Heat-Induced Up-Regulationof NAD(P)H:Quinone ...clincancerres.aacrjournals.org/content/clincanres/11/24/8866.full.pdf · Heat-Induced Up-Regulationof NAD(P)H:Quinone Oxidoreductase

Heat-Induced Up-Regulation of NAD(P)H:Quinone OxidoreductasePotentiates Anticancer Effects of B-LapachoneHeon JooPark,1,2 EunKyungChoi,3 JihyungChoi,1Ki-JungAhn,1Eun JungKim,2 In-MiJi,2 YeonHeeKook,2

Seung-Do Ahn,3Brent Williams,1RobertGriffin,1David A.Boothman,4ChungK. Lee,1andChang W.Song1

Abstract Purpose: The purpose of the present study was to evaluate the efficacy of mild hyperthermiato potentiate the anticancer effects of h-lapachone (3,4-dihydro-2,2-dimethyl-2H-naph-thol[1,2-b]pyran-5,6-dione) by up-regulating NAD(P)H:quinone oxidoreductase (NQO1) incancer cells.Experimental Design: Effects of h-lapachone alone or in combinationwithmild heating on theclonogenic survival of FSaII fibrosarcoma cells of C3H mice and A549 human lung tumor cellsin vitro was determined. Effects of heating on the NQO1level in the cancer cells in vitro wereassessed usingWestern blot analysis for NQO1expression, biochemical determination of NQO1activity, and immunofluorescence microscopy for NQO1expression. Growth of FSaII tumors inthe hind legs of C3H mice was determined after treating the host mice with i.p. injection of45 mg/kg h-lapachone followed by heating the tumors at 42jC for 1hour every other day forfour times.Results: Incubationof FSaII tumor cells andA549 tumor cellswithh-lapachone at 37jC reducedclonogenic survival of the cells in dose-dependent and incubation time ^ dependent manner.NQO1level in the cancer cells in vitro increased within1hour after heating at 42jC for1hour andremained elevated for >72 hours. The clonogenic cell death caused by h-lapachone increased inparallel with the increase in NQO1levels in heated cells. Heating FSaII tumors in the legs of C3Hmice enhanced the effect of i.p.-injected h-lapachone in suppressing tumor growth.Conclusion:Weobserved for the first time thatmildheat shockup-regulatesNQO1in tumor cells.The heat-inducedup-regulation of NQO1enhanced the anticancer effects of h-lapachone in vitroand in vivo.

NAD(P)H:quinone oxidoreductase (NQO1, E.C.1.6.99.2; alsoknown as DT-diaphorase) is a flavoprotein enzyme thatcatalyzes an obligatory two-electron reduction of a broad rangeof quinones to hydroquinones using NADH or NAD(P)H aselectron sources (1–7). In general, the resultant hydroquinones

are stable and are removed from cells after forming conjugateswith glutathione, UDP-glucuronic acid, or other moieties(1–7). Quinones are also converted to semiquinones throughone electron reduction mediated by NADPH:cytochromeP450 reductase, NADH:cytochrome b5 reductase, or xanthineoxidase (3, 4, 8, 9). Unlike the two electron–reduced hydro-quinones, the semiquinones induce redox cycling and generatereactive oxygen species, thereby causing oxidative damageto cells. Thus, with most quinones, their conversion to therelatively redox-stable hydroquinones by NQO1 is regarded asa detoxification process (1, 3, 5–7, 10). In fact, the chemo-preventive activity of a variety of synthetic compounds andvegetables and fruits have been attributed to their ability toup-regulate NQO1 and other phase II detoxifying enzymes incells (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-h-en-a-ol), and RH1 (2,5-diaziri-dinyl-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 RHIare cytotoxic in vitro, the clinical use of these agents has beenshown to be limited (1).h-Lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]

pyran-5,6-dione) is a novel quinone-containing anticanceragent (14–24), originally obtained from the leaves and inner

Cancer Therapy: Preclinical

Authors’Affiliations: 1Radiobiology Laboratory, Department of TherapeuticRadiology/Radiation Oncology, University of Minnesota, Minneapolis, Minnesota;2Department of Microbiology, College of Medicine, Inha University, Inchon,South Korea; 3Department of Therapeutic Radiology, College of Medicine,University of Ulsan, Seoul, South Korea; and 4Laboratory of Molecular StressResponses, Department of Radiation Oncology, CaseWestern Reserve University,Cleveland, OhioReceived 4/12/05; revised 8/10/05; accepted10/6/05.Grant support: NIH/National Cancer Institute grants RO1CA 44114 (C.W. Song)and RO1CA102792 (D.A. Boothman); National R&DProgram for Cancer Control,Ministry of Health andWelfare, Republic of Korea (03203002-2); and 2003 KoreaInstitute of Science andTechnology Evaluation andPlanningandMinistry of Scienceand Technology, Korean government, through its National Nuclear TechnologyProgram (H.J. Park).The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Requests for reprints: ChangW. Song, Radiobiology Laboratory, Department ofTherapeutic Radiology-Radiation Oncology, University of Minnesota MedicalSchool, MMC 494, 420 Delaware Street Southeast, Minneapolis, MN 55455.Phone: 612-626-6852; Fax: 612-626-6245; E-mail: [email protected].

F2005 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-05-0818

www.aacrjournals.orgClin Cancer Res 2005;11(24) December 15, 2005 8866

Cancer Research. on December 26, 2019. © 2005 American Association forclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

bark of the Lapacho tree (Tabebuia avallanedae) in SouthAmerica (14, 15). Cell death caused by h-lapachone (3,4-dihydro-22,2-dimethyl-2H -naphthol[1,2-b]pyran-5,6-dione)was initially attributed to inhibition or activation of top-oisomerase I (25–31) and inhibition of topoisomerases II(32, 33). However, such effects on topoisomerases by h-lapachone observed in cell-free systems in vitro did not seem toplay major role in the anticancer effect of h-lapachone in vivo(25, 26). It has also been suggested that h-lapachone causesapoptosis by affecting cell cycle checkpoints, thereby inhibitingcell cycle progression (34–36). This hypothesis has also beendismissed by more recent studies (21–24), which stronglyindicated that two-electron reduction of h-lapachone mediatedby NQO1 is the major mechanism by which h-lapachone killscancer cells (21–24). Indications are that NQO1 induces afutile cycling between quinone and hydroquinone forms ofh-lapachone [i.e., h-lapachone(HQ)], using NADH or NAD(P)Has electron sources, resulting in severe depletion of intracellularNADH and NAD(P)H. These events lead to depletion of ATP,release of Ca2+ ions from the endoplasmic reticulum intocytosol, depolarization of mitochondrial membrane, and releaseof cytochrome c (21–24). Ultimately, these catastrophic intra-cellular disturbances activate the calcium-dependent A-calpain(22, 37), degrading vital proteins, such as p53 and poly(ADP-ribose) polymerase (22, 23). Another possible mechanismunderlying the cell death caused by h-lapachone is that thetwo electron–reduced h-lapachone is not directly oxidized backto h-lapachone but it is first converted to one electron–reducedsemiquinone form of h-lapachone [i.e., h-lapachone(SQ).�].This active semiquinone may then induce redox cycling, therebygenerating cytotoxic reactive oxygen species (14, 22).

It was previously reported that h-lapachone sensitizes cancercells to ionizing radiation by inhibiting the repair of potentiallylethal radiation damage (26, 27, 38, 39). Our recent results,however, indicated that the synergistic interaction betweenionizing radiation and h-lapachone in causing cell deathin vitro and in vivo is due in part to a marked increase in thesensitivity of cells to h-lapachone as a result of ionizingradiation–induced up-regulation of NQO1 activity (40). Inthe present report, we describe our unprecedented observationthat mild hyperthermic shock at 42jC for 1 hour causes long-lasting elevation of NQO1 expression and enzyme activity incancer cells and that the heat-induced up-regulation of NQO1markedly increases the sensitivity of cancer cells to h-lapachone.

Materials andMethods

Cell lines. FSaII tumor cells, a fibrosarcoma of C3H/HeJ mice (40),and A549 human lung cancer cells were used. The A549 cells wereoriginally 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 (HycloneLaboratories, Inc., Logan, UT), penicillin (50 units/mL), and strepto-mycin (50 Ag/mL) at 37jC in a humidified 95% air-5% CO2

atmosphere and were free of Mycoplasma contamination. Appropriatenumbers 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.

b-Lapachone. h-lapachone was purchased from Sigma ChemicalCo. (St. Louis, MO). For the in vitro study, h-lapachone was dissolved inDMSO at 10 mmol/L and then diluted to desired concentrations inRPMI 1640 immediately before use. For the in vivo study, h-lapachone

was dissolved in h-hydroxypropyl-h-cyclodextrin as previously de-scribed (41). The concentration of h-lapachone was adjusted to injecti.p. at 45 mg/kg h-lapachone in 0.2 mL.

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

Heat treatment in vitro. Culture flasks, in which appropriatenumbers of cells were plated the day before, were tightly closed andthe cap area was sealed with wax paper. The flasks were thenimmersed into a preheated water bath at 42jC for 1 hour (42). Afterheating, the medium in the flask was replaced with fresh completemedium and the clonogenic cell survival was determined by culturingthe cells at 37jC as indicated above. Because oxygen is released fromplastic, sealing the plastic culture flasks for several hours does notsignificantly change the pO2 in the culture medium in the flasks evenat elevated temperatures.5

Effect of NAD(P)H:quinone oxidoreductase inhibitor dicoumarol on cell

death caused by b-lapachone alone or in combination with hyperthermia.

The role of NQO1 in cell death caused by h-lapachone alone or incombination with heating was investigated using dicoumarol [3-3Vmethylene-bis(4-hydroxycoumarin), Sigma Chemical], an inhibitorof NQO1 (22, 43). A549 cells were heated at 42jC for 1 hour,incubated at 37jC for 24 hours, and then treated with 5 Amol/Lh-lapachone and/or 50 Amol/L dicoumarol for 4 hours at 37jC.Unheated cells were also treated with h-lapachone and/or withdicoumarol. After treatment, cells were gently rinsed with freshcomplete culture medium, cultured at 37jC, and the clonogenicsurvival 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 withacetone/methanol (1:1) for 20 minutes. After blocking with 1% bovineserum albumin, cells were incubated with anti-NQO1 antibody (1:100dilution in PBS, Santa Cruz Biotech, Inc., Santa Cruz, CA) for 2 hoursfollowed by incubation with secondary antibody conjugated with FITC(Jackson Immuno Research Laboratory, Inc., West Grove, PA) for1 hour. After washing the labeled cells with PBS four times, cells wereexamined for NQO1 expression with a confocal laser scanningfluorescence microscope (40).

Western blot analysis of NAD(P)H:quinone oxidoreductase expression.

Cells were harvested by trypsinization (0.25% trypsin and 1 mmol/LEDTA), washed twice with ice-cold PBS, and treated with solubilizingbuffer (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 orthovana-date, 1 mmol/L sodium fluoride, 2 mmol/L phenylmethylsulfonylfluoride, 10 mmol/L iodoacetamide, 10 Ag/mL aprotinin, and 10 Ag/mL leupeptin). Aliquots containing 50 Ag of protein were separated by7.5% SDS-PAGE and transblotted onto Hybond-P (Amersham LifeSciences, Inc., Arlington Heights, IL) in transfer buffer (192 mmol/Lglycine, 25 mmol/L SDS, and 10% methanol). Blots were blocked with3% nonfat dry milk in TBST (pH 7.4), incubated with anti-NQO1antibody (1:100 dilution in PBS, Santa Cruz Biotech), and treated withhorseradish peroxidase–conjugated anti-goat IgG secondary antibody(1:1,000 dilution, Santa Cruz Biotech). Immunoreactive bands werevisualized using chemiluminescence (40). Equal sample loading wasconfirmed by reprobing the same blots with mouse monoclonalantiserum against h-tubulin.

NAD(P)H:quinone oxidoreductase activity assays. The enzymaticactivity of NQO1 in cells was determined following methods previouslydescribed (9, 11, 22, 38, 40). Cells were harvested by trypsinization,washed twice with ice-cold PBS and phenol red–free HBSS, and then

5 C.W. Song, unpublished observation.

Enhanced b-Lap Lethality by Heat-Induced NQO1

www.aacrjournals.org Clin Cancer Res 2005;11(24) December 15, 20058867



resuspended in PBS (pH 7.2) containing 10 Ag/AL aprotinin. Cellsuspensions were sonicated four times using 10-second pulses on iceand S9 supernatants were harvested by centrifugation at 14,000 � g for20 minutes. The resulting S9 supernatants were collected and usedfor NQO1 assays or stored at �80jC for future use. Reaction mixturesfor NQO1 assays consisted of S9 supernatant, 77 Amol/L cytochrome c(practical grade, Sigma Chemical) as substrate, NADH (200 Amol/L) asthe immediate electron donor, menadione (10 Amol/L) as theintermediate electron acceptor, Tris-HCl buffer (50 mmol/L, pH 7.5),and 0.14% bovine serum albumin. Each assay was done in the presenceand absence of 20 Amol/L dicoumarol (NQO1 inhibitor), and theactivity, which was inhibited by dicoumarol, was taken as NQO1activity. Enzyme activity was calculated as micromoles of cytochrome creduced per minute per milligram of protein, based on the initial rate ofchange in absorbance at 550 nm and an extinction coefficient forcytochrome c of 21.1 mmol/L/cm, which were read with a BeckmanDU 640 spectrophotometer (Beckman Coulter, Fullerton, CA).

Tumor growth delay. FSaII tumor cells in exponential growth phasein culture were harvested with trypsin treatment, washed, and f2.0 �105 cells suspended in 0.05 mL of serum-free RPMI medium wereinjected 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 h-lapachone dissolved in 0.2 mL of h-hydroxypropyl-h-cyclodextrin (44), and then 30 minutes later tumors were heated with a42jC water bath for 1 hour as described previously (45). The effects ofh-lapachone alone and tumor heating alone were also studied. Thesetreatments were repeated every other day for four times. The tumordiameters were measured with a caliper, and tumor volumes werecalculated using the formula V = a2b/2, where a was the shortest tumordiameter and b was the longest tumor diameter measured. Whentumor size reached f1.5 cm3, the host mice were euthanized byCO2 asphyxiation. All experiments were done following a protocolapproved by the University of Minnesota Institutional Animal Care UseCommittee (protocol number 0112A13064).

Results

Clonogenic cell death caused by b-lapachone. The clonogenicdeaths of FSaII cells and A549 cells after incubation with 5 or

10 Amol/L h-lapachone for varying lengths of time at 37jC areshown in Fig. 1. The treatment with 5 Amol/L h-lapachone for 4hours reduced the clonogenic survival of FSaII cells and A549cells to f12% and 10%, respectively. The cell death caused by10 Amol/L h-lapachone treatment for 4 hours was >10-foldgreater than that caused by 5 Amol/L h-lapachone treatment for4 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

Fig.1. Effects of h-lapachone on the survival of A549 and FSaII tumor cells in vitro.Exponentially growing cells in culturewere treatedwith 5 or10 Amol/L h-lapachonefor 4 hours, washed, cultured for 7 to 9 days at 37jC, and clonogenic survivalwas determined. Points, mean of six to eight experiments with duplicate cultures;bars, SE.

Fig. 2. Induction of NQO1in A549 and FSaII tumor cells by hyperthermia.Exponentially growing cells in culture were heated at 42jC for1hour as describedin Materials andMethods.A,Western blot analyses of NQO1expression.B, assessment of NQO1enzymatic activities. Points, mean of four experiments foreach group; bars, SE. C, assessment of NQO1levels in FsaII tumor cells before andafter hyperthermia by immunofluorescent staining as described in Materials andMethods.





expression in A549 cells and FSaII cells after heating at 42jCfor 1 hour. NQO1 levels in A549 cells increased f2.5-fold by24 hours and remained elevated for 72 hours after heating,the extent of our observation. The NQO1 expression in A549cells also significantly increased after heating and remainedelevated until 72 hours after heating. Changes in theenzymatic activity of NQO1 after heating are shown inFig. 2B. The NQO1 activity in the control FSaII tumor cellswas 2.1 F 0.2 Amol/L/min/mg and it increased to 3.3 F0.2 Amol/L/min/mg at 24 hours after heating at 42jC for1 hour. The NQO1 activity in the FSaII cells remainedelevated until 48 hours after heating and then began todecline. The NQO1 activity in the control A549 cells was12.1 F 0.2 Amol/L/min/mg and it increased to peak value of19.5 F 0.9 Amol/L/min/mg at 24 hours after heating at 42jCfor 1 hour. The immunofluorescence staining for NQO1 ofFSaII cells also markedly increased 24 hours after heatingat 42jC for 1 hour (Fig. 2C), again demonstrating that NQO1level is increased by mild heat shock.Effect of b-lapachone in combination with heating on cell

survival. Heating A549 lung cancer cells at 42jC for 1 hourreduced clonogenic cell survival to 79.9% (Fig. 3). Treatment ofA549 cells with 5 Amol/L h-lapachone for 4 hours at 37jCreduced survival to 11.5%. Importantly, the survival of A549cells decreased to 5.5% when cells were treated with 5 Amol/Lh-lapachone for 4 hours immediately after heating, whereas itfurther decreased to 1.9% when treated with h-lapachone 24hours after heating.

Figure 4 shows that heating FSaII tumor cells at 42jC for 1hour decreased clonogenic survival to 76.5% and incubationwith 5 Amol/L h-lapachone for 4 hours at 37jC decreased thecell survival to 10.2%. Heating the cells during the last hour ofthe 4-hour treatment with h-lapachone (3 hours at 37jC andthen 1 hour at 42jC) reduced clonogenic survival to 4.5%,whereas heating the cells at 42jC during the first hour of the4-hour treatment with h-lapachone (1 hour at 42jC and then3 hours at 37jC) reduced survival to 2.1%. h-Lapachonetreatment immediately after heating and 24 hours after heating

at 42jC for 1 hour resulted in significantly different cell survival(i.e., 2.50% and 0.71%, respectively, P < 0.05). Thus, in bothA549 cells and FSaII cells, cell death caused h-lapachonetreatment applied 24 hours after heating was significantlygreater than that caused by h-lapachone treatment immediatelyafter heating.

Effect of dicoumarol on the cytotoxicity of b-lapachone alone orin combination with hyperthermia. As shown in Fig. 5, a 4-hourincubation of A549 cells with 50 Amol/L dicoumarol at 37jCslightly reduced cell survival. An incubation of A549 cells with5 Amol/L h-lapachone at 37jC for 4 hours decreased survival to9.8%. In contrast, cell survival decreased only to 51.2% whencells were coincubated with 5 Amol/L h-lapachone and 50Amol/L dicoumarol. Cell survival decreased to 88.0% followingheating at 42jC for 1 hour, and a 4-hour treatment with 5Amol/L h-lapachone 24 hours after heating reduced the cellsurvival to 2.2%. On the other hand, 18.2% of cells survivedwhen cells were treated with 5 Amol/L h-lapachone togetherwith 50 Amol/L dicoumarol 24 hours after heating.

Tumor growth delay induced by heating and b-lapachone.Figure 6 shows the effects of heating and h-lapachone treatmentapplied individually or in combination on the growth of FSaIItumors in the legs of C3H mice. The volume of control tumorsincreased 5-fold in 5.6 F 0.2 days. The days required for 5-foldincrease in tumor volume for the tumors heated at 42jC for 1hour every other day for four times was 7.6 F 0.3 days, 2.0 dayslonger than that for the control tumors, whereas that for tumorstreated with i.p. injection of 45 mg/kg h-lapachone every otherday for four times was 7.1 F 0.2 days, 1.5 days longer than thatfor the control tumors. When host mice were injected i.p. with45 Ag/kg h-lapachone and tumors heated 30 minutes later at42jC for 1 hour every other day for four times, the number ofdays required for 5-fold increase in tumor volume was 11.8 F0.4 days, which was 6.2 days longer than that for the controltumors (P < 0.05). The 6.2-day delay is longer than the sum ofthe delays caused by hyperthermia (2.0 days) and h-lapachonetreatment (1.5 days). In this connection, the control tumor

Fig. 3. Combined effect of hyperthermia and h-lapachone on the clonogenicsurvival of A549 cells.A, cells were heated at 42jC for1hour. B, cells were treatedwith 5 Amol/L h-lapachone (b-lap) for 4 hours at 37jC.C, cellswereheated at 42jCfor1hour and immediately treated with 5 Amol/L h-lapachone for 4 hours.D, cellswere heated at 42jC for1hour, incubated at 37jC for 24 hours, and treated with5 Amol/L h-lapachone for 4 hours. Columns, mean of five to six experiments withduplicate cultures; bars, SE.

Fig. 4. Combined effects of hyperthermia and h-lapachone on the clonogenicsurvival of FSaII cells.A, cells were heated at 42jC for1hour. B, cells were treatedwith 5 Amol/L h-lapachone for 4 hours at 37jC. C, cells were heated at 42jC for1hour during the1st hour of 4-hour treatment with 5 Amol/L h-lapachone treatment.D, cells were heated at 42jC for1hour during the last hour of the 4-hour treatmentwith 5 Amol/L h-lapachone treatment. E, cells were heated at 42jC for1hour andimmediately treated with 5 Amol/L h-lapachone for 4 hours. F, cells were heated at42jC for1hour, incubated at 37jC for 24 hours, and treated with 5 Amol/Lh-lapachone for 4 hours. Columns, mean of five to six experiments with duplicatedcultures; bars, SE.





volume increased 14.3 times in 12 days, whereas the volumes oftumors treated with h-lapachone alone, hyperthermia alone, andcombined increased 11.8, 9.6, and 5.3 times, respectively. Theseresults indicated that the combined effect of hyperthermia andh-lapachone in suppressing tumor growth was greater thanadditive.

Discussion

Cellular NQO1 activity has been shown to increase by anumber of structurally dissimilar chemicals (1, 2, 6, 7) and alsoby ionizing radiation (40, 46). The present study is the first toshow that heating up-regulates NQO1 protein and enzymeactivity in cancer cells and that such an up-regulation of NQO1increases the anticancer effect of h-lapachone, a naturallyoccurring quinone-containing compound.

NQO1 has been reported to play a major role in thebioactivation of a number of bioreductive agents (1–7). Overthe past several years, the mechanism underlying h-lapachone-induced cell death has become clearer. In our previous study(40), dicoumarol, a known inhibitor of NQO1 (22, 43),significantly reduced h-lapachone-induced clonogenic death ofFSaII tumor cells, demonstrating the importance of NQO1 in h-lapachone-induced cell death. In this connection, the sensitivityof various cells to h-lapachone was reported to be positivelyrelated to the level of NQO1 activity in the cells (21–23).Furthermore, stable transfection of NQO1-expressing plasmidinto cells lacking NQO1 increased the sensitivity of cells toh-lapachone (21). These observations clearly indicate thatNQO1-mediated reduction is an important upstream ofh-lapachone-induced cell death pathways. However, the down-stream pathway leading to cell death following reduction ofh-lapachone has not yet been clearly delineated althoughdepletion 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 havebeen suggested to be the cause of the h-lapachone-induced celldeath (21–24, 37). On the other hand, it has been proposed thath-lapachone induces apoptosis by causing cell cycle arrest(34–36). How h-lapachone induces cell cycle arrest has not yetbeen elucidated. Another proposed mechanism for the cell deathcaused by h-lapachone is that h-lapachone affects topoisomeraseI and topoisomerase II, thereby causing cell death (25–33).However, these mechanisms did not seem to play significantrole in h-lapachone-induced cell death in vivo (25, 26).

In the present study, Western blot analysis, immunofluores-cence microscopy, and biochemical analysis of enzymaticactivity all showed that heating at 42jC for 1 hour caused along-lasting up-regulation of NQO1 in both A549 cells andFSaII cells (Fig. 2). As shown in Figs. 3 and 4, the clonogenicdeath of A549 cells and FSaII cells caused by h-lapachonetreatment applied 24 hours after heating at 42jC for 1 hour wassignificantly greater than that caused by h-lapachone treatmentapplied immediately after heating. Importantly, inhibition ofNQO1activity by dicoumarol could suppress the cell deathcaused by combination of heating and h-lapachone treatment(Fig. 5). Taken together, we may conclude that the increase inNQO1 activity 24 hours after heating at 42jC for 1 hoursensitized the cells to h-lapachone. Interestingly, heating FSaIIcells during the first 1 hour of the 4-hour h-lapachone treatmentwas more effective than heating the cells during the last 1 hourof the 4-hour h-lapachone treatment (Fig. 4). It seemed that theheating during the first 1 hour of the 4-hour h-lapachonetreatment elevated the NQO1 activity and rendered the cellssensitive to h-lapachone during the remaining 3-hourh-lapachone treatment, whereas an increase in NQO1 activitycaused by the heating during the last 1 hour of the 4-hourh-lapachone treatment exerted relatively little influence on theresponse of cells to h-lapachone. The combined effect of tumorheating and i.p. injection of h-lapachone to host animals insuppressing the FSaII tumors grown in the hind legs of mice wasapparently greater than additive (Fig. 6). In this study, tumors

Fig. 5. Effects of dicoumarol on the clonogenic survival of FSaII tumor cells treatedwith h-lapachone alone or in combinationwith hyperthermia. A, cells were treatedwith 5 Amol/L h-lapachone for 4 hours at 37jC. B, cells were treated with50 Amol/L dicoumarol for 4 hours at 37jC. C, cells were treated with 5 Amol/Lh-lapachone with 50 Amol/L discoumarol for 4 hours at 37jC.D, cells were heatedat 42jC for1hour. E, cells were heated at 42jC for1hour, incubated at 37jC for24 hours, and treated with 5 Amol/L h-lapachone. F, cells were heated at 42jC for1hour, incubated at 37jC for 24 hours, and treated with 5 Amol/L h-lapachonetogether with 50 Amol/L dicoumarol. Columns, mean of five experiments withduplicated cultures; bars, SE.

Fig. 6. Effects of various treatments on the growthof s.c. grown FSaII tumors in thehind legs of C3Hmice.Treatment consisted of i.p. injection of 45 mg/kgh-lapachone to host mice, heating tumors by immersing the tumor-bearing legsinto a 42jCwater bath for1hour, and i.p. injectionof 45mg/kg h-lapachone to hostmice and heating the tumors at 42jC for1hour beginning 30 minutes later.Thetreatments were repeated every other day for four times. Points, average volume ofseven to eight tumors per group; bars, SE.





were heated every other day for four times and h-lapachone wasapplied 30 minutes before each tumor heating. Because heat-induced up-regulation of NQO1 lasted longer than 72 hoursin vitro (Fig. 2), it would be reasonable to assume that theNQO1 activity in the tumors remained increased during theintervals of heating applied every 48 hours and sensitized tumorcells to h-lapachone. Detailed information on the pharmacoki-netics of h-lapachone and that on the kinetics of changes inNQO1 activity following heating in tumors may enable us tobetter exploit the heat-induced activation of NQO1 forpotentiation of antitumor effect of h-lapachone.

It is important to note that the NQO1 content in mosthuman tumors is intrinsically greater than that in adjacentnormal tissues (1, 4, 9, 47–49). A number of divergent agents,

including many dietary components, have been reported to beable to activate NQO1 in cancer cells and investigations are inprogress to identify ideal inducing agents for NQO1 to enhancethe antitumor effects of bioreductive agents (1, 5–7). Theresults of the present study and in a previous report from ourgroup (40) strongly suggest that NQO1 activity in tumors maybe further and selectively elevated using local radiotherapy orhyperthermia, established cancer treatment modalities, toimprove the cytotoxicity of h-lapachone against cancer cells.

Acknowledgments

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



References1. Begleiter A, Fourie J. Induction of NQO1 in cancercells. Methods Enzymol 2004;382:321^51.

2. Joseph P, XieT, XuY, et al. NAD(P)H:quinone oxido-reductase 1 (DT-diaphorase): expression, regulation,and role in cancer. Oncol Res1994;6:525^32.

3. Joseph P, Jaiswal AK. NAD(P)H: quinone oxidore-ductase I (DTdiaphorase) specifically prevents the for-mation of benzo(a)pyrene quinone-DNA adductsgenerated cytochrome P4501A1and P450 reductase.Proc Natl Acad Sci US A1994;91:8413^7.

4. Rauth AM, Goldberg Z, MirsaV. DT-diaphorase: pos-sible roles in cancer chemotherapy and carcinogene-sis. Oncol Res1997;9:339^49.

5. Begleiter A, Leith MK,Thlivenis JA, et al. Dietary in-duction of NQO1 increases the antitumor activity ofmitomycin C inhumancolon tumor in vivo. BrJCancer2004;91:624^31.

6. RossD, Siegel D. NAD(P)H: quinone oxidoreductase1 (NQO1,DT-diaphorase), functions andpharmacoge-netics. Methods Enzymol 2004;382:115^44.

7. Talalay P. Mechanisms or induction of enzymes thatprotect against chemical carcinogenesis. Adv EnzymeRegul1989;28:149^59.

8. HodnickWF, Satorelli AC. Reductive activation ofmitomycin C by NADH: cytochrome b5 reductase.Cancer Res1993;53:4907^12.

9. Main A, Lopez de Cerain A, Hamilton E, et al. DT-diaphorase and cytochrome b5 reductase in humanlung and breast tumors. BrJCancer1997;76:923^9.

10.Talalay P, Dinkova-Kostova AT. Role of nicotinamidequinone oxidoreductase 1 (NQO1) in protectionagainst toxicity of electrophiles and reactive oxygenintermediates. Methods Enzymol 2004;382:355^67.

11. Siegel D, Beall H, Senekowitach C, et al. Bioreduc-tive activation of mitomycin C by DT-diaphorase. BiolChem1992;31:7879^85.

12. Paull K, Camalier R, Fitzsimmons SA, et al. Correla-tions of DT-diaphorase expression with cell sensitivitydata obtained from theNCI human tumor cell line pan-el. Proc AmAssoc Cancer Res1994;35:369.

13.Watanabe N, FormanHJ. Autoxidationof extracellu-lar hydroquinones is a causative event for the cytotox-icity of menadione and DMNQ in A549-S cells. ArchBiochemBiophys 2003;411:145^57.

14. PardeeAB, LiYZ, Li C. Cancer therapy with h-lapa-chone. Curr Cancer DrugTargets 2002;2:227^42.

15. Gupta D, Podar K,Tai Y, et al. h-Lapachone, a novelplant product, overcomes drug resistance in humanmultiple myeloma cells. Exp Hematol 2002;30:711^20.

16. Li YZ, Li CJ,Yu D, et al. Potent induction of apopto-sis by h-lapachone in human multiple myeloma celllines and patient cells. Mol Med 2000;6:1008^15.

17. DubinM, FernandezVillamil SH, Stoppani AO.Cyto-toxicity of h-lapachone, an naphthoquinone with pos-sible therapeutic use. Medicina (B Aires) 2001;61:343^50.

18. Planchone SM,Wuerzberger S, Frydman B, et al. h-Lapachone-mediated apoptosis in human promyelo-cytic leukemia 9 (HL-60) and human prostate cancer

cells: a p53-independent response. Cancer Res1995;55:3706^11.

19. Li CJ,Wang C, PardeeAB. Inductionof apoptosis byh-lapachone in human prostate cancer cells. CancerRes1995;55:3712^5.

20. Lai CC, Lui TJ, Ho LK, et al. h-Lapachone inducedcell death in human hepatoma (HepA2) cells. HistolHistopathol1998;13:89^97.

21. Planchone SM, Pink JJ,Tagliarino C, et al. h-Lapa-chone-induced apoptosis in human prostate cancercells: involvement of Nqo/xip3. Exp Cell Res 2001;267:95^06.

22. Pink JJ, Planchone SM, Tagliarino C, et al.NAD(P)H:quinone oxidoreductase activity is the prin-cipal determinant of h-lapachone cytotoxicity. J BiolChem 2000;275:5416^24.

23. Pink JJ,Wuerzberger-Davis S, Tagliarino C, et al.Activation of cysteine protease in MCF-7 and T47Dbreast cancer cells during h-lapachone mediated apo-ptosis. Exp Cell Res 2000;25:144^55.

24.Tagliarino C, Pink JJ, Dubyak GR, et al. Calcium is akey signaling molecule in h-lapachone mediated celldeath. JBiol Chem 2001;276:19150^9.

25.Wuerzberger SM, Pink JJ, Planchon SM, et al. In-duction of apoptosis in MCF-7:WS8 breast cancercells by h-lapachone. Cancer Res1998;58:1876^85.

26. Miyamoto S, HuangTT,Wuerzberger-Davis S, et al.Cellular and molecular responses to topoisomerase Ipoisons: exploiting synergy for improved radiotherapy.Ann NYAcad Sci 2000;922:274^92.

27. Boothman DA, Pardee AB. Inhibition of radiation-induced neoplastic transformation by h-lapachone.Proc Natl Acad Sci US A1989;86:4963^7.

28. Hueber A, Esser P, Heimann K, et al. The topoiso-merase I inhibitors, camptothecinandh-lapachone, in-duce apoptosis of human retinal pigment epithelialcells. Exp Eye Res1998;67:525^30.

29. Li CJ, Averboukh L, Pardee AB. h-Lapachone, anovel DNA topoisomerase I inhibitor with a mode ofaction different from camptothecin. J Biol Chem1993;268:22463^8.

30.Weeler M,Winter S, Schmidt C, et al. Topoisomer-ase-I inhibitors for human malignant glioma: differen-tial modulation of p53, p21, bax and bel-2 expressionand of CD95-mediated apoptosis by camptothecinand h-lapachone. IntJCancer1997;73:707^14.

31. FuruyaY, Ohta S, Ito H. Apoptosis and androgen-independent mammary and prostate cell lines inducedby topoisomerase inhibitors: common pathways ofgene regulation. Anticancer Res1997;17:2089^93.

32. Frydman B, Marton LJ, Sun JS, et al. Induction ofDNA topoisomerase II-mediated DNA cleavage by h-lapachone and related naphthoquinones. Cancer Res1997;57:620^7.

33. Krishnan P, Bastow KF. Novelmechanism of cellularDNA topoisomerase II inhibition by the pyranonaph-thoquinone derivatives a-lapachone and h-lapachone.Cancer Chemother Pharmacol 2001;47:197^8.

34. Li Y, Li CJ, YuD, et al. Potent inductionofapoptosis

by h-lapachone in human multiple myeloma cell lineand patient care. Mol Med 2000;6:1008^15.

35. LiY, SunX, LaMontJT, et al. Selective killingofcan-cer cells by h-lapachone: direct checkpoint activationas a strategy against cancer. Proc Natl Acad Sci USA2003;100:2674^8.

36. Li CJ, Li YZ, Pinto AV, et al. Potent inhibition of tu-mor survival in vivo by h-lapachone plusTaxol: com-bining drugs imposes different artificial checkpoints.Proc Natl Acad Sci USA1999;96:13369^74.

37. Tagliarino C, Pink JJ, Boothman DA. Calpains andapoptosis. KoreanJBiol Sci 2001;5:267^74.

38. Boothman DA,Trask DK, Pardee AB. Inhibition ofpotentially lethal DNA damage repair in human tumorcells by h-lapachone, an activator of topoisomerase.Cancer Res1989;49:605^12.

39. Boothman DA, GreerA, PardeeAB. Potentiation ofhalogenated pyrimidine radiosensitizers in human car-cinoma cells by h-lapachone (3,4-dihyrdro-2,2-di-methyl-2H-naptho[1,2-b]pyran-5,6-dione), a novelDNA repair inhibitor. Cancer Res1987;47:5361^6.

40. Park HJ, Ahn KJ, Ahn SD, et al. Susceptibility ofcancer cells toh-lapachone is enhancedby ionizing ra-diation. IntJRadiat Oncol Biol Phys 2005;61:212^9.

41. Nasongkla N,Wiedmann AF, Bruening A, et al, En-hancement of solubility and bioavailability of h-lapa-chone using cyclodextrin inclusion in complexes.Pharm Res 2003;20:1626^33.

42. Ahn KJ, Lee CK, Choi EK, et al. Cytotoxicity of per-illyl alcohol against cancer cells is potentiates by hy-perthermia. Int J Radiat Oncol Biol Phys 2003;57:813^9.

43.Winski SL, Swann E, Hargreaves RHJ, et al. Rela-tionship between NAD(P)H:quinone oxidoreductase1 (NQO1) levels in a series of stably transfected celllines and susceptibility to antitumor quinones. Bio-chem Pharmacol 2001;61:1509^16.

44.OgawaA, Griffin RJ, SongCW. Effects of combina-tion of mild temperature hyperthermia and nicotin-amide on radiation response of experimental tumours.Radiat Res 2000;153:327^31.

45. Griffin RJ, Lee SH, Rood LK, et al. Use of arsenictrioxide as an anti-vascular and thermosensitizngagent in solid tumors. Neoplasia 2001;2:555^60.

46. Boothman DA, Meyers M, Fukunaga S, et al. Isola-tion of X-ray-inducible transcripts from radioresistantmelanoma cells. Proc Natl Acad Sci U S A 1993;90:7200^4.

47. Siegel D, FranklinWA, Ross D. Immunohistochemi-cal detection of NAD(P)H:quinone oxidoreductase inhuman lung and lung tumors. Clin Cancer Res1998;4:2065^70.

48. BelinskyM, Jaiswal AK.NAD(P)H: quinoneoxidore-ductase1 (DT-diaphorase) expression innormal and tu-mor tissues. CancerMetastasis Rev1993;12:103^17.

49. Cresteil T, Jaiswal AK. High levels of expression ofthe NAD(P)H:quinone oxidoreductase (NQO1) genein tumor cells compared to normal cells of the sameorigin. Biochem Pharmacol1991;42:1021^7.



2005;11:8866-8871. Clin Cancer Res Heon Joo Park, Eun Kyung Choi, Jihyung Choi, et al. -Lapachone

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

Updated version

http://clincancerres.aacrjournals.org/content/11/24/8866

Access the most recent version of this article at:

Cited articles

http://clincancerres.aacrjournals.org/content/11/24/8866.full#ref-list-1

This article cites 45 articles, 16 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://clincancerres.aacrjournals.org/content/11/24/8866To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/content/11/24/8866.full#ref-list-1

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



Documents

Heat-Induced Up-Regulationof NAD(P)H:Quinone ...clincancerres.aacrjournals.org/content/clincanres/11/24/8866.full.pdf · Heat-Induced Up-Regulationof NAD(P)H:Quinone Oxidoreductase