Cytotoxicity of doxrubicin loaded single-walled carbon nanotubes.

Carbon nanotube (CNTs) is a new alternative for efficient drug delivery and it has a great potential to change drug delivery system profile in pharmaceutical industry. One of the important advantage of CNTs is their needle-like, cylindrical shape. This shape provides a high surface area for multiple connections and adsorption onto for millions of therapeutic molecules. CNTs can be internalized by cells via endocytosis, passive diffusion and phagocytosis and release the drug with different effects like pH and temperature. The acidic nature of cancer cells and the susceptibility of CNTs to release the drug in the acidic environment have made it a promising area of research in cancer drug delivery. In this research, we investigated cell viability, cytotoxicity and drug delivery in breast cancer cell line by designing non-covalent single walled carbon nanotubes (SWNT)-doxorubicin (DOX) supramolecular complex that can be developed for cancer therapy. Applied high concentrations of DOX loaded SWNTs changed the actin structure of the cells and prevented the proliferation of the cells. It was showed that doxorubicin loaded SWNTs were more effective than free doxorubicin at relatively small concentrations. Once we applied same procedure for short and long (short: 1-1.3 µm; long: 2.5-4 µm) SWNTs and compared the results, more disrupted cell structure and reduction in cell proliferation were observed for long CNTs. DOX is bounded more to nanotubes in basic medium, less bound in acidic environment. Cancer cells were also examined for concentration at which they were effective by applying DOX and it was seen that 3.68 µM doxorubicin kills more than 55% of the cells.

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.

Depolymerization of actin filaments by Cucurbitacin I through binding G-actin.

Cucurbitacins have high economic value as they are a major source of food and have pharmacological properties. Cucurbitacin I (CuI) is a plant-derived natural tetracyclic triterpenoid compound that shows an anticancer effect via inhibiting the JAK2-STAT3 signaling pathway. The actin cytoskeleton is the most abundant protein in cells and regulates critical events through reorganization in cells. In this study, it is aimed at determining the direct effect of CuI on actin dynamics. The fluorescence profile of G-actin in the presence of CuI (1-200 nM) shifted to a higher temperature, suggesting that G-actin binds CuI and that G-actin-CuI is more thermally stable than the ligand-free form. CuI dose-dependently inhibited the polymerization of F-actin in vitro and disrupted actin filaments in endothelial cells. Docking and MD simulations suggested that CuI binds to the binding site formed by residues I136, I175, D154, and A138 that are at the interface of monomers in F-actin. The migration ability of cells treated with CuI for 24 h was significantly lower than the control group (p < .001). This study reveals the molecular mechanisms of CuI in the regulation of actin dynamics by binding G-actin. More importantly, this study indicates a novel role of CuI as an actin-targeting drug by binding directly to G-actin and may contribute to the mode of action of CuI on anticancer activities.

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 K(m) = 2.2 nM; V(max) = 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 the presence 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.

Valproic Acid Attenuated PTZ-induced Oxidative Stress, Inflammation, and Apoptosis in the SH-SY5Y Cells via Modulating the TRPM2 Channel.

Valproic acid (VPA) is one of the most widely used antiepileptic drugs. The protective role of VPA and the role of the TRPM2 channel in this mechanism in developing neuronal damage due to increased pentylenetetrazol (PTZ)-induced neurotoxicity in SH-SY5Y cells were not clarified. Here, we investigated the role of VPA via modulation of TRPM2 channel on cell death and oxidative neurotoxicity in SH-SY5Y cells. The SH-SY5Y cell toxicity model was constructed by treating SH-SY5Y cells with PTZ. The VPA and TRPM2 channel antagonist N-(p-amylcinnamoyl) anthranilic acid (ACA) were added to prevent neurotoxicity in PTZ-induced SH-SY5Y cells. The role of the VPA and TRPM2 channel was evaluated using an ELISA kit and patch-clamp. Primarily, antioxidant (GSH and GSH-Px) and oxidative stress (MDA and ROS) levels and inflammatory factors (IL-1ß, IL-6, and TNF-α) in cells were determined by ELISA kits. Then, TRPM2 channel activation in cells was detected using both the ELISA kit and patch-clamp methods. In addition, apoptosis and cell viability levels in cells were determined by performing PARP1, caspase-3, caspase-9, and CCK-8 assays by ELISA kits. Our results showed that the TRPM2 channel is vital in damage formation in PTZ-induced cells. Furthermore, we observed that VPA attenuated PTZ-induced neurotoxicity by suppressing cells' oxidative stress and inflammation, and reducing TRPM2 channel activation. In our study, in which the protective effect of VPA and the role of the TRPM2 channel in PTZ-induced SH-SY5Y cells were investigated for the first time, we can conclude that VPA treatment and TRPM2 channel blockade can suppress PTZ-induced neurotoxicity.

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.

Intracellular trafficking of diphtheria toxin and its mutated form, CRM197, in the endocytic pathway.

OBJECTIVE: Diphtheria toxin (DTx) is a well-characterized bacterial toxin. However, the endocytic pathway of the mutant of DTx, CRM197, which is used as an immunological adjuvant, has not yet been fully explained. The aim of this study was to investigate the intracellular trafficking of CRM197-loaded endosomes. METHODS: Human umbilical vein endothelial cells (HUVECs) were used in a cell culture. The effective incubation time was determined by transmission electron microscopy in toxin-treated cells. Density gradient centrifugation and ADP-ribosylation assay were used to isolate and detect toxin-loaded endosomal fractions. Endosomal fractions from CRM197-treated cells were elicited after 15 minutes of incubation and the presence of fragment A was demonstrated using Western blot. Immunofluorescence microscopy was used to identify endosomes in CRM197-treated endothelial cells. RESULTS: DTx-loaded endosomes were detected as enlarged vesicles in the perinuclear area with 15 minutes of toxin treatment. DTx-loaded endosomal fractions were determined by ADP-ribosyltransferase activity test and Western blot analysis. Enzymatic activity of the toxin-loaded endosomal fraction increased by 20% in actin cytoskeletal-damaged cells treated with cytochalasin D. The steps for the toxin treatment of HUVECs with DTx and obtaining endosomal fractions were repeated for CRM197. In the CRM197-loaded endosomal fraction, actin and Hsp90 were identified in addition to fragment A. Fluorescent images revealed that CRM197-loaded endosomes were co-localized with actin filaments and that Rab11, which signals the return to the plasma membrane, was more prominent than Rab7, the lysosomal pathway indicator. CONCLUSION: These results suggest that CRM197-loaded endosomes participate in the recycling pathway.

Endogenous ADP-ribosylation of eukaryotic elongation factor 2 and its 32 kDa tryptic fragment.

Eukaryotic elongation factor 2 (eEF-2) can undergo ADP-ribosylation in the absence of diphtheria toxin. The binding of free ADP-ribose and endogenous transferase-dependent ADP-ribosylation were distinct reactions for eEF-2, as indicated by different findings. Incubation of eEF-2 tryptic fragment 32/33 kDa (32F) with NAD was ADP-ribosylated and gave rise to the covalent binding of ADP-ribose to eEF-2. 32F was revealed to be at the C-terminal by Edman degradation sequence analysis. In our study, the elution of 32F from SDS-PAGE was ADP-ribosylated both in the presence and absence of diphtheria toxin. These results suggest that endogenous ADP-ribosylation of 32F might be related to protein synthesis. This modification appears to be important for the cell function.

Virtual screening of small molecules databases for discovery of novel PARP-1 inhibitors: combination of in silico and in vitro studies.

Poly(ADP-ribose) polymerase-1 (PARP-1) enzyme has critical roles in DNA replication repair and recombination. Thus, PARP-1 inhibitors play an important role in the cancer therapy. In the current study, we have performed combination of in silico and in vitro studies in order to discover novel inhibitors against PARP-1 target. Structure-based virtual screening was carried out for an available small molecules database. A total of 257,951 ligands from Otava database were screened at the binding pocket of PARP-1 using high-throughput virtual screening techniques. Filtered structures based on predicted binding energy results were then used in more sophisticated molecular docking simulations (i.e. Glide/standard precision, Glide/XP, induced fit docking - IFD, and quantum mechanics polarized ligand docking - QPLD). Potential high binding affinity compounds that are predicted by molecular simulations were then tested by in vitro methods. Computationally proposed compounds as PARP-1 inhibitors (Otava Compound Codes: 7111620047 and 7119980926) were confirmed by in vitro studies. In vitro results showed that compounds 7111620047 and 7119980926 have IC50 values of 0.56 and 63 µM against PARP-1 target, respectively. The molecular mechanism analysis, free energy perturbation calculations using long multiple molecular dynamics simulations for the discovered compounds which showed high binding affinity against PARP-1 enzyme, as well as structure-based pharmacophore development (E-pharmacophore) studies were also studied.

Interleukin-1ß effect on the endogenous ADP-ribosylation and phosphorylation of eukaryotic elongation factor 2.

Eukaryotic elongation factor 2 (eEF2) plays an important role in eukaryotic polypeptide chain elongation. Adenosine diphosphate (ADP)-ribosylation is a post-translational modification reaction that catalyzes the transfer of ADP-ribose group to eEF2 and this causes the inhibition of protein synthesis. Indeed, in the absence of diptheria toxin, endogenous ADP-ribosylation can occur. eEF2 is phosphorylated by eEF2 kinase which prevents binding to ribosomes thus inhibiting its activity. Increase in endogenous ADP-ribosylation level approximately 70-75 % was observed in IL-1ß treated HUVECs. Moreover, a 70 % rise of phosphorylation of eEF2 was measured. Alteration of endogenous ADP-ribosylation of eEF2 activity was related with cellular mono-ADP-ribosyltransferases (ADPrT). Increment of endogenous ADP-ribosylation on eEF2 did not seem to occur as a direct effect of IL-1ß; it arises from the activation of ADPrT. This 2.5 fold increase was abolished by ADPrT inhibitors. Due to these post-translational modifications, global protein synthesis is inhibited. After dephosphorylation of phospho-eEF2, around 20 % increase in protein synthesis was observed. In conclusion, systemic IL-1ß has an important role in the regulation of global protein synthesis.

Effect of oxidative stress on in vivo ADP-ribosylation of eukaryotic elongation factor 2.

Different lines of evidence indicate that eukaryotic elongation factor 2 (eEF2) can be ADP-ribosylated endogenously. The physiological significance of this reaction has, however, remained unclarified. In order to address this issue we investigated the in vivo ADP-ribosylation of eEF2 and the effect of oxidative stress thereon. The investigation revealed that the endogenous ADP-ribosylation of eEF2 is complex and can take place in K562 cell lysates either under the action of endogenous transferase from [adenosine-14C]NAD or by direct binding of free [14C]ADP-ribose. These two types of ADP-ribosylation were distinguished by use of different treatments based on the chemical stability of the respective bonds formed. Under standard culture conditions, in vivo labeling of eEF2 in the presence of [14C]adenosine was reversed to about 65% in the presence of diphtheria toxin and nicotinamide. This finding implied that the modification that took place under physiological circumstances was, mainly, of an enzymic nature. On the other hand, H2O2-promoted oxidative stress gave rise to a nearly two-fold increase in the extent of in vivo labeling of eEF2. This was accompanied by a loss of eEF2 activity in polypeptide chain elongation. Oxidative stress specifically inhibited the subsequent binding of free ADP-ribose to eEF2. The results thus provide evidence that endogenous ADP-ribosylation of eEF2 can also take place by the binding of free ADP-ribose. This nonenzymic reaction appears to account primarily for in vivo ADP-ribosylation of eEF2 under oxidative stress.

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 other's 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.

Endogenous ADP-ribosylation for eukaryotic elongation factor 2: evidence of two different sites and reactions.

Eukaryotic elongation factor 2 can undergo ADP-ribosylation in the absence of diphtheria toxin under the action of an endogenous transferase. The investigation which aimed to gain insight into the nature of endogenous ADP-ribosylation revealed that this reaction may be, in some cases, due to covalent binding of free ADP-ribose to elongation factor 2. Binding of free ADP-ribose, and NAD- and endogenous transferase-dependent ADP-ribosylation were suggested to be distinct reactions by different findings. Free ADP-ribose could bind to elongation factor 2 previously subjected to ADP-ribosylation by diphtheria toxin or endogenous transferase. The binding of free ADP-ribose was inhibited by neutral NH2OH, L-lysine and picrylsulfonate, whereas endogenous ADP-ribosyltransferase was inhibited by NAD glycohydrolase inhibitors and L-arginine. The ADP-ribosyl-elongation factor 2 adduct which formed upon binding of free ADP-ribose was resistant to neutral NH2OH, but decomposed almost completely upon treatment with NaOH. The product of endogenous transferase-dependent ADP- ribosylation was partially resistant to NH2OH and NaOH treatment. Moreover, this reaction was reversed in the presence of diphtheria toxin and nicotinamide. Both types of endogenous ADP-ribosylation gave rise to inhibition of polyphenylalanine synthesis. This study thus provides evidence for the presence of two different types of endogenous ADP-ribosylation of eukaryotic elongation factor 2. The respective sites involved in these reactions are distinct from one another as well as from diphthamide, the site of attack by diphtheria toxin.

Actin--an inhibitor of eukaryotic elongation factor activities.

An inhibitor of diphtheria toxin- and endogenous transferase-dependent ADP-ribosylation of eukaryotic elongation factor 2 (eEF2) has been found in the cytoplasmic fraction from rat liver. We provide evidence that this cytoplasmic inhibitor corresponds to actin, which gives rise also to inhibition of polyphenylalanine (polyPhe) synthesis. Both globular monomeric (G-actin) and filamentous (F-actin) forms of actin appear to be inhibitory on the action of elongation factors 1 and 2 (eEF1 and eEF2) in polyPhe synthesis with the inhibitory effect of G-actin proving to be stronger. Some component(s) in the postribosomal supernatant (S-130) fraction and also DNase I prevent actin-promoted inhibition of polyPhe synthesis.