Anti-neuroinflammatory microRNA-146a-5p as a potential biomarker for neuronavigation-guided rTMS therapy success in medication resistant depression disorder.

Treatment-resistant depression (TRD) is a challenging issue to address. Repetitive transcranial magnetic stimulation (rTMS) is commonly used but shows varying efficacy, necessitating a deeper understanding of depression physiology and rTMS mechanisms. Notably, an increasing amount of recent data has displayed the connection of TRD and its clinical outcome with chronic inflammatory processes. The current study included 19 TRD patients undergoing rTMS and 11 depressed patients responding to medication as a comparison group. We assessed therapeutic efficacy using MADRS, HAM-D-17, GAD-7, and PHQ-9 tests. Inflammatory markers, neurotrophins, and associated miRNAs were measured in patients blood serum before and during treatment. A control group of 18 healthy individuals provided baseline data. The results of our study showed significantly higher levels of pro-inflammatory interleukins-6 and - 8 in TRD patients compared to drug-responders, which also related to more severe symptoms before treatment. In addition, TRD patients, both before and during treatment, exhibited higher average blood serum concentrations of pro-inflammatory interleukin-18 and lower levels of anti-neuroinflammatory miR-146a-5p compared to healthy controls. We also observed that the expression of miR-16-5p, miR-93-5p, and especially miR-146a-5p correlated with clinical changes following rTMS. Our study confirmed that TRD patients possess a higher inflammatory status, while the anti-neuroinflammatory miR-146a-5p was demonstrated to have a considerable potential for predicting their rTMS treatment success.

Effect of 3D Spheroid Culturing on NF-κB Signaling Pathway and Neurogenic Potential in Human Amniotic Fluid Stem Cells.

Human amniotic fluid stem cells (hAFSCs) are known for their advantageous properties when compared to somatic stem cells from other sources. Recently hAFSCs have gained attention for their neurogenic potential and secretory profile. However, hAFSCs in three-dimensional (3D) cultures remain poorly investigated. Therefore, we aimed to evaluate cellular properties, neural differentiation, and gene and protein expression in 3D spheroid cultures of hAFSCs in comparison to traditional two-dimensional (2D) monolayer cultures. For this purpose, hAFSCs were obtained from amniotic fluid of healthy pregnancies and cultivated in vitro, either in 2D, or 3D under untreated or neuro-differentiated conditions. We observed upregulated expression of pluripotency genes OCT4, NANOG, and MSI1 as well as augmentation in gene expression of NF-κB-TNFα pathway genes (NFKB2, RELA and TNFR2), associated miRNAs (miR103a-5p, miR199a-3p and miR223-3p), and NF-κB p65 protein levels in untreated hAFSC 3D cultures. Additionally, MS analysis of the 3D hAFSCs secretome revealed protein upregulation of IGFs signaling the cascade and downregulation of extracellular matrix proteins, whereas neural differentiation of hAFSC spheroids increased the expression of SOX2, miR223-3p, and MSI1. Summarizing, our study provides novel insights into how 3D culture affects neurogenic potential and signaling pathways of hAFSCs, especially NF-κB, although further studies are needed to elucidate the benefits of 3D cultures more thoroughly.

Metabolic Profile and Neurogenic Potential of Human Amniotic Fluid Stem Cells From Normal vs. Fetus-Affected Gestations.

Human amniotic fluid stem cells (hAFSCs) possess some characteristics with mesenchymal stem cells (MSCs) and embryonic stem cells and have a broader differentiation potential compared to MSCs derived from other sources. Although hAFSCs are widely researched, their analysis mainly involves stem cells (SCs) obtained from normal, fetus-unaffected gestations. However, in clinical settings, knowledge about hAFSCs from normal gestations could be poorly translational, as hAFSCs from healthy and fetus-diseased gestations may differ in their differentiation and metabolic potential. Therefore, a more thorough investigation of hAFSCs derived from pathological gestations would provide researchers with the knowledge about the general characteristics of these cells that could be valuable for further scientific investigations and possible future clinical applicability. The goal of this study was to look into the neurogenic and metabolic potential of hAFSCs derived from diseased fetuses, when gestations were concomitant with polyhydramnios and compare them to hAFSCs derived from normal fetuses. Results demonstrated that these cells are similar in gene expression levels of stemness markers (SOX2, NANOG, LIN28A, etc.). However, they differ in expression of CD13, CD73, CD90, and CD105, as flow cytometry analysis revealed higher expression in hAFSCs from unaffected gestations. Furthermore, hAFSCs from "Normal" and "Pathology" groups were different in oxidative phosphorylation rate, as well as level of ATP and reactive oxygen species production. Although the secretion of neurotrophic factors BDNF and VEGF was of comparable degree, as evaluated with enzyme-linked immunosorbent assay (ELISA) test, hAFSCs from normal gestations were found to be more prone to neurogenic differentiation, compared to hAFSCs from polyhydramnios. Furthermore, hAFSCs from polyhydramnios were distinguished by higher secretion of pro-inflammatory cytokine TNFα, which was significantly downregulated in differentiated cells. Overall, these observations show that hAFSCs from pathological gestations with polyhydramnios differ in metabolic and inflammatory status and also possess lower neurogenic potential compared to hAFSCs from normal gestations. Therefore, further in vitro and in vivo studies are necessary to dissect the potential of hAFSCs from polyhydramnios in stem cell-based therapies. Future studies should also search for strategies that could improve the characteristics of hAFSCs derived from diseased fetuses in order for those cells to be successfully applied for regenerative medicine purposes.

Menstrual Blood-Derived Endometrial Stem Cells' Impact for the Treatment Perspective of Female Infertility.

When looking for the causes and treatments of infertility, much attention is paid to one of the reproductive tissues-the endometrium. Therefore, endometrial stem cells are an attractive target for infertility studies in women of unexplained origin. Menstrual blood stem cells (MenSCs) are morphologically and functionally similar to cells derived directly from the endometrium; with dual expression of mesenchymal and embryonic cell markers, they proliferate and regenerate better than bone marrow mesenchymal stem cells. In addition, menstrual blood stem cells are extracted in a non-invasive and painless manner. In our study, we analyzed the characteristics and the potential for decidualization of menstrual blood stem cells isolated from healthy volunteers and women diagnosed with infertility. We demonstrated that MenSCs express CD44, CD166, CD16, CD15, BMSC, CD56, CD13 and HLA-ABC surface markers, have proliferative properties, and after induction of menstrual stem cell differentiation into epithelial direction, expression of genes related to decidualization (PRL, ESR, IGFBP and FOXO1) and angiogenesis (HIF1, VEGFR2 and VEGFR3) increased. Additionally, the p53, p21, H3K27me3 and HyperAcH4 proteins' expression increased during MenSCs decidualization, they secrete proteins that are involved in the regulation of the actin cytoskeleton, estrogen and relaxin signaling pathways and the management of inflammatory processes. Our findings reveal the potential use of MenSCs for the treatment of reproductive disorders.

Pharmaceutical Drug Metformin and MCL1 Inhibitor S63845 Exhibit Anticancer Activity in Myeloid Leukemia Cells via Redox Remodeling.

Metabolic landscape and sensitivity to apoptosis induction play a crucial role in acute myeloid leukemia (AML) resistance. Therefore, we investigated the effect of metformin, a medication that also acts as an inhibitor of oxidative phosphorylation (OXPHOS), and MCL-1 inhibitor S63845 in AML cell lines NB4, KG1 and chemoresistant KG1A cells. The impact of compounds was evaluated using fluorescence-based metabolic flux analysis, assessment of mitochondrial Δψ and cellular ROS, trypan blue exclusion, Annexin V-PI and XTT tests for cell death and cytotoxicity estimations, also RT-qPCR and Western blot for gene and protein expression. Treatment with metformin resulted in significant downregulation of OXPHOS; however, increase in glycolysis was observed in NB4 and KG1A cells. In contrast, treatment with S63845 slightly increased the rate of OXPHOS in KG1 and KG1A cells, although it profoundly diminished the rate of glycolysis. Generally, combined treatment had stronger inhibitory effects on cellular metabolism and ATP levels. Furthermore, results revealed that treatment with metformin, S63845 and their combinations induced apoptosis in AML cells. In addition, level of apoptotic cell death correlated with cellular ROS induction, as well as with downregulation of tumor suppressor protein MYC. In summary, we show that modulation of redox-stress could have a potential anticancer activity in AML cells.

Brain stimulation effects on serum BDNF, VEGF, and TNFα in treatment-resistant psychiatric disorders.

Resistance to pharmacological treatment poses a notable challenge for psychiatry. Such cases are usually treated with brain stimulation techniques, including repetitive transcranial magnetic stimulation (rTMS) and electroconvulsive therapy (ECT). Empirical evidence links treatment resistance to insufficient brain plasticity and chronic inflammation. Therefore, this study encompasses analysis of neurotrophic and inflammatory factors in psychiatric patients undergoing rTMS and ECT in order to refine the selection of patients and predict clinical outcomes. This study enrolled 25 drug-resistant depressive patients undergoing rTMS and 31 drug-resistant schizophrenia patients undergoing ECT. Clinical efficacy of brain stimulation therapies was gauged using MADRS and HAM-D scales in the depression group and PANSS scale in the schizophrenia group. Blood-derived BDNF, VEGF, and TNFα were analysed during the treatment course. For reference, 19 healthy control subjects were also enrolled. After statistical analysis, no significant differences were detected in BDNF, VEGF, and TNFα concentrations among healthy, depressive, and schizophrenic subject groups before the treatment. However, depressive patient treatment with rTMS has increased BDNF concentration, while schizophrenic patient treatment with ECT has lowered the concentration of TNFα. Our findings suggest that a lower initial TNFα concentration could be a marker for treatment success in depressed patients undergoing rTMS, whereas in schizophrenic patient group treated with ECT, a higher concentration of VEGF correlates to milder symptoms post-treatment, especially in the negative scale.

The epigenetic treatment remodel genome-wide histone H4 hyper-acetylation patterns and affect signaling pathways in acute promyelocytic leukemia cells.

Although majority of acute promyelocytic leukemia (APL) patients achieve complete remission after the standard treatment, 5-10% of patients are shown to relapse or develop resistance to treatment. In such cases, medications that target epigenetic processes could become an appealing supplementary approach. In this study, we tested the anti-leukemic activity of histone deacetylase inhibitor Belinostat (PXD101) and histone methyltransferase inhibitor 3-Deazaneplanocin A combined with all-trans retinoic acid in APL cells NB4, promyelocytes resembling HL-60 cells and APL patients' cells. After HL-60 and NB4 cell treatment, ChIP-sequencing was performed using antibodies against hyper-acetylated histone H4. Hyper-acetylated histone H4 distribution peaks were compared in treated vs untreated HL-60 and NB4 cells. Results demonstrated that in treated HL-60 cells, the majority of peaks were distributed within the regions of proximal promoters, whereas in treated NB4 cells, hyper-acetylated histone H4 peaks were mainly localized in gene body regions. Further ChIP-seq data analysis revealed the changes in histone H4 hyper-acetylation in promoter/gene body regions of genes involved in cancer signaling pathways. In addition, quantitative gene expression analysis proved changes in various cellular pathways important for carcinogenesis. Epigenetic treatment down-regulated the expression of MTOR, LAMTOR1, WNT2B, VEGFR3, FGF2, FGFR1, TGFA, TGFB1, TGFBR1, PDGFA, PDGFRA and PDGFRB genes in NB4, HL-60 and APL patients' cells. In addition, effect of epigenetic treatment on protein expression of aforementioned signaling pathways was confirmed with mass spectrometry analysis. Taken together, these results provide supplementary insights into molecular changes that occur during epigenetic therapy application in in vitro promyelocytic leukemia cell model.

Amyloid ß oligomers inhibit growth of human cancer cells.

Effects of amyloid beta (Aß) oligomers on viability and function of cell lines such as NB4 (human acute promyelocytic leukemia), A549 (human lung cancer (adenocarcinomic alveolar basal epithelial tumor)) and MCF-7 (human breast cancer (invasive breast ductal carcinoma)) were investigated. Two types of Aß oligomers were used in the study. The first type was produced in the presence of oligomerization inhibitor, hexafluoroisopropanol (HFIP). The second type of amyloids was assembled in the absence of the inhibitor. The first type preparation was predominantly populated with dimers and trimers, while the second type contained mostly pentadecamers. These amyloid species exhibited different secondary protein structure with considerable amount of antiparallel ß sheet structural elements in HFIP oligomerized Aß mixtures. The effect of the cell growth inhibition, which was stronger in the case of HFIP Aß oligomers, was observed for all cell lines. Tests aiming at elucidating the effects of the amyloid species on cell cycles showed little differences between amyloid preparations. This prompts us to conclude that the effect on the cancer cell proliferation rate is less specific to the biological processes developing inside the cells during the proliferation. Therefore, cell growth inhibition may involve interactions with the peripheral parts of the cancer cells, such as a phospholipid membrane, and only in case of the NB4 cells, where accumulation of amyloid species inside the cells was detected, one may imply the opposite. In general, cancer cells were much less susceptible to the damaging effects of amyloid oligomers compared to earlier observations in mixed neuronal cell cultures.

Anti-leukemic effects of HDACi Belinostat and HMTi 3-Deazaneplanocin A on human acute promyelocytic leukemia cells.

Development of acute myeloid leukemia is usually sustained by deregulated epigenome. Alterations in DNA methylation and histone modifications are common manifestations of the disease. Acute promyelocytic leukemia (APL) is not an exception. Therefore, drugs that target epigenetic processes suggest an appealing strategy for APL treatment. In this study we tested the anti-leukemic activity of histone deacetylase inhibitor (HDACi) Belinostat (PXD101, (2E)-N-Hydroxy-3-[3-(phenylsulfamoyl)phenyl]prop-2-enamide), and histone methyltransferase inhibitor (HMTi) 3-Deazaneplanocin A (DZNep, 5R-(4-amino-1H-imidazo[4,5-c]pyridin-1-yl)-3-(hydroxymethyl)-3-cyclopentene-1S,2R-diol) combined with retinoic acid (RA) in APL cells NB4 and HL-60. We demonstrated that APL cell treatment with combinations of differentiation inductor RA, HDACi Belinostat and HMTi DZNep caused a depletion of leukemia cell growth and viability, initiated apoptosis and exaggerated RA induced granulocytic differentiation. Also an increased expression of transcription factors C/EBPε and PPARγ was demonstrated, while no significant reduction in C/EBPα gene level was detected. Furthermore, combined treatment depleted gene expression levels of EZH2 and SUZ12, especially in HL-60 cells, and diminished protein levels of Polycomb Repressive Complex 2 (PRC2) components EZH2, SUZ12 and EED. In addition, our study has shown that Belinostat and DZNep together with RA caused a depletion in HDAC1 and HDAC2 protein levels, HDAC2 gene expression and increased hyperacetylation of histone H4 in both leukemia cell lines. Using ChIP method we also demonstrated the increased association of hyperacetylated histone H4 with the C/EBPα and C/EBPε promoter regions in HL-60 cells. Summarizing, these findings indicate that combined treatment with RA, Belinostat and 3-Deazaneplanocin A is an effective epigenetic inducer for leukemia cell differentiation.

Histone modifications patterns in tissues and tumours from acute promyelocytic leukemia xenograft model in response to combined epigenetic therapy.

Xenograft models are suitable for in vivo study of leukemia's pathogenesis and the preclinical development of anti-leukemia agents but understanding of epigenetic regulatory mechanisms linking to adult cell functions in pathological conditions during different in vivo treatments is yet unknown. In this study, for the first time epigenetic chromatin modifications were characterized in tissues and tumours from murine xenograft model generated using the human acute promyelocytic leukemia (APL) NB4 cells engrafted in immunodeficient NOG mice. Xenografts were subjected to combined epigenetic treatment by histone deacetylase inhibitor Belinostat, histone methyltransferase inhibitor 3-DZNeaplanocin A and all-trans-retinoic acid based on in vitro model, where such combination inhibited NB4 cell growth and enhanced retinoic acid-induced differentiation to granulocytes. Xenotransplantation was assessed by peripheral blood cells counts, the analysis of cell surface markers (CD15, CD33, CD45) and the expression of certain genes (PML-RAR alpha, CSF3, G-CSFR, WT1). The combined treatment prolonged APL xenograft mice survival and prevented tumour formation. The analysis of the expression of histone marks such as acetylation of H4, trimethylation of H3K4, H3K9 and H3K27 in APL xenograft mice tumours and tissues demonstrated tissue-specific changes in the level of histone modifications and the APL prognostic mark, WT1 protein. In summary, the effects of epigenetic agents used in this study were positive for leukemia prevention and linked to a modulation of the chromatin epigenetic environment in adult tissues of malignant organism.

Human Amniotic Fluid Mesenchymal Stem Cells from Second- and Third-Trimester Amniocentesis: Differentiation Potential, Molecular Signature, and Proteome Analysis.

Human amniotic fluid stem cells have become an attractive stem cell source for potential applications in regenerative medicine and tissue engineering. The aim of this study was to characterize amniotic fluid-derived mesenchymal stem cells (AF-MSCs) from second- and third-trimester of gestation. Using two-stage protocol, MSCs were successfully cultured and exhibited typical stem cell morphological, specific cell surface, and pluripotency markers characteristics. AF-MSCs differentiated into adipocytes, osteocytes, chondrocytes, myocytes, and neuronal cells, as determined by morphological changes, cell staining, and RT-qPCR showing the tissue-specific gene presence for differentiated cell lineages. Using SYNAPT G2 High Definition Mass Spectrometry technique approach, we performed for the first time the comparative proteomic analysis between undifferentiated AF-MSCs from late trimester of gestation and differentiated into myogenic, adipogenic, osteogenic, and neurogenic lineages. The analysis of the functional and expression patterns of 250 high abundance proteins selected from more than 1400 demonstrated the similar proteome of cultured and differentiated AF-MSCs but the unique changes in their expression profile during cell differentiation that may help the identification of key markers in differentiated cells. Our results provide evidence that human amniotic fluid of second- and third-trimester contains stem cells with multilineage potential and may be attractive source for clinical applications.

Synthesis and antiproliferative activity of α-branched α,ß-unsaturated ketones in human hematological and solid cancer cell lines.

A series of α-branched α,ß-unsaturated ketones were prepared via boron trifluoride etherate mediated reaction between arylalkynes and carboxaldehydes. The evaluation of the antiproliferative activity over hematological (NB4) and solid cancer (A549, MCF-7) cell lines provided a structure-activity relationship. 5-Parameter QSAR equations were built which were able to explain 80%-92% of the variance in activity. The resulting selective lead compound showed IC50 value 0.6 µM against the hematological cell line and did not cause apoptosis, but blocked cell cycle in G0/G1. Moreover, it was demonstrated that this compound enhances and accelerates retinoic acid induced granulocytic differentiation.

Belinostat, a potent HDACi, exerts antileukaemic effect in human acute promyelocytic leukaemia cells via chromatin remodelling.

Epigenetic changes play a significant role in leukaemia pathogenesis, therefore histone deacetylases (HDACis) are widely accepted as an attractive strategy for acute promyelocytic leukaemia (APL) treatment. Belinostat (Bel, PXD101), a hydroxamate-type HDACi, has proved to be a promising cure in clinical trials for solid tumours and haematological malignancies. However, insight into molecular effects of Bel on APL, is still lacking. In this study, we investigated the effect of Bel alone and in combination with differentiation inducer retinoic acid (RA) on human promyelocytic leukaemia NB4 and HL-60 cells. We found that treatment with Bel, depending on the dosage used, inhibits cell proliferation, whereas in combination with RA enhances and accelerates granulocytic leukaemia cell differentiation. We also evaluated the effect of used treatments with Bel and RA on certain epigenetic modifiers (HDAC1, HDAC2, PCAF) as well as cell cycle regulators (p27) gene expression and protein level modulation. We showed that Bel in combination with RA up-regulates basal histone H4 hyperacetylation level more strongly compared to Bel or RA alone. Furthermore, chromatin immunoprecipitation assay indicated that Bel induces the accumulation of hyperacetylated histone H4 at the p27 promoter region. Mass spectrometry analysis revealed that in control NB4 cells, hyperacetylated histone H4 is mainly found in association with proteins involved in DNA replication and transcription, whereas after Bel treatment it is found with proteins implicated in pro-apoptotic processes, in defence against oxidative stress and tumour suppression. Summarizing, our study provides some novel insights into the molecular mechanisms of HDACi Bel action on APL cells.

Epigenetic and molecular mechanisms underlying the antileukemic activity of the histone deacetylase inhibitor belinostat in human acute promyelocytic leukemia cells.

Therapeutic strategies targeting histone deacetylase (HDAC) inhibition have become promising in many human malignancies. Belinostat (PXD101) is a hydroxamate-type HDAC inhibitor tested in phase I and II clinical trials in solid tumors and hematological cancers. However, little is known about the use of belinostat for differentiation therapy against acute myelogenous leukemia. Here, we characterize the antileukemia activity of belinostat as a single drug and in combination with all-trans-retinoic acid (RA) in promyelocytic leukemia HL-60 and NB4 cells. Belinostat exerted dose-dependent growth-inhibitory or proapoptotic effects, promoting cell cycle arrest at the G0/G1 or the S transition. Apoptosis was accompanied by activation of caspase 3, degradation of PARP-1, and cell cycle-dependent changes in the expression of survivin, cyclin E1, and cyclin A2. Belinostat induced a dose-dependent reduction in the expression of EZH2 and SUZ12, HDAC-1, HDAC-2, and histone acetyltransferase PCAF (p300/CBP-associated factor). Belinostat increased acetylation of histone H4, H3 at K9 and H3 at K16 residues in a dose-dependent manner, but did not reduce trimethylation of H3 at K27 at proapoptotic doses. Combined treatment with belinostat and RA dose dependently accelerated and reinforced granulocytic differentiation, accompanied by changes in the expression of CD11b, C/EBPα (CCAAT/enhancer binding protein-α), and C/EBPε. Our results concluded the usefulness of belinostat, as an epigenetic drug, for antileukemia and differentiation therapy.

Euchromatic histone methyltransferase 2 inhibitor, BIX-01294, sensitizes human promyelocytic leukemia HL-60 and NB4 cells to growth inhibition and differentiation.

The involvement of histone lysine methyltransferases (HMT) in carcinogenesis is not well understood. Here, we describe a dose-dependent growth and survival inhibitory effects of BIX-01294, a specific inhibitor of euchromatic HMT2, in promyelocytic leukemia HL-60 and NB4 cells. BIX-01294 combined with all-trans retinoic acid or together with histone deacetylase and DNA methyltransferase inhibitors enhanced cell differentiation to granulocytes and induced cell line-specific changes in the expression of cell cycle-, survival- and differentiation regulating genes and proteins in association with histone modification state. Our results suggest that targeting EHMT2 may be of therapeutical benefits in myeloid leukemia.