Histone Methyltransferases as a New Target for Epigenetic Action of Vorinostat.

Epigenetic genome regulation during malignant cell transformation is characterized by the aberrant methylation and acetylation of histones. Vorinostat (SAHA) is an epigenetic modulator actively used in clinical oncology. The antitumor activity of vorinostat is commonly believed to be associated with the inhibition of histone deacetylases, while the impact of this drug on histone methylation has been poorly studied. Using HeLa TI cells as a test system allowing evaluation of the effect of epigenetically active compounds from the expression of the GFP reporter gene and gene knockdown by small interfering RNAs, we showed that vorinostat not only suppressed HDAC1, but also reduced the activity of EZH2, SUV39H1, SUV39H2, and SUV420H1. The ability of vorinostat to suppress expression of EZH2, SUV39H1/2, SUV420H1 was confirmed by Western blotting. Vorinostat also downregulated expression of SUV420H2 and DOT1L enzymes. The data obtained expand our understanding of the epigenetic effects of vorinostat and demonstrate the need for a large-scale analysis of its activity toward other enzymes involved in the epigenetic genome regulation. Elucidation of the mechanism underlying the epigenetic action of vorinostat will contribute to its more proper use in the treatment of tumors with an aberrant epigenetic profile.

Insights into the Mechanism of Curaxin CBL0137 Epigenetic Activity: The Induction of DNA Demethylation and the Suppression of BET Family Proteins.

The development of malignant tumors is caused by a complex combination of genetic mutations and epigenetic alterations, the latter of which are induced by either external environmental factors or signaling disruption following genetic mutations. Some types of cancer demonstrate a significant increase in epigenetic enzymes, and targeting these epigenetic alterations represents a compelling strategy to reverse cell transcriptome to the normal state, improving chemotherapy response. Curaxin CBL0137 is a new potent anticancer drug that has been shown to activate epigenetically silenced genes. However, its detailed effects on the enzymes of the epigenetic system of transcription regulation have not been studied. Here, we report that CBL0137 inhibits the expression of DNA methyltransferase DNMT3a in HeLa TI cells, both at the level of mRNA and protein, and it decreases the level of integral DNA methylation in Ca Ski cells. For the first time, it is shown that CBL0137 decreases the level of BET family proteins, BRD2, BRD3, and BRD4, the key participants in transcription elongation, followed by the corresponding gene expression enhancement. Furthermore, we demonstrate that CBL0137 does not affect the mechanisms of histone acetylation and methylation. The ability of CBL0137 to suppress DNMT3A and BET family proteins should be taken into consideration when combined chemotherapy is applied. Our data demonstrate the potential of CBL0137 to be used in the therapy of tumors with corresponding aberrant epigenetic profiles.

Synephrine and Its Derivative Compound A: Common and Specific Biological Effects.

This review is focused on synephrine, the principal phytochemical found in bitter orange and other medicinal plants and widely used as a dietary supplement for weight loss/body fat reduction. We examine different aspects of synephrine biology, delving into its established and potential molecular targets, as well as its mechanisms of action. We present an overview of the origin, chemical composition, receptors, and pharmacological properties of synephrine, including its anti-inflammatory and anti-cancer activity in various in vitro and animal models. Additionally, we conduct a comparative analysis of the molecular targets and effects of synephrine with those of its metabolite, selective glucocorticoid receptor agonist (SEGRA) Compound A (CpdA), which shares a similar chemical structure with synephrine. SEGRAs, including CpdA, have been extensively studied as glucocorticoid receptor activators that have a better benefit/risk profile than glucocorticoids due to their reduced adverse effects. We discuss the potential of synephrine usage as a template for the synthesis of new generation of non-steroidal SEGRAs. The review also provides insights into the safe pharmacological profile of synephrine.

Perspectives of Cell Sensitivity/Resistance Assay in Soft Tissue Sarcomas Chemotherapy.

Treatment of highly malignant soft tissue sarcomas (STSs) requires multicomponent therapy including surgery, radiotherapy, and chemotherapy. Despite the advancements in targeted cancer therapies, cytostatic drug combinations remain the gold standard for STS chemotherapy. The lack of algorithms for personalized selection of STS chemotherapy leads to unhelpful treatment of chemoresistant tumors, causing severe side effects in patients. The goal of our study is to assess the applicability of in vitro chemosensitivity/resistance assays (CSRAs) in predicting STS chemoresistance. Primary cell cultures were obtained from 148 surgery samples using enzymatic and mechanical disaggregation. CSRA was performed using resazurin-based metabolic activity measurement in cells cultured with doxorubicin, ifosfamide, their combination and docetaxel, gemcitabine, and also their combination for 7 days. Both the clinical data of patients and the CSRA results demonstrated a higher resistance of some cancer histotypes to specific drugs and their combinations. The correlation between the CSRA results for doxorubicin and ifosfamide and clinical responses to the combination chemotherapy with these drugs was demonstrated via Spearman rank order correlation. Statistically significant differences in recurrence-free survival were also shown for the groups of patients formed, according to the CSRA results. Thus, CSRAs may help both practicing physicians to avoid harmful and useless treatment, and researchers to study new resistance markers and to develop new STS drugs.

Effects of G-Quadruplex-Binding Plant Secondary Metabolites on c-MYC Expression.

Guanine-rich DNA sequences tending to adopt noncanonical G-quadruplex (G4) structures are over-represented in promoter regions of oncogenes. Ligands recognizing G4 were shown to stabilize these DNA structures and drive their formation regulating expression of corresponding genes. We studied the interaction of several plant secondary metabolites (PSMs) with G4s and their effects on gene expression in a cellular context. The binding of PSMs with G4s formed by the sequences of well-studied oncogene promoters and telomeric repeats was evaluated using a fluorescent indicator displacement assay. c-MYC G4 folding topology and thermal stability, as well as the PMS influence on these parameters, were demonstrated by UV-spectroscopy and circular dichroism. The effects of promising PSMs on c-MYC expression were assessed using luciferase reporter assay and qPR-PCR in cancer and immortalized cultured cells. The ability of PMS to multi-targeting cell signaling pathways was analyzed by the pathway-focused gene expression profiling with qRT-PCR. The multi-target activity of a number of PSMs was demonstrated by their interaction with a set of G4s mimicking those formed in the human genome. We have shown a direct G4-mediated down regulation of c-MYC expression by sanguinarine, quercetin, kaempferol, and thymoquinone; these effects being modulated by PSM's indirect influence via cell signaling pathways.

Nutritional Sensor REDD1 in Cancer and Inflammation: Friend or Foe?

Regulated in Development and DNA Damage Response 1 (REDD1)/DNA Damage-Induced Transcript 4 (DDIT4) is an immediate early response gene activated by different stress conditions, including growth factor depletion, hypoxia, DNA damage, and stress hormones, i.e., glucocorticoids. The most known functions of REDD1 are the inhibition of proliferative signaling and the regulation of metabolism via the repression of the central regulator of these processes, the mammalian target of rapamycin (mTOR). The involvement of REDD1 in cell growth, apoptosis, metabolism, and oxidative stress implies its role in various pathological conditions, including cancer and inflammatory diseases. Recently, REDD1 was identified as one of the central genes mechanistically involved in undesirable atrophic effects induced by chronic topical and systemic glucocorticoids widely used for the treatment of blood cancer and inflammatory diseases. In this review, we discuss the role of REDD1 in the regulation of cell signaling and processes in normal and cancer cells, its involvement in the pathogenesis of different diseases, and the approach to safer glucocorticoid receptor (GR)-targeted therapies via a combination of glucocorticoids and REDD1 inhibitors to decrease the adverse atrophogenic effects of these steroids.

Analysis of Multiple Drug Resistance Mechanism in Different Types of Soft Tissue Sarcomas: Assessment of the Expression of ABC-Transporters, MVP, YB-1, and Analysis of Their Correlation with Chemosensitivity of Cancer Cells.

Chemotherapy of soft tissue sarcomas (STS) is restricted by low chemosensitivity and multiple drug resistance (MDR). The purpose of our study was the analysis of MDR mechanism in different types of STS. We assessed the expression of ABC-transporters, MVP, YB-1, and analyzed their correlation with chemosensitivity of cancer cells. STS specimens were obtained from 70 patients without metastatic disease (2018-2020). Expression level of MDR-associated genes was estimated by qRT-PCR and cytofluorimetry. Mutations in ABC-transporter genes were captured by exome sequencing. Chemosensitivity (SI) of STS to doxorubicin (Dox), ifosfamide (Ifo), gemcitabine (Gem), and docetaxel (Doc) was analyzed in vitro. We found strong correlation in ABCB1, ABCC1, and ABCG2 expression. We demonstrated strong negative correlations in ABCB1 and ABCG2 expression with SI (Doc) and SI (Doc + Gem), and positive correlation of MVP expression with SI (Doc) and SI (Doc + Gem) in undifferentiated pleomorphic sarcoma. Pgp expression was shown in 5 out of 44 STS samples with prevalence of synovial sarcoma relapses and it is strongly correlated with SI (Gem). Mutations in MDR-associated genes were rarely found. Overall, STS demonstrated high heterogeneity in chemosensitivity that makes reasonable in vitro chemosensitivity testing to improve personalized STS therapy, and classic ABC-transporters are not obviously involved in MDR appearance.

Synthesis of Novel Lipophilic Polyamines via Ugi Reaction and Evaluation of Their Anticancer Activity.

Natural polyamines (PAs) are involved in the processes of proliferation and differentiation of cancer cells. Lipophilic synthetic polyamines (LPAs) induce the cell death of various cancer cell lines. In the current paper, we have demonstrated a new method for synthesis of LPAs via the multicomponent Ugi reaction and subsequent reduction of amide groups by PhSiH3. The anticancer activity of the obtained compounds was evaluated in the A-549, MCF7, and HCT116 cancer cell lines. For the first time, it was shown that the anticancer activity of LPAs with piperazine fragments is comparable with that of aliphatic LPAs. The presence of a diglyceride fragment in the structure of LPAs appears to be a key factor for the manifestation of high anticancer activity. The findings of the study strongly support further research in the field of LPAs and their derivatives.

AML-Associated Mutations in DNA Methyltransferase DNMT3A.

In mammals, DNA methylation is an essential epigenetic modification necessary for the maintenance of genome stability, regulation of gene expression, and other processes. Carcinogenesis is accompanied by multiple changes in the DNA methylation pattern and DNA methyltransferase (DNMT) genes; these changes are often associated with poor disease prognosis. Human DNA methyltransferase DNMT3A is responsible for de novo DNA methylation. Missense mutations in the DNMT3A gene occur frequently at the early stages of tumor development and are often observed in hematologic malignances, especially in acute myeloid leukemia (AML), with a prevalence of the R882H mutation. This mutation is the only one that has been extensively studied using both model DNA substrates and cancer cell lines. Biochemical characterization of other DNMT3A mutants is necessary to assess their potential effects on the DNMT3A functioning. In this review, we describe DNMT3A mutations identified in AML with special emphasis on the missense mutations in the DNMT3A catalytic domain. The impact of R882H and less common missense mutations on the DNMT3A activity toward model DNA substrates and in cancer cell lines is discussed together with the underlying molecular mechanisms. Understanding general features of these mechanisms will be useful for further development of novel approaches for early diagnostics of hematologic diseases and personalized cancer therapy.

Molecular Mechanisms of Anticancer Activity of N-Glycosides of Indolocarbazoles LCS-1208 and LCS-1269.

Novel indolocarbazole derivatives named LCS were synthesized by our research group. Two of them were selected as the most active anticancer agents in vivo. We studied the mechanisms of anticancer activity in accordance with the previously described effects of indolocarbazoles. Cytotoxicity was estimated by MTT assay. We analyzed LCS-DNA interactions by circular dichroism in cholesteric liquid crystals and fluorescent indicator displacement assay. The effect on the activity of topoisomerases I and II was studied by DNA relaxation assay. Expression of interferon signaling target genes was estimated by RT-PCR. Chromatin remodeling was analyzed-the effect on histone H1 localization and reactivation of epigenetically silenced genes. LCS-induced change in the expression of a wide gene set was counted by means of PCR array. Our study revealed the cytotoxic activity of the compounds against 11 cancer cell lines and it was higher than in immortalized cells. Both compounds bind DNA; binding constants were estimated-LCS-1208 demonstrated higher affinity than LCS-1269; it was shown that LCS-1208 intercalates into DNA that is typical for rebeccamycin derivatives. LCS-1208 also inhibits topoisomerases I and IIα. Being a strong intercalator and topoisomerase inhibitor, LCS-1208 upregulates the expression of interferon-induced genes. In view of LCSs binding to DNA we analyzed their influence on chromatin stability and revealed that LCS-1269 displaces histone H1. Our analysis of chromatin remodeling also included a wide set of epigenetic experiments in which LCS-1269 demonstrated complex epigenetic activity. Finally, we revealed that the antitumor effect of the compounds is based not only on binding to DNA and chromatin remodeling but also on alternative mechanisms. Both compounds induce expression changes in genes involved in neoplastic transformation and target genes of the signaling pathways in cancer cells. Despite of being structurally similar, each compound has unique biological activities. The effects of LCS-1208 are associated with intercalation. The mechanisms of LCS-1269 include influence on higher levels such as chromatin remodeling and epigenetic effects.

Novel anticancer drug curaxin CBL0137 impairs DNA methylation by eukaryotic DNA methyltransferase Dnmt3a.

Novel DNA intercalating anticancer drug curaxin CBL0137 significantly inhibited in vitro DNA methylation by eukaryotic DNA methyltransferase Dnmt3a catalytic domain (Dnmt3a-CD) at low micromolar concentrations (IC50 3-9 µM). CBL0137 reduced the binding affinity of Dnmt3a-CD to its DNA target, causing up to four-fold increase in the Kd of the enzyme/DNA complex. Binding of CBL0137 to Dnmt3a-CD was not observed. The observed decrease in methylation activity of Dnmt3a-CD in the presence of CBL0137 can be explained by curaxin's ability to intercalate into DNA.

Toward G-Quadruplex-Based Anticancer Agents: Biophysical and Biological Studies of Novel AS1411 Derivatives.

Certain G-quadruplex forming guanine-rich oligonucleotides (GROs), including AS1411, are endowed with cancer-selective antiproliferative activity. They are known to bind to nucleolin protein, resulting in the inhibition of nucleolin-mediated phenomena. However, multiple nucleolin-independent biological effects of GROs have also been reported, allowing them to be considered promising candidates for multi-targeted cancer therapy. Herein, with the aim of optimizing AS1411 structural features to find GROs with improved anticancer properties, we have studied a small library of AS1411 derivatives differing in the sequence length and base composition. The AS1411 derivatives were characterized by using circular dichroism and nuclear magnetic resonance spectroscopies and then investigated for their enzymatic resistance in serum and nuclear extract, as well as for their ability to bind nucleolin, inhibit topoisomerase I, and affect the viability of MCF-7 human breast adenocarcinoma cells. All derivatives showed higher thermal stability and inhibitory effect against topoisomerase I than AS1411. In addition, most of them showed an improved antiproliferative activity on MCF-7 cells compared to AS1411 despite a weaker binding to nucleolin. Our results support the hypothesis that the antiproliferative properties of GROs are due to multi-targeted effects.

Responses of DNA Mismatch Repair Proteins to a Stable G-Quadruplex Embedded into a DNA Duplex Structure.

DNA mismatch repair (MMR) plays a crucial role in the maintenance of genomic stability. The main MMR protein, MutS, was recently shown to recognize the G-quadruplex (G4) DNA structures, which, along with regulatory functions, have a negative impact on genome integrity. Here, we studied the effect of G4 on the DNA-binding activity of MutS from Rhodobacter sphaeroides (methyl-independent MMR) in comparison with MutS from Escherichia coli (methyl-directed MMR) and evaluated the influence of a G4 on the functioning of other proteins involved in the initial steps of MMR. For this purpose, a new DNA construct was designed containing a biologically relevant intramolecular stable G4 structure flanked by double-stranded regions with the set of DNA sites required for MMR initiation. The secondary structure of this model was examined using NMR spectroscopy, chemical probing, fluorescent indicators, circular dichroism, and UV spectroscopy. The results unambiguously showed that the d(GGGT)4 motif, when embedded in a double-stranded context, adopts a G4 structure of a parallel topology. Despite strong binding affinities of MutS and MutL for a G4, the latter is not recognized by E. coli MMR as a signal for repair, but does not prevent MMR processing when a G4 and G/T mismatch are in close proximity.

FACT is a sensor of DNA torsional stress in eukaryotic cells.

Transitions of B-DNA to alternative DNA structures (ADS) can be triggered by negative torsional strain, which occurs during replication and transcription, and may lead to genomic instability. However, how ADS are recognized in cells is unclear. We found that the binding of candidate anticancer drug, curaxin, to cellular DNA results in uncoiling of nucleosomal DNA, accumulation of negative supercoiling and conversion of multiple regions of genomic DNA into left-handed Z-form. Histone chaperone FACT binds rapidly to the same regions via the SSRP1 subunit in curaxin-treated cells. In vitro binding of purified SSRP1 or its isolated CID domain to a methylated DNA fragment containing alternating purine/pyrimidines, which is prone to Z-DNA transition, is much stronger than to other types of DNA. We propose that FACT can recognize and bind Z-DNA or DNA in transition from a B to Z form. Binding of FACT to these genomic regions triggers a p53 response. Furthermore, FACT has been shown to bind to other types of ADS through a different structural domain, which also leads to p53 activation. Thus, we propose that FACT acts as a sensor of ADS formation in cells. Recognition of ADS by FACT followed by a p53 response may explain the role of FACT in DNA damage prevention.

Transcription blockage by stable H-DNA analogs in vitro.

DNA sequences that can form unusual secondary structures are implicated in regulating gene expression and causing genomic instability. H-palindromes are an important class of such DNA sequences that can form an intramolecular triplex structure, H-DNA. Within an H-palindrome, the H-DNA and canonical B-DNA are in a dynamic equilibrium that shifts toward H-DNA with increased negative supercoiling. The interplay between H- and B-DNA and the fact that the process of transcription affects supercoiling makes it difficult to elucidate the effects of H-DNA upon transcription. We constructed a stable structural analog of H-DNA that cannot flip into B-DNA, and studied the effects of this structure on transcription by T7 RNA polymerase in vitro. We found multiple transcription blockage sites adjacent to and within sequences engaged in this triplex structure. Triplex-mediated transcription blockage varied significantly with changes in ambient conditions: it was exacerbated in the presence of Mn(2+) or by increased concentrations of K(+) and Li(+). Analysis of the detailed pattern of the blockage suggests that RNA polymerase is sterically hindered by H-DNA and has difficulties in unwinding triplex DNA. The implications of these findings for the biological roles of triple-stranded DNA structures are discussed.

Genetic, Epigenetic and Transcriptome Alterations in Liposarcoma for Target Therapy Selection.

Liposarcoma (LPS) is one of the most common adult soft-tissue sarcomas (STS), characterized by a high diversity of histopathological features as well as to a lesser extent by a spectrum of molecular abnormalities. Current targeted therapies for STS do not include a wide range of drugs and surgical resection is the mainstay of treatment for localized disease in all subtypes, while many LPS patients initially present with or ultimately progress to advanced disease that is either unresectable, metastatic or both. The understanding of the molecular characteristics of liposarcoma subtypes is becoming an important option for the detection of new potential targets and development novel, biology-driven therapies for this disease. Innovative therapies have been introduced and they are currently part of preclinical and clinical studies. In this review, we provide an analysis of the molecular genetics of liposarcoma followed by a discussion of the specific epigenetic changes in these malignancies. Then, we summarize the peculiarities of the key signaling cascades involved in the pathogenesis of the disease and possible novel therapeutic approaches based on a better understanding of subtype-specific disease biology. Although heterogeneity in liposarcoma genetics and phenotype as well as the associated development of resistance to therapy make difficult the introduction of novel therapeutic targets into the clinic, recently a number of targeted therapy drugs were proposed for LPS treatment. The most promising results were shown for CDK4/6 and MDM2 inhibitors as well as for the multi-kinase inhibitors anlotinib and sunitinib.

Secreted cyclophilin A is non-genotoxic but acts as a tumor promoter.

Chronic inflammation is associated with malignant transformation and creates the microenvironment for tumor progression. Cyclophilin A (CypA) is one of the major pro-inflammatory mediators that accumulates and persists in the site of inflammation in high doses over time. According to multiomics analyses of transformed cells, CypA is widely recognized as a pro-oncogenic factor. Vast experimental data define the functions of intracellular CypA in carcinogenesis, but findings on the role of its secreted form in tumor formation and progression are scarce. In the studies here, we exploit short-term in vitro and in vivo tests to directly evaluate the mutagenic, recombinogenic, and blastomogenic effects, as well as the promoter activity of recombinant human CypA (rhCypA), an analogue of secreted CypA. Our findings showed that rhCypA had no genotoxicity and, thus, was neither involved in nor influenced the initiation stage of carcinogenesis. At high doses, rhCypA could disrupt gap junctions in rat liver epithelial IAR-2 cells in vitro by decreasing the expression of connexins 26 and 43 in these cells and inhibit A549 cell adhesion. These data suggested that rhCypA could contribute to epithelial-mesenchymal transition in malignant cells. The research presented here elucidated the role of secreted CypA in carcinogenesis, revealing that it is not a tumor initiator but can act as a tumor promoter at high concentrations.

Targeting Features of Curaxin CBL0137 on Hematological Malignancies In Vitro and In Vivo.

The anticancer activity of Curaxin CBL0137, a DNA-binding small molecule with chromatin remodulating effect, has been demonstrated in different cancers. Herein, a comparative evaluation of CBL0137 activity was performed in respect to acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia and multiple myeloma (MM) cultured in vitro. MTT assay showed AML and MM higher sensitivity to CBL0137's cytostatic effect comparatively to other hematological malignancy cells. Flow cytometry cell cycle analysis revealed an increase in subG1 and G2/M populations after CBL0137 cell treatment, but the prevalent type of arrest varied. Apoptosis activation by CBL0137 measured by Annexin-V/PI dual staining was more active in AML and MM cells. RT2 PCR array showed that changes caused by CBL0137 in signaling pathways involved in cancer pathogenesis were more intensive in AML and MM cells. On the murine model of AML WEHI-3, CBL0137 showed significant anticancer effects in vivo, which were evaluated by corresponding changes in spleen and liver. Thus, more pronounced anticancer effects of CBL0137 in vitro were observed in respect to AML and MM. Experiments in vivo also indicated the perspective of CBL0137 use for AML treatment. This in accordance with the frontline treatment approach in AML using epigenetic drugs.