Başak Varol

Iron(III) and nickel(II) complexes as potential anticancer agents: synthesis, physicochemical and structural properties, cytotoxic activity and DNA interactions

Template reactions of 2-hydroxy-R-benzaldehyde-S-methylisothiosemicarbazones (R = 3-methoxy or 4-hydroxy) with the corresponding aldehydes in the presence of FeCl3 and NiCl2 yielded N1,N4-disalicylidene chelate complexes. The compounds were characterized by means of elemental and spectroscopic methods. The structure of complex 1 was determined by X-ray single crystal diffraction. Crystal data (Mo Kα; 296 K) are as follows: monoclinic space group P21/c, a = 12.9857(8) A, b = 7.8019(4) A, c = 19.1976(12) A, β = 101.655(5)°, Z = 4. Cytotoxic effects of the compounds were evaluated by the MTT assay in K562 leukemia, ECV304 endothelial and normal mononuclear cells, and DNA fragmentation analysis using the diphenylamine reaction was performed. The DNA binding capacity of thiosemicarbazones at IC50 and different concentrations was investigated. The DNA fragmentation percentage of compound treated cells was higher than that of non-treated control cells but was higher for compound 3 (84%) compared to the others. The interaction of compounds 1–4 and DNA was investigated voltammetrically by using nucleic acid modified electrodes after the double stranded fish sperm DNA (fsDNA), or poly(dA)·poly(dT), was immobilized onto the surface of pencil graphite electrodes (PGEs). Accordingly, the oxidation signals of DNA bases, guanine and adenine, were measured by using differential pulse voltammetry (DPV). The changes in the signals of guanine and adenine were evaluated before and after the interaction process. The results indicated that compound 3 was cytotoxic at very low concentrations in K562 leukemia cells unlike other cells and that could damage the DNA double stranded form, specifically the adenine base. Therefore, it may have a selective antileukemic effect and drug potential.

Interaction of diphtheria toxin (fragment A) with actin

It was shown by gel filtration and viscosity measurements that N‐terminal fragment (FA) of diphtheria toxin (DT) can interact with both G‐ and F‐actin (filamentous actin). Elution profiles on Sephadex G‐100 indicated the formation of a binary complex of fragment A (FA) with globular actin monomer (G‐actin), which was inhibited by gelsolin. Deoxyribonuclease I (DNase I) in turn appeared to interact with this complex. Tritiated FA was found to bind to F‐actin stoichiometrically. This binding was inhibited again by gelsolin and G‐actin, but not by DNase I. The binding of FA inhibited polymerization of G‐actin and induced a time‐dependent breakdown of F‐actin under polymerization conditions. Inhibition of its ADP‐ribosyltransferase activity did not have any effect on the interactions of FA with actin. FA interacted with actin also in the cell. After treatment of human umbilical vein endothelial cells (HUVEC) with biotin‐labeled DT, Western blot analysis revealed predominantly the presence of actin in affinity‐isolated complexes of the labeled FA. Similarly, FA was found in immunoaffinity‐isolated complexes of actin. Copyright

On diphtheria toxin fragment A release into the cytosol--cytochalasin D effect and involvement of actin filaments and eukaryotic elongation factor 2.

Diphtheria toxin has been well characterized in terms of its receptor binding and receptor mediated endocytosis. However, the precise mechanism of the cytosolic release of diphtheria toxin fragment A from early endosomes is still unclear. Various reports differ regarding the requirement for cytosolic factors in this process. Here, we present data indicating that the distribution of actin filaments due to cytochalasin D action enhances the retention of diphtheria toxin in early endosomes. Treating cells with cytochalasin D reduces the cytosolic fragment A activity and leads to changes in the intracellular distribution and size of early endosomes with toxin cargo. F-actin and eukaryotic elongation factor 2 can promote fragment A release from toxin-loaded early endosomes in an in vitro translocation system. Moreover, these proteins bind to toxin-loaded early endosomes in vitro and promote each others binding. They are thus thought to be involved in the cytosolic release of fragment A. Finally, ADP-ribosylation of eukaryotic elongation factor 2 is shown to inhibit fragment A release and, via a feed-back mechanism, to account for the minute amounts of fragment A normally found in the cytosol.

Clinical significance of serum ADP-ribosylation and NAD glycohydrolase activity in patients with colorectal cancer

The objective of this study was to evaluate the clinical significance of serum ADP-ribosylation and NAD glycohydrolase activity in patients with colorectal cancer (CRC). A total of 108 patients with CRC who underwent curative surgery and 20 healthy volunteers were enrolled in this study. ADP-ribosylation and NAD glycohydrolase activity levels were determined. The association of ADP-ribosylation and NAD glycohydrolase with clinical and laboratory factors and their impact on overall survival (OS) and disease free survival (DFS) were shown. The preoperative ADP-ribosylation and NAD glycohydrolase activity levels were significantly higher in patients with CRC than in the control group (p < 0.001). ADP-ribosylation and NAD glycohydrolase activity levels were correlated with tumor stage (p = 0.05, p = 0.001), stage of disease (p < 0.001, p < 0.001), serum CEA level (p < 0.001, p < 0.001), and site of lesion (p < 0.001, p < 0.001), respectively. Patients with high ADP-ribosylation had significantly unfavorable OS and DFS compared with those with lower levels (p < 0.001, p < 0.001), respectively. Moreover, the patients with high NAD glycohydrolase activity showed significantly worse OS and DFS rates, similar to ADP-ribosylation. Serum levels of ADP-ribosylation and NAD glycohydrolase activity correlate well with tumor stage, stage of disease, serum CEA level, and site of lesion. In conclusion, elevated levels of preoperative ADP-ribosylation and NAD glycohydrolase levels in serum are associated with poor prognosis in patients with CRC.

THE CYTOTOXIC EFFECT OF DIPHTHERIA TOXIN ON THE ACTIN CYTOSKELETON

Diphtheria toxin (DT) and its N-terminal fragment A (FA) catalyse the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) into a covalent linkage with eukaryotic elongation factor 2 (eEF2). DT-induced cytotoxicity is versatile, and it includes DNA cleavage and the depolymerisation of actin filaments. The inhibition of the ADP-ribosyltransferase (ADPrT) activity of FA did not affect the deoxyribonuclease activity of FA or its interaction with actin. The toxin entry rate into cells (HUVEC) was determined by measuring the ADP-ribosyltransferase activity. DT uptake was nearly 80% after 30 min. The efficiency was determined as Km = 2.2 nM; Vmax = 0.25 pmol.min−1. The nuclease activity was tested with hyperchromicity experiments, and it was concluded that G-actin has an inhibitory effect on DT nuclease activity. In thepresence of DT and mutant of diphtheria toxin (CRM197), F-actin depolymerisation was determined with gel filtration, WB and fluorescence techniques. In the presence of DT and CRM197, 60–65% F-actin depolymerisation was observed. An in vitro FA-actin interaction and F-actin depolymerisation were reported in our previous paper. The present study thus confirms the depolymerisation of actin cytoskeleton in vivo.

Toxin Structure, Delivery and Action

Diphtheria toxin (DTx) consists of a 535 amino acids polypeptide and contains the following three domains: the amino terminal fragment A (FA or catalytic C-domain) that catalyses the transfer of an ADP-ribosyl group of NAD+ to a post-translationally modified histidine (diphthamide) residue on eukaryotic elongation factor 2 (eEF2) and inhibits protein synthesis. Fragment B (FB) consist of the carboxy terminal receptor-binding R-domain, and the translocation (or transmembrane) T-domain. Following binding to its cell surface receptor via R-domain, DTx is internalized through the clathrin-dependent endocytosis. The acid pH created in the early endosomes triggers a conformational change in the toxin leading to the insertion of the T and C-domains in the membrane. The catalytic domain is then translocated into the cytosol across the early endosomal membrane and protein synthesis inhibition occurs. DTx-induced cytotoxicity is versatile, and it includes DNA cleavage and the depolymerisation of actin filaments. FA can interact with both G and F-actin. The binding to the latter appears to take place at the plus end of the filament blocking further polymerisation and it was concluded that G-actin has an inhibitory effect on DTx nuclease activity.

Cross-reacting material 197 (CRM197) affects actin cytoskeleton of endothelial cells.

CRM197, cross-reacting material 197, is a mutant of diphtheria toxin (DTx). CRM197 is used in pharmacology as a carrier protein. It has been recently shown that CRM197 causes breakdown in actin filaments. In order to show intracellular localization of CRM197 and visualize cell structure via actin cytoskeleton, endothelial cells were cultured and subjected to CRM197 in vitro. To address the interaction between CRM197 and actin both experimental and theoretical studies were carried out. Colocalization of CRM197 with actin filaments was determined by immunofluorescence microscopy. Following 24-hour incubation, the loss of cell-cell contact between cells was prominent. CRM197 was shown to bind to G-actin by gel filtration chromatography, and this binding was confirmed by Western blot analysis of eluted samples obtained following chromatography. Based on crystal structure, docked model of CRM197-actin complex was generated. Molecular dynamics simulation revealed that Lys42, Cys218, Cys233 of CRM197 interacts with Gly197, Arg62 and Ser60 of G-actin, respectively. CRM197 binding to G-actin, colocalization of CRM197 with actin filament, and actin cytoskeleton rearrangement resulting in the loss of cell-cell contact show that actin comes into sight as target molecule for CRM197.