The causes of sporadic Parkinson's disease (PD) are poorly understood. 6-Hydroxydopamine (6-OHDA), a PD mimetic, is widely used to model this neurodegenerative disorder in vitro and in vivo; however, the underlying mechanisms remain incompletely elucidated. We demonstrate here that 6-OHDA evoked endoplasmic reticulum (ER) stress, which was characterized by an up-regulation in the expression of GRP78 and GADD153 (Chop), cleavage of procaspase-12, and phosphorylation of eukaryotic initiation factor-2 alpha in a human dopaminergic neuronal cell line (SH-SY5Y) and cultured rat cerebellar granule neurons (CGNs). Glycogen synthase kinase-3 beta (GSK3beta) responds to ER stress, and its activity is regulated by phosphorylation. 6-OHDA significantly inhibited phosphorylation of GSK3beta at Ser9, whereas it induced hyperphosphorylation of Tyr216 with little effect on GSK3beta expression in SH-SY5Y cells and PC12 cells (a rat dopamine cell line), as well as CGNs. Furthermore, 6-OHDA decreased the expression of cyclin D1, a substrate of GSK3beta, and dephosphorylated Akt, the upstream signaling component of GSK3beta. Protein phosphatase 2A (PP2A), an ER stress-responsive phosphatase, was involved in 6-OHDA-induced GSK3beta dephosphorylation (Ser9). Blocking GSK3beta activity by selective inhibitors (lithium, TDZD-8, and L803-mts) prevented 6-OHDA-induced cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), DNA fragmentations and cell death. With a tetracycline (Tet)-controlled TrkB inducible system, we demonstrated that activation of TrkB in SH-SY5Y cells alleviated 6-OHDA-induced GSK3beta dephosphorylation (Ser9) and ameliorated 6-OHDA neurotoxicity. TrkB activation also protected CGNs against 6-OHDA-induced damage. Although antioxidants also offered neuroprotection, they had little effect on 6-OHDA-induced GSK3beta activation. These results suggest that GSK3beta is a critical intermediate in pro-apoptotic signaling cascades that are associated with neurodegenerative diseases, thus providing a potential target site amenable to pharmacological intervention.

Glycogen synthase kinase 3B (GSK3B) is a multifunctional serine/threonine kinase. We showed that the expression of GSK3B was drastically down-regulated in human cutaneous squamous cell carcinomas and basal cell carcinomas. Due to its negative regulation of many oncogenic proteins, we hypothesized that GSK3B may function as a tumor suppressor during the neoplastic transformation of epidermal cells. We tested this hypothesis using an in vitro model system, JB6 mouse epidermal cells. In response to epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA), the promotion-sensitive JB6 P+ cells initiate neoplastic transformation, whereas the promotion-resistant JB6 PÀ cells do not. JB6 PÀ cells expressed much higher levels of GSK3B than JB6 P+ cells; JB7 cells, the transformed derivatives of JB6, had the least amount of GSK3B. The activity of GSK3B is negatively regulated by its phosphorylation at Ser 9 . EGF and TPA induced strong Ser 9 phoshorylation in JB6 P+ cells, but phosphorylation was seen at a much lesser extent in JB6 PÀ cells. EGF and TPA-stimulated Ser 9 phosphorylation was mediated by phosphoinositide-3-kinase (PI3K)/Akt and protein kinase C (PKC) pathways. Inhibition of GSK3B activation significantly stimulated activator protein-1 (AP-1) activity. Overexpression of wild-type (WT) and S9A mutant GSK3B in JB6 P+ cells suppressed EGF and TPA-mediated anchorage-independent growth in soft agar and tumorigenicity in nude mice. Overexpression of a kinase-deficient (K85R) GSK3B, in contrast, potentiated anchorage-independent growth and drastically enhanced in vivo tumorigenicity. Together, these results indicate that GSK3B plays an important role in skin tumorigenesis. [Cancer Res 2007;67(16):7756-64]

Both epidemiological and experimental studies indicate that ethanol is a tumor promoter and may promote metastasis of breast cancer. However, the molecular mechanisms underlying ethanol-mediated tumor promotion remain unknown. Overexpression of ErbB proteins in breast cancer patients is generally associated with poor prognosis. The ErbB proteins are a family of receptor kinases that include four closely related members: epidermal growth factor receptor (EGFR/ErbB1), ErbB2/neu, ErbB3, and ErbB4. Particularly, ErbB2 plays a pivotal role in ErbB-mediated activities. Here we demonstrated that amplification of ErbB2 expression sensitized a specific cellular response to ethanol. Human breast cancer cells or mammary epithelial cells with a high expression of ErbB2 exhibited an enhanced response to ethanol-stimulated cell invasion in vitro. Ethanol also stimulated cell proliferation; however, this stimulation was independent of ErbB2 levels. Ethanol triggered divergent intracellular signaling among cells expressing different ErbB2 levels. In the cells overexpressing ErbB2, ethanol was more effective in the activation of c-Jun NH 2 terminal protein kinases (JNKs) and p38 mitogen-activated protein kinase (p38 MAPK) as well as the induction of reactive oxygen species (ROS) than the cells with normal ErbB2 expression. Blockage of either JNKs or p38 MAPK activation eliminated ethanol-mediated cell invasion. In contrast, the reduction of hydrogen peroxide concentration by catalase exposure had little effect on ethanolinduced cell invasion. These results indicated that ethanolinduced cell invasion was primarily mediated by JNKs and p38 MAPK, whereas the involvement of ROS formation might be minimal. Our study suggests that overexpression of ErbB2 may augment ethanol-elicited signaling and promote ethanol-stimulated tumor metastasis.

Ethanol is a tumor promoter and may enhance the metastasis of breast cancer. We have previously demonstrated that over-expression of ErbB2 promoted ethanol-mediated invasion of mammary epithelial cells and breast cancer cells. However, the underlying cellular/molecular mechanisms remain unknown. By gelatin zymography, we showed that over-expression of ErbB2 increased the production of matrix metalloproteinase-2 (MMP-2) and MMP-9 in human mammary epithelial cells (HB2). Transient or stable transfection of ErbB2 cDNA to HB2 cells upregulated the transcripts and the activity of the MMP-2/-9 gene promoter; the upregulation of MMP-2/-9 expression was mediated by p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase (PI3K). Although ethanol, at physiologically relevant concentrations (100-400 mg/dl), did not affect the production of MMP-2/-9, it activated MMP-2 in HB2 cells over-expressing ErbB2 (HB2 Key words: alcohol; ErbB; metastasis; proteinases; signal transduction; oxidative stress Breast cancer is a leading cause of morbidity and mortality in women.1 The endogenous and environmental factors that contribute to its etiology remain elusive. Alcohol is a tumor promoter; there is a positive correlation between heavy alcohol intake and the risk of breast cancer.2-4 Epidemiological studies reveal that alcohol consumption is associated with advanced and invasive breast tumors, [5][6][7] suggesting that alcohol may enhance tumor development and metastasis. These epidemiological results are supported by experimental studies using animal models and cell culture systems, which consistently show that ethanol promotes mammary tumorigenesis and stimulates proliferation, as well as the invasion of breast cancer cells. [8][9][10][11][12][13][14][15][16] The molecular mechanisms underlying ethanol action, however, remain to be determined.The ErbB family of receptor kinases includes 4 closely related members: epidermal growth factor receptor (EGFR or ErbB1), ErbB2, ErbB3 and ErbB4. Among the family, ErbB2 is most directly related to breast cancer. Amplification of ErbB2 is found in 20-30% of breast cancer patients and is associated with poor prognosis.17 Over-expression of ErbB2 is positively correlated with lymph node metastasis in breast cancer patients. 18,19 Although no known ligand has been identified, ErbB2 is the preferred heterodimerization partner for all ErbB family members, and it plays a pivotal role in intracellular signaling mediated by other ErbB receptors. 20 The status of ErbB2 expression in a given cell is critical in determining the cellular response to growth factors or environmental stimuli. [21][22][23] We have previously shown that over-expression of ErbB2 dramatically promoted ethanol-induced invasion of mammary epithelial cells and breast tumor cells.12 The present study was designed to determine the underlying molecular mechanisms.The metastasis of cancer cells involves multiple steps. Tumor cells must first detach from the tumor mass and invade the surrounding extracellular m...

The findings of this first population-based assessment of reproductive health following the massive Wenchuan earthquake may help in shaping public health measures benefiting women surviving large-sale disasters.

The developing central nervous system (CNS) is particularly susceptible to ethanol toxicity. The loss of neurons underlies many of the behavioral deficits observed in fetal alcohol spectrum disorders (FASD). The mechanisms of ethanol-induced neuronal loss, however, remain incompletely elucidated. We demonstrated that glycogen synthase kinase 3β (GSK3β), a multifunctional serine/threonine kinase, was involved in ethanol neurotoxicity. The activity of GSK3β is negatively regulated by its phosphorylation at serine 9 (Ser9). Ethanol induced dephosphorylation of GSK3β at Ser9 and the activation of Bax as well as caspase-3 in the developing mouse brain. These ethanol-induced alterations were ameliorated by the pretreatment of a GSK3β inhibitor, lithium. To determine the role of GSK3β in ethanol neurotoxicity, we overexpressed wild-type (WT), S9A mutant or dominant-negative (DN) mutant GSK3β in a neuronal cell line (SK-N-MC). Ethanol only modestly reduced the viability of parental SK-N-MC cells but drastically induced caspase-3 activation and apoptosis in cells overexpressing WT or S9A GSK3β, indicating that the high levels of GSK3β or the active form of GSK3β increased cellular sensitivity to ethanol. Contrarily, overexpression of DN GSK3β conferred resistance to ethanol toxicity. Lithium and other specific GSK3β inhibitors abolished the hypersensitivity to ethanol caused by WT or S9A overexpression. Bax, a proapoptotic protein, is a substrate of GSK3β. Cells overexpressing WT or S9A GSK3β were much more sensitive to ethanol-induced Bax activation than parental SK-N-MC cells. Our results indicate that GSK3β may be a mediator of ethanol neurotoxicity, and its expression status in a cell may determine ethanol vulnerability.

Ethanol is a tumor promoter and may promote metastasis of breast cancer. However, the underlying cellular/molecular mechanisms remain unknown. Overexpression and high activity of matrix metalloproteinase-2 (MMP-2) are frequently associated with metastatic breast cancers and serve as a prognostic indicator of clinical outcome. MMP-2 is predominantly expressed in stromal fibroblasts and plays a pivotal role in regulating the invasive behavior of breast tumor cells. We hypothesized that ethanol may enhance the invasion of breast tumor cells by modulating the activity of fibroblastic MMP-2. With in vitro models (HS68 and CCD1056SK human fibroblasts), we showed that ethanol at physiologically relevant concentrations (50 -200 mg/dl) activated MMP-2; conversely, at a higher concentration (400 mg/dl), it inhibited the MMP-2 activity. Consistently, conditioned medium collected from ethanol ( Key words: alcohol; ErbB; metastasis; proteinases; signal transductionBreast cancer is a leading cause of morbidity and mortality in women. 1 The endogenous and environmental factors that contribute to its etiology remain elusive. Despite being responsive to hormonal manipulation and chemotherapy, relapse after treatment is common, particularly in patients presenting with metastatic disease. 2 The metastatic process involves the degradation of different macromolecular components of the extracellular matrix (ECM) and basement membranes and is regulated by intrinsic properties of the tumor cells as well as microenvironmental factors. Alcohol is a tumor promoter; there is a positive correlation between alcohol intake and the risk of several human cancers, including mouth/oropharyngeal cancer, oesophageal cancer, liver cancer and breast cancer. [3][4][5][6][7][8][9][10][11] Epidemiologic studies indicate that alcohol consumption is associated with advanced and invasive breast tumors, [12][13][14] suggesting that alcohol may enhance tumor development and metastasis. These epidemiologic results are supported by experimental studies using animal models and cell culture systems, which show that ethanol promotes mammary tumorigenesis and stimulates proliferation as well as invasion of breast cancer cells. [15][16][17][18][19][20][21] The molecular mechanisms underlying ethanol action, however, remain to be determined.Abnormal communication between the mammary epithelium and stromal cells promotes tumorigenesis and development of breast carcinomas. 22 Cancer-stroma interaction is mediated at least in part through the matrix metalloproteinases (MMPs). MMPs are a family of zinc-dependent endopeptidases that collectively are capable of degrading all components of the ECM. MMPs have been implicated in normal matrix remodeling events such as development of the mammary gland 23 and in pathologic conditions, including tumor invasion and metastasis. 24 Coupled with their function in metastasis, the MMPs also have a role in carcinogenesis. 25,26 High levels of MMP-2 and MMP-9 have been found to correlate with enhanced metastasis and poor prognosis in ...

One of the most devastating effects of ethanol exposure during development is the loss of neurons in selected brain areas. The underlying cellular/molecular mechanisms remain unclear. The endoplasmic reticulum (ER) is involved in posttranslational protein processing and transport. The accumulation of unfolded or misfolded proteins in the ER lumen triggers ER stress, which is characterized by translational attenuation, synthesis of ER chaperone proteins such as GRP78, and activation of transcription factors such as ATF4, ATF6, and CHOP. Sustained ER stress ultimately leads to cell death. ER stress response can be induced experimentally by treatment with tunicamycin and thapsigargin. Using SH-SY5Y neuroblastoma cells and primary cerebellar granule neurons as in vitro models, we demonstrated that exposure to ethanol alone had little effect on the expression of markers for ER stress; however, ethanol drastically enhanced the expression of GRP78, CHOP, ATF4, ATF6, and phosphorylated PERK and elF2α when induced by tunicamycin and thapsigargin. Consistently, ethanol promoted tunicamycin-and thapsigargininduced cell death. Ethanol rapidly caused oxidative stress in cultured neuronal cells; antioxidants blocked ethanol's potentiation of ER stress and cell death, suggesting that the ethanol-promoted ER stress response is mediated by oxidative stress. CHOP is a proapoptotic transcription factor. We further demonstrated that CHOP played an important role in ethanol-promoted cell death. Thus, the effect of ethanol may be mediated by the interaction between oxidative stress and ER stress. Keywordsalcohol; apoptosis; cerebellum; development; fetal alcohol syndrome Alcohol exposure alters the structure and physiology of the brain in many ways. During development, alcohol-induced structural and physiological alterations contribute to the brain dysfunction of children with fetal alcohol syndrome (FAS). FAS is the most common nonhereditary cause of mental retardation (May and Gossage, 2001). One of the most prominent alcohol-induced neuropathological changes in the developing brain is the loss of neurons (West et al., 1990;Luo and Miller, 1998;Ikonomidou et al., 2000; Goodlet and © 2007 Horn, 2001). In adults, heavy alcohol consumption also causes neuronal loss in selected brain regions (Kril et al., 1997;Gotz et al., 2001;Ikegami et al., 2003). The mechanisms underlying ethanol-induced neuronal loss, however, remain incompletely elucidated. Oxidative stress is caused by the disruption of intracellular redox homeostasis. The generation of reactive oxygen species (ROS) initiates this process and may cause neuronal death (Mattson et al., 2001;Watts et al., 2005;Loh et al., 2006). Alcohol exposure may induce oxidative stress in the central nervous system (Marino et al., 2004;Kumral et al., 2005;Chu et al., 2007), and the brain is particularly susceptible to ROS-induced damage (Annunziato et al., 2003). Oxidative stress has been proposed to be a potential mechanism for ethanol-induced damage (Sun et al., 2001;Chu et al., 2...