Dragana Nikitovic

Glycosaminoglycans: key players in cancer cell biology and treatment

Glycosaminoglycans are natural heteropolysaccharides that are present in every mammalian tissue. They are composed of repeating disaccharide units that consist of either sulfated or non‐sulfated monosaccharides. Their molecular size and the sulfation type vary depending on the tissue, and their state either as part of proteoglycan or as free chains. In this regard, glycosami‐noglycans play important roles in physiological and pathological conditions. During recent years, cell biology studies have revealed that glycosaminoglycans are among the key macromolecules that affect cell properties and functions, acting directly on cell receptors or via interactions with growth factors. The accumulated knowledge regarding the altered structure of glycosaminoglycans in several diseases indicates their importance as biomarkers for disease diagnosis and progression, as well as pharmacological targets. This review summarizes how the fine structural characteristics of glycosaminoglycans, and enzymes involved in their biosynthesis and degradation, are involved in cell signaling, cell function and cancer progression. Prospects for glycosaminoglycan‐based therapeutic targeting in cancer are also discussed.

The Biology of Small Leucine-rich Proteoglycans in Bone Pathophysiology

The class of small leucine-rich proteoglycans (SLRPs) is a family of homologous proteoglycans harboring relatively small (36–42 kDa) protein cores compared with the larger cartilage and mesenchymal proteoglycans. SLRPs have been localized to most skeletal regions, with specific roles designated during all phases of bone formation, including periods relating to cell proliferation, organic matrix deposition, remodeling, and mineral deposition. This is mediated by key signaling pathways regulating the osteogenic program, including the activities of TGF-β, bone morphogenetic protein, Wnt, and NF-κB, which influence both the number of available osteogenic precursors and their subsequent development, differentiation, and function. On the other hand, SLRP depletion is correlated with degenerative diseases such as osteoporosis and ectopic bone formation. This minireview will focus on the SLRP roles in bone physiology and pathology.

Role of oxidative stress in chemical allergens induced skin cells activation

Allergic contact dermatitis (ACD) is an important occupational and environmental disease caused by topical exposure to chemical allergens. It describes the adverse effects that may results when exposure to a chemical elicits a T cell-mediated inflammatory skin disease. The ability of contact sensitizers to induce the oxidative stress pathway in keratinocytes and dendritic cells has been confirmed by several authors. Reactive oxygen species (ROS) can serve as essential second messengers mediating cellular responses resulting in immune cells activation. Oxidative stress may be the starter point, as it leads to the activation of transcription factors and signaling pathways, including NF-kB and p38 MAPK, which leads to the release of cytokines and chemokines. ROS are also involved in the activation of the NLRP3/NALP3 inflammasome, which is required to direct the proteolytic maturation of inflammatory cytokines such as IL-1β and IL-18, which are all integral to the process of dendritic cells mobilization, migration and functional maturation. Moreover, emerging evidence correlates ROS to changes in the constitution of the extracellular microenvironment found to facilitate ACD. The purpose of this review is to provide both conceptual and technical frameworks on the role of oxidative stress in chemical allergy.

Lumican, a small leucine-rich proteoglycan.

Lumican belongs to the family of small leucine‐rich repeat proteoglycans. Recent studies have shown that lumican participates in the maintenance of tissue homeostasis and modulates cellular functions including cell proliferation, migration, and differentiation. The expression of lumican has been correlated to the growth and metastasis of various malignancies; however, its exact role in tumorogenesis remains elusive. This review focuses upon the role of lumican in cell biology, providing insights into molecular mechanisms that lumican likely utilizes to control processes relevant to tumorogenesis.

Role of Receptor for Hyaluronic Acid-mediated Motility (RHAMM) in Low Molecular Weight Hyaluronan (LMWHA)-mediated Fibrosarcoma Cell Adhesion

Background: Hyaluronan (HA) modulates key cancer cell functions through interaction with its CD44 and RHAMM receptors. Results: Low molecular weight HA (LMWHA) significantly increased (p ≤ 0.01) the adhesion capacity of HT1080 cells in a RHAMM-dependent manner. Conclusion: RHAMM/HA interaction regulates fibrosarcoma cell adhesion via the activation of FAK and ERK1/2 signaling pathways. Significance: Identification of a novel HA-signaling pathway. Hyaluronan (HA) modulates key cancer cell functions through interaction with its CD44 and receptor for hyaluronic acid-mediated motility (RHAMM) receptors. HA was recently found to regulate the migration of fibrosarcoma cells in a manner specifically dependent on its size. Here, we investigated the effect of HA/RHAMM signaling on the ability of HT1080 fibrosarcoma cells to adhere onto fibronectin. Low molecular weight HA (LMWHA) significantly increased (p ≤ 0.01) the adhesion capacity of HT1080 cells, which high molecular weight HA inhibited. The ability of HT1080 RHAMM-deficient cells, but not of CD44-deficient ones, to adhere was significantly decreased (p ≤ 0.001) as compared with control cells. Importantly, the effect of LMWHA on HT1080 cell adhesion was completely attenuated in RHAMM-deficient cells. In contrast, adhesion of RHAMM-deficient cells was not sensitive to high molecular weight HA treatment, which identifies RHAMM as a specific conduit of the LMWHA effect. Western blot and real time-PCR analyses indicated that LMWHA significantly increased RHAMM transcript (p ≤ 0.05) and protein isoform levels (53%, 95 kDa; 37%, 73 kDa) in fibrosarcoma cells. Moreover, Western blot analyses showed that LMWHA in a RHAMM-dependent manner enhanced basal and adhesion-dependent ERK1/2 and focal adhesion kinase (FAK) phosphorylation in HT1080 cells. Utilization of a specific ERK1/2 inhibitor completely inhibited (p ≤ 0.001) LMWHA-dependent adhesion, suggesting that ERK1/2 is a downstream effector of LMWHA/RHAMM signaling. Likewise, the utilization of the specific ERK1 inhibitor resulted in a strong down-regulation of FAK activation in HT1080 cells, which identifies ERK1/2 as a FAK upstream activator. In conclusion, our results suggest that RHAMM/HA interaction regulates fibrosarcoma cell adhesion via the activation of FAK and ERK1/2 signaling pathways.

Lumican expression is positively correlated with the differentiation and negatively with the growth of human osteosarcoma cells

Osteosarcoma is the most common primary bone tumour associated with childhood and adolescence. The possible role of the small leucine‐rich proteoglycan, lumican, in the growth and metastasis of various cancer types has recently been investigated. In this study, the expression of lumican was examined in moderately differentiated (MG‐63) and well‐differentiated (Saos 2) human osteosarcoma cell lines of high and low metastatic capability, respectively. Real‐time PCR, western blotting with antibodies against the protein core and keratan sulfate, and specific enzymatic digestions were the methods employed. The two human osteosarcoma cell lines were found to express and secrete lumican partly substituted with keratan sulfate glycosaminoglycans. Importantly, the non‐metastatic, well‐differentiated Saos 2 cells produced lumican at rates that were up to sevenfold higher than those of highly metastatic MG‐63 cells. The utilization of short interfering RNA specific for the lumican gene resulted in efficient down‐regulation of its mRNA levels in both cell lines. The growth of Saos 2 cells was inhibited by lumican, whereas their migration and chemotactic response to fibronectin were found to be promoted. Lumican expression was negatively correlated with the basal level of Smad 2 activation in these cells, suggesting that lumican may affect the bioavailability of Smad 2 activators. By contrast, these cellular functions of highly aggressive MG‐63 cells were demonstrated not to be sensitive to a decrease in their low endogenous lumican levels. These results suggest that lumican expression may be positively correlated with the differentiation and negatively correlated with the progression of osteosarcoma.

Transforming Growth Factor‐β as a key molecule triggering the expression of versican isoforms v0 and v1, Hyaluronan Synthase‐2 and synthesis of Hyaluronan in Malignant Osteosarcoma cells

Versican, a large sized chondroitin‐sulphate proteoglycan (PG), and its binding partner, hyaluronan (HA), are extracellular matrix (ECM) components that play an essential role in transformed cell behavior. Expression of certain versican isoforms has been implicated in cell migration and proliferation of cancer cells and, on the other hand, disruption of HA synthesis by inhibiting hyaluronan synthase‐2 (HAS2) expression in osteosarcoma cells by suppressing cell proliferation, invasiveness and motility. Considering that growth factors, such as TGF‐β, bFGF and PDGF‐BB, are important regulators for the expression of the ECM macromolecules, in this study we examined the effect of these growth factors on the expression of the various versican isoforms, HA synthases as well as HA synthesis by MG‐63 osteosarcoma cells and normal human osteoblastic periodontal ligament cells (hPDL). Real‐time PCR and metabolic labelling followed by fine HPLC analysis coupled to radiochemical detection were the methods utilized. It was found that, contrary to normal hPDL cells, osteosarcoma MG‐63 cells do not constitutively express the versican isoforms V0 and V1. Exogenous addition of TGF‐β2 stimulated the versican transcript levels mainly by forcing osteosarcoma cells to express V1 and V0 isoforms. PDGF‐BB and bFGF had only minor effects in these cells. In hPDL cells a strong stimulation of the V3 transcript by all growth factors was observed. TGF‐β2 was also the major stimulator of HAS2 isoform expression as well as hyaluronan synthesis in osteosarcoma cells, while PDGF‐BB exerted dominant influence on HAS2 isoform expression and hyaluronan biosynthesis by osteoblasts. The obtained results show for the first time that TGF‐β2 triggers the malignant phenotype pattern of versican and hyaluronan expression in human osteosarcoma cells and indicate that this growth factor may account for the metastatic potential of these cells. IUBMB Life, 58: 47 ‐ 53, 2006

Protein bio-corona: critical issue in immune nanotoxicology.

Abstract With the expansion of the nanomedicine field, the knowledge focusing on the behavior of nanoparticles in the biological milieu has rapidly escalated. Upon introduction to a complex biological system, nanomaterials dynamically interact with all the encountered biomolecules and form the protein “bio-corona.” The decoration with these surface biomolecules endows nanoparticles with new properties. The present review will address updates of the protein bio-corona characteristics as influenced by nanoparticle’s physicochemical properties and by the particularities of the encountered biological milieu. Undeniably, bio-corona generation influences the efficacy of the nanodrug and guides the actions of innate and adaptive immunity. Exploiting the dynamic process of protein bio-corona development in combination with the new engineered horizons of drugs linked to nanoparticles could lead to innovative functional nanotherapies. Therefore, bio-medical nanotechnologies should focus on the interactions of nanoparticles with the immune system for both safety and efficacy reasons.

Early Effects of Simvastatin versus Atorvastatin on Oxidative Stress and Proinflammatory Cytokines in Hyperlipidemic Subjects

The authors investigated the time-dependent action of atorvastatin and simvastatin on oxidative stress and cytokine levels immediately after the start of treatment. These factors play a role in endothelial dysfunction. Hyperlipidemic patients (n=132) were assigned to treatment with 40 mg atorvastatin, 40 mg simvastatin, or placebo. Blood samples were taken before, 2 hours, 24 hours, 7 days, and 3 weeks after the administration of the statin or placebo to evaluate serum concentrations of total peroxides (TP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-a) and soluble intercellular vascular adhesion molecule 1 (sICAM 1). In the atorvastatin group the TP changes were significantly different at 2 hours and 24 hours (p=0.005), whereas in the simvastatin group there was a gradual, more or less linear decline in TP until 7 days (p=0.006) and then a plateau. Simvastatin exhibited a faster statistically significant decrease over time in IL-6 and sICAM 1 levels (at 7 days, p=0.014 and p=0.001, respectively). TNF-a demonstrated a faster linear trend in the simvastatin group, but the significant effect appeared late (p=0.006). Both simvastatin and atorvastatin exerted early beneficial effects on oxidative stress, proinflammatory cytokines, and endothelial activation in hyperlipidemic subjects. These effects became significant 2 hours following the initiation of therapy.

Cross-talk between estradiol receptor and EGFR/IGF-IR signaling pathways in estrogen-responsive breast cancers: Focus on the role and impact of proteoglycans

In hormone-dependent breast cancer, estrogen receptors are the principal signaling molecules that regulate several cell functions either by the genomic pathway acting directly as transcription factors in the nucleus or by the non-genomic pathway interacting with other receptors and their adjacent pathways like EGFR/IGFR. It is well established in literature that EGFR and IGFR signaling pathways promote cell proliferation and differentiation. Moreover, recent data indicate the cross-talk between ERs and EGFR/IGFR signaling pathways causing a transformation of cell functions as well as deregulation on normal expression pattern of matrix molecules. Specifically, proteoglycans, a major category of extracellular matrix (ECM) and cell surface macromolecules, are modified during malignancy and cause alterations in cancer cell signaling, affecting eventually functional cell properties such as proliferation, adhesion and migration. The on-going strategies to block only one of the above signaling effectors result cancer cells to overcome such inactivation using alternative signaling pathways. In this article, we therefore review the underlying mechanisms in respect to the role of ERs and the involvement of cross-talk between ERs, IGFR and EGFR in breast cancer cell properties and expression of extracellular secreted and cell bound proteoglycans involved in cancer progression. Understanding such signaling pathways may help to establish new potential pharmacological targets in terms of using ECM molecules to design novel anticancer therapies.