TFP5, a Peptide Derived from Cdk5 Activator p35, Protects Pancreatic ß Cells from Glucose Toxicity.

We studied the effect of TFP5 on MIN6 cells (cultured mouse islet ß cells) treated with different concentrations of glucose (5 or 25 mM). The results were verified in C57BL/6J mice (control; n=12) and db/db mice with type 2 diabetes mellitus (n=12). To synthesize TFP5, peptide p5 (a derivative of p35 protein, activator of cyclin-dependent kinase 5, Cdk5) was conjugated with a FITC tag at the N-terminus and an 11-amino acid TAT protein transduction domain at the C-terminus. TFP5 was employed to inhibit Cdk5 activity and then to evaluate its efficiency in treating experimental type 2 diabetes mellitus. TFP5 effectively inhibited the pathological hyperactivity of Cdk5, enhanced insulin secretion, and protected pancreatic ß cells from apoptosis in vitro and in vivo. In addition, TFP5 inhibited inflammation in pancreatic islets by reducing the expression of inflammatory cytokines TGF-ß1, TNFα, and IL-1ß. These novel data indicates that TFP5 is a promising candidate for treatment of type 2 diabetes mellitus.

Vascular Endothelial Barrier Protection Prevents Atrial Fibrillation by Preserving Cardiac Nanostructure.

BACKGROUND: Atrial fibrillation (AF), the most common cardiac arrhythmia, is widely associated with inflammation, vascular dysfunction, and elevated levels of the vascular leak-inducing cytokine, vascular endothelial growth factor (VEGF). Mechanisms underlying AF are poorly understood and current treatments only manage this progressive disease, rather than arresting the underlying pathology. The authors previously identified edema-induced disruption of sodium channel (NaV1.5)-rich intercalated disk nanodomains as a novel mechanism for AF initiation secondary to acute inflammation. Therefore, we hypothesized that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. OBJECTIVES: In this study the authors tested the hypothesis that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. They identified 2 molecular targets for vascular barrier protection, connexin43 (Cx43) hemichannels and pannexin-1 (Panx1) channels, which have been implicated in cytokine-induced vascular leak. METHODS: The authors undertook in vivo electrocardiography, electron microscopy, and super-resolution light microscopy studies in mice acutely treated with a clinically relevant level of VEGF. RESULTS: AF incidence was increased in untreated mice exposed to VEGF relative to vehicle control subjects. VEGF also increased the average number of AF episodes. VEGF shifted NaV1.5 signal to longer distances from Cx43 gap junctions, measured by a distance transformation-based spatial analysis of 3-dimensional confocal images of intercalated disks. Similar effects were observed with NaV1.5 localized near mechanical junctions composed of neural cadherin. Blocking connexin43 hemichannels (αCT11 peptide) or Panx1 channels (PxIL2P peptide) significantly reduced the duration of AF episodes compared with VEGF alone with no treatment. Concurrently, both peptide therapies preserved NaV1.5 distance from gap junctions to control levels and reduced mechanical junction-adjacent intermembrane distance in these hearts. Notably, similar antiarrhythmic efficacy was also achieved with clinically-relevant small-molecule inhibitors of Cx43 and Panx1. CONCLUSIONS: These results highlight vascular barrier protection as an antiarrhythmic strategy following inflammation-induced vascular leak.

Alleviation of cadmium-induced photoinhibition and oxidative stress by melatonin in Chlamydomonas reinhardtii.

As one of the most threatening challenges to the natural environment and human health, cadmium (Cd) pollution has seriously impacted natural organisms. Green algae, such as Chlamydomonas reinhardtii (C. reinhardtii), can provide a safer, lower cost, and more effective ecological approach to the treatment of heavy metal ions in wastewater due to their sorption properties. However, heavy metal ions affect C. reinhardtii when adsorbed. Melatonin is able to protect the plant body from damage when the plant is under biotic/abiotic stress. Therefore, we investigated the effects of melatonin on the cell morphology, chlorophyll content, chlorophyll fluorescence parameters, enzymatic activity of the antioxidant system, gene expression, and the ascorbic acid (AsA)-glutathione (GSH) cycle of C. reinhardtii under the stress of Cd (13 mg/L). Our results indicated that Cd significantly induced photoinhibition and overaccumulation of reactive oxygen species (ROS). By application with the concentration of 1.0 µM melatonin, the algal solute of C. reinhardtii under the Cd stress gradually regained its green color, the cell morphology became intact, and the photosynthetic electron transport function was retained. However, in the melatonin-silenced strain, there was a significant decrease in all of the above indicators. In addition, the use of exogenous melatonin or the expression of endogenous melatonin genes could enhance the intracellular enzyme activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR). It also upregulated the expression of active enzyme genes such as SOD1, CAT1, FSD1, GSH1, GPX5, and GSHR1. These results indicate that the presence of melatonin effectively protects the activity of photosynthetic system II in C. reinhardtii, enhances antioxidant activity, upregulates gene expression in the AsA-GSH cycle, and reduces the level of ROS, thereby alleviating the damage caused by Cd toxicity.

AdipoAI suppresses osteoclastogenesis by activating AdipoR1/APPL1: An in vivo experimental study in diabetes-associated peri-implantitis.

AIM: Diabetics experience severe peri-implant inflammatory bone damage. We aimed to provide powerful evidence supporting the novel adiponectin receptor agonist AdipoAI in treating diabetes-associated peri-implantitis. MATERIALS AND METHODS: Twenty-four ZDF-Leprfa/Crl rats were randomly allocated to three groups (N = 8). After feeding with a high-fat diet to establish diabetic rats, experimental peri-implantitis was induced by implanting titanium rods (1.5 mm diameter and 20 mm length) contaminated with Staphylococcus aureus into the femurs. Radiographic evaluation, microCT, histological analyses and qRT-PCR were used to detect inflammatory infiltration and bone destruction. In vitro, the inhibition by AdipoAI of osteoclastogenesis, including the number and function of osteoclasts, was investigated by TRAP staining, immunofluorescence, qRT-PCR and Western blotting. Immunofluorescence, qRT-PCR and Western blotting were also utilized to explore AdipoR1, APPL1, NF-κB and Wnt5a-Ror2 signalling molecules in this process. One-way ANOVA with Tukey's post hoc test was used to compare the data. RESULTS: AdipoAI reduced inflammation and bone destruction caused by peri-implantitis in diabetic rats, which were manifested by a reduction in F4/80-positive macrophage infiltration by 72%, the number of osteoclasts by 58% and the levels of cytokines (p < .05) in disease group. In vitro, 1 µM AdipoAI decreased the number of osteoclasts to 51%, inhibited F-actin ring formation and reduced the levels of related markers (p < .05). Mechanistically, AdipoAI activated AdipoR1/APPL1 and conversely suppressed the phosphorylation of IκB-α, nuclear translocation of P65 and the Wnt5a-Ror2 signalling pathway (p < .05). CONCLUSIONS: AdipoAI suppressed osteoclastogenesis in diabetes-associated peri-implantitis by inhibiting the NF-κB and Wnt5a-Ror2 pathways via the AdipoR1/APPL1 axis.

Neurog1-Derived Peptides RMNE1 and DualPep-Shine Penetrate the Skin and Inhibit Melanin Synthesis by Regulating MITF Transcription.

Anti-pigmentation peptides have been developed as alternative skin-lightening agents to replace conventional chemicals that have adverse effects on the skin. However, the maximum size of these peptides is often limited by their low skin and cell penetration. To address this issue, we used our intra-dermal delivery technology (IDDT) platform to identify peptides with hypo-pigmenting and high cell-penetrating activity. Using our cell-penetrating peptides (CPPs) from the IDDT platform, we identified RMNE1 and its derivative RMNE3, "DualPep-Shine", which showed levels of α-Melanocyte stimulating hormone (α-MSH)-induced melanin inhibition comparable to the conventional tyrosinase inhibitor, Kojic acid. In addition, DualPep-Shine was delivered into the nucleus and regulated the gene expression levels of melanogenic enzymes by inhibiting the promoter activity of microphthalmia-associated transcription factor-M (MITF-M). Using a 3D human skin model, we found that DualPep-Shine penetrated the lower region of the epidermis and reduced the melanin content in a dose-dependent manner. Furthermore, DualPep-Shine showed high safety with little immunogenicity, indicating its potential as a novel cosmeceutical ingredient and anti-pigmentation therapeutic agent.

Tilianin alleviates lipid deposition and fibrosis in mice with nonalcoholic steatohepatitis by activating the PPARα/Nnat axis.

The understanding and treatment of nonalcoholic steatohepatitis (NASH) are still very limited. This study reports the therapeutic effect of tilianin on mice with NASH and further explores its possible molecular mechanisms. A mice model of NASH was established using low-dose streptozotocin combined with a high-fat diet and tilianin treatment. Liver function was assessed by determining serum aspartate aminotransferase and alanine aminotransferase in serum. Interleukin (IL)-1ß, IL-6, transforming growth factor ß1 (TGF-ß1), and tumor necrosis factor α (TNF-α) levels in serum were determined. Hepatocyte apoptosis was assessed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling staining. Oil Red O staining and boron dipyrrin staining were used to determine lipid deposition in liver tissues. Masson staining was used to evaluate liver fibrosis, and immunohistochemistry and western blot analysis were used to determine the expression of target proteins. Tilianin treatment significantly ameliorated liver function, inhibited hepatocyte apoptosis, and reduced lipid deposition and liver fibrosis in mice with NASH. The expression of neuronatin (Nnat) and peroxisome proliferator-activated receptor (PPAR) α was upregulated, whereas that of sterol regulatory element-binding protein 1 (SREBP-1), TGF-ß1, nuclear factor (NF)-κB p65, and phosphorylated p65 was downregulated in the liver tissues of mice with NASH after tilianin treatment. The above effects of tilianin were significantly reversed after Nnat knock-down, but its effect on PPARα expression was unaffected. Thus, the natural drug tilianin shows potential in treatig NASH. Its mechanism of action may be related to the targeted activation of PPARα/Nnat, thereby inhibiting the activation of the NF-κB signaling pathway.

Bin1 targeted immunotherapy alters the status of the enteric neurons and the microbiome during ulcerative colitis treatment.

Ulcerative colitis (UC) is a common chronic disease of the large intestine. Current anti-inflammatory drugs prescribed to treat this disease have limited utility due to significant side-effects. Thus, immunotherapies for UC treatment are still sought. In the DSS mouse model of UC, we recently demonstrated that systemic administration of the Bin1 monoclonal antibody 99D (Bin1 mAb) developed in our laboratory was sufficient to reinforce intestinal barrier function and preserve an intact colonic mucosa, compared to control subjects which displayed severe mucosal lesions, high-level neutrophil and lymphocyte infiltration of mucosal and submucosal areas, and loss of crypts. A dysbiotic microbiome may lead to UC. We determined the effects of Bin1 mAb on the gut microbiome and colonic neurons and correlated the benefits of immunotherapeutic treatment. In the DSS model, we found that induction of UC was associated with disintegration of enteric neurons and elevated levels of glial cells, which translocated to the muscularis at distinct sites. Further, we characterized an altered gut microbiome in DSS treated mice associated with pathogenic proinflammatory characters. Both of these features of UC induction were normalized by Bin1 mAb treatment. With regard to microbiome changes, we observed in particular, increase in Enterobacteriaceae; whereas Firmicutes were eliminated by UC induction and Bin1 mAb treatment restored this phylum including the genus Lactobacillus. Overall, our findings suggest that the intestinal barrier function restored by Bin1 immunotherapy in the DSS model of UC is associated with an improvement in the gut microbiome and preservation of enteric neurons, contributing overall to a healthy intestinal tract.

Luteolin loaded on zinc oxide nanoparticles ameliorates non-alcoholic fatty liver disease associated with insulin resistance in diabetic rats via regulation of PI3K/AKT/FoxO1 pathway.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a worldwide health problem with high prevalence and morbidity associated with obesity, insulin resistance, type 2 diabetes mellitus (T2DM), and dyslipidemia. Nano-formulation of luteolin with Zn oxide in the form of Lut/ZnO NPs may improve the anti-diabetic property of each alone and ameliorate the insulin resistance thus management of NAFLD. This study aimed to measure the efficiency of Lut/ZnO NPs against insulin resistance coupled with NAFLD and T2DM. METHODS: A diabetic rat model with NAFLD was induced by a high-fat diet and streptozotocin (30 mg/kg I.P). Serum diabetogenic markers levels, lipid profile, and activity of liver enzymes were measured beside liver oxidative stress markers. Moreover, the hepatic expressions of PI3K/AKT/FoxO1/SERBP1c as well as heme oxygenase-1 were measured beside the histopathological examination. RESULTS: Lut/ZnO NPs treatment effectively reduced hyperglycemia, hyperinsulinemia, and ameliorated insulin resistance. Additionally, Lut/ZnO NPs improved the hepatic functions, the antioxidant system, and reduced the oxidative stress markers. Furthermore, the lipid load in the liver, as well as the circulating TG and TC, was minified via the suppression of lipogenesis and gluconeogenesis. Moreover, Lut/ZnO NPs activated the PI3K/AKT signaling pathway, hence inactivating FoxO1, therefore enhancing the hepatic cells' insulin sensitivity. CONCLUSION: Lut/ZnO NPs have a hepatoprotective effect and may relieve the progression of NAFLD by alleviating insulin resistance, ameliorating the antioxidant status, and regulating the insulin signal pathway.

Melatonin Inhibits 17ß-Estradiol-Induced Epithelial-Mesenchymal Transition in Endometrial Adenocarcinoma Cells via Upregulating Numb Expression.

OBJECTIVES: Melatonin (MLT) shows antitumor effects in various tumor types, including endometrial carcinoma. However, the molecular mechanism involved is unclear. In the current study, we investigated the effect of MLT on the estrogen-induced epithelial-mesenchymal transition (EMT) in endometrial adenocarcinoma cells and explored the pathway that might be involved. DESIGN: Laboratory study was via cultured endometrial cancer cells. Design refers only to in vitro experiments. METHODS: In cell culture experiments, cell growth was examined using CCK-8 assays. The expression of Numb and EMT markers in Ishikawa cells was examined using Western blot analysis and real-time PCR. Cell invasion was examined using transwell assays. Cell migration was examined using wound-healing assays and transwell assays. Using immunohistochemistry analysis, the expression of Numb in human endometrial cancers was examined. RESULTS: In immunohistochemistry experiments, we found that 15.2% of atypical endometrial hyperplasia and 15.6% of endometrial carcinoma did not express Numb. In cell culture experiments, MLT inhibited cell proliferation, invasion, and migration induced by 17ß-estradiol (E2) in endometrial cancer cells. MLT decreased the expression of vimentin and Slug and increased the expression of Numb and E-cadherin in Ishikawa cells. Numb knockdown in cancer cells significantly increased cell proliferation, invasion, and migration. LIMITATIONS: No animal experiments were performed. CONCLUSIONS: MLT blocked E2-induced cell growth and EMT in endometrial cancer cells via upregulating Numb expression.

Plexin-A4 Mediates Cytotoxic T-cell Trafficking and Exclusion in Cancer.

Cytotoxic T cell (CTL) infiltration of the tumor carries the potential to limit cancer progression, but their exclusion by the immunosuppressive tumor microenvironment hampers the efficiency of immunotherapy. Here, we show that expression of the axon guidance molecule Plexin-A4 (Plxna4) in CTLs, especially in effector/memory CD8+ T cells, is induced upon T-cell activation, sustained in the circulation, but reduced when entering the tumor bed. Therefore, we deleted Plxna4 and observed that Plxna4-deficient CTLs acquired improved homing capacity to the lymph nodes and to the tumor, as well as increased proliferation, both achieved through enhanced Rac1 activation. Mice with stromal or hematopoietic Plxna4 deletion exhibited enhanced CTL infiltration and impaired tumor growth. In a melanoma model, adoptive transfer of CTLs lacking Plxna4 prolonged survival and improved therapeutic outcome, which was even stronger when combined with anti-programmed cell death protein 1 (PD-1) treatment. PLXNA4 abundance in circulating CTLs was augmented in melanoma patients versus healthy volunteers but decreased after the first cycle of anti-PD-1, alone or in combination with anti-cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4), in those patients showing complete or partial response to the treatment. Altogether, our data suggest that Plxna4 acts as a "checkpoint," negatively regulating CTL migration and proliferation through cell-autonomous mechanisms independent of the interaction with host-derived Plxna4 ligands, semaphorins. These findings pave the way toward Plxna4-centric immunotherapies and propose Plxna4 detection in circulating CTLs as a potential way to monitor the response to immune checkpoint blockade in patients with metastatic melanoma.

Slit2-Mediated Metabolic Reprogramming in Bone Marrow-Derived Macrophages Enhances Antitumor Immunity.

Slit2 exerts antitumor effects in various cancers; however, the underlying mechanism, especially its role in regulating the immune, especially in the bone marrow niche, system is still unknown. Elucidating the behavior of macrophages in tumor progression can potentially improve immunotherapy. Using a spontaneous mammary tumor virus promoter-polyoma middle T antigen (PyMT) breast cancer mouse model, we observed that Slit2 increased the abundance of antitumor M1 macrophage in the bone marrow upon differentiation in vitro. Moreover, myeloablated PyMT mice injected with Slit2-treated bone marrow allografts showed a marked reduction in tumor growth, with enhanced recruitment of M1 macrophage in their tumor stroma. Mechanistic studies revealed that Slit2 significantly enhanced glycolysis and reduced fatty acid oxidation in bone marrow-derived macrophages (BMDMs). Slit2 treatment also altered mitochondrial respiration metabolites in macrophages isolated from healthy human blood that were treated with plasma from breast cancer patients. Overall, this study, for the first time, shows that Slit2 increases BMDM polarization toward antitumor phenotype by modulating immune-metabolism. Furthermore, this study provides evidence that soluble Slit2 could be developed as novel therapeutic strategy to enhance antitumor immune response.

c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) attenuates curcumin-induced cell death differently from its family member, JNK-associated leucine zipper protein (JLP).

Curcumin, a major component of turmeric, is known to exhibit multiple biological functions including antitumor activity. We previously reported that the mitogen-activated protein kinase (MAPK) scaffold protein c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP) reduces curcumin-induced cell death by modulating p38 MAPK and autophagy through the regulation of lysosome positioning. In this study, we investigated the role of JNK/stress-activated protein kinase-associated protein 1 (JSAP1), a JLP family member, in curcumin-induced stress, and found that JSAP1 also attenuates curcumin-induced cell death. However, JSAP1 knockout showed no or little effect on the activation of JNK and p38 MAPKs in response to curcumin. In addition, small molecule inhibitors of JNK and p38 MAPKs did not increase curcumin-induced cell death. Furthermore, JSAP1 depletion did not impair lysosome positioning and autophagosome-lysosome fusion. Instead, we noticed substantial autolysosome accumulation accompanied by an inefficient autophagic flux in JSAP1 knockout cells. Taken together, these results indicate that JSAP1 is involved in curcumin-induced cell death differently from JLP, and may suggest that JSAP1 plays a role in autophagosome degradation and its dysfunction results in enhanced cell death. The findings of this study may contribute to the development of novel therapeutic approaches using curcumin for cancer.

Ethacrynic acid, a loop diuretic, suppresses epithelial-mesenchymal transition of A549 lung cancer cells via blocking of NDP-induced WNT signaling.

Lung cancer is one of the leading causes of death in cancer patients. Epithelial-mesenchymal transition (EMT) plays an important role in lung cancer progression. Therefore, for lung cancer treatment, it is crucial to find substances that inhibit EMT. Ethacrynic acid (ECA) is a diuretic that inhibits cellular ion flux and exerts anticancer effects. However, the effects of ECA on EMT in lung cancer remain unclear. We examined the effects of ECA on sphingosylphosphorylcholine (SPC) or TGF-ß1-induced EMT process in A549 and H1299 cells via reverse transcription polymerase chain reaction and Western blotting. We found that ECA inhibited SPC-induced EMT and SPC-induced WNT signalling in EMT. We observed that SPC induces the expression of NDP [Norrie disease protein] and WNT-2, whereas ECA suppressed their expression. SPC-induced WNT activation, EMT, migration, and invasion were suppressed by NDP small-interfering RNA (siNDP), but NDP overexpression (pNDP) enhanced these events in A549 and H1299 cells. Accordingly, NDP expression may influence lung cancer prognosis. In summary, our results revealed that ECA inhibited SPC or TGF-ß1-induced EMT in A549 and H1299 lung cancer cells by downregulating NDP expression and inhibiting WNT activation. Therefore, ECA might be a new drug candidate for lung cancer treatment.

Human splenic TER cells: A relevant prognostic factor acting via the artemin-GFRα3-ERK pathway in pancreatic ductal adenocarcinoma.

Splenectomy is routinely performed during distal or total pancreatectomy (DP or TP) for pancreatic ductal adenocarcinoma (PDAC), but information about its oncological value is limited. TER cells, nonimmune cells discovered in the spleens of tumour-bearing mice, are elicited by tumours and promote tumour progression, while their role in the clinical outcomes of patients with PDAC remains unclear. In our study, postoperative specimens from 622 patients who underwent DP or TP with splenectomy were analysed by flow cytometry or immunofluorescence, and the relationship between splenic TER cell count and clinical parameters was calculated. We also purified human TER cells for functional experiments and mechanistic studies. We found that TER cell numbers were increased only in the spleens of patients with PDAC but not in PDAC tissue and adjacent pancreatic tissue. High splenic TER cell counts independently predicted poor prognosis (P < .001) and indicated large tumour size, lymph node metastasis, advanced 8th AJCC/mAJCC stage and high CA19-9 classification (all P < .050) in patients with PDAC. Mechanistic analysis showed that TER cells express artemin, which facilitates the proliferation and invasion of PDAC cells by activating GFRα3-ERK signalling. Our study reveals that TER cell count is an indicator of poor prognosis of PDAC, while splenectomy during pancreatic surgery might provide oncological benefits in addition to ensuring the radical resection of PDAC.

Identification of Abnormal Spindle Microtubule Assembly as a Promising Therapeutic Target for Osteosarcoma.

OBJECTIVE: To demonstrate the expression of abnormal spindle microtubule assembly (ASPM) in clinical osteosarcoma tissue specimens collected in our hospital, and to explore the function of ASPM in osteosarcoma in vitro and in vivo. METHODS: Tissue specimens from 82 cases of osteosarcoma were collected and analyzed by immunohistochemistry assay. We also investigated the relationship between ASPM expression and clinicopathological characteristics in the patients. We transfected shASPM plasmid and the empty control plasmid, respectively, and then used quantitative polymerase chain reaction and western blot analysis to detect ASPM expression. Cell colony assay and MTT were used to observe the proliferation ability. In vivo study was undertaken to explore the ASPM function further. RESULTS: In this study, ASPM showed high expression in osteosarcoma tissue samples compared with non-tumor normal tissues. ASPM was positively correlated with clinical pathological characteristics, including tumor size (P = 0.024) and clinical stage (P = 0.045). Our results further showed that ASPM depletion dramatically inhibited the proliferation of osteosarcoma cells (with fewer cells in the sh-RNA-ASPM group compared with the control group(P < 0.05, respectively), and the in vivo assays further confirmed that ASPM ablation markedly blocked tumor growth compared with control (P < 0.05). CONCLUSION: Our data provides strong evidence that the high expression of ASPM in osteosarcoma promotes proliferation in vitro and in vivo, indicating its potential role as an osteosarcoma therapeutic target.

Tenascin-C Induces Phenotypic Changes in Fibroblasts to Myofibroblasts with High Contractility through the Integrin αvß1/Transforming Growth Factor ß/SMAD Signaling Axis in Human Breast Cancer.

Tenascin-C (TNC) is strongly expressed by fibroblasts and cancer cells in breast cancer. To assess the effects of TNC on stromal formation, we examined phenotypic changes in human mammary fibroblasts treated with TNC. The addition of TNC significantly up-regulated α-smooth muscle actin (α-SMA) and calponin. TNC increased the number of α-SMA- and/or calponin-positive cells with well-developed stress fibers in immunofluorescence, which enhanced contractile ability in collagen gel contraction. The treatment with TNC also significantly up-regulated its own synthesis. Double immunofluorescence of human breast cancer tissues showed α-SMA- and/or calponin-positive myofibroblasts in the TNC-deposited stroma. Among several receptors for TNC, the protein levels of the αv and ß1 integrin subunits were significantly increased after the treatment. Immunofluorescence showed the augmented colocalization of αv and ß1 at focal adhesions. Immunoprecipitation using an anti-αv antibody revealed a significant increase in coprecipitated ß1 with TNC in lysates. The knockdown of αv and ß1 suppressed the up-regulation of α-SMA and calponin. The addition of TNC induced the phosphorylation of SMAD2/3, whereas SB-505124 and SIS3 blocked myofibroblast differentiation. Therefore, TNC enhances its own synthesis by forming a positive feedback loop and increases integrin αvß1 heterodimer levels to activate transforming growth factor-ß signaling, which is followed by a change to highly contractile myofibroblasts. TNC may essentially contribute to the stiffer stromal formation characteristic of breast cancer tissues.

PCP4/PEP19 downregulates neurite outgrowth via transcriptional regulation of Ascl1 and NeuroD1 expression in human neuroblastoma M17 cells.

Purkinje cell protein 4/peptide 19 (PCP4/PEP19) is 7.6 kDa peptide originally found in Purkinje cells. PCP4/PEP19 is a differentiation maker of Purkinje cells, where it functions as an antiapoptotic factor. Cerebral neuronal cells also express PCP4/PEP19, which may be related to neuronal cell survival. However, evidence suggests that PCP4/PEP19 may also be involved in neuronal differentiation. Here, we investigated the effects of PCP4/PEP19 expression on neuronal differentiation by analyzing neurite outgrowth, and expression of neuronal differentiation markers in cultured human neuroblastoma M17 cells. When PCP4/PEP19 expression was reduced by siRNA-mediated knockdown, neurite outgrowth was significantly increased. Among many differentiation markers tested, expression of NeuroD1 was increased, while that of Ascl1 was decreased upon PCP4/PEP19 knockdown. Furthermore, luciferase reporter assays revealed that PCP4/PEP19 knockdown upregulated NeuroD1 and downregulated Ascl1 expression, at the transcriptional level. These results suggest a new function of PCP4/PEP19, which suppresses neurite outgrowth and neuronal differentiation through the regulation of NeuroD1 and Ascl1 expression in M17 cells. Furthermore, immunohistochemical studies showed that PCP4/PEP19 localizes in the nuclei of human neuroblastoma cells. Therefore, PCP4/PEP19 may also be an intranuclear negative regulator of neuronal differentiation and may thus be a potential therapeutic target to promote cellular differentiation in human neuroblastoma.

Presynaptic Caytaxin prevents apoptosis via deactivating DAPK1 in the acute phase of cerebral ischemic stroke.

Death-associated protein kinase 1 (DAPK1) is a key protein that mediates neuronal death in ischemic stroke. Although the substrates of DAPK1 and molecular signal in stroke have been gradually discovered, the modulation of DAPK1 itself is still unclear. Here we first reveal that Caytaxin, a brain-specific member of BCL2/adenovirus E1B -interacting protein (BNIP-2), increases and interacts with DAPK1 as early as 2 h after middle cerebral artery occlusion (MCAO) in the penumbra area of mouse brain. Furthermore, Caytaxin binds to DAPK1 at the presynaptic site and inhibits DAPK1 catalytic activity. Silencing Caytaxin by Caytaxin shRNA (Sh-Caytaxin) enhances DAPK1 activity, deteriorates neuronal apoptosis and brain injuries both in vivo and in vitro. Thus, elevating presynaptic Caytaxin could prevent neuronal apoptosis by inhibiting DAPK1 activation in the acute stage of ischemic stroke. Caytaxin may physiologically protect neuronal cells and represent a potential prevention and therapeutic target in the early phase of cerebral ischemic stroke.

Norrin Protects Retinal Ganglion Cells from Excitotoxic Damage via the Induction of Leukemia Inhibitory Factor.

PURPOSE: To investigate whether and how leukemia inhibitory factor (Lif) is involved in mediating the neuroprotective effects of Norrin on retinal ganglion cells (RGC) following excitotoxic damage. Norrin is a secreted protein that protects RGC from N-methyl-d-aspartate (NMDA)-mediated excitotoxic damage, which is accompanied by increased expression of protective factors such as Lif, Edn2 and Fgf2. METHODS: Lif-deficient mice were injected with NMDA in one eye and NMDA plus Norrin into the other eye. RGC damage was investigated and quantified by TUNEL labeling 24 h after injection. Retinal mRNA expression was analyzed by quantitative real-time polymerase chain reaction following retinal treatment. RESULTS: After intravitreal injection of NMDA and Norrin in wild-type mice approximately 50% less TUNEL positive cells were observed in the RGC layer when compared to NMDA-treated littermates, an effect which was lost in Lif-deficient mice. The mRNA expression for Gfap, a marker for Müller cell gliosis, as well as Edn2 and Fgf2 was induced in wild-type mice following NMDA/Norrin treatment but substantially blocked in Lif-deficient mice. CONCLUSIONS: Norrin mediates its protective properties on RGC via Lif, which is required to enhance Müller cell gliosis and to induce protective factors such as Edn2 or Fgf2.

Inhibitory effects of Semaphorin 3F as an alternative candidate to anti-VEGF monoclonal antibody on angiogenesis.

Vascular endothelial growth factor (VEGF) inhibition forms the basis for anti-angiogenic therapies. With the methods based on the monoclonal antibody-mediated typical VEGF blockade, pathological angiogenesis in the tumor microenvironment is inhibited and the limitation of tumor growth is provided; however, the existing tumor tissue cannot be intervened. In this study, the anti-angiogenic effects of Semaphorin (SEMA) 3F, which has frequently been reported to have tumor suppressive properties, on a chick chorioallantoic membrane model as well as in vitro cell-cell interactions were investigated and comparatively assessed using anti-VEGF antibody. Vascular endothelial cells and chick embryos were stimulated with 10-16 ng/mL VEGF165 prior to SEMA 3F administration in order to generate pathological vascularization conditions. Both in vitro and in ovo results revealed that SEMA 3F suppressed VEGF165-induced abnormal vascularization more effectively than anti-VEGF. Moreover, the required dose of SEMA 3F was significantly lower than that of anti-VEGF (103 times less under in ovo conditions). In light of these results, SEMA 3F is recommended as an important therapeutic agent for the prevention of pathological angiogenesis. SEMA 3F may offer an effective and efficient anti-angiogenic intervention that can be administered at a lower dose alternative to typical VEGF blocking agents.