Regal Kellie M, Mercer Susan L, Deweese Joseph E
Department of Pharmaceutical Sciences, Lipscomb University College of Pharmacy and Health Sciences , Nashville, Tennessee 37204-3951, United States.
Chem Res Toxicol. 2014 Dec 15;27(12):2044-51. doi: 10.1021/tx500245m. Epub 2014 Dec 3.
Topoisomerases are essential enzymes that are involved in DNA metabolism. Topoisomerase II generates transient DNA strand breaks that are stabilized by anticancer drugs, such as doxorubicin, causing an accumulation of DNA damage. However, doxorubicin causes cardiac toxicity and, like etoposide and other topoisomerase II-targeted agents, can induce DNA damage, resulting in secondary cancers. The cannabinoid quinone HU-331 has been identified as a potential anticancer drug that demonstrates more potency in cancer cells with less off-target toxicity than that of doxorubicin. Reports indicate that HU-331 does not promote cell death via apoptosis, cell cycle arrest, caspase activation, or DNA strand breaks. However, the precise mechanism of action is poorly understood. We employed biochemical assays to study the mechanism of action of HU-331 against purified topoisomerase IIα. These assays examined DNA binding, cleavage, ligation, relaxation, and ATPase activities of topoisomerase IIα. Our results demonstrate that HU-331 inhibits topoisomerase IIα-mediated DNA relaxation at micromolar levels. We find that HU-331 does not induce DNA strand breaks in vitro. When added prior to the DNA substrate, HU-331 blocks DNA cleavage and relaxation activities of topoisomerase IIα in a redox-sensitive manner. The action of HU-331 can be blocked, but not reversed, by the presence of dithiothreitol. Our results also show that HU-331 inhibits the ATPase activity of topoisomerase IIα using a noncompetitive mechanism. Preliminary binding studies also indicate that HU-331 decreases the ability of topoisomerase IIα to bind DNA. In summary, HU-331 inhibits relaxation activity without poisoning DNA cleavage. This action is sensitive to reducing agents and appears to involve noncompetitive inhibition of the ATPase activity and possibly inhibition of DNA binding. These studies provide a promising foundation for the exploration of HU-331 as a catalytic inhibitor of topoisomerase IIα.
拓扑异构酶是参与DNA代谢的必需酶。拓扑异构酶II产生短暂的DNA链断裂，这些断裂可被阿霉素等抗癌药物稳定下来，从而导致DNA损伤的积累。然而，阿霉素会引起心脏毒性，并且与依托泊苷和其他靶向拓扑异构酶II的药物一样，会诱导DNA损伤，进而导致继发性癌症。大麻素醌HU - 331已被鉴定为一种潜在的抗癌药物，与阿霉素相比，它在癌细胞中表现出更强的效力且脱靶毒性更小。报告表明，HU - 331不会通过凋亡、细胞周期停滞、半胱天冬酶激活或DNA链断裂来促进细胞死亡。然而，其确切的作用机制尚不清楚。我们采用生化分析方法来研究HU - 331对纯化的拓扑异构酶IIα的作用机制。这些分析检测了拓扑异构酶IIα的DNA结合、切割、连接、松弛和ATP酶活性。我们的结果表明，HU - 331在微摩尔水平上抑制拓扑异构酶IIα介导的DNA松弛。我们发现HU - 331在体外不会诱导DNA链断裂。当在DNA底物之前添加时，HU - 331以氧化还原敏感的方式阻断拓扑异构酶IIα的DNA切割和松弛活性。二硫苏糖醇的存在可以阻断但不能逆转HU - 331的作用。我们的结果还表明，HU - 331使用非竞争性机制抑制拓扑异构酶IIα的ATP酶活性。初步的结合研究也表明，HU - 331降低了拓扑异构酶IIα结合DNA的能力。总之，HU - 331抑制松弛活性而不会使DNA切割中毒。这种作用对还原剂敏感，似乎涉及对ATP酶活性的非竞争性抑制以及可能对DNA结合的抑制。这些研究为探索HU - 331作为拓扑异构酶IIα的催化抑制剂提供了有前景的基础。
