Ruthenium metallodendrimer against triple-negative breast cancer in mice.

Carbosilane metallodendrimers, based on the arene Ru(II) complex (CRD13) and integrated to imino-pyridine surface groups have been investigated as an anticancer agent in a mouse model with triple-negative breast cancer. The dendrimer entered into the cells efficiently, and exhibited selective toxicity for 4T1 cells. In vivo investigations proved that a local injection of CRD13 caused a reduction of tumour mass and was non-toxic. ICP analyses indicated that Ru(II) accumulated in all tested tissues with a greater content detected in the tumour.

Triple negative breast cancer metastasis is hindered by a peptide antagonist of F11R/JAM­A protein.

BACKGROUND: The F11R/JAM-A cell adhesion protein was examined as the therapeutic target in triple negative breast cancer (TNBC) with the use of the peptide antagonist to F11R/JAM-A, that previously inhibited the early stages of breast cancer metastasis in vitro. METHODS: The online in silico analysis was performed by TNMPlot, UALCAN, and KM plotter. The in vitro experiments were performed to verify the effect of peptide 4D (P4D) on human endothelial cell lines EA.hy926 and HMEC-1 as well as on human TNBC cell line MDA-MB-231. The cell morphology upon P4D treatment was verified by light microscopy, while the cell functions were assessed by colony forming assay, MTT cell viability assay, BrdU cell proliferation assay, and Transepithelial/Endothelial Electrical Resistance measurements. The in vivo experiments on 4T1 murine breast cancer model were followed by histopathological analysis and a series of quantitative analyses of murine tissues. RESULTS: By in silico analysis we have found the elevated gene expression in breast cancer with particular emphasis on TNBC. The elevated F11R expression in TNBC was related with poorer survival prognosis. Peptide 4D has altered the morphology and increased the permeability of endothelial monolayers. The colony formation, viability, and proliferation of MDA-MB-231 cells were decreased. P4D inhibited the metastasis in 4T1 breast cancer murine model in a statistically significant manner that was demonstrated by the resampling bootstrap technique. CONCLUSIONS: The P4D peptide antagonist to F11R/JAM-A is able to hinder the metastasis in TNBC. This assumption needs to be confirmed by additional 4T1 mouse model study performed on larger group size, before making the decision on human clinical trials.

Carbosilane ruthenium metallodendrimer as alternative anti-cancer drug carrier in triple negative breast cancer mouse model: A preliminary study.

The carbosilane metallodendrimer G1-[[NCPh(o-N)Ru(η6- p-cymene)Cl]Cl]4 (CRD13), based on an arene Ru(II) complex coordinated to imino-pyridine surface groups, has been conjugated with anti-cancer drugs. Ruthenium in the positively-charged dendrimer structure allows this nanoparticle to be considered as an anticancer drug carrier, made more efficient because ruthenium has anticancer properties. The ability of CRD13 to form complexes with Doxorubicin (DOX), 5-Fluorouracil (5-Fu), and Methotrexate (MTX) has been evaluated using zeta potential measurement, transmission electron microscopy (TEM) and computer simulation. The results show that it forms stable nanocomplexes with all those drugs, enhancing their effectiveness against MDA-MB-231 cancer cells. In vivo tests indicate that the CRD13/DOX system caused a decrease of tumor weight in mice with triple negative breast cancer. However, the tumors were most visibly reduced when naked dendrimers were injected.

SARS-CoV-2 Spike Protein and Neutralizing Anti-Spike Protein Antibodies Modulate Blood Platelet Function.

Several studies report elevated blood platelet activation and altered platelet count in COVID-19 patients, but the role of the SARS-CoV-2 spike protein in this process remains intriguing. Additionally, there is no data that anti-SARS-CoV-2 neutralizing antibodies (nAb) may attenuate spike protein activity toward blood platelets. Our results indicate that under in vitro conditions, the spike protein increased the collagen-stimulated aggregation of isolated platelets and induced the binding of vWF to platelets in ristocetin-treated blood. The spike protein also significantly reduced collagen- or ADP-induced aggregation or decreased GPIIbIIIa (fibrinogen receptor) activation in whole blood, depending on the presence of the anti-spike protein nAb. Our findings suggest that studies on platelet activation/reactivity in COVID-19 patients or in donors vaccinated with anti-SARS-CoV-2 and/or previously-infected COVID-19 should be supported by measurements of spike protein and IgG anti-spike protein antibody concentrations in blood.

Protein disulfide isomerase-A1 regulates intraplatelet reactive oxygen species-thromboxane A2 -dependent pathway in human platelets.

BACKGROUND: Platelet-derived protein disulfide isomerase 1 (PDIA1) regulates thrombus formation, but its role in the regulation of platelet function is not fully understood. AIMS: The aim of this study was to characterize the role of PDIA1 in human platelets. METHODS: Proteomic analysis of PDI isoforms in platelets was performed using liquid chromatography tandem mass spectometry, and the expression of PDIs on platelets in response to collagen, TRAP-14, or ADP was measured with flow cytometry. The effects of bepristat, a selective PDIA1 inhibitor, on platelet aggregation, expression of platelet surface activation markers, thromboxane A2 (TxA2 ), and reactive oxygen species (ROS) generation were evaluated by optical aggregometry, flow cytometry, ELISA, and dihydrodichlorofluorescein diacetate-based fluorescent assay, respectively. RESULTS: PDIA1 was less abundant compared with PDIA3 in resting platelets and platelets stimulated with TRAP-14, collagen, or ADP. Collagen, but not ADP, induced a significant increase in PDIA1 expression. Bepristat potently inhibited the aggregation of washed platelets induced by collagen or convulxin, but only weakly inhibited platelet aggregation induced by TRAP-14 or thrombin, and had the negligible effect on platelet aggregation induced by arachidonic acid. Inhibition of PDIA1 by bepristat resulted in the reduction of TxA2 and ROS production in collagen- or thrombin-stimulated platelets. Furthermore, bepristat reduced the activation of αIIbß3 integrin and expression of P-selectin. CONCLUSIONS: PDIA1 acts as an intraplatelet regulator of the ROS-TxA2 pathway in collagen-GP VI receptor-mediated platelet activation that is a mechanistically distinct pathway from extracellular regulation of αIIbß3 integrin by PDIA3.

Adenosine Receptor Agonist HE-NECA Enhances Antithrombotic Activities of Cangrelor and Prasugrel in vivo by Decreasing of Fibrinogen Density in Thrombus.

Blood platelets' adenosine receptors (AR) are considered to be a new target for the anti-platelet therapy. This idea is based on in vitro studies which show that signaling mediated by these receptors leads to a decreased platelet response to activating stimuli. In vivo evidence for the antithrombotic activity of AR agonists published to date were limited, however, to the usage of relatively high doses given in bolus. The present study was aimed at verifying if these substances used in lower doses in combination with inhibitors of P2Y12 could serve as components of dual anti-platelet therapy. We have found that a selective A2A agonist 2-hexynyl-5'-N-ethylcarboxamidoadenosine (HE-NECA) improved the anti-thrombotic properties of either cangrelor or prasugrel in the model of ferric chloride-induced experimental thrombosis in mice. Importantly, HE-NECA was effective not only when applied in bolus as other AR agonists in the up-to-date published studies, but also when given chronically. In vitro thrombus formation under flow conditions revealed that HE-NECA enhanced the ability of P2Y12 inhibitors to decrease fibrinogen content in thrombi, possibly resulting in their lower stability. Adenosine receptor agonists possess a certain hypotensive effect and an ability to increase the blood-brain barrier permeability. Therefore, the effects of anti-thrombotic doses of HE-NECA on blood pressure and the blood-brain barrier permeability in mice were tested. HE-NECA applied in bolus caused a significant hypotension in mice, but the effect was much lower when the substance was given in doses corresponding to that obtained by chronic administration. At the same time, no significant effect of HE-NECA was observed on the blood-brain barrier. We conclude that chronic administration of the A2A agonist can be considered a potential component of a dual antithrombotic therapy. However, due to the hypotensive effect of the substances, dosage and administration must be elaborated to minimize the side-effects. The total number of animals used in the experiments was 146.

Time-dependent interactions of blood platelets and cancer cells, accompanied by extramedullary hematopoiesis, lead to increased platelet activation and reactivity in a mouse orthotopic model of breast cancer - implications for pulmonary and liver metastasis.

Aging has become a significant risk factor for several diseases, including breast cancer.Platelet activation and platelet-cancer cell aggregate fractions were found to increase with tumor progression in a mouse model of breast cancer. At advanced stages of tumor development, platelets from mice with breast cancer were hyperreactive to low agonist concentrations and hyporeactive to high ones. Platelet activation and reactivity were strongly associated with breast cancer metastasis in the lungs and extramedullary hematopoiesis in the liver. A greater fraction of platelet aggregates was observed in 4T1-injected mice at the advanced stages of breast cancer. In vitro, platelet activation was elevated after incubation with 4T1 cells, and thrombin-stimulated platelets formed aggregates with 4T1 cells. Neither GPIbα, nor GPIIb/IIIa blocking antibodies, were able to affect platelet-cancer cell aggregation in vitro.The primed circulating platelets became more sensitive to subthreshold stimuli at advanced stages of tumor development, and the formation of platelet-cancer cell aggregates increased with cancer progression. Our findings demonstrate that the age-associated progression of breast cancer cells is connected with increased platelet functioning, and that it can be manifested by the increased number of metastases and extramedullary hematopoiesis in a time-dependent-manner.

Functional inhibition of F11 receptor (F11R/junctional adhesion molecule-A/JAM-A) activity by a F11R-derived peptide in breast cancer and its microenvironment.

PURPOSE: To examine the involvement of the F11R/JAM-A protein in breast cancer metastasis, we utilized the F11R/JAM-A antagonistic peptide 4D (P4D) in experiments of transendothelial migration (TEM) of breast cancer cells. METHODS: Experiments were conducted in the mouse 4T1 breast cancer model utilizing the human mammary epithelial cell and endothelial cell lines. The levels of soluble F11R/JAM-A (sJAM-A) in the murine plasmas were measured by ELISA. Levels of F11R/JAM-A mRNA and protein in cell lines were assessed by qRT-PCR and Western blot, respectively. Cell surface expression of F11R/JAM-A was demonstrated by flow cytometry. Functional tests included the TEM of breast cancer cells and adhesion of breast cancer cells to the endothelium. The endothelial permeability was studied by fluorescent tracer assay and by the Real-Time Cell Analysis (RTCA). RESULTS: The tumor inducers Tß4 and TGF-ß1 reduced the levels of sJAM-A in murine plasma, and reduced the F11R/JAM-A protein levels in the human microvascular endothelial cell line HMEC-1. The adhesion and TEM measured between breast cancer cells and inflamed or Tß4-treated endothelium were inhibited by P4D. The presence of P4D did not destabilize the pre-existing tight junctions in the endothelial monolayer. The barrier-protecting effect of P4D was stronger than that of forskolin, when a booster dose of P4D was applied to the inflamed endothelium. CONCLUSIONS: F11R/JAM-A protein can be considered as a novel target in the treatment of breast cancer metastasis. In vivo and clinical studies are needed to further investigate the effectiveness of F11R/JAM-A-derived peptide as a possible anti-metastatic drug.

Interleukin 17A Promotes Lymphocytes Adhesion and Induces CCL2 and CXCL1 Release from Brain Endothelial Cells.

The nature of the interaction between Th17 cells and the blood-brain barrier (BBB) is critical for the development of autoimmune inflammation in the central nervous system (CNS). Tumor necrosis factor alpha (TNF-α) or interleukin 17 (IL-17) stimulation is known to enhance the adherence of Th17 cells to the brain endothelium. The brain endothelial cells (bEnd.3) express Vascular cell adhesion molecule 1 (VCAM-1), the receptor responsible for inflammatory cell adhesion, which binds very late antigen 4 (VLA-4) on migrating effector lymphocytes at the early stage of brain inflammation. The present study examines the effect of the pro-inflammatory cytokines TNF-α and IL-17 on the adherence of Th17 cells to bEnd.3. The bEnd.3 cells were found to increase production of CCL2 and CXCL1 after stimulation by pro-inflammatory cytokines, while CCL2, CCL5, CCL20 and IL17 induced Th17 cell migration through a bEnd.3 monolayer. This observation may suggest potential therapeutic targets for the prevention of autoimmune neuroinflammation development in the CNS.

The impact of multiple sclerosis relapse treatment on migration of effector T cells--Preliminary study.

UNLABELLED: Migration of inflammatory cells from the blood to the central nervous system (CNS) is crucial for development of multiple sclerosis (MS). Inhibition of this process would allow to control disease activity. The first step confirming this approach would be the analysis of the impact of effective MS relapse therapy on migration of effector T cells. The aim of the study was to analyze the influence of methylprednisolone (MP) on the migratory activity of effector CD4+ T cells from MS patients. Moreover, to study the potential mechanism of this process we studied expression of chemokine receptors on migrating cells. MATERIAL AND METHODS: Peripheral blood samples were obtained from relapsing-remitting MS (RR-MS) patients during relapse (n=23) and from control group (n=23). After isolation CD4+ T cells were incubated with various concentrations of MP. Then they were stimulated in chemotaxis assay with chemokines CCL3 or CXCL10 or were used to CCR1 and CXCR3 expression analysis. RESULTS: CXCL10- and CCL3-stimulated migration of CD4+ T cells was significantly increased in MS. MP was able to reduce in vitro migration of effector T cells induced by CXCL10, but not by CCL3. Inhibition by MP was dose-dependent. Expression of analyzed chemokine receptors was unaltered after MP incubation. CONCLUSIONS: MP reduced CD4+ T cells migration induced by CXCL10 without affecting CXCR3 expression. These observations demonstrate one of the potential mechanisms of MP action in MS, distinct from inducing cell apoptosis, and suggests the new targets for development of more effective MS treatments.

Interactions between neutrophils, Th17 cells, and chemokines during the initiation of experimental model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS) in which activated T cell and neutrophil interactions lead to neuroinflammation. In this study the expression of CCR6, CXCR2, and CXCR6 in Th17 cells and neutrophils migrating to the brain during EAE was measured, alongside an evaluation of the production of IL-17, IL-23, CCL-20, and CXCL16 in the brain. Next, inflammatory cell subpopulations accumulating in the brain after intracerebral injections of IL-17 or CXCL1, as well as during modulation of EAE with anti-IL-23R or anti-CXCR2 antibodies, were analyzed. Th17 cells upregulate CXCR2 during the preclinical phase of EAE and a significant migration of these cells to the brain was observed. Neutrophils upregulated CCR6, CXCR2, and CXCR6 during EAE, accumulating in the brain both prior to and during acute EAE attacks. Production of IL-17, IL-23, CCL20, and CXCL16 in the CNS was increased during both preclinical and acute EAE. Intracerebral delivery of CXCL1 stimulated the early accumulation of neutrophils in normal and preclinical EAE brains but reduced the migration of Th17 cells to the brain during the preclinical stage of EAE. Modulation of EAE by anti-IL-23R antibodies ameliorated EAE by decreasing the intracerebral accumulation of Th17 cells.