As the most common and aggressive primary malignant brain tumor, glioblastoma is still lacking a satisfactory curative approach. The standard management consisting of gross total resection followed by radiotherapy and chemotherapy with temozolomide only prolongs patients’ life moderately. In recent years, many therapeutics have failed to give a breakthrough in GBM treatment. In the search for new treatment solutions, we became interested in the repurposing of existing medicines, which have established safety profiles. We focused on the possible implementation of well-known drugs, metformin, and arginine. Metformin is widely used in diabetes treatment, but arginine is mainly a cardiovascular protective drug. We evaluated the effects of metformin and arginine on total DNA methylation, as well as the oxidative stress evoked by treatment with those agents. In glioblastoma cell lines, a decrease in 5-methylcytosine contents was observed with increasing drug concentration. When combined with temozolomide, both guanidines parallelly increased DNA methylation and decreased 8-oxo-deoxyguanosine contents. These effects can be explained by specific interactions of the guanidine group with m5CpG dinucleotide. We showed that metformin and arginine act on the epigenetic level, influencing the foreground and potent DNA regulatory mechanisms. Therefore, they can be used separately or in combination with temozolomide, in various stages of disease, depending on desired treatment effects.

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.

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.

In the present study, we continue our work related to the synthesis of 1,8-naphthalimide and carborane conjugates and the investigation of their anticancer activity and DNA-binding ability. For this purpose, a series of 4-carboranyl-1,8-naphthalimide derivatives, mitonafide, and pinafide analogs were synthesized using click chemistry, reductive amination, amidation, and Mitsunobu reactions. The calf thymus DNA (ct-DNA)-binding properties of the synthesized compounds were investigated by circular dichroism (CD), UV–vis spectroscopy, and thermal denaturation experiments. Conjugates 54–61 interacted very strongly with ct-DNA (∆Tm = 7.67–12.33 °C), suggesting their intercalation with DNA. They were also investigated for their in vitro effects on cytotoxicity, cell migration, cell death, cell cycle, and production of reactive oxygen species (ROS) in a HepG2 cancer cell line as well as inhibition of topoisomerase IIα activity (Topo II). The cytotoxicity of these eight conjugates was in the range of 3.12–30.87 µM, with the lowest IC50 value determined for compound 57. The analyses showed that most of the conjugates could induce cell cycle arrest in the G0/G1 phase, inhibit cell migration, and promote apoptosis. Two conjugates, namely 60 and 61, induced ROS production, which was proven by the increased level of 2′-deoxy-8-oxoguanosine in DNA. They were specifically located in lysosomes, and because of their excellent fluorescent properties, they could be easily detected within the cells. They were also found to be weak Topo II inhibitors.

We synthesized a series of novel 3-carboranyl-1,8-naphthalimide derivatives, mitonafide and pinafide analogs, using click chemistry, reductive amination and amidation reactions and investigated their in vitro effects on cytotoxicity, cell death, cell cycle, and the production of reactive oxygen species in a HepG2 cancer cell line. The analyses showed that modified naphthalic anhydrides and naphthalimides bearing ortho- or meta-carboranes exhibited diversified activity. Naphthalimides were more cytotoxic than naphthalic anhydrides, with the highest IC50 value determined for compound 9 (3.10 µM). These compounds were capable of inducing cell cycle arrest at G0/G1 or G2M phase and promoting apoptosis, autophagy or ferroptosis. The most promising conjugate 35 caused strong apoptosis and induced ROS production, which was proven by the increased level of 2′-deoxy-8-oxoguanosine in DNA. The tested conjugates were found to be weak topoisomerase II inhibitors and classical DNA intercalators. Compounds 33, 34, and 36 fluorescently stained lysosomes in HepG2 cells. Additionally, we performed a similarity-based assessment of the property profile of the conjugates using the principal component analysis. The creation of an inhibitory profile and descriptor-based plane allowed forming a structure–activity landscape. Finally, a ligand-based comparative molecular field analysis was carried out to specify the (un)favorable structural modifications (pharmacophoric pattern) that are potentially important for the quantitative structure–activity relationship modeling of the carborane–naphthalimide conjugates.

Recently, small compound-based therapies have provided new insights into the treatment of glioblastoma multiforme (GBM) by inducing oxidative impairment. Kinetin riboside (KR) and newly designed derivatives (8-azaKR, 7-deazaKR) selectively affect the molecular pathways crucial for cell growth by interfering with the redox status of cancer cells. Thus, these compounds might serve as potential alternatives in the oxidative therapy of GBM. The increased basal levels of reactive oxygen species (ROS) in GBM support the survival of cancer cells and cause drug resistance. The simplest approach to induce cell death is to achieve the redox threshold and circumvent the antioxidant defense mechanisms. Consequently, cells become more sensitive to oxidative stress (OS) caused by exogenous agents. Here, we investigated the effect of KR and its derivatives on the redox status of T98G cells in 2D and 3D cell culture. The use of spheroids of T98G cells enabled the selection of one derivative-7-deazaKR-with comparable antitumor activity to KR. Both compounds induced ROS generation and genotoxic OS, resulting in lipid peroxidation and leading to apoptosis. Taken together, these results demonstrated that KR and 7-deazaKR modulate the cellular redox environment of T98G cells, and vulnerability of these cells is dependent on their antioxidant capacity.

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-β-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.

DNA modifications can be used to monitor pathological processes. We have previously shown that estimating the amount of the main DNA epigenetic mark, 5-methylcytosine (m5C), is an efficient and reliable way to diagnose brain tumors, hypertension, and other diseases. Abnormal increases of reactive oxygen species (ROS) are a driving factor for mutations that lead to changes in m5C levels and cancer evolution. 8-oxo-deoxyguanosine (8-oxo-dG) is a specific marker of ROS-driven DNA-damage, and its accumulation makes m5C a hotspot for mutations. It is unknown how m5C and 8-oxo-dG correlate with the malignancy of gliomas. We analyzed the total contents of m5C and 8-oxo-dG in DNA from tumor tissue and peripheral blood samples from brain glioma patients. We found an opposite relationship in the amounts of m5C and 8-oxo-dG, which correlated with glioma grade in the way that low level of m5C and high level of 8-oxo-dG indicated increased glioma malignancy grade. Our results could be directly applied to patient monitoring and treatment protocols for gliomas, as well as bolster previous findings, suggesting that spontaneously generated ROS react with m5C. Because of the similar mechanisms of m5C and guanosine oxidation, we concluded that 8-oxo-dG could also predict glioma malignancy grade and global DNA demethylation in cancer cells.

The development of 1,8-naphthalimide derivatives as DNA-targeting anticancer agents is a rapidly growing area and has resulted in several derivatives entering into clinical trials. One of original recent developments is the use of boron clusters: carboranes and metallacarboranes in the design of pharmacologically active molecules. In this direction several naphthalimide-carborane and metallacarborane conjugates were synthesized in the present study. Their effect on a cancer cell line - cytotoxicity, type of cell death, cell cycle, and ROS production were investigated. The tested conjugates revealed different activities than the leading members of the naphthalimides family, namely mitonafide and pinafide. These derivatives could induce GO/G1 arrest and promote mainly apoptosis in HepG2 cell line. Our investigations demonstrated that the most promising molecule is N-{[2-(3,3'commo-bis(1,2-dicarba-3-cobalta(III)-doso-dodecaborate-1-yeethyll-1'-aminoethyl)}-1,8-naphthalimidel (17). It was shown that 17 exhibited cytotoxicity against HepG2 cells, activated cell apoptosis, and caused cell cycle arrest in HepG2 cells. Further investigations in HepG2 cells revealed that compound 17 can also induce ROS generation, particularly mitochondrial ROS (mtROS), which was also proved by increased 8-oxo-dG level in DNA. Additionally to biological assays the interaction of the new compounds with ct-DNA was studied by CD spectra and melting temperature, thus demonstrating that these compounds were rather weak classical DNA intercalators.

Tyrosyl-tRNA synthetases (TyrRSs) as essential enzymes for all living organisms are good candidates for therapeutic target in the prevention and therapy of microbial infection. We examined the effect of various polyphenols, alkaloids, and terpenes-secondary metabolites produced by higher plants showing many beneficial properties for the human organism, on bacterial aminoacylation reaction. The most potent inhibitors of Escherichia coli TyrRS are epigallocatechin gallate, acacetin, kaempferide, and chrysin, whereas the enzymes from Staphylococcus aureus and Pseudomonas aeruginosa are inhibited mainly by acacetin and chrysin. Most of them act as competitive inhibitors. Structure-activity relationship showed that the most potent flavonoid inhibitors contain hydroxyl group at position 5 and 7 of A ring and OCH3 group at position 4' of B ring.

Reactions of 6,6″-dimethyl-2,2′:6′,2″-terpyridine ligand L and VX3 (X = Cl, Br) in aerobic conditions result in formation of two new mononuclear coordination compounds: [VOLCl2] 1 and [VO2L]2Br2·3H2O 2, as revealed by means of single crystal X-ray analyses. The final form of vanadium complex in the solid state, either oxido (V(IV)) or dioxido (V(V)), was found to be contingent on the size of applied halide, as demonstrated by direct V–X interaction for small chloride (1) or ionic interaction for bulkier bromide (2). Depending on crystallization conditions, [VOLCl2] 1 forms polymorphic green or yellow crystals from MeCN or MeOH respectively. Compounds were characterized by elemental analysis, spectrometric (ESI-MS), electrochemical, (cyclic voltammetry) spectroscopic methods (UV–VIS, FTIR, EPR) and evaluated in terms of biological properties. Their cytotoxic activity was investigated in MTT assay and supported by absorption titration spectra suggesting the possible mechanism of action going through intercalative binding mode. On the basis of high binding constants Kbind = 4.4 × 105 M−1 (1) and Kbind = 1.0 × 106 M−1 (2) the relationship between structure of the complex and DNA interaction could be established, highlighting indirect impact of counterions on the final structure formation and thus biological activity. Flow cytometry analysis further confirmed the DNA binding based mechanism of action. What is more, compounds 1 and 2 are first reported cytotoxically active vanadium systems with 2,2′:6′,2″-terpyridine scaffolded family towards T47D cell line.

Methylation at position 5 of cytosine (Cyt) at the CpG sequences leading to formation of 5-methyl-cytosine (m(5)Cyt) is an important element of epigenetic regulation of gene expression. Modification of the normal methylation pattern, unique to each organism, leads to the development of pathological processes and diseases, including cancer. Therefore, quantification of the DNA methylation and analysis of changes in the methylation pattern is very important from a practical point of view and can be used for diagnostic purposes, as well as monitoring of the treatment progress. In this paper we present a new method for quantification of 5-methyl-2'deoxycytidine (m(5)C) in the DNA. The technique is based on conversion of m(5)C into fluorescent 3,N(4)-etheno-5-methyl-2'deoxycytidine (εm(5)C) and its identification by reversed-phase high-performance liquid chromatography (RP-HPLC). The assay was used to evaluate m(5)C concentration in DNA of calf thymus and peripheral blood of cows bred under different conditions. This approach can be applied for measuring of 5-methylcytosine in cellular DNA from different cells and tissues.

6,6″-Dimethyl-2,2':6',2″-terpyridine ligand (L) reacts in equimolar ratio with Ag(I) ions what results in formation of dinuclear double helicates, which differ in terms of framework and complexity in accordance to counterions and solvent applied. Obtained complexes were thoroughly studied in terms of their biological activity, with the positive antiproliferative outcome on three human cancer cell lines: human breast cancer (T47D), human cervical carcinoma (HeLa) and human lung cancer (A-549). Performed DNA binding experiments showed that given Ag(I) species specifically interact with DNA double helix via intercalation and were visualized by confocal microscopy to specifically bind to the nuclei. All newly synthesized helical systems exhibit promising antimicrobial activity against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus bacterial strains. Spectrophotometric properties were described as fulfilment of structural studies of newly presented complexes confirming their helical structure in solution.

The tertiary structure of nucleic acids results from an equilibrium between electrostatic interactions of phosphates, stacking interactions of bases, hydrogen bonds between polar atoms and water molecules. Water interactions with ribonucleic acid play a key role in its structure formation, stabilization and dynamics. We used high hydrostatic pressure and osmotic pressure to analyze changes in RNA hydration. We analyzed the lead catalyzed hydrolysis of tRNAPhe from S. cerevisiae as well as hydrolytic activity of leadzyme. Pb(II) induced hydrolysis of the single phosphodiester bond in tRNAPhe is accompanied by release of 98 water molecules, while other molecule, leadzyme releases 86.

DNA cytosine methylation catalyzed by DNA methyltransferase 1 (DNMT1) is an epigenetic method of gene expression regulation and development. Changes in methylation pattern lead to carcinogenesis. Inhibition of DNMT1 activity could be a good strategy of safe and efficient epigenetic therapy. In this work, we present a novel group of cytosine analogs as inhibitors of DNA methylation. We show new methods of synthesis and their effect on in vitro reaction of DNA methylation. Almost all of analyzed compounds inhibit DNA methyltransferase activity in the competitive manner. K(i) values for the most potent compound 4-N-furfuryl-5,6-dihydroazacytosines is 0.7 μM. These compounds cause also a decrease of 5-methylcytosine (m(5)C) level in DNA of mammalian HeLa and HEK293 cells.

Lead is the most common naturally occurring heavy metal and one of the few that are found in a pure form. Because of its chemical properties it has already been used by the oldest civilizations, early people experienced its toxic properties. In this paper, we present the influence of lead on the human body and its interaction with macromolecules (proteins and nucleic acids) and cellular structures. We discuss as well the mechanism of its toxicity at the molecular level.