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Nutrition and Cancer

Dietary Isothiocyanates Inhibit Caco-2 Cell Proliferation and Induce G₂/M Phase Cell Cycle Arrest, DNA Damage, and G₂/M Checkpoint Activation¹

Section of Gastrointestinal Science, University of Manchester, Manchester, UK and ^* The Rowett Research Institute, Aberdeen, UK

²To whom correspondence should be addressed. E-mail: ppadfiel{at}fs1.ho.man.ac.uk.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Benzyl isothiocyanate and phenethyl isothiocyanate, two aromatic phytochemicals present in substantial concentrations in edible vegetables of the genus Brassica, were investigated for their effects on Caco-2 cell proliferation. Benzyl and phenethyl isothiocyanate inhibited DNA synthesis, with 50% inhibitory concentrations of 5.1 and 2.4 µmol/L, respectively, and significantly increased the doubling times of Caco-2 cells from 32 h to 220 and 120 h, respectively. There was no adverse effect of either chemical on cell viability in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, but benzyl isothiocyanate and phenethyl isothiocyanate both caused an accumulation of cells in the G₂/M phase of the cell cycle, which was maintained for at least 48 h in cells synchronized at prometaphase with nocodazole and subsequently treated with 10 µmol/L benzyl isothiocyanate or phenethyl isothiocyanate. Both benzyl and phenethyl isothiocyanate increased DNA strand breakage, increased phosphorylation of the G₂/M checkpoint enforcer Chk2, and induced p21 expression. These results suggest that the antiproliferative effects of benzyl and phenethyl isothiocyanates toward Caco-2 cells are due at least in part to the activation of the G₂/M DNA damage checkpoint, and that sustained G₂/M phase cell cycle arrest in response to benzyl and phenethyl isothiocyanates may be maintained through upregulation of p21. This study indicates that some dietary isothiocyanates may exert an antiproliferative effect through activation of the G₂/M DNA damage checkpoint.

KEY WORDS: • isothiocyanates • proliferation • DNA damage • checkpoints

The human diet exerts a profound influence on the risk of cancer, particularly in the gastrointestinal system (1), and there is currently considerable interest in the application of dietary phytochemicals, including isothiocyanates (ITCs),³ in cancer chemoprevention, that is, the long-term pharmacologic management of cancer risk (2–4).

ITCs are a diverse group of phytochemicals present in substantial quantities in Brassica vegetables (5). They are of particular interest because of their abundance in the human diet, and the observation that Brassica vegetable consumption is associated with reduced overall cancer risk (6), whereas the effect of total fruit and vegetable consumption on cancer risk is less clear (7). Animal studies provide further evidence for a potential cancer chemopreventive effect of ITCs; for example, benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) inhibit lung and esophageal tumorigenesis by tobacco carcinogens in rats and mice (8–10). ITCs modulate activity of the phase I and II detoxifying enzymes, and competitively inhibit cytochromes P₄₅₀ (11); these activities are thought to be responsible at least in part for their chemopreventive effects.

More recently, further possible cellular mechanisms for the chemopreventive effects of ITCs were investigated. Several ITCs induce apoptosis, which is an important event in protection against tumorigenesis in the gastrointestinal system and elsewhere, with a variety of mechanisms involving p53-dependent and -independent pathways (12,13). Furthermore, some ITCs affect the cell cycle and may thereby impede cell proliferation. Indole-3-carbinol disrupts CDK6 transcription to induce G₁ arrest (14), and sulforaphane induces G₂/M arrest, with increased levels of cyclins A and B in HT29 cells (15). Allyl ITC was also shown to inhibit cell proliferation through the induction of G₂/M phase cell cycle arrest, although not all cell lines were equally susceptible to allyl ITC (16).

Despite the growing body of information on the cell cycle effects of some ITCs, nothing is known about the induction of cell cycle checkpoint mechanisms by ITCs. Cell cycle checkpoints are important growth arrest mechanisms that ensure the orderly progression of cell-cycle events and prevent aberrant mitosis in response to a range of events, including DNA damage. The aim of this study was to investigate the antiproliferative effects of BITC and PEITC, two common dietary ITCs, and to establish whether the G₂/M phase DNA damage checkpoint was involved in inhibition of proliferation by BITC and PEITC.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Materials. Nocodazole, aprotinin, pepstatin, and leupeptin were purchased from Calbiochem. Antibodies to Chk2 were purchased from Santa Cruz Biotechnology. Antibodies to p21 and the Thr68 phosphorylated form of Chk2 were purchased from Cell Signaling Technology. Horseradish peroxidase conjugated secondary antibodies were purchased from Bio-Rad. Tissue culture media were purchased from Invitrogen. All other reagents were purchased from Sigma unless stated otherwise.

    Cell culture, proliferation and cytotoxicity assays. Caco-2 cells were maintained in DMEM supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine, 50 kU/L penicillin G, 50 mg/L streptomycin and 1X nonessential amino acids (Invitrogen) in a tissue-culture incubator at 37°C, 5% CO₂ and subcultured every 7–10 d. For DNA synthesis assays, 12-well tissue culture plates were grown to 60% confluence. Cells were treated for 3 or 21 h with 0.1–10 µmol/L BITC or PEITC in culture medium. For the final 3 h, 1 µCi [³H]thymidine (Amersham Pharmacia Biotech) was added to each well; then the cells were washed with PBS and 5% trichloroacetic acid. Each treatment was performed in triplicate and data shown derive from 3 or more independent experiments. For cell proliferation assays, 25-cm² tissue culture flasks were inoculated with Caco-2 cells and incubated for 24 h before exposure to 10 µmol/L BITC or PEITC. Cells were harvested from treated and untreated flasks every 24 h and cell density calculated using a hemacytometer. Culture media and treatments were replaced every 48 h. Cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, Caco-2 cells were cultured to confluence in 96-well plates and treated with BITC or PEITC for 24 or 48 h. MTT was then added to each well to a final concentration of 0.2 g/L and the cells incubated for a further 5 h; during that period, incorporation of MTT by Caco-2 cells was linear. Cell culture supernatants were then aspirated and the cells solubilized in 200 µL dimethyl sulfoxide and optical density at 570 nm measured. For all experiments, BITC and PEITC were added to culture media as 50 mmol/L solutions in dimethyl sulfoxide and controls were matched for dimethyl sulfoxide concentration.

    Western blotting. Western blotting was performed as described previously (17).

    Flow cytometric determination of cell cycle phase. Cells were treated for up to 48 h with 10 µmol/L BITC or PEITC, harvested using trypsin, and fixed in 1 mL ice-cold 70% ethanol. Fixed cells were resuspended in 0.5 mL PBS containing 35 mg/L propidium iodide and 35 mg/L RNase A. Samples were analyzed on a Beckton Dickinson FACSvantage flow sorter measuring forward and side scatter, peak width, and area of fluorescence at 488 nm. Events were gated for peak width and area to exclude subcellular debris and aggregates. A minimum of 5000 gated events were recorded for each sample, and a frequency histogram of peak area was generated and analyzed using Modfit LT software (Verity).

Where appropriate, cells were synchronized at prometaphase by treatment with 0.2 µmol/L nocodazole for 24 h before exposure to BITC or PEITC. After nocodazole treatment, cells were washed twice with PBS to release the nocodazole-induced G₂/M arrest, and subsequently treated with BITC or PEITC.

    Single-cell alkaline gel electrophoresis. Caco-2 cells were treated for 24 h with 10 µmol/L BITC or PEITC before assessment of DNA strand breakage by single-cell alkaline gel electrophoresis (SCGE) as described previously (18). Etoposide, a potent inducer of DNA strand breakage, was used as a positive control. Cells were scored by fluorescence microscopy by a trained and experienced observer who was unaware of the treatment. A minimum of 100 cells from each sample were analyzed visually on the basis of comet tail intensity, and placed in 1 of 5 classes reflecting the proportion of DNA in the comet tail relative to the head. Visual scoring shows a clear relation to the percentage DNA in the tail measured by computer image analysis (19). The mean comet score (normalized to 100 cells) was calculated for each treatment and control sample from class (0, 1, 2, 3, or 4) of a minimum of 100 cells/sample from each of 3 separate experiments.

    Statistical analysis. Results of [³H]thymidine incorporation assays, cell cycle studies, and SCGE were analyzed by Student’s t test. Doubling times for cell populations were calculated using a linear least-squares best fit of log-transformed data and differences between control and treated cell doubling times analyzed using Student’s t test. Results of MTT assays were analyzed using 1-way ANOVA. All tests were 1-tailed and P = 0.05 was taken as the limit of significance. Data are presented as means ± SE.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    ITCs inhibit Caco-2 cell proliferation. To establish whether BITC or PEITC (Fig. 1A) inhibited Caco-2 cell proliferation, the influence of both ITCs on [³H]thymidine incorporation and cell doubling was determined. Both BITC and PEITC produced a concentration-dependent decrease in thymidine incorporation (Fig 1B). The 50% inhibitory concentrations were 5.1 µmol/L for BITC and 2.4 µmol/L for PEITC. Exposure to BITC or PEITC (10 µmol/L) also significantly increased cell doubling time. For control cells, the doubling time was 32 h. This increased to 220 h (P < 0.0001) and 120 h (P < 0.0001) for cells exposed to 10 µmol/L BITC and PEITC, respectively.

View larger version (17K): [in this window] [in a new window]   FIGURE 1 Chemical structures of BITC and PEITC (A) and inhibition of Caco-2 cell proliferation by BITC and PEITC (B). Caco-2 cells were treated with increasing concentrations of BITC or PEITC for 21 h before proliferation was measured by [3H]thymidine incorporation. The data are expressed relative to control, untreated cells and are the means ± SE of 3 independent experiments. *P 0.05 compared with untreated cells.

    ITCs induce cell cycle arrest after prometaphase. Next, we determined whether either ITC arrested cells in a specific phase of the cell cycle. BITC and PEITC both caused a gradual accumulation of cells in G₂/M phase at the expense of G₀/G₁ phase (Fig. 2). After 48 h, the percentage of cells in G₂/M phase was increased from 12.4 ± 0.5% among controls to 48.3 ± 4.1% (P < 0.001) and 36.0 ± 2.5% (P = 0.001) for cells treated with 10 µmol/L BITC or PEITC, respectively. The slight accumulation of cells at S phase after 24 h of treatment with BITC was not significant.

View larger version (17K): [in this window] [in a new window]   FIGURE 2 BITC and PEITC induce G2/M phase cell cycle arrest in Caco-2 cells. Caco-2 cells were incubated with or without BITC or PEITC (10 µmol/L) for up to 48 h. At set times, cells were harvested and analyzed by flow cytometry to determine the percentage of cells in each phase of the cell cycle. The data are the means ± SE of 3 independent experiments. *P 0.05 compared with untreated cells.

View larger version (14K): [in this window] [in a new window]   FIGURE 3 BITC and PEITC prevent prometaphase-synchronized cells from exiting mitosis and reentering G0/G1 phase. Caco-2 cells blocked in prometaphase by exposure to nocodazole were released from arrest and incubated with or without BITC or PEITC (10 µmol/L) for up to 48 h. At set times, cells were harvested and analyzed by flow cytometry to determine the percentage of cells in each phase of the cell cycle. The data are the means ± SE of 3 independent experiments. *P 0.05 compared with untreated cells.

View larger version (36K): [in this window] [in a new window]   FIGURE 4 BITC and PEITC induce DNA strand breaks in Caco-2 cells. (A) Representative micrographs of ITC-treated Caco-2 cells after SCGE showing the different classes of comet scoring. Comets were assigned to a class between 0 and 4 on the basis of visual inspection of the relative amount of DNA in the comet tail to the comet head. (B) Cells were treated for 24 h with 10 µmol/L BITC or PEITC, or with 5 µmol/L etoposide as positive control, and the extent of DNA damage measured by comet assay. The mean proportion of cells in each class is shown for 3 independent experiments.

View larger version (46K): [in this window] [in a new window]   FIGURE 5 BITC and PEITC induce phosphorylation of Chk2 (A) and cause an increase in p21 protein levels (B). Caco-2 cells were treated for up to 12 h with or without BITC or PEITC (10 µmol/L) before being harvested. Cell lysates were then analyzed by Western blot for Chk2 and phospho-Chk2 (A) or actin and p21 (B).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The DNA damage checkpoint mediated by the ataxia-telangiectasia mutated and/or ataxia-telangiectasia mutated and rad3-related kinases and the resultant activation of Chk1 and/or Chk2 likely represent the most common mechanism for the induction of G₂/M arrest. Our findings that BITC and PEITC cause both DNA damage and phosphorylation (activation) of Chk2 strongly imply that both BITC and PEITC induce arrest via activation of the DNA damage checkpoint enforced by Chk2. To date, only one other dietary phytochemical, genistein, was shown to cause induction of the G₂/M checkpoint and cell cycle arrest through Chk2 (24,25).

We also observed increased protein levels of p21 in response to treatment with BITC and PEITC. Because Caco-2 cells are p53 negative, the ITC-induced expression of p21 would appear to be independent of the p53 pathway. p21 is a broad-specificity CDK inhibitor that acts to prevent cell cycle progression in response to a wide range of stimuli, including DNA damage and other forms of cell stress. Its classical function is to prevent cell cycle progression at G₁ phase; however, it now has established functions at other phases of the cell cycle. In particular, p21 is required for sustained cell cycle arrest at G₂/M phase (20–22) and inhibition of apoptosis (26–28). The upregulation of p21 levels by BITC and PEITC (Fig. 5B) is therefore consistent with the failure of BITC and PEITC to affect cell viability adversely in the MTT assay. Also consistent with the established cell cycle effects of p21 upregulation, we found that cell cycle arrest induced by BITC and PEITC in cells previously synchronized at prometaphase with the microtubule inhibitor nocodazole was sustained for 48 h (Fig. 3). Enforcement of the G₂/M DNA damage checkpoint by p21 involves inhibition of a stimulatory phosphorylation (at Thr161) of cdc2, the cyclin B1 kinase partner (20). Because cyclin B1/cdc2 activity is required for passage through mitosis until anaphase, the observation that G₂/M arrest is sustained after prometaphase in response to BITC and PEITC is consistent with the involvement of p21 in the cell cycle arrest induced by BITC and PEITC in these experiments.

DNA strand breakage is undoubtedly a procarcinogenic, rather than an anticarcinogenic phenomenon; thus, the induction of DNA strand breakage by BITC and PEITC seems inconsistent with the epidemiologic evidence, which indicates a reduction of cancer risk with increasing consumption of ITC-rich Brassica vegetables. Nevertheless, previous reports also suggested that ITCs may induce DNA strand breakage or have mutagenic effects (29,30). Most recently, glucoraphanin, the precursor of the ITC sulforaphane, was found to increase activity of phase I (procarcinogen-activating) enzymes, increase oxidative stress, and damage DNA (31). Substances that appear capable of exerting both "pro-" and "anti-" mutagenic and -carcinogenic effects have been termed "Janus mutagens" (32), and the data presented here, taken together with results from other laboratories (29,30), strongly suggest that BITC and PEITC at least, and possibly a wider range of structurally related ITCs, should be considered potential Janus mutagens. Animal models provided further evidence for the possible carcinogenic effects of ITCs, in particular suggesting that ITCs may in fact be cocarcinogenic with common experimental chemical carcinogens, rather than simply opposing their action as suggested in previous studies (9); in fact, some studies showed that ITCs may promote urinary bladder carcinogenesis in rodents (33,34).

In conclusion, these results demonstrate that BITC and PEITC are potent antiproliferative agents toward Caco-2 cells in vitro, and that this antiproliferative effect may be mediated through the G₂/M DNA damage checkpoint. To the best of our knowledge, the induction of this checkpoint by any dietary phytochemical is, with the exception of genistein, unprecedented (24,25). Both agents induced sustained G₂/M cell cycle arrest, which was associated with increased phosphorylation of Chk2, and upregulation of p21 levels. Both BITC and PEITC also induced DNA strand breakage, suggesting that their antiproliferative effects result from G₂/M cell cycle arrest enforced by at least 2 distinct checkpoint mechanisms, namely, upregulation of Chk2 activity and upregulation of p21 protein levels. In addition to demonstrating novel effects of these ITCs, and novel mechanisms of cell cycle arrest-induction by these ITCs, these observations indicate the need for further toxicological studies of these and other ITCs, before they are employed in doses in excess of normal dietary levels as part of a long-term pharmacologic approach to the management of cancer risk (35).

	ACKNOWLEDGMENTS

 
We thank Michael Jackson for excellent technical assistance.

	FOOTNOTES

 
¹ Supported by the Biotechnology and Biological Sciences Research Council and the Scottish Executive Environment and Rural Affairs Department.

³ Abbreviations used: BITC, benzyl isothiocyanate; ITC, isothiocyanate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PEITC, phenethyl isothiocyanate; SCGE, single-cell gel electrophoresis.

Manuscript received 19 May 2004. Initial review completed 4 July 2004. Revision accepted 17 August 2004.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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