The co-expression of Neogenin with SOX2 in hippocampal neurons.

Dementia has been shown to be closely related with neuronal degeneration and/or a decrease in the activity of neural stem cells in many brain regions, including the hippocampus. It has been recently established that Neogenin is involved in the cell fate determination by regulating Oct3/4, SOX and Nanog, notable embryonic cell markers, expressions in pre-implantation mouse embryos. Further, Neogenin expression at both mRNA and protein levels is manifest in many brain regions in mice, but it remains unclear whether Neogenin expression is prerequisite for the maintenance of neural stem cells, particularly, playing a critical role in the hippocampus, a brain region known to be involved in memory generation and consolidation. Here, we provide evidence that supports that Neogenin is implicated in the maintenance of neural stem cells in the hippocampus by enhancing PCNA expressions. We have performed RT-PCR analysis, Western blotting, and immunohistochemistry with fetal rat brain tissues at E18 for Neogenin mRNA and protein profiling. Neuronal cells obtained from the hippocampus were subjected to FACS analysis for the identification of Neogenin-positive and/or neuronal stem cell marker-positive cells. Western blotting results showed that Neogenin expression was higher in the hippocampal region compared to the cortical region. FACS analysis results indicated that a significant population of fetal rat neuronal cells exhibiting Neogenin expression also displayed SOX2 expression, implying co-expression of Neogenin and SOX2 in the hippocampus. Next, we investigated the role of Neogenin through gain- and loss-of-function studies with cultured rat hippocampal neurons. Neogenin down-regulation by small hairpin RNAs led to a dramatic decrease in SOX2 expression while its up-regulation by overexpression caused an increase in PCNA expression, a cell proliferation marker, compared with none-transfected cells. From this study, we propose a model whereby Neogenin could maintain neural stem cell population and control cell proliferation.

Effects of L- and T-type Ca²(+) channel blockers on spermatogenesis and steroidogenesis in the prepubertal mouse testis.

PURPOSE: To assess the involvement of L-type and T-type Ca²(+) channel blockers in inducing male infertility. METHODS: Prepubertal male mice were fed Ca²(+) channel blockers nifedipine and ethosuximide for 20 days at dosages below maximum tolerated dose (MTD) and assayed for gross morphological changes in the testis such as body weight, testis size and weight. Sperm and Leydig cell counting were conducted concomitantly with serum testosterone level measurement by radioimmunoassay (RIA) and StAR protein mRNA measurement by reverse transcription and polymerase chain reaction (RT-PCR). RESULTS: A chronic exposure to nifedipine or ethosuximide caused a significant reduction in body weight, testis size/weight and sperm production in a dose-dependent fashion associated with a spermatogenic arrest largely at the elongating spermatid stage. The number of Leydig cells, the serum testosterone level but not the luteinizing hormone level, and the content of StAR protein mRNA were also drastically reduced relative to the controls. CONCLUSIONS: Both T- and L-type Ca²(+) channel blockers play an adverse role in normal spermatogenesis and steroidogenesis partly by blocking postmeiotic germ cell maturation and/or by abrogating StAR protein expression, contributing to male sterility. Therefore, any therapeutic application of Ca²(+) channel blockers must be used with caution due to its potential adverse side effects on male infertility.

Ginsenoside 20(S)-protopanaxadiol induces cell death in human endometrial cancer cells via apoptosis.

BACKGROUND: 20(S)-protopanaxadiol (20(S)-PPD), one of the aglycone derivatives of major ginsenosides, has been shown to have an anticancer activity toward a variety of cancers. This study was initiated with an attempt to evaluate its anti-cancer activity toward human endometrial cancer by cell and xenograft mouse models. METHODS: Human endometrial cancer (HEC)-1A cells were incubated with different 20(S)-PPD concentrations. 20(S)-PPD cytotoxicity was evaluated using MTT assay. Apoptosis was detected using the annexin V binding assay and cell cycle analysis. Cleaved poly (ADP-ribose) polymerase (PARP) and activated caspase-9 were assessed using western blotting. HEC-1A cell tumor xenografts in athymic mice were generated by inoculating HEC-1A cells into the flank of BALB/c female mice and explored to validate 20(S)-PPD anti-endometrial cancer toxicity. RESULTS: 20(S)-PPD inhibited HEC-1A cell proliferation in a dose-dependent manner with an IC50 value of 3.5 µM at 24 h. HEC-1A cells morphologically changed after 20(S)-PPD treatment, bearing resemblance to Taxol-treated cells. Annexin V-positive cell percentages were 0%, 10.8%, and 58.1% in HEC-1A cells when treated with 0, 2.5, and 5 µM of 20(S)-PPD, respectively, for 24 h. 20(S)-PPD subcutaneously injected into the HEC-1A cell xenograft-bearing mice three times a week for 17 days manifested tumor growth inhibition by as much as 18% at a dose of 80 mg/kg, which sharply contrasted to controls that showed an approximately 2.4-fold tumor volume increase. These events paralleled caspase-9 activation and PARP cleavage. CONCLUSION: 20(S)-PPD inhibits endometrial cancer cell proliferation by inducing cell death via a caspase-mediated apoptosis pathway. Therefore, the 20(S)-PPD-like ginsenosides are endowed with ample structural information that could be utilized to develop other ginsenoside-based anticancer agents.

Syndecan-1 overexpression promotes tumor growth and angiogenesis in an endometrial cancer xenograft model.

OBJECTIVES: Upregulation of syndecan-1, a member of the transmembranous proteoglycans that serves as a coreceptor for a wide pool of extracellular ligands, has been well documented in enabling the promotion of growth and invasion of endometrial cancer. As a step toward understanding a potential role for syndecan-1 in this process, we questioned whether syndecan-1 upregulates tumor-promoting characteristics, particularly, angiogenesis in an in vivo human xenograft tumor model. METHODS: Human syndecan-1 was stably transfected into human endometrial adenocarcinoma 1A cells, and resulting transfectants were subcutaneously grafted into athymic mice; their outcomes were examined with respect to the enhancement of tumor growth and angiogenesis by immunohistochemistry, immunoblotting, and zymography. RESULTS: Overexpression of syndecan-1 promoted tumor growth concomitant with increased angiogenesis in tumor xenografts as evidenced by an increase in immunoreactivity for vascular endothelial growth factor and vascular endothelial cell marker CD34. Furthermore, zymographic studies revealed that syndecan-1 overexpression markedly enhanced activities of matrix metalloproteinases 2 and 9. CONCLUSIONS: This is the first in vivo xenograft analysis providing evidence that supports that syndecan-1 has a critical role in carcinogenic progression, particularly, contributing to the development of angiogenesis and invasive phenotype in association with matrix metalloproteinases 2 and 9 activations in endometrial cancer.

Oleanolic acid 3-acetate, a minor element of ginsenosides, induces apoptotic cell death in ovarian carcinoma and endometrial carcinoma cells via the involvement of a reactive oxygen species-independent mitochondrial pathway.

OBJECTIVES: Oleanolic acid, a minor element of ginsenosides, and its derivatives have been shown to have cytotoxicity against some tumor cells. The impact of cytotoxic effect of oleanolic acid 3-acetate on ovarian cancer SKOV3 cells and endometrial cancer HEC-1A cells were examined both in vivo and in vitro to explore the underlying mechanisms. METHODS: Cytotoxic effects of oleanolic acid 3-acetate were assessed by cell viability, phosphatidylserine exposure on the cell surface, mitochondrial release of cytochrome C, nuclear translocation of apoptosis-inducing factor, depolarization of mitochondrial transmembrane potential (ΔΨm), and generation of reactive oxygen species (ROS). In vivo inhibition of tumor growth was also assessed with xenografts in immunocompromised mice. RESULTS: Oleanolic acid 3-acetate exhibited potent cytotoxicity toward SKOV3 and HEC-1A cells by decreasing cell viability in a concentration-dependent manner. Importantly, oleanolic acid 3-acetate effectively suppressed the growth of SKOV3 cell tumor xenografts in immunocompromised mice. Furthermore, oleanolic acid 3-acetate induced apoptotic cell death as revealed by loss of ΔΨm, release of cytochrome c, and nuclear translocation of apoptosis-inducing factor with a concomitant activation of many proapoptotic cellular components including poly(ADP-ribose) polymerase, Bcl-2, and caspases-8, caspase-3, and caspase-7. Oleanolic acid 3-acetate, however, caused a decrease in ROS production, suggesting the involvement of an ROS-independent pathway in oleanolic acid 3-acetate-induced apoptosis in SKOV3 and HEC-1A cells. CONCLUSION: These findings support the notion that oleanolic acid 3-acetate could be used as a potent anticancer supplementary agent against ovarian and endometrial cancer. Oleanolic acid 3-acetate exerts its proapoptotic effects through a rather unique molecular mechanism that involves an unconventional ROS-independent but mitochondria-mediated pathway.

Endometrial cancer invasion depends on cancer-derived tumor necrosis factor-alpha and stromal derived hepatocyte growth factor.

Cancer invasion is an outcome of interactions of the cancer and the host cell. It is now becoming increasingly clear that ovarian hormones have a huge influence on such intercommunications in various types of cancers. Estrogen is known to aggravate the aggressiveness of the endometrial cancer whereas progesterone seems to act as a negative factor. Insight into the mode of ovarian hormonal actions could come from the studies of its regulation of the paracrine interactions between the endometrial cancer and the normal stromal cells during the cancer invasion. In this context, we report here that estrogen promotes the endometrial cancer invasion by inducing humoral interactions between the cancer and the stromal cells, i.e., estrogen stimulates tumor necrosis factor-alpha expression from the endometrial cancer cells, which, in turn, induces the stromal expression of hepatocyte growth factor (HGF), conferring the enhanced NK4 (HGF-antagonist/angiogenesis inhibitor)-sensitive invasion characteristic of the endometrial cancer cells. Additionally, we demonstrate a close correlation of the invasion of endometrial cancer cells with the expression and dimerization of integrin alpha(v)beta(5) as well as the activation of focal adhesion kinase as the consequences of paracrine interactions. Thus, understanding of paracrine interactions of cancer cells with host stromal cells can yield new insight into the architecture and function of cancer invasion and metastasis, leading to a development of a new cancer therapeutic intervention.

Syndecan-1 enhances the endometrial cancer invasion by modulating matrix metalloproteinase-9 expression through nuclear factor kappaB.

OBJECTIVES: Up-regulated expression of syndecan-1, a member of the transmembranous proteoglycans that serves as a co-receptor for a wide pool of extracellular ligands, has been ascribed to the promotion of growth of various cancers including breast, ovarian, and endometrial cancers. Here, we have extended these observations to gain insight into correlation between the expression level of syndecan-1 and its tumor-promoting characteristics, particularly, cancer invasion, in endometrial cancer. METHODS: Human syndecan-1 was stably transfected into three human endometrial cancer cell lines, and its effects were examined with respect to cell survival/proliferation and invasion. In addition, the activation of underlying signaling components, including integrins, focal adhesion kinase (FAK), and nuclear factor kappaB (NF-kappaB) was examined. The activity of NF-kappaB as a transcription factor for matrix metalloproteinase (MMP)-9 was assessed. RESULTS: The innate expression level of syndecan-1 was moderate to high in all endometrial cancer cell lines. Overexpression of syndecan-1 promoted tumor cell proliferation concomitant with the activation of NF-kappaB. Furthermore, overexpression of syndecan-1 markedly enhanced the cancer invasion accompanied by enhanced expression of integrin alphav/beta5 and enhanced phosphorylation of FAK. The transcriptional activation of MMP-9 by NF-kappaB was up-regulated in syndecan-1 overexpression. CONCLUSION: These findings provide evidence that supports that syndecan-1 may have a critical role in carcinogenic progression, particularly, contributing to the development of proliferative and invasive phenotype through NF-kappaB-mediated MMP-9 gene expression in endometrial 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.

A well-defined in vitro three-dimensional culture of human endometrium and its applicability to endometrial cancer invasion.

A three-dimensional (3-D) endometrium culture was established, in which human endometrial stromal cells embedded in a mixture of collagen I, a major component of extracellular matrix, and matrigel, a basement membrane material, supports the epithelial cells seeded on top of the collagen/matrigel matrix. The biological growth and differentiation of the epithelial cells were studied microscopically and immunohistochemically. Transmission electron microscopy showed a polarized columnar epithelium in monolayer with basally positioned nuclei. Scanning electron microscopy revealed a confluent epithelium with an abundance of microvilli and cilia as well as pinopodes on the apical surface. An immunohistochemical staining showed that integrin alpha1, alpha4, and beta3 were co-localized with cytokeratin, confirming the epithelial origin of the cells. In contrast, immunoreactivity against cyclooxygenase-1 or -2 was positive in both epithelial and stromal cells. When epithelial cells were replaced by KLE cells, an endometrial cancer cell of epithelial origin, invasion of KLE cells into the stromal fraction was observed. The invasion was closely correlated to expression of matrix metalloproteinases and their tissue inhibitors of metalloproteinases in a manner consistent with paracrine fashion. The present 3-D culture imitates the normal endometrium physiologically as well as morphologically, thus provides an excellent in vitro tissue suitable for reproducing in vivo physiological processes, including endometrial cancer invasion.

ATP-induced apoptosis of human granulosa luteal cells cultured in vitro.

OBJECTIVE: To investigate the role of extracellular adenosine triphosphatase (ATP) as an inducer of apoptotic cell death in human granulosa cells and to elucidate its underlying mechanism. DESIGN: Prospective study. SETTING: Gynecologic clinic and human reproduction research laboratory. PATIENT(S): Twenty-five patients undergoing IVF or IVF-ET. INTERVENTION(S): ATP treatment of granulosa luteal cells subjected to primary culture. MAIN OUTCOME MEASURE(S): Apoptosis was assessed by the annexin V binding assay and the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling assay. The receptor type that binds ATP, thus mediating ATP-induced apoptosis, was determined by using the calcium imaging and patch-clamp techniques. Mitochondrial depolarization was assessed by staining with the membrane potential-sensitive dye 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolyl carbocyanide iodide (JC-1). RESULT(S): ATP elevated [Ca(2+)](i) by mobilizing intracellularly stored Ca(2+). An ionic membrane current evoked by ATP was mediated by the Ca(2+)-activated K(+) channel. ATP induced a mitochondrial depolarization with a concomitant increase in cellular apoptosis. Treatment with hCG eliminated both ATP-induced mitochondrial depolarization and apoptosis. CONCLUSION(S): Extracellular ATP recognized by P(2Y) type purinoceptor triggers apoptosis in human granulosa luteal cells, and the downstream apoptotic cascade may act at least in part through mitochondria. The antiapoptotic effect of hCG has possible clinical implications in the treatment of disorders involving granulosa cell degeneration (such as follicular atresia).