Guo Lei, Liu Xiaojun, Nishikawa Kiyohiro, Plunkett William
Department of Experimental Therapeutics, Box 71, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
Mol Cancer Ther. 2007 May;6(5):1501-8. doi: 10.1158/1535-7163.MCT-06-0780.
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.
NK314是一种新型合成苯并[c]菲啶生物碱，基于其在临床前模型中的显著活性，最近已作为一种抗肿瘤化合物进入临床试验阶段。目前的研究旨在确定该药物的作用机制。NK314在几种细胞系中诱导了显著的G(2)期细胞周期停滞，与p53状态无关，这表明存在一种共同的检查点激活机制。在ML-1人急性髓系白血病细胞中，Chk1-Cdc25C-Cdk1 G(2)检查点通路在100 nmol/L NK314的作用下被激活。这与组蛋白变体H2AX的磷酸化有关，这种作用在G(2)期群体中占主导地位，表明双链DNA断裂导致细胞激活检查点通路。当G(2)检查点被7-羟基星孢菌素消除时，双链DNA断裂表现为染色体畸变。体外试验表明，NK314抑制拓扑异构酶IIα使超螺旋DNA松弛的能力，并将拓扑异构酶IIα捕获在其切割复合物中间体中。由于拓扑异构酶IIα突变而对依托泊苷耐药的CEM/VM1细胞对NK314也产生交叉耐药。然而，由于拓扑异构酶I突变而对喜树碱耐药的CEM/C2细胞仍保持敏感性。这些发现支持了NK314的主要作用机制是抑制拓扑异构酶IIα的结论，这种作用导致双链DNA断裂的产生，从而激活G(2)期DNA损伤检查点通路。
