[Epigenetic effects affecting etoposide distribution and metabolism in the human body]. / Az etopozid szervezeten belüli eloszlásában és metabolizmusában szerepet játszó epigenetikai hatások.

Etoposide is a topoisomerase II inhibitor antitumor agent which is widely used in the treatment of several hematologic malignancies and solid tumors. The therapeutic index of etoposide is quite high, thus its application causes several short-term and long-term side effects which can decrease the chance to cure patients. Drug dosing is based on body surface area calculation; recommendations for individual dosing do not exist yet. The biotransformation and transportation of etoposide are carried out by enzymes and transporters as reported in pharmacogenomic studies published in this area. Nowadays pharmacoepigenetics research has come to the fore. The authors wish to give an insight into the research of the epigenetical changes of the etoposide pathways, especially focusing on published findings on enzymes and transporters with pharmacokinetic relevance. In the future, epigenetical changes of the etoposide pathway might have a great role in diagnostics, prognostics and personalized medicine. Orv Hetil. 2018; 159(32): 1295-1302.

Antiferromagnetically Coupled Dimeric Dodecacopper Supramolecular Architectures of Macrocyclic Ligands with a Symmetrical µ6-BO3(3-) Central Moiety.

Reactions between 2,6-diformyl-4-alkyl(R)-phenol (R = CH3 or C(CH3)3) and 1,3-diamino-2-hydroxypropane (1,3-DAP) in the presence of copper(II) salts (Cu(BF4)2·6H2O, Cu(ClO4)2·6H2O/H3BO3/Ar) and triethylamine (TEA) in a single pot result in self-assembly of dimeric dodecacopper supramolecular architectures of 30-membered hexatopic macrocyclic ligands (H6L4 and H6L5) with unique and fascinating structures having the BO3(3-) anion as the central species bonded to all six copper centers in a symmetrical fashion (µ6-BO3(3-)). A number of closely related macrocyclic hexacopper complexes are reported: {[Cu6(L4)(µ6-BO3)(µ-H2O)(C3H7NO)2(BF4)][BF4]2·3C3H7NO}2 (1) (DMF = C3H7NO), {[Cu6(L4)(µ6-BO3)(µ-C3H7NO)3][ClO4]3·3C3H7NO}2 (2), {[Cu6(L5)(µ6-BO3)(µ-OH)(H2O)3(C3H7NO)][BF4]2·6C3H7NO·4C2H5OH·2H2O}2 (3), {[Cu6(L5)(µ6-BO3)(µ-CH3OH)(CH3OH)2][ClO4]3·10H2O}2 (4), and {[Cu6(L5)(µ6-BO3)(µ-CH3CO2)(µ-CH3O)(CH3OH)][BF4]·13CH3OH·8H2O}2 (5). A polymeric side product {[Cu2(H2L2)(CH3OH)(BF4)][BF4]}n (6), involving a 2 + 2 macrocyclic ligand, was also isolated and structurally characterized. Complex 6 involves dinuclear copper(II) units linked through BF4(-) anions to form a novel 1D single-chain polymeric coordination compound. This appears to be the first report in which a central BO3(3-) species is linked to six copper(II) ions held together by a single macrocyclic ligand through three µ1,1-O(BO3(3-)) and three µ1,3-O(BO3(3-)) bridges. In complexes 1-5 the BO3(3-) is present in the center of the macrocyclic cavity and is bonded to all six metal centers arranged in a benzene-like hexagonal array. In the hexagonal array there are alternate double (µ-alkoxide and µ1,3-O(BO3(3-))) and (µ-phenoxide and µ1,1-O(BO3(3-))) bridges between the Cu(II) centers. The symmetrical hexa-bridging nature of µ6-BO3(3-) is unprecedented in transition metal complex chemistry, and along with alkoxide and phenoxide bridges in the equatorial plane provides effective pathways for an overall antiferromagnetic exchange interaction between six copper(II) centers. In 1, 3, and 5 the BO3(3-) moiety is produced in one step (synthetic) by an unusual copper(II)-macrocycle complex catalyzed hydrolysis of BF4(-) ion in methanol. In 2 and 4 the central species (BO3(3-)) comes from boric acid (H3BO3) which is added to reaction mixture of Cu(ClO4)2/H6L4/H6L5 under inert conditions to confirm the identity of the central species.

Antitumoral effects of 9-cis retinoic acid in adrenocortical cancer.

The currently available medical treatment options of adrenocortical cancer (ACC) are limited. In our previous meta-analysis of adrenocortical tumor genomics data, ACC was associated with reduced retinoic acid production and retinoid X receptor-mediated signaling. Our objective has been to study the potential antitumoral effects of 9-cis retinoic acid (9-cisRA) on the ACC cell line NCI-H295R and in a xenograft model. Cell proliferation, hormone secretion, and gene expression have been studied in the NCI-H295R cell line. A complex bioinformatics approach involving pathway and network analysis has been performed. Selected genes have been validated by real-time qRT-PCR. Athymic nude mice xenografted with NCI-H295R have been used in a pilot in vivo xenograft model. 9-cisRA significantly decreased cell viability and steroid hormone secretion in a concentration- and time-dependent manner in the NCI-H295R cell line. Four major molecular pathways have been identified by the analysis of gene expression data. Ten genes have been successfully validated involved in: (1) steroid hormone secretion (HSD3B1, HSD3B2), (2) retinoic acid signaling (ABCA1, ABCG1, HMGCR), (3) cell-cycle damage (GADD45A, CCNE2, UHRF1), and the (4) immune response (MAP2K6, IL1R2). 9-cisRA appears to directly regulate the cell cycle by network analysis. 9-cisRA also reduced tumor growth in the in vivo xenograft model. In conclusion, 9-cisRA might represent a promising new candidate in the treatment of hormone-secreting adrenal tumors and adrenocortical cancer.

Critical role of extracellular vesicles in modulating the cellular effects of cytokines.

Under physiological and pathological conditions, extracellular vesicles (EVs) are present in the extracellular compartment simultaneously with soluble mediators. We hypothesized that cytokine effects may be modulated by EVs, the recently recognized conveyors of intercellular messages. In order to test this hypothesis, human monocyte cells were incubated with CCRF acute lymphoblastic leukemia cell line-derived EVs with or without the addition of recombinant human TNF, and global gene expression changes were analyzed. EVs alone regulated the expression of numerous genes related to inflammation and signaling. In combination, the effects of EVs and TNF were additive, antagonistic, or independent. The differential effects of EVs and TNF or their simultaneous presence were also validated by Taqman assays and ELISA, and by testing different populations of purified EVs. In the case of the paramount chemokine IL-8, we were able to demonstrate a synergistic upregulation by purified EVs and TNF. Our data suggest that neglecting the modulating role of EVs on the effects of soluble mediators may skew experimental results. On the other hand, considering the combined effects of cytokines and EVs may prove therapeutically useful by targeting both compartments at the same time.

One Step Back from Bedside to the Bench-How Do Different Arterial Stiffness Parameters Behave in Relation to Peripheral Resistance?

The investigation of arterial stiffening is a promising approach to estimating cardiovascular risk. Despite the widespread use of different methods, the dynamic nature of measured and calculated stiffness parameters is marginally investigated. We aimed to determine the stability of large artery elasticity parameters assessed via commonly used, ultrasound-based and oscillometric methods in relation to peripheral resistance modulation. A human experimental environment was composed, and fifteen young males were investigated at rest after extremity heating and external compression. Functional vascular parameters were monitored in each session, and several arterial stiffness parameters were analysed. The distensibility coefficient (DC) did not show significant changes during heat provocation and extremity compression, while DC's stability seemed to be acceptable. The same stability of carotid-femoral pulse wave velocity (PWV) was detected with ultrasound measurement (5.43 ± 0.79, 5.32 ± 0.86 and 5.28 ± 0.77, with p = 0.38, p = 0.27 and p = 0.76, respectively) with excellent intersession variability (intraclass correlation coefficient of 0.90, 0.88 and 0.91, respectively). However, the oscillometric PWV (oPWV) did change significantly between the heating and outer compression phase of the study (7.46 ± 1.37, 7.10 ± 1.18 and 7.60 ± 1.21, with p = 0.05, p = 0.68 and p < 0.001, respectively), the alteration of which is closely related to wave reflection, represented by the changes in reflection time. Our results indicate the good stability of directly measured elastic parameters such as DC and PWV, despite the extreme modulation of peripheral resistance. However, the oscillometric, indirectly detected PWV might be altered by physical interventions, which depend on wave reflection. The effective modulation of wave reflection was characterized by changes in the augmentation index, detected using both oscillometry and applanation tonometry. Thus, the environment during oscillometric measurement should be rigorously standardized. Furthermore, our results suggest the dynamic nature of the reflection point, rather than being a fixed anatomical point, proposed previously as aortic bifurcation.

Both freshly prepared and frozen-stored amniotic membrane cells express the complement inhibitor CD59.

Amniotic membrane proved to be very effective tool in the treatment of a number of ocular surface diseases. The amniotic membrane, however, has to be stored before its transplantation onto the ocular surface followed by mandatory serologic control in order to exclude the transmission of certain viruses. Therefore it is most important to study if cryopreservation of the membrane affects cell surface expression of the molecules. We measured cell surface expression of CD59, a membrane-bound complement inhibitor on the cells of freshly prepared and cryopreserved amniotic membrane. Cells of amniotic membrane were separated mechanically. Epithelial and mesenchymal cells were identified by the intracellular expression of nanog and the cell surface ICAM1 positivity, respectively. Multicolor flow cytometric immunophenotyping was used for determination of the CD59 expression. CellQuest-Pro software program (Becton Dickinson) was used both for measurements and analysis. CD59-positive cells could be detected in all investigated samples and in all investigated cell types, although the expression level of CD59 differed. CD59 was expressed both on freshly prepared and frozen-stored samples. Higher level of CD59 was detected on ICAM1+ mesenchymal cells than on nanog+ epithelial cells. Our findings indicate that amniotic membranes maintain their complement inhibiting capacity after cryopreservation.

Identifying the Leader in You.

The leadership journey is often a long and winding road, with speed bumps and unexpected turns. During this session of JADPRO Live Virtual, presenters discussed the leadership qualities that they have found integral, including emotional intelligence, vulnerability, and personal reflection.

Development of a Standardized Bone Marrow Procedure Training and Competency Toolkit for Advanced Practice Providers in a Large Community Oncology Practice.

Bone marrow procedures are a common diagnostic tool utilized in hematology/oncology and can be completed in the office by trained clinicians. Currently, there are limited guidelines for appropriate training and competency for bone marrow procedures performed by advanced practice providers (APPs) in a community oncology practice setting. The need to create a standardized training and competency protocol for APPs in this setting was recognized. A comprehensive, standardized educational and procedural toolkit was created. The creation of a comprehensive training toolkit for APPs in the community oncology practice setting helps to ensure a high standard of procedural proficiency and consistency among individual providers and practices. The creation of such an extensive toolkit is time consuming. By adopting and standardizing toolkits such as this one, community hematology/oncology practices can ensure the delivery of high-quality patient care by highly trained and proficient APPs.

Advanced Practitioner Leadership in Times of Crisis.

JADPRO Live Virtual kicked off with the opening panel on advanced practitioner leadership during the COVID-19 pandemic. The group discussed their institutional emergency protocols, how they leveraged advanced practitioners (APs) to provide care during the crisis peak, and how they responded to the personal issues and anxieties of their AP colleagues.

Telemedicine in Community Cancer Care: How Technology Helps Patients With Cancer Navigate a Pandemic.

COVID-19 places unprecedented demands on the oncology ecosystem. The extensive pressure of managing health care during the pandemic establishes the need for rapid implementation of telemedicine. Across our large statewide practice of 640 practitioners at 221 sites of service, an aggressive multidisciplinary telemedicine strategy was implemented in March by coordinating and training many different parts of our healthcare delivery system. From March to September, telemedicine grew to serve 15%-20% of new patients and 20%-25% of established patients, permitting the practice to implement safety protocols and reduce volumes in clinic while continuing to manage the acute and chronic care needs of our patient population. We surveyed practice leaders, queried for qualitative feedback, and established 76% were satisfied with the platform. The common challenges for patients were the first-time use and technology function, and patients were, in general, grateful and happy to have the option to visit their clinicians on a telemedicine platform. In addition to conducting new and established visits remotely, telemedicine allows risk assessments, avoidance of hospitalization, family education, psychosocial care, and improved pharmacy support. The implementation has limitations including technical complexity; increased burden on patients and staff; and broadband access, particularly in rural communities. For telemedicine to improve as a solution to enhance the longitudinal care of patients with cancer, payment coverage policies need to continue after the pandemic, technologic adoption needs to be easy for patients, and broadband access in rural areas needs to be a policy priority. Further research to optimize the patient and clinician experience is required to continue to make progress.

HtrA1 is a novel mast cell serine protease of mice and men.

Mast cells are rich sources of proteases, such as tryptases and chymases that control many physiological and pathological processes, for example vascular permeability, smooth muscle cell proliferation or extracellular matrix remodeling. Murine mucosal mast cells mature under the influence of TGF-beta and play a role in asthmatic and anti-helminthic immune responses. In an attempt to identify novel genes that are highly upregulated during mucosal mast cell differentiation, we detected HtrA1 protease as a novel protein in mast cells by microarray experiments. HtrA1 level was much higher in murine mucosal than in connective tissue-type mast cells. Furthermore, HtrA1 is not localized in the secretory granules and is constitutively secreted by human mast cells. Although HtrA1 has been attributed a TGF-beta-inhibitory activity, it did not show any influence on TGF-beta-induced mucosal mast cell differentiation. As many extracellular target proteins have been suggested for HtrA1, this protease may participate in the mast cell-induced extracellular remodeling.

Phenotypic profiling of engineered mouse melanomas with manipulated histamine production identifies histamine H2 receptor and rho-C as histamine-regulated melanoma progression markers.

In the present study, the impact of acquired neoplastic L-histidine decarboxylase (HDC) expression, and its direct consequence, the release of histamine in the tumor environment, was assessed on melanoma tumor progression. B16-F10 mouse melanoma cells were manipulated via stable transfection, and nine novel transgenic variants were generated in triplicates, constitutively expressing the full-length sense mouse HDC mRNA, a mock control, and an antisense HDC RNA segment, respectively. Establishing both primary skin tumors and lung metastases in C57BL/6 mice, the nine variants with different histamine-releasing capacities were subjected to a comprehensive comparative progression profiling in vivo. Our analyses showed trends of markedly accelerated tumor growth (P < 0.001), and moderately increased metastatic colony-forming potential (P = 0.010) along with rising levels of local histamine production. Using RNase protection assay for screening of the melanoma progression profile, and Western blotting for subsequent result validation, we looked for molecular progression markers affected by melanoma histamine secretion. Investigation of 21 functionally clustered markers associated with tumor proliferation, angiogenesis, invasivity, metastasis formation, local or systemic immunomodulation, and histamine signaling revealed positive correlations between histamine production, tumor histamine H2 receptor and rho-C expression (P < 0.001, P = 0.002, respectively). These observations confirm the involvement of histamine in the molecular machinery of melanoma progression.

Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA.

Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes.

Soluble interleukin-6 receptor enhanced by oncostatin M induces major changes in gene expression profile of human hepatoma cells.

Interleukin-6 (IL-6) binds to a receptor complex consisting of an 80 kDa binding unit (IL-6R) and gp130 responsible for signal transduction. Due to alternative splicing and/or proteolytic digestion IL-6R occurs in soluble form (sIL-6R), as well. Soluble IL-6R is able to bind to gp130 expressing on nucleated cells, thus sIL-6R makes most cells responsive to IL-6. In this study we found that oncostatin M (OSM), an other gp130 dependent cytokine with proliferation inhibitory potential, increases the expression of both membrane-bound IL-6R and sIL-6R generated by alternative splicing in hepatic and mammary carcinoma cell lines. Furthermore, we studied the functional relevance of the presence and binding of soluble IL-6R to HepG2 cells. Using a cDNA expression array, mRNA levels of about 580 human genes were tested by differential display analysis. Our findings suggest, that elevation of surface density of IL-6R by attachment of sIL-6R induces major modulation in gene expression profile of the hepatoma cells. Soluble IL-6R alone has minor effect, it rather decreases expression of some genes, while incubation with IL-6 and sIL-6R together induces major changes in the mRNA pattern of HepG2 cells. These data strongly suggest that presence and binding of soluble cytokine receptors are important elements of inter-cytokine cross talk and affects actual gene expression profile of responding cells.

High leptin level is accompanied with decreased long leptin receptor transcript in histamine deficient transgenic mice.

Leptin is a multifunctional cytokine and hormone that primarily acts in the hypothalamus and plays a key role in regulation of food intake and energy expenditure. Leptin acts through its receptor (OBR), the product of db gene that activates the Jak/STAT pathway predominantly. To exert its functions, leptin interacts with histamine as well. Histamine is a downstream effector of leptin as its release, metabolism is enhanced by leptin and hypothalamic histamine reduces food intake. In a bi-directional regulatory loop histamine also influences leptin concentration by inhibiting its expression. In this study we demonstrate that histamine deficiency elevates serum leptin level and decreases full-length leptin receptor isoform with a slight increase of the short one and results in mild late onset obesity. These observation can help to elucidate further the bi-lateral interaction of leptin and histamine, and therefore provide useful data to understand the pathomechanism of obesity.

Effect of UVA radiation on membrane fluidity and radical decay in human fibroblasts as detected by spin labeled stearic acids.

The ultraviolet A (UVA) radiation component of sunlight (320-400 nm) has been shown to be a source of oxidative stress to cells via generation of reactive oxygen species. We report here some consequences of the UVA irradiation on cell membranes detected by electron paramagnetic resonance (EPR) spectroscopy. Paramagnetic nitroxide derivatives of stearic acid bearing the monitoring group at different depths in the hydrocarbon chain were incorporated into human fibroblasts membranes to analyze two main characteristics: kinetics of the nitroxide reduction and membrane fluidity. These two characteristics were compared for control and UVA-irradiated (0-250 kJ/m(2)) cells. The term relative redox capacity (RRC) was introduced to characterize and to compare free radical reduction measured by EPR with some well-known viability/clonogenicity tests. Our results showed that UVA-irradiation produces a more rigid membrane structure, especially at higher doses. Furthermore, we found that trends agree in survival measured by neutral red (NR), trypan blue (TB), and clonogenic efficiency compared with RRC values measured by EPR for low and medium exposure doses. Above 100 kJ/m(2), differences between these tests were observed. Antioxidant effect was modeled by alpha-tocopherol-acetate treatment of the cells before UVA irradiation. While NR, TB and clonogenicity tests showed protection at the highest UVA doses (>100 kJ/m(2)), results obtained with EPR measurements, both membrane fluidity and kinetics, or using MTT test did not exhibit this protective effect.

A synthetic corticosteroid, dexamethasone regulates generation of soluble form of interleukin-6 receptor of human lymphocytes, in vitro.

In contrast to most of the soluble cytokine receptor antagonists properties, the soluble IL-6 receptor (sIL-6R) occurring in various body fluids of healthy persons and patients with various diseases is an agonist. The enhancing effect is due to its ability to form complex with IL-6 and to bind to gp130 making constitutively IL-6 receptor negative cells responsive for IL-6. The generation as well as the functional role of soluble IL-6 receptor is poorly understood. Earlier, we found that the sIL-6R levels in sera of patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) were higher than those of the control group measured by ELISA sandwich technology. In the present study we detected different levels of sIL-6R in the supernatants of lymphocyte cultures of healthy persons and patients with RA as well as SLE. Moreover, we found, that in vitro dexamethasone treatment stimulated generation of sIL-6R in both healthy persons and in active SLE, while it strongly suppressed production of sIL-6R in both RA groups. At mRNA level, we found that in SLE both the IL-6R mRNA encoding the membrane spanning and alternatively spliced (soluble) variants increased. Surprisingly, the strong decrease of sIL6R protein in RA was not found at mRNA level.

Myeloid-derived microRNAs, miR-223, miR27a, and miR-652, are dominant players in myeloid regulation.

In the past few years expanding knowledge has been accumulated about the role of microRNAs (miRNAs) not only in hematopoiesis and cancer, but also in inflammatory and infectious diseases. Regarding myeloid cells, our knowledge is relatively insufficient, therefore we intended to collect the available data of miRNA profiles of myeloid cells. In addition to a rather general myeloid regulator miR-223, two other miRNAs seem to be useful subjects in understanding of myeloid miRNA biology: miR-27a and miR-652. We review functions of these three miRNAs and other myeloid miRNAs focusing on their roles in monocytes, neutrophils, eosinophils, basophils and mast cells.

Effects of mitotane on gene expression in the adrenocortical cell line NCI-H295R: a microarray study.

AIM: The adrenolytic agent mitotane is widely used in the treatment of adrenocortical cancer; however, its mechanism of action is poorly elucidated. We have studied mitotane-induced mRNA expression changes in the NCI-H295R adrenocortical cancer cell line. MATERIALS & METHODS: Cell viability and hormone assays were used to select the optimal mitotane concentration effectively inhibiting hormone secretion without affecting cell viability. RNA isolated from cultures treated for 48 and 72 h was subjected to Agilent 4×44K microarray platforms. Microarray results were validated by quantitative reverse-transcription PCR. RESULTS: Altogether, 117 significantly differentially expressed genes were detected at 48 h and 72 h (p < 0.05) in mitotane-treated samples relative to controls. Three significantly underexpressed genes involved in steroid hormone biosynthesis (HSD3B1, HSD3B2 and CYP21A2) and four significantly overexpressed genes (GDF15, ALDH1L2, TRIB3 and SERPINE2) have been validated. CONCLUSION: Gene-expression changes might be involved in the adrenal action of mitotane and in the inhibition of hormone secretion.

Lymphocytes from rheumatoid arthritis patients have elevated levels of intracellular peroxiredoxin 2, and a greater frequency of cells with exofacial peroxiredoxin 2, compared with healthy human lymphocytes.

Peroxiredoxin 2 has immune regulatory functions, but its expression in human peripheral blood lymphocytes and levels in extracellular fluid in healthy subjects and rheumatoid arthritis patients are poorly described. In the present study, the median intracellular peroxiredoxin 2 protein content of lymphocytes from rheumatoid arthritis patients was more than two-fold higher compared with healthy subjects' lymphocytes. Intracellular peroxiredoxin 3 levels were similar in healthy and rheumatoid arthritis lymphocytes. Flow cytometry detected peroxiredoxin 2 on the surface of ca. 8% of T cells and ca. 56% of B cells (median % values) of all subjects analyzed. Exofacial thioredoxin-1 was also observed. In the total lymphocyte population from rheumatoid arthritis patients, few cells (median, 6%) displayed surface peroxiredoxin 2. In contrast, a significantly increased proportion of interleukin-17(+ve) lymphocytes were exofacially peroxiredoxin 2(+ve) (median, 39%). Prdx2 was also detected in human extracellular fluids. We suggest that crucial inflammatory cell subsets, i.e. interleukin-17(+ve) T cells, exhibit increased exofacial redox-regulating enzymes and that peroxiredoxin 2 may be involved in the persistence of pro-inflammatory cells in chronic inflammation.