Leptin Promotes Vasculogenic Mimicry in Breast Cancer Cells by Regulating Aquaporin-1.

Leptin is an obesity-related hormone that plays an important role in breast cancer progression. Vasculogenic mimicry (VM) refers to the formation of vascular channels lined by tumor cells. This study aimed to investigate the relationship between leptin and VM in human breast cancer cells. VM was measured by a 3D culture assay. Signal transducers and activators of transcription 3 (STAT3) signaling, aquaporin-1 (AQP1), and the expression of VM-related proteins, including vascular endothelial cadherin (VE-cadherin), twist, matrix metalloproteinase-2 (MMP-2), and laminin subunit 5 gamma-2 (LAMC2), were examined by Western blot. AQP1 mRNA was analyzed by a reverse transcriptase-polymerase chain reaction (RT-PCR). Leptin increased VM and upregulated phospho-STAT3, VE-cadherin, twist, MMP-2, and LAMC2. These effects were inhibited by the leptin receptor-blocking peptide, Ob-R BP, and the STAT3 inhibitor, AG490. A positive correlation between leptin and AQP1 mRNA was observed and was confirmed by RT-PCR. Leptin upregulated AQP1 expression, which was blocked by Ob-R BP and AG490. AQP1 overexpression increased VM and the expression of VM-related proteins. AQP1 silencing inhibited leptin-induced VM and the expression of VM-related proteins. Thus, these results showed that leptin facilitates VM in breast cancer cells via the Ob-R/STAT3 pathway and that AQP1 is a key mediator in leptin-induced VM.

CRIF1 siRNA-Encapsulated PLGA Nanoparticles Suppress Tumor Growth in MCF-7 Human Breast Cancer Cells.

Mitochondrial oxidative phosphorylation (OXPHOS) system dysfunction in cancer cells has been exploited as a target for anti-cancer therapeutic intervention. The downregulation of CR6-interacting factor 1 (CRIF1), an essential mito-ribosomal factor, can impair mitochondrial function in various cell types. In this study, we investigated whether CRIF1 deficiency induced by siRNA and siRNA nanoparticles could suppress MCF-7 breast cancer growth and tumor development, respectively. Our results showed that CRIF1 silencing decreased the assembly of mitochondrial OXPHOS complexes I and II, which induced mitochondrial dysfunction, mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential depolarization, and excessive mitochondrial fission. CRIF1 inhibition reduced p53-induced glycolysis and apoptosis regulator (TIGAR) expression, as well as NADPH synthesis, leading to additional increases in ROS production. The downregulation of CRIF1 suppressed cell proliferation and inhibited cell migration through the induction of G0/G1 phase cell cycle arrest in MCF-7 breast cancer cells. Similarly, the intratumoral injection of CRIF1 siRNA-encapsulated PLGA nanoparticles inhibited tumor growth, downregulated the assembly of mitochondrial OXPHOS complexes I and II, and induced the expression of cell cycle protein markers (p53, p21, and p16) in MCF-7 xenograft mice. Thus, the inhibition of mitochondrial OXPHOS protein synthesis through CRIF1 deletion destroyed mitochondrial function, leading to elevated ROS levels and inducing antitumor effects in MCF-7 cells.

APE1/Ref-1 Inhibits Adipogenic Transcription Factors during Adipocyte Differentiation in 3T3-L1 Cells.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in DNA repair and redox regulation. The redox activity of APE1/Ref-1 is involved in inflammatory responses and regulation of DNA binding of transcription factors related to cell survival pathways. However, the effect of APE1/Ref-1 on adipogenic transcription factor regulation remains unknown. In this study, we investigated the effect of APE1/Ref-1 on the regulation of adipocyte differentiation in 3T3-L1 cells. During adipocyte differentiation, APE1/Ref-1 expression significantly decreased with the increased expression of adipogenic transcription factors such as CCAAT/enhancer binding protein (C/EBP)-α and peroxisome proliferator-activated receptor (PPAR)-γ, and the adipocyte differentiation marker adipocyte protein 2 (aP2) in a time-dependent manner. However, APE1/Ref-1 overexpression inhibited C/EBP-α, PPAR-γ, and aP2 expression, which was upregulated during adipocyte differentiation. In contrast, silencing APE1/Ref-1 or redox inhibition of APE1/Ref-1 using E3330 increased the mRNA and protein levels of C/EBP-α, PPAR-γ, and aP2 during adipocyte differentiation. These results suggest that APE1/Ref-1 inhibits adipocyte differentiation by regulating adipogenic transcription factors, suggesting that APE1/Ref-1 is a potential therapeutic target for regulating adipocyte differentiation.

Resveratrol suppresses serum-induced vasculogenic mimicry through impairing the EphA2/twist-VE-cadherin/AKT pathway in human prostate cancer PC-3 cells.

Vasculogenic mimicry (VM) is closely related to cancer progression and metastasis, contributing to poor prognosis in patients with cancer. Resveratrol (RES) is well known to possess anti-cancer activity. This study explored the new role of RES in VM incidence in human prostate cancer (PCa) PC-3 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, transwell invasion, and three-dimensional culture VM tube formation assays were performed to check the cell viability, invasive ability, and vessel-like networks formation, respectively. VM-related proteins were detected by Western blots. The activity of metalloproteinase-2 (MMP-2) was identified by gelatin zymography. Vascular endothelial cadherin (VE-cadherin) mRNA was assessed by reverse transcriptase-polymerase chain reaction. Nuclear twist expression was observed by immunofluorescence assay. RES reduced serum-induced invasion and VM formation. Serum-induced phosphorylation of erythropoiethin-producing hepatoceullular A2 (EphA2) and the expression of VE-cadherin at the protein and mRNA levels were decreased after RES treatment. RES inhibited serum-induced expression and nuclear localization of twist. Serum-activated AKT signaling pathway, including MMP-2 and laminin subunit 5 gamma-2, was impaired by RES. These results suggested that RES may have an anti-VM effect through suppressing the EphA2/twist-VE-cadherin/AKT signaling cascade in PCa PC-3 cells.

Capsanthin Inhibits Atherosclerotic Plaque Formation and Vascular Inflammation in ApoE-/- Mice.

Capsanthin is a red pigment and the major carotenoid component of red paprika (Capsicum annuum L.). However, its role in atherosclerosis is yet to be fully elucidated. This study investigated the role of dietary capsanthin in vascular inflammation in atherosclerotic mice. We evaluated the anti-atherosclerotic effects of daily oral administration of capsanthin (0.5 mg/kg of body weight/day) in apolipoprotein E-deficient (ApoE-/-) mice fed a Western-type diet (WD). Capsanthin treatment inhibited vascular cell adhesion molecule 1 expression and nuclear factor-κB ser536 phosphorylation in tumor necrosis factor-α-stimulated cultured endothelial cells. Dietary capsanthin significantly inhibited the WD-induced elevation in the plasma levels of total cholesterol, low-density lipoprotein cholesterol (LDL-C), and triglyceride in mice. Interestingly, capsanthin reduced aortic plaque formation and VCAM-1 expression, which is vascular inflammation, in atherosclerotic mice. In addition, the neutrophil-lymphocyte ratio, a systemic inflammatory marker, was inhibited in capsanthin-treated mice. Furthermore, capsanthin significantly reduced the levels of proinflammatory cytokines, such as TNF-α, interleukin-6, and monocyte chemoattractant protein-1, in the plasma of atherosclerotic mice. Collectively, our data demonstrate that dietary capsanthin plays a protective role against atherosclerosis in hyperlipidemic mice. This protective effect could be attributed to the anti-inflammatory properties of capsanthin.

Ceramide NPs derived from natural oils of Korean traditional plants enhance skin barrier functions and stimulate expressions of genes for epidermal homeostasis.

BACKGROUND: New ceramide (CER) NPs were prepared by linking fatty acids derived from oils of Korean traditional plants to phytosphingosine (PHS). The oils of Korean traditional plants were extracted from the seeds of Panax ginseng, Camellia sinensis, Glycine max napjakong, Glycine max seoritae, and Camellia japonica as sources of diverse fatty acids. AIMS: The aim of this study was to investigate signaling bioactivities of HP-C. sinensis ceramide NP that was column purified to remove any residual PHS and to evaluate the skin barrier functions of the HP-C. sinensis ceramide NP in human skin. METHODS: The expressions of genes related to epidermal differentiation were analyzed in vitro by qPCR. Human studies were also performed to determine the skin barrier functions with respect to TEWL and SC cohesion. RESULTS: The HP-C. sinensis CER NP significantly enhanced the expressions of FLG, CASP14, and INV indicates that the signaling biological activities of oil-derived ceramide NPs could be different depend on the natural oils. The control ceramide, C18-CER NP, had no effect on the expression of the three genes. HP-C. sinensis CER NP was selected for the in vivo human studies. Application of 0.5% HP-C. sinensis CER NP cream stimulated significantly faster recovery of a disrupted skin barrier than that of the control C18-CER NP. A significant enhancement of SC cohesion of the skin treated with 0.5% HP-C. sinensis CER NP was also observed. CONCLUSION: Taken all together, our results clearly demonstrate that HP-C. sinensis CER NP, P. ginseng CER NP, and other oil-derived CER NP could be a better choice for developing moisturizers to improve skin barrier function as they more closely mimic the endogenous CER composition of the actual human skin barrier.

Sp1 Plays a Key Role in Vasculogenic Mimicry of Human Prostate Cancer Cells.

Sp1 transcription factor regulates genes involved in various phenomena of tumor progression. Vasculogenic mimicry (VM) is the alternative neovascularization by aggressive tumor cells. However, there is no evidence of the relationship between Sp1 and VM. This study investigated whether and how Sp1 plays a crucial role in the process of VM in human prostate cancer (PCa) cell lines, PC-3 and DU145. A cell viability assay and three-dimensional culture VM tube formation assay were performed. Protein and mRNA expression levels were detected by Western blot and reverse transcriptase-polymerase chain reaction, respectively. The nuclear twist expression was observed by immunofluorescence assay. A co-immunoprecipitation assay was performed. Mithramycin A (MiA) and Sp1 siRNA significantly decreased serum-induced VM, whereas Sp1 overexpression caused a significant induction of VM. Serum-upregulated vascular endothelial cadherin (VE-cadherin) protein and mRNA expression levels were decreased after MiA treatment or Sp1 silencing. The protein expression and the nuclear localization of twist were increased by serum, which was effectively inhibited after MiA treatment or Sp1 silencing. The interaction between Sp1 and twist was reduced by MiA. On the contrary, Sp1 overexpression enhanced VE-cadherin and twist expressions. Serum phosphorylated AKT and raised matrix metalloproteinase-2 (MMP-2) and laminin subunit 5 gamma-2 (LAMC2) expressions. MiA or Sp1 silencing impaired these effects. However, Sp1 overexpression upregulated phosphor-AKT, MMP-2 and LAMC2 expressions. Serum-upregulated Sp1 was significantly reduced by an AKT inhibitor, wortmannin. These results demonstrate that Sp1 mediates VM formation through interacting with the twist/VE-cadherin/AKT pathway in human PCa cells.

17ß-Estradiol Increases APE1/Ref-1 Secretion in Vascular Endothelial Cells and Ovariectomized Mice: Involvement of Calcium-Dependent Exosome Pathway.

Apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that can be secreted, and recently suggested as new biomarker for vascular inflammation. However, the endogenous hormones for APE1/Ref-1 secretion and its underlying mechanisms are not defined. Here, the effect of twelve endogenous hormones on APE1/Ref-1 secretion was screened in cultured vascular endothelial cells. The endogenous hormones that significantly increased APE1/Ref-1 secretion was 17ß-estradiol (E2), 5?-dihydrotestosterone, progesterone, insulin, and insulin-like growth factor. The most potent hormone inducing APE1/Ref-1 secretion was E2, which in cultured endothelial cells, E2 for 24 h increased APE1/Ref-1 secretion level of 4.56 ± 1.16 ng/mL, compared to a basal secretion level of 0.09 ± 0.02 ng/mL. Among the estrogens, only E2 increased APE1/Ref-1 secretion, not estrone and estriol. Blood APE1/Ref-1 concentrations decreased in ovariectomized (OVX) mice but were significantly increased by the replacement of E2 (0.39 ± 0.09 ng/mL for OVX vs. 4.67 ± 0.53 ng/mL for OVX + E2). E2-induced APE1/Ref-1secretion was remarkably suppressed by the estrogen receptor (ER) blocker fulvestrant and intracellular Ca2+ chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), suggesting E2-induced APE1/Ref-1 secretion was dependent on ER and intracellular calcium. E2-induced APE1/Ref-1 secretion was significantly inhibited by exosome inhibitor GW4869. Furthermore, APE1/Ref-1 level in CD63-positive exosome were increased by E2. Finally, fluorescence imaging data showed that APE1/Ref-1 co-localized with CD63-labled exosome in the cytoplasm of cells upon E2 treatment. Taken together, E2 was the most potent hormone for APE1/Ref-1 secretion, which appeared to occur through exosomes that were dependent on ER and intracellular Ca2+. Furthermore, hormonal effects should be considered when analyzing biomarkers for vascular inflammation.

Protective Role of Dietary Capsanthin in a Mouse Model of Nonalcoholic Fatty Liver Disease.

Capsanthin is the main carotenoid compound in red paprika (Capsicum annuum L.). However, little is known about the beneficial effects of capsanthin in nonalcoholic fatty liver disease (NAFLD). In this study, the hepatoprotective activity of capsanthin was investigated in a mouse model of NAFLD. Apolipoprotein-E knockout mice were fed with normal diet, Western-type diet (WD, NAFLD model), WD with capsanthin (0.5 mg/kg of body weight/day, CAP), WD with capsanthin-rich extract (25 mg/kg of body weight/day; CRE), or WD with red paprika powder (25 mg/kg of body weight/day, RPP) for 12 weeks. The carotenoid content in CRE or RPP was analyzed using ultraperformance liquid chromatography. The capsanthin concentration in CRE was 2067 mg/100 g of dry weight, which was 63% of total carotenoids. The oral administration of CRE or capsanthin significantly reduced the WD-induced increase in body weight and lipid accumulation in the liver (vs. the RPP group). In addition, CRE or capsanthin significantly inhibited the WD-induced increase in cholesterol and low-density lipoprotein levels. Furthermore, CRE or capsanthin showed reduced levels of plasma alanine and aspartate aminotransferase (ALT and AST, respectively), suggesting a steatohepatitis protective effect. Capsanthin regulated mRNA levels of peroxisome proliferator-activated receptor alpha (Pparα), carnitine palmitoyltransferase 1A (Cpt1a), acyl-CoA oxidase 1 (Acox1), and sterol regulatory element binding protein-1c (Srebp1c), which are associated with hepatic fatty acid metabolism. Overall, our results suggest that the capsanthin of red paprika plays a protective role against hepatic steatosis/steatohepatitis in NAFLD.

Plasma APE1/Ref-1 Correlates with Atherosclerotic Inflammation in ApoE-/- Mice.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is involved in DNA base repair and reducing activity. However, the role of APE1/Ref-1 in atherosclerosis is unclear. Herein, we investigated the role of APE1/Ref-1 in atherosclerotic apolipoprotein E (ApoE-/-) mice fed with a Western-type diet. We found that serologic APE1/Ref-1 was strongly correlated with vascular inflammation in these mice. Neutrophil/lymphocyte ratio (NLR), endothelial cell/macrophage activation, and atherosclerotic plaque formation, reflected by atherosclerotic inflammation, were increased in the ApoE-/- mice fed with a Western-type diet. APE1/Ref-1 expression was upregulated in aortic tissues of these mice, and was co-localized with cells positive for cluster of differentiation 31 (CD31) and galectin-3, suggesting endothelial cell/macrophage expression of APE1/Ref-1. Interestingly, APE1/Ref-1 plasma levels of ApoE-/- mice fed with a Western-type diet were significantly increased compared with those of the mice fed with normal diet (15.76 ± 3.19 ng/mL vs. 3.51 ± 0.50 ng/mL, p < 0.05), and were suppressed by atorvastatin administration. Correlation analysis showed high correlation between plasma APE1/Ref-1 levels and NLR, a marker of systemic inflammation. The cut-off value for APE1/Ref-1 for predicting atherosclerotic inflammation at 4.903 ng/mL showed sensitivity of 100% and specificity of 91%. We conclude that APE1/Ref-1 expression is upregulated in aortic endothelial cells/macrophages of atherosclerotic mice, and that plasma APE1/Ref-1 levels could predict atherosclerotic inflammation.

Effect of a new herbal composition comprised of red clover and hop extract on human endothelial cell damage and vasorelaxant activity.

Hormone replacement therapy may cause various side effects, including enhancing the risk of cardiovascular disease (CVD) in postmenopausal women. Here, we investigated the effect of red clover and hop extract combination (RHEC) on estrogen receptor (ER) binding and endothelial function of human umbilical vein endothelial cells (HUVECs) to develop an herbal agent for reducing the risk of CVDs. In ER competitor assay, RHEC showed binding affinity toward ERα and ERß with IC50 values of 5.92 µg/ml and 1.66 µg/ml, respectively. In HUVECs, RHEC significantly increased the cell viability and reduced the reactive oxygen species production against oxidative stress-induced damage. We also showed that RHEC increased the NO production through upregulating the endothelial nitric oxide synthase expression via ER activation in estrogen depleted condition. In particular, RHEC showed greater efficacy with increase in NO and decrease in endothelin-1 than red clover or hop treatment alone. Additionally, 0.3-0.5 mg/ml of RHEC-induced vasorelaxation of rat aortic rings precontracted by phenylephrine. PRACTICAL APPLICATIONS: Recently, a large interest has grown in the synergistic effects of phytochemicals for better therapies to treat various diseases. Red clover and hop are well-known edible plants which are widely used to help relieve postmenopausal symptoms including CVD. However, their combination has not been studied so far. For the first time, we demonstrated that RHEC, a new herbal combination comprising the extracts from red clover and hop, appeared to be effective in protection of endothelial function against oxidative stress and estrogen depletion. Therefore, RHEC could be a potent herbal agent for reducing the risk of endothelial damage.

Epigallocatechin-3-Gallate Suppresses Vasculogenic Mimicry through Inhibiting the Twist/VE-Cadherin/AKT Pathway in Human Prostate Cancer PC-3 Cells.

Vasculogenic mimicry (VM) is the alternative process of forming vessel-like networks by aggressive tumor cells, and it has an important role in tumor survival, growth, and metastasis. Epigallocatechin-3-gallate (EGCG) is well known to have diverse bioactivities including anti-cancer effects. However, the efficacy of EGCG on VM is elusive. In this study, we explored whether and how EGCG affects VM in human prostate cancer (PCa) PC-3 cells. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Invasive and VM formation abilities were assessed by an invasion assay and a three-dimensional (3D) culture VM tube formation assay, respectively. Western blots were carried out. An immunofluorescence assay was performed to detect nuclear twist expression. EGCG effectively inhibited the invasive ability, as well as tubular channel formation, without affecting cell viability. EGCG significantly downregulated the expression of vascular endothelial cadherin (VE-cadherin) and its transcription factor, twist, N-cadherin, vimentin, phosphor-AKT, and AKT, but not phospho-erythropoietin-producing hepatocellular receptor A2 (EphA2) and EphA2. In addition, EGCG diminished the nuclear localization of twist. Treatment with SC79, an AKT activator, effectively rescued EGCG-inhibited VM formation. These results demonstrated for the first time that EGCG causes marked suppression of VM through inhibiting the twist/VE-cadherin/AKT pathway in human PCa PC-3 cells.

Lysates of a Probiotic, Lactobacillus rhamnosus, Can Improve Skin Barrier Function in a Reconstructed Human Epidermis Model.

The main function of the skin is to protect the body from the external environment. The barrier function of the skin is mainly provided by the stratum corneum, which consists of corneocytes bound with the corneodesmosomes and lamellar lipids. Skin barrier proteins like loricrin and filaggrin also contribute to the skin barrier function. In various skin diseases, skin barrier dysfunction is a common symptom, and skin irritants like detergents or surfactants could also perturb skin barrier function. Many efforts have been made to develop strategies to improve skin barrier function. Here, we investigated whether the microfluidized lysates of Lactobacillus rhamnosus (LR), one of the most widely used probiotic species for various health benefits, may improve the skin barrier function in a reconstructed human epidermis, Keraskin™. Application of LR lysate on Keraskin™ increased the expression of tight junction proteins; claudin 1 and occludin as determined by immunofluorescence analysis, and skin barrier proteins; loricrin and filaggrin as determined by immunohistochemistry and immunofluorescence analysis and qPCR. Also, the cytotoxicity of a skin irritant, sodium lauryl sulfate (SLS), was alleviated by the pretreatment of LR lysate. The skin barrier protective effects of LR lysate could be further demonstrated by the attenuation of SLS-enhanced dye-penetration. LR lysate also attenuated the destruction of desmosomes after SLS treatment. Collectively, we demonstrated that LR lysate has protective effects on the skin barrier, which could expand the utility of probiotics to skin-moisturization ingredients.

ATP Binding Cassette Transporter A1 is Involved in Extracellular Secretion of Acetylated APE1/Ref-1.

Acetylation of nuclear apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is associated with its extracellular secretion, despite the lack of an N-terminal protein secretion signal. In this study, we investigated plasma membrane targeting and translocation of APE1/Ref-1 in HEK293T cells with enhanced acetylation. While APE1/Ref-1 targeting was not affected by inhibition of the endoplasmic reticulum/Golgi-dependent secretion, its secretion was reduced by inhibitors of ATP-binding cassette (ABC) transporters, and siRNA-mediated down-regulation of ABC transporter A1. The association between APE1/Ref-1 and ABCA1 transporter was confirmed by proximal ligation assay and immunoprecipitation experiments. An APE1/Ref-1 construct with mutated acetylation sites (K6/K7R) showed reduced co-localization with ABC transporter A1. Exposure of trichostatin A (TSA) induced the acetylation of APE1/Ref-1, which translocated into membrane fraction. Taken together, acetylation of APE1/Ref-1 is considered to be necessary for its extracellular targeting via non-classical secretory pathway using the ABCA1 transporter.

The extracellular role of Ref-1 as anti-inflammatory function in lipopolysaccharide-induced septic mice.

Apurinic/apyrimidinic endonuclease/redox factor-1 (Ref-1), a multifunctional protein secreted from stimulated cells, has been identified as a new serological biomarker. Despite recent reports on the role of Ref-1 in inflammation, the biological function of secreted Ref-1 remains unknown, especially in vivo. This study aimed to evaluate the possible roles of secreted Ref-1 in lipopolysaccharide-induced systemic inflammation in vivo. We generated a secretory Ref-1 adenoviral vector system, AdPPT-LS-Ref-1, by conjugation of preprotrypsin leading sequence (PPT-LS) with full-length Ref-1 sequences. Expression of tumor necrosis factor-α (TNF-α)-induced vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells and lipopolysaccharide (LPS)-induced cyclooxygenase-2 in Raw264.7 cells was inhibited by secretory Ref-1, and this inhibitory effect was abrogated following neutralization of Ref-1 with anti-Ref-1 antibody. Plasma Ref-1 levels following administration of AdPPT-LS-Ref-1 (2 × 109 ifu, i.p.) for 24 h were substantially higher than those recorded following administration of Adßgal (84.6 ±â€¯7.2 ng/ml vs. 4.4 ±â€¯1.5 ng/ml). Treatment with LPS (10 mg/kg, i.v. for 6 h) markedly increased VCAM-1 expression, cathepsin or myeloperoxidase activity, which were significantly suppressed by treatment with AdPPT-LS-Ref-1. Furthermore, LPS-induced cytokines, such as TNF-α, interleukin (IL)-1ß, IL-6, and monocyte chemoattractant protein 1, were significantly inhibited in AdPPT-LS-Ref-1-treated mice. However, LPS-induced myeloperoxidase activities were not suppressed by treatment with the redox mutant of secretory Ref-1, AdPPT-LS-Ref-1(C65A/C93A), or wild-type AdRef-1. Collectively, these results suggest that secreted Ref-1 has anti-inflammatory properties and that its redox cysteine residue is associated with the anti-inflammatory activity in vivo. Furthermore, our findings indicate that secretory Ref-1 may be useful as a therapeutic biomolecule against systemic inflammation.

Fomes fomentarius Ethanol Extract Exerts Inhibition of Cell Growth and Motility Induction of Apoptosis via Targeting AKT in Human Breast Cancer MDA-MB-231 Cells.

Fomes fomentarius, an edible mushroom, is known to have anti-cancer, anti-inflammatory, and anti-diabetes effects. However, the underlying anti-cancer mechanism of F. fomentarius is unknown. To determine the molecular mechanism of the anti-cancer effects of F. fomentarius, various methods were used including fluorescence-activated cell sorting, Western blotting, migration, and crystal violet assays. F. fomentarius ethanol extract (FFE) decreased cell viability in six cancer cell lines (MDA-MB-231, MCF-7, A549, H460, DU145, and PC-3). FFE decreased the migration of MDA-MB-231 cells without causing cell toxicity. Furthermore, FFE attenuated the expression of matrix metalloproteinase-9 and phosphorylation of Akt as well as increased E-cadherin in MDA-MB-231 cells. FFE arrested the S and G2/M populations by inhibiting the expression of cell cycle regulatory proteins such as cyclin-dependent kinase 2, cyclin A/E, and S-phase kinase-associated protein 2. FFE increased the sub-G1 population and expression of cleaved caspase-9, -3, and cleaved poly adenosine diphosphate (ADP-ribose) polymerase at 72 h and suppressed B-cell lymphoma 2. Interestingly, FFE and AKT inhibitors showed similar effects in MDA-MB-231 cells. Additionally, FFE contained betulin which inhibited p-AKT in MDA-MB-231 cells. Our findings demonstrate that FFE inhibits cell motility and growth and induces apoptosis by inhibiting the phsphoinositide 3- kinase /AKT pathway and caspase activation.

Serum promotes vasculogenic mimicry through the EphA2/VE-cadherin/AKT pathway in PC-3 human prostate cancer cells.

AIMS: Serum is widely used for in vitro cell culture of eukaryotic cells. Although serum is well known to affect various biological activities in cancer cells, its effect in vasculogenic mimicry (VM) is not yet fully defined. Thus, this study investigated the role of serum in VM in human prostate cancer (PCa) PC-3 cells. MAIN METHODS: Invasion assay and 3D culture VM tube formation assay are performed. VM-related molecules are checked by western blot and reverse transcriptase-polymerase chain reaction. Nuclear twist is detected by confocal after twist-FITC/DAPI double staining. KEY FINDINGS: Serum dramatically induced not only invasion but also VM. Serum increased the phosphorylation of erythropoietin-producing hepatocellular A2 (EphA2) without affecting EphA2 expression. Both the protein and mRNA expression levels of vascular endothelial cadherin (VE-cadherin) are up-regulated by serum. Twist expression was increased in the nucleus by serum. Serum activated AKT through phosphorylation, despite the unchanged AKT expression. Serum caused an increase in matrix metalloproteinase-2 (MMP-2) and laminin subunit 5 gamma-2 (LAMC2) protein expressions. Wortmannin, a phosphoinositide-3-kinase inhibitor, significantly decreased serum-induced invasion and VM. SIGNIFICANCE: These results demonstrated that serum activates EphA2 and up-regulates twist/VE-cadherin, which in turn activate AKT that up-regulates MMP-2 and LAMC2, thereby inducing the invasion and VM of human PCa PC-3 cells.

Comparison of the main components and bioactivity of Rhus verniciflua Stokes extracts by different detoxification processing methods.

BACKGROUND: Rhus verniciflua Stokes is an Asian tree species that is used as a food supplement and traditional medicine in Korea. However, its use is restricted by its potential to cause allergy. Thus, allergen-free R. verniciflua extracts are currently being marketed as a functional health food in Korea. In the present study, three different allergen-free R. verniciflua extracts (DRVE, FRVE, and FFRVE) were produced by detoxification of R. verniciflua, and their properties and constituents were compared. METHODS: The main components and properties (antibacterial, antioxidant, anticancer, and hepatic lipogenesis inhibitory effects) of the three allergen-free extracts were compared. Moreover, the major phenolic constituents of R. verniciflua, including gallic acid, fustin, fisetin, and quercetin, were analyzed in the three extracts. RESULTS: DRVE was superior to the two other extracts with regard to antioxidant activity, while FRVE was superior with regard to antimicrobial activity and suppression of hepatic lipogenesis. FRVE exhibited lipid-lowering effects by lowering sterol regulatory element-binding protein 1 and triglyceride levels, and promoting the activation of peroxisome proliferator-activated receptor and AMP-activated protein kinase in an in vitro model of non-alcoholic fatty liver. CONCLUSIONS: Overall, our findings demonstrate various differences among the three extracts. This suggests that functional and bioactive compounds present in R. verniciflua could be altered by the detoxification process, and this property could be considered in the development of functional health foods in the future.

Luteolin induces caspase-dependent apoptosis via inhibiting the AKT/osteopontin pathway in human hepatocellular carcinoma SK-Hep-1 cells.

AIMS: Luteolin, a naturally occurring flavonoid, possesses anti-cancer effects including induction of apoptosis. This study investigated the involvement of osteopontin (OPN) in luteolin-induced apoptosis in human hepatocellular carcinoma (HCC) SK-Hep-1 cells with high OPN expression. MAIN METHODS: MTT assay was used to determine the cell viability. Cell cycle analysis was performed to identify apoptosis. Apoptosis was confirmed by detecting cytoplasmic histone-associated-DNA-fragments using a cell death detection ELISAPLUS kit. The expression of proteins was evaluated by Western blot. Reverse transcriptase-polymerase chain reaction was employed to detect the expression of mRNA. KEY FINDINGS: Cytotoxic effect of luteolin was higher in cancer cell line SK-Hep-1 cells than in normal cell line AML12 cells. Luteolin led a significantly increase in apoptosis accompanied by activation of caspase 8, -9 and -3 and cleavage of poly (ADP-ribose) polymerase (PARP), which was completely blocked by Z-VAD-FMK, a pan caspase inhibitor. Luteolin significantly downregulated the expression of X-linked inhibitor of apoptosis (XIAP), Mcl-1 and Bid. Furthermore, luteolin effectively decreased OPN expression at both mRNA and protein level. Exogenous OPN markedly blocked apoptosis induction, caspases activation, PARP cleavage, downregulation of XIAP and Mcl-1 in luteolin-treated cells. Luteolin impaired the AKT pathway by inhibiting the phosphorylation of AKT. SC79, an AKT activator, blocked apoptosis induction, caspases activation, PARP cleavage, downregulation of OPN, XIAP, Mcl-1 and Bid in luteolin-treated cells. SIGNIFICANCE: These results demonstrated that luteolin inhibits the AKT/OPN pathway, thereby inducing caspase-dependent apoptosis in human HCC SK-Hep-1 cells with little toxicity.

Anthocyanin-Rich Extract from Red Chinese Cabbage Alleviates Vascular Inflammation in Endothelial Cells and Apo E-/- Mice.

Anthocyanins, the most prevalent flavonoids in red/purple fruits and vegetables, are known to improve immune responses and reduce chronic disease risks. In this study, the anti-inflammatory activities of an anthocyanin-rich extract from red Chinese cabbage (ArCC) were shown based on its inhibitory effects in cultured endothelial cells and hyperlipidemic apolipoprotein E-deficient mice. ArCC treatment suppressed monocyte adhesion to tumor necrosis factor-α-stimulated endothelial cells. This was validated by ArCC's ability to downregulate the expression and transcription of endothelial adhesion molecules, determined by immunoblot and luciferase promoter assays, respectively. The regulation of adhesion molecules was accompanied by transcriptional inhibition of nuclear factor-κB, which restricted cytoplasmic localization as shown by immunocytochemistry. Administration of ArCC (150 or 300 mg/kg/day) inhibited aortic inflammation in hyperlipidemic apolipoprotein E-deficient mice, as shown by in vivo imaging. Immunohistochemistry and plasma analysis showed that the aortas from these mice exhibited markedly lower leukocyte infiltration, reduced plaque formation, and lower concentrations of blood inflammatory cytokines than those observed in the control mice. The results suggest that the consumption of anthocyanin-rich red Chinese cabbage is closely correlated with lowering the risk of vascular inflammatory diseases.