2-[4-(7-Chloro-2-quinoxalinyloxyphenoxy]-propionic Acid (XK469), an Inhibitor of Topoisomerase (Topo) II , Up-Regulates Topo II and Enhances Topo II -mediated Cytotoxicity

Topoisomerase (Topo) II has proven to be an adequate anticancer target for tumors expressing this enzyme. In this study, we elucidated the effect of 2-[4(7-chloro-2-quinoxalinyloxyphenoxy]-propionic acid (XK469; a new Topo II inhibitor) in the modulation of Topo II levels and sensitivity to Topo II poisons. We demonstrate by Western blot analysis that indolent Bcell tumors express undetectable levels of this enzyme and are refractory to the effects of Topo II poisons such as VP16. Using the Waldenstrom’s macroglobulinemia (WM) cell line WSU-WM, we show that XK469 induced the expression of Topo II protein by 24 h compared with control. Immunofluorescence studies by confocal microscopy using a specific monoclonal antibody against Topo II supported the immunoblot findings with high intensity staining in XK469-exposed cells. To determine the effect of upregulating Topo II on sensitivity of Topo II -directed inhibitors, WSU-WM cells were exposed to simultaneous, sequential, and reverse order XK469 and VP16. We demonstrate that 24 h of exposure to XK469 before VP16 resulted in a maximum synergistic response. In contrast, simultaneous or reverse order exposure resulted in an antagonistic effect. A similar trend was observed with cells obtained from chronic lymphocytic leukemia patients, but not in normal lymphocytes. This increase in VP16 sensitivity after 24 h of XK469 exposure was associated with VP16dependent DNA cleavage, as demonstrated by formation of a smeared DNA band in a SDS-KCL DNA cleavage assay. From this study, we concluded that XK469 up-regulates Topo II levels and consequently sensitizes indolent malignant B cells to the cytotoxic effect of VP16 in a schedule-dependent manner.