Diagnostic and Prognostic Markers for Pancreatitis and Pancreatic Ductal Adenocarcinoma.

Diagnostic markers are desperately needed for the early detection of pancreatic ductal adenocarcinoma (PDA). We describe sets of markers expressed in temporal order in mouse models during pancreatitis, PDA initiation and progression. Cell type specificity and the differential expression of PDA markers were identified by screening single cell (sc) RNAseq from tumor samples of a mouse model for PDA (KIC) at early and late stages of PDA progression compared to that of a normal pancreas. Candidate genes were identified from three sources: (1) an unsupervised screening of the genes preferentially expressed in mouse PDA tumors; (2) signaling pathways that drive PDA, including the Ras pathway, calcium signaling, and known cancer genes, or genes encoding proteins that were identified by differential mass spectrometry (MS) of mouse tumors and conditioned media from human cancer cell lines; and (3) genes whose expression is associated with poor or better prognoses (PAAD, oncolnc.org). The developmental progression of PDA was detected in the temporal order of gene expression in the cancer cells of the KIC mice. The earliest diagnostic markers were expressed in epithelial cancer cells in early-stage, but not late-stage, PDA tumors. Other early markers were expressed in the epithelium of both early- and late-state PDA tumors. Markers that were expressed somewhat later were first elevated in the epithelial cancer cells of the late-stage tumors, then in both epithelial and mesenchymal cells, or only in mesenchymal cells. Stromal markers were differentially expressed in early- and/or late-stage PDA neoplasia in fibroblast and hematopoietic cells (lymphocytes and/or macrophages) or broadly expressed in cancer and many stromal cell types. Pancreatitis is a risk factor for PDA in humans. Mouse models of pancreatitis, including caerulein treatment and the acinar-specific homozygous deletion of differentiation transcription factors (dTFs), were screened for the early expression of all PDA markers identified in the KIC neoplasia. Prognostic markers associated with a more rapid decline were identified and showed differential and cell-type-specific expression in PDA, predominately in late-stage epithelial and/or mesenchymal cancer cells. Select markers were validated by immunohistochemistry in mouse and human samples of a normal pancreas and those with early- and late-stage PDA. In total, we present 2165 individual diagnostic and prognostic markers for disease progression to be tested in humans from pancreatitis to late-stage PDA.

Landscape of Druggable Molecular Pathways Downstream of Genomic CDH1/Cadherin-1 Alterations in Gastric Cancer.

Loss of CDH1/Cadherin-1 is a common step towards the acquisition of an abnormal epithelial phenotype. In gastric cancer (GC), mutation and/or downregulation of CDH1/Cadherin-1 is recurrent in sporadic and hereditary diffuse GC type. To approach the molecular events downstream of CDH1/Cadherin-1 alterations and their relevance in gastric carcinogenesis, we queried public databases for genetic and DNA methylation data in search of molecular signatures with a still-uncertain role in the pathological mechanism of GC. In all GC subtypes, modulated genes correlating with CDH1/Cadherin-1 aberrations are associated with stem cell and epithelial-to-mesenchymal transition pathways. A higher level of genes upregulated in CDH1-mutated GC cases is associated with reduced overall survival. In the diffuse GC (DGC) subtype, genes downregulated in CDH1-mutated compared to cases with wild type CDH1/Cadherin-1 resulted in being strongly intertwined with the DREAM complex. The inverse correlation between hypermethylated CpGs and CDH1/Cadherin-1 transcription in diverse subtypes implies a common epigenetic program. We identified nonredundant protein-encoding isoforms of 22 genes among those differentially expressed in GC compared to normal stomach. These unique proteins represent potential agents involved in cell transformation and candidate therapeutic targets. Meanwhile, drug-induced and CDH1/Cadherin-1 mutation-related gene expression comparison predicts FIT, GR-127935 hydrochloride, amiodarone hydrochloride in GC and BRD-K55722623, BRD-K13169950, and AY 9944 in DGC as the most effective treatments, providing cues for the design of combined pharmacological treatments. By integrating genetic and epigenetic aspects with their expected functional outcome, we unveiled promising targets for combinatorial pharmacological treatments of GC.

cAMP Response Element-Binding Protein Controls the Appearance of Neuron-Like Traits in Chorion Mesenchymal Cells.

BACKGROUND: Mesenchymal stromal cells (MSC) from bone marrow have been reported to undergo the initial phases of neural differentiation in response to an increase of intracellular cAMP. We investigated the possibility that a similar effect applies to chorion-derived MSC. METHODS: The intracellular concentration of cAMP was increased either by forskolin, to promote its synthesis, or by inhibitors of its degradation. The consequent reduction in the expression of mesenchymal markers was associated with the appearance of neuron-like morphology in a subset of cells. The effect was measured and characterized using biomarkers and an inhibitor of cAMP response element-binding protein (CREB). RESULTS: The dramatic morphological change induced by all the treatments that promoted intracellular cAMP was transient and peaked on the third day. After that, cells returned to the typical fibroblast-like appearance within 24 hours. The distinctive morphology was associated to the expression of neuregulin 1, doublecortin, neuron-specific class III ß-tubulin, and required cAMP response element-binding protein activity. Basic-fibroblast growth factor (b-FGF) treatment increased both the timeframe and number of cells undergoing the morphological change induced by the effect of forskolin. As opposite, arginine-vasopressin (AVP) and sphingosine-1-phosphate (S1P) reduced it. CONCLUSIONS: We conclude that cAMP and the ensuing CREB activation trigger a preliminary step towards neuronal differentiation of chorion-derived MSC. However, likewise other MSC, the stimulus is not sufficient to promote stable differentiation.

High Values of Drain Fluid Epidermal Growth Factor and Transforming Growth Factor-Beta Are Associated with the Development of Pancreatic Fistula after Pancreatoduodenectomy.

INTRODUCTION: Postoperative inflammatory response may act as a major determinant of anastomotic failure after pancreaticoduodenectomy. In this pilot study, we investigated the potential role of drain fluid cytokines in predicting postoperative pancreatic fistula (POPF). METHODS: Drain fluid TGF-ß, IGF-1, EGF, and IL-6, together with serum amylase and drain fluid amylase, were measured on POD1 and correlated with the development of POPF. RESULTS: The study population consisted of 66 patients. POPF and Clavien-Dindo ≥3 morbidity rates were 12.1% and 9.1%, respectively. Patients developing POPF presented significantly higher values of POD1 serum amylase level (477 vs. 54 UI/L, p < 0.001), drain fluid amylase (7,500 vs. 127 UI/L, p < 0.001), TGFß (94 vs. 40 pg/g, p = 0.045), and EGF (17 vs. 13, p = 0.015). There were no differences in terms of IGF-1 and IL-6 values. CONCLUSION: Assessing the local inflammatory response after pancreatoduodenectomy could represent a promising field of research since both TGFß and EGF seem to be associated with the occurrence of POPF.

Circulating tumour DNA: a challenging innovation to develop "precision onco-surgery" in pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the third leading cause of cancer-related mortality within the next decade. Management of PDAC remains challenging with limited effective treatment options and a dismal long-term prognosis. Liquid biopsy and circulating biomarkers seem to be promising to improve the multidisciplinary approach in PDAC treatment. Circulating tumour DNA (ctDNA) is the most studied blood liquid biopsy analyte and can provide insight into the molecular profile and individual characteristics of the tumour in real-time and in advance of standard imaging modalities. This could pave the way for identifying new therapeutic targets and markers of tumour response to supplement diagnostic and provide enhanced stratified treatment. Although its specificity seems excellent, the current sensitivity of ctDNA remains a limitation for clinical use, especially in patients with a low tumour burden. Increasing evidence suggests that ctDNA is a pertinent candidate biomarker to assess minimal residual disease after surgery but also a strong independent prognostic biomarker. This review explores the current knowledge and recent developments in ctDNA as a screening, diagnostic, prognostic and predictive biomarker in the management of resectable PDAC but also technical and analytical challenges that must be overcome to move toward "precision onco-surgery."

Mitoxantrone-Loaded Nanoferritin Slows Tumor Growth and Improves the Overall Survival Rate in a Subcutaneous Pancreatic Cancer Mouse Model.

Pancreatic cancer (PC) represents an intriguing topic for researchers. To date, the prognosis of metastasized PC is poor with just 7% of patients exceeding a five-year survival period. Thus, molecular modifications of existing drugs should be developed to change the course of the disease. Our previously generated nanocages of Mitoxantrone (MIT) encapsulated in human H-chain Ferritin (HFt), designated as HFt-MP-PASE-MIT, has shown excellent tumor distribution and extended serum half-life meriting further investigation for PC treatment. Thus, in this study, we used the same nano-formulation to test its cytotoxicity using both in vitro and in vivo assays. Interestingly, both encapsulated and free-MIT drugs demonstrated similar killing capabilities on PaCa44 cell line. Conversely, in vivo assessment in a subcutaneous PaCa44 tumor model of PC demonstrated a remarkable capability for encapsulated MIT to control tumor growth and improve mouse survival with a median survival rate of 65 vs. 33 days for loaded and free-MIT, respectively. Interestingly, throughout the course of mice treatment, MIT encapsulation did not present any adverse side effects as confirmed by histological analysis of various murine tissue organs and body mass weights. Our results are promising and pave the way to effective PC targeted chemotherapy using our HFt nanodelivery platforms.

Gα15 in early onset of pancreatic ductal adenocarcinoma.

The GNA15 gene is ectopically expressed in human pancreatic ductal adenocarcinoma cancer cells. The encoded Gα15 protein can promiscuously redirect GPCR signaling toward pathways with oncogenic potential. We sought to describe the distribution of GNA15 in adenocarcinoma from human pancreatic specimens and to analyze the mechanism driving abnormal expression and the consequences on signaling and clinical follow-up. We detected GNA15 expression in pre-neoplastic pancreatic lesions and throughout progression. The analysis of biological data sets, primary and xenografted human tumor samples, and clinical follow-up shows that elevated expression is associated with poor prognosis for GNA15, but not any other GNA gene. Demethylation of the 5' GNA15 promoter region was associated with ectopic expression of Gα15 in pancreatic neoplastic cells, but not in adjacent dysplastic or non-transformed tissue. Down-modulation of Gα15 by shRNA or CRISPR/Cas9 affected oncogenic signaling, and reduced adenocarcimoma cell motility and invasiveness. We conclude that de novo expression of wild-type GNA15 characterizes transformed pancreatic cells. The methylation pattern of GNA15 changes in preneoplastic lesions coincident with the release a transcriptional blockade that allows ectopic expression to persist throughout PDAC progression. Elevated GNA15 mRNA correlates with poor prognosis. In addition, ectopic Gα15 signaling provides an unprecedented mechanism in the early steps of pancreas carcinogenesis distinct from classical G protein oncogenic mutations described previously in GNAS and GNAQ/GNA11.

Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor-mediated chemotaxis.

G protein-coupled receptor signaling is required for the navigation of immune cells along chemoattractant gradients. However, chemoattractant receptors may couple to more than one type of heterotrimeric G protein, each of which consists of a Gα, Gß, and Gγ subunit, making it difficult to delineate the critical signaling pathways. Here, we used knockout mouse models and time-lapse microscopy to elucidate Gα and Gß subunits contributing to complement C5a receptor-mediated chemotaxis. Complement C5a-mediated chemokinesis and chemotaxis were almost completely abolished in macrophages lacking Gnai2 (encoding Gαi2), consistent with a reduced leukocyte recruitment previously observed in Gnai2-/- mice, whereas cells lacking Gnai3 (Gαi3) exhibited only a slight decrease in cell velocity. Surprisingly, C5a-induced Ca2+ transients and lamellipodial membrane spreading were persistent in Gnai2-/- macrophages. Macrophages lacking both Gnaq (Gαq) and Gna11 (Gα11) or both Gna12 (Gα12) and Gna13 (Gα13) had essentially normal chemotaxis, Ca2+ signaling, and cell spreading, except Gna12/Gna13-deficient macrophages had increased cell velocity and elongated trailing ends. Moreover, Gnaq/Gna11-deficient cells did not respond to purinergic receptor P2Y2 stimulation. Genetic deletion of Gna15 (Gα15) virtually abolished C5a-induced Ca2+ transients, but chemotaxis and cell spreading were preserved. Homozygous Gnb1 (Gß1) deletion was lethal, but mice lacking Gnb2 (Gß2) were viable. Gnb2-/- macrophages exhibited robust Ca2+ transients and cell spreading, albeit decreased cell velocity and impaired chemotaxis. In summary, complement C5a-mediated chemotaxis requires Gαi2 and Gß2, but not Ca2+ signaling, and membrane protrusive activity is promoted by G proteins that deplete phosphatidylinositol 4,5-bisphosphate.

A Potential Role of RUNX2- RUNT Domain in Modulating the Expression of Genes Involved in Bone Metastases: An In Vitro Study with Melanoma Cells.

Ectopic expression of RUNX2 has been reported in several tumors. In melanoma cells, the RUNT domain of RUNX2 increases cell proliferation and migration. Due to the strong link between RUNX2 and skeletal development, we hypothesized that the RUNT domain may be involved in the modulation of mechanisms associated with melanoma bone metastasis. Therefore, we evaluated the expression of metastatic targets in wild type (WT) and RUNT KO melanoma cells by array and real-time PCR analyses. Western blot, ELISA, immunofluorescence, migration and invasion ability assays were also performed. Our findings showed that the expression levels of bone sialoprotein (BSP) and osteopontin (SPP1) genes, which are involved in malignancy-induced hypercalcemia, were reduced in RUNT KO cells. In addition, released PTHrP levels were lower in RUNT KO cells than in WT cells. The RUNT domain also contributes to increased osteotropism and bone invasion in melanoma cells. Importantly, we found that the ERK/p-ERK and AKT/p-AKT pathways are involved in RUNT-promoted bone metastases. On the basis of our findings, we concluded that the RUNX2 RUNT domain is involved in the mechanisms promoting bone metastasis of melanoma cells via complex interactions between multiple players involved in bone remodeling.

Publisher Correction: Runx2 stimulates neoangiogenesis through the Runt domain in melanoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cocoa Flavonoids Reduce Inflammation and Oxidative Stress in a Myocardial Ischemia-Reperfusion Experimental Model.

: Consumption of flavonoid-rich nutraceuticals has been associated with a reduction in coronary events. The present study analyzed the effects of cocoa flavonols on myocardial injury following acute coronary ischemia-reperfusion (I/R). A commercially available cocoa extract was identified by chromatographic mass spectrometry. Nineteen different phenolic compounds were identified and 250 mg of flavan-3-ols (procyanidin) were isolated in 1 g of extract. Oral administration of cocoa extract in incremental doses from 5 mg/kg up to 25 mg/kg daily for 15 days in Sprague Dawley rats (n = 30) produced a corresponding increase of blood serum polyphenols and become constant after 15 mg/kg. Consequently, the selected dose (15 mg/kg) of cocoa extract was administered orally daily for 15 days in a treated group (n = 10) and an untreated group served as control (n = 10). Both groups underwent surgical occlusion of the left anterior descending coronary artery and reperfusion. Cocoa extract treatment significantly reversed membrane peroxidation, nitro-oxidative stress, and decreased inflammatory markers (IL-6 and NF-kB) caused by myocardial I/R injury and enhanced activation of both p-Akt and p-Erk1/2. Daily administration of cocoa extract in rats is protective against myocardial I/R injury and attenuate nitro-oxidative stress, inflammation, and mitigates myocardial apoptosis.

Methylation Dynamics of RASSF1A and Its Impact on Cancer.

5-methyl cytosine (5mC) is a key epigenetic mark entwined with gene expression and the specification of cellular phenotypes. Its distribution around gene promoters sets a barrier for transcriptional enhancers or inhibitor proteins binding to their target sequences. As a result, an additional level of regulation is added to the signals that organize the access to the chromatin and its structural components. The tumor suppressor gene RASSF1A is a microtubule-associated and multitasking scaffold protein communicating with the RAS pathway, estrogen receptor signaling, and Hippo pathway. RASSF1A action stimulates mitotic arrest, DNA repair and apoptosis, and controls the cell cycle and cell migration. De novo methylation of the RASSF1A promoter has received much attention due to its increased frequency in most cancer types. RASSF1A methylation is preceded by histones modifications and could represent an early molecular event in cell transformation. Accordingly, RASSF1A methylation is proposed as an epigenetic candidate marker in many cancer types, even though an inverse correlation of methylation and expression remains to be fully ascertained. Some findings indicate that the epigenetic abrogation of RASSF1A can promote the alternative expression of the putative oncogenic isoform RASSF1C. Understanding the complexity and significance of RASSF1A methylation is instrumental for a more accurate determination of its biological and clinical role. The review covers the molecular events implicated in RASSF1A methylation and gene silencing and provides a deeper view into the significance of the RASSF1A methylation patterns in a number of gastrointestinal cancer types.

Runx2 stimulates neoangiogenesis through the Runt domain in melanoma.

Runx2 is a transcription factor involved in melanoma cell migration and proliferation. Here, we extended the analysis of Runt domain of Runx2 in melanoma cells to deepen understanding of the underlying mechanisms. By the CRISPR/Cas9 system we generated the Runt KO melanoma cells 3G8. Interestingly, the proteome analysis showed a specific protein signature of 3G8 cells related to apoptosis and migration, and pointed out the involvement of Runt domain in the neoangiogenesis process. Among the proteins implicated in angiogenesis we identified fatty acid synthase, chloride intracellular channel protein-4, heat shock protein beta-1, Rho guanine nucleotide exchange factor 1, D-3-phosphoglycerate dehydrogenase, myosin-1c and caveolin-1. Upon querying the TCGA provisional database for melanoma, the genes related to these proteins were found altered in 51.36% of total patients. In addition, VEGF gene expression was reduced in 3G8 as compared to A375 cells; and HUVEC co-cultured with 3G8 cells expressed lower levels of CD105 and CD31 neoangiogenetic markers. Furthermore, the tube formation assay revealed down-regulation of capillary-like structures in HUVEC co-cultured with 3G8 in comparison to those with A375 cells. These findings provide new insight into Runx2 molecular details which can be crucial to possibly propose it as an oncotarget of melanoma.

New Insights into the Runt Domain of RUNX2 in Melanoma Cell Proliferation and Migration.

The mortality rate for malignant melanoma (MM) is very high, since it is highly invasive and resistant to chemotherapeutic treatments. The modulation of some transcription factors affects cellular processes in MM. In particular, a higher expression of the osteogenic master gene RUNX2 has been reported in melanoma cells, compared to normal melanocytes. By analyzing public databases for recurrent RUNX2 genetic and epigenetic modifications in melanoma, we found that the most common RUNX2 genetic alteration that exists in transcription upregulation is, followed by genomic amplification, nucleotide substitution and multiple changes. Additionally, altered RUNX2 is involved in unchecked pathways promoting tumor progression, Epithelial Mesenchymal Transition (EMT), and metastasis. In order to investigate further the role of RUNX2 in melanoma development and to identify a therapeutic target, we applied the CRISPR/Cas9 technique to explore the role of the RUNT domain of RUNX2 in a melanoma cell line. RUNT-deleted cells showed reduced proliferation, increased apoptosis, and reduced EMT features, suggesting the involvement of the RUNT domain in different pathways. In addition, del-RUNT cells showed a downregulation of genes involved in migration ability. In an in vivo zebrafish model, we observed that wild-type melanoma cells migrated in 81% of transplanted fishes, while del-RUNT cells migrated in 58%. All these findings strongly suggest the involvement of the RUNT domain in melanoma metastasis and cell migration and indicate RUNX2 as a prospective target in MM therapy.

An integrated approach identifies new oncotargets in melanoma.

Melanoma is an aggressive skin cancer; an early detection of the primary tumor may improve its prognosis. Despite many genes have been shown to be involved in melanoma, the full framework of melanoma transformation has not been completely explored. The characterization of pathways involved in tumor restraint in in vitro models may help to identify oncotarget genes. We therefore aimed to probe novel oncotargets through an integrated approach involving proteomic, gene expression and bioinformatic analysis We investigated molecular modulations in melanoma cells treated with ascorbic acid, which is known to inhibit cancer growth at high concentrations. For this purpose a proteomic approach was applied. A deeper insight into ascorbic acid anticancer activity was achieved; the discovery of deregulated processes suggested further biomarkers. In addition, we evaluated the expression of identified genes as well as the migration ability in several melanoma cell lines. Data obtained by a multidisciplinary approach demonstrated the involvement of Enolase 1 (ENO1), Parkinsonism-associated deglycase (PARK7), Prostaglansin E synthase 3 (PTGES3), Nucleophosmin (NPM1), Stathmin 1 (STMN1) genes in cell transformation and identified Single stranded DNA binding protein 1 (SSBP1) as a possible onco-suppressor in melanoma cancer.

The curious case of Gαs gain-of-function in neoplasia.

BACKGROUND: Mutations activating the α subunit of heterotrimeric Gs protein are associated with a number of highly specific pathological molecular phenotypes. One of the best characterized is the McCune Albright syndrome. The disease presents with an increased incidence of neoplasias in specific tissues. MAIN BODY: A similar repertoire of neoplasms can develop whether mutations occur spontaneously in somatic tissues during fetal development or after birth. Glands are the most "permissive" tissues, recently found to include the entire gastrointestinal tract. High frequency of activating Gαs mutations is associated with precise diagnoses (e.g., IPMN, Pyloric gland adenoma, pituitary toxic adenoma). Typically, most neoplastic lesions, from thyroid to pancreas, remain well differentiated but may be a precursor to aggressive cancer. CONCLUSIONS: Here we propose the possibility that gain-of-function mutations of Gαs interfere with signals in the microenvironment of permissive tissues and lead to a transversal neoplastic phenotype.

Immunomodulation after radiofrequency ablation of locally advanced pancreatic cancer by monitoring the immune response in 10 patients.

OBJECTIVE/BACKGROUND: RFA of pancreatic cancer has been demonstrated to be feasible and safe with a positive impact on survival. The aim was to investigate whether an immune reaction is activated after locally advanced pancreatic cancer (LAPC) ablation. METHODS: Peripheral Blood samples were obtained preoperatively and on post-operative days 3-30. Evaluated parameters were: cells [CD4+, CD8+ and activated subsets, T-Reg, Monocytes, myeloid and plasmocytoid Dendritic cells (mDC and pDC)] and cytokines [Interleukin (IL)-6, Stromal-cells derived factor (SDF)-1, IL-1ß, Tumour-Necrosis Factor (TNF)-α, Interferon (IFN)-γ, Vascular Endothelial Growth Factor (VEGF), chemokine (C-C motif) ligand 5 (CCL-5), Transforming-Growth Factor (TGF)-ß]. RESULTS: Ten patients were enrolled. CD4+, CD8+ and TEM increased from day 3 suggesting the activation of the adaptive response. Immunosuppressive T-Reg cells were stable despite the possibility that laparotomy and heating might favour their expansion. Myeloid DCs, that present tumour-associated antigens, increased at day 30. RFA dramatically increased circulating IL-6 at day 3 but this decreased to baseline by day 30, consistent with the supposed anti-tumour effect. RFA did not significantly modulate essential chemokines, such as CCL-5 and SDF1, VEGF, TGF-ß and TNF-α, that favour tumour-growth by sustaining cancer angiogenesis and fuelling tumour-associated inflammation. CONCLUSIONS: This study provides the first evidence of RFA-based immunomodulation in LAPC. We observed a general activation of adaptive response along with a decrease of immunosuppression. Furthermore, most cells showed prolonged activation some weeks after the procedure, suggesting true immunomodulation rather than a normal inflammatory response.

Pleiotropic effects of sphingosine-1-phosphate signaling to control human chorionic mesenchymal stem cell physiology.

Chorionic stem cells represent a promising opportunity for regenerative medicine. A deeper understanding of the stimuli that regulate their physiology, could lead to innovative clinical approaches. We revealed the presence of multiple sphingosine-1-phosphate (S1P) receptor isoforms in chorion-derived mesenchymal stem cells (CMSCs). Their activation simultaneously propagated from the plasma membrane through Gi and other heterotrimeric G proteins and further diverged toward extracellular-signal-regulated kinase 1/2 (ERK1/2), p38 and protein kinase D 1. At a functional level, S1P signaling inhibited CMSC migration, while promoting proliferation. Instead, a reduction of cell density was obtained when S1P was combined to treatments that increased cAMP intracellular concentration. Such surprising reduction of cell viability was relatively specific as it was not observed with stromal stem cells from bone marrow. Neither it was observed by activating analogous G proteins with bradykinin nor by inducing cell death via a cAMP-independent pathway. S1P could thus reveal novel keys to improve CMSC differentiation programs acting on cAMP concentration. Furthermore, S1P receptor agonists/antagonists could become instrumental in favoring CMSC engraftment by controlling cell motility.

Characterization and Expression of Sphingosine 1-Phosphate Receptors in Human and Rat Heart.

Aim: Sphingosine 1-phosphate (S1P), sphingolipid derivatives are known anti-inflammatory, anti-apoptotic, and anti-oxidant agent. S1P have been demonstrated to have a role in the cardiovascular system. The purpose of this study was to understand the precise expression and distribution of S1P receptors (S1PRs) in human and rat cardiovascular tissues to know the significance and possible implementation of our experimental studies in rat models. Methods and Results: In this study, we investigated the localization of S1PRs in human heart samples from cardiac surgery department, University of Verona Hospital and rat samples. Immunohistochemical investigation of paraffin-embedded sections illustrated diffused staining of the myocardial samples from human and rat. The signals of the human heart were similar to those of the rat heart in all chambers of the heart. The immunohistochemical expression levels correlated well with the results of RT-PCR-based analysis and western blotting. We confirmed by all techniques that S1PR1 expressed strongly as compared to S1PR3, and are uniformly distributed in all chambers of the heart with no significant difference in human and rat myocardial tissue. S1PR2 expression was significantly weak while S1PR4 and S1PR5 were not detectable in RT-PCR results in both human and rat heart. Conclusion: These results indicate that experimental studies using S1PR agonists on rat models are more likely to have a potential for translation into clinical studies, and second important information revealed by this study is, S1P receptor agonist can be used for cardioprotection in global ischemia-reperfusion injury.

Runx2 downregulation, migration and proliferation inhibition in melanoma cells treated with BEL ß-trefoil.

Malignant melanoma is a lethal form of skin cancer and highly metastatic tumor with poor prognosis; BEL ß-trefoil, a lectin, obtained by our group, possesses the ability to act specifically on malignant cells. Therefore, the aim of our study was to investigate the effects of BEL ß-trefoil in melanoma cells in an attempt to evaluate its potential usage as anticancer agent. BEL ß-trefoil was purified by chromatography and A375 and MeWo melanoma cells were treated. Viability and proliferation were evaluated as well as apoptosis, RUNX2 gene expression and migration ability. The treated tumor cells decreased viability as well as proliferative ability. Flow cytometry analysis showed a lessen effect of the treatment on apoptosis. The gene expression analysis showed a reduction of RUNX2 expression in a dose-dependent manner and migration ability was reduced significantly in both treated cell lines. Our findings suggest that BEL ß-trefoil can be considered a useful tool against malignancy due to its effect based on the simultaneous proliferation ability reduction as well as the inhibition of migration capacity on melanoma tumor cells.