Fluvoxamine, an anti-depressant, inhibits human glioblastoma invasion by disrupting actin polymerization.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor with a median survival time about one year. Invasion of GBM cells into normal brain is the major cause of poor prognosis and requires dynamic reorganization of the actin cytoskeleton, which includes lamellipodial protrusions, focal adhesions, and stress fibers at the leading edge of GBM. Therefore, we hypothesized that inhibitors of actin polymerization can suppress GBM migration and invasion. First, we adopted a drug repositioning system for screening with a pyrene-actin-based actin polymerization assay and identified fluvoxamine, a clinically used antidepressant. Fluvoxamine, selective serotonin reuptake inhibitor, was a potent inhibitor of actin polymerization and confirmed as drug penetration through the blood-brain barrier (BBB) and accumulation of whole brain including brain tumor with no drug toxicity. Fluvoxamine inhibited serum-induced ruffle formation, cell migration, and invasion of human GBM and glioma stem cells in vitro by suppressing both FAK and Akt/mammalian target of rapamycin signaling. Daily treatment of athymic mice bearing human glioma-initiating cells with fluvoxamine blocked tumor cell invasion and prolonged the survival with almost same dose of anti-depressant effect. In conclusion, fluvoxamine is a promising anti-invasive treatment against GBM with reliable approach.

Tumor-specific delivery of BSH-3R for boron neutron capture therapy and positron emission tomography imaging in a mouse brain tumor model.

Glioblastoma, a malignant brain tumor with poor disease outcomes, is managed in modern medicine by multimodality therapy. Boron neutron capture therapy (BNCT) is an encouraging treatment under clinical investigation. In malignant cells, BNCT consists of two major factors: neutron radiation and boron uptake. To increase boron uptake in cells, we created a mercapto-closo-undecahydrododecaborate ([B12HnSH](2-)2Na(+), BSH) fused with a short arginine peptide (1R, 2R, 3R) and checked cellular uptake in vitro and in vivo. In a mouse brain tumor model, only BSH with at least three arginine domains could penetrate cell membranes of glioma cells in vitro and in vivo. Furthermore, to monitor the pharmacokinetic properties of these agents in vivo, we fused BSH and BSH-3R with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); DOTA is a metal chelating agent for labeling positron emission tomography (PET) probe with (64)Cu. We administered BSH-DOTA-(64)Cu and BSH-3R-DOTA-(64)Cu to the tumor model through a mouse tail vein and determined the drugs' pharmacokinetics by PET imaging. BSH-3R showed a high uptake in the tumor area on PET imaging. We concluded that BSH-3R is the ideal boron compound for clinical use during BNCT and that in developing this compound for clinical use, the BSH-3R PET probe is essential for pharmacokinetic imaging.

The transdermal inhibition of melanogenesis by a cell-membrane-permeable peptide delivery system based on poly-arginine.

Topical therapy is the most favored form of treatment for whitening against hyper-pigmentation and sunburn because it lends itself to self-administration, patient compliance and an absence of systemic adverse effects. However, high-molecular-weight, hydrophilic chemicals are difficult to use as transdermal delivery drugs and the use of topical drugs has been highly limited. There are now many potent tyrosinase inhibitors, for example, sulfite or kojic acid, but the efficacy of their skin transduction remains a big problem. Furthermore, melanogenesis inhibitors from natural sources have great potential, as they are considered to be safe and largely free from adverse side effects. We applied 11-arginine (11R), a cell-membrane-permeable peptide, as a transdermal delivery system with a skin delivery enhancer, pyrenbutyrate. We performed intracellular screening for melanogenesis inhibitors with 11R fused with several kinds of tyrosinase inhibitory peptides from natural sources. Of 28 tyrosinase peptides, 13 melanin synthesis inhibitory peptides were selected. Peptide No. 10 found in gliadin protein, a wheat component, most strongly inhibited melanin production. This No. 10 peptide, of only 8 amino acids, fused to 11R showed no cytotoxicity and inhibited melanin synthesis as determined through melanin content measured using an absorption spectrometer and observation with a transmission electron microscope. Next, we transduced this 11R-No. 10 into skin with an 11R transdermal delivery system after previous treatment with pyrenbutyrate and performed daily repetitive topical application for two weeks against a UV-induced sun-tanning guinea pig model. We observed a whitening effect in a model skin sample by Masson-Fontana staining and the 11R-No. 10 peptide-applied area showed significant melanogenesis inhibition. These results show that 11R using a transdermal drug delivery system with melanogenesis inhibitory peptide is a very safe and promising method for applications from cosmetics to the pharmaceutical industry.

The acceleration of boron neutron capture therapy using multi-linked mercaptoundecahydrododecaborate (BSH) fused cell-penetrating peptide.

New anti-cancer therapy with boron neutron capture therapy (BNCT) is based on the nuclear reaction of boron-10 with neutron irradiation. The median survival of BNCT patients with glioblastoma was almost twice as long as those receiving standard therapy in a Japanese BNCT clinical trial. In this clinical trial, two boron compounds, BPA (boronophenylalanine) and BSH (sodium borocaptate), were used for BNCT. BPA is taken up into cells through amino acid transporters that are expressed highly in almost all malignant cells, but BSH cannot pass through the cell membrane and remains outside the cell. We simulated the energy transfer against the nucleus at different locations of boron from outside the cell to the nuclear region with neutron irradiation and concluded that there was a marked difference between inside and outside the cell in boron localization. To overcome this disadvantage of BSH in BNCT, we used a cell-penetrating peptide system for transduction of BSH. CPP (cell-membrane penetrating peptide) is very common peptide domains that transduce many physiologically active substances into cells in vitro and in vivo. BSH-fused CPPs can penetrate the cell membrane and localize inside a cell. To increase the boron ratio in one BSH-peptide molecule, 8BSH fused to 11R with a dendritic lysine structure was synthesized and administrated to malignant glioma cells and a brain tumor mouse model. 8BSH-11R localized at the cell nucleus and showed a very high boron value in ICP results. With neutron irradiation, the 8BSH-11R administrated group showed a significant cancer killing effect compared to the 100 times higher concentration of BSH-administrated group. We concluded that BSH-fused CPPs were one of the most improved and potential boron compounds in the next-stage BNCT trial and 8BSH-11R may be applied in the clinical setting.

Cyclin G2 promotes hypoxia-driven local invasion of glioblastoma by orchestrating cytoskeletal dynamics.

Microenvironmental conditions such as hypoxia potentiate the local invasion of malignant tumors including glioblastomas by modulating signal transduction and protein modification, yet the mechanism by which hypoxia controls cytoskeletal dynamics to promote the local invasion is not well defined. Here, we show that cyclin G2 plays pivotal roles in the cytoskeletal dynamics in hypoxia-driven invasion by glioblastoma cells. Cyclin G2 is a hypoxia-induced and cytoskeleton-associated protein and is required for glioblastoma expansion. Mechanistically, cyclin G2 recruits cortactin to the juxtamembrane through its SH3 domain-binding motif and consequently promotes the restricted tyrosine phosphorylation of cortactin in concert with src. Moreover, cyclin G2 interacts with filamentous actin to facilitate the formation of membrane ruffles. In primary glioblastoma, cyclin G2 is abundantly expressed in severely hypoxic regions such as pseudopalisades, which consist of actively migrating glioma cells. Furthermore, we show the effectiveness of dasatinib against hypoxia-driven, cyclin G2-involved invasion in vitro and in vivo. Our findings elucidate the mechanism of cytoskeletal regulation by which severe hypoxia promotes the local invasion and may provide a therapeutic target in glioblastoma.

Combining poly-arginine with the hydrophobic counter-anion 4-(1-pyrenyl)-butyric acid for protein transduction in transdermal delivery.

Topical therapy is the most favored form of treatment for whitening against hyperpigmentation and sunburn because it lends itself to self-administration, patient compliance, and absence of systemic adverse effects. However, transdermal delivery of hydrophilic chemicals is difficult. The main purpose of this study is to develop a delivering system of hydrophilic drugs and proteins across the skin. Hydroquinone (HQ), a well-known tyrosinase inhibitor and antimelanogenesis compound, and enhanced green fluorescent protein (EGFP) were fused with eleven poly-arginine (11R). Both HQ-11R and EGFP-11R were efficiently delivered in B16 cells, a mouse melanoma cell line. HQ-11R was as effective as HQ alone at inhibiting melanin synthesis in B16 cells. EGFP-11R was efficiently delivered into cells of the epidermis with 4-(1-pyrenyl)-butyric acid (PB), a counteranion bearing an aromatic hydrophobic moiety, in vivo, but EGFP alone or EGFP-11R without PB was not. Finally, topical application of HQ-11R with PB significantly inhibited UV irradiation-induced pigmentation in guinea pigs compared with HQ alone. These results suggest that topical therapy using poly-arginine in combination with PB is useful for the delivery of hydrophilic drugs and proteins by the transdermal route.

A protein transduction method using oligo-arginine (3R) for the delivery of transcription factors into cell nuclei.

Protein transduction with cell-penetrating peptides such as poly-arginine and HIV TAT peptides is widely used to deliver proteins, peptides, siRNA and biologically active compounds. It has been thought that poly-arginine peptides transduce proteins in a manner dependent on the number of arginine residues and oligo-peptides such as three arginines (3R) are ineffective. Here we showed that 3R-fused proteins were effectively delivered and functioned in cells co-treated with pyrenebutyrate, a counteranion bearing an aromatic hydrophobic moiety. Little 3R was transduced in glioma cells without pyrenebutyrate whereas the oligo-arginine was effectively delivered with pyrenebutyrate. Enhanced green fluorescence protein (eGFP) fused with 3R was effectively delivered into various kinds of cells including primary cultured cells and suspended cells in the presence of pyrenebutyrate. p53 fused with 3R (3R-p53) was delivered into glioma cells without pyrenebutyrate but could not be translocated into the nucleus. In contrast, 3R-p53 was observed in nuclei of glioma cells when co-applied with pyrenebutyrate. Although 3R-p53 was delivered less effectively than 11R-p53 with pyrenebutyrate, its transcriptional activity was higher than that of 11R-p53. Moreover, a single administration of 3R-p53 with pyrenebutyrate significantly inhibited the growth of cancer cells. These results suggest protein transduction using an oligo-arginine (3R) with pyrenebutyrate to be a good tool for the delivery of functional transcription factors and a promising method of treating cancer.

RGS2 mediates the anxiolytic effect of oxytocin.

The neuropeptide oxytocin (OT) has been shown to exert multiple functions in both males and females, and to play a key role in the regulation of emotionality in the central nervous system (CNS). OT has an anxiolytic effect in the CNS of rodents and humans. However, the molecular mechanisms of this effect are unclear. Here we show that OT induced the expression of regulator of G-protein signaling 2 (RGS2), a regulatory factor for anxiety, in the central amygdala (CeA) of female mice. Bath application of OT increased RGS2 levels in slices of the amygdala of virgin mice. RGS2 levels in the CeA were higher in lactating mice than in virgin mice. In contrast, RGS2 levels in mice that had given birth did not increase when the pups were removed. Acute restraint stress for 4h induced RGS2 expression within the CeA, and local administration of an OT receptor antagonist inhibited this expression. Behavioral experiments revealed that transient restraint stress had an anxiolytic effect in wild-type females, and RGS2 levels in the CeA correlated with the anxiolytic behavior. By contrast, in the OT receptor-deficient mice, restraint stress neither increased RGS2 levels in the CeA nor had an anxiolytic effect. These results suggest that OT displays an anxiolytic effect through the induction of RGS2 expression in the CNS.

Ca(2+)-independent syntaxin binding to the C(2)B effector region of synaptotagmin.

Although synaptotagmin I, which is a calcium (Ca(2+))-binding synaptic vesicle protein, may trigger soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated synaptic vesicle exocytosis, the mechanisms underlying the interaction between these proteins remain controversial, especially with respect to the identity of the protein(s) in the SNARE complex that bind(s) to synaptotagmin and whether Ca(2+) is required for their highly effective binding. To address these questions, native proteins were solubilized, immunoprecipitated from rat brain extracts, and analyzed by immunoblotting. SNARE complexes comprising syntaxin 1, 25-kDa synaptosomal-associated protein (SNAP-25), and synaptobrevin 2 were coprecipitated with synaptotagmin I in the presence of ethylene glycol tetraacetic acid. The amount of coprecipitated proteins was significantly unaltered by the addition of Ca(2+) to the brain extract. To identify the component of the SNARE complex that bound to synaptotagmin, SNARE was coexpressed with synaptotagmin in HEK293 cells and immunoprecipitated. Syntaxin, but not SNAP-25 and synaptobrevin, bound to synaptotagmin in a Ca(2+)-independent manner, and the binding was abolished in the presence of 1M NaCl. Synaptotagmin contains 2 Ca(2+)-binding domains (C(2)A, C(2)B). Mutating the positively charged lysine residues in the putative effector-binding region of the C(2)B domain, which are critical for transmitter release, markedly inhibited synaptotagmin-syntaxin binding, while similar mutations in the C(2)A domain had no effect on binding. Synaptotagmin-syntaxin binding was reduced by mutating multiple negatively charged glutamate residues in the amino-terminal half of the syntaxin SNARE motif. These results indicate that synaptotagmin I binds to syntaxin 1 electrostatically through its C(2)B domain effector region in a Ca(2+)-independent fashion, providing biochemical evidence that synaptotagmin I binds SNARE complexes before Ca(2+) influx into presynaptic nerve terminals.

Expression of a constitutively active calcineurin encoded by an intron-retaining mRNA in follicular keratinocytes.

Hair growth is a highly regulated cyclical process. Immunosuppressive immunophilin ligands such as cyclosporin A (CsA) and FK506 are known as potent hair growth modulatory agents in rodents and humans that induce active hair growth and inhibit hair follicle regression. The immunosuppressive effectiveness of these drugs has been generally attributed to inhibition of T cell activation through well-characterized pathways. Specifically, CsA and FK506 bind to intracellular proteins, principally cyclophilin A and FKBP12, respectively, and thereby inhibit the phosphatase calcineurin (Cn). The calcineurin (Cn)/NFAT pathway has an important, but poorly understood, role in the regulation of hair follicle development. Here we show that a novel-splicing variant of calcineurin Aß CnAß-FK, which is encoded by an intron-retaining mRNA and is deficient in the autoinhibitory domain, is predominantly expressed in mature follicular keratinocytes but not in the proliferating keratinocytes of rodents. CnAß-FK was weakly sensitive to Ca(2+) and dephosphorylated NFATc2 under low Ca(2+) levels in keratinocytes. Inhibition of Cn/NFAT induced hair growth in nude mice. Cyclin G2 was identified as a novel target of the Cn/NFATc2 pathway and its expression in follicular keratinocytes was reduced by inhibition of Cn/NFAT. Overexpression of cyclin G2 arrested the cell cycle in follicular keratinocytes in vitro and the Cn inhibitor, cyclosporin A, inhibited nuclear localization of NFATc2, resulting in decreased cyclin G2 expression in follicular keratinocytes of rats in vivo. We therefore suggest that the calcineurin/NFAT pathway has a unique regulatory role in hair follicle development.

Regulation of mitochondrial dynamics and neurodegenerative diseases.

Mitochondria are important cellular organelles in most metabolic processes and have a highly dynamic nature, undergoing frequent fission and fusion. The dynamic balance between fission and fusion plays critical roles in mitochondrial functions. In recent studies, several large GTPases have been identified as key molecular factors in mitochondrial fission and fusion. Moreover, the posttranslational modifications of these large GTPases, including phosphorylation, ubiquitination and SUMOylation, have been shown to be involved in the regulation of mitochondrial dynamics. Neurons are particularly sensitive and vulnerable to any abnormalities in mitochondrial dynamics, due to their large energy demand and long extended processes. Emerging evidences have thus indicated a strong linkage between mitochondria and neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and Huntington's disease. In this review, we will describe the regulation of mitochondrial dynamics and its role in neurodegenerative diseases.

SNARE protein recycling by αSNAP and ßSNAP supports synaptic vesicle priming.

Neurotransmitter release proceeds by Ca(2+)-triggered, SNARE-complex-dependent synaptic vesicle fusion. After fusion, the ATPase NSF and its cofactors α- and ßSNAP disassemble SNARE complexes, thereby recycling individual SNAREs for subsequent fusion reactions. We examined the effects of genetic perturbation of α- and ßSNAP expression on synaptic vesicle exocytosis, employing a new Ca(2+) uncaging protocol to study synaptic vesicle trafficking, priming, and fusion in small glutamatergic synapses of hippocampal neurons. By characterizing this protocol, we show that synchronous and asynchronous transmitter release involve different Ca(2+) sensors and are not caused by distinct releasable vesicle pools, and that tonic transmitter release is due to ongoing priming and fusion of new synaptic vesicles during high synaptic activity. Our analysis of α- and ßSNAP deletion mutant neurons shows that the two NSF cofactors support synaptic vesicle priming by determining the availability of free SNARE components, particularly during phases of high synaptic activity.

Oxytocin mediates the antidepressant effects of mating behavior in male mice.

A significant association between plasma oxytocin (OT) levels and depression has been demonstrated. A recent study found that sexual activity and mating with a female induced the release of OT in the central nervous system of male rats. Here we examined the effect of mating behavior on depression-related behavior in wild-type (WT) and OT receptor-deficient (OTR KO) male mice. The WT males showed a reduction in depression-related behavior after mating behavior, but the OTR KO mice did not. Application of an OTR antagonist inhibited mating behavior-induced antidepressant effect in WT males. OT may mediate the antidepressant effects of mating behavior.

A Cdk5 inhibitor enhances the induction of insulin secretion by exendin-4 both in vitro and in vivo.

Exendin-4 (Ex4) is a peptide found in the lizard Heloderma suspectum, and it has a high similarity to glucagon-like peptide 1 (GLP-1). It induces insulin secretion without the risk of hypoglycemic episodes. Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that is predominantly expressed in neurons. Recent studies have shown that this kinase regulates glucose-stimulated insulin secretion. Cdk5 inhibition enhances insulin secretion under conditions of stimulation by high glucose, but not low glucose. In the present study, we examined whether R-roscovitine (R-ros), a Cdk5 inhibitor, enhances insulin secretion induced by Ex4. R-ros induced Ex4-dependent insulin secretion under conditions of high glucose, but not low glucose in MIN6B1 cells. The enhancement by R-ros was also observed in db/db mice, a mouse model of type 2 diabetes. Moreover, long-term treatment with Ex4 and R-ros significantly improved HbA1c compared with treatment using only Ex4. These results suggest that a co-application of R-ros and Ex4 may become a promising therapy for the treatment of type 2 diabetes.

Development of bionanocapsules targeting brain tumors.

Bionanocapsules (BNCs) are hollow nanoparticles that are composed of L protein (the hepatitis B virus surface antigen) and show specific affinity for human hepatocytes. The pre-S1 peptide displayed on the surface of BNCs is the specific ligand for binding to the receptor on human hepatocytes. Therefore, BNCs are not delivered to other tissues, such as the brain. The aim of the present study was to develop a novel drug delivery system (DDS) targeting brain tumors using BNCs that selectively targeted brain tumors. Epidermal growth factor receptor (EGFR), especially a constitutively active genomic sequence deletion variant of EGFR (EGFRvIII), is overexpressed in human glioblastoma. In the present study, we replaced the pre-S1 peptide with the antibody affinity motif of protein A and made hybrid BNCs conjugated with anti-human EGFR antibody recognizing EGFRvIII. The hybrid BNCs were efficiently delivered to glioma cells but not normal glial cells. Moreover, we confirmed the specific delivery of the hybrid BNCs to brain tumors in an in vivo brain tumor model. These results suggest that this new approach using BNCs is a promising system for brain tumor-targeted drug delivery.

Truncations of amphiphysin I by calpain inhibit vesicle endocytosis during neural hyperexcitation.

Under normal physiological conditions, synaptic vesicle endocytosis is regulated by phosphorylation and Ca(2+)-dependent dephosphorylation of endocytic proteins such as amphiphysin and dynamin. To investigate the regulatory mechanisms that may occur under the conditions of excessive presynaptic Ca(2+) influx observed preceding neural hyperexcitation, we examined hippocampal slices following high-potassium or high-frequency electrical stimulation (HFS). In both cases, three truncated forms of amphiphysin I resulted from cleavage by the protease calpain. In vitro, the binding of truncated amphiphysin I to dynamin I and copolymerization into rings with dynamin I were inhibited, but its interaction with liposomes was not affected. Moreover, overexpression of the truncated form of amphiphysin I inhibited endocytosis of transferrin and synaptic vesicles. Inhibiting calpain prevented HFS-induced depression of presynaptic transmission. Finally, calpain-dependent amphiphysin I cleavage attenuated kainate-induced seizures. These results suggest that calpain-dependent cleavage of amphiphysin I inhibits synaptic vesicle endocytosis during neural hyperexcitation and demonstrate a novel post-translational regulation of endocytosis.

Dual roles of the C2B domain of synaptotagmin I in synchronizing Ca2+-dependent neurotransmitter release.

Although the vesicular protein synaptotagmin I contains two Ca2+-binding domains (C2A and C2B), Ca2+ binding to the C2B domain is more important for triggering synchronous neurotransmitter release. We have used point mutagenesis to determine the functional contributions of the five negatively charged aspartate (Asp) residues that constitute the Ca2+-binding sites in the C2B domain of synaptotagmin I. Transfecting wild-type synaptotagmin I DNA into cultured hippocampal neurons from synaptotagmin I knock-out mice rescued Ca2+-dependent synchronous transmitter release and reduced a slower, asynchronous component of release, indicating that synaptotagmin I suppresses asynchronous release. Mutating either the second or third Asp residues of the C2B domain potently inhibited the ability of synaptotagmin I to rescue synchronous release but did not change its ability to suppress asynchronous release. Synaptotagmin I with mutations in the first or fourth Asp residues of the C2B domain partially rescued synchronous release and partially suppressed asynchronous release, whereas neutralizing the fifth Asp residue had no effect on the ability of synaptotagmin I to rescue transmitter release. Thus, we conclude that the C2B domain of synaptotagmin I regulates neurotransmitter release in at least two ways. Synchronous release absolutely requires binding of Ca2+ to the second and third Asp residues in this domain. For the suppression of asynchronous release, Ca2+ binding to the C2B domain of synaptotagmin I apparently is not necessary because mutation of the second Asp residue inhibits Ca2+ binding, yet still allows this protein to suppress asynchronous release.

Synaptotagmin I synchronizes transmitter release in mouse hippocampal neurons.

We have asked whether loss of the Ca2+ sensor protein synaptotagmin I influences the total amount of neurotransmitter released after a presynaptic action potential. Hippocampal neurons from synaptotagmin I knock-out mice had a greatly reduced fast synchronous component of glutamate release, as reported previously. However, the amount of glutamate released during the slow asynchronous component increased in these knock-out neurons. As a result of these changes in the kinetics of release, there was no significant difference between wild-type and knock-out neurons in the total amount of transmitter released within 400 msec after a presynaptic stimulus. Fluorescence imaging experiments demonstrated that wild-type and knock-out neurons take up and release similar amounts of FM dye after depolarization, indicating normal amounts of synaptic vesicle trafficking in the knock-out neurons. These results indicate that synaptotagmin I knock-out neurons are fully capable of releasing neurotransmitter, with the increased slow component of release serving to compensate for loss of the fast component. Thus, synaptotagmin I synchronizes the rapid release of neurotransmitters after Ca2+ entry into presynaptic terminals and also appears to suppress the slower, asynchronous form of transmitter release.