I2 Catalyzed and TBHP/Ammonium-Promoted Conversion of Arylethanone to Nitriles via ß-Scission of Iminyl Radicals.

Herein, we report a general I2-catalyzed and TBHP/ammonium-promoted conversion of arylethanone to aromatic nitriles under air. This procedure proceeded with the ß-scission of iminyl radical, which was facilitated via quenching the released alkyl radical by tert-butyl peroxyl radical leading to peroxide followed with Kornblum-DeLaMare rearrangement. A series of aryl methyl ketone and alkyl aryl ketone worked well with good functional group tolerance in high yields. As such, this metal-free procedure represents a facile, safe, green, and practical procedure in conversion of arylethanone to aromatic nitriles.

Pyridine-catalyzed ring-opening reaction of cyclopropenone with bromomethyl carbonyl compounds toward furan-2(5H)-ones.

We developed a pyridine-catalyzed annulation of diaryl cyclopropenone with bromomethyl carbonyl compounds leading to 5-carbonyl furan-2(5H)-ones. Pyridinium, derived from the reaction of bromomethyl carbonyl and pyridine, triggered the reaction by the inter-molecular Michael addition to cyclopropenone. This procedure was sensitive neither to air nor moisture and proceeded at room temperature with broad substrate scopes and good functional group tolerance in moderate-to-good yields. As such, it represents a facile and practical pathway leading to 5-carbonyl furan-2(5H)-one derivatives.

Phosphine-catalyzed ring-opening reaction of cyclopropenones with dicarbonyl compounds.

We developed a phosphine-catalyzed ring-opening reaction of cyclopropenones with dicarbonyl compounds as C-nucleophiles, leading to 1,3,3'-tricarbonyl compounds. During this neutral procedure, C-acylation is more dominant than O-acylation. This transition-metal free procedure features mild and neutral reaction conditions with good atom economy. As such, it represents a facile pathway to access 1,3,3'-tricarbonyl derivatives.

Mfn2 ablation causes an oxidative stress response and eventual neuronal death in the hippocampus and cortex.

BACKGROUND: Mitochondria are the organelles responsible for energy metabolism and have a direct impact on neuronal function and survival. Mitochondrial abnormalities have been well characterized in Alzheimer Disease (AD). It is believed that mitochondrial fragmentation, due to impaired fission and fusion balance, likely causes mitochondrial dysfunction that underlies many aspects of neurodegenerative changes in AD. Mitochondrial fission and fusion proteins play a major role in maintaining the health and function of these important organelles. Mitofusion 2 (Mfn2) is one such protein that regulates mitochondrial fusion in which mutations lead to the neurological disease. METHODS: To examine whether and how impaired mitochondrial fission/fusion balance causes neurodegeneration in AD, we developed a transgenic mouse model using the CAMKII promoter to knockout neuronal Mfn2 in the hippocampus and cortex, areas significantly affected in AD. RESULTS: Electron micrographs of neurons from these mice show swollen mitochondria with cristae damage and mitochondria membrane abnormalities. Over time the Mfn2 cKO model demonstrates a progression of neurodegeneration via mitochondrial morphological changes, oxidative stress response, inflammatory changes, and loss of MAP2 in dendrites, leading to severe and selective neuronal death. In this model, hippocampal CA1 neurons were affected earlier and resulted in nearly total loss, while in the cortex, progressive neuronal death was associated with decreased cortical size. CONCLUSIONS: Overall, our findings indicate that impaired mitochondrial fission and fusion balance can cause many of the neurodegenerative changes and eventual neuron loss that characterize AD in the hippocampus and cortex which makes it a potential target for treatment strategies for AD.

Inhibition of mitochondrial fragmentation protects against Alzheimer's disease in rodent model.

Mitochondrial dysfunction is an early prominent feature in susceptible neurons in the brain of patients with Alzheimer's disease, which likely plays a critical role in the pathogenesis of disease. Increasing evidence suggests abnormal mitochondrial dynamics as important underlying mechanisms. In this study, we characterized marked mitochondrial fragmentation and abnormal mitochondrial distribution in the pyramidal neurons along with mitochondrial dysfunction in the brain of Alzheimer's disease mouse model CRND8 as early as 3 months of age before the accumulation of amyloid pathology. To establish the pathogenic significance of these abnormalities, we inhibited mitochondrial fragmentation by the treatment of mitochondrial division inhibitor 1 (mdivi-1), a mitochondrial fission inhibitor. Mdivi-1 treatment could rescue both mitochondrial fragmentation and distribution deficits and improve mitochondrial function in the CRND8 neurons both in vitro and in vivo. More importantly, the amelioration of mitochondrial dynamic deficits by mdivi-1 treatment markedly decreased extracellular amyloid deposition and Aß1-42/Aß1-40 ratio, prevented the development of cognitive deficits in Y-maze test and improved synaptic parameters. Our findings support the notion that abnormal mitochondrial dynamics plays an early and causal role in mitochondrial dysfunction and Alzheimer's disease-related pathological and cognitive impairments in vivo and indicate the potential value of restoration of mitochondrial dynamics as an innovative therapeutic strategy for Alzheimer's disease.

The Effects and Molecular Mechanisms of MiR-106a in Multidrug Resistance Reversal in Human Glioma U87/DDP and U251/G Cell Lines.

Chemotherapy resistance is one of the major obstacles to effective glioma therapy. Currently, the mechanism underlying chemotherapy resistance is unclear. A recent study showed that miR-106a is an important molecule involved in chemotherapy resistance. To explore the effects and mechanisms of miR-106a on multidrug resistance reversal in human glioma cells, we silenced miR-106a expression in the cisplatin-resistant U87 (U87/DDP) and the gefitinib-resistant U251 (U251/G) glioma cell lines and measured the resulting drug sensitivity, cell apoptosis rate and rhodamine 123 content. In addition, we detected decreased expression of P-glycoprotein, MDR1, MRP1, GST-π, CDX2, ERCC1, RhoE, Bcl-2, Survivin and Topo-II, as well as reduced production of IL-6, IL-8 and TGF-ß in these cell lines. Furthermore, we found decreased expression of p-AKT and transcriptional activation of NF-κB, Twist, AP-1 and Snail in these cell lines. These results suggest that miR-106a is a promising therapeutic target for the treatment of human multidrug resistant glioma.

Targeted deletion of the murine Lgr4 gene decreases lens epithelial cell resistance to oxidative stress and induces age-related cataract formation.

Oxidative stress contributes to the formation of cataracts. The leucine rich repeat containing G protein-coupled receptor 4 (LGR4, also known as GPR48), is important in many developmental processes. Since deletion of Lgr4 has previously been shown to lead to cataract formation in mice, we sought to determine the specific role that Lgr4 plays in the formation of cataracts. Initially, the lens opacities of Lgr4(-/-) mice at different ages without ocular anterior segment dysgenesis (ASD) were evaluated with slit-lamp biomicroscopy. Lenses from both Lgr4(-/-) and wild-type mice were subjected to oxidation induced protein denaturation to assess the ability of the lens to withstand oxidation. The expression of antioxidant enzymes was evaluated with real-time quantitative PCR. Phenotypically, Lgr4(-/-) mice showed earlier onset of lens opacification and higher incidence of cataract formation compared with wild-type mice of similar age. In addition, Lgr4(-/-) mice demonstrated increased sensitivity to environmental oxidative damage, as evidenced by altered protein expression. Real-time quantitative PCR showed that two prominent antioxidant defense enzymes, catalase (CAT) and superoxidase dismutase-1 (SOD1), were significantly decreased in the lens epithelial cells of Lgr4(-/-) mice. Our results suggest that the deletion of Lgr4 can lead to premature cataract formation, as well as progressive deterioration with aging. Oxidative stress and altered expression of several antioxidant defense enzymes contribute to the formation of cataracts.

Inhibitory effect of microRNA-34a on retinal pigment epithelial cell proliferation and migration.

PURPOSE: Retinal pigment epithelial (RPE) cells play important roles in ophthalmologic diseases such as proliferative vitreoretinopathy, AMD, and diabetic retinopathy. MicroRNA-34a (miR-34a) has been reported to be important in the regulation of cell proliferation, migration, differentiation, and apoptosis. In this study, we explored the effects of miR-34a on RPE cells. METHODS: The expression level of miR-34a in subconfluent and postconfluent ARPE-19 cells was investigated with quantitative real-time PCR. MicroRNA mimic and small interfering RNA (siRNA) were transiently transfected into RPE cells. Transfected RPE cells were analyzed with WST-1 proliferation assay, and their migration was analyzed with transwell assay and in vitro scratch study. The expression or activation of target proteins was detected by Western blotting. RESULTS: MicroRNA-34a was significantly downregulated in subconfluent ARPE-19 cells compared with postconfluent cells. Introduction of miR-34a inhibited the proliferation and migratory ability of RPE cells without obvious cell apoptosis. In miR-34a transfected cells, many important proliferation and/or migration related molecules such as c-Met, CDK2, CDK4, CDK6, E2F1, and phosphorylated-Cdc2 (p-Cdc2) were downregulated. Small interfering RNA designed to target c-Met also inhibited the proliferation and migration of RPE cells and downregulated CDK2, CDK6, E2F1, and p-Cdc2. CONCLUSIONS: MicroRNA-34a is downregulated in subconfluent RPE cells. MicroRNA-34a can inhibit the proliferation and migration of RPE cells through downregulation of its targets c-Met and other cell cycle-related molecules. Our results indicated that miR-34a is involved in the regulation of RPE cells.

GPR48-Induced keratinocyte proliferation occurs through HB-EGF mediated EGFR transactivation.

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.

STAT3 activation induced by Epstein-Barr virus latent membrane protein1 causes vascular endothelial growth factor expression and cellular invasiveness via JAK3 And ERK signaling.

The principal Epstein-Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1), has been suggested to contribute to the highly invasive nature of nasopharyngeal carcinoma (NPC). Signal transducer and activator of transcription 3 (STAT3) is a master transcriptional regulator in proliferation and apoptosis and is newly implicated in angiogenesis and invasiveness, which, in turn, are likely to contribute to the highly invasive character of NPC. The fundamental molecular mechanisms of LMP1-regulated STAT3 activation in NPC cell invasion have not been completely explored. Here, we showed that LMP1 signals the Janus kinase 3 (JAK3) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways upon the activation of STAT3 as well as STAT transactivation activity. LMP1 induces vascular endothelial growth factor (VEGF) expression via the JAK/STAT and mitogen-activated protein kinase (MAPK)/ERK signalling pathways. Induction of STAT3 by the human viral oncoprotein LMP1 may contribute to the invasion of NPC.

LMP1-augmented kappa intron enhancer activity contributes to upregulation expression of Ig kappa light chain via NF-kappaB and AP-1 pathways in nasopharyngeal carcinoma cells.

BACKGROUND: Expression of kappa gene is under the control of distinct cis-regulatory elements, including the kappa intron enhancer (iE kappa) and the kappa 3' enhancer (3'E kappa). The active enhancers and expression of immunoglobulin is generally considered to be restricted to B lymphocytes. However, accumulating evidence indicated that epithelial cancer cells, including nasopharyngeal carcinoma (NPC) cell lines, express immunoglobulins. The mechanisms underlying the expression of Igs in nonlymphoid cells remain unknown. On the basis of our previous finding that expression of kappa light chain in NPC cells can be upregulated by EBV-encoded latent membrane protein 1(LMP1) through the activation of NF-kappaB and AP-1 signaling pathways, we thus use NPC cells as model to further explore the molecular mechanisms of nonlymphoid cells expressing Ig kappa. RESULTS: In this study, luciferase reporter plasmid containing human wild-type iE kappa, and its derivative plasmids containing mutant binding sites for transcription factor NF-kappaB or AP-1 were constructed. Luciferase reporter assays demonstrate iE kappa is active in Ig kappa-expressing NPC cells and LMP1 expression can upregulate the activity of iE kappa in NPC cells. Mutation of the NF-kappaB or AP-1 site within and downstream the iE kappa, inhibition of the NF-kappaB and AP-1 pathways by their respective chemical inhibitor Bay11-7082 and SP600125 as well as stable or transient expression of dominant-negative mutant of I kappaB alpha (DNMI kappaB alpha) or of c-Jun (TAM67) indicate that both sites are functional and LMP1-enhanced iE kappa activity is partly regulated by these two sites. Gel shift assays show that LMP1 promotes NF-kappaB subunits p52 and p65 as well as AP-1 family members c-Jun and c-Fos binding to the kappa NF-kappaB and the kappa AP-1 motifs in vitro, respectively. Both chemical inhibitors and dominant negative mutants targeting for NF-kappaB and AP-1 pathways can attenuate the LMP1-enhanced bindings. Co-IP assays using nuclear extracts from HNE2-LMP1 cells reveal that p52 and p65, c-Jun and c-Fos proteins interact with each other at endogenous levels. ChIP assays further demonstrate p52 and p65 binding to the kappaB motif as well as c-Jun and c-Fos binding to the AP-1 motif of Ig kappa gene in vivo. CONCLUSION: These results suggest that human iE kappa is active in Ig kappa-expressing NPC cells and LMP1-stimulated NF-kappaB and AP-1 activation results in an augmenting activation of the iE kappa. LMP1 promotes the interactions of heterodimeric NF-kappaB (p52/p65) and heterodimeric AP-1 (c-Jun/c-Fos) transcription factors with the human iE kappa enhancer region are important for the upregulation of kappa light chain in LMP1-positive nasopharyngeal carcinoma cells.

GPR48 regulates epithelial cell proliferation and migration by activating EGFR during eyelid development.

PURPOSE: Eyelid development is a dynamic process involving cell proliferation, differentiation, and migration regulated by a number of growth factors and cytokines. Mice deficient in the orphan G protein-coupled receptor 48 (GPR48) showed an eye open at birth (EOB) phenotype. In this study, the authors attempted to clarify the role of GPR48 in eyelid development and the molecular mechanisms leading to the EOB phenotype. METHODS: Phenotypic analysis of the eyelids of Gpr48(-/-) mice was carried out using histology and scanning electron microscopy. GPR48 expression pattern was determined using X-gal staining. In vitro scratch assay was used to determine cell motility defects in Gpr48(-)(/)(-) keratinocytes. The molecular mechanism underlying GPR48-mediated eyelid closure was explored using Western blot and immunostaining analyses. Expression levels of EGFR and its phosphorylated counterpart were examined in Gpr48(-/-) and wild-type keratinocytes and in eyelids. RESULTS: GPR48 is highly expressed in the epithelium and apical mesenchymal cells of eyelids during embryonic development. Detailed analysis revealed that Gpr48(-/-) mice exhibited delayed leading-edge extension, reduced filopodia formation, and decreased rounded periderm cell formation around eyelid margins. Keratinocytes lacking GPR48 are defective in cell proliferation and migration with reduced F-actin staining. In addition, the phosphorylation of EGFR was dramatically decreased in cultured keratinocytes and developing eyelids in the absence of GPR48. CONCLUSIONS: Inactivation of GPR48 induces the EOB phenotype by reducing epithelial cell proliferation and migration, indicating that GPR48 plays an essential role in eyelid development. Furthermore, GPR48 contributes to eyelid development through the regulation of the EGFR signaling pathway.

Epstein-Barr virus latent membrane protein 1 mediates serine 25 phosphorylation and nuclear entry of annexin A2 via PI-PLC-PKCalpha/PKCbeta pathway.

We have previously elucidated that Epstein-Barr-virus-encoded latent membrane protein 1 (LMP1) can increase the serine phosphorylation level of annexin A2 by activating the protein kinase C (PKC) signaling pathway and that LMP1 induces the nuclear entry of annexin A2 in an energy- and temperature-dependent manner. Here, we further confirm that LMP1 increases the serine phosphorylation level of annexin A2 by activating the phosphoinositide-specific phospholipase C (PI-PLC)-PKC alpha/PKC beta pathway, mainly through the activation of the PKCbeta pathway. Additionally, active recombinant PKC alpha, PKC beta I, and PKC beta II kinases are able to phosphorylate annexin A2 in vitro. Annexin A2 in the nucleus plays an important role in DNA synthesis and cell proliferation. By site-specific substitution of glutamic acid in the place of serine 11 and 25 in the N-terminus, we show that serine 25 phosphorylation of annexin A2 was associated with the nuclear entry of annexin A2, DNA synthesis and cell proliferation, whereas serine 11 has no obvious influence. We demonstrate for the first time that the PI-PLC-PKCalpha/PKCbeta pathway plays an important role in serine phosphorylation and in the nuclear entry of annexin A2 mediated by LMP1. In addition, we show that annexin A2 is the substrate protein of PKC alpha, PKC betaI, and PKC betaII kinases. Serine 25 phosphorylation of annexin A2 is shown to be associated with its nuclear entry, DNA synthesis, and cell proliferation.

DNAzymes targeted to EBV-encoded latent membrane protein-1 induce apoptosis and enhance radiosensitivity in nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) is involved in the carcinogenesis of several types of cancers such as nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma. The latent membrane protein (LMP1) encoded by EBV is expressed in the majority of EBV-associated human malignancies and has been suggested to be one of the major oncogenic factors in EBV-mediated carcinogenesis. Therefore, genetic manipulation of LMP1 expression may provide a novel strategy for the treatment of the EBV-associated human cancers. Deoxyribozymes (DNAzymes) are catalytic nucleic acids that bind and cleave a target RNA in a highly sequence-specific manner. We have designed several LMP1-specific DNAzymes and tested their effect on cell proliferation and apoptosis in LMP1-positive cells. Here, we show that active DNAzymes down-regulated the expression of the EBV oncoprotein LMP1 and inhibited cellular signal transduction pathways abnormally activated by LMP1. This down-regulation of the LMP1 expression was shown to be associated with a decrease in the level of antiapoptotic Bcl-2 and an increase in Caspase-3 and -9 activities in the nasopharyngeal carcinoma cell line CNE1-LMP1, which constitutively expresses the LMP1. When combined with radiation treatment, the DNAzymes significantly induced apoptosis in CNE1-LMP1 cells, leading to an increased radiosensitivity both in cells and in a xenograft NPC model in mice. The results suggest that LMP1 may represent a molecular target for DNAzymes and provide a basis for the use of the LMP1 DNAzymes as potential radiosensitizers for treatment of the EBV-associated carcinomas.

Phosphorylation and nuclear translocation of STAT3 regulated by the Epstein-Barr virus latent membrane protein 1 in nasopharyngeal carcinoma.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) oncoprotein has been shown to mediate activation of the signal transducer and activator of transcription 3 (STAT3). In the present study, we delineated the mechanism by which LMP1 stimulates STAT3 in a human nasopharyngeal carcinoma (NPC) cell line. LMP1 stimulated STAT3 Tyr 705-dependent nuclear accumulation, as well as the phosphorylation of STAT3 at both Tyr 705 and Ser 727. Treatment of cells with interleukin-6 neutralizing antibody inhibited the phosphorylation of STAT3 Tyr 705 and Ser 727. The differential phosphorylation of STAT3 was found to be a result of activation of Janus kinase 3 (JAK3) and extracellular signal-regulated kinase (ERK). The biological significance of JAK3-mediated activation of STAT3 Tyr 705 phosphorylation was further assessed by treating the cells with an inhibitor (WHI-P131) of JAK3. Inhibition of ERK activity by an inhibitor (PD98059) of MAPK/extracellular signal-regulated kinase kinase (MEK1) decreased the LMP1-induced activation of STAT3 Ser 727. Furthermore, immunohistochemical analysis showed an increased nuclear STAT3 Tyr 705 staining in LMP1-positive cells and STAT3 Tyr 705 phosphorylation related to NPC stages III and IV. Demonstration of the involvement of different kinases in LMP1-induced STAT3 activation supports the involvement of the JAK/STAT and mitogen-activated protein kinase (MAPK)/ERK signaling pathways in the regulation of STAT3 activation by LMP1.

Latent membrane protein 1 of Epstein-Barr virus regulates p53 phosphorylation through MAP kinases.

The Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1), an oncogenic protein, plays an important role in the carcinogenesis of nasopharyngeal carcinoma (NPC). Phosphorylation of p53 protein is likely to play the key role in regulating its activity. p53 protein accumulates but mutation of p53 gene is not common in NPC. The molecular mechanisms of p53 augmentation have not been completely elucidated. Here, the role of MAP kinases in the phosphorylation of p53 modulated by LMP1 was determined. p53 could be activated and phosphorylated clearly at Ser15, Ser20, Ser392, and Thr81 modulated by LMP1. Furthermore, LMP1-induced phosphorylation of p53 at Ser15 was directly by ERKs; at Ser20 and Thr81 by JNK, at Ser 15 and Ser392 by p38 kinase. The phosphorylation of p53 was associated with its transcriptional activity and stability modulated by LMP1. These results strongly suggest that MAP kinases have a direct role in LMP1-induced phosphorylation of p53 at multiple sites, which provide a novel view for us to understand the mechanism of the activation of p53 in the carcinogenesis of nasopharyngeal carcinoma.

Blockade of AP-1 activity by dominant-negative TAM67 can abrogate the oncogenic phenotype in latent membrane protein 1-positive human nasopharyngeal carcinoma.

Although activating protein-1 (AP-1) transcription factors play an important role in mediating metastasis for nasopharyngeal carcinoma (NPC), the biological and physiological functions of AP-1, in relation to the oncogenic phenotype of NPC, are not fully understood. Our previous study showed that the latent membrane protein 1 (LMP1) mediated a primary dimer form of c-jun and jun B. In this study, we used a NPC cell line that express a specific inhibitor of AP-1, a dominant-negative c-jun mutant (TAM67), to investigate the role of AP-1 in regulating the NPC oncogenic phenotype. First, we observed that TAM67 inhibited cell growth in vitro and in vivo. Next, with Western blotting, we discovered that TAM67 impaired the cyclin D1/cdk4 complex but had little effect on the cyclin E/cdk2 complex, concomitantly with inhibiting Rb phosphorylation. RT-PCR and luciferase assay results demonstrated that the levels of cyclin D1 mRNA and the promoter activity in TAM67 transfectants were reduced as compared with control cells. Thereby, we show that blockade of AP-1 transcriptional activity has a negative impact on cyclin D1 transcription. We obtained the first evidence that TAM67 prevented NPC growth both in vitro and in vivo. AP-1 appears to be a novel target for treating or preventing LMP1-positive NPC effectively.