Christina J. Malavaki

Imatinib as a key inhibitor of the platelet‐derived growth factor receptor mediated expression of cell surface heparan sulfate proteoglycans and functional properties of breast cancer cells

Cell surface heparan sulfate proteoglycans (HSPGs), syndecans and glypicans, play crucial roles in the functional properties of cancer cells, such as proliferation, adhesion, migration and invasion. Platelet‐derived growth factor (PDGF)/PDGF receptor (PDGF‐R) mediated signaling, on the other hand, is highly associated with cancer progression. Specifically, PDGF‐Rα and PDGF‐Rβ expressions documented in breast cancer tissue specimens as well as breast cancer cell lines are correlated with tumor aggressiveness and metastasis. Imatinib (Glivec®) is a tyrosine kinase inhibitor specific for PDGF‐Rs, c‐ΚΙΤ and BCR‐ABL. In this study we evaluated the effects of imatinib on the properties of breast cancer cells as well as on the expression of HSPGs in the presence and absence of PDGF‐BB. These studies have been conducted in a panel of three breast cancer cell lines of low and high metastatic potential. Our results indicate that imatinib exerts a significant inhibitory effect on breast cancer cell proliferation, invasion and migration as well as on the cell surface expression of HSPGs even after exposure of PDGF. These effects depend on the aggressiveness of breast cancer cells and the type of HSPG. It is suggested that imatinib may be of potential therapeutic usefulness in breast cancer regimes.

Heparan sulfate: biological significance, tools for biochemical analysis and structural characterization

Heparan sulfate (HS) and heparin (HP) are functionally important glycosaminoglycans, which interact with a plethora of proteins and participate in several cellular events. They form specific proteoglycans, which are ubiquitously distributed at both extracellular and cellular levels. HS and HP chains vary in the sulfation pattern and the degree of C-5 epimerization of d-glucuronic acid to l-iduronic acid. These modifications are not uniformly distributed within the chain, providing functional oligomeric domains interacting specifically with various effective proteins. The utilization of specific lyases and chemical depolymerization are the commonest procedures used for structural analysis. Di- and oligosaccharide composition of HS can be accurately and sensitively determined by HPLC, CE and MS. Ultraviolet detection is satisfactory enough for unsaturated saccharides and pre-column derivatization with fluorophores and detection with laser-induced fluorescence results in even higher sensitivity. Solid-phase assays can also be used for monitoring interactions with other molecules. In this article the biological significance of HS and HP in health and disease as well as the portfolio of analytical methods that may help to a deeper understanding of their roles in various pathological processes is presented. Such methodologies are of crucial importance for disease diagnosis and the design of novel synthetic sugar-based drugs.

A comparative biochemical analysis of glycosaminoglycans and proteoglycans in human orthotopic and heterotopic bone

Glycosaminoglycans and proteoglycans are macromolecules of the bone and are involved in the assembly, maturation, mineralization, and maintenance of the extracellular matrix. Heterotopic ossification is the rapid development of calcified bone tissue at ectopic sites of the body, mainly in soft tissues that normally do not ossify. The aim of this study was to characterize the molecular profiles of glycosaminoglycans and proteoglycans in normal and heterotopic bone samples to assess whether differences exist between orthotopic and heterotopic bone. Heterotopic bone tissues contained lower amounts of glycosaminoglycans compared to normal femoral bone. Structural analysis of chondroitin sulfate (CS) revealed that both heterotopic and normal femoral bones were composed mainly of 6‐sulfated disaccharides. Quantitative differences in the disaccharide composition of CS, such as the decrease of 6‐sulfated disaccharides in heterotopic bone with a concurrent increase of 4‐sulfated and nonsulfated disaccharides, were found between ectopic bone and normal femoral bone. The proteoglycans decorin and aggrecan were both detected in all bone samples using specific antibodies. The detection of minor amounts of aggrecan in mature human bone, such as femoral bone, as well as in ectopic bone is described for the first time. These results may elucidate the phenomenon of ectopic bone formation and assist in early detection.

Wild blueberry (V. angustifolium)-enriched diets alter aortic glycosaminoglycan profile in the spontaneously hypertensive rat

Glycosaminoglycans (GAGs) are essential polysaccharide components of extracellular matrix and cell surface with key roles on numerous vascular wall functions. Previous studies have documented a role of wild blueberries on the GAG profile of the Sprague-Dawley rat with a functional endothelium as well as in the vascular tone of the spontaneously hypertensive rat (SHR) with endothelial dysfunction. In the present study, the effect of wild blueberries on the composition and structure of aortic GAGs was examined in 20-week-old SHRs after 8 weeks on a control (C) or a wild blueberry-enriched diet (WB). Aortic tissue GAGs were isolated following pronase digestion and anion-exchange chromatography. Treatment of the isolated populations with specific GAG-degrading lyases and subsequent electrophoretic profiling revealed the presence of three GAG species, i.e., hyaluronic acid (HA), heparan sulfate (HS) and galactosaminoglycans (GalAGs). A notable reduction of the total sulfated GAGs and a redistribution of the aortic GAG pattern were recorded in the WB as compared to the C group: a 25% and 10% increase in HA and HS, respectively, and an 11% decrease in GalAGs. Fine biochemical analysis of GalAGs at the level of constituent disaccharides with high-performance capillary electrophoresis revealed a notable increase of nonsulfated (18.0% vs. 10.7%) and a decrease of disulfated disaccharides (2.2% vs. 5.3%) in the WB aorta. This is the first study to report the redistribution of GAGs at the level of composition and their fine structural characteristics with implications for the endothelial dysfunction of the SHR.

Increased β-glucuronidase activity in bronchoalveolar lavage fluid of children with bacterial lung infection: A case-control study.

β‐Glucuronidase is a lysosomal enzyme released into the extracellular fluid during inflammation. Increased β‐glucuronidase activity in the cerebrospinal and peritoneal fluid has been shown to be a useful marker of bacterial inflammation. We explored the role of β‐glucuronidase in the detection of bacterial infection in bronchoalveolar lavage fluid (BALF) of paediatric patients.

A modified protocol for isolation and purity evaluation of a staphylococcal acidic polysaccharide by chromatography and capillary electrophoresis

The extracellular slime of Staphylococcus epidermidis contains, amongst various macromolecules, an acidic polysaccharide (PS) of a molecular mass of 20 kDa with significant antigenic and biological properties. The isolation procedure used so far includes multiple fractionations in anion-exchange chromatographic columns before its final purification by gel filtration chromatography. This protocol is laborious, time-consuming and includes the risk of unnecessary loss of PS quantities. Because of the significance of this PS, a modified protocol resulting in an easier and quicker isolation procedure was developed. Furthermore, identification, purity, charge density and molecular integrity of the isolated polysaccharide were evaluated by a reverse-polarity capillary electrophoresis method.

Validated Capillary Electrophoretic Assays for Disaccharide Composition Analysis of Galactosaminoglycans in Biologic Samples and Drugs/Nutraceuticals

Capillary electrophoresis is a separation technique with high resolving power and sensitivity with applications in glycosaminoglycan analysis. In this chapter, we present validated protocols for determining the variously sulfated chondroitin or dermatan sulfate-derived disaccharides. These approaches involve degradation of the polysaccharides with specific chondro/dermato-lyases and electrophoretic analysis with capillary zone electrophoresis in a low pH operating buffer and reversed polarity. This methodology has been applied to drug/nutraceutical formulations or to biologic samples (blood serum, lens capsule) and has been validated. Analysis of biologic tissue samples is often more demanding in terms of detection sensitivity, and thus concentration pretreatment steps and/or a derivatization step with 2-aminoacridone are often advisable.

Investigation on Toxicity and Teratogenicity in Rats of a Retinoid-Polyamine Conjugate with Potent Anti-Inflammatory Properties

Previous studies have shown that N(1),N(12)-bis(all-trans-retinoyl)spermine (RASP), a retinoid analog, inhibits RNase P activity and angiogenesis in the chicken embryo chorioallantoic membrane, demonstrates anti-tumor activity on prostate cancer cells, and acts as anti-inflammatory agent, being more effective and less toxic than all-trans retinoic acid. In an attempt to further characterize the biological profile of RASP, we tested its effects on organ toxicity and teratogenicity by daily oral gavage of RASP at a level of 50 mg/Kg of body weight in two generations of rats. We found that this compound does not induce changes to the body growth, the appearance of physical features, and the animals reflexes. Additionally, no substantial histopathological lesions were found in brain, heart, lung, thymus, liver, thyroid gland, adrenal gland, pituitary gland, kidneys, spleen, skin, femora, prostate, testis, epididymis, vagina, uterus, and ovaries of RASP-treated animals. These results suggest RASP, as a promising lead compound for the treatment of several dermatological disorders and certain cancer types, has apparently minimal toxic side-effects as revealed in this two-generation reproduction study in rats.