Extra Virgin Olive Oil-Based Formulations: A "Green" Strategy against Chlamydia trachomatis.

In recent decades, antibiotic misuse has emerged as an important risk factor for the appearance of multi-drug-resistant bacteria, and, recently, antimicrobial resistance has also been described in Chlamydia trachomatis as the leading cause of bacterial sexually transmitted diseases worldwide. Herein, we investigated, for the first time, the antibacterial activity against C. trachomatis of a polyphenolic extract of extra virgin olive oil (EVOO), alongside purified oleocanthal and oleacein, two of its main components, in natural deep eutectic solvent (NaDES), a biocompatible solvent. The anti-chlamydial activity of olive-oil polyphenols (OOPs) was tested in the different phases of chlamydial developmental cycle by using an in vitro infection model. Transmission and scanning electron microscopy analysis were performed for investigating potential alterations of adhesion and invasion, as well as morphology, of chlamydial elementary bodies (EBs) to host cells. The main result of our study is the anti-bacterial activity of OOPs towards C. trachomatis EBs down to a total polyphenol concentration of 1.7 µg/mL, as shown by a statistically significant decrease (93.53%) of the total number of chlamydial-inclusion-forming units (p < 0.0001). Transmission and scanning electron microscopy analysis supported its anti-chlamydial effect, suggesting that OOP might damage the chlamydial outer layers, impairing their structural integrity and hindering EB capability to infect the host cell. In conclusion, OOPs may represent an interesting alternative therapeutic option toward C. trachomatis, although further studies are necessary for exploring its clinical applications.

Interaction of Drug-Sensitive and -Resistant Human Melanoma Cells with HUVEC Cells: A Label-Free Cell-Based Impedance Study.

Cancer cell extravasation is a crucial step in cancer metastasis. However, many of the mechanisms involved in this process are only now being elucidated. Thus, in the present study we analysed the trans-endothelial invasion of melanoma cells by a high throughput label-free cell impedance assay applied to transwell chamber invasion assay. This technique monitors and quantifies in real-time the invasion of endothelial cells by malignant tumour cells, for a long time, avoiding artefacts due to preparation of the end point measurements. Results obtained by impedance analysis were compared with endpoint measurements. In this study, we used human melanoma M14 wild type (WT) cells and their drug resistant counterparts, M14 multidrug resistant (ADR) melanoma cells, selected by prolonged exposure to doxorubicin (DOX). Tumour cells were co-cultured with monolayers of human umbilical vein endothelial cells (HUVEC). Results herein reported demonstrated that: (i) the trans-endothelial migration of resistant melanoma cells was faster than sensitive ones; (ii) the endothelial cells appeared to be strongly affected by the transmigration of melanoma cells which showed the ability to degrade their cytoplasm; (iii) resistant cells preferentially adopted the transcellular invasion vs. the paracellular one; (iv) the endothelial damage mediated by tumour metalloproteinases seemed to be reversible.

How stereochemistry of lipid components can affect lipid organization and the route of liposome internalization into cells.

Though liposome-based drugs are in clinical use, the mechanism of cell internalization of liposomes is yet an object of controversy. The present experimental investigation, carried out on human glioblastoma cells, indicated different internalization routes for two diastereomeric liposomes. Molecular dynamics simulations of the lipid bilayers of the two formulations indicated that the different stereochemistry of a lipid component controls some parameters such as area per lipid molecule and fluidity of lipid membranes, surface potential and water organization at the lipid/water interface, all of which affect the interaction with biomolecules and cell components.

Structurally related glucosylated liposomes: Correlation of physicochemical and biological features.

Liposomes functionalized on their surface with carbohydrates (glycoliposomes) represent an optimal approach for targeting of drugs to diseased tissues in vivo, thanks to biocompatibility, low toxicity and easy manufacturing of these lipid nanoparticles. Here we report on the study of liposomes including a novel glycosylated amphiphile and on the comparison of their features with those of glycosylated analogues described previously. Further, the capability of the different glucosylated formulations to interact with three breast cancer cell lines was investigated. Our results show that the hydrophobic portion of the lipid bilayer strongly influences both the properties and the internalization of glycosylated liposomes.

The proneural gene ASCL1 governs the transcriptional subgroup affiliation in glioblastoma stem cells by directly repressing the mesenchymal gene NDRG1.

Achaete-scute homolog 1 gene (ASCL1) is a gene classifier for the proneural (PN) transcriptional subgroup of glioblastoma (GBM) that has a relevant role in the neuronal-like differentiation of GBM cancer stem cells (CSCs) through the activation of a PN gene signature. Besides prototypical ASCL1 PN target genes, the molecular effectors mediating ASCL1 function in regulating GBM differentiation and, most relevantly, subgroup specification are currently unknown. Here we report that ASCL1 not only promotes the acquisition of a PN phenotype in CSCs by inducing a glial-to-neuronal lineage switch but also concomitantly represses mesenchymal (MES) features by directly downregulating the expression of N-Myc downstream-regulated gene 1 (NDRG1), which we propose as a novel gene classifier of MES GBMs. Increasing the expression of ASCL1 in PN CSCs results in suppression of self-renewal, promotion of differentiation and, most significantly, decrease in tumorigenesis, which is also reproduced by NDRG1 silencing. Conversely, both abrogation of ASCL1 expression in PN CSCs and enforcement of NDRG1 expression in either PN or MES CSCs induce proneural-to-mesenchymal transition (PMT) and enhanced mesenchymal features. Surprisingly, ASCL1 overexpression in MES CSCs increases malignant features and gives rise to a neuroendocrine-like secretory phenotype. Altogether, our results propose that the fine interplay between ASCL1 and its target NDRG1 might serve as potential subgroup-specific targetable vulnerability in GBM; enhancing ASCL1 expression in PN GBMs might reduce tumorigenesis, whereas repressing NDRG1 expression might be actionable to hamper the malignancy of GBM belonging to the MES subgroup.

Metabolic Heterogeneity Evidenced by MRS among Patient-Derived Glioblastoma Multiforme Stem-Like Cells Accounts for Cell Clustering and Different Responses to Drugs.

Clustering of patient-derived glioma stem-like cells (GSCs) through unsupervised analysis of metabolites detected by magnetic resonance spectroscopy (MRS) evidenced three subgroups, namely clusters 1a and 1b, with high intergroup similarity and neural fingerprints, and cluster 2, with a metabolism typical of commercial tumor lines. In addition, subclones generated by the same GSC line showed different metabolic phenotypes. Aerobic glycolysis prevailed in cluster 2 cells as demonstrated by higher lactate production compared to cluster 1 cells. Oligomycin, a mitochondrial ATPase inhibitor, induced high lactate extrusion only in cluster 1 cells, where it produced neutral lipid accumulation detected as mobile lipid signals by MRS and lipid droplets by confocal microscopy. These results indicate a relevant role of mitochondrial fatty acid oxidation for energy production in GSCs. On the other hand, further metabolic differences, likely accounting for different therapy responsiveness observed after etomoxir treatment, suggest that caution must be used in considering patient treatment with mitochondria FAO blockers. Metabolomics and metabolic profiling may contribute to discover new diagnostic or prognostic biomarkers to be used for personalized therapies.

The product of the human AHI-1 (Abelson helper integration site) gene: experimental in vitro data point to its involvement in tumor cell invasion.

INTRODUCTION: Each of the steps involved in invasion of tumors requires specific molecular program in which the modulation of adhesive and migratory properties of disseminating cells plays an essential role. The improvement in the knowledge of these mechanisms can lead to discovery of new target candidates in drug development. In this study we focused attention on the product of the human AHI-1 (Abelson helper integration site) gene Jouberin (Jbn). METHODS: In particular, we explore by in vitro invasion assay, AHI-1 knockdown and electron microscopy, if Jbn is involved in the signaling machinery that regulates tumor invasion. To this purpose tumor cells of different histological derivation (brain, breast, skin) were employed. RESULTS: We found that Jbn expression correlates with the proliferation, invasive potential and invasion strategy of the tested tumor cells, and that its downregulation reduces their capability of migrating and invading the extracellular matrix. CONCLUSIONS: The results obtained in this study for the first time point to Jbn as a new candidate involved in the invasion process of tumor cells, and as potential molecular target in anticancer therapy.

Targeting CXCR4 by a selective peptide antagonist modulates tumor microenvironment and microglia reactivity in a human glioblastoma model.

BACKGROUND: The CXCL12/CXCR4 pathway regulates tumor cell proliferation, metastasis, angiogenesis and the tumor-microenvironment cross-talk in several solid tumors, including glioblastoma (GBM), the most common and fatal brain cancer. In the present study, we evaluated the effects of peptide R, a new specific CXCR4 antagonist that we recently developed by a ligand-based approach, in an in vitro and in vivo model of GBM. The well-characterized CXCR4 antagonist Plerixafor was also included in the study. METHODS: The effects of peptide R on CXCR4 expression, cell survival and migration were assessed on the human glioblastoma cell line U87MG exposed to CXCL12, by immunofluorescence and western blotting, MTT assay, flow cytometry and transwell chamber migration assay. Peptide R was then tested in vivo, by using U87MG intracranial xenografts in CD1 nude mice. Peptide R was administered for 23 days since cell implantation and tumor volume was assessed by magnetic resonance imaging (MRI) at 4.7 T. Glioma associated microglia/macrophage (GAMs) polarization (anti-tumor M1 versus pro-tumor M2 phenotypes) and expressions of vascular endothelial growth factor (VEGF) and CD31 were assessed by immunohistochemistry and immunofluorescence. RESULTS: We found that peptide R impairs the metabolic activity and cell proliferation of human U87MG cells and stably reduces CXCR4 expression and cell migration in response to CXCL12 in vitro. In the orthotopic U87MG model, peptide R reduced tumor cellularity, promoted M1 features of GAMs and astrogliosis, and hindered intra-tumor vasculature. CONCLUSIONS: Our findings suggest that targeting CXCR4 by peptide R might represent a novel therapeutic approach against GBM, and contribute to the rationale to further explore in more complex pre-clinical settings the therapeutic potential of peptide R, alone or in combination with standard therapies of GBM.

Characterization of the cell penetrating properties of a human salivary proline-rich peptide.

Saliva contains hundreds of small proline-rich peptides most of which derive from the post-translational and post-secretory processing of the acidic and basic salivary proline-rich proteins. Among these peptides we found that a 20 residue proline-rich peptide (p1932), commonly present in human saliva and patented for its antiviral activity, was internalized within cells of the oral mucosa. The cell-penetrating properties of p1932 have been studied in a primary gingival fibroblast cell line and in a squamous cancer cell line, and compared to its retro-inverso form. We observed by mass-spectrometry, flow cytometry and confocal microscopy that both peptides were internalized in the two cell lines on a time scale of minutes, being the natural form more efficient than the retro-inverso one. The cytosolic localization was dependent on the cell type: both peptide forms were able to localize within nuclei of tumoral cells, but not in the nuclei of gingival fibroblasts. The uptake was shown to be dependent on the culture conditions used: peptide internalization was indeed effective in a complete medium than in a serum-free one allowing the hypothesis that the internalization could be dependent on the cell cycle. Both peptides were internalized likely by a lipid raft-mediated endocytosis mechanism as suggested by the reduced uptake in the presence of methyl-ß-cyclodextrin. These results suggest that the natural peptide may play a role within the cells of the oral mucosa after its secretion and subsequent internalization. Furthermore, lack of cytotoxicity of both peptide forms highlights their possible application as novel drug delivery agents.

Migratory behaviour of tumour cells: a scanning electron microscopy study.

BACKGROUND: Tumour cells utilize different migration strategies to invade surrounding tissues and elude anticancer treatments. It is therefore important to understand the mechanisms underlying migration process, in order to aid the development of therapies aimed at blocking the dissemination of cancer cells. AIMS: In this study tumour cell lines of different histological origin were analysed by combining 2D and 3D in vitro assays, biochemical tests and high resolution imaging by scanning electron microscopy (SEM) in order to look insight strategies adopted by tumour cells to invade extracellular matrix. RESULTS: Quantitative (computer-assisted colour camera equipped-light microscopy) and qualitative analysis (SEM) indicated that the most aggressive tumour cells adopt an "individual" behaviour. The analysis of intracellular signalling demonstrated that the highest invasive potential was associated with the activation of AKT, ERK, FAK and ERM proteins. The "individual" behaviour was positively related to the expression of VLA-2 and inversely related with the E-cadherin expression. CONCLUSIONS: The combination of 2D and 3D in vitro assays, biochemical tests and ultrastructural investigations proved to be a suitable test for the investigation of tumour cell migration and invasion. The high resolution imaging by SEM highlighted the interrelationships between cells in different migratory behaviours of tumour cells.

Cyr61 as mediator of Src signaling in triple negative breast cancer cells.

SFKs are involved in tumorigenesis and metastasis. Here we analyzed c-Src contribution to initial steps of metastasis by tetracycline-dependent expression of a specific shRNA-c-Src, which suppressed c-Src mRNA and protein levels in metastatic MDA-MB-231 cells. c-Src suppression did not alter cell proliferation or survival, but it significantly reduced anchorage-independent growth. Concomitantly with diminished tyrosine-phosphorylation/activation of Fak, caveolin-1, paxillin and p130CAS, c-Src depletion also inhibited cellular migration, invasion and transendothelial migration. Quantitative proteomic analyses of the secretome showed that Cyr61 levels, which were detected in the exosomal fraction, were diminished upon shRNA-c-Src expression. In contrast, Cyr61 expression was unaltered inside cells. Cyr61 partially colocalized with cis-Golgi gp74 marker and with exosomal marker CD63, but c-Src depletion did not alter their cellular distribution. In SUM159PT cells, transient c-Src suppression also reduced secreted exosomal Cyr61 levels. Furthermore, conditional expression of a c-Src dominant negative mutant (SrcDN, c-Src-K295M/Y527F) in MDA-MB-231 and in SUM159PT diminished secreted Cyr61 as well. Cyr61 transient suppression in MDA-MB-231 inhibited invasion and transendothelial migration. Finally, in both MDA-MB-231 and SUM159PT, a neutralizing Cyr61 antibody restrained migration. Collectively, these results suggest that c-Src regulates secreted proteins, including the exosomal Cyr61, which are involved in modulating the metastatic potential of triple negative breast cancer cells.

Brain tumor stem cell dancing.

BACKGROUND: Issues regarding cancer stem cell (CSC) movement are important in neurosphere biology as cell-cell or cell-environment interactions may have significant impacts on CSC differentiation and contribute to the heterogeneity of the neurosphere. AIMS: Despite the growing body of literature data on the biology of brain tumor stem cells, floating CSC-derived neurospheres have been scarcely characterized from a morphological and ultrastructural point of view. RESULTS: Here we report a morphological and ultrastructural characterization performed by live imaging and scanning electron microscopy. Glioblastoma multiforme (GBM) CSC-derived neurospheres are heterogeneous and are constituted by cells, morphologically different, capable of forming highly dynamic structures. These dynamic structures are regulated by not serendipitous cell-cell interactions, and they synchronously pulsate following a cyclic course made of "fast" and "slow" alternate phases. Autocrine/paracrine non canonical Wnt signalling appears to be correlated with the association status of neurospheres. CONCLUSIONS: The results obtained suggest that GBM CSCs can behave both as independents cells and as "social" cells, highly interactive with other members of its species, giving rise to a sort of "multicellular organism".

The combined treatment with chloroquine and the enzymatic oxidation products of spermine overcomes multidrug resistance of melanoma M14 ADR2 cells: a new therapeutic approach.

It has been confirmed that multidrug resistant (MDR) melanoma cells (M14 ADR2) are more sensitive than their wild-type counterparts (M14 WT) to H2O2 and aldehydes, the products of bovine serum amine oxidase (BSAO)-catalyzed oxidation of spermine. The metabolites formed by BSAO and spermine are more toxic, in M14 cells, than exogenous H2O2 and acrolein, even though their concentration is lower during the initial phase of incubation due to their more gradual release than the exogenous products. Binding of BSAO to the cell membrane and release of the reaction products of spermine into the immediate vicinity of the cells, or directly into the cells, may explain the apparently paradoxical phenomenon. Both WT and MDR cells, after pre-treatment for 24 h, or longer, with the lysosomotropic compound chloroquine (CQ), show to be sensitized to subsequent exposure to BSAO/spermine enzymatic system. Evidence of ultrastructural aberrations and acridine orange release from lysosomes is presented in this study that is in favor of the permeabilization of the lysosomal membrane as the major cause of sensitization by CQ. Pre-treatment with CQ amplifies the ability of the metabolites formed from spermine by oxidative deamination to induce cell death. Melanocytes, differently from melanoma cells, were unaffected by the enzymatic system, even when preceded by CQ treatment. Since it is conceivable that combined treatment with a lysosomotropic compound and BSAO/spermine would be effective against tumour cells, it is of interest to search for such novel compounds, which might be promising for application in a therapeutic setting.

Morphological transformation induced by multiwall carbon nanotubes on Balb/3T3 cell model as an in vitro end point of carcinogenic potential.

In this work we investigated the toxicological effects of nude and chemically functionalised (-NH(2), -OH and -COOH groups) multiwall carbon nanotubes (mwCNTs) using immortalised mouse fibroblasts cell line (Balb/3T3) as in vitro model, alternative to the use of animals, to assess basal cytotoxicity, carcinogenic potential, genotoxicity and cell interaction of nanomaterials (NM). Combining in vitro tests such as cell transformation assay and micronucleus with physicochemical and topological analysis, we obtained results showing no cytotoxicity and genotoxicity. Carcinogenic potential and mwCNTs interaction with cells were instead evident. We stressed the importance that different toxicological end points have to be considered when studying NM, therefore, assays able to detect long-term effects, such as carcinogenicity, must be taken into account together with a panel of tests able to detect more immediate effects like basal cytotoxicity or genotoxicity.

Immune surveillance properties of human NK cell-derived exosomes.

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.

Src kinases catalytic activity regulates proliferation, migration and invasiveness of MDA-MB-231 breast cancer cells.

SFKs are frequently deregulated in cancer where they control cellular proliferation, migration, survival and metastasis. Here we study the role of SFKs catalytic activity in triple-negative/basal-like and metastatic human breast cancer MDA-MB-231 cells employing three well-established inhibitors: Dasatinib, PP2 and SU6656. These compounds inhibited migration and invasion. Concomitantly, they reduced Fak, paxillin, p130CAS, caveolin-1 phosphorylation and altered cytoskeletal structures. They also inhibited cell proliferation, but in different manners. Dasatinib and PP2 increased p27(Kip1) expression and reduced c-Myc levels, restraining G1­S transition. In contrast, SU6656 did not modify p27(Kip1) expression, slightly altered c-Myc levels and generated polyploid multinucleated cells, indicating inhibition of cytokinesis. These later effects were also observed in SYF fibroblasts, suggesting a SFKs-independent action. ZM447439, an Aurora B kinase inhibitor, produced similar cell cycle and morphological alterations in MDA-MB-231 cells, indicating that SU6656 blocked Aurora B kinase. This was confirmed by inhibition of histone H3 phosphorylation, the canonical Aurora B kinase substrate. Furthermore, hierarchical clustering analysis of gene expression profiles showed that SU6656 defined a set of genes that differed from Dasatinib and PP2. Additionally, Gene Set Enrichment Analyses revealed that SU6656 significantly reduces the Src pathway. Together, these results show the importance of SFKs catalytic activity for MDA-MB-231 proliferation, migration and invasiveness. They also illustrate that SU6656 acts as dual SFKs and Aurora B kinase inhibitor, suggesting its possible use as a therapeutic agent in breast cancer.

Inhibition of phosphatidylcholine-specific phospholipase C results in loss of mesenchymal traits in metastatic breast cancer cells.

INTRODUCTION: Acquisition of mesenchymal characteristics confers to breast cancer (BC) cells the capability of invading tissues different from primary tumor site, allowing cell migration and metastasis. Regulators of the mesenchymal-epithelial transition (MET) may represent targets for anticancer agents. Accruing evidence supports functional implications of choline phospholipid metabolism in oncogene-activated cell signaling and differentiation. We investigated the effects of D609, a xanthate inhibiting phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelin synthase (SMS), as a candidate regulator of cell differentiation and MET in the highly metastatic BC cell line MDA-MB-231. METHODS: PC-PLC expression and activity were investigated using confocal laser scanning microscopy (CLSM), immunoblotting and enzymatic assay on human MDA-MB-231 compared with MCF-7 and SKBr3 BC cells and a nontumoral immortalized counterpart (MCF-10A). The effects of D609 on PC-PLC and SMS activity, loss of mesenchymal markers and changes in migration and invasion potential were monitored in MDA-MB-231 cells by enzymatic assays, CLSM, immunoblotting and transwell chamber invasion combined with scanning electron microscopy examinations. Cell proliferation, formation and composition of lipid bodies and cell morphology were investigated in D609-treated BC cells by cell count, CLSM, flow-cytometry of BODIPY-stained cells, nuclear magnetic resonance and thin-layer chromatography. RESULTS: PC-PLC (but not phospholipase D) showed 2- to 6-fold activation in BC compared with nontumoral cells, the highest activity (up to 0.4 pmol/µg protein/min) being detected in the poorly-differentiated MDA-MB-231 cells. Exposure of the latter cells to D609 (50 µg/mL, 24-72 h) resulted into 60-80% PC-PLC inhibition, while SMS was transiently inhibited by a maximum of 21%. These features were associated with progressive decreases of mesenchymal traits such as vimentin and N-cadherin expression, reduced galectin-3 and milk fat globule EGF-factor 8 levels, ß-casein formation and decreased in vitro cell migration and invasion. Moreover, proliferation arrest, changes in cell morphology and formation of cytosolic lipid bodies typical of cell differentiation were induced by D609 in all investigated BC cells. CONCLUSIONS: These results support a critical involvement of PC-PLC in controlling molecular pathways responsible for maintaining a mesenchymal-like phenotype in metastatic BC cells and suggests PC-PLC deactivation as a means to promote BC cell differentiation and possibly enhance the effectiveness of antitumor treatments.

Adult human circulating CD34⁻Lin⁻CD45⁻CD133⁻ cells can differentiate into hematopoietic and endothelial cells.

A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast, we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34, lineage markers, CD45 and CD133 (CD34⁻Lin⁻CD45⁻CD133⁻ cells), endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34⁻Lin⁻CD45⁻CD133⁻ cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo, CD34⁻Lin⁻CD45⁻CD133⁻ cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34⁻Lin⁻D45⁻CD133⁻ cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials, characterized by the expression of c-Kit and CXCR4 as well as EphB4, EphB2, and ephrinB2. Further molecular and functional characterization of CD34⁻Lin⁻CD45⁻CD133⁻ cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life.

Tea tree oil might combat melanoma.

In this study we present new data from experiments focused on the antitumor activity of tea tree oil (TTO), an essential oil distilled from Melaleuca alternifolia. TTO proved to be capable of inhibiting the growth of melanoma cells and of overcoming multidrug resistance (MDR), as we reported in our previous study. Moreover, the survival role of the MDR-marker P-glycoprotein appears to be involved in the mechanism of invasion of melanoma cells. The results reported herein indicate that TTO and its main active component, terpinen-4-ol, can also interfere with the migration and invasion processes of drug-sensitive and drug-resistant melanoma cells.

Urotensin II receptor predicts the clinical outcome of prostate cancer patients and is involved in the regulation of motility of prostate adenocarcinoma cells.

Urotensin II (UT-II) is a potent vasoconstrictor peptide and its receptor (UTR) was correlated with human cortico-adrenal carcinoma proliferation. In this study, we have evaluated the correlation between UTR expression and prognosis of human prostate adenocarcinoma and the involvement of this receptor in the regulation of biological properties on both in vivo and in vitro models. UTR mRNA and protein, evaluated by real-time PCR and Western blotting, respectively, were expressed at high levels only in androgen-dependent LNCaP cells. In order to investigate UTR changes occurring in human prostate tumorigenesis, we have also evaluated the expression of UTR in vivo in 195 human prostate tissue samples. UTR was always expressed at low intensity in hyperplastic tissues and at high intensity in well-differentiated carcinomas (Gleason 2-3). Moreover, we have evaluated the effects of an antagonist of UTR, urantide on migration and invasion of LNCaP cells. Urantide induced a dose-dependent decrease of motility and invasion of LNCaP cells whose characteristic ameboid movement seems to be advantageous for their malignancy. These effects were paralleled by down-regulating the autophosphorylation of focal adhesion kinase and the integrin surface expression on LNCaP cells. The effects on cell motility and invasion were likely due to the inhibition of RhoA activity induced by both urantide and shRNA UTR. These data suggest that UTR can be considered a prognostic marker in human prostate adenocarcinoma patients.