Topoisomerase IIβ Research Articles

A phase II clinical study of 13‐deoxy, 5‐iminodoxorubicin (GPX‐150) with metastatic and unresectable soft tissue sarcoma

Background13‐Deoxy, 5‐iminodoxorubicin (GPX‐150) is a doxorubicin (DOX) analog synthesized to reduce the formation of reactive oxygen species and the cardiotoxic metabolite, doxorubiciniol, the two pathways that are linked to the irreversible, cumulative dose‐dependent cardiotoxicity of DOX. In a preclinical chronic models and a phase I clinical study of GPX‐150, no irreversible, cumulative dose‐dependent cardiotoxicity was demonstrated. Recent studies suggest that DOX cardiotoxicity may be mediated, at least in part, by the poisoning of topoisomerase IIβ.Patients and MethodsAn open‐label, single‐arm phase II clinical study in metastatic and unresectable soft tissue sarcoma (STS) patients was initiated to further evaluate the efficacy and safety of GPX‐150, including cardiac function, specifically left ventricular ejection fraction (LVEF).ResultsGPX‐150 was administered at 265 mg/m2 every 3 weeks for up to 16 doses with prophylactic G‐CSF until progression, death, or patient withdrawal from the study. GPX‐150 exhibited efficacy assessed as progression‐free survival (PFS) rates of 38% and 12% at 6 and 12 months and an overall survival rate of 74% and 45% at 6 and 12 months. GPX‐150–treated patients did not develop any evidence of irreversible, cumulative dose‐dependent chronic cardiotoxicity. Toxicities included grade 3 anemia, neutropenia, and one grade 4 leukopenia. Correlative analysis demonstrated that GPX‐150 was more selective than DOX for the inhibition of topoisomerase IIα over IIβ in vitro.ConclusionThese results suggest future studies are warranted to further evaluate the clinical efficacy of GPX‐150 in STS, perhaps at doses higher than 265 mg/m2.

Examination of the Effect of Copper (II) α‐(N)‐Heterocyclic Thiosemicarbazone Complexes on DNA Topoisomerase IIβ

During replication, transcription, and cell division, knots and tangles can form in DNA. To relieve this stress, type II topoisomerases are employed, which use a transient enzyme‐linked double strand break to remove the knots and tangles formed within the DNA. Furthermore, because the cell has an immediate need for topoisomerase II activity in order to properly perform cellular functions, topoisomerase II has become a target of interest for anticancer therapy. Over the past several years, a class of compounds termed α‐(N)‐heterocyclic thiosemicarbazones have been identified as a possible anticancer therapy option due to their ability to impact topoisomerase II activity. Moreover, literature evidence suggests that copper complexes [Cu(II)] of α‐(N)‐heterocyclic thiosemicarbazones act as catalytic inhibitors of topoisomerase II similar to the topoisomerase II inhibitor Dexrazoxane. Previously, our lab demonstrated activity against human topoisomerase IIα, but there is a second isoform in humans, topoisomerase IIβ, which has been largely unexplored with thiosemicarbazones. Therefore, we set out to determine the mechanism of two Cu(II) complexes of α‐(N)‐heterocyclic thiosemicarbazones as inhibitors of topoisomerase IIβ. The Cu(II) complexes, copper(II) acetylpyridine‐ethylthiosemicarbazone [Cu(APY‐ETSC)Cl] and copper(II)benzoylpyridine‐ethylthiosemicarbazone [Cu(BZP‐ETSC)Cl] were examined for their ability to alter the catalytic activity of topoisomerase IIβ. Both Cu(II) complexes were effective at inhibiting DNA relaxation at around 10–25 μM. Additionally, both Cu(II) complexes increased double‐stranded DNA cleavage peaking around 25–50 μM. When narrowing the focus to just Cu(APY‐ETSC)Cl and Cu(BZP‐ETSC)Cl, it was determined that both compounds interfere with ATP hydrolysis of topoisomerase IIβ, which is required for strand passage. Additional experiments determined that ATP cannot outcompete Cu(APY‐ETSC)Cl or Cu(BZP‐ETSC)Cl for binding to topoisomerase IIβ, which suggests that these compounds bind outside the ATP binding pocket but allosterically impact ATP hydrolysis. Lastly, we examined whether the Cu(II) complexes could stabilize the N‐terminal ATPase domain in a closed conformation similar to ATP. Both compounds stabilize the N‐terminal closed conformation of the ATPase domain, suggesting a mechanism for topoisomerase II inhibition by these compounds. Taken together, these results provide evidence that Cu(II) complexes of α‐(N)‐heterocyclic thiosemicarbazones catalytically inhibit topoisomerase IIβ similar to topoisomerase IIα. Additionally, the mechanism involves binding to the ATPase domain outside of the ATP pocket and inducing a closed N‐terminal gate of topoisomerase II. These results also provide a possible explanation for the increase in topoisomerase II‐mediated DNA cleavage observed in the presence of Cu(II) thiosemicarbazone complexes.Support or Funding InformationJMK and JED were supported by LUCOPHS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NMDA Receptor Signaling Mediates cFos Expression via Top2β-Induced DSBs in Glioblastoma Cells.

The activation of Ca2+-permeable N-methyl-D-aspartic acid (NMDA) receptor channels (NMDARs) is crucial for the development and survival of neurons, but many cancers use NMDAR-mediated signaling as well, enhancing the growth and invasiveness of tumors. Thus, NMDAR-dependent pathways emerge as a promising target in cancer therapy. Here, we use the LN229 and U-87MG glioblastoma multiforme (GBM) cells and immunofluorescence staining of 53BP1 to analyze NMDAR-induced DNA double-strand breaks (DSBs), which represent an important step in the NMDAR signaling pathway in neurons by facilitating the expression of early response genes. Our results show that NMDAR activation leads to the induction of DSBs in a subpopulation of glioma cells. In a further analogy to neurons, our results demonstrate that the induction of DSBs in LN229 cells is dependent on the activity of topoisomerase IIβ (Top2β). Western blot analysis revealed that the inhibition of NMDARs, cAMP-responsive element binding transcription factor (CREB) and Top2β decreased the expression of the proto-oncogene cFos. Knockdown of Top2β with siRNAs resulted in a downregulation of cFos and increased the radiosensitivity of LN229 cells in clonogenic survival. We also observed impaired cFos expression upon NMDAR and Top2β inhibition in a primary GBM cell line, suggesting that NMDAR signaling may be widely used by GBMs, demonstrating the potential of targeting NMDAR signaling proteins for GBM therapy.

Examining the Impact of Antimicrobial Fluoroquinolones on Human DNA Topoisomerase IIα and IIβ

Fluoroquinolones are a class of widely prescribed antibiotics with a broad range of activity against Gram-positive, Gram-negative, and some atypical microbes. Unfortunately, these drugs are associated with significant adverse events including neuropathy, tendinopathy, cardiac rhythm abnormalities, and mental health side effects. The mechanism by which fluoroquinolones cause many of these toxicities is unknown. The antibacterial mechanism of action involves disruption of the catalytic mechanism of type-II topoisomerases in bacteria, namely topoisomerase IV and DNA gyrase. Fluoroquinolones inhibit the ability of the enzymes to ligate cleaved DNA and result in single- and double-stranded DNA breaks. Thus, there is an interest in investigating whether human topoisomerase II is involved in mediating the adverse events associated with quinolones. Previous studies demonstrate some response of human topoisomerase IIα and IIβ to high levels of ciprofloxacin. However, it is not clear whether the concentration of ciprofloxacin utilized in those studies corresponds to concentrations that would be routinely achievable in patients. Therefore, this study set out to examine three clinically relevant fluoroquinolones along with two older agents to determine whether these compounds display activity against topoisomerase IIα and IIβ at drug concentrations that more closely approximate typical patient plasma values. On the basis of our evidence, none of the quinolones studied were able to poison DNA cleavage by either human enzyme. Ciprofloxacin, desethylene-ciprofloxacin, and the recently removed from market gemifloxacin were able to inhibit topoisomerase II-mediated DNA relaxation at concentrations of 200–300 μM. On the basis of these data, we propose that human topoisomerase II is not likely to be the main cause of these adverse events and that additional targets need to be identified to clarify the mechanisms underlying quinolone toxicities.

BRCA1-BARD1 Regulates Topoisomerase IIβ Stability in Transcription

RNA polymerase II (Pol II) dependent transcription in stimulus-inducible genes requires topoisomerase IIβ-mediated DNA strand break and the activation of DNA damage response signaling. Here we report a novel function of DNA repair proteins, involving a breast cancer 1 (BRCA1)-BRCA1 associated RING domain 1 (BARD1) complex, in transcriptional activation of stimulus-inducible genes in humans. We have found that BRCA1 is phosphorylated at S1524 by the kinases ATM and ATR during gene activation and that this phosphorylation event is essential for productive transcription. The major TOPIIβ binding site in the EGR1 TSS was mapped in the promoter between −132 and −14. We showed that BARD1 interacts with and could ubiquitinate TOPIIβ, and that BRCA1 regulates this interaction. Together, our findings indicate that the BRCA1-BARD1 complex controls TOPIIβ stability and gene association during Pol II transcription.

Novel nalidixic acid derivatives targeting topoisomerase II enzyme; Design, synthesis, anticancer activity and effect on cell cycle profile

AimDesign and synthesis of novel nalidixic acid derivatives of potent anticancer and topoisomerase II inhibitory activities were our major aim. Materials & methodsAll the newly synthesized nalidixic acid derivatives were submitted to the National Cancer Institute (NCI), Bethesda, USA and were accepted for single dose screening. Further investigation via IC50 determination of the most potent compound 6a against K-562 and SR leukemia cell lines. Finally, the topoisomerase II inhibitory activity, the cell cycle analysis and molecular docking of 6a were performed in order to identify the possible mechanism of the anticancer activity. ResultsCompound 6a showed interesting selectivity against leukemia especially K-562 and SR subpanels with IC50 35.29 µM and 13.85 µM respectively. Moreover, compound 6a revealed potent topoisomerase IIα and topoisomerase IIβ inhibitory activity compared with known topoisomerase inhibitors such as doxorubicin and topotecan with IC50 1.30 µM and 0.017 µM respectively. Cell cycle analysis indicated that compound 6a induced cell cycle arrest at G2-M phase leading to inhibition of cell proliferation and apoptosis. Molecular modeling demonstrated that the potent topoisomerase inhibitory activity of 6a was due to the interaction with the topoisomerase II enzyme through coordinate bonding with the magnesium ion Mg2+, hydrogen bonding with Asp 545 and arene cation interaction with His 759.

TOP2B: The First Thirty Years.

Type II DNA topoisomerases (EC 5.99.1.3) are enzymes that catalyse topological changes in DNA in an ATP dependent manner. Strand passage reactions involve passing one double stranded DNA duplex (transported helix) through a transient enzyme-bridged break in another (gated helix). This activity is required for a range of cellular processes including transcription. Vertebrates have two isoforms: topoisomerase IIα and β. Topoisomerase IIβ was first reported in 1987. Here we review the research on DNA topoisomerase IIβ over the 30 years since its discovery.

Polyamine-containing etoposide derivatives as poisons of human type II topoisomerases: Differential effects on topoisomerase IIα and IIβ

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.

The Roles of DNA Topoisomerase IIβ in Transcription

Type IIA topoisomerases allow DNA double helical strands to pass through each other by generating transient DNA double strand breaks βDSBs), and in so doing, resolve torsional strain that accumulates during transcription, DNA replication, chromosome condensation, chromosome segregation and recombination. Whereas most eukaryotes possess a single type IIA enzyme, vertebrates possess two distinct type IIA topoisomerases, Topo IIα and Topo IIβ. Although the roles of Topo IIα, especially in the context of chromosome condensation and segregation, have been well-studied, the roles of Topo IIβ are only beginning to be illuminated. This review begins with a summary of the initial studies surrounding the discovery and characterization of Topo IIβ and then focuses on the insights gained from more recent studies that have elaborated important functions for Topo IIβ in transcriptional regulation.

Synthesis and molecular docking studies of some novel Schiff bases incorporating 6-butylquinolinedione moiety as potential topoisomerase IIβ inhibitors.

A series of novel pyranoquinolinone-based Schiff's bases were designed and synthesized. They were evaluated for topoisomerase IIβ (TOP2B) inhibitory activity, and cytotoxicity against breast cancer cell line (MCF-7) for the development of novel anticancer agents. A molecular docking study was employed to investigate their binding and functional properties as TOP2B inhibitors, using the Discovery Studio 2.5 software, where they showed very interesting ability to intercalate the DNA–topoisomerase complex. Compounds 2a, 2c and 2f showed high docking score values (82.36% −29.98 kcal mol−1 for compound 2a, 78.18% −26.98 kcal mol−1 for compound 2c and 78.65, −28.11 kcal mol−1 for compound 2f) and revealed the highest enzyme inhibition activity. The best hit compounds exhibited highly potent TOP2B inhibitors with submicromolar IC50 at 5 µM compared to the reference doxorubicin.

Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences.

Etoposide and other topoisomerase II-targeted drugs are important anticancer therapeutics. Unfortunately, the safe usage of these agents is limited by their indiscriminate induction of topoisomerase II-mediated DNA cleavage throughout the genome and by a lack of specificity toward cancer cells. Therefore, as a first step toward constraining the distribution of etoposide-induced DNA cleavage sites and developing sequence-specific topoisomerase II-targeted anticancer agents, we covalently coupled the core of etoposide to oligonucleotides centered on a topoisomerase II cleavage site in the PML gene. The initial sequence used for this ‘oligonucleotide-linked topoisomerase inhibitor’ (OTI) was identified as part of the translocation breakpoint of a patient with acute promyelocytic leukemia (APL). Subsequent OTI sequences were derived from the observed APL breakpoint between PML and RARA. Results indicate that OTIs can be used to direct the sites of etoposide-induced DNA cleavage mediated by topoisomerase IIα and topoisomerase IIβ. OTIs increased levels of enzyme-mediated cleavage by inhibiting DNA ligation, and cleavage complexes induced by OTIs were as stable as those induced by free etoposide. Finally, OTIs directed against the PML-RARA breakpoint displayed cleavage specificity for oligonucleotides with the translocation sequence over those with sequences matching either parental gene. These studies demonstrate the feasibility of using oligonucleotides to direct topoisomerase II-mediated DNA cleavage to specific sites in the genome.

HU-331 and Oxidized Cannabidiol Act as Inhibitors of Human Topoisomerase IIα and β

Topoisomerase II is a critical enzyme in replication, transcription, and the regulation of chromatin topology. Several anticancer agents target topoisomerases in order to disrupt cell growth. Cannabidiol is a major non-euphoriant, pharmacologically active component of cannabis. Previously, we examined the cannabidiol derivative HU-331 in order to characterize the mechanism of the compound against topoisomerase IIα. In this current work, we explore whether cannabidiol (CBD) impacts topoisomerase II activity, and we additionally examine the activity of these compounds against topoisomerase IIβ. CBD does not appear to strongly inhibit DNA relaxation and is not a poison of topoisomerase II DNA cleavage. However, oxidation of CBD allows this compound to inhibit DNA relaxation by topoisomerase IIα and β without poisoning DNA cleavage. Additionally, we found that oxidized CBD, similar to HU-331, inhibits ATP hydrolysis and can result in inactivation of topoisomerase IIα and β. We also determined that oxidized CBD and HU-331 are both able to stabilize the N-terminal clamp of topoisomerase II. Taken together, we conclude that while CBD does not have significant activity against topoisomerase II, both oxidized CBD and HU-331 are active against both isoforms of topoisomerase II. We hypothesize that oxidized CBD and HU-331 act against the enzyme through interaction with the N-terminal ATPase domain. According to the model we propose, topoisomerase II inactivation may result from a decrease in the ability of the enzyme to bind to DNA when the compound is bound to the N-terminus.

Topoisomerase IIβ Selectively Regulates Motor Neuron Identity and Peripheral Connectivity through Hox/Pbx-Dependent Transcriptional Programs.

Vital motor functions, such as respiration and locomotion, rely on the ability of spinal motor neurons (MNs) to acquire stereotypical positions in the ventral spinal cord and to project with high precision to their peripheral targets. These key properties of MNs emerge during development through transcriptional programs that dictate their subtype identity and connectivity; however, the molecular mechanisms that establish the transcriptional landscape necessary for MN specification are not fully understood. Here, we show that the enzyme topoisomerase IIβ (Top2β) controls MN migration and connectivity. Surprisingly, Top2β is not required for MN generation or survival but has a selective role in columnar specification. In the absence of Top2β, phrenic MN identity is eroded, while other motor columns are partially preserved but fail to cluster to their proper position. In Top2β-/- mice, peripheral connectivity is impaired as MNs exhibit a profound deficit in terminal branching. These defects likely result from the insufficient activation of Hox/Pbx-dependent transcriptional programs as Hox and Pbx genes are downregulated in the absence of Top2β. Top2β mutants recapitulate many aspects of Pbx mutant mice, such as MN disorganization and defects in medial motor column (MMC) specification. Our findings indicate that Top2β, a gene implicated in neurodevelopmental diseases such as autism spectrum disorders, plays a critical, cell-specific role in the assembly of motor circuits.

Molecular modeling of cationic porphyrin-anthraquinone hybrids as DNA topoisomerase IIβ inhibitors

Human DNA Topoisomerase II has been regarded as a promising target in anticancer drug discovery. In the present study, we designed six porphyrin-anthraquinone hybrids bearing pyrazole or pyridine group as meso substituents and evaluated their potentials as DNA Topoisomerase IIβ inhibitor. First, we investigated the binding orientation of porphyrin hybrids into DNA topoisomerase IIβ employing AutoDock 4.2 and then performed 20-ns molecular dynamics simulations to see the dynamic stability of each porphyrin-Topo IIβ complex using Amber 14. We found that the binding of porphyrin hybrids occured through intercalation and groove binding mode in addition interaction with the amino acid residues constituting the active cavity of Topo IIβ. Each porphyrin-Topo IIβ complex was stabilized during 20-ns dynamics simulations. The MM-PBSA free energy calculation shows that the binding affinities of porphyrin hybrids were modified with the number of meso substituent. Interestingly, the affinity of all porphyrin hybrids to Topo IIβ was stronger than that of native ligand (EVP), indicating the potential of the designed porphyrin to be considered in experimental research.

Investigation of novel dexrazoxane analogue JR-311 shows significant cardioprotective effects through topoisomerase IIbeta but not its iron chelating metabolite

Novel dexrazoxane derivative JR-311 was prepared to investigate structure-activity relationships and mechanism(s) of protection against anthracycline cardiotoxicity. Its cardioprotective, antiproliferative, iron (Fe) chelation and inhibitory and/or depletory activities on topoisomerase IIbeta (TOP2B) were examined and compared with dexrazoxane. While in standard assay, JR-311 failed in both cardioprotection and depletion of TOP2B, its repeated administration to cell culture media led to depletion of TOP2B and significant protection of isolated rat neonatal ventricular cardiomyocytes from daunorubicin-induced damage. This effect was explained by a focused analytical investigation that revealed rapid JR-311 decomposition, resulting in negligible intracellular concentrations of the parent compound but high exposure of cells to the decomposition products, including Fe-chelating JR-H2. Although chemical instability is an obstacle for the development of JR-311, this study identified a novel dexrazoxane analogue with preserved pharmacodynamic properties, contributed to the investigation of structure-activity relationships and suggested that the cardioprotection of bis-dioxopiperazines is likely attributed to TOP2B activity of the parent compound rather than Fe chelation of their hydrolytic metabolites/degradation products. Moreover, this study highlights the importance of early stability testing during future development of novel dexrazoxane analogues.

Structural Insight into BLM Recognition by TopBP1

Topoisomerase IIβ binding protein 1 (TopBP1) is a critical protein-protein interaction hub in DNA replication checkpoint control. It was proposed that TopBP1 BRCT5 interacts with Bloom syndrome helicase (BLM) to regulate genome stability through either phospho-Ser304 or phospho-Ser338 of BLM. Here we show that TopBP1 BRCT5 specifically interacts with the BLM region surrounding pSer304, not pSer338. Our crystal structure of TopBP1 BRCT4/5 bound to BLM reveals recognition of pSer304 by a conserved pSer-binding pocket, and interactions between an FVPP motif N-terminal to pSer304 and a hydrophobic groove on BRCT5. This interaction utilizes the same surface of BRCT5 that recognizes the DNA damage mediator, MDC1; however the binding orientations of MDC1 and BLM are reversed. While the MDC1 interactions are largely electrostatic, the interaction with BLM has higher affinity and relies on a mix of electrostatics and hydrophobicity. We suggest that similar evolutionarily conserved interactions may govern interactions between TopBP1 and 53BP1.

Jadomycins Inhibit Type II Topoisomerases and Promote DNA Damage and Apoptosis in Multidrug-Resistant Triple-Negative Breast Cancer Cells

Jadomycins are natural products that kill drug-sensitive and multidrug-resistant (MDR) breast cancer cells. To date, the cytotoxic activity of jadomycins has never been tested in MDR breast cancer cells that are also triple negative. Additionally, there is only a rudimentary understanding of how jadomycins cause cancer cell death, which includes the induction of intracellular reactive oxygen species (ROS). We first created a paclitaxel-resistant, triple-negative breast cancer cell line [paclitaxel-resistant MDA-MB-231 breast cancer cells (231-TXL)] from drug-sensitive control MDA-MB-231 cells (231-CON). Using thiazolyl blue methyltetrazolium bromide cell viability-measuring assays, jadomycins B, S, and F were found to be equipotent in drug-sensitive 231-CON and MDR 231-TXL cells; and using ROS-detecting assays, these jadomycins were determined to increase ROS activity in both cell lines by up to 7.3-fold. Jadomycins caused DNA double-strand breaks in 231-CON and 231-TXL cells as measured by γH2AX Western blotting. Coincubation with the antioxidant N-acetyl cysteine or pro-oxidant auranofin did not affect jadomycin-mediated DNA damage. Jadomycins induced apoptosis in 231-CON and 231-TXL cells as measured by annexin V affinity assays, a process that was retained when ROS were inhibited. This indicated that jadomycins are capable of inducing MDA-MB-231 apoptotic cell death independently of ROS activity. Using quantitative polymerase chain reaction, Western blotting, and direct topoisomerase inhibition assays, it was determined that jadomycins inhibit type II topoisomerases and that jadomycins B and F selectively poison topoisomerase IIβ We therefore propose novel mechanisms through which jadomycins induce breast cancer cell death independently of ROS activity, through inhibition or poisoning of type II topoisomerases and the induction of DNA damage and apoptosis.

Development of pyridine dicoumarols as potent anti HIV-1 leads, targeting HIV-1 associated topoisomeraseIIβ kinase.

A structural study of a series of pyridine dicoumarol derivatives with potential activity against a novel Topoisomerase IIβ kinase which was identified in the HIV-1 viral lysate, compounds were designed and synthesized based on a 3D-QSAR study. Based on QSAR model we have designed and synthesized a series of pyridine dicoumarol derivatives and characterized by spectral studies, all the molecules are biologically evaluated by kinase assay, cytotoxicity assay, ELISA and PCR method. We demonstrated the achievement of water soluble disodium pyridine dicoumarate derivatives showing high anti-HIV-1 activity (IC50 <25nM) which provides a crucial point for further development of pyridine dicoumarol series as HIV-1-associated topoisomerase IIβ kinase inhibitors for clinical application against AIDS. A new class of anti-HIV-1 lead compounds have been designed and tested. Further studies would result in development ofnovel and potential drugs.

Calcein-acetoxymethy ester enhances the antitumor effects of doxorubicin in nonsmall cell lung cancer by regulating the TopBP1/p53RR pathway.

Calcein acetoxymethyl ester (calcein-AM) treatment has been reported to exert antitumor effects in certain cancer cells; however, the detailed mechanism of action of calcein-AM in cancers remains unclear, especially in nonsmall cell lung cancer (NSCLC). This study focused on the function and mechanism of action of calcein-AM in NSCLC. We used cell viability assays, western blotting, and EdU proliferation assay combined with calcein-AM treatment or siRNA interference to investigate the role of topoisomerase IIβ binding protein 1 (TopBP1) and p53 in NSCLC chemotherapy. We found that calcein-AM has antitumor effects in lung cancer and enhances the antitumor effects of doxorubicin in NSCLC. Furthermore, we found that TopBP1, which we previously showed was involved in doxorubicin resistance through upregulation of aberrant p53, was involved in calcein-AM-mediated increased doxorubicin sensitivity. Doxorubicin upregulated the expression of aberrant p53. Calcein-AM repressed the expression of TopBP1, which resulted in reduced expression of aberrant p53 and disrupted the antiapoptotic activity mediated by the TopBP1/mutp53 pathway in NSCLC. Together, our findings show that calcein-AM, the cell-permeable derivative of calcein, exerts significant antitumor effects in NSCLC, and can enhance the antitumor effect of doxorubicin by regulating the TopBP1/mutp53 pathway. These findings provide novel insight into lung cancer treatment.

Abstract 510: Topoisomerase IIβ silencing increases R loops at specific genomic loci associated with an increase of γH2AX and cell cycle progression delay in human cancer cells

Abstract DNA topoisomerases (Top) are important players in maintaining the genome stability of cells by removing negative and positive DNA supercoils during transcription, replication and other DNA transactions. Recent evidence demonstrated that Top 1 silencing and poisoning by camptothecin affect the formation of R-loops, which are RNA/DNA hybrid structures involved in genome instability and are favored by negative supercoils of the DNA. The findings overall demonstrate that Top 1 reduces R loop levels during transcription whereas it favors R loop formation at early origins of DNA replication. Here, we address the question of whether DNA topoisomerase IIβ can also contribute to steady-state levels of R loop structures in the genome of U2OS cancer cells. We have then investigated the effects of Top2β silencing and Top2 poisons, doxorubicin and etoposide, on R loops and DNA damage in human U2OS cancer cells. Similarly to published data with camptothecin (Marinello et al., Nucleic Acids Research, 2013), Top2 poisons increase cellular R loops by IF after short treatment times and reduce them after 1 hour of treatment. The bi-phasic effect of poisons is mainly dependent on transcription. Moreover, we have determined the changes of R-loop levels by IF after Top2β silencing and the findings demonstrate that the enzyme can strongly modulate the formation of R loops as a full Top2β depletion increases nuclear R-loop levels. In addition, Top2β depletion leads to a slight increase of phosphorylation of H2AX histone along with cell cycle delay and eventually cell death. Thus, Top2β depletion can trigger genomic DNA breakage through alterations of R-loops. In order to establish which are the genomic regions of altered R loop levels, we have mapped Top2β-dependent R loop alterations by the DRIP method showing that specific genomic sites are affected by Top2β. Bioinformatic analyses of R loop maps in U2OS cells will be presented and discussed at the meeting. Altogether the findings demonstrate a critical role of Top2β in governing R loop structures in human cancer cells indicating that DNA torsional tension is a main driving factor of R loop formation and hence genome instability in cancer cells. Citation Format: Maria Delcuratolo, Jessica Marinello, Giovanni Capranico. Topoisomerase IIβ silencing increases R loops at specific genomic loci associated with an increase of γH2AX and cell cycle progression delay in human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 510. doi:10.1158/1538-7445.AM2017-510