Azacyanines as Novel Topoisomerase II Alpha Inhibitors

Introduction: Topoisomerase II alpha (Topo IIα) has become one of the extensively exploited targets in chemotherapy due to its role in regulating the topological constraints of DNA during replication and transcription. Small molecules targeting Topo IIα’s activity such as etoposide (VP-16) and doxorubicin are extensively used in the treatment of many different types of cancer. Objective: Here, the effects of three small molecules, named as azacyanines, on Topo IIα have been assessed. Methods: In-vitro Topoisomerase IIα drug screening kit and agarose gel imaging were used for the assessment of Topo IIα’s activity. Results: Our results revealed that all the azacyanines investigated decreased the catalytic activity of Topo IIα dramatically. More importantly, the decrease in the catalytic activity of Topo IIα in the presence of azacyanines was higher than the presence of VP-16, which is a commercially available chemotherapy drug. Upon further investigation, it has been observed that Azamethyl’s catalytic inhibition of Topo IIα was concentration dependent and the catalytic activity of Topo IIα was almost completely abolished in the presence of 100.0 μM of Azamethyl. Conclusion: These findings reveal the potential of azacyanines as effective Topo IIα inhibitors and chemotherapeutic agents.

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Methods for addressing the protein-protein interaction between histone deacetylase 6 and ubiquitin

10.1101/203372 ◽

2017 ◽

Author(s):

Carolina dos S. Passos ◽

Nathalie Deschamps ◽

Yun Choi ◽

Robert E. Cohen ◽

Remo Perozzo ◽

...

Keyword(s):

Catalytic Activity ◽

Stress Response ◽

Small Molecules ◽

Histone Deacetylase ◽

Protein Interaction ◽

Potential Role ◽

Histone Deacetylase 6 ◽

Protein Protein Interaction ◽

Binding Function

AbstractHistone deacetylase 6 (HDAC6) is a cytoplasmic HDAC isoform able to remove acetyl groups from cellular substrates such as α-tubulin. In addition to the two deacetylase domains, HDAC6 has a C-terminal zinc-finger ubiquitin (Ub)-binding domain (ZnF-UBP) able to recognize free Ub. HDAC6-Ub interaction is thought to function in regulating the elimination of misfolded proteins during stress response through the aggresome pathway. Small molecules inhibiting deacetylation by HDAC6 were shown to reduce aggresomes, but the interplay between HDAC6 catalytic activity and Ub-binding function is not fully understood. Here we describe two methods to measure the HDAC6-Ub interaction in vitro using full-length HDAC6. Both methods were effective for screening inhibitors of the HDAC6-Ub protein-protein interaction independently of the catalytic activity. Our results suggest a potential role for the HDAC6 deacetylase domains in modulating HDAC6-Ub interaction. This new aspect of HDAC6 regulation can be targeted to address the roles of HDAC6-Ub interaction in normal and disease conditions.

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CD26/Dipeptidyl Peptidase IV (DPPIV) Regulates p38 Phosphorylation and Topoisomerase II Alpha Expression in the B-Lymphoma Line Jiyoye, Associated with Enhanced In Vitro and In Vivo Sensitivity to Doxorubicin.

Blood ◽

10.1182/blood.v104.11.3407.3407 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3407-3407

Author(s):

Toshiko Yamochi ◽

Tadanori Yamochi ◽

Ugur Aytac ◽

Tsutomu Sato ◽

Chikao Morimoto ◽

...

Keyword(s):

T Cell ◽

Topoisomerase Ii ◽

Low Dose ◽

Topoisomerase Ii Alpha ◽

In Vitro Sensitivity ◽

Alpha Level ◽

Lymphoma Line ◽

Cd26 Expression

Abstract CD26 is a 110 kDa surface glycoprotein with diverse functional properties, including having a key role in normal T-cell biology, being a marker of aggressive disease for selected T-cell malignancies and being involved in the development of certain cancers. Its extracellular domain encodes a membrane-associated dipeptidyl peptidase IV (DPPIV) activity capable of processing biological factors to alter their functional profiles. We have shown previously that expression of CD26 on the T cell line Jurkat is associated with increased topoisomerase II alpha level and in vitro sensitivity to topoisomerase II inhibitors (Aytac U et al. Cancer Res61:7204, 2001; Aytac U et al. Br J Cancer88:455, 2003; Sato K et al. Br J Cancer89:1366, 2003). We now show that CD26 expression, particularly its DPPIV enzyme activity, on the B-lymphoma line Jiyoye results in increased topoisomerase II alpha level and in vitro sensitivity to doxorubicin-induced apoptosis. Examining the molecular mechanisms involved in CD26-associated signaling, our present findings also indicate that CD26/DPPIV expression on Jiyoye cells is associated with increased phosphorylation of p38 and its upstream regulators MKK3/6 and ASK1. Importantly, inhibition of p38 phosphorylation decreases topoisomerase II alpha expression, suggesting a role for p38 in the regulation of topoisomerase II alpha. Finally, studies using a SCID mouse xenograft model with CD26 Jiyoye transfectants show that CD26 expression is associated with enhanced survival following treatment with low doses of doxorubicin. In particular, treatment with low-dose doxorubicin of SCID mice injected with CD26-negative parental Jiyoye cells does not lead to a statistically significant survival advantage over those treated with saline. On the other hand, SCID mice injected with CD26 Jiyoye transfectants show significantly greater survival when treated with low-dose doxorubicin than with saline alone, indicating that CD26 presence renders tumor cells more sensitive to doxorubicin in an in vivo model. Our data thus characterize the biochemical linkage among CD26 and other key intracellular molecules, while demonstrating that CD26 may have a role in tumor sensitivity to antineoplastic agents targeting topoisomerase II alpha. In addition, our work suggests that CD26/DPPIV may be an appropriate target for therapy for selected hematological malignancies of both B- and T-cell lineages.

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Extracellular Signal-Regulated Kinase Activates Topoisomerase IIα through a Mechanism Independent of Phosphorylation

Molecular and Cellular Biology ◽

10.1128/mcb.19.5.3551 ◽

1999 ◽

Vol 19 (5) ◽

pp. 3551-3560 ◽

Cited By ~ 71

Author(s):

Paul S. Shapiro ◽

Anne M. Whalen ◽

Nicholas S. Tolwinski ◽

Julie Wilsbacher ◽

Stacie J. Froelich-Ammon ◽

...

Keyword(s):

Topoisomerase Ii ◽

Map Kinases ◽

Specific Activity ◽

Nucleic Acid Metabolism ◽

Topoisomerase Iiα ◽

Potential Target ◽

Nuclear Extracts ◽

Extracellular Signal

ABSTRACT The mitogen-activated protein (MAP) kinases, extracellular signal-related kinase 1 (ERK1) and ERK2, regulate cellular responses by mediating extracellular growth signals toward cytoplasmic and nuclear targets. A potential target for ERK is topoisomerase IIα, which becomes highly phosphorylated during mitosis and is required for several aspects of nucleic acid metabolism, including chromosome condensation and daughter chromosome separation. In this study, we demonstrated interactions between ERK2 and topoisomerase IIα proteins by coimmunoprecipitation from mixtures of purified enzymes and from nuclear extracts. In vitro, diphosphorylated active ERK2 phosphorylated topoisomerase IIα and enhanced its specific activity by sevenfold, as measured by DNA relaxation assays, whereas unphosphorylated ERK2 had no effect. However, activation of topoisomerase II was also observed with diphosphorylated inactive mutant ERK2, suggesting a mechanism of activation that depends on the phosphorylation state of ERK2 but not on its kinase activity. Nevertheless, activation of ERK by transient transfection of constitutively active mutant MAP kinase kinase 1 (MKK1) enhanced endogenous topoisomerase II activity by fourfold. Our findings indicate that ERK regulates topoisomerase IIα in vitro and in vivo, suggesting a potential target for the MKK/ERK pathway in the modulation of chromatin reorganization events during mitosis and in other phases of the cell cycle.

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A noncatalytic function of the topoisomerase II CTD in Aurora B recruitment to inner centromeres during mitosis

The Journal of Cell Biology ◽

10.1083/jcb.201511080 ◽

2016 ◽

Vol 213 (6) ◽

pp. 651-664 ◽

Cited By ~ 22

Author(s):

Heather Edgerton ◽

Marnie Johansson ◽

Daniel Keifenheim ◽

Soumya Mukherjee ◽

Jeremy M. Chacón ◽

...

Keyword(s):

Catalytic Activity ◽

Binding Site ◽

Chromosome Segregation ◽

Topoisomerase Ii ◽

Histone H3 ◽

Aurora B ◽

Topoisomerase Iiα ◽

Dna Topoisomerase ◽

Topo Ii ◽

Dna Topoisomerase Iiα

Faithful chromosome segregation depends on the precise timing of chromatid separation, which is enforced by checkpoint signals generated at kinetochores. Here, we provide evidence that the C-terminal domain (CTD) of DNA topoisomerase IIα (Topo II) provides a novel function at inner centromeres of kinetochores in mitosis. We find that the yeast CTD is required for recruitment of the tension checkpoint kinase Ipl1/Aurora B to inner centromeres in metaphase but is not required in interphase. Conserved CTD SUMOylation sites are required for Ipl1 recruitment. This inner-centromere CTD function is distinct from the catalytic activity of Topo II. Genetic and biochemical evidence suggests that Topo II recruits Ipl1 via the Haspin–histone H3 threonine 3 phosphorylation pathway. Finally, Topo II and Sgo1 are equally important for Ipl1 recruitment to inner centromeres. This indicates H3 T3-Phos/H2A T120-Phos is a universal epigenetic signature that defines the eukaryotic inner centromere and provides the binding site for Ipl1/Aurora B.

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mTOR Activation Is Necessary for Primary AML Cells to Survive Genotoxic Stress.

Blood ◽

10.1182/blood.v104.11.91.91 ◽

2004 ◽

Vol 104 (11) ◽

pp. 91-91 ◽

Cited By ~ 4

Author(s):

Qing Xu ◽

J. Thompson ◽

Martin Carroll

Keyword(s):

Stem Cells ◽

Cell Survival ◽

Topoisomerase Ii ◽

Mammalian Target Of Rapamycin ◽

Genotoxic Stress ◽

Chemotherapeutic Agents ◽

Pi3 Kinase ◽

Leukemic Cells ◽

Vitro Assay

Abstract The signaling pathways that regulate the survival of AML cells and whether those pathways can be targeted therapeutically are still poorly defined. We have previously demonstrated that AML cells require PI3 kinase activation for survival but clinical targeting of PI3 kinase itself is not currently feasible. Therefore, we have chosen to study the role of the mammalian target of rapamycin or mTOR protein in the survival of leukemic cells. mTOR mediates the effects of PI3 kinase and can be inhibited by the immunosuppressant, rapamycin. For these studies, patient samples were collected from patients seen at the Hospital of the University of Pennsylvania and we selected samples with greater than 75% blast cells for analysis. Here we show that mTOR is activated in over 90% of patient samples examined as shown by constitutive phosphorylation of the mTOR target protein, p70 S6 kinase (p70S6K). A second substrate, 4EBP1, is phosphorylated in the majority of AML samples but less consistently than p70S6K. Inhibition of mTOR with rapamycin caused only a modest 20% decrease in cell survival based on a 48 hour in vitro assay. Strikingly, however, when AML blasts were incubated with a combination of a topoisomerase II inhibitor, etoposide, and rapamycin, the dose response to etoposide was shifted to the left with the ED50 decreased by at least one log in each of 5 tested samples. Rapamycin inhibited mTOR as shown by inhibition of p70S6K phosphorylation and this inhibition was not altered in the presence of etoposide. To determine if combinations of rapamycin and etoposide would have combined effects on leukemic stem cells, AML cells were incubated in etoposide alone or etoposide with rapamycin for 16 hours and then transplanted into NOD/SCID mice for analysis of stem cell survival. Engraftment of AML cells into NOD/SCID animals was decreased by etoposide and this inhibition was increased by the combination of etoposide and rapamycin. These studies suggest that mTOR regulates a critical cell survival pathway in AML stem cells and this pathway is necessary for survival in the presence of a distinct apoptotic signal. Furthermore, these studies suggest that combinations of rapamycin with chemotherapy may enhance the efficacy of chemotherapeutic agents in the treatment of AML and, therefore, we have recently initiated a Phase I trial of rapamycin in combination with MEC chemotherapy for the treatment of relapsed AML.

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