Baicalein, a natural flavonoid obtained from the rhizome of Scutellaria baicalensis Georgi, has been reported to have anticancer activities in several human cancer cell lines. However, its antimetastatic effects and associated mechanisms in melanoma cells have not been extensively studied. The current study examined the effects of baicalein on cell motility and anti-invasive activity using mouse melanoma B16F10 cells. Within the noncytotoxic concentration range, baicalein significantly inhibited the cell motility and invasiveness of B16F10 cells in a concentration-dependent manner. Baicalein also reduced the activity and expression of matrix metalloproteinase (MMP)-2 and -9; however, the levels of tissue inhibitor of metalloproteinase-1 and -2 were concomitantly increased. The inhibitory effects of baicalein on cell motility and invasiveness were found to be associated with its tightening of tight junction (TJ), which was demonstrated by an increase in transepithelial electrical resistance and downregulation of the claudin family of proteins. Additionally, treatment with baicalein markedly reduced the expression levels of lipopolysaccharide-induced phosphorylated Akt and the invasive activity in B16F10 cells. Taken together, these results suggest that baicalein inhibits B16F10 melanoma cell migration and invasion by reducing the expression of MMPs and tightening TJ through the suppression of claudin expression, possibly in association with a suppression of the phosphoinositide 3-kinase/Akt signaling pathway.
Animals ; Cell Line ; Cell Movement ; Down-Regulation ; Electric Impedance ; Humans ; Matrix Metalloproteinases ; Melanoma* ; Mice* ; Rhizome ; Scutellaria baicalensis ; Tight Junctions ; Tissue Inhibitor of Metalloproteinase-1

In order to study the character of periodontal ligament cells (PDLCs) attaching on commercially pure titanium (cpTi) by morphology and metrology on the early stage (24 h), 1 x 10(5)/ml PDLCs in 2 ml culture medium were seeded on cpTi discs fixed in 24-well culture plates. Morphology of cell attachment was observed by contrast phase microscope, scanning electron microscope (SEM) and fluroscence microscopy. Cell adhesion was analyzed by MTT at 0.5, 1, 2, 4 h respectively. PDLCs could attach and spread on cpTi discs. SEM showed that PDLCs had pseudopod-like protuberance. PDLCs showed different attaching phases and reached saturation in cell number at 2 h. It was concluded that PDLCs had good biocompatibility with cpTi, and showed a regular and dynamic pattern in the process of attaching to cpTi.
Biocompatible Materials ; pharmacology ; Cell Adhesion ; Cell-Matrix Junctions ; Cells, Cultured ; Dental Implants ; Humans ; Periodontal Ligament ; cytology ; Surface Properties ; Time Factors ; Titanium ; pharmacology

In order to study the character of periodontal ligament cells (PDLCs) attaching on commercially pure titanium (cpTi) by morphology and metrology on the early stage (24 h), 1 x 10(5)/ml PDLCs in 2 ml culture medium were seeded on cpTi discs fixed in 24-well culture plates. Morphology of cell attachment was observed by contrast phase microscope, scanning electron microscope (SEM) and fluroscence microscopy. Cell adhesion was analyzed by MTT at 0.5, 1, 2, 4 h respectively. PDLCs could attach and spread on cpTi discs. SEM showed that PDLCs had pseudopod-like protuberance. PDLCs showed different attaching phases and reached saturation in cell number at 2 h. It was concluded that PDLCs had good biocompatibility with cpTi, and showed a regular and dynamic pattern in the process of attaching to cpTi.
Biocompatible Materials/pharmacology ; Cell Adhesion ; Cell-Matrix Junctions ; Cells, Cultured ; *Dental Implants ; Periodontal Ligament/*cytology ; Surface Properties ; Time Factors ; Titanium/*pharmacology

OBJECTIVETo investigate the effect of cell surface sialic acid and its linkage on the cell-cell and cell-matrix adhesion of mammary carcinoma cells MD-MB-435.

METHODSMD-MB-435 cells were sense-transfected with ST6Gal I cDNA or antisense-transfected with part of the ST6Gal I sequence inserted in pcDNA 3.1 vector, with mock transfection with pcDNA3.1 vector as the control. The cell surface alpha2, 6-linked sialylation was determined by fluorescence-activated cell sorting (FACS) using lectin SNA (Sambucus nigra agglutinin specific to alpha2, 6-linked sialic acid on N-linked glycoprotein). A significantly increased alpha2, 6-sialylation subclone in sense-transfectants and a decreased alpha2, 6-sialylation subclone in antisense-transfectants were selected for further examination of cell-cell and cell-matrix (collagen IV) adhesion. The transfectants were also treated with sialidase to compare the capacity of cell adhesion affected by cell surface sialylation.

RESULTSSense-transfection subclone showed a reduced cell-cell aggregation but enhanced cell-matrix adhesion. In contrast, the antisense-transfection subclone exhibited increased cell-cell aggregation and decreased cell-matrix adhesion. After treatment with sialidase, the cell-matrix adhesion of all the transfectants and the parental MDA-MB-435 cells were significantly reduced to the level of 31%-57% of untreated cells.

CONCLUSIONCell surface sialic acid and alpha2, 6-linked sialylation play an important role in cell-cell and cell-matrix adhesion of mammary carcinoma cell MDA-MB-435.

Antigens, CD ; genetics ; metabolism ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Adhesion ; Cell Line, Tumor ; Cell Membrane ; metabolism ; Cell-Matrix Junctions ; metabolism ; Collagen Type IV ; metabolism ; Extracellular Matrix ; metabolism ; Humans ; N-Acetylneuraminic Acid ; metabolism ; Sialyltransferases ; genetics ; metabolism ; Transfection

The importance of soft tissue response to implant abutments has become one of the major issues in current implant dentistry. To date, numerous studies have emphasized on maintaining connective tissue barriers in quantity, as well as in quality for the long term success of dental implants. The cells mainly consisting the soft tissue around dental implants are fibroblasts and epithelial cells. The mechanism of the fibroblasts'adhesions to certain substrata can be explained by the 'focal adhesion'theory. On the other hand, epithelial cells adhere to the substratum via hemidesmosomes. The typical integrin-mediated adhesions of cells to certain matrix are called 'cell-matrix adhsions'. The focal adhesion complex of fibroblasts, in relation to the cell-matrix adhsions, consists of the extracellular matrix(ECM) such as fibronectin, the transmembrane proteins such as integrins, the intracellular cytoplasmic proteins such as vinculin, talin, and more, and the cytoskeletal structures such as filamentous actin and microtubules. The mechanosensory function of integrins and focal adhesion complexes are considered to play a major role in the cells'adhesion, migration, proliferation, differentiation, division, and even apoptosis. The '3-D matrix adhesions'defined by Cukierman et al. makes a promising future for the verification of the actual process of the cell-matrix adhesions in vivo and can be applied to the field of implant dentistry in relation to obtaining strong soft tissue attachment to the implant abutments.
Actins ; Apoptosis ; Cell-Matrix Junctions* ; Connective Tissue ; Cytoplasm ; Dental Implants* ; Dentistry ; Epithelial Cells ; Fibroblasts ; Fibronectins ; Focal Adhesions ; Hand ; Hemidesmosomes ; Integrins ; Microtubules ; Talin ; Vinculin

The importance of soft tissue response to implant abutments has become one of the major issues in current implant dentistry. To date, numerous studies have emphasized on maintaining connective tissue barriers in quantity, as well as in quality for the long term success of dental implants. The cells mainly consisting the soft tissue around dental implants are fibroblasts and epithelial cells. The mechanism of the fibroblasts'adhesions to certain substrata can be explained by the 'focal adhesion'theory. On the other hand, epithelial cells adhere to the substratum via hemidesmosomes. The typical integrin-mediated adhesions of cells to certain matrix are called 'cell-matrix adhsions'. The focal adhesion complex of fibroblasts, in relation to the cell-matrix adhsions, consists of the extracellular matrix(ECM) such as fibronectin, the transmembrane proteins such as integrins, the intracellular cytoplasmic proteins such as vinculin, talin, and more, and the cytoskeletal structures such as filamentous actin and microtubules. The mechanosensory function of integrins and focal adhesion complexes are considered to play a major role in the cells'adhesion, migration, proliferation, differentiation, division, and even apoptosis. The '3-D matrix adhesions'defined by Cukierman et al. makes a promising future for the verification of the actual process of the cell-matrix adhesions in vivo and can be applied to the field of implant dentistry in relation to obtaining strong soft tissue attachment to the implant abutments.
Actins ; Apoptosis ; Cell-Matrix Junctions* ; Connective Tissue ; Cytoplasm ; Dental Implants* ; Dentistry ; Epithelial Cells ; Fibroblasts ; Fibronectins ; Focal Adhesions ; Hand ; Hemidesmosomes ; Integrins ; Microtubules ; Talin ; Vinculin

PURPOSE: Despite the well-known anti-inflammatory effects of vitamin D in periodontal health, its mechanism has not been fully elucidated. In the present study, the effect of vitamin D on strengthening E-cadherin junctions (ECJs) was explored in human gingival keratinocytes (HGKs). ECJs are the major type of intercellular junction within the junctional epithelium, where loose intercellular junctions develop and microbial invasion primarily occurs. METHODS: HOK-16B cells, an immortalized normal human gingival cell line, were used for the study. To mimic the inflammatory environment, cells were treated with tumor necrosis factor-alpha (TNF-α). Matrix metalloproteinases (MMPs) in the culture medium were assessed by an MMP antibody microarray and gelatin zymography. The expression of various molecules was investigated using western blotting. The extent of ECJ development was evaluated by comparing the average relative extent of the ECJs around the periphery of each cell after immunocytochemical E-cadherin staining. Vitamin D receptor (VDR) expression was examined via immunohistochemical analysis. RESULTS: TNF-α downregulated the development of the ECJs of the HGKs. Dissociation of the ECJs by TNF-α was accompanied by the upregulation of MMP-9 production and suppressed by a specific MMP-9 inhibitor, Bay 11-7082. Exogenous MMP-9 decreased the development of ECJs. Vitamin D reduced the production of MMP-9 and attenuated the breakdown of ECJs in the HGKs treated with TNF-α. In addition, vitamin D downregulated TNF-α-induced nuclear factor kappa B (NF-κB) signaling in the HGKs. VDR was expressed in the gingival epithelium, including the junctional epithelium. CONCLUSIONS: These results suggest that vitamin D may avert TNF-α-induced downregulation of the development of ECJs in HGKs by decreasing the production of MMP-9, which was upregulated by TNF-α. Vitamin D may reinforce ECJs by downregulating NF-κB signaling, which is upregulated by TNF-α. Strengthening the epithelial barrier may be a way for vitamin D to protect the periodontium from bacterial invasion.
Bays ; Blotting, Western ; Cadherins ; Cell Line ; Down-Regulation ; Epithelial Attachment ; Epithelium ; Gelatin ; Humans ; Intercellular Junctions ; Keratinocytes ; Matrix Metalloproteinase 9 ; Matrix Metalloproteinases ; NF-kappa B ; Periodontium ; Receptors, Calcitriol ; Tumor Necrosis Factor-alpha ; Up-Regulation ; Vitamin D ; Vitamins

OBJECTIVE: Immunofluorescence microscopy including confocal laser scanning microscopy and electron microscopy were used to study the production of fibronectin, tenascin, and laminin in the cumulus-corona (CC) cells surrounding mature, unfertilized oocytes after ovulation in view of their presumptive importance in the coordination of the processes leading to fertilization and early embryo cleavage, including the final maturation of the ovum, the sperm-egg interaction, and the complex biochemical mechanism between the ovum and the oviduct. METHODS: Mature oocyte-cumulus complex (OCC) was cultured for 24 and 48 hour and fixed in 3.7% formaldehyde. Specimens were incubated with a mixture of primary monoclonal antibodies recognizing different epitopes of fibronectin, tenascin, and laminin, and then with a mixture of secondary antibodies containing FITC, TRITC, and Cy-5 conjugated antibodies. Observation was made by confocal laser scanning microscope equipped with epifluorescece optics. Transmission electron microscopy were used to observe the OCC at 24 and 48 hours after cultrue. RESULTS: The immunocytochemical date demonstrated that CC masses are capable of producing fibronectin and tenascin but their production is heterogeneous in the CC population. Immunoreactivity to fibronectin and tenascin was shown mostly by inner corona cells, and the intensity of immunofluorescence decreased from the central corona cells to the peripheral cumulus cells. Colocalization of fibronectin and tenascin was evident in most CC cells. Moreover, fibronectin and tenascin immunoreactive material was observed in the intracytoplasmic areas, at the plasma membrane level as well as in the extracellular matrix. Whereas, laminin immunofluorescence was found around plasma membrane and extracellular area, but a intracytoplasmic reaction was rarely observed. The distribution of laminin immunofluorescence was similar to that of fibronectin and tenascin, but in some cumulus cells, colocalization between them was not found. Ultrastructurally, cumulus cells projected numerous long, thin microvilli into the intercellular area and some micovilli penetrated into zona pellucida. The inner layer of the cumulus mass was loose arrangement of relatively uniform, small cells with widened intercellular spaces, whereas in the outer layer, cumulus cells are rather larger in size and compact arrangement by narrow, irregular spaces. A small and large linear gap junctions were easily found at cell contacts. The cytoplasm of most cells had abundant organelles typical of steroidogenesis: numerous mitochondrias, a well-developed smooth endoplasmic reticulum, electron dense lipid droplets, and bundles of microtubules and microfilaments. Rudimentary disrupted basal lamina along the cytoplasmic border was rarely seen in a few inner conora cells. CONCLUSION: Even though the functional role of these extracellular matrix proteins remains still unclear, it is reasonable to suggest that they are necessary in various steps of the reproductive process. Cumulus cells appears to be a heterogeneous and dynamic system for suitable microenviroment of fertilization. And functional differences between corona and cumulus cells during the oocyte denudation may be accounted for particular distribution of these adhesive proteins and steroidogenesis-related organelles.
Actin Cytoskeleton ; Adhesives ; Animals ; Antibodies ; Antibodies, Monoclonal ; Basement Membrane ; Cell Membrane ; Cumulus Cells ; Cytoplasm ; Embryonic Structures ; Endoplasmic Reticulum, Smooth ; Epitopes ; Extracellular Matrix ; Extracellular Matrix Proteins ; Extracellular Space ; Female ; Fertilization ; Fibronectins* ; Fluorescein-5-isothiocyanate ; Fluorescent Antibody Technique ; Formaldehyde ; Gap Junctions ; Immunohistochemistry ; Laminin* ; Microscopy, Confocal ; Microscopy, Electron* ; Microscopy, Electron, Transmission ; Microscopy, Fluorescence ; Microtubules ; Microvilli ; Mitochondria ; Oocytes ; Organelles ; Oviducts ; Ovulation ; Ovum ; Sperm-Ovum Interactions ; Tenascin* ; Zona Pellucida