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.

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.

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.

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.

Impedimetric Analysis of the Effect of Decellularized Porcine Heart Scaffold on Human Fibrosarcoma, Endothelial, and Cardiomyocyte Cell Lines.

BACKGROUND Experiments on porcine heart scaffold represent significant assays in development of immunoneutral materials for cardiac surgery. Characterization of cell-cell and cell-scaffold interactions is essential to understand the homing process of cardiac cells into the scaffolds. MATERIAL AND METHODS In the present study, the highly sensitive and real-time impedimetric technique of xCELLigence SP was used to monitor cell adhesion, which is the key process of recellularization in heart scaffolds. Our objectives were: (i) to characterize the effect of decellularized porcine heart scaffold on cell adhesion of human cardiovascular cells potentially used in the recellularization process; and (ii) to investigate cell-extracellular matrix element interactions for building artificial multi-layer systems, applied as cellular models of recellularization experiments. Human fibrosarcoma, endothelial, and cardiomyocyte cells were investigated and the effect of decellularized porcine heart scaffold (HS) and fibronectin on cell adhesion was examined. Adhesion was quantified as slope of curves. RESULTS Heart scaffold had neutral effect on cardiomyocytes as well as on endothelial cells. Adhesion of cardiomyocytes was increased by fibronectin (1.480±0.021) compared to control (0.745±0.029). The combination of fibronectin and HS induced stronger adhesion of cardiomyocytes (2.407±0.634) than fibronectin alone. Endothelial and fibrosarcoma cells showed similarly strong adhesion profiles with marked enhancer effect by fibronectin. CONCLUSIONS Decellularized porcine HS does not inhibit adhesion of human cardiovascular cells at the cell biological level, while fibronectin has strong cell adhesion-inducer effect, as well as an enhancer effect on activity of HS. Consequently, decellularized porcine hearts could be used as scaffolds for recellularization with cardiomyocytes and endothelial cells with fibronectin acting as a regulator, leading to construction of working bioartificial hearts.

Metastasis-associated S100A4 is a specific amine donor and an activity-independent binding partner of transglutaminase-2.

Transglutaminase-2 (TG2) is best known as a Ca(2+)-dependent cross-linking enzyme; however, some of its extracellular matrix-related functions are independent of its catalytic activity and include matrix remodelling, adhesion and migration. S100A4 belongs to the Ca(2+)-binding EF-hand S100 protein family and acts both intra- and extra-cellularly through binding to various partners. It regulates cell migration and its overexpression is strongly associated with metastasis and poor survival in various cancers. It has recently been suggested that TG2 mediates S100A4-dependent tumour cell migration. In the present study we provide evidence that S100A4 is an interacting partner and also a specific amine donor of TG2. TG2 incorporates a glutamine donor peptide to Lys(100) in the C-terminal random coil region of S100A4. Importantly, the enzyme activity is not necessary for the interaction: S100A4 also binds to TG2 in the presence of a specific inhibitor that keeps the enzyme in an open conformation, or to an enzymatically inactive mutant. We also found that S100A4 considerably enhances TG2-mediated adhesion of A431 epithelial carcinoma cells to the extracellular matrix. This role is independent of enzyme activity and requires the open conformation of TG2. We propose that S100A4 stabilizes the open conformation of TG2, which binds to its cell-surface receptor in this state and increases cell adhesion.

Effect of vasoactive peptides in Tetrahymena: chemotactic activities of adrenomedullin, proadrenomedullin N-terminal 20 peptide (PAMP) and calcitonin gene-related peptide (CGRP).

Adrenomedullin (AMD), proadrenomedullin N-terminal 20 peptide (PAMP) and calcitonin gene-related peptide (CGRP) were studied for chemotaxis, chemotactic selection and G-actin/F-actin transition in Tetrahymena. The aim of the experiments was to study the effects of two different peptides encoded by the same gene compared to a peptide related to one of the two, but encoded by a different gene, at a low level of phylogeny. The positive, chemotactic effect of ADM and the strong negative, chemorepellent effect of PAMP suggest that in Tetrahymena, the two peptides elicit their chemotactic effects via different signalling mechanisms. The complexity of swimming behaviour modulated by the three peptides underlines that chemotaxis, chemokinesis and some characteristics of migratory behaviour (velocity, tortuosity) are working as a sub-population level complex functional unit. Chemotactic responsiveness to ADM and CGRP is short-term, in contrast to PAMP, which as a chemorepellent ligand, has the ability to select sub-populations with negative chemotactic responsiveness. The different effects of ADM and PAMP on the polymerization of actin networks show that the microtubular structure of cilia is more essential to chemotactic response than are transitions of the actin network. The results draw attention to the characteristic effects of vasoactive peptides at this low level of phylogeny.

Effect of SXWS/WSXWS peptides on chemotaxis and adhesion of the macrophage-like cell line J774.

WSXWS motif is a conserved amino acid sequence that is present in type I cytokine receptors. This motif that can be found both in the ligand binding chains and signal transducer molecule of the receptors with different amino acids at the position "X" plays a role in the receptor folding, ligand binding and signal transduction as well. Structural analysis proved that WSEWS motif of IL-6R is located in a highly accessible location in the protein. Structural properties and chemotaxis of a tetrapeptide library with SXWS sequence, where X was the 19 proteinogenic amino acids except cystein were systematically studied earlier. It has been proved that C-terminal amidation and the identity of amino acid X had a pronounced influence on the chemotactic properties but less of the structure of the peptides. Here, we present our findings on the effect of a tetrapeptide and a pentapeptide library with the sequence of SXWS and WSXWS on the chemotaxis and adhesion of J774 murine macrophage cell line. We studied the effect of the presence/absence of N-terminal tryptophan and the different amino acids at the X position on these physiological responses. Results indicated that amino acid X had a marked influence on chemotaxis, adhesion as well as on proliferation induced by (W)SXWS peptides. Elongation of SXWS sequence with a tryptophan at the N terminus also altered pronouncedly all the physiological responses of the cells studied. A good correlation could be observed between the chemotaxis and the proliferation and physicochemical parameters of the amino acid X.

Growth and differentiation factor-15: A link between inflammaging and cardiovascular disease.

Age-related disorders are closely linked to the accumulation of senescent cells. The senescence-associated secretory phenotype (SASP) sustains and progresses chronic inflammation, which is involved in cellular and tissue dysfunction. SASP-related growth and differentiation factor-15 (GDF-15) is an immunoregulatory cytokine that is coupled to aging and thus may have a regulatory role in the development and maintenance of atherosclerosis, a major cause of cardiovascular disease (CVD). Although the effects of GDF-15 are tissue-specific and dependent on microenvironmental changes such as inflammation, available data suggest that GDF-15 has a significant role in CVD. Thus, GDF-15 is a promising biomarker and potential therapeutic target for atherosclerotic CVD.

Association of growth and differentiation factor-15 with coronary artery calcium score and ankle-brachial index in a middle-aged and elderly Caucasian population sample free of manifest cardiovascular disease.

Growth and differentiation factor-15 (GDF-15) is a stress-associated cytokine of the transforming growth factor-ß superfamily. The inflammatory and angiogenic effects of GDF-15 in atherosclerosis are controversial, and its correlation with the long asymptomatic phase of the disease is not well understood. Coronary artery calcium score (CACS) and ankle-brachial index (ABI) are sensitive markers of subclinical atherosclerosis. To date, only a few studies have examined the impact of GDF-15 on coronary artery calcification, and the association between GDF-15 and ABI has not been evaluated. Therefore, we aimed to investigate the possible relationship between serum GDF-15 concentrations and CACS and ABI in a Caucasian population sample of middle-aged (35-65 years) and elderly (> 65 years) people. In addition to recording demographic and anthropometric characteristics, atherosclerotic risk factors, and laboratory tests including serum HDL-cholesterol, LDL-cholesterol, hemoglobin A1c (HbA1c), high-sensitivity C-reactive protein, and N-terminal pro-B-type natriuretic peptide (NT-proBNP); GDF-15 level, cardiac computed tomography, and ABI measurements were also performed. A total of 269 asymptomatic individuals (men, n = 125; median age, 61.5 [IQR, 12.7] years) formed the basis of this study. Participants were divided into two groups according to their age (middle-aged, n = 175 and elderly, n = 94). Hypertension and diabetes mellitus were significantly more prevalent and CACS values and HbA1c, NT-proBNP, and GDF-15 levels were significantly higher (all p < 0.001) in the elderly group compared to the middle-aged group. Multivariate ridge regression analysis revealed a significant positive association between GDF-15 and CACS (middle-aged group: ß = 0.072, p = 0.333; elderly group: ß = 0.148, p = 0.003), and between GDF-15 and ABI (middle-aged group: ß = 0.062, p = 0.393; elderly group: ß = 0.088, p = 0.041) only in the elderly group. Our results show that GDF-15 is not only a useful biomarker of inflammation but can also predict early signs of asymptomatic atherosclerosis, especially in elderly people with chronic systemic inflammation associated with aging (inflammaging).

Chemotaxis induced by SXWS tetrapeptides in Tetrahymena--overlapping chemotactic effects of SXWS sequences and their identical amino acids.

The chemotactic potential of SXWS peptides and the components of the extracellular domain of cytokine receptors were investigated in Tetrahymena as a functional index of substitution with different amino acids in the position 'X' of the tetrapeptide. Data obtained demonstrate that position X plays a special determining role in the ligand, SEWS and STWS possess extremely strong chemoattractant ability, and aromatic amino acids result in chemorepellent ligands. Diverse effects of structurally related molecules, for example, SNWS-SDWS, demonstrate a highly sensitive discrimination potential in the applied model system. Physicochemical characteristics (hydropathy, residue size, and solvent-exposed area) of the amino acids were correlated with the chemotactic activity. Data obtained by computer-assisted conformation analysis of SXWS peptides and the highly overlapping chemotactic effects of the investigated SXWS peptides as well as the presence of the amino acids in the 'X' position indicate that member 'X' of the SXWS sequence performs a special role in interactions with the chemotaxis receptors in the membrane.

Basic cell physiological activities (cell adhesion, chemotaxis and proliferation) induced by selegiline and its derivatives in Mono Mac 6 human monocytes.

Selegiline (R-deprenyl), a monoamine oxidase-B (MAO-B) inhibitor, has complex pharmacological effect that contributes to treatment of neurodegenerative diseases such as Parkinson's and presumably Alzheimer's disease and might work as an inhibitor of tumor growth. In respect of tumorigenesis and metastasis formation, the controlled modifications of adhesion and migration have high therapeutic significance. In the present study, our purpose was to investigate cell physiological responses (adhesion, chemotaxis and proliferation) induced by selegiline, its metabolites and synthetic derivatives and to find some correlations between the molecular structure and the reported antitumor behavior of the derivatives. Our results demonstrated that both R- and S-deprenyls have the potency to elicit increased adhesion and a chemorepellent activity in monocyte model (Mono Mac 6 cell line derived from monoblastic leukemia); however, only the R-enantiomer proved to be cytotoxic. Among the metabolites R-amphetamine has retained the adhesion inducer and the chemorepellent effect of the parent drug on the most significant level. In contrast, a reversed chemotactic effect and an improved cytotoxic character were detected in the presence of fluoro group (p-fluoro-S-deprenyl). In summary, the adhesion inducer activity, chemorepellent and advantageous cytotoxic effects of selegiline and some derivatives indicate that these drug molecules might have inhibitory effects in metastasis formation in primary tumors.

Investigation of the Antitumor Effects of Tamoxifen and Its Ferrocene-Linked Derivatives on Pancreatic and Breast Cancer Cell Lines.

Tamoxifen is a long-known anti-tumor drug, which is the gold standard therapy in estrogen receptor (ER) positive breast cancer patients. According to previous studies, the conjugation of the original tamoxifen molecule with different functional groups can significantly improve its antitumor effect. The purpose of this research was to uncover the molecular mechanisms behind the cytotoxicity of different ferrocene-linked tamoxifen derivates. Tamoxifen and its ferrocene-linked derivatives, T5 and T15 were tested in PANC1, MCF7, and MDA-MB-231 cells, where the incorporation of the ferrocene group improved the cytotoxicity on all cell lines. PANC1, MCF7, and MDA-MB-231 express ERα and GPER1 (G-protein coupled ER 1). However, ERß is only expressed by MCF7 and MDA-MB-231 cells. Tamoxifen is a known agonist of GPER1, a receptor that can promote tumor progression. Analysis of the protein expression profile showed that while being cytotoxic, tamoxifen elevated the levels of different tumor growth-promoting factors (e.g., Bcl-XL, Survivin, EGFR, Cathepsins, chemokines). On the other hand, the ferrocene-linked derivates were able to lower these proteins. Further analysis showed that the ferrocene-linked derivatives significantly elevated the cellular oxidative stress compared to tamoxifen treatment. In conclusion, we were able to find two molecules possessing better cytotoxicity compared to their unmodified parent molecule while also being able to counter the negative effects of the presence of the GPER1 through the ER-independent mechanism of oxidative stress induction.

Daunomycin-polypeptide conjugates with antitumor activity.

We have developed a group of water-soluble drug conjugates in which daunomycin (Dau) is coupled to cationic, amphoteric or anionic branched polypeptides and a new conjugate containing a cationic polypeptide carrier modified with a cell penetrating octaarginine. We investigated in vitro physiological activity of these conjugates in several aspects: in vitro cytotoxicity and cytostatic effect, adhesion and cellular uptake were examined on murine (J774 and L1210) and human (MonoMac6 and HL-60) leukemia cell lines and on murine bone marrow derived macrophages. We found that these processes are dependent on the properties of the carrier, on experimental conditions like concentration and incubation time. We found that attachment of polypeptide and cell penetrating peptide to the bioactive agent, depending on the cell line, could significantly improve the antitumor activity of the drug.

Effect of dental antiseptic agents on the viability of human periodontal ligament cells.

OBJECTIVES: We aimed to study whether or not various dental antiseptic agents affect the viability and proliferation of human periodontal ligament cells (PDLCs). MATERIALS AND METHODS: Human PDLCs were isolated from a total of 10 surgically extracted impacted third molars and were.cultured in-vitro. The cells were exposed to commonly used dental antiseptics, including chlorhexidine, cetylpyridinium chloride, triclosan, povidone-iodine and sodium bicarbonate for ultra-short-term (10, 20, 30 sec), short-term (10, 20, 30 min) and long-term (24, 48 h) at various concentrations. Cell morphology was observed with light microscopy. Cell viability was studied with impedimetric real-time xCELLigence and resazurin-based alamarBlue® assays. We used one-way ANOVA with Tukey's and Bonferroni test (p < 0.05) for statistical analysis. RESULTS: Both alamarBlue® and xCELLigence analysis results were in agreement that ultra-short-term contact with cetylpyridinium chloride ≥ 0.01 mg/ml, chlorhexidine ≥ 1 mg/ml, triclosan ≥ 1 mg/ml and povidone-iodine ≥ 1 mg/ml as well as long-term exposure to cetylpyridinium chloride ≥ 0.001 mg/ml, chlorhexidine ≥ 0.01 mg/ml, triclosan ≥ 1 mg/ml, povidone-iodine ≥ 1 mg/ml and sodium bicarbonate ≥ 10 mg/ml was able to reduce the viability of human PDLCs significantly. According to the half-maximal inhibitory concentration (IC50) the rank of cytotoxicity was cetylpyridinium chloride > chlorhexidine > triclosan > povidone-iodine > sodium bicarbonate. CONCLUSIONS: Our findings suggest that the tested antiseptic agents were cytotoxic to human PDLCs at lower than practically applied concentrations in dental interventions.