The majority of CAKUT-associated CNVs overlap at least one miRNA gene, thus affecting the cellular levels of the corresponding miRNA. We aimed to investigate the potency of restitution of ...CNV-affected miRNA levels to remediate the dysregulated expression of target genes involved in kidney physiology and development in vitro.
Heterozygous MIR484 knockout HEK293 and homozygous MIR185 knockout HEK293 cell lines were used as models depicting the deletion of the frequently affected miRNA genes by CAKUT-associated CNVs. After treatment with the corresponding miRNA mimics, the levels of the target genes have been compared to the non-targeting control treatment. For both investigated miRNAs, MDM2 and PKD1 were evaluated as common targets, while additional 3 genes were investigated as targets of each individual miRNA (NOTCH3, FIS1 and APAF1 as hsa-miR-484 targets and RHOA, ATF6 and CDC42 as hsa-miR-185-5p targets).
Restitution of the corresponding miRNA levels in both knockout cell lines has induced a change in the mRNA levels of certain candidate target genes, thus confirming the potential to alleviate the CNV effect on miRNA expression. Intriguingly, HEK293 WT treatment with investigated miRNA mimics has triggered a more pronounced effect, thus suggesting the importance of miRNA interplay in different genomic contexts.
Dysregulation of multiple mRNA targets mediated by CNV-affected miRNAs could represent the underlying mechanism behind the unresolved CAKUT occurrence and phenotypic variability observed in CAKUT patients. Characterizing miRNAs located in CNVs and their potential to become molecular targets could eventually help in understanding and improving the management of CAKUT.
Rare copy number variants (CNVs) are among the most common genomic disorders underlying CAKUT. miRNAs located in rare CNVs represent well-founded functional variants for human CAKUT research. The ...study aimed to identify and functionally interpret miRNAs most frequently affected by rare CNVs in CAKUT and to estimate the overall burden of rare CNVs on miRNA genes in CAKUT. The additional aim of this study was to experimentally confirm the effect of a rare CNV in CAKUT on candidate miRNA's expression and the subsequent change in mRNA levels of selected target genes. A database of CAKUT-associated rare CNV regions, created by literature mining, was used for mapping of the miRNA precursors. miRNAs and miRNA families, most frequently affected by rare CAKUT-associated CNVs, have been subjected to bioinformatic analysis. CNV burden analysis was performed to identify chromosomes with over/underrepresentation of miRNA genes in rare CNVs associated with CAKUT. A functional study was performed on HEK293 MIR484
KO and HEK293 WT cell lines, followed by the analysis of relative miRNA and mRNA target gene levels. 80% of CAKUT patients with underlying rare CNV had at least one miRNA gene overlapping the identified CNV. Network analysis of the most frequently affected miRNAs has revealed the dominant regulation of the two miRNAs, hsa-miR-484 and hsa-miR-185-5p. Additionally, miR-548 family members have shown substantial enrichment in rare CNVs in CAKUT. An over/underrepresentation of miRNA genes in rare CNVs associated with CAKUT was observed in multiple chromosomes, such as chr16, chr20, and chr21. A significant 0.37 fold downregulation of hsa-miR-484, followed by a notable upregulation of MDM2 and APAF1 and downregulation of NOTCH3 was detected in HEK293 MIR484
KO compared to HEK293 WT cell lines, supporting the study hypothesis. miRNA genes are frequently affected by rare CNVs in CAKUT patients. Understanding the potential of CNV-affected miRNAs to participate in CAKUT as genetic drivers represent a crucial implication for the development of novel therapeutic approaches.
Two new dinuclear bimetallic complexes, {PdCl(bipy)}{μ-(pyrazine)}{PtCl(bipy)}Cl(ClO4) (1) (bipy is 2,2'-bipyridine) and {PdCl(en)}{μ-(pyrazine)}{PtCl(en)}Cl(ClO4) (2) (en is ethylenediamine), have ...been synthesized and characterized by elemental microanalysis, IR, (1)H NMR spectroscopy and MALDI-TOF mass spectrometry. The pKa values of the coordinated water molecules of the diaqua species were determined as well. Substitution reactions of complexes (1) and (2) with thiourea (Tu), l-methionine (l-Met), l-cysteine (l-Cys), l-histidine (l-His) and guanosine-5'-monophosphate (5'-GMP) were studied under the pseudo-first order conditions as a function of nucleophile concentration and temperature. The order of reactivity of nucleophiles was: Tu > l-Met > l-Cys > l-His > 5'-GMP. Substitution reactions with Tu, l-Cys and l-His were followed by decomposition of bimetallic complexes to the corresponding substituted mononuclear complexes Pd(N-N)(Nu)2 and Pt(N-N)(Nu)2 (N-N = bipy, en), releasing the bridging ligand. However, the structures of starting bimetallic complexes were preserved during the reactions with l-Met and 5'-GMP. The absorption spectroscopic study of interactions of calf-thymus DNA (CT-DNA) with complexes (1), (2) and {PdCl(bipy)}{μ-(NH2(CH2)6H2N)} {PtCl(bipy)}Cl(ClO4) (3), has shown that all the complexes exhibit high intrinsic binding constants (Kb = 10(4)-10(5) M(-1)). DNA-ethidium bromide (DNA-EB) fluorescence was quenched after addition of complexes (1), (2) or (3), indicating displacement of intercalating EB by complexes. All complexes have shown good binding affinity to bovine serum albumin protein (BSA). Chemosensitivity of A375 (human melanoma) and HeLa (human cervical cancer) cell lines toward complexes (1), (2) and (3) was analyzed by SRB assay. Complex (1) displayed significant inhibitory effect on the growth of both cell lines.
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•Non-cancerous MRC-5 cells are more sensitive to ZnO NPs than cancerous HeLa cells.•Differences in Raman spectra of two types of NP-treated cells are registered.•Raman markers of cell ...damage are determined using non-negative PCA.•Lipid saturation level plays a role in cell-ZnO interaction.
Selective cytotoxicity of ZnO nanoparticles among different cell types and cancer and non-cancerous cells has been demonstrated earlier. In the view of anticancer potential of ZnO nanoparticles and their presence in numerous industrial products, it is of great importance to carefully evaluate their effects and mechanisms of action in both cancerous and healthy cells. In this paper, the effects of ZnO nanoparticles on cancerous HeLa and non-cancerous MRC-5 cells are investigated by studying the changes in the vibrational properties of the cells using Raman spectroscopy. Both types of cells were incubated with ZnO nanoparticles of average size 40 nm in the doses from the range 10–40 µg/ml for the period of 48 h, after which Raman spectra were collected. Raman modes’ intensity ratios I1659/I1444, I2855/I2933 and I1337/I1305 were determined as spectral markers of the cytotoxic effect of ZnO in both cell types. Non-negative principal component analysis was used instead of standard one for analysis and detection of spectral features characteristic for nanoparticle-treated cells. The first several non-negative loading vectors obtained in this analysis coincided remarkably well with the Raman spectra of particular biomolecules, showing increase of lipid and decrease of nucleic acids and protein content. Our study pointed out that Raman spectral markers of lipid unsaturation, especially I1270/I1300, are relevant for tracing the cytotoxic effect of ZnO nanoparticles on both cancerous and non-cancerous cells. The change of these spectral markers is correlated to the dose of applied nanoparticles and to the degree of cellular damage. Furthermore, great similarity of spectral features of increasing lipids to spectral features of phosphatidylserine, one of the main apoptotic markers, was recognized in treated cells. Finally, the results strongly indicated that the degree of lipid saturation, presented in the cells, plays an important role in the interaction of cells with nanoparticles.
Objects of the present study are improved fullerene C
drug carrier properties trough encapsulation by microbial polysaccharides, levan (LEV), pullulan (PUL), and their hydrophobized ...cholesterol-derivatives (CHL and CHP), that show better interaction with cancer cells. The zeta potential, polydispersity index, and the diameter of particles were determined, and their cytotoxicity against three cancer cell lines were tested. Biochemical changes in HeLa cells are analyzed by synchrotron radiation (SR) FTIR spectro-microscopy combined with the principal component analysis (PCA). The most significant changes occur in HeLa cells treated with LEV-C
and correspond to the changes in the protein region, i.e. Amide I band, and the changes in the structure of lipid bodies and membrane fluidity are evident. The highest cytotoxicity was also induced by LEV-C
. In HeLa cells, cytotoxicity could not be strictly associated with biochemical changes in lipids, proteins and nucleic acids, but these findings are significant contribution to the study of the mechanism of interaction of C
-based nanoparticles with cellular biomolecules. In conclusion, LEV, PUL, CHL, and CHP enhanced fullerene C
potential to be used as target drug delivery system with the ability to induce specific intracellular changes in HeLa cancer cells.
Apart from cytotoxicity, inhibitors of the COX-2 enzyme have demonstrated additional effects important for cancer treatment (such as radiosensitization of tumor cells and cell antimigratory effects); ...however, the relationship between the inhibition of other inflammation-related enzyme 5-LOX inhibitors and anticancer activity is still not well understood. In our study, the cytotoxicity of thirteen COX-2 and 5-LOX inhibitors previously presented by our group (1–13) was tested on three cancer cell lines (HCT 116, HT-29 and BxPC-3) and one healthy cell line (MRC-5). Compounds 3, 5, 6 and 7 showed moderate cytotoxicity, but good selectivity towards cancer cell lines. IC50 values were in the range of 22.99–51.66 µM (HCT 116 cell line), 8.63–41.20 µM (BxPC-3 cell line) and 24.78–81.60 µM (HT-29 cell line; compound 7 > 100 µM). In comparison to tested, commercially available COX-2 and 5-LOX inhibitors, both cytotoxicity and selectivity were increased. The addition of compounds 6 and 7 to irradiation treatment showed the most significant decrease in cell proliferation of the HT-29 cell line (p < 0.001). The antimigratory potential of the best dual COX-2 and 5-LOX inhibitors (compounds 1, 2, 3 and 5) was tested by a wound-healing assay using the SW620 cell line. Compounds 1 and 3 were singled out as compounds with the most potent effect (relative wound closure was 3.20% (24 h), 5,08% (48 h) for compound 1 and 3.86% (24 h), 7.68% (48 h) for compound 3). Considering all these results, compound 3 stood out as the compound with the most optimal biological activity, with the best dual COX-2 and 5-LOX inhibitory activity, good selectivity towards tested cancer cell lines, significant cell antimigratory potential and a lack of toxic effects at therapeutic doses.
Herein, the stability, lipophilicity, in vitro cytotoxicity, and influence on acetylcholinesterase of five dinuclear platinum(II) complexes with the general formula {Pt(en)Cl}2(μ-L)2+ (L is a ...different aromatic nitrogen-containing heterocyclic bridging ligands pyrazine (pz, Pt1), pyridazine (pydz, Pt2), quinoxaline (qx, Pt3), phthalazine (phtz, Pt4) and quinazoline (qz, Pt5), while en is bidentate coordinated ethylenediamine) were evaluated. The most active analyzed platinum complexes induced time-dependent growth inhibition of A375, HeLa, PANC-1, and MRC-5 cells. The best efficiency was achieved on HeLa and PANC-1 cells for Pt1, Pt2, and Pt3 at the highest concentration, while Pt1 was significantly more potent than cisplatin at a lower concentration. Additionally, a lower effect on normal cells was observed compared to cisplatin, which may indicate potentially fewer side effects of these complexes. Selected complexes induce reactive oxygen species and apoptosis on tumor cell lines. The most potent reversible acetylcholinesterase (AChE) inhibitors were Pt2, Pt4, and Pt5. Pt1 showed similar inhibitory potential toward AChE as cisplatin, but a different type of inhibition, which could contribute to lower neurotoxicity. Docking studies revealed that Pt2 and Pt4 were bound to the active gorge above the catalytic triad. In contrast, the other complexes were bound to the edge of the active gorge without impeding the approach to the catalytic triad. According to this, Pt1 represents a promising compound with potent anticancer properties, high selectivity, and low neurotoxicity.
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•Pt1, Pt2, and Pt3 have shown similar effects as cisplatin on HeLa and PANC-1.•Pt1 was significantly more potent than cisplatin at low concentrations.•Pt1 shows a significantly lower impact on MRC-5 cells than cisplatin.•Pt1 is the low-potency reversible inhibitor of AChE.
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