Characterisation of the cell and molecular biological effect of peptide-based daunorubicin conjugates developed for targeting pancreatic adenocarcinoma (PANC-1) cell line.

Pancreatic adenocarcinoma is one of the tumours with the worst prognosis, with a 5-year survival rate of 5-10%. Our aim was to find and optimise peptide-based drug conjugates with daunorubicin (Dau) as the cytotoxic antitumour agent. When conjugated with targeting peptides, the side effect profile and pharmacokinetics of Dau can be improved. The targeting peptide sequences (e.g. GSSEQLYL) we studied were originally selected by phage display. By Ala-scan technique, we identified that position 6 in the parental sequence (Dau=Aoa-LRRY-GSSEQLYL-NH2, ConjA) could be modified without the loss of antitumour activity (Dau=Aoa-LRRY-GSSEQAYL-NH2, Conj03: 14. 9% viability). Our results showed that the incorporation of p-chloro-phenylalanine (Dau=Aoa-LRRY-GSSEQF(pCl)YL-NH2, Conj16) further increased the antitumour potency (10-5 M: 9.7% viability) on pancreatic adenocarcinoma cells (PANC-1). We found that conjugates containing modified GSSEQLYL sequences could be internalised to PANC-1 cells and induce cellular senescence in the short term and subsequent apoptotic cell death. Furthermore, the cardiotoxic effect of Dau was markedly reduced in the form of peptide conjugates. In conclusion, Conj16 had the most effective antitumor activity on PANC-1 cells, which makes this conjugate promising for developing new targeted therapies without cardiotoxic effects.

Photoinduced Hydrogel-Forming Caged Peptides with Improved Solubility.

Self-assembling peptides are attractive alternatives in the field of biomaterial science due to their variability and biocompatibility. Unfortunately, such peptides have poor solubility, and their purification, synthesis, and overall handling are challenging. Our main objective was to develop a cage peptide design with full control over self-assembly. Theoretically, aggregation can be suppressed by temporally masking the amino acid side chains at critical positions. Taking into account several biological and synthetic requirements, a photosensitive protecting group, p-hydroxy-phenacyl (pHP), was chosen as the "masking" moiety. To test our theory, EAK16-II was chosen as a model self-assembling peptide, and a caged derivative containing photosensitive pHP groups was synthesized. Both spectroscopic and in vitro experiments on A2058 melanoma cells confirmed our hypothesis that the caged-EAK16-II peptide has good solubility and that the hydrogel formed after photolysis results in similar viability and cell aggregate formation of melanoma cells as the native EAK16-II-based hydrogel.

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.

PCSK9, A Promising Novel Target for Age-Related Cardiovascular Dysfunction.

Cardiovascular diseases (CVDs) are the leading cause of death among elderly people. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important regulator of cholesterol metabolism. Herein, we investigated the role of PCSK9 in age-related CVD. Both in humans and rats, blood PCSK9 level correlated positively with increasing age and the development of cardiovascular dysfunction. Age-related fatty degeneration of liver tissue positively correlated with serum PCSK9 levels in the rat model, while development of age-related nonalcoholic fatty liver disease correlated with cardiovascular functional impairment. Network analysis identified PCSK9 as an important factor in age-associated lipid alterations and it correlated positively with intima-media thickness, a clinical parameter of CVD risk. PCSK9 inhibition with alirocumab effectively reduced the CVD progression in aging rats, suggesting that PCSK9 plays an important role in cardiovascular aging.

Three-dimensional, PEG-based hydrogels induce spheroid formation and enhance viability of A2058 melanoma cells.

Traditional drug screening methods use monolayer (2D) tumor cell cultures, which lack basic features of tumor complexity. As an alternative, 3D hydrogels have begun to emerge as simple, time-, and cost-saving systems. One of the most promising candidates, synthetic alkoxysilane-PEG (polyethylene glycol)-based hydrogels, are formed by "sol-gel" polymerization in an aqueous medium, which allows control over the incorporated elements. Our aims were to optimize siloxane-PEG hydrogels for three different cell lines of skin origin and utilize these 3D hydrogels as a feasible drug (e.g., daunorubicin) screening assay. A drastic increase in survival and the formation of cellular aggregates (spheroids) could be observed in A2058 melanoma cells, but not in keratinocyte and endothelial cell lines. A deep-learning neural network was trained to recognize and distinguish between the cellular formations and allowed the fast processing of hundreds of microscopic images. We developed an artificial intelligence (AI)-assisted application (https://github.com/enyecz/CancerDetector2), which indicated that, in terms of average area of the spheroids treated with daunorubicin, A2058 melanoma cell 3D aggregates have better survival in a hydrogel containing 15% bis-mono-ethoxysilane-PEG.

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.

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.

Chemotaxis in Leishmania (Viannia) braziliensis: Evaluation by the two-chamber capillary assay.

Chemotactic responses play a significant role during Leishmania (V.) braziliensis differentiation through its life cycle and during infection. The aim of this description has been to portray the modified "two-chamber capillary chemotaxis assay" as a technique useful for quantitative in vitro evaluation of Leishmania chemotaxis after reviewing the methods described until now to assess chemotaxis in vitro in Leishmania sp. This valued simple and reproducible method convenient for parasite migration determination, was tested by the use of controlled changes in monosaccharide (D-glucose and D-fructose) concentrations as referent ligands. The validation of the method demonstrates that this technique is useful to evaluate the relationship existing between parasite migration towards the monosaccharides and sugar concentration. This means that within specific ranges, parasites attracted by the monosaccharide migrate towards more concentrated solutions and accumulate (higher number of parasites) at that spot. Interestingly, both the time course of the experiment and the osmolality of the solution influence parasite migration capacity. Our validation suggests that this improved methodology quantitatively evaluates taxis of Leishmania towards/against different substances. On the basis of our herein presented data, we conclude that this technique is a novel, rapid and reliable screening method to evaluate chemotaxis in Leishmania.•The two-chamber capillary chemotaxis assay was standardized for Leishmania.•The technique is useful to quantitatively evaluate in vitro chemotaxis in Leishmania.•Parasite migration was characterized by monosaccharide chemical gradients.•This assay is a novel, rapid and reliable screening method to evaluate chemotaxis. Contain between 1 and 3 bullet points highlighting the customization rather than the steps of the procedure.

The Synergistic Activity of Bortezomib and TIC10 against A2058 Melanoma Cells.

Combination antitumor treatments are essential parts of modern tumor therapy as-compared to monotherapies-(i) they are more effective; (ii) the dose of the compounds can be reduced; and (iii) therefore the side effects are improved. Our research group previously demonstrated the antitumor character of bortezomib (BOZ) in A2058 melanoma cells. Unfortunately, dose-related side effects are common during BOZ therapy, which could be prevented by reducing the dose of BOZ. This study aimed to characterize synergistic combinations of BOZ with a TRAIL (TNF-related apoptosis-inducing ligand) -inducing compound (TIC10), where the doses can be cut down but the efficacy is preserved. Endpoint cell viability assays were performed on A2058 cells, and synergism of BOZ and TIC10 was observed after 72 h. Synergism was further validated in a real-time impedimetric assay, and our results showed that BOZ-treated melanoma cells survived the treatment, an effect not registered in the co-treatments. Treatment with the combinations resulted in increased apoptosis, which was not accompanied by enhanced LDH release. Nevertheless, the expression of death receptor 5 (DR5) was increased on the cell surface without transcriptional regulation. In summary, our findings support the theory that the application of BOZ and TIC10 in combination could provide higher efficacy in vitro.

Alpha-lipoic acid alters the antitumor effect of bortezomib in melanoma cells in vitro.

Bortezomib (BOZ) is a proteasome inhibitor chemotherapeutic agent utilized to treat multiple myeloma and recently offered to cure melanoma. Bortezomib-induced neuropathy is one of the dose-limiting side-effects, which can be treated with antioxidants (e.g. alpha-lipoic acid-ALA and Vitamin B1-vit B1). We hypothesized that these antioxidants may counteract the antitumor activity by disrupting the BOZ-induced pathways (e.g. proteasome inhibition or reactive oxygen species generation). The objectives were: (i) to verify the anti-proliferative effect of BOZ; (ii) to compare the influence of the antioxidants on the antitumor effect of BOZ in melanoma (A2058) and myeloma (U266) cells. At first, the reduction in the anti-proliferative effect of BOZ by ALA was proved in melanoma cells. Analysis of p53 phosphorylation and the cell cycle progression revealed that ALA failed to counteract these effects of BOZ. Nevertheless, a good correlation was found between the inhibition of the anti-proliferative effect, the anti-proteasome activity and the oxidative stress level after the co-treatment with 20 ng/mL BOZ + 100 µg/mL ALA. Downregulation of apoptotic proteins such as HO-1 and Claspin along with the inhibition of the cleavage of Caspase-3 indicated the proteomic background of the altered responsiveness of the melanoma cells exposed to BOZ + ALA. This phenomenon draws attention to the proper application of cancer supportive care to avoid possible interactions.

Phage Display-Based Homing Peptide-Daunomycin Conjugates for Selective Drug Targeting to PANC-1 Pancreatic Cancer.

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.

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.

[Comparison of therapeutic peptides targeting pancreatic cancer]. / Hasnyálmirigytumort célzó terápiás irányítópeptidek összehasonlítása.

Despite the small number of cases, pancreatic cancer is one of the biggest challenges in tumor therapy as its treatment is not yet resolved and the expected 5-year survival rate is only 5%. Therefore, innovative solutions for pancreatic cancer are of great importance. Targeted tumor therapy might provide new possibilities in this field. In our research, we focused on finding peptide-based homing molecules and modified their structure to achieve better targeting properties. We compared several peptides that efficiently recognize receptors that are specific for or overexpressed by pancreatic cancer cells. Their structure-effect relationship was determined that can be useful during drug designing in the future. The antitumor effect of Dau=Aoa-GFLG-K(Dau=Aoa) SKAAKN-OH conjugate, which turned out to be the most efficient one during in vitro studies, were analyzed in vivo in female SCID mice. The obtained 30% inhibition, beside the low toxic side effects, might be a good starting point to develop further, more powerful conjugates.

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.

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.

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.

Comparative cell biological study of in vitro antitumor and antimetastatic activity on melanoma cells of GnRH-III-containing conjugates modified with short-chain fatty acids.

Background: Peptide hormone-based targeted tumor therapy is an approved strategy to selectively block the tumor growth and spreading. The gonadotropin-releasing hormone receptors (GnRH-R) overexpressed on different tumors (e.g., melanoma) could be utilized for drug-targeting by application of a GnRH analog as a carrier to deliver a covalently linked chemotherapeutic drug directly to the tumor cells. In this study our aim was (i) to analyze the effects of GnRH-drug conjugates on melanoma cell proliferation, adhesion and migration, (ii) to study the mechanisms of tumor cell responses, and (iii) to compare the activities of conjugates with the free drug. Results: In the tested conjugates, daunorubicin (Dau) was coupled to 8Lys of GnRH-III (GnRH-III(Dau=Aoa)) or its derivatives modified with 4Lys acylated with short-chain fatty acids (acetyl group in [4Lys(Ac)]-GnRH-III(Dau=Aoa) and butyryl group in [4Lys(Bu)]-GnRH-III(Dau=Aoa)). The uptake of conjugates by A2058 melanoma model cells proved to be time dependent. Impedance-based proliferation measurements with xCELLigence SP system showed that all conjugates elicited irreversible tumor growth inhibitory effects mediated via a phosphoinositide 3-kinase-dependent signaling. GnRH-III(Dau=Aoa) and [4Lys(Ac)]-GnRH-III(Dau=Aoa) were shown to be blockers of the cell cycle in the G2/M phase, while [4Lys(Bu)]-GnRH-III(Dau=Aoa) rather induced apoptosis. In short-term, the melanoma cell adhesion was significantly increased by all the tested conjugates. The modification of the GnRH-III in position 4 was accompanied by an increased cellular uptake, higher cytotoxic and cell adhesion inducer activity. By studying the cell movement of A2058 cells with a holographic microscope, it was found that the migratory behavior of melanoma cells was increased by [4Lys(Ac)]-GnRH-III(Dau=Aoa), while the GnRH-III(Dau=Aoa) and [4Lys(Bu)]-GnRH-III(Dau=Aoa) decreased this activity. Conclusion: Internalization and cytotoxicity of the conjugates showed that GnRH-III peptides could guard Dau to melanoma cells and promote antitumor activity. [4Lys(Bu)]-GnRH-III(Dau=Aoa) possessing the butyryl side chain acting as a "second drug" proved to be the best candidate for targeted tumor therapy due to its cytotoxicity and immobilizing effect on tumor cell spreading. The applicability of impedimetry and holographic phase imaging for characterizing cancer cell behavior and effects of targeted chemotherapeutics with small structural differences (e.g., length of the side chain in 4Lys) was also clearly suggested.

Skin-homing CD8+ T cells preferentially express GPI-anchored peptidase inhibitor 16, an inhibitor of cathepsin K.

This study sought to identify novel CD8+ T cell homing markers by studying acute graft versus host disease (aGvHD), typically involving increased T cell homing to the skin and gut. FACS-sorted skin-homing (CD8ß+ /CLA+ ), gut-homing (CD8ß+ /integrinß7+ ), and reference (CD8ß+ /CLA- /integrinß7- ) T cells were compared in patients affected by cutaneous and/or gastrointestinal aGVHD. Microarray analysis, qPCR, and flow cytometry revealed increased expression of peptidase inhibitor 16 (PI16) in skin-homing CD8+ T cells. Robust association of PI16 with skin homing was confirmed in all types of aGvHD and in healthy controls, too. PI16 was not observed on CLA+ leukocytes other than T cells. Induction of PI16 expression on skin-homing T cells occurred independently of vitamin D3. Among skin-homing T cells, PI16 expression was most pronounced in memory-like CD45RO+ /CD127+ /CD25+ /CD69- /granzyme B- cells. PI16 was confined to the plasma membrane, was GPI-anchored, and was lost upon restimulation of memory CD8+ T cells. Loss of PI16 occurred by downregulation of PI16 transcription, and not by Phospholipase C (PLC)- or Angiotensin-converting enzyme (ACE)-mediated shedding, or by protein recycling. Inhibitor screening and pull-down experiments confirmed that PI16 inhibits cathepsin K, but may not bind to other skin proteases. These data link PI16 to skin-homing CD8+ T cells, and raise the possibility that PI16 may regulate cutaneous cathepsin K.

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.