The glioma-associated oncogene homolog 1 (GLI1) family of zinc finger transcription factors is the nuclear mediator of the Hedgehog pathway that regulates genes essential for various stages of tumor development and progression. However, the role and mechanism by which high expression of GLI1 contributes to the invasion and metastasis of human esophageal squamous cell cancer (ESCC) has not been fully elucidated. In the present study, we demonstrated that GLI1 was over-expressed in human ESCC tissues, especially in ESCC tissues with deep invasion and lymph-node metastasis. Moreover, GLI1 was also over-expressed in ESCC cell lines and correlated with the aggressiveness of ESCC cell lines. In addition, GLI signaling pathway agonist purmorphamine could increase the invasion and metastasis ability of ESCC cells in vitro. There is increasing evidence for the contribution of epithelial-mesenchymal transition (EMT) to ESCC invasion and metastasis, therefore we investigated GLI1's role in EMT. Our results showed that high expression of GLI1 dampened expression of E-cadherin and enhanced the expression of Vimentin, and it also improved the expression of Snail, indicative of its role in EMT occurrence. Mechanistic studies showed that down-expression of Snail reversed GLI1 activation-regulated expression of EMT markers, suggesting the role of Snail in GLI1-mediated EMT. Taken together, our results had revealed that GLI1 could participate in the invasion and metastasis of ESCC through EMT. These studies indicated that in ESCC, GLI1 could be a useful target for cancer prevention and therapy.

BackgroundNon-small cell lung cancer (NSCLC) is difficult to treat successfully. This intractability is mainly due to the cancer progressing through invasion, metastasis, chemotherapeutic resistance and relapse. Stemness has been linked to the various steps of cancer progression in a variety of tumors, yet little is known regarding its role in NSCLC.PurposeIn this study, we sought to determine the role of SOX2, a master regulator of pluripotency, in the growth of extracellular matrix (ECM)-detached cells during cancer progression.MethodsWe established a three-dimensional (3D) Poly-2-hydroxyethyl methacrylate (poly-HEMA) culture of lung adenocarcinoma (LUAD) A549 cells as an ECM-detached cell growth model and examined the role of stemness genes using siRNA and small molecule inhibitor in comparison to standard two dimensional (2D) culture.ResultsIn poly-HEMA culture, A549 cells formed substratum-detached spheroids with characteristics of intermediate epithelial to mesenchymal transition (EMT) and exhibited greater expression of SOX2 than did control 2D cells. Knockdown of SOX2 markedly suppressed the growth of A549 cell aggregates in poly-HEMA culture conditions and furthermore increased their sensitivity to the anticancer drug vinblastine with concomitant downregulation of the activity of the anti-apoptotic AKT kinase. Interestingly, a small molecule, RepSox, which replaces SOX2, stimulated A549 cell growth in poly-HEMA 3D culture condition.ConclusionOur findings strongly indicate that SOX2 contributes to anchorage-independent growth and chemoresistance via its downstream signaling mediator AKT kinase during the disease progression of NSCLC. SOX2 may therefore be an invaluable therapeutic target of NSCLC.

Biological systems consist of a variety of distinct cell types that form functional networks. Superresolution imaging of individual cells is required for better understanding of these complex systems. Direct visualization of 3D subcellular and nano-scale structures in cells is helpful for the interpretation of biological interactions and system-level responses. Here we introduce a modified magnified analysis of proteome (MAP) method for cell super-resolution imaging (Cell-MAP) which preserves cell fluorescence. Cell-MAP expands cells more than four-fold while preserving their overall architecture and three-dimensional proteome organization after hydrogel embedding. In addition, Optimized-Cell-MAP completely preserves fluorescence and successfully allows for the observation of tagged small molecular probes containing peptides and microRNAs. Optimized-Cell-MAP further successfully applies to the study of structural characteristics and the identification of small molecules and organelles in mammalian cells. These results may give rise to many other applications related to the structural and molecular analysis of smaller assembled biological systems. Biological systems are stunningly complex, often consisting of millions of individual cells. Each cell can be classified as belonging to one of a number of distinct cell types, and is part of one or more closely interconnected functional networks. Cells are the basic structures of all living things, and individual cells often show clear heterogenicity. Studies using advanced techniques of subcellular and nano-scale imaging are essential for understanding the individual characteristics of cells. Over the past few decades, cell imaging analysis systems have been developed to observe cells in three-dimensions due to technological advances in confocal microscopy. However, further resolution via current imaging analysis through confocal microscopy is technically limited due to lens magnification, point spread function, and diffraction limitations 1-3. It is therefore necessary to develop new technologies for single cell imaging that are capable of super-resolution. Two approaches have been developed for the super-resolution imaging of cells. One approach includes super-resolution optical techniques, such as photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and stimulated emission depletion microscopy (STED) 4-6. The other approach is super-resolution imaging by physical tissue expansion, including expansion microscopy (ExM), and magnified analysis of the proteome (MAP) 7,8. Both super-resolution microscopy and tissue expansion techniques have advantages and disadvantages. Super-resolution microscopy can be applied to living cells, but is not easily applied, as the equipment is very expensive and cannot be used with thick tissue and conventional immunostaining 9. In contrast, tissue expansion techniques may not be applied to living cells, but are usually less expensive and

Salivary gland stem cells (SGSCs) are potential cell sources for the treatment of salivary gland diseases. The control of cell survival is an essential factor for applying stem cells to regenerative medicine or stem cell-based research. The purpose of this study was to investigate the effects of the ROCK inhibitor Y-27632 on the survival of SGSCs and its underlying mechanisms. SGSCs were isolated from mouse submandibular glands and cultured in suspension. Treatment with Y-27632 restored the viability of SGSCs that was significantly decreased during isolation and the subsequent culture. Y-27632 upregulated the expression of anti-apoptotic protein BCL-2 in SGSCs and, in the apoptosis assay, significantly reduced apoptotic and necrotic cell populations. Matrigel was used to mimic the extracellular environment of an intact salivary gland. The expression of genes regulating apoptosis and the ROCK signaling pathway was significantly reduced when SGSCs were embedded in Matrigel. SGSCs cultured in Matrigel and treated with Y-27632 showed no difference in the total numbers of spheroids and expression levels of apoptosis-regulating genes. Matrigel-embedded SGSCs treated with Y-27632 increased the number of spheroids with budding structures and the expression of acinar cell-specific marker AQP5. We demonstrate the protective effects of Y-27632 against dissociation-induced apoptosis of SGSCs during their culture in vitro.

Cell-penetrating peptides (CPPs) are short amino acid sequences known to act as a vehicle for enhancing the intracellular translocating efficiency of extracellular molecules. Although many groups have attempted to develop peptides with high cellpenetrating efficiencies, very few have demonstrated efficient cellular uptake of CPPs at low concentrations. Here, we describe a newly synthesized peptide derived from Arabidopsis, Ara-27, which exhibits significant improvement in cell-penetrating efficiency compared to existing CPPs. The cell-penetrating efficiency of Ara-27 was compared with the commonly used Tat-protein transduction domain (Tat-PTD) and membrane translocating sequence (MTS) in human dermal fibroblast (HDF) and human dental pulp stem cells (hDPSC). Cell-penetrating efficiency of fluorescein isothiocyanate (FITC)-labeled CPPs were assessed by flow cytometry and visualized by confocal microscopy. Flow cytometric analysis revealed >99% cell-penetrating efficiency for 2 μM Ara-27 in both HDF and hDPSC. In contrast, 2 μM Tat-PTD and MTS showed <10% cell-penetrating efficiency in both cells. In support, relative fluorescence intensities of FITC-labeled Ara-27 were around 8 to 22 times higher than those of Tat-PTD and MTS in both cells. Confocal analysis revealed internalization of 0.2 and 2 μM Ara-27 in both human cells, which was not observed for Tat-PTD and MTS at either concentration. In conclusion, this study describes a novel CPP, Ara-27, which exhibit significant improvement in intracellular uptake compared to conventional CPPs, without affecting cell viability. Thus, development of Ara-27 based peptides may lead to improved delivery of functional cargo such as small molecules, siRNA, and drugs for in vivo studies.