Kim S A, Kwon Y, Kim J H, Muller M T, Chung I K
Department of Biology, Bioproducts Research Center, Yonsei University, Seoul, Korea.
Biochemistry. 1998 Nov 17;37(46):16316-24. doi: 10.1021/bi9810961.
Topoisomerase II is the cytotoxic target for a number of clinically relevant antitumor drugs. Berberrubine, a protoberberine alkaloid which exhibits antitumor activity in animal models, has been identified as a specific poison of topoisomerase II in vitro. Topoisomerase II-mediated DNA cleavage assays showed that berberrubine poisons the enzyme by stabilizing topoisomerase II-DNA cleavable complexes. Subsequent proteinase K treatments revealed that berberrubine-induced DNA cleavage was generated solely by topoisomerase II. Topoisomerase II-mediated DNA religation with elevated temperature revealed a substantial reduction in DNA cleavage induced by berberrubine, to the extent comparable to that of other prototypical topoisomerase II poison, etoposide, suggesting that DNA cleavage involves stabilization of the reversible enzyme-DNA cleavable complex. However, the step at which berberrubine induces cleavable complex may differ from that of etoposide as revealed by the difference in the formation of the intermediate product, nicked DNA. This suggests that berberrubine's primary mode of linear formation may involve trapping nicked molecules, formed at transition from linear to covalently closed circular DNA. Unwinding of the duplex DNA by berberrubine is consistent with an intercalative binding mode for this compound. In addition to the ability to induce the cleavable complex mediated with topoisomerase II, berberrubine at high concentrations was shown to specifically inhibit topoisomerase II catalytic activity. Berberrubine, however, did not inhibit topoisomerase I at concentrations up to 240 microM. Cleavage sites induced by topoisomerase II in the presence of berberrubine and etoposide were mapped in DNA. Berberrubine induces DNA cleavage in a site-specific and concentration-dependent manner. Comparison of the cleavage pattern of berberrubine with that of etoposide revealed that they share many common sites of cleavage. Taken together, these results indicate that berberrubine represents a new class of antitumor agent which exhibits the topoisomerase II poison activity as well as catalytic inhibition activity and may have a potential clinical value in cancer treatment.
拓扑异构酶II是多种临床相关抗肿瘤药物的细胞毒性靶点。小檗红碱是一种原小檗碱生物碱，在动物模型中表现出抗肿瘤活性，在体外已被确定为拓扑异构酶II的特异性毒物。拓扑异构酶II介导的DNA切割试验表明，小檗红碱通过稳定拓扑异构酶II-DNA可切割复合物来毒害该酶。随后的蛋白酶K处理表明，小檗红碱诱导的DNA切割仅由拓扑异构酶II产生。拓扑异构酶II介导的高温下的DNA重新连接显示，小檗红碱诱导的DNA切割大幅减少，其程度与其他典型的拓扑异构酶II毒物依托泊苷相当，这表明DNA切割涉及可逆的酶-DNA可切割复合物的稳定。然而，小檗红碱诱导可切割复合物的步骤可能与依托泊苷不同，这从中间产物切口DNA形成的差异中可以看出。这表明小檗红碱线性形成的主要模式可能涉及捕获在从线性DNA转变为共价闭合环状DNA时形成的切口分子。小檗红碱对双链DNA的解旋与该化合物的嵌入结合模式一致。除了能够诱导拓扑异构酶II介导的可切割复合物外，高浓度的小檗红碱还被证明能特异性抑制拓扑异构酶II的催化活性。然而，小檗红碱在浓度高达240微摩尔时不抑制拓扑异构酶I。在DNA中绘制了拓扑异构酶II在小檗红碱和依托泊苷存在下诱导的切割位点。小檗红碱以位点特异性和浓度依赖性方式诱导DNA切割。小檗红碱与依托泊苷的切割模式比较表明，它们有许多共同的切割位点。综上所述，这些结果表明小檗红碱代表了一类新的抗肿瘤药物，它具有拓扑异构酶II毒物活性以及催化抑制活性，在癌症治疗中可能具有潜在的临床价值。
