Upregulation of NAD(P)H:Quinone Oxidoreductase By Radiation Potentiates the Effect of Bioreductive β-Lapachone on Cancer Cells

Abstract

We found that beta-lapachone (beta-lap), a novel bioreductive drug, caused rapid apoptosis and clonogenic cell death in A549 human lung epithelial cancer cells in vitro in a dose-dependent manner. The clonogenic cell death caused by beta-lap could be significantly inhibited by dicoumarol, an inhibitor of NAD(P)H:quinone oxido-reductase (NQO1), and also by siRNA for NQO1, demonstrating that NQO1-induced bioreduction of beta-lap is an essential step in beta-lap-induced cell death. Irradiation of A549 cells with 4 Gy caused a long-lasting upregulation of NQO1, thereby increasing NQO1-mediated beta-lap-induced cell deaths. Although the direct cause of beta-lap-induced apoptosis is not yet clear, beta-lap treatment reduced the expression of p53 and NF-kappaB, whereas it increased cytochrome C release, caspase-3 activity, and gammaH2AX foci formation. Importantly, beta-lap treatment immediately after irradiation enhanced radiation-induced cell death, indicating that beta-lap sensitizes cancer cells to radiation, in addition to directly killing some of the cells. The growth of A549 tumors induced in immunocompromised mice could be markedly suppressed by local radiation therapy when followed by beta-lap treatment. This is the first study to demonstrate that combined radiotherapy and beta-lap treatment can have a significant effect on human tumor xenografts.

β-Lapachone suppresses radiation-induced activation of nuclear factor-κB

“…In good agreement with previous reports (Boothman et al, 1993;Planchon et al, 1995Planchon et al, , 2001Wuerzberger et al, 1998;Pink et al, 2000;Park et al, 2005a;Suzuki et al, 2006;Choi et al, 2007;Song et al, 2008), we have observed in the present study that not only the apoptosis and clonogenic cell death caused by β-lap but also the synergistic interaction between β-lap and irradiation in causing cell death occurred preferentially in the NQO1-positive A549 cells relative to NQO1-null shNQO1 A549 cells (Figure 1). These results clearly demonstrated that the effect of β-lap and NQO1 activity is closely related.…”

“…The cells were stained with propidium iodide (PI, Sigma) and apoptosis was assessed using the FACSCalibur flow cytometer (Beckman Dickinson) (Choi et al, 2007).…”

“…In addition to killing a variety of cancer cells, β-lap has been shown to sensitize cancer cells to radiation by inhibiting the repair of sublethal radiation damage (Boothman et al, 1993;Park et al, 2005b;Suzuki et al, 2006;Choi et al, 2007). The anti-tumor activity and the radiosensitizing activity of β-lap are dependent on the cytosolic flavoenzyme NAD(P)H:quinone oxidoreductase (NQO1, E＞C＞1.6.99.2), which catalyzes the two-electron reduction of endogenous as well as exogenous quinone compounds to their corresponding hydroquinone forms using H + from NADH or NADPH (Boothman et al, 1993;Trush et al, 1996;Pink et al, 2000;Planchon et al, 2001;Tagliarino et al, 2001;Park et al, 2005aPark et al, , 2005bSuzuki et al, 2006;Bey et al, 2007;Choi et al, 2007;Song et al, 2008). Boothman and his colleagues (Trush et al, 1996;Pink et al, 2000;Planchon et al, 2001;Tagliarino et al, 2001;Park et al, 2005b;Bentle et al, 2006;Bey et al, 2007) proposed that NQO1 mediates the two-electron reduction of β-lap and that the reduced form of β-lap is unstable and thus immediately oxidized to the original oxidized form.…”

“…In addition, the futile cycling between the oxidized and reduced forms of β-lap causes depletion of ATP, the elevation of cytosolic Ca 2+ and the release of cytochrome C, thereby inducing cell death. Treatment of cells with dicoumarol, an inhibitor of NQO1 (Boothman et al, 1993;Park et al, 2005b;Suzuki et al, 2006; Song et al, 2008), or with the small interfering RNA (siRNA)-NQO1 (Choi et al, 2007) have been shown to effectively suppress the cytotoxic and radiosensitizing effects of β-lap. Furthermore, NQO1-null-cells were resistant to β-lap and this resistance could be reversed by administration of exogenous NQO1 (Pink et al, 2000).…”

“…Importantly, the NQO1 content in various human cancer cells is significantly greater than that observed in normal tissues implying that tumors may be preferentially damaged by β-lap. Furthermore, NQO1 expression in cancer cells can be increased by a number of different types of stresses such as ionizing radiation (Choi et al, 2007), heat shock (Park et al, 2005b) and certain chemotherapeutic drugs such as cisplatin (Terai et al, 2009). These observations strongly indicated that conventional cancer treatments may increase the sensitivity of the cancer to β-lap treatment by increasing NQO1 activity in the target tumor cells (Park et al, 2005b;Suzuki et al, 2006;Choi et al, 2007;Song et al, 2008).…”