Effects of nutrients, mainly from mediterranean dietary foods, on mesenchymal stem derived cells: growth or differentiation

During the last decade the interest for the mesenchymal cells is growing due to their possible uses in therapies to treat certain degenerative pathologies. Mesenchymal stem cells have been found in the bone marrow and they have been shown to be responsible for bone repair and fat cells production. Mesenchymal stromal cells can be obtained from a wide variety of tissues in addition to bone marrow and can differentiate into many other cell types. The study of cell differentiation and programming provides new models for drug discovery and cell therapy that now overcomes gene therapy. Senescence, cancer development and degenerative diseases depend on mesenchymal cells contribution to tissue homeostasis. On the other hand, diet and life style are included among risk factors, which can contribute to the success of pharmacological treatments. This review focuses on nutrients from Mediterranean diet and supplements, which have been shown to influence mesenchymal stem cells and cells derived from them. Dietary intake of nutrients impairs both in vitro and in vivo observations, this review aims to gather the results about the effects of food compounds on mesenchymal cells from which adipocytes and osteoblasts derive. Amino acids and proteins, carbohydrates, lipids, fatty acids and vegetable secondary metabolites, differently act on mesenchymal cells bearing on modulation of gene expression and controlling the fate of cell lineages. Remarkable, the analysis of literature shows that the main effect of nutrients on mesenchymal cells is the stimulation of transcription factors which address the cells toward proliferation or differentiation. For instance, carbohydrates, simple or complex, and lipids appear to stimulate the PPAR receptors, whereas proteins and amino acids result to act on the mTOR system and they can also stimulate the MyoD-1 transcription factor and cooperating proteins. In conclusion, nutrients can promote cell growth and differentiation of mesenchymal cells

The induction of Maspin expression by a glucosamine-derivative has an antiproliferative activity in prostate cancer cell lines

Mammary serine protease inhibitor or Maspin has been characterized as a class II tumor suppressor gene in several cancer types, among them prostate cancer (CaP). Androgen ablation is an effective therapy for CaP, but with short-term effectiveness, thus new therapeutic strategies are actively sought. The present study is aimed to explore the effects of a glucosamine derivative, 2-(N-Carbobenzyloxy)L-phenylalanylamido-2-deoxy-β-D-glucose (NCPA), on two CaP cell lines, PC3 and LNCaP. In particular we analyzed the impact of NCPA on Maspin production, cell viability and cell cycle progression and apoptosis/necrosis pathway activation has been determined in PC3 and LNCaP cell lines. NCPA is able to stimulate Maspin production in PC3 and not in LNCaP cell lines. NCPA blocks the PC3 cell cycle in G1 phase, by inhibiting Cyclin D1 production and induces the apoptosis, therefore interfering with aggressiveness of this androgen-insensitive cell line. Moreover, NCPA is able to induce the expression of Maspin in LNCaP cell line treated with androgen receptor inhibitor, Bicalutamide, and in turn to stimulate the apoptosis of these cells. These findings suggest that NCPA, stimulating the endogenous production of a tumor suppressor protein, could be useful in the design of new therapeutic strategies for treatment of CaP

Biochemical and computational studies of the interaction between a glucosamine derivative, NAPA, and the IKKα kinase

The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose (NAPA), was shown to inhibit the kinase activity of IKKα, one of the two catalytic subunits of IKK complex, decreasing the inflammatory status in osteoarthritis chondrocytes. In the present work we have investigated the inhibition mechanism of IKKα by NAPA by combining computational simulations, in vitro assays and Mass Spectrometry (MS) technique. The kinase in vitro assay was conducted using a recombinant IKKα and IKKtide, a 20 amino acid peptide substrate derived from IkBα kinase protein and containing the serine residues Ser32 and Ser36. Phosphorylated peptide production was measured by Ultra Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS), and the atomic interaction between IKKα and NAPA has been studied by molecular docking and Molecular Dynamics (MD) approaches. Here we report that NAPA was able to inhibit the IKKα kinase activity with an IC50 of 0.5 mM, to decrease the Km value from 0.337 mM to 0.402 mM and the Vmax from 0.0257 mM·min-1 to 0.0076 mM·min-1. The computational analyses indicate the region between the KD, ULD and SDD domains of IKKα as the optimal binding site explored by NAPA. Biochemical data indicate that there is a non-significant difference between Km and Ki whereas there is a statistically significant difference between the two Vmax values. This evidence, combined with computational results, consistently indicates that the inhibition is non-competitive, and that the NAPA binding site is different than that of ATP or IKKtide

Platelet rich fibrin (PRF) and its related products: biomolecular characterization of the liquid fibrinogen

Liquid fibrinogen is an injectable platelet concentrate rich in platelets, leukocytes, and fibrinogen obtained by blood centrifugation. The aim of this study was to analyze the release of different growth factors in the liquid fibrinogen at different times and to assess possible correlations between growth factors and cell counts. The concentration of transforming growth factor beta 1 (TGF-β1), platelet-derived growth factor-AB (PDGF-AB), platelet-derived growth factor-BB (PDGF-BB), bone morphogenetic protein 2 (BMP-2), fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor (VEGF) released by liquid fibrinogen were examined with ELISA at three time points (T0, time of collection; T7, 7 days; T14, 14 days). The cellular content of the liquid fibrinogen and whole blood was also calculated for each volunteer. A mean accumulation of platelets of almost 1.5-fold in liquid fibrinogen compared to whole blood samples was found. An increase of TGF-β1, PDGF-AB, FGF-2, and VEGF levels was detected at T7. At T14, the level of TGF-β1 returned to T0 level; PDGF-AB amount remained high; the levels of FGF-2 and VEGF decreased with respect to T7, but remained higher than the T0 levels; PDGF-BB was high at all time points; BMP-2 level was low and remained constant at all time points. TGF-β1, PDGF-AB, and PDGF-BB showed a correlation with platelet amount, whereas BMP-2, FGF-2, and VEGF showed a mild correlation with platelet amount. Due to the high concentration of platelets, liquid fibrinogen does contain important growth factors for the regeneration of both soft and hard tissue. The centrifugation protocol tested in this study provides a valid solution to stimulate wound healing in oral and periodontal surgery

A glucosamine derivative affects metastatic activity in two prostate cancer cell lines by stimulating Maspin expression

Prostate Cancer (CaP) is the most common male tumor and is the third leading cause of cancer death, with an incidence of 1.28 million cases worldwide, according to the data collected by the Global Cancer Observatory in 2018. PC3 and LNCaP cell lines represent suitable models to study CaP development, due to their different metastatic origin and their distinct sensitiveness to androgen signaling. PC3 is a hormone-insensitive cell line isolated from a vertebral metastatic prostatic tumor. It lacks of androgen receptor (AR) and its abnormal growth could be attributed to enhanced expression levels of TGF-α, EGF, and EGF-R. LNCaP cell line was isolated from a human metastatic prostate adenocarcinoma found in a lymphnode. It expresses a T877A mutated AR form, which results in an enhanced binding affinity for several steroid compounds. Both cell lines present very low basal levels of Maspin expression. Maspin, an unusual member of the Serine Proteases superfamily, has been characterized as a class II tumor suppressor gene in many cancer types, among them CaP, due to its ability to inhibit cell invasiveness and proliferation and to increase apoptosis, thus inhibiting metastasis. In normal prostate epithelial cells, Maspin is highly expressed, whereas in prostate cancer cells its expression is almost completely suppressed. Previously in our laboratory, a glucosamine derivative, NCPA, has been proved to be effective in stimulating Maspin expression and to induce its nuclear localization in an osteosarcoma cell line, 143B. The aim of my PhD project was to evaluate the ability of NCPA to affect metastatic activity in two prostate cancer cell lines, the hormone-insensitive PC3 and the hormone-sensitive LNCaP cells, respectively representative of late-stage and early-stage CaP

Surface functionalized PLLA for scaffold preparation

Biocompatible and degradable poly(alpha-hydroxy acids) are among the more widely used materials in scaffolds for tissue engineering, although they often need surface modification to improve their interaction with the cells. In the present research Poly(L-lactide) 3D scaffold were prepared by salt-leaching method, with a porosity of 80 % and interconnected pores. In order to increase the hydrophilicity of the PLLA scaffolds surface, taurine was grafted through aminolysis reaction. The reaction enriched the surface with sulfonate groups increasing PLLA hydrophilicity and electrostatic interaction with collagen. In vitro biological tests with chondrocytes or fibroblasts showed that taurine grafting and collagen absorption improved cell viability and adhesion compared to the unmodified scaffold, suggesting that these modifications make PLLA substrate suitable for cartilage repair

Nanostructured TiC Layer: a suitable surface for osteointegration

In recent years, the number of patients undergoing arthroplasty surgery for joint problems, such as osteoarthritis and accidental fractures, has grown considerably. Wide research has been conducted to study the possible use of biomaterials in orthopedic surgery that would provide bone fixation or able to induce new bone tissue formation and osteointegration. Titanium is the gold standard material used for permanent implants in contact with bone, thanks to its biocompatibility, resistance to corrosion and mechanical properties. In our laboratory, nanostructured titanium-derivative surfaces have been analysed with the aim to find a surface with the best osseointegration features. Titanium carbide (TiC) layer was produced by IPPA deposition directly on glass slides, obtaining surfaces with 25% light transmittance ability. This feature allows to perform several kinds of experiments on cells, retaining the good characteristics of nanostructured titanium. We studied the adhesion, proliferation, and morphology of cells on nanostructured TiC surfaces, comparing them to both cell-culture-treated polystyrene dishes and poly-d-lysinated glass slides (poly-d-Lys). For a more reliable investigation, we chose to use human primary cells, isolated from patients undergoing arthroplasty surgery. Three different types of cells were studied, dermal fibroblasts (FBs), human osteoblasts (hOBs) and human chondrocytes (HPCs). To study the effect of the different surfaces on cell adhesion and morphology, an immunofluorescence experiment was performed evaluating the actin filament organization. Very interestingly, the cells cultivated on TiC showed an actin structure more similar to the tissue disposition. The FBs were arranged in 3D structure, showing filaments disposed on different planes. HPCs formed a particular structure with the nucleus on one side and the cytoplasm on the other side and the hOBs showed a complex network, with a larger number of contact points among cells. These differences in adhesion are also confirmed by the Atomic Force Microscopy (AFM) images, where the cells grown on TiC substrates show well-defined actin filaments which are not evident on the membrane of cells grown on polystyrene and poly-d-Lys. Moreover, the presence of these stress fibers and the overall height of the cells over the substrate indicate that the cells have a better attachment on TiC. In order to analyse the effect of TiC surface on cellular metabolism involved factors release, an ELISA assay was performed. The amount of Fibroblast Growth Factor-2 (FGF-2), Bone Morphogenetic Protein-2 (BMP-2) and Osteocalcin (OC) was measured in cell culture medium of FBs, HPCs and hOBs, respectively. FBs cultivated on TiC produced a higher amount of FGF-2 compared to polystyrene and poly-d-Lys, while HPCs cultured on poly-d-Lys and TiC produced a higher amount of BMP-2 compared to polystyrene. Osteocalcin is used as a serum marker of bone formation and as indicator of the proliferative and differentiated state of osteoblasts in cell culture. In hOBs cultivated on TiC a higher amount of OC compared to polystyrene and poly-d-Lys was obtained. Considering the involvement of FBs, HPCs and hOBs in the production of extracellular matrix components in the in vivo tissues, the effect of TiC surface on Collagen protein expression was evaluate by immunofluorescence. In order to allow cells to produce a detectable amount of collagens, cells were cultivated for seven days before analysis. Collagen type I in hFBs and hOBs, and Collagen type II in HPCs resulted increased in cells seeded on TiC compared to the other two substrates. All these results suggest how TiC is an excellent additional layer to cell culture and that can be considered as a biomaterial useful for in vivo osteointegration. [1] Lopreiato M., Mariano A. and Cocchiola R., Condensed matter, 2020, 5, 29

Effects of a glucosamine-derivative on prostate cancer cell line PC3

Prostate Cancer (CaP) is the most common form of male tumor and is the second leading cause of cancer death. Androgen ablation has proved to be an effective therapy for metastatic prostate cancer, but the regression of metastatic lesions lasts only 18 to 24 months. Mammary serine protease inhibitor or Maspin is a 42 kDa, a non-inhibitory member of the serine protease inhibitor superfamily, and has been characterized as a class II tumor suppressor gene in several cancer types, among them prostate cancer (CaP), due to its ability to inhibit metastasis. In normal prostate epithelial cells, Maspin is highly expressed whereas in prostate cancer cell lines its expression is almost completely suppressed. Previously, it has been demonstrated that NCPA, a glucosamine-derivative synthetized in our laboratory, was able to inhibit IKKα nuclear translocation and to stimulate the production and nuclear localization of Maspin in an osteosarcoma cell line: 143B. IKKα, one of two catalytic subunits of NF-B transcriptional factors, enhances tumor promotion by repressing, among other mechanisms, maspin promoter. Tumor-suppressing and anti-metastatic activities of Maspin have been attributed to its ability to inhibit both invasiveness and cell cycle progression and to stimulate apoptosis of tumor cells. Aim of this presentation is to analyze the ability of NCPA to affect metastatic and proliferation activity of PC3, which is an androgen-insensitive prostate cancer cell line

Glucosamine and its peptidyl-derivative NAPA: novel therapeutic strategy for chondrocytes matrix remodeling

Cartilage degradation, due to an imbalance between anabolic and catabolic rate of chondrocyte metabolism, is the main feature of Osteoarthritis (OA). To date, OA is mainly treated with Non Steroidal Anti-Inflammatory Drugs (NSAIDs), in order to reduce arthritis-related symptoms. In the last decades an increasing number of patients have started to use supplements, such as Glucosamine (GlcN) and chondroitin sulfate, as potential chondroprotective agents. Several in vivo clinical trials as well as in vitro experiments have been performed reporting inconsistent outcomes. Previously, in our lab we analyzed the anabolic effects of GlcN and its N-acetyl-phenylalanine derivative (NAPA) in a rabbit OA model, finding that intra-articular administration of GlcN and NAPA was very effective in reducing cartilage changes in injured rabbit knee. GlcN and NAPA intra-articular administration allows higher concentrations to be reached in the joints compared to oral administration, thus providing an explanation for the ability of both molecules to interfere with OA progression. We also studied the effects of GlcN and NAPA on inflammatory pathways, finding that both molecules can interfere with MAP kinase and NF-kB pathways, by interfering with IKK activity. Finally, we studied the effectiveness of GlcN and NAPA one the biosynthetic activity and hence the matrix production of human primary chondrocytes cultured in micromasses, which represent a good tridimensional culture model. We explored the ability of GlcN and NAPA to stimulate the synthesis of collagen type II (Coll II), Aggrecan (ACAN) and Small Leucine-Rich Proteoglycans (SLRPs). After 6 weeks, micromasses stimulated with GlcN + NAPA still showed a large amount of ECM compared to untreated cells. Moreover, Collagen type II was more abundant and better organized compared to that produced by untreated cells. Finally, cells resulted viable in both treated and untreated micromasses, even if in the middle of untreated micromasses, few dead cells were observed, whereas in the treated micromasses only viable cells and cells completely surrounded by ECM were detected

NAPA, a glucosamine derivative: biochemical analysis of interaction with IKKα Kinase

The IKB kinase (IKK) complex, involved in several cellular pathways, comprises two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, NEMO. Globally, the organization of the two kinases is similar, even if significant differences can be observed. IKKβ, associated with NEMO, activates the canonical pathway of NF-κB, while IKKα mainly activates the non-canonical pathway. NAPA, 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-b-D-glucose, is a glucosamine derivative synthetized and tested in vitro in our lab on human primary chondrocytes isolated from cartilage of patients with Osteoarthritis (OA) the most common inflammatory joints disease. The NF-κB kinases have a prominent role in the activation of inflammatory processes in OA such as the stimulation of several molecules, matrix metalloproteinases (MMPs) and transcriptional factors. Considering that the IKKα kinase activity resulted inhibited by NAPA, as shown in our previous work, the aim of this study is to explore the interaction between NAPA and IKKα through in vitro kinase assay. The IKKα enzymatic activity was studied performing an in vitro assay using a recombinant IKKα kinase and a synthetic peptide, IKKtide, containing the two serine residues. Interestingly, in relation of these reaction conditions, IKKα was able to phosphorylate the IKKtide preferable on one serine only, as pIKKtide was largely exceeding the ppIKKtide amount revealed by UPLC/MS. To evaluate the inhibitory effect of NAPA on the IKKtide phosphorylation, different concentrations of NAPA were incubated with IKKα, ATP and IKKtide, as substrate. The NAPA IC50 was found to be 0.5 ± 0.086 mM. that could seem high, anyway, it has to be considered that a strong inhibition of IKKα could result in a detrimental effect for cells and could display side effects in humans. To verify if NAPA inhibited IKKα interacting with the active site of the kinase, the assay was performed using different amount of ATP, but all these concentrations were unable to revert the inhibition of NAPA, suggesting that NAPA did not interact with the ATP binding site of IKKα. To confirm this hypothesis, a kinetic assay was performed using a fixed amount of enzyme and increasing concentrations of substrate, IKKtide. The same experiment was conducted in presence of different concentrations of NAPA. The difference between Km and Ki obtained resulted not statistically significant, in contrast, the difference between the Vmax values obtained in absence or in the presence of NAPA strongly suggested that the inhibition could be non-competitive. It can be concluded that this inhibition of IKKα kinase activity makes NAPA a very appealing molecule considering that phosphorylation has a central role in biological regulation of intracellular pathways. Our findings demonstrated that NAPA does not bind to the ATP binding site and made this molecule extremely interesting and particularly suitable for long-term treatments, such as those required for OA