Under physiological and pathological conditions, extracellular vesicles (EVs) are present in the extracellular compartment simultaneously with soluble mediators. We hypothesized that cytokine effects may be modulated by EVs, the recently recognized conveyors of intercellular messages. In order to test this hypothesis, human monocyte cells were incubated with CCRF acute lymphoblastic leukemia cell line-derived EVs with or without the addition of recombinant human TNF, and global gene expression changes were analysed. EVs alone regulated the expression of numerous genes related to inflammation and signaling. In combination, the effects of EVs and TNF were additive, antagonistic or independent. The differential effects of EVs and TNF or their simultaneous presence were also validated by Taqman assays, ELISA and testing different populations of purified EVs. In the case of the paramount chemokine IL-8, we were able to demonstrate a synergistic up-regulation by purified EVs and TNF. Our data suggest that neglecting the modulating role of EVs on the effects of soluble mediators may skew experimental results. On the other hand, considering the combined effects of cytokines and EVs may prove therapeutically useful by targeting both compartments at the same time.
AbstractIn drug targeting NGR-peptides recognized by CD13 receptors on tumor neovasculature have got improved interest. Here we present the synthesis and structure analysis of novel thioether linked cyclic NGR-peptides. We found that chemo-stability (resistance against spontaneous decomposition forming isoAsp and Asp derivatives) strongly depends both on sample handling conditions and structural properties. Significant correlation was found between chemo-stability and structural measures: e.g. NH Gly …CO Asn-sc distances. Side chain orientation of Asn is the key determining factor, if turned away from HN Gly chemo-stability increases. Structure stabilizing factors (e.g. H-bond(s)) lower their internal dynamics and thus macromolecules become even more resistant against spontaneous decomposition. Effect of cyclic NGR-peptides on cell adhesion was examined on A2058 melanoma cell lines. It was found that some of them gradually increased the cell adhesion with long term characteristics indicating the time-dependent formation of integrin binding isoAsp derivatives, responsible for the adhesion inducing effect.
The Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most aggressive and dangerous cancerous diseases, leading to a high rate of mortality. Therefore, the development of new, more efficient treatment approaches is necessary to cure this illness. Peptide-based drug targeting provides a new tool for this purpose. Previously, a hexapeptide Cys-Lys-Ala-Ala-Lys-Asn (CKAAKN) was applied efficiently as the homing device for drug-loaded nanostructures in PDAC cells. In this research, Cys was replaced by Ser in the sequence and this new SKAAKN targeting moiety was used in conjugates containing daunomycin (Dau). Five different structures were developed and tested. The results indicated that linear versions with one Dau were not effective on PANC-1 cells in vitro; however, branched conjugates with two Dau molecules showed significant antitumor activity. Differences in the antitumor effect of the conjugates could be explained with the different cellular uptake and lysosomal degradation. The most efficient conjugate was Dau=Aoa-GFLG-K(Dau=Aoa)SKAAKN-OH (conjugate 4) that also showed significant tumor growth inhibition on s.c. implanted PANC-1 tumor-bearing mice with negligible side effects. Our novel results suggest that peptide-based drug delivery systems could be a promising tool for the treatment of pancreatic cancers.
BackgroundAlizarin and purpurin are di- and trihydroxyanthraquinones derived from Rubia tinctorum L. Previous pharmacological studies have demonstrated that they exhibit certain degree of selective inhibitory effects towards cancer cells suggesting their application as a targeted drug for cancer. Our present work was aimed to investigate the suitability of hydroxyanthraquinones of Rubia tinctorum L. for targeted tumor therapy. The effects of alizarin, purpurin and an aqueous extract from transformed hairy root culture of Rubia tinctorum L. were examined on (1) cell proliferation, (2) apoptosis, (3) cell adhesion/morphology and (4) migration (chemotaxis, chemokinesis) of human melanoma cell lines (A2058, HT168-M1) and human fibroblast cells (MRC-5), as well as (5) the aqueous extract was analytically characterized.MethodsThe aqueous extract was prepared from R. tinctorum hairy root culture and qualitatively analyzed by HPLC and ESI–MS methods. The cell growth inhibitory activity of anthraquinones was evaluated by MTT-assay and by flow cytometry. The effect of anthraquinones on cell adhesion was measured by an impedance based technique, the xCELLigence SP. For the chemotaxis assay NeuroProbe® chamber was used. Computer based holographic microscopy was applied to analyze chemokinetic responses as well as morphometry. Statistical significance was determined by the one-way ANOVA test.ResultsIn the aqueous extract, munjistin (Mr = 284, tR = 18.4 min) as a principal component and three minor anthraquinones (pseudopurpurin, rubiadin and nordamnacanthal) were identified. The purpurin elicited a stronger but not apoptosis-mediated antitumor effect in melanoma cells (A2058: 10−6–10−5 M: 90.6–64.1 %) than in normal fibroblasts (10−6–10−5 M: 97.6–84.8 %). The aqueous extract in equimolar concentrations showed the most potent cytotoxicity after 72 h incubation (A2058: 10−6–10−5 M: 87.4–55.0 %). All tested substances elicited chemorepellent effect in melanoma cells, while in MRC-5 fibroblasts, only the alizarin exhibited such a repellent character. Indices of chemokinesis measured by holographic microscopy (migration, migration directness, motility and motility speed) were significantly enhanced by alizarin and purpurin as well, while morphometric changes were weak in the two melanoma cell lines.ConclusionsOur results highlight the effective and selective inhibitory activity of purpurin towards melanoma cells and its possible use as a targeted anticancer agent. The anthraquinones of the cytotoxic extract are suggested to apply in drug delivery systems as an anticancer drug.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-015-0271-4) contains supplementary material, which is available to authorized users.
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