Resveratrol Regulates Insulin-Like Growth Factor-II in Breast Cancer Cells
http://www.100md.com
《内分泌学杂志》
Departments of Anatomy (S.V.) and Physiology (Y.A., D.D.D.L.), Loma Linda University, Loma Linda, California 92350
Abstract
IGF-II is a potent mitogen and inhibitor of apoptosis in breast cancer. Regulation of IGF-II is complex and includes inhibition by tumor suppressors, stimulation by oncogenes, and imprinting and hormonal regulation by estrogens. Resveratrol (RSV) is a phytoestrogen that displays estrogen-like agonistic and antagonistic activity. Recent studies have shown that RSV inhibits the growth of breast cancer cells and may represent a potent agent in chemopreventive therapy. Because 17-estradiol regulates IGF-II, we hypothesized that RSV may have a similar effect on IGF-II. The present study was designed to examine whether: 1) RSV modulates IGF-II in breast cancer cells; 2) regulation of IGF-II by RSV is dependent on the ER status; and 3) IGF-II (not IGF-I) mediates RSV effects on breast cancer cells. Treatment of MCF-7 and T47D cells with RSV (10–6 M) caused stimulation of precursor IGF-II mRNA and protein; this effect was blocked by coincubation with 17-estradiol (10–9 M). Cell growth stimulated by RSV (10–6 M) was blocked by addition of a blocking IGF-I receptor antibody, or the antiestrogen tamoxifen (10–7 M). In contrast, RSV treatment (10–4 M) inhibited IGF-II secretion and cell growth in MCF-7 and T47D cells. No increase in IGF-II levels is seen in estrogen receptor (–) MCF-10 cells, even though cell growth was inhibited by RSV 10–4 M and precursor IGF-II blocked the inhibitory effect of resveratrol. No change in IGF-I was observed with RSV treatment (10–6 to 10–4 M). Our study demonstrates that RSV regulates IGF-II and that IGF-II mediates RSV effect on cell survival and growth in breast cancer cells.
Introduction
IGF-II IS A POTENT mitogen that stimulates cell proliferation and inhibits apoptosis of breast cancer cells. IGF-II gene expression is paternally imprinted and regulated by tumor suppressor genes such as p53, WT1 (Wilms’ tumor gene), and guanine-cytosine factor (1, 2, 3). Furthermore, the complex regulation of IGF-II expression involves both developmentally regulated promoters (P1-P4) and alternative splicing of different untranslated leader sequences (4). IGF-II is translated as a prohormone that is cleaved and glycosylated to produce isoforms ranging from 21 [precursor IGF-II (proIGF-II)] to 7.5 kDa (mature IGF-II) (5). ProIGF-II is the predominant form secreted by most tumors and it is biologically active (5, 6, 7, 8). This growth factor binds several receptors including the IGF-II/mannose-6 phosphate receptor (M6P), IGF-I receptor (IGF-IR) and the insulin receptor isoform A (IR-A). IGF-II mitogenic effects are mediated through the IGF-IR and the IR-A, whereas insulin binding to IR-A stimulates metabolic pathways (9, 10, 11). IGF-II binding to the IGF-II/mannose-6 phosphate receptor results in intracellular degradation of IGF-II and decreased signaling through the IGF-I and IR-A receptors (12). IGF-II is also regulated by 17-estradiol (E2). E2 stimulates breast cancer cell proliferation and tumor development, and use of antiestrogens blocks this stimulatory response. Of interest, IGF-II signaling through the IGF-IR enhances estrogen receptor (ER) activation in human breast cancer cells (13).
Resveratrol (RSV), a phytoestrogen found mainly in grapes, has a molecular structure similar to E2 (14, 15) and works as an agonist/antagonist of E2 activity. RSV exhibits a variety of pharmacological effects, some of which may be associated with cardioprotective effects; but increasing interest in this compound is due to its potential as a chemopreventive agent (16, 17, 18, 19, 20, 21, 22, 23, 24). BecauseE2 regulates IGF-II, we hypothesized that RSV may have a similar effect on IGF-II. Thus, our hypothesis is that RSV inhibitory and stimulatory effects are mediated by IGF-II.
The present study was designed to examine whether: 1) RSV modulates IGF-II in breast cancer cells; 2) regulation of IGF-II by RSV is dependent on the ER status; and 3) IGF-II (not IGF-I) mediates RSV effects on breast cancer cells.
Materials and Methods
Cell culture
MCF-7, T47D breast carcinoma cell lines, and MCF-10 breast epithelial cells were obtained from the American Type Culture Collection (Manassas, VA). MCF-7 cells were maintained in a 5% CO2 incubator at 37 C, using DMEM/F12 media (Cellgro; Mediatech Inc., Herndon, VA) supplemented with 10 ml of 5000 U penicillin/streptomycin (100 U/ml penicillin and 100 U/ml streptomycin sulfate, Cellgro), 4 mM L-glutamine (Cellgro), 3 μg/ml -amphotericin, and 5% fetal bovine serum (Hyclone, Logan, UT). T47D cells were grown in RPMI 1640 media supplemented with 10 μg/ml insulin, 2 mM L-glutamine (Cellgro), 5000 U penicillin/streptomycin (100 U/ml penicillin and 100 U/ml streptomycin sulfate, Cellgro), and 10% fetal bovine serum (Hyclone). MCF-10 DMEM/F12 media was supplemented with 10 ml of 5000 U penicillin/streptomycin (100 U/ml penicillin and 100 U/ml streptomycin sulfate, Cellgro), 4 mM L-glutamine (Cellgro), 3 μg/ml -amphotericin, 10 μg/ml insulin (Sigma, St. Louis, MO), 0.5 μg/ml hydrocortisone (Sigma), 20 ng/ml murine epidermal growth factor (Gibco-BRL, Gaithersburg, MD), 100 ng/ml cholera toxin (Sigma), and 5% equine serum. Cells were detached by trypsinization (1x trypsin EDTA, Cellgro). Recombinant human proIGF-II (amino acids 1–156) was purchased from GroPep (Adelaide, Australia). RSV, E2, and tamoxifen (TAM) were purchased from Sigma and dissolved in dimethyl sulfoxide (Fischer Scientific, Pittsburgh, PA). Media from RSV-treated cells (conditioned media) was collected (24 and 48 h), centrifuged (800 rpm for 5 min), and frozen (–20 C) until assayed.
Western blot analysis
Total protein concentration (30 μg) of serum-free medium (SFM) collected after 24 and 48 h of RSV treatment (10–7 to 10–4) and control group (SFM with vehicle) was used to load 10–20% polyacrylamide-sodium dodecyl sulfate gradient gels and transferred to a nitrocellulose membrane Bio-Trace NT (Life Sciences, Ann Arbor, MI) using a semidry electrophoretic transfer cell (Bio-Rad Laboratories, Hercules, CA). Protein concentration was measured using the Coomassie Plus protein assay reagent (Pierce Biotechnology, Rockford, IL) Nitrocellulose membranes were blocked with 2% BSA IgG free (Sigma) in PBS/0.05% Tween 20 for 2 h. Membranes were then incubated with Amano IGF-II monoclonal antibody, clone S1-F2 (1:1000 Amano, Mitsubishi, Troy, VA). The Amano antibody is able to detect both, the mature and precursor forms of IGF-II. Similarly, the IGF-I polyclonal antibody (1:1000; Oncogene Research Products, San Diego, CA) was used to detect IGF-I. The blots were also probed with -actin antibody (1:10,000, Sigma) and used as a protein loading control. After 3 x 10 min washes in PBS/0.05% Tween, the corresponding secondary antibodies (1:1000, Amersham, Arlington Heights, IL) were added to the membranes (1 h at RT), followed by 3 x 10 min washes and horseradish-peroxidase (1:1000 Amersham, Arlington Heights, IL). Protein visualization was achieved by using enhanced chemiluminescence (ECL) and autoradiography with Hyperfilm ECL film (Amersham). The signals on the x-ray films were quantified using ChemiImager 4000 (Alpha Innotech Corp., San Leandro, CA).
Northern blot analysis
Total RNA was extracted using Tri reagent (Molecular Research Center, Cincinnati, OH), after 12 and 24 h incubation of MCF-7 and MCF-10 cells in SFM in the presence of 10–6 or 10–4 M RSV. RNA was then precipitated using isopropanol and solubilized in 0.5% sodium dodecyl sulfate. Ten micrograms of each sample RNA were then electrophoresed on a 1% agarose-formaldehyde gel and transferred to a nylon membrane (Hybond-N, Amersham) followed by UV cross-linking. The membranes were prehybridized (expressHyb solution; CLONTECH Laboratories, Mountain View, CA) for 1 h at 68 C before 32P-labeled IGF-II cDNA was added. Blots were hybridized at 42 C. The membranes were then washed and exposed to film (Kodak, Rochester, NY) with intensifying screens at –80 C for 2–3 d. Subsequently the membranes were stripped and reprobed with 32P-labeled cyclophilin cDNA, which was used as a loading control. The signals were quantitated using densitometric analysis of the autoradiographs by Chemi-Imager 4000 (Alpha Innotech).
Cell growth studies
Cell growth was measured by bromodeoxyuridine (BrdU) incorporation assay (Oncogene Research Products), after 24 and 48 h of RSV treatment. MCF-7 cells (1 x 104/well) were plated in 96-well tissue culture plates and grown in SFM. After 6 h incubation, 10–6 or 10–4 M RSV was added to the wells. Cells were then incubated with BrdU label for 20 h (5% CO2, 37 C incubator). BrdU incorporation was detected immunochemically after partial denaturation of double-stranded DNA. In this assay, horseradish-peroxidase catalyzes the conversion of the chromogenic substrate tetramethylbenzidine to a blue solution, the intensity of which is proportional to the amount of incorporated BrdU in the cells. The colored reaction product is then quantified using a spectrophotometer (405–595 nm). To evaluate at what extent the concentration-dependent effect of RSV on cell growth was due to IGF-II, BrdU incorporation was measured in the presence of 2 μg/ml anti-IGF-IR antibody (Santa Cruz Biotechnology, Santa Cruz, CA), which inhibits binding as well as basal and IGF-I/IGF-II-stimulated DNA synthesis. The effect of different concentrations of proIGF-II (10–100 ng/ml) on RSV (10–4 M)-treated MCF-7 cells was also assessed by BrdU incorporation after 24 and 48 h.
Cell viability assay
Cell viability was measured by the 3-[4,5-dimethylthiazol2-yl]2,5-diphenyltetrazolium bromide (MTT) assay; cells (1 x 104/well) were seeded in 96-well plates and grown in SFM. Cell viability was assessed at 24 and 48 h by measuring the rate of tetrazolium salts reduction to formazan (MTT, Sigma), which is proportional to the number of living cells. At the end of incubation, the absorbance was read at 540 nm. To evaluate at what extent the concentration-dependent effect of resveratrol on cell survival is due to proIGF-II signaling through the IGF-IR, parallel wells containing MCF-7 cells treated with 10–6 M RSV were incubated simultaneously with 2 μg/ml anti-IGF-IR antibody.
Statistical analysis
Values are expressed as the mean ± SEM. Statistical differences between mean values were determined by one-way ANOVA (SPSS 11.0 software; SPSS, Inc., Chicago, IL). A level of P < 0.05 was considered significant.
Results
Effect of resveratrol on IGF-II protein secretion
First, we screened the effect of different RSV concentrations (10–7 to 10–4 M) on IGF-II protein secretion. As seen in Fig. 1A, the only changes in IGF-II protein were detected with RSV treatment of 10–4 and 10–6 M; thus, we expanded our experiments using only RSV 10–4 and 10–6 M for further characterization. Figure 1B shows a Western blot of IGF-II secreted at 24 and 48 h after RSV treatment at 10–4 and 10–6 M. Our results show a differential effect on IGF-II secretion, which is RSV concentration dependent. Treatment with 10–6 M RSV induced a 2-fold increase in proIGF-II secretion (17 kDa), whereas higher RSV concentration (10–4 M) reduced IGF-II secretion by 30% at 24 h and 40% at 48 h. Figure 1 (A and B, lower panels) shows bar graphs of densitometric analysis of IGF-II densitometry units [integrated density units (IDVs)] normalized to -actin densitometry units on three separate experiments. Please note that although only one Western blot is depicted as a representative experiment, the bar graphs represent three separate experiments done in triplicate (three Western blots per experiment).
Because estradiol regulates both, IGF-I and IGF-II mRNA (both genes have the ER consensus sequence), we measured IGF-I protein secretion into the media of MCF-7 cells to determine whether RSV also regulated IGF-I. No IGF-I (proIGF-I or mature) was detected in media from control or RSV-treated cells at any studied concentration (Fig. 1C). Thus, even though IGF-I and IGF-II are regulated by estradiol, only IGF-II is regulated by RSV.
We also studied RSV (10–6 or 10–4 M) effect on IGF-II secretion in the presence of TAM (10–7 M) or E2 (10–9 M). Figure 2 shows that RSV (10–6 M) and also E2 induced a 2-fold increase in IGF-II secretion, whereas RSV (10–4 M) and TAM decreased IGF-II secretion by 30 and 20% respectively, when compared with control. MCF-7 cells treated with RSV (10–6) in combination with E2 (10–9) showed a decrease in IGF-II secretion by 40%, when compared with either RSV (10–6) or E2-treated cells. A similar effect was observed when cells were cotreated with E2 and TAM. RSV (10–6) and TAM cotreatment decreased IGF-II secretion by 25%, whereas RSV (10–4) and E2 cotreatment decreased IGF-II secretion by 40%. Figure 2 (lower panel) shows bar graphs of densitometric analysis of IGF-II densitometry units (IDVs) normalized to -actin densitometry units on three separate experiments.
Modulation of IGF-II mRNA by RSV
The concentration-dependent effect of RSV on IGF-II gene expression was assessed by Northern blot analysis after12 (Fig. 3A) and 24 (Fig. 3B) h treatment. Because IGF-II protein levels changed at 24 and 48 h, we reasoned that mRNA changes may occur earlier and chose to analyze mRNA levels at 12 and 24 h after treatment. Figure 3 (A and B) shows increased IGF-II mRNA, when cells were treated with 10–6 M RSV. In contrast, a decrease in IGF-II mRNA was observed when cells were treated with 10–4 M RSV. Figure 3 (A and B, lower panels) shows bar graph of densitometric analysis of IGF-II densitometry units (IDVs) normalized to cyclophilin mRNA densitometry units on three separate experiments. Please note that only one Northern blot is shown for representation purposes, whereas data from three separate membranes of three different experiments were used in the bar graphs below for the statistical analysis. The increase in IGF-II mRNA when cells were treated with RSV (10–6 M) and compared with control was 3-fold at 12 h (P < 0.01) and 2.5-fold at 24 h (P < 0.01). In contrast, IGF-II mRNA decreased by 50% (P < 0.05) at 12 h and 40% (P < 0.05) at 24 h when cells were treated with 10–4 M RSV. The most significant differences (P < 0.001) were seen between the IGF-II mRNA of cells treated with RSV 10–6, compared with cells treated with RSV 10–4, a 6-fold difference at 12 h and 4.5-fold at 24 h.
RSV effect on cell growth and survival
To assess cell proliferation, MCF-7 cells were treated with RSV and examined by BrdU incorporation. Figure 4A shows that BrdU incorporation by MCF-7 cells treated with 10–6 M RSV was significant when compared with control (P < 0.01), inducing a 2-fold increase at 24 and 48 h after treatment. RSV 10–4 (P < 0.05) induced a significant decrease in MCF-7 cell survival at 24 and 48 h (by 50 and 70%, respectively). Because IGF-II exerts its effects by binding mainly to the IGF-IR, we blocked the IGF-IR on cells treated with 10–6 M RSV. Our results show that IGF-IR antibody significantly decreased BrdU incorporation in MCF-7 cells treated with RSV 10–6 M (P < 0.05).
Increasing concentrations of proIGF-II (10–100 ng/ml) were added to MCF-7 cells treated with RSV (10–4 M) to determine whether proIGF-II could prevent cell death. Figure 4B shows that addition of proIGF-II at 24 and 48 h significantly prevented cell death even at the lowest IGF-II concentration used (10 and 25 ng/ml: 2- and 4-fold increase at 24 and 48 h, respectively) when compared with RSV (10–4 M)-treated cells. The IGF-II response plateau at 50 ng/ml (2.5- and 4.5-fold increase 24 and 48 h after treatment). These data indicate that proIGF-II is able to rescue MCF-7 cells (P < 0.01) from the growth-inhibitory effect of RSV (10–4 M).
The MTT cell viability assay was used to determine whether RSV effect on IGF-II modulates cell survival. Figure 5A shows that treatment with RSV (10–4 M) caused a significant decrease in cell viability at 24 and 48 h. In contrast, treatment with RSV (10–6 M) increases cell survival, but this increase is statistically significant at 48 h (Fig. 5A, lower panel). Addition of the IGF-IR antibody blocks the effect of RSV (10–6) at 24 and 48 h (Fig. 5A).
To determine whether the observed effects of RSV on IGF-II are mediated through the ER, MCF-7 cells treated with RSV (10–6 and 10–4 M) were coincubated with or without the estrogen antagonist TAM. TAM (10–7 M) completely abolished the increase in cell number induced by RSV (10–6 M) (Fig. 5B) 24 and 48 h after treatment (P < 0.001). No significant difference was observed between the group treated with RSV (10–4 M) and the group treated with TAM and RSV (10–4) (Fig. 5B).
We also assessed the effect of RSV on IGF-II protein levels and cell growth in the ER-positive (ER+) T47D and ER-negative MCF-10 cells. Figure 6A (upper panel) shows that RSV (10–4 M) inhibits IGF-II, and RSV (10–6 M) stimulates IGF-II in T47D breast cancer cells at 24 and 48 h. Figure 6A (lower panel) shows bar graph representations of densitometric analysis of IGF-II densitometry units (IDVs) normalized to -actin protein units on three separate experiments. As seen, treatment with RSV (10–4 M) significantly (*, P < 0.05) inhibited IGF-II (by 60 and 40% 24 and 48 h, respectively, after treatment), whereas RSV (10–6 M) significantly increased IGF-II protein levels by 2- and 4-fold 24 and 48 h after treatment (**, P < 0.01). A similar effect on cell growth, as seen on MCF-7 cells, was observed on T47D cells, was a RSV (10–6 M)-induced increase in cell number (*, P < 0.05), whereas the opposite effect was seen with RSV (10–4 M) (**, P < 0.01).
Discussion
RSV inhibits the growth of cancer cells of different origins (9, 10, 11, 12). However, the effect of RSV on breast cancer cell growth and ER-mediated gene activation is a controversial subject under intense investigation. Several studies reported that treatment with RSV stimulated cell proliferation of ER-positive breast cancer cell lines, T47D and MCF-7 (14, 23). In contrast, other reports have shown that RSV treatment inhibited cell growth of both ER-positive and ER-negative breast cancer cells (22, 24, 25, 26). The wide range in the concentration of RSV used in the studies referenced above is the most likely explanation as to what caused the contrasting results. In addition, the use of unstripped (estrogen containing) serum in the culture media (26) may have masked the proliferative effect of RSV resulting in conflicting results.
Our study demonstrated that RSV regulates IGF-II gene expression in a dose-dependent manner in MCF-7 and T47D breast cancer cell lines. At low concentrations (10–6 M), RSV stimulated IGF-II gene expression leading to an increase in proIGF-II secretion. Furthermore, the increase in proIGF-II levels correlated with an increase in MCF-7 and T47D cell growth, suggesting that IGF-II mediated RSV stimulatory effects.
Our previous studies have shown that both MCF-7 and T47D cells are very sensitive to IGF-II and express IGF-I receptors and that IGF-II actions are mediated through the IGF-IR (27, 28). Because the IGF-IR antibody blocked the stimulatory effect of RSV treatment (10–6 M) in this study, we conclude that the IGF-II effect is mediated primarily through the IGF-IR. In addition, the blocking effect of the antibody on both the MTT (used to estimate cell number and viability) and BrdU assays (DNA synthesis) also demonstrated that IGF-II regulates both cell survival and growth.
Even though our study demonstrated that RSV does not regulate IGF-II in the ER-negative MCF-10 breast epithelial cell line, exogenously added proIGF-II was able to rescue these cells as well as the ER+ from RSV (10–4 M)-induced growth inhibition and cell death. These results demonstrate that all cell lines studied are responsive to IGF-II, but only ER+ cells are stimulated by RSV (10–6 M). Thus, RSV stimulatory effect requires the ER, whereas the inhibitory effect of RSV is ER independent.
Our study also shows that E2 (10–9 M), like RSV (10–6 M), stimulates IGF-II secretion by MCF-7 cells. Of note, cotreatment of MCF-7 cells with RSV (10–6) and E2 led to a decrease in IGF-II secretion, suggesting that RSV can act as an ER agonist but can also exert ER antagonist activity in the presence of E2. A similar effect was observed when cells where cotreated with RSV (10–4) and E2 (10–9). RSV inhibits the binding of E2 to the ER and induces ER-dependent transcriptional activation of genes with an estrogen-response element. This effect can be inhibited by specific estrogen antagonists (14). TAM, an estrogen antagonist, suppresses transcriptional activity by blocking E2 coactivator binding and promoting corepressor recruitment (29). Our data demonstrated that TAM inhibits RSV (10–6)-induced increase in IGF-II secretion as well as MCF-7 cell growth, thus confirming that the ER is required in mediating RSV stimulatory effect on IGF-II levels.
Of note, RSV did not stimulate IGF-I. Both, IGF-I and IGF-II are regulated by E2 because both genes have the estrogen-response element (13). Because our study showed that RSV regulated IGF-II, not IGF-I, we propose that RSV regulation of IGF-II is distinct from E2 regulation of these peptides.
Many reports from other laboratories have shown a biphasic effect of estrogen or RSV on many different systems, as we observed with RSV in our study. Amara and Dannies (30) reported that estradiol stimulated GH cell growth at low concentration but significantly inhibited growth at higher concentrations. Likewise, Kuwajerwala et al. (31) demonstrated that RSV exerted opposing effects on cell cycle progression in prostate cancer cells. RSV induced the cell cycle S phase at low concentrations, but it inhibited DNA synthesis at high concentrations.
The specific signaling pathways activated by RSV are mostly unknown. Because this estrogen antagonist binds the ER, it will induce genomic effects through the ER pathway as seen with proIGF-II regulation in our study. Other reports have also shown that RSV can stimulate nongenomic effects binding to the estrogen membrane receptor (32). The changes in IGF-II induced by RSV in ER+ cells will affect gene targets in the IGF-IR pathway as well as genes in the insulin receptor (isoform A) pathway. To our knowledge, there are no published data showing RSV stimulation of IGF-II or its downstream targets. Preliminary studies in our laboratory indicate that IGF-II regulates many antiapoptotic proteins. Work is in progress to further characterize the stimulatory effect of RSV on proIGF-II.
In summary, our study demonstrates that RSV effects on cell growth are mediated by proIGF-II. Furthermore, proIGF-II effects are mediated through the IGF-IR. In addition, because RSV regulates proIGF-II in ER+ (not ER negative) breast cancer cells and this effect is blocked by TAM, we conclude that RSV regulation of IGF-II is mediated through the ER. In contrast, the inhibitory effect of RSV is independent of the ER presence because RSV (10–4 M) inhibited proliferation and cell survival of ER cells.
Thus, we propose that proIGF-II is a key player in the mechanism of action of RSV and a promising target to develop new therapies for breast cancer treatment and chemoprevention.
Footnotes
This work was supported by California Breast Cancer Program Grant 0315-8818-03 and National Cancer Institute (NCI) Grant R01 CA71823-05.
Abbreviations: BrdU, Bromodeoxyuridine; E2, 17-estradiol; ECL, enhanced chemiluminescence; ER, estrogen receptor ; IDV, integrated density unit; IGF-IR, IGF-I receptor; IR-A, insulin receptor isoform A; MTT, 3-[4,5-dimethylthiazol2-yl]2,5-diphenyltetrazolium bromide; proIGF-II, precursor IGF-II; RSV, resveratrol; SFM, serum-free medium; TAM, tamoxifen.
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Pozo-Guisado E, Lorenzo-Benayas MJ, Fernandez-Salguero PM 2004 Resveratrol modulates the phosphoinositide 3-kinase pathway through an estrogen receptor -dependent mechanism: relevance in cell proliferation. Int J Cancer. 109:167–173(Sharda Vyas, Yayesh Asmer)
Abstract
IGF-II is a potent mitogen and inhibitor of apoptosis in breast cancer. Regulation of IGF-II is complex and includes inhibition by tumor suppressors, stimulation by oncogenes, and imprinting and hormonal regulation by estrogens. Resveratrol (RSV) is a phytoestrogen that displays estrogen-like agonistic and antagonistic activity. Recent studies have shown that RSV inhibits the growth of breast cancer cells and may represent a potent agent in chemopreventive therapy. Because 17-estradiol regulates IGF-II, we hypothesized that RSV may have a similar effect on IGF-II. The present study was designed to examine whether: 1) RSV modulates IGF-II in breast cancer cells; 2) regulation of IGF-II by RSV is dependent on the ER status; and 3) IGF-II (not IGF-I) mediates RSV effects on breast cancer cells. Treatment of MCF-7 and T47D cells with RSV (10–6 M) caused stimulation of precursor IGF-II mRNA and protein; this effect was blocked by coincubation with 17-estradiol (10–9 M). Cell growth stimulated by RSV (10–6 M) was blocked by addition of a blocking IGF-I receptor antibody, or the antiestrogen tamoxifen (10–7 M). In contrast, RSV treatment (10–4 M) inhibited IGF-II secretion and cell growth in MCF-7 and T47D cells. No increase in IGF-II levels is seen in estrogen receptor (–) MCF-10 cells, even though cell growth was inhibited by RSV 10–4 M and precursor IGF-II blocked the inhibitory effect of resveratrol. No change in IGF-I was observed with RSV treatment (10–6 to 10–4 M). Our study demonstrates that RSV regulates IGF-II and that IGF-II mediates RSV effect on cell survival and growth in breast cancer cells.
Introduction
IGF-II IS A POTENT mitogen that stimulates cell proliferation and inhibits apoptosis of breast cancer cells. IGF-II gene expression is paternally imprinted and regulated by tumor suppressor genes such as p53, WT1 (Wilms’ tumor gene), and guanine-cytosine factor (1, 2, 3). Furthermore, the complex regulation of IGF-II expression involves both developmentally regulated promoters (P1-P4) and alternative splicing of different untranslated leader sequences (4). IGF-II is translated as a prohormone that is cleaved and glycosylated to produce isoforms ranging from 21 [precursor IGF-II (proIGF-II)] to 7.5 kDa (mature IGF-II) (5). ProIGF-II is the predominant form secreted by most tumors and it is biologically active (5, 6, 7, 8). This growth factor binds several receptors including the IGF-II/mannose-6 phosphate receptor (M6P), IGF-I receptor (IGF-IR) and the insulin receptor isoform A (IR-A). IGF-II mitogenic effects are mediated through the IGF-IR and the IR-A, whereas insulin binding to IR-A stimulates metabolic pathways (9, 10, 11). IGF-II binding to the IGF-II/mannose-6 phosphate receptor results in intracellular degradation of IGF-II and decreased signaling through the IGF-I and IR-A receptors (12). IGF-II is also regulated by 17-estradiol (E2). E2 stimulates breast cancer cell proliferation and tumor development, and use of antiestrogens blocks this stimulatory response. Of interest, IGF-II signaling through the IGF-IR enhances estrogen receptor (ER) activation in human breast cancer cells (13).
Resveratrol (RSV), a phytoestrogen found mainly in grapes, has a molecular structure similar to E2 (14, 15) and works as an agonist/antagonist of E2 activity. RSV exhibits a variety of pharmacological effects, some of which may be associated with cardioprotective effects; but increasing interest in this compound is due to its potential as a chemopreventive agent (16, 17, 18, 19, 20, 21, 22, 23, 24). BecauseE2 regulates IGF-II, we hypothesized that RSV may have a similar effect on IGF-II. Thus, our hypothesis is that RSV inhibitory and stimulatory effects are mediated by IGF-II.
The present study was designed to examine whether: 1) RSV modulates IGF-II in breast cancer cells; 2) regulation of IGF-II by RSV is dependent on the ER status; and 3) IGF-II (not IGF-I) mediates RSV effects on breast cancer cells.
Materials and Methods
Cell culture
MCF-7, T47D breast carcinoma cell lines, and MCF-10 breast epithelial cells were obtained from the American Type Culture Collection (Manassas, VA). MCF-7 cells were maintained in a 5% CO2 incubator at 37 C, using DMEM/F12 media (Cellgro; Mediatech Inc., Herndon, VA) supplemented with 10 ml of 5000 U penicillin/streptomycin (100 U/ml penicillin and 100 U/ml streptomycin sulfate, Cellgro), 4 mM L-glutamine (Cellgro), 3 μg/ml -amphotericin, and 5% fetal bovine serum (Hyclone, Logan, UT). T47D cells were grown in RPMI 1640 media supplemented with 10 μg/ml insulin, 2 mM L-glutamine (Cellgro), 5000 U penicillin/streptomycin (100 U/ml penicillin and 100 U/ml streptomycin sulfate, Cellgro), and 10% fetal bovine serum (Hyclone). MCF-10 DMEM/F12 media was supplemented with 10 ml of 5000 U penicillin/streptomycin (100 U/ml penicillin and 100 U/ml streptomycin sulfate, Cellgro), 4 mM L-glutamine (Cellgro), 3 μg/ml -amphotericin, 10 μg/ml insulin (Sigma, St. Louis, MO), 0.5 μg/ml hydrocortisone (Sigma), 20 ng/ml murine epidermal growth factor (Gibco-BRL, Gaithersburg, MD), 100 ng/ml cholera toxin (Sigma), and 5% equine serum. Cells were detached by trypsinization (1x trypsin EDTA, Cellgro). Recombinant human proIGF-II (amino acids 1–156) was purchased from GroPep (Adelaide, Australia). RSV, E2, and tamoxifen (TAM) were purchased from Sigma and dissolved in dimethyl sulfoxide (Fischer Scientific, Pittsburgh, PA). Media from RSV-treated cells (conditioned media) was collected (24 and 48 h), centrifuged (800 rpm for 5 min), and frozen (–20 C) until assayed.
Western blot analysis
Total protein concentration (30 μg) of serum-free medium (SFM) collected after 24 and 48 h of RSV treatment (10–7 to 10–4) and control group (SFM with vehicle) was used to load 10–20% polyacrylamide-sodium dodecyl sulfate gradient gels and transferred to a nitrocellulose membrane Bio-Trace NT (Life Sciences, Ann Arbor, MI) using a semidry electrophoretic transfer cell (Bio-Rad Laboratories, Hercules, CA). Protein concentration was measured using the Coomassie Plus protein assay reagent (Pierce Biotechnology, Rockford, IL) Nitrocellulose membranes were blocked with 2% BSA IgG free (Sigma) in PBS/0.05% Tween 20 for 2 h. Membranes were then incubated with Amano IGF-II monoclonal antibody, clone S1-F2 (1:1000 Amano, Mitsubishi, Troy, VA). The Amano antibody is able to detect both, the mature and precursor forms of IGF-II. Similarly, the IGF-I polyclonal antibody (1:1000; Oncogene Research Products, San Diego, CA) was used to detect IGF-I. The blots were also probed with -actin antibody (1:10,000, Sigma) and used as a protein loading control. After 3 x 10 min washes in PBS/0.05% Tween, the corresponding secondary antibodies (1:1000, Amersham, Arlington Heights, IL) were added to the membranes (1 h at RT), followed by 3 x 10 min washes and horseradish-peroxidase (1:1000 Amersham, Arlington Heights, IL). Protein visualization was achieved by using enhanced chemiluminescence (ECL) and autoradiography with Hyperfilm ECL film (Amersham). The signals on the x-ray films were quantified using ChemiImager 4000 (Alpha Innotech Corp., San Leandro, CA).
Northern blot analysis
Total RNA was extracted using Tri reagent (Molecular Research Center, Cincinnati, OH), after 12 and 24 h incubation of MCF-7 and MCF-10 cells in SFM in the presence of 10–6 or 10–4 M RSV. RNA was then precipitated using isopropanol and solubilized in 0.5% sodium dodecyl sulfate. Ten micrograms of each sample RNA were then electrophoresed on a 1% agarose-formaldehyde gel and transferred to a nylon membrane (Hybond-N, Amersham) followed by UV cross-linking. The membranes were prehybridized (expressHyb solution; CLONTECH Laboratories, Mountain View, CA) for 1 h at 68 C before 32P-labeled IGF-II cDNA was added. Blots were hybridized at 42 C. The membranes were then washed and exposed to film (Kodak, Rochester, NY) with intensifying screens at –80 C for 2–3 d. Subsequently the membranes were stripped and reprobed with 32P-labeled cyclophilin cDNA, which was used as a loading control. The signals were quantitated using densitometric analysis of the autoradiographs by Chemi-Imager 4000 (Alpha Innotech).
Cell growth studies
Cell growth was measured by bromodeoxyuridine (BrdU) incorporation assay (Oncogene Research Products), after 24 and 48 h of RSV treatment. MCF-7 cells (1 x 104/well) were plated in 96-well tissue culture plates and grown in SFM. After 6 h incubation, 10–6 or 10–4 M RSV was added to the wells. Cells were then incubated with BrdU label for 20 h (5% CO2, 37 C incubator). BrdU incorporation was detected immunochemically after partial denaturation of double-stranded DNA. In this assay, horseradish-peroxidase catalyzes the conversion of the chromogenic substrate tetramethylbenzidine to a blue solution, the intensity of which is proportional to the amount of incorporated BrdU in the cells. The colored reaction product is then quantified using a spectrophotometer (405–595 nm). To evaluate at what extent the concentration-dependent effect of RSV on cell growth was due to IGF-II, BrdU incorporation was measured in the presence of 2 μg/ml anti-IGF-IR antibody (Santa Cruz Biotechnology, Santa Cruz, CA), which inhibits binding as well as basal and IGF-I/IGF-II-stimulated DNA synthesis. The effect of different concentrations of proIGF-II (10–100 ng/ml) on RSV (10–4 M)-treated MCF-7 cells was also assessed by BrdU incorporation after 24 and 48 h.
Cell viability assay
Cell viability was measured by the 3-[4,5-dimethylthiazol2-yl]2,5-diphenyltetrazolium bromide (MTT) assay; cells (1 x 104/well) were seeded in 96-well plates and grown in SFM. Cell viability was assessed at 24 and 48 h by measuring the rate of tetrazolium salts reduction to formazan (MTT, Sigma), which is proportional to the number of living cells. At the end of incubation, the absorbance was read at 540 nm. To evaluate at what extent the concentration-dependent effect of resveratrol on cell survival is due to proIGF-II signaling through the IGF-IR, parallel wells containing MCF-7 cells treated with 10–6 M RSV were incubated simultaneously with 2 μg/ml anti-IGF-IR antibody.
Statistical analysis
Values are expressed as the mean ± SEM. Statistical differences between mean values were determined by one-way ANOVA (SPSS 11.0 software; SPSS, Inc., Chicago, IL). A level of P < 0.05 was considered significant.
Results
Effect of resveratrol on IGF-II protein secretion
First, we screened the effect of different RSV concentrations (10–7 to 10–4 M) on IGF-II protein secretion. As seen in Fig. 1A, the only changes in IGF-II protein were detected with RSV treatment of 10–4 and 10–6 M; thus, we expanded our experiments using only RSV 10–4 and 10–6 M for further characterization. Figure 1B shows a Western blot of IGF-II secreted at 24 and 48 h after RSV treatment at 10–4 and 10–6 M. Our results show a differential effect on IGF-II secretion, which is RSV concentration dependent. Treatment with 10–6 M RSV induced a 2-fold increase in proIGF-II secretion (17 kDa), whereas higher RSV concentration (10–4 M) reduced IGF-II secretion by 30% at 24 h and 40% at 48 h. Figure 1 (A and B, lower panels) shows bar graphs of densitometric analysis of IGF-II densitometry units [integrated density units (IDVs)] normalized to -actin densitometry units on three separate experiments. Please note that although only one Western blot is depicted as a representative experiment, the bar graphs represent three separate experiments done in triplicate (three Western blots per experiment).
Because estradiol regulates both, IGF-I and IGF-II mRNA (both genes have the ER consensus sequence), we measured IGF-I protein secretion into the media of MCF-7 cells to determine whether RSV also regulated IGF-I. No IGF-I (proIGF-I or mature) was detected in media from control or RSV-treated cells at any studied concentration (Fig. 1C). Thus, even though IGF-I and IGF-II are regulated by estradiol, only IGF-II is regulated by RSV.
We also studied RSV (10–6 or 10–4 M) effect on IGF-II secretion in the presence of TAM (10–7 M) or E2 (10–9 M). Figure 2 shows that RSV (10–6 M) and also E2 induced a 2-fold increase in IGF-II secretion, whereas RSV (10–4 M) and TAM decreased IGF-II secretion by 30 and 20% respectively, when compared with control. MCF-7 cells treated with RSV (10–6) in combination with E2 (10–9) showed a decrease in IGF-II secretion by 40%, when compared with either RSV (10–6) or E2-treated cells. A similar effect was observed when cells were cotreated with E2 and TAM. RSV (10–6) and TAM cotreatment decreased IGF-II secretion by 25%, whereas RSV (10–4) and E2 cotreatment decreased IGF-II secretion by 40%. Figure 2 (lower panel) shows bar graphs of densitometric analysis of IGF-II densitometry units (IDVs) normalized to -actin densitometry units on three separate experiments.
Modulation of IGF-II mRNA by RSV
The concentration-dependent effect of RSV on IGF-II gene expression was assessed by Northern blot analysis after12 (Fig. 3A) and 24 (Fig. 3B) h treatment. Because IGF-II protein levels changed at 24 and 48 h, we reasoned that mRNA changes may occur earlier and chose to analyze mRNA levels at 12 and 24 h after treatment. Figure 3 (A and B) shows increased IGF-II mRNA, when cells were treated with 10–6 M RSV. In contrast, a decrease in IGF-II mRNA was observed when cells were treated with 10–4 M RSV. Figure 3 (A and B, lower panels) shows bar graph of densitometric analysis of IGF-II densitometry units (IDVs) normalized to cyclophilin mRNA densitometry units on three separate experiments. Please note that only one Northern blot is shown for representation purposes, whereas data from three separate membranes of three different experiments were used in the bar graphs below for the statistical analysis. The increase in IGF-II mRNA when cells were treated with RSV (10–6 M) and compared with control was 3-fold at 12 h (P < 0.01) and 2.5-fold at 24 h (P < 0.01). In contrast, IGF-II mRNA decreased by 50% (P < 0.05) at 12 h and 40% (P < 0.05) at 24 h when cells were treated with 10–4 M RSV. The most significant differences (P < 0.001) were seen between the IGF-II mRNA of cells treated with RSV 10–6, compared with cells treated with RSV 10–4, a 6-fold difference at 12 h and 4.5-fold at 24 h.
RSV effect on cell growth and survival
To assess cell proliferation, MCF-7 cells were treated with RSV and examined by BrdU incorporation. Figure 4A shows that BrdU incorporation by MCF-7 cells treated with 10–6 M RSV was significant when compared with control (P < 0.01), inducing a 2-fold increase at 24 and 48 h after treatment. RSV 10–4 (P < 0.05) induced a significant decrease in MCF-7 cell survival at 24 and 48 h (by 50 and 70%, respectively). Because IGF-II exerts its effects by binding mainly to the IGF-IR, we blocked the IGF-IR on cells treated with 10–6 M RSV. Our results show that IGF-IR antibody significantly decreased BrdU incorporation in MCF-7 cells treated with RSV 10–6 M (P < 0.05).
Increasing concentrations of proIGF-II (10–100 ng/ml) were added to MCF-7 cells treated with RSV (10–4 M) to determine whether proIGF-II could prevent cell death. Figure 4B shows that addition of proIGF-II at 24 and 48 h significantly prevented cell death even at the lowest IGF-II concentration used (10 and 25 ng/ml: 2- and 4-fold increase at 24 and 48 h, respectively) when compared with RSV (10–4 M)-treated cells. The IGF-II response plateau at 50 ng/ml (2.5- and 4.5-fold increase 24 and 48 h after treatment). These data indicate that proIGF-II is able to rescue MCF-7 cells (P < 0.01) from the growth-inhibitory effect of RSV (10–4 M).
The MTT cell viability assay was used to determine whether RSV effect on IGF-II modulates cell survival. Figure 5A shows that treatment with RSV (10–4 M) caused a significant decrease in cell viability at 24 and 48 h. In contrast, treatment with RSV (10–6 M) increases cell survival, but this increase is statistically significant at 48 h (Fig. 5A, lower panel). Addition of the IGF-IR antibody blocks the effect of RSV (10–6) at 24 and 48 h (Fig. 5A).
To determine whether the observed effects of RSV on IGF-II are mediated through the ER, MCF-7 cells treated with RSV (10–6 and 10–4 M) were coincubated with or without the estrogen antagonist TAM. TAM (10–7 M) completely abolished the increase in cell number induced by RSV (10–6 M) (Fig. 5B) 24 and 48 h after treatment (P < 0.001). No significant difference was observed between the group treated with RSV (10–4 M) and the group treated with TAM and RSV (10–4) (Fig. 5B).
We also assessed the effect of RSV on IGF-II protein levels and cell growth in the ER-positive (ER+) T47D and ER-negative MCF-10 cells. Figure 6A (upper panel) shows that RSV (10–4 M) inhibits IGF-II, and RSV (10–6 M) stimulates IGF-II in T47D breast cancer cells at 24 and 48 h. Figure 6A (lower panel) shows bar graph representations of densitometric analysis of IGF-II densitometry units (IDVs) normalized to -actin protein units on three separate experiments. As seen, treatment with RSV (10–4 M) significantly (*, P < 0.05) inhibited IGF-II (by 60 and 40% 24 and 48 h, respectively, after treatment), whereas RSV (10–6 M) significantly increased IGF-II protein levels by 2- and 4-fold 24 and 48 h after treatment (**, P < 0.01). A similar effect on cell growth, as seen on MCF-7 cells, was observed on T47D cells, was a RSV (10–6 M)-induced increase in cell number (*, P < 0.05), whereas the opposite effect was seen with RSV (10–4 M) (**, P < 0.01).
Discussion
RSV inhibits the growth of cancer cells of different origins (9, 10, 11, 12). However, the effect of RSV on breast cancer cell growth and ER-mediated gene activation is a controversial subject under intense investigation. Several studies reported that treatment with RSV stimulated cell proliferation of ER-positive breast cancer cell lines, T47D and MCF-7 (14, 23). In contrast, other reports have shown that RSV treatment inhibited cell growth of both ER-positive and ER-negative breast cancer cells (22, 24, 25, 26). The wide range in the concentration of RSV used in the studies referenced above is the most likely explanation as to what caused the contrasting results. In addition, the use of unstripped (estrogen containing) serum in the culture media (26) may have masked the proliferative effect of RSV resulting in conflicting results.
Our study demonstrated that RSV regulates IGF-II gene expression in a dose-dependent manner in MCF-7 and T47D breast cancer cell lines. At low concentrations (10–6 M), RSV stimulated IGF-II gene expression leading to an increase in proIGF-II secretion. Furthermore, the increase in proIGF-II levels correlated with an increase in MCF-7 and T47D cell growth, suggesting that IGF-II mediated RSV stimulatory effects.
Our previous studies have shown that both MCF-7 and T47D cells are very sensitive to IGF-II and express IGF-I receptors and that IGF-II actions are mediated through the IGF-IR (27, 28). Because the IGF-IR antibody blocked the stimulatory effect of RSV treatment (10–6 M) in this study, we conclude that the IGF-II effect is mediated primarily through the IGF-IR. In addition, the blocking effect of the antibody on both the MTT (used to estimate cell number and viability) and BrdU assays (DNA synthesis) also demonstrated that IGF-II regulates both cell survival and growth.
Even though our study demonstrated that RSV does not regulate IGF-II in the ER-negative MCF-10 breast epithelial cell line, exogenously added proIGF-II was able to rescue these cells as well as the ER+ from RSV (10–4 M)-induced growth inhibition and cell death. These results demonstrate that all cell lines studied are responsive to IGF-II, but only ER+ cells are stimulated by RSV (10–6 M). Thus, RSV stimulatory effect requires the ER, whereas the inhibitory effect of RSV is ER independent.
Our study also shows that E2 (10–9 M), like RSV (10–6 M), stimulates IGF-II secretion by MCF-7 cells. Of note, cotreatment of MCF-7 cells with RSV (10–6) and E2 led to a decrease in IGF-II secretion, suggesting that RSV can act as an ER agonist but can also exert ER antagonist activity in the presence of E2. A similar effect was observed when cells where cotreated with RSV (10–4) and E2 (10–9). RSV inhibits the binding of E2 to the ER and induces ER-dependent transcriptional activation of genes with an estrogen-response element. This effect can be inhibited by specific estrogen antagonists (14). TAM, an estrogen antagonist, suppresses transcriptional activity by blocking E2 coactivator binding and promoting corepressor recruitment (29). Our data demonstrated that TAM inhibits RSV (10–6)-induced increase in IGF-II secretion as well as MCF-7 cell growth, thus confirming that the ER is required in mediating RSV stimulatory effect on IGF-II levels.
Of note, RSV did not stimulate IGF-I. Both, IGF-I and IGF-II are regulated by E2 because both genes have the estrogen-response element (13). Because our study showed that RSV regulated IGF-II, not IGF-I, we propose that RSV regulation of IGF-II is distinct from E2 regulation of these peptides.
Many reports from other laboratories have shown a biphasic effect of estrogen or RSV on many different systems, as we observed with RSV in our study. Amara and Dannies (30) reported that estradiol stimulated GH cell growth at low concentration but significantly inhibited growth at higher concentrations. Likewise, Kuwajerwala et al. (31) demonstrated that RSV exerted opposing effects on cell cycle progression in prostate cancer cells. RSV induced the cell cycle S phase at low concentrations, but it inhibited DNA synthesis at high concentrations.
The specific signaling pathways activated by RSV are mostly unknown. Because this estrogen antagonist binds the ER, it will induce genomic effects through the ER pathway as seen with proIGF-II regulation in our study. Other reports have also shown that RSV can stimulate nongenomic effects binding to the estrogen membrane receptor (32). The changes in IGF-II induced by RSV in ER+ cells will affect gene targets in the IGF-IR pathway as well as genes in the insulin receptor (isoform A) pathway. To our knowledge, there are no published data showing RSV stimulation of IGF-II or its downstream targets. Preliminary studies in our laboratory indicate that IGF-II regulates many antiapoptotic proteins. Work is in progress to further characterize the stimulatory effect of RSV on proIGF-II.
In summary, our study demonstrates that RSV effects on cell growth are mediated by proIGF-II. Furthermore, proIGF-II effects are mediated through the IGF-IR. In addition, because RSV regulates proIGF-II in ER+ (not ER negative) breast cancer cells and this effect is blocked by TAM, we conclude that RSV regulation of IGF-II is mediated through the ER. In contrast, the inhibitory effect of RSV is independent of the ER presence because RSV (10–4 M) inhibited proliferation and cell survival of ER cells.
Thus, we propose that proIGF-II is a key player in the mechanism of action of RSV and a promising target to develop new therapies for breast cancer treatment and chemoprevention.
Footnotes
This work was supported by California Breast Cancer Program Grant 0315-8818-03 and National Cancer Institute (NCI) Grant R01 CA71823-05.
Abbreviations: BrdU, Bromodeoxyuridine; E2, 17-estradiol; ECL, enhanced chemiluminescence; ER, estrogen receptor ; IDV, integrated density unit; IGF-IR, IGF-I receptor; IR-A, insulin receptor isoform A; MTT, 3-[4,5-dimethylthiazol2-yl]2,5-diphenyltetrazolium bromide; proIGF-II, precursor IGF-II; RSV, resveratrol; SFM, serum-free medium; TAM, tamoxifen.
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Pozo-Guisado E, Lorenzo-Benayas MJ, Fernandez-Salguero PM 2004 Resveratrol modulates the phosphoinositide 3-kinase pathway through an estrogen receptor -dependent mechanism: relevance in cell proliferation. Int J Cancer. 109:167–173(Sharda Vyas, Yayesh Asmer)