Search

Natural killer/T‐cell lymphoma (NKTL) is an uncommon malignancy with poor prognosis and limited therapeutic options. Activating mutations of signal transducer and activator of transcription 3 (STAT3) are frequently found in patients with NKTL, suggesting that targeted inhibition of STAT3 is a potential therapeutic option for this disease. Here, we have developed a small molecule drug WB737 as a novel and potent STAT3 inhibitor that directly binds to the STAT3‐Src homology 2 domain with high affinity. In addition, the binding affinity of WB737 to STAT3 is 250‐fold higher than STAT1 and STAT2. Interestingly, WB737 is more selective for NKTL with STAT3‐activating mutations in terms of growth inhibition and apoptotic induction when compared with Stattic. Mechanistically, WB737 inhibits both canonical and noncanonical STAT3 signaling via suppression of STAT3 phosphorylation at Tyr705 and Ser727, respectively, thereby inhibiting the expression of c‐Myc and mitochondria‐related genes. Moreover, WB737 inhibited STAT3 more potently than Stattic, resulting in a significant antitumor effect with undetectable toxicity, followed by almost complete tumor regression in an NKTL xenograft model harboring a STAT3‐activating mutation. Taken together, these findings provide preclinical proof‐of‐concept for WB737 as a novel therapeutic strategy for the treatment of NKTL patients with STAT3‐activating mutations. [ABSTRACT FROM AUTHOR]

Optogenetic genome engineering is a powerful technology for high‐resolution spatiotemporal genetic manipulation, especially for in vivo studies. It is difficult to generate stable transgenic animals carrying a tightly regulated optogenetic system, as its long‐term expression induces high background activity. Here, the generation of an enhanced photoactivatable Cre recombinase (ePA‐Cre) transgenic mouse strain with stringent light responsiveness and high recombination efficiency is reported. Through serial optimization, ePA‐Cre is developed to generate a transgenic mouse line that exhibits 175‐fold induction upon illumination. Efficient light‐dependent recombination is detected in embryos and various adult tissues of ePA‐Cre mice crossed with the Ai14 tdTomato reporter. Importantly, no significant background Cre activity is detected in the tested tissues except the skin. Moreover, efficient light‐inducible cell ablation is achieved in ePA‐Cre mice crossed with Rosa26‐LSL‐DTA mice. In conclusion, ePA‐Cre mice offer a tightly inducible, highly efficient, and spatiotemporal‐specific genome engineering tool for multiple applications. [ABSTRACT FROM AUTHOR]

Emerging evidence suggests that physiological distress is highly correlated with cancer incidence and mortality. However, the mechanisms underlying psychological challenges‐mediated tumor immune evasion are not systematically explored. Here, it is demonstrated that acute restraint (AR) increases the level of the plasma neuropeptide hormones, kisspeptin, and the expression levels of its receptor, Gpr54, in the hypothalamus, splenic and tumor‐infiltrating T cells, suggesting a correlation between the neuroendocrine system and tumor microenvironment. Accordingly, administration of kisspeptin‐10 significantly impairs T cell function, whereas knockout of Gpr54 in T cells inhibits lung tumor progression by suppressing T cell dysfunction and exhaustion with or without AR. In addition, Gpr54 defective OT‐1 T cells show superior antitumor activity against OVA peptide‐positive tumors. Mechanistically, ERK5‐mediated NR4A1 activation is found to be essential for kisspeptin/GPR54‐facilitated T cell dysfunction. Meanwhile, pharmacological inhibition of ERK5 signaling by XMD8‐92 significantly reduces the tumor growth by enhancing CD8+ T cell antitumor function. Furthermore, depletion of GPR54 or ERK5 by CRISPR/Cas9 in CAR T cells intensifies the antitumor responses to both PSMA+ and CD19+ tumor cells, while eliminating T cell exhaustion. Taken together, these results indicate that kisspeptin/GPR54 signaling plays a nonredundant role in the stress‐induced tumor immune evasion. [ABSTRACT FROM AUTHOR]

Proper regulation of B‐cell function is essential for effective humoral immunity and maintenance of immune tolerance. Here, we found that FBW7 (F‐box/WD40 repeat‐containing protein 7) is highly expressed in germinal centre B and B1 cells, and confirmed that it has an intrinsic role in maintaining homeostasis of mature B cells and B‐1 cells. FBW7 deletion led to an impairment of antibody response, and although germinal centre formation was not affected, antibody class‐switch recombination and affinity maturation processes were defective. Likewise, memory immune response was severely impaired. Moreover, FBW7 ablation ameliorated the pathogenesis of an autoimmune disease model, collagen‐induced arthritis, by reducing the production of anti‐collagen II autoantibodies. Taken together, these data suggest that FBW7 may be an attractive target for developing new therapeutics for the treatment of autoimmune diseases. [ABSTRACT FROM AUTHOR]

Oncolytic virus therapy is perhaps the next major breakthrough in cancer treatment following the success in immunotherapy using immune checkpoint inhibitors. However, the potential oncolytic ability of the recombinant newcastle disease virus (NDV) Anhinga strain carried with tumor necrosis factor‐related apoptosis inducing ligand (TRAIL) has not been fully explored at present. In the present study, the recombinant NDV/Anh‐TRAIL that secretes soluble TRAIL was constructed and the experiment results suggested NDV/Anh‐TRAIL as a promising candidate for glioma therapy. Growth kinetic and TRAIL secreted quantity of recombinant NDV/Anh‐TRAIL virus were measured. Cytotoxic and cell apoptosis were analyzed for its anti‐glioma therapy in vitro. Nude mice were used for the in vivo evaluation. Both tumor volume and mice behavior after injection were observed. The recombinant virus replicated with the same kinetics as the parental virus and the highest expression of TRAIL (77.8 ng/L) was found at 48 hours. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide, a tetrazole and flow cytometry data revealed that the recombinant NDV/Anh‐TRAIL (56.1 ± 8.2%) virus could induce a more severe apoptosis rate, when compared with the NDV wild type (37.2 ± 7.0%) and mock (7.0 ± 1.8%) groups (P <.01), in U251 cells. Furthermore, in the present animal study, the average tumor volume was smaller in the NDV/Anh‐TRAIL group (97.21 mm3), when compared with the NDV wild type (205.03 mm3, P <.05) and PBS (310.30 mm3, P <.01) groups. Highlights: NDV/Anh‐TRAIL exerts an obvious result in U251 glioma therapeutic effect in vitro and in vivo. Anhinga strain could be an effectual vector for tumor therapy. NDV/Anh‐TRAIL could become a potent candidate for clinical treatment especially for glioma. [ABSTRACT FROM AUTHOR]

Intestinal tumors mainly originate from transformed crypt stem cells supported by Wnt signaling, which functions through downstream critical factors enriched in the intestinal stem/progenitor compartment. Here, we show Uhrf2 is predominantly expressed in intestinal crypts and adenomas in mice and is transcriptionally regulated by Wnt signaling. Upregulated UHRF2 correlates with poor prognosis in colorectal cancer patients. Although loss of Uhrf2 did not affect intestinal homeostasis and regeneration, tumor initiation and progression were inhibited, leading to a markedly prolonged life span in Uhrf2 null mice on an ApcMin background. Uhrf2 deficiency also strongly reduced primary tumor organoid formation suggesting impairment of tumor stem cells. Moreover, ablation of Uhrf2 suppressed tumor cell proliferation through downregulation of the Wnt/β‐catenin pathway. Mechanistically, Uhrf2 directly interacts with and sumoylates Tcf4, a critical intranuclear effector of the Wnt pathway. Uhrf2 mediated SUMOylation stabilized Tcf4 and further sustained hyperactive Wnt signaling. Together, we demonstrate that Wnt‐induced Uhrf2 expression promotes tumorigenesis through modulation of the stability of Tcf4 for maintaining oncogenic Wnt/β‐catenin signaling. This is a new reciprocal feedforward regulation between Uhrf2 and Wnt signaling in tumor initiation and progression. What's new? UHRF2 has been shown to be enriched in intestinal stem cells and upregulated in colorectal cancer. However, the role of UHRF2 in intestinal homeostasis and tumorigenesis has not been explored in vivo. This study shows that Uhrf2 is expressed in mouse intestinal crypts and adenomas and is transcriptionally regulated by Wnt signaling. Uhrf2 further maintains oncogenic Wnt/β−catenin signaling through modulation of Tcf4 protein stability to enhance the stemness and tumorigenicity of intestinal progenitors. The findings reveal reciprocal feedforward regulation between Uhrf2 and Wnt signaling in tumor initiation and progression, and highlight Uhrf2 as a potential therapeutic target in colorectal cancer. [ABSTRACT FROM AUTHOR]

Natural product corynoline is a unique isoquinoline alkaloid extracted from traditional Chinese medicine Corydalis bungeana Turcz, whereas its anticancer properties have not been investigated. In this study, we found that corynoline potently impairs the growth of melanoma cells, B16F10, and A375 in a concentration-dependent manner. Treatment of melanoma cells with corynoline results in G2 cell arrest accompanied by reduced cdc2 activation. Furthermore, corynoline triggers apoptosis of melanoma cells, which is associated with increased expression of Bax and cleaved caspase-3. Mechanistic study indicates that corynoline strongly induces reactive oxygen species (ROS) generation and subsequent DNA damage as evidenced by γ-H2AX accumulation. Notably, the effect of corynoline on melanoma cell cycle and apoptosis is abolished by a ROS scavenger N-acetyl cysteine (NAC), indicating a ROS-dependent mechanism. Finally, corynoline significantly inhibits in vivo B16F10 melanoma tumor growth accompanied by reduced expression of Ki-67 in tumor tissue. Taken together, our data suggest that corynoline suppresses melanoma cell growth in vitro and in vivo by inducing oxidative stress and represents a potential therapeutic agent for melanoma patients. [ABSTRACT FROM AUTHOR]

Rapid metabolism differentiates cancer cells from normal cells and relies on anaplerotic pathways. However, the mechanisms of anaplerosis‐associated enzymes are rarely understood. The lack of potent and selective antimetabolism drugs restrains further clinical investigations. A small molecule ZY‐444 ((N4‐((5‐(4‐(benzyloxy)phenyl)‐2‐thiophenyl)methyl)‐N2‐isobutyl‐2,4‐pyrimidinediamine) is discovered to inhibit cancer cell proliferation specifically, having potent efficacies against tumor growth, metastasis, and recurrence. ZY‐444 binds to cellular pyruvate carboxylase (PC), a key anaplerotic enzyme of the tricarboxylic acid cycle, and inactivates its catalytic activity. PC inhibition suppresses breast cancer growth and metastasis through inhibiting the Wnt/β‐catenin/Snail signaling pathway. Lower PC expression in patient tumors is correlated with significant survival benefits. Comparative profiles of PC expression in cancer versus normal tissues implicate the tumor selectivity of ZY‐444. Overall, ZY‐444 holds promise therapeutically as an anti‐cancer metabolism agent. [ABSTRACT FROM AUTHOR]

Summary: Ethyl‐N‐dodecanoyl‐l‐arginate hydrochloride (LAE, ethyl lauroyl arginate HCl) is a cationic surfactant used as a food preservative with broad‐spectrum antibacterial activities. However, its resistance development, influences on gut microbiome and molecular target are unclear. In this study, bacteria were stimulated by LAE for 30 days to test the bacterial resistance. Several infected animal models were used to evaluate the antibacterial effect of LAE in vivo. Mice were orally treated with LAE to test its effect on animal growth. The influence of LAE on mice gut microbiome was analysed by 16S rDNA sequencing. The results indicated that Escherichia coli did not develop resistance to LAE. LAE significantly combats bacterial infection in mice, ducklings and piglets. Moreover, LAE promotes mouse weight gain without changing body composition or reducing animal vitality, and induces lower hepatotoxicity than ampicillin. In the mouse gut microbiome assessment and characterization, LAE modifies host gut microbiota structure. Mechanistically, LAE specifically binds to acidic phospholipids including phosphatidylserine, depolarizes the membrane and disrupts the bacterial membrane followed by bacterial growth inhibition. This study investigates the molecular mechanism of LAE as well as its antibacterial functions in poultry and livestock. Our data suggest LAE is a potential antibacterial agent in animal health. [ABSTRACT FROM AUTHOR]

The B‐cell CLL/lymphoma 6 (Bcl6) oncogenic repressor is a master regulator of humoral immunity and B‐cell lymphomagenesis. Although much research has focused on its regulation and function of GC B cells and T cells, the role of Bcl6 in regulating the functions of innate immunity is not well defined. Here, we demonstrated that EAE is exacerbated in LysM Cre+/−Bcl6fl/fl mice. Although other cells such as neutrophils might be involved in this conditional mutant mouse model, we found that the disease pathology is mainly associated with a biased M1 macrophage activity and an enhanced encephalitogenic CD4+ Th17 cell response. In addition, LPS‐induced sepsis mice exhibited an enhanced M1 and inhibited M2 response, further confirming that Bcl6 has an important role in regulating macrophage polarization. Mechanistically, Bcl6 interacts with IκBζ and interferes its binding to the interleukin‐6 (Il‐6) promoter in macrophages, leading to a suppressed transcription of Il‐6. These findings have demonstrated that Bcl6 exerts its regulatory function mainly by repressing Il‐6 expression in macrophages. Thus, our study presents a novel role for Bcl6 in regulating immune response and inflammation. Interaction between Bcl6 and IκBζ in macrophages may provide a potential therapeutic target for autoimmune inflammatory disease. [ABSTRACT FROM AUTHOR]

The key signaling networks regulating bone marrow mesenchymal stem cells (BMSCs) are poorly defined. Lgr4, which belongs to the leucine‐rich repeat‐containing G protein‐coupled receptor (LGR) family, is widely expressed in multiple tissues from early embryogenesis to adulthood. We investigated whether Lgr4 functions in BMSCs and in osteogenesis, adipogenesis, and skeletal myoblasts, using mice with a β‐geo gene trap inserted into the Lgr4 gene. Abundant Lgr4 expression was detected in skeletal, adipose and muscular tissue of Lgr4+/– mice at E16.5 by β‐gal staining, and Lgr4‐deficiency promoted BMSC proliferation (16 ± 4 in wild‐type [WT] and 28 ± 2 in Lgr4−/−) using colony forming units‐fibroblast assay, while suppressing BMSC migration (from 103 ± 18 in WT to 57 ± 10 in Lgr4−/−) by transwell migration assay and apoptosis ratio (from 0.0720 ± 0.0123 to 0.0189 ± 0.0051) by annexin V staining assay. Deletion of Lgr4 decreased bone mass (BV/TV from 19.16 ± 2.14 in WT mice to 10.36 ± 1.96 in KO) and fat mass through inhibiting BMSC differentiation to osteoblasts or adipocytes. Furthermore, LGR4‐regulated osteogenic, adipogenic, and myogenic gene expression. Importantly, our data showed that loss of Lgr4‐inhibited fracture healing by suppressing osteoblast differentiation. Moreover, deletion of Lgr4 in BMSCs‐delayed fracture healing following stem cell therapy by BMSC transplantation. Together, our results demonstrated that LGR4 is essential for mesoderm‐derived tissue development and BMSC differentiation, demonstrating that LGR4 could be a promising drug target for related diseases and a critical protein for stem cell therapy. Lgr4 promoted bone mesenchymal stem cell (BMSC) migration and apoptosis and inhibited BMSC proliferation. Lgr4 deficiency led to reduced bone mass and fat mass through inhibiting BMSCs differentiation to osteoblasts or adipocytes. Furthermore, Lgr4 deficiency impaired osteoblast mineralization and healing in bone fracture model, demonstrating that LGR4 could be a promising drug target for related diseases and a critical protein for stem cell therapy. [ABSTRACT FROM AUTHOR]

Background and Purpose: Intrinsic and/or acquired resistance of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) commonly occurs in patients with non-small-cell lung cancer (NSCLC). Here, we developed a combined therapy of histone deacetylase inhibition by a novel HDAC inhibitor, YF454A, with erlotinib to overcome EGFR-TKI resistance in NSCLC.Experimental Approach: The sensitization of the effects of erlotinib by YF454A was examined in a panel of EGFR-TKI-resistant NSCLC cell lines in vitro and two different erlotinib-resistant NSCLC xenograft mouse models in vivo. Western blotting and Affymetrix GeneChip expression analysis were further performed to determine the underlying mechanisms for the effects of the combination of erlotinib and YF454A.Key Results: YF454A and erlotinib showed a strong synergy in the suppression of cell growth by blocking the cell cycle and triggering cell apoptosis in EGFR-TKI-resistant NSCLC cells. The combined treatment led to a significant decrease in tumour growth and tumour weight compared with single agents alone. Mechanistically, this combination therapy dramatically down-regulated the expression of several crucial EGFR-TKI resistance-related receptor tyrosine kinases, such as Her2, c-Met, IGF1R and AXL, at both the transcriptional and protein levels and consequently blocked the activation of downstream molecules Akt and ERK. Transcriptomic profiling analysis further revealed that YF454A and erlotinib synergistically suppressed the cell cycle pathway and decreased the transcription of cell-cycle related genes, such as MSH6 and MCM7.Conclusion and Implications: Our preclinical study of YF454A provides a rationale for combining erlotinib with a histone deacetylase inhibitor to treat NSCLC with EGFR-TKI resistance. [ABSTRACT FROM AUTHOR]

As the most important host defence against viral infection, interferon ( IFN) stimulates hundreds of antiviral genes ( ISGs) that together establish an 'antiviral state'. However, the antiviral function of most ISGs in viral infection still need further exploration. Here, we demonstrated that the expression of G-protein-coupled receptor 146 ( GPR146) is highly increased by both IFN- β and IFN- γ in a signal transducer and activator of transcription 1-dependent signalling pathway. Most importantly, overexpression of GPR146 protects the host cells from vesicular stomatitis virus and Newcastle disease virus infection but not from infection by herpes simplex virus. In contrast, the virus-induced IFN- β production changed little in Gpr146-knockout cells. Furthermore, the Gpr146-deficient mice showed similar susceptibility to wild-type mice with vesicular stomatitis virus infection. Interestingly, the expression of GPR146 in virus-infected cells was strikingly reduced and can partially explain why the viral infection was little influenced in Gpr146-knockout mice. Surprisingly, virus-activated IFN regulatory factor 3 ( IRF3) signalling not only induces the expression of IFN but also represses GPR146 expression through HES1 (hairy and enhancer of split-1)-mediated transcriptional activity to establish a dynamic equilibrium between pro-viral and antiviral stages in host cells. Taken together, these data reveal the antiviral role of GPR146 in fighting viral infection although the GPR146-mediated protection is eliminated by IRF3/ HES1-signalling, which suggests a potential therapeutic significance of both GPR146 and HES1 signalling in viral infection. [ABSTRACT FROM AUTHOR]

The clearance of oxidative stress compounds is critical for the protection of the organism from malignancy, but how this key physiological process is regulated is not fully understood. Here, we found that the expression of GPRC5A, a well-characterized tumor suppressor in lung cancer, was elevated in colorectal cancer tissues in patients. In both cancer cell lines and a colitis-associated cancer model in mice, we found that GPRC5A deficiency reduced cell proliferation and increased cell apoptosis as well as inhibited tumorigenesis in vivo. Through RNA-Seq transcriptome analysis, we identified oxidative stress associated pathways were dysregulated. Moreover, in GPRC5A deficient cells and mouse tissues, the oxidative agents were reduced partially due to increased glutathione (GSH) level. Mechanistically, GPRC5A regulates NF-κB mediated Vanin-1 expression which is the predominant enzyme for cysteamine generation. Administration of cystamine (the disulfide form of cysteamine) in GPRC5A deficient cell lines inhibited γ-GCS activity, leading to reduction of GSH level and increase of cell growth. Taken together, our studies suggest that GPRC5a is a potential biomarker for colon cancer and promotes tumorigenesis through stimulation of Vanin-1 expression and oxidative stress in colitis associated cancer. This study revealed an unexpected oncogenic role of GPRC5A in colorectal cancer suggesting there are complicated functional and molecular mechanism differences of this gene in distinct tissues. [ABSTRACT FROM AUTHOR]

P-glycoprotein (P-gp), an important efflux transporter in intestine, regulates the bioavailability of orally taken drugs. To develop an in vitro model that preferably mimics the physiological microenvironment of human intestine, we employed the three-dimensionally (3D) cultured organoids from human normal small intestinal epithelium. It was observed that the intestinal crypts could efficiently form cystic organoid structure with the extension of culture time. Furthermore, the physiological expression of ABCB1 was detected at both mRNA and protein levels in cultured organoids. Rhodamine 123 (Rh123), a typical substrate of P-gp, was actively transported across 3D organoids and accumulated in the luminal space. This transport process was also inhibited by verapamil and mitotane. In summary, the above-mentioned model based on human small intestinal 3D organoids is suitable to imitate the small intestinal epithelium and could be used as a novel in vitro model especially for P-gp inhibitor screening. [ABSTRACT FROM AUTHOR]

A photo fuel cell (PFC) offers an attractive way to simultaneously convert solar and biomass energy into electricity. Photocatalytic biomass oxidation on a semiconductor photoanode combined with dark electrochemical reduction of oxygen molecules on a metal cathode (usually Pt) in separated compartments is the common configuration for a PFC. Herein, we report a membrane-free PFC based on a dual electrode, including a W-doped BiVO4 photoanode and polyterthiophene photocathode for solar-stimulated biomass-to-electricity conversion. Air- and water-soluble biomass derivatives can be directly used as reagents. The optimal device yields an open-circuit voltage ( VOC) of 0.62 V, a short-circuit current density ( JSC) of 775 μA cm−2, and a maximum power density ( Pmax) of 82 μW cm−2 with glucose as the feedstock under tandem illumination, which outperforms dual-photoelectrode PFCs previously reported. Neither costly separating membranes nor Pt-based catalysts are required in the proposed PFC architecture. Our work may inspire rational device designs for cost-effective electricity generation from renewable resources. [ABSTRACT FROM AUTHOR]

P-glycoprotein (P-gp), as the most important efflux transporter in intestines, plays the key role to determine the bioavailability of many drugs. The three-dimensional (3D) organoid model is suitable to imitate small intestinal epithelium. In this study, a rapid, sensitive and efficient method to measure rhodamine 123 (Rh123, P-gp substrate) in 3D organoids was developed to analyse P-gp-mediated drug transport. Ultrasonic cell disruptor was used to smash the organoid, and automatic microplate reader was used for detecting the concentration of Rh123 (λex/λem = 485/520 nm). Moreover, verapamil, quinidine and mitotane were used to make validation about this newly developed approach. All three P-gp inhibitors significantly inhibited the transport of Rh123 into 3D organoids. Therefore, the above-mentioned method could serve as a new model for P-gp inhibitor screening in a high-throughput way. [ABSTRACT FROM AUTHOR]

The X-linked genetic bleeding disorder caused by deficiency of coagulator factor IX, hemophilia B, is a disease ideally suited for gene therapy with genome editing technology. Here, we identify a family with hemophilia B carrying a novel mutation, Y371D, in the human F9 gene. The CRISPR/Cas9 system was used to generate distinct genetically modified mouse models and confirmed that the novel Y371D mutation resulted in a more severe hemophilia B phenotype than the previously identified Y371S mutation. To develop therapeutic strategies targeting this mutation, we subsequently compared naked DNA constructs versus adenoviral vectors to deliver Cas9 components targeting the F9 Y371D mutation in adult mice. After treatment, hemophilia B mice receiving naked DNA constructs exhibited correction of over 0.56% of F9 alleles in hepatocytes, which was sufficient to restore hemostasis. In contrast, the adenoviral delivery system resulted in a higher corrective efficiency but no therapeutic effects due to severe hepatic toxicity. Our studies suggest that CRISPR/Cas-mediated in situ genome editing could be a feasible therapeutic strategy for human hereditary diseases, although an efficient and clinically relevant delivery system is required for further clinical studies. [ABSTRACT FROM AUTHOR]

Background and Purpose Compounds targeting epigenetic events of tumours are likely to be an important addition to anticancer therapy. Histone deacetylase inhibitors ( HDACI) have emerged as a promising novel class for therapeutic interventions associated with cancer, and many of them are currently in clinical investigation. Here, we assessed a novel hydroxamate-based HDACI, LW479, in breast cancer progression and explored its underlying mechanism(s). Experimental Approach LW479 was identified using the HDACI screening kit. Western blot and flow cytometry were used to analyse the biological effects of LW479 as a novel HDACI. The effects of LW479 were assessed in mouse models of spontaneous and experimental breast cancer. Co-immunoprecipitation, immunofluorescent staining and chromatin immunoprecipitation assays along with immunohistochemical analysis, were used to elucidate the molecular basis of the actions of LW479. Key Results LW479 was identified as a novel HDACI and showed marked cytotoxicity and induced apoptosis, as well as cell cycle arrest, in a panel of breast cancer cell lines. Intraperitoneal injections of LW479 markedly suppressed breast tumour growth and pulmonary metastasis in nude mice. LW479 also decreased levels of EGF receptors ( EGFR) by blocking the binding of the transcription factor Sp1 and HDAC1 to the EGFR promoter region. Conclusions and Implications Our data have elucidated the mechanisms underlying the inhibition by LW479 of tumour growth and metastasis, in models of breast cancer with aberrant EGFR expression. LW479 could be a candidate drug for breast cancer prevention. [ABSTRACT FROM AUTHOR]

This study firstly investigated the effects of cucurbitacin E (CuE), a tetracyclic triterpene compound from Cucurbitaceae, on the pharmacokinetics ( PK) and pharmacodynamics ( PD) of warfarin, a model CYP2C probe substrate, in the rat. In PK studies, the concentration of warfarin in blood samples was determined by HPLC- DAD, and the PK parameters were analysed using non-compartmental methods. In PD studies, the prothrombin time ( PT) in blood plasma at each sample point was measured via thromboplastin reagents. CuE treatment (50, 100 and 200 μg/kg, i.p.) decreased warfarin clearance (28-32%), increased the area under the curve ( AUC0-∞; 55-62%) and prolonged plasma half-life ( t1/2; 58-72%). At the same time, the anticoagulation effect of warfarin ( PTmax) was also significantly increased in the presence of CuE. These data demonstrated that CuE affected the PK and PD of warfarin, and these effects may be due to the inhibition of CYP2C activity by CuE. Hence, careful monitoring should be carried out during concomitant use of herbal products containing CuE with drugs that are metabolized by CYP2C enzymes. [ABSTRACT FROM AUTHOR]

Cell migration and invasion are key processes in the metastasis of cancer, and suppression of these steps is a promising strategy for cancer therapeutics. The aim of this study was to explore small molecules for treating colorectal cancer ( CRC) and to investigate their anti-metastatic mechanisms. In this study, six CRC cell lines were used. We showed that YH-306 significantly inhibited the migration and invasion of CRC cells in a dose-dependent manner. In addition, YH-306 inhibited cell adhesion and protrusion formation of HCT116 and HT-29 CRC cells. Moreover, YH-306 potently suppressed uninhibited proliferation in all six CRC cell lines tested and induced cell apoptosis in four cell lines. Furthermore, YH-306 inhibited CRC colonization in vitro and suppressed CRC growth in a xenograft mouse model, as well as hepatic/pulmonary metastasis in vivo. YH-306 suppressed the activation of focal adhesion kinase ( FAK), c-Src, paxillin, and phosphatidylinositol 3-kinases ( PI3K), Rac1 and the expression of matrix metalloproteases ( MMP) 2 and MMP9. Meanwhile, YH-306 also inhibited actin-related protein (Arp2/3) complex-mediated actin polymerization. Taken together, YH-306 is a candidate drug in preventing growth and metastasis of CRC by modulating FAK signalling pathway. [ABSTRACT FROM AUTHOR]

The class of adhesion G protein-coupled receptors (aGPCRs), with 33 human homologs, is the second largest family of GPCRs. In addition to a seven-transmembrane α-helix-a structural feature of all GPCRs-the class of aGPCRs is characterized by the presence of a large N-terminal extracellular region. In addition, all aGPCRs but one (GPR123) contain a GPCR autoproteolysis-inducing (GAIN) domain that mediates autoproteolytic cleavage at the GPCR autoproteolysis site motif to generate N- and a C-terminal fragments (NTF and CTF, respectively) during protein maturation. Subsequently, the NTF and CTF are associated noncovalently as a heterodimer at the plasma membrane. While the biological function of the GAIN domain-mediated autocleavage is not fully understood, mounting evidence suggests that the NTF and CTF possess distinct biological activities in addition to their function as a receptor unit. We discuss recent advances in understanding the biological functions, signaling mechanisms, and disease associations of the aGPCRs. [ABSTRACT FROM AUTHOR]

By tuning the chemical modalities on gold nanoparticle surface using hexapeptide ligands the responses of immune cells (dendritic cells) to nanoparticles can be remarkably manipulated from minimum reaction (in the presence of negatively charged peptide ligands) to activation (in the presence of peptide ligands with aromatic rings containing amino acids). [ABSTRACT FROM AUTHOR]

Podosomes are adhesive structures on the ventral surface of cells that invade and degrade the extracellular matrix. Recently, we reported that phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator, induced podosome formation in normal human bronchial epithelial (NHBE) cells, and atypical PKCζ regulated MMP-9 recruitment to podosomes for its release and activation. The objective of this study was to explore signaling pathways that are involved in PKC activation-induced podosome formation and matrix degradation. Herein, we found that PDBu increased phosphorylation of PI3K p85, Akt, Src, ERK1/2, and JNK. Inhibitors for PI3K, Akt, and Src suppressed PDBu-induced podosome formation and matrix degradation. In contrast, blockers for MEK/ERK or JNK did not inhibit podosome formation but reduced proteolytic activity of podosomes. Inhibition of PKCζ activity with its pseudosubstrate peptide (PS)-inhibited PDBu-induced phosphorylation of MEK/ERK and JNK. On the other hand, inhibition of MEK/ERK or JNK pathway did not affect PKCζ phosphorylation, but reduced the recruitment of PKCζ and MMP-9 to podosomes. We conclude that PKCζ may regulate MEK/ERK and JNK phosphorylation and in turn activated MEK/ERK and JNK may regulate the proteolytic activity of PDBu-induced podosomes by influencing the recruitment of PKCζ and MMP-9 to podosomes. J. Cell. Physiol. 228: 416-427, 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

In this study, we expanded regulatory T cells (Tregs) ex vivo from CD4+ CD25+ T cells from cord blood (CB) and CD4+ CD25+ CD127− T cells from adult peripheral blood (APB) and compared the suppressive functions of the newly generated Tregs. The Tregs from CB and APB were expanded either in two cycles with a polyclonal stimulus or in two cycles with an alloantigen stimulus in the first cycle and a polyclonal stimulus in the second cycle. Cell yield after Treg expansion with polyclonal stimulation was greater than that of Tregs expanded with combined alloantigen and polyclonal stimulation. The expanded Tregs expressed high levels of Foxp3, CD39 and cytotoxic T-lymphocyte antigen-4 and low levels of CD127, interleukin-2 and interferon-γ. After two cycles of expansion, the CB Tregs maintained expression of the GARP gene and showed greater suppressive function than APB Tregs. The CB Tregs that were expanded with two cycles of polyclonal stimulation suppressed not only the polyclonal antigen-driven responder T (Tresp) cell proliferation but also the HLA mismatched dendritic cell-driven Tresp cell proliferation. When CB and APB Tregs were expanded with a primary alloantigen stimulus followed by a secondary polyclonal stimulus, the Tregs showed a potent, antigen-specific suppressive capacity. The Tregs expanded with two cycles of polyclonal stimulation from both CB and APB alleviated acute graft-versus-host disease symptoms and prolonged survival in a murine model of graft-versus-host disease. In conclusion, CB Tregs expanded with two cycles of polyclonal stimulation had a stronger immunosuppressive function than APB Tregs. It is feasible to obtain human functional alloantigen-specific Tregs expanded ex vivo from CB and APB in large numbers. [ABSTRACT FROM AUTHOR]

BACKGROUND: Human cord blood (CB) is a superior source of regulatory T cells (Tregs) compared with peripheral blood. Initial studies have shown that CB-derived Tregs can be effectively expanded ex vivo. However, in vitro suppressor activity of expanded CB-Tregs and their efficacy in the prevention of acute graft-versus-host disease (aGVHD) in vivo are poorly understood. STUDY DESIGN AND METHODS: In vitro, human CB CD4+CD25+ T cells expanded with anti-CD3/CD28 beads plus interleukin (IL)-2 and the phenotypes, expression of cytokines, and suppression of expanded cells were analyzed after two cycles of stimulation. In vivo, the addition of human CB-Tregs was transferred in the major histocompatibility complex-mismatched aGVHD mouse model. Survival, body weight, GVHD scoring, histopathologic specimens, serum cytokines, and Th subsets were analyzed in CB-Treg-treated mice and untreated controls. RESULTS: After being expanded ex vivo, human CB-derived Tregs with potent suppressor function could meet clinical demands. Up to 85% of mice with CB-Tregs treatment survived beyond Day 63 after bone marrow transplantation; however, all aGVHD mice died within 18 days. In the serum of the CB-Treg-treated mice, the production of transforming growth factor-β increased continuously, as opposed to IL-17, which decreased quickly. Consistent with the changes of cytokines, the percentage of mouse CD4+ forkhead box protein 3+ Tregs increased while that of Th17 cells decreased. CONCLUSION: Ex vivo expanded human CB-Tregs significantly prevented allogeneic aGVHD in vivo by modulating various cytokine secretion and polarizing the Treg/Th17 balance toward Treg, which suggests the potential use of expanded CB-Tregs as a therapeutic approach for GVHD. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE Angiogenesis-based therapy is an effective anti-tumour strategy and previous reports have shown some beneficial effects of a naturally occurring bioactive compound plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone). Here, we sought to determine the biological effects of plumbagin on signalling mechanisms during tumour angiogenesis. EXPERIMENTAL APPROACH The effects of plumbagin were evaluated in various in vitro assays which utilised human umbilical vein endothelial cells (HUVEC) proliferation, migration and tube formation. Plumbagin was also evaluated in vivo using chicken embryo chorioallantoic membrane (CAM) and mouse corneal micropocket models., Human colon carcinoma and prostate cancer xenograft mouse models were used to evaluate the effects of plumbagin on angiogenesis. Immunofluorescence, GST pull-down and Western blotting were employed to explore the underlying mechanisms of VEGF receptor (VEGFR)2-mediated Ras signalling pathways. KEY RESULTS Plumbagin not only inhibited endothelial cell proliferation, migration and tube formation but also suppressed chicken chorioallantoic membrane neovascularzation and VEGF-induced mouse corneal angiogenesis. Moreover, plumbagin suppressed tumour angiogenesis and tumour growth in human colon carcinoma and prostate cancer xenograft mouse models. At a molecular level, plumbagin blocked the Ras/Rac/cofilin and Ras/MEK signalling pathways mediated by VEGFR2 in HUVECs. CONCLUSIONS AND IMPLICATIONS Plumbagin inhibited tumour angiogenesis and tumour growth by interference with the VEGFR2-mediated Ras signalling pathway in endothelial cells. Our findings demonstrate a molecular basis for the effects of plumbagin and suggest that this compound might have therapeutic ant-tumour effects. [ABSTRACT FROM AUTHOR]

Loss of WASF3 function in breast cancer cells results in loss of invasion phenotypes and reduced metastatic potential. By using oligonucleotide arrays, we now demonstrate that knockdown of WASF3 leads to the upregulation of the KISS1 metastasis suppressor gene with concomitant reduced invasion and loss of matrix metalloproteinases (MMP)-9 activity. Using a luciferase reporter, KISS1 transcription is significantly increased in the absence of WASF3. Knockdown of KISS1 in WASF3-silenced cells resulted in the recovery of the invasion phenotype. WASF3 knockdown also resulted in elevated IκBα levels in the cytoplasm and reduced levels of nuclear factor-kappa-B (NF-κB) p65/50 subunits in the nucleus. Tumor necrosis factor-alpha (TNF-α) has been associated with cell invasion through induction of MMP-9 production via KISS1 regulation of the NF-κB pathway. When WASF3 knockdown cells are treated with TNF-α, no effect is seen on invasion or nuclear translocation of NF-κB. Thus, coordinated expression patterns of the WASF3 metastasis promoter gene and the KISS1 metastasis suppressor gene appear to exert their influence through inhibition of NF-κB signaling, which in turn regulates MMP-9 production facilitating invasion. [ABSTRACT FROM AUTHOR]

Tumor angiogenesis is one of the hallmarks of the development in malignant neoplasias and metastasis. Many angiogenesis inhibitors are small molecules from natural products. Indirubin, the active component of a traditional Chinese herbal medicine, Banlangen, has been shown to exhibit antitumor and anti-inflammation effects. But its roles in tumor angiogenesis, the key step involved in tumor growth and metastasis, and the involved molecular mechanism is unknown. Here, we identified that indirubin inhibited prostate tumor growth through inhibiting tumor angiogenesis. Using chick chorioallantoic membrane (CAM) assay and mouse corneal model, we found that indirubin inhibited angiogenesis in vivo. We also showed the inhibition activity of indirubin in endothelial cell migration, tube formation and cell survival in vitro. Furthermore, indirubin suppressed vascular endothelial growth factor receptor 2-mediated Janus kinase (JAK)/STAT3 signaling pathway but had little effects on the activity of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase in endothelial cell. Our study provided the first evidence for antitumor angiogenesis activity of indirubin and the related molecular mechanism. Our investigations suggested that indirubin was a potential drug candidate for angiogenesis related diseases. [ABSTRACT FROM AUTHOR]

Osteoclastogenesis is associated with aging and various age-related inflammatory chronic diseases, including cancer. Receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL), a member of the tumor necrosis factor superfamily, has been implicated as a major mediator of bone resorption, suggesting that agents that can suppress RANKL signaling might inhibit osteoclastogenesis, a process closely linked to bone resorption. We therefore investigated whether butein, a tetrahydroxychalcone, could inhibit RANKL signaling and suppress osteoclastogenesis induced by RANKL or tumor cells. We found that human multiple myeloma cells (MM.1S and U266), breast tumor cells (MDA-MB-231) and prostate tumor cells (PC-3) induced differentiation of macrophages to osteoclasts, as indicated by tartrate-resistant acid phosphatase (TRAP)-positive cells, and that butein suppressed this process. The chalcone also suppressed the expression of RANKL by the tumor cells. We further found that butein suppressed RANKL-induced NF-κB activation and that this suppression correlated with the inhibition of IκBα kinase and suppression of phosphorylation and degradation of IκBα, an inhibitor of NF-κB. Finally, butein also suppressed the RANKL-induced differentiation of macrophages to osteoclasts in a dose-dependent and time-dependent manner. Collectively, our results indicate that butein suppresses the osteoclastogenesis induced by tumor cells and by RANKL, by suppression of the NF-κB activation pathway. [ABSTRACT FROM AUTHOR]

Abstract: AFAP is an adaptor protein involved in cytoskeletal organization and intracellular signaling. AFAP binds and activates c-Src; however, the downstream signals of this interaction remain unknown. Here we show that co-expression of AFAP and c-Src induce transcriptional activation of SRE and AP-1 in a c-Src activity dependent fashion. Structural-functional studies suggest that the proline-rich motif in the N-terminus of AFAP is critical for c-Src activation, and subsequent SRE/AP-1 transactivation and the actin-binding domain in the AFAP C-terminus is negatively involved in the regulation of AFAP/c-Src mediated SRE/AP-1 transactivation. Selective deletion of this domain enhances transactivation of SRE. We conclude that in addition to its role in the regulation of cytoskeletal structures, AFAP may also be involved in the c-Src related transcriptional activities. Structured summary: Src physically interacts with AFAP by anti bait coimmunoprecipitation (View interaction) [Copyright &y& Elsevier]

Hispidulin, an active component from Artemisia vestita, a traditional Tibetan medicinal plant, has been shown to possess anti-inflammatory and anti-oxidative activities. However, the functional role of hispidulin on tumor growth and angiogenesis has not been elucidated. We found that hispidulin significantly inhibited human pancreatic tumor growth in xenograft mice when s.c. treated at a dosage of 20 mg/kg daily, and this effect was accompanied with a potent inhibition on angiogenesis. When examining the cytotoxicity of hispidulin on HUVECs and pancreatic cancer cells in vitro, we found that HUVECs were more susceptible to the treatment, suggesting angiogenesis might be the primary target of hispidulin. Our results further showed that hispidulin inhibited vascular endothelial growth factor (VEGF)-induced cell migration, invasion, and capillary-like structure formation of HUVECs in a dose-dependent manner. In ex vivo and in vivo angiogenesis assays, we showed that hispidulin suppressed VEGF-induced microvessel sprouting of rat aortic rings and corneal neovascularization in C57/BL6 mice . To understand the underlying molecular basis, we next examined the effects of hispidulin on different molecular components in treated HUVECs, and found that hispidulin suppressed the VEGF-triggered activation of VEGF receptor 2, PI3K, Akt, mTOR, and ribosomal protein S6 kinase, but had little effect on focal adhesion kinase or extracellular signal-regulated kinase at an effective concentration. Taken together, our results indicate that hispidulin targets the VEGF receptor 2-mediated PI3K/Akt/mTOR signaling pathway in endothelial cells, leading to the suppression of pancreatic tumor growth and angiogenesis. ( Cancer Sci 2011; 102: 219-225) [ABSTRACT FROM AUTHOR]

Abstract: GPR48 can mediate keratinocyte proliferation and migration. Our investigations showed that AG1478, an inhibitor of EGFR tyrosine kinase, could block GPR48-mediated cellular processes. AG1478 treatment of Gpr48 +/+ cells also decreased phosphorylation of EGFR, ERK and STAT3. Subsequent screening using conditioned media immunodepleted of EGFR ligands identified HB-EGF as the ligand responsible for phosphorylation of EGFR, ERK and STAT3. HB-EGF was reduced in Gpr48 −/− cell culture medium, but its addition restored the phosphorylation of EGFR, ERK, STAT3, as well as cell proliferation. Confirmation that GPR48 mediates EGFR signaling pathway through HB-EGF was subsequently performed using an inhibitor of HB-EGF. [ABSTRACT FROM AUTHOR]

Isoalvaxanthone (IAX) is a bioactive xanthone isolated from Cudrania cochinchinensis (Lour.). However, the function and mechanism of this compound in cancer migration and invasion have not been elucidated to date. In this study, we found that IAX could suppress various steps of tumor metastasis including proliferation, migration and invasion in a dose-dependent manner on colorectal cancer cells. Especially matrix metalloproteinase 2 (MMP-2), the pivotal factor in cancer invasion, was suppressed both on activation and expression after treated with IAX. To understand the underlying mechanism of IAX on the inhibitory effect of proliferation, migration and invasion, we demonstrated that IAX could significantly inhibit the activation of Rac1 but has undetectable effect on GTP-RhoA, GTP-Cdc42 and the phosphorylation of ERK1/2, p38 MAPK and JNK. Moreover, IAX showed little influence on the transcriptional activity of nuclear transcription factor κB (NF-κB) but strongly inhibited that of activator protein-1 (AP-1), which is the downstream transcriptional factor of Rac1. Together, our results indicate that IAX exerts anticancer effect in SW620 cells by targeting MMP-2 via regulating the activity of Rac1 and AP-1. These results are the first to reveal the function of IAX in tumor metastasis and its underlying molecular mechanism, thus suggest IAX to be a promising antimetastatic agent. [ABSTRACT FROM AUTHOR]

Spreading and migration of the basal cells neighboring a wound is essential for airway epithelial repair. To gain insight into the molecular mechanisms that govern these cellular processes, we asked whether normal human airway epithelial cells can form podosomes, a cellular structure discovered from cancer and mesenchymal cells that controls migration and invasion. Herein, we report that phorbol-12, 13-dibutyrate (PDBu), a protein kinase C activator, induced reorganization of cytoskeletal structure in primary normal human bronchial epithelial cells, and in normal human airway epithelial BEAS2B cells. Z-stack scanning confocal microscopy showed that PDBu-induced podosome-like structures contain actin-rich columns that arise from the ventral surface of the cell, and also revealed the presence of circular ruffles/waves at the dorsal cell surface. The molecular components of these cytoskeletal structures were determined with immunofluorescent staining. Using in situ zymography, we demonstrated that PDBu-induced podosomes were capable of degrading fibronectin-gelatin-sucrose matrix. PDBu also increased epithelial cell invasion across Transwell chamber. Podosomes and circular dorsal ruffles may be important for epithelial cell migration and invasion, thus contributing to respiratory epithelial repair and regeneration. J. Cell. Physiol. 218: 366–375, 2009. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The G-protein coupled receptors (GPCRs) recognize a large variety of extracellular molecules (such as hormones, neurotransmitters, growth and developmental factors) and several sensory messages (such as light, odors and pain). GPCRs and their signal transduction pathway represent important specific targets for a variety of human diseases. To investigate the potential roles of GPCRs in human normal prostate and prostate cancers, we identified and characterized a novel human G-protein coupled receptor, PSGR2, which is highly overexpressed in human prostate cancers. Although PSGR2 shares sequence homology with human olfactory G-protein coupled receptors, the expression of PSGR2 is highly restricted to human prostate tissue, and no expression was detected in 22 normal and 10 tumor tissues examined using Northern blot and PCR analysis. Furthermore, we investigated the expression levels of PSGR2 in 133 human prostate samples with real-time quantitative reverse transcription-PCR and in situ hybridization method. We demonstrated that PSGR2 expression increased significantly in human high grade prostate intraepithelial neoplasia (PIN) and prostate cancers (approximately 10-fold) as compared to normal and BPH (benign prostatic hyperplasia) tissues ( p < 0.001), suggesting PSGR2 may play an important role in human prostate cancer development and progression. Together, our results suggest that PSGR2 is a novel prostate specific G-protein coupled receptor and may be useful as a tissue marker and molecular target for the early detection and treatment of human prostate cancers. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Cdx-2 is a transactivator for the proglucagon gene in pancreatic and intestinal endocrine cells. Cdx-2 is also expressed in differentiated intestinal epithelia of nonendocrine origin. Cdx-2–/– mice are embryonic lethal, while Cdx-2+/– mutants show multiple malfunctions including the formation of intestinal polyps. Within the polyps, the remaining wild type Cdx-2 allele ceases its expression, while the expression of both Cdx-2 and proglucagon in the endocrine cells remains unaltered, indicating that Cdx-2 could be haplo-insufficient for nonendocrine cells, but not for proglucagon producing endocrine cells. We propose that mechanisms underlying Cdx-2 expression and auto-regulation[Xu F, Li H&Jin T (1999),J Biol Chem274,34310–34316] differ in these two types of cells. We show here that forskolin and cAMP upregulate Cdx-2 expression in proglucagon producing cells, but not in colon cancer cells and primary intestinal cell cultures. It is unlikely that the activation is mainly mediated by PKA, because the activation was observed in a PKA deficient cell line. Cotransfecting a dominant negative Ras expression plasmid substantially repressed the Cdx-2 promoter, in contrast to a previous finding that Ras is a negative factor for Cdx-2 expression in colon cancer cells. Furthermore, forskolin activated ERK1/2 phosphorylation in the endocrine cells, and attenuation of ERK1/2 phosphorylation by its inhibitor is associated with attenuated Cdx-2 expression. Finally, an Epac pathway specific cAMP analogue stimulated both ERK1/2 phosphorylation and Cdx-2 expression. Taken together, our observations suggest that Cdx-2 expression is regulated by the second messenger cAMP, cell-type specifically, via the Epac pathway. [ABSTRACT FROM AUTHOR]

The G-protein-coupled receptors and signal transduction pathways represent important specific targets for a variety of human diseases, ranging from the control of blood pressure, allergic response, hormonal disorders and neurologic diseases to tumorigenesis. Most recently, we and others have identified a novel human prostate-specific G-protein coupled receptor (PSGR). To investigate the potential roles of PSGR in human normal prostate and prostate cancers, we examined the expression level of PSGR in 146 human prostate samples with real-time quantitative reverse transcription-PCR and in situ hybridization method. We significantly extended previous studies and demonstrated that PSGR is specifically expressed in human prostate tissues, not in any other normal and tumor samples tested. Compared to normal and benign prostatic hyperplasia tissues, the expression of PSGR increased significantly in human prostate intraepithelial neoplasia (PIN) and prostate tumors (approximately 10-fold), especially in early prostate tumors, suggesting PSGR may play an important role in early prostate cancer development and progression. The sensitivity and specificity estimates for PSGR expression were calculated as the area under the receiver-operating characteristics curve (0.902), indicating high-level sensitivity and specificity for discriminating benign prostate tissues from malignant prostate tissues. The association of PSGR expression with clinical parameters (clinical stages, Gleason scores, recurrent status and metastasis) was also investigated in this study. Our data suggest that overexpression of PSGR in human PIN and prostate cancers have the potential for early prostate cancer detection and diagnosis. © 2004 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract: The Rho-family of small GTPases consists of essential regulators of neurite outgrowth, axonal pathfinding, and dendritic arborization. Previous work has demonstrated in non-neuronal cell types that Smurf1, an E3 ubiquitin ligase, regulates cell polarity and protrusive activity via PKCζ-dependent recruitment to cellular protrusion sites, and subsequent ubiquitination and proteasomal degradation of RhoA. In this study, we show that Smurf1 enhances neurite outgrowth in Neuro2a neuroblastoma cells. We demonstrate that RhoA is ubiquitinated, and that Smurf1 and RhoA physically interact in vivo. Interestingly, Smurf1 overexpression in Neuro2a cells dramatically reduces RhoA protein levels during dibutyric cyclic AMP, but not retinoic acid induced neurite outgrowth. This Smurf1-dependent reduction in RhoA protein levels was abrogated using the general proteasome inhibitor MG132, suggesting that RhoA is targeted for ubiquitination and degradation via Smurf1. Together, our data suggest that localized regulation of different subsets of Rho GTPases by specific guidance signals results in an intracellular asymmetry of RhoA activity, which could regulate neurite outgrowth and guidance. [Copyright &y& Elsevier]

Src kinase regulation of N‐methyl‐D‐aspartate (NMDA) subtype glutamate receptors in the central nervous system (CNS) has been found to play an important role in processes related to learning and memory, ethanol sensitivity and epilepsy. However, little is known regarding the mechanisms underlying the regulation of Src family kinase activity in the control of NMDA receptors. Here we report that the distal phosphatase domain (D2) of protein tyrosine phosphatase α (PTPα) binds to the PDZ2 domain of post‐synaptic density 95 (PSD95). Thus, Src kinase, its activator (PTPα) and substrate (NMDA receptors) are linked by the same scaffold protein, PSD95. Removal of PTPα does not affect the association of Src with NMDA receptors, but turns off the constitutive regulation of NMDA receptors by the kinase. Further more, we found that application of the PTPα catalytic domains (D1 + D2) into neurones enhances NMDA receptor‐mediated synaptic responses. Conversely, the blockade of endogenous PTPα inhibits NMDA receptor activity and the induction of long‐term potentiation in hippocampal neurones. Thus, PTPα is a novel up‐regulator of synaptic strength in the CNS. [ABSTRACT FROM AUTHOR]

Src kinase regulation of N-methyl-D-aspartate (NMDA) subtype glutamate receptors in the central nervous system (CNS) has been found to play an important role in processes related to learning and memory, ethanol sensitivity and epilepsy. However, little is known regarding the mechanisms underlying the regulation of Src family kinase activity in the control of NMDA receptors. Here we report that the distal phosphatase domain (D2) of protein tyrosine phosphatase a (PTPα) binds to the PDZ2 domain of post-synaptic density 95 (PSD95). Thus, Src kinase, its activator (PTPα) and substrate (NMDA receptors) are linked by the same scaffold protein, PSD95. Removal of PTPα does not affect the association of Src with NMDA receptors, but turns off the constitutive regulation of NMDA receptors by the kinase. Further- more, we found that application of the PTPα catalytic domains (D1 + D2) into neurones enhances NMDA receptor-mediated synaptic responses. Conversely, the blockade of endogenous PTPα inhibits NMDA receptor activity and the induction of tong-term potentiation in hippocampal neurones. Thus, PTPα is a novel up-regulator of synaptic strength in the CNS. [ABSTRACT FROM AUTHOR]