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1.  Activation of the NF-κB pathway as a mechanism of alcohol enhanced progression and metastasis of human hepatocellular carcinoma 
Molecular Cancer  2015;14(1):10.
Background
Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is the third leading cause of cancer-related death in human. Alcohol is a known risk factor for HCC. However it is still unclear whether and how alcohol enhances the progression and metastasis of existing HCC.
Methods and results
We first retrospectively investigated 52 HCC patients (24 alcohol-drinkers and 28 non-drinkers), and found a positive correlation between alcohol consumption and advanced Tumor-Node-Metastasis (TNM) stages, higher vessel invasion and poorer prognosis. In vitro and in vivo experiments further indicated that alcohol promoted the progression and migration/invasion of HCC. Specifically, in a 3-D tumor/endothelial co-culture system, we found that alcohol enhanced the migration/invasion of HepG2 cells and increased tumor angiogenesis. Consistently, higher expression of VEGF, MCP-1 and NF-κB was observed in HCC tissues of alcohol-drinkers. Alcohol induced the accumulation of intracellular reactive oxygen species (ROS) and the activation of NF-κB signaling in HepG2 cells. Conversely, blockage of alcohol-mediated ROS accumulation and NF-κB signaling inhibited alcohol-induced expression of VEGF and MCP-1, the tumor growth, angiogenesis and metastasis.
Conclusion
This study suggested that chronic moderate alcohol consumption may promote the progression and metastasis of HCC; the oncogenic effect may be at least partially mediated by the ROS accumulation and NF-ĸB-dependent VEGF and MCP-1 up-regulation.
doi:10.1186/s12943-014-0274-0
PMCID: PMC4320626  PMID: 25622857
Alcohol; Angiogenesis; Human hepatocellular cancer; Metastasis; Reactive oxygen species
2.  Efficacy of capecitabine-based combination therapy and single-agent capecitabine maintenance therapy in patients with metastatic breast cancer 
Objective
The purpose of this study was to observe the efficacy and toxicities of capecitabine-based chemotherapy and capecitabine monotherapy as maintenance therapy in the treatment of metastatic breast cancer (MBC).
Patients and methods
A total of 98 MBC patients were treated with capecitabine combined with vinorelbine (NX).
Results
The median number of treatment was 6 cycles (1-7 cycles). There were two cases of complete remission (CR), 58 partial remission, 27 stable disease (SD), 11 progression disease. The overall response rate (ORR) (CR + PR) was 61.2%. The clinical benefit rate (CBR) was 75.5%. Fifty of effective patients received with capecitabine monotherapy as maintenance therapy. The ORR (CR + PR) was 4%. The CBR was 48%. The median progression-free survival (PFS) was 12 months. In maintenance therapy or not, the median post metastasis survival rate (MSR) was 63 and 28 months, respectively. In the combination therapy group, the major grade 3/4 toxicities included hand-foot syndrome (3.1%), skin pigmentation (2.0%), diarrhoea and abdominal distension (5.1%), stomatitis (1.0%), and leukopenia (20.4%).
Conclusions
Capecitabine-based combination therapy and single-agent capecitabine maintenance therapy were well tolerated and effective to MBC.
doi:10.3978/j.issn.1000-9604.2014.12.13
PMCID: PMC4279202  PMID: 25561767
Metastatic breast cancer (MBC); capecitabine; maintenance therapy
3.  SOX1 down-regulates β-catenin and reverses malignant phenotype in nasopharyngeal carcinoma 
Molecular Cancer  2014;13(1):257.
Background
Aberrant activation of the Wnt/β-catenin signaling pathway is an important factor in the development of nasopharyngeal carcinoma (NPC). Previous studies have demonstrated that the developmental gene sex-determining region Y (SRY)-box 1 (SOX1) inhibits cervical and liver tumorigenesis by interfering with the Wnt/β-catenin signaling pathway. However, the role of SOX1 in NPC remains unclear. This study investigates the function of SOX1 in NPC pathogenesis.
Results
Down-regulation of SOX1 was detected in NPC cell lines and tissues. Besides, quantitative methylation-specific polymerase chain reaction revealed that SOX1 promoter was hypermethylated in NPC cell lines. Ectopic expression of SOX1 in NPC cells suppressed colony formation, proliferation and migration in vitro and impaired tumor growth in nude mice. Restoration of SOX1 expression significantly reduced epithelial-mesenchymal transition, enhanced cell differentiation and induced cellular senescence. Conversely, transient knockdown of SOX1 by siRNA in these cells partially restored cell proliferation and colony formation. Notably, SOX1 was found to physically interact with β-catenin and reduce its expression independent of proteasomal activity, leading to inhibition of Wnt/β-catenin signaling and decreased expression of downstream target genes.
Conclusions
SOX1 decreases the expression of β-catenin in a proteasome-independent manner and reverses the malignant phenotype in NPC cells.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-257) contains supplementary material, which is available to authorized users.
doi:10.1186/1476-4598-13-257
PMCID: PMC4326525  PMID: 25427424
SOX1; β-catenin; Methylation; Differentiation; NPC
4.  CEACAM6 Promotes Gastric Cancer Invasion and Metastasis by Inducing Epithelial-Mesenchymal Transition via PI3K/AKT Signaling Pathway 
PLoS ONE  2014;9(11):e112908.
Overexpressed CEACAM6 in tumor tissues plays important roles in invasion, metastasis and anoikis resistance in a variety of human cancers. We recently reported that CEACAM6 expression is upregulated in Gastric cancer (GC) tissues and promoted GC metastasis. Here, we report that CEACAM6 promotes peritoneal metastases in vivo and is negatively correlated with E-cadherin expression in GC tissues. Overexpressed CEACAM6 induced epithelial-mesenchymal transition (EMT) in GC, as measured by increases in the EMT markers N-cadherin, Vimentin and Slug while E-cadherin expression was decreased in CEACAM6-overexpressing GC cells; opposing results were observed in CEACAM6-silenced cells. Furthermore, E-cadherin expression was negatively correlated with depth of tumor invasion, lymph node metastasis and TNM stage in GC tissues. Additionally, CEACAM6 elevated matrix metalloproteinase-9 (MMP-9) activity in GC, and anti-MMP-9 antibody could reverse the increasing invasion and migration induced by CEACAM6. CEACAM6 also increased the levels of phosphorylated AKT, which is involved in the progression of a variety of human tumors. We further observed that LY294002, a PI3K inhibitor, could reverse CEACAM6-induced EMT via mesenchymal-epithelial transition. These findings suggest that CEACAM6 enhances invasion and metastasis in GC by promoting EMT via the PI3K/AKT signaling pathway.
doi:10.1371/journal.pone.0112908
PMCID: PMC4232574  PMID: 25398131
5.  Testosterone regulates keratin 33B expression in rat penis growth through androgen receptor signaling 
Asian Journal of Andrology  2014;16(6):817-823.
Androgen therapy is the mainstay of treatment for the hypogonadotropic hypogonadal micropenis because it obviously enhances penis growth in prepubescent microphallic patients. However, the molecular mechanisms of androgen treatment leading to penis growth are still largely unknown. To clarify this well-known phenomenon, we successfully generated a castrated male Sprague Dawley rat model at puberty followed by testosterone administration. Interestingly, compared with the control group, testosterone treatment stimulated a dose-dependent increase of penis weight, length, and width in castrated rats accompanied with a dramatic recovery of the pathological changes of the penis. Mechanistically, testosterone administration substantially increased the expression of androgen receptor (AR) protein. Increased AR protein in the penis could subsequently initiate transcription of its target genes, including keratin 33B (Krt33b). Importantly, we demonstrated that KRT33B is generally expressed in the rat penis and that most KRT33B expression is cytoplasmic. Furthermore, AR could directly modulate its expression by binding to a putative androgen response element sequence of the Krt33b promoter. Overall, this study reveals a novel mechanism facilitating penis growth after testosterone treatment in precastrated prepubescent animals, in which androgen enhances the expression of AR protein as well as its target genes, such as Krt33b.
doi:10.4103/1008-682X.129935
PMCID: PMC4236322  PMID: 24994782
androgen receptor; keratin 33B; penis; testosterone
6.  Androgen receptor promotes gastric cancer cell migration and invasion via AKT-phosphorylation dependent upregulation of matrix metalloproteinase 9 
Oncotarget  2014;5(21):10584-10595.
Androgen receptor (AR) plays an important role in many kinds of cancers. However, the molecular mechanisms of AR in gastric cancer (GC) are poorly characterized. Here, we investigated the role of AR in GC cell migration, invasion and metastatic potential. Our data showed that AR expression was positively correlated with lymph node metastasis and late TNM stages. These findings were accompanied by activation of AKT and upregulation of matrix metalloproteinase 9 (MMP9). AR overexpression induced increases in GC cell migration, invasion and proliferation in vitro and in vivo. These effects were attenuated by inhibition of AKT, AR and MMP9. AR overexpression upregulated MMP9 protein levels, whereas this effect was counteracted by AR siRNA. Inhibition of AKT by siRNA or an inhibitor (MK-2206 2HC) decreased AR protein expression in both stably transfected and parental SGC-7901 cells. Luciferase reporter and chromatin immunoprecipitation assays demonstrated that AR bound to the AR-binding sites of the MMP9 promoter. In summary, AR overexpression induced by AKT phosphorylation upregulated MMP9 by binding to its promoter region to promote gastric carcinogenesis. The AKT/AR/MMP9 pathway plays an important role in GC metastasis and may be a novel therapeutic target for GC treatment.
PMCID: PMC4279395  PMID: 25301736
Androgen receptor; AKT; MMP9; Gastric cancer
7.  PI3K/AKT Pathway Involvement in the Osteogenic Effects of Osteoclast Culture Supernatants on Preosteoblast Cells 
Tissue Engineering. Part A  2013;19(19-20):2226-2232.
This study aimed to investigate the ability of osteoclasts during bone resorption activities to regulate the differentiation and calcification of osteoblast precursor cells. The bone resorption model was established using in vitro cortical bone slices and mouse RAW264.7 cells, which were differentiated into osteoclasts by stimulation with the receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor. Tartrate-resistant acid phosphatase (TRAP) staining, reverse transcriptase–polymerase chain reaction (RT-PCR), and scanning electron microscopy (SEM) were used to detect osteoclast differentiation. The osteoblast precursor cell line MC3T3-E1 was cultured with the bone resorption supernatant (BRS). Involvement of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in osteogenesis was evaluated by Western blotting, RT-PCR, and ELISA analysis of markers of the early (runt-related transcription factor-2 and alkaline phosphatase) and late (osteocalcin [OCN]) stages of osteogenesis, and Alizarin Red S staining of matrix mineralization. TRAP staining, RT-PCR, and SEM analysis demonstrated the successful establishment of the bone resorption model. Osteoclast BRS effectively increased the differentiation and calcification of MC3T3-E1 cells. Western blot analysis indicated that the BRS enhanced AKT and p-AKT expression levels in MC3T3-E1 cells. Following AKT2 knockdown and treatment with the PI3K/AKT pathway inhibitor LY294002, the expression of OCN in MC3T3-E1 cells was decreased (p<0.05), as was the calcification area (p<0.05). The data obtained in this study indicated that the osteoclast bone resorption medium promoted the differentiation and calcification of MC3T3-E1 cells and that the PI3K/AKT pathway played a role in this process.
doi:10.1089/ten.tea.2012.0469
PMCID: PMC3761438  PMID: 23617625
8.  Essential roles for GSK-3s and GSK-3 primed substrates in neurotrophin-induced and hippocampal axon growth 
Neuron  2006;52(6):981-996.
Summary
Glycogen synthase kinase-3β (GSK-3β) is thought to mediate morphological responses to a variety of extracellular signals. Surprisingly we found no gross morphological deficits in nervous system development in GSK-3β null mice. We therefore designed an shRNA that targeted both GSK-3 isoforms. Strong knockdown of both GSK-3α and β markedly reduced axon growth in dissociated cultures and slice preparations. We then assessed the role of different GSK-3 substrates in regulating axon morphology. Elimination of activity towards primed substrates only using the GSK-3 R96A mutant was associated with a defect in axon polarity (axon branching) compared to an overall reduction in axon growth induced by a kinase dead mutant. Consistent with this finding, moderate reduction of GSK-3 activity by pharmacological inhibitors induced axon branching and was associated primarily with effects on primed substrates. Our results suggest that GSK-3 is a downstream convergent point for many axon growth regulatory pathways and that differential regulation of primed versus all GSK-3 substrates is associated with a specific morphological outcome.
doi:10.1016/j.neuron.2006.10.031
PMCID: PMC4167845  PMID: 17178402
GSK-3; axon; neurotrophin; hippocampal neurons; MAP1B; APC; CRMP2
9.  Aberrant neuronal synaptic connectivity in CA1 area of the hippocampus from pilocarpine-induced epileptic rats observed by fluorogold 
In this study, we observed synaptic connectivity among neurons in CA1 region of pilocarpine-induced chronic seizures in rats. Twenty healthy male Sprague-Dawley rats were divided randomly into an epilepsy group (n = 10) and a control group (n = 10). Approximately 60 days after status epilepticus (SE) , Fluorogold (FG) was injected into the CA1 area of the hippocampus in vivo. Somatostatin (SS) expression was observed using immunofluorescence. The distribution of FG-positive and FG/SS double-labeled neurons was observed using a confocal microscope. FG-labeled pyramidal cells could be seen remotely from the FG-injected site in the CA1 area and in the subiculum in the experimental group. FG/SS double-labeled interneurons were distributed remotely from the FG-injected site in the CA1 area in the epileptic rats. These changes suggest aberrant neuronal connectivity in CA1 region, which may lead to the formation of aberrant excitatory and inhibitory circuitry, and may play an important role in the generation or compensation for temporal lobe epilepsy.
PMCID: PMC4211776  PMID: 25356126
Temporal lobe epilepsy (TLE); fluorogold (FG); somatostatin (SS); neuronal connectivity; circuit rearrangement; interneuron; lithium chloride; pilocarpin
10.  Gold nanoparticle transfer through photothermal effects in a metamaterial absorber by nanosecond laser 
Scientific Reports  2014;4:6080.
A non-complicated, controllable method of metallic nanoparticle fabrication at low operating light power is proposed. The method is based on laser-induced forward transfer, using a metamaterial absorber as the donor to significantly enhance the photothermal effect and reduce the operating light fluence to 35 mJ/cm2, which is much lower than that in previous works. A large number of metallic nanoparticles can be transferred by one shot of focused nanosecond laser pulses. Transferred nanoparticles exhibit good size uniformity and the sizes are controllable. The optical properties of transferred particles are characterized by dark-field spectroscopy and the experimental results agree with the simulation results.
doi:10.1038/srep06080
PMCID: PMC4143792  PMID: 25156404
11.  Value of segmental coronary calcium score on diagnosis and interventional treatment of coronary lesions by 320-slice DVCT 
The global coronary calcium score has been widely used in the evaluation of coronary plaque burden and cardiovascular disease events. In this study, we investigated the value of segmental coronary calcium score (SCCS) on the diagnosis and interventional treatment. We studied 87 patients with coronary angiography (CAG) and coronary CT angiography (CTA) by 320-slice dynamic volume CT (DVCT). SCCS was determined for each segmental separately. All lesions which SCCS was greater than 0 were enrolled, and were divided into three groups, mild calcification group (SCCS were less than 80), Moderate calcification group (SCCS were more than 80 and less than 200) and Severe calcification group (SCCS were more than 200). From above three groups, lesions received the intervention treatment were elected as subgroup. The position of lesions, plaque morphology, calcification proportion and interventional treatment data were analyzed. Severe calcification group were more frequent in the proximal lesions, stenosis with lesser extent, nubbly and nodular types of plaque, and the inconsistency with CAG was higher than the other two groups (P < 0.05). In the subgroup, more pre-dilatation and post-dilatation balloon were used in severe calcification group, with higher expansion pressure of balloon and stent (P < 0.05), but the diameter of stents was no difference between the three groups. Conclusion: SCCS is better than GCCS in the evaluation of coronary calcification, and play an important role in the judgment of stenosis by coronary CT and in the choice of interventional therapeutic devices.
PMCID: PMC4161572  PMID: 25232412
Segmental coronary calcium score; plaque morphology; calcification; dynamic volume CT; coronary angiography
12.  Increased methylation of the MOR gene proximal promoter in primary sensory neurons plays a crucial role in the decreased analgesic effect of opioids in neuropathic pain 
Molecular Pain  2014;10:51.
Background
The analgesic potency of opioids is reduced in neuropathic pain. However, the molecular mechanism is not well understood.
Results
The present study demonstrated that increased methylation of the Mu opioid receptor (MOR) gene proximal promoter (PP) in dorsal root ganglion (DRG) plays a crucial role in the decreased morphine analgesia. Subcutaneous (s.c.), intrathecal (i.t.) and intraplantar (i.pl.), not intracerebroventricular (i.c.v.) injection of morphine, the potency of morphine analgesia was significantly reduced in nerve-injured mice compared with control sham-operated mice. After peripheral nerve injury, we observed a decreased expression of MOR protein and mRNA, accompanied by an increased methylation status of MOR gene PP, in DRG. However, peripheral nerve injury could not induce a decreased expression of MOR mRNA in the spinal cord. Treatment with 5-aza-2′-deoxycytidine (5-aza-dC), inhibited the increased methylation of MOR gene PP and prevented the decreased expression of MOR in DRG, thereby improved systemic, spinal and periphery morphine analgesia.
Conclusions
Altogether, our results demonstrate that increased methylation of the MOR gene PP in DRG is required for the decreased morphine analgesia in neuropathic pain.
doi:10.1186/1744-8069-10-51
PMCID: PMC4137045  PMID: 25118039
Neuropathic pain; Epigenetics; Opioids
13.  Comparison of the clinical features and outcomes in two age-groups of elderly patients with atrial fibrillation 
Background
Atrial fibrillation (AF) disproportionately affects older adults. However, direct comparison of clinical features, medical therapy, and outcomes in AF patients aged 65–74 and ≥75 years is rare. The objective of the present study was to evaluate the differences in clinical characteristics and prognosis in these two age-groups of geriatric patients with AF.
Materials and methods
A total of 1,336 individuals aged ≥65 years from a Chinese AF registry were assessed in the present study: 570 were in the 65- to 74-year group, and 766 were in the ≥75-year group. Multivariable Cox hazards regression was performed to analyze the major adverse cardiac events (MACEs) between groups.
Results
In our population, the older group were more likely to have coronary artery disease, hypertension, previous stroke, cognitive disorder, or chronic obstructive pulmonary disease, and the 65- to 74-year group were more likely to have valvular heart disease, left ventricular systolic dysfunction, or sleep apnea. The older patients had 1.2-fold higher mean CHADS2 (congestive heart failure, hypertension, age ≥75 years, diabetes, stroke) scores, but less probability of being prescribed drugs. Compared with those aged 65–74 years, the older group had a higher risk of death (hazard ratio 2.881, 95% confidence interval 1.981–4.189; P<0.001) or MACE (hazard ratio 2.202, 95% confidence interval 1.646–2.945; P<0.001) at the 1-year follow-up. In multivariable Cox analyses, secondary AF diagnosis, a history of chronic obstructive pulmonary disease, and left ventricular systolic dysfunction were independent predictors of MACE in the older group.
Conclusion
Patients aged ≥75 years had a worse prognosis than those aged 65–74 years, and were associated with a higher risk of both death and MACE.
doi:10.2147/CIA.S67123
PMCID: PMC4136954  PMID: 25143720
atrial fibrillation; geriatric patients; mortality; major adverse cardiac events (MACE)
14.  Maintenance treatment of trastuzumab for patients with advanced breast cancer to achieve long term survival: two case reports and literature review 
Molecular subtyping of breast cancer may provide additional prognostic information regarding patient outcome. The epidermal growth factor receptor (HER2) overexpressing breast cancers are designated as HER2-postive (HER2+) breast cancer and carry a particularly unfavorable prognosis. We present two cases of HER2-postive metastatic breast cancer (MBC) who are found to be a challenge to treat, especially due to the occurrence of brain metastasis. Trastuzumab-based therapy improves clinical outcomes, even if the patient has undergone multi-line treatment. These case reports also emphasize the importance of retesting HER2 status because it can be discordance in receptor status between primary and recurrent breast cancer.
doi:10.3978/j.issn.1000-9604.2014.08.10
PMCID: PMC4153919  PMID: 25232224
Breast cancer; trastuzumab; brain metastasis
15.  E3-ligase Skp2 predicts poor prognosis and maintains cancer stem cell pool in nasopharyngeal carcinoma 
Oncotarget  2014;5(14):5591-5601.
Nasopharyngeal carcinoma (NPC) is one of the severe head and neck carcinomas, which is rare in west countries but has high incidence in Southern Asia especially South China. Although NPC is relatively sensitive to radiotherapy, the prognosis of patients is poor due to the advanced stage at the time of diagnosis. Therefore, it is important to understand the mechanisms involved in tumorigenesis and develop early diagnostic techniques. S-phase kinase associated protein 2 (Skp2) is overexpressed in several human cancers and associates with poor prognosis. However, its function in NPC has not been fully addressed. In this study we found Skp2 was highly expressed in NPC specimen and correlated with poor prognosis. We generated Skp2 knockdown cells to further delineate its role in NPC development. Knockdown of Skp2 partially reduced cell proliferation, promoted cellular senescence, and decreased the population of stem cell like aldehyde dehydrogenase1 positive cells as well as their self-renewal ability. Our study not only interprets the predictive role of Skp2 in the poor prognosis of NPC patients, but also reveals that Skp2 regulates the NPC cancer stem cell maintenance, which shed lights on the target therapy and early diagnosis of NPC in clinical application.
PMCID: PMC4170633  PMID: 25015320
S-phase kinase associated protein 2 (Skp2); nasopharyngeal carcinoma (NPC); aldehyde dehydrogenase1 (ALDH1); cancer stem cell (CSC)
16.  MicroRNA-34A inhibits the growth, invasion and metastasis of gastric cancer by targeting PDGFR and MET expression 
Bioscience Reports  2014;34(3):e00112.
Within the family of RTKs (receptor tyrosine kinases), PDGFR (platelet-derived growth factor receptor) has been implicated in carcinogenesis and tumour development. miRNAs (microRNAs), which can target the mRNAs (messenger RNAs) of cancer-associated genes, are abnormally expressed in various cancers. In this study, our aim was to identify the miRNAs that target PDGFR-α/β and to study the functions of these miRNAs. miR-34a was predicted to target PDGFR, and luciferase reporter assays showed that miR-34a could directly target PDGFR. Meanwhile, we found that miR-34a was down-regulated in gastric cancer tissues and was associated with metastasis. Our findings showed that miR-34a could inhibit gastric cancer cell migration, invasion and proliferation, but these tumourigenic properties were only partially restored when PDGFR-α/β was overexpressed. In subsequent experiments, we found that the overexpression of both PDGFR and MET could completely restore the gastric cancer tumourigenic properties. Moreover, the cancer-associated cell signalling pathway was studied, and we found that miR-34a could inhibit Akt [PKB (protein kinase B)] phosphorylation, which was restored by the overexpression of both PDGFR and MET. In conclusion, miR-34a may act as a potential tumour suppressor in gastric cancer and is associated with the mechanisms of gastric cancer metastasis; miR-34a can inhibit gastric cancer tumourigenesis by targeting PDGFR and MET through the PI3K (phosphoinositide 3-kinase)/Akt pathway.
doi:10.1042/BSR20140020
PMCID: PMC4069683  PMID: 24837198
miR-34a; PDGFR; MET; gastric cancer; Akt, PKB (protein kinase B); HRP, horseradish peroxidase; miRNA, microRNA; mRNA, messenger RNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; PDGFR, platelet-derived growth factor receptor; PI3K, phosphoinositide 3-kinase; RTK, receptor tyrosine kinase
17.  Dysfunction of annexin A2 contributes to hyperglycaemia-induced loss of human endothelial cell surface fibrinolytic activity 
Thrombosis and haemostasis  2013;109(6):1070-1078.
Summary
Hyperglycaemia impairs fibrinolytic activity on the surface of endothelial cells, but the underlying mechanisms are not fully understood. In this study, we tested the hypothesis that hyperglycaemia causes dysfunction of the endothelial membrane protein annexin A2, thereby leading to an overall reduction of fibrinolytic activity. Hyperglycaemia for 7 days significantly reduced cell surface fibrinolytic activity in human brain microvascular endothelial cells (HBMEC). Hyperglycaemia also decreased tissue type plasminogen activator (t-PA), plasminogen, and annexin A2 mRNA and protein expression, while increasing plasminogen activator inhibitor-1 (PAI-1). No changes in p11 mRNA or protein expression were detected. Hyperglycaemia significantly increased AGE-modified forms of total cellular and membrane annexin A2. The hyperglycemia-associated reduction in fibrinolytic activity was fully restored upon incubation with recombinant annexin A2 (rA2), but not AGE-modified annexin A2 or exogenous t-PA. Hyperglycaemia decreased t-PA, upregulated PAI-1 and induced AGE-related disruption of annexin A2 function, all of which contributed to the overall reduction in endothelial cell surface fibrinolytic activity. Further investigations to elucidate the underlying molecular mechanisms and pathophysiological implications of A2 derivatisation might ultimately lead to a better understanding of mechanisms of impaired vascular fibrinolysis, and to development of new interventional strategies for the thrombotic vascular complications in diabetes.
doi:10.1160/TH12-12-0944
PMCID: PMC4066328  PMID: 23572070
Annexin A2; hyperglycaemia; fibrinolytic activity; advanced glycation end-product; human brain microvascular endothelial cells
18.  Structural Insights into SraP-Mediated Staphylococcus aureus Adhesion to Host Cells 
PLoS Pathogens  2014;10(6):e1004169.
Staphylococcus aureus, a Gram-positive bacterium causes a number of devastating human diseases, such as infective endocarditis, osteomyelitis, septic arthritis and sepsis. S. aureus SraP, a surface-exposed serine-rich repeat glycoprotein (SRRP), is required for the pathogenesis of human infective endocarditis via its ligand-binding region (BR) adhering to human platelets. It remains unclear how SraP interacts with human host. Here we report the 2.05 Å crystal structure of the BR of SraP, revealing an extended rod-like architecture of four discrete modules. The N-terminal legume lectin-like module specifically binds to N-acetylneuraminic acid. The second module adopts a β-grasp fold similar to Ig-binding proteins, whereas the last two tandem repetitive modules resemble eukaryotic cadherins but differ in calcium coordination pattern. Under the conditions tested, small-angle X-ray scattering and molecular dynamic simulation indicated that the three C-terminal modules function as a relatively rigid stem to extend the N-terminal lectin module outwards. Structure-guided mutagenesis analyses, in addition to a recently identified trisaccharide ligand of SraP, enabled us to elucidate that SraP binding to sialylated receptors promotes S. aureus adhesion to and invasion into host epithelial cells. Our findings have thus provided novel structural and functional insights into the SraP-mediated host-pathogen interaction of S. aureus.
Author Summary
Staphylococcus aureus is an important pathogen that causes a range of human diseases, such as infective endocarditis, osteomyelitis, septic arthritis and sepsis. The increasing resistance of S. aureus to most of the current antibiotics emphasizes the need to develop new approaches to control staphylococcal infections. As a surface-exposed serine-rich repeat glycoprotein (SRRP), S. aureus SraP is involved in the pathogenesis of infective endocarditis via its ligand-binding region (BR) adhering to human platelets. However, little is known about how SraP interacts with its host receptor(s). Through structural and functional analyses of the BR domain, we have discovered a specific binding of SraP to N-acetylneuraminic acid (Neu5Ac), in agreement with a recent report of the trisaccharide ligand of SraP. Further mutagenesis analysis showed that SraP binding to Neu5Ac and the trisaccharide promotes S. aureus adhesion to and invasion into host epithelial cells. These findings increase our knowledge of surface protein mediated interaction of S. aureus with host epithelial cells.
doi:10.1371/journal.ppat.1004169
PMCID: PMC4047093  PMID: 24901708
19.  Tumor suppressor miR-24 restrains gastric cancer progression by downregulating RegIV 
Molecular Cancer  2014;13:127.
Background
microRNAs are small noncoding RNAs that modulate a variety of cellular processes by regulating multiple targets, which can promote or inhibit the development of malignant behaviors. Accumulating evidence suggests miR-24 plays important roles in human carcinogenesis. However, its precise biological role remains largely elusive. This study examined the role of miR-24 in gastric cancer (GC).
Methods
The expression of miR-24 in GC tissues compared with matched non-tumor tissues and GC cells was detected by qRT-PCR. Synthetic short single or double stranded RNA oligonucleotides and lentiviral vectors were used to regulate miR-24 expression in GC cells to investigate its function in vitro and in vivo.
Results
miR-24 was significantly downregulated in GC tissues compared with matched non-tumor tissues and was associated with tumor differentiation. Ectopic expression of miR-24 in SGC-7901 GC cells suppressed cell proliferation, migration and invasion in vitro as well as tumorigenicity in vivo by inducing cell cycle arrest in G0/G1 phase and promoting cell apoptosis. Furthermore, we identified RegIV as a target of miR-24 and demonstrated that miR-24 regulated RegIV expression via binding its 3′ untranslated region.
Conclusions
miR-24 functions as a novel tumor suppressor in GC and the anti-oncogenic activity may involve its inhibition of the target gene RegIV. These findings suggest the possibility for miR-24 as a therapeutic target in GC.
doi:10.1186/1476-4598-13-127
PMCID: PMC4041902  PMID: 24886316
miR-24; RegIV; Gastric cancer; Proliferation; Invasion; Metastasis
20.  Pregnenolone, a cholesterol metabolite, induces glioma cell apoptosis via activating extrinsic and intrinsic apoptotic pathways 
Oncology Letters  2014;8(2):645-650.
Gliomas are one of the most common types of malignant tumors worldwide, however, an effective therapeutic strategy not yet been fully determined. The present study aimed to investigate the anti-glioma activity and underlying mechanisms of pregnenolone, which originates from cholesterol and is metabolized into important steroid hormones in the body. The results demonstrated that 100 μM pregnenolone induced a significant loss of cell viability in various malignant glioma cell lines. In the U-87 MG, LN-18 and C6 cell lines, the loss of cell viability resulted from cell apoptosis, which was evidenced by apoptotic nuclear morphology changes and caspase 3 activation. Moreover, the increased activities of caspase 8 and 9 strongly indicated that pregnenolone activated the extrinsic and intrinsic pathways of apoptosis. Additionally, glioma cell apoptosis was prevented by the general caspase inhibitor, Z-VAD-FMK. In the C6 cells, upregulation of Fas and Fas ligand triggered the activation of the extrinsic pathway, whereas knockdown of Fas significantly abrogated the cell apoptosis that was induced by pregnenolone. Furthermore, downregulation of the anti-apoptotic protein, B-cell lymphoma 2 and upregulation of pro-apoptotic proteins, such as Bax and Bak, activated the intrinsic pathway. In conclusion, pregnenolone induced glioma cell apoptosis in a caspase-dependent manner, which was mediated by activation of the extrinsic and intrinsic apoptotic pathways.
doi:10.3892/ol.2014.2147
PMCID: PMC4081362  PMID: 25013479
pregnenolone; glioma; apoptosis
21.  Synthetic triterpenoid induces 15-PGDH expression and suppresses inflammation-driven colon carcinogenesis 
The Journal of Clinical Investigation  2014;124(6):2472-2482.
Colitis-associated colon cancer (CAC) develops as a result of inflammation-induced epithelial transformation, which occurs in response to inflammatory cytokine-dependent downregulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and subsequent suppression of prostaglandin metabolism. Agents that both enhance 15-PGDH expression and suppress cyclooxygenase-2 (COX-2) production may more effectively prevent CAC. Synthetic triterpenoids are a class of small molecules that suppress COX-2 as well as inflammatory cytokine signaling. Here, we found that administration of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-C28-methyl ester (CDDO-Me) suppresses CAC in mice. In a spontaneous, inflammation-driven intestinal neoplasia model, deletion of Smad4 specifically in T cells led to progressive production of inflammatory cytokines, including TNF-α, IFN-γ, iNOS, IL-6, IL-1β; as well as activation of STAT1 and STAT3; along with suppression of 15-PGDH expression. Oral administration of CDDO-Me to mice with SMAD4-deficient T cells increased survival and suppressed intestinal epithelial neoplasia by decreasing production of inflammatory mediators and increasing expression of 15-PGDH. Induction of 15-PGDH by CDDO-Me was dose dependent in epithelial cells and was abrogated following treatment with TGF-β signaling inhibitors in vitro. Furthermore, CDDO-Me–dependent 15-PGDH induction was not observed in Smad3–/– mice. Similarly, CDDO-Me suppressed azoxymethane plus dextran sodium sulfate–induced carcinogenesis in wild-type animals, highlighting the potential of small molecules of the triterpenoid family as effective agents for the chemoprevention of CAC in humans.
doi:10.1172/JCI69672
PMCID: PMC4089461  PMID: 24837432
22.  Maternal embryonic leucine zipper kinase enhances gastric cancer progression via the FAK/Paxillin pathway 
Molecular Cancer  2014;13:100.
Background
Elevated MELK expression is featured in multiple tumors and correlated with tumorigenesis and tumor development. This study is aimed to investigate the mechanisms of MELK-mediated development of gastric cancer.
Methods
MELK expression levels in human gastric cancer were determined by quantitative-PCR and immunohistochemistry. The effect of MELK on cell activity was explored by knockdown and overexpression experiments. Cell growth was measured using the CCK-8 assay. Apoptosis and cell cycle distributions were analyzed by flow cytometry. Migration and invasion were tested using a transwell migration assay. Cytoskeletal changes were analyzed by immunofluorescence. To explore the molecular mechanism and effect of MELK on migration and invasion, Western blotting was used to analyze the FAK/Paxillin pathway and pull down assays for the activity of small Rho GTPases. In vivo tumorigenicity and peritoneal metastasis experiments were performed by tumor cell engraftment into nude mice.
Results
MELK mRNA and protein expression were both elevated in human gastric cancer, and this was associated with chemoresistance to 5-fluorouracil (5-FU). Knockdown of MELK significantly suppressed cell proliferation, migration and invasion of gastric cancer both in vitro and in vivo, decreased the percentages of cells in the G1/G0 phase and increased those in the G2/M and S phases. Moreover, knockdown of MELK decreased the amount of actin stress fibers and inhibited RhoA activity. Finally, knockdown of MELK decreased the phosphorylation of the FAK and paxillin, and prevented gastrin-stimulated FAK/paxillin phosphorylation. By contrast, MELK overexpression had the opposite effect.
Conclusions
MELK promotes cell migration and invasion via the FAK/Paxillin pathway, and plays an important role in the occurrence and development of gastric cancer. MELK may be a potential target for treatment against gastric cancer.
doi:10.1186/1476-4598-13-100
PMCID: PMC4113179  PMID: 24885567
MELK; Gastric cancer; Tumor migration; Tumor invasion; FAK; Paxillin
23.  Relationships among androgen receptor CAG repeat polymorphism, sex hormones and penile length in Han adult men from China: a cross-sectional study 
Asian Journal of Andrology  2014;16(3):478-481.
This study aimed to investigate the correlations among androgen receptor (AR) CAG repeat polymorphism, sex hormones and penile length in healthy Chinese young adult men. Two hundred and fifty-three healthy men (aged 22.8 ± 3.1 years) were enrolled. The individuals were grouped as CAG short (CAGS) if they harbored repeat length of ≤20 or as CAG long (CAGL) if their CAG repeat length was >20. Body height/weight, penile length and other parameters were examined and recorded by the specified physicians; CAG repeat polymorphism was determined by the polymerase chain reaction (PCR) method; and the serum levels of the sex hormones were detected by radioimmunoassay. Student's t-test or linear regression analysis was used to assess the associations among AR CAG repeat polymorphism, sex hormones and penile length. This investigation showed that the serum total testosterone (T) level was positively associated with the AR CAG repeat length (P = 0.01); whereas, no significant correlation of T or AR CAG repeat polymorphism with the penile length was found (P = 0.593). Interestingly, an inverse association was observed between serum prolactin (PRL) levels and penile length by linear regression analyses (β= −0.024, P = 0.039, 95% confidence interval (CI): −0.047, 0). Collectively, this study provides the first evidence that serum PRL, but not T or AR CAG repeat polymorphism, is correlated with penile length in the Han adult population from northwestern China.
doi:10.4103/1008-682X.124560
PMCID: PMC4023382  PMID: 24589466
androgen receptor; CAG repeat; testosterone; prolactin; penile length
24.  Overexpression of lncRNA H19 enhances carcinogenesis and metastasis of gastric cancer 
Oncotarget  2014;5(8):2318-2329.
Long non-coding RNAs (lncRNAs) play key roles in the progression and metastasis of some carcinomas. We previously showed that the expression of lncRNA H19 (H19) was higher in gastric cancer (GC) tissues than that in paired noncanerous tissues. However, the underlying mechanisms remain unclear.
In this study, H19/miR-675 knockdown models in the MKN45 cell line and ectopic expression models in the SGC7901 cell line were established, and a co-expression network of H19 was generated to identify target genes by RIP and DLR. The results showed that overexpression of H19 promoted the features of GC including proliferation, migration, invasion and metastasis. An H19 co-expression network identified ISM1 as a binding protein of H19, and its expression was positively correlated with that of H19. CALN1 was identified as a target gene of miR-675 and its expression was negatively correlated with that of miR-675. H19 and MiR-675 function in a similar manner. However, H19 RNA actively binds to ISM1 and miR-675 targets CALN1. These differences suggest that H19 plays other roles besides encoding miR-675 in GC. Our results suggest that the effect of H19 in GC is mediated by the direct upregulation of ISM1 and the indirect suppression of CALN1 expression via miR-675.
PMCID: PMC4039165  PMID: 24810858
gastric cancer; long non-coding RNA; H19; microRNA; miR-675
25.  Intravenous infusion of mesenteric lymph from severe intraperitoneal infection rats causes lung injury in healthy rats 
AIM: To investigate whether mesenteric lymph from rats with severe intraperitoneal infection (SII) induces lung injury in healthy rats.
METHODS: Twenty adult male specific pathogen-free Wistar rats were divided into two groups. Animals in the SII group received intraperitoneal injection of Escherichia coli (E. coli) at a dose of 0.3 mL/100 g. Control rats underwent the same procedure, but were injected with normal saline rather than E. coli. We ligated and drained the mesenteric lymphatic vessels and collected the mesenteric lymph. Mesenteric lymph collected from SII or control rats was infused intravenously into male healthy rats at a rate of 1 mL/h for 4 h. At the end of the infusion, all rats were sacrificed. Lungs were removed and examined histologically, and wet-to-dry weight (W/D) ratio and myeloperoxidase (MPO) activity were determined. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the levels of the proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6. We performed Western blot to investigate the activation of Toll-like receptor (TLR)-4, and nuclear factor (NF)-κB p65.
RESULTS: Compared with the control infusion group, there were obvious pathological changes in the SII group. The W/D ratio was significantly increased in the SII compared to control infusion group (5.86 ± 0.06 vs 5.37 ± 0.06, P < 0.01). MPO activity significantly increased in the SII infusion rats with a mean level of 0.86 ± 0.02 U/g compared to 0.18 ± 0.05 U/g in the control group (P < 0.01). The concentrations of TNF-α and IL-6 were significantly increased in the SII infusion group. The concentration of TNF-α was significantly increased in the SII infusion rats compared to control infusion rats (2104.46 ± 245.91 vs 1475.13 ± 137.82 pg/mL, P < 0.01). The concentration of IL-6 was significantly increased in the SII infusion rats with a mean level of 50.56 ± 2.85 pg/mL compared to 43.29 ± 2.02 pg/mL (P < 0.01). The expression levels of TLR-4 (7496.68 ± 376.43 vs 4589.02 ± 233.16, P < 0.01) and NF-κB (8722.19 ± 323.96 vs 6498.91 ± 338.76, P < 0.01) were significantly increased in the SII infusion group compared to the control infusion group. The infusion of SII lymph, but not control lymph, caused lung injury.
CONCLUSION: The results indicate that SII lymph is sufficient to induce acute lung injury.
doi:10.3748/wjg.v20.i16.4771
PMCID: PMC4000515  PMID: 24782631
Severe intraperitoneal infection; Mesenteric lymph; Acute lung injury; Toll-like receptor 4; Nuclear factor κB

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