μM zinc sulfate-supplemented medium. Resistant cells specifically accumulate high zinc levels in intracellular vesicles. These resistant cells also exhibit high expression of metallothioneins (MT), mainly located in the cytoplasm. Exposure of HZR to Zn-depleted medium for 3 or 7 d decreases the intracellular zinc content, but only slightly reduces MT levels of resistant cells. No changes of the intracellular redox status were detected, but zinc resistance enhanced H2O2-mediated cytotoxicity. Conversely, zinc-depleted resistant cells were protected against H2O2-induced cell death. Basal- and oxidant-induced DNA damage was increased in zinc resistant cells. Moreover, measurement of DNA damage on zinc-depleted resistant cells suggests that cytoplasmic metal-free MT ensures an efficient protection against oxidative DNA damage, while Zn-MT does not. This newly developed Zn-resistant HeLa model demonstrates that high intracellular concentrations of zinc enhance oxidative DNA damage and subsequent cell death. Effective protection against oxidative damage is provided by metallothionein under nonsaturating zinc conditions. Thus, induction of MT by zinc may mediate the main cellular protective effect of zinc against oxidative injury.