TOP2A Research Articles

Retracted: DNA Topoisomerase II-α Regulated by miR-22-5p Promotes Hepatocellular Carcinoma Invasion and Migration through the Hippo Pathway.

[This retracts the article DOI: 10.1155/2022/4277254.].

Novel bacterial topoisomerase inhibitors: unique targeting activities of amide enzyme-binding motifs for tricyclic analogs.

Antimicrobial resistance has made a sizeable impact on public health and continues to threaten the effectiveness of antibacterial therapies. Novel bacterial topoisomerase inhibitors (NBTIs) are a promising class of antibacterial agents with a unique binding mode and distinct pharmacology that enables them to evade existing resistance mechanisms. The clinical development of NBTIs has been plagued by several issues, including cardiovascular safety. Herein, we report a sub-series of tricyclic NBTIs bearing an amide linkage that displays promising antibacterial activity, potent dual-target inhibition of DNA gyrase and topoisomerase IV (TopoIV), as well as improved cardiovascular safety and metabolic profiles. These amide NBTIs induced both single- and double-strand breaks in pBR322 DNA mediated by Staphylococcus aureus DNA gyrase, in contrast to prototypical NBTIs that cause only single-strand breaks. Unexpectedly, amides 1a and 1b targeted human topoisomerase IIα (TOP2α) causing both single- and double-strand breaks in pBR322 DNA, and induced DNA strand breaks in intact human leukemia K562 cells. In addition, anticancer drug-resistant K/VP.5 cells containing decreased levels of TOP2α were cross-resistant to amides 1a and 1b. Together, these results demonstrate broad spectrum antibacterial properties of selected tricyclic NBTIs, desirable safety profiles, an unusual ability to induce DNA double-stranded breaks, and activity against human TOP2α. Future work will be directed toward optimization and development of tricyclic NBTIs with potent and selective activity against bacteria. Finally, the current results may provide an additional avenue for development of selective anticancer agents.

The abscission checkpoint senses chromatin bridges through Top2α recruitment to DNA knots.

In response to chromatin bridges, the abscission checkpoint delays completion of cytokinesis to prevent chromosome breakage or tetraploidization. Here, we show that spontaneous or replication stress-induced chromatin bridges exhibit "knots" of catenated and overtwisted DNA next to the midbody. Topoisomerase IIα (Top2α) forms abortive Top2-DNA cleavage complexes (Top2ccs) on DNA knots; furthermore, impaired Top2α-DNA cleavage activity correlates with chromatin bridge breakage in cytokinesis. Proteasomal degradation of Top2ccs is required for Rad17 localization to Top2-generated double-strand DNA ends on DNA knots; in turn, Rad17 promotes local recruitment of the MRN complex and downstream ATM-Chk2-INCENP signaling to delay abscission and prevent chromatin breakage. In contrast, dicentric chromosomes that do not exhibit knotted DNA fail to activate the abscission checkpoint in human cells. These findings are the first to describe a mechanism by which the abscission checkpoint detects chromatin bridges, through generation of abortive Top2ccs on DNA knots, to preserve genome integrity.

Topoisomerase IIα immunoexpression in feline mammary carcinomas: A correlation with Ki67 immunoexpression and the mitotic count

The aim of the study was to compare the immunohistochemical expression of topoisomerase IIα protein (Topo IIα) with Ki67 expression and mitotic count in feline mammary carcinomas (FMCs). Topo IIα is considered as a proliferation indicator as well as a molecular target of anthracycline chemotherapy. The studied material included 70 FMCs from female cats treated with mastectomy. Primary mouse monoclonal antibodies directed against Topo IIα and Ki67 were used in immunohistochemical reactions. The number of mitotic figures was counted at 400× magnification in a field of 2.37 mm2. Immunohistochemical reaction for Topo IIα occurred in cell nuclei. The Topo IIα index ranged from 6.12% to 54.60% and was positively correlated with the values of the Ki67 index (r = 0.7193) and the mitotic count (r = 0. 2858). This indicates the potential possibility of use of the immunohistochemical expression of Topo IIα to assess the rate of proliferation in FMCs. The wide range of expression of Topo IIα in individual tumorus found in the conducted studies allows us to hypothesize that its assessment could be used as a predictive marker in chemotherapy of FMCs with the use of anthracyclines. However, this requires confirmation in clinical trials.

Screening and validation of pivotal genes in hepatitis B virus-associated hepatocellular carcinoma

Objective: To screen the pivotal genes involved in the occurrence and development of HBV-associated HCC. Additionally, perform validation and biological function analysis to evaluate changes in the expression of pivotal genes and their prognostic value in patients with hepatocellular carcinoma. Methods: The GSE121248 gene expression profile data of HBV-HCC patients were searched and downloaded from the GEO database. The R language was used to compare the differences in gene expression between hepatocellular carcinoma and paracancerous tissues. KEGG and GO function enrichment analyses were performed on the differential genes. PPI plots and pivotal gene screening were carried out through online tools like STRING and Cytoscape software. 369 cases of hepatocellular carcinoma and 160 healthy controls in TCGA and GTEx were used as validation cohorts to verify the expression levels of the pivotal genes. A Kaplan-Meier plot was drawn to evaluate the prognostic value of the pivotal gene. Results: A total of 120 differentially expressed genes were screened, of which 89 were up-regulated and 31 were down-regulated. Differential genes were mainly enriched in the metabolic pathways related to retinol metabolism, cytochrome P450 metabolism, and the p53 signaling pathway. The top 10 differential genes were selected as pivotal genes by the Cytoscape plug-in cytoHubba. There were significant differences in the expression levels of four types of CCNB1, CDK1, RRM2, and TOP2A genes in the validation cohort. All four types of genes were up-regulated. Survival analysis showed that patients with elevated expression levels of four genes had a poorer prognosis, with statistical differences in results. Conclusion: Four types of genes, CCNB1, CDK1, RRM2, and TOP2A, have high expression levels in patients with HBV-HCC and are correlated to shorter survival times, making them a potential target for diagnosis, prognosis, and treatment.

Evaluating the expression of heat shock protein 27 and topoisomerase II α in a retrospective cohort of patients diagnosed with locally advanced breast cancer and treated with neoadjuvant anthracycline-based chemotherapies.

Neoadjuvant anthracycline-based chemotherapy (NAC) is a major regimen for the treatment of local advanced breast cancer (LABC), while resistance to NAC remains a paramount clinical obstacle. To investigate the role of heat shock protein 27 (Hsp27) and/or topoisomerase IIα (TopoIIα) in LABC patients treated with NAC, we performed this retrospective study. Associations of Hsp27 transcripts with clinic-pathological characteristics, survival and drug response were investigated in public databases. Hsp27-related genes were identified, followed by functional enrichment analyses. Besides, two protein-protein interaction networks were built. Then, tumors from 103 patients who were diagnosed with LABC and received NAC were collected, and Hsp27 and TopoIIα were examined by Immunohistochemistry (IHC). Chi-square or Fisher's exact tests were performed, as well as survival analyses. Either at the transcriptional level in public databases or at the protein level tested by IHC, a high level of Hsp27 was associated with aggressive tumor characteristics such as lymph node invasion and chemotherapy resistance. Hsp27-related genes mostly involved in the metabolic pathway and the gamete generation biological process. An elevated Hsp27 indicated a poor prognosis in patients with breast cancer (log-rank test P = 0.002 and 0.004 for disease-free survival [DFS] and overall survival [OS], respectively), while it might not be an independent predictor. Of note, tumors with high TopoIIα expression (TopoIIα+) was less likely to express Hsp27 (Hsp27+), in contrast to those with TopoIIα negativity (31.1% vs. 86.2%, P<0.001), and survival analyses revealed that patients with Hsp27+ and TopoIIα- tumors had a significantly lower DFS and OS (log-rank test P < 0.001 and 0.001, respectively), in contrast to the other three groups. Hsp27 was associated with aggressive breast cancers and more predictable for the prognosis of LABC patients treated with NAC when concomitantly considering TopoIIα expression.

Polyheterocyclic peptidomimetics: Parallel solid phase synthesis of oligo cyclic guanidines and their inhibition activity against Mycobacterium tuberculosis DNA gyrase

Polyheterocycles are one of the most desired synthetic targets due to their numerous and valuable applications in various fields. We report the design and the parallel synthesis of novel linear oligocyclic guanidine peptidomimetics from predesigned reduced polyamides. A screening of these compounds identified active Mycobacterium tuberculosis DNA gyrase inhibitors which do not inhibit human DNA topoisomerase IIα and topoisomerase I.

Based on network pharmacology and experiments to explore the underlying mechanism of Mahonia bealei (Fortune) Carrière for treating alcoholic hepatocellular carcinoma

Ethnopharmacological relevanceMahonia bealei (Fortune) Carrière (M. bealei) is a traditional medicine widely used by the Hmong community in Guizhou. It possesses diverse biological activities and shows promise in cancer treatment; however, contemporary pharmacological research in this area is lacking. Aims of the studyThis study aimed to investigate the effects and underlying mechanisms of M. bealei on alcoholic hepatocellular carcinoma (HCC). Materials and methodsWe initially employed the LC-MS/MS method to identify the compounds present in M. bealei serum. Subsequently, its potential targets were predicted using public databases. Bioinformatics and network pharmacology approaches, such as univariate Cox regression and random forest (RF) algorithms, were utilized to identify differentially expressed genes (DEGs) associated with the prognosis of alcoholic HCC. Survival curve and receiver operating characteristic (ROC) analyses were conducted using alcoholic HCC-related data from TCGA and GEO to determine the diagnostic value of the identified DEGs. Molecular docking using the CDOCKER approach based on CHARMm was performed to validate the affinity between the predictive compounds and targets. Additionally, we evaluated the impact of M. bealei on cell proliferation, migration, and conducted western blot assays. ResultsThe LC-MS/MS approach identified 17 therapeutic components and predicted 483 component-related targets, of which 63 overlapped with alcoholic HCC targets and were considered potential therapeutic targets. GO and KEGG pathway analysis revealed significant associations between the 63 overlapping targets and alcoholic HCC progression. Through various approaches in the Cytoscape 3.9.0 software, we confirmed 9 hub genes (CDK1, CXCR4, DNMT1, ESR1, KIT, PDGFRB, SERPINE1, TOP2A, and TYMS) as core targets. TOP2A and CDK1 genes were identified as advantageous for diagnosing alcoholic HCC using univariate Cox regression, RF, survival curve, and ROC analysis. Molecular docking analysis demonstrated strong binding affinity between key bioactive components cyclamic acid, perfluoroalkyl carboxylic acid, perfluorosulfonic acid, alpha-linolenic acid, adenosine receptor antagonist (CGS 15943), and Prodigiosin and TOP2A and CDK1. In vitro experiments confirmed that M. bealei significantly suppressed cell proliferation and migration of HepG2 cells, while downregulating TOP2A and CDK1 expression. ConclusionThis study highlights the potential of M. bealei as a natural medicine for the treatment of alcoholic HCC. Six compounds (cyclamic acid, perfluoroalkylic carboxylic acids, perfluorosulfonic acid, alpha-linolenic acid, adenosine receptor antagonist (CGS 15943), and Prodigiosin) present in M. bealei serum may exhibit therapeutic effects against alcoholic HCC by downregulating CDK1 and TOP2A expression levels in vitro.

The role of TOP2A in immunotherapy and vasculogenic mimicry in non-small cell lung cancer and its potential mechanism

Type IIA topoisomerase (TOP2A) is significantly associated with malignant tumor development, invasion, treatment and its prognosis, and has been shown to be a therapeutic target against cancer. In contrast, the role of TOP2A in the immunotherapy of non-small cell lung cancer as well as in Vasculogenic mimicry (VM) formation and its potential mechanisms are unclear. The aim of this study was to investigate the role of TOP2A in proliferation, skeleton regulation, motility and VM production in non-small cell lung cancer and its mechanisms by using bioinformatics tools and molecular biology experiments. Subgroup analysis showed that the low-risk group had a better prognosis, while the high-risk group was positively correlated with high tumor mutational load, M1-type macrophage infiltration, immune checkpoint molecule expression, and immunotherapy efficacy. As confirmed by further clinical specimens, the presence of TOP2A and VM was significantly and positively correlated with poor prognosis. Our study established a model based on significant co-expression of TOP2A genes, which significantly correlated with mutational load and immunotherapy outcomes in patients with non-small cell lung cancer. Further mechanistic exploration suggests that TOP2A plays an important role in immunotherapy and VM formation in NSCLC through upregulation of Wnt3a and PD-L1 expression.

Cytotoxic flavone-C-glycosides from the leaves of Dypsis pembana (H.E.Moore) Beentje & J.Dransf., Arecaceae: in vitro and molecular docking studies

BackgroundCancer poses a health threat, with an increased incidence worldwide. Thus, it is essential to develop new natural anticancer agents. Dypsis pembana (H.E.Moore) Beentje & J.Dransf (DP) is an ornamental plant belonging to the family Arecaceae. This study aimed to isolate and identify phytoconstituents from the leaves of this plant and evaluate their in vitro cytotoxic activities.MethodsDifferent chromatographic techniques were applied to fractionate the hydro-alcoholic extract of DP and separate the major phytoconstituents. The isolated compounds were structurally elucidated based on their physical and spectroscopic data. The in vitro cytotoxic activities of the crude extract and fractions thereof were evaluated against human colon carcinoma (HCT-116), human breast carcinoma (MCF-7), and human hepatocellular carcinoma (HepG-2) cell lines via MTT assay. Moreover, selected isolates were tested against HepG-2 cell line. Molecular docking analysis was performed to investigate the interactions of these compounds with two potential targets, the human topoisomerase IIα and cyclin-dependent kinase 2 enzymes.ResultsThirteen diverse compounds were reported for the first time from DP, providing significant chemotaxonomic biomarkers. Among tested compounds, vicenin-II (7) was the most cytotoxic against HepG-2 cell line, with an IC50 value of 14.38 µg/mL, followed by isovitexin (13) (IC50 of 15.39 µg/mL). These experimental findings were complemented by molecular docking, which demonstrated that vicenin-II exhibited superior enzyme-binding affinities to the studied vital targets and shed light on the structure–activity relationships among the investigated flavone-C-glycosides members.ConclusionThe phytochemical profile of DP was characterized for the first time, reflecting chemotaxonomic data about the concerned species, genus, or even the family. Biological and computational findings revealed that vicenin-II and isovitexin are possible lead structures as inhibitors of the human topoisomerase IIα and cyclin-dependent kinase 2 enzymes.

Expression of microRNAs and their target genes in melanomas originating from gynecologic sites.

Melanomas from gynecologic sites (MOGS) are rare and have poor survival. MicroRNAs (miRs) regulate gene expression and are dysregulated in cancer. We hypothesized that MOGS would display unique miR and mRNA expression profiles. The miR and mRNA expression profile in RNA from formalin fixed, paraffin embedded vaginal melanomas (relative to vaginal mucosa) and vulvar melanomas (relative to cutaneous melanoma) were measured with the Nanostring Human miRNA assay and Tumor Signaling mRNA assay. Differential patterns of expression were identified for 21 miRs in vaginal and 47 miRs in vulvar melanoma (fold change >2, p<0.01). In vaginal melanoma, miR-145-5p (tumor suppressor targeting TLR4, NRAS) was downregulated and miR-106a-5p, miR-17-5p, miR-20b-5p (members of miR-17-92 cluster) were upregulated. In vulvar melanoma, known tumor suppressors miR-200b-3p and miR-200a-3p were downregulated, and miR-20a-5p and miR-19b-3p, from the miR-17-92 cluster, were upregulated. Pathway analysis showed an enrichment of "proteoglycans in cancer". Among differentially expressed mRNAs, topoisomerase IIα (TOP2A) was upregulated in both MOGS. Gene targets of dysregulated miRs were identified using publicly available databases and Pearson correlations. In vaginal melanoma, suppressor of cytokine signaling 3 (SOCS3) was downregulated, was a validated target of miR-19b-3p and miR-20a-5p and trended toward a significant inverse Pearson correlation with miR-19b-3p (p = 0.093). In vulvar melanoma, cyclin dependent kinase inhibitor 1A (CDKN1A) was downregulated, was the validated target of 22 upregulated miRs, and had a significant inverse Pearson correlation with miR-503-5p, miR-130a-3p, and miR-20a-5p (0.005 < p < 0.026). These findings support microRNAs as mediators of gene expression in MOGS.

Extensive Bioinformatics Analyses Reveal a Phylogenetically Conserved Winged Helix (WH) Domain (Zτ) of Topoisomerase IIα, Elucidating Its Very High Affinity for Left-Handed Z-DNA and Suggesting Novel Putative Functions

The dynamic processes operating on genomic DNA, such as gene expression and cellular division, lead inexorably to topological challenges in the form of entanglements, catenanes, knots, “bubbles”, R-loops, and other outcomes of supercoiling and helical disruption. The resolution of toxic topological stress is the function attributed to DNA topoisomerases. A prominent example is the negative supercoiling (nsc) trailing processive enzymes such as DNA and RNA polymerases. The multiple equilibrium states that nscDNA can adopt by redistribution of helical twist and writhe include the left-handed double-helical conformation known as Z-DNA. Thirty years ago, one of our labs isolated a protein from Drosophila cells and embryos with a 100-fold greater affinity for Z-DNA than for B-DNA, and identified it as topoisomerase II (gene Top2, orthologous to the human UniProt proteins TOP2A and TOP2B). GTP increased the affinity and selectivity for Z-DNA even further and also led to inhibition of the isomerase enzymatic activity. An allosteric mechanism was proposed, in which topoII acts as a Z-DNA-binding protein (ZBP) to stabilize given states of topological (sub)domains and associated multiprotein complexes. We have now explored this possibility by comprehensive bioinformatic analyses of the available protein sequences of topoII representing organisms covering the whole tree of life. Multiple alignment of these sequences revealed an extremely high level of evolutionary conservation, including a winged-helix protein segment, here denoted as Zτ, constituting the putative structural homolog of Zα, the canonical Z-DNA/Z-RNA binding domain previously identified in the interferon-inducible RNA Adenosine-to-Inosine-editing deaminase, ADAR1p150. In contrast to Zα, which is separate from the protein segment responsible for catalysis, Zτ encompasses the active site tyrosine of topoII; a GTP-binding site and a GxxG sequence motif are in close proximity. Quantitative Zτ-Zα similarity comparisons and molecular docking with interaction scoring further supported the “B-Z-topoII hypothesis” and has led to an expanded mechanism for topoII function incorporating the recognition of Z-DNA segments (“Z-flipons”) as an inherent and essential element. We further propose that the two Zτ domains of the topoII homodimer exhibit a single-turnover “conformase” activity on given G(ate) B-DNA segments (“Z-flipins”), inducing their transition to the left-handed Z-conformation. Inasmuch as the topoII-Z-DNA complexes are isomerase inactive, we infer that they fulfill important structural roles in key processes such as mitosis. Topoisomerases are preeminent targets of anti-cancer drug discovery, and we anticipate that detailed elucidation of their structural–functional interactions with Z-DNA and GTP will facilitate the design of novel, more potent and selective anti-cancer chemotherapeutic agents.

Targeted sequencing of cancer-related genes reveals a recurrent TOP2A variant which affects DNA binding and coincides with global transcriptional changes in glioblastoma.

High-grade gliomas are aggressive, deadly primary brain tumors. Median survival of patients with glioblastoma (GBM, WHO grade 4) is 14 months and <10% of patients survive 2 years. Despite improved surgical strategies and forceful radiotherapy and chemotherapy, the prognosis of GBM patients is poor and did not improve over decades. We performed targeted next-generation sequencing with a custom panel of 664 cancer- and epigenetics-related genes, and searched for somatic and germline variants in 180 gliomas of different WHO grades. Herein, we focus on 135 GBM IDH-wild type samples. In parallel, mRNA sequencing was accomplished to detect transcriptomic abnormalities. We present the genomic alterations in high-grade gliomas and the associated transcriptomic patterns. Computational analyses and biochemical assays showed the influence of TOP2A variants on enzyme activities. In 4/135 IDH-wild type GBMs we found a novel, recurrent mutation in the TOP2A gene encoding topoisomerase 2A (allele frequency [AF] = 0.03, 4/135 samples). Biochemical assays with recombinant, wild type (WT) and variant proteins demonstrated stronger DNA binding and relaxation activity of the variant protein. GBM patients carrying the altered TOP2A had shorter overall survival (median OS 150 vs 500 days, P = .0018). In the GBMs with the TOP2A variant we found transcriptomic alterations consistent with splicing dysregulation.luA novel, recurrent TOP2A mutation, which was found exclusively in four GBMs, results in the TOP2A E948Q variant with altered DNA binding and relaxation activities. The deleterious TOP2A mutation resulting in transcription deregulation in GBMs may contribute to disease pathology.

The root essential oil from the Tunisian endemic plant Ferula tunetana: Chemical composition, biological evaluation, molecular docking analysis and drug-likeness prediction

Oxidative stress is closely related to cancer aspects, such as the induction of gene mutations resulting from cellular injury and the effects on transcription and signal transduction factors. In addition, antibiotic resistance is also linked with oxidative stress, which could contribute to the selection of resistant bacterial strains. With this in mind, and considering that essential oils are well known to display antioxidant, antimicrobial, and cytotoxic activities, this study was destined to investigate the chemical composition and to screen these properties for the root essential oil (REO) of the Tunisian endemic species Ferula tunetana Pomel ex Batt. The REO GC/MS analysis led to the identification of nine compounds, representing 94.5% of the total oil composition. The phytochemical profile of this essential oil (EO) was characterized by the dominance of sesquiterpenes, comprising 11.7% of sesquiterpene hydrocarbons and 82.8% of oxygenated sesquiterpenes. The three major constituents of the EO were caryophyllene oxide (33.9%), α-cyperone (13.9%), and 14-hydroxy-9-epi-(E)-caryophyllene (12.3%). REO showed a good antioxidant potential against DPPH (IC50 = 30.13 ± 0.28 μg/mL), O2•- (IC50 = 42.87 ± 0.81 μg/mL) and H2O2 (IC50 = 48.03 ± 1.21 μg/mL). Additionally, the antimicrobial activity results showed that REO had a strong antibacterial potential against all target microbial strains, including five Gram-negative, six Gram-positive bacteria, and two Candida species (MICs = 0.039–0.625 mg/mL). Furthermore, the extracted EO was found to have good cytotoxic properties against five human cell lines viz. HT-29, HCT-116, HeLa, A549 and U937, with IC50 values ranging from 3.37 ± 0.02 to 46.66 ± 1.22 μg/mL. The main REO constituents were docked to the human DNA topoisomerase IIα enzyme and the in vitro cellular toxicities were rationalized. The drug-likeness of the main compounds identified in the studied EO was predicted. Overall, the results of the current study prove that the EO of F. tunetana roots has a noteworthy antioxidant potential and represents an interesting candidate to treat infectious diseases and cancer.

O-GlcNAcylation promotes topoisomerase IIα catalytic activity in breast cancer chemoresistance.

DNA topoisomerase IIα (TOP2A) plays a vital role in replication and cell division by catalytically altering DNA topology. It is a prominent target for anticancer drugs, but clinical efficacy is often compromised due to chemoresistance. In this study, we investigate the role of TOP2A O-GlcNAcylation in breast cancer cells and patient tumor tissues. Our results demonstrate that elevated TOP2A, especially its O-GlcNAcylation, promotes breast cancer malignant progression and resistance to adriamycin (Adm). O-GlcNAcylation at Ser1469 enhances TOP2A chromatin DNA binding and catalytic activity, leading to resistance to Adm in breast cancer cells and xenograft models. Mechanistically, O-GlcNAcylation-modulated interactions between TOP2A and cell cycle regulators influence downstream gene expression and contribute to breast cancer drug resistance. These results reveal a previously unrecognized mechanistic role for TOP2A O-GlcNAcylation in breast cancer chemotherapy resistance and provide support for targeting TOP2A O-GlcNAcylation in cancer therapy.

Casticin as potential anticancer agent: recent advancements in multi-mechanistic approaches.

Plants, with their range of pharmacologically active molecules, represent the most promising source for the production of new anticancer drugs and for the formulation of adjuvants in chemotherapy treatments to reduce drug content and/or counteract the side effects of chemotherapy. Casticin is a major bioactive flavonoid isolated from several plants, mainly from the Vitex species. This compound is well known for its anti-inflammatory and antioxidant properties, which are mainly exploited in traditional medicine. Recently, the antineoplastic potential of casticin has attracted the attention of the scientific community for its ability to target multiple cancer pathways. The purpose of this review is, therefore, to present and critically analyze the antineoplastic potential of casticin, highlighting the molecular pathways underlying its antitumor effects. Bibliometric data were extracted from the Scopus database using the search strings "casticin" and "cancer" and analyzed using VOSviewer software to generate network maps to visualize the results. Overall, more than 50% of the articles were published since 2018 and even more recent studies have expanded the knowledge of casticin's antitumor activity by adding interesting new mechanisms of action as a topoisomerase IIα inhibitor, DNA methylase 1 inhibitor, and an upregulator of the onco-suppressive miR-338-3p. Casticin counteracts cancer progression through the induction of apoptosis, cell cycle arrest, and metastasis arrest, acting on several pathways that are generally dysregulated in different types of cancer. In addition, they highlight that casticin can be considered as a promising epigenetic drug candidate to target not only cancer cells but also cancer stem-like cells.

Methylated histones on mitotic chromosomes promote topoisomerase IIα function for high fidelity chromosome segregation

DNA Topoisomerase IIα (TopoIIα) decatenates sister chromatids, allowing their segregation in mitosis. Without the TopoIIα Strand Passage Reaction (SPR), chromosome bridges and ultra-fine DNA bridges (UFBs) arise in anaphase. The TopoIIα C-terminal domain is dispensable for the SPR invitro but essential for mitotic functions invivo. Here, we present evidence that the Chromatin Tether (ChT) within the CTD interacts with specific methylated nucleosomes and is crucial for high-fidelity chromosome segregation. Mutation of individual αChT residues disrupts αChT-nucleosome interaction, induces loss of segregation fidelity and reduces association of TopoIIα with chromosomes. Specific methyltransferase inhibitors reducing histone H3 or H4 methylation decreased TopoIIα at centromeres and increased segregation errors. Methyltransferase inhibition did not further increase aberrant anaphases in the ChT mutants, indicating a functional connection. The evidence reveals novel cellular regulation whereby TopoIIα specifically interacts with methylated nucleosomes via the αChT to ensure high-fidelity chromosome segregation.

Exploration of anti-leukemic effect of soft coral-derived 13-acetoxysarcocrassolide: Induction of apoptosis via oxidative stress as a potent inhibitor of heat shock protein 90 and topoisomerase II.

13-Acetoxysarcocrassolide (13-AC) is a marine cembranoid derived from the aquaculture soft coral of Lobophytum crassum. The cytotoxic effect of 13-AC against leukemia cells was previously reported but its mechanism of action is still unexplored. In the current study, we showed that 13-AC induced apoptosis of human acute lymphoblastic leukemia Molt4 cells, as evidenced by the cleavage of PARP and caspases, phosphatidylserine externalization, as well as the disruption of mitochondrial membrane potential. The use of N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, attenuated the cytotoxic effect induced by 13-AC. Molecular docking and thermal shift assay indicated that the cytotoxic mechanism of action of 13-AC involved the inhibition of heat shock protein 90 (Hsp 90) activity by eliciting the level of Hsp 70 and topoisomerase IIα in Molt4 cells. 13-AC also exhibited potent antitumor activity by reducing the tumor volume (48.3%) and weight (72.5%) in the in vivo Molt4 xenograft mice model. Our findings suggested that the marine cembranoid, 13-AC, acted as a dual inhibitor of Hsp 90 and topoisomerase IIα, exerting more potent apoptotic activity via the enhancement of ROS generation.

Abstract 4386: Comparison of cytotoxicity of anthracycline based antineoplastic drugs in breast cancer

Abstract Mitoxantrone (MTX) is used for the chemotherapeutic treatment of breast cancer, but it has a dose-limiting cardiotoxicity. One of the mechanisms of MTX cytotoxicity is by inhibiting the topoisomerase II enzyme, which is crucial for maintaining cellular processes like replication. The aim of the study is to investigate if an alternative analog of MTX, named KP71 would be a suitable antineoplastic drug with reduced cardiovascular side effects. Cytotoxicity of both drugs in breast cancer cell lines MDA-MB-468, MDA-MB-231, MCF7, the non-neoplastic breast cell line MCF10, cardiac human fibroblasts (HFB), and human umbilical vein endothelial cells (HUVEC) was assessed by MTT at 96h. The IC50 values (nM) for MTX/KP71 were as shown in table below. DNA decatenation assay demonstrated topoisomerase II inhibition with values of IC50 = 2.9/12.3 µM for topoisomerase IIα, and 2.1/7.0 µM for topoisomerase IIβ for MTX/KP71 respectively. Western blotting showed DNA damage by the gradation in γH2AX expression at different concentrations of MTX and KP71 at 6 h and 24 h treatment. KP71 was found to retain the ability to inhibit the proliferation of neoplastic cell lines in vitro with a slower profile of cytotoxicity on non-neoplastic cell lines as compared to MTX. KP71 partly exerted cytotoxicity by damaging DNA and inhibiting topoisomerase II enzyme activity. Organotypic HFB-HUVEC co-cultures were used to investigate the effects of anthracycline drugs in angiogenesis. Although both drugs reduced the number of tubules (and branches) compared to control (absence of drug), there were not significant differences between MTX and KP71. MTT IC50 (nM) MTX KP71 MDA-MB-468 27 50 MDA-MB231 38 169 MCF7 63 150 MCF10 35 90 HFB 160 1177 HUVEC 56 93 Citation Format: Mitali Singhal, Jacobo Elies Gomez, Sanjit Nayak, Kirsten Riches Suman, Amalia Ruiz Estrada. Comparison of cytotoxicity of anthracycline based antineoplastic drugs in breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4386.

Basis for the discrimination of supercoil handedness during DNA cleavage by human and bacterial type II topoisomerases.

To perform double-stranded DNA passage, type II topoisomerases generate a covalent enzyme-cleaved DNA complex (i.e. cleavage complex). Although this complex is a requisite enzyme intermediate, it is also intrinsically dangerous to genomic stability. Consequently, cleavage complexes are the targets for several clinically relevant anticancer and antibacterial drugs. Human topoisomerase IIα and IIβ and bacterial gyrase maintain higher levels of cleavage complexes with negatively supercoiled over positively supercoiled DNA substrates. Conversely, bacterial topoisomerase IV is less able to distinguish DNA supercoil handedness. Despite the importance of supercoil geometry to the activities of type II topoisomerases, the basis for supercoil handedness recognition during DNA cleavage has not been characterized. Based on the results of benchtop and rapid-quench flow kinetics experiments, the forward rate of cleavage is the determining factor of how topoisomerase IIα/IIβ, gyrase and topoisomerase IV distinguish supercoil handedness in the absence or presence of anticancer/antibacterial drugs. In the presence of drugs, this ability can be enhanced by the formation of more stable cleavage complexes with negatively supercoiled DNA. Finally, rates of enzyme-mediated DNA ligation do not contribute to the recognition of DNA supercoil geometry during cleavage. Our results provide greater insight into how type II topoisomerases recognize their DNA substrates.