Survivin overexpression in head and neck squamous cell carcinomas as a new therapeutic target (Review).

Head and neck squamous cell carcinoma (HNSCC) is the sixth most commonly diagnosed cancer worldwide. It has poor clinical outcome due to intrinsic or acquired drug resistance. Deregulation of both apoptosis and autophagy contributes to chemotherapy resistance and disease progression. A new member of the inhibitors of apoptosis protein (IAP) family, namely survivin, is selectively overexpressed in tumors, including HNSCC, but not in normal tissues. Thus, it is considered a tumor biomarker. Here, we reviewed survivin expression and function in tumor progression focusing on its nodal role in the regulation of cell apoptosis and autophagy. Based on literature data, survivin targeting may be envisaged as a novel therapeutic strategy.

Autophagy: A New Mechanism of Prosurvival and Drug Resistance in Multiple Myeloma.

Autophagy is an intracellular self-degradative process that balances cell energy source and regulates tissue homeostasis. In physiological condition, autophagy funnels cytoplasmic constituents to autophagolysosomes for degradation and is an alternative way for cell-death behavior. Here, we inspected autophagy as a prosurvival mechanism essential for drug resistance in multiple myeloma (MM). Accordingly, autophagy inhibitors used in association to conventional anti-MM drugs might enforce the effect against resistant MM plasma cells and render autophagy a new therapeutic target.

Transient increases in intracellular calcium and reactive oxygen species levels in TCam-2 cells exposed to microgravity.

The effects of microgravity on functions of the human body are well described, including alterations in the male and female reproductive systems. In the present study, TCam-2 cells, which are considered a good model of mitotically active male germ cells, were used to investigate intracellular signalling and cell metabolism during exposure to simulated microgravity, a condition that affects cell shape and cytoskeletal architecture. After a 24 hour exposure to simulated microgravity, TCam-2 cells showed 1) a decreased proliferation rate and a delay in cell cycle progression, 2) increased anaerobic metabolism accompanied by increased levels of intracellular Ca2+, reactive oxygen species and superoxide anion and modifications in mitochondrial morphology. Interestingly, all these events were transient and were no longer evident after 48 hours of exposure. The presence of antioxidants prevented not only the effects described above but also the modifications in cytoskeletal architecture and the activation of the autophagy process induced by simulated microgravity. In conclusion, in the TCam-2 cell model, simulated microgravity activated the oxidative machinery, triggering transient macroscopic cell events, such as a reduction in the proliferation rate, changes in cytoskeleton-driven shape and autophagy activation.

Altered Kv2.1 functioning promotes increased excitability in hippocampal neurons of an Alzheimer's disease mouse model.

Altered neuronal excitability is emerging as an important feature in Alzheimer's disease (AD). Kv2.1 potassium channels are important modulators of neuronal excitability and synaptic activity. We investigated Kv2.1 currents and its relation to the intrinsic synaptic activity of hippocampal neurons from 3xTg-AD (triple transgenic mouse model of Alzheimer's disease) mice, a widely employed preclinical AD model. Synaptic activity was also investigated by analyzing spontaneous [Ca(2+)]i spikes. Compared with wild-type (Non-Tg (non-transgenic mouse model)) cultures, 3xTg-AD neurons showed enhanced spike frequency and decreased intensity. Compared with Non-Tg cultures, 3xTg-AD hippocampal neurons revealed reduced Kv2.1-dependent Ik current densities as well as normalized conductances. 3xTg-AD cultures also exhibited an overall decrease in the number of functional Kv2.1 channels. Immunofluorescence assay revealed an increase in Kv2.1 channel oligomerization, a condition associated with blockade of channel function. In Non-Tg neurons, pharmacological blockade of Kv2.1 channels reproduced the altered pattern found in the 3xTg-AD cultures. Moreover, compared with untreated sister cultures, pharmacological inhibition of Kv2.1 in 3xTg-AD neurons did not produce any significant modification in Ik current densities. Reactive oxygen species (ROS) promote Kv2.1 oligomerization, thereby acting as negative modulator of the channel activity. Glutamate receptor activation produced higher ROS levels in hippocampal 3xTg-AD cultures compared with Non-Tg neurons. Antioxidant treatment with N-Acetyl-Cysteine was found to rescue Kv2.1-dependent currents and decreased spontaneous hyperexcitability in 3xTg-AD neurons. Analogous results regarding spontaneous synaptic activity were observed in neuronal cultures treated with the antioxidant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Our study indicates that AD-related mutations may promote enhanced ROS generation, oxidative-dependent oligomerization, and loss of function of Kv2.1 channels. These processes can be part on the increased neuronal excitability of these neurons. These steps may set a deleterious vicious circle that eventually helps to promote excitotoxic damage found in the AD brain.

Responses of peripheral blood mononuclear cells to moderate exercise and hypoxia.

The purpose of this study was to analyze the physiological features of peripheral blood mononuclear cells (PBMCs) isolated from healthy female trekkers before and after physical activity carried out under both normoxia (low altitude, < 2000 m a.s.l.) and hypobaric hypoxia (high altitude, > 3700 m a.s.l.). The experimental design was to differentiate effects induced by exercise and those related to external environmental conditions. PBMCs were isolated from seven female subjects before and after each training period. The PBMCs were phenotypically and functionally characterized using fluorimetric and densitometric analyses, to determine cellular activation, and their intracellular Ca(2+) levels and oxidative status. After a period of normoxic physical exercise, the PBMCs showed an increase in fully activated T lymphocytes (CD3(+) CD69(+) ) and a reduction in intracellular Ca(2+) levels. On the other hand, with physical exercise performed under hypobaric hypoxia, there was a reduction in T lymphocytes and an increase in nonactivated B lymphocytes, accompanied by a reduction in O2 (-) levels in the mitochondria. These outcomes reveal that in women, low- to moderate-intensity aerobic trekking induces CD69 T cell activation and promotes anti-stress effects on the high-altitude-induced impairment of the immune responses and the oxidative balance.

Specific association of growth-associated protein 43 with calcium release units in skeletal muscles of lower vertebrates.

Growth-associated protein 43 (GAP43), is a strictly conserved protein among vertebrates implicated in neuronal development and neurite branching. Since GAP43 structure contains a calmodulin-binding domain, this protein is able to bind calmodulin and gather it nearby membrane network, thus regulating cytosolic calcium and consequently calcium-dependent intracellular events. Even if for many years GAP43 has been considered a neuronal-specific protein, evidence from different laboratories described its presence in myoblasts, myotubes and adult skeletal muscle fibers. Data from our laboratory showed that GAP43 is localized between calcium release units (CRUs) and mitochondria in mammalian skeletal muscle suggesting that, also in skeletal muscle, this protein can be a key player in calcium/calmodulin homeostasis. However, the previous studies could not clearly distinguish between a mitochondrion- or a triad-related positioning of GAP43. To solve this question, the expression and localization of GAP43 was studied in skeletal muscle of Xenopus and Zebrafish known to have triads located at the level of the Z-lines and mitochondria not closely associated with them. Western blotting and immunostaining experiments revealed the expression of GAP43 also in skeletal muscle of lower vertebrates (like amphibians and fishes), and that the protein is localized closely to the triad junction. Once more, these results and GAP43 structural features, support an involvement of the protein in the dynamic intracellular Ca2+ homeostasis, a common conserved role among the different species.

Influence of titanium laser surface geometry on proliferation and on morphological features of human mandibular primary osteoblasts.

The aim of this study is to assess in vitro the proliferation and the morphological changes of primary osteoblast-like cells (HOst) seeded on titanium dish grade 4 and 5 with different roughness and different titanium grade: machined (M), sandblasted (SBT), laser-treated with pitches of 20-microm diameter and 30-microm interpore distance. The titanium disks were divided into two groups: group A (titanium grade 4) and Group B (titanium Grade 5), respectively. Proliferation rate of attached cells was evaluated at different time (24, 48, 72 h and 1 week) by the quantitative colorimetric MTT assay. Our results showed a cell growth decrease evident in M titanium surfaces in both Groups A and B, while the cells seeded on the STB and laser disks displayed an increase of cells growth, more evident in laser titanium surfaces in groups A and B. Morphological changes of the biocomplex cells/titanium was assessed by light, scanning and confocal microscopy. In fact, the microscopic analysis helped to clarify the behavior of the cells in contact with the titanium surfaces, in particular the M surface induced significant morphological changes, which were less evident in the SBT surfaces. Laser-engineered porous titanium surfaces promoted viability and proliferation of the osteoblasts. In particular, hemispherical porosity of 20 microm could be responsible for the higher HOst activation, in terms of cells proliferation, adhesion and morphological features.

Quantitative shape analysis of chemoresistant colon cancer cells: correlation between morphotype and phenotype.

Morphological, qualitative observations allow pathologists to correlate the shape the cells acquire with the progressive, underlying neoplastic transformation they are experienced. Cell morphology, indeed, roughly scales with malignancy. A quantitative parameter for characterizing complex irregular structures is the Normalized Bending Energy (NBE). NBE provides a global feature for shape characterization correspondent to the amount of energy needed to transform the specific shape under analysis into its lowest energy state. We hypothesized that a chemotherapy resistant cancer cell line would experience a significant change in its shape, and that such a modification might be quantified by means of NBE parameterization. We checked out the usefulness of a mathematical algorithm to distinguish wild and 5-fluorouracil (5-FU)-resistant colon cancer HCT-8 cells (HCT-8FUres). NBE values, as well as cellular and molecular parameters, were recorded in both cell populations. Results demonstrated that acquisition of drug resistance is accompanied by statistically significant morphological changes in cell membrane, as well as in biological parameters. Namely, NBE increased progressively meanwhile cells become more resistant to increasing 5-FU concentrations. These data indicate how tight the relationships between morphology and phenotype is, and they support the idea to follow a cell transition toward a drug-resistant phenotype by means of morphological monitoring.

Neurogenic potential of mesenchymal-like stem cells from human amniotic fluid: the influence of extracellular growth factors.

Amniotic fluids contain human stem cells, among which mesenchymal stem cells could be isolated. These cells have multipotent differentiation ability and no tumorigenic potential after transplantation in mice. These features make them good candidates for in vitro studies and for therapeutic purposes. The aim of this study was to isolate mesenchymal stem cell-like cultures from different amniotic fluids in order to study in vitro their neurogenic potential and assess if this process could be reproducible and standardized. We focused attention on the possible differential effects of soluble growth factors. Immunophenotypical and molecular characterization showed that the 31 amniotic fluid-derived cultures expressed mesenchymal markers as well as some stemness properties. These cells also appeared to be responsive to purines or acetylcholine showing an intracellular calcium increase, also reported for mesenchymal stem cells derived from other sources. Interestingly, in the presence of retinoic acid, these cells assumed a neuronal-like morphology. In addition, functional and molecular analyses revealed that retinoic acid-treated cells showed immature electric functional properties, the expression of neuronal markers and stemness genes. In conclusion, even if further investigations are required, the results presented here contribute to support the finding that amniotic fluid contains cells able to differentiate in vitro towards neural-like lineage in the presence of retinoic acid. The ability of retinoic acid to induce a possible neuronal progenitor culture makes the model useful to study a possible in vivo transplantation of these cells and to contribute to define the protocols for cell therapy.

Effect of pre-breathing oxygen at different depth on oxidative status and calcium concentration in lymphocytes of scuba divers.

AIM: In-water pre-breathing oxygen at various depths reduces decompression-induced bubble formation and platelet activation, but it could induce side effects such as oxidative stress. The aim of this study was to investigate the effect of in-water pre-breathing oxygen, at different depths, on the oxidative status and intracellular calcium ([Ca(2+) ]i) of peripheral blood lymphocytes isolated from six divers. They participated in a 4-diving protocol. Two week recovery time was allowed between successive dives. Before diving, all divers, for 20 min, breathed normally at sea level (dive 1), 100% oxygen at sea level (dive 2), 100% oxygen at 6 msw (dive 3), 100% oxygen at 12 msw (dive 4). Then they dived to 30 msw for 20 min with air tank. METHODS: Blood samples were collected before and after each dive. Hydrogen peroxide (H(2) O(2) ) levels, catalase (CAT) activity, mRNA expression of CAT, glutathione peroxidase (GPx) and superoxide dismutase (SOD), and the [Ca(2+) ]i in lymphocytes were measured. RESULTS: The dives slightly decreased lymphocyte number and significantly reduced lymphocyte H(2) O(2) levels. CAT activity was higher after scuba diving and, dive 3 enhanced mRNA gene expression of CAT, GPx and SOD. The [Ca(2+) ]i was higher after dive 1 and 2 than pre-diving, while was maintained at pre-diving value after dive 3 and 4. CONCLUSION: Our results suggest that pre-breathing oxygen, in particular at 12 msw, may enhance lymphocyte antioxidant activity and reduce reactive oxygen species levels. Pre-breathing oxygen in water may also preserve calcium homeostasis, suggesting a protective role in the physiological lymphocyte cell functions.

Functional characterization of calcium-signaling pathways of human skin-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) derived from adult human tissues are able to differentiate into various specialized cell types. In research, they can therefore be used like embryonic cells but without the ethical restrictions. Among the various human tissues, skin as a source is characterized by great accessibility and availability using noninvasive procedures and is without the risk of oncogenesis after transplantation. The recent isolation of MSCs has shown the lack of knowledge regarding their specific features, including the calcium-signaling pathways. METHODS: In this study, we isolated MSCs from human skin biopsies (S-MSCs) and characterized them phenotypically and their calcium-signaling pathways by the means of Ca2+ imaging and video microscopic experiments. RESULTS: The cytofluorimetric analysis of the expression of surface markers on S-MSCs revealed that they express the normal pattern present on MSCs. Interestingly, these cells appeared to be successfully cryopreserved at early passages. Calcium imaging on single S-MSCs shows that these cells did not display significant spontaneous activity or a response to a depolarizing agent. However, ATP or acetylcholine-induced intracellular calcium increase via ionotropic or metabotropic receptors, respectively. CONCLUSION: The results presented here reveal that S-MSCs show morphological and functional features that make them useful as an in vitro model to study cell differentiation.

Enhancement of fibroblast proliferation, collagen biosynthesis and production of growth factors as a result of combining sodium hyaluronate and aminoacids.

Fibroblasts play a key role in tissue healing by producing the majority of extracellular matrix components, favouring granulation tissue formation, and stimulating re-epithelialization. Hyaluronan is a component of ECM and its anti-inflammatory effects and properties in enhancing wound closure are well known. In this study, we examined the effects of Aminogam gel, a new pharmacological preparation suggested to improve wound healing, composed of hyaluronic acid, proline, lysine, glycine and leucine, on human fibroblasts. Results show that fibroblasts treated with hyaluronic acid plus aminoacid solution increased their proliferative activity, collagen I and III, and fibronectin synthesis. Moreover, HA plus aminoacid solution increased the expression of transforming growth factor beta, connective tissue growth factor, interleukin-6 and -8, assayed by RT-PCR. These results suggested that Aminogam gel, involved in several stages of wound healing, as fibroblast proliferation, granulation tissue formation, ECM component deposition, and production of cytokines, may be a useful device to favour and accelerate wound closure.

Extracellular guanosine and GTP promote expression of differentiation markers and induce S-phase cell-cycle arrest in human SH-SY5Y neuroblastoma cells.

SH-SY5Y neuroblastoma cells, a model for studying neuronal differentiation, are able to differentiate into either cholinergic or dopaminergic/adrenergic phenotypes depending on media conditions. Using this system, we asked whether guanosine (Guo) or guanosine-5'-triphosphate (GTP) are able to drive differentiation towards one particular phenotype. Differentiation was determined by evaluating the frequency of cells bearing neurites and assessing neurite length after exposure to different concentrations of Guo or GTP for different durations. After 6 days, 0.3 mM Guo or GTP induced a significant increase in the number of cells bearing neurites and increased neurite length. Western blot analyses confirmed that purines induced differentiation; cells exposed to purines showed increases in the levels of GAP43, MAP2, and tyrosine hydroxylase. Proliferation assays and cytofluorimetric analyses indicated a significant anti-proliferative effect of purines, and a concentration-dependent accumulation of cells in S-phase, starting after 24 h of purine exposure and extending for up to 6 days. A transcriptional profile analysis using gene arrays showed that an up-regulation of cyclin E2/cdk2 evident after 24 h was responsible for S-phase entry, and a concurrent down-regulation of cell-cycle progression-promoting cyclin B1/B2 prevented S-phase exit. In addition, patch-clamp recordings revealed that 0.3 mM Guo or GTP, after 6 day incubation, significantly decreased Na(+) currents. In conclusion, we showed Guo- and GTP-induced cell-cycle arrest in neuroblastoma cells and suggest that this makes these cells more responsive to differentiation processes that favor the dopaminergic/adrenergic phenotype.

Accelerated wound healing of oral soft tissues and angiogenic effect induced by a pool of aminoacids combined to sodium hyaluronate (AMINOGAM).

In this study we investigated the property of a new medical substance, in the form of a gel compound containing four aminoacids (glycine, leucine, proline, lysine) and sodium hyaluronate (AMINOGAM), to accelerate the wound healing process of the soft oral tissues and to promote angiogenesis in vivo in the vascular proliferation in chick embryo chorioallantoic membrane (CAM) assay. Furthermore, we investigated the capacity of AMINOGAM to induce the expression of an angiogenic cytokine, namely vascular endothelial growth factor (VEGF) in human fibroblasts in vitro. Results showed that AMINOGAM promoted wound healing in post-surgical wounds (after teeth extraction, oral laser surgery with secondary healing without direct suture of the surgical wound, and after dental implant insertion). Stimulated angiogenesis in vivo in the CAM assay and the response was similar to that obtained with vascular endothelial growth factor, a well-known angiogenic cytokine, tested in the same assay, and confirmed by clinical outcomes, which showed reduction of the healing time of oral soft tissues after three different kinds of surgery and also the absence of post-operative infections.

A histochemical approach to the evaluation of the in vivo cytotoxicity of the nitrobutadienes (1E,3E)-1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene and methyl (2Z,4E)-2-methylsulfanyl-5-(1-naphthyl)-4-nitro-2,4-pentadienoate in mice liver and kidney.

Two new molecules (1E,3E)-1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene (1-Naph-DNB) and (2Z,4E)-2-methylsulfanyl-5-(1-naphthyl)-4-nitro-2,4-pentadienoate (1-Naph-NMCB) in previous studies showed interesting antiproliferative activity in vitro. Furthermore, toxicological tests and histological analysis provided promising results, in particular for 1-Naph-NMCB that displayed lower toxic activity both in terms of lethal effect and tissue damage of the main organs. Finally, studies of the antitumour activity in vivo confirmed the efficacy of both molecules, though with some differences in tumour selectivity and levels of activity. In this investigation the activities of some specific enzymes, acid phosphatase (AcPase), alkaline phosphatase (AlkPase), catalase (Cat), succinic dehydrogenase (SDH), glucose-6-phosphatase (G6Pase) and K+ p-nitrophenyl phosphatase (K+ pNPPase) were studied in the liver and kidney as histopathological biomarkers, to assess the effects of the two compounds in organs generally involved in the metabolism and excretion of different drugs. As oxidative stress may also develop as a consequence of the toxic effect of chemicals, reactive oxygen species (ROS) production was evaluated by a histochemical method. The results indicated that some enzyme activities and ROS expression changed in a dose-related manner. Nevertheless, neither in the liver nor in the kidney were dramatic toxic effects evident. By contrast, the variations of some enzyme activities (AlkPase, AcPase, Cat, K+ pNPPase) were interpreted as possible defensive mechanisms for tolerating high dosage of the compounds.

Organ-specific manganese toxicity: a comparative in vitro study on five cellular models exposed to MnCl(2).

Manganese (Mn) is both an essential nutrient and a toxicant, with specific effects on liver and kidney (acute exposure) and on central nervous system (CNS) (chronic exposure). Mn neurotoxicity includes neurobehavioral disorders and extra-pyramidal motor dysfunctions (manganism), possibly due to focal injuries to the basal ganglia. Even if widely investigated, the molecular mechanisms responsible for Mn toxicity remain to be clarified. Aim of this study was to identify suitable in vitro models to investigate these molecular pathways. To this purpose we compared the effect of manganese chloride on four cell lines, representative of the main target organs of Mn toxicity in vivo. HepG2 and MDCK cell lines were selected for liver and kidney, respectively; glial GL15 and neuronal SHSY5Y cells were used as models of CNS components. To complete the "motor system" model, skeletal muscle C2C12 cells were also included. Our results demonstrate that hepatic, renal, glial and neuronal cell types differently react to Mn, mirroring the specific in vivo response of the tissue they represent. This confirms their value as suitable in vitro models to study Mn-related toxic events. Interestingly, also muscle C2C12 cells showed a noticeable sensitivity to Mn, preferential targets being differentiated myotubes.

Signal transduction events induced by extracellular guanosine 5' triphosphate in excitable cells.

A better understanding of the physiological effects of guanosine-based purines should help clarify the complex subject of purinergic signalling. We studied the effect of extracellular guanosine 5' triphosphate (GTP) on the differentiation of two excitable cell lines that both have specific binding sites for GTP: PC12 rat pheochromocytoma cells and C2C12 mouse skeletal muscle cells. PC12 cells can be differentiated into fully functional sympathetic-like neurons with 50-100 ng ml⁻¹ of nerve growth factor, whereas serum starvation causes C2C12 cells to differentiate into myotubes showing functional excitation-contraction coupling, with the expression of myosin heavy chain proteins. Our results show that GTP enhances the differentiation of both of these excitable cell lines. The early events in guanosine-based purine signal transduction appear to involve an increase in intracellular Ca²âº levels and membrane hyperpolarization. We further investigated the early activation of extracellular-regulated kinases and phosphoinositide 3-kinase in GTP-stimulated PC12 and C2C12 cells, respectively. We found that GTP promotes the activation of both kinases. Together, our results suggest that, even if there are some differences in the signalling pathways, GTP-induced differentiation in both cell lines is dependent on an increase in intracellular Ca²âº.

Beta-catenin gene analysis in oral squamous cell carcinoma.

The molecular mechanisms involved in the development of oral squamous cell carcinomas (OSCC) are not yet well understood. Evidence of recent studies suggests that aberrant beta-catenin signalling may participate in the neoplastic transformation and that it is implicated in the development of several tumours. Beta-catenin is a component of the catenin family and plays a crucial role in cadherin mediated cell adhesion. However, it has recently been shown that beta-catenin is also involved in other functions such as intracellular signalling and the regulation of gene transcription. The aim of this study is to evaluate the presence of mutation in exon 3 of the beta-catenin gene in 20 OSCC cell lines. DNA was extracted using Qiagen Qiamp DNA minikit and a region encompassing the exon 3 of beta-catenin gene was amplified using a single PCR assay. The PCR products were analysed by SSCP and direct sequencing to detect any mutation of the gene. Most of the cell lines examined showed, by immunofluorescence, a beta-catenin delocalization. SSCP and sequence analysis of the PCR products did not show any mutation of the beta-catenin gene in any of the cell lines. In conclusion, although aberrant expressions or abnormal localization of beta-catenin have been detected in several OSCC cells, it appears that this finding has no relationship with beta-catenin gene mutations.

Cooperation in signal transduction of extracellular guanosine 5' triphosphate and nerve growth factor in neuronal differentiation of PC12 cells.

Guanosine 5' triphosphate (GTP), acting synergistically with the nerve growth factor (NGF), enhances the proportion of neurite-bearing cells in cultures of PC12 rat pheochromocytoma cells. We studied the transduction mechanisms activated by GTP in PC12 cells and found that addition of GTP (100 microM) increased intracellular calcium concentration ([Ca(2+)](i)) in cells that were between 60 and 70% confluent. Addition of GTP also enhanced activation of NGF-induced extracellular regulated kinases (ERKs) and induced Ca(2+) mobilization. This mobilization, due to the activation of voltage-sensitive and ryanodine-sensitive calcium channels, as well as pertussis toxin-sensitive purinoceptors, modulates Ca(2+)-activated K(+) channels not involved in activation of ERKs. The results presented here indicate that GTP-triggered [Ca(2+)](i) increase may be a key event in GTP signal transduction, which can modulate activity of ERKs. The physiological importance of the GTP effect lies in its capacity to interact with the NGF-activated pathway to enhance neurite outgrowth from PC12 cells.

P-cadherin expression predicts clinical outcome in oral squamous cell carcinomas.

P-cadherin, a transmembrane molecule similar to E-cadherin involved in the cell-cell adhesion, and catenins form complexes between its cytoplasmic domain and the cytoskeleton. Five cell lines, 108 specimens of oral squamous cell carcinomas (OSCC), 9 metastasis and 10 of normal oral mucosa were examined to evaluate P-cadherin expression and cellular localization by immunohistochemistry and western-blotting. In normal oral mucosa there was a membranous expression only in basal and parabasal layers. 91 cases (84%) showed membranous/cytoplasmic positivity, whereas 17 cases (16%) were negative. In particular, while well-differentiated carcinomas showed P-cadherin upregulation, the protein was homogeneously hypo- or unexpressed in low-differentiated carcinomas. There was a statistically significant correlation between P-cadherin expression and tumour grading: G3 tumours had a lower score than G1-G2 tumours (P<0.05). When analysed for prognostic significance, patients with no P-cadherin expression (score 0) had poorer overall and diseases-free survival rates than the P-cadherin-expressing group (score 1) (P=0.0463 and P=0.0471, respectively). Western blotting analysis of cell lines and tissue samples confirmed immunohistochemical findings. When cell staining pattern of positive cases was examined, 52 cases showed a prevalent membranous pattern, while 39 had a prevalent cytoplasmic pattern. Cases with prevalent cytoplasmic staining showed high rates of lymph node metastases (P>0.05), and regional relapse (P <0.05) and poorer survival rates than the group with prevalent membranous expression (P<0.0001). An absent P-cadherin expression could constitute a hallmark of aggressive biological behaviour in oral squamous cell carcinoma.