Extracellular matrix components affect cell migration and invasive potential of cultured human pancreatic ductal adenocarcinoma cells

The tumor microenvironment influences cancer cell behavior in relation to tumor progression, as well as cell proliferation and invasion. Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense desmoplastic reaction and extracellular matrix (ECM) components in the tumor microenvironment are involved in a cross-talk between tumor cells, stromal fibroblasts and ECM components, influencing tumor cell behavior. We aimed at analyzing in vitro the effect of the crosstalk between PDAC cells and the ECM of the microenvironment by culturing PDAC cells on different ECM proteins used as a substrate, in order to better understand the relationship between cancer cell phenotype and the proteins occurring in the desmoplastic tissue. For this purpose, we analyzed some epithelial-to-mesenchymal transition (EMT) markers and the migration and invasive potential in human HPAF-II, HPAC and PL45 PDAC cells cultured on collagen type I (COL), laminin (LAM) and fibronectin (FN). Interestingly, the expression of E-cadherin was not significantly affected, but some differences were revealed by the wound healing assay. In fact, migration of HPAF-II and PL45 cells was decreased on FN and LAM, and increased on COL, compared to control cells grown on plastic (NC). By contrast, HPAC was very rapid and unaffected by the substrate. SDS-zymography showed that COL induced a strong upregulation of MMP-2 activity in HPAF-II and HPAC cells, and of MMP-9 in HPAF-II and PL45 cells, compared to NC. These preliminary results suggest that ECM components could differently affect PDAC migration and invasion, possibly depending on the differentiation grade. The characterization of the mutual effects elicited by the tumor-stroma interplay on the cancer cell will contribute to better understand the influence of the stroma on PDAC cancer cell phenotype, in order to develop new therapeutic strategies

Exploitation of Tartary Buckwheat as Sustainable Ingredient for Healthy Foods Production

AbstractTartary buckwheat (Fagopyrum tataricum Gaertn) is a minor crop belonging to the Polygonaceae family that can be considered as sustainable crop thanks to its low input requirements. It is a pseudo-cereal known for its high healthy value related to antioxidant compounds present in its grains. For this reason it could be employed for the production of functional foods. This paper as well as reviewing about the agronomical and nutritional traits of buckwheat also provides the latest experimental results achieved by ENEA research activities

Characterization of an in vitro model to study the role of human Polyomavirus BK in prostate cancer

Prostate cancer (PCa) is one of the most common male neoplasm in the western world, being the most commonly diagnosed non-skin cancer and the second leading cause of cancer death. Various potential risk factors exist for the initial triggering events, including exposure to infectious agents, such as the human Polyomavirus BK (BKV). BKV is a good candidate as risk factor of PCa because it naturally infects the human reno-urinary tract, it establishes latency, and encodes oncoproteins that interfere with tumor suppressors pathways, thus altering the normal progression of cell cycle. Previous studies suggested a potential association between BKV and PCa, revealing that the prevalence of BKV was significantly higher in cancer than in control tissues, with a significant association between viral expression and cancer. However, this hypothesis is controversial because BKV is not restricted to tumor tissues but also infects healthy individuals in a high percentage. Moreover, an in vitro model of BKV infection in prostate cells is not available to understand the role for BKV in pathogenesis of PCa. Our aims were to determine whether BKV a) could infect normal epithelial prostate cells, b) affects cell phenotype and c) affects the phenotype of human prostate tumor cell line PC3. For this purpose normal epithelial prostate cell line RWPE-1 and prostate cancer cells PC3 were infected with BKV for 21 days. Cell proliferation, epithelial-to-mesenchymal markers (EMT) and invasion potential were analyzed by, respectively, MTT, immunofluorescence and SDS-zymography. Our results show that cell proliferation was increased or decreased by BKV, respectively, in RWPE-1 and PC3 cells. BKV induced E-cadherin downregulation and vimentin expression in both control and BKV-infected cells RWPE-1, suggesting that uninfected cells underwent EMT. Matrix metalloproteinase-2 and 9 activity was increased in RWPE-1 cells after BKV infection. By contrast, BKV did not significantly modified the phenotype of PC3 cells. These preliminary results suggest that normal epithelial prostate cells RWPE-1 and PC3 are susceptible and permissive to BKV infection. However, RWPE-1 cells exhibit some phenotype modifications related to EMT, possibly induced by the papilloma virus used to obtain their immortalization, thus suggesting that further experiments will be necessary to define if they represent a good experimental model to study prostate cancer

Effect of extracellular matrix components on the expression of epithelial-to-mesenchymal transition markers in cultured human pancreatic ductal adenocarcinoma cells

Epithelial-to-mesenchymal transition (EMT) is a step-wise process leading to the phenotypic switch of epithelial to mesenchymal cells, providing these cells with a metastatic phenotype. During EMT epithelial cells loose adhesion by down regulation of E-cadherin and express N-cadherin, display cytoskeleton reorganization by expressing vimentin and α-smooth muscle actin (αSMA), acquire motile properties and become invasive by secretion of matrix metalloproteinases (MMPs). Cancer cell phenotype is influenced by the tumor microenvironment in relation to tumor progression, as well as to cell proliferation and invasion. The role of the extracellular matrix (ECM) in the microenvironment is particularly relevant in pancreatic ductal adenocarcinoma (PDAC) since this carcinoma is characterized by an intense desmoplastic reaction, representing the environment where the complex interplay between tumor cells, stromal fibroblasts and ECM components occurs. We aimed at analyzing in vitro the effect of the crosstalk between PDAC cells and their microenvironment by characterizing PDAC cell phenotype in cells cultured on different ECM proteins used as a substrate, in order to better understand the relationship between cancer cell behaviour and the proteins occurring in the desmoplastic tissue. We analyzed by immunofluorescence the expression of the main EMT markers such as E-cadherin, N-cadherin, β-catenin, αSMA, vimentin and collagen type I (COL-I) in PDAC cells cultured on laminin, fibronectin, COL-I and without coating (NC). Moreover, we investigated cell proliferation and MMPs activity in cell culture supernatants by SDS-zymography. Cell morphology was similar in PDAC cells cultured on laminin, fibronectin, COL-I, and in NC, as well as the E-cadherin/β-catenin complex, αSMA and COLI expression; by contrast, vimentin was undetectable in all the experimental conditions. N-cadherin was slightly detectable in cells cultured on fibronectin, COL-I, and laminin, and at lower extent in NC cells. Cell proliferation resulted similar in NC and in cells cultured on fibronectin, decreased on laminin and increased on COL-I. MMP-9 activity exhibited a similar trend, resulting similar on fibronectin, decreased on laminin and stimulated on COL-I. These preliminary results provide new insights in the characterization of the mutual effects elicited by the tumor-stroma interplay on the cancer cell, and will contribute to better understand the influence of the stroma on PDAC cancer cell phenotype, in order to develop new therapeutic strategies

Essential role for acid sphingomyelinase-inhibited autophagy in melanoma response to cisplatin

In advanced stages, melanoma is still a therapeutic challenge, despite the large number of chemotherapeutic regimens so far developed. Single drug chemotherapy is in many cases ineffective and combinations of chemotherapeutic drugs have demonstrated response rates only marginally higher, and at the cost of systemic toxicity. The new targeted therapies and immunotherapies have shown better efficacy and have supplanted chemotherapy as first-and second-line therapy. However, since melanoma cells eventually become resistant also to these novel therapies, the quest for new, more effective and possibly less toxic approaches is still open. The sphingolipid metabolising enzyme Acid Sphingomyelinase (A-SMase) has been recently shown to inhibit melanoma progression and correlate inversely to tumour grade [1]. We have investigated the role of A-SMase in the chemo-resistance to anticancer treatment using mice with melanoma allografts and melanoma cells differing in terms of expression/activity of A-SMase. Furthermore, as autophagy is a crucial determinant of the melanoma sensitivity to chemotherapeutic drugs, we have also investigated whether an action of A-SMase in autophagy can explain its role [2]. Melanoma sensitivity to chemotherapeutic agent cisplatin in terms of cell viability/apoptosis, tumour growth, and animal survival depended directly on the A-SMase levels in tumoural cells. A-SMase action was due to inhibition of autophagy through activation of Akt/mammalian target of rapamycin (mTOR) pathway. Treatment of melanoma-bearing mice with the autophagy inhibitor chloroquine restored sensitivity to cisplatin of tumours expressing low levels of A-SMase while no additive effects were observed in tumours characterised by sustained A-SMase levels. In conclusion A-SMase, affecting mTOR-regulated autophagy and playing a central role in cisplatin efficacy, is an attractive target in anti-tumour strategy for melanomas and our data encourage pre-clinical testing of the modulation of A-SMase levels/activity as possible novel anti-neoplastic strategy

In vivo morphological evaluation of the efficacy of an hydrogel conduit as scaffold for peripheral nerve regeneration

Autologous nerve grafting is considered the gold standard for bridging nerve lesion, although in several cases the use of artificial tubular guides is needed. Nowadays, the research is addressed to identify the ideal treatment able to support complete nerve regeneration and functional recovery. Therefore, the use of artificial synthetic or natural guides is required but, despite numerous studies on several kinds of conduits, the clinicians are still waiting for an alternative scaffolds giving the best regenerative properties. In this light, we evaluated in vivo the efficacy of a polyamidoamine-based hydrogel, shaped as small tubing for nerve regeneration. Studies were done on 3 experimental groups consisting of injured guide-implanted, sham operated and autograft rats (in which sciatic nerve was excised, inverted and re-implanted). In the implanted rats the sciatic nerve was transected and 5 mm gap between proximal and distal stumps was left in the middle of the conduit. Animals were then analyzed at 1, 3, 6, months post-surgery. The gait analysis and the thermal sensitivity analysis demonstrated movement improvement and sensitive recovery. The repaired, control and autograft nerves from all groups analyzed (implanted, sham and autograft) were processed for light microscopy and morphometric analysis. The axon size, myelin thickness and fiber density showed a complete nerve regeneration covering the gap in the central part of the conduit. In particular, the medial segment of the nerve tended to turn similar to sham operated (control) animals, while the autograft rats showed a less extensive regenerative process. Moreover, immunofluorescence analysis of longitudinal sections of the conduits were performed by labeling the axonal neurofilaments. The immunopositivity observed further confirmed that the regenerative process was complete, being the fibres present through the entire conduit between the proximal and the distal stumps. Overall, our data demonstrate that polyamidoamine-based hydrogel conduits are an implantable material providing a good support for the peripheral nerve regeneration. These conduits are biocompatible and biodegradable over the time to non toxic product metabolites. Finally our findings are promising for the achievement of implantable poliamidoamine- based hydrogel conduits functionalized for drug delivery with growth factors. This work has been supported by Fondazione CARIPLO under the Project “Functional Polymeric Hydrogels for Tissue Regeneration” (no. 2010-0501), call for the Scientific Research on Advanced Materials, 2010

Epithelial-to-mesenchymal transition in pancreas adenocarcinoma cells: effect of 2D versus 3D arrangement

It was shown that three-dimensional (3D) cell cultures allow mimicking the functions of living tissues and provide pivotal information encoded in the tissue architecture [1]. Considered the primary role of epithelial-to-mesenchymal transition (EMT) in carcinoma progression [2], we aimed at investigating the effect of the 3D arrangement on the expression of some key markers of EMT in cultured human pancreas adenocarcinoma cells (PDAC). HPAC cells were cultured in both two-dimensional (2D) monolayers and in 3D spheroids, and were analyzed by morphological and molecular approaches. Immunofluorescence analysis for E-cadherin, β-catenin, actin, vimentin and collagen type I was performed on cells grown on 12 mm coverslips or on free-floating spheroids after 4% paraformaldehyde fixation. E-cadherin gene expression was assessed by real time PCR. Fluorescence and confocal microscopy analysis revealed that the E-cadherin/β-catenin complex was similarly expressed at the cell boundaries on the plasma membrane in 2D monolayers as well as in the 3D spheroids. Phalloidin-stained F-actin was mainly arranged into cortical actin filaments while vimentin was undetectable, suggesting an epithelial-like phenotype for HPAC cells in 2D and 3D arrangement. Interestingly, after 3D arrangement decreased E-cadherin mRNA levels and some cells expressing collagen type I were observed in spheroids. Our findings suggest that the 3D arrangement induced the expression of mesenchymal phenotype-related markers in HPAC cells, providing a model to better understand the biology of PDAC

Short-Term Variations in Neutrophil-to-Lymphocyte and Urea-to-Creatinine Ratios Anticipate Intensive Care Unit Admission of COVID-19 Patients in the Emergency Department

Background: Timely assessment of COVID-19 severity is crucial for the rapid provision of appropriate treatments. Definitive criteria for the early identification of severe COVID-19 cases that require intensive care unit admission are lacking. Methods: This was a single-center, retrospective case-control study of 95 consecutive adults admitted to the intensive care unit (cases) or a medical ward (controls) for laboratory-confirmed COVID-19. Clinical data were collected and changes in laboratory test results were calculated between presentation at the emergency department and admission. Univariate and multivariable logistic regression was performed to calculate odds ratios for intensive care unit admission according to changes in laboratory variables. Results: Of the 95 adults with COVID-19, 25 were admitted to intensive care and 70 to a medical ward after a median 6 h stay in the emergency department. During this interval, neutrophil counts increased in cases and decreased in controls (median, 934 vs. −295 × 106/L; P = 0.006), while lymphocyte counts decreased in cases and increased in controls (median, −184 vs. 109 × 106/L; P < 0.001). In cases, the neutrophil-to-lymphocyte ratio increased 6-fold and the urea-to-creatinine ratio increased 20-fold during the emergency department stay, but these ratios did not change in controls (P < 0.001 for both comparisons). By multivariable logistic regression, short-term increases in the neutrophil-to-lymphocyte ratio (OR = 1.43; 95% CI, 1.16–1.76) and urea-to-creatinine ratio (OR = 1.72; 95% CI, 1.20–2.66) were independent predictors of intensive care unit admission. Conclusion: Short-time changes in neutrophil-to-lymphocyte ratio and urea-to-creatinine ratio emerged as stand-alone parameters able to identify patients with aggressive disease at an early stage

Adipocyte-Derived Extracellular Vesicles Promote Prostate Cancer Cell Aggressiveness by Enabling Multiple Phenotypic and Metabolic Changes

Background: In recent decades, obesity has widely emerged as an important risk factor for prostate cancer (PCa). Adipose tissue and PCa cells have been shown to orchestrate a complex interaction network to support tumor growth and evolution; nonetheless, the study of this communication has only been focused on soluble factors, although increasing evidence highlights the key role of extracellular vesicles (EVs) in the modulation of tumor progression. Methods and Results: In the present study, we found that EVs derived from 3T3-L1 adipocytes could affect PC3 and DU145 PCa cell traits, inducing increased proliferation, migration and invasion. Furthermore, conditioning of both PCa cell lines with adipocyte-released EVs resulted in lower sensitivity to docetaxel, with reduced phosphatidylserine externalization and decreased caspase 3 and PARP cleavage. In particular, these alterations were paralleled by an Akt/HIF-1α axis-related Warburg effect, characterized by enhanced glucose consumption, lactate release and ATP production. Conclusions: Collectively, these findings demonstrate that EV-mediated crosstalk exists between adipocytes and PCa, driving tumor aggressiveness

Activity of Experimental Mouthwashes and Gels Containing DNA-RNA and Bioactive Molecules against the Oxidative Stress of Oral Soft Tissues: The Importance of Formulations. A Bioreactor-Based Reconstituted Human Oral Epithelium Model

Background: DNA-RNA compounds have shown promising protection against cell oxidative stress. This study aimed to assess the cytotoxicity, protective, or preventive effect of different experimental formulations on oral epithelia’s oxidative stress in vitro. Methods: Reconstituted human oral epithelia (RHOE) were grown air-lifted in a continuous-flow bioreactor. Mouthwashes and gels containing DNA-RNA compounds and other bioactive molecules were tested on a model of oxidative stress generated by hydrogen peroxide treatment. Epithelia viability was evaluated using a biochemical MTT-based assay and confocal microscopy; structural and ultrastructural morphology was evaluated by light microscopy and TEM. Results: DNA-RNA showed non-cytotoxic activity and effectively protected against oxidative stress, but did not help in its prevention. Gel formulations did not express adequate activity compared to the mouthwashes. Excipients played a fundamental role in enhancing or even decreasing the bioactive molecules’ effect. Conclusion: A mouthwash formulation with hydrolyzed DNA-RNA effectively protected against oxidative stress without additional enhancement by other bioactive molecules. Active compounds, such as hyaluronic acid, β-Glucan, allantoin, bisabolol, ruscogenin, and essential oils, showed a protective effect against oxidative stress, which was not synergistic with the one of DNA-RNA. Incorporation of surfactant agents showed a reduced, yet significant, cytotoxic effect