Alexis J. Aletras

Inhibition of Angiogenesis by Anthracyclines and Titanocene Dichloridea

The anthracycline antibiotics, daunorubicin, doxorubicin, and epirubicin, which are widely used for treatment of malignancies, have been evaluated for their effect on angiogenesis in relation to the inhibition of collagenase type IV reported previously. In the chick chorioallantoic membrane (CAM) system of angiogenesis, anthracyclines inhibited vascular density at doses of 5-20 micrograms/disc as well as collagenous protein biosynthesis, which is a reliable index of angiogenesis. Similarly, all three anthracyclines inhibited tube formation in the in vitro system of angiogenesis using human umbilical vein endothelial cells (HUVECs) plated on Matrigel. The inhibition was dose-dependent and caused 50% inhibition at concentrations of 2.5-15 micrograms/mL. At concentrations of anthracyclines which prevented tube formation and angiogenesis, there were no cytotoxic effects, as evidenced by methylene blue uptake, and the growth of these endothelial cells was not inhibited. The experimental antitumor agent titanocene dichloride inhibited collagenase type IV from Walker 256 carcinosarcoma with IC50 approximately 0.2 mM. Titanocene also prevented angiogenesis in the CAM and tube formation by HUVECs on Matrigel at concentrations that were without effect on growth or cytotoxicity of endothelial cells or Walker 256 cells in culture. The antiangiogenic effect of the aforementioned antitumor agents at therapeutically attainable concentrations may explain, at least in part, their antitumor properties because angiogenesis is an essential process for tumor growth and metastasis. The antiangiogenic effect is, however, unrelated to metalloproteinase inhibition because higher concentrations are required for that effect than for inhibition of angiogenesis.

Extraction and fractionation of proteoglycans from squid skin

The extractability of squid skin proteoglycans with solutions of varying concentrations of guanidine-HCl, urea and SDS was studied; 4 M guanidine-HCl, being the best extractant, removed 95% of the tissue proteoglycans (glycosaminoglycan uronic acid). The proteoglycans in the 4 M guanidine-HCl extract were fractionated by repeated ion exchange and gel chromatography on Sepharose CL-4B to give three main populations, all being present in about equal proportions. Two populations (Kd 0.34 and 0.56) contained only chondroitin (proteochondroitin) and the other (Kd 0.50) only oversulphated chondroitin sulphate (oversulphated proteochondroitin sulphate). Two minor populations, one containing chondroitin and chondroitin sulphate and the other chondroitin sulphate and oversulphated chondroitin sulphate, were also identified.

In situ interaction of cartilage proteoglycans with matrix proteins

The interaction of proteoglycans with other matrix proteins via thiol-disulphide interchange was explored. Chick sternal cartilage was extracted with 4 M guanidine hydrochloride in the presence and absence of N-ethylmaleimide and the proteoglycans from the centrifugation A2 fractions were isolated. Those from extracts without N-ethylmaleimide were linked with reducible bonds with 10-15 proteins-glycoproteins including the link proteins, the 148 kDa and 36 kDa proteins. The same was observed with extracts of pig laryngeal and sheep nasal cartilage. The linked proteoglycans from sheep amounted to 2-3% of the extractable uronic acid and belonged to two populations. The major fraction was included by Sepharose 6B (Mr 110 000) had twice as long chondroitin sulphate chains, higher 4-sulphated residues and a high content of aspartic acid and leucine-rich protein. The larger proteoglycans had a size and composition similar to those of aggregating proteoglycans.

Degradation of intact basement membranes by human and murine tumor enzymes

Homogenates from malignant tumors, obtained from surgery specimens or from transplants of Walker 256 carcinosarcoma in rats, contained an enzyme activity capable of degrading intact 3H-acetylated basement membranes from bovine lens. The enzyme activity from murine tumor was purified about 7500-fold by (NH4)2SO4 fractionation, ion exchange and gel chromatography. The apparent molecular weight of the purified enzyme was approximately 50,000. The rate of degradation of 3H-labelled basement membrane by the murine tumor enzyme was reduced by addition of excess type IV collagen, but not of excess type I, type III or type V collagen. These results suggested specificity of this enzyme for type IV collagen. Inhibitors of serine proteinases, thiol proteinases and soybean trypsin inhibitor were without effect on the enzyme activity. Chelators such as 1,10-phenanthroline or EDTA reduced the activity to control levels, indicating that the enzyme activity was due to a metalloproteinase. Chromatographic and electrophoretic separation of the enzymatic products from 3H-labelled basement membrane and type IV collagen indicated that the enzyme activity was due to a type IV collagenase. The use of basement membrane in the native physiological state as a substrate for the study of basement membrane-degrading activity by homogenates of solid malignant tumors offers an in vitro model for the investigation of the metastatic potential of these tumors.

A solid-phase assay for quantitative analysis of sulfated glycosaminoglycans at the nanogram level. Application to tissue samples.

A sensitive and accurate solid-phase methodology for the quantitative analysis of glycosaminoglycans is described. Chondroitin-4-sulfate (CSA) was labelled with biotin hydrazide after the reaction of its carboxyl groups with it in the presence of carbodiimide. Polystyrene plates modified with sequential reaction with glutaraldehyde (GH) and spermine to possess amino groups were used to immobilize electrostatically the biotin labelled CSA. Exogenously added sulfated glycosaminoglycans (GAGS) [variously sulfated chondroitin sulfates and heparan sulfate (HS)] were found to compete to this immobilization in a concentration dependent mode, within a concentration range from 10 up to 300 ng/ml. Glycosaminoglycan-derived oligosaccharides competed to a degree similar to that of intact molecules. Hyaluronan (HA) and keratan sulfate (KS) did not compete the immobilization. The procedure was applied for the rapid and reproducible determination of the sulfated glycosaminoglycans in proteinase digests of small tissue samples or cell cultures with high sensitivity and accuracy.

Determination of the HNK-1 epitope (3-sulphated glucuronic acid) in intact chondroitin sulphates by ELISA. Application to squid skin proteoglycans and their oversulphated carbohydrate structures

The reactivity of the HNK-1 monoclonal antibody to chondroitin sulphates and derived disaccharides was studied using an ELISA inhibition test. The antibody readily reacted with its specific epitope (3-sulphated glucuronic acid) in intact chondroitin sulphates as well as with the equivalent oversulphated delta 4-disaccharides obtained by chondroitinase digestion and identified as sulphated at C-3 of the hexuronate. It is showed that by using the oversulphated delta 4-disaccharides as standards in an ELISA inhibition test, the amount of 3-sulphated glucuronic acid can be estimated also in the polymer preparations. When applying this ELISA test to the PG populations isolated from squid skin, most of the oversulphation seen in HPLC analyses of these preparations was found to be associated with 3-sulphation of the glucuronic acid.

ANALYSIS OF N-ACETYL AND N-GLYCOLYLNEURAMINIC ACID IN RAT SERUM AND TISSUES WITH WALKER 256 CARCINOMA BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Serum and tissue specimens from healthy Wistar rats and from rats with Walker 256 carcinoma were analysed for N-acetyl and N-glycolylneuraminic acid by high performance liquid chromatography (HPLC) as per-O-benzoylated derivatives. Both neuraminic acids were identified, while N-acetylneuraminic acid was the predominant sialic acid. Samples from rats with generalized metastasis showed a significant increase (45-80%) of total sialic acids. This phenomenon in serum is caused by the overproduction of sialic acids, as a result of synthesis of both types of neuraminic acids to a similar molar ratio. The increase of sialic acids in rat bones with metastatic cancer is mainly because of increased N-acetylneuraminic acid synthesis. These results suggest that the molecular mechanisms responsible for cancer metastasis in different tissues may be closely associated with increased synthesis of dominating neuraminic acid.

Proteoglycans from squid cranial cartilage: extraction and characterization

Abstract 1. 1. The majority of proteoglycans (85% of uronic acid) were extracted from squid cranial cartilage with 2% SDS. 2. 2. Conventional methods with 4 M Gdn HCl alone or with 4% Zwittergent, extracted only 30%. 3. 3. The proteoglycans were isolated by density gradient centrifugation. 4. 4. At least 25% of the extracted proteoglycans with 2% SDS do not form aggregates with hyaluronate. 5. 5. The proteoglycan monomers are of varying hydrodynamic size, those extracted with 4 M Gdn HCl being the smallest of average molecular weight 750,000.

Glutamate-like immunoreactivity in chick cerebellum and optic tectum

Glutamate was coupled via glutaraldehyde to bovine serum albumin. The conjugate was used for raising specific anti-glutamate antibodies. The purified antibody was used for immunostaining of chick cerebellum and optic tectum. Staining was intense in the molecular layer and in cell bodies of the granule cell layer. In the optic tectum a diffuse staining was detected in the superficial layers of stratum griseum fibrosum superficiale and in cell bodies especially in the layers a and e. Large cell bodies located in the stratum griseum centrale were also stained.

Identification of L-glutamate binding sites in chick brain by photoaffinity labeling

The photoaffinity cross-linker, N-hydroxysuccinimidyl-4-azido benzoate ester, was used to attach L-[3H]glutamate irreversibly to chick brain membranes. Electrophoretic analysis with sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed a major radioactive protein band with an apparent molecular weight of 45,600 +/- 300 Da. A second band with a smaller amount of radioactivity and with an apparent molecular weight of 28,300 +/- 500 Da was also detected. Photolabeling was inhibited by quisqualic acid.