nk314 and Neoplasms

Topoisomerase II (Top2) is a ubiquitous nuclear enzyme that relieves torsional stress in chromosomal DNA during various cellular processes. Agents that target Top2, involving etoposide, doxorubicin, and mitoxantrone, are among the most effective anticancer drugs used in the clinic. Mammalian cells possess two genetically distinct Top2 isoforms, both of which are the target of these agents. Top2alpha is essential for cell proliferation and is highly expressed in vigorously growing cells, whereas Top2beta is nonessential for growth and has recently been implicated in treatment-associated secondary malignancies, highlighting the validity of a Top2alpha-specific drug for future cancer treatment; however, no such agent has been hitherto reported. Here we show that NK314, a novel synthetic benzo[c]phenanthridine alkaloid, targets Top2alpha and not Top2beta in vivo. Unlike other Top2 inhibitors, NK314 induces Top2-DNA complexes and double-strand breaks (DSBs) in an alpha isoform-specific manner. Heterozygous disruption of the human TOP2alpha gene confers increased NK314 resistance, whereas TOP2beta homozygous knock-out cells display increased NK314 sensitivity, indicating that the alpha isoform is the cellular target. We further show that the absence of Top2beta does not alleviate NK314 hypersensitivity of cells deficient in non-homologous end-joining, a critical pathway for repairing Top2-mediated DSBs. Our results indicate that NK314 acts as a Top2alpha-specific poison in mammalian cells, with excellent potential as an efficacious and safe chemotherapeutic agent. We also suggest that a series of human knock-out cell lines are useful in assessing DNA damage and repair induced by potential topoisomerase-targeting agents.

Topics: Antigens, Neoplasm; Antineoplastic Agents; Cell Proliferation; DNA Breaks, Double-Stranded; DNA Topoisomerases, Type II; DNA-Binding Proteins; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Gene Deletion; HeLa Cells; Humans; Isoenzymes; Neoplasm Proteins; Neoplasms; Phenanthrenes; Poly-ADP-Ribose Binding Proteins; Topoisomerase II Inhibitors

NK314 is a novel synthetic benzo[c]phenanthridine alkaloid that shows strong antitumor activity. It inhibited topoisomerase II activity and stabilized topoisomerase II-DNA cleavable complexes. The DNA breaks occurred within 1h after treatment with NK314 even without digestion of topoisomerase II by proteinase K, whereas etoposide required digestion of the enzyme protein in cleavable complex to detect DNA breaks. Pretreatment with topoisomerase II catalytic inhibitors, ICRF-193 and suramin, reduced both cleavable complex-mediated DNA breaks and proteinase K-independent DNA breaks, but protease inhibitors and nuclease inhibitors only decreased the latter. These results indicate that NK314 might affect topoisomerase II in the different manner from cleavable complex formation and activate intracellular proteinase and nuclease to produce DNA fragmentation. As a result of this unique mechanism of DNA breakage, NK314 showed substantial growth inhibition of topoisomerase II inhibitor-resistant tumors.

Topics: Animals; Antigens, Neoplasm; Antineoplastic Agents; Cell Proliferation; DNA Breaks, Double-Stranded; DNA Topoisomerases, Type II; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Endopeptidase K; Enzyme Inhibitors; Female; HL-60 Cells; Humans; Leukemia P388; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms; Phenanthrenes; Time Factors; Topoisomerase II Inhibitors; Xenograft Model Antitumor Assays