Distribution Of Focal Adhesion Kinase Research Articles

A chalcone-syringaldehyde hybrid inhibits triple-negative breast cancer cell proliferation and migration by inhibiting CKAP2-mediated FAK and STAT3 phosphorylation

BackgroundAlthough triple-negative breast cancer (TNBC) accounts for only 15% of breast cancer cases, it is associated with a high relapse rate and poor outcome after standard treatment. Currently, the effective drugs and treatment strategies for TNBC remain limited, and thus, developing effective treatments for TNBC is pressing. Several studies have demonstrated that both chalcone and syringaldehyde have anticancer effect, but their potential anti-TNBC bioactivity are still unknown. PurposeThe present study aimed to synthesize a chalcone-syringaldehyde hybrid (CSH1) and explore its potential anti-TNBC effects and the underlying molecular mechanism. MethodsCell cytotoxicity was determined by 3-(4,5-dimethythiazol)-2,5-diphenyltetrazolium bromide (MTT). The activity of cell proliferation was measured by colony formation assay and 5-ethynyl-2’-deoxyuridine (EdU) staining assay. Cell cycle distribution and cell apoptosis were determined by fluorescence-activated cell sorter (FACS). The situation of DNA damage was observed using fluorescence microscopy. The ability of cell-matrix adhesion, migration and invasion was detected using cell adhesion assay and transwell assay. Transcriptome sequencing was performed to find out the changed genes. Levels of various signaling proteins were assessed by western blotting. ResultsCSH1 treatment triggered DNA damage and inhibited DNA replication, cell cycle arrest, and cell apoptosis via suppressing signal transducer and activator of transcription 3 (STAT3) phosphorylation. Whole genome RNA-seq analysis suggested that 4% of changed genes were correlated to DNA damage and repair, and nearly 18% of changed genes were functionally related to cell adhesion and migration. Experimental evidence indicated that CSH1 treatment significantly affected the distribution of focal adhesion kinase (FAK) and its phosphorylation, resulting in cell-matrix-adhesion reduction and migration inhibition of TNBC cells. Further mechanistic studies indicated that CSH1 inhibited TNBC cell proliferation, adhesion, and migration by inhibiting cytoskeleton-associated protein 2 (CKAP2)-mediated FAK and STAT3 phosphorylation signaling. ConclusionThese results suggest that CKAP2-mediated FAK and STAT3 phosphorylation signaling is a valuable target for TNBC treatment, and these findings also reveal the potential of CSH1 as a prospective TNBC drug.

Piperlongumine inhibits migration and proliferation of castration-resistant prostate cancer cells via triggering persistent DNA damage

BackgroundMetastatic castration-resistant prostate cancer (CRPC) is the leading cause of death among men diagnosed with prostate cancer. Piperlongumine (PL) is a novel potential anticancer agent that has been demonstrated to exhibit anticancer efficacy against prostate cancer cells. However, the effects of PL on DNA damage and repair against CRPC have remained unclear. The aim of this study was to further explore the anticancer activity and mechanisms of action of PL against CRPC in terms of DNA damage and repair processes.MethodsThe effect of PL on CRPC was evaluated by MTT assay, long-term cell proliferation, reactive oxygen species assay, western blot assay, flow cytometry assay (annexin V/PI staining), β-gal staining assay and DAPI staining assay. The capacity of PL to inhibit the invasion and migration of CRPC cells was assessed by scratch-wound assay, cell adhesion assay, transwell assay and immunofluorescence (IF) assay. The effect of PL on DNA damage and repair was determined via IF assay and comet assay.ResultsThe results showed that PL exhibited stronger anticancer activity against CRPC compared to that of taxol, cisplatin (DDP), doxorubicin (Dox), or 5-Fluorouracil (5-FU), with fewer side effects in normal cells. Importantly, PL treatment significantly decreased cell adhesion to the extracellular matrix and inhibited the migration of CRPC cells through affecting the expression and distribution of focal adhesion kinase (FAK), leading to concentration-dependent inhibition of CRPC cell proliferation and concomitantly increased cell death. Moreover, PL treatment triggered persistent DNA damage and provoked strong DNA damage responses in CRPC cells.ConclusionCollectively, our findings demonstrate that PL potently inhibited proliferation, migration, and invasion of CRPC cells and that these potent anticancer effects were potentially achieved via triggering persistent DNA damage in CRPC cells.

Geometric constraints of endothelial cell migration on electrospun fibres

Biomaterial scaffolds that can form a template for tissue growth and repair forms the basis of many tissue engineering paradigms. Cell migration and colonisation is an important, and often overlooked, first step. In this study, fibrous guidance structures were produced via electrospinning and the effect of physical features such as fibre diameter (ranging from 500 nm to 10 μm) on endothelial cell migration was assessed. Using a modified wound healing assay, fibre diameter was found to have a significant effect on the rate of wound closure and the peak migration velocity of the cells with scaffold diameter shown to influence both morphology and alignment of the migrating cells. The expression, phosphorylation and distribution of focal adhesion kinase (FAK) was disrupted on the different scaffolds with small-diameter scaffolds exhibiting increased FAK phosphorylation with the kinase present in the cytosol whereas on large-diameter scaffolds FAK was largely restricted to focal adhesions at the cell periphery. This study demonstrates that electrospun scaffolds can be used to model cell migration on fibrous substrates, and particularly for the studying effects of physical features of the substrate, and that FAK is a key mediator of cell-scaffold interactions on migrating cells.

Investigation of cell adhesion to structured surfaces using total internal reflection fluorescence and confocal laser scanning microscopy

Adhesion of adherent cells on structured surfaces is influenced by the surface pattern given. Here, we designed a structured gold relief surface based on cell adhesion patterns we had previously observed. We analysed the geometric parameters and the overall distribution of focal adhesion kinase in focal adhesions on unstructured glass surfaces using optical microscopy. The basic structural elements obtained from this analysis were arranged in regular clusters that resembled the shape of a polarised migratory cell. In time-lapse studies we observed that the cells adhere preferentially to the gold pads and adopt the shape of the clusters. Staining of the actin cytoskeleton revealed that the actin filaments are aligned to the gold pads of the elementary structure.

Projection model and calibration method for oblique-viewing endoscope

Skeletal homeostasis is partly regulated by the mechanical environment and specific signals generated by a cell's adhesion to the matrix. Previous studies demonstrated that osteopontin (OPN) expression is stimulated in response to both cellular adhesion and mechanical stimulation. The present studies examine if specific integrin ligands mediate osteoblast selective adhesion and whether opn mRNA expression is induced in response to these same ligands. Embryonic chicken calvaria osteoblastic cells were plated on bacteriological dishes coated with fibronectin (FN), collagen type I (Col1), denatured collagen/gelatin (G), OPN, vitronectin (VN), laminin (LN) or albumin (BSA). Osteoblastic cells were shown to selectively adhere to FN, Col1, G and LN, yet not to VN, OPN or BSA. Opn mRNA expression was induced by adhesion to Col1, FN, LN and G, but neither OPN nor VN induced this expression. Examination of the activation of the protein kinases A and C second signaling systems showed that only adhesion to FN induced protein kinase A and protein kinase C (PKC) activity while adherence to Col1 induced PKC. Evaluation of the intracellular distribution of focal adhesion kinase (FAK) and p-tyrosine within cells after adherence to FN, VN or BSA demonstrated that adherence to FN stimulated FAK translocation from the nucleus to the cytoplasm and high levels of p-tyrosine localization at the cell surface. However, cell adherence to VN or BSA did not show these morphological changes. These data illustrate that osteoblast selective adhesion is mediated by specific integrin ligands, and induction of intracellular second signal kinase activity is related to the nature of the ligand.

Selective adhesion of osteoblastic cells to different integrin ligands induces osteopontin gene expression.

Skeletal homeostasis is partly regulated by the mechanical environment and specific signals generated by a cell's adhesion to the matrix. Previous studies demonstrated that osteopontin (OPN) expression is stimulated in response to both cellular adhesion and mechanical stimulation. The present studies examine if specific integrin ligands mediate osteoblast selective adhesion and whether opn mRNA expression is induced in response to these same ligands. Embryonic chicken calvaria osteoblastic cells were plated on bacteriological dishes coated with fibronectin (FN), collagen type I (Col1), denatured collagen/gelatin (G), OPN, vitronectin (VN), laminin (LN) or albumin (BSA). Osteoblastic cells were shown to selectively adhere to FN, Col1, G and LN, yet not to VN, OPN or BSA. Opn mRNA expression was induced by adhesion to Col1, FN, LN and G, but neither OPN nor VN induced this expression. Examination of the activation of the protein kinases A and C second signaling systems showed that only adhesion to FN induced protein kinase A and protein kinase C (PKC) activity while adherence to Col1 induced PKC. Evaluation of the intracellular distribution of focal adhesion kinase (FAK) and p-tyrosine within cells after adherence to FN, VN or BSA demonstrated that adherence to FN stimulated FAK translocation from the nucleus to the cytoplasm and high levels of p-tyrosine localization at the cell surface. However, cell adherence to VN or BSA did not show these morphological changes. These data illustrate that osteoblast selective adhesion is mediated by specific integrin ligands, and induction of intracellular second signal kinase activity is related to the nature of the ligand.

Expression and Cytoskeletal Association of Integrin Subunits Is Selectively Increased in Rat Perivenous Hepatocytes After Chronic Ethanol Administration

For normal function and survival, hepatocytes require proper cell-extracellular matrix (ECM) contacts mediated by integrin receptors and focal adhesions. Previous studies have shown that chronic ethanol consumption selectively impairs perivenous (PV) hepatocyte attachment and spreading on various ECM substrates but increases expression of the beta1 integrin subunit, the common beta subunit for two major hepatocyte-ECM receptors, alpha1beta1 and alpha5beta1 integrins. This study examined the effects of ethanol treatment on the expression and cytoskeletal distribution of alpha1, alpha5, and beta1 integrin subunits, the epidermal growth factor receptor (EGF-R), and the cytoskeletal proteins focal adhesion kinase, paxillin, vinculin, and actin in periportal and PV hepatocytes. Periportal and PV hepatocytes were isolated from control and ethanol-fed rats. For expression analysis, lysates were examined by SDS-PAGE and immunoblotting procedures. For cytoskeletal distribution studies, Triton-soluble and -insoluble (cytoskeletal) fractions from hepatocytes cultured on collagen IV were analyzed by SDS-PAGE and immunoblotting. Chronic ethanol administration caused PV-specific increases in expression and cytoskeletal association of the integrin subunits. Although ethanol treatment did not affect expression of the EGF-R in either cell type, it did increase the association of the EGF-R with the cytoskeleton selectively in PV hepatocytes. Ethanol treatment had no significant effect on either the expression or the cytoskeletal distribution of focal adhesion kinase, paxillin, vinculin, or actin in either cell type. The increases in integrin expression and cytoskeletal association observed after chronic ethanol administration suggest that a process downstream of integrin-ECM interactions is impaired selectively in PV hepatocytes, possibly involving altered focal adhesion assembly or turnover, processes essential for efficient cell-ECM adhesion. Alterations in these processes could contribute to the impaired hepatocyte function and structure observed after chronic ethanol administration.

Substrate affects the initial attachment and subsequent behavior of human osteoblastic cells (Saos-2)

Interaction between implant materials and bone cells contributes to the clinical success of dental implants. The object of this study was to investigate the initial attachment and subsequent behavior of human osteoblastic cells (Saos-2) to pure titanium (Ti), hydroxyapatite (HA), and glass. We, therefore, performed a time-course study for examining the area, attachment rate, distribution of focal adhesion kinase (FAK) vinculin, and actin, and the motility of Saos-2 cells on the materials. On Ti, cell area increased gradually, whereas on HA, cells spread quickly, but quitted spreading at 12 h after cell seeding. The number of cells on HA was greater than on the other materials. On Ti, the numbers of FAK- and vinculin-positive focal adhesions increased continuously. On HA, although the number of FAK-positive focal adhesions also increased continuously, the number of vinculin-positive focal adhesions decreased. Furthermore, actin staining showed that the cells on HA poorly formed stress fibers with weak polarity, whereas the cell on Ti possessed well-defined polarized stress fibers. On HA, cells started extension earlier than on Ti, motility was inactive, and the cells settled on the materials. These results suggest that the earlier settling of osteoblasts on HA might result in earlier osteogenesis on HA than other materials.

Concentration of pp125 focal adhesion kinase (FAK) at the myotendinous junction.

Focal adhesion kinase is a recently characterized tyrosine kinase that is concentrated at focal contacts in cultured cells. It is thought to play an important role in the regulation of the integrin-based signal transduction mechanism involved in the assembly of this membrane specialization. In this study, we examined the immunocytochemical distribution of focal adhesion kinase in Xenopus skeletal muscle and its role in the formation of two sarcolemmal specializations, the myotendinous junction and the neuromuscular junction, using a monoclonal antibody (2A7) against this protein. Immunoprecipitation of Xenopus embryonic tissues with this antibody demonstrated a single band at a relative molecular mass of 116 kDa. A distinct concentration of immunolabeling for focal adhesion kinase was observed at the myotendinous junction of muscle fibers in vivo. At this site, the labeling for this protein is correlated with an accumulation of phosphotyrosine immunolabeling. Focal adhesion kinase was not concentrated at the neuromuscular junction in muscle cells either in vivo or in vitro. However, it was localized at spontaneously formed acetylcholine receptor clusters in cultured Xenopus myotomal muscle cells, although its distribution was not exactly congruent with that of the receptors. In these cells, the accumulation focal adhesion kinase was induced by polystyrene microbeads. In addition, beads also induce the formation of acetylcholine receptor clusters and myotendinous junction-like specializations. By following the appearance of the focal adhesion kinase relative to the formation of these sarcolemmal specializations at bead-muscle contacts in cultured muscle cells, we conclude that the accumulation of this protein was in pace with the development of the myotendinous junction, but occurred well after the clustering of acetylcholine receptors. These results suggest that focal adhesion kinase may be involved in the development and/or maintenance of the myotendinous junction through an integrin-based signaling system. Although it can accumulate at acetylcholine receptor clusters formed in culture, it does not appear to be involved in the development of the neuromuscular junction.