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 IIalpha to relax
supercoiled DNA and trapped topoisomerase IIalpha in its cleavage complex intermediate. CEM/VM1 cells, which are resistant to
etoposide due to mutations in topoisomerase IIalpha, 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 IIalpha, an action that leads to the generation of double-strand DNA breaks, which activate the G(2) DNA damage checkpoint pathway.