When cells were treated with 100?M PPNO, there was a significant decrease in m-AMSA-mediated cell killing (IC50=7.50.510?6?M), which represented a 4C5-fold increase in resistance to m-AMSA (Fig. an NO donor, resulted in inhibition of the catalytic activity of topo II. Furthermore, PPNO significantly inhibited topo II-dependent ATP hydrolysis. ?NO-induced inhibition of these topo II ( and ) functions resulted in a decrease in cleavable complex formation in MCF-7 cells in the presence of m-AMSA and XK469 and induced significant resistance to both drugs in MCF-7 cells. Conclusion PPNO treatment resulted in the nitrosation of the topo II protein in MCF-7 cancer cells and inhibited both catalytic-, and ATPase activities of topo II. Furthermore, PPNO significantly affected the DNA damage and cytotoxicity of m-AMSA and XK469 in MCF-7 tumor cells. General significance As tumors express nitric oxide synthase and generate ?NO, inhibition of topo II functions by ?NO/?NO-derived species could render tumors resistant to certain topo II-poisons in the clinic. strong class=”kwd-title” Keywords: Topoisomerase, Nitric oxide, ATPase inhibition, m-AMSA, XK469, Resistance 1.?Introduction Nitric Oxide (?NO) is a small gaseous free radical molecule which easily diffuses in cells and tissues. Extensive research has now shown that ?NO acts as a cellular signaling molecule and is involved in many biological processes, including cell survival, cell death, cancer progression, and the AN-3485 innate immune response , , , , . It has Rabbit Polyclonal to DQX1 been shown that the effects of ?NO are biphasic: At high concentrations it induces DNA damage, apoptosis, and cell death while at low concentrations it induces cell survival and tumor progression , . ?NO and/or its reactive metabolites (NO+, N2O3, -OONO) are known to induce nitrosation (nitrosylation) of many proteins by reacting with free thiol groups, resulting in altered protein functions , . Furthermore, it is believed that nitrosation plays a significant role in the signaling functions of ?NO. em In vivo /em , ?NO is formed from l-arginine by nitric oxide synthase (NOS). Three forms of NOS have been identified, including neuronal (nNOS), endothelial (eNOS), and a Ca2+-independent inducible isoform (iNOS). High manifestation of iNOS and improved production of ?NO have been described in many human being tumors, AN-3485 including breast, prostate and colorectal cancers , , , . Recent studies from our laboratory have shown that ?NO and/or its reactive metabolites, delivered via an NO donor (PPNO), induce significant resistance to both topoisomerase I and II poisons , . Topoisomerases (topo) are nuclear enzymes responsible for keeping the topology of DNA and many of DNA’s functions in cells , , , . Inhibition of topoisomerase enzyme functions results in the inhibition of cellular synthesis and ultimately cellular death. It has been demonstrated that the resistance to the clinically active topo poisons results from mutation of the topo gene, and/or decreases in activity of the proteins , , , . We have found that ?NO nitrosylates both topo I and II in human being breast MCF-7 tumor and colon tumor HT-29 cells when treated with an NO-donor , . Interestingly, this nitrosylation of the topo I had developed no significant effects upon the activity of the protein, nor did it decrease topo I-dependent DNA damage in tumor cells. We have also found that ?NO reacts directly with etoposide, a topo II poison, in the presence of molecular O2, and the products of the reaction are significantly less toxic to tumor cells than the parent drug . Furthermore, we have recently demonstrated that ?NO also causes nitrosylation of topo II-SH functional organizations in cells . Unlike topo I, nitrosylation of topo II prospects to a decrease in its catalytic and relaxation activities both in vitro and in tumor cells . An ?NO-induced decrease in topo II activity leads to a decrease in DNA damage and induction of significant resistance to etoposide in MCF-7 breast tumor cells. While ?NO-derived species nitrosylated free thiol groups of both topo I and II similarly, only the activity of topo II was significantly compromised, resulting in decreased DNA damage. In the present study, we have further characterized the relationships of ?NO (via PPNO, an NO donor) with topo II ( and ) both in vitro and in human being MCF-7 breast tumor cells. We display here that ?NO significantly modulates the cytotoxicity of XK469, a topo II selective drug , and m-AMSA, used clinically for the treatment of lymphoma and adult acute leukemia . With this study we found that ?NO significantly AN-3485 inhibits the ATPase activity of.