Fibulins (FBLNs), a family of extracellular matrix proteins, have recently been shown to act as tumor suppressors or activators in different cancers, and the underlying molecular mechanisms of their action in cancer remain unclear. We have previously shown that the expression of FBLN3 is suppressed by promoter hypermethylation and is associated with invasiveness in aggressive non-small cell lung cancer. In this study, we evaluated the roles and signaling mechanism of FBLN3 in lung cancer stem cells (CSCs). Forced expression of FBLN3 suppressed invasion and migration of lung adenocarcinoma cells and decreased the expression of epithelial-to-mesenchymal transition (EMT) activators, including N-cadherin and Snail. Stemness activities of lung adenocarcinoma cells were also suppressed by FBLN3 as indicated by a decrease in spheroid formation and the levels of stemness markers such as Sox2 and β-catenin. These effects of FBLN3 were mediated by the glycogen synthase kinase-3β, GSK3β/β-catenin pathway, and the upstream regulators of GSK3β, including phosphoinositide 3-kinase (PI3K)/AKT and insulin-like growth factor-1 receptor (IGF1R), were inactivated by FBLN3. Moreover, IGF1R was shown to be a direct target of FBLN3, which competitively inhibited insulin-like growth factor (IGF) action. To confirm the effect of FBLN3 on lung CSCs, aldehyde dehydrogenase-positive (ALDH+) A549 lung CSCs were sorted and treated with recombinant FBLN3 protein. FBLN3 clearly suppressed EMT, stemness activity and the over-activated IGF1R/PI3K/AKT/GSK3β pathway of the ALDH+ CSC subpopulation. In addition, injection of recombinant FBLN3 protein around subcutaneous xenografts established with ALDH+ CSCs in athymic nude mice significantly suppressed tumor growth and progression. Overall, our results show that FBLN3 suppresses both EMT and self-renewal of the lung CSCs by modulating the IGF1R/PI3K/AKT/GSK3β pathway and that FBLN3 would be useful as an alternative CSC therapy.

Glycogen synthase kinase 3β (GSK3β) has been shown to be a crucial mediator of the intensity and direction of the innate immune system response to bacterial stimuli. This review focuses on: (i) the central role of GSK3β in the regulation of pathogen-induced inflammatory responses through the regulation of pro- and anti-inflammatory cytokine production, (ii) the extensive ongoing efforts to exploit GSK3β for its therapeutic potential in the control of infectious diseases, and (iii) the increasing evidence that specific pathogens target GSK3β-related pathways for immune evasion. A better understanding of complex bacteria-GSK3β interactions is likely to lead to more effective anti-inflammatory interventions and novel targets to circumvent pathogen colonization and survival.

We have previously shown that 17beta-estradiol (E(2)) increases vascular endothelial growth factor A (Vegfa) gene expression in the rat uterus, resulting in increased microvascular permeability, and that this involves the simultaneous recruitment of hypoxia-inducible factor 1 (HIF1) and estrogen receptor alpha (ESR1) to the Vegfa gene promoter. Both events require the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. However, those studies were carried out using whole uterine tissue, and while most evidence indicates that the likely site of E(2)-induced Vegfa expression is luminal epithelial (LE) cells, other studies have identified stromal cells as the site of that expression. To address this question, the pathway regulating Vegfa expression was reexamined using LE cells rapidly isolated after E(2) treatment. In addition, we further characterized the nature of the receptor through which E(2) triggers the signaling events that lead to Vegfa expression using the specific ESR1 antagonist ICI 182,780. In agreement with previous results in the whole uterus, E(2) stimulated Vegfa mRNA expression in LE cells, peaking at 1 h (4- to 14-fold) and returning to basal levels by 4 h. Treatment with E(2) also increased phosphorylation of AKT in LE cells, as well as of the downstream mediators FRAP1 (mTOR), GSK3B, and MDM2. The alpha subunit of HIF1 (HIF1A) was present in LE cells before E(2) treatment, was unchanged 1 h after E(2), but was >2-fold higher by 4 h. Chromatin immunoprecipitation analysis showed that HIF1A was recruited to the Vegfa promoter by 1 h and was absent again by 4 h. The E(2) activation of the PI3K/AKT pathway, HIF1A recruitment to the Vegfa promoter, and Vegfa expression were all blocked by ICI 182,780. In summary, the rapid E(2)-induced signaling events that lead to the expression of Vegfa observed previously using the whole uterus occur in LE cells and appear to be initiated via a membrane form of ESR1.

Abnormally hyperphosphorylated tau is often caused by tau kinases, such as GSK3β and Cdk5. Such occurrence leads to neurofibrillary tangle formation and neuronal degeneration in tauopathy, including Alzheimer's disease (AD). However, little is known about the signaling cascade underlying the pathologic phosphorylation of tau by Aβ(42). In this study, we show that adenylate kinase 1 (AK1) is a novel regulator of abnormal tau phosphorylation. AK1 expression is markedly increased in the brains of AD patients and AD model mice and is significantly induced by Aβ(42) in the primary neurons. Ectopic expression of AK1 alone augments the pathologic phosphorylation of tau at PHF1, CP13 and AT180 epitopes and enhances the formation of tau aggregates. Inversely, downregulation of AK1 alleviates Aβ(42)-induced hyperphosphorylation of tau. AK1 plays a role in Aβ(42)-induced impairment of AMPK activity and GSK3β activation in the primary neurons. Pharmacologic studies show that treatment with an AMPK inhibitor activates GSK3β, and a GSK3β inhibitor attenuates AK1-mediated tau phosphorylation. In a Drosophila model of human tauopathy, the retinal expression of human AK1 severely exacerbates rough eye phenotype and increases abnormal tau phosphorylation. Further, neural expression of AK1 reduces the lifespan of tau transgenic files. Taken together, these observations indicate that the neuronal expression of AK1 is induced by Aβ(42) to increase abnormal tau phosphorylation via AMPK-GSK3β and contributes to tau-mediated neurodegeneration, providing a new upstream modulator of GSK3β in the pathologic phosphorylation of tau.

The breast cancer stem cells (BCSC) play important roles in breast cancer occurrence, recurrence and metastasis. However, the role of estrogen signaling, a signaling pathway important in development and progression of breast cancer, in regulation of BCSC has not been well established. Previously, we identified and cloned a variant of estrogen receptor α, ER-α36, with a molecular weight of 36 kDa. ER-α36 lacks both transactivation domains AF-1 and AF-2 of the 66 kDa full-length ER-α (ER-α66) and mediates rapid estrogen signaling to promote proliferation of breast cancer cells. In this study, we aim to investigate the function and the underlying mechanism of ER-α36-mediated rapid estrogen signaling in growth regulation of the ER-positive breast cancer stem/progenitor cells. ER-positive breast cancer cells MCF7 and T47D as well as the variants with different levels of ER-α36 expression were used. The effects of estrogen on BCSC's abilities of growth, self-renewal, differentiation and tumor-seeding were examined using tumorsphere formation, flow cytometry, indirect immunofluorence staining and in vivo xenograft assays. The underlying mechanisms were also studied with Western-blot analysis. We found that 17-β-estradiol (E2β) treatment increased the population of ER-positive breast cancer stem/progenitor cells while failed to do so in the cells with knocked-down levels of ER-α36 expression. Cells with forced expression of recombinant ER-α36, however, responded strongly to E2β treatment by increasing growth in vitro and tumor-seeding efficiency in vivo. The rapid estrogen signaling via the AKT/GSK3β pathway is involved in estrogen-stimulated growth of ER-positive breast cancer stem/progenitor cells. We concluded that ER-α36-mediated rapid estrogen signaling plays an important role in regulation and maintenance of ER-positive breast cancer stem/progenitor cells.

OBJECTIVE

Evasion from chemotherapy-induced apoptosis due to p53 loss strongly contributes to drug resistance. Identification of specific targets for the treatment of drug-resistant p53-null tumors would therefore increase the effectiveness of cancer therapy.

METHODS

By using a kinase-directed short hairpin RNA library and HCT116p53KO drug-resistant colon carcinoma cells, glycogen synthase kinase 3 beta (GSK3B) was identified as a target whose silencing bypasses drug resistance due to loss of p53. p53-null colon cancer cell lines with different sets of mutations were used to validate the role of GSK3B in sustaining resistance and to characterize cell death mechanisms triggered by chemotherapy when GSK3B is silenced. In vivo xenograft studies were conducted to confirm resensitization of drug-resistant cells to chemotherapy upon GSK3 inhibition. Colon cancer samples from a cohort of 50 chemotherapy-treated stage II patients were analyzed for active GSK3B expression.

RESULTS

Downregulation of GSK3B in various drug-resistant p53-null colon cancer cell lines abolished cell viability and colony growth after drug addition without affecting cell proliferation or cell cycle in untreated cells. Cell death of 5-fluorouracil (5FU)-treated p53-null GSK3B-silenced colon carcinoma cells occurred via PARP1-dependent and AIF-mediated but RIP1-independent necroptosis. In vivo studies showed that drug-resistant xenograft tumor mass was significantly reduced only when 5FU was given after GSK3B inhibition. Tissue microarray analysis of colon carcinoma samples from 5FU-treated patients revealed that GSK3B is significantly more activated in drug-resistant versus responsive patients.

CONCLUSIONS

Targeting GSK3B, in combination with chemotherapy, may represent a novel strategy for the treatment of chemotherapy-resistant tumors.

Intracerebral-ventricular (ICV) injection of streptozotocin (STZ) induces an insulin-resistant brain state that may underlie the neural pathogenesis of sporadic Alzheimer disease (AD). Our previous work showed that prior ICV treatment of glucagon-like peptide-1 (GLP-1) could prevent STZ-induced learning memory impairment and tau hyperphosphorylation in the rat brain. The Chinese herbal medicine geniposide is known to relieve symptoms of type 2 diabetes. Because geniposide is thought to act as a GLP-1 receptor agonist, we investigated the potential therapeutic effect of geniposide on STZ-induced AD model in rats. Our result showed that a single injection of geniposide (50 μM, 10 μL) to the lateral ventricle prevented STZ-induced spatial learning deficit by about 40% and reduced tau phosphorylation by about 30% with Morris water maze test and quantitative immunohistochemical analysis, respectively. It has been known that tau protein can be phosphorylated by glycogen synthase kinase-3 (GSK3) and STZ can increase the activity of GSK3β. Our result with Western blot analysis showed that central administration of geniposide resulted in an elevated expression of GSK3β(pS-9) but suppressed GSK3β(pY-216) indicating that geniposide reduced STZ-induced GSK3β hyperactivity. In addition, ultrastructure analysis showed that geniposide averted STZ-induced neural pathology, including paired helical filament (PHF)-like structures, accumulation of vesicles in synaptic terminal, abnormalities of endoplasmic reticulum (ER) and early stage of apoptosis. In summary, our study suggests that the water soluble and orally active monomer of Chinese herbal medicine geniposide may serve as a novel therapeutic agent for the treatment of sporadic AD.

BACKGROUND

N-Myc Interactor is an inducible protein whose expression is compromised in advanced stage breast cancer. Downregulation of NMI, a gatekeeper of epithelial phenotype, in breast tumors promotes mesenchymal, invasive and metastatic phenotype of the cancer cells. Thus the mechanisms that regulate expression of NMI are of potential interest for understanding the etiology of breast tumor progression and metastasis.

METHODS

Web based prediction algorithms were used to identify miRNAs that potentially target the NMI transcript. Luciferase reporter assays and western blot analysis were used to confirm the ability of miR-29 to target NMI. Quantitive-RT-PCRs were used to examine levels of miR29 and NMI from cell line and patient specimen derived RNA. The functional impact of miR-29 on EMT phenotype was evaluated using transwell migration as well as monitoring 3D matrigel growth morphology. Anti-miRs were used to examine effects of reducing miR-29 levels from cells. Western blots were used to examine changes in GSK3β phosphorylation status. The impact on molecular attributes of EMT was evaluated using immunocytochemistry, qRT-PCRs as well as Western blot analyses.

RESULTS

Invasive, mesenchymal-like breast cancer cell lines showed increased levels of miR-29. Introduction of miR-29 into breast cancer cells (with robust level of NMI) resulted in decreased NMI expression and increased invasion, whereas treatment of cells with high miR-29 and low NMI levels with miR-29 antagonists increased NMI expression and decreased invasion. Assessment of 2D and 3D growth morphologies revealed an EMT promoting effect of miR-29. Analysis of mRNA of NMI and miR-29 from patient derived breast cancer tumors showed a strong, inverse relationship between the expression of NMI and the miR-29. Our studies also revealed that in the absence of NMI, miR-29 expression is upregulated due to unrestricted Wnt/β-catenin signaling resulting from inactivation of GSK3β.

CONCLUSIONS

Aberrant miR-29 expression may account for reduced NMI expression in breast tumors and mesenchymal phenotype of cancer cells that promotes invasive growth. Reduction in NMI levels has a feed-forward impact on miR-29 levels.

Cancer initiation and progression have been attributed to newly discovered subpopulations of self-renewing, highly tumorigenic, drug-resistant tumor cells termed cancer stem cells. Recently, we and others reported a new phenotypic plasticity wherein highly tumorigenic, drug-resistant cell populations could arise not only from pre-existing cancer stem-like populations but also from cancer cells lacking these properties. In the current study, we hypothesized that this newfound phenotypic plasticity may be mediated by PI3K/Akt and Wnt/β-catenin signaling, pathways previously implicated in carcinogenesis, pluripotency and drug resistance. Using GFP expression, Hoechst dye exclusion and fluorescence activated cell sorting (FACS) of cancer cell lines, we identified and tracked cancer stem-like side populations (SP) of cancer cells characterized by high tumorigenicity and drug resistance. We found that pharmacological inhibition or genetic depletion of PI3K and AKT markedly reduced the spontaneous conversion of nonside population (NSP) cells into cancer stem-like SP cells, whereas PI3K/Akt activation conversely enhanced NSP to SP conversion. PI3K/AKT signaling was mediated through downstream phosphorylation of GSK3β, which led to activation and accumulation of β-catenin. Accordingly, pharmacological or genetic perturbation of GSK3β or β-catenin dramatically impacted conversion of NSP to SP. Further downstream, β-catenin's effects on NSP-SP equilibrium were dependent upon its interaction with CBP, a KAT3 family coactivator. These studies provide a mechanistic model wherein PI3K/Akt/β-catenin/CBP signaling mediates phenotypic plasticity in and out of a drug-resistant, highly tumorigenic state. Therefore, targeting this pathway has unique potential for overcoming the therapy resistance and disease progression attributed to the cancer stem-like phenotype.

Excitotoxicity and cell death induced by glutamate are involved in many neurodegenerative disorders. We have previously demonstrated that excitotoxicity induced by millimolar concentrations of glutamate in hippocampal slices involves apoptotic features and glutamate-induced glutamate release. Guanosine, an endogenous guanine nucleoside, prevents excitotoxicity by its ability to modulate glutamate transport. In this study, we have evaluated the neuroprotective effect of guanosine against glutamate-induced toxicity in hippocampal slices and the mechanism involved in such an effect. We have found that guanosine (100 μM) was neuroprotective against 1 mM glutamate-induced cell death through the inhibition of glutamate release induced by glutamate. Guanosine also induced the phosphorylation and, thus, activation of protein kinase B (PKB/Akt), a downstream target of phosphatidylinositol-3 kinase (PI3K), as well as phosphorylation of glycogen synthase kinase 3β, which has been reported to be inactivated by Akt after phosphorylation at Ser9. Glutamate treated hippocampal slices showed increased inducible nitric oxide synthase (iNOS) expression that was prevented by guanosine. Slices preincubated with SNAP (an NO donor), inhibited the protective effect of guanosine. LY294002 (30 μM), a PI3K inhibitor, attenuated guanosine-induced neuroprotection, guanosine prevention of glutamate release, and guanosine-induced GSK3β(Ser9) phosphorylation but not guanosine reduction of glutamate-induced iNOS expression. Taken together, the results of this study show that guanosine protects hippocampal slices by a mechanism that involves the PI3K/Akt/GSK3β(Ser9) pathway and prevention of glutamate-induced glutamate release. Furthermore, guanosine also reduces glutamate-induced iNOS by a PI3K/Akt-independent mechanism.

OBJECTIVE

The present study was designed to examine the impact of chronic Akt activation on endoplasmic reticulum (ER) stress-induced cardiac mechanical anomalies, if any, and the underlying mechanism involved.

RESULTS

Wild-type and transgenic mice with cardiac-specific overexpression of the active mutant of Akt (Myr-Akt) were subjected to the ER stress inducer tunicamycin (1 or 3 mg/kg). ER stress led to compromised echocardiographic (elevated left ventricular end-systolic diameter and reduced fractional shortening) and cardiomyocyte contractile function, intracellular Ca(2+) mishandling, and cell survival in wild-type mice associated with mitochondrial damage. In vitro ER stress induction in murine cardiomyocytes upregulated the ER stress proteins Gadd153, GRP78, and phospho-eIF2α, and promoted reactive oxygen species production, carbonyl formation, apoptosis, mitochondrial membrane potential loss, and mitochondrial permeation pore (mPTP) opening associated with overtly impaired cardiomyocyte contractile and intracellular Ca(2+) properties. Interestingly, these anomalies were mitigated by chronic Akt activation or the ER chaperon tauroursodeoxycholic acid (TUDCA). Treatment with tunicamycin also dephosphorylated Akt and its downstream signal glycogen synthase kinase 3β (GSK3β) (leading to activation of GSK3β), the effect of which was abrogated by Akt activation and TUDCA. The ER stress-induced cardiomyocyte contractile and mitochondrial anomalies were obliterated by the mPTP inhibitor cyclosporin A, GSK3β inhibitor SB216763, and ER stress inhibitor TUDCA.

METHODS

This research reported the direct relationship between ER stress and cardiomyocyte contractile and mitochondrial anomalies for the first time.

CONCLUSIONS

Taken together, these data suggest that ER stress may compromise cardiac contractile and intracellular Ca(2+) properties, possibly through the Akt/GSK3β-dependent impairment of mitochondrial integrity.

Traf2 and NcK interacting kinase (TNiK) contains serine-threonine kinase and scaffold domains and has been implicated in cell proliferation and glutamate receptor regulation in vitro. Here we report its role in vivo using mice carrying a knock-out mutation. TNiK binds protein complexes in the synapse linking it to the NMDA receptor (NMDAR) via AKAP9. NMDAR and metabotropic receptors bidirectionally regulate TNiK phosphorylation and TNiK is required for AMPA expression and synaptic function. TNiK also organizes nuclear complexes and in the absence of TNiK, there was a marked elevation in GSK3β and phosphorylation levels of its cognate phosphorylation sites on NeuroD1 with alterations in Wnt pathway signaling. We observed impairments in dentate gyrus neurogenesis in TNiK knock-out mice and cognitive testing using the touchscreen apparatus revealed impairments in pattern separation on a test of spatial discrimination. Object-location paired associate learning, which is dependent on glutamatergic signaling, was also impaired. Additionally, TNiK knock-out mice displayed hyperlocomotor behavior that could be rapidly reversed by GSK3β inhibitors, indicating the potential for pharmacological rescue of a behavioral phenotype. These data establish TNiK as a critical regulator of cognitive functions and suggest it may play a regulatory role in diseases impacting on its interacting proteins and complexes.

Activation of AKT signaling by PTEN loss or PIK3CA mutations occurs frequently in human cancers, but targeting AKT has been difficult due to the mechanism-based toxicities of inhibitors that target the inactive conformation of AKT. Ipatasertib (GDC-0068) is a novel selective ATP-competitive small-molecule inhibitor of AKT that preferentially targets active phosphorylated AKT (pAKT) and is potent in cell lines with evidence of AKT activation. In this phase I study, ipatasertib was well tolerated; most adverse events were gastrointestinal and grade 1-2 in severity. The exposures of ipatasertib ≥200 mg daily in patients correlated with preclinical TGI90, and pharmacodynamic studies confirmed that multiple targets (i.e., PRAS40, GSK3β, and mTOR) were inhibited in paired on-treatment biopsies. Preliminary antitumor activity was observed; 16 of 52 patients (30%), with diverse solid tumors and who progressed on prior therapies, had radiographic stable disease, and many of their tumors had activation of AKT.

Potent inhibition of AKT signaling with ipatasertib was associated with a tolerable safety profile and meaningful disease control in a subgroup of patients. Targeting pAKT with an ATP-competitive inhibitor provides a greater therapeutic window than allosteric inhibitors. Further investigation with ipatasertib is ongoing in phase II studies. Cancer Discov; 7(1); 102-13. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1.

Neural crest cells (NCCs) are an embryonic migratory cell population with the ability to differentiate into a wide variety of cell types that contribute to the craniofacial skeleton, cornea, peripheral nervous system, and skin pigmentation. This ability suggests the promising role of NCCs as a source for cell-based therapy. Although several methods have been used to induce human NCCs (hNCCs) from human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), further modifications are required to improve the robustness, efficacy, and simplicity of these methods. Chemically defined medium (CDM) was used as the basal medium in the induction and maintenance steps. By optimizing the culture conditions, the combination of the GSK3β inhibitor and TGFβ inhibitor with a minimum growth factor (insulin) very efficiently induced hNCCs (70-80%) from hPSCs. The induced hNCCs expressed cranial NCC-related genes and stably proliferated in CDM supplemented with EGF and FGF2 up to at least 10 passages without changes being observed in the major gene expression profiles. Differentiation properties were confirmed for peripheral neurons, glia, melanocytes, and corneal endothelial cells. In addition, cells with differentiation characteristics similar to multipotent mesenchymal stromal cells (MSCs) were induced from hNCCs using CDM specific for human MSCs. Our simple and robust induction protocol using small molecule compounds with defined media enabled the generation of hNCCs as an intermediate material producing terminally differentiated cells for cell-based innovative medicine.

The epithelial-mesenchymal transition (EMT) plays a fundamental role in the early stages of breast cancer invasion. Snail, a zinc finger transcriptional repressor, is an important regulator of EMT. Snail is phosphorylated by GSK3β and is subsequently degraded by βTrCP-mediated ubiquitination. We identified an additional kinase, DYRK2, that regulates Snail stability. Knockdown of DYRK2 promoted EMT and cancer invasion in vitro and in vivo. Consistent with these results, DYRK2 was found to be down-regulated in human breast cancer tissue. Patients with low DYRK2-expressing tumors had a worse outcome than those with high DYRK2-expressing tumors. These findings revealed that DYRK2 regulates cancer invasion and metastasis by degrading Snail.

Higher plasma urate level is reported to be associated with a reduced risk and slower progression of Parkinson's disease (PD). In this study, we explored the effects of urate on dopaminergic neurons in nigrostriatal pathway in the 6-hydroxydopamine (6-OHDA) unilaterally lesioned rats. Uric acid (UA), when given twice daily at 200 mg/kg intraperitoneally for 10 consecutive days, elevated urate (the anionic form of UA) in plasma and striatum by 55% and 36.8%, respectively, as compared with vehicle group. This regimen of UA was found to ameliorate the behavioral deficits, dopaminergic neuron loss as well as dopamine depletion in the nigrostriatal system. Moreover, UA administration was capable of increasing glutathione level and superoxide dismutase activity while decreasing malondialdehyde accumulation in striatum. In addition, the phosphorylation of both protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK3β) in the lesioned striata of 6-OHDA-lesioned rats was dramatically reduced as compared with sham-operated rats. This reduction was attenuated in the Parkinsonian rats receiving UA treatment. Similarly, in vitro findings showed that UA alleviated the decrease in Akt activation and the increase in GSK3β activity caused by 6-OHDA. Furthermore, neuroprotection by urate and its regulation on GSK3β phosphorylation at Ser9 was found to be abolished in the presence of PI3K inhibitor. Therefore, our findings demonstrated that urate was able to protect dopaminergic neurons in rat nigrostriatal pathway against the neurotoxicity of 6-OHDA, and showed that its beneficial effects may be related to its regulation on Akt/GSK3β signaling.

Differentiation of 3T3-L1 cells into adipocytes involves a highly orchestrated series of complex events in which microRNAs might play an essential role. In this study, we found that the overexpression of microRNA-344 (miR-344) inhibits 3T3-L1 cell differentiation and decreases triglyceride accumulation after MDI stimulation. We demonstrated that miR-344 directly targets the 3' UTR of GSK3β (Glycogen synthase kinase 3 beta). Knockdown of GSK3β with siRNA results in inhibiting 3T3-L1 differentiation, while its overexpression restores the effect of miR-344. In addition, miR-344 elevates the level of active β-catenin, which is the downstream effector of GSK3β in the Wnt/β-catenin signaling pathway. These data indicate that miR-344 inhibits adipocyte differentiation via targeting GSK3β and subsequently activating the Wnt/β-catenin signaling pathway.

Abnormal tau accumulation can lead to the development of neurodegenerative diseases. P301S mice overexpress the human tau mutated gene, resulting in tau hyperphosphorylation and tangle formation. Mice also develop synaptic deficits and microglial activation prior to any neurodegeneration and tangles. Oxidative stress can also affect tauopathy. We studied the role of oxidative stress in relationship to behavioral abnormalities and disease progression in P301S mice at 2, 7, and 10 mo of age. At 7 mo of age, P301S mice had behavioral abnormalities, such as hyperactivity and disinhibition. At the same age, we observed increased carbonyls in P301S mitochondria (∼215 and 55% increase, males/females), and deregulation in the activity and content of mitochondrial enzymes involved in reactive oxygen species formation and energy metabolism, such as citrate synthase (∼19 and ∼5% decrease, males/females), MnSOD (∼16% decrease, males only), cytochrome C (∼19% decrease, females only), and cytochrome C oxidase (∼20% increase, females only). These changes in mitochondria proteome appeared before tau hyperphosphorylation and tangle formation, which were observed at 10 mo and were associated with GSK3β activation. At that age, mitochondria proteome deregulation became more apparent in male P301S mitochondria. The data strongly suggest that oxidative stress and mitochondrial abnormalities appear prior to tau pathology.

OBJECTIVE

Epidermal growth factor plays a critical role in breast malignancies by enhancing cell proliferation, invasion, angiogenesis and metastasis. Epithelial-mesenchymal transition (EMT) is a crucial process by which epithelial cells lose polarity and acquire migratory mesenchymal properties. Gold nanoparticles are an efficient drug delivery vehicle for carrying chemotherapeutic agents to target cancer cells and quercetin is an anti-oxidative flavonoid known with potent anti-malignant cell activity.

METHODS

Cell viability was assessed by MTT assay, and protein expression was examined by Western blotting and immunocytochemistry. Cell invasion was monitored using invasion chambers, and cell migration was analysed by scratch wound-healing assay. In vitro and ex vivo angiogenesis studies were performed by capillary-like tube formation assay and chick embryo angiogenesis assay (CEA). 7,12-dimethylbenz(a)anthracene (DMBA) induced mammary carcinoma in Sprague-Dawley rats.

RESULTS

We observed a significant reduction in protein expression of vimentin, N-cadherin, Snail, Slug, Twist, MMP-2, MMP-9, p-EGFR, VEGFR-2, p-PI3K, Akt and p-GSK3β, and enhanced E-cadherin protein expression in response to AuNPs-Qu-5 treatment. AuNPs-Qu-5 inhibited migration and invasion of MCF-7 and MDA-MB-231 cells compared to free quercetin. AuNPs-Qu-5-treated HUVECs had reduced cell viability and capillary-like tube formation. In vitro and in vivo angiogenesis assays showed that AuNPs-Qu-5 suppressed tube and new blood vessel formation. Treatment with AuNPs-Qu-5 impeded tumour growth in DMBA-induced mammary carcinoma in SD rats compared to treatment with free quercetin.

CONCLUSIONS

Our results suggest that AuNPs-Qu-5 inhibited EMT, angiogenesis and metastasis of the breast cancer cells tested by targeting the EGFR/VEGFR-2 signalling pathway.

BACKGROUND

Glycogen synthase kinase-3β (GSK3β), cAMP-response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) play critical roles in neuronal survival, synaptic plasticity and memory and participate in the pathophysiology of both depressive disorder and Alzheimer's disease (AD).

METHODS

This study was designed to determine the association of GSK3β activity, CREB activity and BDNF concentration in peripheral blood of patients with AD with or without depressive symptoms and in depressive patients without AD. GSK3β activity in platelets, CREB activity in lymphocytes and BDNF concentration in plasma, platelet-rich plasma or platelets were measured in 85 AD patients (36 of whom displayed co-morbid depressive symptoms), 65 non-AD patients with depressive disorder and 96 healthy controls. AD patients were clinically assessed for stage of dementia, cognitive impairment and severity of depressive symptoms. Depressive patients were clinically assessed for severity of depression.

RESULTS

We observed increased CREB activity and GSK3β activity in AD with depressive symptoms or in AD at mild stage of dementia. Decreased BDNF concentration was found in platelet-rich plasma of AD patients at moderate to severe stages of dementia or in AD without depressive symptoms. An association was revealed of the severity of cognitive impairment with the increase of GSK3β in the platelets of AD patients with mild dementia. In depressive patients, a lower concentration of phosphorylated GSK3β was associated with a higher severity of depression. Association was confirmed between severity of depression, CREB activation, and BDNF concentration in drug-naïve depressive patients.

CONCLUSIONS

Our data demonstrated that AD is accompanied by increased CREB activity in lymphocytes and a decreased concentration of BDNF in platelet-rich plasma. The decreased BDNF concentration appears to correlate with moderate to severe stages of dementia in AD. Observation of decreased phosphorylation of GSK3β in platelets of both AD patients with depressive symptoms and depressive patients after treatment confirms the role of increased GSK3β activity in the pathophysiology of both AD and depressive disorder. Associations were confirmed between AD and platelet GSK3β activity, lymphocyte CREB activity and plasma BDNF. CREB activity and platelet BDNF concentration seems to be related to depressive disorder.

Experimental autoimmune encephalomyelitis (EAE) is a rodent model of multiple sclerosis (MS), a debilitating autoimmune disease of the CNS, for which only limited therapeutic interventions are available. Because MS is mediated in part by autoreactive T cells, particularly Th17 and Th1 cells, in the current study, we tested whether inhibitors of glycogen synthase kinase-3 (GSK3), previously reported to reduce Th17 cell generation, also alter Th1 cell production or alleviate EAE. GSK3 inhibitors were found to impede the production of Th1 cells by reducing STAT1 activation. Molecularly reducing the expression of either of the two GSK3 isoforms demonstrated that Th17 cell production was sensitive to reduced levels of GSK3β and Th1 cell production was inhibited in GSK3α-deficient cells. Administration of the selective GSK3 inhibitors TDZD-8, VP2.51, VP0.7, or L803-mts significantly reduced the clinical symptoms of myelin oligodendrocyte glycoprotein35-55-induced EAE in mice, nearly eliminating the chronic progressive phase, and reduced the number of Th17 and Th1 cells in the spinal cord. Administration of TDZD-8 or L803-mts after the initial disease episode alleviated clinical symptoms in a relapsing-remitting model of proteolipid protein139-151-induced EAE. Furthermore, deletion of GSK3β specifically in T cells was sufficient to alleviate myelin oligodendrocyte glycoprotein35-55-induced EAE. These results demonstrate the isoform-selective effects of GSK3 on T cell generation and the therapeutic effects of GSK3 inhibitors in EAE, as well as showing that GSK3 inhibition in T cells is sufficient to reduce the severity of EAE, suggesting that GSK3 may be a feasible target for developing new therapeutic interventions for MS.

Green tea has antidiabetic, antiobesity, and anti-inflammatory activities in animal models, but the molecular mechanisms of these effects have not been fully understood. Quantitative real-time polymerase chain reaction (PCR) was used to investigate the relative expression levels and the effects of green tea (1 and 2 g solid extract/kg diet) on the expression of glucose transporter family genes (Glut1/Slc2a1, Glut2/Slc2a2, Glut3/Slc2a3, and Glut4/Slc2a4) and insulin signaling pathway genes (Ins1, Ins2, Insr, Irs1, Irs2, Akt1, Grb2, Igf1, Igf2, Igf1r, Igf2r, Gsk3b, Gys1, Pik3cb, Pik3r1, Shc1, and Sos1) in liver and muscle of rats fed a high-fructose diet known to induce insulin resistance and oxidative stress. Glut2 and Glut4 were the major Glut mRNAs in rat liver and muscle, respectively. Green tea extract (1 g) increased Glut1, Glut4, Gsk3b, and Irs2 mRNA levels by 110, 160, 30, and 60% in the liver, respectively, and increased Irs1 by 80% in the muscle. Green tea extract (2 g) increased Glut4, Gsk3b, and Pik3cb mRNA levels by 90, 30, and 30% but decreased Shc1 by 60% in the liver and increased Glut2, Glut4, Shc1, and Sos1 by 80, 40, 60, and 50% in the muscle. This study shows that green tea extract at 1 or 2 g/kg diet regulates gene expression in the glucose uptake and insulin signaling pathway in rats fed a fructose-rich diet.

Klotho is an anti-aging protein predominantly expressed in renal tubular epithelial cells. Although Klotho was recently identified as a tumor suppressor gene in a variety of cancers, the potential role and molecular events for Klotho in renal cell carcinoma (RCC) remain obscure. In the present study, immunohistochemical staining in tissue microarrays containing 125 RCC samples showed that intratumoral Klotho levels were negatively correlated with tumor size, TNM stage and nuclear grade. The overall survival rate of RCC patients with high Klotho expression was significantly higher than that of patients with low Klotho expression. Functional analysis after gain and loss of Klotho expression revealed that Klotho blunted epithelial-mesenchymal transition and cellular migration and invasion in RCC. Also, no alteration of α-2,6-sialidase activity was found after Klotho overexpression in RCC. The molecular signals for this phenomenon involved the Klotho-mediated inhibition of PI3K/Akt/GSK3β/Snail pathway. Importantly, compared to localized RCC tissues, advanced RCC tissues exhibited low Klotho expression accompanied with high pAkt and Snail expression. These results indicate Klotho acts as a tumor suppressor by inhibiting PI3K/Akt/GSK3β/Snail signaling, thus suppressing epithelial-mesenchymal transition and tumor migration and invasion during RCC progression. As a result, Klotho might be used as a potential therapy for advanced RCC.

The Wnt/β-catenin signaling pathway, commonly hyperactivated in pancreatic cancer, has been reported to play an important role in the maintenance of stemness of cancer stem cells (CSCs), which is closely related to the progression of pancreatic cancer. Therefore, exploring the regulatory mechanism in Wnt/β-catenin signaling may provide valuable clinical targets for cancer therapy. In the current study, we demonstrated that upregulation of miR-744 in pancreatic cancer promoted Wnt/β-catenin signaling by directly targeting secreted frizzled-related protein 1 (SFRP1), glycogen synthase kinase 3β (GSK3β), and transducin-like enhancer of split 3 (TLE3), important negative modulators of Wnt/β-catenin signaling. Expression of miR-744 was markedly upregulated in pancreatic cancer and positively correlated with poor patient survival. Furthermore, we found that overexpressing miR-744 enhanced, while inhibiting miR-744 reduced, the stem cell-like phenotype of pancreatic cancer cells in vitro. Importantly, in vivo model of human-derived pancreatic xenografts showed that miR-744 upregulation enhanced the tumorigenicity of pancreatic cancer cells. These findings suggest that miR-744 plays a vital role in promoting the stem cell-like phenotype of pancreatic cancer cells, and may represent a novel prognostic biomarker and therapeutic target.