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...