Heat-Induced Up-Regulation of NAD(P)H:Quinone Oxidoreductase Potentiates Anticancer Effects of β-Lapachone

NAD(P)H:quinone oxidoreductase (NQO1, E.C.1.6.99.2; also known as DT-diaphorase) is a flavoprotein enzyme that catalyzes an obligatory two-electron reduction of a broad range of quinones to hydroquinones using NADH or NAD(P)H as electron sources (1–7). In general, the resultant hydroquinones are stable and are removed from cells after forming conjugates with glutathione, UDP-glucuronic acid, or other moieties (1–7). Quinones are also converted to semiquinones through one electron reduction mediated by NADPH:cytochrome P450 reductase, NADH:cytochrome b₅ reductase, or xanthine oxidase (3, 4, 8, 9). Unlike the two electron–reduced hydroquinones, the semiquinones induce redox cycling and generate reactive oxygen species, thereby causing oxidative damage to cells. Thus, with most quinones, their conversion to the relatively redox-stable hydroquinones by NQO1 is regarded as a detoxification process (1, 3, 5–7, 10). In fact, the chemopreventive activity of a variety of synthetic compounds and vegetables and fruits have been attributed to their ability to up-regulate NQO1 and other phase II detoxifying enzymes in cells (1).

On the other hand, certain natural or synthetic quinones, such as mitomycin C, EO9 (3-hydroxy-5-aziridinyl-1-methyl-2[indol-4,7-dionel]-prop-β-en-α-ol), and RH1 (2,5-diaziridinyl-3-3[hydroxymethyl]-6-methyl-1,4-benzoquinone), become cytotoxic when they are reduced by NQO1 (1, 2, 4–6, 8–14). Although the two electron–reduced EO9 and RHI are cytotoxic in vitro, the clinical use of these agents has been shown to be limited (1).

β-Lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione) is a novel quinone-containing anticancer agent (14–24), originally obtained from the leaves and inner bark of the Lapacho tree (Tabebuia avallanedae) in South America (14, 15). Cell death caused by β-lapachone (3,4-dihydro-22,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione) was initially attributed to inhibition or activation of topoisomerase I (25–31) and inhibition of topoisomerases II (32, 33). However, such effects on topoisomerases by β-lapachone observed in cell-free systems in vitro did not seem to play major role in the anticancer effect of β-lapachone in vivo (25, 26). It has also been suggested that β-lapachone causes apoptosis by affecting cell cycle checkpoints, thereby inhibiting cell cycle progression (34–36). This hypothesis has also been dismissed by more recent studies (21–24), which strongly indicated that two-electron reduction of β-lapachone mediated by NQO1 is the major mechanism by which β-lapachone kills cancer cells (21–24). Indications are that NQO1 induces a futile cycling between quinone and hydroquinone forms of β-lapachone [i.e., β-lapachone(HQ)], using NADH or NAD(P)H as electron sources, resulting in severe depletion of intracellular NADH and NAD(P)H. These events lead to depletion of ATP, release of Ca²⁺ ions from the endoplasmic reticulum into cytosol, depolarization of mitochondrial membrane, and release of cytochrome c (21–24). Ultimately, these catastrophic intracellular disturbances activate the calcium-dependent μ-calpain (22, 37), degrading vital proteins, such as p53 and poly(ADP-ribose) polymerase (22, 23). Another possible mechanism underlying the cell death caused by β-lapachone is that the two electron–reduced β-lapachone is not directly oxidized back to β-lapachone but it is first converted to one electron–reduced semiquinone form of β-lapachone [i.e., β-lapachone(SQ)^•−]. This active semiquinone may then induce redox cycling, thereby generating cytotoxic reactive oxygen species (14, 22).

It was previously reported that β-lapachone sensitizes cancer cells to ionizing radiation by inhibiting the repair of potentially lethal radiation damage (26, 27, 38, 39). Our recent results, however, indicated that the synergistic interaction between ionizing radiation and β-lapachone in causing cell death in vitro and in vivo is due in part to a marked increase in the sensitivity of cells to β-lapachone as a result of ionizing radiation–induced up-regulation of NQO1 activity (40). In the present report, we describe our unprecedented observation that mild hyperthermic shock at 42°C for 1 hour causes long-lasting elevation of NQO1 expression and enzyme activity in cancer cells and that the heat-induced up-regulation of NQO1 markedly increases the sensitivity of cancer cells to β-lapachone.

Cell lines. FSaII tumor cells, a fibrosarcoma of C3H/HeJ mice (40), and A549 human lung cancer cells were used. The A549 cells were originally obtained from the American Type Culture Collection (Manassas, VA). All cells were grown in RPMI 1640 (Life Technologies, Grand Island, NY) containing 10% bovine calf serum (Hyclone Laboratories, Inc., Logan, UT), penicillin (50 units/mL), and streptomycin (50 μg/mL) at 37°C in a humidified 95% air-5% CO₂ atmosphere and were free of Mycoplasma contamination. Appropriate numbers of cells in exponential growth phase were seeded in 25 cm² plastic tissue culture flasks with 5 mL of complete RPMI 1640, incubated overnight, and used for experiments.

β-Lapachone. β-lapachone was purchased from Sigma Chemical Co. (St. Louis, MO). For the in vitro study, β-lapachone was dissolved in DMSO at 10 mmol/L and then diluted to desired concentrations in RPMI 1640 immediately before use. For the in vivo study, β-lapachone was dissolved in β-hydroxypropyl-β-cyclodextrin as previously described (41). The concentration of β-lapachone was adjusted to inject i.p. at 45 mg/kg β-lapachone in 0.2 mL.

Clonogenic cell survival assay. Following treatments with β-lapachone and/or heating individually or combined, cells were cultured under a 95% air-5% CO₂ atmosphere at 37°C for 7 to 8 days. Resultant colonies were fixed with a mixture of methanol and acetic acid (10:1 v/v), stained with 1% crystal violet, and the numbers of colonies containing >50 cells were counted.

Heat treatment in vitro. Culture flasks, in which appropriate numbers of cells were plated the day before, were tightly closed and the cap area was sealed with wax paper. The flasks were then immersed into a preheated water bath at 42°C for 1 hour (42). After heating, the medium in the flask was replaced with fresh complete medium and the clonogenic cell survival was determined by culturing the cells at 37°C as indicated above. Because oxygen is released from plastic, sealing the plastic culture flasks for several hours does not significantly change the pO₂ in the culture medium in the flasks even at elevated temperatures.⁵

Effect of NAD(P)H:quinone oxidoreductase inhibitor dicoumarol on cell death caused by β-lapachone alone or in combination with hyperthermia. The role of NQO1 in cell death caused by β-lapachone alone or in combination with heating was investigated using dicoumarol [3-3′methylene-bis(4-hydroxycoumarin), Sigma Chemical], an inhibitor of NQO1 (22, 43). A549 cells were heated at 42°C for 1 hour, incubated at 37°C for 24 hours, and then treated with 5 μmol/L β-lapachone and/or 50 μmol/L dicoumarol for 4 hours at 37°C. Unheated cells were also treated with β-lapachone and/or with dicoumarol. After treatment, cells were gently rinsed with fresh complete culture medium, cultured at 37°C, and the clonogenic survival of the cells was obtained.

Immunofluorescence microscopy of NAD(P)H:quinone oxidoreductase expression. Cells were cultured on tissue culture chamber slides, heated, and after various intervals rinsed with PBS and fixed with acetone/methanol (1:1) for 20 minutes. After blocking with 1% bovine serum albumin, cells were incubated with anti-NQO1 antibody (1:100 dilution in PBS, Santa Cruz Biotech, Inc., Santa Cruz, CA) for 2 hours followed by incubation with secondary antibody conjugated with FITC (Jackson Immuno Research Laboratory, Inc., West Grove, PA) for 1 hour. After washing the labeled cells with PBS four times, cells were examined for NQO1 expression with a confocal laser scanning fluorescence microscope (40).

Western blot analysis of NAD(P)H:quinone oxidoreductase expression. Cells were harvested by trypsinization (0.25% trypsin and 1 mmol/L EDTA), washed twice with ice-cold PBS, and treated with solubilizing buffer (pH 7.4, 1% Triton X-100, 0.1% SDS, 20 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L sodium orthovanadate, 1 mmol/L sodium fluoride, 2 mmol/L phenylmethylsulfonyl fluoride, 10 mmol/L iodoacetamide, 10 μg/mL aprotinin, and 10 μg/mL leupeptin). Aliquots containing 50 μg of protein were separated by 7.5% SDS-PAGE and transblotted onto Hybond-P (Amersham Life Sciences, Inc., Arlington Heights, IL) in transfer buffer (192 mmol/L glycine, 25 mmol/L SDS, and 10% methanol). Blots were blocked with 3% nonfat dry milk in TBST (pH 7.4), incubated with anti-NQO1 antibody (1:100 dilution in PBS, Santa Cruz Biotech), and treated with horseradish peroxidase–conjugated anti-goat IgG secondary antibody (1:1,000 dilution, Santa Cruz Biotech). Immunoreactive bands were visualized using chemiluminescence (40). Equal sample loading was confirmed by reprobing the same blots with mouse monoclonal antiserum against β-tubulin.

NAD(P)H:quinone oxidoreductase activity assays. The enzymatic activity of NQO1 in cells was determined following methods previously described (9, 11, 22, 38, 40). Cells were harvested by trypsinization, washed twice with ice-cold PBS and phenol red–free HBSS, and then resuspended in PBS (pH 7.2) containing 10 μg/μL aprotinin. Cell suspensions were sonicated four times using 10-second pulses on ice and S9 supernatants were harvested by centrifugation at 14,000 × g for 20 minutes. The resulting S9 supernatants were collected and used for NQO1 assays or stored at −80°C for future use. Reaction mixtures for NQO1 assays consisted of S9 supernatant, 77 μmol/L cytochrome c (practical grade, Sigma Chemical) as substrate, NADH (200 μmol/L) as the immediate electron donor, menadione (10 μmol/L) as the intermediate electron acceptor, Tris-HCl buffer (50 mmol/L, pH 7.5), and 0.14% bovine serum albumin. Each assay was done in the presence and absence of 20 μmol/L dicoumarol (NQO1 inhibitor), and the activity, which was inhibited by dicoumarol, was taken as NQO1 activity. Enzyme activity was calculated as micromoles of cytochrome c reduced per minute per milligram of protein, based on the initial rate of change in absorbance at 550 nm and an extinction coefficient for cytochrome c of 21.1 mmol/L/cm, which were read with a Beckman DU 640 spectrophotometer (Beckman Coulter, Fullerton, CA).

Tumor growth delay. FSaII tumor cells in exponential growth phase in culture were harvested with trypsin treatment, washed, and ∼2.0 × 10⁵ cells suspended in 0.05 mL of serum-free RPMI medium were injected s.c. into the right hind legs of 20 to 23 g female C3H mice. When the tumors grew to 150 to 160 mm³, host mice were injected i.p. with 45 mg/kg β-lapachone dissolved in 0.2 mL of β-hydroxypropyl-β-cyclodextrin (44), and then 30 minutes later tumors were heated with a 42°C water bath for 1 hour as described previously (45). The effects of β-lapachone alone and tumor heating alone were also studied. These treatments were repeated every other day for four times. The tumor diameters were measured with a caliper, and tumor volumes were calculated using the formula V = a²b/2, where a was the shortest tumor diameter and b was the longest tumor diameter measured. When tumor size reached ∼1.5 cm³, the host mice were euthanized by CO₂ asphyxiation. All experiments were done following a protocol approved by the University of Minnesota Institutional Animal Care Use Committee (protocol number 0112A13064).

Clonogenic cell death caused by β-lapachone. The clonogenic deaths of FSaII cells and A549 cells after incubation with 5 or 10 μmol/L β-lapachone for varying lengths of time at 37°C are shown in Fig. 1. The treatment with 5 μmol/L β-lapachone for 4 hours reduced the clonogenic survival of FSaII cells and A549 cells to ∼12% and 10%, respectively. The cell death caused by 10 μmol/L β-lapachone treatment for 4 hours was >10-fold greater than that caused by 5 μmol/L β-lapachone treatment for 4 hours in both cell lines.

Effect of heating on NAD(P)H:quinone oxidoreductase levels in tumor cells.Figure 2A shows Western blot analysis of NQO1 expression in A549 cells and FSaII cells after heating at 42°C for 1 hour. NQO1 levels in A549 cells increased ∼2.5-fold by 24 hours and remained elevated for 72 hours after heating, the extent of our observation. The NQO1 expression in A549 cells also significantly increased after heating and remained elevated until 72 hours after heating. Changes in the enzymatic activity of NQO1 after heating are shown in Fig. 2B. The NQO1 activity in the control FSaII tumor cells was 2.1 ± 0.2 μmol/L/min/mg and it increased to 3.3 ± 0.2 μmol/L/min/mg at 24 hours after heating at 42°C for 1 hour. The NQO1 activity in the FSaII cells remained elevated until 48 hours after heating and then began to decline. The NQO1 activity in the control A549 cells was 12.1 ± 0.2 μmol/L/min/mg and it increased to peak value of 19.5 ± 0.9 μmol/L/min/mg at 24 hours after heating at 42°C for 1 hour. The immunofluorescence staining for NQO1 of FSaII cells also markedly increased 24 hours after heating at 42°C for 1 hour (Fig. 2C), again demonstrating that NQO1 level is increased by mild heat shock.

Effect of β-lapachone in combination with heating on cell survival. Heating A549 lung cancer cells at 42°C for 1 hour reduced clonogenic cell survival to 79.9% (Fig. 3). Treatment of A549 cells with 5 μmol/L β-lapachone for 4 hours at 37°C reduced survival to 11.5%. Importantly, the survival of A549 cells decreased to 5.5% when cells were treated with 5 μmol/L β-lapachone for 4 hours immediately after heating, whereas it further decreased to 1.9% when treated with β-lapachone 24 hours after heating.

Figure 4 shows that heating FSaII tumor cells at 42°C for 1 hour decreased clonogenic survival to 76.5% and incubation with 5 μmol/L β-lapachone for 4 hours at 37°C decreased the cell survival to 10.2%. Heating the cells during the last hour of the 4-hour treatment with β-lapachone (3 hours at 37°C and then 1 hour at 42°C) reduced clonogenic survival to 4.5%, whereas heating the cells at 42°C during the first hour of the 4-hour treatment with β-lapachone (1 hour at 42°C and then 3 hours at 37°C) reduced survival to 2.1%. β-Lapachone treatment immediately after heating and 24 hours after heating at 42°C for 1 hour resulted in significantly different cell survival (i.e., 2.50% and 0.71%, respectively, P < 0.05). Thus, in both A549 cells and FSaII cells, cell death caused β-lapachone treatment applied 24 hours after heating was significantly greater than that caused by β-lapachone treatment immediately after heating.

Effect of dicoumarol on the cytotoxicity of β-lapachone alone or in combination with hyperthermia. As shown in Fig. 5, a 4-hour incubation of A549 cells with 50 μmol/L dicoumarol at 37°C slightly reduced cell survival. An incubation of A549 cells with 5 μmol/L β-lapachone at 37°C for 4 hours decreased survival to 9.8%. In contrast, cell survival decreased only to 51.2% when cells were coincubated with 5 μmol/L β-lapachone and 50 μmol/L dicoumarol. Cell survival decreased to 88.0% following heating at 42°C for 1 hour, and a 4-hour treatment with 5 μmol/L β-lapachone 24 hours after heating reduced the cell survival to 2.2%. On the other hand, 18.2% of cells survived when cells were treated with 5 μmol/L β-lapachone together with 50 μmol/L dicoumarol 24 hours after heating.

Tumor growth delay induced by heating and β-lapachone.Figure 6 shows the effects of heating and β-lapachone treatment applied individually or in combination on the growth of FSaII tumors in the legs of C3H mice. The volume of control tumors increased 5-fold in 5.6 ± 0.2 days. The days required for 5-fold increase in tumor volume for the tumors heated at 42°C for 1 hour every other day for four times was 7.6 ± 0.3 days, 2.0 days longer than that for the control tumors, whereas that for tumors treated with i.p. injection of 45 mg/kg β-lapachone every other day for four times was 7.1 ± 0.2 days, 1.5 days longer than that for the control tumors. When host mice were injected i.p. with 45 μg/kg β-lapachone and tumors heated 30 minutes later at 42°C for 1 hour every other day for four times, the number of days required for 5-fold increase in tumor volume was 11.8 ± 0.4 days, which was 6.2 days longer than that for the control tumors (P < 0.05). The 6.2-day delay is longer than the sum of the delays caused by hyperthermia (2.0 days) and β-lapachone treatment (1.5 days). In this connection, the control tumor volume increased 14.3 times in 12 days, whereas the volumes of tumors treated with β-lapachone alone, hyperthermia alone, and combined increased 11.8, 9.6, and 5.3 times, respectively. These results indicated that the combined effect of hyperthermia and β-lapachone in suppressing tumor growth was greater than additive.

Cellular NQO1 activity has been shown to increase by a number of structurally dissimilar chemicals (1, 2, 6, 7) and also by ionizing radiation (40, 46). The present study is the first to show that heating up-regulates NQO1 protein and enzyme activity in cancer cells and that such an up-regulation of NQO1 increases the anticancer effect of β-lapachone, a naturally occurring quinone-containing compound.

NQO1 has been reported to play a major role in the bioactivation of a number of bioreductive agents (1–7). Over the past several years, the mechanism underlying β-lapachone-induced cell death has become clearer. In our previous study (40), dicoumarol, a known inhibitor of NQO1 (22, 43), significantly reduced β-lapachone-induced clonogenic death of FSaII tumor cells, demonstrating the importance of NQO1 in β-lapachone-induced cell death. In this connection, the sensitivity of various cells to β-lapachone was reported to be positively related to the level of NQO1 activity in the cells (21–23). Furthermore, stable transfection of NQO1-expressing plasmid into cells lacking NQO1 increased the sensitivity of cells to β-lapachone (21). These observations clearly indicate that NQO1-mediated reduction is an important upstream of β-lapachone-induced cell death pathways. However, the downstream pathway leading to cell death following reduction of β-lapachone has not yet been clearly delineated although depletion of NAD(P)H and NADH, increase in cytosolic Ca²⁺, depletion of ATP, release of cytochrome c, activation of Ca²⁺-dependent proteases, such as calpain, and cleavage of p53 have been suggested to be the cause of the β-lapachone-induced cell death (21–24, 37). On the other hand, it has been proposed that β-lapachone induces apoptosis by causing cell cycle arrest (34–36). How β-lapachone induces cell cycle arrest has not yet been elucidated. Another proposed mechanism for the cell death caused by β-lapachone is that β-lapachone affects topoisomerase I and topoisomerase II, thereby causing cell death (25–33). However, these mechanisms did not seem to play significant role in β-lapachone-induced cell death in vivo (25, 26).

In the present study, Western blot analysis, immunofluorescence microscopy, and biochemical analysis of enzymatic activity all showed that heating at 42°C for 1 hour caused a long-lasting up-regulation of NQO1 in both A549 cells and FSaII cells (Fig. 2). As shown in Figs. 3 and 4, the clonogenic death of A549 cells and FSaII cells caused by β-lapachone treatment applied 24 hours after heating at 42°C for 1 hour was significantly greater than that caused by β-lapachone treatment applied immediately after heating. Importantly, inhibition of NQO1activity by dicoumarol could suppress the cell death caused by combination of heating and β-lapachone treatment (Fig. 5). Taken together, we may conclude that the increase in NQO1 activity 24 hours after heating at 42°C for 1 hour sensitized the cells to β-lapachone. Interestingly, heating FSaII cells during the first 1 hour of the 4-hour β-lapachone treatment was more effective than heating the cells during the last 1 hour of the 4-hour β-lapachone treatment (Fig. 4). It seemed that the heating during the first 1 hour of the 4-hour β-lapachone treatment elevated the NQO1 activity and rendered the cells sensitive to β-lapachone during the remaining 3-hour β-lapachone treatment, whereas an increase in NQO1 activity caused by the heating during the last 1 hour of the 4-hour β-lapachone treatment exerted relatively little influence on the response of cells to β-lapachone. The combined effect of tumor heating and i.p. injection of β-lapachone to host animals in suppressing the FSaII tumors grown in the hind legs of mice was apparently greater than additive (Fig. 6). In this study, tumors were heated every other day for four times and β-lapachone was applied 30 minutes before each tumor heating. Because heat-induced up-regulation of NQO1 lasted longer than 72 hours in vitro (Fig. 2), it would be reasonable to assume that the NQO1 activity in the tumors remained increased during the intervals of heating applied every 48 hours and sensitized tumor cells to β-lapachone. Detailed information on the pharmacokinetics of β-lapachone and that on the kinetics of changes in NQO1 activity following heating in tumors may enable us to better exploit the heat-induced activation of NQO1 for potentiation of antitumor effect of β-lapachone.

It is important to note that the NQO1 content in most human tumors is intrinsically greater than that in adjacent normal tissues (1, 4, 9, 47–49). A number of divergent agents, including many dietary components, have been reported to be able to activate NQO1 in cancer cells and investigations are in progress to identify ideal inducing agents for NQO1 to enhance the antitumor effects of bioreductive agents (1, 5–7). The results of the present study and in a previous report from our group (40) strongly suggest that NQO1 activity in tumors may be further and selectively elevated using local radiotherapy or hyperthermia, established cancer treatment modalities, to improve the cytotoxicity of β-lapachone against cancer cells.

We thank Drs. Seymour H. Levitt and Kathryn Dusenbery for their continuous support.