Involvement of the IL-6 Signaling Pathway in the Anti-Anhedonic Effect of the Antidepressant Agomelatine in the Chronic Mild Stress Model of Depression.

Neuroinflammation has emerged as an important factor in the molecular underpinnings of major depressive disorder (MDD) pathophysiology and in the mechanism of action of antidepressants. Among the inflammatory mediators dysregulated in depressed patients, interleukin (IL)-6 has recently been proposed to play a crucial role. IL-6 activates a signaling pathway comprising the JAK/STAT proteins and characterized by a specific negative feedback loop exerted by the cytoplasmic protein suppressor of cytokine signalling-3 (SOCS3). On these bases, here, we explored the potential involvement of IL-6 signaling in the ability of the antidepressant drug agomelatine to normalize the anhedonic-like phenotype induced in the rat by chronic stress exposure. To this aim, adult male Wistar rats were subjected to the chronic mild stress (CMS) paradigm and chronically treated with vehicle or agomelatine. The behavioral evaluation was assessed by the sucrose consumption test, whereas molecular analyses were performed in the prefrontal cortex. We found that CMS was able to stimulate IL-6 production and signaling, including SOCS3 gene and protein expression, but the SOCS3-mediated feedback-loop inhibition failed to suppress the IL-6 cascade in stressed animals. Conversely, agomelatine treatment normalized the stress-induced decrease in sucrose consumption and restored the negative modulation of the IL-6 signaling via SOCS3 expression and activity. Our results provide additional information about the pleiotropic mechanisms that contribute to agomelatine's therapeutic effects.

Multicellular 3D Models to Study Tumour-Stroma Interactions.

Two-dimensional (2D) cell cultures have been the standard for many different applications, ranging from basic research to stem cell and cancer research to regenerative medicine, for most of the past century. Hence, almost all of our knowledge about fundamental biological processes has been provided by primary and established cell lines cultured in 2D monolayer. However, cells in tissues and organs do not exist as single entities, and life in multicellular organisms relies on the coordination of several cellular activities, which depend on cell-cell communication across different cell types and tissues. In addition, cells are embedded within a complex non-cellular structure known as the extracellular matrix (ECM), which anchors them in a three-dimensional (3D) formation. Likewise, tumour cells interact with their surrounding matrix and tissue, and the physical and biochemical properties of this microenvironment regulate cancer differentiation, proliferation, invasion, and metastasis. 2D models are unable to mimic the complex and dynamic interactions of the tumour microenvironment (TME) and ignore spatial cell-ECM and cell-cell interactions. Thus, multicellular 3D models are excellent tools to recapitulate in vitro the spatial dimension, cellular heterogeneity, and molecular networks of the TME. This review summarizes the biological significance of the cell-ECM and cell-cell interactions in the onset and progression of tumours and focuses on the requirement for these interactions to build up representative in vitro models for the study of the pathophysiology of cancer and for the design of more clinically relevant treatments.

Complete neural stem cell (NSC) neuronal differentiation requires a branched chain amino acids-induced persistent metabolic shift towards energy metabolism.

Neural stem cell (NSC) neuronal differentiation requires a metabolic shift towards oxidative phosphorylation. We now show that a branched-chain amino acids-driven, persistent metabolic shift toward energy metabolism is required for full neuronal maturation. We increased energy metabolism of differentiating neurons derived both from murine NSCs and human induced pluripotent stem cells (iPSCs) by supplementing the cell culture medium with a mixture composed of branched-chain amino acids, essential amino acids, TCA cycle precursors and co-factors. We found that treated differentiating neuronal cells with enhanced energy metabolism increased: i) total dendritic length; ii) the mean number of branches and iii) the number and maturation of the dendritic spines. Furthermore, neuronal spines in treated neurons appeared more stable with stubby and mushroom phenotype and with increased expression of molecules involved in synapse formation. Treated neurons modified their mitochondrial dynamics increasing the mitochondrial fusion and, consistently with the increase of cellular ATP content, they activated cellular mTORC1 dependent p70S6 K1 anabolism. Global transcriptomic analysis further revealed that treated neurons induce Nrf2 mediated gene expression. This was correlated with a functional increase in the Reactive Oxygen Species (ROS) scavenging mechanisms. In conclusion, persistent branched-chain amino acids-driven metabolic shift toward energy metabolism enhanced neuronal differentiation and antioxidant defences. These findings offer new opportunities to pharmacologically modulate NSC neuronal differentiation and to develop effective strategies for treating neurodegenerative diseases.

Cross-talk between sphingosine-1-phosphate and EGFR signaling pathways enhances human glioblastoma cell invasiveness.

We show that glioblastoma multiform (GBM) cells overexpressing the constitutively active form of the epidermal growth factor receptor [epidermal growth factor receptor variant III (EGFRvIII) and U87MG human GBM cell line overexpressing EGFRvIII (EGFR+) cells] possess greater invasive properties and have higher levels of extracellular sphingosine-1-phosphate (S1P) and increased sphingosine kinase-1 (SK1) activity than the empty vector-expressing cells. Notably, the inhibition of SK1 or S1P receptors decreases the invasiveness of EGFR+ cells. Moreover, EGFR and MEK1 inhibitors reduce both SK1 activation and cell invasion, suggesting that the enhanced invasiveness observed in the EGFR+ cells depends on the increased S1P secretion, downstream of the EGFRvIII-ERK-SK1-S1P pathway. Altogether, the results of the present study indicate that, in GBM cells, EGFRvIII is connected with the S1P signaling pathway to enhance cell invasiveness and tumor progression.

Hormone-deprived serum impairs angiogenic properties in human endothelial cells regardless of estrogens.

AIMS: In vitro studies on hormone biological activities are commonly performed on cells cultured in nominally hormone-free media consisting of phenol-red-free media supplemented with charcoal-stripped (CS) serum. These media are largely used in almost all cell types, including endothelial cells (ECs). METHODS: Cell number and metabolic activity were measured with standard methods. Angiogenesis was evaluated in a three-dimensional spheroid sprouting assay. RESULTS: When we compared human umbilical vein ECs (HUVECs) cultured in standard conditions (199 medium supplemented with normal serum) with HUVECs grown in the hormone-free medium (phenol-red-free 199 medium supplemented with CS serum), we found that cells stop to grow in the absence of hormones. Notably, neither 17-ß2 estradiol nor dihydrotestosterone reversed this inhibition. Moreover, the presence of the CS serum was sufficient to abrogate the ability of HUVECs to sprout in a three-dimensional spheroid assay, thus affecting a functional property of ECs. CONCLUSIONS: Our results suggest that one or possibly more substances removed by stripping procedure from serum and different from sex hormones are crucial for the maintenance of in vitro ECs distinctive properties. Therefore, caution should be used when ECs are studied in media containing the CS serum.

[A novel standard protocol of long-term follow-up shared with general practitioners after percutaneous coronary intervention: appropriateness and economic impact]. / Il follow-up del paziente sottoposto a rivascolarizzazione coronarica percutanea: impatto potenziale dell'applicazione di un percorso clinico-gestionale strutturato sull'integrazione ospedale-territorio e modulato sul rischio clinico del paziente.

BACKGROUND: Follow-up modalities for patients undergoing percutaneous coronary intervention (PCI) are not well defined and standard protocols have been not established. The purpose of this study was to assess: a) the frequency and patterns of cardiology visits, echocardiographic examinations and stress tests after PCI in clinical practice; b) the impact of a multidisciplinary protocol of long-term follow-up after PCI shared with general practitioners on the appropriateness and reduction in healthcare costs. METHODS: A total of 780 patients who underwent PCI in 2010 in two Italian hospitals were analyzed. The number of cardiological examinations (total, routine and clinically driven) performed during 2 years of follow-up were recorded and stratified according to the patient's risk profile. The latter was defined according to the multidisciplinary protocol. In addition, a simulation of the spread between provided and necessary tests (according to the multidisciplinary protocol) was carried out. RESULTS: The mean number of cardiological examinations per patient provided during follow-up was 5, of which 4.4 were routine tests in asymptomatic patients. Routine tests were performed more frequently in patients at low risk compared to those at higher risk. By applying the multidisciplinary protocol to the case mix and by merging clinical visit and stress test or echocardiographic examination, a reduction of 0.87 tests per patient/year would be expected. This reduction would result in a 39% decrease in follow-up examinations in this specific clinical setting. CONCLUSIONS: This observational study demonstrates that unnecessary cardiological clinical and functional tests are often performed in long-term follow-up of patients submitted to PCI. The application of a standard protocol of follow-up shared with general practitioners may help avoiding unnecessary consultations, thus reducing healthcare costs.

Silencing of Eps8 inhibits in vitro angiogenesis.

AIMS: Eps8 is an actin-binding protein which has been proposed as a regulator of cancer cell motility and invasion. However, nothing much is known about its contribution to the invasive properties of endothelial cells (ECs), and more generally to angiogenesis. MAIN METHODS: Expression and silencing of Eps8 were evaluated by western blot analysis. The effect of Eps8 silencing on cell number and VEGF-induced signaling was tested with standard methods. Migration was evaluated by scratch wound assay and morphogenesis with 2-dimensional (2-D) tube formation and 3-dimensional (3-D) sprouting assays. Actin cytoskeleton was visualized by immunofluorescence. KEY FINDINGS: We found that silencing of Eps8 profoundly affected the ability of human ECs to migrate and to undergo tube formation and sprouting in 2-D and 3-D in vitro assays, respectively. Notably, capillary-like outgrowth was strictly depending on Eps8 expression also in human tumor-derived ECs. SIGNIFICANCE: Our data demonstrate for the first time the involvement of Eps8 in the morphological processes required for in vitro angiogenesis, and suggest that this protein might represent a common target for the design of new anticancer drugs, acting at the same time on both tumor and endothelial cells.

Human umbilical endothelial cells (HUVECs) have a sex: characterisation of the phenotype of male and female cells.

BACKGROUND: Human umbilical endothelial cells (HUVECs) are widely used to study the endothelial physiology and pathology that might be involved in sex and gender differences detected at the cardiovascular level. This study evaluated whether HUVECs are sexually dimorphic in their morphological, proliferative and migratory properties and in the gene and protein expression of oestrogen and androgen receptors and nitric oxide synthase 3 (NOS3). Moreover, because autophagy is influenced by sex, its degree was analysed in male and female HUVECs (MHUVECs and FHUVECs). METHODS: Umbilical cords from healthy, normal weight male and female neonates born to healthy non-obese and non-smoking women were studied. HUVEC morphology was analysed by electron microscopy, and their function was investigated by proliferation, viability, wound healing and chemotaxis assays. Gene and protein expression for oestrogen and androgen receptors and for NOS3 were evaluated by real-time PCR and Western blotting, respectively, and the expression of the primary molecules involved in autophagy regulation [protein kinase B (Akt), mammalian target of rapamycin (mTOR), beclin-1 and microtubule-associated protein 1 light chain 3 (LC3)] were detected by Western blotting. RESULTS: Cell proliferation, migration NOS3 mRNA and protein expression were significantly higher in FHUVECs than in MHUVECs. Conversely, beclin-1 and the LC3-II/LC3-I ratio were higher in MHUVECs than in FHUVECs, indicating that male cells are more autophagic than female cells. The expression of oestrogen and androgen receptor genes and proteins, the protein expression of Akt and mTOR and cellular size and shape were not influenced by sex. Body weights of male and female neonates were not significantly different, but the weight of male babies positively correlated with the weight of the mother, suggesting that the mother's weight may exert a different influence on male and female babies. CONCLUSIONS: The results indicate that sex differences exist in prenatal life and are parameter-specific, suggesting that HUVECs of both sexes should be used as an in vitro model to increase the quality and the translational value of research. The sex differences observed in HUVECs could be relevant in explaining the diseases of adulthood because endothelial dysfunction has a crucial role in the pathogenesis of cardiovascular diseases, diabetes mellitus, neurodegeneration and immune disease.

Silencing of Eps8 blocks migration and invasion in human glioblastoma cell lines.

Glioblastoma multiforme (GBM) is the most malignant human primary brain tumor, and its infiltrative nature represents the leading cause for the failure of therapies and tumor recurrences. It is therefore crucial the knowledge of the molecular mechanisms underlying GBM invasion to identify novel therapeutic targets to limit motility. In this study, we evaluated the role of Epidermal growth factor receptor Pathway Substrate 8 (Eps8), a crucial regulator of the actin cytoskeleton dynamics accompanying cell motility and invasion, in GBM migration and invasiveness. We found that silencing of the protein by small interfering RNAs (siRNAs) abrogated the migratory and invasive capacity of three different human GBM cell lines both in 2-dimensional (2-D) and 3-dimensional (3-D) in vitro assays. The inhibitory effect on invasion was maintained independently by the migration mode utilized by the cells in our 3-D model, and was accompanied by an impaired formation of actin-based cytoskeletal protrusive structures. Our data propose Eps8 as a key molecule involved in the control of the intrinsic invasive behavior of GBM cells, and suggest that this protein might represent a useful target for the design of new drugs for the treatment of these tumors.

Chronic deficiency of nitric oxide affects hypoxia inducible factor-1α (HIF-1α) stability and migration in human endothelial cells.

BACKGROUND: Endothelial dysfunction in widely diffuse disorders, such as atherosclerosis, hypertension, diabetes and senescence, is associated with nitric oxide (NO) deficiency. Here, the behavioural and molecular consequences deriving from NO deficiency in human umbilical vein endothelial cells (HUVECs) were investigated. RESULTS: Endothelial nitric oxide synthase (eNOS) was chronically inhibited either by N(G)-Nitro-L-arginine methyl ester (L-NAME) treatment or its expression was down-regulated by RNA interference. After long-term L-NAME treatment, HUVECs displayed a higher migratory capability accompanied by an increased Vascular Endothelial Growth Factor (VEGF) and VEGF receptor-2 (kinase insert domain receptor, KDR) expression. Moreover, both pharmacological and genetic inhibition of eNOS induced a state of pseudohypoxia, revealed by the stabilization of hypoxia-inducible factor-1α (HIF-1α). Furthermore, NO loss induced a significant decrease in mitochondrial mass and energy production accompanied by a lower O(2) consumption. Notably, very low doses of chronically administered DETA/NO reverted the HIF-1α accumulation, the increased VEGF expression and the stimulated migratory behaviour detected in NO deficient cells. CONCLUSION: Based on our results, we propose that basal release of NO may act as a negative controller of HIF-1α levels with important consequences for endothelial cell physiology. Moreover, we suggest that our experimental model where eNOS activity was impaired by pharmacological and genetic inhibition may represent a good in vitro system to study endothelial dysfunction.

Oxytocin stimulates in vitro angiogenesis via a Pyk-2/Src-dependent mechanism.

We previously reported that the hypothalamic hormone oxytocin (OT), best known for its uterotonic activity, also stimulates migration and invasion in human umbilical vein endothelial cells (HUVECs), thus suggesting a possible role for the peptide in the regulation of angiogenesis. We identified the Gq coupling of OT receptors (OTRs) and phospholipase C (PLC) as the main effectors of OT's action in HUVECs. Moreover, the pro-migratory effect of OT required the OTR-induced activation of the phosphatidylinositol-3-kinase (PI-3-K)/AKT/endothelial nitric oxide synthase (eNOS) pathway. To better characterize the proposed pro-angiogenic effect of OT in HUVECs, we have now utilized a three-dimensional (3-D) in vitro angiogenesis assay, and demonstrated that OT stimulates the outgrowth of capillary-like structures from HUVEC spheroids to an extent comparable to that of vascular endothelial growth factor (VEGF). This OT effect was abolished by inhibitors of PLC, PI-3-K and Src kinase. It was also found that OT phosphorylates proline-rich tyrosine kinase-2 (Pyk-2) and Src kinase in a PLC- and calcium-dependent manner. Furthermore, knockdown of Pyk-2 expression by RNA interference markedly impaired Src phosphorylation, migration and endothelial cell sprouting induced by OT. In conclusion, by using a pharmacological and genetic approach, the OT pro-angiogenic action and the cascade of intracellular signals responsible for it were defined by showing for the first time that OT, by interacting with its Gq-coupled receptor, induces HUVEC capillary outgrowth via Pyk-2 phosphorylation, which activates Src which in turn activates the PI-3-K/AKT pathway.

Basal nitric oxide release attenuates cell migration of HeLa and endothelial cells.

Nitric oxide (NO) generated by endothelial NO synthase (eNOS) is a key regulator of endothelial cell (EC) migration. Whereas the effects of acute NO generation are generally stimulatory, the role of chronic basal NO release has not been explored so far. Here, we addressed this question both in HeLa and in human endothelial cells. In stably transfected HeLa cells, inducibly expressing eNOS, expression of the enzyme per se blunted the phosphorylation of Akt/PKB in response to serum and strongly inhibited chemotaxis, an effect partially blocked by eNOS- and soluble guanylyl cyclase (sGC) inhibitors. Likewise, long-term pre-treatment of non-transfected HeLa cells with nanomolar concentrations of an NO donor inhibited subsequent migration, an effect blocked by sGC inhibition and mimicked by a cGMP analog. Finally, EC migration was stimulated by chronic pre-treatment with an eNOS inhibitor. Thus, in addition to its well-known stimulatory role, eNOS attenuates migration through basal long-term NO release.

Deregulated human glioma cell motility: inhibitory effect of somatostatin.

Malignant gliomas are highly invasive tumors which are lethal despite aggressive therapy. The motility behavior of two human glioma cell lines i.e. T98G and U87-MG cells was analysed. The glioma cells showed a high degree of basal motility (especially U87-MG cells) that may be related to the considerable local invasiveness of such tumours even in the absence of exogenous factors. The two cell lines responded equally well to platelet-derived growth factor (PDGF) as chemoattractant factor. The phosphatidylinositol 3-kinase (PI3-K) signaling, but not the extracellular signal-related kinase (ERK) signaling, was strongly involved in the PDGF-stimulated glioma cell motility. Somatostatin was capable of inhibiting the migration in both glioma cell lines without affecting crucial targets for motility control like PI3-K and Rac activity. These data suggest that somatostatin, by interfering with a target further downstream to Rac, negatively affects glioma cell motility, and may thus offer a pharmacological approach to controlling the deregulated motility of these aggressive tumoral cells.

Expression studies in gliomas and glial cells do not support a tumor suppressor role for LGI1.

Disruptions of LGI1 in glioblastoma (GBM) cell lines and LGI1 mutations in families with autosomal dominant epilepsy imply a role for LGI1 in glial cells as well as in neurons. Although we and others could not find LGI1 mutations in malignant gliomas, our initial studies appeared to support the idea that LGI1 is poorly expressed or absent in these tumors. Microarray data suggested that LGI1 could be involved in the control of matrix metalloproteinases, and we found that tumors derived from U87 glioblastoma cells overexpressing LGI1 were less aggressive than U87 control tumors. To our surprise, we observed that LGI1 expression after differentiation of murine neural stem cells was robust in neurons but negligible in glial cells, in agreement with immunohistochemistry studies on rodent brain. This observation could suggest that the variable levels of LGI1 expression in gliomas reflect the presence of neurons entrapped within the tumor. To test this hypothesis, we investigated LGI1 expression in parallel with expression of the neuronal marker NEF3 by real-time PCR on 30 malignant gliomas. Results showed a strong, positive correlation between the expression levels of these two genes (P < 0.0001). Thus, our data confirm that LGI1 is involved in cell-matrix interactions but suggest that its expression is not relevant in glial cells, implying that its role as a tumor suppressor in gliomas should be reconsidered.

Studies on the structure-activity relationship of endostatin: synthesis of human endostatin peptides exhibiting potent antiangiogenic activities.

The aim of the present research was to study the relationship between chemical structure and antiangiogenic activity of endostatin. Four peptides, containing about 40 amino acid residues, designed to cover nearly the whole sequence of endostatin, were synthesized by the solid-phase method. They were termed Fragment I (sequence 6-49), II (sequence 50-92), III (sequence 93-133), and IV (sequence 134-178), with the latter bearing the original disulfide bond Cys135-Cys165. These peptides were tested for their ability to inhibit endothelial cell proliferation, migration, and both in vitro and in vivo angiogenesis assays in matrigel. Fragments I and IV inhibited cell proliferation and cell migration with a potency and an efficacy higher than that of the full length endostatin. Fragment I was also active in inhibiting in vitro the formation of tubules and in vivo the vascularization of the matrigel. Fragments II and III were devoid of antiangiogenic activity. We propose to use the peptides 6-49 and 134-178 as angiogenesis inhibitors in substitution of full length endostatin, in therapeutic applications for cancer, rheumatoid arthritis, and retinopathies.

Anti-migratory and anti-invasive effect of somatostatin in human neuroblastoma cells: involvement of Rac and MAP kinase activity.

Cell motility and invasion are crucial events for the spread of cancer and, consequently, the metastatic process. Platelet-derived growth factor (PDGF) is not only capable of stimulating the proliferation of SH-SY5Y human neuroblastoma cells, but also their migration and invasion through an extracellular matrix barrier. Experiments using wortmannin and PD98059, specific inhibitors of the phosphatidylinositol 3-kinase (PI3-K) and of the mitogen-activated protein kinases (ERK 1 and 2) signaling, respectively, show that the activation of both pathways is required for the PDGF-induced cell motility responses. We have previously shown that somatostatin inhibits cell division and ERK 1/2 and Ras activity in SH-SY5Y cells. We report here that it is also capable of potently and effectively inhibiting their PDGF-stimulated migration and invasion. The inhibitory effect of somatostatin is sensitive to pertussis toxin. Although somatostatin does not affect PI3-K, it inhibits ERK 1/2 and the small G-protein Rac activation and ruffle formation induced by PDGF. These results indicate that somatostatin can be considered an anti-migratory and anti-invasive agent that acts by inhibiting ERK 1/2 signaling and the PI3-K pathway via the inhibition of Rac in SHSY5Y cells.

Human endostatin-derived synthetic peptides possess potent antiangiogenic properties in vitro and in vivo.

Pharmacological control of the angiogenic process (i.e., the neovascularization necessary for the growth and progression of tumors and metastases) is considered to be one of the most promising approaches to antineoplastic therapy. Endostatin, a 20-kDa protein derived from collagen XVIII, is one of the first recently discovered endogeneous antiangiogenic substances, but its cell targets and mechanism(s) of action are still unknown. We thought it would be interesting to test whether shorter peptides derived from endostatin might preserve its antiangiogenic activity. Four synthetic peptides corresponding to the sequences 6-49 (I), 50-92 (II), 93-133 (III), and 134-178 (IV) of human endostatin were tested for their ability to inhibit endothelial cell proliferation, migration, and both in vitro and in vivo angiogenesis. Fragment I (and fragment IV in the tests performed) was found to be fully biologically active in all of the angiogenesis assays, and sometimes showed even greater potency and efficacy than full-length human endostatin itself.

Alprostadil suppresses angiogenesis in vitro and in vivo in the murine Matrigel plug assay.

1. Prostaglandin E(1) (PGE(1), alprostadil) is used as a vasodilator for the treatment of peripheral vascular diseases. 2. Previous reports suggested a pro-angiogenic effect for PGE(1). 3. We studied the in vitro and in vivo effect of PGE(1), complexed with alpha-cyclodextrin, on the angiogenic process. Contrary to what was expected, we found that, in human umbilical vein endothelial cells (HUVECs), PGE(1) inhibited proliferation, migration and capillary-like structure formation in Matrigel. 4. By RT-PCR studies, the expression of the EP(2) and EP(3) subtypes of the PG receptor was detected in HUVECs. 5. PGE(1) alone stimulated adenylate cyclase activity at micromolar concentrations, while at nanomolar concentrations potentiated the forskolin-induced cAMP accumulation. 6. 8-Bromoadenosine-3':5'-cyclic monophosphate (Br-cAMP) mimicked the inhibitory effect of PGE(1) on endothelial cell growth, motility and tube formation. 7. Sulprostone, an agonist at the EP(3) subtype of PG receptors, mimicked the in vitro anti-angiogenic effects of PGE(1), while butaprost, an EP(2) receptor agonist, had no effect. 8. Finally, in the plug assay model of angiogenesis in mice, PGE(1) showed a strong inhibitory effect on Matrigel neovascularization. 9. Thus, PGE(1) possesses strong anti-angiogenic activity in vitro and in vivo.

Invasive behaviour of glioblastoma cell lines is associated with altered organisation of the cadherin-catenin adhesion system.

As little is known about the role of cadherin-mediated cell-cell adhesion in astrocytes and its alteration in migrating and invasive glioblastomas, we investigated its molecular composition and organisation in primary cultured astrocytes and the T98G and U373MG glioblastoma cell lines. Biochemical and morphological analysis indicated that all three cell types express all of the structural components of the adhesion system, including the LIN-7 PDZ protein, a novel component involved in the organisation of the junctional domain in epithelia and neurons. However, only the astrocytes and T98G cells generated and maintained mature adhesive junctional domains to which LIN-7 was recruited. Alterations in the junctional domain of U373MG cells were associated with higher motility in a poly-L-lysine migration assay. When the T98G cells were cultured on Matrigel matrix, they acquired invasive properties but, despite unchanged cadherin adhesion system protein levels, the invasive T98G cell-cell contacts failed to accumulate LIN-7 and failed to mature. These results identify the LIN-7 PDZ protein as a marker of cell adhesion maturity and cell invasion and indicate that instability and disorganisation of cadherin-mediated junctions rather than reduced expression of cadherin-catenin system components are required to promote migration and invasiveness in glioblastoma cell lines.