T140 Epigenetically active xenobiotics in cancer prevention and therapy optimization

Nowadays the term “epigenetics” is described as regulation of gene expression persisting from one cell division to the next, despite a lack of changes in the underlying DNA sequence. The “epigenome” refers to different epigenetic states of a cell recognized as heritable environment influence on genome. The main epigenetic phenomena in mammals are DNA methylation and histone modifications, which are tightly interdependent. Many authors classify microRNA regulation as a third epigenetic phenomenon. Moreover, recently the discussion has been open that many different factors modifying DNA conformation represent a new class of epigenetic agents. In response to various environmental stimuli, cells produce different epigenetic changes that determine either an active or a repressed chromatin state.Epigenetic perturbations have been shown to associate with exposure to a range of drugs and toxicants, including non-genotoxic carcinogens. Consequently, on one hand, potential impact of epigenomics on drug development is under consideration as even well-known pharmacological drugs were shown to cause epigenetic changes that may be beneficial or hazardous. In particular, epigenetic effects were described for synthetic estrogens and contraceptives, beta-blockers and fluoroquinolone antibiotics, neuroleptics and anesthetics, chemotherapeutics and statins. Drug influence on gene silencing might have some therapeutic advantage in addition to the unfavourable effects. In particular, valproate, hydralazine and procainamide might be utilized to induce gene expression in cancers, where activation of a methylated gene might be of benefit. Screening of xenobiotics for epigenetic activity might identify new potential drugs for some specific diseases.On the other hand, epigenetic aspects of drug safety are investigated intensively. Many recent reviews in this field of research were devoted to the test elaboration for revealing different short-term and longer-lasting epigenetic changes modifying gene expression. Most of the short-term epigenetic screening tests are based on revealing of xenobiotics to influence on specific enzymes participating in epigenetic modifications, especially DNA methylation and histone modifications. Recently a new model system for the screening of compounds reactivating epigenetically repressed genes was described. It represents HeLa cell population that harbors silent GFP-reporter gene which could be activated via different epigenetic mechanisms.Epigenetic studies of xenobiotic-induced mechanisms will reveal valuable information clarifying both mechanisms of desirable therapy and side effects and open potential drug design directions. The major challenge that remains is to determine how epigenetic control is regulated and how it might be possible to intervene in such processes

P88 Multiple mechanisms of DNA minor groove binding ligand epigenetic effects

PurposeDNA minor groove is the main target of small molecules, which noncovalently and to a certain extent site-specifically bind to appropriate nucleotide sequences. Study of these substances can give rise to understanding the mechanistic relationship between sites of interaction and activity of appropriate enzymes with “houskeeping” function including helicases, topoisomerases, methyltransferases, demethylases and DNA/RNA-polymerases.ResultsWe revealed for the first time that AT-specific minor groove binding ligands (MGBLs), in particular bisbenzimidazoles (Hoechst33258 and its derivatives), widely used in molecular and cell biology for DNA-staining, induce loss of heterozygosity at high frequency while point mutations and chromosome deletions at insignificant levels. Moreover, we demonstrated that the agents realized their genotoxic blastomogenic effects via homologous recombination mechanism exclusively. Lately the same mechanism of genotoxicity has been shown for MGBL carbazole derivative Curaxin, which is toxic for a broad range of tumor cell lines in vitro and inhibit tumor growth in different mouse models of cancer in vivo. Moreover, powerful antitumor activity has been demonstrated for Trabectedin, which binds to the DNA’s minor groove and alkylate guanine residues. All this provided a framework for wide-ranging investigation of cell response to MGBLs exposure, molecular mechanisms of their recombinogenic as also their anticancer activity. A special interest is paid to epigenetic mechanisms of MGBs action.Our study aimed to examine the epigenetic effects of recombinogenic (Hoechst33342, Hoechst33258) and non-recombinogenic (DAPI, Diminazene, Pentamidine and Netropsin) MGBLs.After we unmasked MGBLs’ recombinogenic activity, we hypothesized that their molecular mechanism of indirect DNA damage involves poly(ADP-ribose)polymerase-1 (PARP-1) activation. Surprisingly, we found that all AT-specific MGBLs preventing PARP-1 interaction with DNA inhibit its activation, and hence, the DNA-dependent pathway of PARP-1 activation function. These inhibitors effectively block PARP-1 activity in vivo, as it was demonstrated in a Drosophila experimental system and in human breast cancer-derived BT474 cell line.Further epigenetic effects of these indirect genotoxic carcinogens were analyzed using HeLa cell population with epigenetically suppressed GFP-reporter gene as a model. All compounds had strong GFP- reactivation effect. The obtained results confirm scarce data of previous publications on the ability of DNA minor groove ligands to influence gene transcription process. Statistically significant results of changes of DNA methylation level were detected under 5-azaC and Hoechst 33258 treatment, but it was absent after Hoechst 33342 treatment. For the rest compounds significant loss of promoter region methylation was not observed. The common epigenetic marks of transcription include histone H3 trimethylation in lysine 4 (H3K4me3) and histone H3 and H4 acetylation (acH3/acH4) on promoter regions of genes. We showed that TSA, Hoechst 33342 and DAPI treatment of HeLa-TI cells lead to increased level of histone H3 trimethylation of lysines 4 (H3K4me3), but for all that the level of histone H4 acetylation remains without significant changes. On the contrary, histone H3 acetylation level was stably increased at all samples. The modifications are typical for silent genes and decrease of their amount suggests the transcriptional reactivation. Loss of H4K20me3 mark in comparison with untreated control was demonstrated for MGBLs.ConclusionTaking together, findings of the study will lay the fundamental groundwork for the development of novel anticancer strategies and new chemotherapy effects of small molecules. The mechanism of PARP-1 inhibition by MGBLs and its epigenetic influence on silent genes, via DNA methylation and histone modifications make reasonable further study of these compounds in three prospective directions: (1) as self-acting cytotoxic agents; (2) as a component of combined chemotherapy targeting DNA repair by PARP-1, thereby facilitating DNA damage caused by other anticancer drugs; (3) as an agents reactivating epigenetically repressed genes which silencing occurs during the earliest stages of neoplasia and accumulates with progression toward malignancy

Dosing of antibiotics in patients with sepsis, including those undergoing renal replacement therapy

In critically ill patients the adequacy of starting empirical antimicrobial therapy is a determining factor of the survival of patients with sepsis This article describes the main aspects of the empirical prescription of antibiotics in patients with sepsis who are on renal replacement therapy. Changes in the pharmacokinetic and pharmacodynamic mechanisms that lead to the selection of specific dosing regimens for antibiotics are described. Information on dosing changes for current groups of antibacterial drugs is presented. The purpose of this article is to rationalize antibiotic therapy in a selected group of patients.</jats:p

HeLa TI cell-based assay as a new approach to screen for chemicals able to reactivate the expression of epigenetically silenced genes

Chemicals reactivating epigenetically silenced genes target diverse classes of enzymes, including DNMTs, HDACs, HMTs and BET protein family members. They can strongly influence the expression of genes and endogenous retroviral elements with concomitant dsRNA synthesis and massive transcription of LTRs. Chemicals reactivating gene expression may cause both beneficial effects in cancer cells and may be hazardous by promoting carcinogenesis. Among chemicals used in medicine and commerce, only a small fraction has been studied with respect to their influence on epigenetic silencing. Screening of chemicals reactivating silent genes requires adequate systems mimicking whole-genome processes. We used a HeLa TSA-inducible cell population (HeLa TI cells) obtained by retroviral infection of a GFP-containing vector followed by several rounds of cell sorting for screening purposes. Previously, the details of GFP epigenetic silencing in HeLa TI cells were thoroughly described. Herein, we show that the epigenetically repressed gene GFP is reactivated by 15 agents, including HDAC inhibitors–vorinostat, sodium butyrate, valproic acid, depsipeptide, pomiferin, and entinostat; DNMT inhibitors–decitabine, 5-azacytidine, RG108; HMT inhibitors–UNC0638, BIX01294, DZNep; a chromatin remodeler–curaxin CBL0137; and BET inhibitors–JQ-1 and JQ-35. We demonstrate that combinations of epigenetic modulators caused a significant increase in cell number with reactivated GFP compared to the individual effects of each agent. HeLa TI cells are competent to metabolize xenobiotics and possess constitutively expressed and inducible cytochrome P450 mono-oxygenases involved in xenobiotic biotransformation. Thus, HeLa TI cells may be used as an adequate test system for the extensive screening of chemicals, including those that must be metabolically activated. Studying the additional metabolic activation of xenobiotics, we surprisingly found that the rat liver S9 fraction, which has been widely used for xenobiotic activation in genotoxicity tests, reactivated epigenetically silenced genes. Applying the HeLa TI system, we show that N-nitrosodiphenylamine and N-nitrosodimethylamine reactivate epigenetically silenced genes, probably by affecting DNA methylation. © 2021 Maksimova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited