Novel and efficient method for culturing patient-derived gastric cancer stem cells.

Experimental techniques for patient-derived cancer stem-cell organoids/spheroids can be powerful diagnostic tools for personalized chemotherapy. However, establishing their cultures from gastric cancer remains challenging due to low culture efficiency and cumbersome methods. To propagate gastric cancer cells as highly proliferative stem-cell spheroids in vitro, we initially used a similar method to that for colorectal cancer stem cells, which, unfortunately, resulted in a low success rate (25%, 18 of 71 cases). We scrutinized the protocol and found that the unsuccessful cases were largely caused by the paucity of cancer stem cells in the sampled tissues as well as insufficient culture media. To overcome these obstacles, we extensively revised our sample collection protocol and culture conditions. We then investigated the following second cohort and, consequently, achieved a significantly higher success rate (88%, 29 of 33 cases). One of the key improvements included new sampling procedures for tumor tissues from wider and deeper areas of gastric cancer specimens, which allowed securing cancer stem cells more reproducibly. Additionally, we embedded tumor epithelial pieces separately in both Matrigel and collagen type-I as their preference to the extracellular matrix was different depending on the tumors. We also added a low concentration of Wnt ligands to the culture, which helped the growth of occasional Wnt-responsive gastric cancer stem-cell spheroids without allowing proliferation of the normal gastric epithelial stem cells. This newly improved spheroid culture method may facilitate further studies, including personalized drug-sensitivity tests prior to drug therapy.

Loss of FYCO1 leads to cataract formation.

Autophagy is a degradation process of cytoplasmic proteins and organelles trafficked to degradation vesicles known as autophagosomes. The conversion of LC3-I to LC3-II is an essential step of autophagosome formation, and FYCO1 is a LC3-binding protein that mediates autophagosome transport. The p62 protein also directly binds to LC3 and is degraded by autophagy. In the present study, we demonstrated that disrupting the FYCO1 gene in mice resulted in cataract formation. LC3 conversion decreased in eyes from FYCO1 knockout mice. Further, FYCO1 interacted with αA- and αB-crystallin, as demonstrated by yeast two-hybrid screening and immunoprecipitation analyses. In eyes from knockout mice, the soluble forms of αA- and αB-crystallin, the lens's major protein components, decreased. In addition, p62 accumulated in eyes from FYCO1 knockout mice. Collectively, these findings suggested that FYCO1 recruited damaged α-crystallin into autophagosomes to protect lens cells from cataract formation.

Protamine may have anti-atherogenic potential by inhibiting the binding of oxidized-low density lipoprotein to LOX-1.

Oxidized low-density lipoprotein (ox-LDL) leads to atherosclerosis via lectin-like oxidized lipoprotein receptor-1 (LOX-1), one of the major receptor for ox-LDL. Inhibition of the binding of ox-LDL to LOX-1 decreases the proinflammatory and atherosclerotic events. The aim of the present study was to investigate whether protamine, a polybasic nuclear protein, interferes the binding of ox-LDL to LOX-1. Using sandwich ELISA with newly generated antibody, we measured the blocking effect of protamine on the binding of ox-LDL to LOX-1. Protamine dose-dependently inhibited the binding of ox-LDL to LOX-1. DiI-labeled ox-LDL uptake assay in two types of cultured human endothelial cells was performed with fluorescence microplate reader. Activation of extracellular-signal-regulated kinase (ERK)1/2 by ox-LDL was analyzed by immunoblotting. We found that protamine suppressed uptake of ox-LDL in endothelial cells and inhibited ERK1/2 activation by ox-LDL. These results suggest that protamine may possess anti-atherogenic potential by inhibiting ox-LDL binding to LOX-1 through electrostatic interactions.

Zebularine exerts its antiproliferative activity through S phase delay and cell death in human malignant mesothelioma cells.

Malignant mesothelioma is an asbestos-related aggressive tumor and current therapy remains ineffective. Zebularine as a DNA methyltransferase (DNMT) inhibitor has an anti-tumor effect in several human cancer cells. The aim of the present study was to investigate whether zebularine could induce antiproliferative effect in human malignant mesothelioma cells. Zebularine induced cell growth inhibition in a dose-dependent manner. In addition, zebularine dose-dependently decreased expression of DNMT1 in all malignant mesothelioma cells tested. Cell cycle analysis indicated that zebularine induced S phase delay. Zebularine also induced cell death in malignant mesothelioma cells. In contrast, zebularine did not induce cell growth inhibition and cell death in human normal fibroblast cells. These results suggest that zebularine has a potential for the treatment of malignant mesothelioma by inhibiting cell growth and inducing cell death.

EGCG induces human mesothelioma cell death by inducing reactive oxygen species and autophagy.

Malignant mesothelioma is an asbestos-related fatal disease with no effective cure. We studied whether a green tea polyphenol, epigallocathechin-3-gallate (EGCG), could induce cell death in five human mesothelioma cell lines. We found that EGCG induced apoptosis in all five mesothelioma cell lines in a dose-dependent manner. We further clarified the cell killing mechanism. EGCG induced reactive oxygen species (ROS), and impaired the mitochondrial membrane potential. As treatment with ROS scavengers, catalase and tempol, significantly inhibited the EGCG-induced apoptosis, ROS is considered to be responsible for the EGCG-induced apoptosis. Further, we found that EGCG induced autophagy, and that when autophagy was suppressed by chloroquine, the EGCG-induced cell death was enhanced. Taken together, these results suggest that EGCG has a great potential for the treatment of mesothelioma by inducing apoptosis and autophagy.

High dose of ascorbic acid induces cell death in mesothelioma cells.

Malignant mesothelioma is an asbestos-related fatal disease with no effective cure. Recently, high dose of ascorbate in cancer treatment has been reexamined. We studied whether high dose of ascorbic acid induced cell death of four human mesothelioma cell lines. High dose of ascorbic acid induced cell death of all mesothelioma cell lines in a dose-dependent manner. We further clarified the cell killing mechanism that ascorbic acid induced reactive oxygen species and impaired mitochondrial membrane potential. In vivo experiment, intravenous administration of ascorbic acid significantly decreased the growth rate of mesothelioma tumor inoculated in mice. These data suggest that ascorbic acid may have benefits for patients with mesothelioma.

Intercellular accumulation of type V collagen fibrils in accordance with cell aggregation.

We reported previously that human fibroblasts form clumps when cultured on a dish coated with reconstituted type V collagen fibrils. Essentially all the type V collagen fibrils, initially coated on the dish, were recovered in the cell clumps that had eventually formed during the culture. We interpreted that type V collagen fibrils adhere to cells more strongly than to the dish and are detached by cell movements. In this study, type V collagen was suspended with fibroblasts to examine the fate of the type V collagen fibrils and to determine whether the fibrils affect the behaviour of the cells directly adherent to the dish. The added type V collagen accumulated in the intercellular space concomitantly with the local aggregation of fibroblasts. scanning electron microscope examination indicated that type V collagen fibrils were found in the vicinity of cells in cultures without ascorbic acid where essentially no collagen secretion takes place. These results indicate that type V collagen forms fibrils and the fibrils are accumulated in the intercellular spaces. The accumulated type V collagen fibrils work as a cementing material for cell clump formation. This phenomenon is discussed in relation to the possible involvement of type V collagen fibrils in tissue organization.

Collagen-type specificity of glycoprotein VI as a determinant of platelet adhesion.

Of the two physiologically important platelet collagen receptors, glycoprotein (GP) VI is the receptor responsible for platelet activation. However, its reactivities towards different types of vascular collagen have not been directly and quantitatively analysed with collagen preparations of defined composition, although the other major platelet collagen receptor integrin alpha(2)beta(1) was shown to react with collagen types I-VI and VIII under either static or flow conditions. We analysed the collagen type specificity of GPVI binding to identify the physiological contribution of the various vascular collagens and how platelet reactivity towards the various collagens may be affected by fibril size. We used two methods to analyse the binding of recombinant GPVI (GPVI-Fc(2)) to different types of bovine collagen: binding to collagen microparticles in suspension and binding to immobilized collagen. GPVI-Fc(2) bound to type I-III collagens that can form large fibrils, but not to type V that only forms small fibrils. The apparent GPVI binding to types IV and V could be ascribed to type I collagen that was a contaminant in each of these preparations. Kinetic analyses of the binding data showed that type III collagen fibrils have both a higher Kd and Bmax than types I and II. Flow adhesion studies demonstrated that type III collagen supports the formation of larger platelet aggregates than type I. Our present results suggest that the physiological importance of type III collagen is to induce thrombus formation. Furthermore, these studies indicate that GPVI mainly binds to collagen types that can form large collagen fibrils.

Reconstituted type V collagen fibrils as cementing materials in the formation of cell clumps in culture.

Previous studies have reported that type V collagen is an anti-adhesive substrate for cultured cells in that the cells detach from culture dishes coated with type V collagen molecules or polypeptides derived from them. We have noticed that human fetal lung fibroblasts (TIG-1) initially show no reduction in adherence to and spreading on a dish coated with reconstituted type V collagen fibrils but eventually detach from the dish and form cell clumps. To determine the way in which reconstituted type V collagen fibrils are involved in cell clump formation, we have followed the fate of the fluorescence of type V collagen fibrils pre-labeled with fluorescein isothiocyanate. Essentially, all the fluorescence disappeared from the dish surface as the cells detached and was condensed in the cell clumps. The cells that were recovered from clumps and dissociated into separate cells by trypsin treatment proliferated normally after they were seeded on a bare culture dish. This result and those from gel electrophoresis, fluorescence microscopy, and a cell proliferation assay indicate that the cell detachment from the dish is not caused by cell necrosis or apoptosis but by cellular motility together with the unique features of type V collagen fibrils. Not only the adherence of type V collagen fibrils to TIG-1 cells is much stronger than that to the culture dish, but the fibrils are retained on the cellular surface. The strong adherence of type V collagen fibrils to cells plays a role in cementing TIG-1 cells together.