Lectin-carbohydrate complex evaluation by chemiluminescence.

In order to characterize the affinity between specific carbohydrate-binding proteins such as lectins, a model is proposed to study these interactions using a polysaccharide membrane to simulate such adsorption. Here, lectin-carbohydrate interactions were chemiluminescently investigated using lectins conjugated to acridinium ester (AE) and polysaccharides composed of their respective specific carbohydrates. The lectin-AE conjugates were incubated with discs (0.0314-0.6358 cm2) of phytagel, chitosan and carrageenan. The complex formation chemiluminescently detected followed the Langmuir isotherm from which constants were estimated. The association constant (Ka) and maximum binding sites on the membranes were 2.4 × 10-7 M-1 ±â€¯0.8 × 10-7 M-1 and 1.3 × 10-3 mol. mg-1 ± 0.3 × 10-3 mol. mg-1 (Con A); 0.9 × 10-6 M-1 ±â€¯0.4 × 10-6 M-1 and 0.021 × 10-3 mol. mg-1 ± 0.003 × 10-3 mol. mg-1 (WGA) and 2.0 × 10-6 M-1 ±â€¯0.9 × 10-6 M-1 and 0.069 × 10-3 mol. mg-1 ± 0.010 × 10-3 mol. mg-1 (PNA). The proposed model might be useful to study binding affinity and estimate the amount of binding not limited by the sugar content in the membrane.

Ultrastructural Assessment of 2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide activity on human breast adenocarcinoma cells.

The aim of the present study was to investigate ultrastructural changes induced by (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (APHCA) treatment on human breast adenocarcinoma cancer cells MCF-7, besides the evaluation of phosphatidylserine externalization and DNA fragmentation in treated cells. Cell viability analysis demonstrated concentration and time-manner cytotoxicity. Treated MCF-7 cells did not expose phosphatidylserine residues to the external plasma membrane surface and DNA fragmentation was not visualized by electrophoresis. Light microscopy showed compromised cell density and presence of vacuolization after APHCA treatment with 60µM. Scanning and transmission electron microscopies revealed hallmarks of autophagy, namely the presence of membrane bebbling and autophagosomes, besides shrunken cells and cell debris in treated MCF-7 cells. However, more specific tests such as the quantification of mammalian autophagy proteins are necessary to determine the kind of death that is trigged by APHCA.

Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives.

In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a-h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 10(4) to 1.0 × 10(6) M(-1) and quenching constants from -0.2 × 10(4) to 2.18 × 10(4) M(-1) indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N- (4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties.

Evaluation of glycophenotype in prostatic neoplasm by chemiluminescent assay.

This work aimed to evaluate the glycophenotype in normal prostate, bening prostatic hyperplasia (BPH) and prostatic adenocarcinoma (PCa) tissues by a chemiluminescent method. Concanavalin A (Con A), Ulex europaeus agglutinin (UEA-I) and Peanut agglutinin (PNA) lectins were conjugated to acridinium ester (lectins-AE). These conjugates remained capable to recognize their specific carbohydrates. Tissue samples were incubated with lectins-AE. The chemiluminescence of the tissue-lectin-AE complex was expressed in relative light units (RLU). Transformed tissues (0.25 cm(2) by 8 µm of thickness) showed statistical significant lower α-D-glucose/mannose (BPH: 226,931 ± 17,436; PCa: 239,520 ± 12,398) and Gal-ß(1-3)-GalNAc (BPH: 28,754 ± 2,157; PCa: 16,728 ± 1,204) expression than normal tissues (367,566 ± 48,550 and 409,289 ± 22,336, respectively). However, higher α-L-fucose expression was observed in PCa (251,118 ± 14,193) in relation to normal (200,979 ± 21,318) and BHP (169,758 ± 10,264) tissues. It was observed an expressive decreasing of the values of RLU by inhibition of the interaction between tissues and lectins-AE using their specific carbohydrates. The relationship between RLU and tissue area showed a linear correlation for all lectin-AE in both transformed tissues. These results indicated that the used method is an efficient tool for specific, sensitive and quantitative analyses of prostatic glycophenotype.