Lipoyl-Based Antagonists of Transient Receptor Potential Cation A (TRPA1) Downregulate Osteosarcoma Cell Migration and Expression of Pro-Inflammatory Cytokines.

Osteosarcoma is a heterogeneous tumor intimately linked to its microenvironment, which promotes its growth and spread. It is generally accompanied by cancer-induced bone pain (CIBP), whose main component is neuropathic pain. The TRPA1 ion channel plays a key role in metastasis and is increasingly expressed in bone cancer. Here, a novel TRPA1 inhibitor is described and tested together with two other known TRPA1 antagonists. The novel lipoyl derivative has been successfully assessed for its ability to reduce human osteosarcoma MG-63 cell viability, motility, and gene expression of the CIBP pro-inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α). A putative three-dimensional (3D) model of the inhibitor covalently bound to TRPA1 is also proposed. The in vitro data suggest that the novel inhibitor described here may be highly interesting and stimulating for new strategies to treat osteosarcomas.

Matrix Effectors in the Pathogenesis of Keratinocyte-Derived Carcinomas.

Basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC), referred to as keratinocyte carcinomas, are skin cancer with the highest incidence. BCCs, rarely metastasize; whereas, though generally not characterized by high lethality, approximately 2-4% of primary cSCCs metastasize with patients exhibiting poor prognosis. The extracellular matrix (ECM) serves as a scaffold that provides structural and biological support to cells in all human tissues. The main components of the ECM, including fibrillar proteins, proteoglycans (PGs), glycosaminoglycans (GAGs), and adhesion proteins such as fibronectin, are secreted by the cells in a tissue-specific manner, critical for the proper function of each organ. The skin compartmentalization to the epidermis and dermis compartments is based on a basement membrane (BM), a highly specialized network of ECM proteins that separate and unify the two compartments. The stiffness and assembly of BM and tensile forces affect tumor progenitors' invasion at the stratified epithelium's stromal border. Likewise, the mechanical properties of the stroma, e.g., stiffness, are directly correlated to the pathogenesis of the keratinocyte carcinomas. Since the ECM is a pool for various growth factors, cytokines, and chemokines, its' intense remodeling in the aberrant cancer tissue milieu affects biological functions, such as angiogenesis, adhesion, proliferation, or cell motility by regulating specific signaling pathways. This review discusses the structural and functional modulations of the keratinocyte carcinoma microenvironment. Furthermore, we debate how ECM remodeling affects the pathogenesis of these skin cancers.

In Vitro Biocompatibility Assessment of Nano-Hydroxyapatite.

Hydroxyapatite (HA) is an important component of the bone mineral phase. It has been used in several applications, such as bone regenerative medicine, tooth implants, drug delivery and oral care cosmetics. In the present study, three different batches of a commercial nanohydroxyapatite (nHA) material were physicochemically-characterized and biologically-evaluated by means of cytotoxicity and genotoxicity using appropriate cell lines based on well-established guidelines (ISO10993-5 and OECD 487). The nHAs were characterized for their size and morphology by dynamic light scattering (DLS) and transmission electron microscopy (TEM) and were found to have a rod-like shape with an average length of approximately 20 to 40 nm. The nanoparticles were cytocompatible according to ISO 10993-5, and the in vitro micronucleus assay showed no genotoxicity to cells. Internalization by MC3T3-E1 cells was observed by TEM images, with nHA identified only in the cytoplasm and extracellular space. This result also validates the genotoxicity since nHA was not observed in the nucleus. The internalization of nHA by the cells did not seem to affect normal cell behavior, since the results showed good biocompatibility of these nHA nanoparticles. Therefore, this work is a relevant contribution for the safety assessment of this nHA material.

Contact allergen (PPD and DNCB)-induced keratinocyte sensitization is partly mediated through a low molecular weight hyaluronan (LMWHA)/TLR4/NF-κB signaling axis.

Allergic contact dermatitis (ACD) is caused by topical exposure to chemical allergens. Keratinocytes play a key role in innate immunity, as well as in ACD progression. The transmembrane Toll-like receptor 4 (TLR4), strongly implicated in skin inflammation, has the ability to bind Damage Associated Molecular Patterns (DAMPs), like Low Molecular Weight Hyaluronan (LMWHA). Previously, we had determined that p-phenylenediamine (PPD) and 2,4-dinitrochlorobenzene (DNCB) modulate keratinocyte HA deposition in a manner correlated to their sensitization. In the present study, we aimed to investigate putative co-operation of HA and TLR4 in the process of PPD and DNCB-induced keratinocyte activation. Contact sensitizers were shown to significantly increase the expression of Hyaluronan Synthases (HAS) and TLR4 in NCTC2544 human keratinocytes, as demonstrated by western blot and Real-Time PCR. These data, in correlation to earlier shown enhanced HA degradation suggest that the contact sensitizers facilitate HA turnover of keratinocytes and increase the release of pro-inflammatory, LMWHA fragments. Treatment with exogenous LMWHA enhanced TLR4, HAS levels and Nuclear factor-kappa beta (NF-κΒ) activation. PPD, DNCB and LMWHA-effects were shown to be partly executed through TLR4 downstream signaling as shown by Real-Time, western blot, siRNA and confocal microscopy approaches. Specifically, PPD and DNCB stimulated the activation of the TLR4 downstream mediator NF-κB. Therefore, the shown upregulation of TLR4 expression is suggested to further facilitate the release of endogenous, bioactive HA fragments and sustain keratinocyte activation. In conclusion, keratinocyte contact allergen-dependent sensitization is partly mediated through a LMWHA/TLR4/ NF-κB signaling axis.

Role of the extracellular matrix in cancer-associated epithelial to mesenchymal transition phenomenon.

The epithelial to mesenchymal transition (EMT) program is a crucial component in the processes of morphogenesis and embryonic development. The transition of epithelial to mesenchymal phenotype is associated with numerous structural and functional changes, including loss of cell polarity and tight cell-cell junctions, the acquisition of invasive abilities, and the expression of mesenchymal proteins. The switch between the two phenotypes is involved in human pathology and is crucial for cancer progression. Extracellular matrices (ECMs) are multi-component networks that surround cells in tissues. These networks are obligatory for cell survival, growth, and differentiation as well as tissue organization. Indeed, the ECM suprastructure, in addition to its supportive role, can process and deliver a plethora of signals to cells, which ultimately regulate their behavior. Importantly, the ECM derived signals are critically involved in the process of EMT during tumorigenesis. This review discusses the multilayer interaction between the ECM and the EMT process, focusing on contributions of discrete mediators, a strategy that may identify novel potential target molecules. Developmental Dynamics 247:368-381, 2018. © 2017 Wiley Periodicals, Inc.

HA metabolism in skin homeostasis and inflammatory disease.

Hyaluronan (HA), an unsulfated glycosaminoglycan, is an important component of the complex extracellular matrix network which surrounds and supports cells in tissues. HA is detected in all vertebrate tissues, but the bulk of HA is produced and deposited in the skin. In this review we focus on the role of HA in skin-associated inflammatory disease and wound healing. Properties of HA are directly dependent on its molecular weight. Thus, high molecular weight HA (HMWHA) is deposited in normal tissues during homeostasis and promotes their stability whereas low molecular weight HA fragments (LMWHA), on the other hand, may arise from enzymatic or chemical activities. The degradation of HMWHA to LMWHA fragments, often leads to the generation of biologically active oligosaccharides with different properties and postulated functions in wound scar formation and inflammation. More detailed studies of HA involvement in skin-associated inflammatory disease may result in novel treatment modalities.

Parathyroid hormone/parathyroid hormone-related peptide regulate osteosarcoma cell functions: Focus on the extracellular matrix (Review).

Osteosarcoma (OS) is a primary bone tumor of mesenchymal origin mostly affecting children and adolescents. The OS extracellular matrix (ECM) is extensively altered as compared to physiological bone tissue. Indeed, the main characteristic of the most common osteoblastic subtype of OS is non­mineralized osteoid production. Parathyroid hormone (PTH) is a polypeptide hormone secreted by the chief cells of the parathyroid glands. The PTH-related peptide (PTHrP) may be comprised of 139, 141 or 173 amino acids and exhibits considerate N­terminal amino acid sequence homology with PTH. The function of PTH/PTHrP is executed through the activation of the PTH receptor 1 (PTHR1) and respective downstream intracellular pathways which regulate skeletal development, bone turnover and mineral ion homeostasis. Both PTHR1 and its PTH/PTHrP ligands have been shown to be expressed in OS and to affect the functions of these tumor cells. This review aims to highlight the less well known aspects of PTH/PTHrP functions in the progression of OS by focusing on ECM-dependent signaling.

Hyaluronan/Hyaladherins - a Promising Axis for Targeted Drug Delivery in Cancer.

BACKGROUND: Hyaluronan (HA), a glycosaminoglycan, is a key extracellular matrix (ECM) component, and has been established to contribute to fibrotic, angiogenic, inflammatory as well as processes supporting cancer development. The changes in HA deposition in different tumors have been widely studied. Indeed, a multitude of reports demonstrate that HA expression is increased in different neoplasmatic tissues including lung, colon, prostate and breast cancer. The aims of this paper are to critically and in depth discuss aspects of HA metabolism in cancer and recent developments of its utilization in cancer therapy. METHODS: Up to date research and online content are reviewed. RESULTS: The cellular roles of HA are perpetrated through molecular interactions with HA-binding proteins, called hyaladherins, including CD44 receptor as well as receptor for hyaluronan-mediated motility (RHAMM). HA binding can be followed by receptor-mediated endocytosis. Importantly, hyaladherins show an altered expression in tumor tissues. Indeed, post-translational alterations in CD44 structure have been suggested to regulate the equilibrium between the "inactive" low affinity state and the "active" high affinity state of the HA binding capacity. In this concept HA fragments can be utilized as specific targeting ligands for efficient and safe drug delivery in cancer. CONCLUSION: HA-drug bioconjugates and nanoparticles have emerged as a promising platform for drug delivery during cancer treatment as demonstrated in various pre-clinical studies. Recent developments from clinical trials indicate that the utilization of specific HA-drug bioconjugates might be approved for the medical practice in the nearest future.

Hyaluronan regulates chemical allergen-induced IL-18 production in human keratinocytes.

Interleukin-18 (IL-18) has been shown to play a key proximal role in the induction of allergic contact dermatitis. Low molecular weight hyaluronan (LMWHA), an endogenous molecule and a member of the so-called damage associated molecular patterns (DAMPs), has been suggested to elicit immune-stimulatory effects. The purpose of this study was to examine the role of hyaluronan (HA) degradation in IL-18 production in human keratinocytes following stimulation with the contact sensitizers 2,4-dinitrochlorobenzene (DNCB) and PPD. IL-18 production in the human keratinocyte cell line NCTC2544 was measured by ELISA, whereas changes in HA metabolism were determined by Real-time PCR and immunofluorescence. Both contact allergens were able to enhance hyaluronidase (HYAL) 1 and 2 expression inducing HA degradation. Modulation of HA production, by HYAL or aristolochic acid pre-treatment, resulted in a significant reduction of contact allergen-induced IL-18 production. Oxidative stress appears to be the initial step in KC activation, as all the sequels of events can be blocked using antioxidants. This is the first indication that LMWHA can act as a DAMP in keratinocytes. In conclusion LMWHA fragments are important mediators in the process of contact sensitisation leading to IL-18 dependent responses.