Inhibition of topoisomerase IIα and G2 cell cycle arrest by NK314, a novel benzo[c]phenanthridine currently in clinical trials

Abstract

NK314 is a novel synthetic benzo[c]phenanthridine alkaloid that has recently entered clinical trials as an antitumor compound, based on impressive activities in preclinical models. The present investigations were directed at determining the mechanism of action of this agent. NK314 induced significant G 2 cell cycle arrest in several cell lines, independent of p53 status, suggesting the existence of a common mechanism of checkpoint activation. The Chk1-Cdc25C-Cdk1 G 2 checkpoint pathway was activated in response to 100 nmol/L NK314 in ML-1 human acute myeloid leukemia cells. This was associated with the phosphorylation of the histone variant H2AX, an action that was predominant in the G 2 population, suggesting that double-strand DNA breaks caused cells to activate the checkpoint pathway. Double-strand DNA breaks were visualized as chromosomal aberrations when the G 2 checkpoint was abrogated by 7-hydroxystaurosporine. In vitro assays showed that NK314 inhibited the ability of topoisomerase IIA to relax supercoiled DNA and trapped topoisomerase IIA in its cleavage complex intermediate. CEM/VM1 cells, which are resistant to etoposide due to mutations in topoisomerase IIA, were cross-resistant to NK314. However, CEM/C2 cells, which are resistant to camptothecin due to mutations in topoisomerase I, retained sensitivity. These findings support the conclusion that the major mechanism of NK314 is to inhibit topoisomerase IIA, an action that leads to the generation of double-strand DNA breaks, which activate the G 2 DNA damage checkpoint pathway. [Mol Cancer Ther 2007;6(5):1501 -8]

NK314 potentiates antitumor activity with adult T-cell leukemia-lymphoma cells by inhibition of dual targets on topoisomerase IIα and DNA-dependent protein kinase

“…28,29 NK314, a synthetic derivative of benzo[c]phenanthridine, is a unique anticancer agent possessing specific and potent inhibitory activity against topoisomerase II␣ (Top2␣). [30][31][32] We found that NK314 inhibited ATL cell lines with greater potency than etoposide, a Top2 inhibitor frequently used in practice. Because the range of 50% inhibitory concentration values of NK314 for ATL cell lines and non-ATL cell lines differs from those of etoposide, we investigated other targets of NK314 for induction of apoptosis and inhibition of cell growth of ATL cell lines.…”

“…[30][31][32] Because NK314 inhibits Top2␣ more specifically and more potently than etoposide, which is a key drug for ATL treatment, we investigated the antitumor activity of NK314 in ATL cells. Various ATL-related (6 ATL-derived cell lines and 2 HTLV-1-infected) T-cell lines were cultured with or without IL-2.…”

“…Figure 1A) was synthesized at Nippon Kayaku and dissolved in distilled water before use. [30][31][32] Etoposide was also synthesized at Nippon Kayaku. NU7026 was purchased from Sigma-Aldrich.…”

DNA-Dependent Protein Kinase and Ataxia Telangiectasia Mutated (ATM) Promote Cell Survival in Response to NK314, a Topoisomerase IIα Inhibitor

“…1A) (Guo et al, 2007;Toyoda et al, 2008). The specific topoisomerase II␣-targeting activity of NK314 and lack of activity against topoisomerase II␤ distinguish its actions from other topoisomerase II inhibitors such as etoposide and doxorubicin, in that inhibition of topoisomerase II␤ is associated with secondary malignancies and toxicity (Azarova et al, 2007).…”

“…NK314 also showed activity toward tumors resistant to other topoisomerase II inhibitors (Onda et al, 2008). Because NK314 has been demonstrated to induce rapid and extensive DNA DSBs (Guo et al, 2007;Onda et al, 2008), further investigation of the response of DNA repair pathways to NK314 is needed to understand the cell survival and resistance mechanisms and develop mechanism-based combination strategies.…”