Zoharia Evron

Platinum-resistance in ovarian cancer cells is mediated by IL-6 secretion via the increased expression of its target cIAP-2

Ovarian carcinoma patients are initially responsive to platinum-based therapy, but eventually become refractory to treatment due to the development of platinum chemoresistance. Elevated levels of interleukin-6 (IL-6) in the sera and ascites of these patients predict poor clinical outcome. Our goal was to analyze the interaction between cisplatin and cisplatin-resistant ovarian cancer cells, and to identify means of circumventing platinum resistance. We studied ovarian carcinoma cell lines and cells drawn from ovarian carcinoma patients. Gene array analyses were performed on ovarian carcinoma cells upon treatment with cisplatin, and the results were validated by ELISA and Western blotting (WB). Cytotoxicity assays were performed on anti-IL-6 Ab-, IL-6-, and cellular inhibitor of apoptosis 2 (cIAP-2) siRNA-treated cells, following cisplatin addition. Our results revealed a highly significant increase in IL-6 and cIAP-2 mRNA and protein levels upon treatment with cisplatin. WB analysis of cisplatin-treated cells exhibited decreased cIAP-2 expression level following anti-IL-6 Ab addition. Furthermore, IL-6 by itself, significantly increased cIAP-2 levels in ovarian carcinoma cells. Finally, cytotoxicity assays showed sensitization to cisplatin following the addition of IL-6 and cIAP-2 inhibitors. In conclusion, cisplatin treatment of ovarian carcinoma cells upregulates IL-6 and cIAP-2 levels while their inhibition significantly sensitizes them to cisplatin. Here, we present cIAP-2 as a novel inducer of platinum resistance in ovarian carcinoma cells, and suggest an axis beginning with an encounter between cisplatin and these cells, mediated sequentially by IL-6 and cIAP-2, resulting in cisplatin resistance. Consequently, we propose that combining IL-6/cIAP-2 inhibitors with cisplatin will provide new hope for ovarian carcinoma patients by improving the current treatment.

Changes in proteoglycans of intervertebral disc in diabetic patients. A possible cause of increased back pain.

Study Design. Characterization of the analytic profile of proteoglycans in the intervertebral discs at L4‐L5 of nondiabetic (n = 5) and diabetic (n = 5) age‐matched subjects. The discs used were discarded material from operations. Objectives. To clarify the reason for the higher risk of disc prolapse in diabetic patients. Summary of Background Data. The pathogenesis of diabetes results from a combination of neurologic dysfunctions and a yet undefined metabolic failure, which leads to an abnormal proteoglycan profile. Methods. The following methods were used to determine the proteoglycan profile: the measurement of 35S‐sulfate uptake per gram wet tissue into sulfated glycosaminoglycan using fresh tissue explants; extraction of proteoglycans by 4 M guanidinium chloride containing protease inhibitors, with further purification by ultracentrifugation on cesium chloride buoyant density gradient under dissociative conditions; total uronic acid and protein contents in the various gradient fractions; assessing the length of sugar side chains of isolated 35 Sulfate‐glycosaminoglycan molecules by separation of the glycosaminoglycan molecules on a Sepharose 6B‐CL column; and paper chromatography of the final digest products of glycosaminoglycan molecules obtained by chondroitinase ABC, a glycosaminoglycan‐degrading enzyme. Results. The findings show that discs from normal nondiabetic subjects have 15 times the rate of 35Sulfate incorporation into glycosaminoglycan molecules than do discs of diabetic patients. The proteoglycans of diabetic patients are banded at a lower buoyant density, indicating a lowered glycosylation rate and a lower number of sugar side chains per core protein. In discs of diabetic patients, there is a slight increase in the chain length of chondroitin sulfate. Further analysis of the glycosaminoglycan chains showed a decreased amount of keratan sulfate, compared with that in nondiabetic subjects. However, the total uronic acid content of the disc tissues and the ratio of uronic acid to protein of each fraction were unchanged in diabetic patients versus that in control subjects. Conclusions. Discs in patients with diabetes have proteoglycans with lower buoyant density and substantially undersulfated glycosaminoglycan, which with the specific neurologic damage in these patients, might lead to increased susceptibility to disc prolapse.

Synovial chondromatosis: the possible role of FGF 9 and FGF receptor 3 in its pathology

Primary synovial chondromatosis (PSC) is a rare disorder of the synovium typified by cartilaginous nodule formation within the synovial membrane. Fibroblast growth factor receptor 3 (FGFR3) is a recently described specific marker of mesenchymal precartilaginous stem cells. Expression patterns of FGFR3 and its specific ligand, fibroblast growth factor 9 (FGF 9), were evaluated both in situ and in cell cultures. Histologically, cells at the periphery of the cartilage nodules express FGFR3 and PCNA ( proliferating cell nuclear antigen). Elevated levels of FGF 9, its specific ligand, have been found in synovial fluids of patients with synovial chondromatosis. Synoviocytes but not chondrocytes from affected patients express FGF9 in culture. This pattern is absent in normal synovium and cartilage. Downregulation of FGF9 may provide a possible nonoperative therapy for PSC.

The molecular and cellular basis of exostosis formation in hereditary multiple exostoses

The different clinical entities of osteochondromas, hereditary multiple exostoses (HME) and non‐familial solitary exostosis, are known to express localized exostoses in their joint metaphyseal cartilage. In the current study biopsies of osteochondromas patients were screened with respect to a number of cellular and molecular parameters. Specifically, cartilaginous biopsy samples of nine HME patients, 10 solitary exostosis patients and 10 articular cartilages of control subjects were collected and cell cultures were established. Results obtained showed that one of the two HME samples that underwent DNA sequencing analysis (HME‐1) had a novel mutation for an early stop codon, which led to an aberrant protein, migrating at a lower molecular weight position. The EXT‐1 mRNA and protein levels in chondrocyte cultures derived from all nine HME patients were elevated, compared with solitary exostosis patients or control subjects. Furthermore, cell cultures of HME patients had significantly decreased pericellular heparan sulphate (HS) in comparison with cultures of solitary exostosis patients or control subjects. Immunohistochemical staining of tissue sections and Western blotting of cell cultures derived from HME patients revealed higher levels of heparanase compared with solitary exostosis patients and of control subjects. Further investigations are needed to determine whether the low pericellular HS levels in HME patients stem from decreased biosynthesis of HS, increased degradation or a combination of both. In conclusion, it appears that due to a mutated glycosyltransferase, the low content of pericellular HS in HME patients leads to the anatomical deformations with exostoses formation. Hence, elevation of HS content in the pericellular regions should be a potential molecular target for correction.

The perichondrial ring as a reservoir for precartilaginous cells. In vivo model in young chicks' epiphysis.

Background: The purpose of this study is to illustrate the routes of migration of precartilaginous cells from the perichondrial ring of LaCroix, as a potential reservoir for growth-plate germ cells. Methods: Chondrocytes derived from the ring of LaCroix of young chicks’ proximal tibia were cultured in vitro and transfected with adenovirus vector containing the gene encoding for Escherichia coli (beta)-galactosidase (lacZ) gene, which allows assessment of the migratory routes of these cells. The lacZ- transfected cells were injected back into the perichondrial ring of LaCroix of young chicks’ proximal tibias. Four weeks later the migration root was assessed microscopically. Results: Injection of cells derived from the ring of LaCroix of neonate chicks, transfected in culture with adenoviruses containing LacZ reporter gene, allows the assessment of migratory potential of these cells. Stained cells were found at the outer layer of the epiphysis, particularly in areas adjacent to the perichondrial ring. Further longitudinal histopathological studies along the bone axis demonstrated a condensed layer of the stained cells arranged horizontally along parts of the physis. Conclusion: The perichondrial ring of LaCroix represents a potential reservoir of growth-plate germ cells in young chicks.RésuméLe but de cette étude est d’étudier la circulation des cellules souches précartilagineuses provenant de la virole périchondrale de LaCroix considérée comme un réservoir potentiel de ces cellules. Méthode: les chondrocytes provenant de la virole périchondrale de LaCroix prélevés au niveau de l’extrémité supérieure du tibia chez de jeunes poulets ont été cultivés in vitro et transplantés à l’aide d’un adénovirus codant le gène beta galactosidase (lacZ). Les cellules lacZ ainsi transplantées ont été injectées dans la virole périchondrale de l’extrémité supérieure du tibia des mêmes poulets. Quatre semaines après, les migrations cellulaires ont été étudiées au microscope. Résultats: les migrations de ces cellules ont été vérifiées sur le plan histopathologique. Conclusion: On peut affirmer que la virole périchondrale de LaCroix est un réservoir potentiel de cellules souches de croissance chez les jeunes poulets.

β-d-Xylosides Stimulate GAG Synthesis in Chondrocyte Cultures Due to Elevation of the Extracellular GAG Domains, Accompanied by the Depletion of the Intra–pericellular GAG Pools, with Alterations in the GAG Profiles

The familial disease of hereditary multiple exostoses is characterized by abnormal skeletal deformities requiring extensive surgical procedures. In hereditary multiple exostoses patients there is a shortage in the pericellular glycosaminoglycan (GAG) of heparan sulfate (HS), related to defective activity of HS glycosyltransferases, mainly in the pericellular regions of chondrocytes. This study searched for a novel approach employing xylosides with different aglycone groups priming a variety of GAG chains, in attempting to alter the GAG compositional profile. Cell cultures of patients with osteochondroma responded to p-nitrophenyl β-d-xyloside by a significant increase in total GAG synthesis, expressed mainly in the extracellular domains, limited to chondroitin sulfate). The different β-d-xylosides, in addition to increasing the synthesis of extracellular GAGs, led to a significant depletion of the intracellular GAG domains. In mouse chondrocyte cultures, β-d-xylosides with different aglycones created a unique distribution of the GAG pools. Of special interest was the finding that the naphthalene methanol β-d-xyloside showed the highest absolute levels of HS-GAGs in both extracellular and intra–pericellular moieties compared with other β-d-xylosides and with controls without xyloside. In summary, β-d-xylosides can be utilized in chondrocyte cultures to modify the distribution of GAGs between the extracellular and intracellular compartments. In addition, xylosides may alter the profile of specific GAG chains in each moiety.

Improved regional left ventricular function after successful satellite cell grafting in rabbits with myocardial infarction

To evaluate whether satellite cells injected into infarct areas in rabbits remain viable during 6 weeks follow‐up and can improve cardiac function as assessed by echocardiography.

Targeting anthracycline-resistant tumor cells with synthetic aloe-emodin glycosides.

The cytotoxic activity of aloe-emodin (AE), a natural anthranoid that readily permeates anthracycline-resistant tumor cells, was improved by the attachment of an amino-sugar unit to its anthraquinone core. The new class of AE glycosides (AEGs) showed a significant improvement in cytotoxicity-up to more than 2 orders of magnitude greater than those of AE and the clinically used anthracycline doxorubicin (DOX)-against several cancer cell lines with different levels of DOX resistance. Incubation with the synthetic AEGs induced cell death in less than one cell cycle, indicating that these compounds do not directly target the cell division mechanism. Confocal microscopy provided evidence that unlike DOX, AEGs accumulated in anthracycline-resistant tumor cells in which resistance is conferred by P-glycoprotein efflux pumps. The results of this study demonstrate that AEGs may serve as a promising scaffold for the development of cytotoxic agents capable of overcoming anthracycline resistance in tumor cells.

The effects of transforming growth factor β1, insulin-like growth factor 1 and leptin on the proliferation of fetal chondrocytes

One method to exogenously enhance the repair of articular cartilage is the local application of growth factors. This method is based on the chondrogenic effects of some agents and their potential ability to enhance cell migration. Human fetal chondrocytes were isolated and cultured. Their proliferation under the influence of different agents was microscopically evaluated. Fetal calf serum at 5% and 10% concentrations induced microscopically visible cell proliferation. Transforming growth factor beta one (5 and 10 ng/ml), insulin-like growth factor 1 (5 and 10 nmol/l) and leptin (1 and 2 ng/ml) accelerated proliferation of the cells towards the increasing gradient of the agents. Fibroblast growth factor beta (5 and 10 ng/ml), bone morphogenic protein two (10 ng/ml) and laminin (1 mg/ml) did not affect cell proliferation. This study suggests that different agents can play a role in the proliferation of fetal chondrocytes.