Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Flavone and camptothecin were both shown to potently induce apoptosis in HT-29 human colon cancer cells. Whereas camptothecin acts on the basis of topoisomerase-I inhibition, flavone appears to burst mitochondrial production of reactive oxygen species by increasing respiratory chain activity. In our study, we searched for similarities and differences in the proteome response of HT-29 cells when treated with the two different compounds. The accessible proteome of HT-29 cells was separated subsequent to the exposure to flavone or camptothecin by 2D-polyacrylamide-gel electrophoresis using pH-gradients between 4 and 7 and 6 and 11 in the first dimension and proteins with changed expression level were identified by peptide mass fingerprints of tryptic digests of the protein spots. Whereas there was a high congruence with regard to the identities of regulated proteins and their grade of regulation, a number of spots changed specifically only in response to either flavone or camptothecin. Nuclear envelope proteins were specifically increased by camptothecin indicating the intervention of this drug with cell division processes. Increased levels of coproporphyrinogen III oxidase, involved in cytochrome synthesis, and ubiquinol-cytochrome-c reductase suggest adaptations to flavone in order to enable a higher substrate flux through the respiratory chain. In conclusion, HT-29 cells respond to camptothecin and flavone with regulations of many proteins in a similar manner suggesting those alterations to be caused by apoptosis induction. Some protein regulations, however, were specific for each compound and point to the mechanism of their action.
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PMID:Proteome response in HT-29 human colorectal cancer cells to two apoptosis-inducing compounds with different mode of action. 1821 53

Integrin-mediated adhesion to the extracellular matrix plays a fundamental role in tumor metastasis. Salvicine, a novel diterpenoid quinone compound identified as a nonintercalative topoisomerase II poison, possesses a broad range of antitumor and antimetastatic activity. Here, the mechanism underlying the antimetastatic capacity of salvicine was investigated by exploring the effect of salvicine on integrin-mediated cell adhesion. Salvicine inhibited the adhesion of human breast cancer MDA-MB-435 cells to fibronectin and collagen without affecting nonspecific adhesion to poly-l-lysine. The fibronectin-dependent formation of focal adhesions and actin stress fibers was also inhibited by salvicine, leading to a rounded cell morphology. Furthermore, salvicine down-regulated beta(1) integrin ligand affinity, clustering and signaling via dephosphorylation of focal adhesion kinase and paxillin. Conversely, salvicine induced extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. The effect of salvicine on beta(1) integrin function and cell adhesion was reversed by U0126 and SB203580, inhibitors of MAPK/ERK kinase 1/2 and p38 MAPK, respectively. Salvicine also induced the production of reactive oxygen species (ROS) that was reversed by ROS scavenger N-acetyl-l-cysteine. N-acetyl-l-cysteine additionally reversed the salvicine-induced activation of ERK and p38 MAPK, thereby maintaining functional beta(1) integrin activity and restoring cell adhesion and spreading. Together, this study reveals that salvicine activates ERK and p38 MAPK by triggering the generation of ROS, which in turn inhibits beta(1) integrin ligand affinity. These findings contribute to a better understanding of the antimetastatic activity of salvicine and shed new light on the complex roles of ROS and downstream signaling molecules, particularly p38 MAPK, in the regulation of integrin function and cell adhesion.
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PMID:Salvicine inactivates beta 1 integrin and inhibits adhesion of MDA-MB-435 cells to fibronectin via reactive oxygen species signaling. 1831 80

Vaccinia DNA topoisomerase IB (TopIB) relaxes supercoils by forming and resealing a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate. Here we gained new insights to the TopIB mechanism through "chemical mutagenesis." Meta-substituted analogs of Tyr(274) were introduced by in vitro translation in the presence of a chemically misacylated tRNA. We report that a meta-OH reduced the rate of DNA cleavage 130-fold without affecting the rate of religation. By contrast, meta-OCH(3) and NO(2) groups elicited only a 6-fold decrement in cleavage rate. We propose that the meta-OH uniquely suppresses deprotonation of the para-OH nucleophile during the cleavage step. Assembly of the vaccinia TopIB active site is triggered by protein contacts with a specific DNA sequence 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrowN (where downward arrow denotes the cleavage site). A signature alpha-helix of the poxvirus TopIB ((132)GKMKYLKENETVG(144)) engages the target site in the major groove and thereby recruits catalytic residue Arg(130) to the active site. The effects of 11 missense mutations at Tyr(136) highlight the importance of van der Waals interactions with the 3'-G(+4)pG(+3)p dinucleotide of the nonscissile strand for DNA cleavage and supercoil relaxation. Asn(140) and Thr(142) donate hydrogen bonds to the pro-(S(p))-oxygen of the G(+3)pA(+2) phosphodiester of the nonscissile strand. Lys(133) and Lys(135) interact with purine nucleobases in the major groove. Whereas none of these side chains is essential per se, an N140A/T142A double mutation reduces the rate of supercoil relaxation and DNA cleavage by 120- and 30-fold, respectively, and a K133A/K135A double mutation slows relaxation and cleavage by 120- and 35-fold, respectively. These results underscore functional redundancy at the TopIB-DNA interface.
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PMID:Chemical and traditional mutagenesis of vaccinia DNA topoisomerase provides insights to cleavage site recognition and transesterification chemistry. 1836 46

The combination of doxorubicin and mitomycin C has been shown previously to result in supra-additive tumor cell killing in vitro in both murine and human breast cancer cells and in vivo against murine breast cancer cells. Median effect analysis was used to determine the significance and degree of interaction. The origin of this synergy was sought by evaluating the contribution of membrane efflux pump modulation, formaldehyde production, reactive oxygen species, DNA cross-linking, and DNA double-strand breaks to this effect. The interaction of mitomycin C and doxorubicin in vitro was found to be a true synergy whose mechanism was efflux pump-independent. DNA cross-links were only found to increase additively with co-administration of the drugs; however, a supra-additive increase in DNA double-strand breaks was observed. The results suggest that poisoning of topoisomerase IIalpha by doxorubicin may interact with drug-induced DNA cross-links to enhance the formation of DNA double-strand breaks. This interaction, together with glutathione depletion and mitomycin C-derived formaldehyde, may be the underlying mechanism(s) of the synergy observed between mitomycin C and doxorubicin.
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PMID:On the synergistic effect of doxorubicin and mitomycin C against breast cancer cells. 1844

The mechanism of action and the mechanism of resistance of the 4-quinolones are complex and poorly understood. The first barrier these molecules must cross is the bacterial outer membrane. In gram-negative species, 4-quinolones pass through either the porins or lipopolysaccharides (or both) depending on their chemical nature. The cellular target is the DNA of the bacterial chromosome. 4-Quinolones mainly modify the activity of topoisomerase II or DNA gyrase, but also that of topoisomerase I and IV. Any damage to the bacterial genome will induce a stress response which consists in the SOS response, chaperonin synthesis or the generation of oxygen free radicals. Peptidoglycan synthesis is also modified, as shown by the inhibition of PBP(3) activity. Mechanism of resistance involves mutations on gyrA and gyrB genes. gyrA Mutations on the chromosome are responsible for a high level of resistance due to a modification of the A subunit of DNA gyrase, mutations on gyrB gene are responsible for a low level of resistance; the combination of both mutations leads to a high level of resistance. Other mutations are responsible for increasing the MIC, such as a norA mutation in S. aureus. The antibacterial activity of the various molecules is different and, as a result, there is not a single mechanism of action or resistance, but rather a common trunk on which additional mechanisms are grafted.
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PMID:Fluoroquinolones: mechanisms of action and resistance. 1861 33

Hypoxia-inducible factor-1 (HIF-1) is the central mediator of cellular responses to low oxygen and vital to many aspects of cancer biology. In a search for HIF-1 inhibitors, we identified a quassinoid 6alpha-tigloyloxychaparrinone (TCN) as an inhibitor of HIF-1 activation from Ailantus altissima. We here demonstrated the effect of TCN on HIF-1 activation induced by hypoxia or CoCl2. TCN showed the potent inhibitory activity against HIF-1 activation induced by hypoxia in various human cancer cell lines. This compound markedly decreased the hypoxia-induced accumulation of HIF-1alpha protein dose-dependently, whereas it did not affect the expressions of HIF-1beta and topoisomerase-I. Furthermore, TCN prevented hypoxia-induced expression of HIF-1 target genes for vascular endothelial growth factor (VEGF) and erythropoietin. Further analysis revealed that TCN strongly inhibited HIF-1alpha protein synthesis, without affecting the expression level of HIF-1alpha mRNA or degradation of HIF-1alpha protein. Moreover, the levels of phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2), mitogen-activated protein (MAP) kinase-interacting protein kinase-1 (MNK1) and eukaryotic initiation factor 4E (eIF4E) were significantly suppressed by the treatment of TCN, without changing the total levels of these proteins. Our data suggested that TCN may exhibit anticancer activity by inhibiting HIF-1alpha translation through the inhibition of eIF4E phosphorylation pathway and thus provide a novel mechanism for the anticancer activity of quassinoids. TCN could be a new HIF-1-targeted anticancer agent and be effective on mammalian target of rapamycin (mTOR)-targeted cancer therapy, in which mTOR inhibition increases eIF4E phosphorylation.
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PMID:A quassinoid 6alpha-tigloyloxychaparrinone inhibits hypoxia-inducible factor-1 pathway by inhibition of eukaryotic translation initiation factor 4E phosphorylation. 1863 43

The DNA cleavage reaction of human topoisomerase IIalpha is critical to all of the physiological and pharmacological functions of the protein. While it has long been known that the type II enzyme requires a divalent metal ion in order to cleave DNA, the role of the cation in this process is not known. To resolve this fundamental issue, the present study utilized a series of divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that replaced the 3'-bridging oxygen of the scissile bond with a sulfur atom (i.e. 3'-bridging phosphorothiolates). Rates and levels of DNA scission were greatly enhanced when thiophilic metal ions were included in reactions that utilized sulfur-containing substrates. Based on these results and those of reactions that employed divalent cation mixtures, we propose that topoisomerase IIalpha mediates DNA cleavage via a two-metal-ion mechanism. In this model, one of the metal ions makes a critical interaction with the 3'-bridging atom of the scissile phosphate. This interaction greatly accelerates rates of enzyme-mediated DNA cleavage, and most likely is needed to stabilize the leaving 3'-oxygen.
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PMID:Human topoisomerase IIalpha uses a two-metal-ion mechanism for DNA cleavage. 1865 31

All type II topoisomerases require divalent metal ions to cleave and ligate DNA. To further elucidate the mechanistic basis for these critical enzyme-mediated events, the role of the metal ion in the DNA cleavage reaction of human topoisomerase IIbeta was characterized and compared to that of topoisomerase IIalpha. This study utilized divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that substituted a sulfur atom for the 3'-bridging oxygen or the nonbridging oxygens of the scissile phosphate. On the basis of time courses of DNA cleavage, cation titrations, and metal ion mixing experiments, we propose the following model for the use of divalent metal ions by human type II topoisomerases. First, both enzymes employ a two-metal ion mechanism to support DNA cleavage. Second, an interaction between one divalent metal ion and the 3'-bridging atom of the scissile phosphate greatly enhances enzyme-mediated DNA cleavage, most likely by stabilizing the leaving 3'-oxygen. Third, there is an important interaction between a divalent second metal ion and a nonbridging atom of the scissile phosphate that stimulates DNA cleavage mediated by topoisomerase IIbeta. If this interaction exists in topoisomerase IIalpha, its effects on DNA cleavage are equivocal. This last aspect of the model highlights a difference in metal ion utilization during DNA cleavage mediated by human topoisomerase IIalpha and IIbeta.
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PMID:Use of divalent metal ions in the dna cleavage reaction of human type II topoisomerases. 1922 28

Systemic sclerosis (SSc) is a connective tissue disorder of great clinical heterogeneity. Its pathophysiology remains unclear. Our aim was to evaluate the relative roles of reactive oxygen species (ROS) and of the immune system using an original model of SSc. BALB/c and immunodeficient BALB/c SCID mice were injected s.c. with prooxidative agents (hydroxyl radicals, hypochlorous acid, peroxynitrites, superoxide anions), bleomycin, or PBS everyday for 6 wk. Skin and lung fibrosis were assessed by histological and biochemical methods. Autoantibodies were detected by ELISA. The effects of mouse sera on H(2)O(2) production by endothelial cells and on fibroblast proliferation, and serum concentrations in advanced oxidation protein products (AOPP) were compared with sera from patients with limited or diffuse SSc. We observed that s.c. peroxynitrites induced skin fibrosis and serum anti-CENP-B Abs that characterize limited SSc, whereas hypochlorite or hydroxyl radicals induced cutaneous and lung fibrosis and anti-DNA topoisomerase 1 autoantibodies that characterize human diffuse SSc. Sera from hypochlorite- or hydroxyl radical-treated mice and of patients with diffuse SSc contained high levels of AOPP that triggered endothelial production of H(2)O(2) and fibroblast hyperproliferation. Oxidized topoisomerase 1 recapitulated the effects of whole serum AOPP. SCID mice developed an attenuated form of SSc, demonstrating the synergistic role of the immune system with AOPP in disease propagation. We demonstrate a direct role for ROS in SSc and show that the nature of the ROS dictates the form of SSc. Moreover, this demonstration is the first that shows the specific oxidation of an autoantigen directly participates in the pathogenesis of an autoimmune disease.
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PMID:Selective oxidation of DNA topoisomerase 1 induces systemic sclerosis in the mouse. 1938 Aug 34

Tumor-associated macrophages (TAMs) are found in many solid tumors and have often been shown to accumulate in the hypoxic regions surrounding areas of necrosis. TAMs are the major site of expression of nitric oxide synthase (NOS), a heme-containing homodimeric enzyme consisting of oxygenase and reductase domains. The latter has a high degree of sequence homology to cytochrome P450 reductase and a functional consequence of this is the ability of NOS, under hypoxic conditions, to activate the bioreductive drugs tirapazamine and RSU1069. Banoxantrone (AQ4N) is a bioreductive prodrug activated in hypoxia by an oxygen-dependent two-electron reductive process to yield the topoisomerase II inhibitor AQ4. A feature of this process is that the final product could potentially show bystander cell killing. Thus, in this study, we investigated the ability of inducible NOS (iNOS)-expressing TAMs to activate AQ4N and elicit toxicity in cocultured human tumor cells. Murine macrophages were induced to overexpress iNOS by treatment with a combination of cytokines, mixed with HT1080 and HCT116 human tumor cells, and the toxicity of AQ4N was determined under aerobic or hypoxic conditions. The aerobic toxicity of AQ4N toward tumor cells was not affected through coculturing with macrophages. However, under hypoxic conditions, the induction of iNOS activity in the macrophages was associated with an increase in AQ4N metabolism and a substantial increase in tumor cell toxicity, which was dependent on the proportion of macrophages in the culture. This study is the first demonstration of TAM-mediated prodrug activation to result in bystander killing of human tumor cells.
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PMID:Effects of cytokine-induced macrophages on the response of tumor cells to banoxantrone (AQ4N). 1943 66


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