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The expression and surface distribution of monosialoganglioside GM3 on the plasma membranes of NIH3T3 fibroblasts cultured at semiconfluence were analyzed by immunofluorescence as well as by immunogold electron microscopy on thin sections and surface replicas. The GM3 expression was highly variable from cell to cell and the distribution of the ganglioside on the positive cells appeared punctate. Quantitative immunogold electron microscopy showed the existence of well-defined GM3 clusters of different sizes scattered all over the cell surfaces. Double immunofluorescence analysis of 5-bromo-2’-deoxyuridine incorporation to identify proliferating cells and of GM3 expression indicated that most of the GM3-positive cells appear unable to synthesize DNA and demonstrated a growth-dependent expression of GM3. [ABSTRACT FROM AUTHOR]

The biological relevance of extracellular vesicles (EV) released in an ischemia/reperfusion setting is still unclear. We hypothesized that the inflammatory microenvironment prevents cardioprotection mediated by endothelial cell (EC)-derived extracellular vesicles. The effects of naïve EC-derived EV (eEV) or eEV released in response to interleukin-3 (IL-3) (eEV-IL-3) were evaluated in cardiomyoblasts (H9c2) and rat hearts. In transwell assay, eEV protected the H9c2 exposed to hypoxia/reoxygenation (H/R) more efficiently than eEV-IL-3. Conversely, only eEV directly protected H9c2 cells to H/R-induced damage. Consistent with this latter observation, eEV, but not eEV-IL-3, exerted beneficial effects in the whole heart. Protein profiles of eEV and eEV-IL-3, established using label-free mass spectrometry, demonstrated that IL-3 drives changes in eEV-IL-3 protein cargo. Gene ontology analysis revealed that both eEV and eEV-IL-3 were equipped with full cardioprotective machinery, including the Nitric Oxide Signaling in the Cardiovascular System. eEV-IL-3 were also enriched in the endothelial-nitric oxide-synthase (eNOS)-antagonist caveolin-1 and proteins related to the inflammatory response. In vitro and ex vivo experiments demonstrated that a functional Mitogen-Activated Protein Kinase Kinase (MEK1/2)/eNOS/guanylyl-cyclase (GC) pathway is required for eEV-mediated cardioprotection. Consistently, eEV were found enriched in MEK1/2 and able to induce the expression of B-cell-lymphoma-2 (Bcl-2) and the phosphorylation of eNOS in vitro. We conclude that an inflammatory microenvironment containing IL-3 changes the eEV cargo and impairs eEV cardioprotective action. [ABSTRACT FROM AUTHOR]

Uncontrolled accumulation of methylglyoxal (MG) and reactive oxygen species (ROS) occurs in hyperglycemia-induced endothelial dysfunction associated with diabetes. Resveratrol (RSV) protects the endothelium upon high glucose (HG); however, the mechanisms underlying such protective effects are still debated. Here we identified key molecular players involved in the glycative/oxidative perturbations occurring in endothelial cells exposed to HG. In addition, we determined whether RSV essentially required SIRT1 to trigger adaptive responses in HG-challenged endothelial cells. We used primary human umbilical vein endothelial cells (HUVECs) undergoing a 24-h treatment with HG, with or without RSV and EX527 (i.e., SIRT1 inhibitor). We found that HG-induced glycative stress (GS) and oxidative stress (OS), by reducing SIRT1 activity, as well as by diminishing the efficiency of MG- and ROS-targeting protection. RSV totally abolished the HG-dependent cytotoxicity, and this was associated with SIRT1 upregulation, together with increased expression of GLO1, improved ROS-scavenging efficiency, and total suppression of HG-related GS and OS. Interestingly, RSV failed to induce effective response to HG cytotoxicity when EX527 was present, thus suggesting that the upregulation of SIRT1 is essential for RSV to activate the major antiglycative and antioxidative defense and avoid MG- and ROS-dependent molecular damages in HG environment. [ABSTRACT FROM AUTHOR]

The aim of this prospective study was to evaluate the efficacy of gonadotrophin-releasing hormone antagonist (GnRH) in comparison with the standard long protocol in poor-responder patients. Sixty patients with poor ovarian response in previous treatment cycles were randomized into two groups: group A (n = 30) was stimulated with a standard long protocol, and group B (H = 30) received GnRH antagonist. Vaginal ultrasound was performed to evaluate ovarian response. There was a significantly reduced duration of ovarian stimulation (9.8 ± 0.8 versus 14.6 ± 1.2. P = 0.001) in group B in comparison with group A, and a reduced number of ampoules was used in group B (49.3 ± 4.3 versus 72.6 ± 6.8, P = 0.001). In group B, the number of oocytes retrieved was significantly higher than in group A (5.6 ± 1.6 versus 4.3 ± 2.2, P = 0.02) and there was an increased number of follicles with a diameter >15 mm at human chorionic gonadotrophin administration in group B (P = 0.0001). Fewer cycles were cancelled with the use of an antagonist protocol. Five pregnancies (17% for embryo transfer) were obtained with GnRH antagonist protocol and two (7% for embryo transfer) with GnRH agonist protocol. [ABSTRACT FROM AUTHOR]

The ability of a collagen-based matrix to support cell proliferation, migration, and infiltration has been reported; however, the direct effect of an aqueous collagen suspension on cell cultures has not been studied yet. In this work, the effects of a high-concentration aqueous suspension of a micronized type I equine collagen (EC-I) have been evaluated on a normal mouse fibroblast cell line. Immunofluorescence analysis showed the ability of EC-I to induce a significant increase of type I and III collagen levels, parallel with overexpression of crucial proteins in collagen biosynthesis, maturation, and secretion, prolyl 4-hydroxylase (P4H) and heat shock protein 47 (HSP47), as demonstrated by western blot experiments. The treatment led, also, to an increase of α -smooth muscle actin ( α -SMA) expression, evaluated through western blot analysis, and cytoskeletal reorganization, as assessed by phalloidin staining. Moreover, scanning electron microscopy analysis highlighted the appearance of plasma membrane extensions and blebbing of extracellular vesicles. Altogether, these results strongly suggest that an aqueous collagen type I suspension is able to induce fibroblast myodifferentiation. Moreover, our findings also support in vitro models as a useful tool to evaluate the effects of a collagen suspension and understand the molecular signaling pathways possibly involved in the effects observed following collagen treatment in vivo ., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Francesca Lombardi et al.)

Cancer incidence and mortality are rapidly growing worldwide. The main risk factors for cancer can be associated with aging as well as the growth of the population and socioeconomic condition. Breast cancer, a crucial public health problem, is the second cause of death among women. About 70% of patients with advanced breast cancer have bone metastases. In bone metastasis, cancer cells and osteoclasts form a vicious cycle: cancer cells promote osteoclast differentiation and activation that, in turn, induce cancer cell seeding and proliferation in the bone. Growing evidence shows that extracellular vesicles (EVs) play a key role in carcinogenesis, proliferation, pre-metastatic niche formation, angiogenesis, metastasis, and chemoresistance in several tumors, such as breast, lung, prostate, and liver cancer. Here, we discuss the role of EVs released by breast cancer cells, focusing on bone metastasis induction and their clinical implications as biomarkers.

Background: Cyclooxygenase-2 (COX-2), an inflammation-associated enzyme, has been implicated in tumorigenesis and progression of glioblastoma (GBM). The poor survival of GBM was mainly associated with the presence of glioma stem cells (GSC) and the markedly inflammatory microenvironment. To further explore the involvement of COX-2 in glioma biology, the effects of NS398, a selective COX-2 inhibitor, were evaluated on GSC derived from COX-2 expressing GBM cell lines, i.e., U87MG and T98G, in terms of neurospheres' growth, autophagy, and extracellular vesicle (EV) release., Methods: Neurospheres' growth and morphology were evaluated by optical and scanning electron microscopy. Autophagy was measured by staining acidic vesicular organelles. Extracellular vesicles (EV), released from neurospheres, were analyzed by transmission electron microscopy. The autophagic proteins Beclin-1 and LC3B, as well as the EV markers CD63 and CD81, were analyzed by western blotting. The scratch assay test was used to evaluate the NS398 influence on GBM cell migration., Results: Both cell lines were strongly influenced by NS398 exposure, as showed by morphological changes, reduced growth rate, and appearance of autophagy. Furthermore, the inhibitor led to a functional change of EV released by neurospheres. Indeed, EV secreted by NS398-treated GSC, but not those from control cells, were able to significantly inhibit adherent U87MG and T98G cell migration and induced autophagy in recipient cells, thus leading to effects quite similar to those directly caused by NS398 in the same cells., Conclusion: Despite the intrinsic diversity and individual genetic features of U87MG and T98G, comparable effects were exerted by the COX-2 inhibitor NS398 on both GBM cell lines. Overall, our findings support the crucial role of the inflammatory-associated COX-2/PGE2 system in glioma and glioma stem cell biology., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

The role of platelets in haemostasis has long been known, but understanding of these cells' involvement in wound healing/tissue repair is more recent and has given rise to a multitude of translational studies. Tissue repair processes consist of complex, regulated interactions between cells modulated by biologically active molecules, most of which are growth factors released by activated platelets: this aspect represents the rationale on which the use of platelet derivatives for clinical purposes is based. In the last years, many in vitro studies have focused on the mechanisms of action by which these growth factors affect the biological activities of cells, thus supporting tissue healing. Although limited by some drawbacks (two-dimensional in vitro monocultures cannot replicate the tissue architecture and organisation of organs or the continuous interplay between different cell types), in vitro studies do have the advantages of giving rapid results and allowing precise control of platelet concentrations and other parameters.This review offers an updated overview of the data obtained from the most recent bench-top studies focused on the effects of platelet derivatives on a wide variety of human cells, highlighting their possible impact for in vivo applications. The heterogeneity of the data obtained so far is very evident. This can be explained by the different experimental settings used in each study, which may be the cause of the variability in clinical outcomes. In fact, in vitro studies suggest that the composition of platelet derivatives and the method used for their production and activation (or not) and the platelet concentration used can have profound effects on the final results.

The therapeutic potential of mesenchymal stem cell (MSC) extracellular vesicles (EV) is currently under investigation in many pathological contexts. Both adult and perinatal MSC are being considered as sources of EV. Herein, we address antigen expression of cord blood and bone marrow MSC and released EV to define an identity and quality parameter of MSC EV as a medicinal product in the context of clinical applications. The research focuses on EV-shuttled neural/glial antigen 2 (NG2), which has previously been detected as a promising surface marker to distinguish perinatal versus adult MSC. Indeed, NG2 was significantly more abundant in cord blood than bone marrow MSC and MSC EV. Ultracentrifuge-isolated EV were then challenged for their pro-angiogenic properties on an xCELLigence system as quality control. NG2 + cord blood MSC EV, but not bone marrow MSC EV, promote bFGF and PDGF-AA proliferative effect on endothelial cells. Likewise, they successfully rescue angiostatin-induced endothelial cell growth arrest. In both cases, the effects are NG2-dependent. These results point at NG2 as an identity and quality parameter for cord blood MSC EV, paving the way for their clinical translation.

The relevance of nitric oxide synthase 2 (NOS2) as a prognostic factor in Glioblastoma Multiforme (GBM) malignancy is emerging. We analyzed the effect of NOS2 inhibitor 1400W on the autophagic flux and extracellular vesicle (EV) secretion in U87MG glioma cells. The effects of glioma stem cells (GSC)-derived EVs on adherent U87MG were evaluated. Cell proliferation and migration were examined while using Cell Counting Kit-8 assay (CCK-8) and scratch wound healing assay. Cell cycle profile and apoptosis were analyzed by flow cytometry. Autophagy-associated acidic vesicular organelles were detected and quantified by acridine orange staining. The number and size of EVs were assessed by nanoparticle tracking analysis. EV ultrastructure was verified by transmission electron microscopy (TEM). WB was used to analyze protein expression and acid sphingomyelinase was determined through ceramide levels. 1400W induced autophagy and EV secretion in both adherent U87MG and GSCs. EVs secreted by 1400W-treated GSC, but not those from untreated cells, were able to inhibit adherent U87MG cell growth and migration while also inducing a relevant level of autophagy. The hypothesis of NOS2 expression as GBM profile marker or interesting therapeutic target is supported by our findings. Autophagy and EV release following treatment with the NOS2 inhibitor could represent useful elements to better understand the complex biomolecular frame of GBM.

Background: Only few studies have investigated the effect of topical application of tranexamic acid (TXA) on "minimally" invasive joint surgical procedures in which articular cartilage is preserved; for this reason, actually many surgeons avoid the use of topical TXA even if the disadvantage related to a blood loss can occur. The aim of this study was to evaluate the cytotoxicity, on human chondrocytes, of TXA at different concentrations and times of exposure and the mechanisms of cell death., Methods: Experiments were carried out on isolated human chondrocytes harvested from eight patients who underwent total knee replacement. Cell viability was determined using XTT assay and was assessed at 0, 24 and 48 h intervals after a 10-min-long treatment, followed by thorough washes, or at 24 and 48 h of treatment at TXA concentrations of 20, 50, 70 and 100 mg/ml. Cell cycle alterations and occurrence of cell death for apoptosis or necrosis were assessed by cytofluorimetry. Data were analyzed using Proc Mixed Procedure; LSMEANS was used to compare multiple group means with Tukey's honestly significant difference test., Results: A significant correlation between the controlled for factors (type of treatment, time and concentration) was found in the performed experiment. No significant effect on cell viability was observed when the TXA exposure was limited to 10 min, while for increased exposure, 24 and 48 h, a remarkable reduction was found; cell death occurred by apoptosis and was already appreciable after 24 h, reaching a statistical significance after the 48-h-long treatment., Conclusion: A prolonged exposure to TXA may cause cartilage damage, thus its topical application can be expanded also to clinical scenarios that include retention of native cartilage chondrocytes, only if it is limited to few minutes and used at concentrations of 70 mg/ml or less.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Our previous study on 75 cases of advanced oral squamous cell carcinomas (SCC) treated by neoadjuvant chemotherapy, radical surgery, and radiotherapy showed that overexpression of p53 of more than 50% indicated a strong probability of genetic mutation, and tumours that are characterised by this p53 pattern respond poorly to treatment and have a poor prognosis (p= 0.0001). We have studied the same cohort of patients retrospectively to investigate the incidence of human papillomavirus-16 (HPV16) infection, the relation to the overexpression or mutation of the p53 gene, and the association with overall survival. There were 57 men and 18 women, mean age 67 (range 57-72) years. HPV16 infectivity was found in 66 patients (88%) - 49/57 men (86%) and 17/18 women (94%). There was no significant difference between the sexes (p=0.32). The cumulative survival of the entire group after a follow-up of 38 months was 26% (SE 6.4). The log rank test indicated that the combination of HPV-16 infectivity and p53mutation was significantly related to prognosis (p=0.000). On the other hand HPV16 infectivity alone was not significantly related to prognosis (p=0.78) The incidence of HPV-16 infection decreased with increasing immune p53 expression (p=0.005), whereas that of the HPV16+p53mutation combination increased with increasing immune p53 expression (p=0.000). The results show the importance of the investigation of HPV and p53 expression to define prognosis in oral SCC., (Copyright © 2015 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

Since tendon injuries and tendinopathy are a growing problem, sometimes requiring surgery, new strategies that improve conservative therapies are needed. Platelet-rich plasma (PRP) seems to be a good candidate by virtue of its high content of growth factors, most of which are involved in tendon healing. This study aimed to evaluate if different concentrations of platelets in PRP have different effects on the biological features of normal human tenocytes that are usually required during tendon healing. The different platelet concentrations tested (up to 5 × 10(6) plt/µL) stimulated differently tenocytes behavior; intermediate concentrations (0.5 × 10(6), 1 × 10(6) plt/µL) strongly induced all tested processes (proliferation, migration, collagen, and MMPs production) if compared to untreated cells; on the contrary, the highest concentration had inhibitory effects on proliferation and strongly reduced migration abilities and overall collagen production but, at the same time, induced increasing MMP production, which could be counterproductive because excessive proteolysis could impair tendon mechanical stability. Thus, these in vitro data strongly suggest the need for a compromise between extremely high and low platelet concentrations to obtain an optimal global effect when inducing in vivo tendon healing.

We have analysed concentrations of the p53 protein in advanced oral carcinomas immunohistochemically and genetically to detect the percentage of overexpression of this antioncogene that indicates a high probability of mutation. This would point to it being a useful prognostic factor, if we consider the importance of the relation between genetic alterations of p53 and poor overall survival. Seventy-five non-consecutive patients with oral squamous cell carcinoma and metastatic nodes were enrolled if there was homogeneity in histopathological grading (G2) of their tumours, and they were treated according to a multidisciplinary treatment plan. Monoclonal antibodies, extraction of DNA, and amplification of the polymerase chain reaction (PCR) were used for the immunohistochemical and genetic analyses. There was a significant inverse correlation between p53 overexpression and response to chemotherapy and a stronger association between high P53 overexpression (%) and a genetic mutation of p53 (p=0.0001). More than 50% overexpression indicated a strong probability of genetic mutation. There was no association between response to chemotherapy and age-groups or TNM classification (p=0.2), but there was a significant one between sex and site of tumour (p<0.001). Three prognostic factors were significantly related to prognosis: site of tumour (p=0.01), response to chemotherapy (p=0.002), and immuno p53 (p=0.0001). A tumour that is characterised by p53 overexpression of more than 50% indicates a poor prognosis., (Copyright © 2013 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

DNA methylation might be the earliest somatic genome changes in prostate cancer that also play an important role in the process of tumor invasion, growth and metastasis. In recent years, several inhibitors of DNA methyltransferases (DNMTis) have been developed and evaluated in pre-clinical models and in clinical trials. While these compounds are effective in the treatment of hematological conditions, clinical trials in solid tumors and in prostate cancer have shown limited or no efficacy. This may be attributed to inappropriate dose regimens leading to toxicity-related adverse events. As with other anti-target compounds, one of the obstacles encountered with DNMTis in prostate cancer could be the inability to select patients for the clinical studies as well as the inability to monitor the efficacy of the drug if not the conclusion of the study. Primary cultures derived from human prostatic tissues harvested from patients with benign prostatic hyperplasia (BPH) and prostate cancer (PCa) as well as neoplastic and non-neoplastic prostate cell lines were tested for DNMT expression/activity and to monitor azacitidine molecular efficacy. We observed that in primary cultures the levels of DNMT activity as well as the protein levels of DNMT1, DNMT3a and DNMT3b were higher in cultures derived from PCa compared to BPH tissue samples and significantly higher in cultures derived from PCa with Gleason scores ≥7 compared to those observed in cultures derived from Gleason scores <7. In addition, DNMT activity as well as DNMT1, DNMT3a and DNMT3b levels were higher in PCa cell lines compared to their non-neoplastic counterparts. Although DNMT activity was higher in high tumorigenic/aggressive PCa cell lines compared to low tumorigenic/aggressive cell lines, only the levels of DNMT3a and DNMT3b were significantly higher in the first group of cells, suggesting that DNMT1 activity is related to the transition to non-neoplastic versus neoplastic phenotype whereas the de novo methylation enzymes were mainly related to progression. Nevertheless, the comparison in the more aggressive PC3 cell derivatives (PC3-LN4 cells) also possessed higher levels of DNMT1 compared to PC3 and PC3M from which these cells were derived. Collectively, our results confirm previous data on the increased methylation in more aggressive tumors supporting the use of DNMTis in advanced prostate cancer. In addition, since glutathione S-transferase-π (GSTP1) was re-expressed or its protein levels were increased after treatment with non-toxic azacitidine doses and since GSTP1 can easily be measured in patient sera, the monitoring of this protein may aide in the evaluation of therapy in future clinical trials.

Histone deacetylase inhibitors (HDACi) are promising epigenetic cancer chemotherapeutics rapidly approaching clinical use. In this study, we tested using in vitro and in vivo models the differential biological effects of a novel HDAC inhibitor [belinostat (PXD101)], in a wide panel of androgen-sensitive and androgen-independent tumor cells. Belinostat significantly increased acetylation of histones H3 and H4. Belinostat potently inhibited the growth of prostate cancer cell lines (IC50 range from 0.5 to 2.5 µM) with cytotoxic activity preferentially against tumor cells. This agent induced G2/M arrest and increased significantly the percentage of apoptosis mainly in androgen-sensitive tumor cells confirming its growth-inhibitory effects. The cell death mechanisms were studied in three different prostate cancer cell lines with different androgen dependence and expression of androgen receptor; LAPC-4 and 22rv1 (androgen-dependent and expressing androgen receptor) and PC3 (androgen-independent not expressing androgen receptor). Belinostat induced the expression of p21 and p27, acetylation of p53 and G2/M arrest associated with Bcl2 and Bcl-Xl downmodulation and significant reduction of survivin, IAPs and Akt/pAkt and increased caspase-8 and -9 expression/activity. Belinostat effectiveness was dependent on the androgen receptor (AR), since the stable transfection of AR greatly increased the efficacy of this HDAC inhibitor. These observations were correlated using in vivo models. We demonstrated that belinostat preferentially resulted in antitumor effect in androgen-dependent tumor cells expressing AR. Our findings provide evidence that belinostat may be a promising anticancer drug for prostate cancer expressing AR, supporting its clinical role in prostate cancer.

Breast cancer shows a strong predilection to metastasize to bone. Cell surface glycoprotein extracellular matrix metalloproteinase inducer (EMMPRIN)/CD147 induces metalloproteinases (MMP) and vascular endothelial growth factor (VEGF), which may support osteoclastic activity and increased incidence of breast cancer bone metastases. In support of this hypothesis, we observed that MDA-MB-231 human breast tumor cells engineered to overexpress EMMPRIN strongly induced osteolytic lesions in immunodeficient mice, which was blunted by in vivo treatment with an EMMPRIN blocking antibody. Similarly, these cells exhibited increased expression of MMP-9 and VEGF relative to control cells. Treatment of MDA-MB-231 cells with the osteoclastogenic cytokine receptor activator of NF-kappaB ligand (RANKL) upregulated EMMPRIN expression with a parallel increase of MMP-9 and VEGF. Conditioned medium from osteoblasts similarly increased EMMPRIN, MMP-9, and VEGF expression in cells. Osteoblast treatment with the RANKL decoy receptor osteoprotegerin abolished this effect. EMMPRIN overexpression stimulated MDA-MB-231 cell invasion but not proliferation. Conversely, small interfering RNA-mediated knockdown of EMMPRIN downregulated MMP-9 and VEGF basal expression and RANKL-stimulated expression, and reduced cell invasion. Our results argue that EMMPRIN drives breast cancer-induced osteolytic lesions and that activation of the RANKL pathway increases EMMPRIN in osteotropic tumor cells, in turn enhancing tumor-induced bone resorption.

The term vasculogenic mimicry (VM) indicates the process by which aggressive tumor cells are able to generate in vitro non-endothelial cell-lined channels delimited by extracellular matrix. Although VM has been described in several human malignancies, the molecular basis of this phenomenon is not entirely understood. In the present study, we examined VM in two ovarian cancer cell lines with different invasion capability (CABA I, low invasion activity; SKOV3, high invasion activity). Specifically, we focused on the potential role played by CD147/extracellular MMP inducer, a membrane spanning molecule highly expressed in tumor cells, in VM. Previous studies have shown that CD147 may be involved in the progression of malignancies by regulating the expression of metalloproteinases in peritumoral stromal cells. In this study, we found significant correlations between expression of CD147 in ovarian cancer cell lines and tumor invasiveness, the activity of the proteases and the ability to form vascular channels. The treatment of SKOV3 cells with small interfering RNA against CD147 suppressed the ability of these cells to generate non-endothelial cell-lined channels. In contrast, transfection of CD147 cDNA into the CABA I cell line resulted in an increased tumor invasiveness and enabled the formation of vascular channels. Altogether, our data suggest that CD147 may play a critical role in VM of CABA I and SKOV3, human ovarian cancer cell lines.

Background: Erlotinib is a small-molecule tyrosine kinase inhibitor targeted EGFR, known to be overexpressed in a variety of cancers, including prostate cancer. Clinical trials showed insignificant clinical benefit in patients with castration resistant prostate cancer both when EGFR inhibitors were administered as monotherapy or in association with antiandrogens or chemotherapeutics. Why, differently to other tumors, have EGFR inhibitors been so ineffective in human prostate cancer? This is the question that we have set in this report., Methods: For this purpose, the effectiveness of erlotinib, a selective EGFR inhibitor, in a wide range of prostate cancer cells (wild type or engineered to overexpress peculiar proteins including androgen receptor and PTEN)., Results: We demonstrated that the effectiveness of erlotinib was inversely correlated to the EGFR/Her2 ratio rather than EGFR/p-EGFR or Her2/p-Her2 levels. Chronic treatment with bicalutamide induced overexpression of Her2 and reduction of EGFR/Her2ratio and this was associated with increased Akt and Erk activity. In these conditions of treatment a reduced efficacy of erlotinib was observed. At the same time, an increased efficacy versus erlotinib was documented in cancer cells chronically exposed to DHT. In these culture conditions low levels of Her2 and increased EGFR/Her2 ratio were evidenced., Conclusions: Taken together, our results seem to suggest that a low EGFR/Her2 ratio and PTEN absence are the main factors responsible of erlotinib inefficacy. Therefore the inhibition of EGFR could have important antitumor effects in hormone-naive rather than in hormonally treated patients.

Objectives: To evaluate the role of Her2/neu as a molecular marker predictive of the treatment response to bicalutamide in prostate cancer (PCa)., Methods: Four PCa cell lines with graded Her2/neu expression levels and 63 primary tumor cultures derived from men with PCa and selected according to Her2/neu tumor levels were used. Primary tumor cultures and PCa cell lines were treated with bicalutamide (0.1-10 microM) in the presence of dehydrotestosterone (10(-12) M) for 4 days. The presence of a significant correlation between Her2/nue expression and drug efficacy was used to define the role of Her2/neu as molecular predictor of bicalutamide effectiveness. As an indicator of drug effectiveness we used the concentration that inhibits 50% values determined after bicalutamide treatment., Results: After bicalutamide treatment, no significant differences in the concentration that inhibits 50% were found among the different tumor cell lines (P = .081). In this experimental model, the correlation analysis suggested that the effectiveness of this drug was not influenced by Her2/neu levels (r = 0.053, P = .823). In primary cultures with high Her2/neu levels (43 tumor cultures), the mean value of the concentration that inhibits 50% for bicalutamide was 0.43 +/- 0.13 microM, and in cultures with low Her2/neu levels (20 tumor cultures), the same parameter was 0.5 +/- 0.16 microM (P = .14). The correlation analysis suggested that the effectiveness of this drug was not influenced by Her2/neu levels (r = 0.33, P = .101)., Conclusions: Our biologic data seem to indicate that the antitumor effect of bicalutamide is independent of Her2/neu levels in preclinical models of PCa. Bicalutamide could be configured as a pharmacologic option to treat patients with high or low levels of Her2/neu.

Background: Numerous studies have supported the use of topical blood components to improve wound healing and tissue regeneration. Platelet gel (PG), a hemocomponent obtained from mix of activated platelets (PLTs) and cryoprecipitate, is currently being used clinically in an attempt to improve tissue healing. The present study sought to define the most effective PG concentration to promote angiogenesis in vitro., Study Design and Methods: The effects of PG-released supernatant at different concentrations on human endothelial cells were studied using different in vitro assays (proliferation, migration, invasion, cord formation, and wound healing)., Results: The concentration of PG-released supernatant had a significant influence on the angiogenic potential of endothelial cells. The optimal concentration for the stimulation of angiogenesis was 1.5 x 10(6) PLTs per microL in most of the in vitro experiments used in this study. Lower or higher concentrations of PG displayed a lower angiogenic potential., Conclusion: An optimal concentration of PG to promote angiogenesis in human endothelial cells was identified. Excessively high PG concentrations may inhibit the angiogenic process, thereby being counterproductive for wound healing in a clinical setting.

One of the major obstacles in curing prostate cancer is the development of drug resistance. It is not only imperative to discover the molecular basis of resistance but also to find therapeutic agents that can disrupt the resistant pathways. Tumor necrosis factor TNF-related apoptosis-inducing ligand TRAIL-like ligands or agonist TRAIL-receptor monoclonal antibodies have entered phase I and II clinical trials with a very limited cytotoxic profile when used systemically in a variety of cancers. Therefore, TRAIL-receptor agonists are new proapoptotic pharmaceutical agents with great potential as new cancer therapeutic agents. Although many cancer cells undergo TRAIL-mediated apoptosis, some are resistant to TRAIL. Therefore, we have been investigating mechanisms to overcome TRAIL resistance in cancer cells so that TRAIL-associated compounds can be used effectively in clinical trials. Epigenetic inactivation of proapoptotic genes, or activation of survival signaling, can cause cross-resistance to several anti-tumor therapies and to immune cytotoxic lymphocytes. We hypothesize that 5-aza-2 deoxycytidine aza-dCR, decitabine may render TRAIL-resistant prostate cancer cells sensitive to caspase-8-mediated apoptosis and may, therefore, be therapeutically efficient. We evaluated the antiproliferative effects of decitabine on the following four prostate cancer cell lines: well-differentiated AR positive LnCaP p53(+), PTEN- and 22rv1 p53(+) and PTEN(+)]; poorly-differentiated AR negative PC3 p53-, PTEN- and DU145 p53 mutant, PTEN(+). Here, we provide evidence that treatment with sub-optimal concentrations of decitabine are additive to TRAIL effects in well-differentiated PCa cells whereas the same treatment shows synergistic effects in poorly-differentiated PCa cells through increased caspase-8 expression, down-modulation of Akt activation and through the expression of certain anti-apoptotic molecules including FLIP, PED/PEA-15, survivin and c-IAP-1. Our findings demonstrate that decitabine at relatively low concentrations restores caspase-8 expression and sensitises resistant PCa cells to TRAIL-induced apoptosis leading to important implications in novel therapeutic strategies targeting defective apoptosis pathways in advanced prostate tumors.

Background: PTEN is a well-characterized tumor suppressor that negatively regulates cell growth and survival through the modulation of PI3K/Akt pathway., Methods: In this paper, we investigated the effects of an PI3K/Akt inhibitor, perifosine, in human prostate cancer (PCa) cells analyzing cell proliferation, apoptosis, and the synergy with EGFR inhibitors., Results: Clinically achievable concentrations of perifosine, as well as Akt gene knockdown, induced a G0/G1 arrest and apoptosis in PTEN defective PCa cells. Although PTEN introduction was able to restore the control of Akt activity and to reduce cell proliferation, the manipulation of PTEN gene was not able alone to influence apoptosis. Perifosine induced apoptotic program also in PTEN positive cells when Akt activity was augmented by EGF suggesting the possibility that this drug could be used in combination with EGFR inhibitors. The combination treatment between erlotinib and pharmacological or molecular Akt knockdown, indeed, showed synergistic effects. This is the first demonstration that a pharmacological compound against Akt activity can restore the efficacy against EGFR inhibitors in PCa and has important therapeutic fallout since EGFR inhibitors have demonstrated very low effectiveness in PCa patients., Conclusions: Taken together our data have an important clinical relevance in the treatment of advanced prostate tumors. However, further studies in the setting of combination therapies in advanced PCas are necessary.

Background: Platelet gel is used to facilitate wound healing in virtue of the growth factors released from activated platelets at the site of lesion, but little is known about the specific mechanisms underlying cellular repair., Aims: To evaluate, in vitro, cellular effects of different concentrations of platelet gel -released supernatant on endothelial cells., Material and Methods: Platelet concentrate was produced at the Service of Immunohaematology and Transfusion of San Salvatore Hospital of L'Aquila, using multiple bags. Platelet gel was obtained by adding thrombin and calcium gluconate to the concentrates and then centrifuging to recover the supernatant. Human umbilical vein endothelial cells were isolated from umbilical cord veins and grown in appropriate conditions. To study their viability, cells were treated with different concentrations of supernatant and XTT assays were performed on the 3 days following treatment. Endothelial cell motility and invasiveness were assayed using modified Boyden chambers with filters coated with 0.1% gelatin (for the motility test) or with a thick layer of the reconstituted basement membrane Matrigel (for the invasion test). The supernatant, added at various concentrations to the lower compartment of the chamber, was used as an attractant. Umbilical cells were added to the upper compartment of the chamber. After 4 hours (for the motility test) or 6 hours (for the invasion test), filters were stained and the migrated cells in five high-power fields were counted., Results: When used at specific concentrations, platelet gel-released supernatant is able to induce proliferation and to stimulate motility and invasiveness of endothelial human cells. Higher concentrations induce a reversion of the stimulatory processes., Conclusions: There is a large body of evidence indicating that platelets and their derivatives have the potential for a substantial therapeutic role in tissue regeneration. The results of this in vitro study highlight the need for an in-depth analysis of technical protocols for the most appropriate and effective use of platelet gel for in vivo applications.

The aim of this study is to investigate the effects in vitro induced by androgenic anabolic steroids (AAS) (testosterone, nandrolone, androstenedione, norandrostenedione, and norandrostenediol) used illicitly in sport competitions, on the proliferation ability, apoptosis and the intracellular calcium concentration ([Ca2+]i) in human umbilical vein endothelial cells (HUVECs), selected as a prototype of a biological target system whose structure and function can be affected by steroids. For this purpose, we evaluated the proliferation inhibition by cytotoxic assay expressed as the concentration of drug inducing a 50% decrease in growth (IC50). The IC50 was reached for testosterone at 100 microM, androstenedione at 375 microM, nandrolone at 9 microM, norandrostenedione at 500 microM. The IC50 value for norandrostenediol was not reached until a concentration of 6000 microM. The apoptotic effect was evaluated by flow cytometry at IC50 for each drug. We observed that testosterone induced 31% of apoptotic cells, norandrostenedione 25%, androstenedione 15% and nandrolone 18%. We have analyzed the effects of these drugs on [Ca2+]i both in the immediate and long-term continuous presence of each compound. Our data show a statistically significant increase of [Ca2+]i in the acute condition and in long-term treated cultures, suggesting that androgen steroids modulate intracellular levels of calcium independent of incubation time or compound identity. As a whole, this study demonstrates that AAS might alter endothelial homeostasis, predisposing to the early endothelial cell activation that is responsible for vascular complications observed frequently in AAS users.

Objective: To assess possible differences in the activity of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, and their inhibitors, the tissue inhibitors of MMPs, TIMP-1 and TIMP-2, in follicular fluid (FF) of women undergoing in vitro fertilization (IVF) treatment and of normally ovulating women., Design: Prospective study., Methods: MMP-2 and MMP-9 activity was analyzed by gelatin zymography and MMP-2, MMP-9, TIMP-2, TIMP-1 and 17beta-estradiol levels were measured in FF by ELISA., Results: We found significantly reduced MMP levels in FF of women undergoing IVF treatment when compared with those of normally ovulating women. In contrast, the TIMP-1 levels were found significantly increased in FF from IVF patients vs normally ovulating women. No significant differences were found for TIMP-2 between the two groups., Conclusions: These findings underline a marked difference in MMPs and their inhibitors in the IVF women and the control group. Therefore we assume MMPs depend on hormonal steroidogenesis modulation induced by the gonadotropin protocol for IVF treatment.

In this report we demonstrated that cellular prion protein is strictly associated with gangliosides in microdomains of neural and lymphocytic cells. We preliminarily investigated the protein distribution on the plasma membrane of human neuroblastoma cells, revealing the presence of large clusters. In order to evaluate its possible role in tyrosine signaling pathway triggered by GEM, we analyzed PrPc presence in microdomains and its association with gangliosides, using cholera toxin as a marker of GEM in neuroblastoma cells and anti-GM3 MoAb for identification of GEM in lymphoblastoid cells. In neuroblastoma cells scanning confocal microscopical analysis revealed a consistent colocalization between PrPc and GM1 despite an uneven distribution of both on the cell surface, indicating the existence of PrPc-enriched microdomains. In lymphoblastoid T cells PrPc molecules were mainly, but not exclusively, colocalized with GM3. In addition, PrPc was present in the Triton-insoluble fractions, corresponding to GEM of cell plasma membrane. Additional evidence for a specific PrPc-GM3 interaction in these cells was derived from the results of TLC analysis, showing that prion protein was associated with GM3 in PrPc immunoprecipitates. The physical association of PrPc with ganglioside GM3 within microdomains of lymphocytic cells strongly suggests a role for PrPc-GM3 complex as a structural component of the multimolecular signaling complex involved in T cell activation and other dynamic lymphocytic plasma membrane functions.