Induced Cell Adhesion Research Articles

Cell adhesion-induced transient interaction of ADAM15 with poly(A) binding protein at the cell membrane colocalizes with mRNA translation.

The regulation of temporo-spatial compartmentalization of protein synthesis is of crucial importance for a variety of physiologic cellular functions. Here, we demonstrate that the cell membrane-anchored disintegrin metalloproteinase ADAM15, upregulated in a variety of aggressively growing tumor cells, in the hyperproliferative synovial membrane of inflamed joints as well as in osteoarthritic chondrocytes, transiently binds to poly(A) binding protein 1 (PABP) in cells undergoing adhesion. The cytoplasmic domain of ADAM15 was shown to selectively interact with the proline-rich linker of PABP. Immunostainings of adhesion-triggered cells demonstrate an ADAM15-dependent recruitment of PABP to cell membrane foci coinciding with ongoing mRNA translation as visualized by the detection of puromycin-terminated polypeptides. Moreover, the increase in cell membrane-associated neosynthesis of puromycylated proteins upon induction of cell adhesion was proven linked to ADAM15 expression in HeLa and ADAM15-transfected chondrocytic cells. Thus, down regulation of ADAM15 by siRNA and/or the use of a cell line transfected with a mutant ADAM15-construct lacking the cytoplasmic tail resulted in a considerable reduction in the amount of cell membrane-associated puromycylated proteins formed during induced cell adhesion.These results provide first direct evidence for a regulatory role of ADAM15 on mRNA translation at the cell membrane that transiently emerges in response to triggering cell adhesion and might have potential implications under pathologic conditions of matrix remodeling associated with ADAM15 upregulation.

Cell adhesion properties of human defensins

Effector peptides of innate immunity play an important role in host defense. They act directly by inactivating microbes but also link innate to adaptive immunity. A variety of innate immune functions has been described for these peptides, including chemoattraction and cytokine release. In this study, we describe the effect on cell morphology and cell adhesion of human defensins. We find that Human Defensin 5, the major product of specialized gut epithelial cells, causes changes in cell morphology. HD-5 induces cell adhesion, binds to fibronectin and facilitates binding of T cells to intestinal epithelial cells. These effects were found also for a second prominent defensing, termed Human Neutrophil peptide-1, but not for other human defensins.

Lavender Essential Oil and Its Main Constituents Inhibit the Expression of TNF-α-induced Cell Adhesion Molecules in Endothelial Cells.

Lavender essential oil (Lvn) has anti-inflammatory effects in an ovalbumin-sensitized murine model of asthma, and inhibits inflammatory cell infiltration into the lungs. The anti-inflammatory effects of Lvn on cell adhesion molecules are not clear. Here we evaluated the effects of Lvn and its main constituents, linalyl acetate (LA) and linalool (LO), on the expression of tumor necrosis factor-alpha (TNF-α)-induced cell adhesion molecules in murine brain endothelial bEnd.3 cells and human umbilical vein endothelial cells (HUVECs). The bEnd.3 cells were treated with Lvn, LA, or LO and subsequently stimulated with TNF-α. The mRNA expression levels of cell adhesion molecules were detected using RT-PCR. E-selectin and P-selectin protein and phosphorylated-NF-κB p65 were detected by western blotting. The effects of Lvn on HUVECs were measured by RT-PCR. In bEnd.3 cells, Lvn and LA suppressed TNF-α-induced E-selectin, P-selectin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and phosphorylated-NF-κB p65 in the nucleus; LO did not suppress P-selectin or phosphorylated-NF-κB p65. Lvn inhibited TNF-α-induced E-selectin mRNA in HUVECs. These results indicate that Lvn and LA inhibit TNF-α-induced cell adhesion molecules in endothelial cells through the suppression of NF-κB activation. Consequently, Lvn or other essential oils including LA may be useful as alternative anti-inflammatory medicines.

Structure of Rap1b bound to talin reveals a pathway for triggering integrin activation

Activation of transmembrane receptor integrin by talin is essential for inducing cell adhesion. However, the pathway that recruits talin to the membrane, which critically controls talin’s action, remains elusive. Membrane-anchored mammalian small GTPase Rap1 is known to bind talin-F0 domain but the binding was shown to be weak and thus hardly studied. Here we show structurally that talin-F0 binds to human Rap1b like canonical Rap1 effectors despite little sequence homology, and disruption of the binding strongly impairs integrin activation, cell adhesion, and cell spreading. Furthermore, while being weak in conventional binary binding conditions, the Rap1b/talin interaction becomes strong upon attachment of activated Rap1b to vesicular membranes that mimic the agonist-induced microenvironment. These data identify a crucial Rap1-mediated membrane-targeting mechanism for talin to activate integrin. They further broadly caution the analyses of weak protein–protein interactions that may be pivotal for function but neglected in the absence of specific cellular microenvironments.

Osteopontin and their roles in hematological malignancies: Splice variants on the new avenues

Osteopontin (OPN) is a protein expressed in several tissues, including bone marrow, in which it performs distinct roles, such as modulating hematopoietic stem cell niche and bone remodeling. Most data in hematological malignancies (HMs) refers to total OPN (tOPN), comprehending the sum of distinct OPN splicing isoforms (OPN-SI), while reports describing the expression and roles of each OPN-SI are scarce. This review aims to summarize tOPN roles in HMs and provide evidence that OPN-SIs can also modulate specific functions in HMs biology. We summarize that upregulated tOPN can modulate HMs (leukemia, lymphoma and myeloma) progression, inducing cell adhesion, invasion, angiogenesis, cell differentiation and extramedullary and/or central nervous system infiltration. Based on this expression pattern, tOPN has been pointed out as a biomarker in those HMs, thus providing potential targets for therapeutic approaches. Our group found that OPN-SIs are expressed in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell lines (unpublished data), providing early evidence that OPN-SIs are also expressed in BCP-ALL. Further studies should investigate whether these OPN-SIs can differently modulate HMs biology and their putative application as auxiliary biomarkers for HMs.

Human Placental Hofbauer Cells Maintain an Anti-inflammatory M2 Phenotype despite the Presence of Gestational Diabetes Mellitus.

Hofbauer cells (HBCs) are macrophages of the feto-placental unit. Despite the general view that these cells have an anti-inflammatory M2 phenotype, recent studies have claimed that pregnancy pathologies-e.g., gestational diabetes mellitus (GDM)-cause a switch from an M2 to an M1 pro-inflammatory phenotype in HBCs. The pilot-study presented here challenges this claim, showing that HBCs maintain anti-inflammatory properties in spite of the hyperglycemic, low-grade inflammatory environment of GDM. HBCs were isolated from placentae of healthy women (N = 5) and women with GDM (N = 6) diagnosed in the second trimester. FACS was used to measure surface markers associated with either M1 or M2 phenotype on the cells. In addition, placental tissue sections were subjected to immune histochemical imaging to assess the phenotype within the tissue context. Supernatant from control and GDM HBCs was collected at defined time points and used in a multiplex ELISA-on-beads approach to assess secretion of cytokines, chemokines, and growth factors. The effect of HBC cell culture supernatant on placental endothelial activation was investigated. FACS and immune staining showed that, indeed, M2 markers, such as CD206 and CD209, are increased in HBCs isolated from GDM placentae. Also, the M1 marker CD86 was increased, but only by trend. Secretion of numerous cytokines, chemokines and growth factors was not changed; pro-inflammatory interleukin (IL)-1β and IL-6 release form GDM HBC was increased but not significant. Exposure to GDM HBC supernatant did not induce cell adhesion molecules (VCAM-1, selectins, vascular endothelial-cadherin) in placental endothelial cells compared to supernatant from control HBCs, an induction of intracellular adhesion molecule 1 was observed however. Our study-although performed in a small set of patients-shows that placental macrophages maintain their anti-inflammatory, tissue remodeling M2 phenotype even in pregnancies affected by gestational diabetes. This consistent phenotype might be important for propagation of maternal tolerance toward the fetus and for protection of the fetus from a low-grade inflammatory environment.

A novel biomimetic composite substitute of PLLA/gelatin nanofiber membrane for dura repairing

Objective: Biomimetic design will significantly improve growth and regeneration of dural cells and tissue for better repairing effects and fewer complications in repairing the native dura. This study designed a novel composite, biomimetic substitute based on the characteristics of native dura extracellular matrix.Methods and results: This substitute is expected to rapidly induce cell adhesion, migration, and fast regeneration of neotissue. The material characteristics (contact angle, surface charge, and zeta potential were evaluated), in vitro biological characteristics (cell stretch, connections between cells, cell proliferation) and in vivo tissue regeneration capability of this substitute were evaluated, compared to those of collagen dura substitute, the mostly used dura substitute. The results showed that the surface properties of this composite substitute were more biomimetic to native extracellular matrix than collagen substitute did, together with better cytocompatibility, tissue ingrowth, and neoangiogenesis. This composite substitute further demonstrated in clinical case study its ideal repair effect with no CSF leakage or other adverse reactions.Conclusion: In conclusion, the new biomimetic composite substitute provides alternative substitute for dura repairing.

Cell Adhesion Induced Using Surface Modification with Cell-Penetrating Peptide-Conjugated Poly(ethylene glycol)-Lipid: A New Cell Glue for 3D Cell-Based Structures.

We synthesized a novel material, cell-penetrating peptide-conjugated poly(ethylene glycol)-lipid (CPP-PEG-lipid), that can induce the adhesion of floating cells. Firm cell adhesion with spreading could be induced by cell surface modification with the CPP-PEG-lipids. Cell adhesion was induced by CPPs but not by any other cationic short peptides we tested. Here, we demonstrated adherence using the floating cell line CCRF-CEM as well as primary human T cells, B cells, erythrocytes, and hepatocytes. As compared to cells grown in suspension, adherent cells were more rapidly induced to attach to substrates with the cell-surface modification. The critical factor for attachment was localization of CPPs at the cell membrane by PEG-lipids with PEG > 20 kDa. These cationic CPPs on PEG chains were able to interact with substrate surfaces such as polystyrene (PS) surfaces, glass surfaces, and PS microfibers that are negatively charged, inducing firm cell adhesion and cell spreading. Also, as opposed to normal cationic peptides that interact strongly with cell membranes, CPPs were less interactive with the cell surfaces because of their cell-penetrating property, making them more available for adhering cells to the substrate surface. No effects on cell viability or cell proliferation were observed after the induction of cell adhesion. With this technique, cells could be easily immobilized onto PS microfibers, an important step in fabricating 3D cell-based structures. Cells immobilized onto 3D PS microfibers were alive, and human hepatocytes showed normal production of urea and albumin on the microfibers. This method is novel in inducing firm cell adhesion via a one-step treatment.

Bio-active molecules modified surfaces enhanced mesenchymal stem cell adhesion and proliferation

Surface modification of the substrate as a component of in vitro cell culture and tissue engineering, using bio-active molecules including extracellular matrix (ECM) proteins or peptides derived ECM proteins can modulate the surface properties and thereby induce the desired signaling pathways in cells. The aim of this study was to evaluate the behavior of human bone marrow mesenchymal stem cells (hBM-MSCs) on glass substrates modified with fibronectin (Fn), collagen (Coll), RGD peptides (RGD) and designed peptide (R-pept) as bio-active molecules. The glass coverslips were coated with fibronectin, collagen, RGD peptide and R-peptide. Bone marrow mesenchymal stem cells were cultured on different substrates and the adhesion behavior in early incubation times was investigated using scanning electron microscopy (SEM) and confocal microscopy. The MTT assay was performed to evaluate the effect of different bio-active molecules on MSCs proliferation rate during 24 and 72 h. Formation of filopodia and focal adhesion (FA) complexes, two steps of cell adhesion process, were observed in MSCs cultured on bio-active molecules modified coverslips, specifically in Fn coated and R-pept coated groups. SEM image showed well adhesion pattern for MSCs cultured on Fn and R-pept after 2 h incubation, while the shape of cells cultured on Coll and RGD substrates indicated that they might experience stress condition in early hours of culture. Investigation of adhesion behavior, as well as proliferation pattern, suggests R-peptide as a promising bio-active molecule to be used for surface modification of substrate in supporting and inducing cell adhesion and proliferation.

Daratumumab Impairs Myeloma Cell Adhesion Mediated Drug Resistance through CD38 Internalization

Introduction: Daratumumab (HuMax-CD38, Dara) is an immunoglobulin G1 kappa (IgG1k) human monoclonal antibody that binds a CD38 epitope that has been recently approved by the Food and Drugs Administration as a single agent for the treatment of Multiple Myeloma (MM). Multiple myeloma (MM) is a plasma cell disorder affecting approximately 83,000 US citizens with 30,330 new cases per year in the US. The discovery of intra-clonal heterogeneity strengthens the scientific rationale of using novel therapy combinations to overcome mechanisms of resistance.While CD38 participates in NAD+ hydrolysis generating adenosine and influences intracellular calcium homeostasis through cADPR and NAADP synthesis, CD38 facilitates bone marrow (BM) homing of plasma cells through interaction with CD31 which is highly expressed in MM BM stromal cells (BMSCs) and macrophages (BM-M). Since adhesion of MM cells to stromal cells induces cell adhesion mediated drug resistance (CAM-DR), in this work, tested whether CD38 internalization after Daratumumab treatment impairs stromal cell adhesion, sensitizing MM cells to other drugs including proteasome inhibitors.Methods:Flow cytometry analysis and single cell flow analysis was done to measure the extent of surface CD38 internalization into MM cells (MM cell lines and primary cells) in vitro and ex-vivo. Adhesion assays were performed using MM cell lines treated with Daratumumab and co-cultured with BMSCs and BM-M. Apoptotic assays including cell proliferation and Annexin-V/PI staining were done to assess proteasome inhibitor induced apoptosis (bortezomib, BTZ) of MM cells pretreated with Daratumumab in the presence or absence of BM stroma. Chou-Talalay synergy analysis was used to assess the ability of Daratumumab to synergize with BTZ.Results:Single cell flow analysis revealed surface CD38 internalization into MM cell lines (MM1.S, H929, L363, RPMI-8226) and in primary myeloma cells upon incubation with increasing doses of Daratumumab. Our data show that MM cell lines and primary CD138+ MM plasma cells (MM-PCs) revealed loss of adhesion in a dose and time dependent fashion in co-culture experiments with BMSc. Moreover our data indicate that both BMSCs and BM-M protect MM cells to BTZ treatments. In order to investigate whether loss of adhesion of MM cells deprives them of protection, MM cell lines and primary cells were treated with Daratumumab and co-cultured with BM stroma and then treated with BTZ. Interestingly, it was observed that although stromal cells could protect MM cells from induced apoptosis, it failed to do so when MM cells were pretreated with Daratumumab. A more than two-fold increase in MM cell apoptosis was observed with Daratumumab-BTZ combination compared to the single agent treatments. This indicates that Daratumumab potentiates BTZ killing of MM cells.Conclusion:Daratumumab in combination with both proteasome inhibitors and immune modulators (IMiDs) are synergistic as evidenced by the results of CASTOR and POLLUX trials respectively, but the mechanisms of killing and resistance will likely be different. The main anti-MM effect of Daratumumab has so far been attributed to its antibody-dependent cellular cytotoxicity, complement dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis activities and in promoting T cell expansion in relapsed/refractary MM patients enrolled in Daratumumab monotherapy trials. Our data indicate that Daratumumab potentiates the BTZ killing of MM cells via CD38 internalization, providing rationale to further explore CD38 targeting using other drugs or cell therapies. DisclosuresHofmeister:Celgene: Research Funding; Karyopharm Therapeutics: Research Funding; Arno Therapeutics, Inc.: Research Funding; Incyte, Corp: Membership on an entity’s Board of Directors or advisory committees; Signal Genetics, Inc.: Membership on an entity’s Board of Directors or advisory committees; Janssen: Pharmaceutical Companies of Johnson & Johnson: Research Funding; Takeda Pharmaceutical Company: Research Funding; Teva: Membership on an entity’s Board of Directors or advisory committees.

New β-Lactam Derivatives Modulate Cell Adhesion and Signaling Mediated by RGD-Binding and Leukocyte Integrins

A novel series of β-lactam derivatives that was designed and synthesized to target RGD-binding and leukocyte integrins is reported. The compound library was evaluated by investigating the effects on integrin-mediated cell adhesion and cell signaling in cell lines expressing αvβ3, αvβ5, αvβ6, α5β1, αIIbβ3, α4β1, and αLβ2 integrins. SAR analysis of the new series of azetidinones enabled the recognition of structural elements associated with integrin selectivity. We obtained selective and potent agonists that could induce cell adhesion and promote cell signaling mediated by αvβ3, αvβ5, α5β1, or α4β1 integrin, and antagonists for the integrins αvβ3 and α5β1, as well as α4β1 and αLβ2, preventing the effects elicited by the respective endogenous agonists.

Bovine P-selectin mediates leukocyte adhesion and is highly polymorphic in dairy breeds

Bovine P-selectin (SELP) mediates leukocyte rolling and primes leukocyte adhesion to endothelium, both essential for leukocyte recruitment to an infection site. We investigated SELP-mediated adhesion between bovine peripheral blood leukocytes (PBLs) and cultured bovine aortic endothelial cells pre-activated with lipopolysaccharide (LPS). We examined gene polymorphism for bovine selectins SELP, l-selectin (SELL) and E-selectin (SELE) and compared their SNP frequency between five dairy breeds (Holstein, Friesian, Jersey, Ayrshire and Brown Swiss). LPS treatment caused a rapid (10min) and slower (4h) enhancement of PBL adhesion (P<0.01). Antibody blocking of SELP inhibited LPS induced cell adhesion. SELP was highly polymorphic, with 9 of the 13 SNPs in its exons, whereas only three synonymous SNPs in SELL and one in SELE. The resulting amino acid changes for the three missense SELP SNP were located in the lectin domain and in two consensus repeat (CR) regions, CR2 and CR5. The Val475Met variant locus in the CR4 and CR5 linking region was very close to a predicted N-acetyl-d-glucosamine glycosylation site, which is likely to influence SELP function. The AA genotype was under-represented, only being found in 1% of 373 heifers genotyped from the 5 breeds (P=0.056), suggesting that AA homozygous animals carrying the Val475Met substitution for SELP may have compromised development. Our study thus confirmed that SELP mediates the attachment of PBL to endothelium and provides novel evidence that its high polymorphism is likely to affect biological function. This may potentially influence leukocyte migration and fertility, both key to successful performance in dairy cows.

Biologically and mechanically driven design of an RGD-mimetic macroporous foam for adipose tissue engineering applications.

Despite clinical treatments for adipose tissue defects, in particular breast tissue reconstruction, have certain grades of efficacy, many drawbacks are still affecting the long-term survival of new formed fat tissue. To overcome this problem, in the last decades, several scaffolding materials have been investigated in the field of adipose tissue engineering. However, a strategy able to recapitulate a suitable environment for adipose tissue reconstruction and maintenance is still missing. To address this need, we adopted a biologically and mechanically driven design to fabricate an RGD-mimetic poly(amidoamine) oligomer macroporous foam (OPAAF) for adipose tissue reconstruction. The scaffold was designed to fulfil three fundamental criteria: capability to induce cell adhesion and proliferation, support of invivo vascularization and match of native tissue mechanical properties. Poly(amidoamine) oligomers were formed into soft scaffolds with hierarchical porosity through a combined free radical polymerization and foaming reaction. OPAAF is characterized by a high water uptake capacity, progressive degradation kinetics and ideal mechanical properties for adipose tissue reconstruction. OPAAF's ability to support cell adhesion, proliferation and adipogenesis was assessed invitro using epithelial, fibroblast and endothelial cells (MDCK, 3T3L1 and HUVEC respectively). In addition, invivo subcutaneous implantation in murine model highlighted OPAAF potential to support both adipogenesis and vessels infiltration. Overall, the reported results support the use of OPAAF as a scaffold for engineered adipose tissue construct.

Flagellin based biomimetic coatings: From cell-repellent surfaces to highly adhesive coatings

Biomimetic coatings with cell-adhesion-regulating functionalities are intensively researched today. For example, cell-based biosensing for drug development, biomedical implants, and tissue engineering require that the surface adhesion of living cells is well controlled. Recently, we have shown that the bacterial flagellar protein, flagellin, adsorbs through its terminal segments to hydrophobic surfaces, forming an oriented monolayer and exposing its variable D3 domain to the solution. Here, we hypothesized that this nanostructured layer is highly cell-repellent since it mimics the surface of the flagellar filaments. Moreover, we proposed flagellin as a carrier molecule to display the cell-adhesive RGD (Arg-Gly-Asp) peptide sequence and induce cell adhesion on the coated surface. The D3 domain of flagellin was replaced with one or more RGD motifs linked by various oligopeptides modulating flexibility and accessibility of the inserted segment. The obtained flagellin variants were applied to create surface coatings inducing cell adhesion and spreading to different levels, while wild-type flagellin was shown to form a surface layer with strong anti-adhesive properties. As reference surfaces synthetic polymers were applied which have anti-adhesive (PLL-g-PEG poly(l-lysine)-graft-poly(ethylene glycol)) or adhesion inducing properties (RGD-functionalized PLL-g-PEG). Quantitative adhesion data was obtained by employing optical biochips and microscopy. Cell-adhesion-regulating coatings can be simply formed on hydrophobic surfaces by using the developed flagellin-based constructs. The developed novel RGD-displaying flagellin variants can be easily obtained by bacterial production and can serve as alternatives to create cell-adhesion-regulating biomimetic coatings. Statement of SignificanceIn the present work, we show for the first time that-an oriented and dense monolayer of flagellin molecules mimics the surface of the bacterial flagellar filament. Consequently, the fabricated layer is completely cell repellent.-By genetically modifying flagellin, we incorporate cell adhesion regulating functionalities into this anti-adhesive coating.-We can easily tune the adhesion of living cells from completely cell repellent to highly adhesive.

Salusin-β contributes to vascular inflammation associated with pulmonary arterial hypertension in rats

ObjectiveInflammation is closely linked to pulmonary arterial hypertension (PAH). Salusin-β, a bioactive peptide, has been reported to participate in vascular inflammation. We therefore hypothesized that salusin-β contributes to monocrotaline (MCT)-induced PAH in rats. MethodsMale Sprague-Dawley rats were treated with MCT (60 mg kg−1, single intraperitoneal injection). Salusin-β expression in the lungs of the MCT-treated rats was evaluated using immunofluorescence staining, western blot, and real-time PCR. For salusin-β blockade assay, rats injected with MCT were given a chronic infusion of anti-salusin-β immunoglobulin G (IgG) (salusin-β blocker, 1.0 μg kg−1 h−1) or isotype-matched control IgG. Four weeks after MCT+anti-salusin-β treatment, the effects of salusin-β blockade were determined using hemodynamics, western blot, real-time PCR, and immunohistochemical detection. The effect of salusin-β on human pulmonary arterial endothelial cell (HPAEC) function was detected by adhesion and tube formation experiments in vitro. ResultsSalusin-β expression was significantly increased in the lungs of the MCT-treated rats, and immunofluorescence results showed that salusin-β was predominantly expressed in pulmonary macrophages and vascular endothelial cells. Salusin-β blockade significantly ameliorated PAH by acting against pulmonary vascular remodeling, decreasing macrophage infiltration, and reducing pro-inflammatory cytokine expression and nuclear factor-kappa B (NF-κB) activity in the lungs of the MCT-treated rats. In addition, salusin-β could induce cell adhesion and accelerate angiogenesis by activating the NF-κB pathway and promoting pro-inflammatory cytokine expression in the cultured HPAECs. This effect was suppressed by addition of the NF-κB inhibitor, N-acetyl-L-cysteine. ConclusionsSalusin-β plays a crucial role in the development of MCT-induced PAH models.

Receptor FGFRL1 does not promote cell proliferation but induces cell adhesion.

Fibroblast growth factor receptor (FGFR)-like protein 1 (FGFRL1) is the most recently discovered member of the FGFR family. Owing to the fact that it interacts with FGF ligands, but lacks the intracellular tyrosine kinase domain, several researchers have speculated that it may function as a decoy receptor and exert a negative effect on cell proliferation. In this study, we performed overexpression experiments with TetOn-inducible cell clones and downregulation experiments with siRNA oligonucleotides, and found that FGFRL1 had absolutely no effect on cell growth and proliferation. Likewise, we did not observe any influence of FGFRL1 on ERK1/2 activation and on the phosphorylation of 250 other signaling proteins analyzed by the Kinexus antibody microarray. On the other hand, with bacterial petri dishes, we observed a clear effect of FGFRL1 on cell adhesion during the initial hours after cell seeding. Our results suggest that FGFRL1 is a cell adhesion protein similar to the nectins rather than a signaling receptor similar to FGFR1-FGFR4.

Y-632 inhibits heat shock protein 90 (Hsp90) function by disrupting the interaction between Hsp90 and Hsp70/Hsp90 organizing protein, and exerts antitumor activity in vitro and in vivo.

Heat shock protein 90 (Hsp90) stabilizes a variety of proteins required for cancer cell survival and has been identified as a promising drug target for cancer treatment. To date, several Hsp90 inhibitors have entered into clinical trials, but none has been approved for cancer therapy yet. Thus, exploring new Hsp90 inhibitors with novel mechanisms of action is urgent. In the present study, we show that Y‐632, a novel pyrimidine derivative, inhibited Hsp90 in a different way from the conventional Hsp90 inhibitor geldanamycin. Y‐632 induced degradation of diverse Hsp90 client proteins through the ubiquitin–proteasome pathway, as geldanamycin did; however, it neither directly bound to Hsp90 nor inhibited Hsp90 ATPase activity. Y‐632 inhibited Hsp90 function mainly through inducing intracellular thiol oxidation, which led to disruption of the Hsp90–Hsp70/Hsp90 organizing protein complex and further induced cell adhesion inhibition, G0/G1 cell cycle arrest, and apoptosis. Moreover, Y‐632 efficiently overcame imatinib resistance mediated by Bcr‐Abl point mutations both in vitro and in vivo. We believe that Y‐632, acting as a novel small‐molecule inhibitor of the Hsp90–Hsp70/Hsp90 organizing protein complex, has great potential to be a promising Hsp90 inhibitor for cancer therapy, such as for imatinib‐resistant leukemia.

Modification of β-TCP/PLGA scaffold and its effect on bone regeneration in vivo

In order to look for the best proportion of β-tricalcium phosphate(β-TCP) and poly(lactide-co-glycolide) (PLGA) we fabricated porous composites β-TCP/PLGA scaffold using freeze-drying method. Morphological characterization using scanning electron microscopy showed that the interconnected pore distribution was even and there was no significant difference with the increase of PLGA content. Moreover, the porosity, compressive strength and degradation in vitro were characterized. The fabricated scaffolds with increased PLGA in the composites β-TCP/PLGA scaffolds will get stronger mechanical property and better appearance, furthermore, get suitable environment for cells. According to the evaluation indexes for the tissue engineering scaffold, the group of scaffold (β-TCP/PLGA=6:4) was selected to evaluate the induced cell adhesion and proliferative ability of the scaffolds. Then as transplant embed into the bone critical defect sites on rats femur. The repairing processes of bone defect sites were characterized by X-ray analysis within 12 weeks. X-ray analysis showed that the bone defect sites all displayed the formation of callus obviously, In summary, our data suggest that the scaffold (β-TCP/PLGA=6:4) has a promising clinical future in regeneration of bone critical defects.

Receptor activator of NF-κB ligand induces cell adhesion and integrin α2 expression via NF-κB in head and neck cancers.

Cellular interactions with the extracellular matrix play critical roles in tumor progression. We previously reported that receptor activator of NF-κB ligand (RANKL) specifically facilitates head and neck squamous cell carcinoma (HNSCC) progression in vivo. Here, we report a novel role for RANKL in the regulation of cell adhesion. Among the major type I collagen receptors, integrin α2 was significantly upregulated in RANKL-expressing cells, and its knockdown suppressed cell adhesion. The mRNA abundance of integrin α2 positively correlated with that of RANKL in human HNSCC tissues. We also revealed that RANK-NF-κB signaling mediated integrin α2 expression in an autocrine/paracrine manner. Interestingly, the amount of active integrin β1 on the cell surface was increased in RANKL-expressing cells through the upregulation of integrin α2 and endocytosis. Moreover, the RANK-integrin α2 pathway contributed to RANKL-dependent enhanced survival in a collagen gel and inhibited apoptosis in a xenograft model, demonstrating an important role for RANKL-mediated cell adhesion in three-dimensional environments.

Inhibition of Cellular Adhesion by Immunological Targeting of Osteopontin Neoepitopes Generated through Matrix Metalloproteinase and Thrombin Cleavage.

Osteopontin (OPN), a secreted protein involved in inflammatory processes and cancer, induces cell adhesion, migration, and activation of inflammatory pathways in various cell types. Cells bind OPN via integrins at a canonical RGD region in the full length form as well as to a contiguous cryptic site that some have shown is unmasked upon thrombin or matrix metalloproteinase cleavage. Thus, the adhesive capacity of osteopontin is enhanced by proteolytic cleavage that may occur in inflammatory conditions such as obesity, atherosclerosis, rheumatoid arthritis, tumor growth and metastasis. Our aim was to inhibit cellular adhesion to recombinant truncated proteins that correspond to the N-terminal cleavage products of thrombin- or matrix metalloproteinase-cleaved OPN in vitro. We specifically targeted the cryptic integrin binding site with monoclonal antibodies and antisera induced by peptide immunization of mice. HEK 293 cells adhered markedly stronger to truncated OPN proteins than to full length OPN. Without affecting cell binding to the full length form, the raised monoclonal antibodies specifically impeded cellular adhesion to the OPN fragments. Moreover, we show that the peptides used for immunization were able to induce antisera, which impeded adhesion either to all OPN forms, including the full-length form, or selectively to the corresponding truncated recombinant proteins. In conclusion, we developed immunological tools to selectively target functional properties of protease-cleaved OPN forms, which could find applications in treatment and prevention of various inflammatory diseases and cancers.