Juglone in Combination with Temozolomide Shows a Promising Epigenetic Therapeutic Effect on the Glioblastoma Cell Line.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor and one of the human malignancies with the highest mortality. Standard approaches for GBM, including gross total resection, radiotherapy, and chemotherapy, cannot destroy all the cancer cells, and despite advances in its treatment, the prognosis for GBM remains poor. The problem is that we still do not understand what triggers GBM. Until now, the most successful chemotherapy with temozolomide for brain gliomas is not effective, and therefore new therapeutic strategies for GBM are needed. We found that juglone (J), which exhibits cytotoxic, anti-proliferative, and anti-invasive effects on various cells, could be a promising agent for GBM therapy. In this paper, we present the effects of juglone alone and in combination with temozolomide on glioblastoma cells. In addition to the analysis of cell viability and the cell cycle, we looked at the epigenetics effects of these compounds on cancer cells. We showed that juglone induces strong oxidative stress, as identified by a high increase in the amount of 8-oxo-dG, and decreases m5C in the DNA of cancer cells. In combination with TMZ, juglone modulates the level of both marker compounds. Our results strongly suggest that a combination of juglone and temozolomide can be applied for better GBM treatment.

New N4-Donor Ligands as Supramolecular Guests for DNA and RNA: Synthesis, Structural Characterization, In Silico, Spectrophotometric and Antimicrobial Studies.

The present work reports the synthesis of new N4-donor compounds carrying p-xylyl spacers in their structure. Different Schiff base aliphatic N-donors were obtained synthetically and subsequently evaluated for their ability to interact with two models of nucleic acids: calf-thymus DNA (CT-DNA) and the RNA from yeast Saccharomyces cerevisiae (herein simply indicated as RNA). In more detail, by condensing p-xylylenediamine and a series of aldehydes, we obtained the following Schiff base ligands: 2-thiazolecarboxaldehyde (L1), pyridine-2-carboxaldehyde (L2), 5-methylisoxazole-3-carboxaldehyde (L3), 1-methyl-2-imidazolecarboxaldehyde (L4), and quinoline-2-carboxaldehyde (L5). The structural characterisation of the ligands L1-L5 (X-ray, 1H NMR, 13C NMR, elemental analysis) and of the coordination polymers {[CuL1]PF6}n (herein referred to as Polymer1) and {[AgL1]BF4}n, (herein referred to as Polymer2, X-ray, 1H NMR, ESI-MS) is herein described in detail. The single crystal X-ray structures of complexes Polymer1 and Polymer2 were also investigated, leading to the description of one-dimensional coordination polymers. The spectroscopic and in silico evaluation of the most promising compounds as DNA and RNA binders, as well as the study of the influence of the 1D supramolecular polymers Polymer1 and Polymer2 on the proliferation of Escherichia coli bacteria, were performed in view of their nucleic acid-modulating and antimicrobial applications. Spectroscopic measurements (UV-Vis) combined with molecular docking calculations suggest that the thiazolecarboxaldehyde derivative L1 is able to bind CT-DNA with a mechanism different from intercalation involving the thiazole ring in the molecular recognition and shows a binding affinity with DNA higher than RNA. Finally, Polymer2 was shown to slow down the proliferation of bacteria much more effectively than the free Ag(I) salt.

Cross-reactivity between histone demethylase inhibitor valproic acid and DNA methylation in glioblastoma cell lines.

Currently, valproic acid (VPA) is known as an inhibitor of histone deacetylase (epigenetic drug) and is used for the clinical treatment of epileptic events in the course of glioblastoma multiforme (GBM). Which improves the clinical outcome of those patients. We analyzed the level of 5-methylcytosine, a DNA epigenetic modulator, and 8-oxodeoxyguanosine, an cellular oxidative damage marker, affected with VPA administration, alone and in combination with temozolomide (TMZ), of glioma (T98G, U118, U138), other cancer (HeLa), and normal (HaCaT) cell lines. We observed the VPA dose-dependent changes in the total DNA methylation in neoplastic cell lines and the lack of such an effect in a normal cell line. VPA at high concentrations (250-500 µM) induced hypermethylation of DNA in a short time frame. However, the exposition of GBM cells to the combination of VPA and TMZ resulted in DNA hypomethylation. At the same time, we observed an increase of genomic 8-oxo-dG, which as a hydroxyl radical reaction product with guanosine residue in DNA suggests a red-ox imbalance in the cancer cells and radical damage of DNA. Our data show that VPA as an HDAC inhibitor does not induce changes only in histone acetylation, but also changes in the state of DNA modification. It shows cross-reactivity between chromatin remodeling due to histone acetylation and DNA methylation. Finally, total DNA cytosine methylation and guanosine oxidation changes in glioma cell lines under VPA treatment suggest a new epigenetic mechanism of that drug action.

A new molecular mechanism of RNA circularization and the microRNA sponge formation.

A new mechanism of RNA circularization driven by specific binding of miRNAs is described. We identified the 71 CUUCC pentanucleotide motifs distributed regularly throughout the entire molecule of CDR1as RNA that bind to 71 miRNAs through their seed sequence GGAAG. The sequential binding of miR-7 RNAs (71 molecules) brings both ends of CDR1as RNA (1 molecule) together and stimulate phosphodiester bond formation between nucleotides C1 and A1299 at the 5' and 3' end, respectively. The binding of miRNAs to CDR1as RNA results in the unique complex formation, which shows three specific structural domains: (i) two short helixes with an internal loop, (ii) the hinge, and (iii) the triple-helix. The proposed mechanism explains specific RNA circularization and its function as a miRNAs sponge. Furthermore, the existing wet experimental data on the interaction of CDR1as RNA with miR-7 fully supports our observation. Although miR-671 shows the same seed sequence as miR-7, it forms an almost perfect double helix with CDR1as RNA and induces the cleavage of CDR1as, but does not stimulate circularization. To check how common is the proposed mechanism among circular RNAs, we analyzed the most recent circAtlas database counting almost 1.1 million sequences. It turned out that there are a huge number of circRNAs, which showed miRNAs seed binding sequences distributed through the whole circRNA sequences and prove that circularization of linear transcript is miRNA dependent.Communicated by Ramaswamy H. Sarma.

100 years of RNA. The Diamond Jubilee of Information RNA / 100 lat RNA. Diamentowy jubileusz informacyjnego RNA.

The year 2021 marks not only 60 years since the discovery of messenger RNA and the genetic code. Already 100 yaers passed since RNA was discovered. On the occasion of this special anniversary, we would like to recall the most important events in the history of nucleic acids that led to the above discoveries. We remind the beginning of a new era in science caused by the isolation of nuclein and then nucleic acid, whose components and properties were gradually learned, often by little-known researchers. The distinction of RNA and DNA and the analysis of their occurrence in cells made it possible to formulate the first conclusions about the functions of these compounds. Conclusions on the ratio of nitrogenous bases in DNA led to the knowledge of the structure of the double helix, triggering an avalanche of questions about the essence of transmission of genetic information. Answers began to emerge with the discovery of mRNA, and knowledge of the first three nucleotides encoding an amino acid caused a race to decipher the genetic code. The above discoveries are the foundation of molecular biology. The diamond jubilee coincided with the development of an mRNA-based vaccine against the SARS-CoV-2.

Isolation and Characterization of Phages Active against Paenibacillus larvae Causing American Foulbrood in Honeybees in Poland.

The aim of this study was the isolation and characterization, including the phage effect on honeybees in laboratory conditions, of phages active against Paenibacillus larvae, the causative agent of American Foulbrood-a highly infective and easily spreading disease occurring in honeybee larva, and subsequently the development of a preparation to prevent and treat this dangerous disease. From the tested material (over 2500 samples) 35 Paenibacillus spp. strains were obtained and used to search for phages. Five phages specific to Paenibacillus were isolated and characterized (ultrastructure, morphology, biological properties, storage stability, and genome sequence). The characteristics were performed to obtain knowledge of their lytic potential and compose the final phage cocktail with high antibacterial potential and intended use of future field application. Preliminary safety studies have also been carried out on healthy bees, which suggest that the phage preparation administered is harmless.

The model structure of the hammerhead ribozyme formed by RNAs of reciprocal chirality.

RNA-based tools are frequently used to modulate gene expression in living cells. However, the stability and effectiveness of such RNA-based tools is limited by cellular nuclease activity. One way to increase RNA's resistance to nucleases is to replace its D-ribose backbone with L-ribose isomers. This modification changes chirality of an entire RNA molecule to L-form giving it more chance of survival when introduced into cells. Recently, we have described the activity of left-handed hammerhead ribozyme (L-Rz, L-HH) that can specifically hydrolyse RNA with the opposite chirality at a predetermined location. To understand the structural background of the RNA specific cleavage in a heterochiral complex, we used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy as well as performed molecular modelling and dynamics simulations of homo- and heterochiral RNA complexes. The active ribozyme-target heterochiral complex showed a mixed chirality as well as low field imino proton NMR signals. We modelled the 3D structures of the oligoribonucleotides with their ribozyme counterparts of reciprocal chirality. L- or D-ribozyme formed a stable, homochiral helix 2, and two short double heterochiral helixes 1 and 3 of D- or L-RNA strand thorough irregular Watson-Crick base pairs. The formation of the heterochiral complexes is supported by the result of simulation molecular dynamics. These new observations suggest that L-catalytic nucleic acids can be used as tools in translational biology and diagnostics.

Cancer Stem Cell-Inducing Media Activates Senescence Reprogramming in Fibroblasts.

Cellular senescence is a tumor-suppressive mechanism blocking cell proliferation in response to stress. However, recent evidence suggests that senescent tumor cells can re-enter the cell cycle to become cancer stem cells, leading to relapse after cancer chemotherapy treatment. Understanding how the senescence reprogramming process is a precursor to cancer stem cell formation is of great medical importance. To study the interplay between senescence, stemness, and cancer, we applied a stem cell medium (SCM) to human embryonic fibroblasts (MRC5 and WI-38) and cancer cell lines (A549 and 293T). MRC5 and WI-38 cells treated with SCM showed symptoms of oxidative stress and became senescent. Transcriptome analysis over a time course of SCM-induced senescence, revealed a developmental process overlapping with the upregulation of genes for growth arrest and the senescence-associated secretory phenotype (SASP). We demonstrate that histone demethylases jumonji domain-containing protein D3 (Jmjd3) and ubiquitously transcribed tetratricopeptide repeat, X chromosome (Utx), which operate by remodeling chromatin structure, are implicated in the senescence reprogramming process to block stem cell formation in fibroblasts. In contrast, A549 and 293T cells cultured in SCM were converted to cancer stem cells that displayed the phenotype of senescence uncoupled from growth arrest. The direct overexpression of DNA methyltransferases (Dnmt1 and Dnmt3A), ten-eleven translocation methylcytosine dioxygenases (Tet1 and Tet3), Jmjd3, and Utx proteins could activate senescence-associated beta-galactosidase (SA-ß-gal) activity in 293T cells, suggesting that epigenetic alteration and chromatin remodeling factors trigger the senescence response. Overall, our study suggests that chromatin machinery controlling senescence reprogramming is significant in cancer stem cell formation.

Biological macromolecule binding and anticancer activity of synthetic alkyne-containing L-phenylalanine derivatives.

Herein, we described the synthesis of two L-phenylalanines α-derivatized with a terminal alkyne moiety whose structures differed by phenyl ring halogen substitution (two o-Cl in 1 vs. one p-Br in 2) and investigated their effect on biological macromolecules and living cells. We explored their interaction with quadruplex DNA (G4 DNA), using tel26 and c-myc as models, and bovine serum albumin (BSA). By CD spectroscopy, we found that 1 caused minor tel26 secondary structure changes, leading also to a slight thermal stabilization of this hybrid antiparallel/parallel G4 structure, while the c-myc parallel topology remained essentially unchanged upon 1 binding. Other CD evidences showed the ability of 1 to bind BSA, while molecular docking studies suggested that the same molecule could be housed into the hydrophobic cavity between sub-domains IIA, IIB, and IIIA of the protein. Furthermore, preliminary aggregation studies, based on concentration-dependent spectroscopic experiments, suggested the ability of 1 to aggregate forming noncovalent polymeric systems in aqueous solution. Differently from 1, the bromine-modified compound was able to bind Cu(II) ion, likely with the formation of a CuL2 complex, as found by UV spectroscopy. Finally, cell tests excluded any cytotoxic effect of both compounds toward normal cells, but showed slight antiproliferative effects of 2 on PC3 cancerous cells at 24 h, and of 1 on both T98G and MDA-MB-231 cancer cells at 48 h.

Revealing the epigenetic effect of temozolomide on glioblastoma cell lines in therapeutic conditions.

Temozolomide (TMZ) is a drug of choice in glioblastoma treatment. Its therapeutic applications expand also beyond high grade gliomas. However, a significant number of recurrences and resistance to the drug is observed. The key factor in each chemotherapy is to achieve the therapeutic doses of a drug at the pathologic site. Nonetheless, the rate of temozolomide penetration from blood to cerebrospinal fluid is only 20-30%, and even smaller into brain intestinum. That makes a challenge for the therapeutic regimens to obtain effective drug concentrations with minimal toxicity and minor side effects. The aim of our research was to explore a novel epigenetic mechanism of temozolomide action in therapeutic conditions. We analyzed the epigenetic effects of TMZ influence on different glioblastoma cell lines in therapeutically achieved TMZ concentrations through total changes of the level of 5-methylcytosine in DNA, the main epigenetic marker. That was done with classical approach of radioactive nucleotide post-labelling and separation on thin-layer chromatography. In the range of therapeutically achieved temozolomide concentrations we observed total DNA hypomethylation. The significant hypermethylating effect was visible after reaching TMZ concentrations of 10-50 µM (depending on the cell line). Longer exposure time promoted DNA hypomethylation. The demethylated state of the glioblastoma cell lines was overcome by repeated TMZ applications, where dose-dependent increase in DNA 5-methylcytosine contents was observed. Those effects were not seen in non-cancerous cell line. The increase of DNA methylation resulting in global gene silencing and consecutive down regulation of gene expression after TMZ treatment may explain better glioblastoma patients' survival.

Spectroscopic and SEM evidences for G4-DNA binding by a synthetic alkyne-containing amino acid with anticancer activity.

Herein, we present a spectroscopic (CD and UV) and SEM study of a phenylalanine derivative carrying a terminal alkyne moiety and indicated by us CF3IIIPhe, with particular attention to its interaction with Cu(II) cation and some biological macromolecules, as well as a preliminary evaluation of its effect on cancerous cells. CD spectroscopy evidenced the ability of CF3IIIPhe to interact with tel26 and c-myc, two quadruplex DNA (G4 DNA) models explored in this study. Other CD and UV studies revealed the ability of the unnatural amino acid to form aggregates in aqueous solution, to bind Cu(II) cation, and to interact with bovine serum albumin (BSA). Cellular studies demonstrated CF3IIIPhe antiproliferative activity on PC3 cells. Its ability to bind telomeric DNA was verified with tel26 by CD investigation and SEM analysis, that revealed a noteworthy change in DNA morphology (mainly based on nanosphere structures) by CF3IIIPhe, confirming its G4-DNA binding ability already evidenced by spectroscopy.

[Life as a way of the functioning of matter - Theory of organic being of Jedrzej Sniadecki]. / Zycie jako sposób istnienia materii ­ Teoria jestestw organicznych Jedrzeja Sniadeckiego.

In 2018 we celebrate the 250th anniversary of the Jedrzej Sniadecki's birth. This work aims to show the importance of his thoughts for the development of natural sciences. He studied at some of the largest universities in Europe, where he met great scientists of the enlightenment. The effects can be seen in his works. He was remembered as a founder of Polish biochemistry, anthropology and pathology, also as the author of chemical terminology and language. The essence of his thoughts is "Theory of Organic being", which is an attempt to answer the question: "what is life?". Jedrzej Sniadecki introduced a new definition of life based on the term "organic power". This work shows how import are the thoughts of Jedrzej Sniadecki in the context of the times in which he lived, as well as the following development of natural sciences, what makes him and his theories worth memory.

Inhibition of miR-21 in glioma cells using catalytic nucleic acids.

Despite tremendous efforts worldwide, glioblastoma multiforme (GBM) remains a deadly disease for which no cure is available and prognosis is very bad. Recently, miR-21 has emerged as a key omnipotent player in carcinogenesis, including brain tumors. It is recognized as an indicator of glioma prognosis and a prosperous target for anti-tumor therapy. Here we show that rationally designed hammerhead ribozymes and DNAzymes can target miR-21 and/or its precursors. They decrease miR-21 level, and thus silence this oncomiR functions. We demonstrated that anti-miRNA catalytic nucleic acids show a novel terrific arsenal for specific and effective combat against diseases with elevated cellular miR-21 content, such as brain tumors.

The Sequence and Structure Determine the Function of Mature Human miRNAs.

Micro RNAs (miRNAs) (19-25 nucleotides in length) belong to the group of non-coding RNAs are the most abundant group of posttranscriptional regulators in multicellular organisms. They affect a gene expression by binding of fully or partially complementary sequences to the 3'-UTR of target mRNA. Furthermore, miRNAs present a mechanism by which genes with diverse functions on multiple pathways can be simultaneously regulated at the post-transcriptional level. However, little is known about the specific pathways through which miRNAs with specific sequence or structural motifs regulate the cellular processes. In this paper we showed the broad and deep characteristics of mature miRNAs according to their sequence and structural motifs. We investigated a distinct group of miRNAs characterized by the presence of specific sequence motifs, such as UGUGU, GU-repeats and purine/pyrimidine contents. Using computational function and pathway analysis of their targeted genes, we were able to observe the relevance of sequence and the type of targeted mRNAs. As the consequence of the sequence analysis we finally provide the comprehensive description of pathways, biological processes and proteins associated with the distinct group of characterized miRNAs. Here, we found that the specific group of miRNAs with UGUGU can activate the targets associated to the interferon induction pathway or pathways prominently observed during carcinogenesis. GU-rich miRNAs are prone to regulate mostly processes in neurogenesis, whereas purine/pyrimidine rich miRNAs could be involved rather in transport and/or degradation of RNAs. Additionally, we have also analyzed the simple sequence repeats (SSRs). Their variation within mature miRNAs might be critical for normal miRNA regular activity. Expansion or contraction of SSRs in mature miRNA might directly affect its mRNA interaction or even change the function of that distinct miRNA. Our results prove that due to the specific sequence features, these molecules can also be involved in well-defined cellular processes depending on their sequence contents. The pathway mapping and theoretical gene target identification allowed us to create a biological framework to show the relevance of the specific miRNAs in regulation the distinct type of targets.

Comprehensive analysis of microRNA expression profile in malignant glioma tissues.

Malignant gliomas represent the most devastating group of brain tumors in adults, among which glioblastoma multiforme (GBM) exhibits the highest malignancy rate. Despite combined modality treatment, GBM recurs and is invariably fatal. A further insight into the molecular background of gliomagenesis is required to improve patient outcomes. The primary aim of this study was to gain broad information on the miRNA expression pattern in malignant gliomas, mainly GBM. We investigated the global miRNA profile of malignant glioma tissues with miRNA microarrays, deep sequencing and meta-analysis. We selected miRNAs that were most frequently deregulated in glioblastoma tissues, as well as in peritumoral areas, in comparison with normal human brain. We identified candidate miRNAs associated with the progression from glioma grade III to glioma grade IV. The meta-analysis of miRNA profiling studies in GBM tissues summarizes the past and recent advances in the investigation of the miRNA signature in GBM versus noncancerous human brain and provides a comprehensive overview. We propose a list of 35 miRNAs whose expression is most frequently deregulated in GBM patients and of 30 miRNA candidates recognized as novel GBM biomarkers.

Mature miRNAs form secondary structure, which suggests their function beyond RISC.

The generally accepted model of the miRNA-guided RNA down-regulation suggests that mature miRNA targets mRNA in a nucleotide sequence-specific manner. However, we have shown that the nucleotide sequence of miRNA is not the only determinant of miRNA specificity. Using specific nucleases, T1, V1 and S1 as well as NMR, UV/Vis and CD spectroscopies, we found that miR-21, miR-93 and miR-296 can adopt hairpin and/or homoduplex structures. The secondary structure of those miRNAs in solution is a function of RNA concentration and ionic conditions. Additionally, we have shown that a formation of miRNA hairpin is facilitated by cellular environment.Looking for functional consequences of this observation, we have perceived that structure of these miRNAs resemble RNA aptamers, short oligonucleotides forming a stable 3D structures with a high affinity and specificity for their targets. We compared structures of anti-tenascin C (anti-Tn-C) aptamers, which inhibit brain tumor glioblastoma multiforme (GBM, WHO IV) and selected miRNA. A strong overexpression of miR-21, miR-93 as well Tn-C in GBM may imply some connections between them. The structural similarity of these miRNA hairpins and anti-Tn-C aptamers indicates that miRNAs may function also beyond RISC and are even more sophisticated regulators, that it was previously expected. We think that the knowledge of the miRNA structure may give a new insight into miRNA-dependent gene regulation mechanism and be a step forward in the understanding their function and involvement in cancerogenesis. This may improve design process of anti-miRNA therapeutics.

Squalene monooxygenase - a target for hypercholesterolemic therapy.

Squalene monooxygenase catalyzes the epoxidation of C-C double bond of squalene to yield 2,3-oxidosqualene, the key step of sterol biosynthesis pathways in eukaryotes. Sterols are essential compounds of these organisms and squalene epoxidation is an important regulatory point in their synthesis. Squalene monooxygenase downregulation in vertebrates and fungi decreases synthesis of cholesterol and ergosterol, respectively, which makes squalene monooxygenase a potent and attractive target of hypercholesterolemia and antifungal therapies. Currently some fungal squalene monooxygenase inhibitors (terbinafine, naftifine, butenafine) are in clinical use, whereas mammalian enzymes' inhibitors are still under investigation. Research on new squalene monooxygenase inhibitors is important due to the prevalence of hypercholesterolemia and the lack of both sufficient and safe remedies. In this paper we (i) review data on activity and the structure of squalene monooxygenase, (ii) present its inhibitors, (iii) compare current strategies of lowering cholesterol level in blood with some of the most promising strategies, (iv) underline advantages of squalene monooxygenase as a target for hypercholesterolemia therapy, and (v) discuss safety concerns about hypercholesterolemia therapy based on inhibition of cellular cholesterol biosynthesis and potential usage of squalene monooxygenase inhibitors in clinical practice. After many years of use of statins there is some clinical evidence for their adverse effects and only partial effectiveness. Currently they are drugs of choice but are used with many restrictions, especially in case of children, elderly patients and women of childbearing potential. Certainly, for the next few years, statins will continue to be a suitable tool for cost-effective cardiovascular prevention; however research on new hypolipidemic drugs is highly desirable. We suggest that squalene monooxygenase inhibitors could become the hypocholesterolemic agents of the future.

[Lycopene--occurrence, properties and applications]. / Likopen--wystepowanie, wlasciwosci oraz potencjalne zastosowanie.

Natural products from plants, fungi and higher animals are valuable sources of attractive alternatives for therapeutics. One of them, lycopene is a bright red carotene found in several fruits and vegetables. Tomato, tomato-based sauces and juices are the most abundant sources of this compound for human. There is a positive correlation between lycopene intake and health. It plays an important role in preventing several diseases, inclusing cancers. Lycopene is the most efficient oxygen and free radicals scavenger. Moreover it controls cell cycle and activates phase II detoxification enzymes. Epidemiological studies confirm its significant role in preventing diseases. Constant progress on this field makes lycopene an interesting object of researches.

[aaRS--the etiological factor and the attractive target of many disorders]. / Znaczenie syntetaz aminoacylo-tRNA w patologiach i perspektywa ich wykorzystania w diagnostyce i terapii.

Aminoacyl-tRNA synthetases (aaRS) are essential proteins of all living organisms. It is known that they ensure the fidelity of transfer of genetic information from the DNA into the protein. Not far away it occurred that their role is not confined to catalyze the attachment of amino acids to transfer RNAs and thereby establish the rules of genetic code by virtue of matching the nucleotide triplet of anticodon with cognate amino acid. aaRSs are also engaged in the other crucial cellular processes. So the disturbance of function of any of them often causes serious disorders. Therefore this proteins could be an attractive target of drugs, not only against the mentioned illnesses but also against bacterial, fungal and parasitic infections. Constant progress on this field makes aaRSs still an interesting object of researches.