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1.  Neural mechanisms and potential treatment of epilepsy and its complications 
The factors underlying epilepsy are multifaceted, but recent research suggests that the brain’s neural circuits, which play a key role in controlling the balance between epileptic and antiepileptic factors, may lie at the heart of epilepsy. This article provides a comprehensive review of the neural mechanisms and potential treatment of intractable epilepsy from neural inflammatory responses, melanocortin circuits in brain and pedunculopontine tegmental nucleus. Further studies should be undertaken to elucidate the nature of neural circuits so that we may more effectively apply these new preventive and symptomatic therapies to the patient suffering from medically refractory seizures and its complications.
PMCID: PMC4297332  PMID: 25628775
Intractable epilepsy; neural inflammatory responses; melanocortin circuits; pedunculopontine tegmental nucleus
2.  Extracorporeal shock wave therapy ameliorates cyclophosphamide-induced rat acute interstitial cystitis though inhibiting inflammation and oxidative stress-in vitro and in vivo experiment studies 
Background: We investigated whether extracorporeal shock wave (ECSW) therapy can attenuate cyclophosphamide (CYP)-induced acute interstitial cystitis (AIC) in rats. Methods and Results: Eighteen male-adult Sprague-Dawley rats were equally divided into group 1 (sham control), group 2 (AIC induced by 150 mg/kg CYP by intra-peritoneal injection) and group 3 (AIC + ECSW 200 impulses at 0.11 mJ/mm2 to the urinary bladder at 3 and 24 h after CYP treatment). Smooth-muscle cells co-culture with menadione (25 µM) with and without ECSW treatment was performed. Western-blot results demonstrated that ECSW significant attenuated oxidative stress and inflammatory reactions in this in-vitro studies (all p < 0.001). 24-hour urine amount and microscopic findings of red-blood-cell count (i.e., hematuria) were higher in group 2 than in groups 1 and 3, and significantly higher in group 3 than in group 1 (all p < 0.001). The urine levels of albumin and interleukin-6 showed an identical pattern of hematuria among all three groups (all p < 0.001). The cellular and mRNA expressions of macrophage migration inhibitory factor (MIF)+, CD74+, CD68+, substance p+, and Cox-2+ cells in the bladder tissue exhibited an identical pattern of hematuria among all groups (all p < 0.0001). The integrity of epithelial layer and collagen-deposition area as stained by Sirius red displayed an opposite pattern of hematuria among the three groups (p < 0.0001). The protein expression of IL-12, iNOS, TNF-α, NF-κB, MMP-9, NOX-1, NOX-2, RANTES, and Oxyblot displayed an identical pattern of hematuria among all groups (all p < 0.01). Conclusion: ECSW therapy markedly attenuated CYP-induced AIC through inhibitions of the inflammation and oxidative stress.
PMCID: PMC4297333  PMID: 25628776
Acute interstitial cystitis; cyclophosphamide treatment; extra-corporeal shock wave; inflammatory biomarkers
3.  Activation of Keap1/Nrf2 signaling pathway by nuclear epidermal growth factor receptor in cancer cells 
Nuclear translocation of EGFR has been shown to be important for tumor cell growth, survival, and therapeutic resistance. Previously, we detected the association of EGFR with Keap1 in the nucleus. Keap1 is a Kelch-like ECH-associated protein, which plays an important role in cellular response to chemical and oxidative stress by regulating Nrf2 protein stability and nuclear translocation. In this study, we investigate the role of EGFR in regulating Keap1/Nrf2 cascade in the nucleus and provide evidence to show that nuclear EGFR interacts with and phosphorylates nuclear Keap1 to reduce its nuclear protein level. The reduction of nuclear Keap1 consequently stabilizes nuclear Nrf2 and increases its transcriptional activity in cancer cells, which contributes to tumor cell resistance to chemotherapy.
PMCID: PMC4297334  PMID: 25628777
EGFR; Keap1; Nrf2; cancer; ubiquitination
4.  Fibroblast growth factor 1attenuates 6-hydroxydopamine-induced neurotoxicity: an in vitro and in vivo investigation in experimental models of parkinson’s disease 
Parkinson’s disease (PD) is a degenerative disorder of the central nervous system and is characterized by motor system disorders resulting in loss of dopamine producing brain cells. Acidic fibroblast growth factor, also called FGF1, promotes the survival of neurons. The aims of the present study were to confirm FGF1 could protect neurons cultures from 6-hydroxydopamine (6-OHDA) toxicity in vitro and in vivo. Our results demonstrated FGF1 administration improved the motor function recovery, increased the TH-positive neurons survival and up-regulated the levels of neurotransmitters in PD rats. Meanwhile, FGF1 prevents the death of DA neuron at least in part by reducing the levels of α-synuclein and ER stress. The administration of FGF1 activated downstream signals PI3K/Akt and ERK1/2. In conclusion, FGF1 diminished α-synuclein neurotoxicity by down regulating ER stress mediators and the level of apoptosis, and these effects may underlying the activation of the PI3K/Akt and ERK1/2 signal pathway.
PMCID: PMC4297335  PMID: 25628778
ER stress; α-synuclein; parkinson’s disease; FGF1
5.  Agonistic antibody to angiotensin II type 1 receptor accelerates atherosclerosis in ApoE-/- mice 
This study aimed to investigate the effects of agonistic antibody to angiotensin II type 1 receptor (AT1-AA) on atherosclerosis in male ApoE-/- mice which were employed to establish the animal models of AT1-AA in two ways. In the first group, mice were injected subcutaneously with conjugated AT1 peptide at multiple sites; in the second group, mice were infused with AT1-AA prepared from rabbits that were treated with AT1 peptide intraperitoneally. Mice in each group were further randomly divided into five subgroups and treated with AT1 peptide/AT1-AA, AT1 peptide/AT1-AA plus valsartan, AT1 peptide/AT1-AA plus fenofibrate, AT1 peptide/ AT1-AA plus pyrrolidine dithiocarbamate (PDTC) and control vehicle, respectively. Antibodies were detected in mice (except for mice in control group). Aortic atherosclerotic lesions were assessed by oil red O staining, while plasma CRP, TNF-α, nuclear factor-kappa B (NF-κB) and H2O2 were determined by ELISA. CCR2 (the receptor of MCP-1), macrophages, and smooth muscle cells were detected by immunohistochemistry. P47phox, MCP-1 and eNOS were detected by RT-PCR, while P47phox, NF-κB and MCP-1 were detected by Western blot assay. The aortic atherosclerotic lesions were significantly increased in AT1 peptide/AT1-AA treated mice, along with simultaneous increases in inflammatory parameters. However, mice treated with valsartan, fenofibrate or PDTC showed alleviated progression of atherosclerosis and reductions in inflammatory parameters. Thus, AT1-AA may accelerate aortic atherosclerosis in ApoE-/- mice, which is mediated, at least in part, by the inflammatory reaction involving nicotinamide-adenine dinucleotide phosphate oxidase, reactive oxygen species, and NF-κB. In addition, valsartan, fenofibrate and PDTC may inhibit the AT1-AA induced atherosclerosis.
PMCID: PMC4297336  PMID: 25628779
Agonistic antibody to angiotensin II type 1 receptor; atherosclerosis; inflammation
6.  Co-transplantation with mesenchymal stem cells expressing a SDF-1/HOXB4 fusion protein markedly improves hematopoietic stem cell engraftment and hematogenesis in irradiated mice 
Introduction: Mesenchymal stem cells (MSCs) contribute to the engraftment of transplanted hematopoietic stem cells (HSCs). MSCs also accelerate hematological recovery by secreting SDF-1 and enabling HSCs to enter the bone marrow (BM) via the SDF-1/CXCR4 axis. HOXB4 has been shown to stimulate HSC self-renewal. In this study, we examined whether SDF-1 and HOXB4 expression in MSCs co-transplanted with HSCs could synergistically improve hematopoietic recovery in irradiated mice. Methods: Using recombinant adenoviruses, we generated genetically modified BM-MSCs that expressed SDF-1, HOXB4, and an SDF-1/HOXB4 fusion gene. We then co-transplanted these modified MSCs with HSCs and investigated blood cell counts, BM cellularity, degree of human HSC engraftment, and survival rate in irradiated mice. Results: We found that co-culturing the SDF-1/HOXB4 fusion gene-modified MSCs (SDF-1/HOXB4-MSCs) and human umbilical cord blood CD34+ cells significantly improved HSC cell expansion in vitro. More importantly, co-transplantation of CD34+ cells and SDF-1/HOXB4-MSCs markedly increased the hematopoietic potential of irradiated mice as evidenced by the rapid recovery of WBC, PLT and HGB levels in peripheral blood and of BM cellularity. Co-transplantation also markedly improved engraftment of human CD45+ cells in mouse BM. Conclusions: Our study demonstrates that SDF-1/HOXB4-MSCs markedly accelerate hematopoietic recovery and significantly improve survival among mice treated with a lethal dose of irradiation. Therefore, SDF-1/HOXB4-MSCs could have therapeutic value by improving the efficacy of clinical transplantations in patients with defective hematopoiesis.
PMCID: PMC4297337  PMID: 25628780
Mesenchymal stem cells; hematopoietic stem cells; SDF-1 gene; HOXB4 gene; irradiation; hematopoietic reconstitution; NOD/SCID mice
7.  Angiogenesis effect of therapeutic ultrasound on ischemic hind limb in mice 
Although significant progress in bypass surgery and catheter intervention against peripheral artery disease, the number of severe critical limb ischemia (CLI) patients is increasing. Thus, it is crucial to develop new, non-invasive therapeutic strategies. The purpose of this study was to determine the mechanism of therapeutic ultrasound (TUS) on ischemic angiogenesis using mouse model of hindlimb ischemia and the cellular/molecular mechanisms underlying TUS-related neovascularization. The hindlimb ischemic mice were exposed to extracorporeal TUS for 3, 6, 9 minute per day (1 MHz, 0.3 W/cm2) until day 14 after left femoral artery ligation. Increased blood perfusion and capillary density were determined following 9 min of TUS compared with ischemic group. Moreover, TUS treatment increased the protein levels of vascular endothelial growth factor (VEGF), hypoxic inducible factor-1α (HIF-1α), endothelial nitric oxide synthase (eNOS) and p-Akt in vivo. TUS promoted capillary-like tube formation, migration and motility of human umbilical venous endothelial cells (HUVECs). Furthermore, the protein expressions of VEGF, eNOS and p-Akt were increased after TUS treatment. In conclusion, TUS therapy promotes postnatal neovascularization through multiple angiogenic pathways in mice model of ischemic hindlimb.
PMCID: PMC4297338  PMID: 25628781
Therapeutic ultrasound; angiogenesis; critical limb ischemia; VEGF
8.  Role of PDGFR-β/PI3K/AKT signaling pathway in PDGF-BB induced myocardial fibrosis in rats 
Objective: To investigate the role of PDGFR-β/PI3K/AKT signaling pathway in the myocardial fibrosis. Methods: CFs were divided into following 4 groups: control group (CON), PDGF-BB group (P), PDGF-BB+IMA group (IMA), and PDGF-BB+LY294002 (LY). Results: Immunofluorescence staining showed about 90% of cells were positive for vimentin and 10% for α-SMA. After incubation for 7 days, fluorescence microscopy revealed more than 90% of cells were positive for α-SMA, which was significantly higher than that in CON group (P < 0.01), but markedly lower than that in IMA group and LY group (P < 0.01). The mRNA and protein expression of PDGFR-β, Col I, Col III, PI3K and Akt increased dramatically at 48 h after PDGF-BB treatment when compared with CON group (P < 0.01). However, IMA and LY294002 significantly inhibited the expression of PDGFR-β and p-PI3K (P < 0.05). In addition, the mRNA expression of PDGFR-β, PI3K and Akt in IMA group and LY group was also markedly lower than those in P group (P < 0.01), and the mRNA and protein expression of Col I and Col III reduced remarkably when compared with P group (P < 0.01). Of note, the mRNA expression of PDGFR-α was comparable among 4 groups, and PDGFR-β expression after PDGF-BB treatment increased significantly when compared with PDGFR-α expression (P < 0.01). Conclusion: PDGF-BB may induce CF proliferation and their transformation into myofibroblasts, which leads to increased synthesis of collagen, resulting in myocardial fibrosis. This is closely associated with PDGFR-β, but not PDGFR-α. PDGFR-β/PI3K/Akt signaling pathway is involved in the PDGF-BB induced myocardial fibrosis.
PMCID: PMC4297339  PMID: 25628782
Myocardial fibrosis; myofibroblasts; platelet-derived growth factor; signaling pathway
9.  Multi-cellular interactions sustain long-term contractility of human pluripotent stem cell-derived cardiomyocytes 
Therapeutic delivery of cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) represents a novel clinical approach to regenerate the injured myocardium. However, poor survival and contractility of these cells are a significant bottleneck to their clinical use. To better understand the role of cell-cell communication in enhancing the phenotype and contractile properties of hPSC-CMs, we developed a three-dimensional (3D) hydrogel composed of hPSC-CMs, human pluripotent stem cell-derived endothelial cells (hPSC-ECs), and/or human amniotic mesenchymal stem cells (hAMSCs). The objective of this study was to examine the role of multi-cellular interactions among hPSC-ECs and hAMSCs on the survival and long-term contractile phenotype of hPSC-CMs in a 3D hydrogel. Quantification of spontaneous contractility of hPSC-CMs in tri-culture demonstrated a 6-fold increase in the area of contractile motion after 6 weeks with characteristic rhythmic contraction frequency, when compared to hPSC-CMs alone (P < 0.05). This finding was supported by a statistically significant increase in cardiac troponin T protein expression in the tri-culture hydrogel construct at 6 weeks, when compared to hPSC-CMs alone (P < 0.001). The sustained hPSC-CM survival and contractility in tri-culture was associated with a significant upregulation in the gene expression of L-type Ca2+ ion channel, Cav1.2, and the inward-rectifier potassium channel, Kir2.1 (P < 0.05), suggesting a role of ion channels in mediating these processes. These findings demonstrate that multi-cellular interactions modulate hPSC-CM phenotype, function, and survival, and they will have important implications in engineering cardiac tissues for treatment of cardiovascular diseases.
PMCID: PMC4297340  PMID: 25628783
Induced pluripotent stem cell; differentiation; cardiomyocyte; endothelial cell; mesenchymal stem cell; cardiac patch
10.  Col10a1-Runx2 transgenic mice with delayed chondrocyte maturation are less susceptible to developing osteoarthritis 
Osteoarthritis (OA) is the most common joint disease affecting close to 27 million Americans. The pathological change of OA joint is characterized by cartilage degradation and osteophyte formation that have been associated with OA initiation and progression respectively. Upon OA progression, articular chondrocytes undergo hypertrophic differentiation, a process usually occurs only in growth plate chondrocytes during endochondral ossification, suggesting a role of chondrocyte hypertrophy in OA pathogenesis. However, how altered chondrocyte hypertrophy, i.e. accelerated or delayed chondrocyte hypertrophy, influences OA development has not been fully elucidated. We have previously generated transgenic (TG) mice over-expressing Runx2, an essential transcription factor for chondrocyte hypertrophy, using hypertrophic chondrocyte-specific mouse type X collagen gene (Col10a1) control elements. These Col10a1-Runx2 TG mice show delayed chondrocyte hypertrophy and apoptosis in long bone sections of embryonic and new-born mice compared to their wild-type (WT) littermates. Here, we report further analysis of the skeletal phenotypes of these mice at postnatal stages. We have performed histological analysis of 1-month old TG and WT mice. Delayed chondrocyte hypertrophy was also observed in growth plate of TG mice. In addition, μCT analysis showed that the femur length was significantly shorter in TG mice (p = 0.033). Thinner cortical bone and markedly decreased BV/TV were also detected in TG mice compared to their WT littermates (p = 0.027), suggesting that delayed chondrocyte hypertrophy affects postnatal long bone development. Interestingly, histological analysis detected less articular cartilage absorption, while immunohistochemistry assay detected upregulated Sox9 expression in TG mouse joints compared to WT controls, implying that delayed chondrocyte hypertrophy may be OA protective. Indeed, we have performed Tgf-β1 injection and enforced uphill treadmill running (TTR model) to induce OA in TG and WT littermates. The results showed that WT littermates displayed characteristic pathology of fibrotic remodeling at the joint margins and focal cartilage erosion, while the joints in TG mice were essentially protected from remodeling responses, demonstrating that mice with delayed chondrocyte hypertrophy are not susceptible to developing OA. Further translational studies characterizing the role of chondrocyte hypertrophy during OA progression will facilitate identification of therapeutic targets to stop or slow down this degenerative and progressive human joint disease.
PMCID: PMC4297341  PMID: 25628784
Col10a1; Runx2; transgenic mice; Tgf-β1 injection; treadmill running; chondrocyte hypertrophy or maturation; osteoarthritis
11.  Ethanol extract of Portulaca Oleracea L. reduced the carbon tetrachloride induced liver injury in mice involving enhancement of NF-κB activity 
Acute hepatic injury causes high morbidity and mortality world-wide. Management of severe acute hepatic failure continues to be one of the most challenging problems in clinical medicine. In present study, carbon tetrachloride (CCl4) was used to induce acute liver damage in mice and the protective effects of ethanol extract of Portulaca Oleracea L. (PO) were examined. The aminotransferase activities were biochemical estimated and the liver damage was tested by morphological histological analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. The role of PO on the activity of NF-κB was determined by luciferase reporter gene assay and immunohistochemistry. The level of p-p65 was tested by western blot. Our results showed that PO administration on mice would decrease the serum aminotransferase level and reduced the liver histological damage. We also found that nuclear translocation of p65 was enhanced in liver tissues of mice treated with PO compared with control animals. In addition, in cultured hepatic cells, PO increased the NF-κB luciferase reporter gene activity and upregulated the level of phosphorylation of p65, but had no effects on mice liver SOD activity and MDA level. Collectively, PO attenuated CCl4 induced mice liver damage by enhancement of NF-κB activity.
PMCID: PMC4297342  PMID: 25628785
Carbon tetrachloride (CCl4); ethanol extract of Portulaca Oleracea L (PO); liver injury; NF-κB
12.  STAT3 regulation the expression of VEGF-D in HGC-27 gastric cancer cell 
Objective: To explore the potential mechanism of vascular endothelial growth factor D (VEGF-D) contribution to the lymphangiogenesis was regulated by the signal transducer and activator of transcription 3 (STAT3). Methods: We detected the expression in GC tissue, adjacent non-tumor tissue, GC cell lines (AGS, SUN-1, KATO-III, BGC-823, MGC-803, SGC-7901, and HGC-27), and GES-1 cell line. STAT3 siRNA transfection and genome microarray were applied to demonstrate whether the expression of VEGF-D was mediated by the STAT3 in GC. Results: We showed the STAT3, pSTAT3, and VEGF-D expression in GC tissue was significantly higher than those in adjacent non-tumor tissue, respectively. In addition, both STAT3 and VEGF-D mRNA expression was much higher in each GC cell line than those in GES-1 cell line. With STAT3 siRNA transfection, we demonstrated that VEGF-D expression level decreased significantly in HGC-27 cell by using the genome microarray representing STAT3 potential regulation the VEGF-D expression. Conclusion: STAT3, a novel signal transducer inactivating in the GC cell, can contribute to the lymph node metastasis by promoting lymphangiogenesis via up-regulation expression of VEGF-D.
PMCID: PMC4297343  PMID: 25628786
Stomach; neoplasm; signal transducer and activator of transcription 3; vascular endothelial growth factor; lymphatic metastasis
13.  Selected hemostatic parameters in patients with pancreatic tumors 
Introduction: Recent experimental studies have suggested that various coagulation-related molecules may be important players in the development and progression of pancreatic cancer. However, these findings have not yet been verified in a clinical setting. Methods: In this study, we comprehensively examined the levels of multiple hemostatic substances, including prothrombin, antithrombin, plasminogen, thrombin-anti-thrombin (TAT) and plasmin-anti-plasmin (PAP) complexes, as well as, soluble CD40 (sCD40) in patients diagnosed with pancreatic cancer (n = 37) or other tumors (neuroendocrine neoplasms - NEN [n = 7] or solid pseudopapillary tumors-SPT [n = 3]), and healthy individuals (n = 31). Results: We found significantly higher anti-thrombin, PAP and sCD40 levels in patients with pancreatic cancer compared to healthy controls and patients diagnosed with other types of pancreatic tumors (for all, at least p < 0.05). Cancer patients had lower plasminogen concentrations than individuals from the other analyzed groups (for both, p < 0.05). None of the examined coagulation-related parameters was significantly associated with neither systemic sCD40 concentrations nor clinical staging of malignancy. Levels of analyzed molecules were comparable between pancreatic cancer patients presenting with early and advanced disease. Moreover, our study identified a potential diagnostic value of prothrombin/TAT and anti-thrombin/TAT coefficients in detection of pancreatic cancer in humans. However, both of these were inferior to currently used marker-CA19.9. Conclusions: Subclinical hemostatic alterations (mainly in plasmin-related molecules) i) appear as soon as during the earliest stages of the pancreatic adenocarcinoma development in humans, ii) do not seem to alter within progression of the disease nor are associated with clinical staging, iii) are not observed in patients with other types of pancreatic tumors, as well as, iv) do not seem to be associated with elevated sCD40 concentrations in pancreatic cancer patients. Moreover, examined thrombin- and plasmin-related substances do not appear to possess a sufficient diagnostic value to serve as makers of pancreatic adenocarcinoma in humans.
PMCID: PMC4297344  PMID: 25628787
Hemostasis; neuroendocrine tumors; pancreatic cancer; plasmin; thrombin
14.  Inducible costimulatory molecule deficiency induced imbalance of Treg and Th17/Th2 delays rejection reaction in mice undergoing allogeneic tracheal transplantation 
Objective: This study aimed to investigate the role of inducible costimulatory molecule (ICOS) pathway in the rejection reaction of mice undergoing allogeneic tracheal transplantation. Methods: The bronchus was separated from wide-type (WT) BalB/c mice and transplanted into WT BalB/c mice, C57 mice and icos-/- mice to prepare the obliterative bronchiolitis (OB) animal model. The transplanted bronchus was pathologically examined; flow cytometry was done to detect the T cell subsets and activity of the bronchus and spleen of recipient mice. Results: 21 d after transplantation, evident rejection reaction was observed and the proportion of Th2 and Th17 cells increased significantly in the bronchus and spleen in C57 mice receiving allogeneic tracheal transplantation when compared with mice with autologous transplantation, but the proportion of Treg cells was comparable between them. When compared with WT BalB/c mice, the proportion of Th2, Th17 and Treg cells reduced markedly and rejection reaction was attenuated in icos-/- mice receiving tracheal transplantation, although rejection reaction was still noted. Conclusion: icos knockout may delay the rejection reaction after tracheal transplantation, which might be ascribed to the imbalance among Th2, Th17 and Treg cells.
PMCID: PMC4297345  PMID: 25628788
Lung transplantation; inducible costimulatory molecule; chronic rejection; bronchiolitis obliterans; Th2; Th17
15.  Cloning, expression, and analysis of a cDNA coding for the Dermatophagoides farinae group 21 (Der f 21) allergen 
Domestic mite species like Dermatophagoides farinae induce allergies in people worldwide. Here, the cDNA coding for group 21 allergen of Dermatophagoides farinae (Hughes; Acari: Pyroglyphidae) from China was cloned, sequenced, and expressed in E. coli to aid in the development of diagnostic and treatment options for domestic mite hypersensitivity. First, the Der f 21 cDNA fragment was synthesized by RT-PCR; the confirmed full-length sequence comprised 411 nucleotides. The cDNA was ligated to the vector pCold-TF to construct an expression plasmid, pCold-TF-Der f 21. pCold-Tf-Der f 21 was transformed into E. coli BL21 cells, and its expression was induced by IPTG treatment. SDS-PAGE showed a specific band at the predicted molecular weight of Der f 21, demonstrating its successful expression. The recombinant fusion protein was obtained and its structure and molecular weight were confirmed by MALDI-TOF/TOF. Bioinformatics analysis revealed that the protein contained a signal peptide of 17 amino acids. The molecular weight of the mature Der f 21 allergen was approximately 14.16 kDa with a theoretical pI of 4.87. Its predicted secondary structure comprises a-helix (84.03%), extension chain (1.68%), and random coil (14.29%). The successful cloning of Der f 21 and a basic bioinformatics analysis of the protein provide a foundation for further study of this allergen in diagnosis and treatment of domestic mite hypersensitivity.
PMCID: PMC4297346  PMID: 25628789
Dermatophagoides farinae; recombinant allergen; molecular cloning; domestic mite; Der f 21
16.  Epithelial-mesenchymal transition markers in lymph node metastases and primary breast tumors - relation to dissemination and proliferation 
Epithelial-mesenchymal transition (EMT) was shown to enhance metastatic abilities of cancer cells, but it remains elusive in clinical samples. Moreover, EMT is rarely studied in lymph node metastases (LNM), thus limiting our understanding of its role outside of the primary tumors (PT). We collected a set of samples including triplets - PT, circulating tumor cells (CTCs)-enriched blood samples and LNM from 108 early breast cancer patients. With immunohistochemistry we analyzed levels of EMT effectors – E-cadherin, vimentin and N-cadherin in LNM, central areas and margins of PT. Additionally, expression of EMT core regulators TWIST1, SNAI1, SNAI2 was measured with RT-qPCR. Patients with E-cadherin loss had CTCs in 45% of the cases in comparison to 23% with normal E-cadherin level (P = 0.05). Mesenchymal phenotype of CTCs-enriched blood fractions was five-times more frequent in patients with E-cadherin loss in PT compared to PT with normal E-cadherin levels (P = 0.01). Epithelial/mesenchymal status of matched samples at different stages of dissemination was frequently discordant, especially for pairs involving CTCs, indicating high plasticity of tumor cells. LNM showed increased expression of TWIST1, SNAI1, SNAI2 accompanied by decreased Ki67 labeling index, with median Ki67 of 15% in PT and 10% in LNM (P = 0.0002). Our findings demonstrate that E-cadherin loss, not only in PT margin, might lead to seeding of especially malignant CTCs with mesenchymal phenotype. In comparison to PT, cells in LNM re-express E-cadherin, upregulate EMT transcription factors and reduce cell division rate, which could be viewed as their long-term survival strategy.
PMCID: PMC4297347  PMID: 25628790
Epithelial-mesenchymal transition; epithelial-mesenchymal plasticity; lymph node metastases; circulating tumor cells; Ki67
17.  Effects of low-dose spironolactone combined with irbesartan on cardiac hypertrophy induced by pressure overload in rats 
Background: The mineralocorticoid receptor (MR) blockade in the heart is an attractive therapeutic option for the treatment of heart failure. However, the use of MR antagonist is limited by an increased incidence of hyperkalemia owing to MR blockade in the kidney. This study was designed to evaluate and compare the effectiveness of a low, non-pressure-lowering dose of spironolactone (Sp) with that of a conventional blood pressure-lowering dose combined with irbesartan on pathological cardiac remodeling as well as serum potassium level in pressure-overload rats. Methods: The pressure-overloaded myocardial remodelling was produced by partial abdominal aortic constriction (PAAC) in rats. Four weeks after PAAC, animals were respectively treated with vehicle, irbesartan (15 mg/kg) alone, low-dose Sp (1 mg/kg) or conventional-dose of Sp (20 mg/kg) in combination with irbesartan for consecutive four weeks. Results: The result demonstrated that compared to irbesartan monotherapy, the combination of irbesartan and spironolactone both in low- and conventional-dose exhibited additional cardioprotection against PAAC-induced cardiac remodelling. Low-dose spironolactone was as effective in inhibiting cardiac hypertrophy, fibrosis and in improving diastolic function as high dose. Low-dose spironolactone did not lead to a rise in potassium serum levels, but high dose did. Conclusions: This study suggests that combined low dose of spironolactone and irbesartan may be an effective and safety therapeutic strategy for cardiac hypertrophy and heart failure.
PMCID: PMC4297348  PMID: 25628791
Spironolactone; irbesartan; myocardial remodelling; aldosterone
18.  Post-insult valproate treatment potentially improved functional recovery in patients with acute middle cerebral artery infarction 
Animal stroke models suggest that valproate has multiple neuroprotective mechanisms against ischemic brain damage. This study investigated whether valproate improves functional recovery in patients with acute middle cerebral artery (MCA) infarction. This was an open-label controlled trial. Three to 24 hours after acute MCA infarction, patients were assigned to either the valproate group (n = 17) or the non-valproate group (n = 17). The valproate group received intravenous valproate (400 mg) at enrollment, and then every 12 hours for three days, followed by oral valproate (500 mg) every 12 hours for three months. Neurological function, laboratory data, and brain magnetic resonance imaging were examined at stroke onset, and at two-week and three-month follow-up. No significant differences were observed between the groups with regard to demographics or baseline characteristics. All patients were elderly, had a high pretreatment score on the NIH stroke scale (NIHSS), and slow stroke lesion growth with a final large infarct volume at two-week follow-up. At the three-month follow-up, functional outcome between pre- and post-treatment had improved significantly in the valproate group (NIHSS, p = 0.004; modified Rankin scale (mRS), p = 0.007; Barthel index (BI), p = 0.001). No such improvement was noted in the NIHSS or mRS for the non-valproate group, though mild improvement was seen on the BI (p = 0.022). This open-label trial is the first to demonstrate that valproate treatment markedly improves functional outcome in patients with acute MCA infarction.
PMCID: PMC4297349  PMID: 25628792
Ischemic stroke; valproate; stroke outcome; neuroprotection
19.  Somatostatin receptors in gastrointestinal stromal tumors: new prognostic biomarker and potential therapeutic strategy 
Somatostatin receptors (SSTRs) already act as important roles in gastroenteropancreatic neuroendocrine tumors (GEP-NETs) with high expression levels for prognosis predicting and octreotide LAR treatment purposes but less noticed in gastrointestinal stromal tumors (GISTs). Our study aims to fully evaluate the expression levels and prognostic values of SSTRs in GIST patients. For SSTRs expression detection, qPCR were used in 25 fresh GIST specimens, and then, 453 GIST samples (405 GISTs with operation only and 48 with imatinib adjuvant therapy after surgery) were collected for tissue microarrays (TMAs) construction and confirmed by immunohistochemistry (IHC). Clinicopathological data were confirmed by pathological diagnosis and clinical recorders, recurrence-free survivals (RFS) were evaluated in 453 GIST patients. With IHC performed, SSTR1 and SSTR2 present high positive proportion (81.9% and 87.6%) in 453 GISTs in our study, and positive expression rates of SSTR3, SSTR4 and SSTR5 are 56.1%, 8.8% and 47.2%, respectively. SSTR2 and SSTR5 negative expression are associated with decreased RFS when compared to positive cases by Kaplan-Meier survival analyses with log-rank test and univariate analysis in GISTs, furthermore, SSTR2 was an independent prognostic indicator for GISTs by multivariate analysis. In our study, detection of SSRT2 and SSTR5 expression helps to predict different prognosis in GIST patients. SSTR2 is a novel independent prognostic biomarker for GISTs. With high expression performance of SSTRs in GISTs, new therapeutic strategies such as octreotide or pasireotide LAR could be taken into consideration in selected advanced GIST patients.
PMCID: PMC4297350  PMID: 25628793
Gastrointestinal stromal tumor; somatostatin receptor; octreotide; pasireotide; prognosis
20.  MiR-223 suppresses endometrial carcinoma cells proliferation by targeting IGF-1R 
MicroRNAs were recently found to participate in oncogenesis and growth of various tumors. We hypothesized that microRNA-223 (miR-223) plays a role in endometrial carcinoma growth. In this study, we transfected RL95-2 cells with lentivirus containing miR-223 precursor to establish a miR-223 over-expression model. Proliferation of the cells was greatly inhibited when miR-223 was over-expressed, and cell cycle progress was blocked in G0/G1 phase. To investigate the mechanisms involved, we scanned the putative target genes of miR-223 using bioinformatics, and confirmed that insulin-like growth factor-1 receptor (IGF-1R) was a functional target of miR-223 using quantitative PCR, Western blot and luciferase reporter assay. Meanwhile, over-expressed miR-223 was found to regulate the expression of IGF-1R by repressing protein translation. Silencing IGF-1R with small interfering RNA resulted in similar effect as miR-223 overexpression. Therefore, our data suggest that miR-223 regulates RL95-2 cells proliferation and cell cycle progress by targeting IGF-1R.
PMCID: PMC4297351  PMID: 25628794
MiR-223; insulin-like growth factor-1 receptor; cell proliferation; in vitro; endometrial carcinoma cell
21.  Serum Dickkopf-1 (DKK1) is significantly lower in patients with lung cancer but is rapidly normalized after treatment 
DKK1 is a secreted glycoprotein that inhibits Wnt/β-catenin signaling but may up-regulate the nonconanical Wnt signaling. Consistent with its inhibitory function in Wnt/β-catenin signaling, aberrant DKK1 expression has been observed in many types of human cancers, while contradicting findings have been reported in other studies. There are also several studies on serum DKK1 levels in various cancers with conflicting findings. In the present study, serum DKK1 was determined in 217 non- small cell lung cancer (NSCLC) patients, 35 small cell lung cancer (SCLC) patients and 286 matched healthy controls using a commercially available ELISA assay kit. Compared to healthy controls, serum DKK1 level was significantly lower in NSCLC (p < 10-28) and SCLC (p <10-4) patients. Interestingly, serum DKK1 level was higher in NSCLC patients in stage IV (p < 0.0005), with lymph node involvement (p < 0.0002) or with metastasis (p < 0.0001), suggesting that DKK1 may promote metastasis. After surgery and/or chemotherapy, serum DKK1 level is rapidly increased and reached levels observed in healthy controls in most patients. The degree of post therapeutic DKK1 increase varied in different treatment regimens. Our results thus provide strong evidence for the reduced levels of serum DKK1 in both types of lung cancer. However, in the context of all published studies, DKK1 appears to have a dichotomous role in cancer and its effect in a given cancer type or even a given cancer patient is likely to depend on the molecular context of the patient.
PMCID: PMC4297352  PMID: 25628795
DKK1; biomarker; lung cancer; wnt signaling
22.  Conceptualization and utilization of blood stasis syndrome among doctors of Korean medicine: results of a web-based survey 
This survey aimed to assess the conceptualization of and utilization of blood stasis syndrome within traditional East Asian medicine among Doctors of Korean Medicine (DKMs). A survey was conducted with 22 questions classified into four categories (the present status, problems and requirements of a BSS diagnosis; the concept, diseases and prescriptions regarding BSS; the present status and problems with the Korean standard classification of diseases (KCD) relevant to BSS and the demographic characteristics of the survey respondents). A total of 17,550 DKMs affiliated with the association of Korean medicine were sent surveys via e-mail. Of the 678 respondents, more than half (53%) had difficulties with the diagnosis of BSS because objective measurement methods were not readily available. Most respondents (88%) thought that the development of an objective diagnostic method for BSS was necessary. Regarding the concept of blood stasis, “an abnormal mass in organ and tissue” considered the most significant indicator, followed by extravasated blood, the blood circulating sluggishly and a disorder of the blood cells. According to 606 DKMs, a traumatic injury was the most frequent reason for BSS in the clinic. And the Dangkwisoo-san was most frequently mentioned prescription for BSS in the clinic. The majority of respondents (76%) reported that it was necessary for an additional code of BSS in KCD. Our data suggest the need to develop more objective diagnostic tools for BSS diagnosis. Future research into BSS should consider DKMs’ perceptions of BSS, diseases relevant to BSS and KCD codes. However, we cannot completely discount the possibility that the low response rate could indicate a biased selection of respondents and limit the interpretations of our study results.
PMCID: PMC4297353  PMID: 25628796
Blood stasis; traditional Korean medicine; traditional East Asian medicine; survey
23.  Association between miR-125a rs12976445 and survival in breast cancer patients 
MicroRNAs (miRNAs) act as an oncogene or a tumor suppressor by negatively regulating target genes. Genetic variants in miRNA genes confer susceptibility to cancer and risk of death in cancer patients. The aim of this study was to investigate whether miRNA polymorphisms were associated with survival in breast cancer patients. Five miRNA polymorphisms (miR-26a1 rs7372209, miR-125a rs12976445, miR-218 rs11134527, miR-423 rs6505162, and miR-608 rs4919510) were genotyped in 196 breast cancer patients. We found that miR-125a rs12976445 was significantly associated with survival in codominant, recessive, and dominant models. However, only association under the codominant model remained significant after adjustment for lymph node metastasis, TNM stage, estrogen receptor, and progesterone receptor. Furthermore, this effect remained in stratification analysis. In conclusion, our results provide evidence that miR-125a rs12976445 may serve as a prognostic biomarker for breast cancer. Further large-scale studies are required to confirm these findings.
PMCID: PMC4297354  PMID: 25628797
MicroRNA; miR-125a; polymorphism; breast cancer; prognosis
24.  Role of lumbricus extract in the nerve amplification effect during peripheral nerve regeneration 
Among the methods of the peripheral nerve repair, artificial conduit bridging surgery is superior to epineurium and perineurium neurorrhaphy because of supplying enough space for nerve regeneration. Artificial conduit provides important microenvironment for peripheral nerve regeneration, especially for nerve amplification effect. Amplification phenomenon has been demonstrated in many studies using artificial conduit. When a finer nerve is used as a donor to connect to a distal nerve after injury, the donor nerve regenerates more lateral buds than its own fibers, which grow into distal endoneurial tubes and finally dominate the target organs. In this study, we used artificial conduit to investigate the amplification phenomenon in rats treated with Lumbricus extract as adjuvant treatment. The rats were divided into three groups at random. In the surgical groups, the proximal common peroneal nerve was used as a donor nerve to connect the distal tibial nerve. Rats in the normal group were not performed surgery. Postoperatively, the treatment group was administered Lumbricus extract as adjuvant treatment, while the model group and normal group were not given treatment. The results showed that the nerve conduction velocity, the morphometric measurements, the histological analysis and the amplification ratio in the treatment group were better than in the model group.
PMCID: PMC4297355  PMID: 25628798
Traditional Chinese medicine; lumbricus extract; amplification effect; lateral bud; peripheral nerve regeneration; nerve conduction velocity
25.  Exercise training and immune crosstalk in breast cancer microenvironment: exploring the paradigms of exercise-induced immune modulation and exercise-induced myokines 
Observational research suggests that exercise may reduce the risk of breast cancer and improve survival. One proposed mechanism for the protective effect of aerobic exercise related to cancer risk and outcomes, but has not been examined definitively, is the immune response to aerobic exercise. Two prevailing paradigms are proposed. The first considers the host immune response as modifiable by aerobic exercise training. This exercise-modulated immune-tumor crosstalk in the mammary microenvironment may alter the balance between tumor initiation and progression versus tumor suppression. The second paradigm considers the beneficial role of exercise-induced, skeletal muscle-derived cytokines, termed “myokines”. These myokines exert endocrine-like effects on multiple organs, including the mammary glands. In this systematic review, we i) define the role of macrophages and T-cells in breast cancer initiation and progression; ii) address the two paradigms that support exercise-induced immunomodulation; iii) systematically assessed the literature for exercise intervention that assessed biomarkers relevant to both paradigms in human intervention trials of aerobic exercise training, in healthy women and women with breast cancer; iv) incorporated pre-clinical animal studies and non-RCTs for background discussion of putative mechanisms, through which aerobic exercise training modulates the immunological crosstalk, or the myokine-tumor interaction in the tumor microenvironment; and v) speculated on the potential biomarkers and mechanisms that define an exercise-induced, anti-tumor “signature”, with a view toward developing relevant biomarkers for future aerobic exercise intervention trials.
PMCID: PMC4212920  PMID: 25360210
Exercise; breast cancer; immune; myokines; translational; immunotherapy

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