Structural Patterns Enhancing the Antibacterial Activity of Metallacarborane-Based Antibiotics.

Healthcare systems heavily rely on antibiotics to treat bacterial infections, but the widespread presence of multidrug-resistant bacteria puts this strategy in danger. Novel drugs capable of overcoming current resistances are needed if our ability to treat bacterial infections is to be maintained. Boron clusters offer a valuable possibility to create a new class of antibiotics and expand the chemical space of antibiotics beyond conventional carbon-based molecules. In this work, we identified two promising structural patterns providing cobalta bis(dicarbollide)(COSAN)-based compounds with potent and selective activity toward Staphylococcus aureus (including clinical strains): introduction of the α-amino acid amide and addition of iodine directly to the metallacarborane cage. Furthermore, we found that proper hydrophilic-lipophilic balance is crucial for the selective activity of the tested compounds toward S. aureus over mammalian cells. The patterns proposed in this paper can be useful in the development of metallacarborane-based antibiotics with potent antibacterial properties and low cytotoxicity.

Isothiocyanates as Tubulin Polymerization Inhibitors-Synthesis and Structure-Activity Relationship Studies.

Among the various substances that interfere with the microtubule formation process, isothiocyanates (ITCs) are the group of compounds for which the binding mode and mechanism of action have not yet been explained. To better understand the structure-activity relationship of tubulin-isothiocyanate interactions, we designed and synthesized a series of sixteen known and novel, structurally diverse ITCs, including amino acid ester-derived isothiocyanates, bis-isothiocyanates, analogs of benzyl isothiocyanate, and phosphorus analogs of sulforaphane. All synthesized compounds and selected natural isothiocyanates (BITC, PEITC, AITC, and SFN) were tested in vitro to evaluate their antiproliferative activity, tubulin polymerization inhibition potential, and influence on cell cycle progression. The antiproliferative activity of most of the newly tested compounds exceeded the action of natural isothiocyanates, with four structures being more potent as tubulin polymerization inhibitors than BITC. As a confirmation of anti-tubulin activity, the correlation between polymerization inhibition and cell cycle arrest in the G2/M phase was observed for the most active compounds. In light of the biological results indicating significant differences in the impact of structurally diverse isothiocyanate on tubulin polymerization, in silico analysis was conducted to analyze the possible mode of isothiocyanate-tubulin binding and to show how it can influence the polymerization reaction.

Cobalt bis(dicarbollide) is a DNA-neutral pharmacophore.

Cobalt bis(dicarbollide) (COSAN) is a metallacarborane used as a versatile pharmacophore to prepare biologically active hybrid organic-inorganic compounds or to improve the pharmacological properties of nucleosides, antisense oligonucleotides, and DNA intercalators. Despite these applications, COSAN interactions with nucleic acids remain unclear, limiting further advances in metallacarborane-based drug development. Although some studies showed that COSAN intercalates into DNA, COSAN-containing intercalators do not, and while COSAN shows low cytotoxicity, intercalators are often highly toxic. The present study aimed at comprehensively characterizing interactions between COSAN and DNA using a wide range of techniques, including UV-Vis absorption, circular (CD) and linear (LD) dichroism, nuclear magnetic resonance (NMR) spectroscopy, thermal denaturation, viscosity, differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC), and equilibrium dialysis measurements. Our results showed that COSAN has no effect on DNA structure, length, stability, or hybridization, with no or only faint signs of COSAN binding to DNA. Moreover, DNA is not necessary for COSAN to induce cytotoxicity at high concentrations, as shown by in vitro experiments. These findings demonstrate that COSAN is a DNA-neutral pharmacophore, thus confirming the general safety and biocompatibility of metallacarboranes and opening up new opportunities for further developing metallacarborane-based drugs.

One-Pot Phosphonylation of Heteroaromatic Lithium Reagents: The Scope and Limitations of Its Use for the Synthesis of Heteroaromatic Phosphonates.

A one-pot lithiation-phosphonylation procedure was elaborated as a method to prepare heteroaromatic phosphonic acids. It relied on the direct lithiation of heteroaromatics followed by phosphonylation with diethyl chlorophosphite and then oxidation with hydrogen peroxide. This protocol provided the desired phosphonates with satisfactory yields. This procedure also had some limitations in its dependence on the accessibility and stability of the lithiated heterocyclic compounds. The same procedure could be applied to phosphonylation of aromatic compounds, which do not undergo direct lithiation and thus require the use of their bromides as substrates. The obtained compounds showed weak antiproliferative activity when tested on three cancer cell lines.

Modulation of Fibroblast Activity via Vitamin D3 Is Dependent on Tumor Type-Studies on Mouse Mammary Gland Cancer.

Vitamin D3 and its analogs are known to modulate the activity of fibroblasts under various disease conditions. However, their impact on cancer-associated fibroblasts (CAFs) is yet to be fully investigated. The aim of this study was to characterize CAFs and normal fibroblasts (NFs) from the lung of mice bearing 4T1, 67NR, and E0771 cancers and healthy mice fed vitamin-D3-normal (1000 IU), -deficient (100 IU), and -supplemented (5000 IU) diets. The groups receiving control (1000 IU) and deficient diets (100 IU) were gavaged with calcitriol (+cal). In the 4T1-bearing mice from the 100 IU+cal group, increased NFs activation (increased α-smooth muscle actin, podoplanin, and tenascin C (TNC)) with a decreased blood flow in the tumor was observed, whereas the opposite effect was observed in the 5000 IU and 100 IU groups. CAFs from the 5000 IU group of E0771-bearing mice were activated with increased expression of podoplanin, platelet-derived growth factor receptor ß, and TNC. In the 100 IU+cal group of E0771-bearing mice, a decreased blood flow was recorded with decreased expression of fibroblast growth factor 23 (FGF23) and C-C motif chemokine ligand 2 (CCL2) in tumors and increased expression of TNC on CAFs. In the 67NR model, the impact of vitamin D3 on blood flow or CAFs and lung NFs was not observed despite changes in plasma and/or tumor tissue concentrations of osteopontin (OPN), CCL2, transforming growth factor-ß, vascular endothelial growth factor, and FGF23. In healthy mice, divergent effects of vitamin D3 supplementation/deficiency were observed, which lead to the creation of various body microenvironments depending on the mouse strain. Tumors developing in such microenvironments themselves modified the microenvironments by producing, for example, higher concentrations of OPN and stromal-cell-derived factor 1 (4T1), which influences the response to vitamin D3 supplementation/deficiency and calcitriol administration.

Metallacarborane Derivatives as Innovative Anti-Candida albicans Agents.

Infections caused by Candida species have increased significantly in the past decades and are among the leading causes of morbidity and mortality worldwide, resulting in serious public health problems. Currently, conventional antifungals are often ineffective as Candida spp. have developed growing resistance to systemic drugs. Since inorganic metallacarboranes are known to affect cellular events, new derivatives of these abiotic compounds were tested against Candida albicans. Compounds based on cobalt bis-dicarbollide [COSAN] were studied on Candida albicans strains, including a panel of 100 clinical isolates. The presented data prove that metallacarborane derivatives are effective against clinical isolates of Candida albicans, even those resistant to systemic drugs, and show synergistic potential in combination with amphotericin B, and low toxicity against human cells and Danio rerio embryos. This paper is a consequential step in the investigations of the broad spectrum and valuable future medical applications of metallacarboranes, especially in the fight against drug-resistant pathogens.

Microfluidic-Assisted Human Cancer Cells Culturing Platform for Space Biology Applications.

In the paper, the lab-on-chip platform applicable for the long-term cultivation of human cancer cells, as a solution meeting the demands of the CubeSat biological missions, is presented. For the first time, the selected cancer cell lines-UM-UC-3 and RT 112 were cultured on-chip for up to 50 days. The investigation was carried out in stationary conditions (without medium microflow) in ambient temperature and utilizing the microflow perfusion system in the incubation chamber assuring typical cultivation atmosphere (37 °C). All the experiments were performed to imitate the conditions that are provided before the biological mission starts (waiting for the rocket launch) and when the actual experiment is initialized on a CubeSat board in space microgravity. The results of the tests showed appropriate performance of the lab-on-chip platform, especially in the context of material and technological biocompatibility. Cultured cells were characterized by adequate morphology-high attachment rate and visible signs of proliferation in each of the experimental stage. These results are a good basis for further tests of the lab-on-chip platform in both terrestrial and space conditions. At the end of the manuscript, the authors provide some considerations regarding a potential 3-Unit CubeSat biological mission launched with Virgin Orbit company. The lab-on-chip platform was modelled to fit a 2-Unit autonomous laboratory payload.

Combined anticancer therapy with imidazoacridinone analogue C-1305 and paclitaxel in human lung and colon cancer xenografts-Modulation of tumour angiogenesis.

The acridanone derivative 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) has been described as a potent inhibitor of cancer cell growth. Its mechanism of action in in vitro conditions was attributed, among others, to its ability to bind and stabilize the microtubule network and subsequently exhibit its tumour-suppressive effects in synergy with paclitaxel (PTX). Therefore, the objective of the present study was to analyse the effects of the combined treatment of C-1305 and PTX in vivo. In addition, considering the results of previous genomic analyses, particular attention was given to the effects of this treatment on tumour angiogenesis. Treatment with C-1305 revealed antitumor effect in A549 lung cancer cells, and combined treatment with PTX showed tendency to anticancer activity in HCT116 colon cancer xenografts. It also improved tumour blood perfusion in both tumour models. The plasma level of CCL2 was increased and that of PDGF was decreased after combined treatment with C-1305 and PTX. The experimental results showed that the levels of FGF1, TGF-ß and Ang-4 decreased, whereas the levels of ERK1/2 and Akt phosphorylation increased in HCT116 tumour tissue following combined treatment with both drugs. The results of in vitro capillary-like structure formation assay demonstrated the inhibiting effect of C-1305 on this process. Although previous in vitro and in vivo studies suggested a positive effect of C-1305 on cancer cells, combined treatment of HCT116 human colon and A549 lung cancer cells with both PTX and C-1305 in vivo showed that the antitumor activity was restricted and associated with the modulation of tumour angiogenesis.

Metallacarborane Derivatives Effective against Pseudomonas aeruginosa and Yersinia enterocolitica.

Pseudomonas aeruginosa is an opportunistic human pathogen that has become a nosocomial health problem worldwide. The pathogen has multiple drug removal and virulence secretion systems, is resistant to many antibiotics, and there is no commercial vaccine against it. Yersinia pestis is a zoonotic pathogen that is on the Select Agents list. The bacterium is the deadliest pathogen known to humans and antibiotic-resistant strains are appearing naturally. There is no commercial vaccine against the pathogen, either. In the current work, novel compounds based on metallacarborane cage were studied on strains of Pseudomonas aeruginosa and a Yersinia pestis substitute, Yersinia enterocolitica. The representative compounds had IC50 values below 10 µM against Y. enterocolitica and values of 20-50 µM against P. aeruginosa. Artificial generation of compound-resistant Y. enterocolitica suggested a common mechanism for drug resistance, the first reported in the literature, and suggested N-linked metallacarboranes as impervious to cellular mechanisms of resistance generation. SEM analysis of the compound-resistant strains showed that the compounds had a predominantly bacteriostatic effect and blocked bacterial cell division in Y. enterocolitica. The compounds could be a starting point towards novel anti-Yersinia drugs and the strategy presented here proposes a mechanism to bypass any future drug resistance in bacteria.

Vitamin D Compounds PRI-2191 and PRI-2205 Enhance Anastrozole Activity in Human Breast Cancer Models.

1,25-Dihydroxycholecalciferol, the hormonally active vitamin D3 metabolite, is known to exhibit therapeutic effects against breast cancer, mainly by lowering the expression of estrogen receptors and aromatase activity. Previously, the safety of the vitamin D active metabolite (24R)-1,24-dihydroxycholecalciferol (PRI-2191) and 1,25(OH)2D3 analog PRI-2205 was tested, and the in vitro activity of these analogs against different cancer cell lines was studied. We determined the effect of the two vitamin D compounds on anastrozole (An) activity against breast cancer based on antiproliferative activity, ELISA, flow cytometry, enzyme inhibition potency, PCR, and xenograft study. Both the vitamin D active metabolite and synthetic analog regulated the growth of not only estrogen receptor-positive cells (T47D and MCF-7, in vitro and in vivo), but also hormone-independent cancer cells such as SKBR-3 (HER-2-positive) and MDA-MB-231 (triple-negative), despite their relatively low VDR expression. Combined with An, PRI-2191 and PRI-2205 significantly inhibited the tumor growth of MCF-7 cells. Potentiation of the antitumor activity in combined treatment of MCF-7 tumor-bearing mice is related to the reduced activity of aromatase by both An (enzyme inhibition) and vitamin D compounds (switched off/decreased aromatase gene expression, decreased expression of other genes related to estrogen signaling) and by regulation of the expression of the estrogen receptor ERα and VDR.

Dipeptides of S-Substituted Dehydrocysteine as Artzyme Building Blocks: Synthesis, Complexing Abilities and Antiproliferative Properties.

BACKGROUND: Dehydropeptides are analogs of peptides containing at least one conjugate double bond between α,ß-carbon atoms. Its presence provides unique structural properties and reaction centre for chemical modification. In this study, the series of new class of dipeptides containing S-substituted dehydrocysteine with variety of heterocyclic moieties was prepared. The compounds were designed as the building blocks for the construction of artificial metalloenzymes (artzymes). Therefore, the complexing properties of representative compounds were also evaluated. Furthermore, the acknowledged biological activity of natural dehydropeptides was the reason to extend the study for antiproliferative action of against several cancer cell lines. METHODS: The synthetic strategy involves glycyl and phenylalanyl-(Z)-ß-bromodehydroalanine as a substrate in one pot addition/elimination reaction of thiols. After deprotection of N-terminal amino group the compounds with triazole ring were tested as complexones for copper(II) ions using potentiometric titration and spectroscopic techniques (UV-Vis, CD, EPR). Finally, the antiproliferative activity was evaluated by sulforhodamine B assay. RESULTS AND CONCLUSIONS: A simple and efficient procedure for preparation of dipeptides containing S-substituded dehydrocysteine was provided. The peptides containing triazole appeared to be strong complexones of copper(II) ions. Some of the peptides exhibited promising antiproliferative activities against number of cancer cell lines, including cell lines resistant to widely used anticancer agent.

Synthesis and Anticancer Evaluation of Novel Derivatives of Isoxazolo[4,5-e][1,2,4]triazepine Derivatives and Potential Inhibitors of Protein Kinase C.

In the present study, using Thorpe's reaction with Gewald's modification, 4-acetylamino-5-acetyl or 5-benzoyl 3-carboxamide compounds 3 or 4 were obtained. From these compounds, two series of compounds (5, 7, and 9 and 6, 8, and 10) were obtained with 98% hydrazine. Compounds 6, 7, 8, and 9 were then reacted with the appropriate aldehydes to afford a series of new isoxazole derivatives (11-18, 27-36, and 37-41) and the main compounds, 19-26 and 42-45, were isoxazolo[4,5-e][1,2,4]triazepine derivatives. The anticarcinogenic activities of selected compounds were tested on six lines of cancer cells, and their activities were compared with the relevant concentrations of the anticarcinogenic drugs cisplatin and doxorubicin in IITD PAN. Several compounds were tested on 60 lines of cancer cells by the NCI (Bethesda, MD, USA). The cyclization of compound 12 into derivative 46 was also carried out. Compound 21 showed extremely high antitumor activity.

Phosphonic Analogs of Alanine as Acylpeptide Hydrolase Inhibitors.

Acylpeptide hydrolase is a serine protease, which, together with prolyl oligopeptidase, dipeptidyl peptidase IV and oligopeptidase B, belongs to the prolyl oligopeptidase family. Its primary function is associated with the removal of N-acetylated amino acid residues from proteins and peptides. Although the N-acylation occurs in 50-90 % of eukaryotic proteins, the precise functions of this modification remains unclear. Recent findings have indicated that acylpeptide hydrolase participates in various events including oxidized proteins degradation, amyloid ß-peptide cleavage, and response to DNA damage. Considering the protein degradation cycle cross-talk between acylpeptide hydrolase and proteasome, inhibition of the first enzyme resulted in down-regulation of the ubiquitin-proteasome system and induction of cancer cell apoptosis. Acylpeptide hydrolase has been proposed as an interesting target for the development of new potential anticancer agents. Here, we present the synthesis of simple derivatives of (1-aminoethyl)phosphonic acid diaryl esters, phosphonic analogs of alanine diversified at the N-terminus and ester rings, as inhibitors of acylpeptide hydrolase and discuss the ability of the title compounds to induce apoptosis of U937 and MV-4-11 tumor cell lines.

Hydroxyethylcellulose as a methotrexate carrier in anticancer therapy.

Clinical and experimental cancer therapy is multifaceted; one such facet is the use of drug carriers. Drug carriers are various nano- and macromolecules, e.g., oligosaccharides, proteins, and liposomes. The present study aimed to verify the suitability of cellulose as a carrier for methotrexate (MTX). Hydroxyethylcellulose, with a molecular weight of 90 kDa and soluble in water, was used. Methotrexate was linked to cellulose by methyl ester bonds. A conjugate containing on average 9.5 molecules of MTX per molecule of cellulose was developed. Gel filtration HPLC analysis showed that the conjugate contained approximately 2% free drug. Dynamic light scattering analysis showed an increase in the polydispersity of the conjugate. The degradation of the conjugate in phosphate buffer and plasma followed first-order kinetics. The conjugate showed the lowest stability (half-life 154 h) in plasma. The conjugate showed 10-fold lower cytotoxicity to the 4 T1 mammary tumour cell line than the free drug. In the in vivo experiment to treat orthotopically implanted mammary tumours, the conjugate and the free drug, both applied intravenously, showed maximum inhibition of tumour growth of 48.4% and 11.2%, respectively. In conclusion, cellulose, which is a non-biodegradable chain glucose polymer, can be successfully used as a drug carrier, which opens up new research perspectives.

Design and in Vitro Characterization of Tricyclic Benzodiazepine Derivatives as Potent and Selective Antileukemic Agents.

Currently available chemotherapeutic treatments for blood cancers (leukemia) usually have strong side effects. More selective, efficient, and less toxic anticancer agents are needed. We synthesized seven, new, optically pure (12aS)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione derivatives and examined their cytotoxicity towards eight cancer cell lines, including urinary bladder (TCC-SUP, UM-UC-3, KU-19-9), colon (LoVo), and breast (MCF-7, MDA-MB-231) cancer representatives, as well as two leukemic cell lines (MV-4-11, CCRF-CEM) and normal murine fibroblasts (Balb/3T3) as reference cell line. Three of the seven newly-obtained compounds ((12aS)-8-bromo-2-(3-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione, (12aS)-8,9-dimethoxy-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione and (12aS)-8-nitro-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione, showed enhanced activity and selectivity toward the leukemic MV-4-11 cell lines when compared to our previously reported compounds, with IC50 values in the range of 2.9-5.6 µM. Additionally, (12aS)-9-nitro-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione exhibited a strong cytotoxic effect against the leukemic CCRF-CEM (IC50 =6.1 µM) and MV-4-11 (IC50 =11.0 µM) cell lines, a moderate cytotoxic effect toward other tumor lines (IC50 =31.8-55.0 µM) and very weak cytotoxic effect toward the Balb/3T3 reference cell lines. Selected compounds were further evaluated for their potential to induce apoptotic cell death in MV-4-11 cells by measuring caspase-3 activity. We also established the crystal structure of three products and investigated the effect of 22 derivatives of 1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione on the activity of the cancer-associated enzyme autotaxin. All compounds proved to be weak inhibitors of autotaxin, although some (R) and (S) enantiomers had Ki values of 10-19 µM. The obtained results showed that the tested compounds exhibited a selective antileukemic effect, which appeared not to be related directly to autotaxin. Molecular targets responsible for this effect remain to be identified. The newly obtained compounds can be used in the search for new, selective anticancer therapies.

Biaryl Sulfonamides Based on the 2-Azabicycloalkane Skeleton-Synthesis and Antiproliferative Activity.

In a search for new, selective antitumor agents, we prepared a series of sulfonamides built on bicyclic scaffolds of 2-azabicyclo(2.2.1)heptane and 2-azabicyclo(3.2.1)octane. To this end, aza-Diels-Alder cycloadducts were converted into amines bearing 2-azanorbornane or a bridged azepane skeleton; their treatment with sulfonyl chlorides containing biaryl moieties led to the title compounds. The study of antiproliferative activity of the new agents showed that some of them inhibited the growth of chosen cell lines with the IC50 values comparable with cisplatin, and some derivatives were found considerably less toxic for nonmalignant cells.

Structure-based design, synthesis, and evaluation of the biological activity of novel phosphoroorganic small molecule IAP antagonists.

One of the strategies employed by novel anticancer therapies is to put the process of apoptosis back on track by blocking the interaction between inhibitor of apoptosis proteins (IAPs) and caspases. The activity of caspases is modulated by the caspases themselves in a caspase/procaspase proteolytic cascade and by their interaction with IAPs. Caspases can be released from the inhibitory influence of IAPs by proapoptotic proteins such as secondary mitochondrial activator of caspases (Smac) that share an IAP binding motif (IBM). The main purpose of the present study was the design and synthesis of phosphorus-based peptidyl antagonists of IAPs that mimic the endogenous Smac protein, which blocks the interaction between IAPs and caspases. Based on the structure of the IAP antagonist and recently reported thiadiazole derivatives, we designed and evaluated the biochemical properties of a series of phosphonic peptides bearing the N-Me-Ala-Val/Chg-Pro-OH motif (Chg: cyclohexylglycine). The ability of the obtained compounds to interact with the binding groove of the X-linked inhibitor of apoptosis protein baculovirus inhibitor of apoptosis protein repeat (XIAP BIR3) domain was examined by a fluorescence polarization assay, while their potential to induce autoubiquitination followed by proteasomal degradation of cellular IAP1 was examined using the MDA-MB-231 breast cancer cell line. The highest potency against BIR3 was observed among peptides containing C-terminal phosphonic phenylalanine analogs, which displayed nanomolar Ki values. Their antiproliferative potential as well as their proapoptotic action, manifested by an increase in caspase-3 activity, was examined using various cell lines.

Three-Component Reaction of Diamines with Triethyl Orthoformate and Diethyl Phosphite and Anti-Proliferative and Antiosteoporotic Activities of the Products.

A three-component reaction between diamines (diaminobenzenes, diaminocyclohexanes, and piperazines), triethyl orthoformate, and diethyl phosphite was studied in some detail. In the case of 1,3- and 1,4-diamines and piperazines, products of the substitution of two amino moieties-the corresponding tetraphosphonic acids-were obtained. In the cases of 1,2-diaminobenzene, 1,2-diaminocyclohexanes and 1,2-diaminocyclohexenes, only one amino group reacted. This is most likely the result of the formation of hydrogen bonding between the phosphonate oxygen and a hydrogen of the adjacent amino group, which caused a decrease in the reactivity of the amino group. Most of the obtained compounds inhibited the proliferation of RAW 264.7 macrophages, PC-3 human prostate cancer cells, and MCF-7 human breast cancer cells, with 1, trans-7, and 16 showing broad nonspecific activity, which makes these compounds especially interesting in the context of anti-osteolytic treatment and the blocking of interactions and mutual activation of osteoclasts and tumor metastatic cells. These compounds exhibit similar activity to zoledronic acid and higher activity than incadronic acid, which were used as controls. However, studies of sheep with induced osteoporosis carried out with compound trans-7 did not support this assumption.

Can supernatant from immortalized adipose tissue MSC replace cell therapy? An in vitro study in chronic wounds model.

BACKGROUND: Mesenchymal stem cells (MSCs) secrete a cocktail of growth factors and cytokines, which could promote tissue regeneration and wound healing. Therefore, in clinical practice, post-culture MSC supernatant treatment could be a more attractive alternative to autologous stem cell transplantation. In this study, we compared the regenerative properties of supernatants harvested from four newly established human adipose tissue mesenchymal stem cell lines (HATMSCs) derived from chronic wound patients or healthy donors. METHODS: HATMSC supernatants were produced in a serum-free medium under hypoxia and their content was analyzed by a human angiogenesis antibody array. The regenerative effect of HATMSCs supernatants was investigated in an in vitro model of chronic wound, where cells originating from human skin, such as microvascular endothelial cells (HSkMEC.2), keratinocytes (HaCaT), and fibroblasts (MSU-1.1), were cultured in serum-free and oxygen-reduced conditions. The effect of supernatant treatment was evaluated using an MTT assay and light microscopy. In addition, fibroblasts and HATMSCs were labeled with PKH67 and PKH26 dye, respectively, and the effect of supernatant treatment was compared to that obtained when fibroblasts and HATMSCs were co-cultured, using flow cytometry and fluorescent microscopy. RESULTS: A wide panel of angiogenesis-associated cytokines such as angiogenin, growth-regulated oncogene (GRO), interleukin-6 and 8 (IL-6, IL-8), vascular endothelial growth factor (VEGF), insulin growth factor 1 (IGF-1), and matrix metalloproteinase (MMP) were found in all tested HATMSCs supernatants. Moreover, supernatant treatment significantly enhanced the survival of fibroblasts, endothelial cells, and keratinocytes in our chronic wound model in vitro. Importantly, we have shown that in in vitro settings, HATMSC supernatant treatment results in superior fibroblast proliferation than in the case of co-culture with HATMSCs. CONCLUSIONS: Our results suggest that therapy based on bioactive factors released by the immortalized atMSC into supernatant has important effect on skin-derived cell proliferation and might preclude the need for a more expensive and difficult cell therapy approach to improve chronic wound healing.

EGFR-targeted immunoliposomes as a selective delivery system of simvastatin, with potential use in treatment of triple-negative breast cancers.

A promising strategy for treatment of EGFR-dependent tumours is EGFR signal transduction suppression via inhibition of HMG-CoA reductase using high doses of statins, popular cholesterol-lowering drugs. The main purpose of this study was to obtain targeted long circulating immunoliposomes containing simvastatin (tLCLS) with anti-EGFR antibody attached to their surface and to test whether they can be effective in treatment of TNBC. The designed tLCLS were characterized in terms of physicochemical properties and long-term stability. In vitro experiments conducted on MDA-MB-231 cells demonstrated that tLCLS induced apoptosis and are characterized by IC50 of 7.5 µM. Treatment of studied cells with tLCLS led to a decrease in membrane order and inhibited PI3K/Akt signalling. Analyses of efficacy of the tLCLS in in vivo experiments in model animals indicate that immunoliposomes were effectively delivered to tumours. Our results showed that regardless of whether tLCLS were administered before or after tumour formation, at the tested dose they inhibited tumour growth by an average of 25% in comparison to the control. However, the results were not statistically significant. The experiments described above allowed us to test the possibility of using immunoliposomes as simvastatin carriers delivering increased amounts of the drug to tumour cells.