The correlation study between TOP2A gene expression in circulating tumor cells and chemotherapeutic drug resistance of patients with breast cancer.

BACKGROUND: Patients with breast cancer (BC) at advanced stages have poor outcomes because of high rate of recurrence and metastasis. Biomarkers for predicting prognosis remain to be explored. This study aimed to evaluate the relationships between circulating tumor cells (CTCs) and outcomes of BC patients. PATIENTS AND METHODS: A total of 50 female were enrolled in this study. Their diagnoses were determined by clinical characteristics, image data, and clinical pathology. CTC subtypes and TOP2A gene expression on CTCs were detected by CanPatrol™ technology and triple color in situ RNA hybridization (RNA-ISH), which divided into epithelial CTCs (eCTCs), mesenchymal CTCs (MCTCs), and hybrid CTCs (HCTCs) based on their surface markers. Hormone receptor, including estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) expression, was measured by immunohistochemistry (IHC) method before treatment. The risk factors for predicting recurrence and metastasis were calculated by COX risk regression model. The progression-free survival (PFS) of patients was determined using Kaplan-Meier survival curve. RESULTS: The patients with a large tumor size (≥ 3 cm) and advanced tumor node metastasis (TNM) stages had high total CTCs (TCTCs) (P < 0.05). These patients also had high TOP2A expression level. COX risk regression analysis indicated that TOP2A expression levels in TCTCs, ER + , HER-2 + , and TNM stages were critical risk factors for recurrence and metastasis of patients (P < 0.05). The PFS of patients with ≥ 5 TCTCs, ≥ 3 HCTCs, and positive TOP2A expression in ≥ 3 TCTCs was significantly longer than that in patient with < 5 TCTCs, < 3 HCTCs, and TOP2A expression in < 3 TCTCs (P < 0.05). In contrast, the PFS of patients with positive hormone receptors (ER + , PR + , HER-2 +) also was dramatically lived longer than that in patients with negative hormone receptor expression. CONCLUSIONS: High TCTC, HCTCs, and positive TOP2A gene expression on CTCs were critical biomarkers for predicting outcomes of BC patients. Positive hormone receptor expression in BC patients has significant favor PFS.

Tumor growth-arrest effect of tetrahydroquinazoline-derivative human topoisomerase II-alpha inhibitor in HPV-negative head and neck squamous cell carcinoma.

Oral malignancies continue to have severe morbidity with less than 50% long-term survival despite the advancement in the available therapies. There is a persisting demand for new approaches to establish more efficient strategies for their treatment. In this regard, the human topoisomerase II (topoII) enzyme is a validated chemotherapeutics target, as topoII regulates vital cellular processes such as DNA replication, transcription, recombination, and chromosome segregation in cells. TopoII inhibitors are currently used to treat some neoplasms such as breast and small cells lung carcinomas. Additionally, topoII inhibitors are under investigation for the treatment of other cancer types, including oral cancer. Here, we report the therapeutic effect of a tetrahydroquinazoline derivative (named ARN21934) that preferentially inhibits the alpha isoform of human topoII. The treatment efficacy of ARN21934 has been evaluated in 2D cell cultures, 3D in vitro systems, and in chick chorioallantoic membrane cancer models. Overall, this work paves the way for further preclinical developments of ARN21934 and possibly other topoII alpha inhibitors of this promising chemical class as a new chemotherapeutic approach for the treatment of oral neoplasms.

Entangling and disentangling mitotic chromosomes.

The DNA topological challenges generated by cellular manipulation of extremely long DNA fibers remain poorly understood. In this issue of Molecular Cell, Hildebrand et al.1 describe how mitotic chromosomes are self entangled and that disentanglement requires TOP2 activity in late mitosis.

Knockdown of TOP2A suppresses IL-17 signaling pathway and alleviates the progression of ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory disease of the colonic mucosa, with a gradually increasing incidence. Therefore, it is necessary to actively seek targets for the treatment of UC. METHODS: Common differentially expressed genes (DEGs) were screened from two microarray data sets related to UC. Protein-protein interaction network was constructed to find the hub genes. The UC mouse model and cell model were induced by dextran sulfate sodium (DSS). The pathological changes of colon tissue were observed by hematoxylin-eosin staining. Immunohistochemistry and immunofluorescence were performed to detect the expressions of Ki67 and Claudin-1. The performance of mice was observed by disease activity index (DAI). The effect of TOP2A on proliferation, inflammation, oxidative stress, and interleukin-17 (IL-17) signaling pathway in UC model was measured by cell counting kit-8, enzyme-linked immunosorbent assay, and western blot. RESULTS: Through bioinformatics analysis, 295 common DEGs were screened, and the hub gene TOP2A was selected. In UC model, there was obvious inflammatory cell infiltration in the colon and less goblet cells, while si-TOP2A lessened it. More Ki67 positive cells and less Claudin-1 positive cells were observed in UC model mice. Furthermore, knockdown of TOP2A increased the body weight and colon length of UC mice, while the DAI was decreased. Through in vivo and in vitro experiments, knockdown of TOP2A also inhibited inflammation and IL-17 signaling pathway, and promoted proliferation in DSS-induced NCM460 cells. CONCLUSION: Knockdown of TOP2A alleviated the progression of UC by suppressing inflammation and inhibited IL-17 signaling pathway.

Natural product inspired diastereoselective synthesis of sugar-derived pyrano[3,2-c]quinolones and their in-silico studies.

Herein, we report the development of a diastereoselective and efficient route to construct sugar-derived pyrano[3,2-c]quinolones utilizing 1-C-formyl glycal and 4-hydroxy quinolone annulation. This methodology will open a route to synthesize nature inspired pyrano[3,2-c]quinolones. This is the first report for the stereoselective synthesis of sugar-derived pyrano[3,2-c]quinolones, where 100% stereoselectivity was observed. A total of sixteen compounds have been synthesized in excellent yields with 100% stereoselectivity. The molecular docking of the synthesized novel natural product analogues demonstrated their binding modes within the active site of type II topoisomerase. The results of the in-silico studies displayed more negative binding energies for the all the synthesized compounds in comparison to the natural product huajiosimuline A, indicating their affinity for the active pocket. Ten out of the sixteen novel synthesized compounds were found to have comparative or relatively more negative binding energy in comparison to the standard anti-cancer drug, doxorubicin. Additionally, the scalability and viability of this protocol was illustrated by the gram scale synthesis.

DNA topoisomerase II inhibition potentiates osimertinib's therapeutic efficacy in EGFR-mutant non-small cell lung cancer models.

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.

Biochemical characterization of the meiosis-essential yet evolutionarily divergent topoisomerase VIB-like protein MTOPVIB from Arabidopsis thaliana.

Formation of programmed DNA double-strand breaks is essential for initiating meiotic recombination. Genetic studies on Arabidopsis thaliana and Mus musculus have revealed that assembly of a type IIB topoisomerase VI (Topo VI)-like complex, composed of SPO11 and MTOPVIB, is a prerequisite for generating DNA breaks. However, it remains enigmatic if MTOPVIB resembles its Topo VI subunit B (VIB) ortholog in possessing robust ATPase activity, ability to undergo ATP-dependent dimerization, and activation of SPO11-mediated DNA cleavage. Here, we successfully prepared highly pure A. thaliana MTOPVIB and MTOPVIB-SPO11 complex. Contrary to expectations, our findings highlight that MTOPVIB differs from orthologous Topo VIB by lacking ATP-binding activity and independently forming dimers without ATP. Most significantly, our study reveals that while MTOPVIB lacks the capability to stimulate SPO11-mediated DNA cleavage, it functions as a bona fide DNA-binding protein and plays a substantial role in facilitating the dsDNA binding capacity of the MOTOVIB-SPO11 complex. Thus, we illustrate mechanistic divergence between the MTOPVIB-SPO11 complex and classical type IIB topoisomerases.

DNA fragility at topologically associated domain boundaries is promoted by alternative DNA secondary structure and topoisomerase II activity.

CCCTC-binding factor (CTCF) binding sites are hotspots of genome instability. Although many factors have been associated with CTCF binding site fragility, no study has integrated all fragility-related factors to understand the mechanism(s) of how they work together. Using an unbiased, genome-wide approach, we found that DNA double-strand breaks (DSBs) are enriched at strong, but not weak, CTCF binding sites in five human cell types. Energetically favorable alternative DNA secondary structures underlie strong CTCF binding sites. These structures coincided with the location of topoisomerase II (TOP2) cleavage complex, suggesting that DNA secondary structure acts as a recognition sequence for TOP2 binding and cleavage at CTCF binding sites. Furthermore, CTCF knockdown significantly increased DSBs at strong CTCF binding sites and at CTCF sites that are located at topologically associated domain (TAD) boundaries. TAD boundary-associated CTCF sites that lost CTCF upon knockdown displayed increased DSBs when compared to the gained sites, and those lost sites are overrepresented with G-quadruplexes, suggesting that the structures act as boundary insulators in the absence of CTCF, and contribute to increased DSBs. These results model how alternative DNA secondary structures facilitate recruitment of TOP2 to CTCF binding sites, providing mechanistic insight into DNA fragility at CTCF binding sites.

Mitotic chromosomes are self-entangled and disentangle through a topoisomerase-II-dependent two-stage exit from mitosis.

The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations, and multi-contact 3C and find that, by contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a topoisomerase-II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias topoisomerase II activity toward decatenation. At the second stage, the loops are released, and the formation of new entanglements is prevented by lower topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed in experiments and models, a normal interphase state cannot be acquired.

Design, synthesis and in vitro anticancer activity of some new lomefloxacin derivatives.

Our main goal was to design and synthesize novel lomefloxacin derivatives that inhibit the topoisomerase II enzyme, leading to potent anticancer activity. Lomefloxacin derivatives substituted at position 3 and 7 were synthesized and screened for cytotoxic activity utilizing 60 different human cancer cell lines. Furthermore, compounds 3a,b,c,e that revealed potent broad-spectrum anticancer activity (with mean percent GI more than 47%) were further evaluated using five dose concentrations and calculating the GI50. Compound 3e was then evaluated for cell cycle analysis and demonstrated cell cycle arrest at the G2-M phase. Moreover, the mechanism of action was determined by determining the topoisomerase inhibitory activity and the molecular modeling study. Compounds 3a,b,c,e showed broad spectrum anticancer activity. Lomefloxacin derivative 5f showed selective cytotoxic activity against melanoma SK-MEL-5 cell line. Compound 3e demonstrated comparable topoisomerase II inhibition to doxorubicin with IC50 of 0.98 µM.

New imidazole-2-thiones linked to acenaphythylenone as dual DNA intercalators and topoisomerase II inhibitors: structural optimization, docking, and apoptosis studies.

In this article, a new series of 2-((3,5-disubstituted-2-thioxo-imidazol-1-yl)imino)acenaphthylen-1(2H)-ones were synthesized. Imidazole-2-thione with acenaphthylen-one gave a hybrid scaffold that integrated key structural elements essential for DNA damage via direct DNA intercalation and inhibition of the topoisomerase II enzyme. All the synthesized compounds were screened to detect their DNA damage using a terbium fluorescent probe. Results demonstrated that 4-phenyl-imidazoles 5b and 5e in addition to 4-(4-chlorophenyl)imidazoles 5h and 5j would induce detectable potent damage in ctDNA. The four most potent compounds as DNA intercalators were further evaluated for their antiproliferative activity against HepG2, MCF-7 and HCT-116 utilizing the MTT assay. The highest anticancer activity was recorded with compounds 5b and 5h against the breast cancer cell line MCF-7 which were 1.5- and 3- folds more active than doxorubicin, respectively. Therefore, imidazole-2-thione tethered acenaphthylenone derivatives can be considered as promising scaffold for the development of effective dual DNA intercalators and topoisomerase II inhibitors.

TOP2A modulates signaling via the AKT/mTOR pathway to promote ovarian cancer cell proliferation.

Ovarian cancer (OC) is a form of gynecological malignancy that is associated with worse patient outcomes than any other cancer of the female reproductive tract. Topoisomerase II α (TOP2A) is commonly regarded as an oncogene that is associated with malignant disease progression in a variety of cancers, its mechanistic functions in OC have yet to be firmly established. We explored the role of TOP2A in OC through online databases, clinical samples, in vitro and in vivo experiments. And initial analyses of public databases revealed high OC-related TOP2A expression in patient samples that was related to poorer prognosis. This was confirmed by clinical samples in which TOP2A expression was elevated in OC relative to healthy tissue. Kaplan-Meier analyses further suggested that higher TOP2A expression levels were correlated with worse prognosis in OC patients. In vitro, TOP2A knockdown resulted in the inhibition of OC cell proliferation, with cells entering G1 phase arrest and undergoing consequent apoptotic death. In rescue assays, TOP2A was confirmed to regulate cell proliferation and cell cycle through AKT/mTOR pathway activity. Mouse model experiments further affirmed the key role that TOP2A plays as a driver of OC cell proliferation. These data provide strong evidence supporting TOP2A as an oncogenic mediator and prognostic biomarker related to OC progression and poor outcomes. At the mechanistic level, TOP2A can control tumor cell growth via AKT/mTOR pathway modulation. These preliminary results provide a foundation for future research seeking to explore the utility of TOP2A inhibitor-based combination treatment regimens in platinum-resistant recurrent OC patients.

PRRX1-TOP2A interaction is a malignancy-promoting factor in human malignant peripheral nerve sheath tumours.

BACKGROUND: Paired related-homeobox 1 (PRRX1) is a transcription factor in the regulation of developmental morphogenetic processes. There is growing evidence that PRRX1 is highly expressed in certain cancers and is critically involved in human survival prognosis. However, the molecular mechanism of PRRX1 in cancer malignancy remains to be elucidated. METHODS: PRRX1 expression in human Malignant peripheral nerve sheath tumours (MPNSTs) samples was detected immunohistochemically to evaluate survival prognosis. MPNST models with PRRX1 gene knockdown or overexpression were constructed in vitro and the phenotype of MPNST cells was evaluated. Bioinformatics analysis combined with co-immunoprecipitation, mass spectrometry, RNA-seq and structural prediction were used to identify proteins interacting with PRRX1. RESULTS: High expression of PRRX1 was associated with a poor prognosis for MPNST. PRRX1 knockdown suppressed the tumorigenic potential. PRRX1 overexpressed in MPNSTs directly interacts with topoisomerase 2 A (TOP2A) to cooperatively promote epithelial-mesenchymal transition and increase expression of tumour malignancy-related gene sets including mTORC1, KRAS and SRC signalling pathways. Etoposide, a TOP2A inhibitor used in the treatment of MPNST, may exhibit one of its anticancer effects by inhibiting the PRRX1-TOP2A interaction. CONCLUSION: Targeting the PRRX1-TOP2A interaction in malignant tumours with high PRRX1 expression might provide a novel tumour-selective therapeutic strategy.

Use of CRISPR/Cas9 with Homology-Directed Repair to Gene-Edit Topoisomerase IIß in Human Leukemia K562 Cells: Generation of a Resistance Phenotype.

DNA topoisomerase IIß (TOP2ß/180; 180 kDa) is a nuclear enzyme that regulates DNA topology by generation of short-lived DNA double-strand breaks, primarily during transcription. TOP2ß/180 can be a target for DNA damage-stabilizing anticancer drugs, whose efficacy is often limited by chemoresistance. Our laboratory previously demonstrated reduced levels of TOP2ß/180 (and the paralog TOP2α/170) in an acquired etoposide-resistant human leukemia (K562) clonal cell line, K/VP.5, in part due to overexpression of microRNA-9-3p/5p impacting post-transcriptional events. To evaluate the effect on drug sensitivity upon reduction/elimination of TOP2ß/180, a premature stop codon was generated at the TOP2ß/180 gene exon 19/intron 19 boundary (AGAA//GTAA→ATAG//GTAA) in parental K562 cells (which contain four TOP2ß/180 alleles) by CRISPR/Cas9 editing with homology-directed repair to disrupt production of full-length TOP2ß/180. Gene-edited clones were identified and verified by quantitative polymerase chain reaction and Sanger sequencing, respectively. Characterization of TOP2ß/180 gene-edited clones, with one or all four TOP2ß/180 alleles mutated, revealed partial or complete loss of TOP2ß mRNA/protein, respectively. The loss of TOP2ß/180 protein correlated with decreased (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid)-induced DNA damage and partial resistance in growth inhibition assays. Partial resistance to mitoxantrone was also noted in the gene-edited clone with all four TOP2ß/180 alleles modified. No cross-resistance to etoposide or mAMSA was noted in the gene-edited clones. Results demonstrated the role of TOP2ß/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents. SIGNIFICANCE STATEMENT: Data indicated that CRISPR/Cas9 editing of the exon 19/intron 19 boundary in the TOP2ß/180 gene to introduce a premature stop codon resulted in partial to complete disruption of TOP2ß/180 expression in human leukemia (K562) cells depending on the number of edited alleles. Edited clones were partially resistant to mitoxantrone and XK469, while lacking resistance to etoposide and mAMSA. Results demonstrated the import of TOP2ß/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents.

Structure-function analysis of the ATPase domain of African swine fever virus topoisomerase.

Type II topoisomerase utilizes the energy from ATP hydrolysis to alter DNA topology during genome replication and transcription. The ATPase domain of this enzyme is required for ATP hydrolysis and plays a crucial role in coupling DNA binding and ATP turnover with the DNA strand passage reaction. The African swine fever virus (ASFV) specifically encodes a topoisomerase II (topo II), which is critical for viral replication and an attractive target for antiviral development. Here, we present a high-resolution crystal structure of the ASFV topo II ATPase domain complexed with the substrate analog AMPPNP. Structural comparison reveals that the ASFV topo II ATPase domain shares a conserved overall structure with its homologs from eukaryotes and prokaryotes but also has three characteristic regions, including the intra-molecular interface formed by the ATP-lid and QTK loop as well as helix α9, the K-loop in the transducer domain, and the antennae-like α-helix at the ATP binding domain. Mutating the key residues within these three regions impairs or abolishes the basal and DNA-stimulated ATPase activities and reduces or eliminates the relaxation activity of the holoenzyme. Our data indicate that all three regions are functionally important for the ATPase and relaxation activities and strongly suggest that ATP hydrolysis, DNA binding, and strand passage are highly coupled and managed by the allosteric coordination of multiple domains of the type II topoisomerase. Moreover, we find a promising druggable pocket in the dimeric interface of the ASFV topo II ATPase domain, which will benefit future anti-ASFV drug development. IMPORTANCE: The ATPase domain of type II topoisomerase provides energy by hydrolyzing ATP and coordinates with the DNA-binding/cleavage domain to drive and control DNA transport. The precise molecular mechanisms of how these domains respond to DNA binding and ATP hydrolysis signals and communicate with each other remain elusive. We determine the first high-resolution crystal structure of the ATPase domain of African swine fever virus (ASFV) topo II in complex with AMPPNP and biochemically investigate its function in ATPase and DNA relaxation activities. Importantly, we find that mutations at three characteristic regions of the ASFV ATPase domain produce parallel effects on the basal/DNA-stimulated ATPase and relaxation activities, implying the tight coupling of the ATP hydrolysis and strand passage process. Therefore, our data provide important implications for understanding the strand passage mechanism of the type II topoisomerase and the structural basis for developing ATPase domain-targeting antivirals against ASFV.

Anthracyclines induce cardiotoxicity through a shared gene expression response signature.

TOP2 inhibitors (TOP2i) are effective drugs for breast cancer treatment. However, they can cause cardiotoxicity in some women. The most widely used TOP2i include anthracyclines (AC) Doxorubicin (DOX), Daunorubicin (DNR), Epirubicin (EPI), and the anthraquinone Mitoxantrone (MTX). It is unclear whether women would experience the same adverse effects from all drugs in this class, or if specific drugs would be preferable for certain individuals based on their cardiotoxicity risk profile. To investigate this, we studied the effects of treatment of DOX, DNR, EPI, MTX, and an unrelated monoclonal antibody Trastuzumab (TRZ) on iPSC-derived cardiomyocytes (iPSC-CMs) from six healthy females. All TOP2i induce cell death at concentrations observed in cancer patient serum, while TRZ does not. A sub-lethal dose of all TOP2i induces limited cellular stress but affects calcium handling, a function critical for cardiomyocyte contraction. TOP2i induce thousands of gene expression changes over time, giving rise to four distinct gene expression response signatures, denoted as TOP2i early-acute, early-sustained, and late response genes, and non-response genes. There is no drug- or AC-specific signature. TOP2i early response genes are enriched in chromatin regulators, which mediate AC sensitivity across breast cancer patients. However, there is increased transcriptional variability between individuals following AC treatments. To investigate potential genetic effects on response variability, we first identified a reported set of expression quantitative trait loci (eQTLs) uncovered following DOX treatment in iPSC-CMs. Indeed, DOX response eQTLs are enriched in genes that respond to all TOP2i. Next, we identified 38 genes in loci associated with AC toxicity by GWAS or TWAS. Two thirds of the genes that respond to at least one TOP2i, respond to all ACs with the same direction of effect. Our data demonstrate that TOP2i induce thousands of shared gene expression changes in cardiomyocytes, including genes near SNPs associated with inter-individual variation in response to DOX treatment and AC-induced cardiotoxicity.

Multi-target rational design and synthesis of novel diphenyl-tethered pyrazolopyrimidines targeting EGFR and topoisomerase II with potential DNA intercalation and apoptosis induction.

Herein, we envisioned the design and synthesis of novel pyrazolopyrimidines (confirmed by elemental analysis, 1H and 13C NMR, and mass spectra) as multitarget-directed drug candidates acting as EGFR/TOPO II inhibitors, DNA intercalators, and apoptosis inducers. The target diphenyl-tethered pyrazolopyrimidines were synthesized starting from the reaction of phenyl hydrazine and ethoxymethylenemalononitrile to give aminopyrazole-carbonitrile 2. The latter hydrolysis with NaOH and subsequent reaction with 4-chlorobenzaldhyde afforded the corresponding pyrazolo[3,4-d]pyrimidin-4-ol 4. Chlorination of 4 with POCl3 and sequential reaction with different amines afforded the target compounds in good yields (up to 73 %). The growth inhibition % of the new derivatives (6a-m) was investigated against different cancer and normal cells and the IC50 values of the most promising candidates were estimated for HNO97, MDA-MB-468, FaDu, and HeLa cancer cells. The frontier derivatives (6a, 6i, 6k, 6l, and 6m) were pursued for their EGFR inhibitory activity. Compound 6l decreased EGFR protein concentration by a 6.10-fold change, compared to imatinib as a reference standard. On the other side, compounds (6a, 6i, 6k, 6l, and 6m) underwent topoisomerase II (TOPO II) inhibitory assay. In particular, compounds 6a and 6l exhibited IC50s of 17.89 and 19.39 µM, respectively, surpassing etoposide with IC50 of 20.82 µM. Besides, the DNA fragmentation images described the great potential of both candidates 6a and 6l in inducing DNA degradation at lower concentrations compared to etoposide and doxorubicin. Moreover, compound 6l, with the most promising EGFR/TOPO II inhibition and DNA intercalation, was selected for further investigation for its apoptosis induction ability by measuring caspases 3, 7, 8, and 9, Bax, p53, MMP2, MMP9, and BCL-2 proteins. Additionally, molecular docking was used to explain the SAR results based on the differences in the molecular features of the investigated congeners and the target receptors' topology.

Olympic gels formed through catenation of dsDNA rings regulated by topoisomerase II: A coarse-grained model.

Topological regulation of DNA by topoisomerases in cells is very crucial for life. We propose a coarse-grained model to study the catenation process of double-stranded DNA (dsDNA) rings regulated by topoisomerase II (TOP2) and provide a computational method to characterize the topological structures of the Olympic gels obtained. The function of TOP2 in the catenation of dsDNA rings is implicitly fulfilled by operating the length of a stretchable catch bond in the dsDNA ring. After the catenation reaction of initially noncatenated dsDNA rings in the solution, the Olympic gel is obtained and the interlocked topology of the dsDNA rings can be characterized by a computational method derived from the HOMFLY polynomial, based on which the catenation degree and the complexity of catenation are quantified. Detailed dependence of the catenation degree and the complexity of the catenated topology on key parameters, including the size of the transient broken gap and the duration time of the break on the dsDNA ring during operation by TOP2, the initial molar ratio of TOP2 to the dsDNA rings, and the reaction temperature, has been investigated.

Localization and quantitative distribution of a chromatin structural protein Topoisomerase II on plant chromosome using HVTEM and UHVTEM.

Topoisomerase II (TopoII) is an essential structural protein of the metaphase chromosome. It maintains the axial compaction of chromosomes during metaphase. It is localized at the axial region of chromosomes and accumulates at the centromeric region in metaphase chromosomes. However, little is known about TopoII localization and distribution in plant chromosomes, except for several publications. We used high voltage transmission electron microscopy (HVTEM) and ultra-high voltage transmission electron microscopy (UHVTEM) in conjunction with immunogold labeling and visualization techniques to detect TopoII and investigate its localization, alignment, and density on the barley chromosome at 1.4 nm scale. We found that HVTEM and UHVTEM combined with immunogold labeling is suitable for the detection of structural proteins, including a single molecule of TopoII. This is because the average size of the gold particles for TopoII visualization after silver enhancement is 8.9 ± 3.9 nm, which is well detected. We found that 31,005 TopoII molecules are distributed along the barley chromosomes in an unspecific pattern at the chromosome arms and accumulate specifically at the nucleolus organizer regions (NORs) and centromeric region. The TopoII density were 1.32-fold, 1.58-fold, and 1.36-fold at the terminal region, at the NORs, and the centromeric region, respectively. The findings of TopoII localization in this study support the multiple reported functions of TopoII in the barley metaphase chromosome.

Chromatin damage generated by DNA intercalators leads to degradation of RNA Polymerase II.

In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much interest given their ability to inhibit RNA Polymerase I transcription (BMH-21), evict histones (Aclarubicin) or induce chromatin trapping of FACT (Curaxin CBL0137). Interestingly, these DNA intercalators lack the capacity to induce DNA damage while still retaining cytotoxic effects and stabilize p53. Herein, we report that these DNA intercalators impact chromatin biology by interfering with the chromatin stability of RNA polymerases I, II and III. These three compounds have the capacity to induce degradation of RNA polymerase II and they simultaneously enable the trapping of Topoisomerases TOP2A and TOP2B on the chromatin. In addition, BMH-21 also acts as a catalytic inhibitor of Topoisomerase II, resembling Aclarubicin. Moreover, BMH-21 induces chromatin trapping of the histone chaperone FACT and propels accumulation of Z-DNA and histone eviction, similarly to Aclarubicin and CBL0137. These DNA intercalators have a cumulative impact on general transcription machinery by inducing accumulation of topological defects and impacting nuclear chromatin. Therefore, their cytotoxic capabilities may be the result of compounding deleterious effects on chromatin homeostasis.