Molecular mechanism of rapid cellular accumulation of anticarcinogenic isothiocyanates
Yuesheng Zhang
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA Email: yzhang{at}jhmi.edu
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Abstract
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Isothiocyanates (ITCs) are abundant in foods derived from vegetables, and many ITCs are potent cancer chemoprotective agents in animal systems. We previously showed that many ITCs rapidly accumulated in cells to very high concentrations (up to millimolar levels), and the accumulations appeared to play a critical role in determining their activities in inducing anticarcinogenic Phase 2 enzymes. Subsequent studies showed that ITCs were principally accumulated as glutathione (GSH) conjugates in cells and that cellular GSH might be the major driving force for ITC accumulation by undergoing conjugation with the entering ITCs. To elucidate the molecular mechanism responsible for the accumulation, the dependence of cellular ITC uptake on conjugation with GSH, as well as the role of cellular GSH transferases (GSTs) known to promote the conjugation was investigated. In addition, the role of ITC lipophilicity in ITC uptake was also addressed. All experiments were conducted with four dietary ITCs: allyl-ITC, benzyl-ITC, phenethyl-ITC and sulforaphane [1-isothiocyanato-(4R,S)-(methylsulfinyl)butane]. Initial uptake rates of the four ITCs in human breast cancer cells (MCF-7) closely correlated with the non-enzymatic second-order rate constants of GSH conjugation reaction with the ITCs. Moreover, elevating cellular GSH levels also resulted in nearly proportional increases in cellular ITC uptake. In MCF-7 cells that overexpress human GST P1-1, the initial uptake rates of ITCs also increased linearly with an increase in the specific GST activity. Interestingly, lipophilicity of ITCs did not seem to influence ITC uptake by cells. Taken together, it is concluded that ITCs are taken up by cells predominantly, if not entirely, through GSH conjugation reactions in cells, and that cellular GST promotes ITC uptake by enhancing the conjugation reaction.
Abbreviations: AUC, area under concentrationtime curve; CDNB, 1-chloro-2,4-dinitrobenzene; DTCs, dithiocarbamates; GCS,
-glutamylcysteine synthetase; GSH, glutathione; GST, glutathione S-transferase; HBCP, 2,5-bis(2-hydroxybenzylidene)-cyclopentanone; ITC, isothiocyanate; SF, sulforaphane [1-isothiocyanato-(4R,S)-(methylsulfinyl)butane].
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Introduction
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Over the past three decades there has been increasing interest in the role of isothiocyanates (ITCs) as cancer chemoprotective agents. Many ITCs protect against chemically induced tumors in a variety of animal organs (1,2). ITCs are found in a large number of edible plants, particularly those in the crucifer family (3,4), and are thus present in human diets derived from these plants. Numerous epidemiological studies have repeatedly shown that consumption of vegetables reduces the risk of many types of cancer in humans (5,6), and it is possible that ITCs may be involved in this protective effect. Indeed, in a recent study, human subjects with detectable urinary excretion of total ITCs were found to have much lower incidence of lung cancer (smoking-adjusted relative risk = 0.65) than those with undetectable urinary ITCs (7).
ITCs perturb several steps in the carcinogenic process by: (i) blocking DNA damage by both inhibition of carcinogen activation through inhibition of Phase 1 enzymes (mainly cytochromes P450) and detoxification of reactive carcinogens through induction of Phase 2 enzymes [e.g., glutathione S-transferases (GSTs)]; (ii) inhibiting cell growth by cell cycle arrest; (iii) removing premalignant and malignant cells through activation of apoptosis (1,810). Indeed, ITCs have been shown to inhibit both initiation and post-initiation phases in animal models of chemical carcinogenesis (1113).
We found that many ITCs rapidly accumulate to very high levels (several hundred-fold over the extracellular concentrations) in cell lines from different species, and the intracellular concentration can reach millimolar levels (14). The levels of accumulation may be critical for the anticarcinogenic activity of these compounds. For example, in murine hepatoma cells, the intracellular concentrations [area under concentrationtime curve (AUC)] of ITCs that differ considerably in their structures correlated closely with their potencies as inducers of Phase 2 enzymes (GST and quinone reductase). ITCs that did not accumulate were not inducers (14). Moreover, Conaway et al. also showed that the considerably more potent chemoprotective activity of 6-phenylhexyl-ITC over that of phenethyl-ITC against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumor in F344 rats was closely correlated with the lung tissue AUC of the compounds (15). I further showed (16) that ITCs are accumulated principally as dithiocarbamates (DTCs) derived from conjugation with glutathione (GSH), that the degree of accumulation is related to intracellular levels of GSH and that intact GSHITC conjugates are not taken up by cells. These results suggested that cellular GSH might be responsible for taking ITCs into cells by undergoing rapid conjugation with the entering ITCs and forming the corresponding DTCs. GSH is the most abundant cellular sulfhydryl agent (~110 mM) (17,18), and plays a critical role in cellular defense against a diverse group of exogenous and endogenous toxic species.
The present study was designed to elucidate in detail the molecular mechanism underlying the rapid cellular uptake and accumulation of ITCs with the ultimate goal of developing ITCs as chemoprotective agents in humans. In particular, I wish to further clarify the role of GSH in ITC uptake, to determine the effect of cytosolic GSTs in the uptake and accumulation since GSTs are known to catalyze the conjugation reaction of ITCs with GSH, and to understand the effect of lipophilicity of ITCs on their uptake. The studies were carried out with four ITCs that are common in vegetables: benzyl-ITC, phenethyl-ITC and sulforaphane [1-isothiocyanato-(4R,S)-(methylsulfinyl)butane] (SF), which are anticarcinogens in animal models, and allyl-ITC (1,12).
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Materials and methods
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Chemicals
Synthetic SF was a gift from Dr Gary H.Posner (Department of Chemistry, Johns Hopkins University). Allyl-ITC, benzyl-ITC, phenethyl-ITC, 2,5-bis(2-hydroxybenzylidene)-cyclopentanone (HBCP) and 1-chloro-2,4-dinitrobenzene (CDNB) were purchased from Aldrich (Milwaukee, WI).
Cell culture
MCF-7/wt cells (parent human breast cancer cells), MCF-7/neo cells (stably transfected with G418R empty vector), MCF-7/hGSTA1 cells (stably transfected with human GST A1), MCF-7/hGSTM1 cells (stably transfected with human GST M1) and MCF-7/hGSTP1 cells (stably transfected with human GST P1) were graciously provided by Dr Alan J.Townsend (Department of Biochemistry, Wake Forest University, Winston-Salem, N.C.) (19). These cells were cultured in Dulbecco's modified Eagle medium (DMEM) + 5% (v/v) fetal bovine serum (FBS, Life Technologies, Rockville, MD). The stocks of MCF-7/neo, MCF-7/hGSTA1, MCF-7hGSTM1 and MCF-7/hGSTP1 were maintained in medium containing 0.4 mg/ml of G418 and changed to drug-free medium at least 48 h before ITC treatment, even though we found that G418 did not affect ITC accumulation in cells (results not shown). COS-7 cells (green monkey kidney cells) were maintained in DMEM with 10% (v/v) FBS and gentamicin (50 µg/ml) (20). PE cells (mouse skin papilloma cells) (21) were cultured in Eagle MEM without Ca2+ (BioWhittaker, Walkersville, MD) + 8% FBS treated with Chelex 100 ion-exchange resin (Bio-Rad, Richmond, CA) (21). To remove calcium from serum, 200 g of Chelex resin was first stirred in 500 ml of water for 1 h and the suspension was adjusted to pH 7.5 with HCl before it was filtered through a 0.2 µm filter to remove water. The resin was then mixed with 500 ml of FBS and the mixture was stirred for 1 h at room temperature. The serum was recovered by passing the mixture through a 0.2 µm filter. All cell lines were grown with 10 ml of medium in each 10 cm plate with 5% CO2 in a humidified incubator at 37°C.
Determination of cellular GSH content, the non-enzymatic second-order rate constants of GSH and ITC conjugation reaction, and cellular GST activity
Cellular content of reduced GSH was determined by a fluorometric assay (22) as described previously (14).
The non-enzymatic second-order rate constants (k2) of GSH and ITC conjugation reactions were determined in a 1 ml assay system containing: 100 mM potassium phosphate pH 7.4, 1 mM GSH (with SF and phenethyl-ITC) or 0.5 mM GSH (with benzyl-ITC and allyl-ITC), 920 µM ITC and 0.25% (v/v) acetonitrile. As shown in Figure 1
, GSH reactions with benzyl-ITC and allyl-ITC were much faster than with SF and phenethyl-ITC. The GSH concentration in the assay was thus reduced in the presence of benzyl-ITC or allyl-ITC to allow accurate determination of the k2 values. The bimolecular behavior of GSH and ITC reactions had been confirmed previously (23). The initial absorbance changes at 274 nm (product formation) were measured at 37°C. Each k2 value was calculated based on the initial concentrations of the reactants and the rate of the product formed in the reaction solution. Molar extinction coefficients of each GSHITC conjugate were measured as described previously (23).

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Fig. 1. Correlation of initial ITC uptake rates in MCF-7/wt cells with the non-enzymatic second-order rate constants of ITC conjugation reactions with GSH. To measure initial uptake rates of allyl-ITC, benzyl-ITC, phenethyl-ITC and SF in MCF-7/wt cells, 5x106 cells suspended in 10 ml of medium were incubated with 0.4 µM allyl-ITC, 0.5 µM benzyl-ITC, 1 µM phenethyl-ITC or 4 µM SF for 1.5, 2, 3 or 4 min at 37°C. Cells were harvested and lysed. ITC/DTC contents in cell lysates were measured by the cyclocondensation assay. The initial uptake rates of each ITC in the cell line were calculated based on these measurements and adjusted to 1 µM extracellular ITC concentration. Because the initial uptake rates of each ITC vary greatly, different concentrations of ITCs were used so that the initial uptake rates could be accurately determined, and our previous study indicated that initial uptake rates of ITCs were completely dependent on the extracellular concentration of the ITC (14). The non-enzymatic second-order rate constants of ITC conjugation reactions with GSH were determined at 37°C, pH 7.4.
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To measure cellular GST activity, 1x106 cells were grown in each 10 cm plate for 3 days. Cells were then trypsinized, suspended in medium and centrifuged at 3000 r.p.m. for 2 min at 4°C. After removing the medium, each cell pellet was resuspended in 10 ml of ice-cold Dulbecco's buffer, recentrifuged, resuspended in 200 µl of the same buffer and lysed by sonication. The lysates were centrifuged at 15 000 r.p.m. for 20 min at 4°C. The supernatant fractions were used for determination of GST activity with both CDNB and the four ITCs as substrates. When CDNB was used as substrate, the assay (1 ml) was performed in 100 mM potassium phosphate pH 6.5, 1 mM GSH, 1 mM CDNB, 2% (v/v) ethanol, and 530 µl of the supernatant fractions. The initial absorbance changes at 340 nm (
am 9600 H/cm) were measured at 25°C and corrected for non-enzymatic rate (24). When ITCs were used as substrates, the assay (1 ml) was performed in 100 mM potassium phosphate pH 6.5, 1 mM GSH (for SF and phenethyl-ITC) or 0.5 mM GSH (for allyl-ITC and benzyl-ITC), 0.51% (v/v) acetonitrile and 530 µl of the supernatant fractions. As mentioned above, due to high non-enzymatic reaction rates, GSH concentration in the assay was decreased when benzyl-ITC and allyl-ITC were used as the substrates so that the GST activity could be carefully determined. To start the reaction, each ITC was added to the above mixture at the following concentrations: 0.069 mM SF, 0.075 mM allyl-ITC, 0.075 mM benzyl-ITC or 0.082 mM phenethyl-ITC. The initial absorbance changes at 274 nm (product formation) were measured at 30°C and corrected for non-enzymatic rate.
Measurement of cellular accumulation of ITCs
The procedures involving cell exposure to ITCs, cell harvest, preparation of cell lysates and measurement of cellular uptake and accumulation levels of ITCs/DTCs by the cyclocondensation assay were described previously (14). Since our previous study showed that ITCs were principally accumulated as DTCs derived from conjugation of ITCs with GSH (16), cellular uptake and accumulation of ITCs is referred as `total intracellular contents of ITCs/DTCs' throughout this paper. The protein amounts in cell lysates were measured by the bicinchoninic acid assay (25).
Transient gene expression of
-glutamylcysteine synthetase (GCS)
GCS expression vectors pCMV/GCSh-neo and pCMV/GCSl-neo, containing the full-length cDNAs for human GCS heavy chain (catalytic subunit) and light chain (regulatory subunit), respectively, were gifts from Dr R.Timothy Mulcahy (Department of Human Oncology, University of Wisconsin; 20). The vectors were cotransfected into COS-7 cells at a 1:1 ratio by calcium phosphate precipitation. Transfection and expression of GCS in COS-7 cells have been successfully demonstrated by Mulcahy et al. (20), and the cells were plated in 10 cm culture plates at 1x106 cells per plate 24 h before transfection. At the time of transfection, 25 µg of DNA were added to each dish. To normalize the transfection efficiency, cells from all plates were pooled by trypsin treatment 24 h after transfection and redistributed to the same number of plates. The cells were grown for another 24 h before measurement of cellular GSH levels and exposure to ITCs for determination of ITC accumulation. Control cells were treated the same way except that the DNA was omitted in the transfection process.
Measurement of lipophilicity of ITCs
The lipophilicity of ITCs was measured with 1-octanol and water: 100 nmol of ITC were added to a 10 ml mixture of 10% 1-octanol and 90% water (v/v) or equal volumes of each. The mixtures were vigorously shaken in 10 ml round-bottomed glass flasks with glass stoppers at room temperature for 1 h. ITC content in aliquots of the 1-octanol layer and the water layer was measured by the cyclocondensation assay (26). The partition coefficient (Log P) for each ITC was calculated by dividing the concentration of ITC in 1-octanol by the concentration of the ITC in water and then converting the value to the common logarithm. The Log P values measured in 10% 1-octanol and 90% water were very close to those measured in the equal volume mixture, and the average value is reported in this paper. The Log P values of allyl-ITC, benzyl-ITC and phenethyl-ITC were previously measured by a different system by other investigators (27), and our values for these ITCs are very similar (see Figure 7
).

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Fig. 7. Effect of lipophilicity of ITCs on their uptake in MCF-7/wt cells. The initial cellular uptake rates of allyl-ITC, benzyl-ITC, phenethyl-ITC and SF in MCF-7/wt cells were determined at 37°C as described in the legend to Figure 1 . The lipophilicity (Log P values) of the four ITCs were measured using the 1-octanol and water system as described in Materials and methods.
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Statistics
All values are means of three independent measurements, or means of two measurements made in two separate experiments, each of which was assayed in duplicate. The standard errors of these analyses are generally less than ±10% of the mean.
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Results and discussion
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Initial cellular ITC uptake is nearly completely dependent on its conjugation reaction with GSH
I previously reported that the rank order of the initial cellular ITC uptake rates in murine hepatoma Hepa 1c1c7 cells correlated with the rank order of the non-enzymatic second order rate constants of ITC conjugation reactions with GSH (16). This observation provided the first indication that conjugation of GSH with ITCs was responsible for ITC accumulation in cells. Nevertheless, because the initial uptake rates and the second order rate constants were measured under different conditions, it was not clear to what degree cellular GSH conjugation with ITCs was involved in ITC accumulation. Therefore, in the present study, ITC uptake rates and the second-order rate constants of ITC and GSH conjugation reaction were measured under the same conditions, and a new cell line, MCF-7/wt cells, was used to measure the uptake rate. MCF-7/wt cells provide a desirable system for determining initial ITC uptake rate because they have very high GSH content (75.1 nmol/mg protein) (Table I
), but GST activity is negligible (Table II
). Consequently, the participation of GST in the uptake process can be ignored in this experiment. On the other hand, recombinant MCF-7 cells that overexpress several GST isozymes have become available. Thus, as reported in this paper, the role of GSTs in ITC uptake and accumulation can be examined in the same cell type. The initial cellular uptake rates of allyl-ITC, benzyl-ITC, phenethyl-ITC and SF were measured at 37°C, pH 7.4, and the values were compared with the non-enzymatic second-order rate constants of the conjugation reaction of these compounds with GSH measured under the same conditions. A very close positive correlation between the two indices was found (Figure 1
), suggesting that cellular uptake rates of ITCs were almost entirely dependent on their conjugation reactions with GSH. This result confirms the earlier observation in Hepa 1c1c7 cells as described above and is supported by our previous findings that prior depletion of cellular GSH results in decreased cellular accumulation of ITC (14) and that ITCs are accumulated principally as GSH conjugates (16).
Elevating cellular GSH enhances intracellular accumulation of ITCs
As mentioned above, in our previous study (14) we showed that lowering cellular GSH by inhibiting GSH synthesis led to decreased ITC accumulation in Hepa 1c1c7 cells. This suggests that raising cellular GSH content would result in an increase in cellular ITC uptake and accumulation. Therefore, whether increasing intracellular GSH content would enhance cellular ITC uptake and accumulation was determined by two complementary experimental approaches. First, the cellular level of GSH was raised moderately by transient cotransfection of both catalytic and regulatory subunits of GCS, the rate-limiting enzyme in GSH synthesis, into COS-7 cells, and the transfected cells were then incubated with each ITC. Mulcahy et al. have shown that cotransfection of both GCS heavy and light subunits to COS-7 cells significantly elevated intracellular GSH levels (20). The advantage of this approach is that GSH is probably specifically elevated. I found that cotransfection of GCS heavy and light subunits to COS-7 cells resulted in a 1.7-fold increase in GSH in 48 h. Incubation of these cells with each ITC at 2733 µM for 30 min at 37°C resulted in 1.4- to 1.7-fold increases in cellular ITC accumulation over control cells (Figure 2
). The initial uptake rates in transfected cells were also similarly increased (results not shown). The experiments were not possible in MCF-7 cells because I was unable to significantly elevate intracellular GSH levels by GCS transfection. In the second approach, cellular GSH was raised to much higher levels in PE cells by treating the cells with an inducer of Phase 2 enzymes, HBCP, a Michael reaction acceptor (28). PE cells and HBCP were selected for the experiment because HBCP potently elevates GSH levels in this cell line, presumably by initiating transcriptional activation of genes involved in GSH synthesis (A.Dinkova-Kostova and P.Talalay, personal communication). Again, MCF-7 cells were not used because GSH elevation by HBCP was much less than in PE cells. When PE cells were exposed to 20 µM HBCP for 24 h, followed by replacement with an additional 20 µM HBCP for a further 24 h to ensure an optimal effect, cellular GSH was increased by 5.6-fold. Fortunately, no increase in cellular GST activity was detected when the cell lysates were assayed with allyl-ITC, benzyl-ITC, phenethyl-ITC or SF as substrates during this time. When such HBCP-treated cells and control cells were then incubated with each ITC at 2733 µM for 30 min, there were 3.4- to 4-fold increases in intracellular concentration of these compounds in HBCP-treated cells (Figure 3
). Moreover, when the initial uptake rates of SF were measured in both HBCP-treated cells and control cells, the ratio of SF uptake rates (treated cells over control cells) correlated closely with the ratio of GSH increase in the same cells (results not shown). Hence, these results showed that increasing intracellular GSH levels resulted in nearly proportional increases in intracellular uptake and accumulation of ITCs.

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Fig. 2. Effect of elevating intracellular GSH by transient transfection of GCS genes on cellular accumulation of ITCs. COS-7 cells were co-transfected with GCS heavy chain construct and GCS light chain construct as described in Materials and methods. Transfected cells were suspended in medium, and each 10 ml suspension (~3x106 cells) was incubated with 33 µM allyl-ITC, 31 µM benzyl-ITC, 28 µM phenethyl-ITC or 27 µM SF and 10 µl acetonitrile on 10 cm plates for 30 min at 37°C. Cells were harvested and lysed. ITC/DTC content in the lysates was measured by the cyclocondensation assay. Control cells were treated the same way except that the DNA was omitted in the transfection process. Intracellular GSH levels were measured before ITC treatment in both GCS transfected cells (closed bars, 133 nmol/mg protein) and control cells (open bars, 77 nmol/mg protein).
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Fig. 3. Effect of elevating intracellular GSH levels with HBCP on cellular accumulation of ITCs. PE cells (2x106 cells grown in monolayers in 10 cm plates with 10 ml of medium) were treated with 20 µM HBCP in 5 µl dimethyl sulfoxide for 24 h at 37°C. The medium was then replaced with the same volume of fresh medium containing 20 µM HBCP and the cells were cultured for a further 24 h. Cells from each plate were trypsinized, suspended in 10 ml of medium, and incubated with 33 µM allyl-ITC, 31 µM benzyl-ITC, 28 µM phenethyl-ITC or 27 µM SF in 10 µl of acetonitrile on 10 cm plates for 30 min at 37°C. Cells were harvested and lysed. ITC/DTC contents in lysates were measured by the cyclocondensation assay. Control cells were subjected to the same procedure except that HBCP was omitted. GSH contents were measured in both HBCP-treated cells (closed bars, 243 nmol/mg protein) and control cells (open bars, 43 nmol/mg protein) before ITC exposure.
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Both COS-7 and PE cells showed the same effect of cellular GSH on ITC uptake and accumulation, which is in agreement with the findings of the role of GSH in ITC uptake in MCF-7 cells (Figure 1
), and similar results were also seen in murine hepatoma Hepa 1c1c7 cells (14). These findings not only further support the notion that cellular GSH is the driving force for ITC accumulation, but also strongly suggest that this function of GSH may occur in a wide variety, if not all, cells. However, as we showed previously, the effect of GSH on ITC accumulation was much less or even undetectable in experiments in which ITCs were incubated with cells for 124 h (14). The reason is not yet clearly understood, but the lack of GSH effect in these long-term experiments may be at least partly related to the elimination of the accumulated ITCs/DTCs from cells since the net intracellular accumulation levels of ITCs/DTCs undoubtedly depend on both uptake of ITCs and elimination of accumulated ITCs/DTCs. Our preliminary studies have indicated that the elimination process is also rapid.
Intracellular GST activities enhance cellular ITC uptake
Since others and we had shown that ITCs were substrates of human GSTs (23,29), it seemed likely that cellular GSTs could participate in ITC accumulation by catalyzing ITC conjugation reactions with GSH. To determine the effect of GST on cellular ITC uptake, we selected MCF-7 cells that either had very low GST activity (MCF-7/wt and MCF-7/neo) or overexpressed each of three human GST isozymes (MCF-7/hGSTA1 cells, overexpressing hGST A1-1; MCF-7/hGSTM1 cells, overexpressing hGST M1-1; and MCF-7/hGSTP1 cells, overexpressing hGST P1-1). The specific GST activities differed enormously among lysates prepared from various strains of MCF-7 cells, and the specific activities in the same lysate also differed significantly when assayed with different substrates (Table II
). GST activity in lysates prepared from both MCF-7/wt and MCF-7/neo cells were almost undetectable with all five substrates, and the activities in the lysates of MCF-7/hGSTM1 and MCF-7/hGSTP1 cells were much higher than MCF-7/hGSTA1. CDNB was included as a positive control and was the best substrate for the three isozymes, followed by benzyl-ITC, phenethyl-ITC and allyl-ITC, with SF being the weakest substrate. These results were consistent with our previous results obtained with pure human GST isozymes (23). The dramatic difference in GST activity in these cell lines with respect to ITCs provided an ideal system for investigating its role in cellular uptake and accumulation of ITCs. Moreover, intracellular GSH concentrations in these MCF-7 cells were similar, ranging from 75.1 nmol/mg protein in MCF-7/wt to 85.6 nmol/mg protein in MCF-7/hGSTP1 (Table I
). Therefore, the role of cellular GST in ITC uptake and accumulation could be examined without much confounding effect of different levels of GSH.
First, ITC uptake in MCF-7/wt was compared with that in MCF-7/hGSTP1 cells. Our previous study indicated that human GST P1-1 is one of the most efficient isozymes in catalyzing ITC conjugation with GSH (23). As shown in Table II
, cell lysates prepared from MCF-7/hGSTP1 had very high GST activity, whereas in lysates of MCF-7/wt GST activity was almost undetectable. Initial cellular uptake of each ITC in each cell type was measured for the first 4 min of exposure at 37°C, and the initial uptake rates were calculated with 1 µM ITC in the medium (see legend to Figure 1
). When the ratios of initial uptake rate of each ITC (MCF-7/hGSTP1 cells versus MCF-7/wt cells) were plotted against the ratios of the specific GST activity with the same ITCs in the same cell types, they exhibited a linear correlation (Figure 4
). The ratios of the initial uptake rates among four ITCs differed by >12-fold, and the ratios of specific GST activity differed by >50-fold. Since cellular GSH contents in each cell type were similar, these results show that the initial cellular uptake of ITCs is also strongly dependent on cellular GST activity. More importantly, this finding provided additional evidence that initial cellular uptake of ITCs results from their conjugation with GSH.
Interestingly, the dramatic effect of GST on cellular ITC uptake appeared to be largely limited to the initial phase of ITC accumulation. The enhancing effect of GSTs on cellular accumulation of ITCs was much smaller when cells were incubated with each ITC for 30 min. When each of the five MCF-7 cell lines was incubated with allyl-ITC, benzyl-ITC, phenethyl-ITC or SF at 1620 µM for 30 min at 37°C, the accumulation levels of ITCs/DTCs in cells that overexpress GST A1-1, M1-1 or P1-1 at the highest level were only slightly higher than in MCF-7/wt or MCF-7/neo that have very low GST activity (Figure 5
). For example, benzyl-ITC was accumulated only 1.35-fold higher in MCF-7/hGSTP1 cells than in MCF-7/wt cells, in striking contrast with the 12.5-fold increase in the initial uptake rate (Figures 1 and 5
). MCF-7/neo cells were included in the experiment to ensure that the vector itself did not affect ITC accumulation. Similar results were seen when the extracellular concentrations of ITCs were increased to 100 and 500 µM (results not shown). In order to understand the longer-term effect of GSTs on ITC accumulation, MCF-7/wt and MCF-7/hGSTP1 cells were also each incubated with each of the four ITCs at 410 µM for up to 24 h (higher concentrations were not used for toxicity reasons). The levels of ITCs/DTCs that accumulated in both cell lines were very similar after 1 h, except for sulforaphane, and the intracellular accumulation levels of all four ITCs rapidly decreased over time in both cell lines (Figure 6
). Accumulation of sulforaphane in MCF-7/hGSTP1 cells was notably higher at all time points than in MCF-7/wt cells.

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Fig. 5. Cellular accumulation levels of ITCs in various types of MCF-7 cells with different levels of GST activity. The experiments were carried out in each of the five MCF-7 cell lines: MCF-7/wt (open bars); MCF-7/neo (dotted bars); MCF-7/hGSTA1 (horizontally shaded bars); MCF-7/hGSTM1 (diagonal shaded bars); and MCF-7/hGSTP1 (closed bars). 5x106 cells were suspended in 10 ml of medium and incubated with 33 µM allyl-ITC, 31 µM benzyl-ITC, 28 µM phenethyl-ITC or 27 µM SF in 10 µl of acetonitrile for 30 min at 37°C on 10 cm plates. Cells were harvested and lysed, and ITC/DTC contents in the lysates were measured by the cyclocondensation assay.
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Fig. 6. Accumulation of ITCs in MCF/wt and MCF-7/hGSTP1 cells as a function of time. MCF-7/wt cells (open bars) or MCF-7/hGSTP1 cells (closed bars) (5x106 cells in monolayers on 10 cm plates with 10 ml medium) were incubated with 10 µM allyl-ITC, 4 µM benzyl-ITC, 5 µM phenethyl-ITC or 9 µM SF in 10 µl acetonitrile for 1, 4, 8 and 24 h at 37°C. Cells were harvested and lysed. ITC/DTC contents in lysates were measured by the cyclocondensation assay.
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The results described above clearly show that the enhancing effect of GSTs on ITC uptake and accumulation occurs mainly in the initial ITC uptake phase, and the degree of such an effect depends on the catalytic activity of GSTs with respect to each ITC. The much smaller or even non-detectable GST effect on ITC accumulation when the cells were incubated with ITCs for 0.524 h is very similar to that of GSH observed in Hepa 1c1c7 cells (14), as mentioned above. It is likely that this phenomenon may also arise from the fact that the net intracellular accumulation levels of ITCs/DTCs depend on both uptake of ITCs and elimination of the accumulated ITCs/DTCs, and that the enhancing effect of GSTs on the uptake may be offset by cellular elimination of the accumulated ITCs/DTCs. Interestingly, the rapid decrease of intracellular ITC accumulation levels in both MCF-7/wt and MCF-7/hGSTP1 cells was also observed previously in Hepa 1c1c7 cells (14). Thus, the rapid decrease of intracellular ITC/DTC levels is unlikely to be cell specific and is more likely related to both elimination of ITCs/DTCs from cells and degradation of ITCs in culture medium. Significant degradation of ITCs in a time-dependent fashion in culture medium has been observed (results not shown).
The experiments presented in this paper show for the first time that GSH and GST not only play important roles in cellular defense against many toxic agents, but are also principally responsible for cellular uptake and accumulation of ITCs that can further increase such defense. Since higher GSH level and/or higher GST activity in cells results in more rapid and higher uptake of ITCs, and many ITCs can elevate GSH levels, induce GSTs and other anticarcinogenic Phase 2 enzymes (1), a synergistic mechanism regarding their Phase 2 enzyme inducer activities may have been uncovered. Exposure of cells to ITCs results in rapid uptake and accumulation of ITCs through the GSH conjugation reaction, which is catalyzed by GSTs; such accumulation then leads to an elevation of cellular GSH, GSTs and other Phase 2 enzymes (increase of cellular defense), which in turn cause more rapid and higher accumulation of ITCs in cells. However, the degree of such synergism may depend on specific ITCs. For example, such synergism may be significant for SF because increases of cellular GSH level and GSTs activity result in increases of both initial uptake and long-term accumulation levels of SF (Figures 13 and 6


; 14), and our previous study showed that the total intracellular accumulation levels of ITCs (AUC) are closely correlated with their activities in inducing Phase 2 enzymes in Hepa 1c1c7 cells (14). In contrast, such synergism may be limited for the other three ITCs since the enhancing effects of GSH and GSTs on allyl-ITC, benzyl-ITC and phenethyl-ITC largely appear to be limited to the initial uptake of ITCs.
Initial cellular ITC uptake rates were not correlated with lipophilicity of ITCs
Although several findings presented above confirmed the critical role of cellular GSH and GST in ITC uptake and accumulation, it was not clear whether lipophilicity of ITCs also contributed to the rapid accumulation of these compounds. Since many ITCs are hydrophobic, I reasoned that their affinity with lipid bilayers of cell membranes might facilitate their entry into cells. Our previous observations also indicated that cellular uptake of SF in Hepa 1c1c7 cells was likely to be non-saturable, suggesting that SF, and potentially other ITCs (14), were taken up by simple diffusion, and hinted at the possible involvement of lipophilicity in ITC accumulation. Surprisingly, when the 1-octanol/water partition cofficients of allyl-ITC, benzyl-ITC, phenethyl-ITC and SF were measured, they spanned a range of >700-fold, whereas the initial uptake rates differed by only 4.2-fold. Moreover, when the Log P values were compared with the initial uptake rates of these compounds in MCF-7/wt cells, no apparent correlation was observed (Figure 7
). For example, the uptake rates of SF and phenethyl-ITC were almost the same, yet the Log P values varied by >8-fold (Figure 7
). These results indicate that lipophilicity of ITCs plays a very minor role, if any, in their uptake by cells. Thus, ITCs appear to penetrate the cell membrane freely, and the initial uptake rates of these compounds are predominantly, if not entirely, dependent on their conjugation rates with GSH in cells.
Summary
Four naturally abundant ITCs: allyl-ITC, benzyl-ITC, phenethyl-ITC and SF, three of which are proven anticarcinogens in animal experiments, are rapidly accumulated to very high concentrations in a variety of cell types. These ITCs were taken into cells almost entirely by conjugation with cellular GSH. Altering cellular GSH levels resulted in nearly proportional changes in cellular ITC uptake and accumulation. Cytosolic GST also participated in cellular ITC uptake and accumulation, apparently by catalyzing the GSH and ITC reactions. Although many ITCs are lipophilic, and affinity with the lipid bilayer of cell membranes might facilitate ITC accumulation, our results show that lipophilicity of ITCs plays a very minor role, if any, in cellular ITC uptake and accumulation.
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Acknowledgments
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I would like to thank Professor Paul Talalay for his guidance and support. The excellent editorial advice of Dr Pamela Talalay is gratefully acknowledged. These studies were supported by a grant from the Cancer Research Foundation of America, an RO1 grant (CA80962) and a Program-Project grant (PO1 CA44530) from the National Cancer Institute, Department of Health and Human Services.
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Received August 25, 2000;
revised October 31, 2000;
accepted December 5, 2000.