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,**,**,
,**,,3
* Departments of Biochemistry and Molecular Genetics and Pharmacology and Toxicology, ** Purdue–University of Alabama at Birmingham Botanicals Center for Age-Related Disease and Comprehensive Cancer Center Mass Spectrometry Shared Facility, University of Alabama at Birmingham, AL 35294
3 To whom correspondence should be addressed. E-mail: sbarnes{at}uab.edu.
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • genistein • affinity chromatography • peptide sequence
There are very different rates of death from cancer around the world. These persist even when the mortality data for each country are adjusted for the different distributions of age groups. Studies of the migration of low-risk groups to the U.S. demonstrate that cancer risk in such immigrants either rises to the rates observed in Americans in the same generation (e.g., prostate cancer) or in the next generation (e.g., breast cancer) ( 1, 2). This observation indicates that genetic factors contribute little to the risk of these cancers. Instead, environmental factors (e.g., diet, air and water quality, exercise, etc.) predominate. Considerable effort has been placed on identifying factors that alter cancer risk that are delivered by the diet ( 3). Eating habits in low-risk countries are typically very different from those practiced in the U.S. Regrettably, a reverse immigration of American culture to the formerly low-risk countries is occurring and is causing a sharp elevation in the rates of certain cancers (e.g., breast cancer in Japan) ( 4).
A low risk of breast cancer occurs in most of Southeast Asia ( 1). Foods based on soy represent a distinct dietary difference between Southeast Asians and Americans. The role of soy in breast cancer has been the subject of intense effort for the past decade ( 5– 7). Although several anticancer factors in soy have been proposed ( 5), most of the research has focused on the isoflavones because of their unique occurrence in soy as compared to the rest of the diet ( 8). When the appreciation of their potential anticancer role first began, isoflavones were already well known for their infertility effects on certain animals, principally sheep ( 9) and captive cheetahs ( 10). They also were shown to have weak uterotrophic effects in immature rodents ( 11). Because of this and their structural resemblance to mammalian estrogens, isoflavones were described as phytoestrogens.
Experiments conducted using rodent models of breast cancer reveal that both soy protein ( 12, 13) and genistein ( 14, 15) increase the latency before tumors appear and reduce the number of mammary tumors. Lamartiniere's group ( 16, 17) has shown that exposure of the mammary in rodents to high dietary amounts of genistein before puberty exerts life-long chemopreventive effects. Two epidemiological studies have reported a similar effect in Asian adolescents who consumed a soy diet ( 18, 19): they had a lower risk of breast cancer than those who either did not eat soy foods in adolescence or only did so in adult life ( 19).
The apparently positive benefit of a soy diet is offset by results from another animal model of breast cancer, namely, the ovariectomized nude mouse that has been orthotopically transplanted with human breast cancer MCF-7 cells. In this model, genistein ( 20) and a soy protein diet ( 21, 22) increase the growth rate of the cancer cells, which is an effect analogous to that of estrogens. Although this model has experimental design issues that may account for the observed result (e.g., ovariectomy is not equivalent to the effects of menopause, and the loss of the thymus removes the interaction of steroids with the immune system), it is nonetheless disturbing. Results using this model provoke critical questions such as, What is(are) the molecular target(s) of genistein? and Is genistein an estrogen? A more global question is, What is an estrogen?
In fact, genistein has been reported to have a wide range of nonestrogenic effects on numerous biochemical targets in cells (
23,
24). This arose largely because of the identification of genistein as a protein tyrosine kinase inhibitor (
25). Investigators have used it in many systems because of this property. In most cases, cells are very tolerant of genistein. This has enabled investigators to use very high concentrations (up to 100 µmol/L) in cell culture and in doing so has allowed genistein to interact with low-affinity and probably nonphysiological targets. However, the plasma concentration in those who consume a soy diet rarely exceeds 1 µmol genistein/L (
26,
27). Of that, 
10 nmol of total genistein/L is in the unconjugated or bioactive form. Thus, the long list of existing proposed mechanisms attributed to genistein are unlikely to occur in vivo. Of the mechanisms thus far identified, only the affinity of genistein for estrogen receptors (
28) falls in this physiological concentration range.
New approaches are needed to assess the effects of xenobiotics in foods, specifically, approaches that do not presuppose what the targets might be. Assembly of the genomes of humans and other mammals over the past 10 years has led to a revolution in science. With the introduction of printed arrays of representatives of all the known genes in an organism, it is now possible to assess the effects of specific compounds on global gene expression. Although the quantitative relationship between mRNA expression and changes in protein concentration is not a simple one, it is anticipated that many of the unexpected players in the mechanism of action of a compound will be revealed by DNA microarray analysis. In a precursor to such studies, Diel et al. ( 29) has already shown that the pattern-of-expression changes of five selected genes are different for each of five putative natural, synthetic and plant estrogens.
In this context, a report (
30) on the comparative effects of different estrogens on gene expression in the developing rat uterus is of considerable interest. In this study, 7,000 rat genes were examined. A total of 26 and 35 genes for 17
-ethinyl estradiol (EE)4 and bisphenol A (BPA), respectively, exhibited dose-dependent changes in expression. In contrast, for genistein the number was 227, the majority of which were downregulated. Examination of the genes undergoing the largest changes in expression by genistein (
Table 1) revealed only one that was previously implicated by traditional biochemistry techniques. The increase in expression of the progesterone receptor (PgR) is consistent with the presumed estrogenic effect of genistein and also is observed for EE and BPA. What is more difficult to assess is which of the gene expression changes are direct effects of genistein and which are due to distal actions provoked by genistein. Indeed, it is possible that the critical target may only be affected to a small degree: it may amplify its effect by increasing the rates of expression of other genes or modification of other proteins. Thus, the increase in expression of both PgR and Dermol (a helix-loop-helix nuclear transcription factor) may have profound effects on cell function.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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Synthesis of 2-carboxygenistein
This derivative of genistein was synthesized in accordance with the procedure of Yoder, Cheng and Burroughs ( 33) with minor modifications. The product was a bright-yellow powder. It was > 99% pure as assessed by a reverse-phase high-performance liquid chromatography method ( 8). Two ions, a molecular ion [mass-to-charge ratio (m/z) of 313 [M-H]-] and a fragment ion (m/z of 269 [M-H-CO2]-), were observed when 2-carboxygenistein was analyzed by electrospray ionization on a PE-Sciex API 3 triple quadrupole mass spectrometer (Concord, ONT, Canada).
The 2-carboxygenistein affinity phase ( Fig. 1) was made by first forming an activated ester by reacting 2-carboxygenistein in water at room temperature with a 10-fold molar excess of EDAC until the yellow solid was completely dissolved. The coupling reaction was accomplished by adding AH-Sepharose 4B and rocking the mixture gently at room temperature overnight. The extent of coupling was assessed by conducting the Keiser test (reaction with ninhydrin) before and after coupling. Coupling was repeated until satisfactory efficiency was achieved.
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Human breast cancer MCF-7 cells were obtained from the American Type Culture Collection (Manassas, VA). They were maintained in Eagle's modified essential media (MEM) and buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; 5% (v/v) fetal bovine serum and antibiotics (100 U of penicillin/mL and 100 µg of streptomycin/mL) were also added. For experiments in the absence of fetal calf serum, insulin-selenium-transferrin and epidermal growth factor were added to the cell culture medium. All cells were cultured as monolayers (passed every 6–8 d) in an air/CO2 (95:5%), water-saturated atmosphere. Cells were harvested and placed in a lysis buffer (1% Nonidet P-40, 150 mmol NaCl/L, 50 mmol Tris-HCl/L, pH 7.5 plus phosphatase and proteinase inhibitors). The lysate was passed through a syringe with a 20-gauge needle 10 times to shear nucleic acids and was stored at -80°C until it was used. Lysates were centrifuged at 3,000 x g for 15 min at 4°C to remove cell debris before chromatography.
Chromatography
Cleared lysates were dialyzed with a 10,000-Da cutoff membrane against 20 mmol Tris-HCl/L, pH 8.0 buffer and were passed over a 5-mL DEAE-Sephadex column that was preequilibrated with the same buffer. The DEAE-Sephadex column flow-through was immediately passed over a 1-mL 2-carboxygenistein–agarose affinity column. Unbound proteins were eluted with 20 mmol Tris-HCl/L, pH 8.0 buffer. Further elution of loosely bound proteins was conducted by applying a gradient of 0–100 mmol NaCl/L in the Tris-HCl buffer. Affinity elution was conducted using a 1 mmol 2-carboxygenistein/L solution of 100 mmol NaCl/L with 20 mmol Tris-HCl/L, pH 8.0 buffer. All other proteins that were bound to the affinity phase were eluted with 0–2 mol NaCl/L with 0.5 mol NaSCN/L.
Proteins in the various fractions were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis using a 10% polyacrylamide gel. Samples were boiled for 5 min in Laemmli sample buffer ( 34). After the gel was fixed with methanol/water/acetic acid (50:45:5, v/v/v), protein bands were visualized either with G250 Coomassie blue stain (Pierce Chemical Co., Rockford, IL) or silver staining ( 35).
Protein analysis
The affinity-purified protein band (from electrophoresis of eight aliquots of concentrated solutions of p38) was transferred from a SDS-PAGE gel (unfixed) to a polyvinylidene difluoride membrane using an electroblotting procedure in 3-(cyclohexylamino)-1-propanesulfonic acid/methanol buffer ( 36). The membrane was carefully washed to remove SDS and was sent to Commonwealth Biotechnology (Richmond, VA) for N-terminal peptide sequencing.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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A large number of proteins passed through both the DEAE-Sephadex and the 2-carboxygenistein–Sepharose 4B column matrices. An extensive wash with 100 mmol NaCl/L of Tris-HCl, pH 7.5 eluted a large number of weakly bound proteins. In contrast, with 1 mmol 2-carboxygenistein/L, only two proteins were eluted: p38 and p66 ( Fig. 2).
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/ß-structural class of proteins, representatives of which are dehydrogenases, kinases and rhodanases (
37). Attempts to amplify by PCR the coding sequence of p38 were unsuccessful (M. Belenky, unpublished data). A later BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) of this sequence revealed in 1996 (
38) a corresponding N-terminal sequence of a protein isolated from synovial fluid (see
Table 2). Since then, two other groups of investigators have reported similar sequences (
Table 2) for proteins isolated from fibroblasts (
39) and from rat brain (
40). In the latter case, the protein was isolated using a cotonine-affinity matrix in a method analogous to the one described in the present study. Related proteins have been described in several plant species (
32,
39).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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Despite all the changes in gene expression caused by genistein ( 28), its protein targets are the most important to understand. The use of 2-carboxygenistein to form an affinity phase takes advantage of the introduction of the carboxyl group because it enables the coupling of the genistein molecule to the AH-Sepharose without using the hydroxyl groups thought to be critical for the biological action of genistein ( 41, 42).
Although only p38 was recovered from MCF-7 cell lysate, it should be pointed out that the initial focus of these experiments was on proteins with isoelectric point values > pH 7.5, namely, those that passed through the DEAE-Sephadex column. Future experiments will be conducted on proteins that bind to the DEAE-Sephadex column as well as to other cell types.
The discovery that p38 was a DING protein was an unexpected surprise. Initially, there was no matching peptide sequence in the NCBI or Swiss Prot databases. However, in late 1996 Hain et al. ( 38) published the N-terminal sequence of a p205 synovial T-cell stimulatory protein. From this it became obvious that p38 and p205 were members of the same family (DING proteins). Since then, three other DING proteins have been described. One was isolated from rat brain using a cotinine (a nicotine metabolite)-affinity column ( 40). Another was isolated with a hirudin-agarose–affinity column from skin fibroblasts ( 39). A third is a saccharide-binding protein isolated from turkey air sacs. This latter property has interesting parallels with the beneficial effects of cranberry polyphenols on urinary tract infection by prevention of bacterial adhesion to the bladder wall ( 43).
Adams et al. ( 39) used internal tryptic peptide sequences from human fibroblast p40 to prepare an 840-bp DNA product using PCR with an MRC-5 human fibroblast cDNA library. Although this cDNA could not be cloned, a large part of the PCR product was sequenced and the amino acid sequence was inferred. The 5' 350 bp of the sequence corresponded to the disheveled gene from Drosophila melanogaster. This has some significance in the mechanisms of breast cancer, because the human equivalent of disheveled is Dvl-3, a member of the pathway linking the protooncogene Wnt-1 to gene transcription. Overexpression of Wnt-1 leads to the appearance of breast cancer in mice ( 44). It remains to be seen whether genistein can disrupt Wnt-1 signaling.
The discovery of the DING protein and its absence from any of the current genome databases reveals several issues that are relevant to contemporary biomedical science. First, the concept of complete genomes is a misnomer: complete in this context means that no more than 95% of the expected information is known. For political expediency, the National Institutes of Health and commercial efforts at sequencing the human genome decided to have a tie in their race to publish this genome long before the finish line was reached. Furthermore, what has been sequenced is not error free. Second, the predicted open reading frames derived from genome sequencing are in a way a hypothesis that remains to be properly tested. By the time the discrepancies between the predicted protein sequences and the actual protein sequences in cells/tissues have been resolved, it is likely that new biochemical events regulating the production of the suites of proteins found in each cell will be established.
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FOOTNOTES |
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2 Part of the work described in this article was submitted in partial fulfillment for a master's thesis (for M. Belenky) in the Department of Biochemistry and Molecular Genetics at the University of Alabama at Birmingham in 1996. Funds for this study were provided in part by the National Cancer Institute (grant R01 CA-61668).
4 Abbreviations used: AH, aminohexyl; BPA, bisphenol A; DEAE, diethylaminoethyl; MEM, modified essential media; EE, 17-ethinyl estradiol; EDAC, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride; GEN, genistein; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PgR, progesterone receptor; SDS, sodium dodecyl sulfate.
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LITERATURE CITED |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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