Kathy W. Y. Sham

Identification of a Membrane Estrogen Receptor in Zebrafish with Homology to Mammalian GPER and Its High Expression in Early Germ Cells of the Testis

Abstract To study the rapid action of estrogen on the male reproductive system in teleost, a full-length cDNA homologous to the seven-transmembrane receptor GPER of humans and rodents was cloned from the testis of zebrafish. Biological characterization of this cloned zebrafish gper was performed based on its functional expression in cultured eukaryotic cells. Saturation analysis and Scatchard plotting of [3H]-estradiol binding to plasma membranes of gper-transfected COS-7 cells and cAMP response element transactivation assay demonstrated the biological function of the cloned gper as an estrogen receptor. In addition, treatment of gper-transfected COS-7 cells with 17beta-estradiol increased the phosphorylation of MAPK3/MAPK1. However, the inactivity of Gper in the FOS promoter transactivation study indicated some functional difference between the zebrafish and human receptors. We found gper to be highly expressed in the brain and testis by RT-PCR analysis. Results of in situ hybridization demonstrated the localization of gper in specific brain regions and in early germ cells of the testis, including the spermatogonia, spermatocytes, and somatic cells such as Sertoli cells in adult male zebrafish. Subsequent RT-PCR analysis in cells derived from laser capture microdissection microscopy further confirmed the high expression of gper in early germ cells of the testis. The present study demonstrates the existence of a functionally active Gper in zebrafish and suggests a putative role in mediating the rapid action of estrogen in male reproduction.

Hollow superparamagnetic iron oxide nanoshells as a hydrophobic anticancer drug carrier: intracelluar pH-dependent drug release and enhanced cytotoxicity

With curcumin and doxorubicin (DOX) base as model drugs, intracellular delivery of hydrophobic anticancer drugs by hollow structured superparamagnetic iron oxide (SPIO) nanoshells (hydrodynamic diameter: 191.9 ± 2.6 nm) was studied in glioblastoma U-87 MG cells. SPIO nanoshell-based encapsulation provided a stable aqueous dispersion of the curcumin. After the SPIO nanoshells were internalized by U-87 MG cells, they localized at the acidic compartments of endosomes and lysosomes. In endosome/lysosome-mimicking buffers with a pH of 4.5-5.5, pH-dependent drug release was observed from curcumin or DOX loaded SPIO nanoshells (curcumin/SPIO or DOX/SPIO). Compared with the free drug, the intracellular curcumin content delivered via curcumin/SPIO was 30 fold higher. Increased intracellular drug content for DOX base delivered via DOX/SPIO was also confirmed, along with a fast intracellular DOX release that was attributed to its protonation in the acidic environment. DOX/SPIO enhanced caspase-3 activity by twofold compared with free DOX base. The concentration that induced 50% cytotoxic effect (CC(50)) was 0.05 ± 0.03 μg ml(-1) for DOX/SPIO, while it was 0.13 ± 0.02 μg ml(-1) for free DOX base. These results suggested SPIO nanoshells might be a promising intracellular carrier for hydrophobic anticancer drugs.

Photocytotoxicity and magnetic relaxivity responses of dual-porous γ-Fe2O3@meso-SiO2 microspheres.

Novel high magnetization microspheres with porous γ-Fe(2)O(3) core and porous SiO(2) shell were synthesized using a templating method, whereas the size of the magnetic core and the thickness of the porous shell can be controlled by tuning the experimental parameters. By way of an example, as-prepared γ-Fe(2)O(3)@meso-SiO(2) microspheres (170 nm) display excellent water-dispersity and show photonic characteristics under externally applied a magnetic field. The magnetic property of the γ-Fe(2)O(3) porous core enables the microspheres to be used as a contrast agent in magnetic resonance imaging with a high r(2) (76.5 s(-1) mM(-1) Fe) relaxivity. The biocompatible composites possess a large BET surface area (222.3 m(2)/g), demonstrating that they can be used as a bifunctional agent for both MRI and drug carrier. Because of the high substrate loading of the magnetic, dual-porous materials, only a low dosage of the substrate will be acquired for potential practical applications. Hydrophobic zinc(II) phthalocyanine (ZnPC) photosensitizing molecules have been encapsulated into the dual-porous microspheres to form γ-Fe(2)O(3)@meso-SiO(2)-ZnPC microspheres. Biosafety, cellular uptake in HT29 cells, and in vitro MRI of these nanoparticles have been demonstrated. Photocytotoxicity (λ > 610 nm) of the HT29 cells uptaken with γ-Fe(2)O(3)@meso-SiO(2)-ZnPC microspheres has been demonstrated for 20 min illumination.

Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells.

EEF2K (eukaryotic elongation factor-2 kinase), also known as Ca2+/calmodulin-dependent protein kinase III, functions in downregulating peptide chain elongation through inactivation of EEF2 (eukaryotic translation elongation factor 2). Currently, there is a limited amount of information on the promotion of autophagic survival by EEF2K in breast and glioblastoma cell lines. However, the precise role of EEF2K in carcinogenesis as well as the underlying mechanism involved is still poorly understood. In this study, contrary to the reported autophagy-promoting activity of EEF2K in certain cancer cells, EEF2K is shown to negatively regulate autophagy in human colon cancer cells as indicated by the increase of LC3-II levels, the accumulation of LC3 dots per cell, and the promotion of autophagic flux in EEF2K knockdown cells. EEF2K negatively regulates cell viability, clonogenicity, cell proliferation, and cell size in colon cancer cells. Autophagy induced by EEF2K silencing promotes cell survival and does not potentiate the anticancer efficacy of the AKT inhibitor MK-2206. In addition, autophagy induced by silencing of EEF2K is attributed to induction of protein synthesis and activation of the AMPK-ULK1 pathway, independent of the suppression of MTOR activity and ROS generation. Knockdown of AMPK or ULK1 significantly abrogates EEF2K silencing-induced increase of LC3-II levels, accumulation of LC3 dots per cell as well as cell proliferation in colon cancer cells. In conclusion, silencing of EEF2K promotes autophagic survival via activation of the AMPK-ULK1 pathway in colon cancer cells. This finding suggests that upregulation of EEF2K activity may constitute a novel approach for the treatment of human colon cancer.

Regulation of the two kiss promoters in goldfish (Carassius auratus) by estrogen via different ERα pathways

Kisspeptin stimulates the synthesis and release of gonadotropin via controlling the secretion of gonadotropin releasing hormone in vertebrates. It also mediates the positive or negative feedback regulation of sex steroids on the hypothalamus-gonadotropic axis. In contrast to mammals, two paralogous genes of kisspeptin (kiss1 and kiss2) have been identified in several teleosts, implying the multiplicity of their physiological functions. In the present study, we cloned the promoters of kiss1 and kiss2 genes in goldfish (Carassius auratus), and identified the presence of putative binding sites for estrogen receptors, glucocorticoid receptors, Sp1, AP1, C/EBP and Oct-1. We further demonstrated that the goldfish Kiss neurons co-express the estrogen receptors, with era1 and erb1 in the habenula Kiss1 neurons and era1, era2 and erb1 in the preoptic and hypothalamic Kiss2 neurons. Using transient transfection in HEK293T cells of the two goldfish kiss gene promoters cloned upstream of a luciferase reporter, estrogen (E2, 17β-estradiol) treatment was shown to enhance the promoter activities of the two goldfish kiss genes in the presence of ERα. Deletion analysis of kiss1 promoter indicated that the E2-induced promoter activity was located between position -633 and -317 where no half ERE motifs were found. Point mutation studies on the kiss2 promoter indicated that the E2-stimulated promoter activity was mediated by a half ERE site located at position -57. Results of the present study provide evidence that E2 is capable of exerting positive feedback regulation on the expression of kiss1 and kiss2 in goldfish via ERE-independent or ERE-dependent ERα pathway, respectively.

G-protein-coupled estrogen receptor 1 is involved in brain development during zebrafish (Danio rerio) embryogenesis

G-protein-coupled estrogen receptor 1 (Gper, formerly known as GPR30) is found to be a trophic and protective factor in mediating action of estrogen in adult brain, while its role in developing brain remains to be elucidated. Here we present the expression pattern of Gper and its functions during embryogenesis in zebrafish. Both the mRNA and protein of Gper were detected throughout embryogenesis. Whole mount in situ hybridization (WISH) revealed a wide distribution of gper mRNAs in various regions of the developing brain. Gper knockdown by specific morpholinos resulted in growth retardation in embryos and morphological defects in the developing brain. In addition, induced apoptosis, decreased proliferation of the brain cells and maldevelopment of sensory and motor neurons were also found in the morphants. Our results provide novel insights into Gper functions in the developing brain, revealing that Gper can maintain the survival of the brain cells, and formation and/or differentiation of the sensory and motor neurons.

Nanoparticle–DNA–polymer composites for hepatocellular carcinoma cell labeling, sensing, and magnetic resonance imaging

This paper describes comparative studies and protocols in (1) self-assembling of ultrasmall superparamagnetic iron oxide nanoparticle (NP), circular plasmid DNA, and branched polyethylenimine (PEI) composites; (2) magnetofection; (3) gene delivery, (4) magnetic resonance imaging (MRI), and (5) cytotoxicity of the composites toward hepatocellular carcinoma HepG2 cells.

Increased Efficacies in Magnetofection and Gene Delivery to Hepatocellular Carcinoma Cells with Ternary Organic–Inorganic Hybrid Nanocomposites

In recent years, liver cancer (hepatocellular carcinoma) has been ranked as the leading cancer in Asia together with lung and stomach cancers. Towards the treatment of human hepatocellular carcinoma, a novel theranostic nanomaterial has yet to be developed for potential simultaneous gene transfer, drug delivery, and magnetic resonance imaging (MRI) enhancement. Superparamagnetic iron oxide nano/microparticles such as ferumoxides and ferucarbotran have been used clinically as contrast agents for MRI in liver. In light of treating human hepatocellular carcinoma, we report herein the use of ternary organic–inorganic hybrid nanocomposites PEI/DNA/NP that contain deferoxamine-coated superparamagnetic iron oxide nanoparticles (NP), circular plasmid DNA (pEGFP-C1 and pRL-CMV), and branched polyethylenimine (PEI, 25 kDa, PDI =2.5), which are potentially suitable for the proposed applications together with transfection studies in HepG2 cells. Only a small amount of branched PEI is employed for forming the stable ternary complex to reduce its cytotoxicity. Plasmid pEGFP-C1 (~4.7 kb) encodes a red-shifted variant of wild-type green fluorescence protein (GFP) for expression in mammalian cells, while pRL-CMV (~4.0 kb) encodes Renilla luciferase (RLU) for expression in various cell types. These two DNAs are commonly used as markers for studying transfection efficacies. The fluorescence/luminescence intensity is directly proportional to the amount of GFP/ RLU, which can be easily detected owing to the strong and constitutive expression of the reporters. By using two signaldetection methods, any reduction of the enhancement of PEI/DNA/NP uptake by cells can be estimated. Deferoxamine is used clinically to treat iron poisoning, as it binds ferric ions in the blood stream and enhances its elimination in urine. Ultrasmall NPs of 8–10 nm in diameter (Figure 1) were attached to deferoxamine ligand at their peripheries in three synthetic steps; their hydroxy functional groups can interact with DNA and PEI, mainly through amide hydrogen bonds and electrostatic interactions. Stable ternary organic– inorganic hybrid nanocomposites of approximately 250 nm in diameter with different component ratios have been prepared in phosphate-buffered saline (PBS) and characterized by light scattering, zeta-potential analyses, mobility tests, etc. Typical Prussian blue staining images of HepG2 cells internalized with six ternary complexes that possess different ratios of components are shown in Figure 2. According to the visual assessment of these images, the cellular uptake efficacy increases as the amount of NPs was increased from 0.1 to 1.0 mg per well. By contrast, the PEI amount of 0.2 ng gives the best cellular uptake efficacy among other amounts [a] Prof. Dr. K. C.-F. Leung, Dr. X.-M. Zhu, C.-H. Wong Department of Chemistry The Hong Kong Baptist University Kowloon Tong, Kowloon, Hong Kong SAR (P. R. China) Fax: (+852) 3411-7348 E-mail : cfleung@hkbu.edu.hk [b] Dr. C.-P. Chak, S.-F. Lee Department of Chemistry The Chinese University of Hong Kong Shatin, NT, Hong Kong SAR (P. R. China) [c] J. M. Y. Lai, K. W. Y. Sham, Prof. Dr. C. H. K. Cheng School of Biomedical Sciences and Center of Novel Functional Molecules The Chinese University of Hong Kong Shatin, NT, Hong Kong SAR (P. R. China) [d] Dr. X.-M. Zhu, Prof. Dr. Y.-X. J. Wang Department of Imaging and Interventional Radiology Prince of Wales Hospital, The Chinese University of Hong Kong Shatin, NT, Hong Kong SAR (P. R. China) [e] Prof. Dr. K. C.-F. Leung Institute of Creativity The Hong Kong Baptist University Kowloon Tong, Kowloon, Hong Kong SAR (P. R. China) [f] Prof. Dr. K. C.-F. Leung Institute of Molecular Functional Materials Areas of Excellence, University Grants Committee Hong Kong SAR (P. R. China) Figure 1. Scheme of the synthetic route to obtain PEI/DNA/NP ternary organic–inorganic hybrid nanocomposites from deferoxamine-coated superparamagnetic iron oxide nanoparticles. Reagents and conditions: a) succinic anhydride, DMF, rt, 24 h; b) N,N’-dicyclohexylcarbodiimide, N-hydroxysuccinimide, THF, rt, 24 h; and c) deferoxamine, DMF, rt, 24 h.

GPER mediates the inhibitory actions of estrogen on adipogenesis in 3T3-L1 cells through perturbation of mitotic clonal expansion.

G-protein-coupled estrogen receptor 1 (GPER) mediates non-genomic signaling of estrogenic events. Here we showed for the first time that Gper/GPER is expressed in Swiss 3T3 mouse embryo preadipocytes 3T3-L1, and that Gper/GPER is up-regulated during differentiation of the cells induced by monocyte differentiation-inducing (MDI) cocktail. Activation of GPER by the natural ligand 17β-estradiol (E2), and the specific agonist G1, was shown to inhibit lipid accumulation in 3T3-L1 cells, while such inhibition was reversed upon knockdown of GPER using specific siRNA. GPER was also found to mediate perturbation of mitotic clonal expansion (MCE) in these cells by inhibiting cell cycle arrest during MDI cocktail-induced differentiation. Persistent activation of cell cycle regulating factors cyclin-dependant kinase (CDK) 4, CDK6 and cyclin D1, and phosphorylation of retinoblastoma (Rb) protein at serine 795 was observed in the G1-treated cells. Taken together, our results indicate that E2-GPER signaling leads to an inhibition of adipogenesis in 3T3-L1 cells via perturbation of MCE.

DNA hybridization of pathogenicity island of vancomycin -resistant Enterococcus faecalis with discretely functionalized gold nanoparticles in organic solvent mixtures

Short single-strand (ss) DNA sensors that involve mono-functionalized gold nanoparticles are reported for the first time. By way of an example, the hybridization and detection of a 10 base-pair oligonucleotide (TGACTCTTAA), which relates to the pathogenicity island of vancomycin-resistant Enterococcus faecalis, has been demonstrated in the organic solvent mixture (DMF/CHCl3/H2O/NaCl) at sub-nanomole concentrations. The formation of the resulting double-strand (ds) DNA–AuNP complexes which involve either tail-to-tail or head-to-head alignment, are sensitive to salt (NaCl) and solvent (CHCl3) concentrations. By UV/visible absorption measurements, it has been found that the melting temperature of the dsDNA–AuNP complexes increases with increasing salt concentrations as well as the chloroform ratio, for both tail-to-tail and head-to-head alignments. Transmission electron microscopic images of the dsDNA–AuNP complexes with mono-functionalized AuNPs reveal significant amounts of dimeric AuNP species while that of randomly functionalized AuNPs reveal an agglomerated AuNP nanostructures.