Oxime-Linked Peptide-Daunomycin Conjugates as Good Tools for Selection of Suitable Homing Devices in Targeted Tumor Therapy: An Overview.

Chemotherapy is still one of the main therapeutic approaches in cancer therapy. Nevertheless, its poor selectivity causes severe toxic side effects that, together with the development of drug resistance in tumor cells, results in a limitation for its application. Tumor-targeted drug delivery is a possible choice to overcome these drawbacks. As well as monoclonal antibodies, peptides are promising targeting moieties for drug delivery. However, the development of peptide-drug conjugates (PDCs) is still a big challenge. The main reason is that the conjugates have to be stable in circulation, but the drug or its active metabolite should be released efficiently in the tumor cells. For this purpose, suitable linker systems are needed that connect the drug molecule with the homing peptide. The applied linker systems are commonly categorized as cleavable and non-cleavable linkers. Both the groups possess advantages and disadvantages that are summarized briefly in this manuscript. Moreover, in this review paper, we highlight the benefit of oxime-linked anthracycline-peptide conjugates in the development of PDCs. For instance, straightforward synthesis as well as a conjugation reaction proceed in excellent yields, and the autofluorescence of anthracyclines provides a good tool to select the appropriate homing peptides. Furthermore, we demonstrate that these conjugates can be used properly in in vivo studies. The results indicate that the oxime-linked PDCs are potential candidates for targeted tumor therapy.

Prevalence of Sjögren's syndrome in patients with dry mouth in the region of Central Hungary.

OBJECTIVE: One-third of the Hungarian population suffers from xerostomia. Since there is no evidence of the actual prevalence of Sjögren's syndrome (SS) in Hungary, this study aimed to evaluate the same. MATERIALS AND METHODS: Data were collected from the Faculty of Dentistry, Semmelweis University from 2008 to 2015. A diagnosis of SS was established based on the American College of Rheumatology and European League Against Rheumatism criteria. RESULTS: Of the 1076 patients examined with sicca symptoms, 188 patients had confirmed SS. Primary SS (pSS) was diagnosed in 135 patients and secondary SS (sSS) was confirmed in 53 patients. According to the available statistical records of the public health service of Hungary, there were an average of 16 (0.0014%, 5-26) newly diagnosed SS cases in the entire population and 141 SS patient-practitioner consultations (49-232) per 100,000 inhabitants in the country over the past 10 years (based on the past 10 years: 2011-2020). CONCLUSION: Results revealed that approximately 1/5th-1/6th of patients with sicca symptoms have SS, among whom 72% and 285 have pSS and sSS, respectively. Global Hungarian records simultaneously revealed that the number of both new diagnoses and doctor-SS patient encounters has significantly decreased (by 50%) yearly over the last decade.

Neutrophil-to-Lymphocyte Ratio Is an Independent Risk Factor for Coronary Artery Disease in Central Obesity.

Several inflammatory biomarkers were found to be associated with an increased risk of cardiovascular disease. Neutrophil-to-lymphocyte ratio (NLR) is a marker of subclinical inflammation that increases with the stress response. Visceral adiposity index (VAI) calculated as a combination of anthropometric and metabolic parameters reflects both the extent and function of visceral adipose tissue. Given the association of subclinical inflammation with both obesity and cardiovascular diseases, it is plausible that the inflammation-CVD association is modulated by the amount and function of adipose tissue. Thus, our aim was to examine the association between NLR and coronary artery calcium score (CACS), an intermediate marker of coronary artery disease in asymptomatic patients across VAI tertiles. Methods: Data from 280 asymptomatic participants of a cardiovascular screening program were analysed. In addition to the collection of lifestyle and medical history, a non-contrast cardiac CT scan and laboratory tests were performed on all participants. Multivariate logistic regression was conducted with CACS > 100 as the outcome and with conventional cardiovascular risk factors and NLR, VAI, and NLR by VAI tertile as predictors. Results: We found an interaction between VAI tertiles and NLR; NLR values were similar in the lower VAI tertiles, while they were higher in the CACS > 100 in the 3rd VAI tertile (CACS ≤ 100: 1.94 ± 0.58 vs. CACS > 100: 2.48 ± 1.1, p = 0.008). According to multivariable logistic regression, the interaction between NLR and VAI tertiles remained: NLR was associated with CACS > 100 in the 3rd VAI tertile (OR = 1.67, 95% CI 1.06-2.62, p = 0.03) but not in the lower tertiles even after adjustment for age, sex, smoking, history of hypertension, hyperlipidaemia, and diabetes mellitus, as well as high-sensitivity C-reactive protein. Our findings draw attention to the independent association between subclinical, chronic, systemic inflammation and subclinical coronary disease in obesity.

Level of advanced oxidation protein products is associated with subclinical atherosclerosis.

BACKGROUND: Oxidative stress is an important factor in the pathomechanism of atherosclerosis. Advanced oxidation protein products (AOPPs) are considered markers of oxidative stress. Thickening of the carotid intima-media layers indicates subclinical atherosclerosis and can be detected by carotid ultrasound. OBJECTIVE: Our aim was to examine the association between carotid intima-media thickness (CIMT) and the level of AOPPs. METHODS: Carotid duplex scans and measurements of AOPPs were performed on 476 participants of a cardiovascular population study. The presence of conventional cardiovascular risk factors was investigated with a questionnaire, physical examination, and laboratory tests. RESULTS: There was a positive correlation between maximum CIMT and the level of AOPPs only in the male population (r = 0.219, p = 0.033). Multivariate analysis has revealed that the association between AOPPs and mean or maximum CIMT was independent of cardiovascular risk factors (OR = 1.458, p = 0.004, and OR = 2.038, p < 0.001). CONCLUSIONS: Among males, the elevated level of AOPPs as a marker of oxidative stress may signal the existence of early atherosclerotic alterations.

The effects of mouthwashes in human gingiva epithelial progenitor (HGEPp) cells.

OBJECTIVES: The gingiva epithelium accounts for a significant proportion of the surface around the tooth. An inflammatory reaction occurs in the presence of bacterial biofilm, adhesion is reduced, and the depth of the sulcus gingivalis increases. The most common antiseptic agents in oral rinses are chlorhexidine digluconate (CHX) and cetylpyridinium chloride. We examined long-lasting effects of residual concentrations of eight commercially available rinses. Our main goals were (i) to analyze the effect of different chemical compositions on cell proliferation, (ii) to examine apoptosis, and (iii) cell morphology on human epithelial progenitor cell line (HGEPp). MATERIALS AND METHODS: Cell proliferation was measured in a real-time system (0-48 h) by impedimetry (xCELLigence). Apoptosis was measured with labeled Annexin-V (BD-FACScalibur). RESULTS: Changes in proliferation were measured at certain concentrations: (i) H2O2 proved to be cytotoxic at almost all concentrations; (ii) low concentrations of CHX (0.0001%; 0.0003%) were proliferation inducers, while higher concentrations were cytotoxic; (iii) for ClO2, advantageous proliferative effect was observed over a broad concentration range (0.06-6 ppm). In mouthwashes, additives in the formulation (e.g., allantoin) appeared to influence cellular responses positively. Apoptosis marker assay results suggested a low-level activation by the tested agents. CONCLUSIONS: Mouthwashes and their reference compounds proved to have concentration-dependent cytotoxic effects on human gingival epithelial cells. CLINICAL RELEVANCE: A better understanding of the effects of mouthwashes and their reference compounds is particularly important. These concentration-dependent effects (cytotoxic or proliferation inducing) interfere with human cells physiology while being used in the fight against the pathogenic flora.

Comparative study of hyperpure chlorine dioxide with two other irrigants regarding the viability of periodontal ligament stem cells.

OBJECTIVES: Periodontal ligament stem cells (PDLSCs) have an underlined significance as their high proliferative capacity and multipotent differentiation provide an important therapeutic potential. The integrity of these cells is frequently disturbed by the routinely used irrigative compounds applied as periodontal or endodontic disinfectants (e.g., hydrogen peroxide (H2O2) and chlorhexidine (CHX)). Our objectives were (i) to monitor the cytotoxic effect of a novel dental irrigative compound, chlorine dioxide (ClO2), compared to two traditional agents (H2O2, CHX) on PDLSCs and (ii) to test whether the aging factor of PDLSC cultures determines cellular responsiveness to the chemicals tested. METHODS: Impedimetry (concentration-response study), WST-1 assays (WST = water soluble tetrazolium salt), and morphology analysis were performed to measure changes in cell viability induced by the 3 disinfectants; immunocytochemistry of stem cell markers (STRO-1, CD90, and CD105) measured the induced mesenchymal characteristics. RESULTS: Cell viability experiments demonstrated that the application of ClO2 does not lead to a significant decrease in viability of PLDSCs in concentrations used to kill microbes. On the contrary, traditional irrigants, H2O2, and CHX are highly toxic on PDLSCs. Aging of PLDSC cultures (passages 3 vs. 7) has characteristic effects on their responsiveness to these agents as the increased expression of mesenchymal stem cell markers turns to decreased. CONCLUSIONS AND CLINICAL RELEVANCE: While the active ingredients of mouthwash (H2O2, CHX) applied in endodontic or periodontitis management have a serious toxic effect on PDLSCs, the novel hyperpure ClO2 is less toxic providing an environment favoring dental structure regenerations during disinfectant interventions.

Folate-Targeted Monodisperse PEG-Based Conjugates Made by Chemo-Enzymatic Methods for Cancer Diagnosis and Treatment.

This paper focuses on preliminary in vitro and in vivo testing of new bivalent folate-targeted PEGylated doxorubicin (DOX) made by modular chemo-enzymatic processes (FA2-dPEG-DOX2). A unique feature is the use of monodisperse PEG (dPEG). The modular approach with enzyme catalysis ensures exclusive γ-conjugation of folic acid, full conversion and selectivity, and no metal catalyst residues. Flow cytometry analysis showed that at 10 µM concentration, both free DOX and FA2-dPEG-DOX2 would be taken up by 99.9% of triple-negative breast cancer cells in 2 h. Intratumoral injection to mice seemed to delay tumor growth more than intravenous delivery. The mouse health status, food, water consumption, and behavior remained unchanged during the observation.

Polyisobutylene-New Opportunities for Medical Applications.

This paper presents the results of the first part of testing a novel electrospun fiber mat based on a unique macromolecule: polyisobutylene (PIB). A PIB-based compound containing zinc oxide (ZnO) was electrospun into self-supporting mats of 203.75 and 295.5 g/m2 that were investigated using a variety of techniques. The results show that the hydrophobic mats are not cytotoxic, resist fibroblast cell adhesion and biofilm formation and are comfortable and easy to breathe through for use as a mask. The mats show great promise for personal protective equipment and other applications.

Amphiphilic drug-peptide-polymer conjugates based on poly(ethylene glycol) and hyperbranched polyglycerol for epidermal growth factor receptor targeting: the effect of conjugate aggregation on in vitro activity.

Numerous peptide-drug conjugates have been developed over the years to enhance the specificity and selectivity of chemotherapeutic agents for tumour cells. In our present work, epidermal growth factor receptor targeting drug-peptide conjugates were prepared using GE11 and D4 peptides. To ensure the drug release, the cathepsin B labile GFLG spacer was incorporated between the targeting peptide and the drug molecule (daunomycin), which significantly increased the hydrophobicity and thereby decreased the water solubility of the conjugates. To overcome the solubility problem, drug-peptide-polymer conjugates with systematic structural variations were prepared, by linking poly(ethylene glycol) (PEG) or a well-defined amino-monofunctional hyperbranched polyglycerol (HbPG) directly or via a pentaglycine spacer to the targeting peptides. All the drug-peptide-polymer conjugates were water-soluble as confirmed by turbidimetric measurements. The results of the in vitro cell viability and cellular uptake measurements on HT-29 human colon adenocarcinoma cells proved that the HbPG and the PEG highly influenced the biological activity. The conjugation of the hydrophilic polymer resulted in the amphiphilic character of the conjugates, which led to self-aggregation and nanoparticle formation that decreased the cellular uptake above a specific aggregation concentration. On the other hand, the hydrodynamic volume and the different polymer chain topology of the linear PEG and the compact hyperbranched HbPG also played an important role in the biological activity. Therefore, in similar systems, the investigation of the colloidal properties is inevitable for the better understanding of the biological activity, which can reveal the structure-activity relationship of amphiphilic drug-peptide-polymer conjugates for efficient tumour targeting.

Functional and cell surface characteristics of periodontal ligament cells (PDLCs) on RGD-synthetic polypeptide conjugate coatings.

BACKGROUND AND OBJECTIVE: Periodontal ligament cells (PDLCs) are an important source for periodontal tissue healing and regeneration. Proper cell adhesion is a key for survival of anchorage-dependent cells and also initiates further intracellular signals for essential cellular functions. We aimed to test 3 different synthetic conjugates with integrin-binding RGD sequence (SAK-c[RGDfC], AK-c[RGDfC], and SAK-opn on the adhesion of human PDLCs and subsequent events including proliferation, migration, behavior of cell surface molecules, and osteogenic differentiation. MATERIALS AND METHODS: Synthetic peptides were synthesized by solid-phase technique and attached to branched chain polymeric polypeptides via thioether linkage. Simple adsorption method was used to coat tissue culture plastic or electric arrays. PDLCs were isolated from 24 surgically extracted human third molars. Cell adhesion and proliferation were measured with real-time impedimetric xCELLigence SP system. Cell migration assay was performed with Ibidi® Culture inserts. Cell surface antigens were detected using flow cytometry analysis. Osteogenic differentiation was assessed with alkaline phosphatase (ALP) assay and Alizarin Red S staining, and real-time qPCR was performed to analyze the osteoblast-related gene expression. Osteogenic differentiation and adipogenic differentiation of PDLCs were monitored by real-time Electrical Cell-Substrate Impedance Spectroscopy (ECIS). RESULTS: Primary outcome of this study relies on that all three synthetic RGD peptides improved PDLC adhesion (P < .05). When animal serum is absent in culture medium, SAK-c[RGDfC] and AK-c[RGDfC] elevated cell adhesion (P < .05). Cell migration was enhanced by SAK-c[RGDfC] and AK-c[RGDfC] (P < .05). After 1-week treatment, all synthetic peptides elevated CD105 (1.7- to 2.2-fold) and CD146 (1.3- to 1.5-fold) markers and caused different integrin patterns. ALP activity (1.4-fold) and ARS (1.8- and 2.0-fold) were increased by SAK-c[RGDfC] and AK-c[RGDfC] in absence of osteogenic supplements, and all the peptides supported the mineralization under osteogenic condition (P < .05). RT-qPCR revealed the upregulation of bone sialoprotein (5.0- to 7.8-fold), osteocalcin (2.3- to 2.7-fold), and ALP (1.9- to 2.3-fold) gene expression in osteogenesis-induced PDLCs. ECIS monitoring showed that higher impedance was generated by the osteogenic induction compared with the adipogenic or the non-induced (P < .05). CONCLUSIONS: Our study demonstrates that SAK-c[RGDfC] and AK-c[RGDfC] improved adhesion and migration of PDLCs and supported osteogenic differentiation of PDLCs. These cyclic RGD peptides proved to be applicable biocompatible material in regenerative medicine.

Novel Polycondensed Partly Saturated ß-Carbolines Including Ferrocene Derivatives: Synthesis, DFT-Supported Structural Analysis, Mechanism of Some Diastereoselective Transformations and a Preliminary Study of Their in vitro Antiproliferative Effects.

Use of a Pictet-Spengler reaction of tryptamine and l-tryptophan methyl ester and subsequent reduction of the nitro group followed by further cyclocondensation with aryl aldehydes and formyl-substituted carboxylic acids, including ferrocene-based components, furnished a series of diastereomeric 6-aryl-substituted 5,6,8,9,14,14b-hexahydroindolo[2',3':3,4]pyrido[1-c]-quinazolines and 5,5b,17,18-tetrahydroindolo[2',3':3,4]pyrido[1,2-c]isoindolo[2,1-a]quinazolin-11-(15bH)-ones with the elements of central-, planar and conformational chirality. The relative configuration and the conformations of the novel polycyclic indole derivatives were determined by 1H- and 13C-NMR methods supplemented by comparative DFT analysis of the possible diastereomers. The structure of one of the pentacyclic methyl esters with defined absolute configuration "S" was also confirmed by single crystal X-ray diffraction measurement. Accounting for the characteristic substituent-dependent diastereoselective formation of the products multistep mechanisms were proposed on the basis of the results of DFT modeling. Preliminary in vitro cytotoxic assays of the products revealed moderate-to-significant antiproliferative effects against PANC-1-, COLO-205-, A-2058 and EBC-1 cell lines that proved to be highly dependent on the stereostructure and on the substitution pattern of the pending aryl substituent.

Blood flow kinetics of a xenogeneic collagen matrix following a vestibuloplasty procedure in the human gingiva-An explorative study.

OBJECTIVES: The aim of the present study was to investigate temporal and spatial blood flow patterns following vestibuloplasty procedures using a collagen matrix (CM) to get an insight into the timing and direction of neovascularization in the CM. METHODS: Five patients were treated using a modified apically repositioned flap combined with a CM. Intraoral photographs and blood flow measurements by laser speckle contrast imaging were taken for 12 months. Thirty regions of interest in the graft and the surrounding mucosa were evaluated. The clinical parameters were assessed after 6 and 12 months. VEGF expression was analyzed in the wound fluid on days 2 and 4. RESULTS: At 6 months, the mean width of keratinized gingiva increased, but the thickness was unchanged. Scar formation was observed in all cases. Perfusion in the graft began to increase at the lateral and coronal edges and then spread concentrically toward the center. The apical side showed a significant delay in perfusion, the highest VEGF expression, and wound fluid production as well as the most abundant scar formation. CONCLUSIONS: Neovascularization occurs mainly from the lateral and coronal edges, which may limit the extent of the surgical area. Abundant scar formation may be explained by increased VEGF expression induced by prolonged ischemia in this area.

Effects of Gonadotropin-Releasing Hormone (GnRH) and Its Analogues on the Physiological Behaviors and Hormone Content of Tetrahymena pyriformis.

The unicellular Tetrahymena distinguishes structure-related vertebrate hormones by its chemosensory reactions. In the present work, the selectivity of hormone receptors was evaluated by analyzing the effects of various gonadotropin-releasing hormone (GnRH) analogs (GnRH-I, GnRH-III) as well as truncated (Ac-SHDWKPG-NH2) and dimer derivatives ([GnRH-III(C)]2 and [GnRH-III(CGFLG)]2) of GnRH-III on (i) locomotory behaviors, (ii) cell proliferation, and (iii) intracellular hormone contents of Tetrahymena pyriformis. The migration, intracellular hormone content, and proliferation of Tetrahymena were investigated by microscope-assisted tracking analysis, flow cytometry, and a CASY TT cell counter, respectively. Depending on the length of linker sequence between the two GnRH-III monomers, the GnRH-III dimers had the opposite effect on Tetrahymena migration. [GnRH-III(CGFLG)]2 dimer had a slow, serpentine-like movement, while [GnRH-III(C)]2 dimer had a rather linear swimming pattern. All GnRH-III derivatives significantly induced cell growth after 6 h incubation. Endogenous histamine content was uniformly enhanced by Ac-SHDWKPG-NH2 and GnRH-III dimers, while some differences between the hormonal activities of GnRHs were manifested in their effects on intracellular levels of serotonin and endorphin. The GnRH peptides could directly affect Tetrahymena migration and proliferation in a structure-dependent manner, and they could indirectly regulate these reactions by paracrine/autocrine mechanisms. Present results support the theory that recognition ability and selectivity of mammalian hormone receptors can be deduced from a phylogenetically ancient level like the unicellular Tetrahymena.

Apoptotic Effects of Drug Targeting Conjugates Containing Different GnRH Analogs on Colon Carcinoma Cells.

The wide range of cellular target reactions (e.g., antitumor) of gonadotropin-releasing hormone (GnRH) variants provides the possibility to develop multifunctional GnRH conjugates. The aim of our work was to compare the cytotoxic/apoptotic activity of different GnRH-based, daunorubicin (Dau)-linked conjugates with or without butyrated Lys in position 4 (4Lys(Bu)) at a molecular level in a human colorectal carcinoma cell line. Cell viability was measured by impedimetry, cellular uptake and apoptosis were studied by flow cytometry, and the expression of apoptosis-related genes was analyzed by qRT-PCR. The modification with 4Lys(Bu) resulted in an increased cytotoxic and apoptotic effects and cellular uptake of the GnRH-I and GnRH-III conjugates. Depending on the GnRH isoform and the presence of 4Lys(Bu), the conjugates could regulate the expression of several apoptosis-related genes, especially tumor necrosis factor (TNF), tumor protein p53 (TP53) and the members of growth-factor signaling. The stronger cytotoxicity of GnRH-I and GnRH-III conjugates containing 4Lys(Bu) was associated with a stronger inhibitory effect on the expression of growth-factor signaling elements in comparison with their 4Ser counterparts, in which the upregulation of TP53 and caspases (e.g., CASP9) seemed to play a more important role. We were able to provide further evidence that targeting the GnRH receptor could serve as a successful therapeutic approach in colon cancer, and GnRH-III-[4Lys(Bu),8Lys(Dau=Aoa)] proved to be the best candidate for this purpose.

Unravelling the Role of Trophoblastic-Derived Extracellular Vesicles in Regulatory T Cell Differentiation.

Regulatory T cells (Treg) are mandatory elements in the maintenance of human pregnancy, but their de novo differentiation has not been completely exposed. HSPE1 chaperone expressing trophoblast cells may have a role in it. Trophoblast-derived extracellular vesicles (EVs), either at the feto-maternal interface or in circulation, target CD4+ T cells. We hypothesized that HSPE1-associated trophoblastic cell line (BeWo)-derived EVs are active mediators of Treg cell differentiation. We proved at first that recombinant HSPE1 promote human Treg cell differentiation in vitro. Developing a CRISPR-Cas9 based HSPE1 knockout BeWo cell line we could also demonstrate, that EV-associated HSPE1 induces Treg development. Next-generation sequencing of miRNA cargo of BeWo-EVs characterized the regulatory processes of Treg polarization. By the use of single-cell transcriptomics analysis, seven Treg cell subtypes were distinguished and we demonstrated for the first time that the expression level of HSPE1 was Treg subtype dependent, and CAPG expression is characteristic to memory phenotype of T cells. Our data indicate that HSPE1 and CAPG may be used as markers for identification of Treg subtypes. Our results suggest, that trophoblastic-derived iEVs-associated HSPE1 and miRNA cargo have an important role in Treg cell expansion in vitro and HSPE1 is a useful marker of Treg subtype characterization.

Synthesis, Structure and In Vitro Cytotoxic Activity of Novel Cinchona-Chalcone Hybrids with 1,4-Disubstituted- and 1,5-Disubstituted 1,2,3-Triazole Linkers.

By means of copper(I)-and ruthenium(II)-catalyzed click reactions of quinine- and quinidine-derived alkynes with azide-substituted chalcones a systematic series of novel cinchona-chalcone hybrid compounds, containing 1,4-disubstituted- and 1,5-disubstituted 1,2,3-triazole linkers, were synthesized and evaluated for their cytotoxic activity on four human malignant cell lines (PANC-1, COLO-205, A2058 and EBC-1). In most cases, the cyclization reactions were accompanied by the transition-metal-catalyzed epimerization of the C9-stereogenic centre in the cinchona fragment. The results of the in vitro assays disclosed that all the prepared hybrids exhibit marked cytotoxicity in concentrations of low micromolar range, while the C9-epimerized model comprising quinidine- and (E)-1-(4-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenyl) fragments, connected by 1,5-disubstituted 1,2,3-triazole linker, and can be regarded as the most potent lead of which activity is probably associated with a limited conformational space allowing for the adoption of a relatively rigid well-defined conformation identified by DFT modelling. The mechanism of action of this hybrid along with that of a model with markedly decreased activity were approached by comparative cell-cycle analyses in PANC-1 cells. These studies disclosed that the hybrid of enhanced antiproliferative activity exerts significantly more extensive inhibitory effects in subG1, S and G2/M phases than does the less cytotoxic counterpart.

Kynurenic acid and its derivatives are able to modulate the adhesion and locomotion of brain endothelial cells.

The neuroprotective actions of kynurenic acid (KYNA) and its derivatives in several neurodegenerative disorders [characterized by damage to the cerebral endothelium and to the blood-brain barrier (BBB)] are well established. Cell-extracellular matrix (ECM) adhesion is supposedly involved in recovery of impaired cerebral endothelium integrity (endothelial repair). The present work aimed to investigate the effects of KYNA and its synthetic derivatives on cellular behaviour (e.g. adhesion and locomotion) and on morphology of the GP8 rat brain endothelial cell line, modeling the BBB endothelium. The effects of KYNA and its derivatives on cell adhesion were measured using an impedance-based technique, the xCELLigence SP system. Holographic microscopy (Holomonitor™ M4) was used to analyse both chemokinetic responses and morphometry. The GP8 cells proved to be a suitable model cell line for investigating cell adhesion and the locomotion modulator effects of kynurenines. KYNA enhanced cell adhesion and spreading, and also decreased the migration/motility of GP8 cells at physiological concentrations (10-9 and 10-7 mol/L). The derivatives containing an amide side-chain at the C2 position (KYNA-A1 and A2) had lower adhesion inducer effects compared to KYNA. All synthetic analogues (except KYNA-A5) had a time-dependent inhibitory effect on GP8 cell adhesion at a supraphysiological concentration (10-3 mol/L). The immobilization promoting effect of KYNA and the adhesion inducer activity of its derivatives indicate that these compounds could contribute to maintaining or restoring the protective function of brain endothelium; they also suggest that cell-ECM adhesion and related cell responses (e.g. migration/motility) could be potential new targets of KYNA.

Label-free profiling of cell dynamics: A sequence of impedance-based assays to estimate tumor cell invasiveness in vitro.

Dynamic properties of cancer cells, most notably their ability to migrate, have been correlated successfully with their invasive nature in vivo. To establish a stronger experimental basis for such a correlation we subjected five different cancer cell lines of well-defined metastatic potential to a sequence of three independent assays reporting on three different aspects of cell dynamics, namely (1) the kinetics of cell spreading, (2) cell shape fluctuations, and (3) cell migration. The sequentially applied assays correspond to different measuring modes of the well-established ECIS technique that is based on non-invasive and label-free impedance readings of planar gold-film electrodes that serve as the growth substrate for the cells under study. Every individual assay returned a characteristic parameter describing the behavior of the cell lines in that particular assay quantitatively. The parameters of all three assays were ranked to establish individual profiles of cell dynamics for every cell line that correlate favorably with the cells' invasive properties. The sequence of impedance-based assays described here requires only small cell populations (< 10.000 cells), it is highly automated and easily adapted to 96-well formats. It provides an in-depth dynamic profile of adherent cells that might be useful in other areas besides cancer research as well.

Ferrocene-Containing Impiridone (ONC201) Hybrids: Synthesis, DFT Modelling, In Vitro Evaluation, and Structure⁻Activity Relationships.

Inspired by the well-established clinical evidence about the interplay between apoptotic TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) mechanism and reactive oxygen species (ROS)-mediated oxidative stress, a set of novel ONC201 hybrids containing the impiridone core and one or two differently positioned ferrocenylalkyl groups were synthesised in our present work. These two types of residues have been implicated in the aforementioned mechanisms associated with cytotoxic activity. A straightforward, primary amine-based synthetic approach was used allowing the introduction of a variety of N-substituents into the two opposite regions of the heterocyclic skeleton. Reference model compounds with benzyl and halogenated benzyl groups were also synthesised and tested. The in vitro assays of the novel impiridones on five malignant cell lines disclosed characteristic structure-activity relationship (SAR) featuring significant substituent-dependent activity and cell-selectivity. A possible contribution of ROS-mechanism to the cytotoxicity of the novel metallocenes was suggested by density functional theory (DFT)studies on simplified models. Accordingly, unlike the mono-ferrocenylalkyl-substituted products, the compounds containing two ferrocenylalkyl substituents in the opposite regions of the impiridone core display a much more pronounced long-term cytotoxic effect against A-2058 cell line than do the organic impiridones including ONC201 and ONC212. Furthermore, the prepared bis-metallocene derivatives also present substantial activity against COLO-205- and EBC-1 cell lines.

Drug targeting to decrease cardiotoxicity - determination of the cytotoxic effect of GnRH-based conjugates containing doxorubicin, daunorubicin and methotrexate on human cardiomyocytes and endothelial cells.

Background: Cardiomyopathy induced by the chemotherapeutic agents doxorubicin and daunorubicin is a major limiting factor for their application in cancer therapy. Chemotactic drug targeting potentially increases the tumor selectivity of drugs and decreases their cardiotoxicity. Increased expression of gonadotropin-releasing hormone (GnRH) receptors on the surface of tumor cells has been reported. Thus, the attachment of the aforementioned chemotherapeutic drugs to GnRH-based peptides may result in compounds with increased therapeutic efficacy. The objective of the present study was to examine the cytotoxic effect of anticancer drug-GnRH-conjugates against two essential cardiovascular cell types, such as cardiomyocytes and endothelial cells. Sixteen different previously developed GnRH-conjugates containing doxorubicin, daunorubicin and methotrexate were investigated in this study. Their cytotoxicity was determined on primary human cardiac myocytes (HCM) and human umbilical vein endothelial cells (HUVEC) using the xCELLigence SP system, which measures impedance changes caused by adhering cells on golden electrode arrays placed at the bottom of the wells. Slopes of impedance-time curves were calculated and for the quantitative determination of cytotoxicity, the difference to the control was analysed. Results: Doxorubicin and daunorubicin exhibited a cytotoxic effect on both cell types, at the highest concentrations tested. Doxorubicin-based conjugates (AN-152, GnRH-III(Dox-O-glut), GnRH-III(Dox-glut-GFLG) and GnRH-III(Dox=Aoa-GFLG) showed the same cytotoxic effect on cardiomyocytes. Among the daunorubicin-based conjugates, [4Lys(Ac)]-GnRH-III(Dau=Aoa), GnRH-III(Dau=Aoa-YRRL), {GnRH-III(Dau=Aoa-YRRL-C)}2 and {[4N-MeSer]-GnRH-III(Dau-C)}2 had a significant but decreased cytotoxic effect, while the other conjugates - GnRH-III(Dau=Aoa), GnRH-III(Dau=Aoa-K(Dau=Aoa)), [4Lys(Dau=Aoa)]-GnRH-III(Dau=Aoa), GnRH-III(Dau=Aoa-GFLG), {GnRH-III(Dau-C)}2 and [4N-MeSer]-GnRH-III(Dau=Aoa) - exerted no cytotoxic effect on cardiomyocytes. Mixed conjugates containing methotrexate and daunorubicin - GnRH-III(Mtx-K(Dau=Aoa)) and [4Lys(Mtx)]-GnRH-III(Dau=Aoa) - showed no cytotoxic effect on cardiomyocytes, as well. Conclusion: Based on these results, anticancer drug-GnRH-based conjugates with no cytotoxic effect on cardiomyocytes were identified. In the future, these compounds could provide a more targeted antitumor therapy with no cardiotoxic adverse effects. Moreover, impedimetric cytotoxicity analysis could be a valuable technique to determine the effect of drugs on cardiomyocytes.