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4
*
Molecular Cell and Developmental Biology Section and
Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712
4To whom correspondence should be addressed. E-mail: surangi{at}mail.utexas.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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KEY WORDS: • estrogen signaling • phytoestrogen • breast cancer progression • FAK activity • Akt activity • phosphorylation • antioxidant activity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). The antioxidant activities of polyphenols are well recognized and may be responsible for various health benefits. Free radicals are produced by pollutants in our food, water and air; oxidation-reduction reactions of lipids and metal ions; and continuous mitochondrial electron transport reactions in the body. Catechins in green and black tea and red wine can defuse these aggressive superoxide, alkyl and hydroxyl free radicals by providing an electron, thereby preventing attack on DNA and other cell components. If not deactivated, the aggressive radicals produce free radical chain reactions, potentially leading to heart disease, stroke, memory loss and cancer. In one step, catechins help subvert these health problems by converting damaging free radicals to inactive compounds while themselves becoming low-energy harmless catechin free radicals. In addition to being powerful antioxidants, catechins such as epigallocatechin gallate (EGCG)5
in green tea absorb ultraviolet light in the 280–320-nm range, preventing the promotion of photo-induced skin cancer. EGCG also has substantial inhibitory effects in vivo against a wide variety of tumors and in vitro against cancer cell lines (2
–5
). Another effective antioxidant polyphenol is resveratrol, which is found in high concentration in red wine as well as in peanuts and grape skins (6
). trans-Resveratrol may prevent cancer by inhibiting cancer growth, tumor angiogenesis and cell invasion (7
–15
).
Some of these compounds have estrogenic (and antiestrogenic) activity and are commonly referred to as phytoestrogens. Soybeans are the main dietary source of two isoflavonoids: genistein and daidzein. These compounds may affect cancer progression as a result of their effects on apoptosis, cell cycle progression, growth and differentiation as well as their antioxidant and antiangiogenic effects. Genistein affects cellular function via inhibition of 17ß-steroid oxidoreductase (an enzyme necessary for estrogen synthesis) and tyrosine-specific protein kinases. Genistein also modulates the activity of topoisomerase II, enzymes involved in phosphoinositide turnover and transforming growth factor-ß (TGFß) signaling cascades (16
–22
). The exact effect of phytoestrogens on breast cancer cells and tumors is concentration dependent, where growth is stimulated at low concentrations (0.1–10 µmol/L) and inhibited at high concentrations (20–100 µmol/L) (23
–26
).
Estrogenic compounds regulate gene transcription via two specific intracellular estrogen receptors (ERs): ER
and ERß. The general scheme of estrogen action involves diffusion into the cytosol, binding to ERs and activation of gene expression (27
,28
). Overexpression of ER is considered to be a predictive and prognostic factor in breast cancer (29
,30
). Consequently, inhibition of ER has become a major strategy for preventing and treating breast cancer (31
–35
). Loss of ER expression is common in malignant progression of breast cancer, making traditional therapy with selective ER modifiers ineffectual. Therefore much effort is directed toward designing novel therapeutic strategies to combat alternative signaling pathways, such as epidermal growth factor receptor signaling, that are dysregulated during breast cancer progression (36
).
The mode of action of the common mammalian estrogen 17ß-estradiol (E2) in regulating cell proliferation as well as tumorigenesis via gene transcription is well established. One method to evaluate estrogenic genomic effects of compounds and crude mixtures is to measure the ability of compounds to bind to ER and to activate estrogen-responsive genes (37
). Using a yeast-based ER transactivational reporter system, we investigated the estrogenic properties of compounds, monitored complex plant extracts and isolated novel estrogenic compounds via activity-guided fractionation (38
,39
).
Recent literature, however, indicates that E2 exerts additional nongenomic effects on cell signaling. Such rapid nongenomic effects have been reported for a variety of cell types, such as bone, neuronal, mammary, ovarian and cardiovascular cells (40
–42
). These cells contain plasma membrane ERs that can cross-activate a variety of signaling cascades, including those mediated by G protein–coupled receptors and tyrosine kinase–type growth factor receptors. Rapid cellular responses to estrogen activate both Gs and Gq type G proteins, leading to stimulation of adenylate cyclase and phospholipase C, which in turn activate protein kinase A, protein kinase C and intracellular Ca2+ fluxes (42
–45
). E2-bound ER associates with the regulatory subunit of phosphatidylinositol-3-kinase (PI3-K) and activates the survival factor Akt (protein kinase B) (46
,47
) as well as stimulating growth factor receptor activity (48
). These effects of E2 signaling stimulate cell proliferation via activation of mitogen-activated protein kinase (MAPK) cascades (42
,43
). Activation of the tyrosine kinases Src and Shc by ER has also been linked to direct stimulation of MAPK signaling (49
). Moreover, E2 has been shown to affect the tyrosine phosphorylation status of key signaling intermediates such as c-Src and focal adhesion kinase (FAK) in both ER-positive (+) and ER-negative (-) breast cancer cell lines (50
). Recent studies have shown that MAPK signaling not only affects gene transcription leading to tumorigenesis but also may promote cancer cell invasion (51
,52
). Therefore E2 signaling to MAPK cascades may be relevant for breast cancer malignancy. The true complexity of estrogen signaling is only now beginning to be elucidated, and even less is known about the nongenomic effects of the vast array of related estrogenic compounds.
In this study the soybean phytoestrogens genistein and daidzein and resveratrol from red wine were chosen to investigate the relevance of nongenomic activity of flavonoids in breast cancer progression. Genistein and daidzein bind to and transactivate both ER and ERß and have been extensively studied for their potential health benefits (39
,53
,54
). In addition to attenuating cell growth via ER, genistein has been shown to block the proliferation of normal and cancer cells stimulated by growth factor and cytokine. High concentrations of genistein act as a tyrosine kinase inhibitor; this function may underlie its anticancer effects (17
,18
,37
). However, in one study the antiproliferative effect of genistein was shown to be uncoupled from its effect as a tyrosine kinase inhibitor, possibly acting via TGFß signaling (19
,55
). More studies are needed to fully evaluate the estrogenic, antiestrogenic and tyrosine kinase inhibitory effects of genistein on breast cancer progression.
Resveratrol is structurally similar to the synthetic estrogen diethylstilbestrol and binds to and activates ER ( and ß) to exert both estrogenic and antiestrogenic effects (56
–60
). The antitumor activity of resveratrol is mediated by MAPKs [i.e., extracellular signal-regulated protein kinases, c-jun NH (2
)-terminal kinases and p38 kinase]. Resveratrol-induced p38 MAPK-mediated p53 activation has been implicated in inhibition of cell cycle progression and initiation of apoptotic pathways (61
–64
). Recently, the stimulatory effect of resveratrol on apoptosis was also demonstrated in a novel mitochondrial pathway controlled by Bcl-2 (65
). Moreover, resveratrol inhibits tumor promoting agent- or UV-induced activity of the activator protein 1 transcription factor through inhibition of c-Src nonreceptor tyrosine kinase and MAPK pathways (66
).
These studies implicate phytoestrogens not only in mediation of genomic effects via ER but also in more rapid signaling cascades. In this study, the effects of phytoestrogens on the activation status of signaling proteins known to control breast cancer cell survival, proliferation and invasion were monitored in ER(+) metastatic and ER(-) nonmetastatic human breast cancer cell lines. Activation of PI3-K, which catalyzes the phosphorylation of phosphatidylinositol-4,5-bisphosphate to generate phosphatidylinositol-3,4,5-bisphosphate (PIP3), is a key event during signal transduction via cell surface receptors (67
). PIP3-induced activation of phosphoinositide-dependent kinase mediates the phosphorylation (in Ser 473 and Thr 308) and activation of Akt. Activated Akt initiates downstream signaling cascades that promote cell survival and proliferation potentially leading to cancer malignancy (68
,69
). FAK is a tyrosine kinase that is recruited to the membrane in response to tyrosine kinase–type growth factor and integrin receptor activation. Activation of FAK by phosphorylation in Tyr 397 triggers various kinase signaling cascades, including activity by Src and Shc, nonreceptor tyrosine kinases and MAPKs. Similarly, these events increase cell proliferation, motility and invasion (52
,70
,71
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Human breast cancer cell lines T47D and MDA-MB-231 were maintained in Dulbecco’s modified Eagle’s medium (DMEM) [10% fetal bovine serum (FBS)] at 37°C with 5% CO2. Before stimulation with estrogenic compounds, cells were washed with phenol red–free DMEM. Approximately 3 x 105 cells/well were transferred to 6-well plates and grown for 48 h in phenol red–free DMEM with 10% FBS. Next, cells were starved in phenol red–free DMEM for 24 h before stimulation. Cells were stimulated for 15 min with either E2 (Sigma Chemical Co., St. Louis, MO), genistein, daidzein or trans-resveratrol (LKT Laboratories, St. Paul, MN). E2 was used at a concentration of 10 nmol/L, and phytoestrogens were used at 1, 50, or 100 µmol/L. Dimethylsulfoxide was added as a vehicle for unstimulated controls.
Western blot analysis.
Cells were disrupted in lysis buffer (20 mmol/L Tris-HCl, pH 7.5), 150 mmol/L NaCl, 1 mmol/L EGTA, 1 mmol/L EDTA, 2.5 mmol/L sodium pyrophosphate, 50 mmol/L sodium fluoride, 1 mmol/L dithiothreitol, 10% glycerol, 1% Nonidet P-40, 0.5% deoxycholate, and protease inhibitors) at 4°C. Lysates were centrifuged at 16,000 x g; the proteins in the supernatant were eluted with Laemmli’s sample buffer and separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels. Proteins were transferred to polyvinylidene fluoride membranes, blocked and probed with specific primary antibodies. Detection was accomplished using alkaline phosphatase–conjugated secondary antibody and developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent (Pierce Chemical, Rockford, IL). Anti-Akt and anti–phospho-Akt (Ser-473) antibodies were purchased from Cell Signaling, MA. Anti-FAK or anti–phospho-FAK (Tyr-397) antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). The density of positive bands was quantified using NIH Image software; the results were averaged.
We calculated the the ratio of the amount of phosphorylated-Akt (P-Akt), as detected with anti–phospho-Akt antibody, to the amount of total Akt, as detected with anti–Akt antibody. Similarly, we calculated the ratio of the amount of phosphorylated-FAK (P-FAK), as detected with anti–phospho-FAK antibody, to the amount of total FAK, as detected by anti–FAK antibody. The relative activity for both Akt and FAK was calculated and plotted onto a graph relative to values for unstimulated controls (i.e., the difference between the the ratio of P-Akt to Akt with stimulation and the ratio of P-Akt to Akt without stimulation divided by the the ratio of P-Akt to Akt without stimulation) for each compound tested.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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The nongenomic effects of estrogen on activation of critical oncogenic signaling molecules are just beginning to be examined. Most studies have focused only on the effects of phytoestrogens at the gene transcription level after prolonged (>24 h) exposure (72
–75
). Very little is known about the effects of phytoestrogens on activation of short-term signaling cascades as well as their effect on prevention of breast tumorigenesis and cancer progression. Reversible protein phosphorylation is one of the most prevalent mechanisms for covalent modification of proteins during signal transduction and control of cellular processes (76
). Therefore as a preliminary step toward phosphoproteomic profiling the effects of dietary phytoestrogens in breast cancer progression, we focused on the rapid activation of key signaling proteins by phosphorylation in ER(+) and ER(-) breast cancer cell lines.
The concentrations of estrogenic compounds that we used were in agreement with previous concentrations of phytoestrogens used to induce gene transcription via both ER and ERß (77
). Our experimental design of adding low, medium and high concentrations of phytoestrogens was intended to differentiate the effects of phytoestrogens as estrogenic, antiestrogenic and tyrosine inhibitory compounds. Our approach of using two breast cancer cell lines with differential ER status—ER(+) T47D cells and ER(-) MB-231—that express low levels of ERß (78
–82
) enabled an evaluation of the ER dependency of the observed effects. Comparison of the total phosphoproteomic profiles of unstimulated or stimulated T47D or MB-231 whole-cell lysates by Western blotting with antibodies specific to phosphoserine or phosphotyrosine demonstrated significant differences in the phosphorylation status of a number of proteins (data not shown).
Activation of Akt by phytoestrogens.
The phosphorylation status and thus activation of Akt in response to estrogenic compounds were monitored in breast cancer cells by Western blotting of unstimulated or stimulated cell lysates with antiphosphoserine 473 Akt antibody to determine phospho-Akt levels or an anti-Akt antibody to determine total Akt levels. In ER(+) T47D cells, E2, genistein and daidzein activated Akt. Interestingly, resveratrol decreased Akt phosphorylation in a concentration-dependent fashion (Fig. 1
). In contrast, in ER(-) MDA-MB-231 cells, all estrogenic compounds except genistein at high concentrations (100 µmol/L) activated Akt. Daidzein had a marked effect on Akt activation in MB-231 cells (Fig. 2
). These results agree with a recent report that demonstrated increased Akt activity in response to E2 with the same MB-231 cell line (47
).
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|
–85
) and, more recently, in both ER(+) and ER(-) human breast cancer cell lines (47
,86
). A study on Akt activation in the myocardium reported that E2 and genistein elevated nuclear phospho-Akt activation (87
). Therefore our observation of activated Akt in response to E2 agrees with published reports.
Our results demonstrate that the effects of estrogenic compounds on Akt activation differed depending on the ER status of breast cancer cells. The inhibitory action of resveratrol in the ER(+) T47D cells, but not in ER(-) MB-231 cells, indicates that this effect was probably ER dependent. In MCF-7 ER(+) breast cancer cells, the same range of resveratrol (10–50 µmol/L) antagonizes the E2-mediated gene expression and growth-stimulatory effect (12
) and induces p53-mediated apoptosis (61
). In MCF-7 and MVLN breast cancer cells, resveratrol demonstrated a biphasic effect by increasing growth at medium concentrations (10 and 25 µmol/L) and decreasing growth at a high concentration (50 µmol/L). Low concentrations (0.1 and 1 µmol/L) had no effect on cell growth (88
). These results agree with our observations that low concentrations of resveratrol demonstrate little or no effect on Akt activity, whereas higher (50 and 100 µmol/L) concentrations demonstrate striking inhibitory effects. More studies using concentrations in the 10–25 µmol/L range are necessary to evaluate whether medium concentrations of resveratrol increase Akt activity in ER(+) breast cancer cells.
In one study, resveratrol decreased hepatocyte growth factor–induced cell scattering and invasion in hepatocellular carcinoma cells independent of Akt activation (15
). However, for the first time in breast cancer cells, we demonstrate that resveratrol directly modulates Akt activity. Activated Akt is a strong cellular survival signal that protects cells from apoptosis by phosphorylation of the proapoptotic Bcl-2 family member Bad, which is then sequestered and degraded (68
,69
). Resveratrol induces p53 activity that leads to activation of the proapoptotic Bcl-2 family member Bax (61
–64
). Thus resveratrol-mediated downregulation of Akt activity coupled with activation of proapoptotic signaling is predicted to act as a strong signal to suppress growth and activate apoptosis in ER(+) breast tumors.
Activation of FAK by phytoestrogens.
We also monitored the effect of phytoestrogens on FAK activity in breast cancer cells. In the ER(+) T47D cell line, 15-min stimulation with E2, genistein or daidzein increases phospho-FAK levels, whereas resveratrol decreases the levels (Fig. 3
). The ER(-) MB-231 cell line demonstrated increased FAK phosphorylation in response to all phytoestrogens tested (Fig. 4
). Similar results were obtained by immunoprecipitations of stimulated cell lysates using an antiphosphotyrosine antibody followed by Western blotting with an anti-FAK antibody (data not shown).
|
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, 4)
. This may reflect the general perplexity in the field regarding the exact mechanism of action of genistein as a tyrosine kinase inhibitor or an antiestrogen (89
). Traditional studies using genistein as a tyrosine kinase inhibitor have added genistein to cells in serum or phenol red–containing media, which contain estrogenic compounds (37
,90
). Others have shown that the antiproliferative effects of genistein are estrogen dependent, including inhibition of estrogen-induced protein tyrosine kinase activity (91
). Some studies have concentrated on long-term effects of genistein on the phosphotyrosine status of proteins, where the reported inhibition of protein tyrosine kinases may result from alterations in kinase expression (18
).
Daidzein, the other major isoflavone in soy that differs structurally from genistein only by the lack of a hydroxyl group, was selected to enable the analysis of effects that may be distinct from the tyrosine kinase inhibitory effects of genistein. These two structurally related phytoestrogens are thought to have discrete target sites and mechanisms in their growth inhibitory action on breast cancer cells (75
). In a previous study using MB-468 breast cancer cells, genistein demonstrated a concentration-dependent biphasic effect on cell cycle progression, whereas daidzein was not effective at high concentrations (92
). Our results demonstrate that both daidzein and genistein increased FAK phosphorylation at low, medium and high concentrations. Unstimulated and stimulated cell lysates of ER(+) T47D and ER(-) MB-231 were probed with an antiphosphotyrosine antibody or immunoprecipitated with an antiphosphotyrosine antibody and probed with a different antiphosphotyrosine antibody to detect overall trends of phosphorylation in response to phytoestrogens (data not shown). In these experiments, daidzein increased whereas high concentrations of genistein decreased overall tyrosine phosphorylation. Therefore the enhanced phospho-FAK levels in the presence of high concentrations of daidzein may reflect a general effect of daidzein on activation of a protein tyrosine kinase or inactivation of a protein phosphatase.
In ER(-) MB-231 cells, all phytoestrogens tested increased FAK activity (Fig. 4)
. In contrast, resveratrol reduced FAK phosphorylation in ER(+) T47D cells (Fig. 3)
. This result may indicate that FAK activation by soy phytoestrogens is not dependent on ER. A single study that investigated the effect of estrogen on FAK activity in breast cancer used the nonmetastatic ER(+) MCF-7 human breast cancer cells. These cells were cultured for 7 d in the presence of E2 at 1 nmol/L, which resulted in decreased tyrosine phosphorylation of FAK. The E2-induced effect was blocked by the ER antagonist 4-hydroxytamoxifen at 100 nmol/L, indicating that dephosphorylation of FAK is an ER-mediated event. E2 treatment also resulted in a reduced association between FAK and paxillin, a focal complex protein that mediates contact between integrin receptors and the actin cytoskeleton (93
). Thus, unlike as shown here, long-term exposure to E2 may exert genomic effects on tyrosine kinases or phosphatases that affect FAK activity.
The anti–phospho-FAK antibody that specifically interacts with the phosphotyrosine residue 397 consistently detected two bands: one at 125 kDa and the other at 90 kDa. Our 125 FAK antibody, which was raised against the carboxyl-terminal residues 748-1053, did not detect the amino-terminal 90-kDa fragment. The 125-kDa and 90-kDa phospho-FAK bands probably correspond to calpain digestion of the full-length FAK (125 kDa) to a 90-kDa fragment that still contains the phosphotyrosine 397 residue (94
). Both fragments were quantified for total phospho-FAK analysis. Unfortunately, the reported data only reflect levels of 125-kDa full-length FAK. Ongoing experiments are focusing on the detection of nonphosphorylated FAK using an additional antibody raised against the amino terminus of FAK.
The tyrosine kinase Src activates calpain proteases that digest FAK during transformation and invasion (70
,95
,96
). Calpain-deficient embryonic fibroblasts reduce cell migration, implying that the cleavage products observed in our breast cancer cells have invasive potential (97
). Interestingly, Src and thus Shc and MAPK signaling are activated by nongenomic actions of E2 (86
,98
,99
). We are currently investigating the possible regulation of focal adhesion turnover by estrogenic compounds via Src-mediated calpain activity.
FAK activation by phosphorylation results in cell proliferation, motility and invasion (51
,52
,70
,71
). Decreased phosphorylation levels and reduced association between FAK and paxillin have been suggested as important steps leading to the loss of stable focal contacts and loss of growth inhibition during tumorigenesis (50
). However, a recent study demonstrated that dephosphorylation of FAK and down-regulation of FAK activity by EGFR induction initiated tumor cell invasion (100
). Our results that demonstrate phytoestrogen-mediated FAK phosphorylation and cleavage in ER(+) and ER(-) breast cancer cells (Figs. 3
, 4)
may indicate potential modulation of cell migration by phytoestrogens. Rapid turnover of focal adhesions by regulation of FAK activity via both phosphorylation and calpain cleavage has been implicated in cell migration and invasion through the extracellular matrix (70
,93
,96
,101
). Therefore the observed differences in FAK activity in response to estrogenic compounds may be relevant to control of breast cancer progression.
Summary.
E2 plays a critical role in the initiation and progression of breast and gynecological cancers and has been implicated in modulation of breast cancer cell invasion and metastasis (102
–104
). Phytoestrogens are known to act as agonists or antagonists of E2 and may protect against some cancers, cardiovascular disease and osteoporosis, as well as to prevent the undesirable symptoms of menopause (105
–108
). Interest in the potential benefits of phytoestrogen consumption continues to increase; however, use of phytoestrogens in prevention of breast and uterine cancer or as a "natural" alternative to hormone replacement therapy remains controversial (18
,106
,109
–116
). Although phytoestrogens are thought to protect against breast cancer by regulating ER and growth factor signaling pathways (18
,117
), their exact mechanism of action needs to be evaluated (118
). Moreover, because of the lack of relevant information, the increased use of phytoestrogens in human diet does not consider nongenomic modes of phytoestrogen action at the cellular and molecular level.
The present data indicate that soybean phytoestrogens increase phosphorylation of both Akt and FAK, molecules that promote cancer cell survival and invasion, whereas the red wine phytoestrogen resveratrol inhibits FAK and Akt activity in an ER-dependent manner. Our novel data demonstrate that genistein, in addition to its effect as a tyrosine kinase inhibitor and an antiestrogen via ER, may also have ER-independent effects on breast cancer cells. With a similar panel of ER(+) and ER(-) breast cancer cell lines, the antiproliferative effects of genistein were shown to be dependent on E2, thus leading the authors to conclude that antiestrogenic effects of genistein were only operative in ER(+) breast cancer cell lines (91
). However, others have suggested that genistein can inhibit growth and induce apoptosis in ER(-) highly metastatic MB-435 breast cancer cells via down-regulation of ErbB-2 and Bcl-2 and decreased matrix metalloproteinase secretion (119
). The present study suggests that the soy phytoestrogens genistein and daidzein may exert additional effects on breast cancer cell survival, proliferation and invasion via activation of Akt and FAK.
Our data on the effects of resveratrol in ER(+) and ER(-) breast cancer cells support the well-established properties of resveratrol as an ER-dependent antagonist of estrogenic effects that inhibit cellular events associated with tumor initiation, promotion and progression (9
,59
,120
). However, studies performed using ER-transfected cell lines have shown that resveratrol acts as a mixed agonist and antagonist. In the absence of E2, resveratrol was shown to exert mixed estrogen agonist-antagonist activities in T47D and MCF-7 ER(+) breast cancer cell lines (37
). For the first time, the present study demonstrates ER-dependent and -independent effects of resveratrol on Akt and FAK activity in breast cancer cells. Interestingly, all estrogenic compounds tested increased Akt and FAK activity in the metastatic ER(-) breast cancer cell line. Shorter ERß isoforms have been detected in the ER(-) MDA-MB-231 cells that express low levels of ERß (81
,82
). Thus the stimulatory effects of phytoestrogens on Akt and FAK activity in MB-231 cells may be mediated by ERß. Although the exact mechanism by which phytoestrogens modulate Akt and FAK activity remains to be elucidated, our results are intriguing and may be pertinent to concerns of varied estrogenic effects during breast cancer progression.
A comprehensive evaluation of nongenomic signaling by phytoestrogens will be achieved by confirming our results in a wider range of human breast cancer cell lines at different stages of breast cancer progression. Many kinases and phosphatases involved in modulation of signaling events have been implicated in a variety of pathological conditions including cancer predisposition (121
). Therefore the phosphoproteins identified in our studies may represent a rich source of potential targets for therapeutic intervention in treatment and prevention of breast cancer as well as markers for early detection of malignant breast cancer.
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FOOTNOTES |
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2 Supported by NIH/NCA Grant R21CA83957–01A1 awarded to S. F. D. and Welch Foundation Grant No. F-130 awarded to T. J. M.
3 Delia M. Brownson and Nicolas G. Azios contributed equally to this work.
5 Abbreviations used: DMEM, Dulbecco’s modified Eagle’s medium; E2, 17ß-estradiol; EGCG, epigallocatechin gallate; ER, estrogen receptor; FAK, focal adhesion kinase; FBS, fetal bovine serum; MAPK, mitogen-activated protein kinase; P-FAK, phosphorylated-FAK; PI3-K, phosphatidylinositol-3-kinase; PIP3, phosphatidylinositol-3,4,5-bisphosphate; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TGFß, transforming growth factor-ß.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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