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)

Brefeldin A, an agent that disrupts protein transport from the endoplasmic reticulum to the Golgi, induces the expression of GRP78 and the activation of nuclear factor (NF)-kappaB in cells. Treatment of cells with brefeldin A causes the development of resistance to topoisomerase II-directed agents, such as etoposide and doxorubicin. In this study, we show that treatment of EMT6 mouse mammary tumor cells with brefeldin A strongly induces GRP78 mRNA (8.5-fold) and resistance to teniposide (VM26). Treatment with okadaic acid causes a minor increase in GRP78 mRNA (2.1-fold) yet still induces resistance to VM26 as effectively as brefeldin A. In contrast, cells treated with castanospermine show a moderate increase in GRP78 mRNA (3.9-fold) but no resistance to VM26. These data imply that GRP78 induction does not mediate the development of drug resistance. An alternative mechanism of drug resistance may involve activation of the transcription factor, NF-kappaB, and we show that both brefeldin A and okadaic acid activate NF-kappaB in EMT6 cells. Furthermore, we demonstrate that treatment with the proteasome inhibitor MG-132 blocks the activation of NF-kappaB and prevents the development of resistance to VM26 induced by brefeldin A. Collectively, these results suggest that the resistance to VM26 in EMT6 cells treated with brefeldin A is mediated by the activation of NF-kappaB rather than the induction of GRP78. Our results also suggest that inhibition of NF-kappaB activation in tumor cells may increase the efficacy of topoisomerase II-directed agents in chemotherapy.
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PMID:Prevention of brefeldin A-induced resistance to teniposide by the proteasome inhibitor MG-132: involvement of NF-kappaB activation in drug resistance. 967 71

GRP94 is a 94-kDa chaperone glycoprotein with Ca(2+)-binding properties. We report here that during apoptosis induced by the topoisomerase II inhibitor etoposide, a fraction of GRP94 associated with the endoplasmic reticulum membrane undergoes specific proteolytic cleavage, coinciding with the activation of the caspase CPP32 and initiation of DNA fragmentation. In vivo, inhibitors of caspases able to block etoposide-induced apoptosis can only partially protect GRP94 from proteolytic cleavage, whereas complete inhibition is observed with calpain inhibitor I but not with the proteasome inhibitor. In vitro, GRP94 is not a substrate for CPP32; rather, it can be completely cleaved by calpain, a Ca(2+)-regulated protease. The cleavage of GRP94 by calpain is Ca(2+)-dependent and generates a discrete polypeptide of 80 kDa. In contrast, calpain has no effect on other stress proteins such as GRP78 or HSP70. Further, immunohistochemical staining reveals specific co-localization of GRP94 with calpain in the perinuclear region following etoposide treatment. We further showed that reduction of GRP94 by antisense decreased cell viability in etoposide-treated Jurkat cells. Our studies provide new evidence that the cytoprotective GRP94, as in the case of the antiapoptotic protein Bcl-2, can be targets of proteolytic cleavage themselves during the apoptotic process.
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PMID:The endoplasmic reticulum chaperone glycoprotein GRP94 with Ca(2+)-binding and antiapoptotic properties is a novel proteolytic target of calpain during etoposide-induced apoptosis. 1049 10

Overexpressed MDM2 inactivates wild-type (wt) p53 in various human tumors. However, whether and how the wild-type p53 can be activated by anticancer drug treatment in the presence of excess MDM2 is still unclear. In the present study, we showed that the topoisomerase II inhibitor of widely used anticancer drugs etoposide and doxorubicin activated wt p53 in BL2, a Burkitt's lymphoma cell line which overexpressed MDM2. Activation of p53 was followed by apoptosis in BL2 cells, while the same drug treatment did not induce apoptosis in Raji cells, another Burkitt's lymphoma cell line which carried mutant p53. Activation of p53 was accompanied by phosphorylation of p53 at Ser-15 and elevated p21 and MDM2, both of which were at least partly blocked by wortmannin, a kinase inhibitor against proteins with a PI3 kinase domain. Although MDM2 protein was rapidly cleaved and degraded after anticancer drug treatment, cotreatment with caspase inhibitor Z-VAD blocked degradation, while wt p53 remained activated, suggesting MDM2 degradation not to be essential for the activation of p53. Treatment with proteasome inhibitor stabilized p53 without being further phosphorylated. This p53 was co-immunoprecipitated with MDM2, but p53 activated by etoposide or doxorubicin barely complexed with MDM2. These results suggest that the wild-type p53 in MDM2-overexpressing cells can be activated by anticancer drugs through phosphorylation of p53, alleviating inhibitory action by MDM2, and activating caspases which in turn downregulates MDM2. The activation of p53 in MDM2-overexpressing tumor cells, which does not require the downregulation of MDM2, may have important implications in cancer therapy.
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PMID:Activation of p53 in MDM2-overexpressing cells through phosphorylation. 1054 21

Physiological cell conditions, such as glucose deprivation and hypoxia, play a role in developing drug resistance in solid tumors. These tumor-specific conditions cause decreased expression of DNA topoisomerase IIalpha (topo IIalpha), rendering cells resistant to topo II-targeted drugs, such as etoposide and doxorubicin. We show here that inhibition of proteasome attenuated drug resistance by inhibiting topo IIalpha depletion induced by glucose starvation and hypoxia. topo IIalpha restoration was seen only at the protein levels, indicating that the topo IIalpha protein depletion occurred through a proteasome-mediated degradation mechanism. The stress-induced etoposide resistance was effectively prevented in vitro by the proteasome inhibitor lactacystin in both intrinsically resistant and sensitive tumor cells (colon cancer HT-29 and ovarian cancer A2780 cells, respectively). Furthermore, lactacystin effectively enhanced the antitumor activity of etoposide in the refractory HT-29 xenograft. These results indicate that lactacystin could serve as a new therapeutic agent to circumvent resistance to topo II-targeted chemotherapy in solid tumors.
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PMID:Proteasome inhibition circumvents solid tumor resistance to topoisomerase II-directed drugs. 1081 Nov 20

Activation of signaling pathways after DNA damage induced by topoisomerase (topo) poisons can lead to cell death by apoptosis. Treatment of human nonsmall cell lung carcinoma (NSCLC-3 or NSCLC-5) cells with the topo I poison SN-38 or the topo II poison etoposide (VP-16) leads to activation of NF-kappaB before induction of apoptosis. Inhibiting the degradation of IkappaBalpha by pretreatment with the proteasome inhibitor MG-132 significantly inhibited NF-kappaB activation and apoptosis but not DNA damage induced by SN-38 or VP-16. Transfection of NSCLC-3 or NSCLC-5 cells with dominant negative mutant IkappaBalpha (mIkappaBalpha) inhibited SN-38 or VP-16 induced transcription and DNA binding activity of NF-kappaB without altering drug-induced apoptosis. Regulation of apoptosis by mitochondrial release of cytochrome c and activation of pro-caspase 9 followed by cleavage of poly(ADP-ribose) polymerase by effector caspases 3 and 7 was similar in neo and mIkappaBalpha cells treated with SN-38 or VP-16. In contrast to pretreatment with MG-132, exposure to MG-132 after SN-38 or VP-16 treatment of neo or mIkappaBalpha cells decreased cell cycle arrest in the S/G2 + M fraction and enhanced apoptosis compared with drug alone. In summary, apoptosis induced by topoisomerase poisons in NSCLC cells is not mediated by NF-kappaB but can be manipulated by proteasome inhibitors.
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PMID:Roles of NF-kappaB and 26 S proteasome in apoptotic cell death induced by topoisomerase I and II poisons in human nonsmall cell lung carcinoma. 1111 10

Physiological stress conditions associated with the tumor microenvironment play a role in resistance to anticancer therapy. In this study, treatment of EMT6 mouse mammary tumor cells with hypoxia or the chemical stress agents brefeldin A (BFA) or okadaic acid (OA) causes the development of resistance to the topoisomerase II inhibitor etoposide. The mechanism of physiological stress-induced drug resistance may involve the activation of stress-responsive proteins and transcription factors. Our previous work shows that treatment with BFA or OA causes activation of the nuclear transcription factor NF-kappa B. Pretreatment with the proteasome inhibitor carbobenzyoxyl-leucinyl-leucinyl-leucinal inhibits stress-induced NF-kappa B activation and reverses BFA-induced drug resistance. To test whether NF-kappa B specifically mediates stress-induced drug resistance, an inducible phosphorylation site-deficient mutant of I kappa B alpha (I kappa B alpha M, S32/36A) was introduced into EMT6 cells. In this study, we show that I kappa B alpha M expression inhibits stress-induced NF-kappa B activation and prevents BFA-, hypoxia-, and OA-induced resistance to etoposide. These results indicate that NF-kappa B activation mediates both chemical and physiological drug resistance to etoposide. Furthermore, they imply that coadministration of agents that inhibit NF-kappa B may enhance the efficacy of topoisomerase II inhibitors in clinical cancer chemotherapy.
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PMID:Reversal of physiological stress-induced resistance to topoisomerase II inhibitors using an inducible phosphorylation site-deficient mutant of I kappa B alpha. 1150 88

Hepatocellular carcinoma (HCC) is a common malignancy and often resistant to chemotherapy. Many chemotherapy regimens have been tried to control advanced HCC, but have produced a low response rate and no clear impact. CPT-11, a derivative of camptothecin, works as type-I DNA topoisomerase inhibitor and showed a major objective response rate in patients with metastatic colorectal cancer. In this study, the mechanism underlying chemo-resistance to SN-38, an active form of CPT-11, in HCC was investigated in relation to anti-apoptotic pathways NF-kappaB and PI3K/Akt. Hep3B was the most resistant to SN-38 among three hepatoma cell lines. NF-kappaB was constitutively activated in Hep3B, and SN-38 further enhanced the nuclear translocation of NF-kappaB. However, inactivation of NF-kappaB by adenovirus expressing IkappaB super-repressor or MG-132, proteasome inhibitor, did not sensitize Hep3B to SN-38-induced apoptosis. On the other hand, SN-38 phosphorylated Akt and pretreatment with PI3K inhibitors increased SN-38-induced apoptosis, indicating that resistance to SN-38 in Hep3B occurs partly through the PI3K/Akt not the NF-kappaB pathway. Blocking of PI3K/Akt may thus be helpful for overcoming chemo-resistance of HCC.
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PMID:Blocking of PI3K/Akt pathway enhances apoptosis induced by SN-38, an active form of CPT-11, in human hepatoma cells. 1580 21

We have identified four small molecules that boost transduction of cells by human immunodeficiency virus (HIV) and investigated their mechanism of action. These molecules include etoposide and camptothecin, which induce DNA damage by inhibiting religation of cleaved topoisomerase-DNA complexes, taxol, which interferes with the function of microtubules, and aphidicolin, which inhibits DNA polymerases. All four compounds arrest the cell cycle at G2/M, though in addition high concentrations of aphidicolin arrest in G1. We find that early events of HIV replication, including synthesis of late reverse transcription products, two-long terminal repeat circles, and integrated proviruses, were increased after treatment of cells with concentrations of each compound that arrested in G2/M. Stimulation was seen for both transformed cell lines (293T and HeLa cells) and primary cells (IMR90 lung fibroblasts). Arrest in G1 with high concentrations of aphidicolin boosted transduction, though not much as with lower concentrations that arrested in G2/M. Arrest of IMR90 cells in G1 by serum starvation and contact inhibition reduced transduction. Previously, the proteasome inhibitor MG132 was reported to increase HIV infection-here we investigated the effects of combinations of the cell cycle inhibitors with MG132 and obtained data suggesting that MG132 may also boost transduction by causing G2/M cell cycle arrest. These data document that cell cycle arrest in G2/M boosts the early steps of HIV infection and suggests methods for increasing transduction with HIV-based vectors.
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PMID:Cell cycle arrest in G2/M promotes early steps of infection by human immunodeficiency virus. 1582 84

Despite rapid advances in the field of DNA repair, little is known about the repair of protein-DNA adducts. Previous studies have demonstrated that topoisomerase II (TopII)-DNA adducts (TopII-DNA covalent complexes) are rapidly degraded by the proteasome. It has been hypothesized that proteasomal degradation of TopII-DNA covalent adducts exposes TopII-concealed DNA double-strand breaks (DSBs) for repair. To test this hypothesis, the anticancer drug, VP-16 (etoposide), was employed to induce TopII-DNA covalent complexes in mammalian cells, and the involvement of proteasome in processing TopII-DNA covalent complexes into DSBs was investigated. Consistent with the hypothesis, VP-16-induced DSBs as monitored by neutral comet assay, as well as DNA damage signals (e.g. gamma-H2AX) were significantly reduced in the presence of the proteasome inhibitor, MG132. Using both top2beta knock-out mouse embryonic fibroblasts and Top2beta small interfering RNA knockdown PC12 cells, as well as postmitotic neurons in which TopIIalpha was absent, we showed that VP-16-induced DNA damage signals were attenuated upon proteasome inhibition, suggesting the involvement of proteasome in the repair/processing of both TopIIalpha-DNA and TopIIbeta-DNA adducts. By contrast, hydrogen peroxide-induced gamma-H2AX was unaffected upon proteasome inhibition, suggesting a specific requirement of the proteasome pathway in the processing of TopII-DNA covalent complexes into DNA damage.
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PMID:A protease pathway for the repair of topoisomerase II-DNA covalent complexes. 1697 21

Acridine derivatives, such as amsacrine, represent a well known class of multi-targeted anti-cancer agents that generally interfere with DNA synthesis and inhibit topoisomerase II. But in addition, these tricyclic molecules often display secondary effects on other biochemical pathways including protein metabolism. In order to identify novel anti-cancer drugs, we evaluated the mechanism of action of a novel series of bis- and tetra-acridines. As expected, these molecules were found to interact with DNA and inhibit the topoisomerase II-mediated DNA decatenation. Interestingly when tested on human tumour cells either sensitive (HL-60) or resistant (HL-60/MX2) to topoisomerase II inhibitors, these molecules proved equicytotoxic against the two cell lines, suggesting that they do not only rely on topoisomerase II inhibition to exert their cytotoxic effects. In order to identify alternative targets, we tested the capacity of acridines 1-9 to inhibit the proteasome machinery. Four tetra-acridines inhibited the proteasome in vitro, with IC(50) values up to 40 times lower than that of the reference proteasome inhibitor lactacystin. Moreover, unlike peptide aldehydes used as reference inhibitors for the proteasome, these new acridine compounds demonstrated a good selectivity towards the proteasome, when tested against four unrelated proteases. A cellular assay based on the degradation of a proteasome protein substrate indicated that at least two of the tetra-acridines maintained this proteasome inhibition activity in a cellular context. This is the first report of tetra-acridines that demonstrate dual topoisomerase II and proteasome inhibition properties. This new dual activity could represent a novel anti-cancer approach to circumvent certain forms of tumour resistance.
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PMID:Novel tetra-acridine derivatives as dual inhibitors of topoisomerase II and the human proteasome. 1739 47


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