Search

Etoposide is a widely-used anticancer agent that targets human type II topoisomerases. Evidence suggests that metabolism of etoposide in myeloid progenitor cells is associated with translocations involved in leukemia development. Previous studies suggest halogenation at the C-2' position of etoposide reduces metabolism. Halogens were introduced into the C-2' position by electrophilic aromatic halogenation onto etoposide (ETOP, 1), podophyllotoxin (PPT, 2), and 4-dimethylepipodophyllotoxin (DMEP, 3), and to bridge the gap of knowledge regarding the activity of these metabolically stable analogs. Five halogenated analogs (6-10) were synthesized. Analogs 8-10 displayed variable ability to inhibit DNA relaxation. Analog 9 was the only analog to show concentration-dependent enhancement of Top2-mediated DNA cleavage. Dose response assay results indicated that 8 and 10 were most effective at decreasing the viability of HCT-116 and A549 cancer cell lines in culture. Flow cytometry with 8 and 10 in HCT-116 cells provide evidence of sub-G1 cell populations indicative of apoptosis. Taken together, these results indicate C-2' halogenation of etoposide and its precursors, although metabolically stable, decreases overall activity relative to etoposide., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Etoposide is a topoisomerase II poison that is utilized to treat a broad spectrum of human cancers. Despite its wide clinical use, 2-3% of patients treated with etoposide eventually develop treatment-related acute myeloid leukemias (t-AMLs) characterized by rearrangements of the MLL gene. The molecular basis underlying the development of these t-AMLs is not well understood; however, previous studies have implicated etoposide metabolites (i.e., etoposide quinone) and topoisomerase IIβ in the leukemogenic process. Although interactions between etoposide quinone and topoisomerase IIα have been characterized, the effects of the drug metabolite on the activity of human topoisomerase IIβ have not been reported. Thus, we examined the ability of etoposide quinone to poison human topoisomerase IIβ. The quinone induced ~4 times more enzyme-mediated DNA cleavage than did the parent drug. Furthermore, the potency of etoposide quinone was ~2 times greater against topoisomerase IIβ than it was against topoisomerase IIα, and the drug reacted ~2-4 times faster with the β isoform. Etoposide quinone induced a higher ratio of double- to single-stranded breaks than etoposide, and its activity was less dependent on ATP. Whereas etoposide acts as an interfacial topoisomerase II poison, etoposide quinone displayed all of the hallmarks of a covalent poison: the activity of the metabolite was abolished by reducing agents, and the compound inactivated topoisomerase IIβ when it was incubated with the enzyme prior to the addition of DNA. These results are consistent with the hypothesis that etoposide quinone contributes to etoposide-related leukemogenesis through an interaction with topoisomerase IIβ.

Topoisomerase II regulates DNA topology by generating transient double-stranded breaks. The anticancer drug etoposide targets topoisomerase II and is associated with the formation of secondary leukemias in patients. The quinone and catechol metabolites of etoposide may contribute to strand breaks that trigger leukemic translocations. To further analyze the characteristics of etoposide metabolites, we extend our previous analysis of etoposide quinone to the catechol. We demonstrate that the catechol is ∼2-3-fold more potent than etoposide and under oxidative reaction conditions induces high levels of double-stranded DNA cleavage. These results support a role for etoposide catechol in contributing to therapy-induced DNA damage.

Etoposide is a topoisomerase II poison that is used to treat a variety of human cancers. Unfortunately, 2-3% of patients treated with etoposide develop treatment-related leukemias characterized by 11q23 chromosomal rearrangements. The molecular basis for etoposide-induced leukemogenesis is not understood but is associated with enzyme-mediated DNA cleavage. Etoposide is metabolized by CYP3A4 to etoposide catechol, which can be further oxidized to etoposide quinone. A CYP3A4 variant is associated with a lower risk of etoposide-related leukemias, suggesting that etoposide metabolites may be involved in leukemogenesis. Although etoposide acts at the enzyme-DNA interface, several quinones poison topoisomerase II via redox-dependent protein adduction. The effects of etoposide quinone on topoisomerase IIα-mediated DNA cleavage have been examined previously. Although findings suggest that the activity of the quinone is slightly greater than that of etoposide, these studies were carried out in the presence of significant levels of reducing agents (which should reduce etoposide quinone to the catechol). Therefore, we examined the ability of etoposide quinone to poison human topoisomerase IIα in the absence of reducing agents. Under these conditions, etoposide quinone was ∼5-fold more active than etoposide at inducing enzyme-mediated DNA cleavage. Consistent with other redox-dependent poisons, etoposide quinone inactivated topoisomerase IIα when incubated with the protein prior to DNA and lost activity in the presence of dithiothreitol. Unlike etoposide, the quinone metabolite did not require ATP for maximal activity and induced a high ratio of double-stranded DNA breaks. Our results support the hypothesis that etoposide quinone contributes to etoposide-related leukemogenesis.

Topoisomerase II is an essential nuclear enzyme involved in regulating DNA topology to facilitate replication and cell division. Disruption of topoisomerase II function by chemotherapeutic agents is in use as an effective strategy to fight cancer. Etoposide is an anticancer therapeutic that disrupts the catalytic cycle of topoisomerase II and stabilizes enzyme-bound DNA strand breaks. Etoposide is metabolized into several species including active quinone and catechol metabolites. Our previous studies have explored some of the details of how these compounds act against topoisomerase II. In our present study, we extend those analyses by examining several effects of etoposide quinone on topoisomerase IIα including whether the quinone impacts ATP hydrolysis, DNA ligation, cleavage complex persistence, and enzyme/DNA binding. Our results demonstrate that the quinone inhibits relaxation at 100-fold lower levels of drug when compared to that of etoposide. Further, the quinone inhibits ATP hydrolysis by topoisomerase IIα. The quinone does appear to stabilize single-strand breaks similar to etoposide suggesting a traditional poisoning mechanism. However, there is minimal difference in cleavage complex persistence in the presence of etoposide or etoposide quinone. In contrast to etoposide, we find that etoposide quinone blocks enzyme/DNA binding, which provides an explanation for previous data showing the ability of the quinone to inactivate the enzyme over time. Finally, etoposide quinone is able to stabilize the N-terminal protein clamp implying an interaction between the compound and this portion of the enzyme. Taken together, the evidence supports a two-mechanism model for the effect of the quinone on topoisomerase II: (1) interfacial poison and (2) covalent poison that interacts with the N-terminal clamp and impacts the binding of DNA. [ABSTRACT FROM AUTHOR]

Coordination between the N-terminal gate and the catalytic core of topoisomerase II allows the proper capture, cleavage, and transport of DNA during the catalytic cycle. Because the activities of these domains are tightly linked, it has been difficult to discern their individual contributions to enzyme-DNA interactions and drug mechanism. To further address the roles of these domains, we analyzed the activity of the catalytic core of human topoisomerase IIα. The catalytic core and the wild-type enzyme both maintained higher levels of cleavage with negatively (as compared to positively) supercoiled plasmid, indicating that the ability to distinguish supercoil handedness is embedded within the catalytic core. However, the catalytic core alone displayed little ability to cleave DNA substrates that did not intrinsically provide the enzyme with a transport segment (i.e., substrates that did not contain crossovers). Finally, in contrast to interfacial topoisomerase II poisons, covalent poisons did not enhance DNA cleavage mediated by the catalytic core. This distinction allowed us to further characterize the mechanism of etoposide quinone, a drug metabolite that functions primarily as a covalent poison. Etoposide quinone retained some ability to enhance DNA cleavage mediated by the catalytic core, indicating that it still can function as an interfadal poison. These results further define the distinct contributions of the N-terminal gate and the catalytic core to topoisomerase II function. The catalytic core senses the handedness of DNA supercoils during cleavage, while the N-terminal gate is critical for capturing the transport segment and for the activity of covalent poisons. [ABSTRACT FROM AUTHOR]