The microRNA miR-519 robustly inhibits cell proliferation, in turn triggering senescence and decreasing tumor growth. However, the molecular mediators of miR-519-elicited growth inhibition are unknown. Here, we systematically investigated the influence of miR-519 on gene expression profiles leading to growth cessation in HeLa human cervical carcinoma cells. By analyzing miR-519-triggered changes in protein and mRNA expression patterns and by identifying mRNAs associated with biotinylated miR-519, we uncovered two prominent subsets of miR-519-regulated mRNAs. One subset of miR-519 target mRNAs encoded DNA maintenance proteins (including DUT1, EXO1, RPA2, and POLE4); miR-519 repressed their expression and increased DNA damage, in turn raising the levels of the cyclin-dependent kinase (cdk) inhibitor p21. The other subset of miR-519 target mRNAs encoded proteins that control intracellular calcium levels (notably, ATP2C1 and ORAI1); their downregulation by miR-519 aberrantly elevated levels of cytosolic [Ca(2+)] storage in HeLa cells, similarly increasing p21 levels in a manner dependent on the Ca(2+)-activated kinases CaMKII and GSK3β. The rises in levels of DNA damage, the Ca(2+) concentration, and p21 levels stimulated an autophagic phenotype in HeLa and other human carcinoma cell lines. As a consequence, ATP levels increased, and the level of activity of the AMP-activated protein kinase (AMPK) declined, further contributing to the elevation in the abundance of p21. Our results indicate that miR-519 promotes DNA damage, alters Ca(2+) homeostasis, and enhances energy production; together, these processes elevate the expression level of p21, promoting growth inhibition and cell survival.

Besides the presence of amyloid beta (Aβ) plaques and neurofibrillary tangles, neurogenesis and synaptic plasticity are markedly impaired in Alzheimer's disease (AD) possibly contributing to cognitive impairment. In this context, neurotrophic factors serve as a promising therapeutic approach via utilization of regenerative capacity of brain to shift the balance from neurodegeneration to neural regeneration. However, besides more conventional "bystander" effect, to what extent can neurotrophic compounds affect underlying AD pathology remains questionable. Here we investigated the effect of chronic oral treatment with a ciliary neurotrophic factor (CNTF) derived peptidergic compound, P021 (Ac-DGGL(A)G-NH2), on disease pathology both at moderate and severe stages in a transgenic mouse model of AD. 3xTg-AD and wild type female mice were treated for 12months with P021 or vehicle diet starting at 9-10months of age. A significant reduction in abnormal hyperphosphorylation and accumulation of tau at known major AD neurofibrillary pathology associated sites was observed. The effect of P021 on Aβ pathology was limited to a significant decrease in soluble Aβ levels and a trend towards reduction in Aβ plaque load in CA1 region of hippocampus, consistent with reduction in Aβ generation and not clearance. This disease modifying effect was probably via increased brain derived neurotrophic factor (BDNF) expression mediated decrease in glycogen synthase kinase-3-β (GSK3β) activity we found in P021 treated 3xTg-AD mice. P021 treatment also rescued deficits in cognition, neurogenesis, and synaptic plasticity in 3xTg-AD mice. These findings demonstrate the potential of the neurotrophic peptide mimetic as a disease modifying therapy for AD.

The adenomatous polyposis coli (APC) protein functions as a negative regulator of the Wnt signaling pathway. In this capacity, APC forms a "destruction complex" with Axin, CK1α, and GSK3β to foster phosphorylation of the Wnt effector β-catenin earmarking it for Lys-48-linked polyubiquitylation and proteasomal degradation. APC is conjugated with Lys-63-linked ubiquitin chains when it is bound to Axin, but it is unclear whether this modification promotes the APC-Axin interaction or confers upon APC an alternative function in the destruction complex. Here we identify HectD1 as a candidate E3 ubiquitin ligase that modifies APC with Lys-63 polyubiquitin. Knockdown of HectD1 diminished APC ubiquitylation, disrupted the APC-Axin interaction, and augmented Wnt3a-induced β-catenin stabilization and signaling. These results indicate that HectD1 promotes the APC-Axin interaction to negatively regulate Wnt signaling.

It is well established that mitochondrial fragmentation plays a key role in the pathogenesis of Alzheimer's disease (AD). Mitochondrial fission is mediated by dynamin-related protein 1 (Drp1), which is highly expressed in nervous system and regulated by various posttranslational modifications including phosphorylation. We identified glycogen synthase kinase (GSK)3β-dependent Drp1 phosphorylation at Ser(40) and Ser(44), which increases Drp1 GTPase activity and its mitochondrial distribution and could induce mitochondrial fragmentation. Moreover, neurons transfected with Ser(40)Ser(44) phosphomimic Drp1 showed increased mitochondria fragmentation and were more vulnerable to amyloid-β (Aβ)-induced apoptosis. Therefore, blocking GSK3β-induced Drp1 phosphorylation may be an effective way to protect neurons from Aβ toxicity. To address this, we designed and synthesized an artificial polypeptide named TAT-Drp1-SpS, which could specifically block GSK3β-induced Drp1 phosphorylation. Our results demonstrated that TAT-Drp1-SpS treatment could significantly reduce Aβ-induced neuronal apoptosis in cultured neurons. Notably, TAT-Drp1-SpS administration in hippocampus Cornu Ammonis 1 (CA1) region significantly reduced Aβ burden and rescued the memory deficits in AD transgenic mice. Although Aβ has multiple targets to exert its neurotoxicity, our findings suggested that GSK3β-induced mitochondrial fragmentation was, at least partially, mediated by Aβ toxicity and contribute to the pathogenesis of AD. Taken together, GSK3β-induced Drp1 phosphorylation provides a novel mechanism for mitochondrial fragmentation in AD, and our findings suggested a novel therapeutic strategy for AD.

Aerobic glycolysis, a metabolic hallmark of cancer, is associated with radioresistance in non-small cell lung cancer (NSCLC). Pyruvate kinase M2 isoform (PKM2), a key regulator of glycolysis, is expressed exclusively in cancers. However, the impact of PKM2 silencing on the radiosensitivity of NSCLC has not been explored. Here, we show a plasmid of shRNA-PKM2 for expressing a short hairpin RNA targeting PKM2 (pshRNA-PKM2) and demonstrate that treatment with pshRNA-PKM2 effectively inhibits PKM2 expression in NSCLC cell lines and xenografts. Silencing of PKM2 expression enhanced ionizing radiation (IR)-induced apoptosis and autophagy in vitro and in vivo, accompanied by inhibiting AKT and PDK1 phosphorylation, but enhanced ERK and GSK3β phosphorylation. These results demonstrated that knockdown of PKM2 expression enhances the radiosensitivity of NSCLC cell lines and xenografts as well as may aid in the design of new therapies for the treatment of NSCLC.

Hexokinase (HK), the rate-limiting enzyme in glycolysis, controls cell survival by promoting metabolism and/or inhibiting apoptosis. Since HK isoforms I and II have mitochondrial targeting sequences, we attempted to separate the protective effects of HK on cell metabolism from those on apoptosis. We exposed renal epithelial cells to metabolic stress causing ATP depletion in the absence of glucose and found that this activated glycogen synthase kinase 3β (GSK3β) and Bax caused mitochondrial membrane injury and apoptosis. ATP depletion led to a progressive HK II dissociation from mitochondria, released mitochondrial apoptosis inducing factor and cytochrome c into the cytosol, activated caspase-3, and reduced cell survival. Compared with control, adenoviral-mediated HK I or II overexpression improved cell survival following stress, but did not prevent GSK3β or Bax activation, improve ATP content, or reduce mitochondrial fragmentation. HK I or HK II overexpression increased mitochondria-associated isoform-specific HK content, and decreased mitochondrial membrane injury and apoptosis after stress. In vivo, HK II localized exclusively to the proximal tubule. Ischemia reduced total renal HK II content and dissociated HK II from proximal tubule mitochondria. In cells overexpressing HK II, Bax and HK II did not interact before or after stress. While the mechanism by which HK antagonizes Bax-mediated apoptosis is unresolved by these studies, one possible scenario is that the two proteins compete for a common binding site on the outer mitochondrial membrane.

Premature ovarian failure (POF) is a major side effect of chemotherapy in young cancer patients. To develop pharmaceutical agents for preserving fertility, it is necessary to understand the mechanisms responsible for chemotherapy-induced follicle loss. Here, we show that treatment with cisplatin, a widely used anticancer drug, depleted the dormant follicle pool in mouse ovaries by excessive activation of the primordial follicles, without inducing follicular apoptosis. Moreover, we show that co-treatment with the antioxidant melatonin prevented cisplatin-induced disruption of the follicle reserve. We quantified the various stages of growing follicles, including primordial, primary, secondary, and antral, to demonstrate that cisplatin treatment alone significantly decreased, whereas melatonin co-treatment preserved, the number of primordial follicles in the ovary. Importantly, analysis of the PTEN/AKT/FOXO3a pathway demonstrated that melatonin significantly decreased the cisplatin-mediated inhibitory phosphorylation of PTEN, a key negative regulator of dormant follicle activation. Moreover, melatonin prevented the cisplatin-induced activating phosphorylation of AKT, GSK3β, and FOXO3a, all of which trigger follicle activation. Additionally, we show that melatonin inhibited the cisplatin-induced inhibitory phosphorylation and nuclear export of FOXO3a, which is required in the nucleus to maintain dormancy of the primordial follicles. These findings demonstrate that melatonin attenuates cisplatin-induced follicle loss by preventing the phosphorylation of PTEN/AKT/FOXO3a pathway members; thus, melatonin is a potential therapeutic agent for ovarian protection and fertility preservation during chemotherapy in female cancer patients.

BACKGROUND

Urinary bladder cancer is a common malignancy worldwide, and outcomes for patients with advanced bladder cancer remain poor. Antiproliferative factor (APF) is a potent glycopeptide inhibitor of epithelial cell proliferation that was discovered in the urine of patients with interstitial cystitis, a disorder with bladder epithelial thinning and ulceration. APF mediates its antiproliferative activity in primary normal bladder epithelial cells via cytoskeletal associated protein 4 (CKAP4). Because synthetic asialo-APF (as-APF) has also been shown to inhibit T24 bladder cancer cell proliferation at nanomolar concentrations in vitro, and because the peptide segment of APF is 100% homologous to part of frizzled 8, we determined whether CKAP4 mediates as-APF inhibition of proliferation and/or downstream Wnt/frizzled signaling events in T24 cells.

METHODS

T24 cells were transfected with double-stranded siRNAs against CKAP4 and treated with synthetic as-APF or inactive control peptide; cells that did not undergo electroporation and cells transfected with non-target (scrambled) double-stranded siRNA served as negative controls. Cell proliferation was determined by 3H-thymidine incorporation. Expression of Akt, glycogen synthase kinase 3β (GSK3β), β-catenin, p53, and matrix metalloproteinase 2 (MMP2) mRNA was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Akt, GSK-3β, MMP2, β-catenin, and p53 protein expression, plus Akt, GSK-3β, and β-catenin phosphorylation, were determined by Western blot.

RESULTS

T24 cell proliferation, MMP2 expression, Akt ser473 and thr308 phosphorylation, GSK3β tyr216 phosphorylation, and β-catenin ser45/thr41 phosphorylation were all decreased by APF, whereas p53 expression, and β-catenin ser33,37/thr41 phosphorylation, were increased by APF treatment in non-electroporated and non-target siRNA-transfected cells. Neither mRNA nor total protein expression of Akt, GSK3β, or β-catenin changed in response to APF in these cells. In addition, the changes in cell proliferation, MMP2/p53 mRNA and protein expression, and Akt/GSK3β/β-catenin phosphorylation in response to APF treatment were all specifically abrogated following CKAP4 siRNA knockdown.

CONCLUSIONS

Synthetic as-APF inhibits cell proliferation in T24 bladder carcinoma cells via the CKAP4 receptor. The mechanism for this inhibition involves regulating phosphorylation of specific cell signaling molecules (Akt, GSK3β, and β-catenin) plus mRNA and protein expression of p53 and MMP2.

Skeletal muscle remodeling in response to muscle disuse and unloading is known to be associated with so-called ER stress, which, in turn, activates autophagy and contributes to muscle atrophy. Different molecules are involved in ER stress-induced autophagy, among which PKCθ has recently been described. In this study, we dissected both in vitro and in vivo ER stress-induced autophagy pathways in muscle. Using C2C12 muscle cells in culture, we demonstrated that PKC activation induced autophagy in the absence of ER stress. We further demonstrated that PKCθ was strongly activated in cultured myoblasts and myotubes during ER stress induced by different stimuli, such as TG or TN treatment, and that it localized into Lc3-positive autophagic dots upon TG treatment. Neither Akt dephosphorylation nor Foxo or GSK3β activation was observed in these conditions. Moreover, PKCθ inhibition in myoblasts and myotubes prevented ER stress-induced Lc3 activation and autophagic dot formation, but not ER stress. In vivo, lack of PKCθ prevented both food deprivation- and immobilization-induced autophagy and muscle atrophy, irrespective of Akt pathway inhibition. Taken together, these results demonstrate that PKCθ functions as an ER stress sensor in skeletal muscle, required for ER-stress-dependent autophagy activation, and can be proposed as a novel molecular target to maintain muscle homeostasis in response to external stimuli, such as disuse and unloading, still allowing intracellular clearance.

Repeated injections of cannabidiol (CBD), the major non-psychotomimetic compound present in the Cannabis sativa plant, attenuate the anxiogenic effects induced by Chronic Unpredictable Stress (CUS). The specific mechanisms remain to be fully understood but seem to involve adult hippocampal neurogenesis and recruitment of endocannabinoids. Here we investigated for the first time if the behavioral and pro-neurogenic effects of CBD administered concomitant the CUS procedure (14 days) are mediated by CB1, CB2 or 5HT1A receptors, as well as CBD effects on dendritic remodeling and on intracellular/synaptic signaling (fatty acid amide hydrolase - FAAH, Akt, GSK3β and the synaptic proteins Synapsin Ia/b, mGluR1 and PSD95). After 14 days, CBD injections (30 mg/kg) induced anxiolytic responses in stressed animals in the elevated plus-maze and novelty suppressed feeding tests, that were blocked by pre-treatment with a CB1 (AM251, 0.3 mg/kg) or CB2 (AM630, 0.3 mg/kg), but not by a 5HT1A (WAY100635, 0.05 mg/kg) receptor antagonist. Golgi staining and immunofluorescence revealed that these effects were associated with an increase in hippocampal neurogenesis and spine density in the dentate gyrus of the hippocampus. AM251 and AM630 abolished the effects of CBD on spines density. However, AM630 was more effective in attenuating the pro-neurogenic effects of CBD. CBD decreased FAAH and increased p-GSK3β expression in stressed animals, which was also attenuated by AM630. These results indicate that CBD prevents the behavioral effects caused by CUS probably due to a facilitation of endocannabinoid neurotransmission and consequent CB1/CB2 receptors activation, which could recruit intracellular/synaptic proteins involved in neurogenesis and dendritic remodeling.

Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3β (GSK3β) and 70kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3β at Ser(9) and, to a lesser extent, Thr(390), the dephosphorylation of p70S6K at Thr(389), and the phosphorylation of p70S6K at Thr(421) and Ser(424). The specific p38 inhibitor SB203080 reduced the p-GSK3β(Ser9) and autophagy through the phosphorylation of p70S6K(Thr389); however, it augmented the levels of p-ERK, p-GSK3β(Thr390), and p-70S6K(Thr421/Ser424) induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our data show that proteasome inhibition regulates p38/GSK(Ser9)/p70S6K(Thr380) and ERK/GSK3β(Thr390)/p70S6K(Thr421/Ser424) kinase signaling, which is involved in cell survival and cell death.

Progranulin (PGRN) is an estrogen-inducible growth factor thought to affect multiple processes in the CNS, including brain sexual differentiation, adult neurogenesis in the hippocampus, and development of neurodegenerative diseases. However, the precise physiological functions of PGRN in individual nerve cells are not fully understood. The aim of the present study was to enhance the understanding of PGRN function in the CNS by investigating the effects of PGRN on neural progenitor cells (NPCs). We found that significant amounts of endogenous PGRN were secreted from isolated NPCs in cultures. To assess the bioactivities of endogenous and exogenous PGRN, we studied NPCs derived from wild-type mice (WT-NPCs) and PGRN-deficient mice (KO-NPCs). We found that proliferation of KO-NPCs was significantly enhanced by PGRN treatment; however, PGRN treatment apparently did not affect proliferation of WT-NPCs perhaps because of the high levels of endogenous PGRN expression. NPC death and asymmetric cellular division of KO-NPCs and WT-NPCs, which results in production of neural stem cells, astrocytes, or oligodendrocytes, were not affected by PGRN treatment. We also investigated the signaling mechanism(s) that mediate PGRN-induced NPC proliferation and found that phosphorylation of serine 9 (S9) of glycogen synthase kinase 3-beta (GSK3β), which was dependent on phosphatidylinositol 3-kinase (PI3K) activity, was induced by PGRN treatment. In addition, a GSK3β-specific inhibitor enhanced NPC proliferation. Taken together, our observations indicate that PGRN enhanced NPC proliferation, at least in part, via inducing GSK3β phosphorylation.

IBD syndromes such as Crohn's disease and ulcerative colitis result from the inflammation of specific intestinal segments. Although many studies have reported on the regenerative response of intestinal progenitor and stem cells to tissue injury, very little is known about the response of differentiated lineages to inflammatory cues. Here, we show that acute inflammation of the mouse small intestine is followed by a dramatic loss of Lgr5+ stem cells. Instead, Paneth cells re-enter the cell cycle, lose their secretory expression signature, and acquire stem-like properties, thus contributing to the tissue regenerative response to inflammation. Stem cell factor secretion upon inflammation triggers signaling through the c-Kit receptor and a cascade of downstream events culminating in GSK3β inhibition and Wnt activation in Paneth cells. Hence, the plasticity of the intestinal epithelium in response to inflammation goes well beyond stem and progenitor cells and extends to the fully differentiated and post-mitotic Paneth cells.

Two genes on chromosome 21, namely dual specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) and regulator of calcineurin 1 (RCAN1), have been implicated in some of the phenotypic characteristics of Down syndrome, including the early onset of Alzheimer disease. Although a link between Dyrk1A and RCAN1 and the nuclear factor of activated T cells (NFAT) pathway has been reported, it remains unclear whether Dyrk1A directly interacts with RCAN1. In the present study, Dyrk1A is shown to directly interact with and phosphorylate RCAN1 at Ser(112) and Thr(192) residues. Dyrk1A-mediated phosphorylation of RCAN1 at Ser(112) primes the protein for the GSK3β-mediated phosphorylation of Ser(108). Phosphorylation of RCAN1 at Thr(192) by Dyrk1A enhances the ability of RCAN1 to inhibit the phosphatase activity of calcineurin (Caln), leading to reduced NFAT transcriptional activity and enhanced Tau phosphorylation. These effects are mediated by the enhanced binding of RCAN1 to Caln and its extended half-life caused by Dyrk1A-mediated phosphorylation. Furthermore, an increased expression of phospho-Thr(192)-RCAN1 was observed in the brains of transgenic mice overexpressing the Dyrk1A protein. These results suggest a direct link between Dyrk1A and RCAN1 in the Caln-NFAT signaling and Tau hyperphosphorylation pathways, supporting the notion that the synergistic interaction between the chromosome 21 genes RCAN1 and Dyrk1A is associated with a variety of pathological features associated with DS.

OBJECTIVE

Our previous study showed there was a change of microRNA (miRNA) expression profile, and miR-21 was significantly down regulated in insulin-resistant adipocytes (IR-adipocytes). Phosphatase and tensin homologs deleted on chromosome 10 (PTEN), a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, was identified to be a target gene of miR-21, which suggested miR-21 might be associated with insulin resistance (IR) or diabetes. However, it is not known whether miR-21 play any role in the development of IR in 3T3-L1 adipocytes.

METHODS

Normal adipocytes and adipocytes transfected with pre-miR-21(pmiR-21) or negative control (pNeg) were treated with high glucose and high insulin for 24 h, insulin-stimulated glucose uptake was determined by 2-Deoxyglucose transport assay, miR-21 expression level was measured by using quantitative real-time RT-PCR (qRT-PCR). The protein expression levels of PTEN, Akt, phospho-Akt (Ser473), IRβ, GSK3β, phospho-GSK3β (Ser9) and GLUT4 were detected by western blotting assay.

RESULTS

We further confirmed that miR-21 was down regulated in IR-adipocytes by qRT-PCR. Over-expression of miR-21 significantly increased insulin-induced glucose uptake and decreased PTEN protein expression, while it had no significant effect on PTEN mRNA expression in IR-adipocytes. Moreover, over-expressing miR-21 significantly increased insulin-induced phosphorylation of AKT (Ser473), GSK3β (Ser9) and the translocation of glucose transporter 4 (GLUT4) in IR-adipocytes.

CONCLUSIONS

In this study, our data demonstrate that miR-21 reverses high glucose and high insulin induced IR in 3T3-L1 adipocytes, possibly through modulating the PTEN-AKT pathway, and miR-21 may be a new therapeutic target for metabolic diseases such as T2DM and obesity.

The relative risk for the development of malignancies following solid organ transplantation seems to be decreased in patients treated with the immunosuppressive agent mycophenolic acid (MPA). However, the molecular mechanisms of the antineoplastic effects of MPA are not completely understood. Here, we report that human endothelial cells and fibroblasts are highly sensitive to MPA treatment. We found that U87 glioblastoma cells were resistant to MPA treatment in vitro. However, U87 tumor growth was markedly inhibited in vivo in BALB/c nude mice, suggesting that MPA exerted its antitumor effects via modulation of the tumor microenvironment. Accordingly, microvascular density and pericyte coverage were markedly reduced in MPA-treated tumors in vivo. Using functional in vitro assays, we showed that MPA potently inhibited endothelial cell and fibroblast proliferation, invasion/migration, and endothelial cell tube formation. To identify the genetic participants governing the antiangiogenic and antifibrotic effects of MPA, we performed genome-wide transcriptional analysis in U87, endothelial and fibroblast cells at 6 and 12 h after MPA treatment. Network analysis revealed a critical role for MYC signaling in endothelial cells treated with MPA. Moreover, we found that the antiangiogenic effects of MPA were organized by coordinated communications between MYC and NDRG1, YYI, HIF1A, HDAC2, CDC2, GSK3B, and PRKACB signaling. The regulation of these "hub nodes" was confirmed by real-time quantitative reverse transcription-PCR and protein analysis. The critical involvement of MYC in the antiangiogenic signaling of MPA was further shown by gene knockdown experiments. Together, these data provide a molecular basis for the antiangiogenic and antifibrotic effects of MPA, which warrants further clinical investigations.

Postconditioning (PostC) can be obtained either with brief cycles of ischemia/reperfusion (I-PostC) or with a direct targeting of mitochondria with Diazoxide (pharmacological PostC, P-PostC). I-PostC may induce the activation of RISK and SAFE pathways and may favor nitric oxide production with S-Nitrosylation of proteins and redox signaling. It is not clear whether Diazoxide can lead to similar effects. We compared the effects of I-PostC and P-PostC on (a) kinases of RISK- and SAFE pathway, (b) S-Nitrosylation of mitochondrial proteins and (c) reduction of death signals (PKCδ, cleaved caspase-3 and Beclin-1) in cytosolic and mitochondrial fractions. Isolated rat hearts underwent (1) perfusion without ischemia (Sham), (2) ischemia/reperfusion (30-min ischemia plus 2-h reperfusion), (3) I-PostC (5 intermittent cycles of 10-s reperfusion and 10-s ischemia immediately after the 30-min ischemia), (4) P-PostC (Diazoxide 30 μM in the first of 3-min of reperfusion) or (5) I-PostC + MPG or P-PostC + MPG (MPG, 2-mercaptopropionylglycine 300 μM). Using Western blot and biotin switch assay, we found that P-PostC induced a redox sensible phosphorylation/translocation of Akt, ERK1/2 and GSK3β into the mitochondria, but not of phospho-STAT3, which was translocated into the mitochondria by I-PostC only. Either I-PostC or P-PostC increased mitochondrial S-Nitrosylated proteins (e.g., VDAC) and reduced the levels of phospho-PKCδ, cleaved caspase-3 and Beclin-1. Therefore, direct targeting of mitochondria with Diazoxide (a) activates the RISK pathway via a redox signaling, (b) favors discrete mitochondrial protein S-Nitrosylation, including VDAC and (c) decreases signals of death. Intriguingly, phospho-STAT3 translocation is induced by I-PostC, but not by P-PostC, thus suggesting a redox-independent mechanism in the SAFE pathway.

Caffeine is the most commonly ingested methylxanthine and has anti-cancer effects in several types of cancer. In this study, we examined the anti-cancer effects of caffeine on gliomas, both in vitro and in vivo. In vitro, caffeine treatment reduced glioma cell proliferation through G(0)/G(1)-phase cell cycle arrest by suppressing Rb phosphorylation. In addition, caffeine induced apoptosis through caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage. Caffeine also phosphorylated serine 9 of glycogen synthase kinase 3 beta (GSK3β). Pretreatment with H89, a pharmacological inhibitor of protein kinase A (PKA), was able to antagonize caffeine-induced GSK3β(ser9) phosphorylation, suggesting that the mechanism might involve a cAMP-dependent PKA-dependent pathway. In vivo, caffeine-treated tumors exhibited reduced proliferation and increased apoptosis compared with vehicle-treated tumors. These results suggest that caffeine induces cell cycle arrest and caspase-dependent cell death in glioma cells, supporting its potential use in chemotherapeutic options for malignant gliomas.

In non-small-cell lung cancer (NSCLC) patients, the activation of alternative pathways contributes to the limited efficacy of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. The present study examines a panel of EGFR wild-type, K-Ras mutated, NSCLC lines, which were all intrinsically resistant to EGFR-TKIs, and demonstrates that the histone deacetylase inhibitor vorinostat can improve the therapeutic efficacy of gefitinib or erlotinib, inducing strong synergistic antiproliferative and pro-apoptotic effects that are paralleled by reactive oxygen species accumulation and by increased DNA damage. By knockdown experiments, we suggested that the up-regulation of voltage-dependent anion-selective channel protein 1 (VDAC1), the major mitochondrial porin of the outer mitochondrial membrane, which was induced by vorinostat and further increased by the combination, could be functionally involved in oxidative stress-dependent apoptosis. Significantly, we also observed the attenuation of the expression of both the enzyme hexokinase1, a negative VDAC1 regulator, and the anti-apoptotic porin VDAC2, only in the combination setting, suggesting convergent mechanisms that enhanced mitochondria-dependent apoptosis by targeting VDAC protein functions. Furthermore, the prosurvival capacities of the cells were also inhibited by the combination treatments, as shown by complete pAKT deactivation, increased GSK3β expression, and c-Myc down-regulation. Finally, we observed that the combination treatment of vorinostat and either of the EGFR-TKIs induced the down-regulation of the c-Myc-regulated nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor and the up-regulation of the NRF2 repressor Kelch-like ECH-associated protein 1 regulator (KEAP1). These two genes are crucial for the redox stress response, often dysfunctional in NSCLC, and involved in EGFR-TKI resistance. Taken together, these results are the first to demonstrate that altering redox homeostasis is a new mechanism underlying the observed synergism between vorinostat and EGFR TKIs in NSCLC.

BACKGROUND

Cancer progression is closely linked to the epithelial-mesenchymal transition (EMT) process. Studies have shown that there is increased expression of tissue tranglutaminase (TG2) in advanced invasive cancer cells. TG2 catalyzes the covalent cross-linking of proteins, exhibits G protein activity, and has been implicated in the modulation of cell adhesion, migration, invasion and cancer metastasis. This study explores the molecular mechanisms associated with TG2's involvement in the acquisition of the mesenchymal phenotype using the highly invasive A431-III subline and its parental A431-P cells.

RESULTS

The A431-III tumor subline displays increased expression of TG2. This is accompanied by enhanced expression of the mesenchymal phenotype, and this expression is reversed by knockdown of endogenous TG2. Consistent with this, overexpression of TG2 in A431-P cells advanced the EMT process. Furthermore, TG2 induced the PI3K/Akt activation and GSK3β inactivation in A431 tumor cells and this increased Snail and MMP-9 expression resulting in higher cell motility. TG2 also upregulated NF-κB activity, which also enhanced Snail and MMP-9 expression resulting in greater cell motility; interestingly, this was associated with the formation of a TG2/NF-κB complex. TG2 facilitated acquisition of a mesenchymal phenotype, which was reversed by inhibitors of PI3K, GSK3 and NF-κB.

CONCLUSIONS

This study reveals that TG2 acts, at least in part, through activation of the PI3K/Akt and NF-κB signaling systems, which then induce the key mediators Snail and MMP-9 that facilitate the attainment of a mesenchymal phenotype. These findings support the possibility that TG2 is a promising target for cancer therapy.

Intrauterine growth restriction (IUGR) increases the risk for metabolic disease and diabetes, although the developmental origins of this remain unclear. We measured glucose metabolism during basal and insulin clamp periods in a fetal sheep model of placental insufficiency and IUGR. Compared with control fetuses (CON), fetuses with IUGR had increased basal glucose production rates and hepatic PEPCK and glucose-6-phosphatase expression, which were not suppressed by insulin. In contrast, insulin significantly increased peripheral glucose utilization rates in CON and IUGR fetuses. Insulin robustly activated AKT, GSK3β, and forkhead box class O (FOXO)1 in CON and IUGR fetal livers. IUGR livers, however, had increased basal FOXO1 phosphorylation, nuclear FOXO1 expression, and Jun NH(2)-terminal kinase activation during hyperinsulinemia. Expression of peroxisome proliferator-activated receptor γ coactivator 1α and hepatocyte nuclear factor-4α were increased in IUGR livers during basal and insulin periods. Cortisol and norepinephrine concentrations were positively correlated with glucose production rates. Isolated IUGR hepatocytes maintained increased glucose production in culture. In summary, fetal sheep with IUGR have increased hepatic glucose production, which is not suppressed by insulin despite insulin sensitivity for peripheral glucose utilization. These data are consistent with a novel mechanism involving persistent transcriptional activation in the liver that seems to be unique in the fetus with IUGR.

Previous studies suggest beta-adrenergic receptor (β-AR) antagonists (β-blockers) decrease breast cancer progression, tumor metastasis, and patient mortality; however the mechanism for this is unknown. Immunohistochemical analysis of normal and malignant breast tissue revealed overexpression of β1-AR and β3-AR in breast cancer. A retrospective cross-sectional study of 404 breast cancer patients was performed to determine the effect of β-blocker usage on tumor proliferation. Our analysis revealed that non-selective β-blockers, but not selective β-blockers, reduced tumor proliferation by 66% (p < 0.0001) in early stage breast cancer compared to non-users. We tested the efficacy of propranolol on an early stage breast cancer patient, and quantified the tumor proliferative index before and after treatment, revealing a propranolol-mediated 23% reduction (p = 0.02) in Ki67 positive tumor cells over a three-week period. The anti-proliferative effects of β-blockers were measured in a panel of breast cancer lines, demonstrating that mammary epithelial cells were resistant to propranolol, and that most breast cancer cell lines displayed dose dependent viability decreases following treatment. Selective β-blockers alone or in combination were not as effective as propranolol at reducing breast cancer cell proliferation. Molecular analysis revealed that propranolol treatment of the SK-BR-3 breast cancer line, which showed high sensitivity to beta blockade, led to a reduction in Ki67 protein expression, decreased phosphorylation of the mitogenic signaling regulators p44/42 MAPK, p38 MAPK, JNK, and CREB, increased phosphorylation of the cell survival/apoptosis regulators AKT, p53, and GSK3β. In conclusion, use of non-selective β-blockers in patients with early stage breast cancer may lead to decreased tumor proliferation.

Autophagy is one of the major immune mechanisms engaged to clear intracellular infectious agents. However, several pathogens have evolved strategies to evade autophagy. Here, we demonstrated that Mycobacteria, Shigella, and Listeria but not Klebsiella, Staphylococcus, and Escherichia inhibit IFNG-induced autophagy in macrophages by evoking selective and robust activation of WNT and SHH pathways via MTOR. Utilization of gain- or loss-of-function analyses as well as mir155-null macrophages emphasized the role of MTOR-responsive epigenetic modifications in the induction of Mir155 and Mir31. Importantly, cellular levels of PP2A, a phosphatase, were regulated by Mir155 and Mir31 to fine-tune autophagy. Diminished expression of PP2A led to inhibition of GSK3B, thus facilitating the prolonged activation of WNT and SHH signaling pathways. Sustained WNT and SHH signaling effectuated the expression of anti-inflammatory lipoxygenases, which in tandem inhibited IFNG-induced JAK-STAT signaling and contributed to evasion of autophagy. Altogether, these results established a role for new host factors and inhibitory mechanisms employed by the pathogens to limit autophagy, which could be targeted for therapeutic interventions.

MicroRNAs (miRNAs) emerge as important regulators of stem cell lineage commitment and bone development. MiRNA-26a (miR-26a) is one of the important miRNAs regulating osteogenic differentiation of both bone marrow-derived mesenchymal stem cells (BMSCs) and adipose tissue-derived mesenchymal stem cells (ADSCs). However, miR-26a functions oppositely in osteogenic differentiation of BMSCs and ADSCs, suggesting distinct post-transcriptional regulation of tissue-specific MSC differentiation. However, the molecular basis is largely unknown. Here, we report that the function of miR-26a is largely depended on the intrinsic signaling regulation network of MSCs. Using bioinformatics and functional assay, we confirmed that miR-26a potentially targeted on GSK3β and Smad1 to regulate Wnt and BMP signaling pathway. Overall comparative analysis revealed that Wnt signaling was enhanced more potently and played a more important role than BMP signaling in osteogenic differentiation of BMSCs, whereas BMP pathway was more essential for promoting osteogenic differentiation of ADSCs. The distinct activation pattern and role of signaling pathways determined that miR-26a majorly targeted on GSK3β to activate Wnt signaling for promoting osteogenic differentiation of BMSCs, whereas it inhibited Smad1 to suppress BMP signaling for interfering with the osteogenic differentiation of ADSCs. Taken together, our study demonstrated that BMSCs and ADSCs applied different signaling pathway to facilitate their osteogenic differentiation, which determined the inverse function of miR-26a. The distinct transcriptional regulation and post-transcriptional regulation network suggested the intrinsic molecular differences between tissue-specific MSCs and the complexity in MSC research and MSC-based cell therapy.