Anna Scotto d'Abusco

Glucosamine affects intracellular signalling through inhibition of mitogen-activated protein kinase phosphorylation in human chondrocytes

The aim of this study was to determine the effects of glucosamine on matrix metalloprotease (MMP) production, on mitogen-activated protein kinase (MAPK) phosphorylation, and on activator protein (AP)-1 transcription factor activation in human chondrocytes. The human immortalized cell line lbpva55 and healthy human chondrocytes (obtained from healthy donors) were subjected to challenge with 10 ng/ml IL-1β after pretreatment with 2.5 or 10 mmol/l glucosamine. MMP mRNA expression levels were evaluated using quantitative real-time PCR, and MMP protein production levels were evaluated in the culture supernatant using ELISA. MAPK phosphorylation was evaluated using Western blotting. AP-1 transcription factor activation was evaluated by measuring AP-1 DNA-binding activity. After IL-1β stimulation, levels of MMP-1, MMP-3 and MMP-13 production were markedly increased. Treatment with 2.5 and 10 mmol/l glucosamine reduced expression of these metalloproteases. MMP expression is regulated by transcription factors such as the AP-1 complex, which is activated by phosphorylated MAPKs. IL-1β stimulated phosphorylation of c-jun amino-terminal kinase, p38 MAPK and extracellular signal-regulated kinase-1/2. Glucosamine inhibited c-jun amino-terminal kinase and p38 phosphorylation, and consequently c-jun binding activity. These findings demonstrate, for the first time, that glucosamine inhibits IL-1β-stimulated MMP production in human chondrocytes by affecting MAPK phosphorylation.

DNA sequences acting as binding sites for NM23/NDPK proteins in melanoma M14 cells

We isolated and analyzed by chromatin immunoprecipitation (ChIP) in viable M14 cells DNA sequences bound to the antimetastatic protein nucleoside diphosphate kinase (NM23/NDPK) to shed some light on the nuclear functions of this protein and on the mechanism by which it acts in development and cancer. We assessed the presence of selected sequences from promoters of platelet‐derived growth factor A (PDGF‐A), c‐myc, myeloperoxidase (MPO), CD11b, p53, WT1, CCR5, ING1, and NM23‐H1 genes in the cross‐linked complexes. Quantitative PCR (Q‐PCR) showed a substantial enrichment of the correlated oncosuppressor genes p53, WT1, ING1, and NM23‐H1 in the immunoprecipitated (IP) DNA. This suggests that NM23/NDPK binding is involved in the transcription regulation of these genes. These results reveal new interactions that should help us to disclose the antimetastatic mechanism of NM23. J. Cell. Biochem. 98: 421–428, 2006.

A peptidyl-glucosamine derivative affects IKKα kinase activity in human chondrocytes

IntroductionNuclear factor-κB (NF-κB) transcription factor regulates several cell signaling pathways, such as differentiation and inflammation, which are both altered in osteoarthritis. Inhibitor κB kinase (IKK)α and IKKβ are kinases involved in the activation of the NF-κB transcription factor. The aim of the present study was to determine the effects of glucosamine (GlcN), which is administered in the treatment of osteoarthritis, and of its 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose (NAPA) derivative on IKK kinases and, consequently, on NF-κB activation in human chondrocytes.MethodsThe human chondrosarcoma cell line HTB-94 and human primary chondrocytes were stimulated with tumor necrosis factor (TNF)α after pre-treatment with GlcN or NAPA. Gene mRNA expression level was evaluated by real-time PCR. Inhibitor κB protein (IκB)α phosphorylation and p65 nuclear re-localization were analyzed by Western blotting; IKKα nuclear re-localization was also investigated by immunocytochemistry and Western blotting. IKK kinase activity was studied by in vitro kinase assay.ResultsAfter TNFα stimulation, the mRNA expression level of some of the genes under NF-κB control, such as interleukin (IL)-6 and IL-8, increased, while treatment with GlcN and NAPA reverted the effect. We investigated the possibility that GlcN and NAPA inhibit IKK kinase activity and found that NAPA inhibits the IKKα kinase activity, whereas GlcN does not. Interestingly, both GlcN and NAPA inhibit IKKα nuclear re-localization.ConclusionsOur results demonstrate that glucosamine and its peptidyl derivative can interfere with NF-κB signaling pathway by inhibiting IKKα activity in human chondrocytes. However, the mechanism of action of the two molecules is not completely overlapping. While NAPA can both specifically inhibit the IKKα kinase activity and IKKα nuclear re-localization, GlcN only acts on IKKα nuclear re-localization.

Osseointegration is improved by coating titanium implants with a nanostructured thin film with titanium carbide and titanium oxides clustered around graphitic carbon

Titanium implants coated with a 500nm nanostructured layer, deposited by the Ion Plating Plasma Assisted (IPPA) technology, composed of 60% graphitic carbon, 25% titanium oxides and 15% titanium carbide were implanted into rabbit femurs whilst into the controlateral femurs uncoated titanium implants were inserted as control. At four time points the animals were injected with calcein green, xylenol orange, oxytetracycline and alizarin. After 2, 4 and 8weeks femurs were removed and processed for histology and static and dynamic histomorphometry for undecalcified bone processing into methylmethacrylate, sectioned, thinned, polished and stained with Toluidine blue and Fast green. The overall bone-implant contacts rate (percentage of bone-implant contacts/weeks) of the TiC coated implant was 1.6 fold than that of the uncoated titanium implant. The histomorphometric analyses confirmed the histological evaluations. More precisely, higher Mineral Apposition Rate (MAR, μm/day) (p<0.005) and Bone Formation Rate (BFR, μm2/μm/day) (p<0.0005) as well as Bone Implant Contact (Bic) and Bone Ingrowth values (p<0.0005) were observed for the TiC coated implants compared to uncoated implants. In conclusion the hard nanostructured TiC layer protects the bulk titanium implant against the harsh conditions of biological tissues and in the same time, stimulating adhesion, proliferation and activity of osteoblasts, induces a better bone-implant contacts of the implant compared to the uncoated titanium implant.

IKKα inibition by a glucosamine derivative enhances Maspin expression in osteosarcoma cell line

Chronic inflammation has been associated to cancer development by the alteration of several inflammatory pathways, such as Nuclear Factor-κB pathway. In particular, IκB kinase α (IKKα), one of two catalytic subunit of IKK complex, has been described to be associated to cancer progression and metastasis in a number of cancers. The molecular mechanism by which IKKα affects cancer progression is not yet completely clarified, anyway an association between IKKα and the expression of Maspin (Mammary Serine Protease Inhibitor or SerpinB5), a tumor suppressor protein, has been described. IKKα shuttles between cytoplasm and nucleus, and when is localized into the nuclei, IKKα regulates the expression of several genes, among them Maspin gene, whose expression is repressed by high amount of nuclear IKKα. Considering that high levels of Maspin have been associated with reduced metastatic progression, it could be hypothesized that the repression of IKKα nuclear translocation could be associated with the repression of metastatic phenotype. The present study is aimed to explore the ability of a glucosamine derivative, 2-(N-Carbobenzyloxy)l-phenylalanylamido-2-deoxy-β-d-glucose (NCPA), synthesized in our laboratory, to stimulate the production of Maspin in an osteosarcoma cell line, 143B. Immunofluorescence and Western blotting experiments showed that NCPA is able to inhibit IKKα nuclear translocation, and to stimulate Maspin production. Moreover, in association with stimulation of Maspin production we found the decrease of β1 Integrin expression, the down-regulation of metalloproteases MMP-9 and MMP-13 production and cell migration inhibition. Taking in account that β1 Integrin and MMP-9 and -13 have been correlated with the invasiveness of osteosarcoma, considering that NCPA affects the invasiveness of 143B cell line, we suggest that this molecule could affect the osteosarcoma metastatic ability.

Modulatory Effects of a Nutraceutical Supplement on Saos-2 Cells Reveal Its Phlebotonic Activity

ABSTRACT Objective: Herbal extract compositions are largely used to manage vein diseases. We prepared a new composition of herbs, named FLEBO OK™, that, when administered as a nutraceutical to patients affected by peripheral vascular diseases, was able to improve their health conditions. We analyzed the effects of this nutraceutical composition on in vitro cultured cells with the aim to obtain information about its mechanisms of action. Methods: A culture of human osteoblast cell line Saos-2 was stimulated with tumor necrosis factor (TNF)-α or interleukin (IL)-1β to induce the expression of some chemokines and matrix metalloproteases (MMPs). This cell culture was then exposed to the prepared composition and the amount of expression of the genes coding for the monocyte chemotactic protein (MCP)-1, IL-8, IL-1β, MMP-2, MMP-3, MMP-9 proteins was measured by real-time polymerase chain reaction (RT-PCR). The experiments were repeated exposing the cells to the same amount of the well-known micronized purified flavonoid fraction. Moreover, we describe the effects of the administration of nutraceutical composition to 20 patients affected by peripheral vascular diseases and 20 healthy individuals. Results: The RT-PCR analyses showed that the new composition induces the expression of MMP-3 and MMP-9 and downregulates MMP-2 in cell cultures stimulated with IL-1β, whereas it induces the expression of IL-8 and represses the expression of IL-1β and MCP-1 in cell cultures stimulated with TNF-α. The induction of the expression of MMP-3 and the downregulation of MCP-1 might result in an antiplatelet activity that was not observed for the micronized purified flavonoid fraction. Interviewed patients reported an improvement in their conditions after 1 month of FLEBO OK treatment. Conclusion: These findings could provide a hypothesis for the high efficiency of the identified nutraceutical composition to management of peripheral vascular diseases.

A Nutraceutical Composition Decreases CPK Levels in Saos-2 Cells and in Patients with Elevated Serum Levels of This Enzyme

Objectives: This study aimed to investigate the effects of a nutraceutical composition on the expression of some genes involved in muscle cells and functioning in osteoblast cells. The effects of nutraceutical composition have been compared to the effects of atorvastatin, which induces muscle pain and elevated creatine phosphokinase (CPK) serum level when administered to patients. In particular, we analyzed the MyoD-1 gene, which is responsible for modulation of the CPK gene, which is a marker of muscle pain and damage. Methods: The effects of nutraceutical composition on Saos-2 cells were compared with the effects of atorvastatin. The mRNAs were extracted and the expression levels of mitochondrial and cytoplasmic CPK genes and MyoD-1 were analyzed by real-time polymerase chain reaction (RT-PCR). Moreover, the effects on lactate dehydrogenase (LDH) activity and adenosine triphosphate (ATP) synthesis were measured in the osteoblast cell line. Furthermore, 11 patients with muscle pain or elevated CPK serum levels received a supplementation of the nutraceutical composition to test whether CPK levels could be downregulated. Results: The analysis in Saos-2 cells showed that the nutraceutical composition upregulates the gene expression of MyoD-1 and downregulates the expression of the cytoplasmic isoform of CPK gene expression (p ≤ 0.05); moreover, it slightly increases ATP amount and decreases LDH activity. Conversely, atorvastatin represses the expression of MyoD-1 gene without significant changing into the expression levels of both cytoplasmic and mitochondrial CPK genes. Moreover, atorvastatin does not increase the ATP amount or increase LDH activity. Remarkable, the nutraceutical composition is able to decrease CPK levels in serum of patients and in some cases improve myalgia symptoms. Conclusion: The nutraceutical composition decreases CPK levels both in vitro and in vivo, suggesting that it might be useful to management of nonneurological myalgia symptoms.

Olive Fruit Blends Modulate Lipid-Sensing Nuclear Receptor PPARγ, Cell Survival, and Oxidative Stress Response in Human Osteoblast Cells

ABSTRACT Objective: The aim of the present study was to investigate how different extravirgin olive oils (EVOOs), obtained by blending Olea europea cultivars, could influence the cell growth, the response to inflammatory stimuli, and oxidative stress in a culture of the osteosarcoma cell line Saos-2. Methods: Three different extravirgin olive oils were physicochemically characterized, determining the free acidity, the oxidation status, the polyphenols content, and the antioxidative activity. Moreover, the effects on Saos-2 cell culture were determined, studying the mRNA expression level by real-time polymerase chain reaction (PCR) assays and the antioxidative activity using fluorescent probes. Results: The cultivars used in the south of Italy, yield extravirgin oils with different amount of fatty acids and polyphenols, which counteract induction of proinflammatory cytokines and regulate free radical production in hydrogen peroxide-stimulated cells. In vitro analysis using the human osteoblast cell line Saos-2 showed that the addition of oils to cell culture simulated a hypoxic stress followed by a reoxygenation period, during which the antioxidant activity of extravirgin olive oils protected cells from oxidative damages. On the other hand, the mRNA expression levels of factors involved in inflammatory processes, cell growth recovery, and antioxidant response, as heme oxygenase-1, were differently stimulated by EVOOs. Moreover, peroxisome proliferator activated receptor γ (PPARγ) was differently modulated by EVOOs. Conclusion: These findings show that the blending of different extravirgin olive oil can impact an osteoblast cell line, in particular regarding cell growth recovery and oxidative stress.