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1.
Cell Transplant ; 21(1): 313-32, 2012.
Article in English | MEDLINE | ID: mdl-21669041

ABSTRACT

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the degeneration of dopaminergic (DA) neurons in the midbrain. Induced pluripotent stem (iPS) cells have shown potential for differentiation and may become a resource of functional neurons for the treatment of PD. However, teratoma formation is a major concern for transplantation-based therapies. This study examined whether functional neurons could be efficiently generated from iPS cells using a five-step induction procedure combined with docosahexaenoic acid (DHA) treatment. We demonstrated that DHA, a ligand for the RXR/Nurr1 heterodimer, significantly activated expression of the Nurr1 gene and the Nurr1-related pathway in iPS cells. DHA treatment facilitated iPS differentiation into tyrosine hydroxylase (TH)-positive neurons in vitro and in vivo and functionally increased dopamine release in transplanted grafts in PD-like animals. Furthermore, DHA dramatically upregulated the endogenous expression levels of neuroprotective genes (Bcl-2, Bcl-xl, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor) and protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced apoptosis in iPS-derived neuronal precursor cells. DHA-treated iPS cells significantly improved the behavior of 6-hydroxydopamine (6-OHDA)-treated PD-like rats compared to control or eicosapentaenoic acid-treated group. Importantly, the in vivo experiment suggests that DHA induces the differentiation of functional dopaminergic precursors and improves the abnormal behavior of 6-OHDA-treated PD-like rats by 4 months after transplantation. Furthermore, we found that DHA treatment in iPS cell-grafted rats significantly downregulated the mRNA expression of embryonic stem cell-specific genes (Oct-4 and c-Myc) in the graft and effectively blocked teratoma formation. Importantly, 3 Tesla-magnetic resonance imaging and ex vivo green fluorescence protein imaging revealed that no teratomas were present in transplanted grafts of DHA-treated iPS-derived DA neurons 4 months after implantation. Therefore, our data suggest that DHA plays a crucial role in iPS differentiation into functional DA neurons and that this approach could provide a novel therapeutic approach for PD treatment.


Subject(s)
Docosahexaenoic Acids/pharmacology , Dopaminergic Neurons/cytology , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/transplantation , Parkinsonian Disorders/therapy , Stem Cell Transplantation/methods , Teratoma/prevention & control , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Animals , Apoptosis/drug effects , Cells, Cultured , Dopamine/metabolism , Dopaminergic Neurons/metabolism , Gene Expression Regulation , Induced Pluripotent Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Neurogenesis , Nuclear Receptor Subfamily 4, Group A, Member 2/biosynthesis , Oxidopamine/pharmacology , Parkinsonian Disorders/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Tyrosine 3-Monooxygenase/biosynthesis
2.
Mol Carcinog ; 51(12): 939-51, 2012 Dec.
Article in English | MEDLINE | ID: mdl-21976141

ABSTRACT

Gastric carcinoma is one of the most common and mortal types of malignancy worldwide. To date, the mechanisms controlling its aggressiveness are not yet fully understood. Notch signal pathway can function as either an oncogene or a tumor suppressor in tumorigenesis. Four members (Notch1-4) of Notch receptors were found in mammals and each exhibits distinct roles in tumor progression. Previous study showed that the activated Notch1 receptor promoted gastric cancer progression through cyclooxygenase-2 (COX-2). This study addressed whether Notch2 signal pathway is also involved in gastric cancer progression. Constitutive expression of Notch2 intracellular domain (N2IC), the activated form of Notch2 receptor, promoted both cell proliferation and xenografted tumor growth of human stomach adenocarcinoma SC-M1 cells. The colony formation, migration, invasion, and wound-healing abilities of SC-M1 cells were enhanced by N2IC expression, whereas these abilities were suppressed by Notch2 knockdown. Similarly, Notch2 knockdown inhibited cancer progressions of AGS and AZ521 gastric cancer cells. Expression of N2IC also caused epithelial-mesenchymal transition in SC-M1 cells. Furthermore, N2IC bound to COX-2 promoter and induced COX-2 expression through a CBF1-dependent manner in SC-M1 cells. The ability of N2IC to enhance tumor progression in SC-M1 cells was suppressed by knockdown of COX-2 or treatment with NS-398, a COX-2 inhibitor. Moreover, the suppression of tumor progression by Notch2 knockdown in SC-M1 cells was reversed by exogenous COX-2 or its major enzymatic product PGE(2) . Taken together, this study is the first to demonstrate that the Notch2-COX-2 signaling axis plays an important role in controlling gastric cancer progression.


Subject(s)
Cyclooxygenase 2/metabolism , Receptor, Notch2/physiology , Stomach Neoplasms/pathology , Animals , Base Sequence , Cell Line, Tumor , DNA Primers , Disease Progression , Gene Knockdown Techniques , Humans , Mice , Mice, Nude , Real-Time Polymerase Chain Reaction , Receptor, Notch2/genetics , Stomach Neoplasms/genetics
3.
J Atheroscler Thromb ; 18(11): 966-80, 2011.
Article in English | MEDLINE | ID: mdl-21817823

ABSTRACT

AIM: Mesenchymal stem cells (MSCs) are a multipotent cell type that can differentiate into non-hematopoietic cells, such as adipocytes. Adipocyte tissue is central to the regulation of energy balance. Two functionally different types of fat are present in mammals. White adipose tissue is the primary site for triglyceride storage, while brown adipose tissue is specialized in energy expenditure. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) controls several aspects of mitochondrial biogenesis. In this study, we hypothesized that PGC-1α plays a role in brown fat differentiation of MSCs. METHODS: Immortalized human MSCs were infected with adenovirus carrying PGC-1α cDNA to create PGC-1α-expressing MSCs. RESULTS: The genetic profiling of PGC-1α-expressing MSCs shows the significant increase of genes related to mitochondrial functions and lipid metabolism compared to that of MSCs. When expressed in MSCs, PGC-1α activates robust mitochondrial biogenesis and respiration. The increase of oxygen consumption and reactive oxygen species represents a cellular readout of increased activity of the respiratory chain. The expression of thermogenic markers, such as cytochrome C and complex II, was significantly increased in MSCs with treatment of adenovirus expressing PGC-1α. Moreover, PGC-1α markedly inhibited the osteogenesis of MSCs under osteogenic induction. During adipogenesis, PGC-1α-expressing MSCs showed a significant increase in brown fat markers and a decrease in white fat markers. Notably, PGC-1α knockdown inhibited adipocyte differentiation of MSCs. CONCLUSIONS: In summary, our data reveal an important role of PGC-1α in promoting brown fat differentiation of MSCs, and provide a new therapeutic approach for the treatment of obesity.


Subject(s)
Adipose Tissue, Brown/cytology , Adipose Tissue, Brown/metabolism , Biomarkers/metabolism , Cell Differentiation , Heat-Shock Proteins/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Transcription Factors/metabolism , Adipogenesis , Adipose Tissue, White/cytology , Adipose Tissue, White/metabolism , Cell Respiration , Energy Metabolism , Fluorescent Antibody Technique , Gene Expression Profiling , Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Proteins/genetics , Humans , Mitochondrial Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Oxygen Consumption , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Reactive Oxygen Species/metabolism , Real-Time Polymerase Chain Reaction , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics
4.
J Cell Mol Med ; 15(3): 612-24, 2011 Mar.
Article in English | MEDLINE | ID: mdl-20158571

ABSTRACT

MafA is a pancreatic transcriptional factor that controls ß-cell-specific transcription of the insulin gene. However, the role of MafA in the regulation of pancreatic transdifferentiation and reprogramming in human stem cells is still unclear. In this study, we investigate the role of MafA in placenta-derived multipotent stem cells (PDMSCs) that constitutively expressed Oct-4 and Nanog. PDMSCs were isolated and transfected with MafA using a lentivector. Our results showed that overexpression of MafA in PDMSCs significantly up-regulated the expression of pancreatic development-related genes (Sox17, Foxa2, Pdx1 and Ngn3). Microarray analysis suggested that the gene expression profile of MafA-overexpressing PDMSCs was similar to that of pancreas and islet tissues. MafA increased the expression levels of the mRNAs of NKx2.2, Glut2, insulin, glucagons and somatostatin, and further facilitated the differentiation of PDMSCs into insulin(+) cells. The glucose-stimulated responses to insulin and c-peptide production in MafA-overexpressing PDMSCs were significantly higher than in PDMSCs with vector control. Our results indicated that MafA-overexpressing PDMSCs were more resistant to oxidative damage and oxidative damage-induced apoptosis than PDMSCs carrying the vector control were. Importantly, the expression of MafA in PDMSCs xenotransplanted into immunocompromised mice improved the restoration of blood insulin levels to control values and greatly prolonged the survival of graft cells in immunocompromised mice with STZ-induced diabetes. In summary, these data suggest that MafA plays a novel role in the reprogramming of stem cells into pancreatic ß-progenitors, promotes the islet-like characteristics of PDMSCs, as well as functionally enhances insulin production to restore the regulation of blood glucose levels in transplanted grafts.


Subject(s)
Cell Differentiation/genetics , Insulin-Secreting Cells/metabolism , Islets of Langerhans/metabolism , Maf Transcription Factors, Large/genetics , Multipotent Stem Cells/metabolism , Animals , Blood Glucose/metabolism , Blotting, Western , Cell Survival/genetics , Cells, Cultured , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Experimental/surgery , Female , Fluorescent Antibody Technique , Gene Expression Profiling , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Homeobox Protein Nkx-2.2 , Homeodomain Proteins , Humans , Insulin-Secreting Cells/cytology , Islets of Langerhans/cytology , Maf Transcription Factors, Large/metabolism , Mice , Mice, SCID , Multipotent Stem Cells/cytology , Multipotent Stem Cells/transplantation , Nuclear Proteins , Oligonucleotide Array Sequence Analysis , Placenta/cytology , Pregnancy , Reverse Transcriptase Polymerase Chain Reaction , Stem Cell Transplantation , Transcription Factors , Transfection , Transplantation, Heterologous
5.
J Pharmacol Exp Ther ; 335(3): 817-29, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20855446

ABSTRACT

Studies have demonstrated that mesenchymal stem-like cells can be isolated from endometrium. However, the potential of endometrial-derived stem cells to differentiate into insulin-positive cells and functionally secrete insulin remains undetermined. We isolated endometrial mesenchymal stem-like cells (EMSCs) from human endometrial tissue from six donors. The insulin-secreting function of EMSCs was further analyzed in vitro and in transplanted grafts in vivo. We successfully isolated EMSCs from human endometrium, and our results showed that EMSCs expressed high levels of stemness genes (Nanog, Oct-4, Nestin). Under specific induction conditions for 2 weeks, EMSCs formed three-dimensional spheroid bodies (SBs) and secreted C-peptide. The high insulin content of SB-EMSCs was confirmed by enzyme-linked immunosorbent assay, and glucose responsiveness was demonstrated by measuring glucose-dependent insulin secretion. Using cDNA microarrays, we found that the expression profiles of SB-EMSCs are related to those of islet tissues. Insulin and C-peptide production in response to glucose was significantly higher in SB-EMSCs than in undifferentiated EMSC controls. Furthermore, upon differentiation, SB-EMSCs displayed increased mRNA expression levels of NKx2.2, Glut2, insulin, glucagon, and somatostatin. Our results also showed that SB-EMSCs were more resistant to oxidative damage and oxidative damage-induced apoptosis than fibroblasts from the same patient. It is noteworthy that SB-EMSCs xenotransplanted into immunocompromised mice with streptozotocin-induced diabetes restored blood insulin levels to control values and greatly prolonged the survival of graft cells. These data suggest that EMSCs not only play a novel role in the differentiation of pancreatic progenitors, but also can functionally enhance insulin production to restore the regulation of blood glucose levels in an in vivo transplantation model.


Subject(s)
Cell Differentiation/physiology , Endometrium/cytology , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Mesenchymal Stem Cells/cytology , Adipocytes/cytology , Adipocytes/metabolism , Adult , Animals , Antigens, CD/metabolism , Apoptosis/drug effects , Blood Glucose/metabolism , C-Peptide/metabolism , Chondrocytes/cytology , Chondrocytes/metabolism , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/therapy , Down-Regulation/genetics , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression/genetics , Glucagon/genetics , Glucagon/metabolism , Glucose/pharmacology , Glutathione/metabolism , Homeobox Protein Nkx-2.2 , Homeodomain Proteins , Humans , Insulin/blood , Insulin/genetics , Insulin/metabolism , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/transplantation , Interleukin-1beta/pharmacology , Mesenchymal Stem Cells/metabolism , Mice , Mice, SCID , Middle Aged , Neurons/cytology , Neurons/metabolism , Nuclear Proteins , Osteocytes/cytology , Osteocytes/metabolism , Reactive Oxygen Species/metabolism , Spheroids, Cellular/cytology , Spheroids, Cellular/drug effects , Spheroids, Cellular/metabolism , Spheroids, Cellular/transplantation , Transcription Factors , Transplantation, Heterologous , Up-Regulation/genetics
6.
J Atheroscler Thromb ; 17(9): 970-9, 2010 Sep 30.
Article in English | MEDLINE | ID: mdl-20644332

ABSTRACT

AIM: Silencing information regulator (SirT1), a NAD-dependent histone deacetylase, is an essential mediator of longevity in normal cells by calorie restriction. SirT1 has many biological functions, including transcription regulation, cell differentiation inhibition, cell cycle regulation, and anti-apoptosis. Resveratrol (RV)-induced SirT1 activation also improves endothelial dysfunction and suppresses vascular inflammation. In this study, we investigated the roles of RV-induced SirT1 activation in endothelial cells under oxidative stress. METHODS: SirT1 mRNA expression levels were examined in the endothelium layer (endothelial cells) of cardiac coronary vessels from patients receiving coronary artery bypass graft surgery (CABG) surgery and aged rats using reverse transcriptase polymerase chain reaction (RT-PCR). To further explore the effect of SirT1 activation on oxidative stress-induced aging, senescence-associated ß-galactosidase (SA-ß-gal) expression in RV-treated human umbilical vein endothelial cells (HUVECs) with or without H(2)O(2) treatment was evaluated. RESULTS: SirT1 expression was decreased in aged and atherosclerotic vessels in vivo, and significantly reduced in endothelial cells purified from vessel tissues. Furthermore, SirT1 levels were dose-dependently increased in RV-treated HUVECs. The SA-ß gal assay showed that RV inhibited the senescent phenotype of H(2)O(2)-treated HUVECs. Reactive oxygen species (ROS) production and the percentage of cells positive for SA-ß gal were significantly increased in siRNA-SirT1 (knockdown of SirT1 expression)-treated HUVEC cells. Importantly, the treatment effect of RV was significantly abolished in the oxidative effects of H(2)O(2)-treated HUVECs by siRNA-SirT1. CONCLUSION: Our data suggested that SirT1 could be a crucial factor involved in the endothelial cells of atherosclerotic CAGB patients and aging rats. RV is a potential candidate for preventing oxidative stress-induced aging in endothelial cells. RV may also prevent ROS-induced damage via increased endothelial SirT1 expression.


Subject(s)
Endothelial Cells/drug effects , Endothelial Cells/metabolism , Sirtuin 1/metabolism , Stilbenes/pharmacology , Animals , Antioxidants/pharmacology , Base Sequence , Cells, Cultured , Cellular Senescence/drug effects , Coronary Artery Disease/genetics , Coronary Artery Disease/metabolism , Coronary Artery Disease/pathology , DNA Primers/genetics , Endothelial Cells/pathology , Gene Knockdown Techniques , Humans , Hydrogen Peroxide/toxicity , Oxidative Stress/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Rats , Resveratrol , Sirtuin 1/antagonists & inhibitors , Sirtuin 1/genetics
7.
Stem Cells Dev ; 19(11): 1757-67, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20192839

ABSTRACT

Ischemic stroke is the leading cause of disability in the world. Cell transplantation has emerged in various neurological diseases as a potential therapeutic approach in the postacute stroke phase. Recently, inducible pluripotent stem (iPS) cells showed potential for multilineage differentiation and provide a resource for stem cell-based therapies. However, whether iPS transplantation could improve the function of stroke-like model is still an open question. The aim of this study is to investigate the therapeutic effects of subdural transplantation of iPS mixed with fibrin glue (iPS-FG) on cerebral ischemic rats induced by middle cerebral artery occlusion (MCAO). We demonstrated an efficient method to differentiate iPS into astroglial-like and neuron-like cells which display functional electrophysiological properties. In vivo study firstly showed that the direct injection of iPS into damaged areas of rat cortex significantly decreased the infarct size and improved the motor function in rats with MCAO. Furthermore, we found that the subdural iPS-FG can also effectively reduce the total infarct volume and greatly improve the behavior of rats with MCAO to perform rotarod and grasping tasks. Importantly, analysis of cytokine expression in iPS-FG-treated ischemic brains revealed a significant reduction of pro-inflammatory cytokines and an increase of anti-inflammatory cytokines. Taken together, these results suggest that iPS cells could improve the motor function, reduce infarct size, attenuate inflammation cytokines, and mediate neuroprotection after ischemic stroke. Subdural iPS-FG could be considered as a more safe approach because this method can avoid iatrogenic injury to brain parenchyma and enhance recovering from stoke-induced impairment.


Subject(s)
Brain Ischemia/pathology , Brain Ischemia/therapy , Fibrin Tissue Adhesive/therapeutic use , Induced Pluripotent Stem Cells/physiology , Stem Cell Transplantation/methods , Subdural Space/surgery , Animals , Behavior, Animal , Brain Ischemia/physiopathology , Cell Differentiation/physiology , Cells, Cultured , Cytokines/metabolism , Fibroblasts/cytology , Fibroblasts/physiology , Induced Pluripotent Stem Cells/cytology , Infarction, Middle Cerebral Artery , Male , Mice , Mice, Inbred C57BL , Neural Stem Cells/cytology , Neural Stem Cells/physiology , Neuropsychological Tests , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Rats , Rats, Long-Evans
8.
Childs Nerv Syst ; 26(7): 897-904, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20179950

ABSTRACT

OBJECTS: Medulloblastoma (MB) is the most malignant primary brain tumor in early childhood that contains cellular and functional heterogeneity. Recent evidence has demonstrated that the tumor stem cells (TSC) may explain the radiochemoresistance of brain tumors, including MB. The aim of the present study is to investigate the possible role of TNF-related apoptosis-inducing ligand (TRAIL) in viability and tumorigenicity of MB cells and MB-derived TSC. METHODS: MB-associated TSC were isolated and cultured by serum-free medium with bFGF and EGF. The parental MB cells and MB-TSC cells were treated with TRAIL in different concentrations and assessed for cell viability, invasion ability, colony forming ability, and radiotherapy effect. RESULTS: We enrich a subpopulation of MB-TSC cells using tumor spheroid formation approach. MB-TSC display enhanced self-renewal and highly expressed "stemness" genes (CD133, Sox-2, Bmi1, Nestin). Additionally, MB-TSC showed significant resistance to TRAIL-induced apoptosis and radiosensitivity compared to the parental MB cells due antiapoptotic gene (c-FLIP, Caspase 8, Bcl-2, and Bax) upregulation. CONCLUSIONS: Our data suggest that MB-TSC are resistant to TRAIL-induced apoptosis and tumorigenic properties. Understanding the molecular mechanisms by which to operate the physiological characteristics in MB-TSC cells offers attractive approach for MB treatment.


Subject(s)
Apoptosis/drug effects , Apoptosis/radiation effects , Cerebellar Neoplasms/pathology , Drug Resistance, Neoplasm , Medulloblastoma/pathology , Neoplastic Stem Cells/pathology , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Apoptosis/genetics , Apoptosis Regulatory Proteins/genetics , Cell Line, Tumor , Cell Separation , Cell Survival/drug effects , Cell Survival/radiation effects , Cerebellar Neoplasms/drug therapy , Cerebellar Neoplasms/radiotherapy , Colorimetry , Drug Resistance, Neoplasm/genetics , Flow Cytometry , Humans , Medulloblastoma/drug therapy , Medulloblastoma/radiotherapy , Neoplasm Invasiveness , Reverse Transcriptase Polymerase Chain Reaction
9.
J Oncol ; 2010: 135632, 2010.
Article in English | MEDLINE | ID: mdl-21461395

ABSTRACT

MicroRNAs have emerged as important regulators of cell proliferation, development, cancer formation, stress responses, cell death, and other physiological conditions in the past decade. On the other hand, head and neck cancer is one of the top ten most common cancers worldwide. Recent advances in microRNAs have revealed their prominent role in regulating gene expression and provided new aspects of applications in diagnosis, prognosis, and therapeutic strategies in head and neck squamous carcinoma. In the present paper, we focus on microRNAs showing significant differences between normal and tumor cells or between cells with differential ability of metastasis. We also emphasize specific microRNAs that could modulate tumor cell properties, such as apoptosis, metastasis, and proliferation. These microRNAs possess the potential to be applied on clinical therapy in the future.

10.
Stem Cells Dev ; 19(2): 247-58, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19656070

ABSTRACT

Resveratrol is a natural polyphenol antioxidant that has been shown to facilitate osteogenic differentiation. A recent breakthrough has demonstrated that ectopic expression of four genes is sufficient to reprogram murine and human fibroblasts into induced pluripotent stem (iPS) cells. However, the roles of resveratrol in the differentiation and cytoprotection of iPS cells have never been studied. In this study, we showed that, in addition to cardiac cells, neuron-like cells, and adipocytes, mouse iPS cells could differentiate into osteocyte-like cells. Using atomic force microscopy that provided nanoscale resolution, we monitored mechanical properties of living iPS cells during osteogenic differentiation. The intensity of mineralization and stiffness in differentiating iPS significantly increased after 14 days of osteogenic induction. Furthermore, resveratrol was found to facilitate osteogenic differentiation in both iPS and embryonic stem cells, as shown by increased mineralization, up-regulation of osteogenic markers, and decreased elastic modulus. Dexamethasone-induced apoptosis in iPS cell-derived osteocyte-like cells was effectively prevented by pretreatment with resveratrol. Furthermore, resveratrol significantly increased manganese superoxide dismutase expression and intracellular glutathione level, thereby efficiently decreasing dexamethasone-induced reactive oxygen species (ROS) production and cytotoxicity. Transplantation experiments using iPS cell-derived osteocyte-like cells further demonstrated that oral intake of resveratrol could up-regulate osteopontin expression and inhibit teratoma formation in vivo. In sum, resveratrol can facilitate differentiation of iPS cells into osteocyte-like cells, protect these iPS cell-derived osteocyte-like cells from glucocorticoid-induced oxidative damage, and decrease tumorigenicity of iPS cells. These findings implicate roles of resveratrol and iPS cells in the stem cell therapy of orthopedic diseases.


Subject(s)
Cell Differentiation/drug effects , Dexamethasone/toxicity , Induced Pluripotent Stem Cells/cytology , Osteocytes/cytology , Stilbenes/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Blotting, Western , Cell Line , Cell Survival/drug effects , Cell Transplantation/methods , Cells, Cultured , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Glucocorticoids/toxicity , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/transplantation , Lentivirus/genetics , Mice , Mice, Nude , Multipotent Stem Cells/cytology , Multipotent Stem Cells/metabolism , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Osteocytes/metabolism , Resveratrol , Reverse Transcriptase Polymerase Chain Reaction , Transduction, Genetic
11.
Biochem Biophys Res Commun ; 385(3): 307-13, 2009 Jul 31.
Article in English | MEDLINE | ID: mdl-19450560

ABSTRACT

Aldehyde dehydrogenase 1 (ALDH1) has been considered to be a marker for cancer stem cells. However, the role of ALDH1 in head and neck squamous cell carcinoma (HNSCC) has yet to be determined. In this study, we isolated ALDH1-positive cells from HNSCC patients and showed that these HNSCC-ALDH1+ cells displayed radioresistance and represented a reservoir for generating tumors. Based on microarray findings, the results of Western blotting and immunofluorescent assays further confirmed that ALDH1+-lineage cells showed evidence of having epithelial-mesenchymal transition (EMT) shifting and endogenously co-expressed Snail. Furthermore, the knockdown of Snail expression significantly decreased the expression of ALDH1, inhibited cancer stem-like properties, and blocked the tumorigenic abilities of CD44+CD24(-)ALDH1+ cells. Finally, in a xenotransplanted tumorigenicity study, we confirmed that the treatment effect of chemoradiotherapy for ALDH1+ could be improved by Snail siRNA. In summary, it is likely that ALDH1 is a specific marker for the cancer stem-like cells of HNSCC.


Subject(s)
Aldehyde Dehydrogenase/biosynthesis , Biomarkers, Tumor/metabolism , Carcinoma, Squamous Cell/enzymology , Head and Neck Neoplasms/enzymology , Isoenzymes/biosynthesis , Mesenchymal Stem Cells/enzymology , Neoplastic Stem Cells/enzymology , Aged , Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase 1 Family , Female , Gene Knockdown Techniques , Humans , Isoenzymes/genetics , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , Retinal Dehydrogenase , Snail Family Transcription Factors , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Cells, Cultured
12.
Childs Nerv Syst ; 25(5): 543-50, 2009 May.
Article in English | MEDLINE | ID: mdl-19225784

ABSTRACT

OBJECTIVES: Resveratrol (RV), a natural polyphenol derived from red wine, recently showed the potential of anticancer and radiosensitizing effects. A recent study has suggested that the cancer stem cells (CSCs) may reflect the clinical refractory malignancy of brain tumors, including medulloblastoma (MB). The aim of the present study is to investigate the possible role of RV in radiosensitivity of MB cells and MB-associated CSCs. MATERIALS AND METHODS: MB-associated CSCs were isolated and cultured by serum-free medium with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). The parental MB cells and MB-CSCs were treated with RV in different concentrations and assessed for cell viability. The treatment includes RV alone, radiation alone, or radiation combined with RV. RESULTS: MB-CSCs selected by serum-free medium with bFGF and EGF can form 3D spheroid formation and display enhanced self-renewal and highly co-expressed "stem cell" genes (Oct-4, Nanog, Nestin, and Musashi-1) as well as antiapoptotic genes (Bcl-2 and Bcl-xL). These MB-CSCs showed significant resistance to radiotherapy as compared to the parental MB cells. Importantly, 100 muM RV could effectively inhibit the proliferation of MB-CSCs and significantly enhance the radiosensitivity in RV-treated MB-CSCs. CONCLUSIONS: Our data suggest that RV can effectively inhibit the proliferation and tumorigenicity of MB-CSCs and significantly synergistically enhance radiosensitivity in RV-treated MB-CSCs.


Subject(s)
Anticarcinogenic Agents/therapeutic use , Cell Proliferation/drug effects , Medulloblastoma/drug therapy , Medulloblastoma/radiotherapy , Neoplastic Stem Cells/drug effects , Stilbenes/therapeutic use , Anticarcinogenic Agents/administration & dosage , Cells, Cultured , Chemotherapy, Adjuvant , Humans , Intermediate Filament Proteins/genetics , Neoplastic Stem Cells/metabolism , Nerve Tissue Proteins/genetics , Nestin , Radiotherapy, Adjuvant , Resveratrol , Reverse Transcriptase Polymerase Chain Reaction , Stilbenes/administration & dosage , bcl-2-Associated X Protein/genetics , bcl-X Protein/genetics
13.
J Cell Biochem ; 106(4): 682-92, 2009 Mar 01.
Article in English | MEDLINE | ID: mdl-19160421

ABSTRACT

The Notch signaling pathway plays important roles in a variety of cellular processes. Aberrant transduction of Notch signaling contributes to many diseases and cancers in humans. The Notch receptor intracellular domain, the activated form of Notch receptor, is extremely difficult to detect in normal cells. However, it can activate signaling at very low protein concentration to elicit its biological effects. In the present study, a cell based luciferase reporter gene assay was established in K562 cells to screen drugs which could modulate the endogenous CBF1-dependent Notch signal pathway. Using this system, we found that the luciferase activity of CBF1-dependent reporter gene was activated by baicalin and baicalein but suppressed by niclosamide in both dose- and time-dependent manners. Treatment with these drugs modulated endogenous Notch signaling and affected mRNA expression levels of Notch1 receptor and Notch target genes in K562 cells. Additionally, erythroid differentiation of K562 cells was suppressed by baicalin and baicalein yet was promoted by niclosamide. Colony-forming ability in soft agar was decreased after treatment with baicalin and baicalein, but was not affected in the presence of niclosamide. Thus, modulation of Notch signaling after treatment with any of these three drugs may affect tumorigenesis of K562 cells suggesting that these drugs may have therapeutic potential for those tumors associated with Notch signaling. Taken together, this system could be beneficial for screening of drugs with potential to treat Notch signal pathway-associated diseases.


Subject(s)
Flavanones/pharmacology , Flavonoids/pharmacology , Niclosamide/pharmacology , Receptors, Notch/metabolism , Signal Transduction/drug effects , Antiparasitic Agents , Cell Differentiation/drug effects , Drug Screening Assays, Antitumor , Enzyme Inhibitors , Humans , K562 Cells , Leukemia/drug therapy , Leukemia/etiology , RNA, Messenger/analysis , Receptor, Notch1/genetics
14.
PLoS One ; 3(7): e2637, 2008 Jul 09.
Article in English | MEDLINE | ID: mdl-18612434

ABSTRACT

CD133 (prominin-1), a 5-transmembrane glycoprotein, has recently been considered to be an important marker that represents the subset population of cancer stem-like cells. Herein we report the isolation of CD133-positive cells (LC-CD133(+)) and CD133-negative cells (LC-CD133(-)) from tissue samples of ten patients with non-small cell lung cancer (LC) and five LC cell lines. LC-CD133(+) displayed higher Oct-4 expressions with the ability to self-renew and may represent a reservoir with proliferative potential for generating lung cancer cells. Furthermore, LC-CD133(+), unlike LC-CD133(-), highly co-expressed the multiple drug-resistant marker ABCG2 and showed significant resistance to chemotherapy agents (i.e., cisplatin, etoposide, doxorubicin, and paclitaxel) and radiotherapy. The treatment of Oct-4 siRNA with lentiviral vector can specifically block the capability of LC-CD133(+) to form spheres and can further facilitate LC-CD133(+) to differentiate into LC-CD133(-). In addition, knock-down of Oct-4 expression in LC-CD133(+) can significantly inhibit the abilities of tumor invasion and colony formation, and increase apoptotic activities of caspase 3 and poly (ADP-ribose) polymerase (PARP). Finally, in vitro and in vivo studies further confirm that the treatment effect of chemoradiotherapy for LC-CD133(+) can be improved by the treatment of Oct-4 siRNA. In conclusion, we demonstrated that Oct-4 expression plays a crucial role in maintaining the self-renewing, cancer stem-like, and chemoradioresistant properties of LC-CD133(+). Future research is warranted regarding the up-regulated expression of Oct-4 in LC-CD133(+) and malignant lung cancer.


Subject(s)
Antigens, CD/analysis , Glycoproteins/analysis , Lung Neoplasms/metabolism , Neoplastic Stem Cells/metabolism , Octamer Transcription Factor-3/metabolism , Peptides/analysis , AC133 Antigen , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/metabolism , Antigens, CD/immunology , Apoptosis , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Cell Survival , Fluorescent Antibody Technique , Gene Expression Regulation, Neoplastic , Glycoproteins/immunology , Humans , Neoplasm Proteins/metabolism , Octamer Transcription Factor-3/genetics , Peptides/immunology , RNA, Small Interfering/metabolism , Reverse Transcriptase Polymerase Chain Reaction
15.
Clin Cancer Res ; 14(13): 4085-95, 2008 Jul 01.
Article in English | MEDLINE | ID: mdl-18593985

ABSTRACT

PURPOSE: Oral squamous cell carcinoma (OSCC), like many solid tumors, contains a heterogeneous population of cancer cells. Recent data suggest that a rare subpopulation of cancer cells, termed cancer stem cells (CSC), is capable of initiating, maintaining, and expanding the growth of tumor. Identification and characterization of CSC from OSCC facilitates the monitoring, therapy, or prevention of OSCC. EXPERIMENTAL DESIGN: We enriched oral cancer stem-like cells (OC-SLC) through sphere formation by cultivating OSCC cells from established OSCC cell lines or primary cultures of OSCC patients within defined serum-free medium. Differential expression profile of stemness genes between enriched OC-SLC and parental OSCC was elucidated. Furthermore, immunohistochemical staining of stemness markers on OSCC patient tissues was examined to evaluate the association between stemness genes and prognosis of OSCC. RESULTS: Enriched OC-SLC highly expressed the stem/progenitor cell markers and ABC transporter gene (Oct-4, Nanog, CD117, Nestin, CD133, and ABCG2) and also displayed induced differentiation abilities and enhanced migration/invasion/malignancy capabilities in vitro and in vivo. Elevated expression of CD133 was shown in the enriched OC-SLC from OSCC patients' tumors. Positive correlations of Oct-4, Nanog, or CD133 expression on tumor stage were shown on 52 OSCC patient tissues. Kaplan-Meier analyses exhibited that Nanog/Oct-4/CD133 triple-positive patients predicted the worst survival prognosis of OSCC patients. CONCLUSION: We enriched a subpopulation of cancer stem-like cell from OSCC by sphere formation. The enriched OC-SLC possesses the characteristics of both stem cells and malignant tumors. Additionally, expression of stemness markers (Nanog/Oct-4/CD133) contradicts the survival prognosis of OSCC patients.


Subject(s)
Carcinoma, Squamous Cell/diagnosis , Carcinoma, Squamous Cell/metabolism , Gene Expression Profiling , Homeodomain Proteins/biosynthesis , Mouth Neoplasms/diagnosis , Mouth Neoplasms/metabolism , Neoplastic Stem Cells/cytology , Octamer Transcription Factor-3/biosynthesis , AC133 Antigen , Animals , Antigens, CD/biosynthesis , Carcinoma, Squamous Cell/mortality , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic , Glycoproteins/biosynthesis , Humans , Mice , Mice, Inbred BALB C , Mouth Neoplasms/mortality , Nanog Homeobox Protein , Neoplasm Transplantation , Peptides
16.
PLoS One ; 3(5): e2090, 2008 May 07.
Article in English | MEDLINE | ID: mdl-18509505

ABSTRACT

Atypical teratoid/rhabdoid tumor (AT/RT) is an extremely malignant neoplasm in the central nervous system (CNS) which occurs in infancy and childhood. Recent studies suggested that CD133 could be considered a marker for brain cancer stem-like cells (CSCs). However, the role of CD133 in AT/RT has never been investigated. Herein we report the isolation of CD133-positive cells (CD133(+)), found to have the potential to differentiate into three germ layer tissues, from tissues of nine AT/RT patients. The migration/invasion/malignancy and radioresistant capabilities of CD133(+) were significantly augmented when compared to CD133(-). The clinical data showed that the amount of CD133(+) in AT/RTs correlated positively with the degree of resistance to radiation therapy. Using cDNA microarray analysis, the genotoxic-response profiles of CD133(+) and CD133(-) irradiated with 10 Gy ionizing radiation (IR) were analyzed 0.5, 2, 6, 12 and 24 h post-IR. We then validated these microarray data and showed increased phosphorylation after IR of p-ATM, p-RAD17, and p-CHX2 as well as increased expression of BCL-2 protein in CD133(+) compared to CD133(-). Furthermore, we found that CD133(+) can effectively resist IR with cisplatin- and/or TRAIL-induced apoptosis. Immunohistochemical analysis confirmed the up-regulated expression of p-ATM and BCL-2 proteins in IR-treated CD133(+) xenotransgrafts in SCID mice but not in IR-treated CD133(-). Importantly, the effect of IR in CD133(+) transplanted mice can be significantly improved by a combination of BCL-2 siRNA with debromohymenialdisine, an inhibitor of checkpoint kinases. In sum, this is the first report indicating that CD133(+) AT/RT cells demonstrate the characteristics of CSCs. The IR-resistant and anti-apoptotic properties in CD133(+) may reflect the clinical refractory malignancy of AT/RTs and thus the activated p-ATM pathway and BCL-2 expression in CD133(+) could be possible targets to improve future treatment of deadly diseases like AT/RT.


Subject(s)
Antigens, CD/immunology , Glycoproteins/immunology , Peptides/immunology , Radiation Tolerance , Rhabdoid Tumor/diagnosis , AC133 Antigen , Animals , Antigens, CD/analysis , Apoptosis/radiation effects , Cell Cycle/radiation effects , Child , Child, Preschool , DNA Repair/genetics , Embryonal Carcinoma Stem Cells , Female , Glycoproteins/analysis , Humans , Infant , Male , Mice , Mice, SCID , Neoplastic Stem Cells/immunology , Peptides/analysis , Phosphorylation , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Small Interfering , Radiation, Ionizing , Rhabdoid Tumor/immunology , Rhabdoid Tumor/pathology
17.
Childs Nerv Syst ; 24(9): 987-94, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18470517

ABSTRACT

OBJECTIVES: Caffeic acid phenethyl ester (CAPE), an active component of propolis, was recently reported to have radiosensitizing effects on medulloblastoma (MB) cells. However, the mechanisms of radiosensitivity involved in medulloblastoma cells are still unclear. The specific aim of this study was to investigate the role of CAPE-induced oxidative stress to influence of radiosensitivity and anti-proliferative effects in medulloblastoma cells. MATERIALS AND METHODS: Medulloblastoma (Daoy) cells were treated with CAPE in different concentrations and assessed for cell viability. The following were also evaluated: migratory ability, reduced glutathione (GSH) level, reactive oxygen species (ROS) level, nuclear factor-kappaB (NF-kappaB) activity, and apoptosis in CAPE alone, radiation alone, or radiation combined with CAPE in Daoy cells. RESULTS: The results indicated that CAPE inhibited the growth of Daoy cells. CAPE treatment in Daoy cells could effectively decrease glutathione reductase and significantly increase glutathione peroxidase. Radiation-activated NF-kappaB was reversed by CAPE pretreatment. Finally, the result of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay showed that CAPE treatment can enhance radiation-induced apoptosis in Daoy cells. CONCLUSIONS: Our study demonstrated the anti-proliferative and radiosensitizing effects of CAPE on MB cells, which may be achievable through depleting GSH, increased ROS activity, and inhibiting NF-kappaB activity.


Subject(s)
Caffeic Acids/pharmacology , Medulloblastoma/metabolism , Oxidative Stress/drug effects , Phenylethyl Alcohol/analogs & derivatives , Radiation-Sensitizing Agents/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Glutathione/drug effects , Humans , In Situ Nick-End Labeling , NF-kappa B/drug effects , Phenylethyl Alcohol/pharmacology , Reactive Oxygen Species/metabolism
18.
J Biomed Mater Res A ; 84(3): 622-30, 2008 Mar 01.
Article in English | MEDLINE | ID: mdl-17635011

ABSTRACT

Skin tissue engineering is a possible solution for the treatment of extensive skin defect. The ultimate goal of skin tissue engineering is to restore the complete functions of native skin, but until now the structures and functions of skins are only partially restored. By negative immunoselection (CD45 and glycophorin A), we isolated and cultivated adult human bone marrow stem cells (hBMSCs) that are of multilineage differentiation potential. In this study, we first demonstrated that by using gelatin/thermo-sensitive poly N-isopropylacrylamide (pNIPAAm) and the immunocompromised mice model, the hBMSCs possess the differentiation potential of epidermis and the capability of healing skin wounds. The in vitro observations and the results of the scanning electron microscope showed that the hBMSCs can attach and proliferate in the gelatin/thermo-sensitive pNIPAAm. To further monitor the in vivo growth effect of the hBMSCs in the skin-defected nude mice, the green fluorescence protein (GFP) gene was transduced into the hBMSCs by the murine stem cell viral vector. The results showed that the rates of cell growth and wound recovery in the hBMSC-treated group were significantly higher than those in the control group, which was only treated with the gelatin/pNIPAAm (p < 0.01). More importantly, the re-epithelialization markers of human pan-cytokeratin and E-cadherin were significantly increased on day 7, day 14, and day 21 after the hBMSC-scaffold with the pNIPAAM in the mice with skin defects (p < 0.05). Moreover, the stem cell markers of human CD13 and CD105 were gradually decreased during the period of wound healing. In sum, this novel method provides a transferring system for cell therapies and maintains its temperature-sensitive property of easy-peeling by lower-temperature treatment. In addition, the in vitro and in vivo GFP imaging systems provide a new imaging modality for understanding the differentiation process and the effective expression of stem cells in wound healing.


Subject(s)
Acrylamides , Adult Stem Cells , Bone Marrow Cells , Gelatin , Polymers , Regeneration/physiology , Stem Cell Transplantation , Tissue Scaffolds , Acrylic Resins , Adolescent , Adult , Animals , Bone Marrow Transplantation , Cell Culture Techniques , Humans , Mice , Mice, Nude , Middle Aged , Skin Physiological Phenomena , Wound Healing/physiology
19.
Eur Neuropsychopharmacol ; 18(2): 128-40, 2008 Feb.
Article in English | MEDLINE | ID: mdl-17566715

ABSTRACT

Depression is accompanied by the activation of the inflammatory-response system, and increased production of proinflammatory cytokines may play a role in the pathophysiology of depressive disorders. Imipramine (IM), a tricyclic antidepressant drug, has recently been shown to promote neurogenesis and improve the survival rate of neurons in the hippocampus. However, whether IM elicits a neuroprotective or anti-inflammatory effect, or promotes the differentiation of neural stem cells (NSCs) remains to be elucidated. In this study, we cultured NSCs derived from the hippocampal tissues of adult rats as an in vitro model to evaluate the NSCs drug-modulation effects of IM. Our results showed that 3 microM IM treatment significantly increased the survival rate of NSCs, and up-regulated the mRNA and protein expression of brain-derived neurotrophic factor (BDNF) and Bcl-2 in Day-7 IM-treated NSCs. Similar to BDNF-treated effect, incubation of NSCs with 3 microM IM increased Bcl-2 protein levels and further prevented lipopolysaccharide (LPS)-induced apoptosis through the activation of the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) pathway. Inhibition of BDNF expression with small interfering RNA (siRNA), or blocking the MAPK pathway with U0126 further significantly decreased Bcl-2 protein levels and abrogated the neuroprotective effects of IM against LPS-induced apoptosis in NSCs. In addition, the percentages of serotonin and MAP-2-positive neuronal cells in the Day 7 culture of IM-treated NSCs were significantly increased. By using microdialysis with high performance liquid chromatography-electrochemical detection, the functional release of serotonin in the process of serotoninergic differentiation of IM-treated NSCs was concomitantly increasing and mediated by the activation of the BDNF/MAPK/ERK pathway/Bcl-2 cascades. In sum, the study results indicate that IM can increase the neuroprotective effects, suppress the LPS-induced inflammatory process, and promote serotoninergic differentiation in NSCs via the modulation of the BDNF/MAPK/ERK pathway/Bcl-2 cascades.


Subject(s)
Adrenergic Uptake Inhibitors/pharmacology , Apoptosis/drug effects , Brain-Derived Neurotrophic Factor/metabolism , Imipramine/pharmacology , Mitogen-Activated Protein Kinase Kinases/metabolism , Neurons/drug effects , Signal Transduction/drug effects , Stem Cells/drug effects , Analysis of Variance , Animals , Cell Differentiation/drug effects , Cell Survival/drug effects , Cells, Cultured , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay , Hippocampus/cytology , In Situ Nick-End Labeling , Lipopolysaccharides/pharmacology , Nerve Tissue Proteins/metabolism , Rats , Rats, Sprague-Dawley , Time Factors
20.
Int J Cancer ; 121(11): 2547-55, 2007 Dec 01.
Article in English | MEDLINE | ID: mdl-17680560

ABSTRACT

The major obstacle for the treatment of gastric cancer is recurrence and metastasis; yet, its molecular mechanism is largely unknown. 2-methoxyestradiol (2-ME), a metabolite of the estradiol-17beta, has recently been demonstrated to have multifactorial effects against tumor proliferation and angiogenesis; how these effects are interrelated and act cooperatively is the key question to be elucidated. Akt activation was shown to promote cancer cell invasiveness, and inhibition of Akt phosphorylation by 2-ME was also noted. We herein investigated the significance of PI3K/Akt activation in gastric cancer metastasis and the anti-metastatic effect of 2-ME through attenuation of Akt activity. Immunohistochemistry of PI3K, phosphorylated Akt (p-Akt) and phosphorylated Erk (p-Erk) was performed in tumors from 56 gastric cancer patients, and a significant correlation between PI3K/p-Akt and tumor stage/prognosis was demonstrated (p < 0.05). An in vitro study of 7 gastric cancer cell lines showed a remarkable correlation between PI3K and p-Akt. PI3K/p-Akt overexpression was associated with invasiveness/migration; in contrast, phosphorylation of Erk was not shown to be correlated with invasiveness. In addition, metastatic gastric cancer clones expressed a higher level of PI3K/p-Akt. The anti-metastatic effect of a low dose of 2-ME and inactivation of Akt was demonstrated. 2-ME also exhibited an ability to inhibit gastric cancer cell proliferation and induce G2/M cell cycle arrest at a higher concentration than that required for inhibition of migration. We conclude that the activation of PI3K/Akt pathway is involved in the late-stage progression and metastasis of gastric cancer, and attenuation of p-Akt by 2-ME suppresses metastasis.


Subject(s)
Antineoplastic Agents/pharmacology , Estradiol/analogs & derivatives , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Stomach Neoplasms/drug therapy , Stomach Neoplasms/pathology , 2-Methoxyestradiol , Adult , Aged , Antineoplastic Agents/therapeutic use , Blotting, Western , Cell Cycle/drug effects , Cell Movement/drug effects , Cell Survival/drug effects , Colorimetry , Estradiol/pharmacology , Estradiol/therapeutic use , Female , Flow Cytometry , Gene Expression Regulation, Neoplastic/drug effects , Green Fluorescent Proteins/analysis , Humans , Immunohistochemistry , Male , Middle Aged , Neoplasm Invasiveness , Neoplasm Staging , Phosphatidylinositol 3-Kinases/drug effects , Phosphorylation/drug effects , Prognosis , Proto-Oncogene Proteins c-akt/drug effects , Stomach Neoplasms/metabolism , Stomach Neoplasms/surgery , Tumor Cells, Cultured , Up-Regulation/drug effects
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