LncCCAT1 interaction protein PKM2 upregulates SREBP2 phosphorylation to promote osteosarcoma tumorigenesis by enhancing the Warburg effect and lipogenesis.

Pyruvate kinase M2 (PKM2) plays an important role in the consumption of glucose and the production of lactic acid, the striking feature of cancer metabolism. The association of PKM2 with osteosarcoma (OS) has been reported but its role in OS has yet to be elucidated. To study this, PKM2­bound RNAs in HeLa cells, a type of cancer cells widely used in the study of molecular function and mechanism, were obtained. Peak calling analysis revealed that PKM2 binds to long noncoding RNAs (lncRNAs), which are associated with cancer pathogenesis and development. Validation of the PKM2­lncRNA interaction in the human OS cell line revealed that lncRNA colon cancer associated transcript­1 (lncCCAT1) interacted with PKM2, which upregulated the phosphorylation of sterol regulatory element­binding protein 2 (SREBP2). These factors promoted the Warburg effect, lipogenesis, and OS cell growth. PKM2 appears to be a key regulator in OS by binding to lncCCAT1. This further extends the biological functions of PKM2 in tumorigenesis and makes it a novel potential therapeutic for OS.

Disrupting cannabinoid receptor interacting protein 1 rescues cognitive flexibility in long-term estrogen-deprived female mice.

Hormone therapy (HT) has failed to improve learning and memory in postmenopausal women according to recent clinical studies; however, the reason for failure of HT in improving cognitive performance is unknown. In our research, we found cognitive flexibility was improved by 17ß-Estradiol (E2) in mice 1 week after ovariectomy (OVXST), but not in mice 3 months after ovariectomy (OVXLT). Isobaric tags for relative and absolute quantitation (iTRAQ) revealed increased cannabinoid receptor interacting protein 1 (CNRIP1) in E2-treated OVXLT mice compared with E2-treated OVXST mice. Adeno-associated virus 2/9 (AAV2/9) delivery of Cnrip1 short-hairpin small interfering RNA (Cnrip1-shRNA) rescued the impaired cognitive flexibility in E2 treated OVXLT mice. This effect is dependent on CB1 function, which could be blocked by AM251-a CB1 antagonist. Our results indicated a new method to increasing cognitive flexibility in women receiving HT by disrupting CNRIP1.

1,8 Cineole and Ellagic acid inhibit hepatocarcinogenesis via upregulation of MiR-122 and suppression of TGF-ß1, FSCN1, Vimentin, VEGF, and MMP-9.

Hepatocellular carcinoma (HCC) is one of the most burdened tumors worldwide, with a complex and multifactorial pathogenesis. Current treatment approaches involve different molecular targets. Phytochemicals have shown considerable promise in the prevention and treatment of HCC. We investigated the efficacy of two natural components, 1,8 cineole (Cin) and ellagic acid (EA), against diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) induced HCC in rats. DEN/2-AAF showed deterioration of hepatic cells with an impaired functional capacity of the liver. In addition, the levels of tumor markers including alpha-fetoprotein, arginase-1, alpha-L-fucosidase, and ferritin were significantly increased, whereas the hepatic miR-122 level was significantly decreased in induced-HCC rats. Interestingly, treatment with Cin (100mg/kg) and EA (60mg/kg) powerfully restored these biochemical alterations. Moreover, Cin and EA treatment exhibited significant downregulation in transforming growth factor beta-1 (TGF-ß1), Fascin-1 (FSCN1), vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), and epithelial-mesenchymal transition (EMT) key marker, vimentin, along with a restoration of histopathological findings compared to HCC group. Such effects were comparable to Doxorubicin (DOX) (2mg/kg); however, a little additive effect was evident through combining these phytochemicals with DOX. Altogether, this study highlighted 1,8 cineole and ellagic acid for the first time as promising phytochemicals for the treatment of hepatocarcinogenesis via regulating multiple targets.

A new KSRP-binding compound suppresses distant metastasis of colorectal cancer by targeting the oncogenic KITENIN complex.

BACKGROUND: Distant metastasis is the major cause of death in patients with colorectal cancer (CRC). Previously, we identified KITENIN as a metastasis-enhancing gene and suggested that the oncogenic KITENIN complex is involved in metastatic dissemination of KITENIN-overexpressing CRC cells. Here, we attempted to find substances targeting the KITENIN complex and test their ability to suppress distant metastasis of CRC. METHODS: We screened a small-molecule compound library to find candidate substances suppressing the KITENIN complex in CRC cells. We selected a candidate compound and examined its effects on the KITENIN complex and distant metastasis through in vitro assays, a molecular docking model, and in vivo tumor models. RESULTS: Among several compounds, we identified DKC1125 (Disintegrator of KITENIN Complex #1125) as the best candidate. DKC1125 specifically suppressed KITENIN gain of function. After binding KH-type splicing regulatory protein (KSRP), DKC1125 degraded KITENIN and Dvl2 by recruiting RACK1 and miRNA-124, leading to the disintegration of the functional KITENIN-KSRP-RACK1-Dvl2 complex. A computer docking model suggested that DKC1125 specifically interacted with the binding pocket of the fourth KH-domain of KSRP. KITENIN-overexpressing CRC cells deregulated certain microRNAs and were resistant to 5-fluorouracil, oxaliplatin, and cetuximab. DKC1125 restored sensitivity to these drugs by normalizing expression of the deregulated microRNAs, including miRNA-124. DKC1125 effectively suppressed colorectal liver metastasis in a mouse model. Interestingly, the combination of DKC1125 with 5-fluorouracil suppressed metastasis more effectively than either drug alone. CONCLUSION: DKC1125 targets the KITENIN complex and could therefore be used as a novel therapeutic to suppress liver metastasis in CRC expressing high levels of KITENIN.

Regulation of monoamine levels by typical and atypical antipsychotics in Caenorhabditis elegans mutant for nuclear distribution element genes.

Mammalian nuclear distribution genes encode proteins with essential roles in neuronal migration and brain formation during embryogenesis. The implication of human nuclear distribution genes, namely nudC and NDE1 (Nuclear Distribution Element 1)/NDEL1 (Nuclear Distribution Element-Like 1), in psychiatric disorders including schizophrenia and bipolar disorder, has been recently described. The partial loss of NDEL1 expression results in neuronal migration defects, while ndel1 null knockout (KO) leads to early embryonic lethality in mice. On the other hand, loss-of-function of the orthologs of nuclear distribution element genes (nud) in Caenorhabditis elegans renders viable worms and influences behavioral endophenotypes associated with dopaminergic and serotoninergic pathways. In the present work, we evaluated the role of nud genes in monoamine levels at baseline and after the treatment with typical or atypical antipsychotics. Dopamine, serotonin and octopamine levels were significantly lower in homozygous loss-of-function mutant worms KO for nud genes compared with wild-type (WT) C. elegans at baseline. While treatment with antipsychotics determined significant differences in monoamine levels in WT, the nud KO mutant worms appear to respond differently to the treatment. According to the best of our knowledge, we are the first to report the influence of nud genes in the monoamine levels changes in response to antipsychotic drugs, ultimately placing the nuclear distribution genes family at the cornerstone of pathways involved in the modulation of monoamines in response to different classes of antipsychotic drugs.

HMGCR inhibition stabilizes the glycolytic enzyme PKM2 to support the growth of renal cell carcinoma.

Renal cell carcinoma (RCC) is responsible for most cases of the kidney cancer. Previous research showed that low serum levels of cholesterol level positively correlate with poorer RCC-specific survival outcomes. However, the underlying mechanisms and functional significance of the role of cholesterol in the development of RCC remain obscure. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) plays a pivotal role in RCC development as it is the key rate-limiting enzyme of the cholesterol biosynthetic pathway. In this study, we demonstrated that the inhibition of HMGCR could accelerate the development of RCC tumors by lactate accumulation and angiogenesis in animal models. We identified that the inhibition of HMGCR led to an increase in glycolysis via the regulated HSP90 expression levels, thus maintaining the levels of a glycolysis rate-limiting enzyme, pyruvate kinase M2 (PKM2). Based on these findings, we reversed the HMGCR inhibition-induced tumor growth acceleration in RCC xenograft mice by suppressing glycolysis. Furthermore, the coadministration of Shikonin, a potent PKM2 inhibitor, reverted the tumor development induced by the HMGCR signaling pathway.

Potential of Forsythoside I as a therapeutic approach for acute lung injury: Involvement of TXNIP/NLRP3 inflammasome.

OBJECTIVE: To explore the role of Forsythoside I (FI) in acute lung injury (ALI) mouse and its underling mechanism. METHODS: The cell models of ALI are constructed by LPS induction. After pretreatment with different concentrations of FI, the lung injury is assessed by pathological changes of lung tissues and cell apoptosis. The cell viability, levels of pro-inflammatory cytokines, and the activation of TXNIP/NLRP3 pathway are inspected to investigate whether the effect of FI on inflammatory response is exerted by regulating the TXNIP/NLRP3 pathway. RESULTS: LPS induces inflammatory cell infiltration, tissue necrosis and pulmonary interstitial edema of mouse tissues, and LPS increases the protein concentration and levels of pro-inflammatory factors in mouse BALF. Additionally, enhanced cell apoptotic level, increased W/D ratio and MPO activity, as well as suppressed SOD activity are observed in LPS-induced mouse models. Those inflammation response, oxidative stress and lung injury can be attenuated by FI (12.5 mg/kg, 25 mg/kg, 50 mg/kg) in a dose-dependent manner. Meanwhile, both in vitro and in vivo studies reveal that FI can lead to suppressed TXNIP expression and inactivated NLRP3 inflammasomes. TXNIP is an upstream target of NLRP3, and FI mitigates ALI by decreasing TXNIP to block NLRP3 inflammasomes. CONCLUSION: FI protects against ALI through the mediation of TXNIP/NLRP3 inflammasome axis and therefore has a certain potential for ALI treatment.

Therapeutic potential of russelioside B as anti-arthritic agent in Freund's adjuvant-induced arthritis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Caralluma species are traditional edible herbs used in folkloric medicine as antidiabetic, antioxidant, antipyretic, antirheumatic, anti-inflammatory and anthelmintic agents. C. quadrangula was selected in this study to document the traditional use of the genus as anti-rheumatic treatment and the possible mechanisms of action. AIM OF THE STUDY: The higher mortality rates and shorter survival among the patients suffering from rheumatoid arthritis (RA) led to the increased interest on searching for new treatments for RA. Russelioside B (RB), a major pregnane glycoside found in C. quadrangula, was evaluated as a new anti-rheumatic agent. MATERIALS AND METHODS: The n-butanol fraction of C. quadrangula was chromatographed on a silica gel column to isolate RB. The adjuvant-induced arthritis (AIA) model was established in rats by intradermal injection of complete Freund's adjuvant (CFA) to evaluate its anti-arthritic effect. Ibuprofen was used as a reference drug. Forty rats were randomly divided into 5 groups (n = 8): normal (NOR); CFA model (CFA); ibuprofen, 5 mg/kg; RB, 25 mg/kg and RB, 50 mg/kg. The treatments were initiated from day 16 when AIA model was established and continued up to day 40. Serum diagnostic rheumatoid markers, inflammatory cytokines, oxidative stress biomarkers, cartilage and bone degeneration enzymes were assessed. RESULTS: RB at 50 mg/kg b. wt., showed significant decreases in the activities of hyaluronidase and ß-glucouronidase enzymes as well significant decreases in the levels of proinflammatory cytokines as nuclear factor-kappa-B (NF-κB), tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) compared to the CFA group; 11.04 ± 0.61 pg/mg protein, 4.35 ± 0.25 pg/mg protein, 3.32 ± 0.13 pg/mg protein & 2.75 ± 0.14 pg/mg protein for RB, 50 mg/kg b. wt. group vs. 25.33 ± 2.13 pg/mg protein, 25.65 ± 2.1 pg/mg protein, 22.20 ± 1.34 pg/mg protein & 13.27 ± 1.40 pg/mg protein for the arthritic group, respectively. The total antioxidant capacity (TAC) was significantly restored to normal values in RB, 50 mg/kg treated rats (4.01 ± 0.09 nmol/mL vs. 3.71 ± 0.27 nmol/mL) and the levels of myeloperoxidase (MPO) reduced by 10-folds of the CFA arthritic group. Bone histomorphometry revealed that RB treatment significantly attenuated the CFA-induced bone loss in a dose-dependent manner. CONCLUSIONS: These findings suggested that the anti-arthritic effect of RB was mediated through the reduction of the rheumatoid markers, anti-inflammatory and antioxidant action, inhibition of cartilage and bone degenerative enzymes as well as attenuation of bone loss and osteoclastogenesis.

CRD-733, a Novel PDE9 (Phosphodiesterase 9) Inhibitor, Reverses Pressure Overload-Induced Heart Failure.

BACKGROUND: Augmentation of NP (natriuretic peptide) receptor and cyclic guanosine monophosphate (cGMP) signaling has emerged as a therapeutic strategy in heart failure (HF). cGMP-specific PDE9 (phosphodiesterase 9) inhibition increases cGMP signaling and attenuates stress-induced hypertrophic heart disease in preclinical studies. A novel cGMP-specific PDE9 inhibitor, CRD-733, is currently being advanced in human clinical studies. Here, we explore the effects of chronic PDE9 inhibition with CRD-733 in the mouse transverse aortic constriction pressure overload HF model. METHODS: Adult male C57BL/6J mice were subjected to transverse aortic constriction and developed significant left ventricular (LV) hypertrophy after 7 days (P<0.001). Mice then received daily treatment with CRD-733 (600 mg/kg per day; n=10) or vehicle (n=17), alongside sham-operated controls (n=10). RESULTS: CRD-733 treatment reversed existing LV hypertrophy compared with vehicle (P<0.001), significantly improved LV ejection fraction (P=0.009), and attenuated left atrial dilation (P<0.001), as assessed by serial echocardiography. CRD-733 prevented elevations in LV end diastolic pressures (P=0.037) compared with vehicle, while lung weights, a surrogate for pulmonary edema, were reduced to sham levels. Chronic CRD-733 treatment increased plasma cGMP levels compared with vehicle (P<0.001), alongside increased phosphorylation of Ser273 of cardiac myosin binding protein-C, a cGMP-dependent protein kinase I phosphorylation site. CONCLUSIONS: The PDE9 inhibitor, CRD-733, improves key hallmarks of HF including LV hypertrophy, LV dysfunction, left atrial dilation, and pulmonary edema after pressure overload in the mouse transverse aortic constriction HF model. Additionally, elevated plasma cGMP may be used as a biomarker of target engagement. These findings support future investigation into the therapeutic potential of CRD-733 in human HF.

Effects of psoralen on hepatic bile acid transporters in rats.

Fructus Psoraleae (FP), widely used in traditional medicine, is increasingly reported to cause serious hepatotoxicity in recent years. However, the main toxic constituents responsible for hepatotoxicity and the underlying mechanisms are poorly understood. In the present study, psoralen, a main and quality-control constituent of FP, was intragastrically administered to Sprague-Dawley rats at a dose of 60 mg/kg for 1, 3 and 7 days. Blood and selected tissue samples were collected and analyzed for biochemistry and histopathology to evaluate hepatotoxicity. The results showed that psoralen could induce hepatotoxicity by enhanced liver-to-body weight ratio and alterations of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total cholesterol after administration for 3 days. In addition, histopathological examinations also indicated the hepatotoxicity induced by psoralen. Furthermore, the mRNA and protein levels of hepatic bile acid transporters were significantly changed, in which MRP4, ABCG5 and ABCG8 were repressed, while the protein level of NTCP tended to increase in the rat liver. Taken together, psoralen caused liver injury possibly through affecting bile acid transporters, leading to the disorder of bile acid transport and accumulation in hepatocytes.

Antiviral activity against Middle East Respiratory Syndrome coronavirus by Montelukast, an anti-asthma drug.

Middle East Respiratory Syndrome (MERS) is a respiratory disease caused by a coronavirus (MERS-CoV). Since its emergence in 2012, nosocomial amplifications have led to its high epidemic potential and mortality rate of 34.5%. To date, there is an unmet need for vaccines and specific therapeutics for this disease. Available treatments are either supportive medications in use for other diseases or those lacking specificity requiring higher doses. The viral infection mode is initiated by the attachment of the viral spike glycoprotein to the human Dipeptidyl Peptidase IV (DPP4). Our attempts to screen antivirals against MERS led us to identify montelukast sodium hydrate (MSH), an FDA-approved anti-asthma drug, as an agent attenuating MERS-CoV infection. We showed that MSH directly binds to MERS-CoV-Receptor-Binding Domain (RBD) and inhibits its molecular interaction with DPP4 in a dose-dependent manner. Our cell-based inhibition assays using MERS pseudovirions demonstrated that viral infection was significantly inhibited by MSH and was further validated using infectious MERS-CoV culture. Thus, we propose MSH as a potential candidate for therapeutic developments against MERS-CoV infections.

The inhibitory effect of curcumin via fascin suppression through JAK/STAT3 pathway on metastasis and recurrence of ovary cancer cells.

BACKGROUND: Fascin is an actin-binding protein and highly expressed in ovarian cancer cells. It is associated with metastasis of cancer and may be a useful prognostic factor. Anticancer activity of curcumin is related to its effect on several signaling mechanisms. Although there have been many reports regarding the anticancer properties of curcumin, its inhibitory effects on migration and invasion of ovarian cancer cells, particularly in the context of fascin expression, have not been reported. The purpose of this study was to investigate the effect of curcumin on fascin expression in ovarian cancer cells and to propose a possible mechanism for the anticancer activity of curcumin through reduced fascin expression. METHODS: SKOV3, human epithelial ovary cancer cell line, was cultured with curcumin at various dose and duration. The fascin was quantified using cell viability test and Western blot. To determine the effect of curcumin on the upstream pathway of fascin expression, the signal transducer and activator of transcription 3 (STAT3) was analyzed by sandwich-ELISA. Attachment assay, migration assay and invasion assay were analyzed to approve the change of cellular invasiveness of ovary cancer after curcumin. To determine the morphological changes of ovarian cancer cells by curcumin, immunofluorescence was performed. RESULTS: MTS assays showed that cell viability was different at various concentration of curcumin, and as concentration increased, cell viability tended to decrease. Curcumin appears to suppress fascin expression, even with a minimal concentration and short exposure time. Also, curcumin may suppress fascin expression in ovarian cancer cells through STAT3 downregulation. The attachment assay, migration assay and invasion assay of the ovarian cancer cells exhibited a statistically significant decrease. Immunofluorescence revealed a change of cell shape from a typical form of uninfluenced cells to a more polygonal appearance, with a significant reduction in filopodia formation. CONCLUSIONS: Curcumin reduces fascin expression through JAK/STAT3 pathway inhibition, which interferes with the cellular interactions essential for the metastasis and recurrence of ovarian cancer cells. Higher curcumin concentrations and longer exposure times concomitantly decreased fascin expression.

Rational design of highly potent broad-spectrum enterovirus inhibitors targeting the nonstructural protein 2C.

There is a great need for antiviral drugs to treat enterovirus (EV) and rhinovirus (RV) infections, which can be severe and occasionally life-threatening. The conserved nonstructural protein 2C, which is an AAA+ ATPase, is a promising target for drug development. Here, we present a structure-activity relationship study of a previously identified compound that targets the 2C protein of EV-A71 and several EV-B species members, but not poliovirus (PV) (EV-C species). This compound is structurally related to the Food and Drug Administration (FDA)-approved drug fluoxetine-which also targets 2C-but has favorable chemical properties. We identified several compounds with increased antiviral potency and broadened activity. Four compounds showed broad-spectrum EV and RV activity and inhibited contemporary strains of emerging EVs of public health concern, including EV-A71, coxsackievirus (CV)-A24v, and EV-D68. Importantly, unlike (S)-fluoxetine, these compounds are no longer neuroactive. By raising resistant EV-A71, CV-B3, and EV-D68 variants against one of these inhibitors, we identified novel 2C resistance mutations. Reverse engineering of these mutations revealed a conserved mechanism of resistance development. Resistant viruses first acquired a mutation in, or adjacent to, the α2 helix of 2C. This mutation disrupted compound binding and provided drug resistance, but this was at the cost of viral fitness. Additional mutations at distantly localized 2C residues were then acquired to increase resistance and/or to compensate for the loss of fitness. Using computational methods to identify solvent accessible tunnels near the α2 helix in the EV-A71 and PV 2C crystal structures, a conserved binding pocket of the inhibitors is proposed.

Divergent receptor proteins confer responses to different karrikins in two ephemeral weeds.

Wildfires can encourage the establishment of invasive plants by releasing potent germination stimulants, such as karrikins. Seed germination of Brassica tournefortii, a noxious weed of Mediterranean climates, is strongly stimulated by KAR1, the archetypal karrikin produced from burning vegetation. In contrast, the closely-related yet non-fire-associated ephemeral Arabidopsis thaliana is unusual because it responds preferentially to KAR2. The α/ß-hydrolase KARRIKIN INSENSITIVE 2 (KAI2) is the putative karrikin receptor identified in Arabidopsis. Here we show that B. tournefortii expresses three KAI2 homologues, and the most highly-expressed homologue is sufficient to confer enhanced responses to KAR1 relative to KAR2 when expressed in Arabidopsis. We identify two amino acid residues near the KAI2 active site that explain the ligand selectivity, and show that this combination has arisen independently multiple times within dicots. Our results suggest that duplication and diversification of KAI2 proteins could confer differential responses to chemical cues produced by environmental disturbance, including fire.

Aspirin modulates STOX1 expression and reverses STOX1-induced insufficient proliferation and migration of trophoblast cells.

BACKGROUND: A major cause of preeclampsia is the placental ischemia caused by insufficient trophoblast cells, invading into the spiral artery. Storkhead-box protein 1 (STOX1) is highly associated with preeclampsia. Meanwhile, low-dose aspirin for patients with preeclampsia is effective in reducing the incidence of preeclampsia. The aim of the present study was to explore the underlying mechanism, and the relationship between STOX1 and aspirin in preeclampsia. METHODS: The human choriocarcinoma cell line JEG-3 was employed to mimic trophoblast cells and establish a model for trophoblast cells overexpressing STOX1 and knockdown of JEG cell lines, which were treated with aspirin afterwards. Cell counting kit-8 (CCK-8) assay was utilized to estimate cell proliferation and optimal concentration of aspirin for further experiments. Meanwhile, transwell assay was used to detect migration, and flow cytometry was used to measure apoptosis. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting were applied to analyze the expression levels of STOX1 and related genes. RESULTS: Overexpression of STOX1 inhibited proliferation of JEG-3 cells through epidermal growth factor (EGF), vascular EGF (VEGF), and transforming growth factor beta 1 (TGF-ß1) proteins, while suppressed migration through MMP2, MMP9, and E-cadherin proteins. In contrast, apoptosis of JEG-3 cells was elevated by STOX1 through Bcl-2, Bax, and Cox-2 proteins. Furthermore, we found that aspirin modulated the expression level of STOX1 and reversed proliferation and migration of STOX1-induced insufficient trophoblast cells. CONCLUSION: The present study suggested that inhibition of the expression of STOX1 could promote the effects of aspirin in the treatment of preeclampsia.

Sodium butyrate-activated TRAF6-TXNIP pathway affects A549 cells proliferation and migration.

TNF receptor-associated factor 6 (TRAF6) promotes the development of human lung cancer through bridging RAS and NF-kB pathways; on the other hand, thioredoxin-interacting protein (TXNIP) suppresses the growth of tumors. However, the crosstalk between TRAF6 and TXNIP in non-small cell lung cancer (NSCLC) is currently unclear. Here, we found that TXNIP expression induced by sodium butyrate (NaBu) was TRAF6-dependent. Moreover, TXNIP interacted with TRAF6 via its PPxY motif. Polyubiquitylation analysis with wild-type or mutant (Cysteine70 to Alanine) of TRAF6 further showed TRAF6 ubiquitylated TXNIP. NaBu reinforced the interaction of TRAF6/TXNIP as well as TXNIP' polyubiquitylation. Moreover, treated with NaBu, the A549 cells with TRAF6/TXNIP double knockdown showed an enhanced protein expression of E-cadherin comparing to cells with single gene or negative knockdown. The experimental results of transwell and nude mice xenograft showed that knocking down both TRAF6 and TXNIP in A549 cells affected its migration and proliferation compared to that of single knockdown or negative control cells. On the other hand, TXNIP localization was different depending on the cell types and fused-tag (eg, FLAG or GFP). Our results revealed TRAF6 regulated the expression and polyubiquitylation of TXNIP in a NaBu-dependent manner, alleviating tumorigenesis of TRAF6.

The N-AcetylCysteine and RAMipril in Takotsubo Syndrome Trial (NACRAM): Rationale and design of a randomised controlled trial of sequential N-Acetylcysteine and ramipril for the management of Takotsubo Syndrome.

BACKGROUND: Takotsubo Syndrome(TTS), contrary to historical reports, is now increasingly recognised to be associated with substantial mortality and morbidity, both in the short- and long-term. Although TTS is often precipitated by a catecholamine "pulse", in-hospital hypotension is a common occurrence, increasing the risk of mortality. Furthermore, despite the transient catecholamine stimulus, there is increasing evidence that there are significant long term sequelae, including persistently impaired left ventricular(LV) systolic dysfunction, myocardial oedema with fibrosis, as well as persistent impairment of quality of life. A definitive therapeutic option to limit the extent of initial myocardial injury, and to accelerate recovery in TTS is therefore justified. However to date, there has been a lack of prospective studies in this area. DESIGN AND RATIONALE: NACRAM is a multi-centre, randomised, placebo-controlled trial, sequentially testing early use of intravenous N-acetylcysteine(NAC), followed by/or oral ramipril for 12 weeks. The rationale for utilising these agents is related to their effects on limiting nitrosative stress and expression of the inflammasome activator thioredoxin interacting protein(TXNIP); both processes fundamental to the pathogenesis of TTS. END POINTS: NACRAM is assessing resolution of myocardial oedema on cardiac magnetic resonance imaging(CMR), improvements in LV systolic function as measured by global longitudinal strain(GLS) on echocardiography, quality of life, and inflammatory markers. DISCUSSION: To the best of our knowledge, NACRAM will be the first prospective study to help definitively evaluate a therapeutic option in acute attacks of TTS.

Regulation of GSK3 cellular location by FRAT modulates mTORC1-dependent cell growth and sensitivity to rapamycin.

The mTORC1 pathway regulates cell growth and proliferation by properly coupling critical processes such as gene expression, protein translation, and metabolism to the availability of growth factors and hormones, nutrients, cellular energetics, oxygen status, and cell stress. Although multiple cytoplasmic substrates of mTORC1 have been identified, how mTORC1 signals within the nucleus remains incompletely understood. Here, we report a mechanism by which mTORC1 modulates the phosphorylation of multiple nuclear events. We observed a significant nuclear enrichment of GSK3 when mTORC1 was suppressed, which promotes phosphorylation of several proteins such as GTF2F1 and FOXK1. Importantly, nuclear localization of GSK3 is sufficient to suppress cell proliferation. Additionally, expression of a nuclear exporter of GSK3, FRAT, restricts the nuclear localization of GSK3, represses nuclear protein phosphorylation, and prevents rapamycin-induced cytostasis. Finally, we observe a correlation between rapamycin resistance and FRAT expression in multiple-cancer cell lines. Resistance to Food and Drug Administration (FDA)-approved rapamycin analogs (rapalogs) is observed in many tumor settings, but the underling mechanisms remain incompletely understood. Given that FRAT expression levels are frequently elevated in various cancers, our observations provide a potential biomarker and strategy for overcoming rapamycin resistance.

Amino acid transporter SLC6A14 depends on heat shock protein HSP90 in trafficking to the cell surface.

Plasma membrane transporter SLC6A14 transports all neutral and basic amino acids in a Na/Cl - dependent way and it is up-regulated in many types of cancer. Mass spectrometry analysis of overexpressed SLC6A14-associated proteins identified, among others, the presence of cytosolic heat shock proteins (HSPs) and co-chaperones. We detected co-localization of overexpressed and native SLC6A14 with HSP90-beta and HSP70 (HSPA14). Proximity ligation assay confirmed a direct interaction of overexpressed SLC6A14 with both HSPs. Treatment with radicicol and VER155008, specific inhibitors of HSP90 and HSP70, respectively, attenuated these interactions and strongly reduced transporter presence at the cell surface, what resulted from the diminished level of the total transporter protein. Distortion of SLC6A14 proper folding by both HSPs inhibitors directed the transporter towards endoplasmic reticulum-associated degradation pathway, a process reversed by the proteasome inhibitor - bortezomib. As demonstrated in an in vitro ATPase assay of recombinant purified HSP90-beta, the peptides corresponding to C-terminal amino acid sequence following the last transmembrane domain of SLC6A14 affected the HSP90-beta activity. These results indicate that a plasma membrane protein folding can be controlled not only by chaperones in the endoplasmic reticulum, but also those localized in the cytosol.