DNA topoisomerase IIα (TOP2α) is a prominent target for anticancer drugs whose clinical efficacy is often limited by chemoresistance. Using antibody specific for the N-terminal of TOP2α, immunoassays indicated the existence of two TOP2α isoforms, 170 and 90 kDa, present in K562 leukemia cells and in an acquired etoposide (VP-16)-resistant clone (K/VP.5). TOP2α/90 expression was dramatically increased in etoposide-resistant K/VP.5 compared with parental K562 cells. We hypothesized that TOP2α/90 was the translation product of novel alternatively processed pre-mRNA, confirmed by 3'-rapid amplification of cDNA ends, polymerase chain reaction, and sequencing. TOP2α/90 mRNA includes retained intron 19, which harbors an in-frame stop codon, and two consensus poly(A) sites. The processed transcript is polyadenylated. TOP2α/90 mRNA encodes a 90,076-Da translation product missing the C-terminal 770 amino acids of TOP2α/170, replaced by 25 unique amino acids through translation of the exon 19/intron 19 read-through. Immunoassays, utilizing antisera raised against these unique amino acids, confirmed that TOP2α/90 is expressed in both cell types, with overexpression in K/VP.5 cells. Immunodetection of complex of enzyme-to-DNA and single-cell gel electrophoresis (Comet) assays demonstrated that K562 cells transfected with a TOP2α/90 expression plasmid exhibited reduced etoposide-mediated TOP2α-DNA covalent complexes and decreased etoposide-induced DNA damage, respectively, compared with similarly treated K562 cells transfected with empty vector. Because TOP2α/90 lacks the active site tyrosine (Tyr) of full-length TOP2α, these results strongly suggest that TOP2α/90 exhibits dominant-negative properties. Further studies are underway to characterize the mechanism(s) by which TOP2α/90 plays a role in acquired resistance to etoposide and other TOP2α targeting agents.

DNA topoisomerase II (170 kDa, TOP2/170) is essential in proliferating cells by resolving DNA topological entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated isoform (TOP2/90), detectable in human leukemia K562 cells but more abundantly expressed in a clonal subline, K/VP.5, with acquired resistance to the anticancer agent etoposide. TOP2/90 (786 aa) is the translation product of a TOP2 mRNA that retains a processed intron 19. TOP2/90 lacks the active-site tyrosine-805 required to generate double-strand DNA breaks as well as nuclear localization signals present in the TOP2/170 isoform (1531 aa). Here, we found that TOP2/90, like TOP2/170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Coimmunoprecipitation of endogenous TOP2/90 and TOP2/170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2/90 in K562 cells suppressed, whereas siRNA-mediated knockdown of TOP2/90 in K/VP.5 cells enhanced, etoposide-mediated DNA strand breaks compared with similarly treated cells transfected with empty vector or control siRNAs, respectively. In addition, forced expression of TOP2/90 in K562 cells inhibited etoposide cytotoxicity assessed by clonogenic assays. qPCR and immunoassays demonstrated TOP2/90 mRNA and protein expression in normal human tissues/cells and in leukemia cells from patients. Together, results strongly suggest that TOP2/90 expression decreases drug-induced TOP2-DNA covalent complexes and is a determinant of chemoresistance through a dominant-negative effect related to heterodimerization with TOP2/170. Alternative processing of TOP2 pre-mRNA, and subsequent synthesis of TOP2/90, may be an important mechanism regulating the formation and/or stability of cytotoxic TOP2/170-DNA covalent complexes in response to TOP2-targeting agents.

No abstract

Pixantrone is a new noncardiotoxic aza-anthracenedione anticancer drug structurally related to anthracyclines and anthracenediones, such as doxorubicin and mitoxantrone. Pixantrone is approved in the European Union for the treatment of relapsed or refractory aggressive B cell non-Hodgkin lymphoma. This study was undertaken to investigate both the mechanism(s) of its anticancer activity and its relative lack of cardiotoxicity. Pixantrone targeted DNA topoisomerase IIa as evidenced by its ability to inhibit kinetoplast DNA decatenation; to produce linear double-strand DNA in a pBR322 DNA cleavage assay; to produce DNA double-strand breaks in a cellular phosphohistone gH2AX assay; to form covalent topoisomerase II-DNA complexes in a cellular immunodetection of complex of enzymeto-DNA assay; and to display cross-resistance in etoposideresistant K562 cells. Pixantrone produced semiquinone free radicals in an enzymatic reducing system, although not in a cellular system, most likely due to low cellular uptake. Pixantrone was 10-to 12-fold less damaging to neonatal rat myocytes than doxorubicin or mitoxantrone, as measured by lactate dehydrogenase release. Three factors potentially contribute to the reduced cardiotoxicity of pixantrone. First, its lack of binding to iron(III) makes it unable to induce iron-based oxidative stress. Second, its low cellular uptake may limit its ability to produce semiquinone free radicals and redox cycle. Finally, because the b isoform of topoisomerase II predominates in postmitotic cardiomyocytes, and pixantrone is demonstrated in this study to be selective for topoisomerase IIa in stabilizing enzyme-DNA covalent complexes, the attenuated cardiotoxicity of this agent may also be due to its selectivity for targeting topoisomerase IIa over topoisomerase IIb.

A series of arylnaphthalene lignan lactones based on the structure of the phyllanthusmins, a class of potent natural products possessing diphyllin as the aglycone, has been synthesized and screened for activity against multiple cancer cell lines. SAR exploration was performed on both the carbohydrate and lactone moieties of this structural class. These studies have revealed the importance of functionalization of the carbohydrate hydroxy groups with both acetylated and methylated analogues showing increased potency relative to those with unsubstituted sugar moieties. In addition, the requirement for the presence and position of the C-ring lactone has been demonstrated through reduction and selective re-oxidation of the lactone ring. The most potent compound in this study displayed an IC value of 18 nM in an HT-29 assay with several others ranging from 50 to 200 nM. In an effort to elucidate their potential mechanism(s) of action, the DNA topoisomerase IIa inhibitory activity of the most potent compounds was examined based on previous reports of structurally similar compounds, but does not appear to contribute significantly to their antiproliferative effects.

Capsicodendrin (CPCD, 1), an epimeric mixture of a dimeric drimane-type sesquiterpene, is one of the major compounds present in the three endemic species of Madagascan traditional chemopreventive plants: Cinnamosma species ( C. fragrans, C. macrocarpa, and C. madagascariensis). Despite the popular use of Cinnamosma in Madagascan traditional medicine and the reported antiproliferative properties of CPCD, elucidation of its mechanism(s) of action is still to be accomplished. In the present study, CPCD at low micromolar concentrations was cytotoxic and induced apoptosis in human myeloid leukemia cells in a time- and concentration-dependent manner. The activity of CPCD in HL-60 and K562 cells was modulated by glutathione (GSH), since depletion of this intracellular thiol-based antioxidant with buthionine sulfoximine resulted in significantly ( p < 0.05) greater potency in antiproliferation assays. GSH depletion also significantly potentiated the cytotoxic activity in CPCD-treated human HL-60 cells. Single-cell gel electrophoresis (Comet) assays revealed that GSH depletion in HL-60 cells enhanced the formation of DNA strand breaks in the presence of CPCD. Although CPCD does not contain an obvious Michael acceptor in its structure, H NMR analyses indicated that cinnamodial (2), a monomer of CPCD, was formed within a few hours when dissolved in DMSO- d and interacts with GSH to form a covalent bond via Michael addition at the C-7 carbon. Together the results strongly suggest that 2 is responsible for the DNA-damaging, pro-apoptotic, and cytotoxic effects of CPCD and that depletion of GSH enhances overall activity by diminishing covalent interaction between GSH and this 2-alkenal decomposition product of CPCD.

No abstract

DNA topoisomerase IIá (topo II) is a known cargo protein for exportin 1 (XPO1, CRM1) (Seminars Cancer Biol. 27: 62-73, 2014) for nuclear export. Here we utilized KPT-330, a selective inhibitor of nuclear export, to evaluate its cytotoxicity, its effects on the nuclear content of topo II in human myeloid leukemia HL60 cells and on the subsequent activity of the topo II targeting agent etoposide. Since KPT-330 is a potential michael acceptor and is known to interact with the CRM1 active site Cys528 to inhibit its activity, we first analyzed interaction of KPT-330 with glutathione (GSH)-containing cysteine. In an in vitro binding study KPT-330 was weakly bound to GSH with a Kd = 130+/-24 μM. In contrast, the natural product compound parthenolide, also a michael acceptor, bound to GSH with 30-fold greater affinity. Depletion of cellular GSH with buthionine sulfoximine (BSO) did not significantly alter the 72 hr growth inhibitory effects of KPT-330 yielding I50-values of 109 +/- 19 and 80 +/- 44 nM (p = 0.133) in the absence and presence of BSO. In contrast, GSH depletion significantly enhanced the activity of parthenolide yielding I50-values of 2.59 +/- 0.16 and 1.35 +/- 0.15 μM (p&lt;0.001) in the absence or presence of BSO. KPT-330 (50 μM) did not alter cellular GSH levels in HL60 cells. Using 3’-(p-hydroxyphenyl) fluorescein, KPT-330 was antioxidant in GSH replete and depleted HL60 cells. Together results indicate that conjugation with GSH does not play a significant role in KPT-330 activity. Less than additive cytotoxicity (trypan blue) and apoptosis (Hoescht) were observed using a single fixed ratio of KPT-330 and etoposide incubated either simultaneously or after overnight pre-incubation with KPT-330. Similarly, using the Chou and Talalay technique, KPT-330/etoposide combinations were less than additive in exponentially growing HL60 cells. The mechanism(s) for this apparent antagonism using this combination are under investigation. Using fluorescently labeled topo II antibody, nuclear content of exponentially growing HL60 cells was 85.9 +/- 2.3% of total in the absence of KPT-330 and 86.5+/- 1.8% after an 16 hr incubation with 100 nM KPT-330. In plateau phase cells there was a statistically significant decrease in nuclear topoisomerase IIá to 74.1+/-1.8% compared to exponentially growing cells (p&lt;0.05). In these plateau phase cells, a similar 16 hr incubation with 100 nM KPT-330 resulted in a significant increase in nuclear topoisomerase IIá to 82.6+/-2.5% of total compared to controls (p&lt;0.05). Results are in accord with the known CRM1-mediated shuttling of topo II from the nucleus only in cells approaching or in plateau phase (Exp. Cell Res. 313: 627-637, 2007). Studies are underway to evaluate etoposide-mediated DNA damage and topo II-covalent complexes in HL60 cells in various growth phases after KPT-330 treatment as a correlate with nuclear topo II residency. Citation Format: Erica Godley, Ragu Kanagasabai, Soumendrakrishna Karmahapatra, Jack C. Yalowich. The effects of KPT-330, a selective inhibitor of nuclear export, on nuclear topoisomerase II alpha levels and etoposide activity in human myeloid leukemia HL-60 cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4792.