Addition Of Reactive Oxygen Species Scavengers Research Articles

A novel ruthenium-silver based antimicrobial potentiates aminoglycoside activity against Pseudomonas aeruginosa.

The rapid dissemination of antibiotic resistance combined with the decline in the discovery of novel antibiotics represents a major challenge for infectious disease control that can only be mitigated by investments in novel treatment strategies. Alternative antimicrobials, including silver, have regained interest due to their diverse mechanisms of inhibiting microbial growth. One such example is AGXX, a broad-spectrum antimicrobial that produces highly cytotoxic reactive oxygen species (ROS) to inflict extensive macromolecular damage. Due to the connections identified between ROS production and antibiotic lethality, we hypothesized that AGXX could potentially increase the activity of conventional antibiotics. Using the gram-negative pathogen Pseudomonas aeruginosa, we screened possible synergistic effects of AGXX on several antibiotic classes. We found that the combination of AGXX and aminoglycosides tested at sublethal concentrations led to a rapid exponential decrease in bacterial survival and restored the sensitivity of a kanamycin-resistant strain. ROS production contributes significantly to the bactericidal effects of AGXX/aminoglycoside treatments, which is dependent on oxygen availability and can be reduced by the addition of ROS scavengers. Additionally, P. aeruginosa strains deficient in ROS detoxifying/repair genes were more susceptible to AGXX/aminoglycoside treatment. We further demonstrate that this synergistic interaction was associated with a significant increase in outer and inner membrane permeability, resulting in increased antibiotic influx. Our study also revealed that AGXX/aminoglycoside-mediated killing requires an active proton motive force across the bacterial membrane. Overall, our findings provide an understanding of cellular targets that could be inhibited to increase the activity of conventional antimicrobials. IMPORTANCE The emergence of drug-resistant bacteria coupled with the decline in antibiotic development highlights the need for novel alternatives. Thus, new strategies aimed at repurposing conventional antibiotics have gained significant interest. The necessity of these interventions is evident especially in gram-negative pathogens as they are particularly difficult to treat due to their outer membrane. This study highlights the effectiveness of the antimicrobial AGXX in potentiating aminoglycoside activities against P. aeruginosa. The combination of AGXX and aminoglycosides not only reduces bacterial survival rapidly but also significantly re-sensitizes aminoglycoside-resistant P. aeruginosa strains. In combination with gentamicin, AGXX induces increased endogenous oxidative stress, membrane damage, and iron-sulfur cluster disruption. These findings emphasize AGXX's potential as a route of antibiotic adjuvant development and shed light on potential targets to enhance aminoglycoside activity.

Reactive oxygen species derived from NADPH oxidase regulate astaxanthin and total fatty acid accumulation in Chromochloris zofingiensis

Many abiotic stresses can promote astaxanthin and total fatty acid (TFA) accumulation in microalgae, but their signal molecules remain undetermined. The reactive oxygen species (ROS) derived from NADPH oxidase has been reported to act as signals and regulate many cellular responses in cell responses to biotic and abiotic stresses in higher plants. The present study explored the regulatory effects of NADPH oxidase derived ROS on astaxanthin and TFA accumulation in the green alga Chromochloris zofingiensis under nitrogen starvation. Diphenylene iodonium (DPI) inhibition of NADPH oxidase significantly decreased the cellular ROS level under nitrogen starvation. This was accompanied with the decreasing of astaxanthin and secondary carotenoids content, and the increasing of primary carotenoids content. Molecular studies showed that DPI depressed the expression of PSY, LCYb, and BKT, but elevated that of LCYe, which were coordinated with carotenoids content. Meanwhile, the TFA and neutral lipid contents, and the expression of their key biosynthesis genes, ACCase and PDAT, were also reduced in the DPI-treated cells. Additionally, the addition of ROS scavenger, N-acetylcysteine (NAC), decreased the astaxanthin and TFA contents. Taken together, our results revealed that NADPH oxidase-derived ROS was the key regulator in astaxanthin and fatty acid accumulation under nitrogen starvation.

A New Antifungal Agent (4-phenyl-1, 3-thiazol-2-yl) Hydrazine Induces Oxidative Damage in Candida albicans.

A gradual rise in immunocompromised patients over past years has led to the increasing incidence of invasive fungal infections. Development of effective fungicides can not only provide new means for clinical treatment, but also reduce the occurrence of fungal resistance. We identified a new antifungal agent (4-phenyl-1, 3-thiazol-2-yl), hydrazine (numbered as 31C) which showed high-efficiency, broad-spectrum and specific activities. The minimum inhibitory concentration of 31C against pathogenic fungi was between 0.0625-4 μg/ml in vitro, while 31C had no obvious cytotoxicity to human umbilical vein endothelial cells with the concentration of 4 μg/ml. In addition, 31C of 0.5 μg/ml could exhibit significant fungicidal activity and inhibit the biofilm formation of C. albicans. In vivo fungal infection model showed that 31C of 10 mg/kg significantly increased the survival rate of Galleria mellonella. Further study revealed that 31C-treatment increased the reactive oxygen species (ROS) in C. albicans and elevated the expression of some genes related to anti-oxidative stress response, including CAP1, CTA1, TRR1, and SODs. Consistently, 31C-induced high levels of intracellular ROS resulted in considerable DNA damage, which played a critical role in antifungal-induced cellular death. The addition of ROS scavengers, such as glutathione (GSH), N-Acetyl-L-cysteine (NAC) or oligomeric proanthocyanidins (OPC), dramatically reduced the antifungal activities of 31C and rescued the 31C-induced filamentation defect. Collectively, these results showed that 31C exhibited strong antifungal activity and induced obvious oxidative damage, which indicated that compounds with a structure similar to 31C may provide new sight for antifungal drug development.

Involvement of the mitochondrial nuclease EndoG in the regulation of cell proliferation through the control of reactive oxygen species

The apoptotic nuclease EndoG is involved in mitochondrial DNA replication. Previous results suggested that, in addition to regulate cardiomyocyte hypertrophy, EndoG could be involved in cell proliferation. Here, by using in vivo and cell culture models, we investigated the role of EndoG in cell proliferation. Genetic deletion of Endog both in vivo and in cultured cells or Endog silencing in vitro induced a defect in rodent and human cell proliferation with a tendency of cells to accumulate in the G1 phase of cell cycle and increased reactive oxygen species (ROS) production. The defect in cell proliferation occurred with a decrease in the activity of the AKT/PKB-GSK-3β-Cyclin D axis and was reversed by addition of ROS scavengers. EndoG deficiency did not affect the expression of ROS detoxifying enzymes, nor the expression of the electron transport chain complexes and oxygen consumption rate. Addition of the micropeptide Humanin to EndoG-deficient cells restored AKT phosphorylation and proliferation without lowering ROS levels. Thus, our results show that EndoG is important for cell proliferation through the control of ROS and that Humanin can restore cell division in EndoG-deficient cells and counteracts the effects of ROS on AKT phosphorylation.

Identification of a novel oxidative stress induced cell death by Sorafenib and oleanolic acid in human hepatocellular carcinoma cells

The lack of effective chemotherapies in hepatocellular carcinoma (HCC) is still an unsolved problem and underlines the need for new strategies in liver cancer treatment. In this study, we present a novel approach to improve the efficacy of Sorafenib, today’s only routinely used chemotherapeutic drug for HCC, in combination with triterpenoid oleanolic acid (OA). Our data show that cotreatment with subtoxic concentrations of Sorafenib and OA leads to highly synergistic induction of cell death. Importantly, Sorafenib/OA cotreatment triggers cell damage in a sustained manner and suppresses long-term clonogenic survival. Sorafenib/OA cotreatment induces DNA fragmentation and caspase-3/7 cleavage and the addition of the pan-caspase inhibitor zVAD.fmk shows the requirement of caspase activation for Sorafenib/OA-triggered cell death. Furthermore, Sorafenib/OA co-treatment stimulates a significant increase in reactive oxygen species (ROS) levels. Most importantly, the accumulation of intracellular ROS is required for cell death induction, since the addition of ROS scavengers (i.e. α-tocopherol, MnTBAP) that prevent the increase of intracellular ROS levels completely rescues cells from Sorafenib/OA-triggered cell death. In conclusion, OA represents a novel approach to increase the sensitivity of HCC cells to Sorafenib via oxidative stress.

Toxicity of perfluorooctane sulfonate and perfluorooctanoic acid to Escherichia coli: Membrane disruption, oxidative stress, and DNA damage induced cell inactivation and/or death

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are two widely used polyfluorinated compounds (PFCs) and are persistent in the environment. This study for the first time systematically investigated their toxicities and the underlying mechanisms to Escherichia coli. Much higher toxicity was observed for PFOA than PFOS, with the 3 h half growth inhibition concentrations (IC50) determined to be 10.6 ± 1.0 and 374 ± 3 mg L−1, respectively, while the bacterial accumulation of PFOS was much greater than that of PFOA. The PFC exposures disrupted cell membranes as evidenced by the dose-dependent variations of cell structures (by transmission electron microscopy observations), surface properties (electronegativity, hydrophobicity, and membrane fluidity), and membrane compositions (by gas chromatogram and Fourier transform infrared spectroscopy analyses). The increases in the contents of intracellular reactive oxygen species (ROS) and malondialdehyde and the activity of superoxide dismutase indicated the increment of oxidative stress induced by the PFCs in the bacterial cells. The fact that the cell growth inhibition was mitigated by the addition of ROS scavenger (N-acetyl cysteine) further evidenced the important role of oxidative damage in the toxicities of PFOS and PFOA. Eighteen genes involved in cell division, membrane instability, oxidative stress, and DNA damage of the exposed cells were up or down expressed, indicating the DNA damage by the PFCs. The toxicities of PFOS and PFOA to E. coli were therefore ascribed to the membrane disruption, oxidative stress, and DNA damage induced cell inactivation and/or death. The difference in the bactericidal effect between PFOS and PFOA was supposed to be related to their different dominating toxicity mechanisms, i.e., membrane disruption and oxidative damage, respectively. The outcomes will shed new light on the assessment of ecological effects of PFCs.

Abstract 5541: Proteasome inhibitor synergizes with histone deacetylase inhibitor to trigger ROS- and ER stress-induced apoptosis of nasopharyngeal carcinoma independent of aggresome disruption

Abstract Proteasome inhibitor and histone deacetylase inhibitor (HDACi) can synergistically induce apoptosis of cancer cells through aggresome disruption or induction of endoplasmic reticulum (ER) stress. We previously reported that suberoylanilide hydroxamic acid (SAHA), a pan-HDACi, and bortezomib, a proteasome inhibitor can synergistically induce apoptosis of nasopharyngeal carcinoma (NPC) cells through reactive oxygen species (ROS)-driven caspase-dependent mechanism. Here, we wish to investigate the role of aggresome disruption or ER stress in the process. We first tested the effect of bortezomib combining with a more specific HDACi, including MS-275, apicidin or romidepsin, on proliferation of NPC cells. Bortezomib could synergize with each of the specific HDACis to induce killing of the NPC cells. Strong apoptosis was demonstrated by the proteolytic cleavage of PARP and caspase-3, -8 and -9 as well as the high percentage of annexin V/propidium iodide (AV/PI)-positive NPC cells upon treatment with combination of bortezomib at 7.5 to 15 nM and romidepsin at 2.5 to 5 nM. Consistently, the apoptosis was ROS- and caspase-dependent. Interestingly, α-tubulin, a key substrate of histone deacetylase 6, was not acetylated, arguing against the involvement of aggresome disruption. In contrast, ER stress induction might be involved because ATF-4 and CHOP, which are markers of ER stress, were up-regulated by combined bortezomib/romidepsin and inhibition of caspase-4 could suppress the apoptosis. Furthermore, addition of ROS scavenger, N-acetyl-cysteine, suppressed the expression of ATF-4 and CHOP. We conclude that proteasome inhibitor can synergize with HDACi to trigger ROS- and ER stress-induced apoptosis of NPC cells, independent of aggresome disruption. This project is funded by NPC Area of Excellence (AoE/M 06/08 Center for Nasopharyngeal Carcinoma Research), CRCG (#10401264) and Epstein-Barr virus research (# 20004525) grants of A.K.S. Chiang. Citation Format: Alan K. Chiang, Kwai Fung Hui. Proteasome inhibitor synergizes with histone deacetylase inhibitor to trigger ROS- and ER stress-induced apoptosis of nasopharyngeal carcinoma independent of aggresome disruption. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5541. doi:10.1158/1538-7445.AM2014-5541

Ouabain Suppresses the Migratory Behavior of Lung Cancer Cells

The migratory capability of cancer cells is one of the most important hallmarks reflecting metastatic potential. Ouabain, an endogenous cardiac glycoside produced by the adrenal gland, has been previously reported to have anti-tumor activities; however, its role in the regulation of cancer cell migration remains unknown. The present study has revealed that treatment with ouabain at physiological concentrations is able to inhibit the migratory activities of human lung cancer H292 cells. The negative effects of ouabain were found to be mediated through the suppression of migration regulatory proteins, such as focal adhesion kinase (FAK), ATP-dependent tyrosine kinase (Akt), and cell division cycle 42 (Cdc42). We found that the observed actions of ouabain were mediated via a reactive oxygen species (ROS)-dependent mechanism because the addition of ROS scavengers (N-acetylcysteine and glutathione) could reverse the effect of ouabain on cell migration. Furthermore, ouabain was shown to inhibit the spheroidal tumor growth and decrease the cancer cell adhesion to endothelial cells. However, the compound had no significant effect on anoikis of the cells. Together, these findings shed light on the understanding of cancer cell biology by exploring the novel function of this endogenous human substance.

Hydroxytyrosol Promotes Superoxide Production and Defects in Autophagy Leading to Anti-proliferation and Apoptosis on Human Prostate Cancer Cells

Hydroxytyrosol, an important polyphenolic compound found in olive oil, has shown anti-tumor activity both in vitro and in vivo. However, effects of hydroxytyrosol on prostate cancer are largely unkown. We found that hydroxytyrosol preferentially reduces the viability of human prostate cancer cells (PC-3, DU145) compared to an immortalized non-malignant prostate epithelial cell line (RWPE-1). Exposure of PC-3 cells to 80 µmol/L hydroxytyrosol resulted in significant increases in both superoxide production and activation of apoptosis. These increases were accompanied by mitochondrial dysfunction, defects in autophagy, and activation of MAP kinases. Moreover, N-acetylcysteine (NAC), an efficient reactive oxygen species (ROS) scavenger, was able to reverse the hydroxytyrosol-induced effects of cell viability loss, defects in autophagy, and activation of apoptosis. This evidence suggests that ROS play a vital role in the loss of PC-3 cell viability. However, MAPK inhibitors including U0126 (for Erk1/2), SB203580 (for p38MAPK) and SP600125 (for JNK) did not decrease hydroxytyrosol-induced growth inhibition, suggesting that these kinases may not be required for the growth inhibitory effect of hydroxytyrosol. Moreover, addition of ROS scavengers (i.e. NAC, catalase, pyruvate, SOD) in the growth media can prevent hydroxytyrosol induced cell viability loss, suggesting that extracellular ROS (superoxide and hydrogen peroxide) facilitate the anti-proliferation effect of hydroxytyrosol in prostate cancer cells. The present work firstly shows that hydroxytyrosol induces apoptotic cell death and mitochondrial dysfunction by generating superoxide in PC-3 cells. This research presents preliminary evidence on the in vitro chemopreventive effect of hydroxytyrosol, and will contribute to further investigation of hydroxytyrosol as an anti-cancer agent.

Abstract 2278: Smac mimetic primes FADD- or caspase-8-deficient leukemia cells for TNFα-induced necroptosis and overcomes apoptosis resistance

Abstract Evasion of apoptosis contributes to treatment resistance, one of the major, yet unresolved obstacles in oncology. Searching for new strategies to bypass apoptosis resistance we investigated the potential of Smac mimetic in acute lymphoblastic leukemia (ALL) cells deficient in FADD or caspase-8. Here, we demonstrate for the first time that Smac mimetic primes apoptosis-resistant leukemia cells for TNFα-induced necroptosis as an alternative mode of cell death. The interaction of Smac mimetic and TNFα occurs is highly synergistic as calculated by combination index (CI <0.1). Kinetic analysis reveals that Smac mimetic and TNFα cause cell death rapidly within few hours. Interestingly, Smac mimetic- and TNFα-mediated cell death in FADD-deficient cells occurs without characteristic features of apoptosis, namely without caspase activation or DNA fragmentation, pointing to non-apoptotic cell death. In sharp contrast, Smac mimetic and TNFα trigger activation of caspase-8, -9 and -3 and DNA fragmentation in apoptosis-proficient wildtype cells that express FADD and caspase-8. Consistently, the caspase inhibitor zVAD.fmk protects wildtype cells against Smac mimetic- and TNFα-triggered cell death, whereas zVAD.fmk fails to block cell death in FADD-deficient cells. Furthermore, production of reactive oxygen species (ROS) precedes Smac mimetic- and TNFα-induced cell death in FADD-deficient cells. In line with the notion that ROS contributes to cell death induction, the addition of ROS scavengers (i.e. butylated hydroxyanisole (BHA) and trolox) or the addition of the NADPH oxidase inhibitor diphenyliodonium (DPI) significantly reduce cell death upon combination treatment. Moreover, Smac mimetic- and TNFα-triggered cell death is accompanied by enhanced RIP1 kinase activity in FADD-deficient cells. Of note, necrostatin-1, a RIP1 kinase inhibitor, reduces Smac mimetic- and TNFα-induced ROS generation and completely abolishes cell death in FADD-deficient cells, in accordance with necroptotic cell death. Importantly, Smac mimetic also sensitizes apoptosis-resistant, primary leukemic blasts derived from patients with ALL to TNFα-induced necroptosis, underscoring the clinical relevance of these findings. In conclusion, Smac mimetic primes leukemia cells to TNFα-induced apoptosis or necroptosis in a context-dependent manner. In apoptosis-proficient cells which express both FADD and caspase-8, Smac mimetic enhances TNFα-induced, caspase-dependent apoptosis. However in apoptosis-resistant cells lacking either FADD or caspase-8, Smac mimetic sensitizes cells for TNFα-initiated necroptosis that critically depends on RIP1 and ROS production. These findings have important implications for the therapeutic exploitation of necroptosis as an alternative cell death program to overcome apoptosis resistance of cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2278. doi:1538-7445.AM2012-2278

Alterations in the Proangiogenic Functions of Adipose Tissue–Derived Stromal Cells Isolated from Diabetic Rats

Cardiovascular complications in diabetic patients have been reported to be related to the impaired proangiogenic actions of endothelial progenitor cells. In this study, we investigated the functions of adipose tissue-derived stromal cells (ASCs) in diabetes. We induced type I diabetes in rats by a intraperitoneal injection of 60 mg/kg of streptozotocin (STZ) and type II diabetes by the combined treatment of high fat diet and 45 mg/kg of STZ. Rat ASCs (rASCs) isolated from the adipose tissues in the interscapular and abdominal region of type I or type II diabetic rats showed lower proliferating ability than those of control rats. Diabetic rASCs showed lower blood flow recovery than those of control rats in a hindlimb ischemia model of nude mouse. When ASCs isolated from rat and human were exposed to high glucose concentrations, their proliferating abilities and improved blood flow in a hindlimb ischemia model were compromised, compared with ASCs that were maintained at control glucose concentrations. However, the same concentrations of mannitol did not affect these characteristics. Exposure of human ASCs (hASCs) to high glucose concentrations increased reactive oxygen species (ROS) production, and the addition of ROS scavengers [N-acetylcysteine (NAC) or catalase] to high glucose media partially decreased the high glucose-induced inhibitory effect on proliferating ability in hASCs. However, hASCs treated with high glucose medium for 6 days showed lower proliferation in control culture medium, which was not recovered by the addition of NAC or catalase. These data indicate that ASCs isolated from diabetic rats and exposed at high concentration of glucose have an impaired proangiogenic action and that the functional impairment is partly due to ROS generated by chronic exposure to high glucose concentrations.

Analytical Studies on the Prediction of Photosensitive/Phototoxic Potential of Pharmaceutical Substances

Phototoxic responses after administration of photosensitive pharmaceutics have been recognized as undesirable side effects, and predicting potential hazardous side effects is gaining importance as new drugs are introduced to the market. In this work, we characterize the photochemical/photobiological properties of model compounds to develop an effective screening method for the prediction of phototoxic/photosensitive potential. Twenty-one known photosensitive/phototoxic compounds and five weak/nonphototoxic compounds were subjected to ultraviolet (UV) spectral analyses and photochemical evaluation including the determination of produced reactive oxygen species (ROS) and photostability study. The photooxidation of linoleic acid was also monitored in the presence of tested compounds, guided on the formation of thiobarbituric acid reactive substances. Most photosensitive/phototoxic drugs tested, even weak UV absorbers, at a concentration of 200 microM showed significant production of ROS under 18 h light exposure (30,000 lx). On the other hand, ROS generated from weak/nonphototoxic compounds, including strong UV absorber benzocaine, were low or negligible. Although exposure of quinine to light resulted in significant degradation (half-life, t1/2=6.4 h), it was dramatically attenuated by the addition of ROS scavengers, especially sodium azide (t1/2=122.6 h). Furthermore, concomitant exposure of photosensitive/phototoxic compounds (200 microM) and linoleic acid (1 mM) for 18 h led to the marked formation of lipoperoxide. Results indicated that known photosensitive/phototoxic compounds tested have the ability to generate ROS under light exposure, and this photochemical reaction could be associated with their photoinstability and/or phototoxic responses. Based on these findings, determination of ROS, generated from photoirradiated compounds, may be an effective predictive model in recognizing their photosensitive/phototoxic potential.

Photochemical effects on microbial activity in natural waters: the interaction of reactive oxygen species and dissolved organic matter

Bacterial utilization of dissolved organic matter (DOM) in surface waters is closely linked to photochemical transformations of DOM. Photochemically produced reactive oxygen species (ROS) play a central role in many photochemical reactions, but the role of ROS for the photochemical facilitation of bacterial utilization of DOM is previously not known. We exposed lake water with high DOM concentrations to simulated sunlight, with and without the addition of ROS scavengers, and quantified the effect on the production of CO 2, the loss of DOM absorbance, and bacterial growth. The photodegradation of DOM through microbial–photochemical interactions was dependent on the action of ROS. The use of ROS scavengers in irradiations of the lake water revealed that photobleaching below 300 nm and the production of CO 2 are highly dependent on the action of ROS. Photobleaching and CO 2 production in irradiated waters decreased significantly with the addition of ROS scavengers, but post-irradiation bacterial growth in the samples containing an ROS scavenger increased significantly above those without. The decrease in ROS activity (CO 2 production) likely caused an accumulation of bioavailable DOM and enhanced microbial processes. Rapid degradation of DOM through the action of ROS would be especially important in high DOM systems. The high photochemical ROS activity may counterbalance the positive effects on bacterial activity of DOM photolysis into bioavailable molecules.