The movement of leukocytes from the blood into peripheral tissues is a central feature of immune surveillance, but also contributes to the pathogenesis of inflammatory and autoimmune diseases. Integrins are a family of adhesion and signaling molecules made up of paired alpha and beta subunits, and the integrin alpha4beta1 plays a prominent role in the trafficking of mononuclear leukocytes. We have previously described the direct interaction of the signaling adaptor molecule paxillin with the cytoplasmic domain of the alpha4 integrin subunit. This interaction is critical for alpha4beta1 integrin dependent cell adhesion under shear flow conditions as it provides a needed connection to the actin cytoskeleton. Furthermore, the alpha4-paxillin interaction is required for effective alpha4beta1 dependent leukocyte migration and does so through the temporal and spatial regulation of the small GTPase Rac. These findings make the alpha4-paxillin interaction a potentially attractive therapeutic target in controlling leukocyte trafficking.
Protein Binding ; Paxillin/*metabolism/physiology ; Models, Biological ; Leukocytes/cytology/*metabolism ; Integrin alpha4beta1/metabolism/physiology ; Integrin alpha4/*metabolism/physiology ; Humans ; Cell Movement/*physiology ; Cell Adhesion/physiology

OBJECTIVETo study the expressions of key assemblies of cytoskeleton, Fascin-1, Ezrin and Paxillin, in laryngeal squamous cell carcinoma (LSCC) and their correlation with clinicopathologic characteristics, cancer recurrence and survival of patients with LSCC.

METHODSThe expressions of Fascin-1, Ezrin and Paxillin proteins were detected by immunohistochemistry in 199 cases of LSCC. Unconditional Logistic regression model or Cox proportional hazards model was used for the analyses of recurrent risks and prognostic factors.

RESULTSSignificantly increased expression of Fascin-1, Ezrin or Paxillin expression was showed in the LSCC with poorly differentiated, positively cervical lymph nodal metastasis, and clinical stage III + IV respectively (P < 0.05). The expressions of three kinds of proteins in the recurrent cases were higher than those in non-recurrent cases respectively (χ(2) were 42.479, 43.673 and 22.261, P < 0.05). The highest recurrence rate (69.1%) was observed in group of cases with the highly co-expression of the three kinds of proteins (P < 0.05). The expression of Fascin-1 (OR = 7.89, 95%CI 2.26 - 27.53, P = 0.001), or Ezrin (OR = 2.51, 95%CI 1.18 - 5.32, P < 0.001) was independent risk for recurrence. Five-year disease-free survival rates of patients with high expression of Fascin-1, Ezrin or Paxillin were lower than those of patients with negative or low expressions for the proteins (P < 0.05). Patients with highly co-expression of three kinds of proteins showed the poorest survival prognosis, with a 5-year disease free survival (DFS) of only 26.4% (P < 0.05), and expressions of three proteins were independent prognostic factors for 5-year DFS (P < 0.05).

CONCLUSIONFascin-1, Ezrin, and Paxillin were correlative with LSCC progression and might be potential predictors for cancer recurrence and survival of patients with LSCC, as well as therapeutic targets for LSCC.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; diagnosis ; metabolism ; pathology ; Carrier Proteins ; metabolism ; Cytoskeletal Proteins ; metabolism ; Cytoskeleton ; metabolism ; Female ; Humans ; Laryngeal Neoplasms ; diagnosis ; metabolism ; pathology ; Male ; Microfilament Proteins ; metabolism ; Middle Aged ; Neoplasm Recurrence, Local ; Paxillin ; metabolism ; Prognosis

Tumor angiogenesis induced by vascular endothelial cells (VECs) migration is a necessary condition for tumor growth and metastasis. The purpose of this study is to investigate the effect of focal adhesion kinase (FAK) inhibitor (50nmol/mL) on the adhesion and migration of endothelial cells(ECs) and the expression of focal adhesion proteins vinculin, talin and paxillin. Scratch wound migration assay was performed to examine the effect of FAK inhibitor with 50nmol/mL on ECs migration at 0, 5, 10, 30, 60 and 120min, respectively. And immunofluorescence analysis was performed to detect the expression of F-actin in ECs treated with FAK inhibitor within 2h. Western blot was carried out to determine the effect of FAK inhibitor on expression of vinculin, talin and paxillin proteins. The results showed that the migration distance and the expression of F-actin in ECs treated with FAK inhibitor decreased significantly compared with that of the controls, and the level of vinculin showed no significant difference with increasing of treated time of FAK inhibitor. However, the talin and paxillin showed an identical decreasing tendency in 5-10min, but slowly going up in 30min and then after subsequently decreasing. The results of this study proved that blocking phosphorylation of FAK could inhibit VECs adhesion and migration by downregulating focal adhesion proteins so that it may inhibit tumor angiogenesis. This may provide a new approach for tumor therapy.
Cell Adhesion ; Cell Movement ; physiology ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Focal Adhesion Protein-Tyrosine Kinases ; antagonists & inhibitors ; metabolism ; Focal Adhesions ; metabolism ; physiology ; Humans ; Neoplasms ; blood supply ; Neovascularization, Pathologic ; Paxillin ; metabolism ; Talin ; metabolism ; Vinculin ; metabolism

Carcinoma-associated fibroblasts (CAFs) are the main cellular components of the tumor microenvironment and promote cancer progression by modifying the extracellular matrix (ECM). The tumor-associated ECM is characterized by collagen crosslinking catalyzed by lysyl oxidase (LOX). Small extracellular vesicles (sEVs) mediate cell-cell communication. However, the interactions between sEVs and the ECM remain unclear. Here, we demonstrated that sEVs released from oral squamous cell carcinoma (OSCC)-derived CAFs induce collagen crosslinking, thereby promoting epithelial-mesenchymal transition (EMT). CAF sEVs preferably bound to the ECM rather than being taken up by fibroblasts and induced collagen crosslinking, and a LOX inhibitor or blocking antibody suppressed this effect. Active LOX (αLOX), but not the LOX precursor, was enriched in CAF sEVs and interacted with periostin, fibronectin, and bone morphogenetic protein-1 on the surface of sEVs. CAF sEV-associated integrin α2β1 mediated the binding of CAF sEVs to collagen I, and blocking integrin α2β1 inhibited collagen crosslinking by interfering with CAF sEV binding to collagen I. CAF sEV-induced collagen crosslinking promoted the EMT of OSCC through FAK/paxillin/YAP pathway. Taken together, these findings reveal a novel role of CAF sEVs in tumor ECM remodeling, suggesting a critical mechanism for CAF-induced EMT of cancer cells.
Humans ; Paxillin/metabolism* ; Protein-Lysine 6-Oxidase/metabolism* ; Carcinoma, Squamous Cell/pathology* ; Epithelial-Mesenchymal Transition ; Integrin alpha2beta1/metabolism* ; Mouth Neoplasms/pathology* ; Collagen/metabolism* ; Fibroblasts ; Extracellular Vesicles/metabolism* ; Cell Line, Tumor ; Tumor Microenvironment

BACKGROUNDColorectal carcinoma is one of the most common malignant tumors. Despite advances in therapy, mortality is still very high. The aim of this study was to evaluate the expression of paxillin in the human colon adenocarcinoma cell line SW480 and its role in cell cycle and apoptosis. We also investigated the expression of paxillin in colorectal carcinoma tissues and its relationship to clinicopathological features and survival.

METHODSPaxillin short hairpin RNA (shRNA) was constructed and transfected into the colon adenocarcinoma cell line SW480. The influence of paxillin shRNA on the cell cycle and cell apoptosis was analyzed by flow cytometry. Immunohistochemistry staining was used to assess the expression of paxillin and its association with the expression of carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9, p53 and Bcl-2 in 102 patients with primary colorectal carcinoma. Western blotting was also used to investigate the expression of paxillin. Medical records were reviewed and a clinicopathological analysis was performed.

RESULTSIn vitro, the percentage of cells in S phase was (45.23±1.05)%, (43.53±1.23)%, and (36.13±0.57)% in the blank control group, negative control group, and paxillin shRNA group respectively. It was significantly decreased in the paxillin shRNA group (P = 0.000). The early apoptosis index of the paxillin shRNA group (17.2±1.18%) was significantly increased compared to the control shRNA group ((13.17±1.15)%, P = 0.013). Paxillin was positive in 71 (69.6%) patients, and it was found to be overexpressed in tumor tissues compared with normal adjacent tissues. Paxillin positive rate was higher in patients who are less than 50-years old (100.0% vs. 65.6%, P = 0.016). Paxillin expression was associated with a high histologic grade of carcinoma (81.4% vs. 61.0%, P = 0.031), a high rate of regional lymph node metastasis (22.5% vs. 13.0%, P = 0.031), mesenteric artery lymph node metastasis (100.0% vs. 64.8%, P = 0.008), distant metastasis (94.1% vs. 64.7%, P = 0.016) and a high Tumor Node Metastasis (TNM) stage (94.1%, 73.2%, 60.0%, and 50%, P = 0.030). Multivariate analyses revealed that recurrence was associated with the rate of regional lymph node metastasis (P = 0.001) and paxillin expression (P = 0.024). Multivariate analysis indicated that the overall survival is related to the TNM stage (P = 0.000).

CONCLUSIONSIn vitro, paxillin may promote cell proliferation and inhibit apoptosis in SW480 cells. Paxillin may be a potential metastasis predictor, and an independent prognosis factor of recurrence. It may also be related to poor patient outcomes, but was not an independent predictor of survival.

Apoptosis ; genetics ; physiology ; Biomarkers, Tumor ; genetics ; metabolism ; Carcinoembryonic Antigen ; metabolism ; Cell Cycle ; genetics ; physiology ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; genetics ; metabolism ; Female ; Humans ; Immunohistochemistry ; In Vitro Techniques ; Male ; Paxillin ; genetics ; metabolism ; RNA, Small Interfering ; genetics

OBJECTIVETo investigate changes in the adherent ability, the expression of adhesion related proteins Pyk2 and paxillin during HL-60 cells differentiation into granulocyte-monocyte induced by low-dose (LD) bufalin in combination with all-trans retinoic acid (ATRA), and to explore the effects of bortezomib on cellular adhesion and the expression of Pyk2 and paxillin.

METHODSThe expression of CD11b was detected by flow cytometry, cellular adherence ability by MTT assay, and the expressions of Pyk2, paxillin and tubulin by Western blot.

RESULTSThe combination of 5 nmol/L bufalin and 30 nmol/L ATRA induced HL-60 cells differentiation in a time-dependent manner, the percentages of CD11b positive cells treated for 2 d and 4 d being (20.0 +/- 2.8)% and (75.0 +/- 5.3)%, respectively, with the increasing of cellular adherence ability. Meanwhile the expressions of Pyk2 and Paxillin were also up-regulated in a time-dependent manner. Bortezomib suppressed HL-60 cell adhesion in a dose-dependent manner. At concentrations of 1 nmol/L and 10 nmol/L the adherence level were (7.8 +/- 0.1)% and (5.3 +/- 0.3)%, respectively, with down-regulation of Pyk2 but not Paxillin.

CONCLUSIONPyk2 is involved in the regulation of cellular adherence function. Bortezomib might inhibit HL-60 cells adhension function by down-regulation of Pyk2 expression.

Boronic Acids ; pharmacology ; Bortezomib ; Bufanolides ; pharmacology ; Cell Adhesion ; drug effects ; Cell Proliferation ; drug effects ; Focal Adhesion Kinase 2 ; metabolism ; HL-60 Cells ; Humans ; Paxillin ; metabolism ; Pyrazines ; pharmacology ; Tretinoin ; pharmacology

Metastasis is responsible for the majority of cancer-related deaths and prevention of metastasis remains a big challenge for cancer therapy. Cucurbitacin B (Cuc B) is a natural triterpenoid with potent anticancer activities while its effect on metastasis remains unclear. In the present study, the inhibitory effect and mechanisms of Cuc B on metastasis were investigated in MDA-MB-231 breast cancer cells. The cells were treated with or without Cuc B, and the cytotoxicity was determined by MTT assay. The effect of Cuc B on metastasis was evaluated with wound healing, transwell, and adhesion assays. Furthermore, the adhesion of cancer cells to endothelial cells was determined. The protein expression was determined by Western blotting. Cuc B (< 100 nmol·L) showed no obvious cytotoxicity to MDA-MB-231 cells, but significantly inhibited migration, invasion, and adhesion to Matrigel, fibronectin, type I collagen, and endothelial cells. Cuc B dramatically inhibited the phosphorylation of focal adhesion kinase (FAK) and paxillin in dose- and time-dependent manners. Furthermore, Cuc B induced intracellular reactive oxygen species (ROS) generation, which could be reduced by N-acetyl-l-cysteine (NAC). In addition, NAC pretreatment could reverse Cuc B-induced suppression of migration and adhesion, expression of FAK, but showed no effect on paxillin expression. In summary, Cuc B suppressed ROS-dependent metastasis through FAK pathway in breast cancer MDA-MB-231 cells, demonstrating novel mechanisms for the anticancer effects of Cuc B.
Acetylcysteine ; pharmacology ; Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; enzymology ; metabolism ; pathology ; physiopathology ; Cell Adhesion ; drug effects ; Cell Line, Tumor ; Cell Movement ; drug effects ; Collagen Type I ; metabolism ; Dose-Response Relationship, Drug ; Down-Regulation ; drug effects ; Female ; Fibronectins ; metabolism ; Focal Adhesion Kinase 1 ; metabolism ; Humans ; Neoplasm Invasiveness ; pathology ; Neoplasm Metastasis ; pathology ; Paxillin ; metabolism ; Phosphorylation ; drug effects ; Reactive Oxygen Species ; metabolism ; Triterpenes ; antagonists & inhibitors ; chemistry ; pharmacology

Adhesion and migration of vascular smooth muscle cells (VSMCs) play an important role in the pathogenesis of atherosclerosis. These processes involve the interaction of VSMCs with extracellular matrix proteins. Here, we investigated integrin isoforms and signaling pathways mediating the adhesion and migration of VSMCs on betaig-h3, a transforming growth factor (TGF)-beta-inducible extracellular matrix protein that is elevated in atherosclerotic plaques. Adhesion assays showed that the alphavbeta5 integrin is a functional receptor for the adhesion of aortic VSMCs to betaig-h3. An YH18 motif containing amino acids between 563 and 580 of betaig-h3 was an essential motif for the adhesion and growth of VSMCs. Interaction between the YH18 motif and the alphavbeta5 integrin was responsible for the migration of VSMCs on betaig-h3. Inhibitors of phosphatidylinositide 3-kinase, extracellular signal-regulated kinase (ERK), and Src kinase reduced the adhesion and migration of VSMCs on betaig-h3. betaig-h3 triggered phosphorylation and activation of AKT, ERK, focal adhesion kinase, and paxillin mediating the adhesion and migration of VSMCs. Taken together, these results suggest that betaig-h3 and alphavbeta5 integrin play a role in the adhesion and migration of VSMCs during the pathogenesis of atherosclerosis.
src-Family Kinases/antagonists & inhibitors ; Transforming Growth Factor beta/genetics/*physiology ; Signal Transduction/*physiology ; Receptors, Vitronectin/genetics/*physiology ; Protein-Tyrosine Kinases/antagonists & inhibitors ; Paxillin/metabolism ; Myocytes, Smooth Muscle/drug effects/metabolism ; Muscle, Smooth, Vascular/cytology/drug effects/*metabolism ; Morpholines/pharmacology ; Molecular Sequence Data ; Integrins/genetics/*physiology ; Humans ; Flavonoids/pharmacology ; Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors ; Extracellular Matrix Proteins/genetics/*physiology ; Enzyme Inhibitors/pharmacology ; Chromones/pharmacology ; Cells, Cultured ; Cell Movement/*physiology ; Cell Adhesion/physiology ; Animals ; Amino Acid Sequence ; Amino Acid Motifs/genetics ; 1-Phosphatidylinositol 3-Kinase/antagonists & inhibitors