Karboksilesteraza djelomice hidrolizira estere gama-butirobetaina u serumu štakora

Although described some time ago, gamma-butyrobetaine esters and related compounds have not gained much attention from researchers, and their physiological function remains obscure. Formerly we detected GBB-esterase enzymatic activity in rat blood serum using phenylated gamma-butyrobetaine as an artificial substrate of the enzyme and HPLC. The aim of the present work was to develop an assay that would enable spectrophotometric or colorimetric determination of the reaction products of GBB-esterase activity and to reveal individual proteins performing GBB-esterase activity in rat blood serum. For this purpose gammabutyrobetaine 1-naphthyl ester was synthesised. Hydrolysis of this ester releases 1-naphthol, which increases the optical absorbance at 322 nm. We have shown that the enzymatic hydrolysis of GBB 1-naphthyl ester to 1-naphthol in rat blood serum is due to GBB-esterase activity. An attempt was done to purify the enzyme from rat blood serum. By combining DEAE Sepharose at pH 4.2 and affinity chromatography with procainamide we achieved a 68-fold enrichment of GBB-esterase activity in our preparations. Separation of fraction proteins in 2D protein electrophoresis with following mass-spectrometry indicated that GBB esterase activity in rat blood serum is performed in part by carboxylesterase.Premda su poznati već neko vrijeme, esteri gama-butirobetaina (GBB) i srodni spojevi nisu dobili mnogo pozornosti u znanstvenoistraživačkoj zajednici, a njihova fiziološka funkcija i dalje je nerazjašnjena. U ranijem smo istraživanju HPLC-metodom otkrili enzimsku aktivnost GBB-esteraze u serumu štakora rabeći umjetni supstrat ovoga enzima, tj. fenilirani gama-butirobetain. Cilj ovoga istraživanja bio je razviti pretragu koja će omogućiti spektrofotometrijsko ili kolorimetrijsko određivanje reakcijskih produkata nastalih aktivnošću GBB-esteraze te utvrditi koji pojedinačni proteini u serumu štakora posjeduju aktivnost GBB-esteraze. U tu je svrhu sintetiziran gama-butirobetain 1-naftil ester. Njegovom hidrolizom otpušta se 1-naftol koji povećava optičku apsorbanciju na 322 nm. Pokazali smo da u hidrolizi GBB 1-naftil estera u 1-naftol u serumu štakora sudjeluje GBB-esteraza. Pokušali smo pročistiti enzim iz štakorskoga seruma. Kombinacijom DEAE sefaroze pri pH 4,2 i afinitetne kromatografije s prokainamidom dobili smo 68-erostruko povećanje aktivnosti ovoga enzima. Razdvajanjem bjelančevina iz frakcije s pomoću 2D elektroforeze te naknadnom spektrometrijom masa utvrđeno je da u serumu štakora esteraznu aktivnost prema gama-butirobetainu kao supstratu djelomice provodi karboksilesteraza

General consensus on multimodal functions and validation analysis of perinatal derivatives for regenerative medicine applications.

Perinatal tissues, such as placenta and umbilical cord contain a variety of somatic stem cell types, spanning from the largely used hematopoietic stem and progenitor cells to the most recently described broadly multipotent epithelial and stromal cells. As perinatal derivatives (PnD), several of these cell types and related products provide an interesting regenerative potential for a variety of diseases. Within COST SPRINT Action, we continue our review series, revising and summarizing the modalities of action and proposed medical approaches using PnD products: cells, secretome, extracellular vesicles, and decellularized tissues. Focusing on the brain, bone, skeletal muscle, heart, intestinal, liver, and lung pathologies, we discuss the importance of potency testing in validating PnD therapeutics, and critically evaluate the concept of PnD application in the field of tissue regeneration. Hereby we aim to shed light on the actual therapeutic properties of PnD, with an open eye for future clinical application. This review is part of a quadrinomial series on functional/potency assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer, anti-inflammation, wound healing, angiogenesis, and regeneration

Effects of human placenta cryopreservation on molecular characteristics of placental mesenchymal stromal cells

Cryopreservation of placenta tissue for long-term storage provides the opportunity in the future to isolate mesenchymal stromal cells that could be used for cell therapy and regenerative medicine. Despite being widely used, the established cryopreservation protocols for freezing and thawing still raise concerns about their impact on molecular characteristics, such as epigenetic regulation. In our study, we compared the characteristics of human placental mesenchymal stromal cells (hPMSCs) isolated from fresh (native) and cryopreserved (cryo) placenta tissue. We assessed and compared the characteristics of native and cryo hPMSCs such as morphology, metabolic and differentiation potential, expression of cell surface markers, and transcriptome. No significant changes in immunophenotype and differentiation capacity between native and cryo cells were observed. Furthermore, we investigated the epigenetic changes and demonstrated that both native and cryo hPMSCs express only slight variations in the epigenetic profile, including miRNA levels, DNA methylation, and histone modifications. Nevertheless, transcriptome analysis defined the upregulation of early-senescence state-associated genes in hPMSCs after cryopreservation. We also evaluated the ability of hPMSCs to improve pregnancy outcomes in mouse models. Improved pregnancy outcomes in a mouse model confirmed that isolated placental cells both from native and cryo tissue have a positive effect on the restoration of the reproductive system. Still, the native hPMSCs possess better capacity (up to 66%) in comparison with cryo hPMSCs (up to 33%) to restore fertility in mice with premature ovarian failure. Our study demonstrates that placental tissue can be cryopreserved for long-term storage with the possibility to isolate mesenchymal stromal cells that retain characteristics suitable for therapeutic use

The Histone Deacetylase Inhibitor BML-210 Influences Gene and Protein Expression in Human Promyelocytic Leukemia NB4 Cells via Epigenetic Reprogramming

Today, cancer is understood as an epigenetic as well as genetic disease. The main epigenetic hallmarks of the cancer cell are DNA methylation and histone modifications. Proteins such as histone deacetylases (HDACs) that cause modifications of histones and other proteins can be targets for novel anticancer agents. Recently, interest in compounds that can inhibit HDACs increased, and now there are many HDACs inhibitors (HDACIs) available with different chemical structures, biological and biochemical properties; hopefully some of them will succeed, probably in combination with other agents, in cancer therapies. In our study we focused on the novel HDACI–BML-210. We found that BML-210 (N-phenyl-Nʹ-(2-Aminophenyl)hexamethylenediamide) inhibits the growth of NB4 cells in dose- and time-dependent manner. In this study we also examined how expression and activity of HDACs are affected after leukemia cell treatment with BML-210. Using a mass spectrometry method we identified proteins that changed expression after treatment with BML-210. We prepared RT-PCR analysis of these genes and the results correlated with proteomic data. Based on these and other findings from our group, we suggest that HDACIs, like BML-210, can be promising anticancer agents in promyelocytic leukemia treatment

The histone deacetylase inhibitor BML-210 influences on gene and protein expression in human promyelocytic leukemia NB4 cells via epigenetic reprogramming

Background: Today, cancer is understood as an epigenetic as well as genetic disease. The main epigenetic hallmarks of the cancer cell are DNA methylation and histone modifications. Proteins such as histone deacetylases (HDACs) that cause modifications of histones and other proteins can be targets for novel anticancer agents. Recently interest in compounds that can inhibit HDACs increased and now there are so many HDACIs available with different chemical structures, biological and biochemical properties that hopefully some of them will succeed, probably in combination with other agents, in cancer therapies. Results: In our study we focused ourselves on a novel HDACI – BML-210. We found that BML-210 inhibits the growth of NB4 cells in dose and time-dependent manner. In this study we also examined how expression and activity of HDACs are affected after leukemia cell treatment with BML-210. Using mass spectrometry method we identified proteins that changed expression after treatment with BML-210. We prepared RT-PCR analysis of these genes and results correlated with proteomic data. Conclusion: Based on these and other our group finding, we suggest that HDACIs, like BML-210, can be promising anticancer agents in promyelocytic leukemia treatment

Anti-leukemic activity of DNA methyltransferase inhibitor procaine targeted on human leukaemia cells

Chromatin remodeling in DNA is fundamental to gene expression, DNA replication and repair processes. Methylation of promoter regions of tumor-suppressor genes and histone deacetylation leads to gene silencing and transcriptionally repressive chromatin. For the past few decades DNA methylation agents became very attractive as the targets for cancer therapy. The purpose of this work was to examine the effects of DNMT inhibitor procaine on growth inhibition, apoptosis and differentiation of human leukaemia cells. The changes in expression of genes, proteins and histone modifications caused by procaine were evaluated under different treatments. We demonstrated that procaine arrests growth of human leukaemia cells and in combination with all-trans retinoic acid (ATRA) induces cancer cell differentiation. Procaine causes reduction of expression of DNA methyltransferases as well. The treatment of human leukaemia cells with procaine increase the expression of molecules associated with differentiation (CD11b, E-cadherin, G-CSF) and apoptosis (PPARγ). Moreover, the examined DNMT inhibitor enhances certain gene transcription activation via chromatin remodelling – the changes in histone H3K4(Me)3 and H3K9Ac/S10P modifications were detected. Our results suggest, that DNMT inhibitor – procaine, can be used for further investigations on epigenetic differentiation therapy of leukaemia cells especially when used in combination with retinoic acid

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

Potential Prognostic Markers for Relapsed/Refractory vs. Responsive Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease. A significant proportion of AML patients is refractory to clinical treatment or relapses. Our aim is to determine new potential AML clinical treatment prognosis markers. We investigated various cell fate and epigenetic regulation important gene level differences between refractory and responsive AML patient groups at diagnosis stage and after clinical treatment using RT-qPCR. We demonstrated that oncogenic MYC and WT1 and metabolic IDH1 gene expression was significantly higher and cell cycle inhibitor CDKN1A (p21) gene expression was significantly lower in refractory patients’ bone marrow cells compared to treatment responsive patients both at diagnosis and after clinical treatment. Moreover, we determined that, compared to clinical treatment responsive patients, refractory patients possess a significantly higher gene expression of histone deacetylase 2 (HDAC2) and epigenetic DNA modulator TET1 and a significantly lower gene expression of lysine acetyltransferase 6A (KAT6A) and nucleosome remodeling and deacetylase (NuRD) complex component GATAD2A. We suggest that MYC, WT1, IDH1, CDKN1A, HDAC2, TET1, KAT6A and GATAD2A gene expression changes might characterize refractory AML. Thus, they might be useful for AML prognosis. Additionally, we suggest that epigenetic modulation might be beneficial in combination with standard treatment

Small molecule treatments improve differentiation potential of human amniotic fluid stem cells

Human amniotic fluid stem cells (AFSC) are an exciting and very promising source of stem cells for therapeutic applications. In this study we investigated the effects of short-term treatments of small molecules to improve stem cell properties and differentiation capability. For this purpose, we used epigenetically active compounds, such as histone deacetylase inhibitors Trichostatin A (TSA) and sodium butyrate (NaBut), as well as multifunctional molecules of natural origin, such as retinoic acid (RA) and vitamin C (vitC). We observed that combinations of these compounds triggered upregulation of genes involved in pluripotency (KLF4, OCT4, NOTCH1, SOX2, NANOG, LIN28a, CMYC), but expression changes of these proteins were mild with only significant downregulation of Notch1. Also, some alterations in cell surface marker expression was established by flow cytometry with the most explicit changes in the expression of CD105 and CD117. Analysis of cellular energetics performed using Seahorse analyzer and assessment of gene expression related to cell metabolism and respiration (NRF1, HIF1 alpha, PPARGC1A, ERR alpha, PKM, PDK1, LDHA, NFKB1, NFKB2, RELA, RELB, REL) revealed that small molecule treatments stimulate AFSCs toward a more energetically active phenotype. To induce cells to differentiate toward neurogenic lineage several different protocols including commercial supplements N2 and B27 together with RA were used and compared to the same differentiation protocols with the addition of a pre-induction step consisting of a combination of small molecules (vitC, TSA and RA). During differentiation the expression of several neural marker genes was analyzed (Nestin, MAP2, TUBB3, ALDH1L1, GFAP, CACNA1D, KCNJ12, KCNJ2, KCNH2) and the beneficial effect of small molecule treatment on differentiation potential was observed with upregulated gene expression. Differentiation was also confirmed by staining TUBB3, NCAM1, and Vimentin and assessed by secretion of BDNF. The results of this study provide valuable insights for the potential use of short-term small molecule treatments to improve stem cell characteristics and boost differentiation potential of AFSCs

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 cell