ABSTRACT
DNA topoisomerases play a key role in tumor proliferation. Chemotherapeutics targeting topoisomerases have been widely used in clinical oncology, but resistance and side effects, particularly cardiotoxicity, usually limit their application. Clinical data show that a decrease in topoisomerase (top) levels is the primary factor responsible for resistance, but in cells there is compensatory effect between the levels of top1 and top2α. Here, we validated cyclizing-berberine A35, which is a dual top inhibitor and preferentially targets top2α. The impact on the top2α catalytic cycle indicated that A35 could intercalate into DNA but did not interfere with DNA-top binding and top2α ATPase activity. A35 could facilitate DNA-top2α cleavage complex formation by enhancing pre-strand and post-strand cleavage and inhibiting religation, suggesting this compound can be a topoisomerase poison and had a district mechanism from other topoisomerase inhibitors. TARDIS and comet assays showed that A35 could induce cell DNA breakage and DNA-top complexes but had no effect on the cardiac toxicity inducer top2ß. Silencing top1 augmented DNA break and silencing top2α decreased DNA break. Further validation in H9c2 cardiac cells showed A35 did not disturb cell proliferation and mitochondrial membrane potency. Additionally, an assay with nude mice further demonstrated A35 did not damage the heart. Our work identifies A35 as a novel skeleton compound dually inhibits topoisomerases, and predominantly and specially targets top2α by interfering with all cleavage steps and its no cardiac toxicity was verified by cardiac cells and mice heart. A35 could be a novel and effective targeting topoisomerase agent.
Subject(s)
Berberine/pharmacology , Carcinoma, Hepatocellular/pathology , DNA Damage/drug effects , DNA Topoisomerases, Type I/chemistry , DNA-Binding Proteins/antagonists & inhibitors , Liver Neoplasms/pathology , Topoisomerase Inhibitors/pharmacology , Animals , Antigens, Neoplasm , Apoptosis/drug effects , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/enzymology , Carcinoma, Hepatocellular/genetics , Cell Proliferation/drug effects , Comet Assay , DNA Topoisomerases, Type II , Electrophoretic Mobility Shift Assay , Fluorescent Antibody Technique , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/enzymology , Liver Neoplasms/genetics , Membrane Potential, Mitochondrial , Mice , Mice, Nude , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
The DNA religation reaction of yeast type II topoisomerase (topo II) was investigated to elucidate its metal-dependent general acid/base catalysis. Quantum mechanical/molecular mechanical calculations were performed for the topo II religation reaction, and the proton transfer pathway was examined. We found a substrate-mediated proton transfer of the topo II religation reaction, which involves the 3' OH nucleophile, the reactive phosphate, water, Arg781, and Tyr782. Metal A stabilizes the transition states, which is consistent with a two-metal mechanism in topo II. This pathway may be required for the cleavage/religation reaction of topo IA and II and will provide a general explanation for the catalytic mechanism in the topo IA and II.