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,3
,**
* Institute of Hygiene,
Center of Biotechnology,
** Institute of Biochemistry "G. Fornaini" University of Urbino, 61029 Urbino, Italy and
Department of Experimental Oncology, Unit 10, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy
3To whom correspondence should be addressed. E-mail: brandi{at}uniurb.it.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • breast cancer • Brassica juice • CDK6 • p27 • chemoprevention
In recent years, chemoprevention has attracted considerable attention as a means of blocking malignant transformation in its early stages and disease progression in later stages. The presence of a large number of potentially anticarcinogenic compounds in fruits and vegetables was suggested, particularly in those belonging to the genus Brassica (e.g., cauliflower and broccoli) (1–3). These cruciferous plants contain high quantities of glucosinolates (GLS),4 whose hydrolytic products were shown to prevent, delay, or reverse carcinogenesis induced by various chemical compounds (4–6). In fresh plants, GLS are present in a stable form, compartmentalized in different sites from the endogenous enzyme myrosinase; when tissues are destroyed, however, myrosinase comes into contact with and hydrolyzes GLS into a series of breakdown products such as isothiocyanates (ITCs; isothiocyanates sulforaphane, benzylisothiocyanate and prophylisothiocyanate, phenethyl isothiocyanates), and indoles (3,3'-diindolylmethane, ascorbagen, indole-3-carbinol, indolo-[3,2-b]carbazole) (7).
Although the mechanism(s) by which cruciferous vegetables exert their cancer chemopreventive action is largely unclear, several hypotheses were proposed; first among these is that some agents contained in these vegetables exert a protective effect by modulating the activity of Phase II and Phase I drug metabolism enzymes. In particular, ITCs and indole-3-carbinol were shown to induce the so-called phase-II enzymes, (i.e., glutathione S-transferase, quinone reductase, glucoronosyl transferase), which play an important role in the detoxification of toxins and carcinogens (4,8–11) and inhibit phase I enzymes, which are responsible for metabolic activation of most chemical carcinogens (10,12).
Recently, several other mechanisms were proposed to explain the protective effect associated with Brassica consumption in breast cancer, such as interactions between agents contained in this vegetable and estrogen metabolism (or signal transduction pathways) (13,14), the induction of apoptotic cell death (15–18), and interference with cell cycle regulatory proteins (19–22). In a previous study (22) we demonstrated that a cyclic tetrameric derivative (tetramer) of indole-3-carbinol (I3C), a naturally occurring compound found in Brassica vegetables, is able to inhibit the growth of breast cancer cells. It does so by inducing their arrest in the G1 phase of the cell cycle as a consequence of inhibition of CDK6 expression and activity, an increase in the p27 expression level, and a decrease in retinoblastoma protein (pRb) expression and phosphorylation.
Most of the evidence concerning the anticarcinogenic effects of the products of hydrolysis of GLS comes from animal experiments, in which indoles and ITCs reduced the incidence and the multiplicity of experimentally induced tumors (4,6,23–26). On the contrary, in humans, the role of cruciferous consumption in preventing cancer is still controversial (6). Many epidemiologic studies indicated a reduction in the incidence of various types of human cancer in subjects consuming large amount of vegetables rich in GLS, such as those of the cruciferous family (2,3,27–31); the degree of protection that resulted was proportional to the level of cruciferous vegetable consumption (6). In other cohort studies, weaker or no associations were found between total vegetable or fruit intake and colorectal cancer (32–34). However, the effects of a diet rich in Brassica vegetables in cancer prevention may differ depending on the cancer type and sites (24,35). In addition, the association between Brassica consumption and breast cancer in humans remains unclear because some studies showed significant protective effects associated with the consumption of Brassica vegetables, whereas others revealed no relation whatsoever (31,36–40). In some cases, these discrepancies may depend on the different methods utilized in these studies or on the end points tested (41). Thus, the role of naturally occurring compounds found in Brassicaceae as cancer chemopreventive agents warrants further investigation.
Although a great number of studies dealing with the anticarcinogenic activity of indoles or ITCs or other single components have been conducted, very few studies were performed using whole Brassica vegetables. It cannot be excluded that the cancer chemopreventive properties of whole Brassica vegetables may be more effective than those of a single compound due to possible interactions among compounds in the whole vegetable.
In this study, we investigated the effect and the mechanism of action of whole fresh cauliflower juice on the growth and viability of human breast cancer cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Preparation of cauliflower juice. Fresh cauliflower leaves were obtained from local producers during the winter and spring months. Whole juice was prepared by mechanical breaking of the leaves followed by homogenization. The homogenate was centrifuged at 8000 x g for 10 min at 4°C, the supernatant decanted, sterilized by filtration on 0.22-µm filters, and used immediately for treatments.
Preparation of dried extracts from cauliflower juice.
Fresh cauliflower leaves were washed with a solution of 0.4 mg/L of chlorine, rinsed with sterile water, and dehydrated at 40°C under air ventilation. Leaves were chopped and processed for ethanolic or aqueous extractions. The dried extracts contained 
5% water. The extract was prepared by processing the juice obtained from the leaves by spray drying (Model B-191-Buchi, Milan) at 180°C inlet temperature and 90°C outlet temperature.
Cell culture. The human breast adenocarcinoma ER+ (MCF-7 and BT474) and ER– (MDA-MB-231 and BT 20) cell lines were obtained from the Centro di Biotecnologie Avanzate. Cells were cultured in DMEM supplemented with 10% FCS, 2 mmol/L L-glutamine, 10 g/L nonessential amino acids, 50 mg/L streptomycin, 1000 U/L penicillin, with (MCF-7) or without 10 mg/L insulin.
VERO African green monkey fibroblastoid kidney cells, permanent ECV304 cells, Hep2 human epithelial cells, 3T3 mouse embryo fibroblast cells, and nontumorigenic human breast MCF-10A cells were cultured in their appropriate growth media supplemented with 10% FCS, 50 mg/L streptomycin, and 1000 U/L penicillin. All cell lines were grown as monolayers in a humidified atmosphere at 37°C with 5% CO2. The experiments were performed with cells in the logarithmic phase of growth.
[3H]thymidine incorporation. Cells were seeded at a density of 30,000/well in 24-well tissue culture dishes and allowed to attach overnight. Duplicate samples of growing cells were treated for 48 h (ECV304, VERO, Hep2, and 3T3) or 72 h (MCF-7, MDA-MB-231, and MCF-10A) with increasing concentrations of juice (from 0.5 to 60 mL/L). During the last 4 h of treatment, cells were pulsed with 111 MBq/L of [3H]Thymidine (962 Bq/mmol) and processed as reported by Brandi et al. (22). The results were expressed as the mean dpm value in juice-treated samples compared with control samples.
Assay for cell viability. MCF-7 cells were seeded at the density of 6 x 105 cells/60-mm dish and allowed to attach overnight. Cells were treated with increasing concentrations of cauliflower juice; at 72 h from the beginning of treatment, total cells (floating and attached cells harvested by trypsinization) were stained with 0.4% trypan blue and counted using a hematocytometer.
Evaluation of apoptotic cell morphology. Cells were harvested at different intervals after juice treatment; floating and adherent cells were collected separately and apoptosis was assessed by staining the cells with Hoechst 33342 (2 mg/L; Sigma) for 15 min at 37°C and costained with ethidium bromide (5 mg/L) added for 5 min just before observation. After staining, the slides were observed under fluorescence microscopy. The percentage of cells with an apoptotic nuclear morphology was determined by scoring at least 300 cells for each sample.
Flow cytometric analyses of DNA content. Cells were seeded onto 75-cm2 plastic flasks and treated with increasing concentrations of juice. At different intervals (24, 48, and 72 h) from the beginning of juice treatment, cells were harvested and samples of 1 x 106 cells were fixed in 70% ethanol, washed in PBS, and stained with a solution containing 50 mg/L propidium iodide, 50 g/L RNAse, and 0.05% NP40 for 30 min at 4°C and then analyzed with a FACScan flow cytometer (Becton Dickinson). The cell cycle distribution was evaluated on DNA plots by CellFit software according to the SOBR model (Becton Dickinson).
Caspase activity measurement. Caspase catalytic activity was determined on cells exposed to 10 mL/L of cauliflower juice for 72 h by means of the Caspase-9/Mch6 Fluorometric Protease Assay Kit (MBL) and the Caspase-3 Assay Kit (BD Biosciences, Pharmingen), respectively. Total protein and the specific fluorogenic substrate (leu-glu-his-asp-7-amino-4-trifluoromethylcoumarin, LEHD-AFC, for caspase-9 and N-acetyl-Asp-Glu-Val-Asp-aldehyde-7-amino-4-methylcoumarin, Ac-DEVD-AMC, for caspase-3) were mixed for 1 h at 37°C. In the assay for caspase-3 activity, a negative control was obtained by incubating each sample in the presence of the inhibitor Ac-DEVD-CHO. The hydrolysis of substrates for caspase-9 and caspase-3 was monitored by spectrofluorometry at 505 and 440 nm, respectively.
Western blot analysis. CDK6, p27kip1, and pRb protein expression was assessed by Western blot. Untreated and juice-treated cells were lysed for 20 min on ice as described by Brandi et al. (22). From the total protein extracted, 25 µg were fractionated on SDS-PAGE, electrically transferred to nitrocellulose membranes, and incubated with anti-CDK6 (1:200), anti-p27kip1 (1:500), and anti-pRb (1:1000) antibodies (Santa Cruz Biotechnology). Proteins detected with enhanced chemiluminescence reagents were quantitated by ChemiDoc System (Bio-Rad). Actin was used to confirm equal protein loading.
RNA isolation, hybridization, and macroarray data analysis.
Gene expression was analyzed using a commercially available nucleic acid array, the PanoramaTM Human Apoptosis Gene Array (Sigma Genosys), consisting of 198 different human apoptosis-related genes. Total RNA was isolated by Trizol LS Reagent from cauliflower juice treated or untreated MCF-7 cells untreated. Complex probes were then prepared by reverse transcription in presence of 740 kBq[
-32P] dCTP, Unincorporated nucleotides were removed by Sephadex G-25 spin column.
The cDNA generated was incubated in hybridization solution and denatured at 95°C for 10 min. The membrane was hybridized overnight and finally exposed to a phosphor-imager screen. The results were normalized by dividing the intensity of the signal gene by the mean intensity of the signals of the 5 housekeeping genes that had a relatively constant expression level among the different samples.
Statistical analysis. The results are presented as means ± SEM of at least 3 separate experiments, each performed in duplicate. Data were analyzed using 1- or 2-way ANOVA as appropriate followed by Tukey’s post hoc test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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50% at a concentration of 3.5 mL/L and >90% at 25 mL/L. Juice administered for 72 h at the same concentration as for MCF-7 cells dose dependently inhibited the growth of ER– cells (Fig. 1B). To confirm these data, we repeated the juice treatment on other ER+ (BT20) and ER– (BT474) human breast cancer cell lines. The results obtained (not shown) were similar to those in Figure 1, and the ranking of sensitivity to juice-induced inhibition of [3H]thymidine incorporation, evaluated as the 50% inhibitory concentration (IC50), was as follows: BT474 > MCF-7 = MDA-MB-231 > BT20 (BT474 vs. MCF-7, P < 0.05; MCF-7 vs. BT20, P < 0.05).
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IC50), whereas a proportional increase in the percentage of necrotic cells (up to 25% at 20 mL/L of juice, P < 0.05), as shown by loss of viability and increase of debris, occurred even at concentrations above the IC50 (Fig. 5). Because flow-cytometric analyses conducted in MCF-7 and in MDA-MB-231 cells after treatment with concentrations of juice that were able to reduce cell proliferation (10 mL/L) did not indicate the presence of a detectable pre-G1 apoptotic cell population (Table 1), we analyzed apoptosis; we also evaluated nuclear chromatin condensation and nuclei fragmentation, both common features in apoptotic cells. After 48 and 72 h of treatment, the analyses of fluorescent nuclei revealed that only a very small number (<3%) of juice-treated cells displayed the typical apoptotic features. Moreover, at the molecular level, there was no evidence of treatment-induced apoptosis. In fact, exposure of MDA-MB-231 cells to 10 mL/L of cauliflower juice for 72 h did not increase the caspase-9 and caspase-3 catalytic activities compared with untreated samples (not shown). Similarly, caspase-9 activity was not enhanced by juice treatment in MCF-7 cells (not shown). In these cells the analysis was limited to caspase-9 because they do not express caspase-3 (42). These results indicate that growth arrest in juice-treated cells is associated with a cytostatic mechanism and with necrotic cell death that occurs at the higher concentrations utilized.
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Expression of cell cycle and apoptosis-related genes. In the treated cells, the expression of several apoptosis-related genes was upregulated (data not shown). Among genes whose products have antiapoptotic functions, we found overexpression of Survivin (150% increase). Among genes whose products have a proapoptotic function, we found overexpression of tumor necrosis factor receptor associated death domain (TRADD, 180% increase), tumor necrosis factor receptor associated factor 2 (TRAF 2, 120% increase), and BID (170% increase), and a decrease in the expression of Fas-associated death domain (FAAD, 50% decrease).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Our data demonstrate that the juice was able to block the proliferation of both ER+ and ER– breast cancer cells and suggest that distinct growth-inhibitory pathways may be involved, with a unique common target represented by the marked decrease in endogenous Rb phosphorylation.
In fact, at the molecular level, juice selectively reduces the levels of CDK6 and increases the level of the CDK6 inhibitor p27 in ER+ cells. Because the endogenous substrate for G1 CDK-6 is the Rb, the reduced level of CDK-6 decreases the amount of Rb phosphorylation; as a consequence, Rb in the hypophosphorylate form binds and inactivates E2F transcription, thus arresting the cell cycle. In ER– cells, CDK6 and p27 remain unchanged after treatment, suggesting that other proteins and/or kinases may be involved in reducing the level of Rb phosphorylation and consequently the block of cell proliferation. Hong et al. (47) reported that 3,3'-diindolylmethane, an indole derived from Brassica, induced G1 cell cycle arrest in breast cancer cells through a mechanism involving p21 and that the induction of p21 expression is independent of the estrogen receptor.
A mechanism of growth arrest in breast cancer cells involving the G1 cell cycle proteins p27 and CDK6 was reported by Cover et al. (19) for I3C and by us for the I3C tetrameric derivative (22). These compounds induced the overexpression of p27 and also reduced the levels of CDK6 in ER– cells. Furthermore, cell proliferation arrest was associated with a block of cells in the G1 phase of the cell cycle, a situation that was not observed in juice-treated cells. It is important to mention, however, that in our previous study, we also observed that a pRb-negative breast cancer cell line (BT-549) was highly sensitive to the tetramer even in the absence of any G1 phase cell accumulation (22). Therefore, in agreement with the data obtained with the tetramer in BT-549 cells, it is unlikely that CDK6 is the only important molecular target for the activity of Brassica juice.
The antiproliferative activity exerted by juice appears to be due to both cytostatic and cytotoxic mechanisms, depending on juice concentrations. At lower concentrations (IC50), juice induced an appreciable inhibition of proliferation without any significant loss of cell viability, suggesting growth arrest via a cytostatic mechanism, whereas at the highest concentrations (>IC50), juice induced significant cell death. In this study, we also utilized concentrations of juice > IC90 and found that an appreciable fraction of cells accumulated in the S and G2/M phases. Cell death at the highest concentrations, however, may also be due to the specific toxic effect of juice, as reported for ITCs (48). Several studies indicated that some compounds naturally present in cauliflower, such as indoles, ITC, and sulforaphane, induce apoptosis in various types of cancer cells (15,17,48–53) including breast cancer cells (15,17,50). In our study, we demonstrated that raw cauliflower juice is unable to induce apoptosis in ER+ and ER– breast cancer cells, at least during the observation period used in this work, and that cell death in our experimental conditions is caused by necrotic events. The lack of apoptosis induction observed after cauliflower juice treatment is also compatible with data obtained from macroarray analysis in MCF-7 cells, indicating an increased expression of both anti- and pro-apoptotic genes.
Taken together these results suggest that several mechanisms may be responsible in inducing cell growth inhibition in breast cancer cells exposed to cauliflower juice, and the anticarcinogenic activity of juice differs at least in part from that suggested for I3C and its tetramer (19,22).
An important finding in this study is that cauliflower juice displays a preferential activity against breast cancer cells. Indeed, comparing the IC50 for MCF-7 cells with those obtained in the other mammalian cell lines tested, breast cells were from 3.7 to 8.7 times more susceptible to juice action. Because we used cell lines with a high replicative rate, this observation strongly suggests that, at the concentration that blocks the growth of breast cancer cells, it is unlikely that the juice could damage normal cells in vivo. However, because different culture media were use to grow different cell lines, we cannot exclude the possibility that an interaction between extract and component(s) of specific media can contribute at least in part to the different sensitivity observed among cell lines. The evidence that a minor antiproliferative effect was observed in the nontumorigenic human breast cell line only at a high extract concentration, one that is not physiologically relevant, corroborates the selective activity of cauliflower juice for breast cancer cells.
Many cruciferous vegetables, including cauliflower, are cooked before consumption. However, cooking destroys myrosinase, thus reducing bioavailability of ITC and other potential chemopreventive compounds. To avoid this problem, we processed cauliflower leaves to obtain a dry extract and demonstrated that the dry powder obtained with a procedure conducted at a low temperature, preserves the antiproliferative activity, which remains stable over an extended time period. Thus, this extract could be utilized by the nutraceutical industry for chemoprevention. In contrast, the spray-drying technique destroyed this activity. We do not know which substance is destroyed by that treatment. However, several compounds, including those responsible for antioxidant activity, were lost (not shown).
This study, to our knowledge, reports for the first time that cauliflower acts as a potent inhibitor of both ER+ and ER– breast cancer cell growth. Although the mechanisms by which growth inhibition occurs must be investigated further, results suggest that cauliflower contains compounds having anticarcinogenic properties that are likely to act through both ER-independent and ER-dependent pathways. Whole juice rather than a single purified compound was used in this study to take advantage of all of the active compounds present in cauliflower. The possibility of using this edible plant, widely present in nature, for the chemoprevention of some types of cancer in subjects who are considered to be at high risk or as a chemotherapeutic agent is of great interest and must be examined further.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by Banca di Credito Cooperativo di Fano and Fanoateneo.
4 Abbreviation used: CDK, cyclin-dependent kinase; DMSO, dimethyl sulfoxide; ER, estrogen receptor; FCS, fetal calf serum; GLS, glucosinolates; I3C, indole-3-carbinol; IC50, 50% inhibitory concentration; ITC, isothiocyanate; pRb, retinoblastoma protein.
Manuscript received 12 May 2004. Initial review completed 29 June 2004. Revision accepted 11 March 2005.
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