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University of Illinois, Department of Food Science and Human Nutrition, Urbana, IL 61801
2To 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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KEY WORDS: • genistein • p38 • mitogen-activated protein kinases
Genistein (4',5,7-trihydroxyisoflavone) is an isoflavonoid present in soybeans that exhibits anticarcinogenic effects in breast, colon and prostate cancer cells (1
–4
). Genistein inhibits several adenosine triphosphate (ATP3)–binding enzymes in vitro, such as protein tyrosine kinases (PTK), and is a very potent inhibitor of the PTK activity of the EGF receptor, in vitro (5
). However, the biological effects of genistein are not always related to its PTK-inhibiting properties. In intact cells, genistein and other PTK inhibitors, at concentrations sufficient to inhibit cell growth, do not block growth factor-stimulated tyrosine phosphorylation (6
–8
). Genistein has also been reported to modulate many nuclear events including G2/M cell cycle arrest (9
,10
), induction of the cell cycle inhibitor p21cip1/waf1 (10
,11
), inhibition of CDK1 (Cdc2) activity (12
) and inhibition of Cdc25C protein expression (13
). Cdc2 is required for transition from the G2 to the M phase of the cell cycle (14
). Genistein interferes with the passage through the G2 to the M cell cycle checkpoint by altering the level of phosphorylation of the cyclin B–Cdc2 complex, negatively regulated by the phosphorylation state of Cdc2 on Tyr15, which can be the result of the action of the kinases Wee1 and Myt1, and/or by the phosphatase Cdc25C (15
,16
).
In light of genistein’s capacity to influence various cell-signaling pathways, interest in its mechanisms of action has expanded to include several protein kinase pathways. The mitogen-activated protein kinase superfamily is composed of several signaling pathways involved in growth regulation (17
). They include extracellular signal-regulated kinase 1 (ERK1) and ERK2 (or p42MAPK and p44MAPK), c-jun NH2-terminal kinases (JNKs), ERK5 (or BMK) and p38 MAPKs, including p38
(or CSBP-1 or RK), p38ß, p38
[or stress-activated protein kinase (SAPK)3 or ERK6] and p38
(or SAPK4). These MAPKs can themselves be phosphorylated by MAPK kinases (MAPKK or MEK) (18
). The p38 MAPK pathway is a primary signaling pathway that is activated by agents that induce cell cycle arrest.
We recently demonstrated that genistein treatment of the immortalized but nonmalignant human mammary epithelial cell line MCF-10F resulted in growth arrest of MCF-10F cells in the G2 phase of the cell cycle, with an IC50 value of 
19 µmol/L (13
). The MCF-10F cells are spontaneously immortalized human breast epithelial cells derived from mortal, diploid mammary epithelial cells obtained from a mastectomy performed on a premenopausal woman with no family history of breast cancer (19
). These MCF-10F cells are not karyotypically normal and, similar to the malignant breast cell lines MCF-7 and MBA-MD-231, show homozygous deletion of the CDKN2 gene that encodes for the cdk inhibitor p16INK4 (20
,21
). Immortal transformation is considered to be an important early transition in malignant progression of human breast cells (22
,23
). Thus, the MCF-10F cells represent a valuable tool for examining early events in mammary carcinogenesis (24
) and a novel system to characterize the impact of the phytoestrogen, genistein, on these events.
Although considerable attention has been focused on examining the capacity of genistein to inhibit proliferation of malignant breast cells, much less is known about its effects on breast epithelial cells at earlier stages in the oncogenic process. With this in mind, we attempted to characterize genistein’s antiproliferative actions toward MCF-10F cells. In previous studies, we observed that growth arrest by genistein was accompanied by a large induction of the Tyr15 phosphorylation of Cdc2 and was associated with a decrease in the activity of Cdc2 (13
). In addition, genistein induced the expression of p21waf1/cip1 and decreased the expression of the cell cycle phosphatase Cdc25C. In light of evidence that MAPK families can influence cell cycle progression, we decided to determine the effect of genistein treatment on MAPK, JNK and p38, particularly as they participate in the genistein-induced G2/M cell cycle block. Involvement of MAPK and SAPK/JNK signaling components in mediating genistein’s antiproliferative influence is poorly understood. In addition, we characterized genistein’s action on multiple signaling pathways concurrently because cross-talk and signal integration among MAPK pathways have been reported, and because the biological effect of a compound like genistein may be best understood by examining combined responses of several pathways (25
–28
).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Human nontransformed mammary epithelial MCF-10F cells (19
) (Michigan Cancer Foundation, Detroit, MI) were grown in DMEM/F12 (GIBCO, Gaithersburg, MD) medium supplemented with horse serum, 50 g/L; hydrocortisone, 0.5 mg/L; cholera toxin, 0.1 mg/L; insulin, 10 mg/L; epidermal growth factor, 0.02 mg/L; and penicillin-streptomycin, 100,000 IU/L. Cells were grown at 37°C, 5% CO2, and 100% relative humidity.
[3H]Thymidine incorporation.
For the measurement of [3H]thymidine (NEN, Boston, MA) incorporation, cells were plated at 1 x 104 cells/cm2 in 24-well plates. Two days later, when the cells were in exponential growth phase, the medium was changed to medium containing genistein or DMSO vehicle. Cells were incubated with genistein or vehicle for an additional 24 h and [3H]thymidine incorporation was quantitated.
Western blotting.
Asynchronous cultures of MCF-10F cells were plated in growth media. Two days later, cells were incubated with medium containing genistein or DMSO for various times (5 min to 24 h), at which time cells were harvested as previously described (45
). Briefly, cells were washed twice in cold TBS and lysed in cold lysis buffer (Na deoxycholate, 10 g/L; Triton X-100, 10 g/L; SDS, 0.1 g/L; NaCl, 150 mmol/L; Tris, 50 mmol/L, pH 7.5; EDTA, 0.05 mmol/L; NaF, 50 mmol/L; Na pyrophosphate, 10 mmol/L; NaVO4, 0.5 mmol/L; PMSF, 1 mmol/L; aprotinin, leupeptin and pepstatin, 20 mg/L each), and sonicated for 10 s on ice. Cell lysates were centrifuged at 14,000 x g at 4°C for 5 min. Total protein was determined and equal amounts of protein were separated by SDS–polyacrylamide gel electrophoresis. After electrophoresis, the gel was stained with zinc stain (BioRad, Hercules, CA) and photographed to confirm equal protein loading. The gel was then destained and transferred to a nitrocellulose membrane. Phospho-Cdc2, phospho-p38 and total p38 were detected by immunoblotting, stripping and reprobing nitrocellulose membranes with rabbit polyclonal phosphospecific Cdc2 (Tyr15, catalogue no. 9111), phosphospecific p38 (Thr180/Tyr182, catalogue no. 9211) and nonphosphospecific p38 (catalogue no. 9212) antibodies (New England Biolabs, Beverly, MA), respectively. Membranes were then incubated with rabbit IgG horseradish peroxidase conjugate (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 25°C. Proteins were visualized by enhanced chemiluminescence (Amersham, Buckinghamshire, UK). Densitometric analyses were conducted through use of Scion Images for Windows Beta 4.0.2 (NIH Image for MacIntosh; Wayne Rasband, wayne @helix.nih.gov). To determine the requirement for p38 in phosphorylation of Cdc2 and expression of Cdc25C, cells were preincubated in SB203580 (5–20 µmol/L) for 1 h, incubated with genistein (45 µmol/L) for various times and subjected to phosphospecific Cdc2 and Cdc25C Western blotting. SB 203580 has been reported to be a specific p38 MAPK inhibitor (30
–32
).
p38, SAPK/JNK and ERK1/2 activity assays.
Kinase activity was measured by use of the MAPK assay kits (New England Biolabs), and MCF-10F cells in 75-cm2 culture flasks were or were not treated with genistein for the indicated times at 37°C. Cells were washed two times with TBS and lysed with lysis buffer [Tris-HCl, 20 mmol/L (pH 7.5), containing NaCl, 150 mmol/L; EDTA, 1 mmol/L; EGTA, 1 mmol/L; Triton X-100, 10 g/L; Na3VO4, 1 mmol/L; sodium pyrophosphate, 2.5 mmol/L; glycerol phosphate, 1 mmol/L; leupeptin, 2 mg/L]. Cells were scraped from dishes, and lysates were transferred to microcentrifuge tubes and sonicated for 1 min. Insoluble material was removed by centrifugation at 13,000 x g for 5 min. Supernatants (300 µg protein) were used for immunoprecipitation and in vitro kinase assay according to the manufacturer’s instructions. Briefly, lysates were immunoprecipitated with phosphospecific p38 or phosphospecific p42/44 MAPK monoclonal antibodies conjugated to Sepharose beads at 4°C with rotation overnight. For SAPK/JNK activity, lysates were incubated with c-Jun fusion protein complexed to Sepharose beads. Immunoprecipitates were pelleted, washed twice in lysis buffer and twice in kinase assay buffer [Tris–HCl, 25 mmol/L (pH 7.5); Na3VO4, 1 mmol/L; glycerol phosphate, 1 mmol/L; dithiothreitol, 2 mmol/L; MgCl2, 10 mmol/L]. Immune complexes were resuspended in 50 µL of kinase assay buffer containing ATP (200 µmol/L) and 2 µg of ATF-2 or 2 µg Elk-1 fusion protein and incubated at 30°C for 30 min. Reactions were terminated by addition of 25 µL of SDS sample buffer. Samples were resolved on SDS–polyacrylamide gel (75 g/L), transferred to nitrocellulose membrane and subsequently incubated with phospho-ATF-2, phospho-c-Jun or phospho-Elk-1 antibodies. Phosphorylated ATF-2, c-Jun and Elk-1 were determined by the enhanced chemiluminescence (ECL) method.
Statistical methods.
Statistical differences (P < 0.05) were determined by use of Student’s t test or ANOVA (with LSD for post hoc analysis) for comparisons between two and among three or more groups, respectively.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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After 24 h, genistein treatment inhibited [3H]thymidine incorporation into DNA in MCF-10F cells (Fig. 1
). Maximal inhibition of DNA synthesis in the MCF-10F cell line was 90%, and occurred at a genistein concentration of 45 µmol/L. The IC50 for inhibition of DNA synthesis by genistein was 18 µmol/L.
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Figure 2A
depicts the kinetics for the phosphorylation of p38 in genistein-treated cells. The lower panel of Figure 2
A depicts the gel loading of total p38 MAPK, as determined by immunoblotting of the membrane in the top panel with a nonphosphospecific p38 antibody. The results of densitometric analysis (Fig. 2
A, D) indicated that phosphorylated p38 increased 2.5-fold within 60 min and was sustained at a level at least threefold above baseline values for 90 to 120 min.
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B, over a concentration range of 0.5–45 µmol/L, the MCF-10F cells displayed a dose-dependent increase in p38 phosphorylation with an EC50 ranging from 1.5 to 2.0 µmol/L. For the genistein concentrations of 0.5, 1.0, 5.0, 10 and 45 µmol/L, p38 phosphorylation was 109, 115, 159, 182 and 227%, respectively, of controls.
To determine whether genistein also increased the activity of p38, MCF-10F cells were treated with genistein for various times, and the cell lysates subjected to an in vitro kinase assay followed by phosphospecific immunoblotting for phospho-ATF-2. As presented in Figure 2
C, genistein treatment increased the phosphorylation of ATF-2 three- to fourfold.
Further studies indicated that ERK activity (as determined by Elk1 phosphorylation) decreased significantly and ERK phosphorylation decreased significantly by 60% in cells treated with genistein, indicating that ERK activity is downregulated by genistein (Fig. 3
). In addition, we determined that the SAPK/JNK pathway is not affected by genistein in MCF-10F cells (data not shown). However, treatment of MCF-10F cells with the JNK inducer cyclohexamide (150 µmol/L) (46
) for 30 min increased the activity of SAPK/JNK, indicating that MCF-10F cells do express SAPK/JNK and that SAPK/JNK is able to be activated in MCF-10F cells (data not shown).
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, genistein induced the activity of p38, which was completely inhibited by SB203580 (at 10 and 20 µmol/L). Subsequently, as depicted in Figure 4
B, we determined that the Tyr15 phosphorylation of Cdc2 was increased by 2.9-fold after genistein treatment for 4 h and was reversed by pretreatment with SB203580 (at 20 µmol/L) for 1 h to a level 1.2-fold greater than that of controls, indicating that p38 participates in the genistein-induced phosphorylation of Cdc2. Genistein treatment for 24 h also decreased the level of Cdc25C by 75%, and pretreatment of MCF-10F cells with SB203580 (at 20 µmol/L) partially reversed the downregulation of Cdc25C level by genistein (Fig. 5
). In contrast, although genistein induced p21waf1/cip1 expression, SB203580 did not alter this action of genistein (Fig. 5)
.
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In addition to examining the ability of the p38 inhibitor to alter genistein induction of Cdc2 phosphorylation (an event directly associated with the suppression of cellular proliferation by genistein), we also examined whether p38 was involved in genistein-associated inhibition of DNA synthesis. MCF-10F cells treated with genistein in the presence of SB203580 exhibited an 84% increase in [3H]thymidine incorporation compared to cells treated with genistein alone (data not shown). Thus, addition of SB203580 to the cells decreased the ability of genistein to inhibit DNA synthesis.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The present findings that genistein activated the p38 pathway and that p38 is involved in the phosphorylation of Tyr15 on Cdc2 and the expression of Cdc25C is of interest in lieu of the reports that genistein inhibits the transition from the G2 to the M phase (9
,10
). Our results with Cdc25C and Cdc2 are similar to those recently reported for human melanoma cells, in which genistein induced a G2 arrest by impairing the Cdc25C-dependent Tyr15 dephosphorylation of Cdc2, possibly through a genistein-induced activation of Chk2 (33
,34
). Furthermore, it was reported that genistein downregulates Cdc25C and induces the degradation of Cdc25C in HN4 squamous carcinoma cells (35
). Findings from others implicate p38 and the phosphorylation of Cdc25C in the control of the G2 to M phase transition. In the report by Wang et al. (36
) two kinases, Chk1 and Cds1, were shown to phosphorylate Cdc25C and prevent it from dephosphorylating and activating Cdc2. The Cds1 kinase was activated in an MKK6-p38–dependent manner and together with Chk1, downregulated Cdc25C activity. This is supported by a recent publication of Bulavin et al. (37
), which demonstrated that p38 and p38ß play a critical role in initiating G2 arrest after UV-irradiation through phosphorylation of Cdc25B and Cdc25C. They also observed that activation of the G2/M checkpoint after hypertonic stress in renal epithelial cells requires p38 kinase (38
). Thus, p38 participation in downregulation of the Cdc25 level may be an important way to impair its actions and an important event in G2/M checkpoint regulation (39
).
Other MAPKs also may be modulated by genistein. Our findings that genistein did not activate SAPK/JNK in MCF-10F cells are not in agreement with the finding that genistein increases the activity of the SAPK/JNK pathway in A431 cells (40
). Cyclohexamide did increase the activity of SAPK/JNK in our system, indicating that SAPK/JNK is expressed in MCF-10F cells. Thus, cell-specific differences possibly relating to transformation status might partly explain the lack of effect of genistein on SAPK/JNK activity in MCF-10F cells compared to that in A431 cells. There is also variability in the reported effects of genistein on ERK activity. Treatment of platelets with pharmacological (500 µmol/L) concentrations of genistein was reported to be correlated with activation of ERK, apparently attributable to upstream inhibition of the phosphorylation and activity of c-src (41
). Our findings are similar to those of Zhang et al. (42
), who reported that genistein opposed the hydrogen peroxide-induced stimulation of ERK activity in vascular smooth muscle cells. Again, cell-specific differences in the control of ERK activity may contribute to these differences in response to genistein. Our findings suggest that examination of the mechanisms underlying an effect of genistein or other agents on proliferation (and related actions such as differentiation and apoptosis) will need to take into account responses of multiple MAPK pathways, especially when comparing different cells or tissues. This approach has been underscored by others (25
,43
) and is illustrated by recent reports describing the effects of drugs and growth factors on MAPK activities (44
,45
).
In summary we demonstrated that genistein rapidly and strongly activates p38, inactivates ERK1/ERK2 and has no effect on SAPK/JNK activity in human nontransformed mammary epithelial cells that are sensitive to the growth inhibitory effects of genistein. In addition, we demonstrated that genistein treatment initially increases the phosphorylation of Cdc2 on Tyr15, and that p38 MAPK is required for Cdc2 phosphorylation at Tyr15, whereas after longer periods (24 h) of genistein treatment, the phosphorylation of Cdc2 is decreased. We also showed that p38 is involved in genistein-induced changes in Cdc2 phosphorylation and that the downregulation of Cdc25C expression by genistein involves the p38 pathway. Finally, we provided evidence that the p38 pathway is involved in genistein-inhibited cell proliferation. These data suggest an important interplay between the p38 pathway and G2/M cell cycle checkpoint control and provide insights into how this isoflavone may suppress the growth of preneoplastic mammary cells.
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FOOTNOTES |
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3 Abbreviations used: ATP, adenosine triphosphate; ECL, enhanced chemiluminescence; EGF, epidermal growth factor; EC50, concentration at 50% effectiveness; ERK, extracellular signal-regulated kinase; IC50, concentration at 50% inhibition; JNK, c-jun NH2-terminal kinase; MAPK, mitogen-activated protein kinases; MEK, MAPK kinase; PTK, protein tyrosine kinases; SAPK, stress-activated protein kinases; TBS, Tris-buffered saline.
Manuscript received 6 June 2002. Initial review completed 3 July 2002. Revision accepted 1 October 2002.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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