T-type calcium channel:from physiological function to therapeutical targeting – Exploring neuronal development

Tämän tutkimuksen tarkoitus oli tutkia T-tyypin kalsiumkanavan toimintaa ja sen mahdollista roolia neuronaalisten kantasolujen migraatiossa. T-tyypin kalsiumkanavan tehtävän kehittyneissä aivoissa tiedetään olevan elektroenkefalografisten oskillaatioiden tuottaminen. Nämä taas ovat eräiden fysiologisten ja patofysiologisten tapahtumien säätelyssä avainasemassa. Tällaisia tapahtumia ovat uni, muisti, oppiminen ja epileptiset poissaolokohtaukset. Näiden lisäksi T-tyypin kalsiumkanavalla on myös periferaalisia vaikutuksia, mutta tämä tutkielma keskittyy sen neuronaalisiin toimintoihin. Tämän matalan jännitteen säätelemän kanavan toiminta neurogeneesin aikana on vähemmän tutkittua ja tunnettua kuin sen vaikutukset kehittyneissä aivoissa. T-tyypin kalsiumkanavan tiedetään edistävän kantasolujen proliferaatiota ja erilaistumista neurogeneesiksen aikana, mutta vaikutukset niiden migraatioon ovat vähemmän tunnetut. Tämä tutkimus näyttää T-tyypin kalsiumkanavan todennäköisesti osallistuvan neuronaaliseen migraatioon hiiren alkion subventrikkeli alueelta eristetyillä kanta- tai progeniittorisoluilla tehdyissä kokeissa. Selektiiviset T-tyypin kalsiumkanavan antagonistit, etosuksimidi, nikkeli ja skorpionitoksiini, kurtoxin hidastivat migraatiota erilaistuvissa progeniittorisoluissa. Tämä tutkimus koostuu kirjallisuuskatsauksesta ja kokeellisesta osasta. Tämän tutkimuksen toinen tarkoitus oli esitellä vaihtoehtoinen lähestymistapa invasiiviselle kantasoluterapialle, joka vaatii kantasolujen viljelyä ja siirtämistä ihmiseen. Tämä toinen tapa on endogeenisten kantasolujen eiinvasiivinen stimulointi, jolla ne saadaan migratoitumaan kohdekudokseen, erilaistumaan siellä ja tehtävänsä suoritettuaan lopettamaan jakaantumisen. Non-invasiivinen kantasoluterapia on vasta tiensä alussa, ja tarvitsee farmakologista osaamista kehittyäkseen. Joitain onnistuneita ei-invasiivisia hoitoja on jo tehty selkärangan vaurioiden korjaamisessa. Vastaavanlaisia menetelmiä voitaisiin käyttää myös keskushermoston vaurioiden ja neurodegeneratiivisten sairauksien hoidossa. Näiden menetelmien kehittäminen vaatii endogeenisten kantasoluja inhiboivien ja indusoivien mekanismien tuntemista. Yksi tärkeä kantasolujen erilaistumista stimuloiva tekijä on kalsiumioni. Jänniteherkät kalsiumkanavat osallistuvat kaikkiin neurogeneesiksen eri vaiheisiin. T-tyypin kalsiumkanava, joka ekspressoituu suuressa määrin keskushermoston kehityksen alkuvaiheessa ja vähenee neuronaalisen kehityksen edetessä, saattaa olla oleellisessa asemassa progeniittorisolujen ohjaamisessa.

Defining the pathway to insulin-like growth factor system targeting in cancer

The insulin-like growth factors (IGEs; IGF-1 and IGF-2) play central roles in cell growth, differentiation, survival, transformation and metastasis. The biologic effects of the IGFs are mediated by the IGF-1 receptor (IGF-1R), a receptor tyrosine kinase with homology to the insulin receptor (IR). Dysregulation of the ICE system is well recognized as a key contributor to the progression of multiple cancers, with IGF-1R activation increasing the tumorigenic potential of breast, prostate, lung, colon and head and neck squamous cell carcinoma (HNSCC). Despite this relationship, targeting the IGF-1R has only recently undergone development as a molecular cancer therapeutic. As it has taken hold, we are witnessing a robust increase and interest in targeting the inhibition of IGF-1R signaling. This is accentuated by the list of over 30 drugs, including monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs) that are under evaluation as single agents or in combination therapies 1]. The ICE-binding proteins (IGFBPs) represent the third component of the ICE system consisting of a class of six soluble secretory proteins. They represent a unique class of naturally occurring ICE-antagonists that bind to and sequester IGF-1 and IGF-2, inhibiting their access to the IGF-1R. Due to their dual targeting of the IGFs without affecting insulin action, the IGFBPs are an untapped ``third'' class of IGF-1R inhibitors. in this commentary, we highlight some of the significant aspects of and prospects for targeting the IGF-1R and describe what the future may hold. (C) 2010 Elsevier Inc. All rights reserved.

Preparation and characterization of immunoliposomes for targeting of antiviral agents

Antibodies specific to avian myeloblastosis virus envelope glycoprotein gp80 were raised. Immunoliposomes were prepared using anti-avian myeloblastosis virus envelope glycoprotein gp80 antibody. The antibody was palmitoylated to facilitate its incorporation into lipid bilayers of liposomes. The fluorescence emission spectra of palmitoylated IgG have exhibited a shift in emission maximum from 330 to 370 nm when it was incorporated into the liposomes. At least 50% of the incorporated antibody molecules were found to be oriented towards the outside in the liposomes. The average size of the liposome was found to be 300 A, and on an average, 15 antibody molecules were shown to be present in a liposome. When adriamycin encapsulated in immunoliposomes was incubated in a medium containing serum for 72 h, about 75% of the drug was retained in liposomes. In vivo localization studies, revealed an enhanced delivery of drug encapsulated in immunoliposomes to the target tissue, as compared to free drug or drug encapsulated in free liposomes. These data suggest a possible use of the drugs encapsulated in immunoliposomes to deliver the drugs in target areas, thereby reducing side effects caused by antiviral agents.

Basic and translational studies on species B adenoviruses

Human adenoviruses (Ads) have been classified into six species (A to F) currently containing 55 serotypes. For almost 2 decades vectors derived from group C serotype Ad5 have been extensively used for gene transfer studies. These Ad5 based vectors are able to efficiently infect many mammalian cell types (including both mitotic and post-mitotic cells) through interaction with a primary attachment receptor, the coxsackie and adenovirus receptor (CAR). Despite the many advantages of Ad5 based vectors a number of limitations have affected their therapeutic application to many diseases. Although they can transduce many tissue types, Ad5 based vectors are unable to efficiently transduce several potential disease target cell types, including hematopoietic stem cells and malignant tumor cells. Therefore, newer vectors have been developed based on Ad serotypes other than Ad5. This thesis focuses on species B Ads. Species B Ads are comprised of three groups based on their receptor usage. Group 1 of species B Ads (Ad16, 21, 35, 50) nearly exclusively utilize CD46 as a receptor; Group 2 (Ad3, Ad7, 14) share a common, unidentified receptor/s, which is not CD46 and which was tentatively named receptor X; Group 3 (Ad11) preferentially interacts with CD46, but also utilizes receptor X if CD46 is blocked. Species B group Ads are important human pathogens. Species B group 2 serotypes are isolated from patients with respiratory tract infections, whereas the Group 1 viruses are described as causing kidney and urinary tract infections. B-group Ad infections often occur in immunocompromised patients, including AIDS patients, recipients of bone marrow transplants, or chemotherapy patients. Recent studies performed in U.S. military training facilities indicate an emergence of diverse species B serotypes at the majority of sites. This included the group 1 serotype 21 and the group 2 serotypes 3, 7, and 14. CD46-targeting vectors derived from Ad35 and Ad11 are important tools for in vitro gene transfer into human stem cells, including hematopoietic stem cells and induced pluripotent stem cells. Ad35 and Ad11 have been used as tools for cancer therapy, because CD46 appears to be uniformely overexpressed on many cancers. Furthermore, receptor X-targeting vectors, i.e vectors derived from Ad3 or vectors containing Ad3 fibers have shown superior in the transduction of tumor cells both in vitro and in vivo and are currently being used clinically in cancer patients. While extensive basic virology studies have been done on Ad5, the information of species B group 1 interaction with CD46 is limited. Furthermore, the receptor for a major subgroup of species B Ads (receptor X) is unknown. The goal of this thesis was it therefore to better understand virological and translational aspects of species B Ads. The specific findings described in this thesis include i) the identification of CD46 binding sites within the Ad35 fiber knob, ii) the study of the in vitro and in vivo properties of Ad vectors with increased affinity to CD46. iii) the study of the receptor usage of a newly emergent Ad14a, iv) the identification of desmoglein 2 as the receptor for Ad3, Ad7, Ad11, and Ad14, v) the delineation of structural details of Ad3 virus interaction with DSG2, and vi) the analysis of functional consequences of Ad3-DSG2 interaction. As a result of these basic virology studies two Ad-derived recombinant proteins have been generated that can be used to enhance cancer therapy by monoclonal antibodies.

Transcription factors that mediate epithelial-mesenchymal transition lead to multidrug resistance by upregulating ABC transporters

Development of multidrug resistance (MDR) is a major deterrent in the effective treatment of metastatic cancers by chemotherapy. Even though MDR and cancer invasiveness have been correlated, the molecular basis of this link remains obscure. We show here that treatment with chemotherapeutic drugs increases the expression of several ATP binding cassette transporters (ABC transporters) associated with MDR, as well as epithelial-mesenchymal transition (EMT) markers, selectively in invasive breast cancer cells, but not in immortalized or non-invasive cells. Interestingly, the mere induction of an EMT in immortalized and non-invasive cell lines increased their expression of ABC transporters, migration, invasion, and drug resistance. Conversely, reversal of EMT in invasive cells by downregulating EMT-inducing transcription factors reduced their expression of ABC transporters, invasion, and rendered them more chemosensitive. Mechanistically, we demonstrate that the promoters of ABC transporters carry several binding sites for EMT-inducing transcription factors, and overexpression of Twist, Snail, and FOXC2 increases the promoter activity of ABC transporters. Furthermore, chromatin immunoprecipitation studies revealed that Twist binds directly to the E-box elements of ABC transporters. Thus, our study identifies EMT inducers as novel regulators of ABC transporters, thereby providing molecular insights into the long-standing association between invasiveness and MDR. Targeting EMT transcription factors could hence serve as novel strategies to curb both metastasis and the associated drug resistance. Cell Death and Disease (2011) 2, e179; doi:10.1038/cddis.2011.61; published online 7 July 2011

RAD51 as a potential biomarker and therapeutic target for pancreatic cancer

Chemotherapy is a very important therapeutic strategy for cancer treatment. The failure of conventional and molecularly targeted chemotherapeutic regimes for the treatment of pancreatic cancer highlights a desperate need for novel therapeutic interventions. Chemotherapy often fails to eliminate all tumor cells because of intrinsic or acquired drug resistance, which is the most common cause of tumor recurrence. Overexpression of RAD51 protein, a key player in DNA repair/recombination has been observed in many cancer cells and its hyperexpression is implicated in drug resistance. Recent studies suggest that RAD51 overexpression contributes to the development, progression and drug resistance of pancreatic cancer cells. Here we provide a brief overview of the available pieces of evidence in support of the role of RAD51 in pancreatic tumorigenesis and drug resistance, and hypothesize that RAD51 could serve as a potential biomarker for diagnosis of pancreatic cancer. We discuss the possible involvement of RAD51 in the drug resistance associated with epithelial to mesenchymal transition and with cancer stem cells. Finally, we speculate that targeting RAD51 in pancreatic cancer cells may be a novel approach for the treatment of pancreatic cancer. (C) 2011 Elsevier B.V. All rights reserved.

C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C

Phosphoinositide-specific phospholipase C (PLC) is involved in Ca2+ mediated signalling events that lead to altered cellular status. Using various sequence-analysis methods, we identified two conserved motifs in known PLC sequences. The identified motifs are located in the C2 domain of plant PLCs and are not found in any other protein. These motifs are specifically found in the Ca2+ binding loops and form adjoining beta strands. Further, we identified certain conserved residues that are highly distinct from corresponding residues of animal PLCs. The motifs reported here could be used to annotate plant-specific phospholipase C sequences. Furthermore, we demonstrated that the C2 domain alone is capable of targeting PLC to the membrane in response to a Ca2+ signal. We also showed that the binding event results from a change in the hydrophobicity of the C2 domain upon Ca2+ binding. Bioinformatic analyses revealed that all PLCs from Arabidopsis and rice lack a transmembrane domain, myristoylation and GPI-anchor protein modifications. Our bioinformatic study indicates that plant PLCs are located in the cytoplasm, the nucleus and the mitochondria. Our results suggest that there are no distinct isoforms of plant PLCs, as have been proposed to exist in the soluble and membrane associated fractions. The same isoform could potentially be present in both subcellular fractions, depending on the calcium level of the cytosol. Overall, these data suggest that the C2 domain of PLC plays a vital role in calcium signalling.

Higher topoisomerase 2 alpha gene transcript levels predict better prognosis in GBM patients receiving temozolomide chemotherapy: identification of temozolomide as a TOP2A inhibitor

The search for molecular markers which predict response to chemotherapy is an important aspect of current neuro-oncology research. MGMT promoter methylation is the only proved marker of glioblastoma. The purpose of this study was to assess the effect of topoisomerase expression on glioblastoma survival and study the mechanisms involved. The transcript levels of all isoforms of the topoisomerase family in all grades of diffuse astrocytoma were assessed. A prospective study of patients with glioblastoma treated by a uniform treatment procedure was performed with the objective of correlating outcome with gene expression. The ability of TOP2A enzyme to relax the super coiled plasmid DNA in the presence of temozolomide was evaluated to assess its effect on TOP2A. The temozolomide cyctotoxicity of TOP2A-silenced U251 cells was assessed. The transcript levels of TOP2A, TOP2B, and TOP3A are upregulated significantly in GBM in comparison with lower grades of astrocytoma and normal brain samples. mRNA levels of TOP2A correlated significantly with survival of the patients. Higher TOP2A transcript levels in GBM patients predicted better prognosis (P = 0.043; HR = 0.889). Interestingly, we noted that temozolomide inhibited TOP2A activity in in-vitro enzyme assays. We also noted that siRNA knock down of TOP2A rendered a glioma cell line resistant to temozolomide chemotherapy. We demonstrated for the first time that temozolomide is also a TOP2A inhibitor and established that TOP2A transcript levels determine the chemosensitivity of glioblastoma to temozolomide therapy. Very high levels of TOP2A are a good prognostic indicator in GBM patients receiving temozolomide chemotherapy.

Targeting ribosome assembly on the HCV RNA using a small RNA molecule

Translation initiation of hepatitis C virus (HCV) RNA is the initial obligatory step of the viral life cycle, mediated through the internal ribosome entry site (IRES) present in the 5'-untranslated region (UTR). Initiation on the HCV IRES is mediated by multiple structure-specific interactions between IRES RNA and host 40S ribosomal subunit. In the present study we demonstrate that the SLIIIef domain, in isolation from other structural elements of HCV IRES, retain the ability to interact with 40S ribosome subunit. A small RNA SLRef, mimicking the SLIIIef domain was found to interact specifically with human La protein and the ribosomal protein S5 and selectively inhibit HCV RNA translation. More importantly, SLRef RNA showed significant suppression of replication in HCV monocistronic replicon and decrease of negative strand synthesis in HCV cell culture system. Finally, using Sendai virus based virosome, the targeted delivery of SLRef RNA into mice liver succeeded in selectively inhibiting HCV IRES mediated translation in vivo.

DNA targeting polyaza macrobicyclic dizinc(II) complexes promoting high in vitro caspase dependent anti-proliferative activity against human carcinoma cancer cells

A series of macrobicyclic dizinc(II) complexes Zn2L1-2B](ClO4)(4) (1-6) have been synthesized and characterized (L1-2 are polyaza macrobicyclic binucleating ligands, and B is the N,N-donor heterocyclic base (viz. 2,2'-bipyridine (bipy) and 1,10-phenanthroline (phen)). The DNA and protein binding, DNA hydrolysis and anticancer activity of these complexes were investigated. The interactions of complexes 1-6 with calf thymus DNA were studied by spectroscopic techniques, including absorption, fluorescence and CD spectroscopy. The DNA binding constant values of the complexes were found to range from 2.80 x 10(5) to 5.25 x 10(5) M-1, and the binding affinities are in the following order: 3 > 6 > 2 > 5 > 1 > 4. All the dizinc(II) complexes 1-6 are found to effectively promote the hydrolytic cleavage of plasmid pBR322 DNA under anaerobic and aerobic conditions. Kinetic data for DNA hydrolysis promoted by 3 and 6 under physiological conditions give observed rate constants (k(obs)) of 5.56 +/- 0.1 and 5.12 +/- 0.2 h(-1), respectively, showing a 10(7)-fold rate acceleration over the uncatalyzed reaction of dsDNA. Remarkably, the macrobicyclic dizinc(II) complexes 1-6 bind and cleave bovine serum albumin (BSA), and effectively promote the caspase-3 and caspase-9 dependent deaths of HeLa and BeWo cancer cells. The cytotoxicity of the complexes was further confirmed by lactate dehydrogenase enzyme levels in cancer cell lysate and content media.

Nuclear targeting terpyridine iron(II) complexes for cellular imaging and remarkable photocytotoxicity

Iron(II) complexes Fe(L)(2)](2+) as perchlorate (1-3) and chloride (1a-3a) salts, where L is 4'-phenyl-2,2':6',2 `'-terpyridine (phtpy in 1, 1a), 4'-(9-anthracenyl)-2,2':6',2 `'-terpyridine (antpy in 2, 2a) and 4'-(1-pyrenyl)-2,2':6',2 `'-terpyridine (pytpy in 3, 3a), were prepared and their photocytotoxicity studied. The diamagnetic complexes 1-3 having an FeN6 core showed an Fe(III)-Fe(II) redox couple near 1.0 V vs. saturated calomel electrode in MeCN-0.1 M tetrabutylammonium perchlorate. Complexes 2 and 3, in addition, displayed a quasi-reversible ligand-based redox process near 0.0 V. The redox and spectral properties are rationalized from the theoretical studies. The complexes bind to DNA in a partial intercalative mode. The pytpy complex efficiently photo-cleaves DNA in green light via superoxide and hydroxyl radical formation. The antpy and pytpy complexes exhibited a remarkable photocytotoxic effect in HeLa cancer cells (IC50, similar to 9 mu M) in visible light (400-700 nm), while remaining essentially nontoxic in dark (IC50, similar to 90 mu M). Formation of reactive oxygen species (ROS) inside the HeLa cells was evidenced from the fluorescence enhancement of dichlorofluorescein upon treatment with the pytpy complex followed by photo-exposure. The antpy and pytpy complexes were used for cellular imaging. Confocal imaging and dual staining study using propidium iodide (PI) showed nuclear localization of the complexes. (c) 2012 Elsevier Inc. All rights reserved.

miR-219-5p inhibits receptor tyrosine kinase pathway by targeting EGFR in glioblastoma

Glioblastoma is one of the common types of primary brain tumors with a median survival of 12-15 months. The receptor tyrosine kinase (RTK) pathway is known to be deregulated in 88% of the patients with glioblastoma. 45% of GBM patients show amplifications and activating mutations in EGFR gene leading to the upregulation of the pathway. In the present study, we demonstrate that a brain specific miRNA, miR-219-5p, repressed EGFR by directly binding to its 3'-UTR. The expression of miR-219-5p was downregulated in glioblastoma and the overexpression of miR-219-5p in glioma cell lines inhibited the proliferation, anchorage independent growth and migration. In addition, miR-219-5p inhibited MAPK and PI3K pathways in glioma cell lines in concordance with its ability to target EGFR. The inhibitory effect of miR-219-5p on MAPK and PI3K pathways and glioma cell migration could be rescued by the overexpression of wild type EGFR and vIII mutant of EGFR (both lacking 3'-UTR and thus being insensitive to miR-219-5p) suggesting that the inhibitory effects of miR-219-5p were indeed because of its ability to target EGFR. We also found significant negative correlation between miR-219-5p levels and total as well as phosphorylated forms of EGFR in glioblastoma patient samples. This indicated that the downregulation of miR-219-5p in glioblastoma patients contribute to the increased activity of the RTK pathway by the upregulation of EGFR. Thus, we have identified and characterized miR-219-5p as the RTK regulating novel tumor suppressor miRNA in glioblastoma.

Biotin-conjugated tumour-targeting photocytotoxic iron(III) complexes

Iron(III) complexes FeL(B)] (1-4) of a tetradentate phenolate-based ligand (H3L) and biotin-conjugated dipyridophenazine bases (B), viz. 7-aminodipyrido 3,2-a: 2',3'-c]-phenazine (dppza in 1), (N-dipyrido3,2-a: 2',3'-c]-phenazino) amidobiotin (dppzNB in 2), dipyrido 3,2-a: 2',3'-c]-phenazine-11-carboxylic acid (dppzc in 3) and 2-((2-biotinamido) ethyl) amidodipyrido 3,2-a: 2',3'-c]-phenazine (dppzCB in 4) are prepared, characterized and their interaction with streptavidin and DNA and their photocytotoxicity and cellular uptake in various cells studied. The high-spin iron(III) complexes display Fe(III)/Fe(II) redox couple near -0.7V versus saturated calomel electrode in dimethyl sulfoxide-0.1M tetrabutylammonium perchlorate. The complexes show non-specific interaction with DNA as determined from the binding studies. Complexes with appended biotin moiety show similar binding to streptavidin as that of free biotin, suggesting biotin conjugation to dppz does not cause any loss in its binding affinity to streptavidin. The photocytotoxicity of the complexes is tested in HepG2, HeLa and HEK293 cell lines. Complex 2 shows higher photocytotoxicity in HepG2 cells than in HeLa or HEK293, forming reactive oxygen species. This effect is attributed to the presence of overexpressed sodium-dependent multi-vitamin transporters in HepG2 cells. Microscopic studies in HepG2 cells show internalization of the biotin complexes 2 and 4 essentially occurring by receptor-mediated endocytosis, which is similar to that of native biotin and biotin fluorescein isothiocyanate conjugate.

Investigational drugs for schizophrenia targeting the dopamine receptor: phase II trials

Introduction: For over half a century now, the dopamine hypothesis has provided the most widely accepted heuristic model linking pathophysiology and treatment in schizophrenia. Despite dopaminergic drugs being available for six decades, this system continues to represent a key target in schizophrenia drug discovery. The present article reviews the scientific rationale for dopaminergic medications historically and the shift in our thinking since, which is clearly reflected in the investigational drugs detailed. Areas covered: We searched for investigational drugs using the key words `dopamine,' `schizophrenia,' and `Phase II' in American and European clinical trial registers (clinicaltrials. gov; clinicaltrialsregister.eu), published articles using National Library of Medicine's PubMed database, and supplemented results with a manual search of cross-references and conference abstracts. We provide a brief description of drugs targeting dopamine synthesis, release or metabolism, and receptors (agonists/partial agonists/antagonists). Expert opinion: There are prominent shifts in how we presently conceptualize schizophrenia and its treatment. Current efforts are not as much focused on developing better antipsychotics but, instead, on treatments that can improve other symptom domains, in particular cognitive and negative. This new era in the pharmacotherapy of schizophrenia moves us away from the older `magic bullet' approach toward a strategy fostering polypharmacy and a more individualized approach shaped by the individual's specific symptom profile.