Baicalin induces apoptosis in human osteosarcoma cell through ROS-mediated mitochondrial pathway

Baicalin is extracted from a traditional Chinese herb, Scutellaria baicalensis. In this study, the anticancer activity and underlying mechanisms of baicalin towards human osteosarcoma cell (HOS) were investigated. Baicalin could inhibit HOS cell proliferation in a concentration-dependent manner. Mitochondrial membrane potential decreased obviously after treated with different concentration of baicalin by flow cytometry assay and revealed that baicalin triggered a significant generation of reactive oxygen species (ROS). Western blotting assay further revealed that baicalin-induced cell apoptosis by suppressing Bcl-2 level, then activating caspase-9 and caspase-3. In vivo experiment, baicalin significantly suppressed tumour growth in female BALB/C nude mice bearing HOS tumours. In addition, baicalin did show toxicity to treated animal by comparing the body weight increase and mortality. In general, the present results demonstrated that baicalin-induced apoptosis in human osteosarcoma cell via a ROS-mediated mitochondrial pathway. The paper indicated that baicalin is a promising candidate for the treatment of HOS.     

Linifanib induces apoptosis in human ovarian cancer cells via activation of FOXO3 and reactive oxygen species

Linifanib is known as an inhibitor of receptor tyrosine kinase. Even though it has been widely recognized as efficient inhibitor of receptor tyrosine kinases, anti-carcinogenic effect has not been investigated enough in ovarian cancer. In this study, we investigated the anti-cancer effect of linifanib on human ovary cancer SKOV3 cells. WST-1, cell counting assay, and observation of morphological changes were performed to evaluate the cytotoxic effect of linifanib in SKOV3 cells. We analyzed SKOV3 cells treated with linifanib using Muse cell analyzer. We focused on investigating the effect of linifanib on DNA damage in nucleus. Additionally, intracellular reactive oxygen species (ROS) level was measured through Muse cell analyzer. Western blotting was performed to evaluate the protein expression level related to apoptosis. We found that linifanib inhibited proliferation of SKOV3 cells. Our results showed that linifanib induced apoptosis in SKOV3 cells. Additionally, linifanib induced DNA damage in SKOV3 cells. We found that intracellular ROS level increased after treatment of linifanib in SKOV3 cells. Interestingly, FOXO3 was transferred from cytosol into nucleus after linifanib treatment. Taken together, our results supported that linifanib inhibited the proliferation of human ovary cancer SKOV3 cells, which suggested that linifanib might have the potential to be developed as drugs for ovarian cancer treatment.     

Reactive oxygen species are involved in lysophosphatidic acid-induced apoptosis in rat cerebellar granule cells

Lysophosphatidic acid (LPA) induced apoptosis in primary rat cerebellar granule cells, which was characterized morphologically by chromatin condensation and the formation of apoptotic bodies. With redox-sensitive fluorescence probes DCFH-DA and DHR123, the formation of endogenous reactive oxygen species (ROS) inside cells during the apoptosis process was monitored by laser confocal scanning microscopy (LCSM). Pretreatment with the antioxidant tetramethylpyrazine (TMP) could effectively inhibit the formation of endogenous ROS and protect neurons from apoptosis. The results suggest that ROS might be involved in LPA-induced apoptosis in neurons.     

Hydroxydibenzoylmethane induces apoptosis through repressing ornithine decarboxylase in human promyelocytic leukemia HL-60 cells

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. Hydroxydibenzoylmethane (HDB), a derivative of dibenzoylmethane of licorice, is a promising chemopreventive agent. In this paper, we investigated whether HDB would inhibit the ODC pathway to enhance apoptosis in human promyelocytic leukemia HL-60 cells. We found ODC enzyme activity was reduced during HDB treatment. Overexpression of ODC in HL-60 parental cells could reduce HDB-induced apoptosis, which leads to loss of mitochondrial membrane potential (Δψ(m)), through lessening intracellular ROS. Furthermore, ODC overexpression protected cytochrome c release and the activation of caspase-3 following HDB treatment. The results demonstrated HDB-induced apoptosis was through a mechanism of down-regulation of ODC and occurred along a ROS-dependent mitochondria-mediated pathway.     

EPR study on the photosensitized generation of reactive oxygen species by actinomycin D

Actinomycin D (AMD) is an anticancer antibiotic that can bind selectively to both double-stranded and single-stranded DNA, and this binding greatly enhances DNA photosensitization. Using electron paramagnetic resonance (EPR) in combination with spin trapping techniques, a systematic study was carried out on the reactive oxygen species generated in the photosensitization process of AMD. It was found that 1O2 and O2- are important reactive intermediates either insolution or in DNA complexes, and the generation of these species is in competition. This finding suggests that the photodynamic action of AMD proceeds via two pathways: energy transfer (type Ⅰ mechanism) and electron transfer (type Ⅱ mechanism). 1O2 is the main product formed via energy transfer reaction in solution while electron transfer between the excited states of AMD and DNA becomes the predominant pathway in DNA complexes.     

Removal of lipopolysaccharide and reactive oxygen species using sialic acid immobilized polysulfone dialyzer

Sialic acid (N‐acetylneuraminic acid, NANA) was covalently immobilized onto the surface of a polysulfone (PSF) hollow fiber membrane. Prior to the immobilization, the surface of PSF was treated with ozone, followed by grafting with acrylic acid, and then the esterification of NANA. The surface concentration of NANA was determined by 2‐thiobarbituric acid (TBA) test. Hemocompatibility, the capability of suppressing oxidative stress, and clearance of lipopolysaccharide (LPS) from the resulting hollow fiber membrane were evaluated. The results show that by immobilizing NANA onto PSF hollow fiber, the adhesion of platelet was reduced, while both APTT and PT were little affected. Furthermore, oxidative stress was suppressed by NANA‐immobilized PSF hollow fibers. The level of LPS was also greatly reduced. Copyright © 2009 John Wiley & Sons, Ltd.     

Rutin inhibits osteoclast formation by decreasing reactive oxygen species and TNF-alpha by inhibiting activation of NF-kappaB

Rutin, a glycoside of flavonol, inhibits osteoclast formation induced by receptor activator of NF-kappaB ligand (RANKL) in bone marrow-derived macrophages. It reduces reactive oxygen species produced by RANKL and its inhibitory effect results from reduced levels of TNF-alpha. Rutin also lowers NF-kappaB activation in response to RANKL.     

Synthesis of new bicarbazole-linked triazoles as non-cytotoxic reactive oxygen species (ROS) inhibitors

Abstract

Carbazole analogs 3 and 4 and a new library of bicarbazole-linked triazoles 6–11 were prepared via new synthetic methodology. Metal-catalyzed oxidative coupling reaction was utilized for the synthesis of bicarbazole acetylene 4 and different metals (Zn⁺², Co⁺², Fe⁺³, Ni⁺², Cu⁺², Mn⁺²) as catalyst were screened. Only Fe-catalyzed reaction was found to be excellent and gave homocoupled product 4. Cu-catalyzed 1,3-dipolar cycloaddition was also utilized to install triazole moiety for the synthesis of hybrid molecules 6–11. Based on reported anti-inflammatory activity of carbazole and triazole scaffolds, all compounds were screened for their reactive oxygen species (ROS) inhibitory potential. Results from these studies revealed triazole 9 as most active compound (IC₅₀ value of 7.6?±?0.1?µg/mL on human whole blood and 2.7?±?0.09?µg/mL on isolated neutrophils) using ibuprofen as a standard. Therefore, class described herein can serve as attractive structural element for further studies on ROS inhibition.     

Analysis of intracellular reactive oxygen species by micellar electrokinetic capillary chromatography with laser-induced-fluorescence detector

Reactive Oxygen Species (ROS) are an important part of the normal cell growth cycle and play essential roles in many biological functions. Many techniques have been developed to detect and measure the amount of ROS present in cells. These techniques include spectrophotometry, high performance liquid chromatography (HPLC), capillary electrophoresis with laser-induced-fluorescence detector (CE-LIF) and capillary electrophoresis-on-a-chip with LIF. As ROS has a short half-life outside of the cell, various fluorescent probes, such as dihydrorhodamine 123 (DHR 123), that are membrane permeable have been used in the detection of intracellular ROS. In this paper, micellar electrokinetic capillary chromatography (MEKC) was coupled with LIF detector. Fluorescent probe, 3′-(p-aminophenyl)-fluorescein (APF), was used to detect specific ROS in Chinese Hamster Ovary (CHO) suspension cells as well as attached mouse bone marrow-derived dendritic cells (BMDCs). Separation buffer composition was optimized at a concentration of 25?mM tetraborate buffer with 50?mM sodium dodecyl sulfate at pH 9.3 and lysis of the cells was done successfully with a buffer of 70% ethanol and 0.1mM sodium dodecyl sulfate using a cell amount of 1?×?10⁷. ROS in cells was successfully analyzed by MEKC-LIF method developed, with acceptable RSD for time at 1.54% and area at 2.81%.     

The effect of covalent immobilization of sialic acid on the removal of lipopolysaccharide and reactive oxygen species for polyethylene terephthalate

Polyethylene terephthalate (PET) was aminolyzed with 1,6‐diaminohexane (DAH) and then sialic acid (NANA) was immobilized via amidation onto the surface. The surface concentration of NANA was determined by 2‐thiobarbituric acid (TBA) test. The hemocompatibility of the resulting PET fabrics was evaluated based on complete blood count (CBC), coagulating times, and protein adsorption. The ability to remove lipopolysaccharide (LPS) was also determined. In addition, the effect of contacting NANA‐immobilizing PET on the suppression of reactive oxygen species (ROS) production was measured by the chemiluminescence (CL) method. The results show that by immobilizing NANA onto PET, the adhesion of platelet (PLt) was reduced, and oxidative stress was suppressed. The level of LPS was also greatly reduced. Copyright © 2010 John Wiley & Sons, Ltd.     

TDAG51 deficiency promotes oxidative stress-induced apoptosis through the generation of reactive oxygen species in mouse embryonic fibroblasts

Apoptosis has an important role in maintaining tissue homeostasis in cellular stress responses such as inflammation, endoplasmic reticulum stress, and oxidative stress. T-cell death-associated gene 51 (TDAG51) is a member of the pleckstrin homology-like domain family and was first identified as a pro-apoptotic gene in T-cell receptor-mediated cell death. However, its pro-apoptotic function remains controversial. In this study, we investigated the role of TDAG51 in oxidative stress-induced apoptotic cell death in mouse embryonic fibroblasts (MEFs). TDAG51 expression was highly increased by oxidative stress responses. In response to oxidative stress, the production of intracellular reactive oxygen species was significantly enhanced in TDAG51-deficient MEFs, resulting in the activation of caspase-3. Thus, TDAG51 deficiency promotes apoptotic cell death in MEFs, and these results indicate that TDAG51 has a protective role in oxidative stress-induced cell death in MEFs.     

Nanozymes for regulation of reactive oxygen species and disease therapy

With high catalytic activity and stability, nanozymes have huge advantage in generating or eliminating the reactive oxygen species (ROS) due to their intrinsic enzyme-mimicking abilities, therefore attracting wide attention in ROS-related disease therapy. To better design nanozyme-based platforms for ROS-related biological application, we firstly illustrate the catalytic mechanism of different activities, and then introduce different strategies for using nanozymes to augment or reduce ROS level for the applications in cancer therapy, pathogen infection, neurodegeneration, etc. Finally, the challenges and future opportunities are proposed for the development and application of nanozymes.     

Regulation of pro-inflammatory responses by lipoxygenases via intracellular reactive oxygen species in vitro and in vivo

Reactive oxygen species (ROS) performs a pivotal function as a signaling mediator in receptor-mediated signaling. However, the sources of ROS in this signaling have yet to be determined, but may include lipoxygenases (LOXs) and NADPH oxidase. The stimulation of lymphoid cells with TNF-alpha, IL-1beta, and LPS resulted in significant ROS production and NF-kappaB activation. Intriguingly, these responses were markedly abolished via treatment with the LOXs inhibitor nordihydroguaiaretic acid (NDGA). We further examined in vivo anti-inflammatory effects of NDGA in allergic airway inflammation. Both intraperitoneal and intravenous NDGA administration attenuated ovalbumin (OVA)-induced influx into the lungs of total leukocytes, as well as IL-4, IL-5, IL-13, and TNF-alpha levels. NDGA also significantly reduced serum levels of OVA-specific IgE and suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine. The results of our histological studies and flow cytometric analyses showed that NDGA inhibits OVA-induced lung inflammation and the infiltration of CD11b+ macrophages into the lung. Collectively, our findings indicate that LOXs performs an essential function in pro-inflammatory signaling via the regulation of ROS regulation, and also that the inhibition of LOXs activity may have therapeutic potential with regard to the treatment of allergic airway inflammation.     

Nanoelectrodes for intracellular measurements of reactive oxygen and nitrogen species in single living cells

Reactive oxygen and nitrogen species (ROS and RNS) play important roles in various physiological processes (e.g. phagocytosis) and pathological conditions (e.g. cancer). The primary ROS/RNS, viz., hydrogen peroxide, peroxynitrite ion, nitric oxide, and nitrite ion, can be oxidized at different electrode potentials and therefore detected and quantified by electroanalytical techniques. Nanometer-sized electrochemical probes are especially suitable for measuring ROS/RNS in single cells and cellular organelles. In this article, we survey recent advances in the localized measurements of ROS/RNS inside single cells and discuss several methodological issues, including optimization of nanoelectrode geometry, precise positioning of an electrochemical probe inside a cell, and interpretation of electroanalytical data.     

Simultaneous determination of glutathione and reactive oxygen species in individual cells by microchip electrophoresis

A microchip electrophoresis method was developed for simultaneous determination of reactive oxygen species (ROS) and reduced glutathione (GSH) in the individual erythrocyte cell. In this method, cell sampling, single-cell loading, docking, lysing, and capillary electrophoretic separation with LIF detection were integrated on a microfluidic chip with crossed channels. ROS was labeled with dihydrorhodamine 123 in the intact cell, while GSH was on-chip labeled with 2,3-naphthalene-dicarboxaldehyde, which was included in the separation medium. On-chip electrical lysis, characterized by extremely fast disruption of the cellular membrane (<40 ms), was exploited to minimize enzymatic effects on analyte concentrations during the determination. The microfluidic network was optimized to prevent cell leaking from the sample reservoir (S) into separation during the separation phase. The structure of the S was modified to avoid blockage of its outlet by deposited cells. Detection limits of 0.5 and 6.9 amol for ROS and GSH, respectively, were achieved. The average cell throughput was 25 cells/h. The effectiveness of the method was demonstrated in the simultaneous determination of GSH and ROS in individual cells and the variations of cellular GSH and ROS contents in response to external stimuli.     

Analysis of oxidation process of cholecystokinin octapeptide with reactive oxygen species by high‐performance liquid chromatography and subsequent electrospray ionization mass spectrometry

The C‐terminal octapeptide of cholecystokinin (CCK8) includes some easily oxidizable amino acids. The oxidation of CCK8 by reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH^?) was investigated using reversed‐phase high performance liquid chromatography (RP‐HPLC) and subsequent electrospray ionization mass spectrometry. The mechanism of oxidation of CCK8 in the H₂O₂ system differed from that of CCK8 in the Fenton system, in which OH^? are produced. In the H₂O₂ system, ²⁸Met and ³¹Met were oxidized to methionine sulfoxide, and no further oxidation or degradation/hydrolysis occurred. On the other hand, in the Fenton system, ²⁸Met and ³¹Met residues were oxidized to methionine sulfone via the formation of methionine sulfoxide. In addition, the oxidized product was observed at the Trp residue but not at the Tyr residue, and small peptide fragments from CCK8 were observed in the Fenton system. From these results, it was concluded that ²⁸Met and ³¹Met residues of CCK8 are susceptible to oxidation by ROS. Copyright © 2009 John Wiley & Sons, Ltd.     

Reactive oxygen species, cell growth, and taxol production of Taxus cuspidata cells immobilized on polyurethane foam

Dynamic changes in reactive oxygen species (ROS) of Taxus cuspidata cells immobilized on polyurethane foam were investigated and the relation between ROS content and taxol production was discussed. Immobilization shortened the lag period of cell growth and moderately increased H₂O₂ and O₂ ^−• contents inside the microenvironment within the first 15 d. After 20 d, excessive production of H₂O₂ and O₂ ^−• was observed accompanied by marked increases in membrane lipid peroxidation and cell membrane permeability. The taxol content of immobilized cells was fourfold that of suspended cells at d 35. The addition of exogenous H₂O₂ barely affected malondialdehyde content and cell membrane permeability but led to an obvious accumulation of taxol. It is inferred that the intracellular and extracellular H₂O₂ inside the microenvironment might be one factor promoting taxol biosynthesis under the immobilization stress.