Promoted ROS Production Research Articles

ITRAQ-based proteomics and in vitro experiments reveals essential roles of ACE and AP-N in the renin–angiotensin system-mediated congenital ureteropelvic junction obstruction

ObjectiveUreteropelvic junction obstruction (UPJO) is a common renal obstructive disorder, but its pathogenic mechanisms remain largely unclear. We aimed to investigate the potential involvement of the renin–angiotensin system in congenital UPJO pathogenesis. MethodsDifferentially expressed proteins in exosomes isolated from amniotic fluid of patients with congenital UPJO were characterized using iTRAQ (isobaric tags for relative and absolute quantification)-based proteomics. The expressions of angiotensin-converting enzyme (ACE) and aminopeptidase N (AP-N) in HK2 cells were inhibited by quinapril and siRNA, respectively. Cell proliferation and reactive oxygen species were measured by EdU staining and flow cytometry, respectively. Gene expression was detected by Western blot or qRT-PCR. The inflammatory factors were measured through ELISA. Mice that underwent unilateral ureteral obstruction were used as the animal model. ResultsThe identity of exosomes from amniotic fluids was confirmed by the expression of CD9 and CD26. In total, 633 differentially expressed proteins were identified in the amniotic fluid-derived exosomes from patients with UPJO, including 376 up- and 257 down-regulated proteins associated with multiple biological processes. Of them, ACE and AP-N were significantly decreased in the amniotic fluid exosomes. Inhibition of ACE and AP-N resulted in suppressed cell proliferation; repressed IARP, AT1R, and MAS1 expression; elevated ROS production; and increased IL-1β, TNF-α, and IL-6 levels in HK2 cells. Decreased ACE expression and elevated IL-1β levels were also observed in the mouse model. ConclusionSuppression of ACE and AP-N expression mediates congenital UPJO pathogenesis by repressing renal tubular epithelial proliferation, promoting ROS production, and enhancing inflammatory factor expression.

Hydroxy-octadecenoic acids instead of phorbol esters are responsible for the Jatropha curcas kernel cake\u2019s toxicity

The toxic kernel cake of Jatropha curcas (KCakeJ) is an emerging health and environmental concern. Although phorbol esters are widely recognized as the major toxin of KCakeJ, convincing evidence is absent. Here, we show that rather than phorbol esters an isomeric mixture of 11-hydroxy-9E-octadecenoic acid, 12-hydroxy-10E-octadecenoic acid and 12-hydroxy-10Z-octadecenoic acid (hydroxy-octadecenoic acids, molecular formula C18H34O3) is the major toxic component. The toxicities of hydroxy-octadecenoic acids on experimental animals, e.g. acute lethality, causing inflammation, pulmonary hemorrhage and thrombi, allergies, diarrhea and abortion, are consistent with those on human/animals caused by Jatropha seed and/or KCakeJ. The hydroxyl group and the double bond are essential for hydroxy-octadecenoic acids’ toxicity. The main pathway of the toxicity mechanism includes down-regulating UCP3 gene expression, promoting ROS production, thus activating CD62P expression (platelet activation) and mast cell degranulation. The identification of the major toxin of KCakeJ lays a foundation for establishing an environmentally friendly Jatropha biofuel industry.

ALCAT1 Overexpression Affects Supercomplex Formation and Increases ROS in Respiring Mitochondria.

Cardiolipin (CL) is a multifunctional dimeric phospholipid that physically interacts with electron transport chain complexes I, III, and IV, and ATP synthase (complex V). The enzyme ALCAT1 catalyzes the conversion of cardiolipin by incorporating polyunsaturated fatty acids into cardiolipin. The resulting CL species are said to be more susceptible to oxidative damage. This is thought to negatively affect the interaction of cardiolipin and electron transport chain complexes, leading to increased ROS production and mitochondrial dysfunction. Furthermore, it is discussed that ALCAT1 itself is upregulated due to oxidative stress. Here, we investigated the effects of overexpression of ALCAT1 under different metabolic conditions. ALCAT1 is located at the ER and mitochondria, probably at contact sites. We found that respiration stimulated by galactose supply promoted supercomplex assembly but also led to increased mitochondrial ROS levels. Endogeneous ALCAT1 protein expression levels showed a fairly high variability. Artificially induced ALCAT1 overexpression reduced supercomplex formation, further promoted ROS production, and prevented upregulation of coupled respiration. Taken together, our data suggest that the amount of the CL conversion enzyme ALCAT1 is critical for coupling mitochondrial respiration and metabolic plasticity.

MOTS-c inhibits Osteolysis in the Mouse Calvaria by affecting osteocyte-osteoclast crosstalk and inhibiting inflammation

The Mitochondrial-derived peptide MOTS-c has recently been reported as a 16-amino acid peptide regulating metabolism and homeostasis in different cells. However, its effects on immune cells and bone metabolism are rarely reported. Here we demonstrate that MOTS-c treatment in ultra-high molecular weight polyethylene (UHMWPE) particle-induced osteolysis mouse model alleviated bone erosion and inflammation. MOTS-c increased osteoprotegerin (OPG)/ receptor activator of nuclear factor kappa-B ligand (RANKL) ratio in osteocytes, leading to inhibition of osteoclastogenesis. In primary bone marrow macrophages (BMMs) MOTS-c alleviated STAT1 and NF-κB phosphorylation triggered by UHMWPE particles. Promoting ROS production or suppressing peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) by adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK) repression blocked these anti-inflammatory effects of MOTS-c treatment. Taken together, these findings provide evidence that the small peptide inhibits osteoclastogenesis by regulating osteocyte OPG/RANKL secretion and suppressing inflammation via restraining NF-κB and STAT1 pathway. Moreover, its effects on NF-κB activation is dependent on the AMPK-PGC-1α-ROS axis, suggesting its potential use in osteolysis and other inflammation disorders.

Aflatoxin B1 impairs leydig cells through inhibiting AMPK/mTOR-mediated autophagy flux pathway

Aflatoxin B1 (AFB1), a potential endocrine disrupter, has been shown to induce hepatotoxicity in animal models, but the effects of AFB1 on Leydig cell function are unclear. In this study, in vivo exposure to AFB1 at 15 and 150 μg/kg/day lowered serum testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels, reduced Leydig cell number, and down-regulated the expression of testosterone biosynthesis-related genes. In vitro study showed that AFB1 (10 μM) significantly increased ROS levels, and decreased T production in Leydig cells by suppressing certain T-biosynthesis gene expressions. Moreover, AFB1 induced Leydig cell apoptosis through lowering pAMPK/AMPK ratio and increasing pmTOR/mTOR ratio, and then further up-regulating autophagy and apoptosis proteins, LC3, BECLIN 1, and BAX, as well as down-regulating autophagy flux protein P62 and anti-apoptosis protein BCL-2. AFB1-induced toxicity in Leydig cells was characterized by inhibiting T-biosynthesis gene expression, reducing Leydig cell number, promoting ROS production, and inducing cell apoptosis via suppressing AMPK/mTOR-mediated autophagy flux pathway.

Effects of advanced glycation end products on endothelial cell senescence and endothelial barrier dysfunction

Objective To investigate the effects and mechanism of advanced glycosylation end products(AGEs)on endothelial cell senescence and endothelial barrier dysfunction. Methods Human umbilical vein endothelial cells(HUVECs)were isolated and cultured.The cells were randomized into three groups: the control group(normal medium), the bovine serum albumin-treated group(BSA control group)and AEGs group(treated with AEGs-BSA). Senescence of HUVECs were detected by senescence-associated β-galactosidase(SA-beta-Gal)staining.The mRNA and protein expressions of senescence-related genes of p53, p21 and p16 in each group were determined by reverse transcription and real-time PCR(RT-qPCR)and Western blot.Reactive oxygen species(ROS)level was determined by dichlorodihdrofluorescence diacetate(DCFH-DA). The transendothelial electric resistance(TER)were measured by endothelial electric resistance meter.The protein levels of myosin light chain kinase(MLCK), phosphorylated myosin light chain(p-MLC), myosin light chain(MLC)were detected by Western blot. Results Compared with the control group and the BSA control group, the AGEs group showed the significantly increased positive rate of senescence-associated SA-beta-Gal staining(67.30±0.75% vs.7.81±0.35% and 7.64±0.91%, respectively, P 0.05), while TER was lower in the AEGs group than in control group and the BSA group[(27.0±4.2)Ω·cm2vs. (48.0±6.3)Ω·cm2 and (42.0±7.8)Ω·cm2, P 0.05], while was increased in the AEGs group versus control group and the BSA group[(69.31±8.47)% vs.(38.36±8.55)% and (41.67±6.93)%, P<0.05). The protein expression levels of MLK and p-MLC/MLC were higher in the AGEs group than in the control group and the BSA group(P<0.05). Conclusions AGEs may lead to endothelial cell senescence and endothelial barrier dysfunction by promoting ROS production and oxidative stress, and by regulating MLCK signaling pathway. Key words: Glycosylation end products, advanced; Endothelial cell; Aging

RETRACTED ARTICLE: Large tumor suppressor kinase 2 overexpression attenuates 5-FU-resistance in colorectal cancer via activating the JNK-MIEF1-mitochondrial division pathway

Background5-Fluorouracil (5-FU) is a standard treatment for colorectal cancer, but most patients develop 5-FU resistance. Here, we conducted experiments to identify an effective approach to augment 5-FU-based treatment in colorectal cancer in vitro.MethodsSW480 cells were in the present study and treated with 5-FU. Besides, LATS2 adenovirus vectors were infected into SW480 cells. Western blotting, immunofluorescence and ELISA were used to evaluate cell death and mitochondrial function. Pathway blocker was used to verify the role of MAPK-JNK pathway in SW480 cell death.ResultsAn obvious drop in large tumor suppressor kinase 2 (LATS2) expression was observed in SW480 cells after treatment with 5-FU. In addition, upregulation of LATS2 expression through infection with LATS2 adenovirus further increased the reduction of SW480 cell viability induced by 5-FU. Functional exploration showed that 5-FU treatment suppressed mitochondrial membrane potential, enhanced cyt-c release into the nucleus, induced an oxidative injury environment by promoting ROS production, and eventually upregulated Bax-related mitochondrial apoptosis. Besides, LATS2 overexpression in combination with 5-FU treatment further perturbed mitochondrial homeostasis, and this effect was achieved by elevating mitochondrial division. Mechanistically, LATS2 overexpression and 5-FU co-treatment amplified mitochondrial division by upregulating MIEF1 expression in a manner dependent on MAPK-JNK axis. Knockdown of MIEF1 using an siRNA-mediated loss of function assay and/or inhibition of the MAPK-JNK pathway using the specific inhibitor SP600125 abolished LATS2/5-FU-mediated deleterious effects on mitochondrial performance and SW480 cell viability.ConclusionsIn light of the above findings, LATS2 downregulation could be a potential mechanism of low response to 5-FU treatment. Overexpression of LATS2 to further disrupt mitochondrial function via the JNK-MIEF1 signalling pathway might be a method to optimize 5-FU-based chemotherapy.

Divergent proteomics response of Apostichopus japonicus suffering from skin ulceration syndrome and pathogen infection

Skin ulceration syndrome (SUS) of sea cucumber is a common and serious disease that affects the stable development of Apostichopus japonicus in the culture industry. The part of sea cucumber that suffers from major injury and is directly observed is the body wall, in which protein variations should be the most direct evidence of the disease. To understand the response mechanisms of A. japonicus in SUS progression, we investigated protein changes in the body wall of diseased A. japonicus induced by Vibrio splendidus and individuals with natural diseases by isobaric tags for relative and absolute quantification (iTRAQ). About 119 proteins were identified in the two iTRAQ groups. A comparison of the protein expression profiles among two SUS conditions revealed that the mode of action induced by V. splendidus (Vs-SUS) was completely different from those in individuals with natural disease (ND-SUS). Most of the differentially expressed proteins (DEPs) (33 in 37 DEPs) were significantly depressed in the Vs-SUS group. Only 13 proteins in 27 DEPs showed similar trend to those in the ND-SUS group. Many important proteins involved in major intercellular signaling pathways associated with SUS disease were identified based on the KEGG and GO database search. Many proteins were located in the mitochondria and mainly involved in the oxidative stress pathway. Glutathione metabolism pathway was associated with reactive oxygen (ROS) production in the ND-SUS group. In the Vs-group, most of the proteins were concentrated in the cytoplasm and were related to immunity and extracellular matrix stability. In the ND-SUS group, the activity of key enzymes (CAT, GPx) that eliminate mitochondrial ROS production and structural stable protein (HSP60, HSP10) decreased, whereas those of complement proteins (C3, C3-2) that promoted ROS production was upregulated. This finding supported that oxidative damage caused by ROS might be the main effector for SUS in the ND-SUS group. The challenge with V. splendidus led to the breakdown of the defense capability of sea cucumber and suppressed the expression of immune-related proteins, such as C-type lectin, caspase, STAT, and cystatin. The downregulation of TIMP led to MMP1 overexpression. Members of the MMP family could directly degrade the extracellular matrix, which may be the main reason for the cell matrix degradation and induced SUS disease in the Vs-SUS group. Hence, ROS and extracellular matrix degradation enzymes could play important roles in the formation of SUS in sea cucumber. Results provide insights into the complex molecular mechanism of SUS in sea cucumber.

Lipopolysaccharide (LPS) Aggravates High Glucose- and Hypoxia/Reoxygenation-Induced Injury through Activating ROS-Dependent NLRP3 Inflammasome-Mediated Pyroptosis in H9C2 Cardiomyocytes.

Diabetes aggravates myocardial ischemia-reperfusion (I/R) injury because of the combination effects of changes in glucose and lipid energy metabolism, oxidative stress, and systemic inflammatory response. Studies have indicated that myocardial I/R may coincide and interact with sepsis and inflammation. However, the role of LPS in hypoxia/reoxygenation (H/R) injury in cardiomyocytes under high glucose conditions is still unclear. Our objective was to examine whether lipopolysaccharide (LPS) could aggravate high glucose- (HG-) and hypoxia/reoxygenation- (H/R-) induced injury by upregulating ROS production to activate NLRP3 inflammasome-mediated pyroptosis in H9C2 cardiomyocytes. H9C2 cardiomyocytes were exposed to HG (30 mM) condition with or without LPS, along with caspase-1 inhibitor (Ac-YVAD-CMK), inflammasome inhibitor (BAY11-7082), ROS scavenger N-acetylcysteine (NAC), or not for 24 h, then subjected to 4 h of hypoxia followed by 2 h of reoxygenation (H/R). The cell viability, lactate dehydrogenase (LDH) release, caspase-1 activity, and intracellular ROS production were detected by using assay kits. The incidence of pyroptosis was detected by calcein-AM/propidium iodide (PI) double staining kit. The concentrations of IL-1β and IL-18 in the supernatants were assessed by ELISA. The mRNA levels of NLRP3, ASC, and caspase-1 were detected by qRT-PCR. The protein levels of NF-κB p65, NLRP3, ASC, cleaved caspase-1 (p10), IL-1β, and IL-18 were detected by western blot. The results indicated that pretreatment LPS with 1 μg/ml not 0.1 μg/ml could efficiently aggravate HG and H/R injury by activating NLRP3 inflammasome to mediate pyroptosis in H9C2 cells, as evidenced by increased LDH release and decreased cell viability in the cells, and increased expression of NLRP3, ASC, cleaved caspase-1 (p10), IL-1β, and IL-18. Meanwhile, Ac-YVAD-CMK, BAY11-7082, or NAC attenuated HG- and H/R-induced H9C2 cell injury with LPS stimulated by reversing the activation of NLRP3 inflammasome-mediated pyroptosis. In conclusion, LPS could increase the sensitivity of H9C2 cells to HG and H/R and aggravated HG- and H/R-induced H9C2 cell injury by promoting ROS production to induce NLRP3 inflammasome-mediated pyroptosis.

Gambogic acid induces autophagy and combines synergistically with chloroquine to suppress pancreatic cancer by increasing the accumulation of reactive oxygen species

BackgroundGambogic acid is a natural component isolated from gamboge that possesses anticancer properties. Our previous study suggested that gambogic acid might be involved in autophagy; however, its role in pancreatic cancer remained unclear.MethodsCell viability and apoptosis of pancreatic cancer cell lines were determined using (4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan and flow cytometry. The effects of gambogic acid on autophagy was assessed by western blot, acridine orange staining, transmission electron microscopy, and measurement of autophagic flux through RFP-GFP-LC3 lentiviral transfection. The mitochondrial membrane potential was assessed by JC-1 staining. The production of reactive oxygen species was measured using CM-H2DCFDA staining. A xenograft tumor model of pancreatic cancer was created to determine the efficacy of gambogic acid and chloroquine.ResultsGambogic acid induced the expression of LC3-II and Beclin-1 proteins in pancreatic cancer cells, whereas the expression of P62 showed a decline. Gambogic acid also increased the formation of both acidic vesicular organelles and autophagosomes, and increased autophagic flux. These findings indicated that gambogic acid induced the autophagic process. Furthermore, inhibition of autophagy by chloroquine or 3-methyladenine, or knockdown of Atg-7 all enhanced the cytotoxicity of gambogic acid, suggesting that gambogic acid-induced autophagy improves the survival of pancreatic cancer cells. Moreover, gambogic acid reduced the mitochondrial membrane potential and promoted ROS production, which contributed to the activation of autophagy. The inhibition of autophagy by chloroquine further reduced the mitochondrial membrane potential and increased the accumulation of ROS. This indicated that the inhibition of autophagy could mitigate the cellular protective effects induced by gambogic acid. The treatment combination of gambogic acid and chloroquine synergistically inhibited tumor growth in the xenograft tumor model.ConclusionsThese results demonstrate that gambogic acid induces cytoprotective autophagy in pancreatic cancer cells. The inhibition of autophagy promotes the cytotoxicity of gambogic acid by increasing the accumulation of ROS in pancreatic cancer cells. Combining chloroquine and gambogic acid may be a promising treatment for pancreatic cancer.

Targeted p53 on Small-Molecules-Induced Ferroptosis in Cancers.

Ferroptosis is a type of programmed cell death characterized by the accumulation of lipid reactive oxygen species (L-ROS) driven by the oxidative degeneration of lipids in an iron-dependent manner. The mechanism by which lipid oxidative degradation drives ROS-ferroptosis involves metabolic dysfunctions that result in impaired intracellular metabolic processes and ROS production. Recent studies have found that p53 acts as a positive regulator of ferroptosis by promoting ROS production. p53 directly regulates the metabolic versatility of cells by favoring mitochondrial respiration, leading to ROS-mediated ferroptosis. In mild stress, p53 protects cell survival via eliminating ROS; additionally, in human colorectal cancer, p53 antagonizes ferroptosis by formation of the DPP4–p53 complex. In short, the mechanisms of p53-mediated ROS production underlying cellular response are poorly understood. In the context of recent research results, the indistinct roles of p53 on ROS-mediated ferroptosis are scrutinized to understand the mechanism underlying p53-mediated tumor suppression.

246 - The immunogenicity in mice of hepatitis C virus core protein delivered as DNA is modulated by its capacity to promote ROS production and activate Nrf2/ARE pathway

Hepatitis C virus (HCV) core protein is an attractive target for HCV vaccine, however little progress was achieved in trials. We aimed to delineate the factors restricting immunogenicity of HCV core, and to improve its performance as DNA immunogen in a mouse model. We investigated the plasmids encoding the full-length protein (1-191 aa), its C-terminally truncated mutants 1-173, 1-152, 1-98, 1-50, 1-36, N-terminally truncated 37-191 form, or harboring the codon-optimized gene (1-152s). They were assessed for the level of protein expression, route of degradation, ability to enhance ROS production and activate expression of heme oxygenase 1 (HO-1) and NAD(P)H:quinone oxidoreductase (Nqo1), regulated through Nrf2/ARE pathway. Cores truncated at the N-terminus and C-terminus beyond aa 152 accumulated at 10-100 times lower level than the full-length protein. Core(1-36) induced ROS production by Nox1 and Nox4, whereas Core(37-191) - by mitochondria, CYP2E1, and Ero1a, and both of them up-regulated the expression of HO-1 and Nqo1 at transcriptional level. The N-terminally truncated core protein upregulated production of ROS. HCV core genes were used to immunize BALB/c mice by intradermal injection with electroporation or C57Bl/6 mice by repeated intramuscular injections. The immunogenicity of HCV core gene in terms of IFN-γ response correlated with levels of ROS production and HO-1/Nqo1 expression. Anti-core immune response inversely correlated with Nqo1/HO-1 expression if recalculated per unit of HCV core protein in a cell. The strongest HCV-specific cellular response was induced by Core(1-152s) gene which supported a modest level of protein expression, moderate ROS production and stress response. Thus, cellular immunogenicity of HCV core is shaped by its ability to promote ROS production and to trigger the stress response.

Abstract 3385: ETNK1 mutations promote ROS production and DNA damage through increased mitochondrial activity

Abstract Atypical chronic myeloid leukemia (aCML) is a clonal disorder belonging to the myelodysplastic/myeloproliferative syndromes. About 13% of aCML cases carry somatic mutations in ETNK1 gene, encoding for H243Y, N244S, and G245V substitutions. We previously showed that ETNK1 mutations cause a decreased catalytic activity of the enzyme. Despite this evidence however, their oncogenic role remained largely unexplained. Since ETNK1 activity is essential for the synthesis of phosphatidylethanolamine (PE) and given that PE is one of the most abundant phospholipids in the inner membrane of mitochondria, we focused our attention on mitochondrial activity. In order to characterize the oncogenic effect of ETNK1 variants we generated CRISPR/Cas9 clones carrying heterozygous N244S mutation and homozygous ETNK1 deletion (KO cells) on the 293 Flp-In™ cell-line. Both N244S and KO cells showed a significant increase in mitochondrial activity (1.78 and 2.13 fold increase, respectively; p= 0.0096 and p=0.0050) compared to WT, as assessed by MitoTracker™ Red. In line with this finding, electron microscopy revealed a significant modification in mitochondria morphology for N244S and KO cells, changing from an elongated, tubular shape to a round, swollen one. ATP (1.67 and 1.68 fold; p&amp;lt;0.0001; ATPlite Luminescence Assay System) and ROS production (1.66 and 1.74 fold increase; p&amp;lt;0.0001; CellROX™ Green Reagent) were similarly increased. Histone H2AX phosphorylation (γ-H2AX) analysis revealed a higher number of foci in N244S and KO cells (2.60±0.22 and 2.89±0.27; p&amp;lt;0.0001) compared to WT (0.56±0.08). A similar increase in γ-H2AX (3.6 fold; p=0.0037) was present in primary samples from aCML patients carrying ETNK1 mutation compared to ETNK1-WT ones. In line with these data, a higher mutation rate was detected in N244S and KO cells (8.09*10-7±9.6*10-8 and 8.20*10-7±1.28*10-7; p=0.0060 and p=0.0264) compared to WT (2.98*10-7±8.2*10-8) by 6-thioguanine assay. The reconstruction of the hierarchy of somatic mutations in ETNK1-mutated aCML patients revealed that ETNK1 variants invariably occur very early in the evolution history of the aCML patients. Taken together, our results show that impairment of ETNK1 function causes an increase in mitochondrial activity, which in turn leads to increased ROS production driving the accumulation of DNA mutations. Since the characterization of aCML subclonal architecture indicates ETNK1 mutations as a very early event in the history of the disease, we hypothesize that ETNK1 could contribute to the onset of aCML through the activation of a mutant phenotype, which in turn would accelerate the accumulation of further oncogenic mutations. Citation Format: Diletta Fontana, Mario Mauri, Antonio Niro, Luca Massimino, Mayla Bertagna, Giovanni Zambrotta, Mario Bossi, Stefania Citterio, Barbara Crescenzi, Giovanni Signore, Vincenzo Piazza, Cristina Mecucci, Guido Cavaletti, Delphine Rea, Carlo Gambacorti-Passerini, Rocco Piazza. ETNK1 mutations promote ROS production and DNA damage through increased mitochondrial activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3385.

Mst1 promotes cardiac ischemia–reperfusion injury by inhibiting the ERK-CREB pathway and repressing FUNDC1-mediated mitophagy

Cardiac ischemia-reperfusion (I/R) injury results mainly from mitochondrial dysfunction and cardiomyocyte death. Mitophagy sustains mitochondrial function and exerts a pro-survival effect on the reperfused heart tissue. Mammalian STE20-like kinase 1 (Mst1) regulates chronic cardiac metabolic damage and autophagic activity, but its role in acute cardiac I/R injury, especially its effect on mitophagy, is unknown. The aim of this study is to explore whether Mst1 is involved in reperfusion-mediated cardiomyocyte death via modulation of FUN14 domain containing 1 (FUNDC1)-related mitophagy. Our data indicated that Mst1 was markedly increased in reperfused hearts. However, genetic ablation of Mst1 in Mst1-knockout (Mst1-KO) mice significantly reduced the expansion of the cardiac infarction area, maintained myocardial function and abolished I/R-mediated cardiomyocyte death. At the molecular level, upregulation of Mst1 promoted ROS production, reduced mitochondrial membrane potential, facilitated the leakage of mitochondrial pro-apoptotic factors into the nucleus, and activated the caspase-9-related apoptotic pathway in reperfused cardiomyocytes. Mechanistically, Mst1 activation repressed FUNDC1 expression and consequently inhibited mitophagy. However, deletion of Mst1 was able to reverse FUNDC1 expression and thus re-activate protective mitophagy, effectively sustaining mitochondrial homeostasis and blocking mitochondrial apoptosis in reperfused cardiomyocytes. Finally, we demonstrated that Mst1 regulated FUNDC1 expression via the MAPK/ERK-CREB pathway. Inhibition of the MAPK/ERK-CREB pathway prevented FUNDC1 activation caused by Mst1 deletion. Altogether, our data confirm that Mst1 deficiency sends a pro-survival signal for the reperfused heart by reversing FUNDC1-related mitophagy and thus reducing cardiomyocyte mitochondrial apoptosis, which identifies Mst1 as a novel regulator for cardiac reperfusion injury via modulation of mitochondrial homeostasis.

Relationship between copper(ii) complexes with FomA adhesin fragments of F. nucleatum and colorectal cancer. Coordination pattern and ability to promote ROS production.

The copper(ii) ion binding of the Ac-KGHGNG-NH2 and Ac-PTVHNE-NH2 fragments of FomA adhesin from Fusobacterium nucleatum was studied using potentiometry, UV-Vis, CD, EPR and DFT techniques. The coordination pattern was described in a wide range of pH values. Ligands begin interactions with metal ions using imidazole nitrogen. At pH 6.8 (a value typical of the large intestine environment), the metal ion was coordinated by the 3N donor atoms {Nim, 2 × N-amide} in both cases. However, the copper(ii) ion was bound more effectively by the Ac-PTVHNE-NH2 peptide. The formation of reactive oxygen species (ROS) was studied by UV-Vis and fluorescence spectroscopy, as well as gel electrophoresis in the presence of H2O2 and/or ascorbic acid. The complexes generated ROS in the highest amounts among all compounds. Moreover, they stimulated the CT26 cell line (mouse colon carcinoma) to produce ROS which lead to oxidative stress. It was also determined that such radicals took part in the plasmid degradation mechanism.

Hace1 Influences zebrafish cardiac development via ROS-dependent mechanisms.

In this study, we reveal a previously undescribed role of the HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1) tumor suppressor protein in normal vertebrate heart development using the zebrafish (Danio rerio) model. We examined the link between the cardiac phenotypes associated with hace1 loss of function to the expression of the Rho small family GTPase, rac1, which is a known target of HACE1 and promotes ROS production via its interaction with NADPH oxidase holoenzymes. We demonstrate that loss of hace1 in zebrafish via morpholino knockdown results in cardiac deformities, specifically a looping defect, where the heart is either tubular or "inverted". Whole-mount in situ hybridization of cardiac markers shows distinct abnormalities in ventricular morphology and atrioventricular valve formation in the hearts of these morphants, as well as increased expression of rac1. Importantly, this phenotype appears to be directly related to Nox enzyme-dependent ROS production, as both genetic inhibition by nox1 and nox2 morpholinos or pharmacologic rescue using ROS scavenging agents restores normal cardiac structure. Our study demonstrates that HACE1 is critical in the normal development and proper function of the vertebrate heart via a ROS-dependent mechanism. Developmental Dynamics 247:289-303, 2018. © 2017 Wiley Periodicals, Inc.

Co-delivery of paclitaxel and tetrandrine via iRGD peptide conjugated lipid-polymer hybrid nanoparticles overcome multidrug resistance in cancer cells

One of the promising strategies to overcome tumor multidrug resistance (MDR) is to deliver anticancer drug along with P-glycoprotein (P-gp) inhibitor simultaneously. To enhance the cancer cellular internalization and implement the controlled drug release, herein an iRGD peptide-modified lipid-polymer hybrid nanosystem (LPN) was fabricated to coload paclitaxel (PTX) and tetrandrine (TET) at a precise combination ratio. In this co-delivery system, PTX was covalently conjugated to poly (D,L-lactide-co-glycolide) polymeric core by redox-sensitive disulfide bond, while TET was physically capsulated spontaneously for the aim to suppress P-gp in advance by the earlier released TET in cancer cells. As a result, the PTX+TET/iRGD LPNs with a core-shell structure possessed high drug loading efficiency, stability and redox-sensitive drug release profiles. Owing to the enhanced cellular uptake and P-gp suppression mediated by TET, significantly more PTX accumulated in A2780/PTX cells treated with PTX+TET/iRGD LPNs than either free drugs or non-iRGD modified LPNs. As expected, PTX+TET/iRGD LPNs presented the highest cytotoxicity against A2780/PTX cells and effectively promoted ROS production, enhanced apoptosis and cell cycle arrests particularly. Taken together, the co-delivery system demonstrated great promise as potential treatment for MDR-related tumors based on the synergistic effects of P-gp inhibition, enhanced endocytosis and intracellular sequentially drug release.

Thioredoxin 1 modulates apoptosis induced by bioactive compounds in prostate cancer cells

Accumulating evidence suggests that natural bioactive compounds, alone or in combination with traditional chemotherapeutic agents, could be used as potential therapies to fight cancer. In this study, we employed four natural bioactive compounds (curcumin, resveratrol, melatonin, and silibinin) and studied their role in redox control and ability to promote apoptosis in androgen sensitive and insensitive prostate cancer cells. Here is shown that curcumin and resveratrol promote ROS production and induce apoptosis in LNCaP and PC-3. An increase in reactive species is a trigger event in curcumin-induced apoptosis and a consequence of resveratrol effects on other pathways within these cells. Moreover, here we demonstrated that these four compounds affect differently one of the main intracellular redox regulator, the thioredoxin system. Exposure to curcumin and resveratrol promoted TRX1 oxidation and altered its subcellular location. Furthermore, resveratrol diminished TRX1 levels in PC-3 cells and increased the expression of its inhibitor TXNIP. Conversly, melatonin and silibinin only worked as cytostatic agents, reducing ROS levels and showing preventive effects against TRX oxidation. All together, this work explores the effect of compounds currently tested as chemo-preventive agents in prostate cancer therapy, on the TRX1 redox state and function. Our work shows the importance that the TRX system might have within the differences found in their mechanisms of action. These bioactive compounds trigger different responses and affect ROS production and redox systems in prostate cancer cells, suggesting the key role that redox-related pathways might play in processes like differentiation or survival in prostate cancer.

Chenodeoxycholic acid activates NLRP3 inflammasome and contributes to cholestatic liver fibrosis.

Accumulation of hydrophobic bile acids in the liver contributes to cholestatic liver injury. Inflammation induced by excessive bile acids is believed to play a crucial role, however, the mechanisms of bile acids triggered inflammatory response remain unclear. Recent studies have highlighted the effect of NLRP3 inflammasome in mediating liver inflammation and fibrosis. In this study, we for the first time showed that chenodeoxycholic acid (CDCA), the major hydrophobic primary bile acid involved in cholestatic liver injury, could dose-dependently induce NLRP3 inflammasome activation and secretion of pro-inflammatory cytokine-IL-1β in macrophages by promoting ROS production and K+ efflux. Mechanistically, CDCA triggered ROS formation in part through TGR5/EGFR downstream signaling, including protein kinase B, extracellular regulated protein kinases and c-Jun N-terminal kinase pathways. Meanwhile, CDCA also induced ATP release from macrophages which subsequently causes K+ efflux via P2X7 receptor. Furthermore, in vivo inhibition of NLRP3 inflammasome with caspase-1 inhibitor dramatically decreased mature IL-1β level of liver tissue and ameliorated liver fibrosis in bile duct ligation (BDL) mouse model. In conclusion, excessive CDCA may represent an endogenous danger signal to activate NLRP3 inflammasome and initiate liver inflammation during cholestasis. Our finding offers a mechanistic basis to ameliorate cholestatic liver fibrosis by targeting inflammasome activation.

Pulse pressure-dependent cerebrovascular eNOS regulation in mice

Arterial blood pressure is oscillatory; whether pulse pressure (PP) regulates cerebral artery myogenic tone (MT) and endothelial function is currently unknown. To test the impact of PP on MT and dilation to flow (FMD) or to acetylcholine (Ach), isolated pressurized mouse posterior cerebral arteries were subjected to either static pressure (SP) or a physiological PP (amplitude: 30 mm Hg; frequency: 550 bpm). Under PP, MT was significantly higher than in SP conditions (p < 0.05) and was not affected by eNOS inhibition. In contrast, under SP, eNOS inhibition increased (p < 0.05) MT to levels observed under PP, suggesting that PP may inhibit eNOS. At a shear stress of 20 dyn/cm2, FMD was lower (p < 0.05) under SP than PP. Under SP, eNOS-dependent production contributed to FMD, while under PP, eNOS-dependent NO was responsible for FMD, indicating that PP favours eNOS coupling. Differences in FMD between pressure conditions were abolished after NOX2 inhibition. In contrast to FMD, Ach-induced dilations were higher (p < 0.05) under SP than PP. Reactive oxygen species scavenging reduced (p < 0.05) Ach-dependent dilations under SP, but increased (p < 0.05) them under PP; hence, under PP, Ach promotes ROS production and limits eNOS-derived NO activity. In conclusion, PP finely regulates eNOS, controlling cerebral artery reactivity.