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1.  Anti-tumor effect of 5-aza-2'-deoxycytidine by inhibiting telomerase activity in hepatocellular carcinoma cells 
AIM: To investigate the effect of the demethylating reagent 5-aza-2’-deoxycitidine (DAC) on telomerase activity in hepatocellular carcinoma (HCC) cell lines, SMMC-7721 and HepG2.
METHODS: The related gene expression in cell lines was examined by real-time reverse transcription-polymerase chain reaction and Western blotting analysis. The telomerase activity was examined by telomeric repeat amplification protocol-enzyme-linked immunosorbent assay and DNA methylation was determined by methylation-specific polymerase chain reaction.
RESULTS: The telomerase activity was significantly reduced in both cell lines treated with DAC, accompanied by downregulation of telomerase reverse transcriptase (hTERT). We also observed the effect of DAC on the methylation status of hTERT promoter and the expression of regulatory genes, such as c-myc, p15, p16, p21, E2F1, and WT1. The methylation status of hTERT promoter could be reversed in SMMC-7721 by DAC, but not in HepG2 cells. However, p16 expression could be reactivated by demethylation of its promoter, and c-Myc expression was repressed in both cell lines. Moreover, DAC could enhance the sensitivity to the chemotherapeutic agents, such as cisplatin, by induction of apoptosis of HCC cells.
CONCLUSION: The DAC exerts its anti-tumor effects in HCC cells by inhibiting the telomerase activity.
PMCID: PMC3353367  PMID: 22654424
5-aza-2’-deoxycitidine; Telomerase; Hepatocellular carcinoma; DNA methylation
2.  Cardiac Spliced BIN1 Folds T-tubule Membrane, Controlling Ion Flux and Limiting Arrhythmia 
Nature medicine  2014;20(6):624-632.
Cardiomyocyte T-tubules are important for regulating ionic flux. Bridging Integrator 1 (BIN1) is a T-tubule protein associated with calcium channel trafficking that is down-regulated in failing hearts. Here we find that cardiac T-tubules normally contain dense protective inner membrane folds that are formed by a cardiac spliced isoform of BIN1. In mice with cardiac Bin1 deletion, T-tubule folding is decreased which does not change overall cardiomyocyte morphology, but frees diffusion of local extracellular calcium and potassium ions, prolonging action potential duration, and increasing susceptibility to ventricular arrhythmias. We also find that T-tubule inner folds are rescued only by the BIN1 isoform BIN1+13+17, which promotes N-WASP dependent actin polymerization to stabilize T-tubule membrane at cardiac Z-discs. In conclusion, BIN1+13+17 recruits actin to fold T-tubule membrane, creating a fuzzy space that protectively restricts ionic flux. When BIN1+13+17 is decreased, as occurs in acquired cardiomyopathy, T-tubule morphology is altered and arrhythmias can result.
PMCID: PMC4048325  PMID: 24836577
3.  Altered regional homogeneity in experimentally induced low back pain: a resting-state fMRI study 
Functional imaging studies have indicated that patients with low back pain can have significant reductions in cerebral cortex grey matter. However, the mechanisms governing the nociceptive pathways in the human brain are unclear. The aim of this study was to use functional magnetic resonance imaging (fMRI) and regional homogeneity (ReHo) to investigate changes in resting-state brain activity in subjects that experienced experimentally induced low back pain.
Healthy subjects (n = 15) underwent fMRI (3.0 T) at baseline and during painful stimulation (intramuscular injection of 3% hypertonic saline).
Compared to the scans conducted at baseline, scans conducted during experimentally induced low back pain showed increased ReHo on the right side in the medial prefrontal cortex, precuneus, insula, parahippocampal gyrus and cerebellum (posterior lobe), but decreased ReHo in the primary somatosensory cortex, anterior cingulate cortex and parahippocampal gyrus on the left side. The right inferior parietal lobule also showed a decreased ReHo (P < 0.05, cluster threshold ≥10).
These findings suggest that abnormally spontaneous resting-state activity in some brain regions may be associated with pain processing. These changes in neural activity may contribute to the recognition, execution, memory and emotional processing of acute low back pain.
PMCID: PMC4237877  PMID: 25080831
Low back pain; Functional magnetic resonance imaging; Resting state; Regional homogeneity
4.  Multilayered Regulation of Cardiac Ion Channels 
Biochimica et biophysica acta  2012;1833(4):876-885.
Essential to beat-to-beat heart function is the ability for cardiomyocytes to propagate electrical excitation and generate contractile force. Both excitation and contractility depend on specific ventricular ion channels, which include the L-type calcium channel (LTCC) and the connexin 43 (Cx43) gap junction. Each of these two channels is localized to a distinct subdomain of the cardiomyocyte plasma membrane. In this review, we focus on regulatory mechanisms that govern the lifecycles of LTCC and Cx43, from their biogenesis in the nucleus to directed delivery to T-tubules and intercalated discs, respectively. We discuss recent findings on how alternative promoter usage, tissue-specific transcription, and alternative splicing determine precise ion channel expression levels within a cardiomyocyte. Moreover, recent work on microtubule and actin-dependent trafficking for Cx43 and LTCC are introduced. Lastly, we discuss how human cardiac disease phenotypes can be attributed to defects in distinct mechanisms of channel regulation at the level of gene expression and channel trafficking.
PMCID: PMC3568256  PMID: 23103513
connexin 43; calcium channel; directed targeting; cytoskeleton; gene expression
5.  Actin Cytoskeleton Rest Stops Regulate Anterograde Traffic of Connexin 43 Vesicles to the Plasma Membrane 
Circulation research  2012;110(7):978-989.
The intracellular trafficking of connexin 43 (Cx43) hemichannels presents opportunities to regulate cardiomyocyte gap junction coupling. Although it is known that Cx43 hemichannels are transported along microtubules to the plasma membrane, the role of actin in Cx43 forward trafficking is unknown.
We explored whether the actin cytoskeleton is involved in Cx43 forward trafficking.
Methods and Results
High-resolution imaging reveals that Cx43 vesicles colocalize with nonsarcomeric actin in adult cardiomyocytes. Live-cell fluorescence imaging reveals Cx43 vesicles as stationary or traveling slowly (average speed 0.09 μm/s) when associated with actin. At any time, the majority (81.7%) of vesicles travel at subkinesin rates, suggesting that actin is important for Cx43 transport. Using Cx43 containing a hemagglutinin tag in the second extracellular loop, we developed an assay to detect transport of de novo Cx43 hemichannels to the plasma membrane after release from Brefeldin A-induced endoplasmic reticulum/Golgi vesicular transport block. Latrunculin A (for specific interference of actin) was used as an intervention after reinitiation of vesicular transport. Disruption of actin inhibits delivery of Cx43 to the cell surface. Moreover, using the assay in primary cardiomyocytes, actin inhibition causes an 82% decrease (P<0.01) in de novo endogenous Cx43 delivery to cell–cell borders. In Langendorff-perfused mouse heart preparations, Cx43/β-actin complexing is disrupted during acute ischemia, and inhibition of actin polymerization is sufficient to reduce levels of Cx43 gap junctions at intercalated discs.
Actin is a necessary component of the cytoskeleton-based forward trafficking apparatus for Cx43. In cardiomyocytes, Cx43 vesicles spend a majority of their time pausing at nonsarcomeric actin rest stops when not undergoing microtubule-based transport to the plasma membrane. Deleterious effects on this interaction between Cx43 and the actin cytoskeleton during acute ischemia contribute to losses in Cx43 localization at intercalated discs.
PMCID: PMC3621031  PMID: 22328533
actin; connexin 43; cytoskeletal dynamics; gap junctions; trafficking
6.  Nutritional status deteriorates as the severity of diabetic foot ulcers increases and independently associates with prognosis 
The prognosis for diabetic foot ulcers (DFUs) remains poor. Nutritional status has not been identified as one of the factors affecting the outcome of DFUs. Therefore, indicators correlated with nutritional status and outcome were analyzed to investigate their relationship. A total of 192 hospitalized patients with Wagner grade 1–5 ulcers and 60 patients with Wagner grade 0 ulcers (all had type 2 diabetes) were assessed by the following: subjective global assessment (SGA), anthropometric measurements, biochemical indicators and physical examinations to evaluate nutritional status, severity of infection and complications. Patient outcome was recorded as healing of the ulcer and the patients were followed up for 6 months or until the wound was healed. The percentage of malnutrition was 62.0% in the DFU patients. The SGA was closely correlated with infection (r=0.64), outcome (r=0.37) and BMI (r=−0.36), all P<0.001. The risk of poor outcome increased with malnutrition [odds ratio (OR), 10.6, P<0.001]. The nutritional status of the DFU patients was independently correlated with the severity of infection and outcome (both P<0.001) and Wagner grades and nutritional status (SGA) were independent risk factors for patient outcome (both P<0.001). Nutritional status deteriorated as the severity of the DFU increased, and malnutrition was a predictor of poor prognosis.
PMCID: PMC3524099  PMID: 23251271
nutritional status; diabetic foot ulcers; complication; infection
7.  Inhibition of autophagy enhances anticancer effects of bevacizumab in hepatocarcinoma 
Angiogenesis inhibitors have long been considered desirable anticancer agents. However, it was found that many tumors could develop resistance to antiangiogenesis inhibitors. Antiangiogenic therapy results in metabolic stress. Autophagy is an important survival mechanism in cancer cells under metabolic stress; however, it remains unknown if autophagy contributes to antiangiogenesis resistance. In this study, we reported that bevacizumab treatment reduced the development of new blood vessels and inhibited cell growth in xenografts of hepatocellular carcinoma (HCC) tumors. Bevacizumab treatment also upregulated expression of the autophagy-related genes (Beclin1 and LC3) and increased autophagosome formation. Our in vitro studies demonstrated that autophagy inhibition significantly increased apoptosis of HCC cells during nutrient starvation or hypoxia. In addition, the combined treatment of an autophagy inhibitor and bevacizumab markedly inhibited the tumor growth of HCC xenografts, led to enhanced apoptosis, and impaired the proliferation of tumor cells compared with treatment with either drug alone. Furthermore, autophagy inhibition led to enhanced reactive oxygen species (ROS) generation in HCC cells exposed to nutrient starvation or hypoxia in vitro and increased DNA oxidative damage in vivo. Antioxidants reduced nutrient starvation or the hypoxia-induced cell death of HCC cells after autophagy inhibition. Our results suggest that autophagy modulates ROS generation and contributes to cell survival under metabolic stress. Therefore, autophagy inhibition may be a novel way of increasing the efficicacy of antiangiogenic agents in the treatment of HCC.
Electronic supplementary material
The online version of this article (doi:10.1007/s00109-012-0966-0) contains supplementary material, which is available to authorized users.
PMCID: PMC3611041  PMID: 23052483
Hepatocarcinoma; Antiangiogenesis; Autophagy; Metabolic stress; Apoptosis
8.  Toll-like receptor 4 signaling promotes epithelial-mesenchymal transition in human hepatocellular carcinoma induced by lipopolysaccharide 
BMC Medicine  2012;10:98.
The endotoxin level in the portal and peripheral veins of hepatocellular carcinoma (HCC) patients is higher and lipopolysaccharide (LPS), a cell wall constituent of gram-negative bacteria, has been reported to inhibit tumor growth. However, in this study, we found that LPS-induced toll-like receptor 4 (TLR4) signaling was involved in tumor invasion and survival, and the molecular mechanism was investigated,
Four HCC cell lines and a splenic vein metastasis of the nude mouse model were used to study the invasion ability of LPS-induced HCC cells and the epithelia-mesenchymal transition (EMT) in vitro and in vivo. A total of 106 clinical samples from HCC patients were used to evaluate TLR4 expression and analyze its association with clinicopathological characteristics
The in vitro and in vivo experiments demonstrated that LPS could significantly enhance the invasive potential and induce EMT in HCC cells with TLR4 dependent. Further studies showed that LPS could directly activate nuclear factor kappa B (NF-κB) signaling through TLR4 in HCC cells. Interestingly, blocking NF-κB signaling significantly inhibited transcription factor Snail expression and thereby inhibited EMT occurrence. High expression of TLR4 in HCC tissues was strongly associated with both poor cancer-free survival and overall survival in patients.
Our results indicate that TLR4 signaling is required for LPS-induced EMT, tumor cell invasion and metastasis, which provide molecular insights for LPS-related pathogenesis and a basis for developing new strategies against metastasis in HCC.
PMCID: PMC3482562  PMID: 22938142
Toll-like receptor 4; Epithelial-mesenchymal transition; Lipopolysaccharide; Human hepatocellular carcinoma
9.  CD133+CXCR4+ colon cancer cells exhibit metastatic potential and predict poor prognosis of patients 
BMC Medicine  2012;10:85.
Colorectal cancer (CRC), which frequently metastasizes to the liver, is one of the three leading causes of cancer-related deaths worldwide. Growing evidence suggests that a subset of cells exists among cancer stem cells. This distinct subpopulation is thought to contribute to liver metastasis; however, it has not been fully explored in CRC yet.
Flow cytometry analysis was performed to detect distinct subsets with CD133 and CXCR4 markers in human primary and metastatic CRC tissues. The 'stemness' and metastatic capacities of different subpopulations derived from the colon cancer cell line HCT116 were compared in vitro and in vivo. The roles of epithelial-mesenchymal transition (EMT) and stromal-cell derived factor-1 (SDF-1) in the metastatic process were also investigated. A survival curve was used to explore the correlation between the content of CD133+CXCR4+ cancer cells and patient survival.
In human specimens, the content of CD133+CXCR4+ cells was higher in liver metastases than in primary colorectal tumors. Clonogenic and tumorigenic cells were restricted to CD133+ cells in the HCT116 cell line, with CXCR4 expression having no impact on the 'stemness' properties. We found that CD133+CXCR4+ cancer cells had a high metastatic capacity in vitro and in vivo. Compared with CD133+CXCR4- cells, CD133+CXCR4+ cancer cells experienced EMT, which contributed partly to their metastatic phenotype. We then determined that SDF-1/CXCL12 treatment could further induce EMT in CD133+CXCR4+ cancer cells and enhance their invasive behavior, while this could not be observed in CD133+CXCR4- cancer cells. Blocking SDF-1/CXCR4 interaction with a CXCR4 antagonist, AMD3100 (1,10-[1,4-phenylenebis(methylene)]bis-1,4,8,11 -tetraazacyclotetradecane octahydrochloride), inhibited metastatic tumor growth in a mouse hepatic metastasis model. Finally, a high percentage of CD133+CXCR4+ cells in human primary CRC was associated with a reduced two-year survival rate.
Strategies targeting the SDF-1/CXCR4 interaction may have important clinical applications in the suppression of colon cancer metastasis. Further investigations on how high expression of CXCR4 and EMT occur in this identified cancer stem cell subset are warranted to provide insights into our understanding of tumor biology.
PMCID: PMC3424958  PMID: 22871210
colorectal cancer; cancer stem cell; CXCR4; epithelial-mesenchymal transition; liver metastasis
10.  Effect of Anti-Obesity Drug on Cardiovascular Risk Factors: A Systematic Review and Meta-Analysis of Randomized Controlled Trials 
PLoS ONE  2012;7(6):e39062.
Anti-obesity drugs are widely used to prevent the complications of obesity, however, the effects of anti-obesity drugs on cardiovascular risk factors are unclear at the present time. We carried out a comprehensively systematic review and meta-analysis to assess the effects of anti-obesity drugs on cardiovascular risk factors.
Methodology and Principal Findings
We systematically searched Medline, EmBase, the Cochrane Central Register of Controlled Trials, reference lists of articles and proceedings of major meetings for relevant literatures. We included randomized placebo-controlled trials that reported the effects of anti-obesity drugs on cardiovascular risk factors compared to placebo. Overall, orlistat produced a reduction of 2.39 kg (95%CI-3.34 to −1.45) for weight, a reduction of 0.27 mmol/L (95%CI: −0.36 to −0.17) for total cholesterol, a reduction of 0.21 mmol/L (95%CI: −0.30 to −0.12) for LDL, a reduction of 0.12 mmol/L (95%CI: −0.20 to −0.04) for fasting glucose, 1.85 mmHg reduction (95%CI: −3.30 to −0.40) for SBP, and a reduction of 1.49 mmHg (95%CI: −2.39 to −0.58) for DBP. Sibutramine only showed effects on weight loss and triglycerides reduction with statistical significances. Rimonabant was associated with statistically significant effects on weight loss, SBP reduction and DBP reduction. No other significantly different effects were identified between anti-obesity therapy and placebo.
We identified that anti-obesity therapy was associated with a decrease of weight regardless of the type of the drug. Orlistat and rimonabant could lead to an improvement on cardiovascular risk factors. However, Sibutramine may have a direct effect on cardiovascular risk factors.
PMCID: PMC3380040  PMID: 22745703
11.  Steroids in the Treatment of IgA Nephropathy to the Improvement of Renal Survival: A Systematic Review and Meta-Analysis 
PLoS ONE  2011;6(4):e18788.
Studies have shown that steroids can improve kidney survival and decrease the risk of proteinuria in patients with Immunoglobulin A nephropathy, but the overall benefit of steroids in the treatment of Immunoglobulin A nephropathy remains controversial. The aim of this study was to evaluate the benefits and risks of steroids for renal survival in adults with Immunoglobulin A nephropathy.
Methodology and Principal Findings
We searched the Cochrane Renal Group Specialized Register, Cochrane Controlled Trial Registry, MEDLINE and EMBASE databases. All eligible studies were measuring at least one of the following outcomes: end-stage renal failure, doubling of serum creatinine and urinary protein excretion. Fifteen relevant trials (n = 1542) that met our inclusion criteria were identified. In a pooled analysis, steroid therapy was associated with statistically significant reduction of the risk in end-stage renal failure (RR: 0.46, 95% CI: 0.27 to 0.79), doubling of serum creatinine (RR = 0.34, 95%CI = 0.15 to 0.77) and reduced urinary protein excretion (MD = −0.47g/day, 95%CI = −0.64 to −0.31).
We identified that steroid therapy was associated with a decrease of proteinuria and with a statistically significant reduction of the risk in end-stage renal failure. Moreover, subgroup analysis also suggested that long-term steroid therapy had a higher efficiency than standard and short term therapy.
PMCID: PMC3075273  PMID: 21533280
12.  Diaqua­(5-methyl-1H-pyrazole-3-carboxyl­ato)(4-nitro­benzoato)copper(II) 
In the title complex, [Cu(C7H4NO4)(C5H5N2O2)(H2O)2], the CuII ion is coordinated in a slightly distorted square-pyramidal enviroment. The basal plane is formed by an N atom and an O atom from a 5-methyl-1H-pyrazole-3-carboxyl­ate ligand and by two O atoms from two water ligands. The apical position is occupied by a carboxylate O atom from a 4-nitro­benzoate ligand. In the crystal structure, inter­molecular O—H⋯O and N—H⋯O hydrogen bonds link complex moleclues, forming extended chains parallel to the a axis.
PMCID: PMC2968408  PMID: 21581806
13.  3-Chloro-6-(3,5-dimethyl-1H-pyrazol-1-yl)picolinic acid–triphenyl­phosphine oxide (1/1) 
In the title 1:1 adduct, C11H10ClN3O2·C18H15OP, the dihedral angle between the pyridine and pyrazole rings is 10.3 (2)°. The two components of the adduct are linked by an O—H⋯O hydrogen bond.
PMCID: PMC2962060  PMID: 21203144
14.  (3,6-Dichloro­pyridin-2-yl)(3,5-dimethyl-1H-pyrazol-1-yl)methanone 
In the crystal structure of the title compound, C11H9Cl2N3O, mol­ecules are held together by short inter­molecular Cl⋯Cl contacts [3.319 (1) Å] and C—H⋯N hydrogen bonds, forming two-dimensional networks parallel to (01).
PMCID: PMC2961666  PMID: 21202861
15.  3,6-Dichloro-N-(4,6-dichloro­pyrimidin-2-yl)picolinamide 
In the title compound, C10H4Cl4N4O, the pyridine and pyrimidine rings are nearly perpendicular to each other, the dihedral angle between them being 86.60 (10)°. In the crystal structure, the N and O atoms in the amide group are involved in inter­molecular hydrogen bonds, forming a one-dimensional chain along the c axis.
PMCID: PMC2961186  PMID: 21202357
16.  Chlorido(1,10-phenanthroline)(1H-1,2,4-triazole-3-carboxyl­ato)copper(II) 
The title complex, [Cu(C3H2N3O2)Cl(C12H8N2)], crystallizes with two independent mol­ecules in the asymmetric unit. Each CuII atom is coordinated by an N atom and an O atom from the bidentate 1H-1,2,4-triazole-3-carboxyl­ate ligand, two N atoms from the 1,10-phenanthroline ligand, and the Cl atom. The coordination geometry is based on a ClN3O square pyramid. In the crystal structure, the mol­ecules are linked by inter­molecular N—H⋯O hydrogen bonds.
PMCID: PMC2914951  PMID: 21200641

Results 1-16 (16)