Multiple low-dose radiation-induced neuronal cysteine transporter expression and oxidative stress are rescued by N-acetylcysteine in neuronal SH-SY5Y cells.

Recently, several studies have demonstrated that low-dose radiation (LDR) therapy has positively impacts on the treatment of Alzheimer's disease (AD). LDR suppresses the production of pro-neuroinflammation molecules and improves cognitive function in AD. However, it is unclear whether direct exposure to LDR causes beneficial effects and what mechanism is involved in neuronal cells. In this study, we first determined the effect of high-dose radiation (HDR) alone on C6 cells and SH-SY5Y cells. We found that SH-SY5Y cells were more vulnerable than C6 cells to HDR. Moreover, in neuronal SH-SY5Y cells exposed to single or multiple LDR, N-type cells showed decreased cell viability with increasing radiation exposure time and frequency, but S-type cells were unaffected. Multiple LDR increased proapoptotic molecules such as p53, Bax and cleaved caspase-3, and decreased anti-apoptotic molecule (Bcl2). Multiple LDR also generated free radicals in neuronal SH-SY5Y cells. We detected a change in the expression of the neuronal cysteine transporter EAAC1. Pretreatment with N-acetylcysteine (NAC) rescued the increased in EAAC1 expression and the generation of ROS in neuronal SH-SY5Y cells after multiple LDR. Furthermore, we verified whether the increased in EAAC1 expression induces cell defense or cell death promotion signaling. We showed that transient overexpression of EAAC1 reduced the multiple LDR-induced p53 overexpression in neuronal SH-SY5Y cells. Our results indicate that neuronal cells can be injured by increased production of ROS not only by HDR but also by multiple LDR, which suggests that combination treatment with anti-free radical agents such as NAC may be useful in multiple LDR therapy.

Neuregulin-1 reverses anxiety-like behavior and social behavior deficits induced by unilateral micro-injection of CoCl2 into the ventral hippocampus (vHPC).

Neuregulin-1 (NRG1) is an epidermal growth factor family member with essential roles in the developing and adult nervous systems. In recent years, establishing evidence has collectively suggested that NRG1 is a new modulator of central nervous system (CNS) injury and disease, with multifaceted roles in neuroprotection, remyelination, neuroinflammation, and other repair mechanisms. NRG1 signaling exerts its effects via the tyrosine kinase receptors ErbB2-ErbB4. The NRG1/ErbB network in CNS pathology and repair has evolved, primarily in recent years. In the present study, we demonstrated that a unilateral microinjection of CoCl2 into the ventral hippocampus (vHPC) induced hypoxic insult and led to anxiety-related behaviors and deficit sociability in mice. NRG1 treatment significantly alleviated the CoCl2-induced increase of hypoxic-related molecules and behavioral abnormalities. Furthermore, NRG1 reduced the CoCl2-induced neuroinflammation and neuronal deficits in the vHPC or primary hippocampal neurons in mice. Collectively, these results suggest that NRG1 ameliorates hypoxia by alleviating synaptic deficits and behavioral abnormalities of the CoCl2-induced vHPC hypoxic model.

A new brain-cutting device and ultraviolet resin-mounted human brain slices as a teaching adjunct for neuroanatomy education.

Although the level of neuroscience research is rapidly developing with the introduction of new technologies, the method of neuroanatomy education remains at the traditional level and requires improvement to meet the needs of educators and trainees. We developed a new three-dimensional (3D) printed device (human brain-cutting mold, HBCM) for creating human brain slices; moreover, we demonstrated a simple method for creating semi-permanent ultraviolet (UV) resin-mounted brain slice specimens for neuroanatomy education. We obtained brain slices of uniform thickness (3 mm) through the HBCM; the resultant brain slices were optimal for assessing morphological details of the human brain. Furthermore, we used an agar-embedding method for brain-slicing with the HBCM, which minimized geometrical distortions of the brain slices. Also, we prepared semi-permanent brain serial specimens using an acrylic brain slice frame and UV-curable resin, which was highly compatible with moist bio-specimens. During UV resin curing, neither air bubble formation nor color change occurred. The resultant UV resin-mounted brain slices produced definite coronal sections with high transparency and morphological accuracy. We also performed 3D modeling by stacking brain slice images that differentiated the cortical area and nine subcortical regions via manual segmentation. This method could be a reliable alternative for displaying high-quality human brain slices and would be helpful for students and trainee to understand anatomical orientation from 2D images to 3D structures. Also, this may present an innovative approach for preparing and preserving coronal sections of the normal or pathological human brain.

Neuregulin 1/ErbB4/Akt signaling attenuates cytotoxicity mediated by the APP-CT31 fragment of amyloid precursor protein.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by neuronal and synaptic loss. The cytoplasmic tail of amyloid precursor protein (APP) undergoes sequential cleavage at a specific intracellular caspase site to generate the cytoplasmic terminal 31 (CT31) fragment. The APP-CT31 fragment is a potent inducer of apoptosis. The cytotoxicity of APP-CT31 in SH-SY5Y cells was evaluated by the lactate dehydrogenase (LDH) assay. TUNEL staining was used to detect apoptotic signals in SH-SY5Y cells and primary cortical neurons. The expression of apoptosis-related proteins, such as p53, PUMA (p53 up-regulated modulator of apoptosis), and cleaved was investigated by immunofluorescence analysis and Western blotting. In this study, we investigated the neuroprotective effect of neuregulin 1 (NRG1) against cytotoxicity induced by APP-CT31. Our data showed that CT31 induced cytotoxicity and apoptosis in SH-SY5Y cells and primary cortical neurons. NRG1 attenuated the neurotoxicity induced by the expression of APP-CT31. We also showed that APP-CT31 altered the expression of p53 and cleaved caspase 3. However, treatment with NRG1 rescued the APP-CT31-induced upregulation of p53 and cleaved caspase 3 expression. The protective effect of NRG1 was abrogated by inhibition of the ErbB4 receptor and Akt. These results indicate an important role of ErbB4/Akt signaling in NRG1-mediated neuroprotection, suggesting that endogenous NRG1/ErbB4 signaling represents a valuable therapeutic target in AD.

Neuregulin-1 inhibits CoCl2-induced upregulation of excitatory amino acid carrier 1 expression and oxidative stress in SH-SY5Y cells and the hippocampus of mice.

Excitatory amino acid carrier 1 (EAAC1) is an important subtype of excitatory amino acid transporters (EAATs) and is the route for neuronal cysteine uptake. CoCl2 is not only a hypoxia-mimetic reagent but also an oxidative stress inducer. Here, we found that CoCl2 induced significant EAAC1 overexpression in SH-SY5Y cells and the hippocampus of mice. Transient transfection of EAAC1 reduced CoCl2-induced cytotoxicity in SH-SY5Y cells. Based on this result, upregulation of EAAC1 expression by CoCl2 is thought to represent a compensatory response against oxidative stress in an acute hypoxic state. We further demonstrated that pretreatment with Neuregulin-1 (NRG1) rescued CoCl2-induced upregulation of EAAC1 and tau expression. NRG1 plays a protective role in the CoCl2-induced accumulation of reactive oxygen species (ROS) and reduction in antioxidative enzyme (SOD and GPx) activity. Moreover, NRG1 attenuated CoCl2-induced apoptosis and cell death. NRG1 inhibited the CoCl2-induced release of cleaved caspase-3 and reduction in Bcl-XL levels. Our novel finding suggests that NRG1 may play a protective role in hypoxia through the inhibition of oxidative stress and thereby maintain normal EAAC1 expression levels.

Early-life stress induces EAAC1 expression reduction and attention-deficit and depressive behaviors in adolescent rats.

Neonatal maternal separation (NMS), as an early-life stress (ELS), is a risk factor to develop emotional disorders. However, the exact mechanisms remain to be defined. In the present study, we investigated the mechanisms involved in developing emotional disorders caused by NMS. First, we confirmed that NMS provoked impulsive behavior, orienting and nonselective attention-deficit, abnormal grooming, and depressive-like behaviors in adolescence. Excitatory amino acid carrier 1 (EAAC1) is an excitatory amino acid transporter expressed specifically by neurons and is the route for the neuronal uptake of glutamate/aspartate/cysteine. Compared with that in the normal control group, EAAC1 expression was remarkably reduced in the ventral hippocampus and cerebral cortex in the NMS group. Additionally, EAAC1 expression was reduced in parvalbumin-positive hippocampal GABAergic neurons in the NMS group. We also found that EAAC1-knockout (EAAC1-/-) mice exhibited impulsive-like, nonselective attention-deficit, and depressive-like behaviors compared with WT mice in adolescence, characteristics similar to those of the NMS behavior phenotype. Taken together, our results revealed that ELS induced a reduction in EAAC1 expression, suggesting that reduced EAAC1 expression is involved in the pathophysiology of attention-deficit and depressive behaviors in adolescence caused by NMS.

Neuregulin-1 Protects Neuronal Cells Against Damage due to CoCl2-Induced Hypoxia by Suppressing Hypoxia-Inducible Factor-1α and P53 in SH-SY5Y Cells.

PURPOSE: Hypoxia-mediated neurotoxicity contributes to various neurodegenerative disorders, including Alzheimer disease. Neuregulin-1 (NRG1) plays an important role in the development and plasticity of the brain. The aim of the present study was to investigate the neuroprotective effect and the regulating hypoxic inducible factor of NRG1 in cobalt chloride (CoCl2) induced hypoxia. METHODS: Hypoxia was induced in SH-SY5Y cells by CoCl2 treatment. SH-SY5Y cells were pretreated with NRG1 and then treated with CoCl2. Western blotting, immunocytochemistry, and lactate dehydrogenase (LDH) release assays were performed to examine neuroprotective properties of NRG1 in SH-SY5Y cells. RESULTS: Our data showed that CoCl2 induced cytotoxicity and changes of hypoxia-inducible factor-1α (HIF-1α) and p53 expression in SH-SY5Y cells. However, pretreatment with NRG1 inhibited CoCl2-induced accumulation of HIF-1α and p53 stability. In addition, NRG1 significantly attenuated cell death of SH-SY5Y induced by CoCl2. CONCLUSION: NRG1 can regulate HIF-1α and p53 to protect neurons against hypoxic damage.

Neuregulin 1/ErbB4 signaling attenuates neuronal cell damage under oxygen-glucose deprivation in primary hippocampal neurons.

The hippocampus is one of the most important brain areas of cognition. This region is particularly sensitive to hypoxia and ischemia. Neuregulin-1 (NRG1) has been shown to be able to protect against focal cerebral ischemia. The aim of the present study was to investigate the neuroprotective effect of NRG1 in primary hippocampal neurons and its underlying mechanism. Our data showed oxygen-glucose deprivation (OGD)-induced cytotoxicity and overexpression of ErbB4 in primary hippocampal neurons. Moreover, pretreatment with NRG1 could inhibit OGD-induced overexpression of ErbB4. In addition, NRG1 significantly attenuated neuronal death induced by OGD. The neuroprotective effect of NRG1 was blocked in ischemic neurons after pretreatment with AG1478, an inhibitor of ErbB4, but not after pretreatment with AG879, an inhibitor of ErbB2. These results indicate an important role of ErbB4 in NRG1-mediated neuroprotection, suggesting that endogenous ErbB4 might serve as a valuable therapeutic target for treating global cerebral ischemia.

Neuregulin1 Attenuates H2O2-Induced Reductions in EAAC1 Protein Levels and Reduces H2O2-Induced Oxidative Stress.

Neuregulin 1 (NRG1) exhibits potent neuroprotective properties. The aim of the present study was to investigate the antioxidative effects and underlying mechanisms of NRG1 against H2O2-induced oxidative stress in primary rat cortical neurons. The expression level of the excitatory amino acid carrier 1 (EAAC1) protein was measured by Western blotting and immunocytochemistry. The levels of lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) generation, superoxide dismutase (SOD) activity, GPx activity, and mitochondrial membrane potential (∆ψm) were determined to examine cell death and the antioxidant properties of NRG1 in primary rat cortical neurons. H2O2 reduced the expression of EAAC1 in a dose-dependent manner. We found that pretreatment with NRG1 attenuated the H2O2-induced reduction in EAAC1 expression. Moreover, NRG1 reduced the cell death and oxidative stress induced by H2O2. In addition, NRG1 attenuated H2O2-induced reductions in antioxidant enzyme activity and ∆ψm. Our data indicate a role for NRG1 in protecting against oxidative stress via the regulation of EAAC1. These observations may provide novel insights into the mechanisms of NRG1 activity during oxidative stress and may reveal new therapeutic targets for regulating the oxidative stress associated with various neurological diseases.

Neuregulin 1 regulates amyloid precursor protein cell surface expression and non-amyloidogenic processing.

The amyloid precursor protein (APP) is a key molecule in Alzheimer's disease. The prevailing view is that APP is initially transported to the plasma membrane as a full-length protein. Its localization at the cell surface can trigger downstream signaling and APP cleavage. Our previous work has shown that Neuregulin 1 (NRG1) has neuroprotective effects in an Alzheimer's disease model. In the present study, we examine whether NRG1 signaling is involved in APP expression and non-amyloidogenic processing in neuronal cells. Here we show that NRG1 increased the cell surface expression of APP without changing the total amount of APP mRNA or protein expression in SH-SY5Y cells and in rat primary cortical neurons. In addition, NRG1 significantly increased the levels of the secreted form of APP, sAPPα, in the conditioned media but did not change the expression of ADAM10 on the cell surface or in the cell lysates. Furthermore, we found that the protein level of NRG1 was reduced in the hippocampus of Alzheimer's disease (AD) patients. Our results demonstrate that NRG1 increased APP expression on the cell surface and sAPPα secretion into the media of neuronal cell cultures. Taken together, these results suggest a role for NRG1 in non-amyloidogenic processing.

Neuroanatomical distribution of galectin-3 in the adult rat brain.

Galectin-3 is a member of the lectin subfamily that enables the specific binding of ß-galactosides. It is expressed in a broad spectrum of species and organs, and is known to have various functions related to cell adhesion, signal transduction, and proinflammatory responses. Although, expression of galectin-3 in some activated neuroglia under neuroinflammation has been well documented in the central nervous system, little is known about the neuronal expression and distribution of galectin-3 in normal brain. To describe the cellular and neuroanatomical expression map of galectin-3, we performed galectin-3 immunohistochemistry on the entire normal rat brain and subsequently analyzed the neuronal distribution. Galectin-3 expression was observed not only in some neuroglia but also in neurons. Neuronal expression of galectin-3 was observed in many functional parts of the cerebral cortex and various other subcortical nuclei in the hypothalamus and brainstem. Neuroanatomical analysis revealed that robust galectin-3 immuno-signals were present in many hypothalamic nuclei related to a variety of physiological functions responsible for mediating anxiety responses, energy balance, and neuroendocrine regulation. In addition, the regions highly connected with these hypothalamic nuclei also showed intense galectin-3 expression. Moreover, multiple key regions involved in regulating autonomic functions exhibited high levels of galectin-3 expression. In contrast, the subcortical nuclei responsible for the control of voluntary motor functions and limbic system exhibited no galectin-3 immunoreactivity. These observations suggest that galectin-3 expression in the rat brain seems to be regulated by developmental cascades, and that functionally and neuroanatomically related brain nuclei constitutively express galectin-3 in adulthood.

Blocking the phosphatidylinositol 3-kinase pathway inhibits neuregulin-1-mediated rescue of neurotoxicity induced by Aß1-42.

OBJECTIVES: Neuregulin-1 (NRG1) has an important role in both the development and the plasticity of the brain as well as neuroprotective properties. In this study, we investigated the downstream pathways of NRG1 signalling and their role in the prevention of Aß1-42 -induced neurotoxicity. METHODS: Lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) generation, superoxide dismutase (SOD) activity and TUNEL staining were assayed to examine the neuroprotective properties in primary rat cortical neurons. KEY FINDINGS: The inhibition of PI3K/Akt activation abolished the ability of NRG1 to prevent Aß1-42 -induced LDH release and increased TUNEL-positive cell count and reactive oxygen species accumulation in primary cortical neurons. CONCLUSIONS: Our results demonstrate that NRG1 signalling exerts a neuroprotective effect against Aß1-42 -induced neurotoxicity via activation of the PI3K/Akt pathway. Furthermore, this suggests that NRG1 has neuroprotective potential for the treatment of AD.

Neuregulin 1 Controls Glutamate Uptake by Up-regulating Excitatory Amino Acid Carrier 1 (EAAC1).

Neuregulin 1 (NRG1) is a trophic factor that is thought to have important roles in the regulating brain circuitry. Recent studies suggest that NRG1 regulates synaptic transmission, although the precise mechanisms remain unknown. Here we report that NRG1 influences glutamate uptake by increasing the protein level of excitatory amino acid carrier (EAAC1). Our data indicate that NRG1 induced the up-regulation of EAAC1 in primary cortical neurons with an increase in glutamate uptake. These in vitro results were corroborated in the prefrontal cortex (PFC) of mice given NRG1. The stimulatory effect of NRG1 was blocked by inhibition of the NRG1 receptor ErbB4. The suppressed expression of ErbB4 by siRNA led to a decrease in the expression of EAAC1. In addition, the ablation of ErbB4 in parvalbumin (PV)-positive neurons in PV-ErbB4(-/-) mice suppressed EAAC1 expression. Taken together, our results show that NRG1 signaling through ErbB4 modulates EAAC1. These findings link proposed effectors in schizophrenia: NRG1/ErbB4 signaling perturbation, EAAC1 deficit, and neurotransmission dysfunction.

Changes of gene expression of Gal3, Hsp27, Lcn2, and Timp1 in rat substantia nigra following medial forebrain bundle transection using a candidate gene microarray.

Neuroinflammation is an early event and important contributor to the pathobiology of neurodegenerative diseases. Neuroglia, especially microglia, are a major central nervous system population that can modulate neuroinflammation. To determine potential key molecules in this process, we employed microarray analysis in the substantia nigra (SN) following medial forebrain bundle (MFB) transection and analyzed the temporal expression profiles of candidate genes implicated in neuroglial activation and functional maturation. The DNA microarray analyzed, 8913 probes. Sixty nine genes were up-regulated and 11 genes were down-regulated at least twofold compared to normal control. Of the 80 genes, 23 were related to cell metabolism, 3 related to apoptosis, 27 related to immunity. Among them, 4 genes (Galectin 3, Heat shock protein 27, Lipocalin 2, Tissue inhibitory metalloproteinase 1) seemed to be related to the neuroglial function. The candidate genes were subjected to quantitative real-time PCR, Western blotting, and immunohistochemical approaches. Expression changes similar to the microarray were evident. In a double immunofluorescence assay, Galectin 3 almost completely co-localized with OX6-positive activated microglia, and Heat shock protein 27 mainly co-localized with glial fibrillary acidic protein (GFAP) positive astrocytes. Lipocalin 2, except for a few matches of GFAP positive astrocytes, did not co-localized with any of neuroglial markers. This is the first study to evaluate gene expression changes in the SN following MFB transection, which has been used as a parkinsonian animal model. Several candidate genes with potential roles in neuroglial activation and functional maturation were identified. The molecular significance of the candidate genes in neuroglial activation and neuroinflammation remains unclear.

Down-regulation of microglial activity attenuates axotomized nigral dopaminergic neuronal cell loss.

BACKGROUND: There is growing evidence that inflammatory processes of activated microglia could play an important role in the progression of nerve cell damage in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease which harbor features of chronic microglial activation, though the precise mechanism is unknown. In this study, we presented in vivo and ex vivo experimental evidences indicating that activated microglia could exacerbate the survival of axotomized dopaminergic neurons and that appropriate inactivation of microglia could be neuroprotective. RESULTS: The transection of medial forebrain bundle (MFB) of a rat induced loss of dopaminergic neurons in a time-dependent manner and accompanied with microglial activation. Along with microglial activation, production of reactive oxygen species (ROS) was upregulated and TH/OX6/hydroethidine triple-immunofluorescence showed that the microglia mainly produced ROS. When the activated microglial cells that were isolated from the substantia nigra of the MFB axotomized animal, were transplanted into the substantia nigra of which MFB had been transected at 7 days ago, the survival rate of axotomized dopaminergic neurons was significantly reduced as compared with sham control. Meanwhile, when the microglial activation was attenuated by administration of tuftsin fragment 1-3 (microglia inhibitory factor) into the lateral ventricle using mini-osmotic pump, the survival rate of axotomized dopaminergic neurons was increased. CONCLUSION: The present study suggests that activated microglia could actively produce and secrete unfavorable toxic substances, such as ROS, which could accelerate dopaminergic neuronal cell loss. So, well-controlled blockade of microglial activation might be neuroprotective in some neuropathological conditions.

Neuregulin-1 exerts protective effects against neurotoxicities induced by C-terminal fragments of APP via ErbB4 receptor.

Neuregulin-1 (NRG1) plays important roles in the development and plasticity of the brain, and it is also reported to have potent neuroprotective properties. We previously reported that NRG1 has neuroprotective actions against Swedish amyloid precursor protein-induced neurotoxicity. In addition to the amyloid beta peptide, other metabolites of amyloid precursor protein (APP) such as the C-terminal fragments of APP (APP-CTs) have been reported to possess cytotoxic effects in neuronal cells. In this study, we investigated whether NRG1 exerts neuroprotective effects against APP-CTs and attempted to determine its neuroprotective mechanisms. NRG1 attenuated the neurotoxicities induced by the expression of APP-CTs in neuronal cells. NRG1 also reduced the accumulation of reactive oxygen species and attenuated mitochondrial membrane potential loss induced by APP-CTs. In addition, NRG1 upregulated the expression of the anti-apoptotic protein Bcl-2. This effect was blocked by the inhibition of ErbB4, a key NRG1 receptor. Taken together, these results demonstrate the neuroprotective potential of NRG1 in Alzheimer's disease.

Evidence of early involvement of apoptosis inducing factor-induced neuronal death in Alzheimer brain.

Apoptosis inducing factor (AIF) has been proposed to act as a putative reactive oxygen species scavenger in mitochondria. When apoptotic cell death is triggered, AIF translocates to the nucleus, where it leads to nuclear chromatin condensation and large-scale DNA fragmentation which result in caspase-independent neuronal death. We performed this study to investigate the possibility that, in addition to caspase-dependent neuronal death, AIF induced neuronal death could be a cause of neuronal death in Alzheimer's disease (AD). We have found that AIF immunoreactivity was increased in the hippocampal pyramidal neurons in the Alzheimer brains compared to those of healthy, age-matched control brains. Nuclear AIF immunoreactivity was detected in the apoptotic pyramidal CA1 neurons at the early stage of AD and CA2 at the advanced stage. Nuclear AIF positive neurons were also observed in the amygdala and cholinergic neurons of the basal forebrain (BFCN) from the early stages of AD. The results of this study imply that AIF-induced apoptosis may contribute to neuronal death within the hippocampus, amygdala, and BFCN in early of AD.

c-Fos immunoreactivity in the neurons of the lateral geniculate nucleus in albino rats by light exposure after dark rearing.

PURPOSE: To investigate the effect of dark rearing immediately after birth on the maturation of the visual relay neurons in the lateral geniculate nucleus. METHODS: Fifty neonatal rats were used. Neonates of the control groups were raised under a normal light/dark cycle. Neonates of the experiment groups were dark reared and isolated from light during the entire experimental period, then exposed to the sun light for 1 hour before sacrifice. RESULTS: In the control groups, the neurons in the dorsal lateral geniculate nucleus developed normally at each age tested. In the experiment groups, the cytoplasm of the large neurons in the dorsal lateral geniculate nucleus of 2-week-old rats contained small vesicles, and the cytoplasm of the large neurons of 4-week-old rats was converted into a vacuole-like space. Moreover, c-Fos immunoreactivity of the large neurons in the dorsal lateral geniculate nucleus in the experiment groups was significantly increased compared to that of the control groups. CONCLUSIONS: We suppose that the maturation of the neurons in the lateral geniculate nucleus might be influenced by light stimulation during the critical period. Furthermore, c-Fos could be a marker of the functional activity of the visual relay neurons of the lateral geniculate nucleus in albino rats.

Constitutive activation of signal transducers and activators of transcription 3 correlates with better prognosis, cell proliferation and hypoxia-inducible factor-1α in human gastric cancer.

OBJECTIVE: We aimed to investigate the biological significance of signal transducers and activators of transcription 3 (STAT3) in gastric carcinoma. METHODS: Immunohistochemistry was performed on tissue array slides containing 285 gastric carcinoma specimens. The relationship between the nuclear expression of phospho-Tyr705-STAT3 (pSTAT3), an active form of STAT3, and prognosis, clinicopathological factors, proliferation, cell cycle regulators, apoptosis regulators, or angiogenesis-related proteins was evaluated. RESULTS: In nonneoplastic gastric mucosa, pSTAT3 was observed primarily in the nuclei of cells in the proliferative zone and intestinal metaplasia. In gastric carcinomas, nuclear STAT3 activation was observed in 36% of cases and was positively correlated with the Ki-67 labeling index and earlier tumor stage, whereas it was inversely correlated with lymphatic metastasis and distant metastasis (p< 0.05). Moreover, survival analyses showed that pSTAT3 expression was an independent prognostic factor of good survival. In addition, the expression of nuclear pSTAT3 positively correlated with that of cyclin D1, p21, p27, hypoxia-inducible factor-1α, or vascular endothelial growth factor (p< 0.05). CONCLUSIONS: STAT3 activation is an early event in gastric tumorigenesis and significantly correlates with better prognosis, proliferation and angiogenesis. Thus, STAT3 activation may be a valuable prognostic variable and therapeutic target in gastric carcinoma.