The Naphthoquinones, Vitamin K3 and Its Structural Analogue Plumbagin, Are Substrates of the Multidrug Resistance–Linked ATP Binding Cassette Drug Transporter ABCG2

Published OnlineFirst December 7, 2007; DOI: 10.1158/1535-7163.MCT-07-0564

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The naphthoquinones, vitamin K3 and its structural analogue plumbagin, are substrates of the multidrug resistance–linked ATP binding cassette drug transporter ABCG2

Suneet Shukla, Chung-Pu Wu, that they are substrates of this transporter. Collectively, Krishnamachary Nandigama, these data show for the first time that vitamin K3 is a and Suresh V. Ambudkar substrate of the ABCG2 transporter. Thus, ABCG2 may have a role in the regulation of vitamin K3 levels in the Laboratory of Cell Biology, Center for Cancer Research, National body. In addition, vitamin K3 and its structural derivative, Cancer Institute, NIH, Department of Health and Human Services, plumbagin, could potentially be used to modulate ABCG2 Bethesda, Maryland function. [Mol Cancer Ther 2007;6(12):3279–86]

Abstract Introduction Vitamin K3 (menadione; 2-methyl-1,4-naphthoquinone) is Multidrug resistance of cancer cells is an obstacle to a structural precursor of vitamins K1 and K2, which are effective chemotherapy of cancer and the ATP binding essential for blood clotting. The naturally occurring struc- cassette (ABC) transporters, including P-glycoprotein tural analogue of this vitamin, plumbagin (5-hydroxy- (ABCB1), MRP1 (ABCC1) and ABCG2, play a major role menadione), is known to modulate cellular proliferation, in the development of this phenotype (1). These ABC apoptosis, carcinogenesis, and radioresistance. We here drug efflux transporters not only play a major role in the report that both vitamin K3 and plumbagin are substrates development of multidrug resistance but also affect the of the multidrug resistance–linked ATP binding cassette disposition of xenobiotics or drugs that are commonly used drug transporter, ABCG2. Vitamin K3 and plumbagin in chemotherapy (1). This leads to either drug resistance specifically inhibited the ABCG2-mediated efflux of or reduced concentrations of the anticancer agents inside mitoxantrone but did not have any effect on the ABCB1- the cells. An attractive approach to overcoming multidrug mediated efflux of rhodamine 123. This inhibition of resistance is by inhibition of the pump action, which would ABCG2 function was due to their interaction at the reinstate the drug accumulation inside the resistant cell substrate-binding site(s). Vitamin K3 and plumbagin 125 to levels similar to those of a drug-sensitive tumor cell. inhibited the binding of [ I]iodoarylazidoprazosin, a Several inhibitors/modulators of these ABC transporters substrate of ABCG2, to this transporter in a concentra- have been developed but their cytotoxic effect and adverse tion-dependent manner with IC50 values of 7.3 and pharmacokinetics have precluded their use (2-6). The M 22.6 mol/L, respectively, but had no effect on the ongoing search for such inhibitors/modulators that can binding of the photoaffinity analogue to ABCB1. Both be applied in the clinic is into its third generation. These compounds stimulated ABCG2-mediated ATP hydrolysis most recent inhibitors are more potent and less toxic than and also inhibited the mitoxantrone-stimulated ATPase the first-generation compounds, yet some are still prone to activity of the ABCG2 transporter, but did not have any adverse effects, poor solubility, and unfavorable changes in significant effect on the ATPase activity of ABCB1. In a pharmacokinetics of the other anticancer drugs. Therefore, cytotoxicity assay, ABCG2-expressing HEK cells were the identification of more specific, more potent, and less 2.8- and 2.3-fold resistant to plumbagin and vitamin K3, toxic inhibitors/modulators for clinical use remains critical respectively, compared with the control cells, suggesting to the possible success of this approach. A number of pharmacologic and natural products have been found to overcome a well-characterized form of experimental drug resistance and have potential in the clinic as inhibitors of Received 8/16/07; revised 10/2/07; accepted 10/15/07. ABC transporters. Grant support: Intramural Research Program of the NIH, National Cancer Here, we identify a set of compounds that have a Institute, Center for Cancer Research. naphthoquinone moiety in their chemical structure, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked which inhibit the function of the multidrug-linked ABC advertisement in accordance with 18 U.S.C. Section 1734 solely to drug transporter ABCG2. Vitamin K3 (2-methyl-1,4- indicate this fact. naphthoquinone), also known as menadione, is one such Requests for reprints: Suresh V. Ambudkar, Laboratory of Cell Biology, naturally occurring naphthoquinones in the body, which National Cancer Institute, MSC 4254, 37 Convent Drive, Bethesda, MD 20892-4256. Phone: 301-402-4178; Fax: 301-435-8188. is necessary for the production of prothrombin and five E-mail: [email protected] other blood clotting factors in humans (7). In the Copyright C 2007 American Association for Cancer Research. intestine, it also assists in converting glucose to glycogen, doi:10.1158/1535-7163.MCT-07-0564 which is then stored in the liver. Vitamins K1 and K2 are

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3280 Vitamin K3 Is a Substrate of ABCG2

the naturally occurring types of vitamin K. Menadione acts have a role in the regulation of vitamin K3 levels in the as a provitamin that gets converted into vitamins K1 and K2 body. in the body; it also regulates bone calcification (7). Vitamin K deficiency can lead to defective blood clotting, resulting in spontaneous or excessive bleeding with trauma or injury. Materials and Methods Vitamin K deficiency in infants can lead to hemorrhagic Chemicals disease of the newborn, also known as vitamin K deficiency DMEM, RPMI medium, fetal bovine serum, penicillin, bleeding. Vitamin K3, in conjunction with ascorbate, has streptomycin, trypsin-EDTA, and PBS were purchased also been reported to have a cytotoxic effect on K562 human from Life Technologies, Inc. Plumbagin, vitamin K3, chronic myelogenous leukemia cells by inducing oxidative mitoxantrone, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte- stress, thereby causing cell death (8–10). In addition, trazolium bromide dye, rhodamine 123, and ouabain were vitamins K2 and K3 were shown to have potent antitumor purchased from Sigma. Radiolabeled [125I]iodoarylazido- effects on hepatocellular carcinoma and tumor growth in doprazosin ([125I]IAAP; 2,200 Ci/mmol) was from Perkin- nude mice (11). Elmer Life Sciences. Vitamin K3 shares structural similarity with another natu- Cell Lines and Culture Conditions rally occurring naphthoquinone, plumbagin (5-hydroxy-2- HEK 293 cells stably transfected with either control methyl-1,4-naphthoquinone; Fig. 1), which is present along pcDNA3.1 vector (pcDNA3.1-HEK 293) or pcDNA3.1 con- with a series of other structurally related naphthoquinones taining ABCG2 were maintained in DMEM supplemented in the roots, leaves, bark, and wood of Juglans regia (English with 10% fetal bovine serum, penicillin, streptomycin, and walnut, Persian walnut, and California walnut), Juglans 2 mg/mL G418 (20). KB-3-1– and ABCB1-overexpressing cinerea (butternut and white walnut), and Juglans nigra KB-V1 cells were maintained in DMEM supplemented with (black walnut; ref. 12). Plumbagin has recently been shown 10% fetal bovine serum, penicillin, and streptomycin, and to have anticancer properties by inhibiting cell proliferation KB-V1 cells were grown in media containing 1 Ag/mL and inducing cells to undergo G -M arrest and autophagic 2 vinblastine (21). MCF-7 FLV1000 cells overexpressing wild- cell death (13, 14). Sandur et al. (15) also showed that type ABCG2 were cultured in RPMI with 10% fetal bovine plumbagin can enhance apoptosis induced by cytokine and serum with 1 Ag/mL flavopiridol (22, 23). The ABCG2- chemotherapeutic agents by inhibiting NF-nB activation. In addition, plumbagin has been shown to exert anticancer expressing cell lines were provided by Dr. Susan Bates and antiproliferative activities in in vitro cultured cells as (Medical Oncology Branch, Center for Cancer Research, well as in an in vivo animal model (16, 17) and is also National Cancer Institute/NIH, Bethesda, MD). MCF-7 reported to possess chemopreventive properties. Besides ADRcells were provided by Dr. Kapil Mehta (Department anticancer effects, plumbagin has also exhibited radio- of Experimental Therapeutics, M.D. Anderson Cancer sensitizing properties in experimental mouse tumors as Center, Houston, TX). well as in tumor cells in vitro (18, 19). Isolation of Crude Membranes In this study, we investigated the interaction of the two Crude membranes from MCF-7, MCF-7 FLV1000, or naphthoquinones, vitamin K3 and plumbagin, with the two High-five insect cells expressing ABCB1 or ABCG2 were major ABC drug transporters, ABCB1 and ABCG2. The prepared as described elsewhere (24, 25). The protein data presented here address the biochemical basis of the content was estimated with the amido black B-dye binding interaction of these naphthoquinones with the ABC drug assay as described earlier (26). transporters, and the results suggest that both vitamin K3 Fluorescent Drug Accumulation Assay by Flow and plumbagin are substrates of ABGC2 and specifically Cytometry inhibit the function of this transporter. Thus, ABCG2 may Accumulation assays with mitoxantrone (5 Amol/L for ABCG2-expressing MCF-7 FLV1000 cells) or rhodamine 123 (0.5 Ag/mL for ABCB1-expressing MCF-7 ADRcells) were done as previously described (23). For all samples, 10,000 events were counted and the analysis was done with Cell Quest software (Becton-Dickinson Immunocy- tometry systems). The mean fluorescence intensity was calculated with the histogram stat program in Cell Quest software. CytotoxicityAssay The cytotoxicity of plumbagin and vitamin K3 was determined by MTT/CCK8 assay as previously described (24). The IC50s of plumbagin and vitamin K3 were calculated from linear regression analysis of the linear portion of the growth curves (24). For the reversal of Figure 1. Chemical structures and names of vitamin K3 and plumbagin. Plumbagin, which is a naturally occurring analogue of vitamin K3, has an cytotoxicity assays, a constant nontoxic concentration of additional hydroxyl group at position 5. either plumbagin (0.5 Amol/L) or vitamin K3 (1 Amol/L)

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Figure 2. Effects of vitamin K3 and plumbagin on the accumulation of fluorescent drug substrates in ABCG2- and ABCB1-expressing cells: MCF-7 (A and C), MCF-7 ADR (B), and MCF-7 FLV1000 (D) cells (300,000 per tube) were incubated with 0.5 Ag/mL rhodamine 123 (A and B)or5Amol/L mitoxantrone (C and D) for 45 min at 37jC in the dark, in the absence (Control; thin line) or presence of 10 Amol/L cyclosporine A (Cys A; bold line; A and B), 10 Amol/L fumitremorgin C (FTC; bold line; C and D), 30 Amol/L vitamin K3 (dotted line), or 30 Amol/L plumbagin (dashed line)as indicated. The cells were then washed and analyzed by flow cytometry as described in Materials and Methods. The histogram derived from the Cell Quest software represents the fluorescence intensity of either untreated cells or cells treated with the above compounds. The traces in each histogram are marked. Representative histogram is shown and similar results were obtained in three additional experiments. E, MCF-7 FLV1000 cells were incubated with 5 Amol/L mitoxantrone for 45 min at 37jC in the presence of varying concentrations (0 – 100 Amol/L) of vitamin K3 (n)or plumbagin (E). The cells were then pelleted, resuspended in PBS with 0.1% bovine serum albumin, and analyzed as described above. The difference in the mean fluorescence intensity in the presence and absence of 10 Amol/L fumitremorgin C was taken as 100% (control) and assigned an arbitrary value of 100. The differences in the mean fluorescence intensity values in the presence of 10 Amol/L fumitremorgin C and the indicated concentration of vitamin K3 or plumbagin were calculated as a percent of the control (Y axis). The data were fitted using the software GraphPad Prism 2.0. Points, mean from three independent experiments done in triplicate; bars, SD. The IC50 values given represent the concentration that inhibited the efflux to 50% of the control values. was added with varying concentrations of mitoxantrone specific activity was recorded as beryllium fluoride– and the extent of reversal was calculated based on the sensitive ATPase activity. changes in the relative resistance values, which were Photoaffinity Labeling of ABCB1 and ABCG2 with 12 5 derived by dividing the IC50 value of the resistant cells by [ I]IAAP the IC50 value of the sensitive control cells. The measure- Crude membranes (1 mg protein/mL) from either ments were carried out in triplicates. ABCB1-expressing High-five cells or ABCG2-expressing ATPase A ssay MCF-7 FLV1000 cells were incubated with 0 to 100 Amol/L Crude membrane protein (100 Ag protein/mL) from of plumbagin or vitamin K3 for 10 min at 21jCto23jCin either High-five cells expressing ABCB1 or ABCG2 was 50 mmol/L Tris-HCl (pH 7.5). [125I]IAAP (2,200 Ci/mmol) incubated at 37jC with varying concentrations of plumba- at 3 to 6 nmol/L was added and incubated for an ginandvitaminK3inthepresenceandabsenceof additional 5 min under subdued light. The samples were beryllium fluoride (0.2 mmol/L beryllium sulfate and illuminated with a UV lamp (365 nm) for 10 min at room 2.5 mmol/L sodium fluoride) in ATPase assay buffer temperature. The labeled ABCG2 was immunoprecipitated (50 mmol/L KCl, 5 mmol/L NaN3, 2 mmol/L EGTA, as previously described (27). Samples were separated on a 10 mmol/L MgCl2, 1 mmol/L DTT, pH 6.8) for 10 min. The 7% Tris-acetate gel at constant voltage and gels were dried reaction was started by the addition of 5 mmol/L ATP and and exposed to X-ray film for 12 to 24 h at À80jC. The incubated for 20 min at 37jC. SDS solution (0.1 mL of 5% incorporation of [125I]IAAP into the ABCG2 or ABCB1 band SDS) was added to terminate the reaction and the amount was quantified using the STORM 860 PhosphorImager of inorganic phosphate released was quantified with a system (Molecular Dynamics) and the software Image- colorimetric reaction, as described previously (25). The QuaNT, as described (28).

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Results compounds at similar binding sites. Taken together, the VitaminK3andPlumbaginInhibitABCG2-Mediated data from Figs. 2 and 3 suggested that plumbagin and Efflux of Substrates vitamin K3 specifically interact with ABCG2 but not with Both vitamin K3 and plumbagin were evaluated for their ABCB1. inhibitory activity against two major ABC drug trans- Effect of Plumbagin andVitamin K3 on ATP Hydrolysis porters, ABCB1 and ABCG2. Accumulation assays with by ABCB1 and ABCG2 different fluorescent substrates (mitoxantrone for ABCG2, It is known that ABC transporters use the energy derived rhodamine 123 for ABCB1) in ABCB1- and ABCG2- from ATP hydrolysis to transport substrates and that the expressing cells were done as described in Materials and transport substrates often stimulate basal ATP hydrolysis by Methods. The control MCF-7 (Fig. 2A and C), ABCB1- these transporters. Therefore, the effect of naphthoquinones expressing MCF-7 ADR(Fig. 2B), or ABCG2-expressing MCF-7 FLV1000 (Fig. 2D) cells were incubated with 0.5 Ag/ mL rhodamine 123 (Fig. 2A and B) or 5 Amol/L mitoxantrone (Fig. 2C and D) in the presence or absence of 30 Amol/L plumbagin, vitamin K3, or their respective specific inhibitors (5 Amol/L cyclosporine A for ABCB1 and 10 Amol/L fumitremorgin C for ABCG2) and incubated at 37jC in the dark for 45 min. The presence of plumbagin and vitamin K3 had no effect on the ABCB1-mediated efflux of rhodamine 123 in MCF-7 ADRcells (Fig. 2A and B) whereas both compounds inhibited ABCG2-mediated efflux of mitoxantrone in MCF-7 FLV1000 cells (Fig. 2C and D), respectively, suggesting that both vitamin K3 and plumbagin block the mitoxantrone efflux from ABCG2-expressing cells. This inhibition was concentration dependent, with A IC50 values of 20.25 and 7.88 mol/L, respectively, as shown in Fig. 2E. VitaminK3andPlumbaginInhibitPhotoCross-Linking of [12 5I]IAAP to ABCG2 To further confirm the specificity of interaction of both vitamin K3 and plumbagin with ABCG2, their effect on photolabeling of [125I]IAAP with ABCB1 and ABCG2 was also studied. [125I]IAAP is known to photolabel both ABCB1 and ABCG2, and this labeling is inhibited by substrates/inhibitors of these transporters (27, 29). The crude membranes from ABCB1-expressing High-five cells (Fig. 3A and C) or ABCG2-expressing MCF-7 FLV1000 cells (Fig. 3B and D) were incubated with varying concentrations (0–75 Amol/L) of plumbagin (Fig. 3A and B) and vitamin K3 (Fig. 3C and D) for 10 min at 21jCto23jC. [125I]IAAP at 3 to 6 nmol/L was added and the membranes were incubated for an additional 5 min under subdued light. The samples were then illuminated with a UV lamp (365 nm) for 10 min and processed as described in Materials and Figure 3. Effect of vitamin K3 and plumbagin on photoaffinity labeling Methods. It was observed that both plumbagin and vitamin 125 125 of ABCG2 and ABCB1 with [ I]IAAP. Crude membranes (500 Ag/mL) K3 inhibited the incorporation of [ I]IAAP into ABCG2 from High-five cells expressing ABCB1 (A and C) or MCF-7 FLV1000 cells (Fig. 3B and D), whereas they did not show any significant (B, D, and E) were incubated with 0 to 75 Amol/L of plumbagin (A and B), effect on the photolabeling of ABCB1 (Fig. 3A and C) up to vitamin K3 (C and D), or mitoxantrone (E) for 5 min at 21jCat23jCin A 125 75 Amol/L. This inhibition of [125I]IAAP binding to ABCG2 50 mol/L Tris-HCl (pH 7.5). [ I]IAAP (2,200 Ci/mmol) at 3 to 6 nmol/L was added and incubated for an additional 5 min under subdued light. The by plumbagin and vitamin K3 was concentration depen- samples were then illuminated with a UV lamp (365 nm) for 10 min and A dent, with IC50 values of 7.3 and 22.6 mol/L, respectively were processed as described in Materials and Methods. Representative (Fig.3F).Itshouldbenotedthatmitoxantronealso autoradiograms from one experiment are shown and similar results were 125 obtained in two additional experiments. Arrows, position of the ABCB1 inhibited the photolabeling of ABCG2 with [ I]IAAP with and ABCG2 bands. F, the incorporation of [125I]IAAP (from autoradiogram; A an IC50 value of 15.7 mol/L (Fig. 3E and F). The IC50 value Y axis) into the ABCB1 (5, o) and ABCG2 (n, ., E) bands was quantified of the inhibition of [125I]IAAP binding by mitoxantrone by estimating the radioactivity of this band using the STORM 860 (Fig. 3F) was comparable to the IC of plumbagin or PhosphorImager system (Molecular Dynamics) and the ImageQuaNT 50 software and plotted as a concentration of plumbagin (5, n), vitamin K3 vitamin K3 for mitoxantrone accumulation in ABCG2- (o, .), and mitoxantrone (E) using the software GraphPad Prism 2.0, as expressing cells (Fig. 2E), suggesting the interaction of these previously described (27).

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varying concentrations of plumbagin and vitamin K3 (0– 20 Amol/L) for 10 min at room temperature and the ATPase activity at 37jC was determined as described in Materials and Methods. As shown in Fig. 4A, both vitamin K3 and plumbagin stimulated the ATPase activity of ABCG2 to 1.5- and 2-fold, respectively, whereas both these compounds did not show any significant effect on the ATP hydrolysis by ABCB1 (data not shown). We further monitored the effect of these compounds on the mitoxantrone-stimulated ATPase activity of ABCG2. Crude membranes expressing ABCG2 were incubated with 10 Amol/L mitoxantrone and 0 to 50 Amol/L of vitamin K3 or mitoxantrone and the ATPase activity was determined as described above. It was observed that both plumbagin and vitamin K3 inhibited the mitoxantrone-stimulated ATPase activity (Fig. 4B) of ABCG2 in a concentration- dependent manner with IC50 values of 15.3 and 42.5 Amol/L, respectively. The fact that both compounds stimulated ATP hydrolysis and inhibited the mitoxantrone-stimulated ATP activity of ABCG2 suggested that they could be transport substrates of this transporter and that they might interact at the same site as mitoxantrone. Cytotoxicity of Plumbagin and Vitamin K3 in ABCB1- and ABCG2-Expressing Cells Figure 4. A, effect of vitamin K3 and plumbagin on beryllium fluoride – As shown in Fig. 2, both plumbagin and vitamin K3 sensitive ATPase activity of ABCG2. Crude membrane protein (100 Ag inhibited the ABCG2-mediated efflux of mitoxantrone but protein/mL) from High-five cells expressing ABCG2-G482 was incubated did not have any effect on the efflux mediated by ABCB1. at 37jC with varying concentrations of plumbagin (n) or vitamin K3 (E)in the presence and absence of beryllium fluoride (BeFx; 0.2 mmol/L The biochemical data presented in Figs. 3 and 4 suggested beryllium sulfate and 2.5 mmol/L sodium fluoride) in ATPase assay buffer that both of these naphthoquinones could also be substrates for 10 min. The reaction was started by the addition of 5 mmol/L ATP at for ABCG2. Therefore, the cytotoxicity of plumbagin and 37jC and was stopped by the addition of 0.1 mL of 5% SDS solution. The amount of inorganic phosphate released and the beryllium fluoride – vitamin K3 in the control (pcDNA3.1-HEK 293 and KB-3-1), sensitive ATPase activity were determined as described in Materials and ABCG2-expressing (R482-HEK 293), and ABCB1-expressing Methods. Points, average from three experiments; bars, SE. B, effect of KB-V1 cells was determined. As shown in Table 1, The plumbagin and vitamin K3 on mitoxantrone-stimulated ATPase activity of ABCG2-expressing R482-HEK cells were 2.8- and 2.3-fold ABCG2. The ATP hydrolysis assay was done as described above except that 10 Amol/L mitoxantrone was added with varying concentrations resistant to plumbagin and vitamin K3, respectively, (0 – 50 Amol/L) of plumbagin (n) and vitamin K3 (E). Points, average from compared with the control pcDNA3.1-HEK 293 cells, three experiments; bars, SE. whereas ABCB1-expressing KB-V1 cells did not show any significant resistance compared with the control KB-3-1 was monitored on the beryllium fluoride–sensitive ATPase cells. This indicated that whereas ABCB1 had a minimal role activity in crude membranes isolated from High-five cells in effluxing these compounds, ABCG2 confers resistance to expressing ABCB1 or ABCG2. The crude membranes both these agents. These results were consistent with the expressing ABCB1 and ABCG2 were incubated with data shown in Figs. 2, and 3 which suggested that these

Table 1. Cytotoxicity of plumbagin and vitamin K3 to ABCB1- and ABCG2-expressing cells

A c Cell line Cytotoxicity IC50 ( mol/L)* Relative resistance

Plumbagin Vitamin K3 Plumbagin Vitamin K3 pcDNA3.1-HEK 1.05 F 0.39 3.70 F 0.84 1 1 R482-HEK 2.96 F 0.87 8.54 F 1.13 2.8b 2.3b KB-3-1 13.02 F 6.43 22.72 F 9.42 1 1 KB-V1 13.51 F 6.62 21.94 F 5.01 1.03 0.96

*The values represent the mean F SD of three independent experiments done in triplicate. c Relative resistance values were obtained by dividing the IC50 value of the resistant R482-HEK cells and KB-V1 cells by the IC50 value of the sensitive control pcDNA3.1-HEK or KB-3-1 cells, respectively. bValues were significantly different from the control cells (P < 0.05).

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compounds did not interact with ABCB1 and further K3 and plumbagin inhibited the photolabeling of ABCG2 indicated that both plumbagin and vitamin K3 are substrates with IAAP but did not show any significant effect on the of ABCG2. binding of the same with ABCB1 (Fig. 3), indicating that Sensitization of ABCG2-Expressing Cells to Mitoxan- both plumbagin and vitamin K3 did not interact at the trone by Plumbagin and Vitamin K3 IAAP binding site of ABCB1. The stimulation of ATP A nontoxic concentration (0.5 Amol/L plumbagin or hydrolysis by ABCG2 and the inhibition of mitoxantrone- 1 Amol/L vitamin K3) was evaluated to augment the stimulated ATPase activity of this transporter by both cytotoxic effect of mitoxantrone for ABCG2-expressing vitamin K3 and plumbagin (Fig. 4) provided additional drug-resistant cells using cytotoxicity assays. The relative evidence of these compounds being substrates of ABCG2. resistance was calculated based on IC50 values in the absence This is the first report, to our knowledge, showing a and presence of plumbagin. Although both plumbagin and specific interaction of vitamin K3 with any multidrug vitamin K3 were able to partially increase the sensitivity resistance–linked ABC transporter. A recent study of the drug-resistant ABCG2-expressing R482-HEK cells showed the role of ABCG2 in the transport of vitamin (data not shown), they did not completely inhibit the drug B2 (riboflavin) into the breast milk using abcg2-knockout resistance mediated by ABCG2 in 3-day assays. Further mice (36). The results of this study suggest that ABCG2 higher concentrations for reversal of cytotoxicity assays could also be involved in the secretion of vitamin K3 in could not be used because they were toxic to the control cells. the breast milk. Although van Herwaarden et al. (36) A possible reason for not observing a complete reversal of found no difference in the levels of vitamin K1 in the the cytotoxicity of mitoxantrone could be that, unlike milk from abcg2-knockout and wild-type mice, it has also fumitremorgin C, these compounds are not true inhibitors been reported that vitamin K levels in breast milk but instead are ABCG2 substrates. Therefore, they may not increase markedly in response to the mother taking be able to maintain intracellular concentrations sufficient vitamin K supplements, which suggests the possible role enough to inhibit the transporter in a long-term 3-day assay. of transporters in transporting these vitamins (37, 38). In In addition, conversion of the plumbagin or vitamin K3 to an addition, the interaction of vitamin K3 with ABCG2 may inactive metabolite in a 3-day assay could also be another have important clinical implications in the therapeutics reason for the observed incomplete reversal of resistance in related to this important molecule involved in blood ABCG2-expressing cells. clotting. A variety of inhibitors of ABC drug transporters have been developed to date with the objective of reversing Discussion resistance against anticancer drugs and chemosensitizing ABCG2 was initially identified in drug-selected breast the resistant cells to anticancer drugs. The fact that both cancer cells and subsequently reported to be expressed at vitamin K3 and plumbagin can inhibit the ABCG2- high levels in the human placenta and to a lesser extent in mediated function suggests that these types of compounds the liver, small intestine and colon, ovary, brain, and can be developed as inhibitors of this transporter to capillary endothelia, kidney, adrenal, and lung, with little overcome ABCG2-mediated multidrug resistance. In fact, to no expression in the heart, stomach, prostate, spleen, and Parekh et al. (39) reported the use of vitamin K3 in cervix (30–34). ABCG2 protein expression has been further overcoming drug resistance by showing that treating the shown to be localized to the apical epithelium of the colon drug-sensitive and multidrug-resistant P388 leukemia and small intestine, to the liver canalicular membrane, and cells simultaneously with doxorubicin and vitamin K3 to the surface of cells in breast ducts and lobules (34, 35). made the drug-resistant cells more sensitive to doxorubicin This suggests that the physiologic role of ABCG2 may be to and also showed that treatment with 1 Amol/L vitamin K3 protect cells from potentially toxic substances and to resulted in an increase in intracellular doxorubicin accu- prevent absorption of xenobiotics ingested in our diet by mulation in drug-resistant cells. Other studies also showed actively transporting compounds from cells. Identification an increase in cytotoxicity when vitamin K3 was combined of the natural physiologic substrates of ABCG2 will provide with different chemotherapeutic agents (40, 41) in cervical additional information about the role of this transporter in and nasopharyngeal carcinoma cells. Earlier, in a phase I the human body. clinical study, Margolin et al. (42) also showed that a The data presented in this study identified vitamin K3 combination of mitomycin C and vitamin K3 led to a 10- to and plumbagin, which is a naturally occurring structural 50-fold reduction in the levels of mitomycin C required for analogue of this vitamin, as substrates for ABCG2. The cells cytotoxicity in solid tumors. The inhibitory potential of expressing ABCG2 showed resistance to vitamin K3 and vitamin K3 in combination with doxorubicin or mitomycin plumbagin whereas ABCB1-expressing cells were sensitive on the cytotoxicity of MCF-7 breast cancer cells was also to these compounds compared with the control cells shown (43), but these studies did not explain the molecular (Table 1), indicating that these two compounds were basis of the synergy of cell killing with vitamin K3 and the substrates of ABCG2. We showed a direct and specific anticancer drugs. The findings presented here may provide interaction of vitamin K3 and plumbagin at the substrate the explanation for the above observations as we show binding site of ABCG2 using the photoaffinity labeling that inhibition of ABCG2 transport function by vitamin K3 assay with a substrate analogue, [125I]IAAP. Both vitamin and plumbagin results in higher accumulation of target

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drug inside the cells (Fig. 1), which may lead to enhanced accumulation in normal liver and drug-resistant tumors after the adminis- tration of the glycoprotein inhibitor, XR9576. Clin Cancer Res 2003;9: cytotoxicity of these drugs in the drug-resistant cells, 650 – 6. although to our knowledge, it is not known if mitomycin 5. Germann UA, Shlyakhter D, Mason VS, et al. Cellular and biochemical C or its metabolite(s) is a substrate of ABCG2. characterization of VX-710 as a chemosensitizer: reversal of P-glycopro- The data presented here suggest that plumbagin and tein-mediated multidrug resistance in vitro. Anticancer Drugs 1997;8: 125 – 40. vitamin K3 interact with ABCG2 but do not show any 6. Hyafil F, Vergely C, Du Vignaud P, Grand-Perret T. In vitro and in vivo interaction with ABCB1 at the concentrations tested. These reversal of multidrug resistance by GF120918, an acridonecarboxamide data are not in concordance with the report by Dolan et al. derivative. Cancer Res 1993;53:4595 – 602. (44), who earlier showed that the ABCB1-expressing KB-V1 7. Shearer MJ. Vitamin K. Lancet 1995;345:229 – 34. cells and KB-3-1 parental cells were equally sensitive to 8. Kassouf W, Highshaw R, Nelkin GM, Dinney CP, Kamat AM. Vitamins tricyclic 1,2-naphthoquinones. In the same study (44), C and K3 sensitize human urothelial tumors to gemcitabine. J Urol 2006; 176:1642 – 7. MCF-7 ADRcells, which were selected for resistance to 9. Verrax J, Stockis J, Tison A, Taper HS, Calderon PB. Oxidative stress Adriamycin, displayed cross-resistance to these naphtho- by ascorbate/menadione association kills K562 human chronic myeloge- quinones. Recent results1 from our group suggest that nous leukaemia cells and inhibits its tumour growth in nude mice. Biochem MCF-7 cells selected with low concentrations of doxoru- Pharmacol 2006;72:671 – 80. bicin overexpress ABCG2. This may explain the anomaly 10. Verrax J, Cadrobbi J, Marques C, et al. Ascorbate potentiates the cytotoxicity of menadione leading to an oxidative stress that kills cancer in the data presented by Dolan et al. because their MCF-7 cells by a non-apoptotic caspase-3 independent form of cell death. ADRcells may have expressed ABCG2, which was not Apoptosis 2004;9:223 – 33. known at that time, and were therefore resistant to these 11. Hitomi M, Yokoyama F, Kita Y, et al. Antitumor effects of vitamins naphthoquinones, whereas the KB-V1 cells, which do not K1, K2 and K3 on hepatocellular carcinoma in vitro and in vivo.IntJ Oncol 2005;26:713 – 20. express ABCG2, did not show any resistance to these 12. Binder RG, Benson ME, Flath RA. Eight 1,4-naphthoquinones from compounds. Juglans. Phytochemistry 1989;28:2799 – 801. Taken together, the experimental data from these studies 13. Kuo PL, Hsu YL, Cho CY. Plumbagin induces G2-M arrest and suggest that plumbagin could potentially be used as a autophagy by inhibiting the AKT/mammalian target of rapamycin pathway competing inhibitor in the drug resistance specifically in breast cancer cells. Mol Cancer Ther 2006;5:3209 – 21. mediated by ABCG2. Additionally, this compound has a 14. Hsu YL, Cho CY, Kuo PL, Huang YT, Lin CC. Plumbagin (5-hydroxy-2- methyl-1,4-naphthoquinone) induces apoptosis and cell cycle arrest in relatively simple chemical structure; therefore, it can be A549 cells through p53 accumulation via c-Jun NH2-terminal kinase- used for structure-activity relationship studies to determine mediated phosphorylation at serine 15 in vitro and in vivo. J Pharmacol the minimal pharmacophore required for ABCG2 inhi- Exp Ther 2006;318:484 – 94. bitory activity, and this could probably serve as a lead 15. Sandur SK, Ichikawa H, Sethi G, Ahn KS, Aggarwal BB. Plumbagin (5- hydroxy-2-methyl-1,4-naphthoquinone) suppresses NF-nB activation and compound for the synthesis of more potent and less NF-nB-regulated gene products through modulation of p65 and InBa kinase cytotoxic ABCG2 inhibitors. activation, leading to potentiation of apoptosis induced by cytokine and We were also able to show that vitamin K3 is a chemotherapeutic agents. J Biol Chem 2006;281:17023 – 33. 16. Naresh RA, Udupa N, Devi PU. Niosomal plumbagin with reduced substrate of ABCG2. The specific interaction of vitamin toxicity and improved anticancer activity in BALB/C mice. J Pharm K3 with this transporter may have physiologic impor- Pharmacol 1996;48:1128 – 32. tance in its distribution and bioavailability in the human 17. Singh UV, Udupa N. Reduced toxicity and enhanced antitumor body. efficacy of betacyclodextrin plumbagin inclusion complex in mice bearing Ehrlich ascites carcinoma. Indian J Physiol Pharmacol 1997; 41:171 – 5. Acknowledgments 18. Devi PU, Rao BS, Solomon FE. Effect of plumbagin on the radiation We thank Dr. Susan E. Bates for providing the cell lines, Dr. Michael M. induced cytogenetic and cell cycle changes in mouse Ehrlich ascites Gottesman for encouragement, Drs. Zuben Sauna and Anna Maria carcinoma in vivo. Indian J Exp Biol 1998;36:891 – 5. Calcagno for comments on the manuscript, and George Leiman for 19. Ganasoundari A, Zare SM, Devi PU. Modification of bone marrow assistance in the preparation of the manuscript. radiosensensitivity by medicinal plant extracts. Br J Radiol 1997;70: 599 – 602. References 20. Robey RW, Honjo Y, Morisaki K, et al. Mutations at amino-acid 482 in the ABCG2 gene affect substrate and antagonist specificity. Br J Cancer 1. Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role 2003;89:1971 – 8. of ATP-dependent transporters. Nat Rev Cancer 2002;2:48 – 58. 21. Shen DW, Fojo A, Chin JE, et al. Human multidrug-resistant cell lines: 2. Stewart A, Steiner J, Mellows G, Laguda B, Norris D, Bevan P. Phase I increased mdr1 expression can precede gene amplification. Science 1986; trial of XR9576 in healthy volunteers demonstrates modulation of P- 232:643 – 5. glycoprotein in CD56+ lymphocytes after oral and intravenous adminis- 22. Robey RW, Medina-Perez WY, Nishiyama K, et al. Overexpression of tration. Clin Cancer Res 2000;6:4186 – 91. the ATP-binding cassette half-transporter, ABCG2 (Mxr/BCrp/ABCP1), in 3. Naito M, Matsuba Y, Sato S, Hirata H, Tsuruo T. MS-209, a quinoline- flavopiridol-resistant human breast cancer cells. Clin Cancer Res 2001;7: type reversal agent, potentiates antitumor efficacy of docetaxel in 145 – 52. multidrug-resistant solid tumor xenograft models. Clin Cancer Res 2002; 23. Robey RW, Honjo Y, van de Laar A, et al. A functional assay for 8:582 – 8. detection of the mitoxantrone resistance protein, MXR (ABCG2). Biochim 4. Agrawal M, Abraham J, Balis FM, et al. Increased 99mTc-sestamibi Biophys Acta 2001;1512:171 – 82. 24. Chearwae W, Anuchapreeda S, Nandigama K, Ambudkar SV, Limtrakul P. Biochemical mechanism of modulation of human P-glycoprotein (ABCB1) by curcumin I, II, and III purified from turmeric powder. Biochem Pharmacol 2004;68:2043 – 52. 1 Calcagno AM, Fostel JM, To KKW, et al., 2007, unpublished data. 25. Ambudkar SV. Drug-stimulatable ATPase activity in crude membranes

Mol Cancer Ther 2007;6(12). December 2007

3286 Vitamin K3 Is a Substrate of ABCG2

of human MDR1-transfected mammalian cells. Methods Enzymol 1998; 35. Jonker JW, Merino G, Musters S, et al. The breast cancer resistance 292:504 – 14. protein BCRP (ABCG2) concentrates drugs and carcinogenic xenotoxins 26. Schaffner W, Weissmann C. A rapid, sensitive, and specific method into milk. Nat Med 2005;11:127 – 9. for the determination of protein in dilute solution. Anal Biochem 1973;56: 36. van Herwaarden AE, Wagenaar E, Merino G, et al. Multidrug 502 – 14. transporter ABCG2/breast cancer resistance protein secretes riboflavin 27. Shukla S, Robey RW, Bates SE, Ambudkar SV. The calcium channel (vitamin B2) into milk. Mol Cell Biol 2007;27:1247 – 53. blockers, 1,4-dihydropyridines, are substrates of the multidrug resistance- 37. Greer FR, Marshall SP, Foley AL, Suttie JW. Improving the vitamin K linked ABC drug transporter, ABCG2. Biochemistry 2006;45:8940 – 51. status of breastfeeding infants with maternal vitamin K supplements. 28. Sauna ZE, Peng XH, Nandigama K, Tekle S, Ambudkar SV. The Pediatrics 1997;99:88 – 92. molecular basis of the action of disulfiram as a modulator of the multidrug 38. Nishiguchi T, Saga K, Sumimoto K, Okada K, Terao T. Vitamin K resistance-linked ATP binding cassette transporters MDR1 (ABCB1) and prophylaxis to prevent neonatal vitamin K deficient intracranial haemor- MRP1 (ABCC1). Mol Pharmacol 2004;65:675 – 84. rhage in Shizuoka prefecture. Br J Obstet Gynaecol 1996;103:1078 – 84. 29. Sauna ZE, Ambudkar SV. Evidence for a requirement for ATP 39. Parekh HK, Mansuri-Torshizi H, Srivastava TS, Chitnis MP. Circum- hydrolysis at two distinct steps during a single turnover of the catalytic vention of Adriamycin resistance: effect of 2-methyl-1,4-naphthoquinone cycle of human P-glycoprotein. Proc Natl Acad Sci U S A 2000;97: (vitamin K3) on drug cytotoxicity in sensitive and MDR P388 leukemia 2515 – 20. cells. Cancer Lett 1992;61:147 – 56. 30. Sarkadi B, Homolya L, Szakacs G, Varadi A. Human multidrug 40. Nutter LM, Cheng AL, Hung HL, Hsieh RK, Ngo EO, Liu TW. resistance ABCB and ABCG transporters: participation in a chemo- Menadione: spectrum of anticancer activity and effects on nucleotide immunity defense system. Physiol Rev 2006;86:1179 – 236. metabolism in human neoplastic cell lines. Biochem Pharmacol 1991;41: 31. Allikmets R, Schriml LM, Hutchinson A, Romano-Spica V, Dean M. A 1283 – 92. human placenta-specific ATP-binding cassette gene (ABCP) on chromo- 41. Liao WC, Wu FY, Wu CW. Binary/ternary combined effects of vitamin some 4q22 that is involved in multidrug resistance. Cancer Res 1998;58: K3 with other antitumor agents in nasopharyngeal carcinoma CG1 cells. 5337 – 9. Int J Oncol 2000;17:323 – 8. 32. Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance 42. Margolin KA, Akman SA, Leong LA, et al. Phase I study of mitomycin transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci C and menadione in advanced solid tumors. Cancer Chemother Pharmacol U S A 1998;95:15665 – 70. 1995;36:293 – 8. 33. Ross DD, Yang W, Abruzzo LV, et al. Atypical multidrug resistance: 43. Tetef M, Margolin K, Ahn C, et al. Mitomycin C and menadione for the breast cancer resistance protein messenger RNA expression in mitoxan- treatment of lung cancer: a phase II trial. Invest New Drugs 1995;13: trone-selected cell lines. J Natl Cancer Inst 1999;91:429 – 33. 157 – 62. 34. Maliepaard M, Scheffer GL, Faneyte IF, et al. Subcellular localization 44. DolanME,FrydmanB,ThompsonCB,etal.Effectsof1,2- and distribution of the breast cancer resistance protein transporter in naphthoquinones on human tumor cell growth and lack of cross-resistance normal human tissues. Cancer Res 2001;61:3458 – 64. with other anticancer agents. Anticancer Drugs 1998;9:437 – 48.

Mol Cancer Ther 2007;6(12). December 2007

The naphthoquinones, vitamin K3 and its structural analogue plumbagin, are substrates of the multidrug resistance − linked ATP binding cassette drug transporter ABCG2

Suneet Shukla, Chung-Pu Wu, Krishnamachary Nandigama, et al.

Mol Cancer Ther 2007;6:3279-3286. Published OnlineFirst December 7, 2007.

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