ABCG2 is not able to catalyze glutathione efflux and does not contribute to GSH-dependent collateral sensitivity

ABCG2 is a key human ATP-binding cassette (ABC) transporter mediating cancer cell chemoresistance. In the case of ABCC1, another multidrug transporter, earlier findings documented that certain modulators greatly increase ABCC1-mediated glutathione (GSH) efflux and, upon depletion of intracellular GSH, induce “collateral sensitivity” leading to the apoptosis of multidrug resistant cells. Recently, it has been suggested that ABCG2 may mediate an active GSH transport. In order to explore if ABCG2-overexpressing cells may be similarly targeted, we first looked for the effects of ABCG2 expression on cellular GSH levels, and for an ABCG2-dependent GSH transport in HEK293 and MCF7 cells. We found that, while ABCG2 overexpression altered intracellular GSH levels in these transfected or drug-selected cells, ABCG2 inhibitors or transport modulators did not influence GSH efflux. We then performed direct measurements of drug-stimulated ATPase activity and 3H-GSH transport in inside-out membrane vesicles of human ABC transporter-overexpressing Sf9 insect cells. Our results indicate that ABCG2-ATPase is not modulated by GSH and, in contrast to ABCC1, ABCG2 does not catalyze any significant GSH transport. Our data suggest no direct interaction between the ABCG2 transporter and GSH, although a long-term modulation of cellular GSH by ABCG2 cannot be excluded.


INTRODUCTION
The development of multidrug resistance (MDR) constitutes a major issue in cancer treatment. Overexpression of the three human ATP-binding cassette (ABC) transporters, ABCB1 (Pglycoprotein/P-gp; Juliano and Ling, 1976), ABCC1 (multidrug resistance protein 1/MRP1; Cole et al., 1992), and ABCG2 (breast cancer resistance protein, BCRP; Allikmets et al., 1998;Doyle et al., 1998;Miyake et al., 1999) has been proposed as one of the main causes of the MDR phenotype in resistant cancer cells. These proteins use ATP hydrolysis as energy source to catalyze the efflux of multiple structurally and functionally diverse chemotherapeutics from cancer cells.
A screening study identified two compounds as potential ABCG2-related CS agents in HEK293 transfected cells, one of them (NSC103054) directly interacting with the transporter (Deeken et al., 2009), and very recently an ABCG2 inhibitor (NP-1250) was reported to induce CS in mitoxantrone-selected MCF7 cancer cells (Ito et al., 2013). Although a mechanism based on extracellular vesicles photodestruction have been shown for another ABCG2-dependent CS (Goler- Baron and Assaraf, 2012), no direct mechanisms have yet been demonstrated; however, these different studies indicate that CS agents, specific for ABCG2, could be developed.
Reduced glutathione (GSH, γ-glutamyl-cysteinyl-glycine) is a tripeptide ubiquitously expressed in cells and involved in many signaling pathways. It has been shown that ABCC1-overexpressing cells were hypersensitive to verapamil through a sharp GSH depletion due to an ABCC1-mediated efflux (Trompier et al., 2004). This phenomenon was further investigated in order to target resistant cancer cells in the frame of a new strategy to overcome the MDR phenotype in cancer (Barattin et al., 2010;Genoux-Bastide et al., 2011). ABCC1 is also known to transport oxidized glutathione disulfide (GSSG) which is however present in low amounts (Keppler et al., 1997). Based on our experience with ABCC1specific CS and on recent reports in which ABCG2 was proposed as a new GSH transporter (Brechbuhl et al., 2009(Brechbuhl et al., , 2010 we aimed at developing new ABCG2-specific modulators able to induce ABCG2-mediated GSH extrusion in order to induce a drastic intracellular GSH depletion leading to cell death. In this study, we focused on searching inducers of ABCG2dependent depletion of intracellular GSH among known death inducers of ABCC1-overexpressing cells, such as verapamil and xanthones (Genoux-Bastide et al., 2011), or known ABCG2 inhibitors (Ahmed-Belkacem et al., 2005;Valdameri et al., 2012). To ascertain the direct role of ABCG2 in GSH efflux, we measured direct transport of radioactive GSH in membrane vesicles.

INTRACELLULAR GLUTATHIONE ASSAY
HEK293 and MCF7 cells were seeded in 96-well plates at respective densities of 1 × 10 4 and 2 × 10 4 cells/well. After 24 h in culture, cells were exposed to the different compounds during 6 or 24 h under normal culture conditions. They were then washed with 200 μl PBS 1X (PAA), stirred during 1 h at 4 • C with 100 μl of 10 mM HCl and freezed at −20 • C overnight, to be lysed. The intracellular total glutathione (reduced GSH and oxidized GSSG) was measured using the method described by Tietze (1969) as modified by Anderson (1985). About 70 μl of the lysate were used to measure intracellular total glutathione and 20 μl for protein quantitation, both being performed in 96-well plates. Total glutathione was assessed by adding 100 μl of a reaction buffer containing 266 μM NADPH, GSH reductase at 10 U/ml and 555 μM DTNB, and the absorbance was read at 412 nm in a microplate reader (PowerWave 340, Biotek) every 30 s during 2 min. The slope for each sample and glutathione standard range was determined to quantify sample glutathione. Protein quantitation was performed using the BCA assay. The results were expressed in nmol glutathione/mg protein and intracellular total glutathione percentages were calculated using the 0 μM samples as 100%.

EXTRACELLULAR GLUTATHIONE ASSAY
HEK293 cells were seeded in 24-well plates at a density of 1.5 × 10 5 cells/well. After 24 h in culture, cells were co-treated with the compound and 0.5 mM acivicin (to block GSH degradation out of the cells) during the 24-h incubation time. Supernatants were collected and cells were washed with 200 μl PBS 1× and treated as for intracellular total glutathione measurement. About 70 μl of the supernatant were used to assess total extracellular glutathione, and protein titration was performed with cell lysate, by the same method as described for intracellular glutathione measurement.

CELL PROLIFERATION AS DETERMINED BY MTT ASSAY
The MTT colorimetric assay, as previously described (Mosmann, 1983), was used to assess the sensitivity of cells to compounds toxicity. HEK293 cells were seeded in 96-well plates at a density of 1 × 10 4 cells/well. After 24 h under normal culture conditions, cells were treated with compounds at increasing concentrations. After 72-h incubation under normal culture conditions a 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium Bromide (MTT) solution was added (0.5 mg/ml final concentration) in wells, and cells were incubated for 4 h at 37 • C. Thereafter, supernatants were carefully withdrawn and 100 μl/well of the buffer ethanol/DMSO (50/50, v/v) were added to solubilize the reduced formazan dye under stirring. Absorbance at 570 and 690 nm were determined by using a microplate reader (PowerWave 340, Biotek). Results were expressed as the difference between OD 570 and OD 690 ; cell survival percentage was calculated using 0 μM sample OD as 100%.

ATPase ACTIVITY ASSAY
The ATP hydrolytic activity of ABCG2 has been determined as described in Ozvegy et al. (2001) and Telbisz et al. (2007). When the effect of GSH was investigated a minor modification in the assay buffer was introduced. About 10 mM DTT was used instead of 2 mM to prevent the oxidation of GSH.

INTRACELLULAR GLUTATHIONE CONCENTRATION IN ABCG2-OVEREXPRESSING CELLS
In order to determine the influence of ABCG2 on cellular glutathione levels, we used two different cell lines overexpressing this transporter. The high level of ABCG2 expression and functionality, through ability to transport a number of substrate drugs, were previously described, in both transfected HEK-ABCG2 cells  and drug-selected MCF7-MX100 cancer cells (Honjo et al., 2001). Moreover, we performed western blot analyses which revealed that all cell lines did not express the ABCC1 protein (data not shown). The intracellular concentration of total glutathione (free GSH + oxidized GSSG) appeared to be significantly modulated by the presence of overexpressed ABCG2 (Figure 1). The glutathione level was lower in ABCG2-transfected HEK293 cells by comparison to the same cells transfected by the pcDNA3.1 empty vector (100 ± 8 versus 130 ± 11 nmol glutathione/mg protein). Interestingly, in drug-selected MCF7 cancer cells, which also overexpress ABCG2, the intracellular glutathione content was significantly higher than in the parental MCF7 cells (154 ± 7 versus 125 ± 10 nmol glutathione/mg protein). These data may indicate a long-term modulation of glutathione levels in various ABCG2overexpressing cell types. Since total glutathione is known to be essentially constituted of free GSH and low amounts of oxidized GSSG, we measured both components separately in the different cell lines, upon incubation with 2-vinylpiridine behaving as a thiol scavenger. In all cases, the remaining oxidized GSSG was too low to be detected (not shown here), indicating no evidence of any change in the ratio between reduced and oxidized forms of glutathione.

INABILITY OF MODULATORS TO STIMULATE AN ACTIVE ABCG2-MEDIATED GLUTATHIONE EFFLUX
Since the 2 ,5 -DHC chalcone was reported to stimulate ABCG2dependent GSH efflux (Brechbuhl et al., 2010), the effects produced by addition of 2 ,5 -DHC at increasing concentrations (up to 40 μM) were analyzed here on the intracellular glutathione levels of both transfected and drug-selected cells. A weak concentration-dependent decrease appeared in ABCG2transfected cells after 6-h incubation with 2 ,5 -DHC (Figure 2A), but not after 24-h incubation where an increase in intracellular glutathione content was observed in both cell lines ( Figure 2B). By contrast, in drug-selected MCF7 cells, no decrease in glutathione content appeared after 6-h incubation ( Figure 2C); a significant difference in glutathione level was observed after 24-h incubation, which was however essentially due to a higher increase in control cells than in ABCG2-overexpressing cells (Figure 2D). The extracellular glutathione content increased after 24-h incubation of ABCG2-transfected cells with increasing 2 ,5 -DHC concentrations (around 40% at 10 μM), but the increase was at least as high in control cells indicating that it was not dependent on ABCG2 ( Figure 2E).
We then studied the effects of verapamil which is known to strongly stimulate GSH efflux in ABCC1-overexpressing cells, leading to a fast and massive intracellular glutathione depletion able to trigger apoptosis (Trompier et al., 2004;Perrotton et al., 2007). A significant decrease of intracellular glutathione was indeed observed in ABCG2-transfected cells with increasing verapamil concentrations, up to 40 μM, which was 25-30% higher than in control HEK293 cells ( Figure 3A). However, no decrease in glutathione content was observed under the same conditions with the ABCG2-overexpressing drug-selected cells, www.frontiersin.org FIGURE 2 | Effects of 2 ,5 -DHC increasing concentrations on total intracellular and extracellular glutathione levels. 2 ,5 -DHC did not induce intracellular GSH depletion in ABCG2 cells (white circles) by comparison to control cells (black circles) in either HEK293 transfected cells during 6 (A) or 24 h (B), or MCF7 cancer cells during 6 (C) or 24 h (D). Moreover, there was no net ABCG2-dependent increase in extracellular GSH (E) induced by 2 ,5 -DHC when comparing HEK-ABCG2 (white bars) and HEK-pcDNA3.1 (black bars) cells after 24-h incubation. The values represent means ± SD corresponding to at least two independent experiments performed in triplicates. Only the differences observed in (D), between MCF7 and MCF7-MX100 cell lines at 24 h, were significant. t -test analysis: *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 3 | Effects of verapamil on intracellular glutathione depletion and cells survival. Verapamil induced a significant intracellular GSH depletion in HEK293 transfected cells during 6-h incubation (A), but not in the MCF7 selected cells (B) when comparing ABCG2-overexpressing cells (white circles) to control cells (black circles)
. This weak effect was not inhibited by Ko143 (C), the difference in intracellular glutathione remaining unchanged. It was not either correlated to any ABCG2-specific collateral sensitivity in MTT cell survival assays (D) with ABCG2-overexpressing cells (white circles) and control cells (black circles). The values represent means ± SD corresponding to at least two independent experiments performed in triplicates. t -test analysis: *p < 0.05, **p < 0.01, and ***p < 0.001. which behaved similarly to control MCF7 cells ( Figure 3B). The ABCG2-related decrease of intracellular glutathione was therefore further characterized in the presence of Ko143, a potent and specific inhibitor of ABCG2 transport activity. Figure 3C shows no significant alteration by comparison to Figure 3A, therefore indicating that such a decrease in intracellular glutathione was not dependent on ABCG2 activity. This was further confirmed by the absence of any CS toward verapamil cytotoxicity, as determined by MTT assays, since the ABCG2-transfected cells were not more sensitive than the control cells ( Figure 3D).
Finally, two other series of compounds were investigated for their ability to modify the intracellular glutathione level. The first series included xanthones (X8, 9, 10, 18, 22, 23) known to induce, similarly as verapamil, a strong depletion in intracellular glutathione in ABCC1-overexpressing cells (Genoux-Bastide et al., 2011), and the second series contained chalcones (C27, 37, 38, 40;Valdameri et al., 2012) and 6-prenylchrysin (6-Pc;Ahmed-Belkacem et al., 2005) known as ABCG2 inhibitors. Figure 4 shows that some xanthones induced a significant decrease in intracellular glutathione, up to around 30% for X8 and X9 and 20% for X23, similarly to the effect observed with verapamil in Figure 3A. By contrast, the ABCG2 inhibitory chalcones, except for C27, and 6-prenylchrysin did not induce any decrease of intracellular glutathione in ABCG2-transfected cells.

NO DETECTABLE INTERACTION BETWEEN GSH AND ABCG2 IN EITHER ATPase OR TRANSPORT ASSAY
We previously demonstrated that the baculovirus-insect cell heterologous expression system is a useful tool for the detection of interactions between a given test compound and ABCG2 (Szakács et al., 2008). Briefly, compounds modifying the ATP hydrolytic activity of ABCG2 interact with the transporter, and can be either transported substrates or inhibitors of the protein. In order to define whether GSH is able to interact with ABCG2, we have tested its effect in the ATPase assay using cholesterol-loaded Sf9 vesicles ensuring higher ABCG2 activity. We found that the ATPase activity of ABCG2 was not affected by GSH addition up to 10 mM, by contrast to a transported substrate such as 1 μM quercetin which www.frontiersin.org stimulated twofold the basal ATPase activity, and the ABCG2specific inhibitor Ko143 which fully inhibited (Figures 5A,B). GSH did not alter the quercetin-stimulated ATPase activity either. Moreover, no effect was produced by the glutathione-conjugate S-(2,4-dinitrophenyl)glutathione (DNP-SG; Figure 5B) known to be actively transported by ABCC1 (Leier et al., 1994).
As the ATPase assay did not give any proof of interaction between GSH and ABCG2, we investigated the ability of GSH to modify the transport of 3 H-methotrexate. As shown in Figure 6, the ABCG2-mediated transport of tritiated methotrexate was not significantly inhibited by GSH addition, up to a 10 mM concentration, by difference with 1 μM Ko143 leading to the low background level observed with inactive mutant ABCG2. This contrasts with the reported prevention by 10 μM methotrexate against the increased extracellular GSH level observed in transformed yeast expressing human ABCG2 (Brechbuhl et al., 2010).

INABILITY OF ABCG2 TO CATALYZE AN ACTIVE TRANSPORT OF GSH
Finally, we measured the direct transport of 3 H-GSH into ABCG2containing membrane vesicles. We found that, in contrast to ABCC1 serving as a positive control, no direct, ATP-dependent and specific inhibitor-sensitive, transport of tritiated GSH by ABCG2 could be detected in insect-cell membrane vesicles (Figure 7). Any ABCG2-mediated GSH transport could not be either determined at other 3 H-GSH concentrations (0.1 or 1 mM, data not shown).

DISCUSSION
The key results of this paper strongly suggest that human ABCG2 is unable to transport GSH. This has been demonstrated by direct measurement of ATP-dependent tritiated GSH uptake in inverted vesicles of insect-cell membranes overexpressing human ABCG2. In contrast, human ABCC1 catalyzed a high level of ATPdependent and MK571-sensitive GSH transport under the same conditions. There was a low level of GSH accumulation in the presence of ABCG2 observed without ATP, which was also observed in the presence of the selective ABCG2 inhibitor Ko143 (Allen et al., 2002), or when the catalytically inactive K86M ABCG2 mutant was expressed. Thus, this background GSH binding could not be attributed to any ABCG2-mediated active transport.
This result is fully consistent with the lack of effect of GSH, even at high concentrations, on both basal and quercetin-stimulated ABCG2-ATPase activity of the insect cell membrane vesicles. Indeed, transported substrates such as prazosin, quercetin, or nilotinib (Telbisz et al., 2012) strongly stimulate the basal ATPase activity, then enhancing "coupled" ATPase activity. Our present results also show the lack of any effect by DNP-SG on the ABCG2 transporter, suggesting that glutathione conjugates are not transported by ABCG2. This is in contrast to various compounds conjugated with either sulfate (Suzuki et al., 2003) or glucuronate (Chen et al., 2003), whereas DNP-SG is actively transported by ABCC1 (Leier et al., 1994). The lack of ABCG2-mediated GSH transport is also consistent with the lack of any antagonism by GSH addition against ABCG2-mediated tritiated-methotrexate transport in inverted vesicles. These results, however disagree with the methotrexate-induced inhibition of GSH efflux reported in transformed yeast cells, expressing human ABCG2 (Brechbuhl et al., 2010).
Our results from experiments using membrane vesicles are quite consistent with those obtained with either transfected or drug-selected ABCG2-overexpressing cells where we did not observe any sharp and rapid decrease of intracellular GSH stimulable by modulators (such as 2 ,5 -DHC, verapamil, or xanthones), or alterable by ABCG2 inactivation (such as using the potent Ko143 inhibitor). In addition, there was no inverse correlation between the observed decrease of intracellular GSH and increase of extracellular GSH, as also noticed in other drugselected cancer cells overexpressing ABCG2 (Brechbuhl et al., Frontiers in Pharmacology | Pharmacology of Anti-Cancer Drugs 2010). This contrasts with the strong effects clearly observed with ABCC1-overexpressing cells (Trompier et al., 2004;Perrotton et al., 2007;Barattin et al., 2010;Genoux-Bastide et al., 2011).
Nevertheless, the intracellular total glutathione concentration appeared to be modulated by overexpressed ABCG2 since, for unknown reasons possibly resulting from different signaling pathways, glutathione was lower in HEK293 transfected cells and higher in the drug-selected MCF7 cancer cells by comparison to their respective control cells. In addition, a significant decrease of intracellular glutathione was actually observed, either in some cases with 2 ,5 -DHC, as previously reported (Brechbuhl et al., 2010), or with known ABCC1 modulators such as verapamil and xanthones. Such a decrease of intracellular glutathione however displayed special characteristics, such as being slow, requiring at least 6-24 h incubation, and not depending on ABCG2 activity since it was not altered by Ko143 inhibition. These results are more likely compatible with the induction of associated signaling pathways, leading to changes in intracellular GSH, than with a direct GSH transport.