Notch2-induced COX-2 expression enhancing gastric cancer progression.

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.

MafA promotes the reprogramming of placenta-derived multipotent stem cells into pancreatic islets-like and insulin+ cells.

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.

Induction of insulin-producing cells derived from endometrial mesenchymal stem-like cells.

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.

Medulloblastoma-derived tumor stem-like cells acquired resistance to TRAIL-induced apoptosis and radiosensitivity.

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.

The autonomous notch signal pathway is activated by baicalin and baicalein but is suppressed by niclosamide in K562 cells.

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.

Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer.

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.

Positive correlations of Oct-4 and Nanog in oral cancer stem-like cells and high-grade oral squamous cell carcinoma.

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.

Evaluation of radiotherapy effect in resveratrol-treated medulloblastoma cancer stem-like cells.

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.

Neuroprotection by Imipramine against lipopolysaccharide-induced apoptosis in hippocampus-derived neural stem cells mediated by activation of BDNF and the MAPK pathway.

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.

2-Methoxyestradiol attenuates phosphatidylinositol 3-kinase/Akt pathway-mediated metastasis of gastric cancer.

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.

Superoxide dismutase and catalase inhibit oxidized low-density lipoprotein-induced human aortic smooth muscle cell proliferation: role of cell-cycle regulation, mitogen-activated protein kinases, and transcription factors.

Several antioxidant enzymes, including copper, zinc-superoxide dismutase (Cu, Zn-SOD) and catalase, have been suggested to be protective against the proliferation of vascular smooth muscle cells exposed to oxidative stress. In the present study, we investigated effects of Cu, Zn-SOD and/or catalase on oxLDL-induced proliferation of, and intracellular signaling in, human aortic smooth muscle cells (HASMCs). HASMCs were transfected with adenovirus carrying the human Cu, Zn-SOD gene and/or the human catalase gene. This resulted in a high level of Cu, Zn-SOD and/or catalase overexpression and decreased oxLDL-induced proliferation. Cu, Zn-SOD and/or catalase also arrested cell cycle progression, which was associated with decreased expression of cyclin D1, cyclin E, CDK2, and CDK4 and upregulation of p21(Cip1) and p27(Kip1). Phosphorylation studies on ERK1/2, JNK, and p38, three major subgroups of mitogen activator protein kinases, demonstrated that Cu, Zn-SOD and/or catalase overexpression suppressed ERK1/2 and JNK phosphorylation. Gel-mobility shift analysis showed that oxLDL caused an increase in the DNA binding activity of activator protein-1 (AP-1) and nuclear factor kappaB (NF-kappaB), which was inhibited by Cu, Zn-SOD and/or catalase overexpression. These results provide the first evidence that overexpression of Cu, Zn-SOD and/or catalase in HASMCs attenuates the cell proliferation caused by oxLDL stimulation and that this inhibitory effect is mediated via downregulation of ERK1/2 and JNK phosphorylation and AP-1 and NF-kappaB inactivation. These observations support the feasibility of the increase of Cu, Zn-SOD and/or catalase expression in human smooth muscle cells as a means of protection against oxidant injury.

Antidepressant administration modulates neural stem cell survival and serotoninergic differentiation through bcl-2.

The hippocampus has long been associated with learning, memory, and modulation of emotional responses. Previous studies demonstrated that stress-induced loss of hippocampal neurons may contribute to the pathogenesis of depression. The recent observations supported that antidepressant drugs increase the production of serotoninergic neurotransmitter and they play a critical role in the initiation of neurogenesis in the hippocampus. In order to explore the possible new mechanism of the treatment of depression, we cultured neural stem cells (NSCs) derived from the hippocampus of adult rats as an in vitro model to evaluate the capabilities of neuroprotection and neural differentiation in NSCs by fluoxetine (FL) treatment. Our results showed that 20 microM FL treatment can significantly increase the proliferation rate of NSCs (p<0.05), and up-regulate the mRNA and protein expressions of Bcl-2 in Day-7 FL-treated NSCs (p<0.01). Using Bcl-2 gene silencing with small interfering RNA, our data verified that FL can prevent Fas ligand-induced caspase-dependent apoptosis in NSCs through the activation of Bcl-2. The in vitro observation and immunofluorescent study further demonstrated that FL treatment can stimulate the neurite development and serotoninergic differentiation of NSCs through the activation of Bcl-2. Using microdialysis with high performance liquid chromatography- electrochemical detection, the functional release of serotonin in the differentiating NSCs with FL treatment was increased and simultaneously regulated by the Bcl-2 expressions. In sum, the study results indicate that antidepressant administration can increase NSCs survival, promote the neurite development, and facilitate NSCs differentiating into the functional serotoninergic neurons via the modulation of Bcl-2 expression.

Enhancement of insulin-producing cell differentiation from embryonic stem cells using pax4-nucleofection method.

AIM: To enhance the differentiation of insulin producing cell (IPC) ability from embryonic stem (ES) cells in vitro. METHODS: Four-day embryoid body (EB)-formatted ES cells were dissociated as single cells for the followed plasmid DNA delivery. The use of Nucleofector electroporator (Amaxa biosystems, Germany) in combination with medium-contained G418 provided a high efficiency of gene delivery for advanced selection. Neucleofected cells were plated on the top of fibronectin-coated Petri dishes. Addition of Ly294002 and raised the glucose in medium at 24 h before examination. The differentiation status of these cells was monitored by semi-quantitative PCR (SQ-PCR) detection of the expression of relative genes, such as oct-4, sox-17, foxa2, mixl1, pdx-1, insulin 1, glucagons and somatostatin. The percentage of IPC population on d 18 of the experiment was investigated by immunohistochemistry (IHC), and the content/secretion of insulin was estimated by ELISA assay. The mice with severe combined immunodeficiency disease (SCID) pretreated with streptozotocin (STZ) were used to eliminate plasma glucose restoration after pax4+ ES implantation. RESULTS: A high efficiency of gene delivery was demonstrated when neucleofection was used in the present study; approximately 70% cells showed DsRed expression 2 d after neucleofection. By selection of medium-contained G418, the percentage of DsRed expressing cells kept high till the end of study. The pancreatic differentiation seemed to be accelerated by pax4 nucleofection. When compared to the group of cells with mock control, foxa2, mixl1, pdx1, higher insulin and somatostatin levels were detected by SQ-PCR 4 d after nucleofection in the group of pax4 expressing plasmid delivery. Approximately 55% of neucleofected cells showed insulin expression 18 d after neucleofection, and only 18% of cells showed insulin expression in mock control. The disturbance was shown by nucleofected pax4 RNAi vector; only 8% of cells expressed insulin 18 d after nucleofection. A higher IPC population was also detected in the insulin content by ELISA assay, and the glucose dependency was demonstrated in insulin secretion level. In the animal model, improvement of average plasma glucose concentration was observed in the group of pax-4 expressed ES of SCID mice pretreated with STZ, but no significant difference was observed in the group of STZ-pretreated SCID mice who were transplanted ES with mock plasmid. CONCLUSION: Enhancement of IPC differentiation from EB-dissociated ES cells can be revealed by simply using pax4 expressing plasmid delivery. Not only more IPCs but also pancreatic differentiation-related genes can be detected by SQ-PCR. Expression of relative genes, such as foxa 2, mixl 1, pdx-1, insulin 1 and somatostatin after nucleofection, suggests that pax4 accelerates the whole differentiation progress. The higher insulin production with glucose dependent modulation suggests that pax4 expression can drive more mature IPCs. Although further determination of the entire mechanism is required, the potential of pax-4-nucleofected cells in medical treatment is promising.

Moclobemide upregulated Bcl-2 expression and induced neural stem cell differentiation into serotoninergic neuron via extracellular-regulated kinase pathway.

1. Moclobemide (MB) is an antidepressant drug that selectively and reversibly inhibits monoamine oxidase-A. Recent studies have revealed that antidepressant drugs possess the characters of potent growth-promoting factors for the development of neurogenesis and improve the survival rate of serotonin (5-hydroxytrytamine; 5-HT) neurons. However, whether MB comprises neuroprotection effects or modulates the proliferation of neural stem cells (NSCs) needs to be elucidated. 2. In this study, firstly, we used the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to demonstrate that 50 microM MB can increase the cell viability of NSCs. The result of real-time reverse transcription-polymerase chain reaction (RT-PCR) showed that the induction of MB can upregulate the gene expressions of Bcl-2 and Bcl-xL. By using caspases 8 and 3, ELISA and terminal dUTP nick-end labeling (TUNEL) assay, our data further confirmed that 50 microM MB-treated NSCs can prevent FasL-induced apoptosis. 3. The morphological findings also supported the evidence that MB can facilitate the dendritic development and increase the neurite expansion of NSCs. Moreover, we found that MB treatment increased the expression of Bcl-2 in NSCs through activating the extracellular-regulated kinase (ERK) phosphorylation. 4. By using the triple-staining immunofluorescent study, the percentages of serotonin- and MAP-2-positive cells in the day 7 culture of MB-treated NSCs were significantly increased (P<0.01). Furthermore, our data supported that MB treatment increased functional production of serotonin in NSCs via the modulation of ERK1/2. In sum, the study results support that MB can upregulate Bcl-2 expression and induce the differentiation of NSCs into serotoninergic neuron via ERK pathway.

Superoxide dismutase inhibits the expression of vascular cell adhesion molecule-1 and intracellular cell adhesion molecule-1 induced by tumor necrosis factor-alpha in human endothelial cells through the JNK/p38 pathways.

OBJECTIVE: Expression of adhesion molecules on endothelial cells and subsequent leukocyte recruitment are critical early events in the development of atherosclerosis. We tried to study possible effects of Cu/Zn superoxide dismutase (SOD) on adhesion molecule expression and its underlying mechanism in the prevention and treatment of cardiovascular disorders. METHODS AND RESULTS: Human aortic endothelial cells (HAECs) were transfected with adenovirus carrying the human SOD gene (AdSOD) to investigate whether SOD expression in HAECs attenuated tumor necrosis factor (TNF)-alpha-induced reactive oxygen species production and adhesion molecule expression and to define the mechanisms involved. SOD expression significantly suppressed TNF-alpha-induced expression of vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1 and reduced the binding of the human neutrophils to TNF-alpha-stimulated HAECs. SOD expression suppressed c-JUN N-terminal kinase and p38 phosphorylation. It also attenuated intracellular superoxide anion production and NADPH oxidase activity in TNF-alpha-treated HAECs. CONCLUSIONS: These results provide evidence that SOD expression in endothelial cells attenuates TNF-alpha-induced superoxide anion production and adhesion molecule expression, and that this protective effect is mediated by decreased JNK and p38 phosphorylation and activator protein-1 and nuclear factor kappaB inactivation. These results suggest that SOD has antiinflammatory properties and may play important roles in the prevention of atherosclerosis and inflammatory response. Superoxide dismutase overexpression in endothelial cells attenuates tumor necrosis factor alpha-induced superoxide anion production and adhesion molecule expression, and this effect is mediated by decreased JNK and p38 phosphorylation and AP-1 and nuclear factor B inactivation. These results suggest that superoxide dismutase may play an important role in the prevention of atherosclerosis and inflammatory response.

Characterization of pancreatic stem cells derived from adult human pancreas ducts by fluorescence activated cell sorting.

AIM: To isolate putative pancreatic stem cells (PSCs) from human adult tissues of pancreas duct using serum-free, conditioned medium. The characterization of surface phenotype of these PSCs was analyzed by flow cytometry. The potential for pancreatic lineage and the capability of beta-cell differentiation in these PSCs were evaluated as well. METHODS: By using serum-free medium supplemented with essential growth factors, we attempted to isolate the putative PSCs which has been reported to express nestin and pdx-1. The Matrigel(TM) was employed to evaluate the differential capacity of isolated cells. Dithizone staining, insulin content/secretion measurement, and immunohistochemistry staining were used to monitor the differentiation. Fluorescence activated cell sorting (FACS) was used to detect the phenotypic markers of putative PSCs. RESULTS: A monolayer of spindle-like cells was cultivated. The putative PSCs expressed pdx-1 and nestin. They were also able to differentiate into insulin-, glucagon-, and somatostatin-positive cells. The spectrum of phenotypic markers in PSCs was investigated; a similarity was revealed when using human bone marrow-derived stem cells as the comparative experiment, such as CD29, CD44, CD49, CD50, CD51, CD62E, PDGFR-alpha, CD73 (SH2), CD81, CD105(SH3). CONCLUSION: In this study, we successfully isolated PSCs from adult human pancreatic duct by using serum-free medium. These PSCs not only expressed nestin and pdx-1 but also exhibited markers attributable to mesenchymal stem cells. Although work is needed to elucidate the role of these cells, the application of these PSCs might be therapeutic strategies for diabetes mellitus.

Elevation of plasma and cerebrospinal fluid osteopontin levels in patients with atypical teratoid/rhabdoid tumor.

Osteopontin, a cancer metastasis-associated gene, is specifically up-regulated in central nervous system (CNS) atypical teratoid/rhabdoid tumor (AT/RT), but its biological behavior in the progression of CNS AT/RT has never been studied. We obtained plasma, cerebrospinal fluid (CSF), and brain tissue specimens from lobectomy or hemispherectomy samples from 39 patients (medulloblastoma, 16; AT/RT, 8; epilepsy, 6; hydrocephalus, 9). By enzyme-linked immunosorbent assay, the median osteopontin levels in plasma and CSF in AT/RT (852.0 and 1,175.0 ng/mL, respectively) were significantly higher than in medulloblastoma (492.5 and 524.5 ng/mL, respectively) and hydrocephalus and epilepsy (208.0 and 168.0 ng/mL, respectively) (P < .05). The results of real-time reverse transcriptase-polymerase chain reaction and immunohistochemical analysis demonstrated that osteopontin expression in AT/RT (n = 5) was significantly higher than in medulloblastoma (n = 8) samples. The differences in osteopontin expression in plasma, CSF, and tumor samples in AT/RT and medulloblastoma correlated with survival differences. In 5 patients with AT/RT, plasma osteopontin levels decreased after treatment but increased with relapse. Osteopontin might be a potential marker to aid in identifying AT/RT recurrence.

Magnolol attenuates VCAM-1 expression in vitro in TNF-alpha-treated human aortic endothelial cells and in vivo in the aorta of cholesterol-fed rabbits.

1. In a previous study, we showed that magnolol, a potent antioxidant derived from a Chinese herb, attenuates monocyte chemotactic protein-1 (MCP-1) expression and intimal hyperplasia in the balloon-injured aorta of cholesterol-fed rabbits. Expression of cell adhesion molecules by the arterial endothelium and the attachment of leukocytes to the endothelium may play a major role in atherosclerosis. In the present study, the effects of magnolol on the expression of endothelial-leukocyte adhesion molecules and the activation of nuclear factor kappa B (NF-kappa B) in tumour necrosis factor-alpha (TNF-alpha)-treated human aortic endothelial cells (HAECs) were investigated. 2. Pretreatment of HAECs with magnolol (5 microM) significantly suppressed the TNF-alpha-induced expression of vascular cell adhesion molecule-1 (VCAM-1) (64.8+/-1.9%), but had no effect on the expression of intercellular cell adhesion molecule-1 and endothelial cell selectin. 3. Magnolol (5 and 10 microM) significantly reduced the binding of the human monocytic cell line, U937, to TNF-alpha-stimulated HAECs (58.4 and 56.4% inhibition, respectively). Gel shift assays using the (32)P-labelled NF-kappa B consensus sequence as probe showed that magnolol pretreatment reduced the density of the shifted bands seen after TNF-alpha-induced activation. Immunoblot analysis and immunofluorescence staining of nuclear extracts demonstrated a 58% reduction in the amount of NF-kappa B p65 in the nuclei in magnolol-treated HAECs. Magnolol also attenuated intracellular H(2)O(2) generation in both control and TNF-alpha treated HAECs. 4. Furthermore, in vivo, magnolol attenuates the intimal thickening and TNF-alpha and VCAM-1 protein expression seen in the thoracic aortas of cholesterol-fed rabbits. 5. Taken together, these data demonstrate that magnolol inhibits TNF-alpha-induced nuclear translocation of NF-kappa B p65 and thereby suppresses expression of VCAM-1, resulting in reduced adhesion of leukocytes. These results suggest that magnolol has anti-inflammatory properties and may play important roles in the prevention of atherosclerosis and inflammatory responses in vivo.

Protective effects of tetramethylpyrazine on kainate-induced excitotoxicity in hippocampal culture.

Tetramethylpyrazine (TMP) is the major component extracted from the Chinese herb, Chuanxiong. This study focuses on the protective effect of tetramethylpyrazine in kainate-induced excitotoxicity in rat hippocampus. Primary neuronal cultures raised from cells isolated from the hippocampi of 7-day old rats were treated with kainate (75-450 microM) for 12, 24, and 48 h. Our results revealed that kainate induced neuronal damage in a dose- and time-dependent manner, reaching maximal damage at 150 microM and 24 h and persisted for higher doses and 48 h. In addition, 1 h of kainate (150 microM) treatment led to significant generation of free radicals and reduction of mitochondrial membrane potential (MMP) which persisted for > or = 4 h on continued exposure. Ten minutes pretreatment with 1 or 5 microM tetramethylpyrazine dose dependently and significantly attenuated the kainate-induced damage. Taken together, the results suggest that multiple mechanisms including protection of mitochondria, decrease in free radical generation and scavenging of free radicals might be involved in TMP's protection against kainate induced cell toxicity.

Docosahexaenoic acid promotes dopaminergic differentiation in induced pluripotent stem cells and inhibits teratoma formation in rats with Parkinson-like pathology.

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.