Ruthenium(II) complex with 2-mercaptothiazoline ligand induces selective cytotoxicity involving DNA damage and apoptosis in melanoma cells.

Melanoma is the most aggressive and lethal type of skin cancer due to its characteristics such as high metastatic potential and low response rate to existing treatment modalities. In this way, new drug prototypes are being studied to solve the problem of treating patients with melanoma. Among these, ruthenium-based metallopharmaceuticals may be promising alternatives due to their antitumor characteristics and low systemic toxicity. In this context, the present study evaluated the antineoplastic effect of the ruthenium complex [Ru(mtz)(dppe)2]PF6-2-mercaptothiazoline-di-1,2-bis(diphenylphosphine) ethaneruthenium(II), namely RuMTZ, on human melanoma (A-375) and murine (B16-F10) cells, considering different approaches. Through XTT colorimetric and clonogenic efficiency assays, the complex revealed the selective cytotoxic activity, with the lowest IC50 (0.4 µM) observed for A375 cells. RuMTZ also induced changes in cell morphology, increased cell population in the sub-G0 phase and inhibiting cell migration. The levels of γH2AX and cleaved caspase 3 proteins were increased in both cell lines treated with RuMTZ. These findings indicated that the cytotoxic activity of RuMTZ on melanoma cells is related, at least in part, to the induction of DNA damage and apoptosis. Therefore, RuMTZ exhibited promising antineoplastic activity against melanoma cells.

Hellebrigenin triggers death of promyelocytic leukemia cells by non-genotoxic ways.

Bufadienolides are digitalis-like aglycones mainly found in skin secretions of toads. Among their biological properties, the mechanisms of antiproliferative action on tumor cells remain unclear for many compounds, including against leukemia cells. Herein, it was evaluated the mechanisms involved in the antiproliferative and genotoxic actions of hellebrigenin on tumor cell lines and in silico capacity to inhibit the human topoisomerase IIa enzyme. Firstly, its cytotoxic action was investigated by colorimetric assays in human tumor and peripheral blood mononuclear cells (PBMC). Next, biochemical and morphological studies were detailed by light microscopy (trypan blue dye exclusion), immunocytochemistry (BrdU uptake), flow cytometry and DNA/chromosomal damages (Cometa and aberrations). Finally, computational modelling was used to search for topoisomerase inhibition. Hellebrigenin reduced proliferation, BrdU incorporation, viability, and membrane integrity of HL-60 leukemia cells. Additionally, it increased G2/M arrest, internucleosomal DNA fragmentation, mitochondrial depolarization, and phosphatidylserine externalization in a concentration-dependent manner. In contrast to doxorubicin, hellebrigenin did not cause DNA strand breaks in HL-60 cell line and lymphocytes, and it interacts with ATPase domain residues of human topoisomerase IIa, generating a complex of hydrophobic and van der Waals interactions and hydrogen bonds. So, hellebrigenin presented potent anti-leukemic activity at concentrations as low as 0.06 µM, a value comparable to the clinical anticancer agent doxorubicin, and caused biochemical changes suggestive of apoptosis without genotoxic/clastogenic-related action, but it probably triggers catalytic inhibition of topoisomerase II. These findings also emphasize toad steroid toxins as promising lead antineoplasic compounds with relatively low cytotoxic action on human normal cells.

Overview of the New Bioactive Heterocycles as Targeting Topoisomerase Inhibitors Useful Against Colon Cancer.

Colorectal cancer (CRC) is the third most common worldwide cancer with high mortality. Factors such as more effective screening programs and improvements in treatment modalities have favored a decrease in the incidence and mortality rate of colorectal cancer in the last three decades. Metastatic CRC is incurable in most cases, and therapy using multiple drugs can increase patients' life expectancy by 2 to 3 years. Chemotherapy is the primary treatment, and fluoropyrimidines correspond to the first treatment line. They can be used in monotherapy or therapeutic schemes of oxaliplatin, FOLFOX (intravenous fluorouracil, leucovorin, and oxaliplatin), and CAPOX (oral capecitabine and oxaliplatin) or regimens based on Irinotecan, such FOLFIRI (fluorouracil, leucovorin, and Irinotecan) and CAPIRI (capecitabine and Irinotecan). Like Camptothecin, irinotecan and other analogs have a mechanism of action based on forming a ternary complex with Topoisomerase I and DNA by reversibly binding, providing DNA damage and consequent cell death. This way, topoisomerases are vital enzymes for DNA maintenance and cell viability. Thus, here we will review the main works demonstrating the correlation between the inhibition of different isoforms of topoisomerases and the in vitro cytotoxic activity in colon cancer. The findings revealed that natural compounds, semi-synthetic and synthetic analogs showed potential cytotoxicity against several colon cancer cell lines in vitro and that this activity was often accompanied by the ability to inhibit type I and II topoisomerases, demonstrating that these enzymes can be promising drug targets for the development of new chemotherapeutics against colon cancer.

Diphenyl ditelluride anticancer activity and DNA topoisomerase I poisoning in human colon cancer HCT116 cells.

Diphenyl ditelluride (DPDT) is an organotellurium (OT) compound with pharmacological properties, including antioxidant, antigenotoxic and antimutagenic activities when applied at low concentrations. However, DPDT as well as other OT compounds also show cytotoxicity against mammalian cells when treatments occur at higher drug concentrations. Considering that the underlying mechanisms of toxicity of DPDT against tumor cells have been poorly explored, the objective of our study was to investigate the effects of DPDT against both human cancer and non-tumorigenic cells. As a model, we used the colonic HCT116 cancer cells and the MRC5 fibroblasts. Our results showed that DPDT preferentially targets HCT116 cancer cells when compared to MRC5 cells with IC50 values of 2.4 and 10.1 µM, respectively. This effect was accompanied by the induction of apoptosis and a pronounced G2/M cell cycle arrest in HCT116 cells. Furthermore, DPDT induces DNA strand breaks at concentrations below 5 µM in HCT116 cells and promotes the occurrence of DNA double strand breaks mostly during S-phase as measured by γ-H2AX/EdU double staining. Finally, DPDT forms covalent complexes with DNA topoisomerase I, as observed by the TARDIS assay, with a more prominent effect observed in HCT116 than in MRC5 cells. Taken together, our results show that DPDT preferentially targets HCT116 colon cancer cells likely through DNA topoisomerase I poisoning. This makes DPDT an interesting molecule for further development as an anti-proliferative compound in the context of cancer.

Cytotoxic Activity, Topoisomerase I Inhibition and In Silico Studies of New Sesquiterpene-aryl Ester Derivatives of (-) Drimenol.

In this study, we aimed to evaluate two sets of sesquiterpene-aryl derivatives linked by an ester bond, their cytotoxic activities, and their capacity to activate caspases 3/7 and inhibit human topoisomerase I (TOP1). A total of 13 compounds were synthesized from the natural sesquiterpene (-)-drimenol and their cytotoxic activity was evaluated in vitro against three cancer cell lines: PC-3 (prostate cancer), HT-29 (colon cancer), MCF-7 (breast cancer), and an immortalized non-tumoral cell line (MCF-10). From the results, it was observed that 6a was the most promising compound due to its cytotoxic effect on three cancer cell lines and its selectivity, 6a was 100-fold more selective than 5-FU in MCF-7 and 20-fold in PC-3. It was observed that 6a also induced apoptosis by caspases 3/7 activity using a Capsase-Glo-3/7 assay kit and inhibited TOP1. A possible binding mode of 6a in a complex with TOP1-DNA was proposed by docking and molecular dynamics studies. In addition, 6a was predicted to have a good pharmacokinetic profile for oral administration. Therefore, through this study, it was demonstrated that the drimane scaffold should be considered in the search of new antitumoral agents.

Study of Genotoxicity, Activities on Caspase 8 and on the Stabilization of the Topoisomerase Complex of Isoeleutherin and Analogues.

This study evaluated the genotoxicity of Ethanol Extract (EEEp), Dichloromethane Fraction (FDCMEp) and isoeleutherin isolated from Eleutherine plicata, using the micronucleus test and the impact of structural alterations on toxicity and molecular docking (topoisomerase II and DNA complex). The extract was obtained by maceration and fractionation in a chromatography column. The genotoxicity was evaluated by the micronucleus test in human hepatoma cells (HepG2). Isoeleutherin was the starting molecule in the search for analogues by structural similarity, using the ZINC and e-Molecules databases. Isoeleutherin and analogues were subjected to in silico toxicity prediction, and compounds free of toxicological risks (CP13, CP14, CP17 and isoeleutherin) were selected for molecular docking in Topoisomerase II (PDB: 1ZXM). In the micronucleus test, isoeleutherin was less genotoxic. Among the 22 isoeleutherin analogues there were variations in the toxicity profile. Molecular docking studies showed that the compounds have good complementarity in the active site with important hydrogens bonds. Therefore, the structural changes of isoeleutherin led to the obtaining of a molecule with a lower mutagenic potential, and the CP13 can be considered a prototype compound for the development of new molecules with pharmacological potential.

Interaction study with DNA/HSA, anti-topoisomerase IIα, cytotoxicity and in vitro antiproliferative evaluations and molecular docking of indole-thiosemicarbazone compounds.

In this work we will discuss the antiproliferative evaluation and the possible mechanisms of action of indole-thiosemicarbazone compounds LTs with anti-inflammatory activity, previously described in the literature. In this perspective, some analyzes were carried out, such as the study of binding to human serum albumin (HSA) and to biological targets: DNA and human topoisomerase IIα (topo). Antiproliferative study was performed with DU-145, Jukart, MCF-7 and T-47D tumor lines and J774A.1, besides HepG2 macrophages and hemolytic activity. In the HSA interaction tests, the highest binding constant was 3.70 × 106 M-1, referring to LT89 and in the fluorescence, most compounds, except for LT76 and LT87, promoted fluorescent suppression with the largest Stern-Volmer constant for the LT88 3.55 × 104. In the antiproliferative assay with DU-145 and Jurkat strains, compounds LT76 (0.98 ± 0.10/1.23 ± 0.32 µM), LT77 (0.94 ± 0.05/1.18 ± 0.08 µM) and LT87 (0.94 ± 0.12/0.84 ± 0.09 µM) stood out, due to their IC50 values mentioned above. With the MCF-7 and T-47D cell lines, the lowest IC50 was presented by LT81 with values of 0.74 ± 0.12 µM and 0.68 ± 0.10 µM, respectively, followed by the compounds LT76 and LT87. As well as the positive control amsacrine, the compounds LT76, LT81 and LT87 were able to inhibit the enzymatic action of human Topoisomerase IIα.

Antitumor copper(II) complexes with hydroxyanthraquinones and N,N-heterocyclic ligands.

Five ternary copper(II) complexes, [Cu2(phen)2(L1)(ClO4)2] (1), [Cu2(phen)2(L1)(DMSO)2](PF6)2 (2), [Cu2(bpy)2(L1)(ClO4)2(H2O)2] (3), [Cu2(dmp)2(L1)(ClO4)2(H2O)2] (4), and [Cu(phen)(L2)]2(ClO4)2 (5), in which phen = 1,10-phenanthroline, bpy = 2,2'-bipyridine, dmp = 2,9-dimethyl-1,10-phenanthroline, H2L1 = 1,4-dihydroxyanthracene-9,10-dione and HL2 = 1-hydroxyanthracene-9,10-dione, DMSO = dimethylsulfoxide, were synthesized and fully characterized. Complex 2 was obtained through the substitution of perchlorate for DMSO. When two hydroxyquinone groups are present, L1 makes a bridge between two Cu(II) ions, which also bind two nitrogens of the respective diimine ligand. The compounds bind to calf thymus DNA and oxidatively cleave pUC19 DNA according to the following order of activity 1 > 4-5 > 3. Furthermore, complexes 1, 3, 4 and 5 inhibit topoisomerase-I activity and the growth of myelogenous leukemia cells with the IC50 values of 1.13, 10.60, 0.078, and 1.84 µmol L-1, respectively. Complexes 1 and 4 are the most active in cancer cells and in DNA cleavage.

Maternal diet in pregnancy and acute leukemia in infants: a case-control study in Mexico City.

Introduction: Epidemiological studies around the world on acute leukemia (AL) and risk factors in infants are scarce. Infant AL has been proposed to originate in utero, which facilitates its study by establishing a short exposure time in pregnant women to environmental and dietary factors that could contribute to the risk of or protection against leukemia. We hypothesized that maternal diet during pregnancy may be an important factor involved in AL in offspring. Methods: We conducted a hospital-based case-control study from 2010 to 2019 on maternal diet during pregnancy in nine high-specialty public hospitals of different health institutions that diagnose and offer treatment to children with AL in Mexico City. Cases (n=109) were children ≤24 months of age with de novo diagnosis of AL, and controls (n=252) were children obtained in hospitals from second-level medical care matched for age, sex, and health institution. Maternal diet during pregnancy was obtained by a semiquantitative food frequency questionnaire. Unconditional logistic regression models were used to assess the association between food groups and infant AL. Potential confounders were assessed by constructing directed acyclic graphs (DAGs) with Dagitty software in which adjusted options were identified for the construction of unconditional logistic regression models. Results: Cases were slightly predominantly female (52.3%). The years of education of the mother in cases and controls was 0-9 on average, and those who reported smoking cigarettes and consuming alcohol during pregnancy did so at a low frequency. Regarding the mother's diet, the main findings were that the consumption of allium vegetables during pregnancy was inversely associated with AL for medium and high consumption (OR=0.26, 95% CI 0.14-0.46; P-trend< 0.001). In contrast, the high consumption of high-fat dairy products had a positive association with AL (OR=2.37, 95% CI 1.30-4.34; P-trend<0.001). No association was found between consumption of topoisomerase II inhibitor foods during pregnancy and AL. Conclusion: The results suggest that maternal intake during pregnancy of allium vegetables, specifically garlic, is inversely associated with the development of AL in children ≤24 months old. On the other hand, consumption of high-fat dairy products is positively associated with AL in children ≤24 months old.

Synthesis and Evaluation of Antiproliferative Activity, Topoisomerase IIα Inhibition, DNA Binding and Non-Clinical Toxicity of New Acridine-Thiosemicarbazone Derivatives.

In this study, we report the synthesis of twenty new acridine-thiosemicarbazone derivatives and their antiproliferative activities. Mechanisms of action such as the inhibition of topoisomerase IIα and the interaction with DNA have been studied for some of the most active derivatives by means of both in silico and in vitro methods, and evaluations of the non-clinical toxicities (in vivo) in mice. In general, the compounds showed greater cytotoxicity against B16-F10 cells, with the highest potency for DL-08 (IC50 = 14.79 µM). Derivatives DL-01 (77%), DL-07 (74%) and DL-08 (79%) showed interesting inhibition of topoisomerase IIα when compared to amsacrine, at 100 µM. In silico studies proposed the way of bonding of these compounds and a possible stereoelectronic reason for the absence of enzymatic activity for CL-07 and DL-06. Interactions with DNA presented different spectroscopic effects and indicate that the compound CL-07 has higher affinity for DNA (Kb = 4.75 × 104 M-1; Ksv = 2.6 × 103 M-1). In addition, compounds selected for non-clinical toxicity testing did not show serious signs of toxicity at the dose of 2000 mg/kg in mice; cytotoxic tests performed on leukemic cells (K-562) and its resistant form (K-562 Lucena 1) identified moderate potency for DL-01 and DL-08, with IC50 between 11.45 and 17.32 µM.

Topoisomerase Enzyme Inhibitors as Potential Drugs Against Cancer: What Makes Them Selective or Dual? - A Review.

Topoisomerase inhibitors are extensively used in cancer chemotherapy. In the process of identifying novel anticancer compounds, biological evaluations are crucial and include, among others, the use of in silico and in vitro approaches. This work aimed to present recent research involving the obtainment and in silico and in vitro evaluation of topoisomerase I, II, and double inhibitors, of synthetic and natural origin, as potential compounds against tumor cells, in addition to proposing the construction of a desirable enzyme catalytic site. Therefore, it was observed that most Topoisomerase I inhibitors presented medium to large structures, with a rigid portion and a flexible region. In contrast, Topoisomerase IIα inhibitors showed medium and large structural characteristics, in addition to the planarity of the aromatic rings, which are mitigated due to flexible rings but may also present elements that restrict conformation. Most compounds that exhibit dual inhibitory activity had relatively long chains, in addition to a flat and rigid portion suggestive of affinity for Topo I and a flexible region characteristic of selective drugs for Topo II. Besides, it is noticed that most compounds that exhibit dual inhibitory showed similarities in the types of interactions and amino acids when compared to the selective compounds of Topo I and II. For instance, selective Topoisomerase I inhibitors interact with Arginine364 residues, and selective Topoisomerase II inhibitors interact with Arginine487 residues, as both residues are targets for dual compounds.

Functions of the CSB Protein at Topoisomerase 2 Inhibitors-Induced DNA Lesions.

Topoisomerase 2 (TOP2) inhibitors are drugs widely used in the treatment of different types of cancer. Processing of their induced-lesions create double-strand breaks (DSBs) in the DNA, which is the main toxic mechanism of topoisomerase inhibitors to kill cancer cells. It was established that the Nucleotide Excision Repair pathway respond to TOP2-induced lesions, mainly through the Cockayne Syndrome B (CSB) protein. In this paper, we further define the mechanism and type of lesions induced by TOP2 inhibitors when CSB is abrogated. In the absence of TOP2, but not during pharmacological inhibition, an increase in R-Loops was detected. We also observed that CSB knockdown provokes the accumulation of DSBs induced by TOP2 inhibitors. Consistent with a functional interplay, interaction between CSB and TOP2 occurred after TOP2 inhibition. This was corroborated with in vitro DNA cleavage assays where CSB stimulated the activity of TOP2. Altogether, our results show that TOP2 is stimulated by the CSB protein and prevents the accumulation of R-loops/DSBs linked to genomic instability.

Three-Component Synthesis of 2-Amino-3-cyano-4H-chromenes, In Silico Analysis of Their Pharmacological Profile, and In Vitro Anticancer and Antifungal Testing.

Chromenes are compounds that may be useful for inhibiting topoisomerase and cytochrome, enzymes involved in the growth of cancer and fungal cells, respectively. The aim of this study was to synthesize a series of some novel 2-amino-3-cyano-4-aryl-6,7-methylendioxy-4H-chromenes 4a-o and 2-amino-3-cyano-5,7-dimethoxy-4-aryl-4H-chromenes 6a-h by a three-component reaction, and test these derivatives for anticancer and antifungal activity. Compounds 4a and 4b were more active than cisplatin (9) and topotecan (7) in SK-LU-1 cells, and more active than 9 in PC-3 cells. An evaluation was also made of the series of compounds 4 and 6 as potential antifungal agents against six Candida strains, finding their MIC50 to be less than or equal to that of fluconazole (8). Molecular docking studies are herein reported, for the interaction of 4 and 6 with topoisomerase IB and the active site of CYP51 of Candida spp. Compounds 4a-o and 6a-h interacted in a similar way as 7 with key amino acids of the active site of topoisomerase IB and showed better binding energy than 8 at the active site of CYP51. Hence, 4a-o and 6a-h are good candidates for further research, having demonstrated their dual inhibition of enzymes that participate in the growth of cancer and fungal cells.

DNA Topoisomerase 3α Is Involved in Homologous Recombination Repair and Replication Stress Response in Trypanosoma cruzi.

DNA topoisomerases are enzymes that modulate DNA topology. Among them, topoisomerase 3α is engaged in genomic maintenance acting in DNA replication termination, sister chromatid separation, and dissolution of recombination intermediates. To evaluate the role of this enzyme in Trypanosoma cruzi, the etiologic agent of Chagas disease, a topoisomerase 3α knockout parasite (TcTopo3α KO) was generated, and the parasite growth, as well as its response to several DNA damage agents, were evaluated. There was no growth alteration caused by the TcTopo3α knockout in epimastigote forms, but a higher dormancy rate was observed. TcTopo3α KO trypomastigote forms displayed reduced invasion rates in LLC-MK2 cells when compared with the wild-type lineage. Amastigote proliferation was also compromised in the TcTopo3α KO, and a higher number of dormant cells was observed. Additionally, TcTopo3α KO epimastigotes were not able to recover cell growth after gamma radiation exposure, suggesting the involvement of topoisomerase 3α in homologous recombination. These parasites were also sensitive to drugs that generate replication stress, such as cisplatin (Cis), hydroxyurea (HU), and methyl methanesulfonate (MMS). In response to HU and Cis treatments, TcTopo3α KO parasites showed a slower cell growth and was not able to efficiently repair the DNA damage induced by these genotoxic agents. The cell growth phenotype observed after MMS treatment was similar to that observed after gamma radiation, although there were fewer dormant cells after MMS exposure. TcTopo3α KO parasites showed a population with sub-G1 DNA content and strong γH2A signal 48 h after MMS treatment. So, it is possible that DNA-damaged cell proliferation due to the absence of TcTopo3α leads to cell death. Whole genome sequencing of MMS-treated parasites showed a significant reduction in the content of the multigene families DFG-1 and RHS, and also a possible erosion of the sub-telomeric region from chromosome 22, relative to non-treated knockout parasites. Southern blot experiments suggest telomere shortening, which could indicate genomic instability in TcTopo3α KO cells owing to MMS treatment. Thus, topoisomerase 3α is important for homologous recombination repair and replication stress in T. cruzi, even though all the pathways in which this enzyme participates during the replication stress response remains elusive.

Inhibition of Aedes aegypti DNA topoisomerase II by etoposide: Impact on survival and morphology of larvae and pupae.

DNA topoisomerase II enzymes maintain DNA stability during vital processes, such as genome replication, transcription and chromosomal segregation during mitosis and meiosis. In the present work, we analyzed functional aspects of the DNA topoisomerase II (AeTopII) enzyme of the mosquito Aedes aegypti. Here, we show that AeTopII mRNA is expressed at all stages of mosquito development. By in situ hybridization, we found that the AeTopII mRNA is concentrated along the ovarian follicular cells as well as in the region of the follicles. The observed expression profiles likely reflect increased topoisomerase II cellular requirements due to the intense ovarian growth and egg production following blood feeding in Ae. aegypti females. The drug etoposide, a classic inhibitor of topoisomerase II, was used for in vivo testing with 2nd stage larvae, in order to investigate the functional importance of this enzyme in Ae. aegypti survival and development. Inhibition of topoisomerase II activity with etoposide concentrations ranging from 10 to 200 µM did not leads to the immediate death of larvae. However, after 10 days of observation, etoposide treatments resulted in 30-40% decrease in survival, in a dose dependent manner, with persisting larvae and pupae presenting incomplete development, as well as morphological abnormalities. Also, approximately 50% of the treated larvae did not reach the pupal stage. Thus, we conclude that AeTopII is a vital enzyme in the development of Ae. aegypti and its sensitivity to inhibitors should be explored for potential chemical agents to be used in vector control.

Structural insights into the biological activity of a thioxopyrimidine derivative.

Bacterial resistance to the main widespread antibiotics, such as those based on quinolones, is a concern of the scientific community, leading to the search for new classes of molecules that can be used as an alternative. Here, we investigate the crystalline and chemical characteristics of a thioxopyrimide to understand its demonstrated biological activity and to identify which portion of the molecule can be used as a framework to develop new antibiotics. For this purpose, structural studies of ethyl 4-methyl-2-phenyl-6-thioxo-1,6-dihydro-5-pyrimidinecarboxylate were carried out aided by Hirshfeld surface analysis, as well as theoretical calculations on frontier molecular orbitals, molecular electrostatic potential, and conformational stability, in addition to docking studies targeting topoisomerase IV. The docking results show a reasonable accommodation of the molecule at the topoisomerase IV binding site and interact mainly by hydrogen bonds between the thioxopyrimidine portion with Glu198, Thr292, and Gly225, aided by hydrophobic interactions involving the rest of the molecule. These results suggest a relationship between the antibacterial activity shown mainly with the 4-thioxopyrimidine portion, leading to the investigation of new compounds that use this scaffold.

Myricitrin from Combretum lanceolatum Exhibits Inhibitory Effect on DNA-Topoisomerase Type IIα and Protective Effect Against In Vivo Doxorubicin-Induced Mutagenicity.

Flavonoids-compounds abundant in balanced daily diets-have been extensively investigated for biological activity. The pronounced antiproliferative effects of flavonoids have prompted studies to elucidate their mode of action against tumor cells. The anticancer properties of myricetin, a 3',4',5'-tri-hydroxylated flavonol, have been confirmed for a number of neoplasms, but myricitrin, its 3-O-rhamnoside derivative found in fruits and other parts of edible plants, has been scarcely investigated as a chemopreventive agent. This study evaluated the antiproliferative potential of myricitrin obtained from Combretum lanceolatum (Combretaceae) against MCF7 (breast), PC-3 (prostate), HT-29 (colon), 786-0 (kidney), and HL-60 (acute promyelocytic leukemia) cancer cell lines, using the sulforhodamine B and tetrazolium salt assays. Myricitrin proved most effective in inhibiting growth of HL-60 cells (GI50 = 53.4 µmol·L-1), yet showed weak antiproliferative activity against other cell lines. Possible cytotoxic mechanisms involving inhibition of topoisomerases I and IIα by myricitrin were also evaluated, revealing inhibitory activity only against topoisomerase IIα. The results suggested that topoisomerase IIα inhibition is the probable mechanism responsible for the antiproliferative activity of myricitrin. In vivo mutagenicity by myricitrin and its possible antimutagenic effect on doxorubicin-induced DNA damage were also investigated by performing the somatic mutation and recombination test (SMART) on Drosophila melanogaster. Myricitrin proved nonmutagenic to the offspring of standard (ST) and high-bioactivation (HB) crosses, while cotreatments with doxorubicin revealed the antimutagenic properties of myricitrin, even under conditions of high metabolic activation.

DNA topoisomerase 3α is involved in homologous recombination repair and replication stress response in Trypanosoma cruzi

DNA topoisomerases are enzymes that modulate DNA topology. Among them, topoisomerase 3α is engaged in genomic maintenance acting in DNA replication termination, sister chromatid separation, and dissolution of recombination intermediates. To evaluate the role of this enzyme in Trypanosoma cruzi, the etiologic agent of Chagas disease, a topoisomerase 3α knockout parasite (TcTopo3α KO) was generated, and the parasite growth, as well as its response to several DNA damage agents, were evaluated. There was no growth alteration caused by the TcTopo3α knockout in epimastigote forms, but a higher dormancy rate was observed. TcTopo3α KO trypomastigote forms displayed reduced invasion rates in LLC-MK2 cells when compared with the wild-type lineage. Amastigote proliferation was also compromised in the TcTopo3α KO, and a higher number of dormant cells was observed. Additionally, TcTopo3α KO epimastigotes were not able to recover cell growth after gamma radiation exposure, suggesting the involvement of topoisomerase 3α in homologous recombination. These parasites were also sensitive to drugs that generate replication stress, such as cisplatin (Cis), hydroxyurea (HU), and methyl methanesulfonate (MMS). In response to HU and Cis treatments, TcTopo3α KO parasites showed a slower cell growth and was not able to efficiently repair the DNA damage induced by these genotoxic agents. The cell growth phenotype observed after MMS treatment was similar to that observed after gamma radiation, although there were fewer dormant cells after MMS exposure. TcTopo3α KO parasites showed a population with sub-G1 DNA content and strong γH2A signal 48 h after MMS treatment. So, it is possible that DNA-damaged cell proliferation due to the absence of TcTopo3α leads to cell death. Whole genome sequencing of MMS-treated parasites showed a significant reduction in the content of the multigene families DFG-1 and RHS, and also a possible erosion of the sub-telomeric region from chromosome 22, relative to non-treated knockout parasites. Southern blot experiments suggest telomere shortening, which could indicate genomic instability in TcTopo3α KO cells owing to MMS treatment. Thus, topoisomerase 3α is important for homologous recombination repair and replication stress in T. cruzi, even though all the pathways in which this enzyme participates during the replication stress response remains elusive.

Anticorpos anti topoisomerase 1 no lúpus eritematoso sistêmico / Anti-topoisomerase 1 antibodies in systemic lupus erythematosus

Antitopoisomerase-1 (ou Scl-70) é um autoanticorpo considerado como biomarcador da forma difusa de esclerodermia. Alguns autores o têm encontrado em pacientes com lúpus. O objetivo deste estudo foi estudar a presença do anticorpo Scl-70 em lúpus eritematoso sistêmico (SLE). É pesquisa com 94 pacientes com LES para anticorpo anti Scl-70 usando o kit comercial de ELISA Virgo™, Columbia, USA. Dados clínicos, epidemiológicos e sorológicos foram obtidos dos prontuários. Como resultado, somente 2 pacientes (2.1%) tinham anticorpos anti Scl-70 em baixos títulos. Nenhum deles tinha características de esclerodermia. Em conclusão, não se confirmam achados anteriores acerca da presença de anti Scl-70 em lúpus. Este anticorpo parece ser específico para esclerodermia.

Antitopoisomerase-1 (or Scl-70) is an autoantibody considered as a biomarker of the diffuse form of scleroderma. Some authors have found it in lupus patients. The aim of this study was to study the presence of the Scl-70 antibody in systemic lupus erythematosus (SLE). It is screened with 94 SLE patients for anti Scl-70 antibody using the commercial Virgo™ ELISA kit, Columbia, USA. Clinical, epidemiological and serological data were obtained from medical records. As a result, only 2 patients (2.1%) had anti-Scl-70 antibodies at low titers. None of them had features of scleroderma. In conclusion, previous findings regarding the presence of anti Scl-70 in lupus are not confirmed. This antibody appears to be specific for scleroderma.

Prostaglandins Isolated from the Octocoral Plexaura homomalla: In Silico and In Vitro Studies Against Different Enzymes of Cancer.

Prostaglandin A2-AcMe (1) and Prostaglandin A2 (2) were isolated from the octocoral Plexaura homomalla and three semisynthetic derivatives (3-5) were then obtained using a reduction protocol. All compounds were identified through one- and two-dimensional (1D and 2D) nuclear magnetic resonance (NMR) experiments. Additionally, evaluation of in vitro cytotoxic activity against the breast (MDA-MB-213) and lung (A549) cancer cell lines, in combination with enzymatic activity and molecular docking studies with the enzymes p38α-kinase, Src-kinase, and topoisomerase IIα, were carried out for compounds 1-5 in order to explore their potential as inhibitors of cancer-related molecular targets. Results showed that prostaglandin A2 (2) was the most potent compound with an IC50 of 16.46 and 25.20 µg/mL against MDA-MB-213 and A549 cell lines, respectively. In addition, this compound also inhibited p38α-kinase in 49% and Src-kinase in 59% at 2.5 µM, whereas topoisomerase IIα was inhibited in 64% at 10 µM. Enzymatic activity was found to be consistent with molecular docking simulations, since compound 2 also showed the lowest docking scores against the topoisomerase IIα and Src-kinase (-8.7 and -8.9 kcal/mol, respectively). Thus, molecular docking led to establish some insights into the predicted binding modes. Results suggest that prostaglandin 2 can be considered as a potential lead for development inhibitors against some enzymes present in cancer processes.