A Biodegradable Magnetic Microrobot Based on Gelatin Methacrylate for Precise Delivery of Stem Cells with Mass Production Capability.

A great deal of research has focused on small-scale robots for biomedical applications and minimally invasive delivery of therapeutics (e.g., cells, drugs, and genes) to a target area. Conventional fabrication methods, such as two-photon polymerization, can be used to build sophisticated micro- and nanorobots, but the long fabrication cycle for a single microrobot has limited its practical use. This study proposes a biodegradable spherical gelatin methacrylate (GelMA) microrobot for mass production in a microfluidic channel. The proposed microrobot is fabricated in a flow-focusing droplet generator by shearing a mixture of GelMA, photoinitiator, and superparamagnetic iron oxide nanoparticles (SPIONs) with a mixture of oil and surfactant. Human nasal turbinate stem cells (hNTSCs) are loaded on the GelMA microrobot, and the hNTSC-loaded microrobot shows precise rolling motion in response to an external rotating magnetic field. The microrobot is enzymatically degraded by collagenase, and released hNTSCs are proliferated and differentiated into neuronal cells. In addition, the feasibility of the GelMA microrobot as a cell therapeutic delivery system is investigated by measuring electrophysiological activity on a multielectrode array. Such a versatile and fully biodegradable microrobot has the potential for targeted stem cell delivery, proliferation, and differentiation for stem cell-based therapy.

Inhibition of Hepatic Stellate Cell Activation Suppresses Tumorigenicity of Hepatocellular Carcinoma in Mice.

Transdifferentiation (or activation) of hepatic stellate cells (HSCs) to myofibroblasts is a key event in liver fibrosis. Activated HSCs in the tumor microenvironment reportedly promote tumor progression. This study analyzed the effect of an inhibitor of HSC activation, retinol-binding protein-albumin domain III fusion protein (R-III), on protumorigenic functions of HSCs. Although conditioned medium collected from activated HSCs enhanced the migration, invasion, and proliferation of the hepatocellular carcinoma cell line Hepa-1c1c7, this effect was not observed in Hepa-1c1c7 cells treated with conditioned medium from R-III-exposed HSCs. In a subcutaneous tumor model, larger tumors with increased vascular density were formed in mice transplanted with Hepa-1c1c7+HSC than in mice transplanted with Hepa-1c1c7 cells alone. Intriguingly, when Hepa-1c1c7+HSC-transplanted mice were injected intravenously with R-III, a reduction in vascular density and extended tumor necrosis were observed. In an orthotopic tumor model, co-transplantation of HSCs enhanced tumor growth, angiogenesis, and regional metastasis accompanied by increased peritumoral lymphatic vessel density, which was abolished by R-III. In vitro study showed that R-III treatment affected the synthesis of pro-angiogenic and anti-angiogenic factors in activated HSCs, which might be the potential mechanism underlying the R-III effect. These findings suggest that the inhibition of HSC activation abrogates HSC-induced tumor angiogenesis and growth, which represents an attractive therapeutic strategy.

Therapeutic Potential of Human Nasal Inferior Turbinate-Derived Stem Cells: Microarray Analysis of Multilineage Differentiation.

INTRODUCTION: Human nasal inferior turbinate-derived stem cells (hNTSCs) are attractive sources of adult stem cells for medical application because they can be easily obtained and cultivated with a highly proliferative capacity. The ability of hNTSCs to differentiate into chondrocytes, osteocytes, and neural cells makes them potential replacement therapeutic candidates in intractable disease. Nevertheless, detailed expression pattern of genes associated with trilineage differentiation (osteogenesis, chondrogenesis, and neurogenesis) in hNTSCs has not been revealed yet. METHODS: In this study, we aimed to evaluate gene expression patterns of various transcription factors and marker genes associated with a particular lineage (osteogenesis, chondrogenesis, and neurogenesis) of differentiation of hNTSCs by DNA microarrays. RESULTS: In microarrays, 36 transcripts such as E2F transcription factor 1, activating transcription factor 5, and AKR1B10 were upregulated and 36 transcripts such as CA9, PPFIA4, HAS2, and COL4A4 were downregulated in osteogenically differentiated hNTSCs. In chondrogenically differentiated hNTSCs, 3 transcripts (NUDT14, CPA4, and heparin-binding epidermal growth factor-like growth factor) were upregulated and 82 transcripts such as PTGS1, CLEC2D, and TET1 were downregulated. In neurally differentiated hNTSCs, 61 transcripts such as insulin-like growth factor-binding protein-1, nerve growth factor receptor, FGF1, OLFML1, and EPGN were upregulated and 98 transcripts such as ACAN, RUNX2, and C21orf96 were downregulated. In gene ontology (GO) analysis, cell signal-related GO terms were highly expressed. By contrast, catalysis GO terms and GO terms related to oxidoreductase were overrepresented in chondrogenically differentiated hNTSCs and osteogenically differentiated hNTSCs, respectively. CONCLUSION: Considering overall results, hNTSCs-specific genetic information may promote further studies on intracellular mechanisms defining key features of these cells.

Defining the endoscopic ultrasound features of chronic pancreatitis in Asians: a multicenter validation study.

BACKGROUND: Although endoscopic ultrasound (EUS) features and criteria have been described in chronic pancreatitis, challenges remain with interoperator variability and ease of adoption. The aim of this study was to define and validate the EUS features of chronic pancreatitis in a multicenter prospective study in Asia. METHOD: The study was divided into two parts: the first part was conducted to derive the EUS features of chronic pancreatitis with adequate interoperator agreement; the second was to prospectively evaluate these features in a multicenter cross-sectional study and determine the optimal combination of features for the diagnosis of chronic pancreatitis. Prospectively enrolled cases had standard internationally validated radiologic or histologic features of chronic pancreatitis, and controls were patients without chronic pancreatitis who underwent EUS examination. RESULTS: The top six EUS features that had good interobserver agreement (mean kappa 0.73, range 0.60 - 0.90) were selected to be further evaluated in part II of the study. These included: hyperechoic foci with shadowing, lobularity with honeycombing, cysts, dilated main pancreatic duct, dilated side branches, and calculi in the main pancreatic duct. A total of 284 subjects (132 cases, 152 controls) were enrolled from 12 centers in Asia. All six features had high accuracy ranging from 63.3 % to 89.1 %. Two or more of these six EUS features accurately defined chronic pancreatitis (sensitivity 94.7 %, specificity 98.0 %), with an area under the receiver operating curve of 0.986. CONCLUSION: This multicenter Asian study characterized and defined the EUS features of chronic pancreatitis. This provides a useful tool in clinical practice and further research in pancreatic cancer surveillance.

Urinary chemokine C-X-C motif ligand 16 and endostatin as predictors of tubulointerstitial fibrosis in patients with advanced diabetic kidney disease.

BACKGROUND: Interstitial fibrosis and tubular atrophy (IFTA) is a well-recognized risk factor for poor renal outcome in patients with diabetic kidney disease (DKD). However, a noninvasive biomarker for IFTA is currently lacking. The purpose of this study was to identify urinary markers of IFTA and to determine their clinical relevance as predictors of renal prognosis. METHODS: Seventy patients with biopsy-proven isolated DKD were enrolled in this study. We measured multiple urinary inflammatory cytokines and chemokines by multiplex enzyme-linked immunosorbent assay in these patients and evaluated their association with various pathologic features and renal outcomes. RESULTS: Patients enrolled in this study exhibited advanced DKD at the time of renal biopsy, characterized by moderate to severe renal dysfunction [mean estimated glomerular filtration rate (eGFR) 36.1 mL/min/1.73 m2] and heavy proteinuria (mean urinary protein:creatinine ratio 7.8 g/g creatinine). Clinicopathologic analysis revealed that higher IFTA scores were associated with worse baseline eGFR (P < 0.001) and poor renal outcome (P = 0.002), whereas glomerular injury scores were not. Among measured urinary inflammatory markers, C-X-C motif ligand 16 (CXCL16) and endostatin showed strong correlations with IFTA scores (P = 0.001 and P < 0.001, respectively), and patients with higher levels of urinary CXCL16 and/or endostatin experienced significantly rapid renal progression compared with other patients (P < 0.001). Finally, increased urinary CXCL16 and endostatin were independent risk factors for poor renal outcome after multivariate adjustments (95% confidence interval 1.070-3.455, P = 0.029). CONCLUSIONS: Urinary CXCL16 and endostatin could reflect the degree of IFTA and serve as biomarkers of renal outcome in patients with advanced DKD.

In vivo Oxygen Condition of Human Nasal Inferior Turbinate-Derived Stem Cells in Human Nose.

BACKGROUND AND OBJECTIVE: Human nasal inferior turbinate-derived stem cells (hNTSCs) have been considered as a potent and useful source for regenerative medicine. To most effectively mimic the native environment of inferior turbinate could be very effective to hNTSCs biology. Thus, the purpose of this study was to evaluate partial pressure of oxygen (ppO2) and temperature in inferior turbinate. METHODS: Ten patients were enrolled who underwent endoscopic endonasal transsphenoidal skull base tumor surgery between January 2014 and December 2015. The commercially available OxyLab pO2 monitor gauges the ppO2 and temperature using a fluorescence quenching technique. Also, hNTSCs were isolated from 10 patients and cultivated under hypercapnic condition (5, 10, and 15%) to mimic hypoxic intranasal conditions. RESULTS: The measured oxygen concentration in submucosa tissue was higher than that at the surface of the inferior turbinate and the temperature in submucosa tissue was higher than the value at the surface of inferior turbinate. The patterns of proliferation were significantly different according to hypercapnic cultivation conditions and there were statistically significant decreased proliferation rates after the exposure of higher CO2 over a period of 5 days. CONCLUSIONS: Intranasal turbinate tissue showed the hypoxia state in concordance with the result of the other tissues or organs. However, indirectly induced hypoxia influenced the influence on the hNTSCs proliferation negatively. Further study is needed to mimic the real hypoxic state, but our results could be used to optimize the culture environment of hNTSCs, thereby producing the stem cells for regenerative therapies.

Nanoscale imaging of rat atrial myocytes by scanning ion conductance microscopy reveals heterogeneity of T-tubule openings and ultrastructure of the cell membrane.

In contrast to ventricular myocytes, the structural and functional importance of atrial transverse tubules (T-tubules) is not fully understood. Therefore, we investigated the ultrastructure of T-tubules of living rat atrial myocytes in comparison with ventricular myocytes. Nanoscale cell surface imaging by scanning ion conductance microscopy (SICM) was accompanied by confocal imaging of intracellular T-tubule network, and the effect of removal of T-tubules on atrial excitation-contraction coupling (EC-coupling) was observed. By SICM imaging, we classified atrial cell surface into 4 subtypes. About 38% of atrial myocytes had smooth cell surface with no clear T-tubule openings and intracellular T-tubules (smooth-type). In 33% of cells, we found a novel membrane nanostructure running in the direction of cell length and named it 'longitudinal fissures' (LFs-type). Interestingly, T-tubule openings were often found inside the LFs. About 17% of atrial cells resembled ventricular myocytes, but they had smaller T-tubule openings and a lower Z-groove ratio than the ventricle (ventricular-type). The remaining 12% of cells showed a mixed structure of each subtype (mixed-type). The LFs-, ventricular-, and mixed-type had an appreciable amount of reticular form of intracellular T-tubules. Formamide-induced detubulation effectively removed atrial T-tubules, which was confirmed by both confocal images and decreased cell capacitance. However, the LFs remained intact after detubulation. Detubulation reduced action potential duration and L-type Ca2+channel (LTCC) density, and prolonged relaxation time of the myocytes. Taken together, we observed heterogeneity of rat atrial T-tubules and membranous ultrastructure, and the alteration of atrial EC-coupling by disruption of T-tubules.

Metformin overcomes resistance to cisplatin in triple-negative breast cancer (TNBC) cells by targeting RAD51.

BACKGROUND: Chemotherapy is a standard therapeutic regimen to treat triple-negative breast cancer (TNBC); however, chemotherapy alone does not result in significant improvement and often leads to drug resistance in patients. In contrast, combination therapy has proven to be an effective strategy for TNBC treatment. Whether metformin enhances the anticancer effects of cisplatin and prevents cisplatin resistance in TNBC cells has not been reported. METHODS: Cell viability, wounding healing, and invasion assays were performed on Hs 578T and MDA-MB-231 human TNBC cell lines to demonstrate the anticancer effects of combined cisplatin and metformin treatment compared to treatment with cisplatin alone. Western blotting and immunofluorescence were used to determine the expression of RAD51 and gamma-H2AX. In an in vivo 4T1 murine breast cancer model, a synergistic anticancer effect of metformin and cisplatin was observed. RESULTS: Cisplatin combined with metformin decreased cell viability and metastatic effect more than cisplatin alone. Metformin suppressed cisplatin-mediated RAD51 upregulation by decreasing RAD51 protein stability and increasing its ubiquitination. In contrast, cisplatin increased RAD51 expression in an ERK-dependent manner. In addition, metformin also increased cisplatin-induced phosphorylation of γ-H2AX. Overexpression of RAD51 blocked the metformin-induced inhibition of cell migration and invasion, while RAD51 knockdown enhanced cisplatin activity. Moreover, the combination of metformin and cisplatin exhibited a synergistic anticancer effect in an orthotopic murine model of 4T1 breast cancer in vivo. CONCLUSIONS: Metformin enhances anticancer effect of cisplatin by downregulating RAD51 expression, which represents a novel therapeutic target in TNBC management.

LIF, a Novel Myokine, Protects Against Amyloid-Beta-Induced Neurotoxicity via Akt-Mediated Autophagy Signaling in Hippocampal Cells.

BACKGROUND: Leukemia inhibitory factor, a novel myokine, is known to be associated with neural function, but the underlying molecular mechanism remains unclear. METHODS: HT-22 mouse hippocampal cells, primary hippocampal cells, and Drosophila Alzheimer's disease model were used to determine the effect of leukemia inhibitory factor on neurons. Immunoblot analysis and immunofluorescence method were used to analyze biological mechanism. RESULTS: Leukemia inhibitory factor increased Akt phosphorylation in a phosphoinositide-3-kinase-dependent manner in hippocampal cells. Leukemia inhibitory factor also increased the phosphorylation of the mammalian target of rapamycin and the downstream S6K. Leukemia inhibitory factor stimulated the phosphorylation of signal transducer and activator of transcription via extracellular signal-regulated kinases. Leukemia inhibitory factor increased c-fos expression through both Akt and extracellular signal-regulated kinases. Leukemia inhibitory factor blocked amyloid ß-induced neural viability suppression and inhibited amyloid ß-induced glucose uptake impairment through the block of amyloid ß-mediated insulin receptor downregulation. Leukemia inhibitory factor blocked amyloid ß-mediated induction of the autophagy marker, microtubule-associated protein 1A/1B-light chain 3. Additionally, in primary prepared hippocampal cells, leukemia inhibitory factor stimulated Akt and extracellular signal-regulated kinase, demonstrating that leukemia inhibitory factor has physiological relevance in vivo. Suppression of the autophagy marker, light chain 3II, by leukemia inhibitory factor was observed in a Drosophila model of Alzheimer's disease. CONCLUSIONS: These results demonstrate that leukemia inhibitory factor protects against amyloid ß-induced neurotoxicity via Akt/extracellular signal-regulated kinase-mediated c-fos induction, and thus suggest that leukemia inhibitory factor is a potential drug for Alzheimer's disease.

TrpA1 Regulates Defecation of Food-Borne Pathogens under the Control of the Duox Pathway.

Pathogen expulsion from the gut is an important defense strategy against infection, but little is known about how interaction between the intestinal microbiome and host immunity modulates defecation. In Drosophila melanogaster, dual oxidase (Duox) kills pathogenic microbes by generating the microbicidal reactive oxygen species (ROS), hypochlorous acid (HOCl) in response to bacterially excreted uracil. The physiological function of enzymatically generated HOCl in the gut is, however, unknown aside from its anti-microbial activity. Drosophila TRPA1 is an evolutionarily conserved receptor for reactive chemicals like HOCl, but a role for this molecule in mediating responses to gut microbial content has not been described. Here we identify a molecular mechanism through which bacteria-produced uracil facilitates pathogen-clearing defecation. Ingestion of uracil increases defecation frequency, requiring the Duox pathway and TrpA1. The TrpA1(A) transcript spliced with exon10b (TrpA1(A)10b) that is present in a subset of midgut enteroendocrine cells (EECs) is critical for uracil-dependent defecation. TRPA1(A)10b heterologously expressed in Xenopus oocytes is an excellent HOCl receptor characterized with elevated sensitivity and fast activation kinetics of macroscopic HOCl-evoked currents compared to those of the alternative TRPA1(A)10a isoform. Consistent with TrpA1's role in defecation, uracil-excreting Erwinia carotovora showed higher persistence in TrpA1-deficient guts. Taken together, our results propose that the uracil/Duox pathway promotes bacteria expulsion from the gut through the HOCl-sensitive receptor, TRPA1(A)10b, thereby minimizing the chances that bacteria adapt to survive host defense systems.

Deficiency of Anoctamin 5/TMEM16E causes nuclear positioning defect and impairs Ca2+ signaling of differentiated C2C12 myotubes.

Anoctamin 5 (ANO5)/TMEM16E belongs to a member of the ANO/TMEM16 family member of anion channels. However, it is a matter of debate whether ANO5 functions as a genuine plasma membrane chloride channel. It has been recognized that mutations in the ANO5 gene cause many skeletal muscle diseases such as limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi muscular dystrophy type 3 (MMD3) in human. However, the molecular mechanisms of the skeletal myopathies caused by ANO5 defects are poorly understood. To understand the role of ANO5 in skeletal muscle development and function, we silenced the ANO5 gene in C2C12 myoblasts and evaluated whether it impairs myogenesis and myotube function. ANO5 knockdown (ANO5-KD) by shRNA resulted in clustered or aggregated nuclei at the body of myotubes without affecting differentiation or myotube formation. Nuclear positioning defect of ANO5-KD myotubes was accompanied with reduced expression of Kif5b protein, a kinesin-related motor protein that controls nuclear transport during myogenesis. ANO5-KD impaired depolarization-induced [Ca2+]i transient and reduced sarcoplasmic reticulum (SR) Ca2+ storage. ANO5-KD resulted in reduced protein expression of the dihydropyridine receptor (DHPR) and SR Ca2+-ATPase subtype 1. In addition, ANO5-KD compromised co-localization between DHPR and ryanodine receptor subtype 1. It is concluded that ANO5-KD causes nuclear positioning defect by reduction of Kif5b expression, and compromises Ca2+ signaling by downregulating the expression of DHPR and SERCA proteins.

Hinokitiol induces DNA demethylation via DNMT1 and UHRF1 inhibition in colon cancer cells.

BACKGROUND: DNA hypermethylation is a key epigenetic mechanism for the silencing of many genes in cancer. Hinokitiol, a tropolone-related natural compound, is known to induce apoptosis and cell cycle arrest and has anti-inflammatory and anti-tumor activities. However, the relationship between hinokitiol and DNA methylation is not clear. The aim of our study was to explore whether hinokitiol has an inhibitory ability on the DNA methylation in colon cancer cells. RESULTS: MTT data showed that hinokitiol had higher sensitivity in colon cancer cells, HCT-116 and SW480, than in normal colon cells, CCD18Co. Hinokitiol reduced DNA methyltransferase 1 (DNMT1) and ubiquitin-like plant homeodomain and RING finger domain 1 (UHRF1) expression in HCT-116 cells. In addition, the expression of ten-eleven translocation protein 1 (TET1), a known DNA demethylation initiator, was increased by hinokitiol treatment. ELISA and FACS data showed that hinokitiol increased the 5-hydroxymethylcytosine (5hmC) level in the both colon cancer cells, but 5-methylcytosine (5mC) level was not changed. Furthermore, hinokitiol significantly restored mRNA expression of O6-methylguanine DNA methyltransferase (MGMT), carbohydrate sulfotransferase 10 (CHST10), and B-cell translocation gene 4 (BTG4) concomitant with reduction of methylation status in HCT-116 cells. CONCLUSIONS: These results indicate that hinokitiol may exert DNA demethylation by inhibiting the expression of DNMT1 and UHRF1 in colon cancer cells.

Endogenous Ligand for GPR120, Docosahexaenoic Acid, Exerts Benign Metabolic Effects on the Skeletal Muscles via AMP-activated Protein Kinase Pathway.

Docosahexaenoic acid (DHA) is an endogenous ligand of G protein-coupled receptor 120 (GPR120). However, the mechanisms underlying DHA action are poorly understood. In this study, DHA stimulated glucose uptake in the skeletal muscles in an AMP-activated protein kinase (AMPK)-dependent manner. GPR120-mediated increase in intracellular Ca(2+) was critical for DHA-mediated AMPK phosphorylation and glucose uptake. In addition, DHA stimulated GLUT4 translocation AMPK-dependently. Inhibition of AMPK and Ca(2+)/calmodulin-dependent protein kinase kinase blocked DHA-induced glucose uptake. DHA and GW9508, a GPR120 agonist, increased GPR120 expression. DHA-mediated glucose uptake was not observed in GPR120 knockdown conditions. DHA increased AMPK phosphorylation, glucose uptake, and intracellular Ca(2+) concentration in primary cultured myoblasts. Taken together, these results indicated that the beneficial metabolic role of DHA was attributed to its ability to regulate glucose via the GPR120-mediated AMPK pathway in the skeletal muscles.

Dehydrozingerone exerts beneficial metabolic effects in high-fat diet-induced obese mice via AMPK activation in skeletal muscle.

Dehydrozingerone (DHZ) exerts beneficial effects on human health; however, its mechanism of action remains unclear. Here, we found that DHZ suppressed high-fat diet-induced weight gain, lipid accumulation and hyperglycaemia in C57BL/6 mice and increased AMP-activated protein kinase (AMPK) phosphorylation and stimulated glucose uptake in C2C12 skeletal muscle cells. DHZ activated p38 mitogen-activated protein kinase (MAPK) signalling in an AMPK-dependent manner. Inhibiting AMPK or p38 MAPK blocked DHZ-induced glucose uptake. DHZ increased GLUT4 (major transporter for glucose uptake) expression in skeletal muscle. Glucose clearance and insulin-induced glucose uptake increased in DHZ-fed animals, suggesting that DHZ increases systemic insulin sensitivity in vivo. Thus, the beneficial health effects of DHZ could possibly be explained by its ability to activate the AMPK pathway in skeletal muscle.

[6]-Gingerol Affects Glucose Metabolism by Dual Regulation via the AMPKα2-Mediated AS160-Rab5 Pathway and AMPK-Mediated Insulin Sensitizing Effects.

[6]-Gingerol has been used to control diabetes and dyslipidemia; however, its metabolic role is poorly understood. In this study, [6]-gingerol increased adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation in mouse skeletal muscle C2C12 cells. Stimulation of glucose uptake by [6]-gingerol was dependent on AMPKα2. Moreover, both Inhibition and knockdown of AMPKα2 blocked [6]-gingerol-induced glucose uptake. [6]-Gingerol significantly decreased the activity of protein phosphatase 2A (PP2A). Inhibition of PP2A activity with okadaic acid enhanced the phosphorylation of AMPKα2. Moreover, the interaction between AMPKα2 and PP2A was increased by [6]-gingerol, suggesting that PP2A mediates the effect of [6]-gingerol on AMPK phosphorylation. In addition, [6]-gingerol increased the phosphorylation of Akt-substrate 160 (AS160), which is a Rab GTPase-activating protein. Inhibition of AMPKα2 blocked [6]-gingerol-induced AS160 phosphorylation. [6]-gingerol increased the Rab5, and AMPKα2 knockdown blocked [6]-gingerol-induced expression of Rab5, indicating AMPK play as an upstream of Rab5. It also increased glucose transporter 4 (GLUT4) mRNA and protein expression and stimulated GLUT4 translocation. Furthermore, insulin-mediated glucose uptake and Akt phosphorylation were further potentiated by [6]-gingerol treatment. This potentiation was not observed in the presence of AMPK inhibitor compound C. In summary, our results suggest that [6]-gingerol plays an important role in glucose metabolism via the AMPKα2-mediated AS160-Rab5 pathway and through potentiation of insulin-mediated glucose regulation.

E3 ubiquitin ligase, WWP1, interacts with AMPKα2 and down-regulates its expression in skeletal muscle C2C12 cells.

It is known that the activity of AMP-activated protein kinase (AMPKα2) was depressed under high glucose conditions. However, whether protein expression of AMPKα2 is also down-regulated or not remains unclear. In this study, we showed that the expression of AMPKα2 was down-regulated in cells cultured under high glucose conditions. Treatment of proteasome inhibitor, MG132, blocked high glucose-induced AMPKα2 down-regulation. Endogenous AMPKα2 ubiquitination was detected by immunoprecipitation of AMPKα2 followed by immunoblotting detection of ubiquitin. The yeast-two hybrid (YTH) approach identified WWP1, an E3 ubiquitin ligase, as the AMPKα2-interacting protein in skeletal muscle cells. Interaction between AMPKα2 and WWP1 was validated by co-immunoprecipitation. Knockdown of WWP1 blocked high glucose-induced AMPKα2 down-regulation. The overexpression of WWP1 down-regulated AMPKα2. In addition, the expression of WWP1 is increased under high glucose culture conditions in both mRNA and protein levels. The level of AMPKα2 was down-regulated in the quadriceps muscle of diabetic animal model db/db mice. Expression of WWP1 blocked metformin-induced glucose uptake. Taken together, our results demonstrated that WWP1 down-regulated AMPKα2 under high glucose culture conditions via the ubiquitin-proteasome pathway.

Alcohol induces cell proliferation via hypermethylation of ADHFE1 in colorectal cancer cells.

BACKGROUND: The hypermethylation of Alcohol dehydrogenase iron containing 1 (ADHFE1) was recently reported to be associated with colorectal cancer (CRC) differentiation. However, the effect of alcohol on ADHFE1 hypermethylation in CRC is still unclear. METHODS: The methylation status and expression levels of ADHFE1 were investigated in primary tumor tissues and adjacent normal tissues of 73 patients with CRC, one normal colon cell line, and 4 CRC cell lines (HT-29, SW480, DLD-1, and LoVo) by quantitative methylation-specific polymerase chain reaction (QMSP) and real-time reverse transcription polymerase chain reaction (real time PCR), respectively. The effect of alcohol on the methylation status of ADHFE1 was analyzed in HT-29, SW480, DLD-1, and CCD18Co cells using QMSP, real-time PCR, immunoblot, and cell proliferation assay. RESULTS: ADHFE1 was hypermethylated in 69 of 73 CRC tissues (95%) compared to adjacent normal tissues (p<0.05). The mRNA expression of ADHFE1 was significantly reduced in CRC compared to adjacent normal tissues (p<0.05) and its expression was decreased in the alcohol consumption group (p<0.05). ADHFE1 was hypermethylated and its expression was decreased in 4 CRC cell lines compared with normal colon cell line. Alcohol induced hypermethylation of ADHFE1, decreased its expression, and stimulated cell proliferation of HT-29, SW480, and DLD-1cells. CONCLUSION: These results demonstrate that the promoter hypermethylation of ADHFE1 is frequently present in CRC and alcohol induces methylation-mediated down expression of ADHFE1 and proliferation of CRC cells.

Electrochemical flow-based solution-solid growth of the Cu2O nanorod array: potential application to lithium ion batteries.

The catalyzed solution-liquid-solid (SLS) growth has been well developed to synthesize semiconductor nanowires with controlled diameters. The SLS growth occurs in the longitudinal direction of nanowires, due to the directional anisotropy driven by the metal catalysts where chemical precursors are introduced. In the present study, we report a selective, template-free, and environmentally-friendly electrochemical flow-based solution-solid (electrochemical flow-SS) growth of the Cu2O nanorod array. The anisotropy for directional growth without any catalysts is generated by the electrical field in a flowing electrolyte of ultra-dilute CuSO4. The filamentary anisotropy originates from electric field enhancement on pyramidal nanocrystals in the electrolyte of low ionic conductivity (13 µS cm(-1)). The Cu2O and Cu nanorods are able to be selectively synthesized by controlling the electrolyte pH and oxygen dissolution into the electrolyte. The synthesized Cu2O nanorod array shows excellent electrochemical properties as an anode material for lithium-ion batteries; the specific capacities increase from 323 to 1206 mA h g(-1) during 500 cycles. The capacity enhancement is due to the phase transformation from Cu2O to CuO, nano-restructuring of nanorods into fragmented nanoparticles, and the progressive generation of an electroactive polymeric gel-like layer on the surface of the nanoparticles. The electrochemical flow-SS growth of Cu2O nanorods is expected to contribute to further development of other functional nanorods.

Rhus verniciflua extract modulates survival of MCF-7 breast cancer cells through the modulation of AMPK-pathway.

Rhus verniciflua STOKES (RVS) is used as an anti-cancer agent in traditional herbal medicine. However, the underlying molecular mechanism of its action is poorly understood. Here, we elucidated the mechanism of the anti-cancer mechanism of RVS in MCF-7 human breast cancer cells. We found that RVS increased the phosphorylation of AMP-activated protein kinase (AMPK) and downstream acetyl-CoA carboxylase (ACC) and suppressed cell viability in an AMPK-dependent fashion. RVS also induced an increase in reactive oxygen species (ROS) levels. RVS-induced AMPK phosphorylation was not observed in the presence of N-acetyl-cysteine (NAC), which indicated that ROS is associated with RVS-induced AMPK phosphorylation. In addition, fluorescent staining (Annexin V/propidium iodide) revealed that RVS increased the expression of Annexin V, which indicates that RVS leads to cancer-induced apoptosis. Moreover, RVS increased the phosphorylation of p53 and the expression of Bax. The inhibition of AMPK blocked RVS-induced p53 phosphorylation and Bax expression, which suggests that AMPK is involved in RVS-induced cancer apoptosis. Taken together, these results demonstrate that RVS has anti-tumor effects on MCF-7 cells through an AMPK-signaling pathway.

Metformin regulates glucose transporter 4 (GLUT4) translocation through AMP-activated protein kinase (AMPK)-mediated Cbl/CAP signaling in 3T3-L1 preadipocyte cells.

Metformin is a leading oral anti-diabetes mellitus medication and is known to stimulate GLUT4 translocation. However, the mechanism by which metformin acts is still largely unknown. Here, we showed that short time treatment with metformin rapidly increased phosphorylation of Cbl in an AMP-activated protein kinase (AMPK)-dependent fashion in 3T3-L1 preadipocytes. Metformin also increased phosphorylation of Src in an AMPK-dependent manner. Src inhibition blocked metformin-mediated Cbl phosphorylation, suggesting that metformin stimulates AMPK-Src-Cbl axis pathway. In addition, long term treatment with metformin stimulated the expression of Cbl-associated protein (CAP) mRNA and protein. Long term treatment with metformin stimulated phosphorylation of c-Jun N-terminal kinase (JNK) and its downstream molecule c-Jun, which is a critical molecule for CAP transcription. Knockdown of AMPK and JNK blocked metformin-induced expression of CAP, implying that metformin stimulates AMPK-JNK-CAP axis pathway. Moreover, AMPK knockdown attenuated metformin-induced Cbl/CAP multicomplex formation, which is critical for GLUT4 translocation. A colorimetric absorbance assay demonstrated that metformin-induced translocation of GLUT4 was suppressed in CAP or Cbl knockdown cells. Furthermore, the promoter activity of CAP was increased by metformin in an AMPK/JNK-dependent fashion. In summary, these results demonstrate that metformin modulates GLUT4 translocation by regulating Cbl and CAP signals via AMPK.