fragment condensation
Dotaz
Zobrazit nápovědu
Glycoconjugation is a powerful tool to improve the anticancer activity of metal complexes. Herein, we modified commercial arylphosphanes with carbohydrate-derived fragments for the preparation of novel glycoconjugated ruthenium(II) p-cymene complexes. Specifically, d-galactal and d-allal-derived vinyl epoxides (VEβ and VEα) were coupled with (2-hydroxyphenyl)diphenylphosphane, affording the 2,3-unsaturated glycophosphanes 1β and 1α. Ligand exchange with [Ru(C2O4)(η6-p-cymene)(H2O)] gave the glycoconjugated complexes Ru1β and Ru1α which were subsequently dihydroxylated with OsO4/N-methylmorpholine N-oxide to Ru2β and Ru2α containing O-benzyl d-mannose and d-gulose units respectively. Besides, aminoethyl tetra-O-acetyl-β-d-glucopyranoside was condensed with borane-protected (4-diphenylphosphanyl)benzoic acid by HATU/DIPEA under MW heating, to afford the amide 3∙BH3. Zemplén deacylation with MeONa/MeOH gave the deprotected d-glucopyranoside derivative 4∙BH3. The glycoconjugated phosphane complexes Ru3 and Ru4 were obtained by reaction of the phosphane-boranes 3∙BH3 and 4∙BH3 with [Ru(C2O4)(η6-p-cymene)(H2O)]. The employed synthetic strategies were devised to circumvent unwanted phosphine oxidation. The compounds were purified by silica chromatography, isolated in high yield and purity and characterized by analytical and spectroscopic (IR and multinuclear NMR) techniques. The behaviour of the six glycoconjugated Ru complexes in aqueous solutions was assessed by NMR and MS measurements. All compounds were screened for their in vitro cytotoxicity against A2780/A2780R human ovarian and MCF7 breast cancer cell lines, revealing a significant cytotoxicity for complexes containing the 2,3-unsaturated glycosyl unit (Ru1β, Ru1α). Additional studies on five other human cancer cells, as well as time-dependent toxicity and cell-uptake analyses on ovarian cancer cells, confirmed the prominent activity of these two compounds - higher than cisplatin - and the better performance of the β anomer. However, Ru1β, Ru1α did not show preferential activity against cancer cells with respect to fetal lung fibroblast and human embryonic kidney cells as models of normal cells. The effects of the two ruthenium glycoconjugated compounds in A2780 ovarian cancer cells were further investigated by cell cycle analysis, induction of apoptosis, intracellular ROS production, activation of caspases 3/7 and disruption of mitochondrial membrane potential. The latter is a relevant factor in the mechanism of action of the highly cytotoxic Ru1β, inducing cell death by apoptosis.
- MeSH
- fosfiny MeSH
- komplexní sloučeniny * chemie farmakologie MeSH
- lidé MeSH
- ligandy MeSH
- nádorové buněčné linie MeSH
- nádory vaječníků * MeSH
- protinádorové látky * chemie MeSH
- ruthenium * chemie farmakologie MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The effects of air pollution on men's reproductive health can be monitored by evaluating semen quality and sperm DNA damage. We used real-time PCR to analyse the effects of air pollution on sperm mitochondrial DNA copy number (mtDNAcn) and deletion (mtDNAdel) rates in semen samples collected from 54 men in two seasons with different levels of industrial and traffic air pollution. MtDNAdel rates were significantly higher following the high exposure period and were positively correlated with mtDNAcn. However, we did not find any difference in mtDNAcn between the two seasons. MtDNAcn was positively correlated with the DNA fragmentation index and the rates of sperm with chromatin condensation defects, previously assessed by sperm chromatin structure assay, and negatively correlated with sperm concentration, progressive motility, viability, and normal morphology. This indicates that mtDNAcn is more closely associated with male fertility than mtDNAdel rates. In contrast, mtDNAdel might be a more sensitive biomarker of air pollution exposure in urban industrial environments.
- MeSH
- analýza spermatu * MeSH
- chromatin MeSH
- lidé MeSH
- mitochondriální DNA genetika MeSH
- motilita spermií MeSH
- spermie MeSH
- variabilita počtu kopií segmentů DNA MeSH
- znečištění ovzduší * škodlivé účinky MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of the present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in the intact cells. We used human hepatoma HepG2 and renal HK-2 cells cultured in 96-well plates treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6-48 h. Afterwards, the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting cell impairment and apoptosis (i.e. WST-1 and glutathione tests, TUNEL, DNA ladder, caspase activity, PARP-1 and JNKs expressions). We found that our developed spectrofluorometric assay provided results of the same sensitivity as the TUNEL assay but with the advantages of being fast processing, low-cost and a high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes.
- MeSH
- apoptóza účinky léků MeSH
- bisbenzimidazol chemie MeSH
- buněčná smrt účinky léků MeSH
- buněčné jádro účinky léků metabolismus MeSH
- buněčné linie MeSH
- buňky Hep G2 MeSH
- cisplatina farmakologie MeSH
- fluorescenční mikroskopie metody MeSH
- fluorescenční spektrometrie metody MeSH
- fragmentace DNA účinky léků MeSH
- kamptothecin farmakologie MeSH
- lidé MeSH
- protinádorové látky farmakologie MeSH
- průtoková cytometrie metody MeSH
- reprodukovatelnost výsledků MeSH
- staurosporin farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Background: Numerous studies have investigated age-based declines in semen traits, but the impact of paternal age on semen parameter values remains inconclusive. Objectives: The aim of this study was to detect an impact of age on semen quality was studied in healthy nonsmoking men exposed to traffic air pollution. Methods: Semen samples from 150 Prague City policemen aged 23 to 63 years were examined for standard semen parameters, sperm DNA fragmentation and high DNA stainability. Results: A significant positive correlation was found between age and %DFI (r = .359, P < .001), and negative correlations were found between age and sperm vitality (r = -.247, P < .001), the % acrosome-intact sperm (r = -.202, P = .013) and the % normal sperm heads (r = -.204, P = .012). A weak but significant negative correlation was found for high DNA stainability (% HDS) vs age (r = -.161, P = .050). No significant correlation was detected between male age and the other investigated semen quality parameters. At ages of 23 to 30, 31 to 40, 41 to 50, and 51 to 63 years, the mean %DFI values were 12.7 ± 7.18, 14.7 ± 7.42, 19.6 ± 11.25, and 34.2 ± 15.08, respectively. Conclusion: Our study shows a strong relationship (P < .001) between the age of men and sperm DNA fragmentation in an occupational cohort at risk of exposure to heavy traffic-related air pollution in a large city center.
- Publikační typ
- časopisecké články MeSH
Reduced fertility of male mouse hybrids relative to their parents, or hybrid sterility, is governed by the hybrid sterility 1 (Hst1) locus. Rescue experiments with transgenes carrying sequences within or near Hst1 manifested that Hst1 contains the gene encoding meiosis-specific histone methyltransferase PRDM9. The Prdm9 gene is responsible for partial meiotic arrest, testicular atrophy, and low sperm count in (C57BL/6J x PWD)F1 mouse hybrids. Here we report that these male hybrids suffer an additional reproductive disadvantage, decreased sperm quality, which is (i) further exacerbated by the introduction of long transgenes carrying sequences from Hst1 with incomplete Prdm9 into their genome and (ii) controlled by the Prdm9 dosage. These transgenic male hybrids displayed the features of severe oligoasthenoteratozoospermia (OAT), a human infertility syndrome characterized by a low number of spermatozoa with poor motility and morphological abnormalities. Analysis of spermiogenesis in these mice revealed acrosome detachment, aberrant elongation and condensation of the nucleus. As a result, the transgenic sperm had acrosome malformations, abnormal chromatin packaging, and fragmented DNA with elevated base oxidation, revealed by using multiple methods. Heterozygosity for one null Prdm9 allele improved meiotic progression and sperm quality of both non- and transgenic hybrids. Our results indicate that genomic analysis of OAT patients should include consideration of allelic variants in PRDM9, and our transgenic models can serve as tools to understand the diverse molecular processes that, when perturbed, can cause this disease.
- MeSH
- histonlysin-N-methyltransferasa fyziologie MeSH
- meióza * MeSH
- motilita spermií * MeSH
- mužská infertilita etiologie metabolismus patologie MeSH
- myši inbrední C57BL MeSH
- myši transgenní MeSH
- myši MeSH
- oligospermie etiologie metabolismus patologie MeSH
- regulace genové exprese * MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Continuing our studies aimed at A-ring modified vitamin D compounds, we designed novel 19-norcalcitriol derivatives bearing at C-2 pegylated chains of different lengths. The terminal fragments of these substituents contain hydroxyls or moieties possessing nitrogen and/or sulfur atoms capable of transition metal ions complexation. Also, two conjugate-type platinum(II) complexes of 19-norcalcitriol were obtained in which l-methionine served as chelating moiety. The convergent synthesis of the target 19-norcalcitriol analogs involved several steps with the crucial one being condensation of A-ring phosphine oxide and the known Grundmann ketone by Wittig-Horner reaction. Further elaboration of the 2-alkylidene substituent provided all final compounds which were then tested to determine their affinity for the vitamin D receptor and cytotoxic activity.
- MeSH
- HL-60 buňky MeSH
- kalcitriol chemická syntéza chemie farmakologie MeSH
- lidé MeSH
- ligandy MeSH
- MFC-7 buňky MeSH
- preklinické hodnocení léčiv MeSH
- protinádorové látky farmakologie MeSH
- racionální návrh léčiv * MeSH
- receptory kalcitriolu účinky léků MeSH
- screeningové testy protinádorových léčiv MeSH
- simulace molekulového dockingu MeSH
- vazebná místa MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Growth and developmental changes in plants induced by pharmaceuticals reflect changes in processes at the cellular and subcellular levels. Due to their growth and cellular characteristics, plant cell suspension cultures can be a suitable model for assessing toxicity. In this study, 10-1000 μg/L of the non-steroidal anti-inflammatory drug diclofenac (DCF) decreased the viability of Nicotiana tabacum BY-2 cells after 24 h of treatment. Further, 0.1-10 mg/L DCF diminished the density of the cell suspension by 9-46% after 96 h of treatment, but at 1 and 10 μg/L, DCF increased the density by 13% and 5%, respectively, after 120 h. These changes were accompanied by increased production of total reactive oxygen species (ROS) and mitochondrial superoxide (up to 17-fold and 5-fold, respectively), and a decrease in the mitochondrial membrane potential (by ∼64%) especially at 1000 μg/L DCF. The increased ROS production was accompanied by decrease in level of reactive nitrogen species (RNS; by 36%) and total thiols (by 61%). Damage to BY-2 cells was evidenced by accumulation of neutral red in acidic compartments (up to 10-fold at 1000 μg/L DCF), and increase of autophagic vacuole formation (up to 8-fold at 1000 μg/L DCF). Furthermore, irregular or stretched nuclei were observed in nearly 27% and 50% of cells at 100 and 1000 μg/L DCF, respectively. Highest levels of chromatin condensation (11% of cells) and apoptotic DNA fragmentation (7%) were found at 10 μg/L DCF. The results revealed a significant effect of DCF on BY-2 cells after 24 h of exposure. Changes in the growth and viability parameters were indisputably related to ROS and RNS production, changes in mitochondrial function, and possible activation of processes leading to cell death.
In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. We simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing had the highest rate of survival after thawing. Our results will facilitate the design of compounds and procedures to decrease injury to cryopreserved cells.
- MeSH
- chromatin účinky léků genetika MeSH
- dimethylsulfoxid farmakologie MeSH
- dvouřetězcové zlomy DNA účinky léků MeSH
- fibroblasty MeSH
- kryoprezervace metody MeSH
- kryoprotektivní látky farmakologie MeSH
- kůže cytologie MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- S fáze účinky léků MeSH
- viabilita buněk účinky léků genetika MeSH
- zmrazování škodlivé účinky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Cinnamtannin B-1 (CNB-1) is a naturally occurring trimeric A-type proanthocyanidin contained in several plants such as cinnamon (Cinnamomum zeylanicum). It is considered to be a potent antioxidant. The protective effect of CNB-1 against oxidative stress was assessed in red deer epididymal sperm incubated at 37 °C. Cryopreserved sperm from six stags were thawed, pooled and extended to 400 × 106 sperm/ml in BGM (bovine gamete medium). After being aliquoted, the samples were supplemented with different concentrations of CNB-1 (0, 0.1, 1, 10 and 100 μg/mL), with or without induced oxidative stress (100 μM Fe2+/ascorbate). The samples were evaluated after 0, 2 and 4 h of incubation at 37 °C. This experiment was replicated six times. Spermmotility (CASA), viability, mitochondrial membrane potential, acrosomal status, lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (TUNEL) were assessed. After 4 h of incubation, CNB-1 prevented the deleterious effects of oxidative stress, thus improved sperm progressivity and velocity (P<0.05). Furthermore, 1 and 10 μM CNB-1 improved sperm linearity, even when compared to those samples that had not been subjected to oxidative stress (P<0.05). The greatest concentration, 100 μM, prevented sperm lipoperoxidation and reduced ROS production in samples subjected to oxidative stress.
- MeSH
- antioxidancia farmakologie MeSH
- fragmentace DNA MeSH
- motilita spermií účinky léků MeSH
- oxidační stres účinky léků MeSH
- peroxidace lipidů MeSH
- proantokyanidiny farmakologie MeSH
- reaktivní formy kyslíku metabolismus MeSH
- spermie účinky léků MeSH
- uchování spermatu veterinární MeSH
- viabilita buněk účinky léků MeSH
- vysoká zvěř * MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
In the present investigation Hibiscus rosa-sinensis (petals), Acorus calamus (rhizome), Moringa oleifera (leaves) and Cucurbita maxima (petals) were screened for their efficacy against osteosarcoma with elucidating a mechanism of their anticancer potentiality. The methanolic plant extracts revealed the presence of all major phytochemicals with quantitative analysis of flavonoids (98.15 ± 2.02 to 12.34 ± 0.57 mg of RUE/g) and total phenolics (26.40 ± 0.11 to 8.54 ± 0.10 mg of GAE/g). The antioxidant activity was assessed by standard DPPH, H2O2 scavenging, NO scavenging assays. The hemolysis, hemagglutination, and erythrocyte aggregation assays unveiled their compatibility with blood components. As most of the opportunistic microbes infect subsequently immunocompromised patients, the antimicrobial activity of the plant extracts showed a zone of inhibition (in mm) against nosocomial strains of Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Vibrio cholerae having MIC between 12.5–50 μg/ml. Through MTT assay the IC50 value was calculated against MG 63 osteosarcoma with detailed studies on DNA fragmentation and chromatin condensation, revealing apoptosis being their primary mode of anticancer effect. Further the migration and colony forming assays supported the anticancer potentials of the methanolic plant extracts. The cell cycle analysis revealed that A. calamus and M. oleifera extracts were capable of arresting the growth of MG 63 osteosarcoma cells.
- MeSH
- antioxidancia MeSH
- Cucurbita chemie MeSH
- fytogenní protinádorové látky * farmakologie chemie MeSH
- fytoterapie MeSH
- Hibiscus chemie MeSH
- léčivé rostliny MeSH
- mikrobiální testy citlivosti MeSH
- Moringa chemie MeSH
- nádorové buňky kultivované účinky léků MeSH
- osteosarkom farmakoterapie MeSH
- puškvorec chemie MeSH
- rostlinné extrakty MeSH
- screeningové testy protinádorových léčiv MeSH
- techniky in vitro MeSH
