Inhibitory effects of extracellular superoxide dismutase on ultraviolet B-induced melanogenesis in murine skin and melanocytes.

AIMS: Several anti-melanogenic molecules have been developed or identified, but their uses are limited due to either adverse effects or instability during the treatment. We aimed to evaluate the effects of extracellular superoxide dismutase (SOD3), a powerful antioxidant, as a candidate anti-melanogenic molecule. MAIN METHODS: UVB-induced reactive oxygen species (ROS) production and proliferation in melan-a cells was evaluated by 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate staining and bromodeoxyuridine incorporation assay, respectively. Quantitative real-time polymerase chain reaction and western blot were performed to detect the melanogenesis-related gene expression and downstream signaling. Anti-melanogenic effects of SOD3 were also evaluated using SOD3 transgenic mice under UVB exposure in-vivo condition. KEY FINDINGS: SOD3 inhibited UVB-induced proliferation, ROS production and melanogenesis in melanocytes. Measurement of melanin content and tyrosinase activity assays showed that SOD3 significantly inhibited melanin synthesis. Moreover, these suppressive effects of SOD3 were dependent on the endothelin-1 (ET-1)/endothelin B receptor, protein kinase C, melanocortin 1 receptor/protein kinase A, Wnt7a/ß-catenin, and mitogen-activated protein kinase pathways, with concomitant downregulation of microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related proteins 1, dopachrome tautomerse. Interestingly, SOD3 was found to inhibit transforming growth factor-beta 1 (TGF-ß1) to inactivate the ET-1 signaling pathway, and finally prevents the production of melanin. SIGNIFICANCE: Our results provide novel insights into the role of SOD3 in melanocyte homeostasis and its uses as a potential biomedicine to treat hyperpigmentary conditions of the skin.

Establishment and Characterization of Immortalized Minipig Neural Stem Cell Line.

Despite the increasing importance of minipigs in biomedical research, there has been relatively little research concerning minipig-derived adult stem cells as a promising research tool that could be used to develop stem cell-based therapies. We first generated immortalized neural stem cells (iNSCs) from primary minipig olfactory bulb cells (pmpOBCs) and defined the characteristics of the cell line. Primary neural cells were prepared from minipig neonate olfactory bulbs and immortalized by infection with retrovirus carrying the v-myc gene. The minipig iNSCs (mpiNSCs) had normal karyotypes and expressed NSC-specific markers, including nestin, vimentin, Musashi1, and SOX2, suggesting a similarity to human NSCs. On the basis of the global gene expression profiles from the microarray analysis, neurogenesis-associated transcript levels were predominantly altered in mpiNSCs compared with pmpOBCs. These findings increase our understanding of minipig stem cells and contribute to the utility of mpiNSCs as resources for immortalized stem cell experiments.

Tumor-specific gene therapy for pancreatic cancer using human neural stem cells encoding carboxylesterase.

Advanced pancreatic cancer is one of the most lethal malignant human diseases lacking effective treatment. Its extremely low survival rate necessitates development of novel therapeutic approach. Human neural stem cells (NSCs) are known to have tumor-tropic effect. We genetically engineered them to express rabbit carboxyl esterase (F3.CE), which activates prodrug CPT-11(irinotecan) into potent metabolite SN-38. We found significant inhibition of the growth of BxPC3 human pancreatic cancer cell line in vitro by F3.CE in presence of CPT-11. Apoptosis was also markedly increased in BxPC3 cells treated with F3.CE and CPT-11. The ligand VEGF and receptor VEGF-1(Flt1) were identified to be the relevant tumor-tropic chemoattractant. We confirmed in vivo that in mice injected with BxPC3 on their skin, there was significant reduction of tumor size in those treated with both F3.CE and BxPC3 adjacent to the cancer mass. Administration of F3.CE in conjunction with CPT-11 could be a new possibility as an effective treatment regimen for patients suffering from advanced pancreatic cancer.

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos.

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

Neurorestorative Role of Stem Cells in Alzheimer's Disease: Astrocyte Involvement.

Neurogenesis is maintained in both neonatal and adult brain, although it is dramatically reduced in aged neurogenic brain region such as the subgranular layer and subventricular zone of the dentate gyrus (DG). Astrocytes play important roles for survival and maintenance of neurons as well as maintenance of neurogenic niche in quiescent state. Aß can induce astrocyte activation which give rise to produce reactive oxygen species (ROS) and cytotoxic cytokines and chemokines, and subsequently induce neuronal death. Unfortunately, the current therapeutic medicines have been limited to reduce the symptoms and delay the pathogenesis of Alzheimer's disease (AD), but not to cure it. Stem cells enhance neurogenesis and Aß clearing as well as improved cognitive impairment. Neurotrophins and growth factors which are produced from both stem cells and astrocytes also have neuroprotective effects via neurogenesis. Secreted factors from both astrocytes and neural stem cells also are influenced in neurogenesis and neuron survival in neurodegenerative diseases. Transplanted stem cells overexpressing neurogenic factors may be an effective and therapeutic tool to enhance neurogenesis for AD.

Human Neural Stem Cells Overexpressing a Carboxylesterase Inhibit Bladder Tumor Growth.

Bladder cancer is a significant clinical and economic problem. Despite intravesical chemotherapy and immunotherapy, up to 80% of patients with non-muscle-invasive bladder cancer develop recurrent tumors, of which 20% to 30% evolve into more aggressive, potentially lethal tumors. Recently, bladder cancer cells are considered to be mediators of resistance to current therapies and therefore represent strong candidates as biologic targets. No effective chemotherapy has yet been developed for advanced bladder cancer. It is desirable that a drug can be delivered directly and specifically to bladder cancer cells. Stem cells have selective migration ability toward cancer cells, and therapeutic genes can be easily transduced into stem cells. In suicide gene therapy for cancer, stem cells carry a gene encoding a carboxylesterase (CE) enzyme that transforms an inert CPT-11 prodrug into a toxic SN-38 product, a topoisomerase 1 inhibitor. In immunodeficient mice, systemically transplanted HB1.F3.CE stem cells migrated toward the tumor implanted by the TCCSUP bladder cancer cell line, and, in combination with CPT-11, the volume of tumors was significantly reduced. These findings may contribute to the development of a new selective chemotherapeutic strategy against bladder cancer. Mol Cancer Ther; 15(6); 1201-7. ©2016 AACR.

Inactivation of NKX6.3 in the stomach leads to abnormal expression of CDX2 and SOX2 required for gastric-to-intestinal transdifferentiation.

Intestinal metaplasia in gastric mucosa is considered a preneoplastic lesion that progresses to gastric cancer. However, the molecular networks underlying this lesion formation are largely unknown. NKX6.3 is known to be an important regulator in gastric mucosal epithelial differentiation. In this study, we characterized the effects of NKX6.3 that may contribute to gastric intestinal metaplasia. NKX6.3 expression was significantly reduced in gastric mucosae with intestinal metaplasia. The mRNA expression levels of both NKX6.3 and CDX2 predicted the intestinal metaplasia risk, with an area under the receiver operating characteristic curve value of 0.9414 and 0.9971, respectively. Notably, the NKX6.3 expression level was positively and inversely correlated with SOX2 and CDX2, respectively. In stable AGS(NKX6.3) and MKN1(NKX6.3) cells, NKX6.3 regulated the expression of CDX2 and SOX2 by directly binding to the promoter regions of both genes. Nuclear NKX6.3 expression was detected only in gastric epithelial cells without intestinal metaplasia. Furthermore, NKX6.3-induced TWSG1 bound to BMP4 and inhibited BMP4-binding activity to BMPR-II. These data suggest that NKX6.3 might function as a master regulator of gastric differentiation by affecting SOX2 and CDX2 expression and the NKX6.3 inactivation may result in intestinal metaplasia in gastric epithelial cells.

Therapeutically targeting neuroinflammation and microglia after acute ischemic stroke.

Inflammation has a pivotal role in the pathogenesis of ischemic stroke, and recent studies posit that inflammation acts as a double-edged sword, not only detrimentally augmenting secondary injury, but also potentially promoting recovery. An initial event of inflammation in ischemic stroke is the activation of microglia, leading to production of both pro- and anti-inflammatory mediators acting through multiple receptor signaling pathways. In this review, we discuss the role of microglial mediators in acute ischemic stroke and elaborate on preclinical and clinical studies focused on microglia in stroke models. Understanding how microglia can lead to both pro- and anti-inflammatory responses may be essential to implement therapeutic strategies using immunomodulatory interventions in ischemic stroke.

Alzheimer's disease and stem cell therapy.

The loss of neuronal cells in the central nervous system may occur in many neurodegenerative diseases. Alzheimer's disease is a common senile disease in people over 65 years, and it causes impairment characterized by the decline of mental function, including memory loss and cognitive impairment, and affects the quality of life of patients. However, the current therapeutic strategies against AD are only to relieve symptoms, but not to cure it. Because there are only a few therapeutic strategies against Alzheimer's disease, we need to understand the pathogenesis of this disease. Cell therapy may be a powerful tool for the treatment of Alzheimer's disease. This review will discuss the characteristics of Alzheimer's disease and various available therapeutic strategies.

Human astrocytes: secretome profiles of cytokines and chemokines.

Astrocytes play a key role in maintenance of neuronal functions in the central nervous system by producing various cytokines, chemokines, and growth factors, which act as a molecular coordinator of neuron-glia communication. At the site of neuroinflammation, astrocyte-derived cytokines and chemokines play both neuroprotective and neurotoxic roles in brain lesions of human neurological diseases. At present, the comprehensive profile of human astrocyte-derived cytokines and chemokines during inflammation remains to be fully characterized. We investigated the cytokine secretome profile of highly purified human astrocytes by using a protein microarray. Non-stimulated human astrocytes in culture expressed eight cytokines, including G-CSF, GM-CSF, GROα (CXCL1), IL-6, IL-8 (CXCL8), MCP-1 (CCL2), MIF and Serpin E1. Following stimulation with IL-1ß and TNF-α, activated astrocytes newly produced IL-1ß, IL-1ra, TNF-α, IP-10 (CXCL10), MIP-1α (CCL3) and RANTES (CCL5), in addition to the induction of sICAM-1 and complement component 5. Database search indicated that most of cytokines and chemokines produced by non-stimulated and activated astrocytes are direct targets of the transcription factor NF-kB. These results indicated that cultured human astrocytes express a distinct set of NF-kB-target cytokines and chemokines in resting and activated conditions, suggesting that the NF-kB signaling pathway differentially regulates gene expression of cytokines and chemokines in human astrocytes under physiological and inflammatory conditions.