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Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL 61801
3To whom correspondence should be addressed. E-mail: kws{at}uiuc.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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10-fold increase over control), and disrupted polymerization of mitotic microtubules in vivo. Subsequent exposure of purified bovine brain tubulin to relatively high doses of SUL significantly (P < 0.05) inhibited both tubulin polymerization rate (51% of control) and total tubulin polymerization (78% of control) in vitro. Additionally, polymerization of purified tubulin exposed to isothiocyanate-containing analogs of SUL was similarly inhibited. Taken together, these findings indicate that SUL has mammary cancer suppressive actions involving mitotic cell cycle arrest and suggest a mechanism linked to the disruption of normal tubulin polymerization and/or more subtle effects on microtubule dynamics.
KEY WORDS: • mitotic arrest • microtubule polymerization • human mammary carcinoma
Sulforaphane [1-isothiocyanato-4-(methyl-sulfinyl)butane] is an isothiocyanate derived from broccoli and other cruciferous vegetables, which is liberated subsequent to hydrolysis of the glucosinolate compound glucoraphanin by the intrinsic plant enzyme thioglucoside glucohydrolase (1). Sulforaphane (SUL)4 has received much attention in the area of nutrition and breast cancer research for several reasons. First, some epidemiologic studies indicate an inverse relation between vegetable intake and breast cancer risk (2–4); SUL is a known component of commonly consumed vegetables, and is especially abundant in broccoli (5). Second, oral SUL was shown to inhibit chemically induced mammary tumor formation in rats (6). Last, SUL was shown to both induce phase II detoxification enzymes (7) and competitively inhibit the phase I enzyme cytochrome P450 isozyme 2E1, an enzyme participating in carcinogen activation (8). Thus considerable attention has focused on the potential of SUL as an inhibitor of cancer initiation. More recently, however, SUL was shown to inhibit neoplastic cell proliferation, block cell cycle progression at G2/M, induce apoptosis, and modulate signal transduction pathways, suggesting that it may thereby also act as an effective inhibitor of cancer promotion/progression (9).
The first reported evidence of SUL’s antiproliferative action was presented in the publication of Gamet-Payrastre et al. (9). In those studies, HT29 human colon cancer cells exposed to 15 µmol/L SUL were shown to both accumulate within the G2/M phase of the cell cycle and express elevated cyclin B1 protein within 24 h. Sulforaphane-treated cells also displayed elevated Bax expression, poly(ADP-ribose) polymerase cleavage, and nuclear chromatin condensation within 48 h, indicating induction of apoptosis. Phenethyl isothiocyanate, which like SUL, exists as a glucosinolate in a number of cruciferous vegetable, was also observed to induce similar cellular responses, including dose- and time-dependent apoptosis associated with caspase activation (10,11).
The activity of the cyclin-dependent kinase p34cdc2 or cdc2 (CDK1) was identified as being key to both the regulation of cell cycle progression from G2 to mitosis, and the initiation of cell death pathways (12–15). Specifically, activation of cdc2 is generally thought to involve phosphorylation at threonine 161 (16–19), binding of cyclin B1 (18,20–22), and dephosphorylation at both threonine 14 and tyrosine 15 (18,21). Phosphorylation and subsequent dephosphorylation of cdc2 at threonine 14 and tyrosine 15 occurs, respectively, via the action of Wee1 (tyrosine kinase) and Myt1 (threonine and tyrosine kinase) kinases (21,23–29) and cdc25C phosphatase (30–32). An active cdc2 then phosphorylates a number of cellular substrates such as H1 histone, nuclear lamins, vimentin, caldesmon, and nucleolin, which allow progression into and through mitosis (33–41). The end of mitosis and return to the G1 phase is marked by inactivation of cdc2, which occurs via dephosphorylation of threonine 161 (catalyzed at least in part by type 1 phosphatases) (42) and degradation of cyclin B1 (43).
Of additional interest is the recognition that inappropriate activation of cdc2 can lead to a form of cell death known as "mitotic catastrophe" (14,15). For example, in murine mammary carcinoma FT210 cells, elevated cdc2 activity was shown to be required for fragmentin-2–induced apoptosis (15). Moreover, Fotedar et al. (44), using a murine T-cell hybridoma (A11 cells), demonstrated an association among activation of apoptosis, elevated cdc2 kinase activity, and arrest of the cell cycle at G2/M. Thus, the activity of cdc2 at the G2/M transition is an important factor in determining whether the cell cycle arrests before or during mitotic progression, and/or whether apoptosis is initiated.
A variety of diverse naturally occurring compounds with heterogeneous chemical structures were observed to arrest cells within the M-phase via dose-dependent mechanisms involving microtubule disruption and to trigger eventual cell death by apoptosis. Compounds such as the Vinca alkaloid vinblastine, at relatively low concentrations, interact directly with tubulin proteins and induce mitotic cell cycle arrest as a result of interference with normal microtubule dynamics. On the other hand, relatively high doses also trigger mitotic arrest, yet the mechanism involves pronounced inhibition of tubulin polymerization and produces gross depolymerization of mitotic microtubules (45). Mitotic arrest triggered by low vinblastine doses was characterized phenotypically as prometaphase inhibition in which 1 or 2 chromosomes fail to congress to the metaphase plate, with kinetochore microtubules shorter, and astral microtubules longer than normal. With increasing vinblastine concentrations, these aberrations of the normal metaphase become more pronounced, and the bipolar spindle is lost to one that is monopolar. Ultimately, at relatively high doses, vinblastine interacts with lower-affinity binding sites on tubulin proteins and produces mitotic figures consisting of condensed chromosomal aggregates essentially devoid of microtubules. The Vinca alkaloid vinblastine is one of the 3 now identified mechanistic classes (i.e., Vinca alkaloids, colchicines, and taxanes) of natural and semisynthetic tubulin-disrupting compounds. Although these classifications are based upon tubulin/microtubule binding characteristics and the subsequent phenotype of mitotic arrest, a fundamental requirement for newly discovered mitosis-arresting compounds to be classified as direct tubulin-disrupting agents is their ability to inhibit the polymerization of purified tubulin in an in vitro system lacking microtubule-associated proteins and serum growth factors.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Some experiments were designed to examine and confirm the effect of SUL on proliferation and cell cycle distribution in other [estrogen receptor positive (ER+) and estrogen receptor negative (ER–)] human mammary cancer cell lines. To prevent potential confounding effects on proliferation resulting from the use of various cell culture media, the ER+ cell lines MCF-7 and ZR-75, and the ER– cell lines BT-20 and MDA-MB-231, were each propagated in the following medium: MEM Eagle (with 10 mmol/L HEPES and Earle’s BSS adjusted to contain 0.1 mmol/L nonessential amino acids and supplemented with 6 mg/L bovine insulin and 3.75 mg/L hydrocortisone) containing 5% heat-inactivated fetal bovine serum. In these experiments, SUL or DMSO vehicle was administered 48–72 h after seeding.
[3H]Thymidine incorporation assay of DNA synthesis. MCF-7 cells were seeded at a density of 1.25 x 104 cells/cm2 in Costar 48-well plates. Cultures were exposed to increasing dosages of SUL or DMSO in the presence of [3H]thymidine (1 µCi). After 48 h, the medium was removed and the cells washed with PBS, treated with 5% trichloroacetic acid (15 min), and the precipitate washed with absolute methanol; after 5 min, it was dissolved in 88% formic acid (Fisher) for 5 min at room temperature. The samples were measured in a Beckman LS6500 Scintillation Counter in the presence of ScintiVerse SX16–4 (Fisher) counting cocktail. To compare the effect of SUL on proliferation of ER+ and ER– human mammary cancer cell lines, MCF-7, ZR-75, BT-20, and MDA-MB-23 cells were each similarly examined in additional experiments (see above).
Cell cycle analysis and quantification of mitotic figures.
MCF-7 cells were seeded (1 x 106 cells/75 cm2 culture flask), treated with either 15 µmol/L SUL or DMSO and, at the indicated times, harvested via trypsinization. The cells were then washed with PBS, fixed in 70% ethanol (at least 12 h, 4°C), washed again with PBS, and resuspended in modified Vindelov’s DNA staining solution (100 mg/L RNase A, 0.1% NP-40, 250 mg/L propidium iodide, in PBS) at a density of 109 cells/L. Fluorescence was then measured 1 h later using a Coulter XL Flow Cytometer. To compare the effect of SUL on cell cycle distribution of ER+ and ER– human mammary cancer cell lines, MCF-7, ZR-75, BT-20, and MDA-MB-23 cells were each similarly examined in additional experiments (see above).
To quantify cells in mitosis, MCF-7 cells were seeded, treated, harvested, and washed (as above), before being resuspended in PBS, and cytospun onto microscope slides. Cells were then stained with Wright-Giemsa dye, and the percentage of mitotic figures determined by analyzing a minimum of 10 cells in each of 6 fields/slide under light microscopy.
Western blot analysis. MCF-7 cells were seeded (1 x 106 cells/75 cm2 culture flask) and treated with either 15 µmol/L SUL or DMSO for the indicated times. Cells were then washed twice with TBS, harvested in ice-cold lysis buffer [1% SDC, 1% Triton X-100, 0.01% SDS, 150 mmol/L NaCl, 50 mmol/L Tris (pH 7.5), 0.5 mmol/L EDTA, 50 mmol/L NaF, 10 mmol/L NaPPi, 0.5 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride, 20 mg/L aprotinin, 20 mg/L leupeptin, 20 mg/L pepstatin], sonicated, and centrifuged (15,000 x g for 15 min at 4°C). The supernatant was assayed for total protein (bicinchoninic acid, Pierce), and aliquots of equal protein concentration were fractionated by SDS-PAGE (7.5%) and transferred to nitrocellulose membranes. The membranes were blocked and then incubated with either a polyclonal antibody to cyclin B1 (Santa Cruz Biotechnology, sc-752) or a polyclonal phospho-specific antibody to histone H1 (Upstate Biotechnology, #06–597). After incubation with an horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology) from the appropriate species, immunodetection was carried out using chemiluminescence (ECL, Amersham Life Science). The exposed X-ray film was then scanned into Adobe Photoshop, and densitometric analysis was performed on an Apple Macintosh G4 computer using the public domain NIH Image program [developed at the U.S. National Institutes of Health and available on the Internet (46)]. Equal loading of total protein was confirmed by reprobing membranes for actin (sc-1616).
Immunofluorescence staining.
MCF-7 cells were sparsely plated on 12-mm round glass coverslips and exposed to 15 µmol/L SUL or DMSO for 24 h. After being washed once with PBS, cells were fixed with glutaraldehyde (1% in PBS, for 10 min at room temperature) followed by sodium borohydride (1 g/L in PBS, twice for 10 min each). Cells were then rinsed 3 times with PBS, permeabilized with wash buffer (0.1% Triton X-100, 1% bovine serum albumin in TBS) for 10 min, and incubated with a monoclonal antibody to 
-tubulin (Oncogene Research Products, CP06) for 30 min at room temperature. After 5 washes with wash buffer, cells were incubated with a fluorescein isothiocyanate–conjugated goat anti-mouse secondary antibody (Santa Cruz Biotechnology, sc-2078) and the DNA counterstained with DAPI (Calbiochem, #268298, 10 mg/L) for 30 min at room temperature. Finally, samples were washed again 5 times with wash buffer, washed once with dH2O, mounted on microscope slides with mounting medium (ICN, #622701), and analyzed by fluorescence microscopy.
In vitro tubulin polymerization assay. The effect of the racemic D, L-sulforaphane, as well as that of the individual D- and L-sulforaphane isomers, sulforaphane nitrile, erucin [1-isothiocyanato-4-(methylthio) butane], and sulforaphene [4-isothiocyanato-1-(methylsulfinyl)-1-butene], on tubulin polymerization in vitro was examined. These compounds were chosen to determine whether a particular isomer or chemical moiety present within SUL might impart the tubulin-disrupting activity observed in whole-cell culture. Bovine brain tubulin (>99% pure, Cytoskeleton, #BK006) was incubated in the presence of D, L-sulforaphane (ICN), sulforaphane nitrile (a generous gift from Dr. Matthew Wallig, University of Illinois College of Veterinary Medicine), D-sulforaphane, L-sulforaphane, erucin, or sulforaphene (each from LKT Labs), or DMSO (vehicle) in a 96-well plate at 37°C. Absorbance readings (n = 3/group) were taken at 340 nm using a Molecular Devices Spectra Max Plus plate reader each minute for 1 h according to kit instructions. The rate of tubulin polymerization (milli-OD340 units/min) was then calculated for each sample from the linear portion of the kinetics curve, and group mean rates were analyzed for statistical significance. In addition, groups were analyzed for differences in total tubulin polymerization (maximum milli-OD340 units) at the end of the 1-h time course.
Statistical analysis.
Data derived from experiments comparing only 2 means were analyzed by an independent t test and the accompanying test for homogeneity of variance. In the case of experiments requiring the comparison of >2 means, ANOVA was performed followed by Tukey’s Studentized Range (HSD) for the pair-wise comparisons. All statistical calculations were completed using the SAS System (SAS Institute). Replicated experiments (each carried out at different times and with n 
3/group) were analyzed independently, and the data presented are those obtained in a single experiment. Significant differences were established at P < 0.05 and are depicted in figures by an asterisk (*).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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15 µmol/L yielded effects that did not differ from one another, the 15 µmol/L dose was chosen for use in cell culture to further characterize the time-dependent actions of SUL in MCF-7 cells. Further, we found that SUL also induced significant (P < 0.05) inhibition of DNA synthesis in both ER+ and ER– cells at doses as low as 1 µmol/L (Fig. 2).
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80% of cells were within the G1 phase of the cell cycle, whereas the controls exited G1 and completed 1 cycle within 45 h. Moreover, control cells peaked within G2/M after 12 h of DMSO vehicle treatment, yet cells exposed to 15 µmol/L SUL did not reach peak G2/M until 18 h later. This SUL-induced delay in G2/M onset was associated with an elevated percentage of cells within the G1 phase after 12 h of treatment (Fig. 4A). After 24 h of treatment, however, SUL induced significant G2/M accumulation (Fig. 4B), and a similar G2/M block was observed at later times (48 and 72 h, data not shown). Asynchronous, randomly cycling MCF-7 cultures were also sensitive to SUL-induced G2/M arrest within 24 h (Fig. 4D), yet the magnitude of the block was less than with synchronized cells. In addition, asynchronous cells treated with SUL for 12 h displayed no accumulation at G1 (Fig. 4C). Furthermore, SUL doses as low as 5 µmol/L induced similar G2/M accumulation in each of the ER+ and ER– cell lines (Fig. 5). Cyclin B1 protein and histone H1 phosphorylation were elevated (P < 0.05) after 24 h of 15 µmol/L SUL treatment in synchronized MCF-7 cultures (Fig. 6), indicating cdc2 kinase activation and cell cycle progression beyond G2. MCF-7 cells exposed to 15 µmol/L SUL and stained with Wright-Giemsa dye displayed, within 24 h, an abundance of mitotic figures in a prophase/prometaphase-like state with chromosomes condensed yet not aligned at the metaphase plate; by 72 h, aberrant nuclear formation and micronucleation were evident (Fig. 7).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Within 24 h, SUL-induced G2/M accumulation in synchronized MCF-7 cells was associated with elevated cyclin B1 protein and evidence of cdc2 kinase activation (Fig. 6). Moreover, SUL-treated cells displayed chromosomes scattered in a prophase- or prometaphase-like state (Fig. 7D), indicating cell cycle arrest early within mitosis yet before metaphase. Interestingly, these early mitotic figures were devoid of normal spindle microtubules (Fig. 8), suggesting a mechanism of SUL-induced mitotic arrest involving perturbation of tubulin polymerization and/or microtubule dynamics, as was reported previously for other naturally occurring antimitotic agents (45). In addition, we also observed that exposure of MCF-7 cells to SUL for periods in excess of 24 h produced aberrant mitoses and micronucleation (Fig. 7F), characteristic of cell death by mitotic catastrophe (48). These findings agree with those from our previous study in which we characterized morphological as well as molecular markers of SUL-induced apoptosis associated with M-phase arrest in mouse mammary carcinoma cells (47).
Experiments conducted in vitro demonstrated that relatively high SUL doses (100 µmol/L) significantly inhibited the rate of polymerization, as well as the maximum polymerization, of purified tubulin proteins (Fig. 9B, Table 1). Although the doses required to produce gross inhibition of tubulin polymerization in vitro are certainly higher than those required to induce Phase II detoxification enzymes (49,50) and likely higher than those achievable via dietary intakes (51), the results obtained here with purified tubulin strongly suggest that microtubule polymerization (or perhaps microtubule dynamics at lower doses) is a mechanistic target that may facilitate SUL’s antiproliferative action. As a chemically reactive thiol, however, SUL (as well as other isothiocyanates) may react and inhibit enzymes other than those involved in tubulin dynamics. Also, it should be noted that some isothiocyanates were reported to trigger DNA damage and may promote carcinogenesis (52); hence, different experimental conditions may likely lead to dichotomous results.
Similar to the results obtained with the racemic D,L-sulforaphane, tubulin polymerization in vitro was significantly inhibited in the presence of the individual D- and L-sulforaphane isomers, as well as by the isothiocyanate-containing SUL analogs, erucin and sulforaphene (Fig. 9C, Table 1). However, sulforaphane nitrile, an alternate breakdown product of the glucoraphanin precursor of SUL, which lacks the isothiocyanate moiety (53), did not significantly alter tubulin polymerization. These results suggest that the isothiocyante group may be responsible for imparting the efficacy of SUL as an inhibitor of tubulin polymerization. Detailed characterization of both the biochemical nature of SUL’s interaction or binding with tubulin proteins and the effect of relatively low doses of SUL on tubulin dynamics in the whole cell is warranted. Also, other naturally occurring isothiocyanate compounds that were reported to induce G2/M arrest (54,55) should be examined for potentially similar mechanisms leading to perturbation of normal tubulin polymerization and/or dynamics, and thereby possibly facilitating mitotic, rather than G2, cell cycle arrest.
In conclusion, this study is the first to report the effectiveness of SUL as an inhibitor of human mammary carcinoma proliferation and to provide confirmatory evidence of a recently identified novel mechanism of SUL action. In addition to SUL’s previously reported inhibitory effects on cancer initiation (6–8), this naturally occurring compound now appears to also affect human breast cancer promotion/progression via mechanisms involving disruption of mitotic microtubules, cdc2 kinase activation, and arrest of the cell cycle within G2/M. It will now be of substantial interest to determine the nature of SUL’s interaction with cellular targets responsible for triggering mitotic cell cycle arrest to ascertain the implications of SUL intake as a result of either the consumption of cruciferous vegetables in the diet or potential chemoprevention strategies directed toward persons deemed to be at high risk for developing malignancy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by the University of Illinois Agricultural Experiment Station and United States Department of Agriculture National Needs Fellowship.
4 Abbreviations used: DMSO, dimethyl sulfoxide; ER, estrogen receptor; SUL, sulforaphane.
Manuscript received 5 January 2004. Initial review completed 30 January 2004. Revision accepted 9 June 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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