Induction Of Intracellular Reactive Oxygen Species Research Articles

The release of polylactic acid nanoplastics (PLA-NPLs) from commercial teabags. Obtention, characterization, and hazard effects of true-to-life PLA-NPLs

This study investigates MNPLs release from commercially available teabags and their effects on both undifferentiated monocultures of Caco-2 and HT29 and in the in vitro model of the intestinal Caco-2/HT29 barrier. Teabags were subjected to mechanical and thermodynamic forces simulating the preparation of a cup of tea. The obtained dispersions were characterized using TEM, SEM, DLS, LDV, NTA, and FTIR. Results confirmed that particles were in the nano-range, constituted by polylactic acid (PLA-NPLs), and about one million of PLA-NPLs per teabag were quantified. PLA-NPLs internalization, cytotoxicity, intracellular reactive oxygen species induction, as well as structural and functional changes in the barrier were assessed. Results show that PLA-NPLs present high uptake rates, especially in mucus-secretor cells, and bio-persisted in the tissue after 72 h of exposure. Although no significant cytotoxicity was observed after the exposure to 100 µg/mL PLA-NPLs during 48 h, a slight barrier disruption could be detected at short-time periods. The present work reveals new insights into the safety of polymer-based teabags, the behavior of true-to-life MNPLs in the human body, as well as new questions on how repeated and prolonged exposures could affect the structure and function of the human intestinal epithelium.

Synergistic activity of pomegranate rind extract and Zn (II) against Candida albicans under planktonic and biofilm conditions, and a mechanistic insight based upon intracellular ROS induction

Candida albicans (C. albicans) is an opportunistic pathogen, which causes superficial infection and can lead to mortal systemic infections, especially in immunocompromised patients. The incidence of C. albicans infections is increasing and there are a limited number of antifungal drugs used in treatment. Therefore, there is an urgent need for new and alternative antifungal drugs. Pomegranate rind extract (PRE) is known for its broad-spectrum antimicrobial activities, including against C. albicans and recently, PRE and Zn (II) have been shown to induce synergistic antimicrobial activity against various microbes. In this study, the inhibitory activities of PRE, Zn (II) and PRE in combination with Zn (II) were evaluated against C. albicans. Antifungal activities of PRE and Zn (II) were evaluated using conventional microdilution methods and the interaction between these compounds was assessed by in vitro checkerboard and time kill assays in planktonic cultures. The anti-biofilm activities of PRE, Zn (II) and PRE in combination with Zn (II) were assessed using confocal laser scanning microscopy, with quantitative analysis of biofilm biomass and mean thickness analysed using COMSTAT2 analysis. In addition, antimicrobial interactions between PRE and Zn (II) were assayed in terms reactive oxygen species (ROS) production by C. albicans. PRE and Zn (II) showed a potent antifungal activity against C. albicans, with MIC values of 4 mg/mL and 1.8 mg/mL, respectively. PRE and Zn (II) in combination exerted a synergistic antifungal effect, as confirmed by the checkerboard and time kill assays. PRE, Zn (II) and PRE and Zn (II) in combination gave rise to significant reductions in biofilm biomass, although only PRE caused a significant reduction in mean biofilm thickness. The PRE and Zn (II) in combination caused the highest levels of ROS production by C. albicans, in both planktonic and biofilm forms. The induction of excess ROS accumulation in C. albicans may help explain the synergistic activity of PRE and Zn (II) in combination against C. albicans in both planktonic and biofilm forms. Moreover, the data support the potential of the PRE and Zn (II) combination as a novel potential anti-Candida therapeutic system.

Effects of Bismuth Oxide Nanoparticles, Cisplatin and Baicalein-rich Fraction on ROS Generation in Proton Beam irradiated Human Colon Carcinoma Cells

Abstract Introduction: Proton beam radiotherapy is an advanced cancer treatment technique, which would reduce the effects of radiation on the surrounding healthy cells. The usage of radiosensitizers in this technique might further elevate the radiation dose towards the cancer cells. Material and methods: The present study investigated the production of intracellular reactive oxygen species (ROS) due to the presence of individual radiosensitizers, such as bismuth oxide nanoparticles (BiONPs), cisplatin (Cis) or baicalein-rich fraction (BRF) from Oroxylum indicum plant, as well as their combinations, such as BiONPs-Cis (BC), BiONPs-BRF (BB), or BiONPs-Cis-BRF (BCB), on HCT-116 colon cancer cells under proton beam radiotherapy. Results: It was found that the ROS in the presence of Cis at 3 Gy of radiation dose was the highest, followed by BC, BiONPs, BB, BRF, and BCB treatments. The properties of bismuth as a radical scavenger, as well as the BRF as a natural compound, might contribute to the lower intracellular ROS induction. The ROS in the presence of Cis and BC combination were also time-dependent and radiation dose-dependent. Conclusions: As the prospective alternatives to the Cis, the BC combination and individual BiONPs showed the capacities to be developed as radiosensitizers for proton beam therapy.

Investigation of the genotoxicity of digested titanium dioxide nanomaterials in human intestinal cells

The widespread use of titanium dioxide nanomaterials (TiO2 NMs) in food and consumer products such as toothpaste or food contact materials, suggests the relevance of human oral exposure to these nanomaterials (NMs) and raises the possibility of adverse effects in the gastrointestinal tract (GIT).We previously showed that the in vitro digestion of TiO2 NMs may increase their toxicity in intestinal cells. In this work, we analyzed the genotoxicity and the intracellular reactive oxygen species induction by physiologically relevant concentrations of three different TiO2 NMs (NM-102, NM-103 and NM-105) in Caco-2 and HT29-MTX-E12 intestinal cells, while considering the potential influence of the digestion process in the NMs’ physiochemical characteristics. The results evidenced a DNA-damaging effect dependent on the NM, more relevant for the rutile/anatase NM-105, possibly due to its lower hydrodynamic size in the cells medium. In addition, the results of the micronucleus assay suggest effects on chromosomal integrity, an indicator of cancer risk, in the HT29-MTX-E12 cells, for all the tested TiO2 NMs, especially after the in vitro digestion. This work supports the evidence for concerns on the use of TiO2 NMs as a food additive, recently reported by EFSA, and for their use in applications in consumer products that may drive human exposure through ingestion.

Synthesis, Characterization, and Preclinical Evaluation of a Small-Molecule Prostate-Specific Membrane Antigen-Targeted Monomethyl Auristatin E Conjugate.

Prostate cancer is the second most common type of cancer among men. Its main method of treatment is chemotherapy, which has a wide range of side effects. One of the solutions to this challenge is targeted delivery to prostate cancer cells. Here we synthesized a novel small-molecule PSMA-targeted conjugate based on the monomethyl auristatin E. Its structure and conformational properties were investigated by NMR spectroscopy. Cytotoxicity, intracellular reactive oxygen species induction, and stability under liver microsomes and P450-cytochrome species were investigated for this conjugate. The conjugate demonstrated 77-85% tumor growth inhibition levels on 22Rv1 (PSMA (+)) xenografts, compared with a 37% inhibition level on PC-3 (PSMA (-)) xenografts, in a single dose of 0.3 mg/kg and a sufficiently high therapeutic index of 21. Acute, chronic, and subchronic toxicities and pharmacokinetics have shown that the synthesized conjugate is a promising potential agent for the chemotherapy of prostate cancer.

Anticancer Activity of Sweroside Nanoparticles in Prostate Cancer Bone Metastasis in PC-3 Cells Involved in Wnt/β-Catenin Signaling Pathway.

Bone metastasis is a significant cause of morbidity and mortality in patients with prostate cancer (PCa). This study is aimed at illustrating the mechanism of sweroside-mediated regulation in bone metastasis in PCa cells. Owing to the limitations of antitumor drugs in terms of their physical and chemical properties, making them into nanomaterials can effectively improve drug stability and bioavailability. Apoptosis was assessed with flow cytometry using the annexin V/propidium iodide binding assay; proteins, including p53, P21, Bcl-2, and Bax; and induction of intracellular reactive oxygen species (ROS). Using colony formation assay, sphere formation assay, and the expression changes in CD133 and CD44, stem cell characteristics were assessed. Epithelial-mesenchymal transition (EMT) activity was accessed by levels of the expression changes of EMT-related markers, vimentin and E-cadherin. Wnt/β-catenin signaling pathway was examined to detect the levels of the expression changes of snail and β-catenin. PC-3 cells were treated with lithium chloride (LiCl), which is an agonist of Wnt/β-catenin signaling, and the levels of CD133, CD44, vimentin, E-cadherin, snail, and β-catenin were detected. T-cell factor/lymphocyte enhancer factor (TCF/LEF) activity in cells overexpressing β-catenin was used to detect the effects on β-catenin transcription, and the expression of c-myc, Cyclin D1, Survivin, and MMP-7 were used to detect Wnt downstream target genes. Our results suggest that sweroside induces apoptosis and intracellular ROS; upregulates apoptotic proteins; and suppresses proliferation, invasion, and migration, preventing stem cell characteristics, including sphere formation, colony formation, and CD133 and CD44 expressions. Furthermore, sweroside nanoparticles exerts inhibitory effects on β-catenin transcription by suppressing TTCF/LEF activity in cells overexpressing β-catenin and downregulation of the expression of Wnt downstream target genes, including c-myc, Cyclin D1, Survivin, and MMP-7. The potential therapeutic effect of sweroside nanoparticles on bone metastatis of PCa was suggested, by these findings.

Copper Sulfide Nanoparticle‐Redirected Macrophages for Adoptive Transfer Therapy of Melanoma

AbstractAdoptive cell therapy (ACT) has achieved landmark advances in treating cancer in clinic. Recent advances in ACT of macrophages engineered to express chimeric antigen receptors (CARs) have shown effectiveness in treating solid tumors. However, the CAR‐macrophage therapy is dependent on tumor antigen recognition and gene editing methods. Herein, an adoptive macrophage therapy is presented through copper sulfide nanoparticle‐regulation that exhibits substantial antitumor effect in melanoma‐bearing mice, without the need for tumor antigen repertoire. Bone marrow derived macrophages (BMDMs) incubated with the nanoparticles promote the cellular production of reactive oxygen species (ROS) through dynamin‐related protein 1 (Drp1)‐mediated mitochodrial fission. The high intracellular ROS level directs BMDMs polarization toward M1 phenotype by classical IKK‐dependent NF‐κB activation. Moreover, the copper sulfide nanoparticle‐stimulated BMDMs (CuS‐MΦ) reduce the expression of programmed death‐1 (PD‐1) and exhibit enhanced phagocytic and digestive ability. Intratumoral transfer of CuS‐MΦ significantly prolongs the median survival time of the tumor‐bearing mice, remodels the tumor microenvironment, and elicits systemic antitumor immunity. These results suggest a cancer therapeutic approach of adoptively transferred macrophages through the induction of intracellular ROS with nanomaterials.

Zataria multiflora Boiss. Essential Oil Induce Apoptosis in Two Human Colon Cancer Cell Lines (HCT116 & SW48)

Zataria multiflora Boiss. is known by the common Persian name "Avishan-e-Shirazi", is one of the best-known medicinal herbs belonging to the Labiatae (Lamiaceae) family. The aim of this study was to evaluate the anticancer effects and the underlying mechanisms of how Z. multiflora Boiss., essential oil induced apoptosis in the human colorectal tumor cell lines (HCT116 & SW48). This study was conducted in National Institute of Genetic Engineering and Biotechnology (NIGEB) (Tehran, Iran) from 2017 to 2019. The cytotoxicity of this essential oil was assessed by 3- (4,5-di-methylthiazol-2-yl)- 2,5-diphenyltetra-zolium bromide (MTT) assay, trypan blue exclusion, and colony formation assays. We assessed apoptosis and measure intracellular reactive oxygen species (ROS) by flow cytometry. Then gene expression was analyzed by Quantitative Real-Time RT-PCR. Z. multiflora Boiss., essential oil time- and dose-dependently inhibits cell proliferation and also induced apoptosis in both cell lines via UCP2-related mitochondrial pathway by the induction of intracellular ROS. Z. multiflora Boiss., essential oil could be a good candidate for use as an inhibitor of the growth of colorectal tumor cells.

Tomentosin Displays Anti-Carcinogenic Effect in Human Osteosarcoma MG-63 Cells via the Induction of Intracellular Reactive Oxygen Species.

Tomentosin is a natural sesquiterpene lactone extracted from various plants and is widely used as a medicine because it exhibits essential therapeutic properties. In this study, we investigated the anti-carcinogenic effects of tomentosin in human osteosarcoma MG-63 cells by performing cell migration/viability/proliferation, apoptosis, and reactive oxygen species (ROS) analysis assays. MG-63 cells were treated with various doses of tomentosin. After treatment with tomentosin, MG-63 cells were analyzed using the MTT assay, colony formation assay, cell counting assay, wound healing assay, Boyden chamber assay, zymography assay, cell cycle analysis, FITC Annexin V apoptosis assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, western blot analysis, and ROS detection analysis. Our results indicated that tomentosin decreased cell viability and migration ability in MG-63 cells. Moreover, tomentosin induced apoptosis, cell cycle arrest, DNA damage, and ROS production in MG-63 cells. Furthermore, tomentosin-induced intracellular ROS decreased cell viability and induced apoptosis, cell cycle arrest, and DNA damage in MG-63 cells. Taken together, our results suggested that tomentosin exerted anti-carcinogenic effects in MG-63 cells by induction of intracellular ROS.

Protection of glucotoxicity by a tripeptide derivative of α-melanocyte-stimulating hormone in human epidermal keratinocytes.

Chronic skin ulcers are a major complication and a therapeutic challenge in patients with diabetes mellitus. Glucose-induced accumulation of reactive oxygen species (ROS) is considered to be an important pathogenetic factor in diabetes. To characterize the impact of high glucose (HG) on normal human keratinocytes (NHKs) and examine if Lys-d-Pro-Thr (KdPT), a tripeptide derived from α-melanocyte-stimulating-hormone, has protective effects. We investigated the key functions of NHKs under HG conditions with or without KdPT in vitro as well as ex vivo employing a skin organ culture model. HG impaired metabolic activity, cell proliferation, viability and migration of NHKs. As shown by atomic force microscopy HG altered the biophysical properties of NHKs, i.e. cell size and elasticity. Glucotoxicity in NHKs was paralleled by the induction of intracellular ROS and endoplasmic reticulum stress. KdPT attenuated HG-induced oxidative stress and antagonized the effects of glucose on cell viability, metabolic activity and migration. Importantly, KdPT also antagonized the suppressive effect of HG on epidermal migration in wounded human skin organ cultures. Our findings highlight a novel effect of KdPT that could be exploited for the future therapy of diabetic skin ulcers.

Genipin induces cell death via intrinsic apoptosis pathways in human glioblastoma cells.

Genipin, a compound derived from Gardenis jasminoides Ellis fruits, was demonstrated to be the specific uncoupling protein 2 (UCP2) inhibitor. UCP2 is a mitochondrial carrier protein that creates proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from adenosine triphosphate (ATP) synthesis. Several studies revealed that UCP2 is broadly over-expressed in leukemia, colorectal, lung, ovarian, prostate, testicular, and bladder cancers. However, the effect of genipin still needs to be elucidated in neurological malignancies. In this study, we investigated the anticancer effect of genipin in U87MG and A172 cell lines. The anticancer effect of genipin on these cell lines was measured by microculture tetrazoliumtest (MTT), Trypan blue exclusion, and colony formation assays, in the presence of various concentrations of genipin at different time intervals. We assessed apoptosis and measure intracellular reactive oxygen species (ROS) by flow cytometry. Expression of UCP2 and some of the genes involved in apoptosis was analyzed by real-time quantitative polymerase chain reaction (PCR). Results of the MTT assay showed that genipin moderately reduced metabolic activity of both cell lines in dose- and time-dependent manner. Result of Trypan blue exclusion test indicated that the viable cell count decreased in the treated group in a concentration-dependent manner. Genipin also significantly decreased colony formation ability of these cells in a concentration-dependent manner. Result of morphological changes showed that there were significant differences in cell number and morphology in treated groups as compared with the untreated groups. Flow cytometric analysis of U87MG and A172 cells with annexin V/propidium iodide staining, 48 hours after treatment with genipin, displays 22.4% and 26.1% apoptotic population, respectively, in treated cells, in comparison to 7.42% and 9.31% apoptotic cells of untreated cells. After treatment, UCP2 and B-cell lymphoma 2 (BCL 2 ) genes are downregulated, and BCL 2 associated X protein, BCL 2 antagonist/killer, BCL 2 interacting killer, and Cytochrome c genes are upregulated. Genipin treatment increased mitochondrial ROS levels and also induced apoptosis through caspase-3 upregulation. In conclusion, the antiproliferative effects of genipin on the growth of both glioblastoma cell lines have been shown in all of these assays, and genipin profoundly induced apoptosis in both cell lines via the UCP2-related mitochondrial pathway through the induction of intracellular ROS.

Elucidating differential nano-bio interactions of multi-walled andsingle-walled carbon nanotubes using subcellular proteomics

Understanding the relationship between adverse exposure events and specific material properties will facilitate predictive classification of carbon nanotubes (CNTs) according to their mechanisms of action, and a safe-by-design approach for the next generation of CNTs. Mass-spectrometry-based proteomics is a reliable tool to uncover the molecular dynamics of hazardous exposures, yet challenges persist with regards to its limited dynamic range when sampling whole organisms, tissues or cell lysates. Here, the simplicity of the sub-cellular proteome was harnessed to unravel distinctive adverse exposure outcomes at the molecular level, between two CNT subtypes. A549, MRC9 and human macrophage cells, were exposed for 24h to non-cytotoxic doses of single-walled or multi-walled CNTs (swCNTs or mwCNTs). Label-free proteomics on enriched cytoplasmic fractions was complemented with analyses of reactive oxygen species (ROS) production and mitochondrial integrity. The extent/number of modulated proteoforms indicated the single-walled variant was more bioactive. Greater enrichment of pathways corresponding to oxido-reductive activity was consistent with greater intracellular ROS induction and mitochondrial dysfunction capacities of swCNTs. Other compromised cellular functions, as revealed by pathway analysis were; ribosome, spliceosome and DNA repair. Highly upregulated proteins (fold change in abundance >6) such as, APOC3, RBP4 and INS are also highlighted as potential markers of hazardous CNT exposure. We conclude that, changes in cytosolic proteome abundance resulting from nano-bio interactions, elucidate adverse response pathways and their distinctive molecular components. Our results indicate that CNT-protein interactions might have a thus far unappreciated significance for protein trafficking, and this warrants further investigation.

Mechanisms of antibiotic resistance in bacteria mediated by silver nanoparticles

ABSTRACT Silver nanoparticles (AgNPs) are widely used in industry, consumer products, and medical appliances due to their efficient antimicrobial properties. However, information on environmental toxicity and bacterial impact of these particles is not completely elucidated. Results showed that AgNPs produced growth inhibition and oxidative stress in bacteria Escherichia coli (gram negative) and Staphylococcus aureus (gram positive), with half-maximal inhibitory concentrations (IC50) of 12 and 7 mg/L, respectively. Surprisingly, bacteria pre-exposed to sublethal dose of AgNPs exhibited increased resistance toward antibiotics (ampicillin and Pen-Strep) with IC50 elevated by 3–13-fold. Further, AgNP pre-exposure raised the minimal inhibitory concentration and minimal biocidal concentration by two- to eightfold when cells were challenged with antibiotics with diverse mechanisms of action (penicillin, chloramphenicol, and kanamycin). Interestingly, we found that upon exposure to ampicillin, strains pretreated with AgNPs exhibited lower levels of membrane damage and oxidative stress, together with elevated levels of intracellular ATP relative to untreated cells. Bacterial reverse mutation assay (Ames test) showed that AgNPs are highly mutagenic, consistent with further assays demonstrating abiotic reactive oxygen species (ROS) generation and intrinsic DNA cleavage activity in vitro of AgNPs. Overall, our results suggest that AgNPs enhance bacterial resistance to antibiotics by promoting stress tolerance through induction of intracellular ROS. Our data suggest potential consequences of incidental environmental exposure of bacteria to AgNPs and indicate the need to regulate use and disposal of AgNPs in industry and consumer products.

P 185 - Low-fluence photodynamic treatment modifies immunogenicity of mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) represent one of the most important component of tissue microenvironment. They can be characterized by high proliferative and regenerative potential as well as low immunogenicity that serves MSC application for regenerative therapy. The same properties make them the key players in the pathogenesis of different diseases. Most of these physiological and pathological processes involving MSCs are accompanied by reactive oxygen species (ROS). However, the ROS effects on MSC functional activity are still poorly investigated. In present study we demonstrated that ROS can modify MSC immunogenicity. MSCs were obtained from stromal vascular fraction of human adipose tissue. Intracellular ROS were induced by low fluence (0.25 J/cm2) photodynamic treatment (PDT) with Al-phthalocynine. We demonstrated that low-fluence PDT significantly increase MSC immunogenicity as manifested by decreased MSC viability after co-culture with human peripheral blood mononuclear cells PBMNs. It was accompanied by enhanced FasL expression in MSCs. In addition, it was found out that intracellular ROS induction resulted in dramatic decrease of CCL2 and increase of IL8 but not IL6 production by MSCs. Low-fluence PDT did not affect Fas, MICA/B, Nectin-2, HLA-ABC expression in MSC as well as co-cultivation with PDT-MSC did not influence the expression of DNAM-1, NKG2D and CD69 in NK.

Comparative oxidative stress elicited by nanosilver in stable HSPA1A promoter-driven luciferase reporter HepG2 and A549 cells.

The use of silver nanoparticles (AgNPs) in consumer products and medical applications is growing rapidly owing to their noted antimicrobial properties. As a consequence, a rapid and reliable toxicology test that allows multiple nanosilver screening in a stress responsive pathway-based assay is needed to ensure consumer safety. Here, novel HSPA1A promoter-driven luciferase reporter gene assays based on two cell models widely used in toxicology testing, HepG2 and A549 cell lines, were developed to analyze the oxidative stress elicited by AgNPs. After AgNP exposure, the luciferase activity, cell viability, and oxidative damage in HepG2- and A549-luciferase cells were determined. The uptake and intracellular localization of AgNPs were measured by transmission electron microscopy. The cellular AgNPs were determined by inductively coupled plasma mass spectrometry. Our results showed that both reporter cells were able to take up nanoparticles and furthermore, exposure to AgNPs resulted in an obviously dose-dependent increase in transcriptional activation of the HSPA1A promoter indicated by luciferase activity. Surprisingly, luciferase activity was greater than 200× the control level in HepG2-luciferase reporter cells treated with 50 μg mL-1 AgNPs. These results were in line with the positive responses in cytotoxicity, intracellular reactive oxygen species induction, glutathione depletion, malondialdehyde concentration, and the cellular silver content. Compared with the A549-luciferase cells, the luciferase signal in the metabolic competent HepG2-luciferase cells is obviously more sensitive and stable. We conclude that these two reporter gene cell systems, especially the HepG2-luciferase cells, demonstrated their utilities in predictive screening of the oxidative stress elicited by nanosilver.

Antioxidant and anti-genotoxic properties of cerium oxide nanoparticles in a pulmonary-like cell system.

Cerium oxide nanoparticles (CeO2-NP) present two different oxidation states what can suppose an auto-regenerative redox cycle. Potential applications of CeO2-NP to quench reactive oxygen species (ROS) in biological systems are currently being investigated. In this context, CeO2-NP may represent a novel agent to protect cells and tissues against oxidative damage by its regenerative free radical-scavenging properties. In this study, we have used a human epithelial lung cell line, BEAS-2B, as a model to study the possible antioxidant and anti-genotoxic effect of CeO2-NP in a pulmonary-like system. We have assessed the protective effect of CeO2-NP pre-treatment in front of a well-defined oxidative stress-inducing agent (KBrO3). Different endpoints like toxicity, intracellular ROS induction, genotoxicity and DNA oxidative damage (comet assay), and gene expression alterations have been evaluated. The obtained results confirmed the antioxidant properties of CeO2-NP. Thus, its pre-treatment significantly reduced the intracellular production of ROS induced by KBrO3. Similarly, a reduction in the levels of DNA oxidative damage, as measured with the comet assay complemented with formamidopyrimidine DNA glycosylase enzyme, was also observed. Pre-treatment of BEAS-2B cells with CeO2-NP (at 2.5µg/mL) slightly increased the viability of cells treated with KBrO3 as well as down-regulated the expression of the Ho1 and Sod2 genes involved in the oxidative Nrf2 pathway. Our finding would support the potential usefulness of CeO2-NP as a pharmacological agent to be used against diseases caused by oxidative stress.

Inhibition of NADPH oxidase-1 preserves beta cell function.

Upregulation of the reactive oxygen species (ROS)-producing enzyme NADPH oxidase (NOX)-1 in islets and beta cells follows acute exposure to inflammatory cytokines and is concomitant with beta cell dysfunction. NOX-1 is a candidate mediator of inflammation-induced beta cell dysfunction. This study aimed to determine whether selective inhibition of NADPH oxidase-1 presents a new strategy to preserve beta cell function. Induced beta cell dysfunction was studied in primary human donor islets, isolated mouse islets and murine beta cell lines. Islets and beta cells were stimulated with inflammatory cytokines (TNF-α, IL-1β, IFN-γ). NOX-1 activity was blocked by the selective inhibitor ML171. Cytokine induction of intracellular ROS was reduced 80% with 1 μmol/l ML171 in murine beta cell lines (p < 0.01). Cytokine-induced apoptosis, measured by caspase-3 activation or quantified fluorescence microscopy, was prevented in islets and beta cell lines up to 100% with ML171 in a concentration-dependent manner (p < 0.05). Functionally, glucose-stimulated insulin secretion was abolished by cytokine exposure but preserved by ML171 in isolated mouse islets and murine beta cell lines. A feed-forward regulation of NOX-1 in islets and beta cell lines was disrupted by ML171. Stimulation of NOX-1 activity is a major component of inflammatory cytokine-induced beta cell dysfunction. Significant protection of beta cells is conferred with selective inhibition of NOX-1. Suppression of NOX-1 activity may present a new therapeutic strategy to preserve and protect beta cell function in diabetes.

Spiruchostatin A and B, novel histone deacetylase inhibitors, induce apoptosis through reactive oxygen species-mitochondria pathway in human lymphoma U937 cells

Spiruchostatin A (SP-A) and spiruchostatin B (SP-B) are the potent histone deacetylase inhibitors (HDACi), that has the potential for chemotherapy of leukemia but the exact mechanism of these compounds remains unclear. In the present study, the role of reactive oxygen species (ROS) production and the mechanism involved in the apoptosis was investigated in human lymphoma U937 cell. When the U937 cells were treated with SP-A and SP-B for 24h at different concentrations, evidence of apoptotic features, including increase in DNA fragmentation and changes in nuclear morphology, were obtained. SP-B showed maximum potency to induce apoptosis, while SP-A was less potent. Apoptosis was also determined by increase in the fraction of sub-G1 cells and Annexin V-FITC staining cells. SP-A and SP-B induced apoptosis was accompanied by significant increase in the formation of intracellular reactive oxygen species (ROS). Pre-treatment with N-acetyl-l-cysteine (NAC), significantly inhibited the SP-A and SP-B mediated apoptosis, suggesting a vital role of ROS involved in the lethality of both agents. Moreover, SP-A and SP-B treatment resulted in the loss of mitochondrial membrane potential (MMP), and Fas, caspase-8 and caspase-3 activation. In addition Bid activation and the release of cytochrome-c to the cytosol was also observed. In this study, we suggest that a marked induction of intracellular ROS mediated mitochondrial pathway and the Fas plays a role in the SP-A and SP-B induced apoptosis. Taken together, our data provides further insights of the mechanism of action of SP-A and SP-B and their potential application as novel chemotherapeutic agents.

Surface molecules on HaCaT keratinocytes after interaction with non‐thermal atmospheric pressure plasma

Non-thermal atmospheric-pressure plasmas have been developed that will be used in future for several purposes, e.g. medicine. Living tissues and cells are at the focus of plasma treatment, e.g. to improve wound healing, or induce apoptosis and growth arrest in tumour cells. Detailed investigations of plasma-cell interactions are needed. Cell surface adhesion molecules as integrins, cadherins or the EGFR (epidermal growth factor receptor) are of importance in wound healing and also for development of cancer metastasis. This study has focused on measurement of cell surface molecules on human HaCaT keratinocytes (human adult low calcium temperature keratinocytes) promoting adhesion, migration and proliferation as one important feature of plasma-cell interactions. HaCaT keratinocytes were treated with plasma by a surface dielectric barrier discharge in air. Cell surface molecules and induction of intracellular ROS (reactive oxygen species) were analysed by flow cytometry 24 h after plasma treatment. Besides a reduction of cell viability a significant down-regulation of E-cadherin and the EGFR expression occurred. The influence on α2- and β1-integrins was less pronounced, and expression of ICAM-1 (intercellular adhesion molecule 1) was unaffected. The extent of effects depended on the exposure time of cells to the plasma and the treatment regimen. Intracellular level of ROS detected by the fluorescent dye H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) increased by plasma treatment, but it was neither dependent on the treatment time nor related to the different treatment regimens. Two-dimensional cultures of HaCaT keratinocytes appear to be a suitable method of investigating plasma-cell interactions.

Downregulation of miR-205 Modulates Cell Susceptibility to Oxidative and Endoplasmic Reticulum Stresses in Renal Tubular Cells

BackgroundOxidative stress and endoplasmic reticulum (ER) stress play a crucial role in tubular damage in both acute kidney injury (AKI) and chronic kidney disease (CKD). While the pathophysiological contribution of microRNAs (miRNA) to renal damage has also been highlighted, the effect of miRNA on renal damage under oxidative and ER stresses conditions remains elusive.MethodsWe assessed changes in miRNA expression in the cultured renal tubular cell line HK-2 under hypoxia-reoxygenation-induced oxidative stress or ER stress using miRNA microarray assay and real-time RT-PCR. The pathophysiological effect of miRNA was evaluated by cell survival rate, intracellular reactive oxygen species (ROS) level, and anti-oxidant enzyme expression in miRNA-inhibited HK-2 or miRNA-overexpressed HK-2 under these stress conditions. The target gene of miRNA was identified by 3′-UTR-luciferase assay.ResultsWe identified 8 and 10 miRNAs whose expression was significantly altered by oxidative and ER stresses, respectively. Among these, expression of miR-205 was markedly decreased in both stress conditions. Functional analysis revealed that decreased miR-205 led to an increase in cell susceptibility to oxidative and ER stresses, and that this increase was associated with the induction of intracellular ROS and suppression of anti-oxidant enzymes. While increased miR-205 by itself made no change in cell growth or morphology, cell viability under oxidative or ER stress conditions was partially restored. Further, miR-205 bound to the 3′-UTR of the prolyl hydroxylase 1 (PHD1/EGLN2) gene and suppressed the transcription level of EGLN2, which modulates both intracellular ROS level and ER stress state.ConclusionsmiR-205 serves a protective role against both oxidative and ER stresses via the suppression of EGLN2 and subsequent decrease in intracellular ROS. miR-205 may represent a novel therapeutic target in AKI and CKD associated with oxidative or ER stress in tubules.