Hesperidin downregulates kinases lrrk2 and gsk3ß in a 6-OHDA induced Parkinson's disease model.

The depletion of dopamine in the striatum region and Lewy bodies are the hallmark characteristics of Parkinson's disease. The pathology also includes the upregulation of various Parkinson's disease (PARK) genes and kinases. Two such kinases, LRRK2 and GSK-3ß have been directly implicated in the formation of tau and alpha-synuclein proteins, causing PD. Hesperidin (HES) is a flavanone glycoside that has multiple therapeutic benefits including neuroprotective effects. In this study, we examined the neuroprotective effects of HES against 6-hydroxydopamine (6-OHDA) induced-neurotoxicity in the in-vitro and in-vivo model. Hesperidin significantly protected the SH-SY5Y cells' stress against 6-OHDA induced toxicity by downregulating biomarkers of oxidative stress. Furthermore, HES downregulated the kinases lrrk2 and gsk3ß along with casp3, casp9, and polg in the zebrafish model. The treatment with HES also improved the locomotor pattern of zebrafish that was affected by 6-OHDA. This study suggests that hesperidin could be a drug of choice in targeting kinases against a 6-OHDA model of PD.

LGK974, a PORCUPINE inhibitor, mitigates cytotoxicity in an in vitro model of Parkinson's disease by interfering with the WNT/ß-CATENIN pathway.

Paraquat (PQ) as an herbicide has been demonstrated to impair dopaminergic (DAergic) neurons and highly correlate with the etiology of Parkinson's disease (PD). WNT/ß-CATENIN signaling is known for the specification and neurogenesis of midbrain DAergic neurons and implicated as a therapeutic target in treating many diseases, such as cancer and degenerative diseases. LGK974, a WNT pathway inhibitor, is currently under clinical trial for patients with malignancies. Since the exact role of WNT/ß-CATENIN signaling in mediating PD is undetermined, LGK974 was used to examine its effect on the PQ-induced cell model of PD. LGK974 attenuated PQ-induced apoptosis and released mitochondrial pro-poptotic molecules in human neuroblastoma SH-SY5Y cell. PQ increased the levels of ß-CATENIN, non-phosphorylated (Ser33/37/Thr41) ß-CATENIN, and phosphorylated glycogen synthase kinase (GSK)-3α/ß. PQ also increased the nuclear translocation of ß-CATENIN, which can be attenuated by LKG974. Furthermore, LGK974 attenuated the PQ-induced release of mitochondrial proapoptotic factors and WNT agonist 1-induced cell death. Taken together, we have shown for the first time that LGK974 mediated through the WNT/ß-CATENIN pathway to prevent PQ-induced cell death.

Impact of silencing hepatic SREBP-1 on insulin signaling.

Sterol Regulatory Element Binding Protein-1 (SREBP-1) is a conserved transcription factor of the basic helix-loop-helix leucine zipper family (bHLH-Zip) that plays a central role in regulating expression of genes of carbohydrate and fatty acid metabolism in the liver. SREBP-1 activity is essential for the control of insulin-induced anabolic processes during the fed state. In addition, SREBP-1 regulates expression of key molecules in the insulin signaling pathway, including insulin receptor substrate 2 (IRS2) and a subunit of the phosphatidylinositol 3-kinase (PI3K) complex, PIK3R3, suggesting that feedback mechanisms exist between SREBP-1 and this pathway. Nevertheless, the overall contribution of SREBP-1 activity to maintain insulin signal transduction is unknown. Furthermore, Akt is a known activator of mTORC1, a sensor of energy availability that plays a fundamental role in metabolism, cellular growth and survival. We have silenced SREBP-1 and explored the impact on insulin signaling and mTOR in mice under fed, fasted and refed conditions. No alterations in circulating levels of insulin were observed. The studies revealed that depletion of SREBP-1 had no impact on IRS1Y612, AktS473, and downstream effectors GSK3αS21 and FoxO1S256 during the fed state. Nevertheless, reduced levels of these molecules were observed under fasting conditions. These effects were not associated with changes in phosphorylation of mTOR. Overall, our data indicate that the contribution of SREBP-1 to maintain insulin signal transduction in liver is modest.

The Dictyostelium GSK3 kinase GlkA coordinates signal relay and chemotaxis in response to growth conditions.

GSK3 plays a central role in orchestrating key biological signaling pathways, including cell migration. Here, we identify GlkA as a GSK3 family kinase with functions that overlap with and are distinct from those of GskA. We show that GlkA, as previously shown for GskA, regulates the cell's cytoskeleton through MyoII assembly and control of Ras and Rap1 function, leading to aberrant cell migration. However, there are both qualitative and quantitative differences in the regulation of Ras and Rap1 and their downstream effectors, including PKB, PKBR1, and PI3K, with glkA- cells exhibiting a more severe chemotaxis phenotype than gskA- cells. Unexpectedly, the severe glkA- phenotypes, but not those of gskA-, are only exhibited when cells are grown attached to a substratum but not in suspension, suggesting that GlkA functions as a key kinase of cell attachment signaling. Using proteomic iTRAQ analysis we show that there are quantitative differences in the pattern of protein expression depending on the growth conditions in wild-type cells. We find that GlkA expression affects the cell's proteome during vegetative growth and development, with many of these changes depending on whether the cells are grown attached to a substratum or in suspension. These changes include key cytoskeletal and signaling proteins known to be essential for proper chemotaxis and signal relay during the aggregation stage of Dictyostelium development.

Inhibition of glycogen synthase kinase-3 (GSK3) promotes the neural differentiation of full-term amniotic fluid-derived stem cells towards neural progenitor cells.

The amniotic fluid has a heterogeneous population of cells. Some human amniotic fluid-derived stem (hAFS) cells have been shown to harbor the potential to differentiate into neural cells. However, the neural differentiation efficiency of hAFS cells remains low. In this study, we isolated CD117-positive hAFS cells from amniotic fluid and then examined the pluripotency of these cells through the formation of embryoid bodies (EBs). Additionally, we induced the neural differentiation of these cells using neuroectodermal medium. This study revealed that the GSK3-beta inhibitor SB216763 was able to stimulate the proliferation of CD117-positive hAFS cells without influencing their undifferentiated state. Moreover, SB216763 can efficiently promote the neural differentiation of CD117-positive hAFS cells towards neural progenitor cells in the presence of DMEM/F12 and N2 supplement. These findings provide an easy and low-cost method to maintain the proliferation of hAFS cells, as well as induce an efficacious generation of neural progenitor cells from hAFS cells. Such induction of the neural commitment of hAFS cells may provide an option for the treatment of neurodegenerative diseases by hAFS cells-based therapies.

The Mechanism by Which Amentoflavone Improves Insulin Resistance in HepG2 Cells.

BACKGROUND: The aim of this study was to explore the mechanism by which amentoflavone (AME) improves insulin resistance in a human hepatocellular liver carcinoma cell line (HepG2). METHODS: A model of insulin resistant cells was established in HepG2 by treatment with high glucose and insulin. The glucose oxidase method was used to detect the glucose consumption in each group. To determine the mechanism by which AME improves insulin resistance in HepG2 cells, enzyme-linked immunosorbent assay (ELISA) and western blotting were used to detect the expression of phosphatidyl inositol 3-kinase (PI3K), Akt, and pAkt; the activity of the enzymes involved in glucose metabolism; and the levels of inflammatory cytokines. RESULTS: Insulin resistance was successfully induced in HepG2 cells. After treatment with AME, the glucose consumption increased significantly in HepG2 cells compared with the model group (MG). The expression of PI3K, Akt, and pAkt and the activity of 6-phosphofructokinas (PFK-1), glucokinase (GCK), and pyruvate kinase (PK) increased, while the activity of glycogen synthase kinase-3 (GSK-3), phosphoenolpyruvate carboxylase kinase (PEPCK), and glucose-6-phosphatase (G-6-Pase) as well as the levels of interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and C reactive protein (CRP) decreased. CONCLUSIONS: The mechanism by which treatment with AME improves insulin resistance in HepG2 cells may involve the PI3K-Akt signaling pathway, the processes of glucose oxygenolysis, glycogen synthesis, gluconeogenesis and inflammatory cytokine expression.

Heme Oxygenase-1 Mediates Neuroprotection Conferred by Argon in Combination with Hypothermia in Neonatal Hypoxia-Ischemia Brain Injury.

BACKGROUND: Hypoxic-ischemic encephalopathy is a major cause of mortality and disability in the newborn. The authors investigated the protective effects of argon combined with hypothermia on neonatal rat hypoxic-ischemic brain injury. METHODS: In in vitro studies, rat cortical neuronal cell cultures were challenged by oxygen and glucose deprivation for 90 min and exposed to 70% Ar or N2 with 5% CO2 balanced with O2, at 33°C for 2 h. Neuronal phospho-Akt, heme oxygenase-1 and phospho-glycogen synthase kinase-3ß expression, and cell death were assessed. In in vivo studies, neonatal rats were subjected to unilateral common carotid artery ligation followed by hypoxia (8% O2 balanced with N2 and CO2) for 90 min. They were exposed to 70% Ar or N2 balanced with oxygen at 33°, 35°, and 37°C for 2 h. Brain injury was assessed at 24 h or 4 weeks after treatment. RESULTS: In in vitro studies, argon-hypothermia treatment increased phospho-Akt and heme oxygenase-1 expression and significantly reduced the phospho-glycogen synthase kinase-3ß Tyr-216 expression, cytochrome C release, and cell death in oxygen-glucose deprivation-exposed cortical neurons. In in vivo studies, argon-hypothermia treatment decreased hypoxia/ischemia-induced brain infarct size (n = 10) and both caspase-3 and nuclear factor-κB activation in the cortex and hippocampus. It also reduced hippocampal astrocyte activation and proliferation. Inhibition of phosphoinositide-3-kinase (PI3K)/Akt pathway through LY294002 attenuated cerebral protection conferred by argon-hypothermia treatment (n = 8). CONCLUSION: Argon combined with hypothermia provides neuroprotection against cerebral hypoxia-ischemia damage in neonatal rats, which could serve as a new therapeutic strategy against hypoxic-ischemic encephalopathy.

Heat shock transcription factor 1-associated expression of slow myosin heavy chain in mouse soleus muscle in response to unloading with or without reloading.

AIM: The effects of heat shock transcription factor 1 (HSF1) deficiency on the fibre type composition and the expression level of nuclear factor of activated T cells (NFAT) family members (NFATc1, NFATc2, NFATc3 and NFATc4), phosphorylated glycogen synthase kinase 3α (p-GSK3α) and p-GSK3ß, microRNA-208b (miR-208b), miR-499 and slow myosin heavy chain (MyHC) mRNAs (Myh7 and Myh7b) of antigravitational soleus muscle in response to unloading with or without reloading were investigated. METHODS: HSF1-null and wild-type mice were subjected to continuous 2-week hindlimb suspension followed by 2- or 4-week ambulation recovery. RESULTS: In wild-type mice, the relative population of slow type I fibres, the expression level of NFATc2, p-GSK3 (α and ß), miR-208b, miR-499 and slow MyHC mRNAs (Myh7 and Myh7b) were all decreased with hindlimb suspension, but recovered after it. Significant interactions between train and time (the relative population of slow type I fibres; P = 0.01, the expression level of NFATc2; P = 0.001, p-GSKß; P = 0.009, miR-208b; P = 0.002, miR-499; P = 0.04) suggested that these responses were suppressed in HSF1-null mice. CONCLUSION: HSF1 may be a molecule in the regulation of the expression of slow MyHC as well as miR-208b, miR-499, NFATc2 and p-GSK3 (α and ß) in mouse soleus muscle.

AMPK interacts with ß-catenin in the regulation of hepatocellular carcinoma cell proliferation and survival with selenium treatment.

Selenium has received much attention as an anticancer agent, although the mechanisms of action underlying its pro-apoptotic properties remain unclear. Tumors that respond well to antioxidant treatments, such as hepatocellular carcinoma (HCC), may benefit from treatment with selenium as this compound also has antioxidant properties. Furthermore, a major oncogenic driver in HCC is the nuclear transcription co-activator, ß-catenin. In the present study, we examined the mechanism by which selenium reduces survival of HCC cells, and whether this was associated with modulation of the ß-catenin pathway. Hep3B cell lines and cancer cell xenografted animals were treated with selenium, and apoptotic events or signals such as AMPK, ß-catenin and GSK3ß were determined. Further interactions among ß-catenin, glycogen synthase kinase 3ß (GSK3ß), and AMPK were explored by applying AMPK small interfering RNA (siRNA) or GSK3ß siRNA with western blotting or immunofluorescence microscopic observation. Selenium activated AMPK, which in turn suppressed ß-catenin. Selenium induced the translocation of AMPK into the nucleus and prevented the accumulation of ß-catenin therein. Upon inactivation of AMPK by AMPK siRNA, selenium no longer modulated ß-catenin, implying that AMPK is an upstream signal for ß-catenin. We found that the binding between AMPK and ß-catenin occurs in the cytosolic fraction, and therefore concluded that the cancer cell antiproliferative effects of selenium are mediated by a GSK3ß-independent AMPK/ß-catenin pathway, although AMPK-mediated GSK3ß regulation was also observed. We primarily discovered that AMPK is a crucial regulator initiating selenium-induced inhibition of ß-catenin expression. Taken together, these novel findings help to illuminate the molecular mechanisms underlying the anticancer effect of selenium and highlight the regulation of ß-catenin by selenium.

MARCKSL1 exhibits anti-angiogenic effects through suppression of VEGFR-2-dependent Akt/PDK-1/mTOR phosphorylation.

Myristoylated alanine-rich C kinase substrate-like 1 (MARCKSL1) plays a pivotal role in the regulation of apoptosis and has been shown to maintain antitumor and metastasis-suppressive properties. In the present study, we examined the effects of MARCKSL1 as a novel anti-angiogenic agent on the inhibition of angiogenesis-mediated cell migration. MARCKSL1 also reduced vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) proliferation, as well as capillary-like tubular structure formation in vitro. MARCKSL1 disrupted phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) in ovarian tumorigenesis. In addition, MARCKSL1 showed potent anti-angiogenic activity and reduced the levels of VEGF and hypoxia-inducible factor 1α (HIF-1α) expression, an essential regulator of angiogenesis. Consistently, MARCKSL1 decreased VEGF­induced phosphorylation of the PI3K/Akt signaling pathway components, including phosphoinositide-dependent protein kinase 1 (PDK-1), mammalian target of rapamycin (mTOR), tuberous sclerosis complex 2 (TSC-2), p70 ribosomal protein S6 kinase (p70S6K), and glycogen synthase kinase 3ß (GSK-3ß) protein. Collectively, our results provide evidence for the physiological/biological function of an endothelial cell system involved in angiogenesis through suppression of Akt/PDK-1/mTOR phosphorylation by interaction with VEGFR-2.

Accelerated cardiac remodeling in desmoplakin transgenic mice in response to endurance exercise is associated with perturbed Wnt/ß-catenin signaling.

Arrhythmogenic ventricular cardiomyopathy (AVC) is a frequent underlying cause for arrhythmias and sudden cardiac death especially during intense exercise. The mechanisms involved remain largely unknown. The purpose of this study was to investigate how chronic endurance exercise contributes to desmoplakin (DSP) mutation-induced AVC pathogenesis. Transgenic mice with overexpression of desmoplakin, wild-type (Tg-DSP(WT)), or the R2834H mutant (Tg-DSP(R2834H)) along with control nontransgenic (NTg) littermates were kept sedentary or exposed to a daily running regimen for 12 wk. Cardiac function and morphology were analyzed using echocardiography, electrocardiography, histology, immunohistochemistry, RNA, and protein analysis. At baseline, 4-wk-old mice from all groups displayed normal cardiac function. When subjected to exercise, all mice retained normal cardiac function and left ventricular morphology; however, Tg-DSP(R2834H) mutants displayed right ventricular (RV) dilation and wall thinning, unlike NTg and Tg-DSP(WT). The Tg-DSP(R2834H) hearts demonstrated focal fat infiltrations in RV and cytoplasmic aggregations consisting of desmoplakin, plakoglobin, and connexin 43. These aggregates coincided with disruption of the intercalated disks, intermediate filaments, and microtubules. Although Tg-DSP(R2834H) mice already displayed high levels of p-GSK3-ß(Ser9) and p-AKT1(Ser473) under sedentary conditions, decrease of nuclear GSK3-ß and AKT1 levels with reduced p-GSK3-ß(Ser9), p-AKT1(Ser473), and p-AKT1(Ser308) and loss of nuclear junctional plakoglobin was apparent after exercise. In contrast, Tg-DSP(WT) showed upregulation of p-AKT1(Ser473), p-AKT1(Ser308), and p-GSK3-ß(Ser9) in response to exercise. Our data suggest that endurance exercise accelerates AVC pathogenesis in Tg-DSP(R2834H) mice and this event is associated with perturbed AKT1 and GSK3-ß signaling. Our study suggests a potential mechanism-based approach to exercise management in patients with AVC.

2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine is an anti-inflammatory TLR-2, -4 and -5 response mediator in human monocytes.

OBJECTIVE AND DESIGN: To elucidate the influence of 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP), a canonical Wnt/ß-catenin pathway activator, on the inflammatory response of TLR-engaged innate cells in vitro. MATERIAL OR SUBJECT: Primary human monocytes. TREATMENT: AMPMB (0-10 µM), LPS (0-1.0 µg/ml), Pam3CSK4, FSL-1, or S. typhimurium flagellin (0-0.25 µg/ml). METHODS: TLR-induced cytokine release (TNF, IL-6, IL-12 p40) was monitored by ELISA while Wnt-related signals (GSK3ß, p65, IκB, ß-catenin) were assessed by Western blot, pharmaceutical inhibition and gene silencing. RESULTS: AMBMP induced the rapid phosphorylation of NFκB p65 at Ser(536) and abrogated total IκB, accompanied by a subsequent increase in pro-inflammatory cytokine production (TNF, IL-6, IL-12 p40) in otherwise naive monocytes. However, in TLR2, -4 and -5-engaged monocytes, AMBMP-suppressed cytokine production. In the context of LPS stimulation, this occurred concomitant with the phosphorylative inactivation of GSK3ß at Ser(9), ß-catenin accumulation and abrogation of NFκB p65 phosphorylation. AMBMP-mediated suppression of the TLR4 -induced inflammatory response was reversed by two pharmaceutical Wnt/ß-catenin pathway inhibitors, IWP-2 and PNU-74654 and by Wnt3a silencing. CONCLUSIONS: Herein, we show that AMBMP induces canonical Wnt signaling events and acts as a suppressor of inflammation in surface TLR-engaged primary human monocytes.

TIPE2 functions as a metastasis suppressor via negatively regulating ß-catenin through activating GSK3ß in gastric cancer.

Tumor necrosis factor (TNF)-α-induced protein 8-like 2 (TNFAIP8L2, TIPE2) is a novel anti-inflammatory factor involved in maintaining immune homeostasis. Accumulating evidence has also shown that TIPE2 displays tumor-suppressive effects in several tumor types. Previous studies revealed that TIPE2 inhibits hepatocellular carcinoma metastasis by repressing Ral and Rac1 GTPases. However, its antimetastatic activity and underlying mechanism in other human cancers is largely unknown. We investigated TIPE2 in AGS, HGC-27 and SGC-7901 human gastric cancer cells compared with GES-1 normal human gastric mucous epithelial cells. We demonstrated that TIPE2 was expressed in GES-1 gastric mucous epithelial cells but lost in all three types of gastric cancer cells. We then performed a gain-of-function study by adenovirus-mediated TIPE2 overexpression (AdVTIPE2) and investigated the effects of TIPE2 on migration and invasion of AGS human gastric cancer cells. Wound healing and Transwell invasion assays showed that forced expression of TIPE2 markedly suppressed the gastric cancer cell migration and invasion in vitro. Mechanistically, TIPE2 remarkably reduced the total levels of pAKT, pGSK3ß and ß-catenin as well as the nuclear level of ß-catenin in gastric cancer cells. The TIPE2-elicited antimetastatic effect in gastric cancer was closely associated with the inhibition of AKT signaling and enhancement of GSK3ß activity followed by the degradation and decreased translocation to nucleus of ß-catenin. These results provide the first compelling evidence that TIPE2 suppresses gastric cancer metastasis via downregulating ß-catenin signaling through inhibiting AKT and activating GSK3ß, indicating that TIPE2 is a promising therapeutic target for human gastric cancer metastasis.

The protective effects of CHIR99021 against oxidative injury in LO2 cells.

Hepatic ischemia-reperfusion injury is one of the most important factors for the prognosis of liver transplantation and hepatic surgery. It was reported that glycogen synthase kinase-3 (GSK-3) regulated injury response during ischemia-reperfusion. In this study, we investigated the protective effects of the GSK-3 inhibitor CHIR99021 against hepatic ischemia-reperfusion injury. A H2O2-induced oxidative injury model using LO2 cells was established. LO2 cells were divided into four groups, including blank control group, CHIR99021 control group treated with CHIR99021 alone, H2O2-injury group treated with H2O2 and protection group treated with H2O2 plus CHIR99021. Cell viability, cell apoptosis or necrosis was determined. Meanwhile, mitochondrial membrane potential, lipid peroxidation, cellular ROS levels, SOD activity, and serum contents of ALS and AST were measured. Protein and mRNA expressions were also detected. The results showed that a cell oxidative injury model was established by treating LO2 cells with 200 µmol/L H2O2 for 6 h. Cells exposed to H2O2 resulted in a significant decrease of cell viability and increase of cell apoptosis, which was accompanied by increasing ROS levels, disruption of mitochondrial membrane potential, excessive lipid peroxidation, reduction of SOD activity, and increased levels of ALT and AST. Treatment with CHIR99021 significantly protected LO2 cells against H2O2-induce oxidative injury by inhibiting the changes of above oxidative injury related indicators. Moreover, CHIR99021 treatment significantly reversed H2O2-induced decrease in p-GSK-3ßSer9 , Bcl-2, Bcl-xl, survivin and ß-catenin expression, whereas it significantly attenuated H2O2-induced increase in caspase-3, cleaved caspase-3 and p-JNK protein expression. In conclusion, CHIR99021 protected LO2 cells against H2O2-induced oxidative injury through reducing GSK-3ß activity and apoptosis, with underlying mechanisms involved in stabilizing mitochondrial membrane potential, attenuating cellular ROS generation, suppressing mitochondria-mediated apoptotic pathway, and activation of GSK-3ß/ß-catenin signaling pathway.

E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation.

Runx2, a master regulator of osteoblast differentiation, is tightly regulated at both transcriptional and post-translational levels. Post-translational modifications such as phosphorylation and ubiquitination have differential effects on Runx2 functions. Here, we show that the reduced expression and functions of Runx2 upon its phosphorylation by GSK3ß are mediated by its ubiquitin-mediated degradation through E3 ubiquitin ligase Fbw7α. Fbw7α through its WD domain interacts with Runx2 both in a heterologous (HEK293T cells) system as well as in osteoblasts. GSK3ß was also present in the same complex as determined by co-immunoprecipitation. Furthermore, overexpression of either Fbw7α or GSK3ß was sufficient to down-regulate endogenous Runx2 expression and function; however, both failed to inhibit endogenous Runx2 when either of them was depleted in osteoblasts. Fbw7α-mediated inhibition of Runx2 expression also led to reduced Runx2 transactivation and osteoblast differentiation. In contrast, inhibition of Fbw7α restored Runx2 levels and promoted osteoblast differentiation. We also observed reciprocal expression levels of Runx2 and Fbw7α in models of bone loss such as lactating (physiological bone loss condition) and ovariectomized (induction of surgical menopause) animals that show reduced Runx2 and enhanced Fbw7α, whereas this was reversed in the estrogen-treated ovariectomized animals. In addition, methylprednisolone (a synthetic glucocorticoid) treatment to neonatal rats showed a temporal decrease in Runx2 with a reciprocal increase in Fbw7 in their calvarium. Taken together, these data demonstrate that Fbw7α negatively regulates osteogenesis by targeting Runx2 for ubiquitin-mediated degradation in a GSK3ß-dependent manner and thus provides a plausible explanation for GSK3ß-mediated bone loss as described before.

Herpes Simplex Virus type-1 infection induces synaptic dysfunction in cultured cortical neurons via GSK-3 activation and intraneuronal amyloid-ß protein accumulation.

Increasing evidence suggests that recurrent Herpes Simplex Virus type 1 (HSV-1) infection spreading to the CNS is a risk factor for Alzheimer's Disease (AD) but the underlying mechanisms have not been fully elucidated yet. Here we demonstrate that in cultured mouse cortical neurons HSV-1 induced Ca(2+)-dependent activation of glycogen synthase kinase (GSK)-3. This event was critical for the HSV-1-dependent phosphorylation of amyloid precursor protein (APP) at Thr668 and the following intraneuronal accumulation of amyloid-ß protein (Aß). HSV-1-infected neurons also exhibited: i) significantly reduced expression of the presynaptic proteins synapsin-1 and synaptophysin; ii) depressed synaptic transmission. These effects depended on GSK-3 activation and intraneuronal accumulation of Aß. In fact, either the selective GSK-3 inhibitor, SB216763, or a specific antibody recognizing Aß (4G8) significantly counteracted the effects induced by HSV-1 at the synaptic level. Moreover, in neurons derived from APP KO mice and infected with HSV-1 Aß accumulation was not found and synaptic protein expression was only slightly reduced when compared to wild-type infected neurons. These data further support our contention that HSV-1 infections spreading to the CNS may contribute to AD phenotype.

Mechanism of Resistance and Novel Targets Mediating Resistance to EGFR and c-Met Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer.

Tyrosine kinase inhibitors (TKIs) against EGFR and c-Met are initially effective when administered individually or in combination to non-small cell lung cancer (NSCLC) patients. However, the overall efficacies of TKIs are limited due to the development of drug resistance. Therefore, it is important to elucidate mechanisms of EGFR and c-Met TKI resistance in order to develop more effective therapies. Model NSCLC cell lines H1975 and H2170 were used to study the similarities and differences in mechanisms of EGFR/c-Met TKI resistance. H1975 cells are positive for the T790M EGFR mutation, which confers resistance to current EGFR TKI therapies, while H2170 cells are EGFR wild-type. Previously, H2170 cells were made resistant to the EGFR TKI erlotinib and the c-Met TKI SU11274 by exposure to progressively increasing concentrations of TKIs. In H2170 and H1975 TKI-resistant cells, key Wnt and mTOR proteins were found to be differentially modulated. Wnt signaling transducer, active ß-catenin was upregulated in TKI-resistant H2170 cells when compared to parental cells. GATA-6, a transcriptional activator of Wnt, was also found to be upregulated in resistant H2170 cells. In H2170 erlotinib resistant cells, upregulation of inactive GSK3ß (p-GSK3ß) was observed, indicating activation of Wnt and mTOR pathways which are otherwise inhibited by its active form. However, in H1975 cells, Wnt modulators such as active ß-catenin, GATA-6 and p-GSK3ß were downregulated. Additional results from MTT cell viability assays demonstrated that H1975 cell proliferation was not significantly decreased after Wnt inhibition by XAV939, but combination treatment with everolimus (mTOR inhibitor) and erlotinib resulted in synergistic cell growth inhibition. Thus, in H2170 cells and H1975 cells, simultaneous inhibition of key Wnt or mTOR pathway proteins in addition to EGFR and c-Met may be a promising strategy for overcoming EGFR and c-Met TKI resistance in NSCLC patients.

miRNA-99b-3p functions as a potential tumor suppressor by targeting glycogen synthase kinase-3ß in oral squamous cell carcinoma Tca-8113 cells.

Dysregulation of microRNAs (miRNAs) has been associated with carcinogenesis in oral squamous cell carcinoma (OSCC). In the present study, we investigated the expression and function of miR-99b-3p in human OSCC. We found that the expression levels of miR-99b-3p decreased in 21 clinical OSCC samples (84%). Furthermore, ectopic expression of miR-99b-3p inhibited OSCC cell proliferation by downregulating glycogen synthase kinase-3ß (GSK3ß), an miR-99b-3p' target gene, at the mRNA and protein levels, both in vitro and in vivo. Moreover, the silencing of GSK3ß recapitulated the cellular and molecular effects in a similar manner to the overexpression of miR-99b-3p, which included inhibition of OSCC cell proliferation and suppression of p65 (RelA) and G1 regulators (cyclin D1, CDK4 and CDK6) in vitro. Our data suggest that miR-99b-3p functions as a tumor suppressor in OSCC via GSK3ß downregulation.

Impaired ILK Function Is Associated with Deficits in Hippocampal Based Memory and Synaptic Plasticity in a FASD Rat Model.

Fetal Alcohol Spectrum Disorder (FASD) is an umbrella term that encompasses a wide range of anatomical and behavioral problems in children who are exposed to alcohol during the prenatal period. There is no effective treatment for FASD, because of lack of complete characterization of the cellular and molecular mechanisms underlying this condition. Alcohol has been previously characterized to affect integrins and growth factor signaling receptors. Integrin Linked Kinase (ILK) is an effector of integrin and growth-factor signaling which regulates various signaling processes. In FASD, a downstream effector of ILK, Glycogen Synthase Kinase 3ß (GSK3ß) remains highly active (reduced Ser9 phosphorylation). GSK3ß has been known to modulate glutamate receptor trafficking and channel properties. Therefore, we hypothesize that the cognitive deficits accompanying FASD are associated with impairments in the ILK signaling pathway. Pregnant Sprague Dawley rats consumed a "moderate" amount of alcohol throughout gestation, or a calorie-equivalent sucrose solution. Contextual fear conditioning was used to evaluate memory performance in 32-33-day-old pups. Synaptic plasticity was assessed in the Schaffer Collateral pathway, and hippocampal protein lysates were used to evaluate ILK signaling. Alcohol exposed pups showed impaired contextual fear conditioning, as compared to control pups. This reduced memory performance was consistent with decrease in LTP as compared to controls. Hippocampal ILK activity and GSK3ß Ser21/9 phosphorylation were significantly lower in alcohol-exposed pups than controls. Increased synaptic expression of GluR2 AMPA receptors was observed with immunoprecipitation of post-synaptic density protein 95 (PSD95). Furthermore, immunoprecipitation of ILK revealed a decreased interaction with GluR2. The ILK pathway appears to play a significant role in memory and synaptic plasticity impairments in FASD rats. These impairments appear to be mediated by reduced GSK3ß regulation and increased synaptic stabilization of the calcium-impermeable GluR2 AMPA receptors.

ERK/GSK3ß signaling is involved in atractylenolide I-induced apoptosis and cell cycle arrest in melanoma cells.

Novel agents need to be developed to overcome the limitations of the current melanoma therapeutics. Atractylenolide I (AT-I) is a sesquiterpene compound isolated from atractylodis macrocephalae rhizoma. Previous findings demonstrated that AT-I exhibited cytotoxic action in melanoma cells. However, the molecular mechanisms of AT­1's anti-melanoma properties remain to be elucidated. In the present study, the cell cycle-arrest and apoptosis-promoting effects as well as the ERK/GSK3ß signaling-related mechanism of action of AT-I were examined. B16 melanoma cells were treated with various concentrations of AT-1 (50, 75 and 100 µM) for 48 or 72 h. Cell cycle and apoptosis were analyzed by flow cytometry. Protein expression levels were detected by western blot analysis. AT-I treatment induced G1 phase arrest, which was accompanied by increased p21 and decreased CDK2 protein expression levels. Apoptosis was observed after AT-I treatment for 72 h, which was accompanied by activated caspase­3 and ­8. AT-I treatment significantly decreased phospho-ERK, phospho-GSK3ß, c-Jun and increased p53 protein expression levels. Lithium chloride (LiCl, 5 mM), a GSK3ß inhibitor, treatment alone did not increase the apoptosis of B16 cells, while pretreatment with LiCl markedly reversed AT-I-induced apoptosis. Additionally, AT-I-induced G1 phase arrest was partially reversed by LiCl pretreatment. In conclusion, ERK/GSK3ß signaling was involved in the apoptotic and G1 phase arrest effects of AT-I in melanoma cells.