DNA Relaxation Assay Research Articles

Deglycobleomycin A 6 analogues modified in the methylvalerate moiety

Previous studies have indicated that the methylvalerate subunit of bleomycin (BLM) plays an important role in facilitating DNA cleavage by BLM and deglycoBLM. Eleven methylvalerate analogues have been synthesized and incorporated into deglycoBLM congeners by the use of solid-phase synthesis. The effect of the valerate moiety in the deglycoBLM analogues has been studied by comparison with the parent deglycoBLM A 5 using supercoiled DNA relaxation and sequence-selective DNA cleavage assays. All of the deglycoBLM analogues were found to effect the relaxation of the plasmid DNA. Those analogues having aromatic C4-substituents exhibited cleavage efficiency comparable to that of deglycoBLM A 5. Some, but not all, of the deglycoBLM analogues were also capable of mediating sequence-selective DNA cleavage.

The topoisomerase II inhibitor voreloxin causes cell cycle arrest and apoptosis in myeloid leukemia cells and acts in synergy with cytarabine

Topoisomerase II is essential for the maintenance of DNA integrity and the survival of proliferating cells. Topoisomerase II poisons, including etoposide and doxorubicin, inhibit enzyme-mediated DNA ligation causing the accumulation of double-stranded breaks and have been front-line drugs for the treatment of leukemia for many years. Voreloxin is a first-in-class anti-cancer quinolone derivative that intercalates DNA and inhibits topoisomerase II. The efficacy and mechanisms of action of voreloxin in acute myeloid leukaemia were addressed in this study. Primary acute myeloid leukemia blasts (n = 88) and myeloid cell lines were used in vitro to study voreloxin through viability assays to assess cell killing and synergy with other drugs. Apoptosis and cell cycling were assessed by flow cytometry. DNA relaxation assays were utilized to determine that voreloxin was active on topoisomerase II. The mean lethal dose 50% (LD(50)) (± standard deviation) of voreloxin for primary acute myeloid leukemia blasts was 2.30 μM (± 1.87). Synergy experiments between voreloxin and cytarabine identified synergism in 22 of 25 primary acute myeloid leukemia samples tested, with a mean combination index of 0.79. Apoptosis was shown to increase in a dose-dependent manner. Furthermore, voreloxin was active in the p53-null K562 cell line suggesting that the action of voreloxin is not affected by p53 status. The action of voreloxin on topoisomerase II was confirmed using a DNA relaxation assay. Voreloxin may provide an interesting addition to the cache of drugs available for the treatment of acute myeloid leukemia, a disease with a poor long-term survival. In addition to its potent action as a single agent in dividing cells, the synergy we demonstrated between voreloxin and cytarabine recommends further investigation of this topoisomerase II inhibitor.

A propionyloxy derivative of 11-keto-β-boswellic acid induces apoptosis in HL-60 cells mediated through topoisomerase I & II inhibition

Boswellic acids have invariably been reported for their antiproliferative potential in various cell systems. In the present study the growth inhibitory effect of propionyloxy derivative of 11-keto-β-boswellic acid (PKBA; a semisynthetic analogue of 11-keto-β-boswellic acid) on HL-60 promyelocytic leukemia cells is being reported for the first time. In the preliminary studies, in vitro cytotoxicity of PKBA was investigated against eight human cancer cell lines viz., IMR-32, SF-295 (both neuroblastoma), PC-3 (prostate), Colo-205 (colon), MCF-7 (breast), OVCAR-5 (ovary), HL-60, Molt-4 (both leukemia) and their respective IC50 values were found to be 5.95, 7.11, 15.2, 14.5, 15, 15.9, 8.7 & 9.5μg/ml, respectively. For determining the mechanism of cell death in HL-60 cells, PKBA was subjected to different mechanistic studies. DNA relaxation assay of PKBA revealed inhibition of both topoisomerases I & II. The fragmentation analysis of DNA revealed typical ladders indicating the cytotoxic effect to be mediated by induction of apoptosis. The morphologic studies of PKBA showed the presence of true apoptotic bodies. Apoptosis was confirmed further by flow-cytometric detection of sub-G1 peaks and enhanced annexin-V-FITC binding of the cells. The activation of apoptotic cascade by PKBA in HL-60 cells was found to be associated with the loss of mitochondrial membrane potential, release of cytochrome c, activation of initiator and executioner caspases and cleavage of poly ADP ribose polymerase (PARP). In vivo studies of PKBA revealed anti-tumoral activity against both ascitic and solid murine tumor models. These studies thus demonstrate PKBA to induce apoptosis in HL-60 cells due to the inhibition of topoisomerases I and II.

Riccardin D, a novel macrocyclic bisbibenzyl, induces apoptosis of human leukemia cells by targeting DNA topoisomerase II

We studied the effect of riccardin D, a macrocyclic bisbibenzyl, which was isolated from the Chinese liverwort plant, on human leukemia cells and the underlying molecular mechanism. Riccardin D had a significant antiproliferative effect on human leukemia cell lines HL-60, K562 and its multidrug resistant (MDR) counterpart K562/A02 cells, but showed no effect on the topoisomerase-II-deficient HL-60/MX2 cells, as measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The pBR322 DNA relaxation assay revealed that riccardin D selectively inhibited the activity of topoisomerase II (topo II). The suppression of topo II activity by riccardin D was stronger than that of etoposide, a known topo II inhibitor. After treatment with riccardin D, nuclear extracts of leukemia K562 and K562/A02 cells left the majority of pBR322 DNA in a supercoiled form. Further examination showed that riccardin D effectively induced HL-60, K562 and K562/A02 apoptosis as evidenced by externalization of phosphatidylserine and formation of DNA ladder fragments. The activation of cytochrome c, caspase-9, caspase-3 and cleaved poly ADP-ribose polymerase (PARP) was also enhanced, as estimated by Western blot analysis. By contrast, riccardin D was unable to induce apoptosis in the topoisomerase-II-deficient HL-60/MX2 cells, indicating that the induction of apoptosis by riccardin D was due to the inhibition of topo II activity. In addition, riccardin D was able to significantly decrease P-glycoprotein (P-gp) expression in K562/A02 cells. Taken together, our data demonstrate that riccardin D is a novel DNA topo II inhibitor which can induce apoptosis of human leukemia cells and that it has therapeutic potential for both regular and MDR strains of leukemia cells.

Structures of Minimal Catalytic Fragments of Topoisomerase V Reveals Conformational Changes Relevant for DNA Binding

Topoisomerase V is an archaeal type I topoisomerase that is unique among topoisomerases due to presence of both topoisomerase and DNA repair activities in the same protein. It is organized as an N-terminal topoisomerase domain followed by 24 tandem helix-hairpin-helix (HhH) motifs. Structural studies have shown that the active site is buried by the (HhH) motifs. Here we show that the N-terminal domain can relax DNA in the absence of any HhH motifs and that the HhH motifs are required for stable protein-DNA complex formation. Crystal structures of various topoisomerase V fragments show changes in the relative orientation of the domains mediated by a long bent linker helix, and these movements are essential for the DNA to enter the active site. Phosphate ions bound to the protein near the active site helped model DNA in the topoisomerase domain and show how topoisomerase V may interact with DNA.

A new ELISA plate based microtiter well assay for mycobacterial topoisomerase I for the direct screening of enzyme inhibitory monoclonal antibody supernatants

Antigen specific monoclonal antibodies present in crude hybridoma supernatants are normally screened by ELISA on plates coated with the relevant antigen. Screening for inhibitory monoclonals to enzymes would require the evaluation of purified antibodies or antibody containing supernatants for their inhibition of enzyme activity in a separate assay. However, screening for inhibitory antibodies against DNA transacting enzymes such as topoisomerase I (topo I) cannot be done using hybridoma supernatants due to the presence of nucleases in tissue culture media containing foetal calf serum which degrade the DNA substrates upon addition. We have developed a simple and rapid screening procedure for the identification of clones that secrete inhibitory antibodies against mycobacterial topo I using 96 well ELISA microtiter plates. The principle of the method is the selective capture of monoclonal antibodies from crude hybridoma supernatants by topo I that is tethered to the plate through the use of plate-bound polyclonal anti-topo I antibodies. This step allows the nucleases present in the medium to be washed off leaving the inhibitor bound to the tethered enzyme. The inhibitory activity of the captured antibody is assessed by performing an in situ DNA relaxation assay by the addition of supercoiled DNA substrate directly to the microtiter well followed by the analysis of the reaction products by agarose gel electrophoresis. The validity of this method was confirmed by purification of the identified inhibitory antibody and its evaluation in a DNA relaxation assay. Elimination of all enzyme-inhibitory constituents of the culture medium from the well in which the inhibitory antibody is bound to the tethered enzyme may make this method broadly applicable to enzymes such as DNA gyrases, restriction enzymes and other DNA transaction enzymes. Further, the method is simple and avoids the need of prior antibody purification for testing its inhibitory activity.

The Mechanism of Vanadium-Mediated Developmental Hypomyelination Is Related to Destruction of Oligodendrocyte Progenitors Through a Relationship with Ferritin and Iron

The second post-natal week in rat is the period of the most intense oligodendrocyte development and myelination. This period coincides with peak iron import by oligodendrocytes. During that time oligodendrocyte progenitors (OPCs) are sensitive to agents that may disturb normal iron homeostasis and assimilation of iron into these cells. One mechanism by which iron homeostasis can be disrupted is by environmental exposure to other metals. Vanadium is a transition metal, and exposure to vanadium during early brain development produces hypomyelination with variety of related neuro-behavioral phenotypes. In the current study, we investigated mechanisms of hypomyelination induced by vanadium exposure in developing rat brain. We demonstrate that both in vivo and in vitro, OPCs are more sensitive to vanadium exposure than astrocytes or mature oligodendrocytes. Vanadium exposure in OPCs resulted in increased ROS generation and increased annexinV labeling suggestive of apoptosis. Because ferritin is a major iron delivery protein for oligodendrocytes, we exposed the cells to recombinant ferritin and iron both of which exacerbated vanadium cytotoxicity, while the iron chelator desferroxamine (DFO) prevented cytotoxic/apoptotic effects of vanadium. To illustrate relationship between ferritin and vanadium, we demonstrate that vanadium exacerbated DNA nicking produced by iron-rich spleen ferritin, but not iron-poor apoferritin, resulting in a single and double strand breaks in a DNA relaxation assay. We propose that developmental exposure to vanadium interferes with normal iron assimilation into oligodendrocytes resulting in oxidative stress and apoptosis. Therefore, depletion of OPCs due to vanadium exposure in early post-natal period may be an important mechanism of vanadium-induced hypomyelination.

Selective inhibition of human leukemia cell growth and induction of cell cycle arrest and apoptosis by pseudolaric acid B

The leukemias account for the largest number of cases of childhood cancer and remain the primary cause of cancer-related mortality among children in the United States. There is a need for novel antileukemia agents due to toxicity and resistant to existing chemotherapeutic agents. In this study, the effects of pseudolaric acid B (PAB) on three human leukemia cell lines, acute promyelocytic leukemia HL-60 cells, acute lymphoblastic leukemia CCRF-CEM cells, and human chronic myeloid leukemia blast-phase K562 cells were investigated in vitro, compared to normal human peripheral blood mononuclear cells (PBMC). Cell viability was determined using CellTiter-Glo luminescent reagent. Colony formation was assessed by Microtitration cloning assay. Cell cycle analysis was carried out by flow cytometry. Tubulin polymerization was measured by recording the increase in absorbance. Inhibition of topoisomerase I (topo I) and topoisomerase II (topo II) enzyme activities was measured by DNA relaxation assay using topo I and II drug screening kit. Apoptosis was observed by DAPI staining assay and Caspase3/7 activities was measured using Caspase-Glo((R)) 3/7 assay kit. Pseudolaric acid B selectively inhibited the growth of human leukemia HL-60, CCRF-CEM and K562 cells, but not normal PBMC. PAB suppressed colony formation in HL-60 cells. Cell cycle analysis showed that PAB blocked the cell cycle at G(2)/M phase in HL-60 cells, suggesting that it suppresses mitosis. DNA topo I and topo II were not inhibited, but tubulin polymerization was inhibited. PAB-induced apoptosis and activated caspase-3/7 activity. This study indicates that PAB has a potential for use against leukemia and its effects might be mediated by inhibiting tubulin polymerization, preventing cell division and activating caspase-3, which leads to apoptosis.

Topoisomerase II Inhibitor Voreloxin Causes Cell Cycle Arrest and Apoptosis in Acute Myeloid Leukaemia Cells and Acts in Synergy with Cytarabine.

Abstract 4152Topoisomerase II is essential for the maintenance of DNA integrity and the survival of proliferating cells. This enzyme functions as a homodimer to modulate DNA supercoiling and the unknotting and untangling of DNA. It acts via the creation of transient double strand breaks in the DNA that allow the resolution of DNA tangles prior to the rejoining of the double strand breaks. In cells with insufficient topoisomerase II activity DNA remains entangled resulting in reduced gene transcription. Conversely if a cell has excessive topoisomerase II activity the cleavage intermediates it forms with DNA can be converted into permanent strand breaks resulting in the loss of DNA integrity. Topoisomerase II poisons, including etoposide and doxorubicin, inhibit enzyme-mediated DNA ligation causing the accumulation of double strand breaks. These agents have been frontline drugs for the treatment of leukaemia for many years. Voreloxin (formerly SNS-595) is a first-in-class anticancer quinolone derivative that intercalates DNA and poisons topoisomerase II, inducing replication-dependent, site-selective DNA double-strand breaks. Primary acute myeloid leukaemia (AML) blasts isolated from patients at diagnosis (n = 88) had a mean LD50 (± SD) for voreloxin of 2.30μM (± 1.87). The mean Ara-C LD50 was 4.90μM (± 5.00) in the same population while the myeloid cell lines, NB4 and HL-60, had LD50 values for voreloxin of 0.23μM and 0.94μM respectively. The lower LD50 values for voreloxin in the cell lines is likely to be due to the fact that they are more actively dividing in culture than primary AML blasts and this agent is, at least to some extent, replication-dependent. Synergy experiments between voreloxin and Ara-C, (voreloxin1:2 Ara-C) identified synergism in 22 of 25 primary AML samples tested, with a mean combination index of 0.79. Apoptosis, measured by increases in Annexin V/propidium iodide (PI) staining and caspase-3 activation, was shown to increase in a dose-dependent manner. Annexin V/PI positivity was significantly increased by concentrations of voreloxin over 0.06μM (P = 0.02) while caspase-3 activation was evident at concentrations of voreloxin greater than 0.25μM (P = 0.0009). Furthermore, voreloxin was active in the p53 null K562 cell line, showing a dose-dependent increase in Annexin V/PI staining and an LD50 0.52μM. These data agree with previous reports suggesting that the action of voreloxin is not affected by p53 status. The action of voreloxin on topoisomerase II was confirmed using a DNA relaxation assay. In the presence of voreloxin the ability of topoisomerase II to relax a supercoiled DNA substrate was reduced in a dose-dependent manner. Voreloxin may provide an interesting addition to the cache of drugs available for the treatment of AML; a disease with poor long term survival. In addition to its potent action as a single agent in dividing cells, the synergy we demonstrated between voreloxin and AraC recommend it for further investigation Disclosures:No relevant conflicts of interest to declare.

Rational Design and Semisynthesis of Betulinic Acid Analogues as Potent Topoisomerase Inhibitors

Chemical transformation studies were conducted on betulinic acid (1), a common plant-derived lupane-type triterpene. Eleven new rationally designed derivatives of 1 (2-5 and 7-13) were synthesized based on docking studies and tested for their topoisomerase I and IIalpha inhibitory activity. Semisynthetic reactions targeted C-3, C-20, and C-28 in 1. Structures of the new compounds were confirmed by spectroscopic methods (1D and 2D NMR and MS). Compound 9, 3-O-[N-(phenylsulfonyl)carbamoyl-17beta-N-(phenylsulfonyl)amide]betulinic acid, showed 1.5-fold the activity of CPT in a topoisomerase I DNA relaxation assay. Four out of 14 betulinic acid analogues (5, 9, 11, and 12) showed 1.5-fold the activity of etoposide in a topoisomerase II assay. The new analogues exhibited better cytotoxic activities against the human colon cancer cells SW948 and HCT-116 and the breast cancer cell line MDA-MB-231 compared to the parent (1). Betulinic acid (1) is a potential scaffold for the design of new topoisomerase I and IIalpha inhibitors.

Kauren‐19‐oic acid induces DNA damage followed by apoptosis in human leukemia cells

This study evaluated the potential cytotoxicity of the natural diterpenoids kauren-19-oic acid (KA), 14-hydroxy-kaurane (1) and xylopic acid (2), and semi-synthetic derivatives of KA (3-5) towards human cancer cell lines (K562, HL60, MDA-MB435 and SF295) and lymphocytes. Mouse erythrocytes were used to verify a possible hemolytic activity Cytotoxicity mechanisms were investigated in HL60 cells. KA showed a moderate antiproliferative effect in MTT assay towards all cancer cells (IC(50), 9.1-14.3 microg ml(-1)). However, KA appeared not selective to cancer cells, since it also inhibited the lymphocytes proliferation (IC(50), 12.6 microg ml(-1)). Unlike KA, compounds 1-5 displayed no cytotoxicity and were also free from antiproliferative and hemolytic effects, suggesting that the exocyclic double bond (C16) unit may be the active pharmacophore of KA cytotoxicity. KA-treated HL60 cells displayed decreased proliferation (5-bromo-2';-deoxyuridine incorporation assay) and topoisomerase I activity (DNA relaxation assay). These assays revealed that KA primarily intercalates with DNA and not with topoisomerase I. Fluorescence microscopy using AO/EB (acridine orange/ethidium bromide) staining indicated that KA can induce both apoptosis and necrosis in HL-60 cell cultures, which corroborate the findings with MTT. From these findings, we conclude that KA, although demonstrating cytotoxic potential, may have a limited or poor therapeutic potential due to lack of selectivity to tumor cells. Further studies on the structure modification of KA and the mechanism of the new derivatives are currently in progress.

Amino Acid Composition and Antioxidative Peptides from Protein Hydrolysates of Yellow Stripe Trevally (Selaroides leptolepis)

Hydrolysates from yellow stripe trevally meat with 15% degree of hydrolysis (DH) obtained using Alcalase (HA) and Flavourzyme (HF) following pH-stat method were characterized. Both hydrolysates, HA and HF, contained glutamic acid and glutamine as the major amino acids, while glycine was the dominant amino acid in fish flesh. Hydrolysates were also rich in aspartic acid, asparagine, alanine, lysine, and leucine. Essential amino acid/nonessential amino acid ratios were 0.55, 0.63, and 0.62 for flesh, HA, and HF, respectively. After separation on a Sephadex G-50 column, antioxidative activity of hydrolysate fractions was determined as Trolox equivalent antioxidant capacity. The fraction of HF with molecular weight (MW) of 1.77 kDa exhibited the strongest antioxidative activity, compared with other fractions. Among all fractions of HA, the one having a MW of 2.44 kDa showed the highest antioxidative activity. Thereafter, the selected Sephadex G-50 fractions from both HA and HF were further chromatographed using a Sephadex C-25 column, followed by HPLC. MW of antioxidative peptides from HA and HF determined by Maldi TOF/TOF was 656 and 617 Da, respectively. However, peptides isolated from both HA and HF possessed a lower antioxidative activity than Trolox at the same concentration (P < 0.05). HA and HF prevented DNA oxidative damage in Fenton reaction system tested by in vitro plasmid DNA relaxation assay. These results indicate that hydrolysates from yellow stripe trevally can be used as an important source of amino acids and serve as alternative natural antioxidants.

Novel Stable Camptothecin Derivatives Replacing the E-Ring Lactone by a Ketone Function Are Potent Inhibitors of Topoisomerase I and Promising Antitumor Drugs

The E-ring lactone is the Achilles' heel of camptothecin derivatives: although it is considered necessary for the inhibition of the enzyme topoisomerase I (topo1), the opening of the lactone into a carboxylate abolishes the generation of topo1-mediated DNA breaks. S38809 is a novel camptothecin analog with a stable 5-membered E-ring ketone; therefore, it lacks the lactone function. DNA relaxation and cleavage assays revealed that S38809 functions as a typical topo1 poison by stimulating DNA cleavage at T downward arrow G sites. The activity was strongly dependent on the stereochemistry of the C-7 carbon atom that bears the hydroxy group. S38809 proved to be a potent cytotoxic agent, with a mean IC50 of 5.4 nM versus 11.6 nM for topotecan and 3.3 nM for SN38 (the active metabolite of irinotecan) on a panel of 31 human tumor cell lines. The cytotoxicity of S38809 and its ability to stabilize cleavable complexes was considerably reduced in camptothecin-resistant cells that express a mutated topo1, confirming that topo1 is its primary target. Cell death induced by topo1 poisoning requires the conversion of DNA single-strand breaks into double-strand breaks that can be detected by the formation of phosphorylated histone H2AX. In HCT116 cells, topotecan, SN38, and S38809 induced histone H2AX phosphorylation in S phase of the cell cycle, with S38809 being as potent as SN38 and 5-fold more potent than topotecan. In vivo, S38809 showed a marked antitumor activity against HCT116 xenografts. These findings open a new route for improving the pharmacological properties of camptothecin derivatives.

Alterations in Linker Flexibility Suppress DNA Topoisomerase I Mutant-induced Cell Lethality

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of a covalent enzyme-DNA intermediate, which is reversibly stabilized by the anticancer agent camptothecin (CPT). Crystallographic studies of the 70-kDa C terminus of human Top1p bound to duplex DNA describe a monomeric protein clamp circumscribing the DNA helix. The structures, which lack the N-terminal domain, comprise the conserved clamp, an extended linker domain, and the conserved C-terminal active site Tyr domain. CPT bound to the covalent Top1p-DNA complex limits linker flexibility, allowing structural determination of this domain. We previously reported that mutation of Ala(653) to Pro in the linker increases the rate of enzyme-catalyzed DNA religation, thereby rendering Top1A653Pp resistant to CPT (Fiorani, P., Bruselles, A., Falconi, M., Chillemi, G., Desideri, A., and Benedetti P. (2003) J. Biol. Chem. 278, 43268-43275). Molecular dynamics studies suggested mutation-induced increases in linker flexibility alter Top1p catalyzed DNA religation. To address the functional consequences of linker flexibility on enzyme catalysis and drug sensitivity, we investigated the interactions of the A653P linker mutation with a self-poisoning T718A mutation within the active site of Top1p. The A653P mutation suppressed the lethal phenotype of Top1T718Ap in yeast, yet did not restore enzyme sensitivity to CPT. However, the specific activity of the double mutant was decreased in vivo and in vitro, consistent with a decrease in DNA binding. These findings support a model where changes in the flexibility or orientation of the linker alter the geometry of the active site and thereby the kinetics of DNA cleavage/religation catalyzed by Top1p.

Regulation of Catalysis by the Smallpox Virus Topoisomerase

The poxvirus type IB topoisomerases catalyze relaxation of supercoiled DNA by cleaving and rejoining DNA strands via a pathway involving a covalent phosphotyrosine intermediate. Recently we determined structures of the smallpox virus topoisomerase bound to DNA in covalent and non-covalent DNA complexes using x-ray crystallography. Here we analyzed the effects of twenty-two amino acid substitutions on the topoisomerase activity in vitro in assays of DNA relaxation, single cycle cleavage, and equilibrium cleavage-religation. Alanine substitutions at 14 positions impaired topoisomerase function, marking a channel of functionally important contacts along the protein-DNA interface. Unexpectedly, alanine substitutions at two positions (D168A and E124A) accelerated the forward rate of cleavage. These findings and further analysis indicate that Asp(168) is a key regulator of the active site that maintains an optimal balance among the DNA cleavage, religation, and product release steps. Finally, we report that high level expression of the D168A topoisomerase in Escherichia coli, but not other alanine-substituted enzymes, prevented cell growth. These findings help elucidate the amino acid side chains involved in DNA binding and catalysis and provide guidance for designing topoisomerase poisons for use as smallpox antivirals.

Imidazoacridinones Are Bifunctional Targeting Agents Active in Leukemia Cells.

C-1311 (Symadex™) is the lead compound in clinical development from a new series of agents, the imidazoacridinones. It was previously reported that C-1311 was solely a topoisomerase II (TOP2) cleavable complex inhibitor and DNA damaging agent. However, contemporary analyses of C-1311 point to alternative structural parallelisms with the molecular scaffolds now seen as common motifs in receptor tyrosine kinase (RTK) inhibitors. To investigate this mechanism of action, a kinase inhibitor screening program was initiated, first in silico by molecular similarity matching, and then in vitro against recombinant RTKs. C-1311 was identified to cluster with RTK inhibitors, especially when sunitinib is used as a molecular “seed” for pattern recognition. The statistical similarity coefficients fall in the 0.7–0.8 positive probability range, compared to 0.2–0.4 for the classical TOP2 inhibitors. C-1311 was found to selectively inhibit wild-type (WT) FLT3 and its D835Y mutant at nanomolar concentrations of 8–15 nM IC50. FGFR2 was also targeted at approximately 100-fold greater IC50 value of 1.2 μM. Inhibition of PDGFR, FGFR1, and KIT was observed at about 3–4 μM IC50; other prominent kinases had values of greater than 10 μM. As a part of the dual mechanism reexamination, TOP2 decatenation of kDNA, DNA relaxation, and competition assays were performed to assess C-1311 interaction with TOP2. Results confirmed that C-1311 is not a cleavable complex inhibitor when compared to etoposide, suggesting that it may not be an inducer of the MLL cleavage. Additionally, the IC50 of C-1311 from the decatenation assay is 44 μM compared to 3, 12, and 149 μM for mitoxantrone, daunorubicin, and merbarone, respectively. Cellular screens were performed to assess the potency of C-1311 in myeloid and lymphoid leukemia cell lines. C-1311 was equally effective in both WT FLT3 expressing (RS4;11) and mutant FLT3 expressing (MV-4–11) cell lines. The dual function of C-1311 may contribute to the potency in these cell lines. These findings warrant further investigation of C-1311 in yet unexplored indications, including other hematological malignancies.

Potent antitumor activity of 10-methoxy-9-nitrocamptothecin

The present data showed that 10-methoxy-9-nitrocamptothecin (MONCPT), a family of camptothecin analogues, possessed high antitumor activity in vitro and in vivo. Cytotoxicity assays showed that MONCPT was a potential and highly efficient antitumor compound with IC50 values of 0.1 to 500 nmol/L in nine tumor cell lines. The high cytotoxic potency of MONCPT was paralleled with its ability to increase the cellular accumulation of DNA damage. DNA relaxation assay also showed that MONCPT exerted high potency as a topoisomerase I inhibitor. Moreover, administration of MONCPT (5-20 mg/kg) for 15 to 17 days significantly inhibited tumor growth in human androgen-independent prostate tumor (PC3) and human non-small cell lung tumor (A549) xenografts; the inhibition rates ranged from 29.6% to 98%. The cytotoxic effect of 1,000 nmol/L of MONCPT in PC3 cells was associated with causing an arrest in G0-G1 phase, whereas that of 10 and 100 nmol/L MONCPT was relative to a persistent block in G2-M phase. Furthermore, down-regulation of CDK2, CDK4, and cyclin D1 was observed in PC3 cells treated with 1,000 nmol/L of MONCPT, whereas overexpression of CDK7, CDK1, and cyclin B1 was seen in PC3 cells treated with 10 and 100 nmol/L of MONCPT. These results suggested that cell cycle regulation might contribute to the anticancer properties of MONCPT and strongly support the further anticancer development of MONCPT.

Cytotoxic and radioprotective effects of Podophyllum hexandrum

Podophyllum hexandrum, a herb thriving in Himalayas has already been reported to exhibit antitumor and radioprotective properties. Present study was undertaken to unravel the possible mechanism responsible for the cytotoxic and radioprotective properties of REC-2001, a fraction isolated from the rhizome of P. hexandrum using murine peritoneal macrophages and plasmid DNA as model systems. Cell death, levels of intracellular reactive oxygen species (ROS) and apoptosis were studied employing trypan blue exclusion assay, dichlorofluorescein diacetate and DNA fragmentation assay, respectively. Superoxide anions, hydroxyl radicals and DNA damage were estimated following nitroblue tetrazolium, 2-deoxyribose degradation and plasmid DNA relaxation assays, respectively. Pre-irradiation administration of REC-2001 to peritoneal macrophages in the concentration range of 25–200 μg/ml significantly reduced radiation induced ROS generation, DNA damage, apoptosis and cell killing in comparison to radiation control group indicating radioprotective potential. Studies with plasmid DNA indicated the ability of REC-2001 to inhibit 20 Gy induced single and double strand breaks further supporting the antioxidative potential. However, REC-2001 in a dose-dependent fashion induced cell death, ROS and DNA fragmentation indicating the cytotoxic nature. REC-2001, in presence of 100 μM copper sulfate, generated significant amount of hydroxyl radicals and superoxide anions indicating ability to act as a pro-oxidant in presence of metal ions. The superoxide anion generation was found to be sensitive to metal chelators like EDTA and deferoxamine mesylate (DFR). These results suggest that the ability of REC-2001 to act as a pro-oxidant in presence of metal ions and antioxidant in presence of free radicals might be responsible for cytotoxic and radioprotective properties.

Assembly of a Polymeric Chain of SUMO1 on Human Topoisomerase I in Vitro

Human (h) DNA topoisomerase I has been identified as a major SUMO1 target in camptothecin-treated cells. In response to TOP1-mediated DNA damage induced by camptothecin, multiple SUMO1 molecules are conjugated to the N-terminal domain of a single TOP1 molecule. To investigate the molecular mechanism of SUMO1 conjugation to TOP1, an in vitro system using purified SAE1/2, Ubc9, SUMO1, and TOP1 peptides was developed. Consistent with results from in vivo studies, multiple SUMO1 molecules were found to be conjugated to the N-terminal domain of a single TOP1 molecule. Systematic analysis has identified a single major SUMO1 conjugation site located between amino acid residues 110 and 125 that contains a single lysine residue at 117 (Lys-117). Using a short peptide spanning this region, we showed that a poly-SUMO1 chain was assembled in this peptide at Lys-117. Interestingly, a Ubc9-poly-SUMO1 intermediate had accumulated to a high level when the sumoylation assay was performed in the absence of hTOP1 substrate, suggesting a possibility that the poly-SUMO1 chain is formed on Ubc9 first and then transferred en bloc onto hTOP1. This is the first definitive demonstration of the assembly of a poly-SUMO1 chain on protein substrate. These results offer new insight into hTOP1 polysumoylation in response to TOP1-mediated DNA damage and may have general implications in protein polysumoylation.

Structure of the N-terminal fragment of topoisomerase V reveals a new family of topoisomerases

Topoisomerases are involved in controlling and maintaining the topology of DNA and are present in all kingdoms of life. Unlike all other types of topoisomerases, similar type IB enzymes have only been identified in bacteria and eukarya. The only putative type IB topoisomerase in archaea is represented by Methanopyrus kandleri topoisomerase V. Despite several common functional characteristics, topoisomerase V shows no sequence similarity to other members of the same type. The structure of the 61 kDa N-terminal fragment of topoisomerase V reveals no structural similarity to other topoisomerases. Furthermore, the structure of the active site region is different, suggesting no conservation in the cleavage and religation mechanism. Additionally, the active site is buried, indicating the need of a conformational change for activity. The presence of a topoisomerase in archaea with a unique structure suggests the evolution of a separate mechanism to alter DNA.