High somatostatin receptor expression and efficacy of somatostatin analogues in patients with metastatic Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive, high-grade, cutaneous neuroendocrine tumour (NET). Agents blocking programmed death 1/programmed death ligand 1 have efficacy in metastatic MCC (mMCC), but half of patients do not derive durable benefit. Somatostatin analogues (SSAs) are commonly used to treat low- and moderate-grade NETs that express somatostatin receptors (SSTRs). OBJECTIVES: To assess SSTR expression and the efficacy of SSAs in mMCC, a high-grade NET. Methods In this retrospective study of 40 patients with mMCC, SSTR expression was assessed radiologically by somatostatin receptor scintigraphy (SRS; n = 39) and/or immunohistochemically when feasible (n = 9). Nineteen patients (18 had SRS uptake in MCC tumours) were treated with SSA. Disease control was defined as progression-free survival (PFS) of ≥ 120 days after initiation of SSA. RESULTS: Thirty-three of 39 patients (85%) had some degree (low 52%, moderate 23%, high 10%) of SRS uptake. Of 19 patients treated with SSA, seven had a response-evaluable target lesion; three of these seven patients (43%) experienced disease control, with a median PFS of 237 days (range 152-358). Twelve of 19 patients did not have a response-evaluable lesion due to antecedent radiation; five of these 12 (42%) experienced disease control (median PFS of 429 days, range 143-1757). The degree of SSTR expression (determined by SRS and/or immunohistochemistry) did not correlate significantly with the efficacy endpoints. CONCLUSIONS: In contrast to other high-grade NETs, mMCC tumours appear frequently to express SSTRs. SSAs can lead to clinically meaningful disease control with minimal side-effects. Targeting of SSTRs using SSA or other novel approaches should be explored further for mMCC.

Control of protein function through oxidation and reduction of persulfidated states.

Irreversible oxidation of Cys residues to sulfinic/sulfonic forms typically impairs protein function. We found that persulfidation (CysSSH) protects Cys from irreversible oxidative loss of function by the formation of CysSSO1-3H derivatives that can subsequently be reduced back to native thiols. Reductive reactivation of oxidized persulfides by the thioredoxin system was demonstrated in albumin, Prx2, and PTP1B. In cells, this mechanism protects and regulates key proteins of signaling pathways, including Prx2, PTEN, PTP1B, HSP90, and KEAP1. Using quantitative mass spectrometry, we show that (i) CysSSH and CysSSO3H species are abundant in mouse liver and enzymatically regulated by the glutathione and thioredoxin systems and (ii) deletion of the thioredoxin-related protein TRP14 in mice altered CysSSH levels on a subset of proteins, predicting a role for TRP14 in persulfide signaling. Furthermore, selenium supplementation, polysulfide treatment, or knockdown of TRP14 mediated cellular responses to EGF, suggesting a role for TrxR1/TRP14-regulated oxidative persulfidation in growth factor responsiveness.

8-Nitro-cGMP is a promoter of osteoclast differentiation induced by RANKL.

Osteoclasts are multinucleated giant cells differentiated from monocyte-macrophage-lineage cells under stimulation of receptor activator of nuclear factor κ-B (RANK) ligand (RANKL) produced by osteoblasts and osteocytes. Although it has been reported that nitric oxide (NO) and reactive oxygen species (ROS) are involved in this process, the mechanism by which these labile molecules promote osteoclast differentiation are not fully understood. In this study, we investigated the formation and function of 8-nitro-cGMP, a downstream molecule of NO and ROS, in the process of osteoclast differentiation in vitro. 8-Nitro-cGMP was detected in mouse bone marrow macrophages and osteoclasts differentiated from macrophages in the presence of RANKL. Inhibition of NO synthase suppressed the formation of 8-nitro-cGMP as well as RANKL-induced osteoclast differentiation from macrophages. On the other hand, RANKL-induced osteoclast differentiation was promoted by addition of 8-nitro-cGMP to the cultures. In addition, 8-nitro-cGMP enhanced the mRNA expression of RANK, the receptor for RANKL. However, 8-bromo-cGMP, a membrane-permeable derivative of cGMP, did not have an effect on either RANKL-induced osteoclast differentiation or expression of the RANK gene. These results suggest that 8-nitro-cGMP is a novel positive regulator of osteoclast differentiation, which might help to explain the roles of NO and ROS in osteoclast differentiation.

Nitric oxide/soluble guanylyl cyclase signaling mediates depolarization-induced protection of rat mesencephalic dopaminergic neurons from MPP⁺ cytotoxicity.

Neuronal electrical activity has been known to affect the viability of neurons in the central nervous system. Here we show that long-lasting membrane depolarization induced by elevated extracellular K(+) recruits nitric oxide (NO)/soluble guanylyl cyclase/protein kinase G signaling pathway, induces 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP)-mediated protein S-guanylation, and confers dopaminergic neuroprotection. Treatment of primary mesencephalic cell cultures with 1-methyl-4-phenylpyridinium (MPP(+)) for 72 h decreased the number of dopaminergic neurons, whereas the cell loss was markedly inhibited by elevated extracellular concentration of K(+) (+40 mM). The neuroprotective effect of elevated extracellular K(+) was significantly attenuated by tetrodotoxin (a Na(+) channel blocker), amlodipine (a voltage-dependent Ca(2+) channel blocker), N(ω)-nitro-l-arginine methyl ester (l-NAME) (a nitric oxide synthase inhibitor), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (a soluble guanylyl cyclase inhibitor), and KT5823 or Rp-8-bromo-ß-phenyl-1,N(2)-ethenoguanosine 3',5'-cyclic monophosphorothioate (Rp-8-Br-PET-cGMPS) (protein kinase G inhibitors). Elevated extracellular K(+) increased 8-nitro-cGMP production resulting in the induction of protein S-guanylation in cells in mesencephalic cultures including dopaminergic neurons. In addition, exogenous application of 8-nitro-cGMP protected dopaminergic neurons from MPP(+) cytotoxicity, which was prevented by zinc protoporphyrin IX, an inhibitor of heme oxygenase-1 (HO-1). Zinc protoporphyrin IX also inhibited the neuroprotective effect of elevated extracellular K(+). On the other hand, KT5823 or Rp-8-Br-PET-cGMPS did not inhibit the induction of HO-1 protein expression by 8-nitro-cGMP, although these protein kinase G inhibitors abrogated the neuroprotective effect of 8-nitro-cGMP. These results suggest that protein S-guanylation (leading to HO-1 induction) as well as canonical protein kinase G signaling pathway plays an important role in NO-mediated, activity-dependent dopaminergic neuroprotection.

Adsorption behaviors of recombinant E-cadherin-IgG Fc fusion protein on polystyrene surface.

Adsorption behaviors of recombinant E-cadherin-IgG Fc (E-cad-Fc) fusion protein and mutated E-cad-Fcs on the polystyrene (PS) surface were investigated using a 27 MHz quartz-crystal microbalance (QCM) and ELISA. The amount of adsorbed E-cad-Fc on PS surface was increased with an increase of E-cad-Fc concentration as a Langmuir-type in a monolayer. Adsorbed E-cad-Fc on PS surface was stable even after washing if calcium ions are absent in the washing solution due to the calcium ion dependence in the adsorption. E-cadherin homophilic adhesion among E-cadherins during adsorption of E-cad-Fc was involved. Deglycosylation of the E-cad in the E-cad-Fc did not affect adsorption of E-cad-Fc on the PS surface although deglycosylation of the E-cad in the E-cad-Fc enhanced cell adhesion compared with E-cad-Fc.

Degradable polyethylenimines as DNA and small interfering RNA carriers.

Gene therapy is a powerful approach in the treatment of a wide range of both inherited and acquired diseases. Nonviral delivery systems have been proposed as safer alternatives to viral vectors because they avoid the inherent immunogenicity and production problems that are seen when viral systems are used. Many cationic polymers, including high-molecular-weight polyethylenimine (PEI) have been widely studied as gene-delivery carriers, both, in vitro and in vivo. However, interest has recently developed in degradable polymeric systems. The advantage of degradable polymer is its low in-vivo cytotoxicity, which is a result of its easy elimination from the cells and body. Degradable polymer also enhances transfection of DNA or small interfering RNA (siRNA) for efficient gene expression or silencing, respectively. This review paper summarizes and discusses the recent advances with degradable PEIs, such as cross-linked and grafted PEIs for DNA and siRNA delivery.

Disrupting actin filaments promotes efficient transfection of a leukemia cell line using cell adhesive protein-embedded carbonate apatite particles.

Tumor cells such as leukemia and lymphoma cells are obvious and attractive targets for gene therapy. Gene transfer and expression for cytokine and immunomodulatory molecules in various kinds of tumor cells have been shown to mediate tumor regression and antimetastatic effects. Moreover, genetically modified leukemia cells expressing costimulatory molecules or cytokines are likely to have significant therapeutic roles for patients with leukemia. One of the major hurdles to the successful implementation of these promising approaches is the lack of a suitable nanocarrier for transgene delivery and expression in a safe and effective manner. Recently, we reported on the development of a safe, efficient nanocarrier system of carbonate apatite that can assist both intracellular delivery and release of DNA, leading to very high level of transgene expression in cancer and primary cells. However, its efficiency in human lymphocytes is poor. We show here that nanocrystals of carbonate apatite, when electrostatically associated with fibronectin and/or E-cadherin-Fc, accelerated transgene delivery in a human T leukemia cell line (Jurkat). Moreover, transgene expression efficiency could be enhanced dramatically with the cell adhesive protein-embedded particles finally up to 150 times by selectively disrupting the actin filaments.

The influence of the cell-adhesive proteins E-cadherin and fibronectin embedded in carbonate-apatite DNA carrier on transgene delivery and expression in a mouse embryonic stem cell line.

Stem cells have the potential to be differentiated to a specific cell type through genetic manipulation and therefore, represent a new and versatile source of cell replacement in regenerative medicine. However, conventional ways of gene transfer to these progenitor cells, suffer from a number of disadvantages particularly involving safety and efficacy issues. We have recently reported on the development of a bio-functionalized DNA carrier of carbonate apatite by embedding fibronectin and E-cadherin chimera on the carrier, leading to its high-affinity interactions with embryonic stem cell surface and accelerated transgene delivery for subsequent expression. Here, we show the molecular basis of synthesizing highly functional composite particles utilizing DNA, cell-adhesive proteins and inorganic crystals, and finally establish a superior transfection system for a mouse stem cell line having potential applications in cell-based therapy.

Galactosylated chitosan-graft-polyethylenimine as a gene carrier for hepatocyte targeting.

Chitosans have been proposed as alternative, biocompatible cationic polymers for nonviral gene delivery. However, the low transfection efficiency and low specificity of chitosan need to be addressed before clinical application. We prepared galactosylated chitosan-graft-polyethylenimine (GC-g-PEI) copolymer by an imine reaction between periodate-oxidized GC and low-molecular-weight PEI. The molecular weight and composition were characterized using gel permeation chromatography column with multi-angle laser scattering and (1)H nuclear magnetic resonance, respectively. The copolymer was complexed with plasmid DNA in various copolymer/DNA (N/P) charge ratios, and the complexes were characterized. GC-g-PEI showed good DNA-binding ability and superior protection of DNA from nuclease attack and had low cytotoxicity compared to PEI 25K. GC-g-PEI/DNA complexes showed higher transfection efficiency than PEI 25K in both HepG2 and HeLa cell lines. Transfection efficiency into HepG2, which has asialoglycoprotein receptors, was higher than that into HeLa, which does not. GC-g-PEI/DNA complexes also transfected liver cells in vivo after intraperitoneal (i.p.) administration more effectively than PEI 25K. These results suggest that GC-g-PEI can be used in gene therapy to improve transfection efficiency and hepatocyte specificity in vitro and in vivo.

Effective delivery with enhanced translational activity synergistically accelerates mRNA-based transfection.

mRNA instead of DNA provides a new and attractive approach for gene therapy and genetic vaccination. Current technologies for mRNA delivery are based on cationic lipids with DOTAP being the most efficient one. We previously reported on the synthesis of an inorganic-organic hybrid carrier by embedding inorganic nano-particles of carbonate apatite onto liposomal carrier DOTAP and demonstrated its high transfection potency of luciferase mRNA both in mitotic and non-mitotic cells. Here we show that in addition to the carrier design for effective endocytosis and release of mRNA to the cytoplasm, enhancement of mRNA translation efficiency is a prerequisite for maximum protein expression. We used the modified cap analog (ARCA) during in vitro transcription of luciferase DNA for proper cap orientation and demonstrated that transfection with ARCA-mRNA resulted in higher protein expression than the mRNA with usual cap structure for both DOTAP and DOTAP-apatite complex. Secondly, exogenous poly(A) was co-delivered with mRNA by the DOTAP-apatite, resulting in very significant expression compared to mRNA delivery only. Finally, when combined both of the effects of smart carrier and the modifications at mRNA translational level, a notable enhancement (100 times) was achieved as compared to the existing DOTAP-based liposome technology. Our findings, therefore, unveiled a novel approach that an effective delivery system can be developed by the improvement of the gene expression level in combination with the enhancement of the carrier potency.

Receptor-mediated gene delivery using chemically modified chitosan.

Chitosan has been investigated as a non-viral vector because it has several advantages such as biocompatibility, biodegradability and low toxicity with high cationic potential. However, the low specificity and low transfection efficiency of chitosan need to be solved prior to clinical application. In this paper, we focused on the galactose or mannose ligand modification of chitosan for enhancement of cell specificity and transfection efficiency via receptor-mediated endocytosis in vitro and in vivo.

Surface functionalization of inorganic nano-crystals with fibronectin and E-cadherin chimera synergistically accelerates trans-gene delivery into embryonic stem cells.

Stem cells holding great promises in regenerative medicine have the potential to be differentiated to a specific cell type through genetic manipulation. However, conventional ways of gene transfer to such progenitor cells suffer from a number of disadvantages particularly involving safety and efficacy issues. Here, we report on the development of a bio-functionalized inorganic nano-carrier of DNA by embedding fibronectin and E-cadherin chimera on the carrier, leading to its high affinity interactions with embryonic stem cell surface and accelerated trans-gene delivery for subsequent expression. While only apatite nano-particles were very inefficient in transfecting embryonic stem cells, fibronectin-anchored particles and to a more significant extent, fibronectin and E-cadherin-Fc-associated particles dramatically enhanced trans-gene delivery with a value notably higher than that of commercially available lipofection system. The involvement of both cell surface integrin and E-cadherin in mediating intracellular localization of the hybrid carrier was verified by blocking integrin binding site with excess free fibronectin and up-regulating both integrin and E-cadherin through PKC activation. Thus, the new establishment of a bio-functional hybrid gene-carrier would promote and facilitate development of stem cell-based therapy in regenerative medicine.

pH-sensing nano-crystals of carbonate apatite: effects on intracellular delivery and release of DNA for efficient expression into mammalian cells.

Two unique and fascinating properties of carbonate apatite which are well-known in hard tissue engineering, have been unveiled, for the first time, for the development of the simplest, but most efficient non-viral gene delivery device - ability of preventing the growth of crystals needed for high frequency DNA transfer across a plasma membrane and a fast dissolution rate for effective release of DNA during endosomal acidification, leading to a remarkably high transgene expression (5 to 100-fold) in mammalian cells compared to the widely used transfecting agents. Moreover, by modulating the crystal dissolution rate of carbonate apatite through incorporation of fluoride or strontium into it, transfection activity could be dramatically controlled, thus shedding light on a new barrier in the non-viral route, which was overlooked so far. Thus we have developed an innovative technology with significant insights, that would come as a promising tool for both basic research laboratories and clinical settings.

Galactose-carrying polymers as extracellular matrices for liver tissue engineering.

Extracellular matrix (ECM) plays important roles in tissue engineering because cellular growth and differentiation, in the two-dimensional cell culture as well as in the three-dimensional space of the developing organism, require ECM with which the cells can interact. Especially, the bioartificial liver-assist device or regeneration of the liver-tissue substitutes for liver tissue engineering requires a suitable ECM for hepatocyte culture because hepatocytes are anchorage-dependent cells and are highly sensitive to the ECM milieu for the maintenance of their viability and differentiated functions. Galactose-carrying synthetic ECMs derived from synthetic polymers and natural polymers bind hepatocytes through a receptor-mediated mechanism, resulting in enhanced hepatocyte functions. Attachment and functions of hepatocytes were affected by physico-chemical properties including ECM geometry as well as the type, density and orientation of galactose. Also, cellular environment, medium composition and dynamic culture system influenced liver-specific functions of hepatocytes beside ECM.

Morphology and metabolism of hepatocytes microencapsulated with acrylic terpolymer-alginate using gelatin and poly(vinyl alcohol) as extracellular matrices.

Microcapsules with good mechanical stability were prepared using an appropriate mixture of alginate and acrylic terpolymer. It was found from the microscopic observation that the microcapsules had a porous structure with interconnected pores, with a size of 50-150 nm. The results of the permeability experiment of microcapsules using FITC-dextrans showed that the capsule had a molecular mass cut-off of 120 kDa. The hepatocytes encapsulated in both alginate and acrylic terpolymer with gelatin and PVA rapidly aggregated in the core. The aggregated cells showed high albumin synthesis and ammonia removal, suggesting good metabolic function.

Integrin-supported fast rate intracellular delivery of plasmid DNA by extracellular matrix protein embedded calcium phosphate complexes.

Extracellular matrix (ECM) proteins, such as collagen and fibronectin, play vital roles in development and maintenance of hard tissue (bone or tooth) and are, consequently, thoroughly investigated for construction of biomimetic scaffolds in combination with calcium phosphate (CaP) material (the major component of hard tissue) for bone or dental tissue engineering. Realizing the natural affinity of ECM components toward inorganic constituents of hard tissue, we successfully constructed the nanohybrids of DNA/CaP particles with either collagen 1 or fibronectin, which finally possessed the capability of specific recognition of integrin receptor for being swiftly internalized across the plasma membrane, leading to remarkably high transgene expression in mammalian cells. This new approach with precise receptor-specific delivery as well as 10- to 50-fold enhanced efficiency level compared to the classical one, has immediate applications for basic research and large scale production of recombinant therapeutic proteins and looks promising for gene therapy.