Chick chorioallantoic membrane (CAM) assay for the evaluation of the antitumor and antimetastatic activity of platinum-based drugs in association with the impact on the amino acid metabolism.

The combination of in ovo and ex ovo chorioallantoic membrane (CAM) assay provides an excellent platform which extends its relevance in studying carcinogenesis to the field of screening of anticancer activity of platinum nanoparticles (PtNPs) and further study of the amino acids' fluctuations in liver and brain. PtNPs are promising candidates for replacing cisplatin (CDDP); however, insufficient data of their antitumor efficiency and activity on the cancer-related amino acid metabolism are available, and the assessment of the in vivo performance has barely scratched the surface. Herein, we used CAM assay as in vivo model for screening of novel therapeutic modalities, and we conducted a comparative study of the effects of CDDP and polyvinylpyrrolidone coated PtNPs on MDA-MB-231 breast cancer xenograft. PtNPs showed a higher efficiency to inhibit the tumor growth and metastasis compared to CDDP. The amino acids profiling in the MDA-MB-231 â€‹cells revealed that the PtNPs had an overall depleting effect on the amino acids content. Noteworthy, more side effects to amino acid metabolism were deduced from the depletion of the amino acids in tumor, brain, and liver upon CDDP treatment. Different sets of enzymes of the tricarboxylic acid (TCA) cycle were targeted by PtNPs and CDDP, and while mRNA encoding multiple enzymes was downregulated by PtNPs, the treatment with CDDP affected only two TCA enzymes, indicating a different mechanism of action. Taken together, CAM assay represents and invaluable model, demonstrating the PtNPs capability of repressing angiogenesis, decrease amino acid contents and disrupt the TCA cycle.

The Anti-Proliferative Activity of Coordination Compound-Based ZnO Nanoparticles as a Promising Agent Against Triple Negative Breast Cancer Cells.

PURPOSE: The present study deals with the in vitro evaluation of the potential use of coordination compound-based zinc oxide (ZnO) nanoparticles (NPs) for the treatment of triple negative breast cancer cells (TNBrCa). As BrCa is one of the most prevalent cancer types and TNBrCa treatment is difficult due to poor prognosis and a high metastasis rate, finding a more reliable treatment option should be of the utmost interest. METHODS: Prepared by reacting zinc carboxylates (formate, acetate, propionate, butyrate, isobutyrate, valerate) and hexamethylenetetramine, 4 distinct coordination compounds were further subjected to two modes of conversion into ZnO NPs - ultrasonication with oleic acid or heating of pure precursors in an air atmosphere. After detailed characterization, the resulting ZnO NPs were subjected to in vitro testing of cytotoxicity toward TNBrCa and normal breast epithelial cells. Further, their biocompatibility was evaluated. RESULTS: The resulting ZnO NPs provide distinct morphological features, size, biocompatibility, and selective cytotoxicity toward TNBrCa cells. They internalize into two types of TNBrCa cells and imbalance their redox homeostasis, influencing their metabolism, morphology, and ultimately leading to their death via apoptosis or necrosis. CONCLUSION: The crucial properties of ZnO NPs seem to be their morphology, size, and zinc content. The ZnO NPs with the most preferential values of all three properties show great promise for a future potential use in the therapy of TNBrCa.

Norepinephrine transporter-derived homing peptides enable rapid endocytosis of drug delivery nanovehicles into neuroblastoma cells.

BACKGROUND: Currently, the diagnosis and treatment of neuroblastomas-the most frequent solid tumors in children-exploit the norepinephrine transporter (hNET) via radiolabeled norepinephrine analogs. We aim to develop a nanomedicine-based strategy towards precision therapy by targeting hNET cell-surface protein with hNET-derived homing peptides. RESULTS: The peptides (seq. GASNGINAYL and SLWERLAYGI) were shown to bind high-resolution homology models of hNET in silico. In particular, one unique binding site has marked the sequence and structural similarities of both peptides, while most of the contribution to the interaction was attributed to the electrostatic energy of Asn and Arg (< - 228 kJ/mol). The peptides were comprehensively characterized by computational and spectroscopic methods showing ~ 21% ß-sheets/aggregation for GASNGINAYL and ~ 27% α-helix for SLWERLAYGI. After decorating 12-nm ferritin-based nanovehicles with cysteinated peptides, both peptides exhibited high potential for use in actively targeted neuroblastoma nanotherapy with exceptional in vitro biocompatibility and stability, showing minor yet distinct influences of the peptides on the global expression profiles. Upon binding to hNET with fast binding kinetics, GASNGINAYLC peptides enabled rapid endocytosis of ferritins into neuroblastoma cells, leading to apoptosis due to increased selective cytotoxicity of transported payload ellipticine. Peptide-coated nanovehicles significantly showed higher levels of early apoptosis after 6 h than non-coated nanovehicles (11% and 7.3%, respectively). Furthermore, targeting with the GASNGINAYLC peptide led to significantly higher degree of late apoptosis compared to the SLWERLAYGIC peptide (9.3% and 4.4%, respectively). These findings were supported by increased formation of reactive oxygen species, down-regulation of survivin and Bcl-2 and up-regulated p53. CONCLUSION: This novel homing nanovehicle employing GASNGINAYLC peptide was shown to induce rapid endocytosis of ellipticine-loaded ferritins into neuroblastoma cells in selective fashion and with successful payload. Future homing peptide development via lead optimization and functional analysis can pave the way towards efficient peptide-based active delivery of nanomedicines to neuroblastoma cells.

Pre-treatment VD levels and VDR receptors as potential predictors of occurrence and overall survival in paediatric patients with solid tumours-a single institution pilot study.

Recently, vitamin D has been recognized as an important player in the immune system, and multiple studies suggested its involvement in cancer, too. The aims of this study were to investigate selected single nucleotide polymorphisms (SNPs) in the VDR gene, BsmI (rs1544410; A > G), FokI (rs 2228570; C > T), TaqI (rs731236; T > C), ApaI (rs 7975232; C > T) and Cdx-2 (rs11568820; A > G), and to evaluate their possible predictive role for outcomes in patients with paediatric solid tumours. A total of 111 children with paediatric solid tumours were enrolled at the Department of Paediatric Oncology, University Hospital Brno (Brno, Czech Republic) along with a control population of 787 adults; all study subjects were available for genotyping of selected SNPs, and the prediagnostic levels of 25-hydroxycholecalciferol (25(OH)D3) and 1,25-dihydroxycholecalciferol (1,25(OH)2D3) were measured in the cases, too. In FokI, the heterozygote CT genotype was weakly associated with a decreased risk of paediatric solid cancer occurrence 0.82 (0.53-1.28), while the CC genotype was associated with a decreased risk of 0.58 (0.30-1.09), p = 0.09. The 1,25(OH)2D3 prediagnostic levels were indicative of the overall survival in the cases (ß = -0.012, HR 0.988, 95 % CI (0.978-0.998), while higher prediagnostic levels of 1,25(OH)2D3 were associated with a statistically significant increase in overall mortality. We observed multiple effects of the alleles of the investigated polymorphisms and of 1,25(OH)2D3 on overall survival, regardless of the underlying disease.

The presence of B-cell activating factor (BAFF) in umbilical cord blood in both healthy and pre-eclamptic pregnancies and in human breast milk.

B-cell activating factor (BAFF) is an important immune regulator that was recently reported to be secreted by placenta. The aim of the study was to investigate the presence of BAFF in umbilical cord blood, maternal serum, and breast milk in normal and in pre-eclamptic pregnancies. Pairs of maternal serum/umbilical cord blood were obtained from 12 pre-eclamptic and 34 physiological pregnancies. Another cohort of 10 healthy lactating women was established that was followed up for 6 months following delivery to investigate BAFF levels in breast milk. BAFF levels in maternal peripheral blood were significantly higher in physiological pregnancies than in pre-eclamptic pregnancies (p < 0.03). Furthermore, we observed a consistent presence of BAFF in breast milk during the 6-month post-partum period of breastfeeding. In this study, we demonstrate that BAFF levels are significantly lower in maternal peripheral blood in pre-eclamptic pregnancies. We also report the consistent presence of BAFF in breast milk in healthy women. More research into the role of BAFF in pregnancy, and during breastfeeding, is imperative.

Period3 VNTR polymorphism influences the time-of-day pain onset of acute myocardial infarction with ST elevation.

It is well established that the incidence and infarct size in acute myocardial infarction (AMI) is subject to circadian variations. At the molecular level, circadian clocks in distinct cells, including cardiomyocytes, generate 24-h cycles of biochemical processes. Possible imbalance or impairment in the cell clock mechanism may alter the cardiac metabolism and function and increase the susceptibility of cardiovascular diseases. One of the key components of the human clock system PERIOD3 (PER3) has been recently demonstrated to affect circadian expression of various genes in different tissues, including the heart. The variable number tandem repeat (VNTR) polymorphism (rs57875989) in gene Period3 (Per3) is related to multiple phenotypic parameters, including diurnal preference, sleep homeostasis, infection and cancer. The aim of our study was to investigate the effect of this polymorphism in AMI with ST elevation (STEMI). The study subjects (314 patients of Caucasian origin with STEMI, and 332 healthy controls) were genotyped for Per3 VNTR polymorphism using an allele-specific polymerase chain reaction. A gender difference in circadian rhythmicity of pain onset was observed with significant circadian pattern in men. Furthermore, the Per3(5/5) variant carriers were associated with higher levels of interleukin-6, B-type natriuretic peptide and lower vitamin A levels. By using cosinor analysis we observed different circadian distribution patterns of AMI onset at the level of genotype and allelic frequencies. Genotypes with at least one 4-repeat allele (Per3(4/5) and Per3(4/4)) (N = 264) showed remarkable circadian activity in comparison with Per3(5/5) (N = 50), especially in men. No significant differences in genotype and/or allele frequencies of Per3 VNTR polymorphism were observed when comparing STEMI cases and controls. Our results indicate that the Per3 VNTR may contribute to modulation of cardiac functions and interindividual differences in development and progression of myocardial infarction.

The prediction role of indexes of circulating adipokines for common anthropometric and nutritional characteristics of obesity in the obese Central European population.

AIMS: This study was designed to investigate the relationship between 8 selected adipokines (leptin, leptin receptor, adiponectin, agouti-related peptide, omentin, visfatin, adipsin and resistin), dietary composition and anthropometric parameters found in the Central European obese population. METHODS: A total of 65 unrelated obese Central European Caucasian individuals were recruited for the study. Phenotypic measurements included weight, height, BMI, lean body mass, fat mass, body fat, waist and hip circumference, waist-hip ratio (WHR) and skinfold thickness. Participants completed standardized self-reported 7-day food records. Plasma levels of leptin, leptin receptor, adiponectin, agouti-related peptide (AgRP), resistin, adipsin, omentin and visfatin were examined using ELISA. RESULTS: Multiple associations (weight, height, percentage of body fat, waist circumference, hip circumference, WHR and sum of skinfold thickness) with the circulation levels of the investigated adipokines were identified. Leptin-Leptin receptor (L-LR) levels were found to correlate with total energy intake and macronutrients while adipsin was found to strongly correlate with multiple adipokines. Furthermore, the L-LR index was found to constitute a more accurate description of the relationship between BMI and body weight than individual measurements and the Ag-LR index was found to strongly correlate with both anthropometric and dietary characteristics. CONCLUSION: Following confirmation on larger population samples and on samples of different ethnicities, the reported adipokine indexes could become a useful tool for estimating nutritional status and predicting the body composition of specific patient groups.

Polymorphisms in HLA-related genes and psoriasis heredity in patients with psoriasis.

BACKGROUND: The aim of this study was to investigate possible associations of the five DNA polymorphic genotypes in the HLA region (transporter associated with antigen processing [TAP1; TAP1 333 a/b, TAP1 637 c/d], the HLA-DRB1*1501-rs3135388, tumor necrosis factor [TNF]α [-238 G/A] and NcoI TNFß) with characteristics of family history in patients with psoriasis vulgaris. MATERIALS AND METHODS: A total of 201 Czech patients with psoriasis were enrolled in the study. The patients were genotyped for the five common polymorphisms in TAP1, TNFα, and TNFß genes (6p21.3) using the polymerase chain reaction-restriction fragment length polymorphism-based methodology. RESULTS: We observed significantly higher prevalence of Ile333Ile TAP1 allele in patients whose first-degree relatives had a positive family history of psoriasis (Pa  = 0.04). No differences related to family history of psoriasis were observed in HLA-DRB1*1501 polymorphism. As for the TNFα (-238 G/A) polymorphism, a significant increase of the GG genotype was observed in patients, especially men with second- and third-degree relatives with psoriasis (Pg  = 0.008). Similarly, the B2B2 genotype of NcoI TNFß polymorphism was more frequent in psoriatic patients, especially women, whose second- and third-degree relatives had psoriasis (Pg  = 0.004). Finally, the haplotype analysis of all five polymorphisms revealed that the frequency of haplotype bcCB1A was different between not only men and women with psoriasis (P = 0.007) but also between men and women without a family history of psoriasis (P = 0.007). CONCLUSIONS: Haplotype association of HLA gene polymorphisms with genealogy aspects of psoriasis facilitates a better understanding of etiopathogenetic aspects of the diseases.

Variability in CNR1 locus influences protein intake and smoking status in the Central-European population.

OBJECTIVES: The endocannabinoid receptor 1 (CB1) is encoded by the CNR1 gene and has been recently recognized to play an important role in the regulation of satiety and feeding behaviour with a huge potential of modulating metabolic response and feeding control. The aim of the study was to investigate the potential of three selected single nucleotide polymorphisms (SNPs) in the CNR1 locus on native dietary composition in the Central-European Caucasian population. METHODS: A total of 258 unrelated individuals originating from the Central-European Caucasian population were enrolled into the study and rs1049353, rs12720071, and rs806368 polymorphisms in CNR1 locus were examined in these individuals using PCR-based methodology. Body composition was assessed using a bioimpedance method, various anthropometric parameters were investigated (waist and hip circumference, skin folds), and native dietary composition was analysed using 7-day food records as well as a food frequency questionnaire. RESULTS: Allelic variations and common haplotypes in the CNR1 gene were associated with the daily intake of proteins, fluids, and fibre, regardless of the physical activity of the individuals. The common haplotype in the CNR1 gene was associated with self-reported smoking (number of cigarettes per day, smoking years). DISCUSSION: Our results indicate that specific genetic variations in the CNR1 gene may act as susceptibility markers for specific dietary composition in the Central-European population.