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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:2004-2010, August 2004

Nutrition and Cancer

Dietary Isothiocyanates Inhibit the Growth of Human Bladder Carcinoma Cells1

Li Tang and Yuesheng Zhang2

Department of Chemoprevention, Roswell Park Cancer Institute, Buffalo, NY 14263

2To whom correspondence should be addressed. E-mail: yuesheng.zhang{at}roswellpark.org.


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Many isothiocyanates (ITCs), some of which are abundant in cruciferous vegetables, have been repeatedly shown to inhibit carcinogenesis in a variety of rodent organs. However, several naturally occurring ITCs also promoted bladder tumorigenesis in rodents, raising the question of whether ITCs behave differently in bladder cells. Alternatively, the observed carcinogenic effects of ITCs may result from prolonged exposure of the bladder epithelium, where the tumors originate, to high concentrations of electrophilic ITCs in the urine. Ingested ITCs are almost exclusively excreted and highly concentrated in the urine as N-acetylcysteine conjugates (NAC-ITC). While several NAC-ITCs also are known anticarcinogens, they are unstable and readily dissociate into parent ITCs. In this study, ITCs, including those that have carcinogenic potential in the rodent bladders, induced apoptosis and/or arrested cell-cycle progression in 2 human bladder carcinoma lines (UM-UC-3 and T24) at 7.5–30 µmol/L. Multiple caspases, including caspase-9, -8, and -3, as well as poly(ADP-ribose)polymerase, were cleaved upon ITC exposure. The ITCs blocked cell-cycle progression at the G2/M and/or S phases in these cells and downregulated several cell-cycle regulators. However, further increases in ITC concentrations abolished their activities, described above. These findings show that urinary ITC concentrations may need to be maintained at low micromolar concentrations for bladder cancer prevention.

KEY WORDS: • isothiocyanate • bladder cancer • chemoprevention • chemopreventive mechanisms of isothiocyanates

Isothiocyanates (ITCs)3 are a family of small molecules characterized by the presence of an –N = C = S group (see Fig. 1 for chemical structures of selected ITCs). Cruciferous vegetables are rich sources of ITCs (1–3). There is strong evidence that many such compounds, both naturally occurring and synthetic, inhibit carcinogen-induced tumorigenesis in a variety of animal organs (4–7). Moreover, several epidemiological studies also have reported an inverse association between human consumption of dietary ITCs and the cancer risk of lung, breast, and colon (8–12).



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  		FIGURE 1 Chemical structures of isothiocyanates.

 
Consumption of ITC-rich cruciferous vegetables was found to inversely correlate with the incidence of human bladder cancer (relative risk = 0.49) (13). However, the effects of ITCs on bladder carcinogenesis in rodent models have been inconsistent. Chronic dietary supplementation of 1-naphthyl ITC (0.6 g/kg diet) or phenylhexyl ITC (PHITC, 0.11 g/kg diet), neither of which is found in nature, inhibited the formation of hyperplasia, papilloma, and carcinoma in the bladder epithelium of rats induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) (14,15). Chronic feeding of benzyl ITC (BITC) at 0.01–1 g/kg diet also reduced the incidence of BBN-induced carcinoma in rats by as much as 74% (16). However, similar BITC treatment at 0.1 g/kg and 1 g/kg diet also caused papillary and nodular hyperplasia in the bladder epithelium without BBN treatment (16), and both BITC and phenethyl ITC (PEITC) at 1 g/kg diet increased carcinogen-induced bladder tumors in other rat models (17). Similarly, chronic oral administration of allyl ITC (AITC) at 12–25 mg/kg body weight 5 times per week also resulted in a slight increase of bladder tumors in male rats (18). Interestingly, Ogawa et al. (19) found that PEITC enhanced or induced rat urinary bladder tumorigenesis only when its levels in the diet exceeded 0.1 or 0.5 g/kg diet, respectively.

Although why ITCs exert both anticarcinogenic and carcinogenic effects in the urinary bladder is not fully understood, it may be related to the metabolism and disposition of ITCs in vivo. ITCs are metabolized principally via the mercapturic acid pathway. Ingested ITCs undergo a rapid conjugation reaction with glutathione (GSH), which takes place spontaneously but which also is enhanced by glutathione transferase (GST) (20–22). The resultant dithiocarbamate conjugates then undergo successive enzymatic modifications to yield N-acetylcysteine conjugates (NAC-ITCs; also called the mercapturic acids), which are disposed of in the urine (23). A considerable body of evidence shows that orally ingested ITCs are efficiently absorbed and then are mainly disposed of and concentrated as NAC-ITCs in the urine of both humans and rats (7,24–31). For example, in one study, when 96.5 µmol BITC was administered orally to each person, 54% of the dose was excreted in the urine as N-acetylcysteine conjugate of BITC (NAC-BITC) within 10 h (25). Should 1 L of urine be produced in 10 h, urinary NAC-BITC concentration would be 52 µmol/L. Although precise information on ITC intake in humans is not available, daily intake of total ITCs, based on a measurement of urinary metabolites or a dietary questionnaire, may commonly be about 10–100 µmol per person (2,32,33), which is 0.14–1.42 µmol/kg body weight, assuming a body weight of 70 kg. However, the ITC doses given to the rodents in the aforementioned experiments were much higher. Akagi et al. (34) estimated that daily ITC intake is 80 mg/kg body weight when rats are fed a diet supplemented with 1 g ITC per 1 kg diet, which is equal to consuming 537 µmol BITC or 491 µmol PEITC per 1 kg body weight daily. These levels of ITC intake may be 2 to 3 orders of magnitude higher than what humans are normally exposed to, suggesting that urinary concentrations of NAC-ITC in these animals may be constantly maintained far beyond 1 mmol/L. Although several NAC-ITCs are known anticarcinogens in a number of rodent organs (35–37), excessive doses of these compounds could be potentially toxic to the bladder epithelium, which is directly exposed to urine and is the site where the bladder tumors originate. NAC-ITCs are unstable and readily dissociate to parent ITCs (38), which are electrophilic and reactive. Indeed, chronic irritation and inflammation induced by urinary ITCs stored in the bladder appear to play very important roles in the development of hyperplasia and neoplasia in the bladder (34,39).

The present study was performed to examine whether ITCs at low concentrations show chemopreventive potential in the bladder. Two human bladder cancer carcinoma cell lines, UM-UC-3 and T24, were used as model cells to evaluate selected ITCs, particularly those shown to induce or to promote carcinogenesis in animal studies. Both cell lines were originally derived from high-grade (G3 and G4) transitional carcinomas. We found that all tested ITCs displayed potent antiproliferative activity in these cells at concentrations < 30 µmol/L. The effects of ITCs on Phase 2 detoxification enzymes, including GST, NAD(P)H:quinone oxidoreductase 1 (NQO1), and UDP-glucuronosyltransferase (UGT) [deficiencies of which were shown to increase the risk of human bladder cancer (26)], as well as the effects of ITCs on GSH, were also examined.


   MATERIALS AND METHODS
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RESULTS
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    Chemicals. Sulforaphane (SF), AITC, BITC, PEITC, phenylpropyl ITC (PPITC), phenylbutyl ITC (PBITC), PHITC, thienylmethyl ITC (TMITC), and thienylbutyl ITC (TBITC) were purchased from LKT Laboratories (see Fig. 1 for chemical structures of the compounds). All other reagents were purchased from Sigma.

    Cell culture. Human bladder cancer UM-UC-3 and T24 cells were purchased from the American Type Culture Collection and were grown in McCOY’s 5A medium with L-glutamine, supplemented with 10% fetal bovine serum (v:v, FBS). The medium and FBS were purchased from Cellgro and Omega Scientific, respectively. All cells were maintained in 75-cm2 flasks in a humidified incubator at 37°C with 5% CO2. Except for the cytotoxicity experiment, which is described below, in all other experiments, 3 x 106 cells were grown in a 10-cm plate with 10 mL medium for 24 h and then were treated with an ITC for 24 h. Each ITC was dissolved in acetonitrile (ACN); the final ACN concentration in the medium was 0.1% (v:v).

    GSH level and the activities of GST, NQO1, and UGT. Cells were treated with an ITC at 1, 3, 7.5, and 15 µmol/L for 24 h. At the end of the ITC treatment, cells were trypsinized and were harvested by centrifugation (500 x g for 5 min at 4°C). The cell pellet from 1 plate was washed with 10 mL ice-cold Dulbecco’s phosphate-buffered saline and was centrifuged again. Each pellet then was lysed by treatment with 200 µL of 0.8 g/L digitonin and 2 mmol/L EDTA (pH 7.8) for 10 min at 37°C, followed by gentle shaking for 10 min at room temperature. The lysates were centrifuged at 10,000 x g for 5 min at 4°C. The supernatant fractions were used for the determination of GSH levels and enzymatic activities. Protein concentration of each sample was quantified by a Pierce’s BCA assay kit.

The GSH level was measured by the GSH reductase-coupled 5,5'-dithiobis-2-nitrobenzoic acid assay (40), by using a 96-well-plate-based procedure (41). The activities of UGT ({rho}-nitrophenol as substrate), GST (1-chloro-2,4-dinitrobenzene as substrate), and NQO1 (menadione as substrate) also were determined as previously described (41,42).

    Cytotoxicity assay. A total of 5 x 103 cells were seeded in each well of a 96-well plate with 150 µL medium for 24 h. An ITC in 50 µL medium then was added to each well. The final concentrations of the ITC in each well were 0.39–100 µmol/L (1-fold serial dilutions). All ITCs were originally dissolved in ACN. After serial dilution, the final ACN concentrations were 0.025% (v:v) or less. For each ITC, 1 set of plates was incubated for 72 h without medium replacement, whereas another set of plates was incubated for only 3 h, followed by incubation of the cells in an ITC-free medium for 69 h (aspirating the original medium and adding back an equal volume of fresh medium). These ITC treatment schedules were selected based on a previous finding that ITCs could rapidly induce cell-growth arrest in a number of human and animal cells of nonbladder origin (43). At the end of the incubation, the cell density in each well was measured by 3-(4,6-dimethylthiazol-2-yl)-2,5-diphenyltetrazolim bromide assay (44). The growth curve of ITC-treated cells then was constructed to determine the ITC concentration required to inhibit cell growth by 50% (IC50).

    Analysis of apoptosis. Apoptotic cells were detected by the terminal deoxynucleotide transferase dUTP nick end labeling assay, by using a flow cytometry-based detection kit (APO-DIRECT) from Phoenix Flow Systems. Cells were plated and were treated as described above, and then were processed with the assay kit and were examined by flow cytometry (10,000 cells from each sample) for apoptotic cells.

The APO-ONE homogeneous caspase-3/7 assay kit from Promega was used to measure the activity of caspase 3/7. After ITC treatment, cells in each plate were pelletized as described above. Each pellet was then suspended in 200 µL of cell lysis buffer from Cell Signaling Technology, was supplemented with 1 mmol/L phenylmethylsulfonyl fluoride, and was sonicated by a Branson’s Model 450 sonifier. The lysates were centrifuged at 10,000 x g for 5 min at 4°C, and the supernatant fractions were used for analysis.

The same supernatant was also used for Western blot analysis. Each sample (50 µg protein) was resolved by SDS-PAGE (8–15%) and was transferred to polyvinylidene difluoride membranes. The membranes were probed by antibodies, and the band of interest was visualized by using an ECL chemiluminescence system from Amersham Biosciences. The antibodies were those specific for the uncleaved and cleaved caspase-3, -7, -8, -9, -12, and poly(ADP-ribose)-polymerase (PARP), purchased from Cell Signaling Technology. An antibody against tubulin from Santa Cruz Biotechnology was used for loading control.

    Analysis of cell-cycle arrest. Cell-cycle arrest was determined by a flow-cytometry-based procedure. After ITC treatment, cells were pelletized as described above. For each sample, 1 x 106 cells were suspended in 1 mL modified Krishan buffer containing 1 g/L sodium citrate, 20 mg/L RNase, 0.3% NP40, and 50 mg propidium iodide (PI) per 1 L (45), and incubated on ice in the dark for at least 1 h, followed by flow-cytometry determination (10,000 cells per sample).

Selected cell-cycle regulators were examined by Western blot analysis by using the same procedure as described above. The antibodies were those specific to cyclin B1, cyclin A, cdc-2, and cdk-2. All the antibodies were purchased from Santa Cruz Biotechnology.

    Statistics. Results are expressed as means ± SD (at least 3 determinations). Data were analyzed by 1-way ANOVA, followed by Dunnett’s t test for separate comparisons with the control group (46). When the comparison involved only 2 groups, the data were analyzed by the Student’s t test. Differences were considered significant at P < 0.05.


   RESULTS
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 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Effect of ITCs on Phase 2 enzymes and the GSH level in human bladder cancer cells. Treatment of UM-UC-3 cells with an ITC at 1, 3, 7.5, or 15 µmol/L for 24 h significantly induced NQO1 (except for TMITC and TBITC). SF appeared to be more efficacious than other ITCs tested, inducing NQO1 maximally 2.4-fold (Fig. 2). Interestingly, the maximal QR1 induction was achieved by SF at 7.5 µmol/L; NQO1 activity tended to decrease (P = 0.07) when the SF concentration was increased to 15 µmol/L. Similar changes occurred with other ITCs, including AITC, BITC, and PEITC (Fig. 2). Although the reason is not fully understood, it may be related to the fact that ITCs at 15 µmol/L strongly inhibited cell growth, as described later. Five ITCs, including PPITC, PBITC, PHITC, TMITC, and TBITC, also were examined at 7.5 µmol/L for the purpose of comparison. There was no apparent association between the methylene chain length of the arylalkyl ITCs [C6H5-(CH2)n-NCS; n = 1, 2, 3, 4, 6] and NQO1-inducing activity, even though PHITC (n = 6) in the series was previously shown to be the most potent inhibitor of rodent-lung carcinogenesis (47). The anticarcinogenic activity of TBITC in the colon and the lungs of rodent models was previously reported (48), but little change in NQO1 activity was detected when UM-UC-3 cells were treated with TBITC or its close analog, TMITC, at 7.5 µmol/L for 24 h. Moreover, in contrast to the frequently reported induction of GST and UGT in other cells and animal tissues by ITCs, neither enzyme in UM-UC-3 cells was significantly affected by the ITCs tested (results not shown). Interestingly, while SF (1–15 µmol/L for 24 h) did not elevate the GSH levels in the cells, AITC, BITC, and PEITC under the same treatment conditions elevated the GSH levels (Fig. 2). BITC appeared to be more efficacious than the other 2 compounds, elevating the total GSH content maximally 2.1-fold at 7.5 µmol/L. Similar effects of the ITCs on the activities of the enzymes and the GSH levels were also detected in another human bladder cancer line of T24 cells (data not shown).



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  		FIGURE 2 Effect of ITCs on GSH level and specific NQO1 activity in UM-UC-3 cells. Cells were incubated with an ITC at the specified concentrations for 24 h at 37°C before harvest for analysis. Each value is a ratio of the level in the treated cells to that in the control cells. Values are means ± SD, n = 3. For GSH, cells treated with the following ITC doses differ from the control (P < 0.5): 1–30 µmol/ L AITC, 1–30 µmol/ L BITC, and 15–30 µmol/ L PEITC. For NQO1, all means differ from the control (P < 0.5) except for cells treated with TMITC or TBITC.

 
    Effect of ITCs on the growth of human bladder cancer cells. All tested ITCs inhibited the growth of UM-UC-3 cells; the IC50 values ranged from 5.1 µmol/L (AITC) to 16.9 µmol/L (PPITC) when the cells were treated with an ITC continuously for 72 h (Table 1). Treatment of the cells by an ITC for only 3 h also inhibited the growth of the cells—the IC50 values increased only 1.3- to 3-fold (Table 1). However, again there was no clear relation between the methylene chain length of the aromatic ITCs (from BITC to PHITC) and their growth-inhibitory activity.


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  		TABLE 1 Growth inhibition of UM-UC-3 cell by ITCs1

 
The IC50 values of these compounds were 1–2 times higher in T24 cells (results not shown) except for AITC, whose IC50 values were 49.4 ± 3.1 µmol/L (3-h treatment) and 35.0 ± 1.3 µmol/L (72-h treatment), which were 3.9–5.9 times higher than those in UM-UC-3 cells. Although the reason for the increased resistance of T24 cells to the ITCs, AITC in particular, is not known, the results suggested that there may be a certain degree of variation among bladder cancer lines in response to the growth inhibitory effect of ITCs.

    Induction of apoptosis by ITCs in human bladder cancer cells. When UM-UC-3 cells were treated with an ITC at 15 µmol/L for 24 h, cellular activity of caspase 3/7 (the executioner caspases) was significantly elevated by all ITCs tested (Fig. 3A), although a higher ITC concentration (30 µmol/L) was uniformly less effective. PEITC was more potent than BITCs (P < 0.05), elevating the caspase activity 5.3- and 4.7-fold, respectively. Apoptotic cells were counted after treatment with PEITC, BITC, AITC, and SF. Treatment of UM-UC-3 cells with 15 µmol/L BITC or PEITC for 24 h significantly increased the number of apoptotic cells (Fig. 3B). Again, PEITC appeared to be more efficacious, increasing the number of apoptotic cells 5.9-fold at 15 µmol/L. Moreover, the rank order of the ITCs in increasing apoptotic cells correlated well with that of their effects on caspase 3/7 activity (Fig. 3A and B). When individual caspases, as well as PARP (the downstream target of activated caspase-3 and -7), were analyzed by a Western blot, cleavage of caspase 3, caspase 8 (death receptor mediated), caspase 9 (mitochondria mediated), and PARP were detected in cells treated with 15 µmol/L BITC or PEITC, while the changes in cells treated with AITC and SF were less clear (Fig. 3C). The ITC-induced caspase cleavage also correlated well with increases of caspase activity and apoptotic cells with respect to the relative potencies of the ITCs. However, our Western blot analysis did not detect the activation of caspase-7 and -12 (endoplasmic reticulum mediated) in cells treated by any ITC. Although the results shown in Figure 3C suggest that the ITCs may be more potent in activating caspase 9 than caspase 8, cleavage of the 2 caspases was similar in ITC-treated T24 cells (results not shown).



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  		FIGURE 3 Effect of ITCs on apoptosis in UM-UC-3 cells. Cells were treated with each ITC at the specified concentrations for 24 h before they were harvested for analysis. Values are means ± SD, n = 3. A, caspase 3/7 activity in cell lysates. Each value with an ITC treatment differs from the control value (P < 0.05). B, percentage of apoptotic cells. The values from cells treated with either 15 µmol/L BITC or 15 µmol/L PEITC each differ from that of vehicle-treated cells (P < 0.05). C, cleavage or activation of various caspases detected in cell lysates by Western blot analysis. The molecular weight for each cleaved and uncleaved caspase is shown in parentheses.

 
    ITC-induced cell-cycle arrest in human bladder cancer cells. AITC, BITC, PEITC, and SF were also examined for their effects on cell-cycle progression in UM-UC-3 and T24 cells. Treatment of UM-UC-3 cells with each ITC at 7.5, 15, or 30 µmol/L for 24 h in many cases led to significant arrest of cells in the G2/M phase and/or the S phase (Table 2). AITC only blocked the cells in the G2/M phase but was the most profound blocker among the ITCs tested, blocking 79.8% of the cells in that phase at 30 µmol/L relative to 8.7% of cells distributed in that phase in control cells. This is in sharp contrast to its weak activity in inducing apoptosis (Fig. 3). BITC, PEITC, and SF arrested cells in both G2/M and S phases, although their effects on the G2/M phase appeared to be more marked (Table 2). Interestingly, for BITC, PEITC, and SF, maximal effects were at 7.5 or 15 µmol/L. Increasing the concentration to 30 µmol/L abolished much of their blocking activity (Table 2). Similarly, while AITC at 30 µmol/L was still more active, further increases of its concentration also diminished blocking activity (result not shown). Although the reason is unknown, this finding suggests that an excessive dose is likely to remove the antiproliferative activity of ITCs.


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  		TABLE 2 Effect of ITCs on UM-UC-3 cell cycle progression1

 
In light of these findings, the expression level of selected cyclins and cdks involved in regulating G2/M and/or S phases were examined. Unlike cdks, which are present at stable levels throughout the cell cycle, each cyclin is known to exist only in a specific cell-cycle phase. As a result, a lack of a corresponding increase in the level of a cyclin when cells accumulate in a phase specific to that cyclin may suggest a downregulation of that cyclin. Cyclin B1 is expressed in the G2/M phase, but its expression level was lower in cells treated by each ITC than in control cells, despite the fact that the ITC-treated cells were significantly arrested in the G2/M phase. The difference in the relative band intensity of cyclin B1 in the 2 concentrations examined for each ITC probably reflects the relative percentage of cells being arrested in the G2/M phase (compare Table 2 and Fig. 4) and suggests that cyclin B1 was not completely eliminated by ITC treatment. In contrast, cdc2, which also is an important regulator of the G2/M phase, appeared to be downregulated by BITC and PEITC but not by AITC and SF. Consistent with the additional arrest of cells in the S phase by BITC, PEITC, and SF, cyclin A, which is expressed in the S phase, was downregulated by all 3 compounds. However, BITC appeared to downregulate both isoforms of cyclin A, whereas PEITC and SF seemed to affect only 1 of the isoforms. No change in the expression level of cdk2, another S phase regulator, was detected in the ITC-treated cells.



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  		FIGURE 4 Western blot analysis of selected cyclins and cdks in ITC-treated UM-UC-3 cells. Cells were exposed to an ITC at 7.5 or 15 µmol/L, or to ACN (vehicle) for 24 h before harvest and preparation of cell lysates for analysis. The 2 bands related to cyclin A probably represent the 2 isoforms: cyclin A and cyclin A1.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Despite previous reports that several ITCs, including AITC, BITC, and PEITC (all naturally occurring), either induced or promoted carcinogenesis in rodent bladder, we found that all 3 compounds, as well as several analogs, including SF, induced apoptosis and cell-cycle arrest in cultured human bladder cancer cells. The ITCs also stimulated NQO1 activity and elevated GSH levels in these cells, likely further contributing to their cancer chemopreventive effects. The current studies also revealed that the concentrations required for the ITCs to exert the observed effects are in the low micromolar range (<30 µmol/L in most cases). Higher ITC concentrations appeared to cause their anticarcinogenic activities to disappear. For example, NQO1 induction levels dropped after the concentration of each ITC was increased beyond 7.5 µmol/L (Fig. 2); the cell-cycle-arresting effects of BITC, PEITC, and SF (Table 2), as well as their effect on caspase 3/7 activation (Fig. 3A) also diminished after their concentrations were raised beyond 15 µmol/L. Even though more cells were arrested in the G2/M phase when AITC concentration was increased from 15 µmol/L to 30 µmol/L, further increases in its concentration also resulted in loss of its activity. On the other hand, since ITCs are electrophilic and elevate reactive oxygen species, excessive doses of these compounds can be potentially toxic (49–55). As discussed in our introduction, total urinary NAC-ITC/ITC concentration may be constantly maintained beyond 1 mmol/L in rats that were fed high doses of ITCs for a prolonged period of time and subsequently had increased bladder carcinogenesis. The epithelia of the urinary bladder in these rats may be chronically exposed to extremely high and toxic concentrations of NAC-ITC/ITC.

Ingested ITCs are rapidly disposed of and concentrated in the urine as NAC-ITCs, several of which are known anticarcinogens in animal studies (nonbladder organs). However, NAC-ITCs are thought to be mere carriers of ITCs, since they are unstable and readily dissociate to parent ITCs (half-life of dissociation of ~3 h, under physiological conditions) (38). Furthermore, unlike ITCs, which rapidly accumulate in cells 100- to 200-fold over the extracellular concentrations (56), including UM-UC-3 cells (our unpublished results), there is little indication that NAC-ITCs themselves could accumulate in cells (57). Therefore, we have focused, in the current study, on the effects of free ITCs. Nevertheless, our preliminary study with synthetic NAC-ITCs indicated that the IC50 values of these conjugates in both UM-UC-3 and T24 cells are close to those of the free ITCs (results not shown).

Exposure of cells to the ITCs for only 3 h subsequently inhibited the growth of the tested cells. The ITCs are potent inducers of apoptosis and cell-cycle arrest (Fig. 3 and Table 2). Caspase-9 and -8 were activated by the ITCs, both of which may be responsible for the activation of caspase 3. Also, multiple cell-cycle regulators, including cyclin B1, cyclin A, and cdc2, appear to be downregulated by ITCs. In contrast, the effect of ITCs on the Phase 2 enzymes examined was limited. There was no change in the activity of Phase 2 enzymes when the cells were treated by an ITC for only 3 h (results not shown). Even when the ITC exposure was continued for 24 h, only NQO1 was induced to some extent, little increase in the activities of GST and UGT. There were some increases in GSH levels in AITC-, BITC-, and PEITC-treated cells, likely due to induction of glutamate cysteine ligase (58) but, surprisingly, not in SF-treated cells (Fig. 2). These results contrasted with previous observations, as described below. Many ITCs are inducers of various carcinogen-detoxifying Phase 2 enzymes in a variety of cells and animal organs of nonbladder origin (4,7,59). Recently, it was reported that oral administration of AITC to Sprague-Dawley rats at 5–200 µmol/kg body weight daily for 5 d elevated NQO1 and GST activities in the bladder up to 4.5- and 2.1-fold, respectively (60). It would be of interest to find out if ITCs are better inducers of these enzymes in normal bladder epithelial cells than these human bladder cancer cell lines.


   ACKNOWLEDGMENTS
 
We would like to thank our colleagues Jun Li for assistance in statistical analysis, Joseph D. Paonessa for critical reading of this manuscript, and Yue Wu for help in preparing some of the figures.


   FOOTNOTES
 
1 This work was supported in part by a grant from the National Cancer Institute (CA80962). Back

3 Abbreviations used: ACN, acetonitrile; AITC, allyl ITC; BBN, N-butyl-N-(4-hydroxybutyl)nitrosamine; BITC, benzyl ITC; FBS, fetal bovine serum; GSH, glutathione; GST, glutathione transferase; IC50, inhibit cell growth by 50%; ITC, isothiocyanate; NAC-ITC, N-acetylcysteine conjugate of ITC; NAC-BITC, N-acetylcysteine conjugate of BITC; PARP, poly(ADP-ribose)polymerase; PBITC, phenylbutyl ITC; PEITC, phenethyl ITC; PHITC, phenylhexyl ITC; PPITC, phenylpropyl ITC; NQO1, NAD(P)H:quinone oxidoreductase 1; SF, sulforaphane (1-isothiocyanato-[4R,S]-[methylsulfinyl]butane); TBITC, thienylbutyl ITC; TMITC, thienylmethyl ITC; UGT, UDP-glucuronosyltransferase. Back

Manuscript received 19 March 2004. Initial review completed 14 April 2004. Revision accepted 28 May 2004.


   LITERATURE CITED
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

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