Tannic acid attenuates the formation of cancer stem cells by inhibiting NF-κB-mediated phenotype transition of breast cancer cells.

Cancer stem cells (CSCs) are innately resistant to standard therapies, which positions CSCs in the focus of anti-cancer research. In this study, we investigated the potential inhibitory effect of tannic acid (TA) on CSCs. Our data demonstrated that TA (10 µM), at the concentration not inhibiting the proliferation of normal mammary cells (MCF10A), inhibited the formation and growth of mammosphere in MCF7, T47D, MDA-MB-231 cells shown as a decrease in mammosphere formation efficiency (MFE), cell number, diameter of mammosphere, and ALDH1 activity. NF-κB pathway was activated in the mammosphere indicated by an up-regulation of p65, a degradation of IκBα, and an increased IL-6. The inhibition of NF-κB pathway via gene silencing of p65 (sip65), NF-κB inhibitor (PDTC), or IKK inhibitor (Bay11-7082) alleviated MFE. Other CSCs markers such as an increase in ALDH1 and CD44high/CD24low ratio were ameliorated by sip65. TA also alleviated TGFß-induced EMT, increase in MFE, and NF-κB activation. In murine xenograft model, TA reduced tumor volume which was associated with a decrease in CD44high/CD24low expression and IKK phosphorylation. These results suggest that TA negatively regulates CSCs by inhibiting NF-κB activation and thereby prevents cancer cells from undergoing EMT and CSCs formation, and may thus be a promising therapy targeting CSCs.

Uric acid induced the phenotype transition of vascular endothelial cells via induction of oxidative stress and glycocalyx shedding.

Recent data suggested a causative role of uric acid (UA) in the development of renal disease, in which endothelial dysfunction is regarded as the key mechanism. Endothelial-to-mesenchymal transition (EndoMT) and shedding of the glycocalyx are early changes of endothelial dysfunction. We investigated whether UA induced EndoMT in HUVECs and an animal model of hyperuricemia fed with 2% oxonic acid for 4 wk. UA induced EndoMT in HUVECs with a generation of reactive oxygen species via the activation of membranous NADPH oxidase (from 15 min) and mitochondria (from 6 h) along with glycocalyx shedding (from 6 h), which were blocked by probenecid. GM6001, an inhibitor of matrix metalloproteinase, alleviated UA-induced glycocalyx shedding and EndoMT. Antioxidants including N-acetyl cysteine, apocynin, and mitotempo ameliorated EndoMT; however, they did not change glycocalyx shedding in HUVECs. In the kidney of hyperuricemic rats, endothelial staining in peritubular capillaries (PTCs) was substantially decreased with a de novo expression of α-smooth muscle actin in PTCs. Plasma level of syndecan-1 was increased in hyperuricemic rats, which was ameliorated by allopurinol. UA caused a phenotypic transition of endothelial cells via induction of oxidative stress with glycocalyx shedding, which could be one of the mechanisms of UA-induced endothelial dysfunction and kidney disease.-Ko, J., Kang, H.-J., Kim, D.-A., Kim, M.-J., Ryu, E.-S., Lee, S., Ryu, J.-H., Roncal, C., Johnson, R. J., Kang, D.-H. Uric acid induced the phenotype transition of vascular endothelial cells via induction of oxidative stress and glycocalyx shedding.

Paricalcitol attenuates TGF-ß1-induced phenotype transition of human peritoneal mesothelial cells (HPMCs) via modulation of oxidative stress and NLRP3 inflammasome.

Phenotype transition of mesothelial cells, such as epithelial-to-mesenchymal transition (EMT), is one of the early mechanisms of peritoneal fibrosis, which is mediated by oxidative stress and inflammation. Nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a multiprotein oligomer that promotes the maturation of IL-1ß and IL-18. Paricalcitol is reported to exert an anti-inflammatory effect; however, there are no studies as to whether paricalcitol modulates the activation of NLRP3 inflammasome. We investigated the role of NLRP3 inflammasome in peritoneal EMT with an exploration of the effect of paricalcitol on oxidative stress, NLRP3 inflammasome, and EMT of mesothelial cells. TGF-ß1-induced EMT in human peritoneal mesothelial cells (HPMCs) was associated with an up-regulation of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and procaspase-1, with an increased production of IL-1ß and IL-18, which was ameliorated by small interfering (si)NLRP3, siASC, caspase inhibitors, or neutralizing antibodies for IL-1ß and IL-18. TGF-ß1 enhanced reactive oxygen species generation with an increase in NADPH oxidase (NOX) activity and mitochondrial NOX4 production. Paricalcitol alleviated TGF-ß1-induced EMT and the NLRP3 inflammasome, which was associated with a down-regulation of NOX activity by interfering with p47phox and p22phox interaction and mitochondrial NOX4 production in HPMCs. Taken together, paricalcitol ameliorated EMT of HPMCs via modulating an NOX-dependent increase in the activity of NLRP3 inflammasome. Paricalcitol could be a novel approach to protect the peritoneum from the development of EMT and peritoneal fibrosis.-Ko, J., Kang, H.-J., Kim, D.-A., Ryu, E.-S., Yu, M., Lee, H., Lee, H. K., Ryu, H.-M., Park, S.-H., Kim, Y.-L., Kang, D.-H. Paricalcitol attenuates TGF-ß1-induced phenotype transition of human peritoneal mesothelial cells (HPMCs) via modulation of oxidative stress and NLRP3 inflammasome.

Metformin ameliorates the Phenotype Transition of Peritoneal Mesothelial Cells and Peritoneal Fibrosis via a modulation of Oxidative Stress.

Phenotype transition of peritoneum is an early mechanism of peritoneal fibrosis. Metformin, 5'-adenosine monophosphate-activated protein kinase (AMPK) activator, has recently received a new attention due to its preventive effect on organ fibrosis and cancer metastasis by inhibiting epithelial-to-mesenchymal transition (EMT). We investigated the effect of metformin on EMT of human peritoneal mesothelial cells (HPMC) and animal model of peritoneal dialysis (PD). TGF-ß1-induced EMT in HPMC was ameliorated by metformin. Metformin alleviated NAPDH oxidase- and mitochondria-mediated ROS production with an increase in superoxide dismutase (SOD) activity and SOD2 expression. Metformin inhibited the activation of Smad2/3 and MAPK, GSK-3ß phosphorylation, nuclear translocalization of ß-catenin and Snail in HPMCs. Effect of metformin on TGF-ß1-induced EMT was ameliorated by either AMPK inhibitor or AMPK gene silencing. Another AMPK agonist, 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide partially blocked TGF-ß1-induced EMT. In animal model of PD, intraperitoneal metformin decreased the peritoneal thickness and EMT with an increase in ratio of reduced to oxidized glutathione and the expression of SOD whereas it decreased the expression of nitrotyrosine and 8-hydroxy-2'-deoxyguanosine. Therefore, a modulation of AMPK in peritoneum can be a novel tool to prevent peritoneal fibrosis by providing a favorable oxidant/anti-oxidant milieu in peritoneal cavity and ameliorating phenotype transition of peritoneal mesothelial cells.

Molecular characterization, transcriptional profiling, and antibacterial potential of G-type lysozyme from seahorse (Hippocampus abdominalis).

Lysozymes are a family of enzymes that catalyze the hydrolysis of bacterial cell wall, acting as antimicrobial effectors of the innate immune system. In the present study, an ortholog of goose-type lysozyme (ShLysG) from the big-belly seahorse (Hippocampus abdominalis) was identified and characterized structurally and functionally. The full-length cDNA sequence (1213 bp) of ShLysG is comprised of an open reading frame made up of 552 bp, encoding a polypeptide of 184 amino acid (aa) with a predicted molecular mass of 20 kDa. In silico analysis of ShLysG revealed the absence of signal peptide and the presence of a characteristic bacterial soluble lytic transglycosylase (SLT) domain bearing three catalytic residues (Glu71, Asp84, and Asp95) and seven N-acetyl-d-glucosamine binding sites (Glu71, Asp95, Tyr98, His99, Ile117, Tyr145, and Asn146). Homology analysis demonstrated that the aa sequence of ShLysG shared 60.7-67.4% identity and 72.6-79.3% similarity with the orthologs of other teleosts. Phylogenetic analysis of ShLysG indicated a closest relationship with the ortholog from Gadus morhua. In healthy seahorse, ShLysG mRNA showed a constitutive expression in all the tissues examined, with the highest expression in kidney and the least expression in liver. The ShLysG mRNA levels were also shown significant elevation upon the bacterial and pathogen-associated molecular pattern (PAMPs) challenges. Furthermore, lytic activities of ShLysG recombinant protein were detected against several Gram-negative and Gram-positive bacterial species. Taken together, these results suggest that ShLysG might possess a potential immune defensive role against invading microbial pathogens in seahorse.

First comparative characterization of three distinct ferritin subunits from a teleost: Evidence for immune-responsive mRNA expression and iron depriving activity of seahorse (Hippocampus abdominalis) ferritins.

Ferritins play an indispensable role in iron homeostasis through their iron-withholding function in living beings. In the current study, cDNA sequences of three distinct ferritin subunits, including a ferritin H, a ferritin M, and a ferritin L, were identified from big belly seahorse, Hippocampus abdominalis, and molecularly characterized. Complete coding sequences (CDS) of seahorse ferritin H (HaFerH), ferritin M (HaFerM), and ferritin L (HaFerL) subunits were comprised of 531, 528, and 522 base pairs (bp), respectively, which encode polypeptides of 177, 176, and 174 amino acids, respectively, with molecular masses of ∼20-21 kDa. Our in silico analyses demonstrate that these three ferritin subunits exhibit the typical characteristics of ferritin superfamily members including iron regulatory elements, domain signatures, and reactive centers. The coding sequences of HaFerH, M, and L were cloned and the corresponding proteins were overexpressed in a bacterial system. Recombinantly expressed HaFer proteins demonstrated detectable in vivo iron sequestrating (ferroxidase) activity, consistent with their putative iron binding capability. Quantification of the basal expression of these three HaFer sequences in selected tissues demonstrated a gene-specific ubiquitous spatial distribution pattern, with abundance of mRNA in HaFerM in the liver and predominant expression of HaFerH and HaFerL in blood. Interestingly, the basal expression of all three ferritin genes was found to be significantly modulated against pathogenic stress mounted by lipopolysaccharides (LPS), poly I:C, Streptococcus iniae, and Edwardsiella tarda. Collectively, our findings suggest that the three HaFer subunits may be involved in iron (II) homeostasis in big belly seahorse and that they are important in its host defense mechanisms.

Trans-1,3-diphenyl-2,3-epoxypropan-1-one, a chalcone derivative, induces apoptosis via ROS-mediated down-regulation of Bcl-xL in human leukemia HL-60 cells.

The anticancer effects of trans-1,3-diphenyl-2,3-epoxypropan-1-one (DPEP), a chalcone derivative, were investigated in human leukemia HL-60 cells. Treatment of HL-60 cells with various concentration of DPEP resulted in a sequence of events characteristic of apoptosis, including loss of cell viability, morphological changes, and increased sub-G1 DNA content. We demonstrated that DPEP elevates reactive oxygen species (ROS) levels in HL-60 cells, and that the ROS scavenger N-acetylcysteine (NAC) could block DPEP-induced ROS generation and apoptosis. Western blot analysis revealed that DPEP inhibits Bcl-xL expression, leading to caspase-3 activation and poly-ADP-ribose polymerase (PARP) cleavage, thereby inducing apoptosis. However, NAC pre-treatment significantly inhibited the activation of caspase-3 and PARP cleavage and reduced Bcl-xL levels. These findings provide the first evidence that DPEP may inhibit the growth of HL-60 cells and induce apoptosis through a ROS-mediated Bcl-xL pathway.

Effects of Fam83h overexpression on enamel and dentine formation.

OBJECTIVE: The aim of this study was to determine if FAM83H over-expression causes dentine or enamel malformations. MATERIALS AND METHODS: The full-length mouse Fam83h cDNA was inserted into the pCAGIG vector between a ß-actin promoter and ß-globin enhancer for ubiquitous expression in transgenic mice. Recombinant mouse FAM83H was expressed and used to generate polyclonal antibodies. Western blots showed enhanced expression of the Fam83h transgene. The effects of transgene expression on tooth development were assessed by microhardness measurements of enamel and dentine. Total thickness of incisor enamel at the level of the alveolar crest was measured and decussating rod patterns were visualized by scanning electron microscopy (SEM). RESULTS: Three transgenic mouse lines were selected based upon their transgene expression levels. There was no statistically significant difference in the Vickers microhardness values of enamel or dentine between the transgenic lines or between the transgenic lines and wild type mice. No statistically significant differences in enamel thickness were observed between the transgenic lines and the wild type mice. SEM analysis revealed no apparent differences in the enamel crystal and rod morphologies. CONCLUSION: Our findings demonstrate that over-expression of FAM83H in mice does not produce a phenotype in dentine or enamel.