Outer membrane vesicles derived from probiotic Escherichia coli Nissle 1917 promote metabolic remodeling and M1 polarization of RAW264.7 macrophages

Successful implantation requires a fine-tuned dialog between the invading embryo and the maternal endometrium. Recently, we discovered that premature senescence of endometrial stromal cells (EnSC) might mediate improper decidual transformation of endometrial tissue and impair endometrial-blastocyst interaction. Here, we show that senescent EnSC are characterized by elevated intracellular reactive oxygen species (ROS) levels that originate from mitochondrial dysfunction and insufficient antioxidant defense. Decidualization of senescent EnSC is defective and is accompanied by the elevated intracellular and mitochondrial ROS levels. Antioxidant defense during decidualization is significantly less efficient in senescent EnSC compared to healthy ones. Senescent EnSC secrete increased amounts of ROS into the extracellular space. Elevated ROS released by senescent EnSC shift the redox balance and induce DNA damage in the neighboring trophoblast-like cells. In an in vitro implantation model, we observed impaired spreading of blastocyst-like spheroids into a monolayer of decidualizing senescent EnSC, which could be compensated by pretreatment of the senescent cells with the antioxidant, Tempol. Hence, we propose a possible mechanism that might be responsible, at least in part, for the defective embryo implantation realized via ROS transmitting from senescent EnSC to trophoblast cells. Such transmission results in the accumulation of ROS and subsequent DNA damage in trophoblastic cells, which might lead to improper migration and invasion of an embryo. In light of these findings, the application of antioxidants prior to implantation might be a promising strategy to improve implantation efficiency.

Real-time detection of intracellular reactive oxygen species and mitochondrial membrane potential in THP-1 macrophages during ultrasonic irradiation for optimal sonodynamic therapy

Like other Gram-negative pathogens, Vibrio cholerae, the causative agent of the diarrheal disease cholera, secretes outer membrane vesicles (OMVs). OMVs are complex entities composed of a subset of envelope lipid and protein components and play a role in the delivery of effector molecules to host cells. We previously showed that V. cholerae O395 cells secrete OMVs that are internalized by host cells, but their role in pathogenesis has not been well elucidated. In the present study, we evaluated the interaction of OMVs with intestinal epithelial cells. These vesicles induced expression of proinflammatory cytokines such as IL-8 and GM-CSF and chemokines such as CCL2, CCL20, and thymic stromal lymphopoietin in epithelial cells through activation of MAPK and NF-κB pathways in NOD1-dependent manner. Epithelial cells stimulated with OMVs activated dendritic cells (DCs) in a direct co-culture system. Activated DCs expressed high levels of co-stimulatory molecules; released inflammatory cytokines IL-1β, IL-6, TNF-α, and IL-23 and chemokines CCL22 and CCL17; and subsequently primed CD4(+) T cells leading to IL-4, IL-13, and IL-17 expression. These results suggest that V. cholerae O395 OMVs modulate the epithelial proinflammatory response and activate DCs, which promote T cell polarization toward an inflammatory Th2/Th17 response.

Gray mold, caused by Botrytis cinerea, poses significant threats to various crops, while it can be remarkably inhibited by ε-poly-L-lysine (ε-PL). A previous study found that B. cinerea extracts could stimulate the ε-PL biosynthesis of Streptomyces albulus, while it is unclear whether the impact of the B. cinerea signal on ε-PL biosynthesis is direct or indirect. This study evaluated the role of elevated reactive oxygen species (ROS) in efficient ε-PL biosynthesis after B. cinerea induction, and its underlying mechanism was disclosed with a transcriptome analysis. The microbial call from B. cinerea could arouse ROS elevation in cells, which fall in a proper level that positively influenced the ε-PL biosynthesis. A systematic transcriptional analysis revealed that this proper dose of intracellular ROS could induce a global transcriptional promotion on key pathways in ε-PL biosynthesis, including the embden-meyerhof-parnas pathway, the pentose phosphate pathway, the tricarboxylic acid cycle, the diaminopimelic acid pathway, ε-PL accumulation, cell respiration, and energy synthesis, in which sigma factor HrdD and the transcriptional regulators of TcrA, TetR, FurA, and MerR might be involved. In addition, the intracellular ROS elevation also resulted in a global modification of secondary metabolite biosynthesis, highlighting the secondary signaling role of intracellular ROS in ε-PL production. This work disclosed the transcriptional mechanism of efficient ε-PL production that resulted from an intracellular ROS elevation after B. cinerea elicitors' induction, which was of great significance in industrial ε-PL production as well as the biocontrol of gray mold disease.

The use of cyclosporine A (CsA) in transplant recipients is limited due to its side effects of causing severe hypertension. We have previously shown that CsA increases the activity of the epithelial sodium channel (ENaC) in cultured distal nephron cells. However, it remains unknown whether ENaC mediates CsA-induced hypertension and how we could prevent hypertension. Our data show that the open probability of ENaC in principal cells of split-open cortical collecting ducts was significantly increased after treatment of rats with CsA; the increase was attenuated by lovastatin. Moreover, CsA also elevated the levels of intracellular cholesterol (Cho), intracellular reactive oxygen species (ROS) via activation of NADPH oxidase p47phox, serum- and glucocorticoid-induced kinase isoform 1 (Sgk1), and phosphorylated neural precursor cell–expressed developmentally downregulated protein 4–2 (p-Nedd4-2) in the kidney cortex. Lovastatin also abolished CsA-induced elevation of α-, ß-, and γ-ENaC expressions. CsA elevated systolic blood pressure in rats; the elevation was completely reversed by lovastatin (an inhibitor of cholesterol synthesis), NaHS (a donor of H2S which ameliorated CsA-induced elevation of reactive oxygen species), or amiloride (a potent ENaC blocker). These results suggest that CsA elevates blood pressure by increasing ENaC activity via a signaling cascade associated with elevation of intracellular ROS, activation of Sgk1, and inactivation of Nedd4-2 in an intracellular cholesterol-dependent manner. Our data also show that NaHS ameliorates CsA-induced hypertension by inhibition of oxidative stress.

<div>Abstract<p>Oncogenic mutations within <i>RAS</i> genes and inactivation of p53 are the most common events in cancer. Earlier, we reported that activated Ras contributes to chromosome instability, especially in p53-deficient cells. Here we show that an increase in intracellular reactive oxygen species (ROS) and oxidative DNA damage represents a major mechanism of Ras-induced mutagenesis. Introduction of oncogenic H- or N-Ras caused elevated intracellular ROS, accumulation of 8-oxo-2′-deoxyguanosine, and increased number of chromosome breaks in mitotic cells, which were prevented by antioxidant <i>N</i>-acetyl-l-cysteine. By using Ras mutants that selectively activate either of the three major targets of Ras (Raf, RalGDS, and phosphatidylinositol-3-kinase) as well as dominant-negative Rac1 and RalA mutants and inhibitors of mitogen-activated protein kinase (MAPK)/extracellular signal–regulated kinases kinase-1 and p38 MAPKs, we have shown that several Ras effectors independently mediate ROS up-regulation. Introduction of oncogenic <i>RAS</i> resulted in repression of transcription from sestrin family genes <i>SESN1</i> and <i>SESN3</i>, which encode antioxidant modulators of peroxiredoxins. Inhibition of mRNAs from these genes in control cells by RNA interference substantially increased ROS levels and mutagenesis. Ectopic expression of <i>SESN1</i> and <i>SESN3</i> from lentiviral constructs interfered with Ras-induced ROS increase, suggesting their important contribution to the effect. The stability of Ras-induced increase in ROS was dependent on a p53 function: in the p53-positive cells displaying activation of p53 in response to Ras, only transient (4–7 days) elevation of ROS was observed, whereas in the p53-deficient cells the up-regulation was permanent. The reversion to normal ROS levels in the Ras-expressing p53-positive cells correlated with up-regulation of p53-responsive genes, including reactivation of <i>SESN1</i> gene. Thus, changes in expression of sestrins can represent an important determinant of genetic instability in neoplastic cells showing simultaneous dysfunctions of Ras and p53. [Cancer Res 2007;67(10):4671–8]</p></div>

<div>Abstract<p>Oncogenic mutations within <i>RAS</i> genes and inactivation of p53 are the most common events in cancer. Earlier, we reported that activated Ras contributes to chromosome instability, especially in p53-deficient cells. Here we show that an increase in intracellular reactive oxygen species (ROS) and oxidative DNA damage represents a major mechanism of Ras-induced mutagenesis. Introduction of oncogenic H- or N-Ras caused elevated intracellular ROS, accumulation of 8-oxo-2′-deoxyguanosine, and increased number of chromosome breaks in mitotic cells, which were prevented by antioxidant <i>N</i>-acetyl-l-cysteine. By using Ras mutants that selectively activate either of the three major targets of Ras (Raf, RalGDS, and phosphatidylinositol-3-kinase) as well as dominant-negative Rac1 and RalA mutants and inhibitors of mitogen-activated protein kinase (MAPK)/extracellular signal–regulated kinases kinase-1 and p38 MAPKs, we have shown that several Ras effectors independently mediate ROS up-regulation. Introduction of oncogenic <i>RAS</i> resulted in repression of transcription from sestrin family genes <i>SESN1</i> and <i>SESN3</i>, which encode antioxidant modulators of peroxiredoxins. Inhibition of mRNAs from these genes in control cells by RNA interference substantially increased ROS levels and mutagenesis. Ectopic expression of <i>SESN1</i> and <i>SESN3</i> from lentiviral constructs interfered with Ras-induced ROS increase, suggesting their important contribution to the effect. The stability of Ras-induced increase in ROS was dependent on a p53 function: in the p53-positive cells displaying activation of p53 in response to Ras, only transient (4–7 days) elevation of ROS was observed, whereas in the p53-deficient cells the up-regulation was permanent. The reversion to normal ROS levels in the Ras-expressing p53-positive cells correlated with up-regulation of p53-responsive genes, including reactivation of <i>SESN1</i> gene. Thus, changes in expression of sestrins can represent an important determinant of genetic instability in neoplastic cells showing simultaneous dysfunctions of Ras and p53. [Cancer Res 2007;67(10):4671–8]</p></div>

Oncogenic mutations within RAS genes and inactivation of p53 are the most common events in cancer. Earlier, we reported that activated Ras contributes to chromosome instability, especially in p53-deficient cells. Here we show that an increase in intracellular reactive oxygen species (ROS) and oxidative DNA damage represents a major mechanism of Ras-induced mutagenesis. Introduction of oncogenic H- or N-Ras caused elevated intracellular ROS, accumulation of 8-oxo-2'-deoxyguanosine, and increased number of chromosome breaks in mitotic cells, which were prevented by antioxidant N-acetyl-L-cysteine. By using Ras mutants that selectively activate either of the three major targets of Ras (Raf, RalGDS, and phosphatidylinositol-3-kinase) as well as dominant-negative Rac1 and RalA mutants and inhibitors of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases kinase-1 and p38 MAPKs, we have shown that several Ras effectors independently mediate ROS up-regulation. Introduction of oncogenic RAS resulted in repression of transcription from sestrin family genes SESN1 and SESN3, which encode antioxidant modulators of peroxiredoxins. Inhibition of mRNAs from these genes in control cells by RNA interference substantially increased ROS levels and mutagenesis. Ectopic expression of SESN1 and SESN3 from lentiviral constructs interfered with Ras-induced ROS increase, suggesting their important contribution to the effect. The stability of Ras-induced increase in ROS was dependent on a p53 function: in the p53-positive cells displaying activation of p53 in response to Ras, only transient (4-7 days) elevation of ROS was observed, whereas in the p53-deficient cells the up-regulation was permanent. The reversion to normal ROS levels in the Ras-expressing p53-positive cells correlated with up-regulation of p53-responsive genes, including reactivation of SESN1 gene. Thus, changes in expression of sestrins can represent an important determinant of genetic instability in neoplastic cells showing simultaneous dysfunctions of Ras and p53.

Regulation of reactive oxygen species and stress fiber formation by calpeptin in Swiss 3T3 fibroblasts

Acute myeloid leukemia (AML) is characterized by multiple mutagenic events that affect proliferation, survival, as well as differentiation. Recently, gain-of-function mutations in the α helical structure within the linker sequence of the E3 ubiquitin ligase CBL have been associated with AML. We identified four novel CBL mutations, including a point mutation (Y371H) and a putative splice site mutation in AML specimens. Characterization of these two CBL mutants revealed that coexpression with the receptor tyrosine kinases FLT3 (Fms-like tyrosine kinase 3) or KIT-induced ligand independent growth or ligand hyperresponsiveness, respectively. Growth of cells expressing mutant CBL required expression and kinase activity of FLT3. In addition to the CBL-dependent phosphorylation of FLT3 and CBL itself, transformation was associated with activation of Akt and STAT5 and required functional expression of the small GTPases Rho, Rac, and Cdc42. Furthermore, the mutations led to constitutively elevated intracellular reactive oxygen species levels, which is commonly linked to increased glucose metabolism in cancer cells. Inhibition of hexokinase with 2-deoxyglucose blocked the transforming activity of CBL mutants and reduced activation of signaling mechanisms. Overall, our data demonstrate that mutations of CBL alter cellular biology at multiple levels and require not only the activation of receptor proximal signaling events but also an increase in cellular glucose metabolism. Pathways that are activated by CBL gain-of-function mutations can be efficiently targeted by small molecule drugs.

Elevated intracellular reactive oxygen species (ROS) and antioxidant defense systems have been recognized as one of the hallmarks of cancer cells. Compared with normal cells, cancer cells exhibit increased ROS to maintain their malignant phenotypes and are more dependent on the "redox adaptation" mechanism. Thus, there are two apparently contradictory but virtually complementary therapeutic strategies for the regulation of ROS to prevent or treat cancer. The first strategy, that is, chemoprevention, is to prevent or reduce intracellular ROS either by suppressing ROS production pathways or by employing antioxidants to enhance ROS clearance, which protects normal cells from malignant transformation and inhibits the early stage of tumorigenesis. The second strategy is the ROS-mediated anticancer therapy, which stimulates intracellular ROS to a toxicity threshold to activate ROS-induced cell death pathways. Therefore, targeting the regulation of intracellular ROS-related pathways by small-molecule candidates is considered to be a promising treatment for tumors. We herein first briefly introduce the source and regulation of ROS, and then focus on small molecules that regulate ROS-related pathways and show efficacy in cancer therapy from the perspective of pharmacophores. Finally, we discuss several challenges in developing cancer therapeutic agents based on ROS regulation and propose the direction of future development.

SIRT3 is a key regulator of mitochondrial reactive oxygen species as well as mitochondrial function. The retina is one of the highest energy-demanding tissues, in which the regulation of reactive oxygen species is critical to prevent retinal neurodegeneration. Although previous reports have demonstrated that SIRT3 is highly expressed in the retina and important in neuroprotection, function of SIRT3 in regulating reactive oxygen species in the retina is largely unknown. In this study, we investigated the role of retinal SIRT3 in a light-induced retinal degeneration model using SIRT3 knockout mice. We demonstrate that SIRT3 deficiency causes acute reactive oxygen species accumulation and endoplasmic reticulum stress in the retina after the light exposure, which leads to increased photoreceptor death, retinal thinning, and decreased retinal function. Using a photoreceptor-derived cell line, we revealed that reactive oxygen species were the upstream initiators of endoplasmic reticulum stress. Under SIRT3 knockdown condition, we demonstrated that decreased superoxide dismutase 2 activity led to elevated intracellular reactive oxygen species. These studies have helped to elucidate the critical role of SIRT3 in photoreceptor neuronal survival, and suggest that SIRT3 might be a therapeutic target for oxidative stress-induced retinal disorders.

The fungal pathogen Sporisorium scitamineum causes sugarcane smut disease. The formation and growth of dikaryotic hypha after sexual mating is critical for S. scitamineum pathogenicity, however regulation of S. scitimineum mating has not been studied in detail. We identified and characterized the core components of the conserved cAMP/PKA pathway in S. scitamineum by reverse genetics. Our results showed that cAMP/PKA signalling pathway is essential for proper mating and filamentation, and thus critical for S. scitamineum virulence. We further demonstrated that an elevated intracellular ROS (reactive oxygen species) level promotes S. scitamineum mating-filamentation, via transcriptional regulation of ROS catabolic enzymes, and is under regulation of the cAMP/PKA signalling pathway. Furthermore, we found that fungal cAMP/PKA signalling pathway is also involved in regulation of host ROS response. Overall, our work displayed a positive role of elevated intracellular ROS in fungal differentiation and virulence.

Cross-linked selenolipoic acid nanoplatform modulates intracellular and extracellular reactive oxygen species for cancer immunotherapy.

Radiation resistance can be overcome by a combination treatment with chemical modifiers. Here, we showed that treatment with 5-bromo-3-(3-hydroxyprop-1-ynyl)-2H-pyran-2-one (BHP), a new 2-pyrone derivative, in combination with ionizing radiation enhances the sensitivity of human cervical cancer cells to ionizing radiation through overproduction of reactive oxygen species (ROS). The combined treatment with BHP and ionizing radiation caused a decrease in clonogenic survival and an increase in apoptotic cell death in cervical cancer cells. The combined treatment promoted conformational activation of Bax and led to mitochondrial apoptotic cell death. The combination treatment also induced a marked increase in intracellular ROS level. Inhibition of ROS attenuated the radiosensitizing effect of BHP, concurrent with a decrease in Bax activation, a decrease in mitochondrial cell death, and an increase in clonogenic survival. These results indicate that BHP synergistically enhances sensitivity of human cervical cancer cells to ionizing radiation through elevation of intracellular ROS and that ROS-dependent Bax activation is critically involved in the increase in apoptotic cell death induced by the combined treatment with BHP and ionizing radiation.