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. Author manuscript; available in PMC: 2008 Dec 1.
Published in final edited form as: Mol Cancer Ther. 2007 Dec 7;6(12 Pt 1):3279–3286. doi: 10.1158/1535-7163.MCT-07-0564

The naphthoquinones, vitamin K3 and its structural analog plumbagin, are substrates of the multidrug resistance-linked ABC drug transporter ABCG2

Suneet Shukla 1, Chung-Pu Wu 1, Krishnamachary Nandigama 1, Suresh V Ambudkar 1
PMCID: PMC2398729  NIHMSID: NIHMS41812  PMID: 18065489

Abstract

Vitamin K3 (Menadione; 2-methyl-1,4-naphthoquinone) is a structural precursor of vitamins K1 and K2 which are essential for blood clotting. The naturally occurring structural analog of this vitamin, plumbagin (5-hydroxy-menadione), is known to modulate cellular proliferation, apoptosis, carcinogenesis, and radioresistance. We, here, report that both vitamin K3 and plumbagin are substrates of the multidrug resistance-linked ATP binding cassette (ABC) drug transporter, ABCG2. Vitamin K3 and plumbagin specifically inhibited the ABCG2-mediated efflux of mitoxantrone, but did not have any effect on the ABCB1-mediated efflux of rhodamine 123. This inhibition of ABCG2 function was due to their interaction at the substrate-binding site(s). They inhibited the binding of [125I]-Iodoarylazidoprazosin (IAAP), a substrate of ABCG2, to this transporter in a concentration-dependent manner with IC50 values of 7.3 and 22.6 μM, respectively, but had no effect on the binding of this photoaffinity analog to ABCB1. Both compounds stimulated ABCG2-mediated ATP hydrolysis and also inhibited the mitoxantrone-stimulated ATPase activity of this transporter, but did not have any significant effect on the ATPase activity of ABCB1. In a cytotoxicity assay, ABCG2-expressing HEK cells were 2.8- and 2.3-fold resistant to plumbagin and vitamin K3, respectively, compared to the control cells, suggesting that they are substrates of this transporter. Collectively, these data demonstrate for the first time that vitamin K3 is a substrate of the ABCG2 transporter. Thus, ABCG2 may have a role in the regulation of vitamin K3 levels in the body. In addition, vitamin K3 and its structural derivative, plumbagin, could potentially be used to modulate ABCG2 function.

Keywords: ABC transporter, ABCG2, multidrug resistance, P-glycoprotein, ATP hydrolysis, chemosensitization, drug transport, photoaffinity labeling

Introduction

Multidrug resistance (MDR) of cancer cells is an obstacle to effective chemotherapy of cancer and the ATP-Binding Cassette (ABC) transporters, including P-glycoprotein (P-gp, ABCB1), MRP1 (ABCC1) and ABCG2, play a major role in the development of this phenotype (1). These ABC drug efflux transporters not only play a major role in the development of MDR but also affect the disposition of xenobiotics or drug which are commonly used in chemotherapy (1). This leads to either drug resistance or reduced concentrations of the anti-cancer agents inside the cells. An attractive approach to overcoming MDR is by inhibition of the pump action, which would re-instate the drug accumulation inside the resistant cell to levels similar to those of a drug-sensitive tumor cell. Several inhibitors/modulators of these ABC transporters have been developed but their cytotoxic effect and adverse pharmacokinetics have precluded their use (2–6). The ongoing search for such inhibitors/modulators that can be applied in the clinic is into its third generation. These most recent inhibitors are more potent and less toxic than the first-generation compounds, yet some are still prone to adverse effects, poor solubility, and unfavorable changes in pharmacokinetics of the other anticancer drugs. Therefore, the identification of more specific, more potent, and less toxic inhibitors/modulators for clinical use remains critical to the possible success of this approach. A number of pharmacological 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 ABC transporters.

Here, we identify a set of compounds which have a napthoquinone moiety in their chemical structure that inhibit the function of the multidrug-linked ABC drug transporter, ABCG2. Vitamin K3 (2-methyl-1,4-naphthoquinone), also known as menadione, is one such naturally occurring napthoquinone in the body which is necessary for the production of prothrombin and five other blood clotting factors in humans (7). In the intestine, it also assists in converting glucose to glycogen, which is then stored in the liver. Vitamins K1 and K2 are the naturally occurring types of vitamin K and menadione acts as a provitamin which gets converted in vitamins K1 and K2 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. Vitamin K deficiency in infants can lead to hemorrhagic disease of the newborn, also known as vitamin K deficiency bleeding (VKDB). Vitamin K3, in conjunction with ascorbate, has also been reported to have a cytotoxic effect on K562 human chronic myelogenous leukemia cells by inducing oxidative stress, thereby causing cell death (8–10). In addition, vitamins K2 and K3 were shown to have potent antitumor effects on hepatocellular carcinoma and tumor growth in nude mice (11).

Vitamin K3 shares structural similarity with another naturally occurring napthoquinone, plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) (Figure 1), which is present along with a series of other structurally related naphthoquinones in the roots, leaves, bark, and wood of Juglans regia (English walnut, Persian walnut, and California walnut), Juglans cinerea (butternut and white walnut), and Juglans nigra (black walnut) (12). Plumbagin has recently been shown to have anticancer properties by inhibiting cell proliferation and inducing cells to undergo G2-M arrest and autophagic cell death (13, 14). Sandur et al. (15) also demonstrated that plumbagin can enhance apoptosis induced by cytokine and chemotherapeutic agents by inhibiting NF-kappaB activation. In addition, plumbagin has been shown to exert anticancer and antiproliferative activities in in vitro cultured cells as well as in an in vivo animal model (16, 17) and is also reported to possess chemopreventive properties. Besides anticancer effects, plumbagin has also exhibited radiosensitizing properties in experimental mouse tumors as well as in tumor cells in vitro (18, 19).

Figure 1. Chemical structures and names of vitamin K3 and plumbagin.

Figure 1

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Plumbagin, which is a naturally occurring analog of vitamin K3, has an additional hydroxyl group at position 5.

In this study, we further investigated the interaction of the two naphthoquinones, vitamin K3 and plumbagin, with the two major ABC drug transporters, ABCB1 and ABCG2. The data presented here addresses the biochemical basis of the interaction of these naphthoquinones with the ABC drug transporters and the results suggest that both vitamin K3 and plumbagin are substrates of ABGC2 and specifically inhibit the function of this transporter. Thus, ABCG2 may have a role in the regulation of vitamin K3 levels in the body.

Materials and Methods

Chemicals

DMEM (Dulbecco’s modified eagle’s medium), RPMI media, fetal bovine serum (FBS), penicillin, streptomycin, trypsin-EDTA and phosphate buffered saline (PBS) were purchased from GIBCO-BRL (Grand Island, NY). Plumbagin, vitamin K3, mitoxantrone, MTT dye, rhodamine 123 and ouabain were purchased from Sigma (St. Louis, MO). Radiolabeled [125I]-IAAP (2200 Ci/mmole) was from Perkin Elmer Life Sciences (Wellesley, MA).

Cell lines and culture conditions

HEK 293 cells stably transfected with either control pcDNA3.1 vector (pcDNA3.1-HEK 293) or pcDNA3.1 containing ABCG2 were maintained in DMEM supplemented with 10% FBS, penicillin, streptomycin and 2 mg/ml of G418 (20). KB-3-1 and ABCB1 overexpressing KB-V1 cells were maintained in DMEM supplemented with 10% FBS, penicillin, streptomycin and KB--V1 cells were grown in media containing 1 μg/ml vinblastine (21). MCF-7 FLV1000 cells overexpressing wild-type ABCG2 were cultured in RPMI with 10% FBS with 1 μg/ml flavopiridol (22, 23). The ABCG2-expressing cell lines were provided by Dr. Susan Bates (NCI/NIH). MCF-7 ADR cells were provided by Dr. Kapil Mehta (M. D. Anderson Cancer Center, Houston)

Isolation of crude membranes

Crude membranes from MCF-7, MCF-7 FLV1000 or High-five insect cells expressing ABCB1 or ABCG2 were prepared as described elsewhere (24, 25). The protein content was estimated using the amido black B-dye binding assay, as described earlier (26).

Fluorescent drug accumulation assay by Flow Cytometry

Accumulation assays with mitoxantrone (5 μM for ABCG2-expressing MCF-7 FLV1000 cells) or rhodamine 123 (0.5 μg/ml for ABCB1-expressing MCF-7 ADR cells) were performed as described previously (23). For all samples, 10,000 events were counted and the analysis was performed with Cell Quest software (Becton-Dickinson Immunocytometry systems). The mean fluorescence intensity was calculated using the histogram stat program in Cell Quest software.

Cytotoxicity assay

Cytotoxicity of plumbagin and vitamin K3 was determined by CCK8 assay, as described previously (24). The concentration of plumbagin and vitamin K3 which produced 50% inhibition of growth (IC50) was calculated from linear regression analysis of the linear portion of the growth curves (24). For the reversal of cytotoxicity assays, a constant non-toxic concentration of either plumbagin (0.5 μM) or vitamin K3 (1 μM) was added with varying concentrations of mitoxantrone and the extent of reversal was calculated based on the changes in the relative resistance values which were derived by dividing the IC50 value of the resistant cells by the IC50 value of the sensitive control cells. The measurements were carried out in triplicates.

ATPase assay

Crude membrane protein (100 μg protein/ml) from either High-five cells expressing ABCB1 or ABCG2 was incubated at 37°C with varying concentrations of plumbagin and vitamin K3 in the presence and absence of BeFx (0.2 mM beryllium sulfate and 2.5 mM sodium fluoride) in ATPase assay buffer (50 mM KCl, 5 mM NaN3, 2 mM EGTA, 10 mM MgCl2, 1 mM DTT pH 6.8) for 10 min. The reaction was started by the addition of 5 mM ATP and incubated for 20 min at 37°C. SDS solution (0.1 ml of 5% SDS) was added to terminate the reaction and the amount of inorganic phosphate released was quantified with a colorimetric reaction, as described previously (25). The specific activity was recorded as BeFx-sensitive ATPase activity.

Photoaffinity labeling of ABCB1 and ABCG2 with [125I]-IAAP

Crude membranes (1 mg protein/ml) from either ABCB1-expressing High-five cells or ABCG2-expressing MCF-7 FLV1000 cells were incubated with 0–100 μM plumbagin or vitamin K3 for 10 min at 21–23°C in 50 mM Tris-HCl, pH 7.5. 3–6 nM [125I]-IAAP (2200 Ci/mmole) was added and incubated for an additional 5 min under subdued light. The samples were illuminated with a UV lamp (365 nm) for 10 min at room temperature. The labeled ABCG2 was immunoprecipitated as described previously (27). Samples were separated on a 7% Tris-acetate gel at constant voltage and gels were dried and exposed to X-ray film for 12–24 h at −80°C. The incorporation of [125I]-IAAP into the ABCG2 or ABCB1 band was quantified using the STORM 860 phosphor imager system (Molecular Dynamics, Sunnyvale, CA, USA) and the software ImageQuaNT, as described (28).

Results

Vitamin K3 and plumbagin inhibit ABCG2-mediated efflux of substrates

Both vitamin K3 and plumbagin were evaluated for their inhibitory activity against two major ABC drug transporters, ABCB1 and ABCG2. Accumulation assays with different fluorescent substrates (mitoxantrone for ABCG2, rhodamine 123 for ABCB1) in ABCB1-and ABCG2-expressing cells were performed as described in the ‘Materials and Methods’ section. The control MCF-7 (Figure 2a, c), ABCB1-expressing MCF-7 ADR (Figure 2b) or ABCG2-expressing MCF-7 FLV1000 (Figure 2d) cells were incubated with 0.5 μg/ml of rhodamine 123 (Figure 2a, b) or 5 μM of mitoxantrone (Figure 2c, d) in the presence or absence of 20 μM plumbagin, vitamin K3 or their respective specific inhibitors (5 μM cyclosporine A (Cys A) for ABCB1 and 10 μM Fumitremorgin C (FTC) for ABCG2) and incubated at 37°C 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 ADR cells (Figure 2a, b) while both compounds inhibited ABCG2-mediated efflux of mitoxantrone in MCF-7 FLV1000 cells (Figure 2c, d), respectively, suggesting that both vitamin K3 and plumbagin block the mitoxantrone efflux from ABCG2-expressing cells. This inhibition was concentration-dependent, with IC50 values of 20.25 and 7.88 μM, respectively as shown in Figure 2e.

Figure 2. Effect of vitamin K3 and plumbagin on the accumulation of fluorescent drug substrates in ABCG2- and ABCB1-expressing cells.

Figure 2

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MCF-7 (a, c), MCF-7 ADR (b), MCF-7 FLV1000 (d) cells (300,000/tube) were incubated with 0.5 μg/ml of rhodamine 123 (a, b) or 5 μM mitoxantrone (c, d) for 45 min at 37°C in the dark, in the absence (control, thin line) or presence of 10 μM cyclosporine A (Cys A, bold line) (a, b), FTC (bold line) (c, d), 30 μM vitamin K3 (dotted line) or 30 μM plumbagin (dashed line) as indicated. The cells were then washed and analyzed by flow cytometer as described in ‘Materials and Methods’. The histogram derived from the Cell Quest software represents the fluorescence intensity of either untreated or 20 μM FTC, Cys A, vitamin K3 or plumbagin treated cells. The traces in each histogram are marked. Shown here is a representative histogram. Similar results were obtained in three additional experiments. (e) MCF-7FLV1000 cells were incubated with 5 μM of mitoxantrone for 45 min at 37°C in the presence of varying concentrations (0–100 μM) of vitamin K3 (■) or plumbagin (▲). The cells were then pelleted, resuspended in PBS with 0.1% BSA, and analyzed as described above. The difference in the mean fluorescence intensity in the presence and absence of 10 μM FTC 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 μM FTC 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 and the mean values from three independent experiments performed in triplicate are plotted (the error bar denote S.D.). The IC50 values given in each panel represent the concentration which inhibited the efflux to 50% of the control values.

Vitamin K3 and plumbagin inhibit photo cross-linking of [125I]-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 (Figure 3a, c) or ABCG2-expressing MCF-7 FLV1000 cells (Figure 3b, d) were incubated with varying concentrations (0–75 μM) of plumbagin (Figure 3a, b) and vitamin K3 (Figure 3c, d) for 10 min at 21–23°C. 3–6 nM [125I]-IAAP 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 Methods’. It was observed that both plumbagin and vitamin K3 inhibited the incorporation of [125I]-IAAP into ABCG2 (Figure 3b, d), while they did not show any significant effect on the photolabeling of ABCB1 (Figure 3a, c) up to 75 μM. This inhibition of [125I]-IAAP binding to ABCG2 by plumbagin and vitamin K3 was concentration-dependent, with IC50 values of 7.3 and 22.6 μM, respectively (Figure 3f). It should be noted that mitoxantrone also inhibited the photolabeling of ABCG2 with [125I]-IAAP with IC50 value of 15.7 μM (Figure 3 e and f). The IC50 value of the inhibition of [125I]-IAAP binding by mitoxantrone (Figure 3f) was comparable to the IC50 of plumbagin or vitamin K3 for mitoxantrone accumulation in ABCG2 expressing cells (Figure 2e) suggesting the interaction of these compounds at similar binding sites. Taken together, the data from Figure 2 and 3 suggested that plumbagin and vitamin K3 specifically interact with ABCG2 but not with ABCB1.

Figure 3. Effect of vitamin K3 and plumbagin on photoaffinity labeling of ABCG2 and ABCB1 with [125I]-IAAP.

Figure 3

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Crude membranes (500 μg/ml) from High-five cells expressing ABCB1 (a, c) or MCF-7 FLV1000 (b, d, e) cells were incubated with 0–75 μM of (a, b) plumbagin, (c, d) vitamin K3 or (e) mitoxantrone for 5 min at 21–23°C in 50 mM Tris-HCl, pH 7.5. 3–6 nM [125I]-IAAP (2200 Ci/mmole) was added and incubated for an additional 5 min under subdued light. The samples were then illuminated with a UV lamp (365 nm) for 10 min and were processed as described in ‘Materials and Methods’. Representative autoradiograms from one experiment are shown and similar results were obtained in two additional experiments. The arrows show the position of the ABCB1 and ABCG2 band. (e) The incorporation of [125I]-IAAP (from autoradiogram, Y-axis) into the ABCB1 (□, ○) and ABCG2 (■, ●, ▲) band was quantified by estimating the radioactivity of this band using the STORM 860 phosphor imager system (Molecular Dynamics, Sunnyvale, CA) and the ImageQuaNT software and plotted as a concentration of plumbagin (□, ■), vitamin K3 (○, ●) and mitoxantrone (▲) using the software GRAPHPAD PRISM 2.0, as described previously (27).

Effect of plumbagin and vitamin K3 on ATP hydrolysis by ABCB1 and ABCG2

It is known that ABC transporters utilize the energy derived from ATP hydrolysis to transport substrates and that the transport substrates often stimulate basal ATP hydrolysis by these transporters. Therefore, the effect of naphthoquinones was monitored on the BeFx-sensitive ATPase activity in crude membranes isolated from High-five cells expressing ABCB1 or ABCG2. The crude membranes expressing ABCB1 and ABCG2 were incubated with varying concentrations of plumbagin and vitamin K3 (0–20 μM) for 10 min at room temperature and the ATPase activity at 37°C was determined as described in ‘Materials and Methods’. As shown in Figure 4a, both vitamin K3 and plumbagin stimulated the ATPase activity of ABCG2 to 1.5- and 2-fold, respectively, while 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 μM mitoxantrone and 0–50 μM of vitamin K3 or mitoxantrone and the ATPase activity was determined as described above. It was observed that both plumbagin and vitamin K3 inhibited mitoxantrone-stimulated ATPase activity (Figure 4b) of this transporter in a concentration-dependent manner with an IC50 of 15.3 and 42.5 μM, 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.

Figure 4. (a) Effect of vitamin K3 and plumbagin on beryllium fluoride-sensitive ATPase activity of ABCG2.

Figure 4

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Crude membrane protein (100 μg protein/ml) from High-five cells expressing ABCG2-G482 was incubated at 37°C with varying concentrations of plumbagin (■) or vitamin K3 (▲) in the presence and absence of BeFx (0.2 mM beryllium sulfate and 2.5 mM sodium fluoride) in ATPase assay buffer for 10 min. The reaction was started by the addition of 5 mM ATP at 37°C and was stopped by the addition of 0.1 ml of 5% SDS solution. The amount of inorganic phosphate released and the BeFx-sensitive ATPase activity was determined as described in ‘Materials and Methods’. The average from three experiments is shown here and the error bars represent SE. (b) Effect of plumbagin and vitamin K3 on mitoxantrone stimulated-ATPase activity of ABCG2: The ATP hydrolysis assay was done as described above except that 10 μM mitoxantrone was added with varying concentrations (0–50 μM) of plumbagin (■) and vitamin K3 (▲). The average from three experiments is shown here and the error bars represent SE.

Cytotoxicity of plumbagin and vitamin K3 in ABCB1- and ABCG2-expressing cells

As shown in Figure 2, both plumbagin and vitamin K3 inhibited the ABCG2-mediated efflux of mitoxantrone but did not have any effect on the efflux mediated by ABCB1. The biochemical data presented in Figures 3 and 4 suggested that both of these naphthoquinones could also be substrates for ABCG2. Therefore, the cytotoxicity of plumbagin and vitamin K3 in the control (pcDNA3.1-HEK 293 and KB-3-1), ABCG2-expressing (R482-HEK 293) and ABCB1-expressing KB-V1 cells was determined. As shown in Table 1, The R482-HEK ABCG2-expressing cells were 2.8 and 2.3-fold resistant to plumbagin and vitamin K3, respectively, compared to the control pcDNA3.1-HEK 293 cells, while ABCB1-expressing KB-V1 cells did not show any significant resistance compared to the control KB-3-1 cells. This indicated that while ABCB1 had a minimal role in effluxing these compounds, ABCG2-expressing cells confer resistance to both these agents. These results were consistent with the data shown in Figure 2 and 3 which suggested that these compounds did not interact with ABCB1 and further indicated that both plumbagin and vitamin K3 are substrates of ABCG2.

Table 1.

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

Cell line Cytotoxicity IC50 (μM)a Relative resistanceb
Plumbagin Vitamin K3 Plumbagin Vitamin K3
pCDNA3.1-HEK 1.05 ± 0.39 3.70 ± 0.84 1 1
R482-HEK 2.96 ± 0.87 8.54 ± 1.13 2.8* 2.3*
KB-3-1 13.02 ± 6.43 22.72 ± 9.42 1 1
KB-V1 13.51 ± 6.62 21.94 ± 5.01 1.03 0.96
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a

The values represent the mean ± SD of three independent experiments performed in triplicate.

b

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.

*

Values were significantly different from the control cells (P< 0.05).

Sensitization of ABCG2 expressing cells to mitoxantrone by plumbagin and vitamin K3

A non toxic concentration (0.5 μM plumbagin or 1 μM vitamin K3) were evaluated to augment the cytotoxic effect of mitoxantrone for ABCG2 expressing drug-resistant cells using cytotoxicity assays. The relative resistance was calculated based on IC50 values in the absence and presence of plumbagin. Although, both plumbagin and vitamin K3 were able to partially increase the sensitivity of the drug-resistant R482-ABCG2 expressing HEK cells (data not shown), they did not completely inhibit the drug resistance mediated by ABCG2 in three day assays. Further higher concentrations for reversal of cytotoxicity assays could not be used as they were cytotoxic to the control cells. A possible reason for not observing a complete reversal of the cytotoxicity of mitoxantrone could be that unlike FTC, these compounds are not true inhibitors, instead are ABCG2 substrates. Therefore, they may not be able to maintain intracellular concentrations sufficient enough to inhibit the transporter in a long term three day assay. In addition, conversion of the plumbagin or vitamin K3 to an inactive metabolite in a three day assay could also be another reason for the observed incomplete reversal of resistance in ABCG2 expressing cells.

Discussion

ABCG2 was initially identified in drug selected breast cancer cells, and subsequently reported to be expressed at high levels in the human placenta and to a lesser extent in the liver, small intestine and colon, ovary, vein and capillary endothelia, kidney, adrenal and lung, with little to no expression in the brain, heart, stomach, prostate, spleen and cervix (30–34). ABCG2 protein expression has been further shown to be localized to the apical epithelium of the colon and small intestine, to the liver canalicular membrane and to the surface of cells in breast ducts and lobules (34, 35). This suggests that the physiological role of ABCG2 may be to protect cells from potentially toxic substances and to prevent absorption of xenobiotics ingested in our diet by actively transporting compounds from cells. Identification of natural physiological substrates of this transporter will provide additional information about the role of this transporter in the human body.

The data presented in this study identified vitamin K3 and plumbagin, which is a naturally occurring structural analog of this vitamin, as substrates for ABCG2. The cells expressing ABCG2 conferred resistance to vitamin K3 and plumbagin while ABCB1-expressing cells were sensitive to these compounds compared to the control cells (Table 1), indicating that these two compounds were substrates of ABCG2. We showed a direct and specific interaction of vitamin K3 and plumbagin at the substrate binding site of ABCG2 using the photoaffinity labeling assay with a substrate analog [125I]-IAAP. Both vitamin K3 and plumbagin inhibited the photolabeling of ABCG2 with IAAP but did not show any significant effect on the binding of the same with ABCB1 (Figure 3), indicating that both plumbagin and vitamin K3 did not interact at the IAAP binding site of ABCB1. The stimulation of ATP hydrolysis by ABCG2 and the inhibition of mitoxantrone-stimulated ATPase activity of this transporter by both these compounds (Figure 4) provided additional evidence of them being substrates of this transporter.

This is the first report to our knowledge showing a specific interaction of vitamin K3 with any multidrug resistance-linked ABC transporter. A recent study demonstrated the role of ABCG2 in the transport of vitamin B2 (riboflavin) into the breast milk using abcg2 knockout mice (36). The results of this study suggest that ABCG2 could also be involved in the secretion of vitamin K3 in the breast milk. Although van Herwaarden et al. (36) found no difference in the levels of vitamin K1 in the milk from abcg2 knockout and wild-type mice, it has also been reported that vitamin K levels in breast milk rise markedly in response to the mother taking vitamin K supplements, which suggests the possible role of transporters in transporting these vitamins (37, 38). In addition, the interaction of vitamin K3 with ABCG2 may have important clinical implications in the therapeutics related to this important molecule involved in blood clotting.

A variety of inhibitors of ABC drug transporters have been developed to date with the objective of reversing resistance against anticancer drugs and chemosensitizing the resistant cells to anticancer drugs. The fact that both vitamin K3 and plumbagin can inhibit the ABCG2-mediated function suggests that these types of compounds can be developed as inhibitors of this transporter to overcome ABCG2-mediated MDR. In fact, Parekh et al. (39) reported the use of vitamin K3 in overcoming drug resistance by showing that treating the drug sensitive and multidrug resistant-P388 leukemia cells simultaneously with doxorubicin and vitamin K3 made the drug-resistant cells more sensitive to doxorubicin and also demonstrated that treatment with 1 μM vitamin K3 resulted in an increase in intracellular doxorubicin accumulation in drug resistant cells. Other studies also demonstrated an increase in cytotoxicity when vitamin K3 was combined with different chemotherapeutic agents (40, 41) in cervical and nasopharyngeal carcinoma cells. Earlier, in a phase I clinical study, Margolin et al. also showed that a combination of mitomycin C and vitamin K3 led to a 10- to 50-fold reduction in the levels of mitomycin C required for cytotoxicity in solid tumors (42). The inhibitory potential of vitamin K3 in combination with doxorubicin or mitomycin on the cytotoxicity of MCF-7 breast cancer cells was also demonstrated (43), but these studies did not explain the molecular basis of the synergy of cell killing with vitamin K3 and the anticancer drugs. The findings presented here may provide the explanation for the above observations as we show that inhibition of ABCG2 transport function by vitamin K3 and plumbagin results in higher accumulation of target drug inside the cells (Figure 1), which may lead to enhanced cytotoxicity of these drugs in the drug-resistant cells although to our knowledge, it is not known if mitomycin C or its metabolite(s) is substrate of ABCG2.

The data presented here suggest that plumbagin and vitamin K3 interact with ABCG2 but do not show any interaction with ABCB1 at the concentrations tested. These data are not in concordance with the report by Dolan et al., who showed earlier that the ABCB1-expressing KB-V1 cells and KB-3-1 parent cells were equally sensitive to tricyclic 1,2-naphthoquinones (44). In the same study, MCF-7 ADR cells, which were selected for resistance to adriamycin, displayed cross-resistance to these naphthoquinones (44). Recent results (Calcagno et al 2007, unpublished data) from our group suggest that MCF-7 cells selected with low concentrations of doxorubicin over express ABCG2. This may explain the anomaly in the data presented by Dolan et al as their MCF-7 ADR cells may have expressed ABCG2, which was not known at that time, and were therefore resistant to these naphthoquinones while the KB-V1 cells which do not express ABCG2, did not show any resistance to these compounds.

Taken together, the experimental data from these studies suggests that plumbagin could be potentially used as a competing inhibitor in the drug resistance specifically mediated by ABCG2. Additionally, this compound has a relatively simple chemical structure, therefore it can be used for structural activity relationship studies to determine the minimal pharmacophore required for ABCG2 inhibitory activity and this could probably serve as a lead compound for the synthesis of more potent and less cytotoxic ABCG2 inhibitors.

We were also able to show that vitamin K3 is a substrate of this transporter. The specific interaction of this vitamin with ABCG2 may have physiological importance in its distribution and bioavailability in the human body.

Acknowledgments

We are grateful to Dr. Susan E. Bates (NCI/NIH) for providing the cell lines. We thank Dr. Michael M. Gottesman for encouragement, and Drs. Zuben Sauna and Anna Maria Calcagno for comments on the manuscript. We also thank George Leiman for assistance in the preparation of the manuscript.

Grant Support: This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

Abbreviations used

ABC

ATP-binding cassette

BeFx

beryllium fluoride

FTC

fumitremorgin C

HEK

human embryonic kidney

IAAP

Iodoarylazidoprazosin

MDR

multidrug resistance

P-gp

P-glycoprotein

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