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Myeloperoxidase (MPO) is found almost exclusively in granulocytes and immature myeloid cells. It plays a key role in the innate immune system, catalysing the formation of reactive oxygen species that are important in anti-microbial action, but MPO also oxidatively transforms the topoisomerase II (TOP2) poison etoposide to chemical forms that have elevated DNA damaging properties. TOP2 poisons such as etoposide are widely used anti-cancer drugs, but they are linked to cases of secondary acute myeloid leukaemias through a mechanism that involves DNA damage and presumably erroneous repair leading to leukaemogenic chromosome translocations. This leads to the possibility that myeloperoxidase inhibitors could reduce the rate of therapy-related leukaemia by protecting haematopoietic cells from TOP2 poison-mediated genotoxic damage while preserving the anti-cancer efficacy of the treatment. We show here that myeloperoxidase inhibition reduces etoposide-induced TOP2B-DNA covalent complexes and resulting DNA double-strand break formation in primary ex vivo expanded CD34 + progenitor cells and unfractionated bone marrow mononuclear cells. Since MPO inhibitors are currently being developed as anti-inflammatory agents this raises the possibility that repurposing of these potential new drugs could provide a means of suppressing secondary acute myeloid leukaemias associated with therapies containing TOP2 poisons., (© 2024 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Development of effective strategies to manage the inevitable acquired resistance to osimertinib, a third-generation EGFR inhibitor for the treatment of EGFR-mutant (EGFRm) non-small cell lung cancer (NSCLC), is urgently needed. This study reports that DNA topoisomerase II (Topo II) inhibitors, doxorubicin and etoposide, synergistically decreased cell survival, with enhanced induction of DNA damage and apoptosis in osimertinib-resistant cells; suppressed the growth of osimertinib-resistant tumors; and delayed the emergence of osimertinib-acquired resistance. Mechanistically, osimertinib decreased Topo IIα levels in EGFRm NSCLC cells by facilitating FBXW7-mediated proteasomal degradation, resulting in induction of DNA damage; these effects were lost in osimertinib-resistant cell lines that possess elevated levels of Topo IIα. Increased Topo IIα levels were also detected in the majority of tissue samples from patients with NSCLC after relapse from EGFR tyrosine kinase inhibitor treatment. Enforced expression of an ectopic TOP2A gene in sensitive EGFRm NSCLC cells conferred resistance to osimertinib, whereas knockdown of TOP2A in osimertinib-resistant cell lines restored their susceptibility to osimertinib-induced DNA damage and apoptosis. Together, these results reveal an essential role of Topo IIα inhibition in mediating the therapeutic efficacy of osimertinib against EGFRm NSCLC, providing scientific rationale for targeting Topo II to manage acquired resistance to osimertinib.

T-cell acute lymphoblastic leukemia (T-ALL) is a serious hematological tumor derived from early T-cell progenitors, which is extremely resistant to chemotherapy. Classically, doxorubicin (DOX) is an effective first-line drug for the treatment of T-ALL; however, DOX resistance limits its clinical effect. The DEK proto-oncogene (DEK) has been involved in neoplasms but remains unexplored in T-ALL. We silenced DEK on Jurkat cells and detected cell proliferation with cell counting and colony formation assay. Then, we detected DEK's drug sensitivity to DOX with CCK-8, cell cycle, and apoptosis with DOX treatment. Western blot analysis was performed to determine protein expression of apoptosis and cell cycle-related genes, including BCL2L1, caspase-3, and cyclin-dependent kinases (CDK). Finally, the tumorigenic ability of DEK was analyzed using a BALB/C nude mouse model. In this study, DEK was highly expressed in Jurkat cells. Inhibition of DEK can lead to decreased cell proliferation and proportion of S-phase cells in the cell cycle and more cell apoptosis, and the effect is more obvious after DOX treatment. Western blot results showed that DOX treatment leads to cell cycle arrest, reduction of cyclin-dependent kinase 6 (CDK6) protein, accumulation of CDKN1A protein, and DOX-induced apoptosis accompanied by reductions in protein levels of BCL2L1, as well as increases in protein level of caspase-3. Furthermore, DEK-silenced Jurkat cells generated a significantly smaller tumor mass in mice. Our study found that DEK is a novel, potential therapeutic target for overcoming DOX resistance in T-ALL., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2022 Xiaoxue Tian et al.)

A new series of fifty-four 2-phenol-4-aryl-6-hydroxyphenylpyridines containing fluorophenyl, trifluoromethylphenyl, and trifluoromethoxy phenyl groups were synthesized and tested for topoisomerase IIα inhibitory and antiproliferative activity against different cancer cell lines in an attempt to look into topoisomerase IIα-targeted prospective anticancer agents to counter the limitations of available treatment options. When compared to positive controls, several compounds 11-12, 37, 50, and 51 showed high antiproliferative activity, while several 4-fluorophenyl substituted compounds 13-14 and 18 showed strong topoisomerase IIα inhibition. Surprisingly, most of the compounds had a significant antiproliferative effect on the HCT15 colorectal adenocarcinoma and T47D breast cancer cell lines. Moreover, compound 12 with para-fluorophenyl at the 4-position and meta-phenolic groups at the 2- and 6-positions inhibited proliferating HeLa cervix adenocarcinoma cells with an IC 50 value of 1.28 μM. Based on biological results, the structure-activity relationships of the synthesized derivatives emphasized the significance of 4-trifluoromethoxyphenyl groups for strong antiproliferative activity and 4-fluorophenyl groups for strong topo IIα inhibition. Furthermore, meta- and para-phenolic groups at the 2- and 4-positions are favorable for strong topo IIα inhibitory and antiproliferative activity. The research findings provide insight into the effect of different fluorine functionalities in the discovery of novel topoisomerase IIα-targeted anticancer agents., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Ku70 protein and topoisomerase IIα (Topo IIα) are promising targets of anticancer drugs, which play critical roles in DNA repair and replication processes. Three platinum(II) complexes, [PtCl(NH 3 ) 2 (9-(pyridin-2-ylmethyl)-9 H -carbazole)]NO 3 (OPPC), [PtCl(NH 3 ) 2 (9-(pyridin-3-ylmethyl)-9 H -carbazole)]NO 3 (MPPC), and [PtCl(NH 3 ) 2 (9-(pyridin-4-ylmethyl)-9 H -carbazole)]NO 3 (PPPC), were designed as inhibitors of Ku70 and Topo IIα. Their antitumor activity and inhibitory efficacy on Ku70 and Topo IIα were investigated on cellular and molecular levels. OPPC exhibited high antiproliferative activity against various cancer cell lines, with acute toxicity to mice being lower than that of cisplatin. Moreover, OPPC could enter cancer cells effectively and cause DNA damage, which was evidenced by the enhanced expression of γ-H2AX, Chk1/2 phosphorylation, p53 and cell cycle arrest. OPPC also downregulated the DNA damage repair protein Ku70 and inhibited the formation of Ku70 foci-the central points or loci of Ku70, which would suppress DNA repair and induce a nonhomologous end joining response in cancer cells. More importantly, these complexes showed inhibition towards Topo IIα; in particular, OPPC was more effective than MPPC and PPPC. In the Topo IIα knockdown cells, Ku70 and Topo IIα were directly associated with the DNA damage and apoptotic response. The molecular docking provided detailed structural insights into the interactions of the complexes with Topo IIα. This study demonstrates that the cytotoxicity of these complexes is associated with the DNA damage and repair pathways mediated by Ku70 and Topo IIα; OPPC is an effective inhibitor of Ku70 and Topo IIα and restrains cancer cells via a mechanism utterly distinct from that of cisplatin.

Aconitine linoleate (1) is a lipo-diterpenoid alkaloid, isolated from Aconitum sinchiangense W. T. Wang. The study aimed at investigating the anti-proliferative efficacy and the underlying mechanisms of 1 against MCF-7 and MCF-7/ADR cells, as well as obvious the safety evaluation in vivo. The cytotoxic activities of 1 were measured in vitro. Also, we investigated the latent mechanism of 1 by cell cycle analysis in MCF-7/ADR cells and topo I and topo IIα inhibition assay. Molecular docking is done by Discovery Studio 3.5 and Autodock vina 1.1.2. Finally, the acute toxicity of 1 was detected on mice. 1 exhibited significant antitumor activity against both MCF-7 and MCF-7/ADR cells, with IC 50 values of 7.58 and 7.02 μM, which is 2.38 times and 5.05 times more active, respectively than etoposide in both cell lines, and being 9.63 times more active than Adriamycin in MCF-7/ADR cell lines. The molecular docking and the topo inhibition test found that it is a selective inhibitor of topoisomerase IIα. Moreover, activation of the damage response pathway of the DNA leads to cell cycle arrest at the G 0 G 1 phase. Furthermore, the in vivo acute toxicity of 1 in mice displayed lower toxicity than aconitine, with LD 50 of 2.2 × 10 5  nmol/kg and only slight pathological changes in liver and lung tissue, 489 times safer than aconitine. In conclusion, compared with aconitine, 1 has more significant anti-proliferative activity against MCF-7 and MCF-7/ADR cells and greatly reduces in vivo toxicity, which suggests this kind of lipo-alkaloids is powerful and promising antitumor compounds for breast cancer., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A series of fluorinated and hydroxylated 2,4-diphenyl indenopyridinols were designed and synthesized using l-proline-catalyzed and microwave-assisted synthetic methods for the development of new anticancer agents. Adriamycin and etoposide were used as reference compounds for the evaluation of topo IIα inhibitory and anti-proliferative activity of the synthesized compounds. Exploring the structure-activity relationships of 36 prepared compounds and biological results, most of the compounds with ortho- and para-fluorophenyl at 4-position of indenopyridinol ring displayed strong topo IIα inhibition. In addition, the majority of the ortho- and meta-fluorophenyl substituted compounds 1-24 displayed strong anti-proliferative activity against DU145 prostate cancer cell line compared to the positive controls. Interestingly, compound 4 possessing ortho-phenolic and ortho-fluorophenyl group at 2- and 4-position, respectively of the central pyridine ring showed high anti-proliferative activity (IC 50  = 0.82 μM) against T47D human breast cancer cell line, while para-phenolic and para-fluorophenyl substituted compound 36 exhibited potent topo IIα inhibitory activity with 94.7% and 88.6% inhibition at 100 μM and 20 μM concentration, respectively. A systematic comparison between the results of this study and the previous study indicated that minor changes in the position of functional groups in the structure affect the topo IIα inhibitory activity and anti-proliferative activity of the compounds. The findings from this study will provide valuable information to the researchers working on the medicinal chemistry of topoisomerase IIα-targeted anticancer agents., (Copyright © 2021 Elsevier Inc. All rights reserved.)

SARS-CoV-2 nucleocapsid (N) protein undergoes RNA-induced phase separation (LLPS) and sequesters the host key stress granule (SG) proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and 2 (G3BP1 and G3BP2) to inhibit SG formation. This will allow viral packaging and propagation in host cells. Based on a genomic-guided meta-analysis, here we identify upstream regulatory elements modulating the expression of G3BP1 and G3BP2 (collectively called G3BP1/2). Using this strategy, we have identified FOXA1, YY1, SYK, E2F-1, and TGFBR2 as activators and SIN3A, SRF, and AKT-1 as repressors of G3BP1/2 genes. Panels of the activators and repressors were then used to identify drugs that change their gene expression signatures. Two drugs, imatinib, and decitabine have been identified as putative modulators of G3BP1/2 genes and their regulators, suggesting their role as COVID-19 mitigation agents. Molecular docking analysis suggests that both drugs bind to G3BP1/2 with a much higher affinity than the SARS-CoV-2 N protein. This study reports imatinib and decitabine as candidate drugs against N protein and G3BP1/2 protein., (Copyright © 2021 Elsevier B.V. All rights reserved.)

DNA topoisomerases II (TOP2) are peculiar enzymes (TOP2α and TOP2β) that modulate the conformation of DNA by momentarily breaking double-stranded DNA to allow another strand to pass through, and then rejoins the DNA phosphodiester backbone. TOP2α and TOP2β play vital roles in nearly all events involving DNA metabolism, including DNA transcription, replication, repair, and chromatin remodeling. Beyond these vital functions, TOP2 enzymes are therapeutic targets for various anticancer drugs, termed TOP2 poisons, such as teniposide, etoposide, and doxorubicin. These drugs exert their antitumor activity by inhibiting the activity of TOP2-DNA cleavage complexes (TOP2ccs) containing DNA double-strand breaks (DSBs), subsequently leading to the degradation of TOP2 by the 26S proteasome, thereby exposing the DSBs and eliciting a DNA damage response. Failure of the DSBs to be appropriately repaired leads to genomic instability. Due to this mechanism, patients treated with TOP2-based drugs have a high incidence of secondary malignancies and cardiotoxicity. While the cytotoxicity associated with TOP2 poisons appears to be TOP2α-dependent, the DNA sequence rearrangements and formation of DSBs appear to be mediated primarily through TOP2β inhibition, likely due to the differential degradation patterns of TOP2α and TOP2β. Research over the past few decades has shown that under various conditions, the ubiquitin-proteasome system (UPS) and the SUMOylation pathway are primarily responsible for regulating the stability and activity of TOP2 and are therefore critical regulators of the therapeutic effect of TOP2-targeting drugs. In this review, we summarize the current progress on the regulation of TOP2α and TOP2β by ubiquitination and SUMOylation. By fully elucidating the basic biology of these essential and complex molecular mechanisms, better strategies may be developed to improve the therapeutic efficacy of TOP2 poisons and minimize the risks of therapy-related secondary malignancy., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Novel mansonone F derivative MSN54 (9-bromo-2,3-diethylbenzo[de]chromene-7,8-dione) exhibited significant cytotoxicity against twelve human tumor cell lines in vitro, with particularly strong potency against HL-60/MX2 cell line resistant to Topo II poisons. MSN54 was found to have IC 50 of 0.69 and 1.43 µM against HL-60 and HL-60/MX2 cells, respectively. The resistance index is 10 times lower than that of the positive control VP-16 (etoposide). Various biological assays confirmed that MSN54 acted as a Topo IIα specific non-intercalative catalytic inhibitor. Furthermore, MSN54 exhibited good antitumor efficacy and low toxicity at a dose of 5 mg/kg in A549 tumor xenograft models. Thus, compound MSN54 is a promising candidate for the development of novel antitumor agents., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A series of triphenylethylene-naphthalimide (TPE-naph) conjugates was synthesized by a molecular hybridization technique, and their anticancer activity was evaluated in vitro on 60 human cancer cell lines through their cytotoxicity. The ratios of E and Z isomers were determined on the basis of HPLC methodology and NMR spectroscopy. The structure-activity relationship for anticancer activity was deduced on the basis of the nature and bulkiness of the amine attached to the C-4 position of the naphthalene ring. Experimental and molecular modeling studies of the most active TPE-naph conjugate bearing a morpholinyl group showed that it was able to inhibit topoisomerase-II (TOPO-II) as a possible intracellular target. Moreover, the transportation behavior of TPE-naph conjugate towards human serum albumin (HSA) indicated efficient binding affinity. The steady-state and time-dependent fluorescent results suggested that this conjugate quenched HSA significantly through static as well as dynamic quenching. Thus, this report discloses the scope of triphenylethylene-naphthalimide (TPE-naph) conjugates as efficient anticancer agents., (© 2021 Wiley-VCH GmbH.)

The objective of this study was to discover potential topoisomerase (topo) targeting anticancer agents. Novel series of hydroxylated and halogenated(-F, -Cl, and -CF 3 ) 2,4-diaryl benzofuro[3,2-b]pyridin-7-ols were systematically designed and synthesized by faster, economic, and environmentally friendly l -proline catalyzed and microwave-assisted one pot reaction method. The synthesized compounds were assessed for topo I and IIα inhibitory and anti-proliferative activities. The in vitroevaluation displayed that most of the compounds have selective topo IIα inhibitoryactivity as well as selectivity towards T47D human cancer cell line. Structure-activity relationship study suggested that the introduction of additional hydroxyl functionality at 7-positon of benzofuro[3,2-b]pyridine skeleton is crucial for selective topo IIα inhibitory activity. Placement of phenolic moiety on the 4-position of the tricyclic system imparts better topo IIα inhibitory and anti-proliferative activity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The detection of intracellular nucleic acids is a fundamental mechanism of host defense against infections. The dysregulated nucleic acid sensing, however, is a major cause for a number of autoimmune diseases. In this study, we report that GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is critical for both intracellular DNA- and RNA-induced immune responses. We found that in both human and mouse cells, the deletion of G3BP1 led to the dampened cGAS activation by DNA and the insufficient binding of RNA by RIG-I. We further found that resveratrol (RSVL), a natural compound found in grape skin, suppressed both intracellular DNA- and RNA-induced type I IFN production through inhibiting G3BP1. Importantly, using experimental mouse models for Aicardi-Goutières syndrome, an autoimmune disorder found in humans, we demonstrated that RSVL effectively alleviated intracellular nucleic acid-stimulated autoimmune responses. Thus, our study demonstrated a broader role of G3BP1 in sensing different kinds of intracellular nucleic acids and presented RSVL as a potential treatment for autoimmune conditions caused by dysregulated nucleic acid sensing., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Accumulation of topological stress in the form of DNA supercoiling is inherent to the advance of RNA polymerase II (Pol II) and needs to be resolved by DNA topoisomerases to sustain productive transcriptional elongation. Topoisomerases are therefore considered positive facilitators of transcription. Here, we show that, in contrast to this general assumption, human topoisomerase IIα (TOP2A) activity at promoters represses transcription of immediate early genes such as c-FOS, maintaining them under basal repressed conditions. Thus, TOP2A inhibition creates a particular topological context that results in rapid release from promoter-proximal pausing and transcriptional upregulation, which mimics the typical bursting behavior of these genes in response to physiological stimulus. We therefore describe the control of promoter-proximal pausing by TOP2A as a layer for the regulation of gene expression, which can act as a molecular switch to rapidly activate transcription, possibly by regulating the accumulation of DNA supercoiling at promoter regions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

A cannabinoid anticancer para-quinone, HU-331, which was synthesized by our group five decades ago, was shown to have very high efficacy against human cancer cell lines in-vitro and against in-vivo grafts of human tumors in nude mice. The main mechanism was topoisomerase IIα catalytic inhibition. Later, several groups synthesized related compounds. In the present presentation, we review the publications on compounds synthesized on the basis of HU-331, summarize their published activities and mechanisms of action and report the synthesis and action of novel quinones, thus expanding the structure-activity relationship in these series.

Alternative lengthening of telomeres (ALT) is mediated by break-induced replication (BIR), but how BIR is regulated at telomeres is poorly understood. Here, we show that telomeric BIR is a self-perpetuating process. By tethering PML-IV to telomeres, we induced telomere clustering in ALT-associated PML bodies (APBs) and a POLD3-dependent ATR response at telomeres, showing that BIR generates replication stress. Ablation of BLM helicase activity in APBs abolishes telomere synthesis but causes multiple chromosome bridges between telomeres, revealing a function of BLM in processing inter-telomere BIR intermediates. Interestingly, the accumulation of BLM in APBs requires its own helicase activity and POLD3, suggesting that BIR triggers a feedforward loop to further recruit BLM. Enhancing BIR induces PIAS4-mediated TRF2 SUMOylation, and PIAS4 loss deprives APBs of repair proteins and compromises ALT telomere synthesis. Thus, a BLM-driven and PIAS4-mediated feedforward loop operates in APBs to perpetuate BIR, providing a critical mechanism to extend ALT telomeres., Competing Interests: Declaration of interests The authors declare no competing interests. L.Z. is a member of the Advisory Board of Molecular Cell., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A549 human lung carcinoma cell lines were treated with a series of new drugs with both tacrine and coumarin pharmacophores (derivatives 1a - 2c ) in order to test the compounds' ability to inhibit both cancer cell growth and topoisomerase I and II activity. The ability of human topoisomerase I ( h TOPI) and II to relax supercoiled plasmid DNA in the presence of various concentrations of the tacrine-coumarin hybrid molecules was studied with agarose gel electrophoresis. The biological activities of the derivatives were studied using MTT assays, clonogenic assays, cell cycle analysis and quantification of cell number and viability. The content and localization of the derivatives in the cells were analysed using flow cytometry and confocal microscopy. All of the studied compounds were found to have inhibited topoisomerase I activity completely. The effect of the tacrine-coumarin hybrid compounds on cancer cells is likely to be dependent on the length of the chain between the tacrine and coumarin moieties ( 1c , 1d = tacrine-(CH 2 ) 8-9 -coumarin). The most active of the tested compounds, derivatives 1c and 1d , both display longer chains.

Zygophyllum coccineum , an edible halophytic plant, is part of the traditional medicine chest in the Mediterranean region for symptomatic relief of diabetes, hypertension, wound healing, burns, infections, and rheumatoid arthritis pain. The current study aimed to characterize Z. coccineum phytoconstituents, and the evaluations of the anti-microbial-biofilm, and anti-cancers bioactivities of the plant's mother liquor, i.e., aqueous-ethanolic extract, and its subsequent fractions. The in silico receptors interaction feasibility of Z. coccineum major constituents with Staph GyraseB, and human topoisomerase-IIβ ( h -TOP-IIβ) were conducted to confirm the plant's anti-microbial and anti-cancer biological activities. Thirty-eight secondary metabolites of flavonoids, stilbene, phenolic acids, alkaloids, and coumarin classes identified by LC-ESI-TOF-MS spectrometric analysis, and tiliroside (kaempferol-3-O-(6''''- p -coumaroyl)-glucoside, 19.8%), zygophyloside-F (12.78%), zygophyloside-G (9.67%), and isorhamnetin-3-O-glucoside (4.75%) were identified as the major constituents. A superior biofilm obliteration activity established the minimum biofilm eradication concentration (MBEC) for the chloroform fraction at 3.9-15.63 µg/mL, as compared to the positive controls (15.63-31.25 µg/mL) against all the microbial strains that produced the biofilm under study, except the Aspergillus fumigatus . The aqueous-ethanolic extract showed cytotoxic effects with IC 50 values at 3.47, 3.19, and 2.27 µg/mL against MCF-7, HCT-116, and HepG2 cell-lines, respectively, together with the inhibition of h- TOP-IIβ with IC 50 value at 45.05 ng/mL in comparison to its standard referral inhibitor (staurosporine, IC 50 , 135.33 ng/mL). This conclusively established the anti-cancer activity of the aqueous-ethanolic extract that also validated by in silico receptor-binding predicted energy levels and receptor-site docking feasibility of the major constituents of the plant's extract. The study helped to authenticate some of the traditional phytomedicinal properties of the anti-infectious nature of the plant.

Pyrimido-pyrimidine derivatives have been developed as rigid merbarone analogues. In a previous study, these compounds showed potent antiproliferative activity and efficiently inhibited topoisomerase IIα. To further extend the structure-activity relationships on pyrimido-pyrimidines, a novel series of analogues was synthesized by a two-step procedure. Analogues 3 - 6 bear small alky groups at positions 1 and 3 of the pyrimido-pyrimidine scaffold whereas at position 6a (4-chloro)phenyl substituent was inserted. The basic side chains introduced at position 7 were selected on the basis of the previously developed structure-activity relationships. The antiproliferative activity of the novel compounds proved to be affected by both the nature of the basic side chain and the substituents on the pyrimido-pyrimidine moiety. Derivatives 5d and 5e were identified as the most promising molecules still showing reduced antiproliferative activity in comparison with the previously prepared pyrimido-pyrimidine analogues. In topoisomerase IIα- 5d docking complex, the ligand would poorly interact with the enzyme and assume a different orientation in comparison with 1d bioactive conformation.

Novel bacterial type II topoisomerase inhibitors (NBTIs) stabilize single-strand DNA cleavage breaks by DNA gyrase but their exact mechanism of action has remained hypothetical until now. We have designed a small library of NBTIs with an improved DNA gyrase-binding moiety resulting in low nanomolar inhibition and very potent antibacterial activity. They stabilize single-stranded cleavage complexes and, importantly, we have obtained the crystal structure where an NBTI binds gyrase-DNA in a single conformation lacking apparent static disorder. This directly proves the previously postulated NBTI mechanism of action and shows that they stabilize single-strand cleavage through asymmetric intercalation with a shift of the scissile phosphate. This crystal stucture shows that the chlorine forms a halogen bond with the backbone carbonyls of the two symmetry-related Ala68 residues. To the best of our knowledge, such a so-called symmetrical bifurcated halogen bond has not been identified in a biological system until now.

This study is to investigate the inhibitory effects and mechanisms of DEK-targeting aptamer (DTA-64) on epithelial mesenchymaltransition (EMT)-mediated airway remodelling in mice and human bronchial epithelial cell line BEAS-2B. In the ovalbumin (OVA)-induced asthmatic mice, DTA-64 significantly reduced the infiltration of eosinophils and neutrophils in lung tissue, attenuated the airway resistance and the proliferation of goblet cells. In addition, DTA-64 reduced collagen deposition, transforming growth factor 1 (TGF-β1) level in BALF and IgE levels in serum, balanced Th1/Th2/Th17 ratio, and decreased mesenchymal proteins (vimentin and α-SMA), as well as weekend matrix metalloproteinases (MMP-2 and MMP-9) and NF-κB p65 activity. In the in vitro experiments, we used TGF-β1 to induce EMT in the human epithelial cell line BEAS-2B. DEK overexpression (ovDEK) or silencing (shDEK) up-regulated or down-regulated TGF-β1 expression, respectively, on the contrary, TGF-β1 exposure had no effect on DEK expression. Furthermore, ovDEK and TGF-β1 synergistically promoted EMT, whereas shDEK significantly reduced mesenchymal markers and increased epithelial markers, thus inhibiting EMT. Additionally, shDEK inhibited key proteins in TGF-β1-mediated signalling pathways, including Smad2/3, Smad4, p38 MAPK, ERK1/2, JNK and PI3K/AKT/mTOR. In conclusion, the effects of DTA-64 against EMT of asthmatic mice and BEAS-2B might partially be achieved through suppressing TGF-β1/Smad, MAPK and PI3K signalling pathways. DTA-64 may be a new therapeutic option for the management of airway remodelling in asthma patients., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Breast cancer represents the most frequently diagnosed malignancy in women worldwide. Various therapeutics are currently used in order to halt the progression of breast tumor, even though certain side effects may limit the beneficial effects. In recent years, many efforts have been addressed to the usefulness of natural compounds as anticancer agents due to their low toxicity. Resveratrol, a stilbene found in grapes, berries, peanuts and soybeans, has raised a notable interest for its antioxidant, anti-inflammatory, and antitumor properties. Here, we report the design, the synthesis and the characterization of the anticancer activity of a small series of imino N -aryl-substituted compounds that are analogues of resveratrol. In particular, the most active compound, named 3 , exhibited anti-tumor activity in diverse types of breast cancer cells through the inhibition of the human topoisomerase II and the induction of apoptotic cell death. Therefore, the abovementioned compound maybe considered as a promising agent in more comprehensive treatments of breast cancer.

Cellular senescence is a known driver of carcinogenesis and age-related diseases, yet senescence is required for various physiological processes. However, the mechanisms and factors that control the negative effects of senescence while retaining its benefits are still elusive. Here, we show that the rasGAP SH3-binding protein 1 (G3BP1) is required for the activation of the senescent-associated secretory phenotype (SASP). During senescence, G3BP1 achieves this effect by promoting the association of the cyclic GMP-AMP synthase (cGAS) with cytosolic chromatin fragments. In turn, G3BP1, through cGAS, activates the NF-κB and STAT3 pathways, promoting SASP expression and secretion. G3BP1 depletion or pharmacological inhibition impairs the cGAS-pathway preventing the expression of SASP factors without affecting cell commitment to senescence. These SASPless senescent cells impair senescence-mediated growth of cancer cells in vitro and tumor growth in vivo. Our data reveal that G3BP1 is required for SASP expression and that SASP secretion is a primary mediator of senescence-associated tumor growth.

Inducing necroptosis in cancer cells is an effective approach to circumvent drug-resistance. Metal-based triggers have, however, rarely been reported. Ruthenium(II) complexes containing 1,1-(pyrazin-2-yl)pyreno[4,5-e][1,2,4]triazine were developed with a series of different ancillary ligands (Ru1-7). The combination of the main ligand with bipyridyl and phenylpyridyl ligands endows Ru7 with superior nucleus-targeting properties. As a rare dual catalytic inhibitor, Ru7 effectively inhibits the endogenous activities of topoisomerase (topo) I and II and kills cancer cells by necroptosis. The cell signaling pathway from topo inhibition to necroptosis was elucidated. Furthermore, Ru7 displays significant antitumor activity against drug-resistant cancer cells in vivo. To the best of our knowledge, Ru7 is the first Ru-based necroptosis-inducing chemotherapeutic agent., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rare or orphan diseases affect only small populations, thereby limiting the economic incentive for the drug development process, often resulting in a lack of progress towards treatment. Drug repositioning is a promising approach in these cases, due to its low cost. In this approach, one attempts to identify new purposes for existing drugs that have already been developed and approved for use. By applying the process of drug repositioning to identify novel treatments for rare diseases, we can overcome the lack of economic incentives and make concrete progress towards new therapies. Adrenocortical Carcinoma (ACC) is a rare disease with no practical and definitive therapeutic approach. We apply Heter-LP, a new method of drug repositioning, to suggest novel therapeutic avenues for ACC. Our analysis identifies innovative putative drug-disease, drug-target, and disease-target relationships for ACC, which include Cosyntropin (drug) and DHCR7, IGF1R, MC1R, MAP3K3, TOP2A (protein targets). When results are analyzed using all available information, a number of novel predicted associations related to ACC appear to be valid according to current knowledge. We expect the predicted relations will be useful for drug repositioning in ACC since the resulting ranked lists of drugs and protein targets can be used to expedite the necessary clinical processes.

Topoisomerase IIα (Topo IIα), a well-conserved double-stranded DNA (dsDNA)-specific decatenase, processes dsDNA catenanes resulting from DNA replication during mitosis. Topo IIα defects lead to an accumulation of ultrafine anaphase bridges (UFBs), a type of chromosome non-disjunction. Topo IIα has been reported to resolve DNA anaphase threads, possibly accounting for the increase in UFB frequency upon Topo IIα inhibition. We hypothesized that the excess UFBs might also result, at least in part, from an impairment of the prevention of UFB formation by Topo IIα. We found that Topo IIα inhibition promotes UFB formation without affecting the global disappearance of UFBs during mitosis, but leads to an aberrant UFB resolution generating DNA damage within the next G1. Moreover, we demonstrated that Topo IIα inhibition promotes the formation of two types of UFBs depending on cell cycle phase. Topo IIα inhibition during S-phase compromises complete DNA replication, leading to the formation of UFB-containing unreplicated DNA, whereas Topo IIα inhibition during mitosis impedes DNA decatenation at metaphase-anaphase transition, leading to the formation of UFB-containing DNA catenanes. Thus, Topo IIα activity is essential to prevent UFB formation in a cell-cycle-dependent manner and to promote DNA damage-free resolution of UFBs.

Cholangiocarcinoma (CCA) is a malignant tumor that arises from the epithelial cells of the bile duct and is notorious for its poor prognosis. The clinical outcome remains disappointing, and thus more effective therapeutic options are urgently required. Cordycepin, a traditional Chinese medicine, provides multiple pharmacological strategies in antitumors, but its mechanisms have not been fully elucidated. In this study, we reported that cordycepin inhibited the viability and proliferation capacity of CCA cells in a time- and dose-dependent manner determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and colony formation assay. Flow cytometry and Hoechst dye showed that cordycepin induced cancer cell apoptosis via extracellular signal-regulated kinase (ERK) 1/2 deactivation. Moreover, cordycepin significantly reduced the angiogenetic capabilities of CCA in vitro as examined by tube formation assay. We also discovered that cordycepin inhibited DEK expression by using Western blot assay. DEK serves as an oncogenic protein that is overexpressed in various gastrointestinal tumors. DEK silencing inhibited CCA cell viability and angiogenesis but not apoptosis induction determined by Western blot and flow cytometry. Furthermore, cordycepin significantly inhibited tumor growth and angiogenic capacities in a xenograft model by downregulating the expression of DEK, phosphorylated ERK1/2 CD31 and von Willebrand factor (vWF). Taken together, we demonstrated that cordycepin inhibited CCA cell proliferation and angiogenesis with a DEK interaction via downregulation in ERK signaling. These data indicate that cordycepin may serve as a novel agent for CCA clinical treatment and prognosis improvement. SIGNIFICANCE STATEMENT: Cordycepin provides multiple strategies in antitumors, but its mechanisms are not fully elucidated, especially on cholangiocarcinoma (CCA). We reported that cordycepin inhibited the viability of CCA cells, induced apoptosis via extracellular signal-regulated kinase 1/2 deactivation and DEK inhibition, and reduced the angiogenetic capabilities of CCA both in vivo and in vitro., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Topoisomerase IIα (topo2α) is an essential nuclear enzyme involved in DNA replication, transcription, recombination, chromosome condensation, and highly expressed in many tumors. Thus, topo2α-targeting has become a very efficient and well-established anticancer strategy. Herein, we investigate the cytotoxic and DNA-damaging activity of thiomaltol-containing ruthenium-, osmium-, rhodium- and iridium-based organometallic complexes in human mammary carcinoma cell lines by means of several biological assays, including knockdown of topo2α expression levels by RNA interference. Results suggest that inhibition of topo2α is a key process in the cytotoxic mechanism for some of the compounds, whereas direct induction of DNA double-strand breaks or other DNA damage is mostly rather minor. In addition, molecular modeling studies performed for two of the compounds (with Ru(II) as the metal center) evinces that these complexes are able to access the DNA-binding pocket of the enzyme, where the hydrophilic environment favors the interaction with highly polar complexes. These findings substantiate the potential of these compounds for application as antitumor metallopharmaceuticals.

Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases ( POLA1, POLE , and POLE2 ) whose silencing sensitized the human A549 non-small cell lung cancer (NSCLC) and SW620 colorectal cancer cell lines to SRA737. B-family polymerases were validated using multiple siRNAs in a panel of NSCLC and colorectal cancer cell lines. Replication stress, DNA damage, and apoptosis were increased in human cancer cells following depletion of the B-family DNA polymerases combined with SRA737 treatment. Moreover, pharmacologic blockade of B-family DNA polymerases using aphidicolin or CD437 combined with CHK1 inhibitors led to synergistic inhibition of cancer cell proliferation. Furthermore, low levels of POLA1, POLE, and POLE2 protein expression in NSCLC and colorectal cancer cells correlated with single-agent CHK1 inhibitor sensitivity and may constitute biomarkers of this phenotype. These findings provide a potential basis for combining CHK1 and B-family polymerase inhibitors in cancer therapy. SIGNIFICANCE: These findings demonstrate how the therapeutic benefit of CHK1 inhibitors may potentially be enhanced and could have implications for patient selection and future development of new combination therapies., (©2020 American Association for Cancer Research.)

The benzo[ c ]phenanthridine P8-D6 was recently found to suppress the catalytic activity of both human topoisomerase (Topo) I and II. Concomitantly, potent cytotoxic activity was observed in different human tumor cell lines, raising questions about the underlying mechanisms in vitro. In the present study, we addressed the question of whether P8-D6 acts as a so-called Topo poison, stabilizing the covalent Topo-DNA intermediate, thus inducing fatal DNA strand breaks in proliferating cells. In HT-29 colon carcinoma cells, fluorescence imaging revealed P8-D6 to be taken up by the cells and to accumulate in the perinuclear region. Confocal microscopy demonstrated that the compound is partially located inside the nuclei, thus reaching the potential target. In the "in vivo complex of enzyme" (ICE) bioassay, treatment of HT-29 cells with P8-D6 for 1 h significantly enhanced the proportion of Topo I and II covalently linked to the DNA in concentrations ≥1 µM, indicating effective dual Topo poisoning. Potentially resulting DNA damage was analyzed by single-cell gel electrophoresis ("comet assay"). Already at 1 h of incubation, significant genotoxic effects were observed in the comet assay in concentrations as low as 1 nM. Taken together, the present study demonstrates the high Topo-poisoning and genotoxic potential of P8-D6 in human tumor cells.

Small molecules can affect many cellular processes. The disambiguation of these effects to identify the causative mechanisms of cell death is extremely challenging. This challenge impacts both clinical development and the interpretation of chemical genetic experiments. CX-5461 was developed as a selective RNA polymerase I inhibitor, but recent evidence suggests that it may cause DNA damage and induce G-quadraplex formation. Here we use three complimentary data mining modalities alongside biochemical and cell biological assays to show that CX-5461 exerts its primary cytotoxic activity through topoisomerase II poisoning. We then show that acquired resistance to CX-5461 in previously sensitive lymphoma cells confers collateral resistance to the topoisomerase II poison doxorubicin. Doxorubicin is already a frontline chemotherapy in a variety of hematopoietic malignancies, and CX-5461 is being tested in relapse/refractory hematopoietic tumors. Our data suggest that the mechanism of cell death induced by CX-5461 is critical for rational clinical development in these patients. Moreover, CX-5461 usage as a specific chemical genetic probe of RNA polymerase I function is challenging to interpret. Our multimodal data-driven approach is a useful way to detangle the intended and unintended mechanisms of drug action across diverse essential cellular processes., Competing Interests: Competing interest statement: M.J.L. and J.R.P. are coinvestigators on a submitted grant.

Background: RAS GTPase-activating protein-binding protein (G3BP1) is an RNA-binding protein that is essential for assembling stress granules. Many functions related to the survival and progression of cancer have been reported. The current study aimed to investigate the role of G3BP1 in radio-sensitisation of cancer cells., Materials and Methods: Radiation sensitivity and chemosensitivity were analysed in A549 and H460 cells transfected with G3BP1 siRNAs, and N-acetyl-L-cysteine (NAC) was used to elucidate the involvement of reactive oxygen species (ROS)., Results: G3BP1 depletion sensitised lung cancer cell lines to radiation, and the effect was related to ROS. G3BP1 depletion impaired the intracellular ROS scavenging system and NAC abolished the radiation-sensitive phenotypes caused by G3BP1 depletion., Conclusion: The study suggested G3BP1 as a promising target for radio- and chemosensitisation of lung cancer., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Anthracyclines are a highly effective component of curative breast cancer chemotherapy but are associated with substantial morbidity 1,2 . Because anthracyclines work in part by inhibiting topoisomerase-II (TOP2) on accessible DNA 3,4 , we hypothesized that chromatin regulatory genes (CRGs) that mediate DNA accessibility might predict anthracycline response. We studied the role of CRGs in anthracycline sensitivity in breast cancer through integrative analysis of patient and cell line data. We identified a consensus set of 38 CRGs associated with anthracycline response across ten cell line datasets. By evaluating the interaction between expression and treatment in predicting survival in a metacohort of 1006 patients with early-stage breast cancer, we identified 54 CRGs whose expression levels dictate anthracycline benefit across the clinical subgroups; of these CRGs, 12 overlapped with those identified in vitro. CRGs that promote DNA accessibility, including Trithorax complex members, were associated with anthracycline sensitivity when highly expressed, whereas CRGs that reduce accessibility, such as Polycomb complex proteins, were associated with decreased anthracycline sensitivity. We show that KDM4B modulates TOP2 accessibility to chromatin, elucidating a mechanism of TOP2 inhibitor sensitivity. These findings indicate that CRGs mediate anthracycline benefit by altering DNA accessibility, with implications for the stratification of patients with breast cancer and treatment decision making.

Stress granules (SGs) are ribonucleoprotein aggregates that form in response to stress conditions. The regulation of SG dynamics is not fully understood. Permanent pathological SG-like structures were reported in neurodegenerative diseases such as amyotrophic lateral sclerosis. The Ras GTPase-activating protein-binding protein G3BP1 is a central regulator of SG dynamics. We found that the lysine 376 residue (K376) of G3BP1, which is in the RRM RNA binding domain, was acetylated. Consequently, G3BP1 RNA binding was impaired by K376 acetylation. In addition, the acetylation-mimicking mutation K376Q impaired the RNA-dependent interaction of G3BP1 with poly(A)-binding protein 1 (PABP1), but its RNA-independent interactions with caprin-1 and USP10 were little affected. The formation of G3BP1 SGs depended on G3BP1 RNA binding; thus, replacement of endogenous G3BP1 with the K376Q mutant or the RNA binding-deficient F380L/F382L mutant interfered with SG formation. Significant G3BP1 K376 acetylation was detected during SG resolution, and K376-acetylated G3BP1 was seen outside SGs. G3BP1 acetylation is regulated by histone deacetylase 6 (HDAC6) and CBP/p300. Our data suggest that the acetylation of G3BP1 facilitates the disassembly of SGs, offering a potential avenue to mitigate hyperactive stress responses under pathological conditions., (Copyright © 2019 American Society for Microbiology.)

Topoisomerase II is a nuclear enzyme involved in the maintenance of DNA and is an effective anticancer drug target. However, several clinical topoisomerase II-targeted agents display significant off-target toxicities and adverse events. Thus, it is important to continue characterizing compounds with activity against topoisomerase II. We previously analyzed α-(N)-heterocyclic thiosemicarbazone copper(II) complexes against human topoisomerase IIα (TOP2A), but humans also express topoisomerase IIβ (TOP2B), which has distinct functional roles. Therefore, we examined two α-(N)-heterocyclic thiosemicarbazone copper [Cu(II)] complexes for activity against TOP2B in a purified system. The Cu(II) complexes, Cu(APY-ETSC)Cl and Cu(BZP-ETSC)Cl, were examined using plasmid DNA cleavage, supercoiled DNA relaxation, enzyme inactivation, protein cross-linking, DNA ligation, and ATP hydrolysis assays with TOP2B to determine whether these compounds act similarly against both enzymes. Both of the Cu(II) thiosemicarbazone (Cu-TSC) complexes we tested disrupted the function of TOP2B in a way similar to the effect on TOP2A. In particular, TOP2B DNA cleavage activity is increased in the presence of these compounds, while the relaxation and ATPase activities are inhibited. Further, both Cu-TSCs stabilize the N-terminal DNA clamp of TOP2A and TOP2B and rapidly inactivate TOP2B when the compounds are present before DNA. Our data provide evidence that the Cu-TSC complexes we tested utilize a similar mechanism against both isoforms of the enzyme. This mechanism may involve interaction with the ATPase domain of TOP2A and TOP2B outside of the ATP binding pocket. Additionally, these data support a model of TOP2 function where the ATPase domain communicates with the DNA cleavage/ligation domain.

Rapidly dividing cells maintain chromatin supercoiling homeostasis via two specialized classes of enzymes, DNA topoisomerase type 1 and 2 (TOP1/2). Several important anticancer drugs perturb this homeostasis by targeting TOP1/2, thereby generating genotoxic DNA damage. Our recent studies indicated that the oncofetal chromatin structuring high-mobility group AT-hook 2 (HMGA2) protein plays an important role as a DNA replication fork chaperone in coping with DNA topological ramifications that occur during replication stress, both genomewide and at fragile sites such as subtelomeres. Intriguingly, a recent large-scale clinical study identified HMGA2 expression as a sole predicting marker for relapse and poor clinical outcomes in 350 acute myeloid leukemia (AML) patients receiving combinatorial treatments that targeted TOP2 and replicative DNA synthesis. Here, we demonstrate that HMGA2 significantly enhanced the DNA supercoil relaxation activity of the drug target TOP2A and that this activator function is mechanistically linked to HMGA2's known ability to constrain DNA supercoils within highly compacted ternary complexes. Furthermore, we show that HMGA2 significantly reduced genotoxic DNA damage in each tested cancer cell model during treatment with the TOP2A poison etoposide or the catalytic TOP2A inhibitor merbarone. Taken together with the recent clinical data obtained with AML patients targeted with TOP2 poisons, our study suggests a novel mechanism of cancer chemoresistance toward combination therapies administering TOP2 poisons or inhibitors. We therefore strongly argue for the future implementation of trials of HMGA2 expression profiling to stratify patients before finalizing clinical treatment regimes., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Introduction: Glioblastoma multiforme (GBM) is the most lethal form of gliomas. New therapies are currently in development to tackle treatment limitations such as chemotherapy resistance. One mechanism of resistance may be the stress granules (SG) assembly, a stress-related cellular response that allows cells to recruit and protect mRNAs during stress. SG are composed of various proteins, being G3BP1 a core element that enucleates and results in SG assembly. Here, we aimed to evaluate the effects of inhibiting the G3PB1 expression in the chemotherapeutical-induced cell death of the U87 glioblastoma cell line., Materials and Methods: G3BP1 mRNA and protein expression were modulated with short-interference RNA (siRNA). The viability of U87 cells after Bortezomib (BZM), a proteasome inhibitor, and Temozolomide (TMZ), an alkylating agent, was assessed by MTT assay. Apoptosis was evaluated by staining cells with Annexin-V/7-AAD and analyzing by flow cytometry. Caspase-3 activation was evaluated by immunoblotting. The chorioallantoic membrane in vivo assay was used to evaluate angiogenesis., Results: When G3BP1 was knocked-down, the SG assembly was reduced and the BZM-treated cells, but not TMZ-treated cells, had a significant increase in the apoptotic response. Corroborating this data, we observed increased Caspase-3 activation in the BZM-treated and G3BP1-knocked-down cells when compared to vehicle-treated and scramble-transfected cells. Worth mentioning, the conditioned culture medium of G3BP1-knocked-down BZM-treated cells inhibited angiogenesis when compared to controls., Conclusion: Our data suggest G3BP1 knockdown diminishes SG formation and stimulates BZM-induced apoptosis of U87 cells in vitro, in addition to inhibiting glioblastoma-induced angiogenesis in vivo.

The aim of this study was to investigate the effect of the cell differentiation status on the sensitivity to genotoxic insults. For this, we utilized the comet assay to test the DNA damage after treatment with 5 different substances with different mechanism of action in human promyelocytic HL60 cells with or without cell differentiation. A 4-hour MMS treatment induced a significant and concentration-dependent increase in DNA damage for both differentiated and undifferentiated cells, but the difference in sensitivity was only significant at the highest concentration. A 4-hour doxorubicin treatment did not induce DNA damage in differentiated HL60 cells, while it did in undifferentiated cells with its highest tested concentration. A one-hour etoposide treatment caused significant increase in DNA damage concentration dependently in both cell variants. This DNA damage was significantly higher in undifferentiated HL60 cells with several tested concentrations of etoposide. The treatment with the oxidizing substances hydrogen peroxide and potassium bromate yielded significant DNA damage induction in both undifferentiated and differentiated cells with no difference according to the differentiation status. Doxorubicin and etoposide are known to inhibit topoisomerase II. The activity of this enzyme has been shown to be higher in undifferentiated actively proliferating cells than in differentiated cells. This may be of relevance when exposures to topoisomerase-inhibiting compounds or the genotoxicity of compounds with unknown mechanism of action are assessed in routine testing., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cytosporolide (Cytos) A-C, isolated from the fungus Cytospora sp., have anti-microbial activity, but their molecular targets in mammalian cells are unknown. We have previously reported the total synthesis of Cytos A by biomimetic hetero-Diels-Alder reaction. In this study, to examine the novel bioactivity of Cytos, we synthesized Cytos C and measured cell growth-inhibiting activities of 7 compounds, including Cytos A and C, in several human cancer cell lines. Among these compounds, Cytos C and tetradeoxycytosporolide A (TD-Cytos A), a model compound for the synthesis of Cytos A, had anti-proliferative effects on cancer cells, and TD-Cytos A exhibited stronger activity than Cytos C. In vitro topoisomerase-mediated DNA relaxing experiments showed that TD-Cytos A inhibited the activities of topoisomerase I and II, whereas Cytos C targeted only topoisomerase I. These data suggest that the anti-proliferative activities of Cytos correlate with the inhibition of topoisomerases and implicated TD-Cytos A as a novel anti-cancer drug that suppresses the activities of topoisomerase I and II., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Bladder cancer progression has been associated with dysfunctional repair of double-strand breaks (DSB), a deleterious type of DNA lesions that fuel genomic instability. Accurate DSB repair relies on two distinct pathways, homologous recombination (HR) and classical non-homologous end-joining (c-NHEJ). The transcription factor E2F1 supports HR-mediated DSB repair and protects genomic stability. However, invasive bladder cancers (BC) display, in contrast to non-invasive stages, genomic instability despite their high E2F1 levels. Hence, E2F1 is either inefficient in controlling DSB repair in this setting, or rewires the repair apparatus towards alternative, error-prone DSB processing pathways., Methods: RT-PCR and immunoblotting, in combination with bioinformatics tools were applied to monitor c-NHEJ factors status in high-E2F1-expressing, invasive BC versus low-E2F1-expressing, non-invasive BC. In vivo binding of E2F1 on target gene promoters was demonstrated by ChIP assays and E2F1 CRISPR-Cas9 knockdown. MIR888-dependent inhibition of APLF by E2F1 was demonstrated using overexpression and knockdown experiments, in combination with luciferase assays. Methylation status of MIR888 promoter was monitored by methylation-specific PCR. The changes in invasion potential and the DSB repair efficiency were estimated by Boyden chamber assays and pulse field electrophoresis, correspondingly., Results: Herein, we show that E2F1 directly transactivates the c-NHEJ core factors Artemis, DNA-PKcs, ligase IV, NHEJ1, Ku70/Ku80 and XRCC4, but indirectly inhibits APLF, a chromatin modifier regulating c-NHEJ. Inhibition is achieved by miR-888-5p, a testis-specific, X-linked miRNA which, in normal tissues, is often silenced via promoter methylation. Upon hypomethylation in invasive BC cells, MIR888 is transactivated by E2F1 and represses APLF. Consequently, E2F1/miR-888/APLF rewiring is established, generating conditions of APLF scarcity that compromise proper c-NHEJ function. Perturbation of the E2F1/miR-888/APLF axis restores c-NHEJ and ameliorates cell invasiveness. Depletion of miR-888 can establish a 'high E2F1/APLF/DCLRE1C' signature, which was found to be particularly favorable for BC patient survival., Conclusion: Suppression of the 'out-of-context' activity of miR-888 improves DSB repair and impedes invasiveness by restoring APLF.

The Topo2a-dependent arrest is associated with faithful segregation of sister chromatids and has been identified as dysfunctional in numerous tumour cell lines. This genome-protecting pathway is poorly understood and its characterization is of significant interest, potentially offering interventional opportunities in relation to synthetic lethal behaviours in arrest-defective tumours. Using the catalytic Topo2a inhibitor ICRF193, we have performed a genome-wide siRNA screen in arrest-competent, non-transformed cells, to identify genes essential for this arrest mechanism. In addition, we have counter-screened several DNA-damaging agents and demonstrate that the Topo2a-dependent arrest is genetically distinct from DNA damage checkpoints. We identify the components of the SMC5/6 complex, including the activity of the E3 SUMO ligase NSE2, as non-redundant players that control the timing of the Topo2a-dependent arrest in G2. We have independently verified the NSE2 requirement in fibroblasts from patients with germline mutations that cause severely reduced levels of NSE2. Through imaging Topo2a-dependent G2 arrested cells, an increased interaction between Topo2a and NSE2 is observed at PML bodies, which are known SUMOylation hotspots. We demonstrate that Topo2a is SUMOylated in an ICRF193-dependent manner by NSE2 at a novel non-canonical site (K1520) and that K1520 sumoylation is required for chromosome segregation but not the G2 arrest., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Computational quantum chemical study and biological evaluation of a synthesized novel sulfur heterocyclic thiophene derivative containing 1,2,3-triazole and pyridine moieties namely BTPT [2-(1-benzyl-5-methyl-1H-1,2,3-triazol-4-yl)-6-methoxy-4-(thiophen-2-yl) pyridine] was presented in this study. The crystal structure was determined by SCXRD method. For the title compound BTPT, spectroscopic characterization like 1 H NMR, 13 C NMR, FTIR, UV-vis were carried out theoretically by computational DFT method and compared with experimental data. Druglikeness parameters of BTPT were found through in silico pharmacological ADMET properties estimation. The molecular docking investigation was performed with human topoisomerase IIα (PDB ID:1ZXM) targeting ATP binding site. In vitro cytotoxicity activity of BTPT/doxorubicin were examined by MTT assay procedure against three human cancer cell lines A549, PC-3, MDAMB-231 with IC50 values of 0.68/0.70, 1.03/0.77 and 0.88/0.98 μM, respectively. Our title compound BTPT reveals notable cytotoxicity against breast cancer cell (MDAMB-231), moderate activity with human lung cancer cell (A-549) and less inhibition with human prostate cancer cell (PC-3) compared to familiar cancer medicine doxorubicin. From the results, BTPT could be observed as a potential candidate for novel anticancer drug development process., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Phytochemical naphtho[1,2-b] furan-4,5‑dione (NFD) presenting in Avicennia marina exert anti-cancer effects, but little is known regarding about DNA damage-mediated apoptosis in non-small-cell lung carcinoma (NSCLC)., Purpose: To examine whether NFD-induced apoptosis of NSCLC cells is correlated with the induction of DNA damage, and to investigate its underlying mechanism., Study Design: The anti-proliferative effects of NFD were assessed by MTS Assay Kit FACS assay, and in vivo nude mice xenograft assay. The DNA damage related proteins, the Bcl-2 family and pro-apoptotic factors were examined by immunofluorescence assay, q-PCR, and western blotting. The activity of NF-κB p65 in nuclear extracts was detected using a colorimetric DNA-binding ELISA assay. The inhibitory activity of topoisomerase II (TOPO II) was evaluated by molecular docking and TOPO II catalytic assay., Results: NFD exerted selective cytotoxicity against NSCLC H1299, H1437 and A549 cells rather than normal lung-embryonated cells MRC-5. Remarkably, we found that NFD activated the hull marker and modulator of DNA damage repairs such as γ-H2AX, ATM, ATR, CHK1, and CHK2 probably caused by the accumulation of intracellular reactive oxygen species (ROS) and inhibition of TOPO II activity. Furthermore, the suppression of transcription factor NF-κB by NFD resulted in significantly decreased levels of pro-survival proteins including Bcl-2 family Bcl-2, Bcl-xL and Mcl-1 and the endogenous inhibitors of apoptosis XIAP and survivin in H1299 cells. Moreover, the nude mice xenograft assay further validated the suppression of H1299 growth by NFD, which is the first report for evaluating the anti-cancer effect of NFD in vivo., Conclusion: These findings provide a novel mechanism indicating the inhibition of TOPO II activity and NF-κB signaling by NFD, leading to DNA damage and apoptosis of NSCLC tumor cells., (Copyright © 2018. Published by Elsevier GmbH.)

Background: The major attention has been received by the natural products in the prevention of diseases due to their pharmacological role., Objective: The major focus of the study was to search for highly potential anti-cancer compounds from marine Streptomyces sp. VITJS4 (NCIM No. 5574)., Methods: Cytotoxic assay was examined by MTT assay on HepG2 cells. Bioassay-guided fractionation of the ethyl acetate extract from the fermented broth led to the isolation of the compound. The lead compound structure was elucidated by combined NMR and MS analysis, and the absolute configuration was assigned by extensive spectroscopic analysis., Results: On the basis of spectroscopic data, the compound was identified as 1, 2 benzenedicarboxylic acid, mono 2-ethylhexyl (BMEH). The compound exhibited in vitro anticancer potential against liver (HepG2) cancer cells. Based on the flow cytometric analysis, it was evident that the BMEH was also effective in arresting the cell cycle at G1 phase. Further, the Western blotting analysis confirmed the down-regulation of Bcl-2 family proteins, and activation of caspase-9 and 3. The molecular docking and dynamics simulation were performed to reveal the activity of the compound over a time period of 10ns. From the molecular dynamics studies, it was found that the stability and compactness were attained by the protein by means of the compound interaction., Conclusion: This study highlights our collaborative efforts to ascertain lead molecules from marine actinomycete. This is the first and foremost report to prove the mechanistic studies of the purified compound 1, 2-benzene dicarboxylic acid, mono(2-ethylhexyl) ester isolated from marine Streptomyces sp.VITJS4 against HepG2 cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Cyclic GMP-AMP synthase (cGAS) is a key sensor responsible for cytosolic DNA detection. Here we report that GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is critical for DNA sensing and efficient activation of cGAS. G3BP1 enhanced DNA binding of cGAS by promoting the formation of large cGAS complexes. G3BP1 deficiency led to inefficient DNA binding by cGAS and inhibited cGAS-dependent interferon (IFN) production. The G3BP1 inhibitor epigallocatechin gallate (EGCG) disrupted existing G3BP1-cGAS complexes and inhibited DNA-triggered cGAS activation, thereby blocking DNA-induced IFN production both in vivo and in vitro. EGCG administration blunted self DNA-induced autoinflammatory responses in an Aicardi-Goutières syndrome (AGS) mouse model and reduced IFN-stimulated gene expression in cells from a patient with AGS. Thus, our study reveals that G3BP1 physically interacts with and primes cGAS for efficient activation. Furthermore, EGCG-mediated inhibition of G3BP1 provides a potential treatment for cGAS-related autoimmune diseases.

Background: Chromatin modification at mitosis is closely related to transcriptional reactivation in the subsequent cell cycle. We reasoned this process is deregulated by oncogenic signals, which would contribute to mitotic stress resistance in pancreatic cancer. Here, we show DMAP1/Bub3 complex mediates mitotic stress-induced cellular apoptosis, while this effect is counteracted by c-Src in pancreatic cancer cells. Our study aims to uncover an unidentified mechanism underlying the distinct response to mitotic stress between normal cells and pancreatic cancer cells., Methods: The interaction between Bub3 and DMAP1 upon mitotic stress signaling was determined through molecular and cell biological methods. The inhibitory effect of c-Src on DMAP1/Bub3-mediated DNA methylation and gene transcription profile was investigated. The association between c-Src-mediated DMAP1 phosphorylation and paclitaxel activity in vivo and clinicopathologic characteristics were analyzed., Results: Mitotic arrest induced p38-dependent phosphorylation of Bub3 at Ser211, which promotes DMAP1/Bub3 interaction. DMAP1/Bub3 complex is recruited by TAp73 to the promoter of anti-apoptotic gene BCL2L1, thus mediates the DNA methylation and represses gene transcription linked to cell apoptosis. Meanwhile, DMAP1 was highly phosphorylated at Tyr 246 by c-Src in pancreatic cancer cells, which impedes DMAP1/Bub3 interaction and the relevant cellular activites. Blocking DMAP1 pTyr-246 potentiates paclitaxel-inhibited tumor growth. Clinically, DMAP1 Tyr 246 phosphorylation correlates with c-Src activity in human pancreatic cancer specimens and poor prognosis in pancreatic cancer patients., Conclusions: Our findings reveal a regulatory role of Bub3 in DMAP1-mediated DNA methylation upon mitotic stress and provide the relevance of DMAP1 pTyr-246 to mitotic stress resistance during pancreatic cancer treatment.

Etoposide is an anticancer drug that acts by inducing topoisomerase II-mediated DNA cleavage. Despite its wide use, etoposide is associated with some very serious side-effects including the development of treatment-related acute myelogenous leukemias. Etoposide targets both human topoisomerase IIα and IIβ. However, the contributions of the two enzyme isoforms to the therapeutic vs. leukemogenic properties of the drug are unclear. In order to develop an etoposide-based drug with specificity for cancer cells that express an active polyamine transport system, the sugar moiety of the drug has been replaced with a polyamine tail. To analyze the effects of this substitution on the specificity of hybrid molecules toward the two enzyme isoforms, we analyzed the activity of a series of etoposide-polyamine hybrids toward human topoisomerase IIα and IIβ. All of the compounds displayed an ability to induce enzyme-mediated DNA cleavage that was comparable to or higher than that of etoposide. Relative to the parent drug, the hybrid compounds displayed substantially higher activity toward topoisomerase IIβ than IIα. Modeling studies suggest that the enhanced specificity may result from interactions with Gln778 in topoisomerase IIβ. The corresponding residue in the α isoform is a methionine., (Published by Elsevier Ltd.)

Inhibition of poly(ADP-ribose) polymerase 1 (PARP1) is one of the most promising strategies for cancer chemotherapy, and a number of inhibitors possessing nicotinamide-like structures are being developed. To discover new types of PARP1 inhibitors, we screened a large number of substances of plant origin and isolated two inhibitory substances from the leaves of Syzygium samarangense (Blume) Merrill & L.M. Perry. The inhibitory substances were identified as vescalagin and its epimer castalagin by analyses using nuclear magnetic resonance and mass spectrometry. The IC 50 of purified vescalagin and castalagin for PARP1 inhibition were 2.67 and 0.86 μM, respectively. Unlike most of synthetic PARP1 inhibitors, castalagin showed a mixed type inhibition, of which Ki was 1.64 μM. When SH-SY5Y cells were treated with these ellagitannins at concentrations of less than 5 μM, cellular poly(ADP-ribosyl)ation was obviously attenuated. Castalagin and vescalagin also possessed inhibitory activity against DNA topoisomerase II, implying that they function as dual inhibitors in cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

DNA topoisomerase II (TOP2) activity involves a normally transient double-strand break intermediate in which the enzyme is coupled to DNA via a 5'-phosphotyrosyl bond. However, etoposide and other topoisomerase drugs poison the enzyme by stabilising this enzyme-bridged break, resulting in the accumulation of TOP2-DNA covalent complexes with cytotoxic consequences. The phosphotyrosyl diesterase TDP2 appears to be required for efficient repair of this unusual type of DNA damage and can remove 5'-tyrosine adducts from a double-stranded oligonucleotide substrate. Here, we adapt the trapped in agarose DNA immunostaining (TARDIS) assay to investigate the role of TDP2 in the removal of TOP2-DNA complexes in vitro and in cells. We report that TDP2 alone does not remove TOP2-DNA complexes from genomic DNA in vitro and that depletion of TDP2 in cells does not slow the removal of TOP2-DNA complexes. Thus, if TDP2 is involved in repairing TOP2 adducts, there must be one or more prior steps in which the protein-DNA complex is processed before TDP2 removes the remaining 5' tyrosine DNA adducts. While this is partly achieved through the degradation of TOP2 adducts by the proteasome, a proteasome-independent mechanism has also been described involving the SUMOylation of TOP2 by the ZATT E3 SUMO ligase. The TARDIS assay was also adapted to measure the effect of TDP2 knockdown on levels of SUMOylated TOP2-DNA complexes, which together with levels of double strand breaks were unaffected in K562 cells following etoposide exposure and proteasomal inhibition., Competing Interests: The authors declare no conflict of interest.

Anthracycline chemotherapy causes dose-related cardiomyocyte injury and death leading to left ventricular dysfunction. Clinical heart failure may ensue in up to 5% of high-risk patients. Improved cancer survival together with better awareness of the late effects of cardiotoxicity has led to growing recognition of the need for surveillance of anthracycline-treated cancer survivors with early intervention to treat or prevent heart failure. The main mechanism of anthracycline cardiotoxicity is now thought to be through inhibition of topoisomerase 2β resulting in activation of cell death pathways and inhibition of mitochondrial biogenesis. In addition to cumulative anthracycline dose, age and pre-existing cardiac disease are risk markers for cardiotoxicity. Genetic susceptibility factors will help identify susceptible patients in the future. Cardiac imaging with echocardiographic measurement of global longitudinal strain and cardiac troponin detect early myocardial injury prior to the development of left ventricular dysfunction. There is no consensus on how best to monitor anthracycline cardiotoxicity although guidelines advocate quantification of left ventricular ejection fraction before and after chemotherapy with additional scanning being justified in high-risk patients. Patients developing significant left ventricular dysfunction with or without clinical heart failure should be treated according to established guidelines. Liposomal encapsulation reduces anthracycline cardiotoxicity. Dexrazoxane administration with anthracycline interferes with binding to topoisomerase 2β and reduces both cardiotoxicity and subsequent heart failure in high-risk patients. Angiotensin inhibition and β-blockade are also protective and appear to prevent the development of left ventricular dysfunction when given prior or during chemotherapy in patients exhibiting early signs of cardiotoxicity., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)