Marialucia Gallorini

Cyclin-Dependent Kinase Modulators and Cancer Therapy

The cell cycle of eukaryotic cells varies greatly from species to species and tissue to tissue. Since an erroneous control of the cell cycle can have disastrous consequences for cellular life, there are genetically programmed signals, so-called cell cycle checkpoints, which ensure that all events of each stage are completed before beginning the next phase. Among the numerous molecules involved in this process, the most important are the cyclin-dependent kinases (CDKs), proteins that are activated only when bound to cyclins (regulatory proteins with fluctuating concentrations).In general, more CDKs are overexpressed in cancer cells than in normal cells, which explains why cancer cells divide uncontrollably. Succeeding in modulating CDK activity with pharmacological agents could result in decreasing the abnormal proliferation rate of cancer cells.This review offers an overview of CDK-cyclin complexes in relation to different cell cycle phases, an analysis of CDK activation and inhibition of molecular mechanisms, and an extensive report, including clinical trials, regarding four new drugs acting as CDK modulators: alvocidib, P276-00, SNS-032 and seliciclib.

HEMA‐induced cytotoxicity: oxidative stress, genotoxicity and apoptosis

Dental resin composites consist of organic polymers with inorganic fillers used as bonding resins and direct filling materials in dentine adhesives and as sealing agents for inlays, crowns and orthodontic brackets. Despite various modifications in the formulation, the chemical composition of composite resins includes inorganic filler particles and additives, which are incorporated into a mixture of an organic resin matrix. Among them, 2-hydroxyethylmethacrylate (HEMA) is one of the most frequently used. Several studies have attempted to clarify the mechanisms underlying HEMA cytotoxicity. Most of them support the hypothesis that this compound, once released in the oral environment, increases reactive oxygen species (ROS) production and oxidative DNA damage through double-strand breaks evidenced by in vitro presence of micronuclei. As a consequence, the glutathione detoxifying intracellular pool forms adducts with HEMA through its cysteine motif and inflammation begins to occur: transcription of early genes of inflammation such as tumour necrosis factor α or inducible cyclooxygenase up to the secretion of prostaglandins 2. These phenomena are counteracted by N-acetylcysteine (NAC), a nonenzymatic antioxidant, but not by vitamin E or other antioxidant. Consequently, NAC prevents HEMA-induced apoptosis acting as a direct ROS scavenger. This minireview collects the most significant papers on HEMA and tries to make an overview of its cytotoxicity on different cell types and experimental models.

Biological Responses of Human Gingival Fibroblasts (HGFs) in an Innovative Co-Culture Model with Streptococcus mitis to Thermosets Coated with a Silver Polysaccharide Antimicrobial System

This study sought to evaluate the in vitro biological response of human gingival fibroblasts (HGFs) co-coltured with Streptococcus mitis to bisphenol A glycidylmethacrylate/triethylene glycol dimethacrylate (BisGMA/TEGDMA) thermosets coated with Chitlac-nAg, a nanocomposite system with antimicrobial properties. To avoid bacterial adhesion to dental devices and to reduce cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilizing silver nanoparticles, which have well-known antimicrobial properties, with a polyelectrolyte solution containing Chitlac. Cytotoxicity, cell morphology, cell migration and inflammatory interleukine-6 (IL-6) and prostaglandin E2 (PGE2) secretion were evaluated. Our results showed that the cytotoxicity exerted on HGFs by our nanocomposite material was absent in our co-culture model, where fibroblasts are able to adhere and migrate. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release rises up 20%. Moreover the presence of S. mitis reduced this release in a greater amount with Chitlac-nAg coated thermosets. The secretion of IL-6 was significant in both Chitlac and Chitlac-nAg coated thermosets, but PGE2 production was minimal, suggesting that the IL-6 production was not related to an inflammatory response. Co-culture and the addiction of saliva did not influence IL-6 and PGE2 secretion. Data obtained in the present work suggest that Chitlac n-Ag coated thermosets could significantly improve the success rates of restorative dentistry, since they limit bacterial adhesion and are not toxic to HGFs.

Involvement of mitochondrial signalling pathway in HGFs/S. mitis coculture response to TEGDMA treatment.

Although triethylene glycol dimethacrylate (TEGDMA), a resin monomer widely used in dental practice, has been shown to have cytotoxic effects on eukaryotic cells, little is known about how the oral environment influences the cytotoxicity of this biomaterial. The aim of this study was to evaluate eukaryotic cell reaction to TEGDMA in terms of the production of reactive oxygen species (ROS), the expression of Bax, the disturbance of mitochondrial membrane potential (MMP), and the occurrence of apoptosis in an in vitro coculture model of human gingival fibroblasts (HGFs) and Streptococcus mitis strain in presence of saliva. We found that S. mitis and saliva reduced the production of ROS (from 2.2 to 1.8 fold), the occurrence of apoptosis (from 11.3 to 4.7%), and the decrease of MMP (from 0.75 to 0.9 fold) induced by TEGDMA treatment. Addition of N-acetylcysteine, a well known antioxidant, improved cell viability in all experimental conditions. The results obtained in this study suggest that the presence of S. mitis and saliva in the periodontal environment could protect cells against TEGDMA toxicity. These results, shedding more light on the biological and molecular events that occur in conjuction with TEGDMA treatment in vitro in a coculture model that mimics the environment of the oral cavity, confirm the key role played by oral bacteria and saliva in preventing toxic events that can occur in vivo in HGFs.

Cell-protection mechanism through autophagy in HGFs/S. mitis co-culture treated with Chitlac-nAg

Silver-based products have been proven to be effective in retarding and preventing bacterial growth since ancient times. In the field of restorative dentistry, the use of silver ions/nanoparticles has been explored to counteract bacterial infections, as silver can destroy bacterial cell walls by reacting with membrane proteins. However, it is also cytotoxic towards eukaryotic cells, which are capable of internalizing nanoparticles. In this work, we investigated the biological effects of Chitlac-nAg, a colloidal system based on a modified chitosan (Chitlac), administered for 24–48 h to a co-culture of primary human gingival fibroblasts and Streptococcus mitis in the presence of saliva, developed to mimic the microenvironment of the oral cavity. We sought to determine its efficiency to combat oral hygiene-related diseases without affecting eukaryotic cells. Cytotoxicity, reactive oxygen species production, apoptosis induction, nanoparticles uptake, and lysosome and autophagosome metabolism were evaluated. In vitro results show that Chitlac-nAg does not exert cytotoxic effects on human gingival fibroblasts, which seem to survive through a homoeostasis mechanism involving autophagy. That suggests that the novel biomaterial Chitlac-nAg could be a promising tool in the field of dentistry.

Combined supplementation of ascorbic acid and thyroid hormone T3 affects tenocyte proliferation. The effect of ascorbic acid in the production of nitric oxide

BACKGROUND Tissue engineering is now increasingly focusing on cell-based treatments as promising tools to improve tendon repair. However, many crucial aspects of tendon biology remain to be understood before adopting the best experimental approach for cell-tissue engineering. METHODS The role played by Ascorbic Acid (AA) alone and in combination with thyroid hormone T3 in the viability and proliferation of primary human tendon-derived cells was investigated. Human tenocyte viability was detected by Trypan blue exclusion test and cellular proliferation rate was evaluated by CFSE CellTrace™. In addition, the potential role of the AA in the production of Nitric Oxide (NO) was also examined. RESULTS In this in vitro model, an increase in tenocyte proliferation rate was observed as a consequence of progressively increased concentrations of AA (from 10 to 50 µg/ml). The addition of the T3 hormone to the culture further increased tenocyte proliferation rate. In detail, the most evident effect on cellular growth was achieved using the combined supplementation of 50 µg/ml AA and 10-7 M T3. CONCLUSION We showed that the highest concentration of AA (100 and 500 µg/ml) caused cytotoxicity to human tenocytes. Moreover, it was shown that AA reduces NO synthesis. These results show that AA is a cell proliferation inducer that triggers tenocyte growth, while it reduces NO synthesis.

A dual role for β1 integrin in an in vitro Streptococcus mitis/human gingival fibroblasts co‐culture model in response to TEGDMA

AIM To evaluate the effect of TEGDMA on human gingival fibroblasts (HGFs) in vitro co-cultured with Streptococcus mitis, focusing on the signalling pathways underlying cell tissue remodelling and inflammatory response processes. METHODOLOGY β1 integrin expression was evaluated by means of imaging flow cytometry. The Western blot technique was used to investigate the expression of protein kinase C (PKC), extracellular signal-regulated kinase (ERK), matrix metalloproteinase 9 (MMP9) and 3 (MMP3). RT-PCR was performed to quantify nuclear factor-kb subunits (Nf-kb1, ReLa), IkB kinase β (IkBkB), cyclooxygenase II (COX-2) and tumour necrosis factor-α (TNF-α) mRNA levels. Statistical analysis was performed using the analysis of variance (anova). RESULTS When HGFs are co-cultured with S. mitis, β1 integrin intensity, phosphorylated PKC (p-PKC), activated ERK (p-ERK), IkBkB mRNA level and MMP9 expression increased (for all molecules P < 0.05 HGFs versus HGFs co-cultured with S. mitis). A higher level of MMP3 in HGFs treated with TEGDMA was recorded (P < 0.05 HGFs versus HGFs exposed to TEGDMA). COX-2 inflammatory factor mRNA level appeared higher in HGFs exposed to 1 mmol L(-1) TEGDMA (P < 0.01 HGFs versus HGFs exposed to TEGDMA), whereas TNF-α gene expression was higher in HGFs co-cultured with S. mitis (P < 0.05 HGFs versus HGFs co-cultured with S. mitis). CONCLUSIONS β1 integrin triggered the signalling pathway, transduced by p-PKCα and involving ERK 1 and 2 and MMPs. This pathway resulted in an unbalanced equilibrium in tissue remodelling process, along with inflammatory response when HGFs are exposed to bacteria or biomaterial alone. On the contrary, the TEGDMA/S. mitis combination restored the balance between extracellular matrix deposition and degradation and prevented an inflammatory response.

Alginate/Hydroxyapatite-Based Nanocomposite Scaffolds for Bone Tissue Engineering Improve Dental Pulp Biomineralization and Differentiation

Tissue engineering is widely recognized as a promising approach for bone repair and reconstruction. Several attempts have been made to achieve materials that must be compatible, osteoconductive, and osteointegrative and have mechanical strength to provide a structural support. Composite scaffolds consisting in biodegradable natural polymers are very promising constructs. Hydroxyapatite (HAp) can support alginate as inorganic reinforcement and osteoconductive component of alginate/HAp composite scaffolds. Therefore, HAp-strengthened polymer biocomposites offer a solid system to engineer synthetic bone substitutes. In the present work, HAp was incorporated into an alginate solution and internal gelling was induced by addition of slowly acid-hydrolyzing D-gluconic acid delta-lactone for the direct release of calcium ions from HAp. It has been previously demonstrated that alginate-based composites efficiently support adhesion of cancer bone cell lines. Human dental pulp stem cells (DPSCs) identified in human dental pulp are clonogenic cells capable of differentiating in multiple lineage. Thus, this study is aimed at verifying the mineralization and differentiation potential of human DPSCs seeded onto scaffolds based on alginate and nano-hydroxyapatite. For this purpose, gene expression profile of early and late mineralization-related markers, extracellular matrix components, viability parameters, and oxidative stress occurrence were evaluated and analyzed. In summary, our data show that DPSCs express osteogenic differentiation-related markers and promote calcium deposition and biomineralization when growing onto Alg/HAp scaffolds. These findings confirm the use of Alg/HAp scaffolds as feasible composite materials in tissue engineering, being capable of promoting a specific and successful tissue regeneration as well as mineralized matrix deposition and sustaining natural bone regeneration.

The Extracellular Matrix, Growth Factors and Morphogens in Biomaterial Design and Tissue Engineering

Cells, morphogens, growth factors, and custom scaffolds are the critical ingredients for successful tissue regeneration in which morphogens and growth factors function sequentially. Extensive studies, in vitro and in vivo, have been made to explore the mechanisms and the roles played by these molecules. As a consequence, precise, localized control over the signaling of these factors and appropriate strategy selection, depending on the tissue or organ to be repaired or regenerated, is known to permit specific management of regenerative processes. The first part of the chapter examines natural ECMs which are a set of molecules secreted by cells that provide structural and biochemical support to the surrounding cells. ECMs also perform many other functions, such as actively regulating cell function through the control of biochemical gradients, cell density, spatial organization, and ligand attachment, thus influencing various types of cell processes. Subsequently, growth factors and morphogens are examined in greater depth to clarify to what degree progress has been made into improving methodologies and functionality and, perhaps, to hint at what remains to be done for the future of tissue engineering.

Functions of transcription factors NF-κB and Nrf2 in the inhibition of LPS-stimulated cytokine release by the resin monomer HEMA

OBJECTIVE Resin monomers like 2-hydroxyethyl methacrylate (HEMA) interfere with effects induced by stressors such as lipopolysaccharide (LPS) released from cariogenic microorganisms. In this study, mechanisms underlying monomer-induced inhibition of the LPS-stimulated secretion of inflammatory cytokines from immunocompetent cells were investigated. METHODS Secretion of pro-inflammatory cytokines TNF-α, IL-6 and the anti-inflammatory IL-10 from RAW264.7 mouse macrophages exposed to LPS and HEMA (0-6-8mM) was determined by ELISA. The formation of reactive oxygen (ROS) and nitrogen species (RNS) was determined by flow cytometry (FACS) after staining of cells with specific fluorescent dyes. Cell viability was analyzed by FACS, and protein expression was detected by Western blotting. RESULTS Secretion of TNF-α, IL-6 and IL-10 from LPS-stimulated cells increased after a 24h exposure. A HEMA-induced decrease in cytokine secretion resulted from the inhibition of LPS-stimulated NF-κB activation. Nuclear translocation of NF-κB was inhibited possibly as a result of enhanced levels of hydrogen peroxide (H2O2) and nitric oxide (NO) in HEMA-exposed cells. Oxidative stress caused by HEMA-induced formation of H2O2 and LPS-stimulated peroxynitrite (ONOO) also enhanced nuclear expression of Nrf2 as the major regulator of redox homeostasis, as well as Nrf2-controlled stress protein HO-1 to inhibit NF-κB activity. HEMA inhibited the LPS-stimulated expression of NOS (nitric oxide synthase) to produce NO but counteracted the expression of Nox2, which forms superoxide anions that combine with NO to peroxynitrite. CONCLUSIONS Resin monomers like HEMA inhibit LPS-stimulated NF-κB activation essential for cytokine release as a crucial response of immunocompetent cells of the dental pulp to invading cariogenic pathogens.