Novel Very Long-Chain α-Methoxylated Δ5,9 Fatty Acids from the Sponge Asteropus niger Are Effective Inhibitors of Topoisomerases IB.

The novel fatty acids (2R,5Z,9Z)-2-methoxy-25-methyl-5,9-hexacosadienoic acid (1a) and (2R,5Z,9Z)-2-methoxy-24-methyl-5,9-hexacosadienoic acid (1b) were isolated in 80 % purity from the Caribbean sponge Asteropus niger by chloroform/methanol extraction followed by solvent partitioning and silica gel column chromatography. The compounds were characterized by utilizing a combination of gas chromatography-mass spectrometry, nuclear magnetic resonance, and circular dichroism. Acids 1a and 1b were not detected in the phospholipids (PtdCho and PtdIns) of the sponge, but rather as free FA and possibly in glycosylceramides. The mixtures of 1a and 1b displayed cytotoxicity towards THP-1 and HepG2 cells with EC50's between 41 and 35 µg/mL. Apoptosis was not the preferred mode of cell death induced by 1a-1b in the THP-1 cells. This implies other types of cytotoxicity mechanisms, such as membrane disruption and/or the inhibition (EC50 = 1.8 µg/mL) of the human topoisomerase IB enzyme (hTopIB), with a mechanism of inhibition different from the one displayed by camptothecin (CPT). In a separate experiment, the mixture of 1a and 1b also displayed cytotoxicity towards ex vivo mouse splenocytes infected with Leishmania infantum amastigotes (IC(50) = 0.17 mg/mL) and free living promastigotes (IC(50) = 0.34 mg/mL). It was also found that the FA were inhibitory of the Leishmania topoisomerase IB (LTopIB) with an EC(50) = 5.1 µg/mL. Taken together, 1a and 1b represent a new class of FA with potential as TopIB inhibitors that preferentially inhibit hTopIB over LTopIB.

GS-2, a pyrazolo[1,5-a]indole derivative with inhibitory activity of topoisomerases, exerts its potent cytotoxic activity by ROS generation.

Pyrazolo[1,5-a]indole derivatives, a new type of topoisomerase (topo) inhibitor, demonstrate a broad spectrum of antitumor activities. However, the mechanism underlying the induced cytotoxicity remains unclear. In this study, we investigated whether GS-2, one of the derivatives, altered the levels of ROS in breast cancer MDA-231 cells and whether these ROS contributed to the observed antitumoral activity. Our data revealed that GS-2 caused a time- and dose-dependent elevation of intracellular ROS level in MDA-231 cells. GS-2 subsequently elicited notable inhibition on the expression of topos, DNA damage, activation of caspase-3, -9. The loss of mitochondrial membrane potential (MMP) was observed during the induction. The addition of N-acetyl cysteine (NAC, a well-known antioxidant) could effectively attenuate the GS-2-induced ROS enhancement and subsequent apoptosis. NAC attenuated the induced inhibition on expression of topos, indicating that topos might be the target of GS-2-induced ROS. The finding of the induced ROS provides new evidence for the molecular mechanisms of antitumor activity of pyrazolo[1,5-a]indole derivatives.

New molecular biologist perspective and insight: DNA topoisomerases production by recombinant DNA technology for medical laboratory application and pharmaceutical industry

DNA topoisomerases are essential enzymes that control the topological state of DNA replication during mitosis. These enzymes are classified based on their mechanisms and physical properties. During mitosis, superhelical DNA must be unwound or relaxed by DNA topoisomerases prior to a decoding step by DNA processing enzymes, such as DNA polymerase and RNA polymerase. By blocking the reaction of resealing the breaks in the DNA ultimately can result in cellular death. Compounds that inhibit the catalytic function of these enzymes can serve as potential anticancer agents. DNA topoisomerases are found in nature and used as high quality and well-validated targets for the screening of potential anticancer agents. Our current work focuses on determining potential anticancer agents from natural resources using DNA topoisomerases as the screening targets. Large scale production of these enzymes using recombinant DNA technology in our academic laboratory is utilised to avoid dependence on expensive commercially available enzymes. The in-house produced enzymes can also be used to enhance our research in the field of molecular medicine by providing an enzyme source that can be used to screen potential anticancer agents, and for other newly developed diagnostic and medical research projects in the near future as well as a step in moving our efforts into the industrial sector.

Mechanisms of ellipticine-mediated resistance in UKF-NB-4 neuroblastoma cells.

Most high-risk neuroblastomas develop resistance to cytostatics and therefore there is a need to develop new drugs. In previous studies, we found that ellipticine induces apoptosis in human neuroblastoma cells. We also investigated whether ellipticine was able to induce resistance in the UKF-NB-4 neuroblastoma line and concluded that it may be possible after long-term treatment with increasing concentrations of ellipticine. The aim of the present study was to investigate the mechanisms responsible for ellipticine resistance. To elucidate the mechanisms involved, we used the ellipticine-resistant subline UKF-NB-4(ELLI) and performed comparative genomic hybridization, multicolor and interphase FISH, expression microarray, real-time RT-PCR, flow cytometry and western blotting analysis of proteins. On the basis of our results, it appears that ellipticine resistance in neuroblastoma is caused by a combination of overexpression of Bcl-2, efflux or degradation of the drug and downregulation of topoisomerases. Other mechanisms, such as upregulation of enzymes involved in oxidative phosphorylation, cellular respiration, V-ATPases, aerobic respiration or spermine synthetase, as well as reduced growth rate, may also be involved. Some changes are expressed at the DNA level, including gains, amplifications or deletions. The present study demonstrates that resistance to ellipticine is caused by a combination of mechanisms.

A DNA-binding surface of SPO11-1, an Arabidopsis SPO11 orthologue required for normal meiosis.

Meiotic recombination is initiated by DNA double-stranded breaks introduced by the SPO11 protein. Despite a decade of research, the biochemical functions of SPO11 remain largely unknown, perhaps because of difficulties in studying the functionally active SPO11. Arabidopsis thaliana encodes three SPO11-related proteins, two of which (SPO11-1 and SPO11-2) are required for, and cooperate in, meiosis. We isolated soluble SPO11-1, fused with or free of a trigger factor-tag at its N terminus. The tag-free SPO11-1 needed to interact physically with soluble SPO11-1 to maintain its solubility, suggesting a multimeric active form including a solubilizing protein cofactor. An N-terminal fragment of PRD1, a SPO11-1-interacting protein required for normal meiosis, but not SPO11-2, forms a soluble complex with trigger factor-tagged SPO11-1, but the trigger factor-tag was required for the solubility. Formation of the complex is not sufficient to express endonuclease activity. Trigger factor-tagged SPO11-1 exhibited DNA-binding activities: Glu substitutions of the invariant Gly215 and Arg222 and of the nonconserved Arg223 and Arg226 in a conserved motif (G215E, R222E, R223E, R226E) reduced the DNA-binding ability in vitro, but substitutions of the conserved Arg130 and invariant Tyr103 (a residue in the putative endonuclease-active center) and of Arg residues outside conserved motifs by Glu or Phe (R130E, Y103F, R207E and R254E), did not. Tests for the ability of mutant spo11-1 proteins to complement the silique-defective phenotype of a spo11-1-homozygous mutant in vivo revealed that R222E and G215E induced serious deficiencies, while R130E caused a partial defect in silique formation. Thus, the Gly215, Arg222 and Arg223 residues of SPO11-1 form a DNA-binding surface that is functional in meiosis.

(-)-Menthol inhibits DNA topoisomerases I, II alpha and beta and promotes NF-kappaB expression in human gastric cancer SNU-5 cells.

It has been reported that (-)-Menthol can inhibit the growth of rat liver epithelial tumor cells and is a potent chemopreventive agent. The purpose of the present experiment was to examine and identify cellular processes leading to cell death which are affected by (-)-Menthol in human gastric SNU-5 cancer cells. Cell death (cytotoxicity) was examined and analyzed by trypan blue stain and flow cytometric methods. It was shown that (-)-Menthol inhibited the proliferation of the cells in a dose- and time-dependent manner, inhibited topoisomerase I, IIa and IIbeta, but promoted the levels of NF-kappaB gene expression based on the Western blot and polymerase chain reaction (PCR) and cDNA microarray methods. These data suggest that (-)-Menthol may induce cytotoxicity through inhibiting gene expression of topoisomerase I, IIalpha and IIbeta and promoting the gene expression of NF-kappaB in SNU-5 cells.

[Clinical implications of DNA-topoisomerases examination in renal biopsies from patients with nephritis]. / Klinicheskoe znachenie issledovaniia DNK-topoizomeraz v bioptatakh pochek bol'nykh nefritami.

AIM: To study expression of topoisomerases (TI) I and II alpha (DNA-bound enzymes involved in transcription and replication) in renal tissue as markers of activity and prognosis of glomerulonephritis (GN) decisive for choice of immunodepressive therapy. MATERIAL AND METHODS: TI expression was studied immunohistochemically in renal biopsies from 177 patients with different morphological variants of GN and in the samples of unaffected kidney tissue removed in 12 patients for local tumors. RESULTS: There are definite differences between proliferative and non-proliferative GN variants--elevation of TI levels and monocytic infiltration in proliferative GN. Focal-segmental glomerulosclerosis is characterized by a high TI II alpha level in mesangial cells and monocytic infiltration of the glomeruli which are typical for inflammation. A statistical relationship between TI levels in mesangial cells and glomerular epithelium suggests a pathogenetic relation between these links of the pathological process. Molecular markers of activation and proliferation of cells and direct inductors of the inflammatory process (cells of monocytic infiltrate) closely correlated with the activity index--an integral indicator of inflammatory activity, as well as with the integral indicator of sclerotic processes in renal tissue--sclerosis index. Monocytic infiltration in the interstitium correlated both with morphological manifestations of activity, progression of nephritis and their clinical equivalents. In high TI expression GN resistance to immunodepressive therapy rose. To overcome the resistance, immunodepressive therapy must be more active--large doses and duration of treatment. In patients with lupus nephritis and mesangiocapillary GN renal prognosis was worse in the presence of high TI expression in mesangial cells and epithelium of the renal canaliculi. CONCLUSION: The authors are the first to demonstrate TI expression in renal tissue of GN patients, correlation of its level with activity of renal process as well as its role in prediction of response to treatment and the rate of renal failure progression. It is suggested that high TI expression entails a progressive course of GN.

New carbocyclic analogues of netropsin: synthesis and inhibition of topoisomerases.

A series of carbocyclic analogues of netropsin were synthesized and evaluated for their capacity to inhibit human topoisomerases I and II in vitro. The compounds are oligopeptides containing 1,4-di- and 1,2,5-trisubstituted benzene rings and unsubstituted N-terminal NH2 groups. Compounds 4-7 consist of two netropsin-like units linked by aliphatic (tetra- and hexamethylene) chains. In the topoisomerase I and II assay, the relaxation of pBR322 plasmid was inhibited by compounds 4-7 at 100 microM concentration.