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Smad pathway
BBRC Biochemical and Biophysical Research Communications 333 (2005) 827–832 www.elsevier.com/locate/ybbrc
Inhibitory effect of genistein on mouse colon cancer MC-26 cells involved TGF-b1/Smad pathway Zengli Yu a,*, Yunan Tang b, Dongsheng Hu a, Juan Li a a
b
School of Public Health, Zhengzhou University, Zhengzhou 450052, China Laboratory of Neuroimmunology, Department of Neurology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7025, USA Received 24 May 2005 Available online 13 June 2005
Abstract TGF-b1/signaling has been shown to be associated with proapoptotic and antimitotic activities in epithelial tissues. Genistein, a major component of soybean isoflavone, has multiple functions resulting in anticancer proliferation. We herein showed that genistein dose-dependently increased TGF-b1 mRNA expression in mouse colon cancer MC-26 cells. A mouse monoclonal anti-TGF-b1 neutralizing antibody partially, but not completely, blocked the growth inhibition by genistein. By using adenoviral vector, we demonstrated that Smad7 overexpression attenuated genistein-induced growth inhibition and apoptosis as determined by MTT and apoptosis ELISA. Smad7 overexpression also inhibited upregulation of p21 and caspase-3 activity by geinistein. To further confirm inhibitory effect of genistein in MC-26 cells require TGF-b1/Smad signaling, we employed Western blot and electrophoretic mobility shift assay to detect formation of Smad–DNA complexes and phosphorylation of Smad2 and Smad3, respectively. Data revealed that genistein induced an evident formation of Smad–DNA complexes and phosphorylation of Smad2 and Smad3, indicating increased TGF-b1 signaling. Taken together, these findings first provided insights into possible molecular mechanisms of growth inhibition by genistein that required Smad signaling, which could aid in its evaluation for colon tumor prevention. 2005 Published by Elsevier Inc. Keywords: Colon cancer; MC-26 cells; Genistein; TGF-b1; Smad proteins
Colon cancer is a major cause of morbidity and mortality in the Western world. The process of colon carcinogenesis involves a sequential transformation of ‘‘normal’’ colonic epithelial cells into precancerous lesions identified as ACF, which then progress into adenomas, carcinomas, and, finally, metastatic tumors [1]. Transforming growth factor-b1 (TGF-b1) belongs to a family of multifunctional cytokines that regulate a variety of biological responses such as proliferation, differentiation, apoptosis, and development [2,3]. Recent studies demonstrated that TGF-b1 signaling might play
*
Corresponding author. E-mail address: [email protected] (Z. Yu).
0006-291X/$ - see front matter 2005 Published by Elsevier Inc. doi:10.1016/j.bbrc.2005.05.177
a critical role in early and late stages of colon carcinogenesis [1]. TGF-b1 exerts its biological effects by interacting with two transmembrane receptors, type I and type II, that have serine/threonine kinase domains in the intracellular region. Binding of ligand to TGF-b receptors induces formation of a heteromeric complex of TbRI and TbRII receptors [4]. Formation of this heteromeric complex enables the TbRII to phosphorylate TbRI, resulting in activation of TbRI kinase [5]. TbRI phosphorylates and thereby activates transcription factors Smad2 and Smad3 [6,7]. Phosphorylated Smad2 and/ or Smad3 then bind their common partner, Smad4, to form a heteromeric complex, which then translocates to and accumulates in the nucleus, where it acts as a
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transcription factor [8,6]. The DNA-binding ability of Smad proteins is achieved mainly by their MH1 and MH2 (Mad homologous domain, MH) domains [9]. For example, Smad3 and Smad4 have been shown to associate with the palindromic sequence GTCTAGAC [10], as well as with GTCT or AGAC motifs in many promoters [11]. The actions of TGF-b1 are antagonized by Smad7, which interacts stably with TbRI to prevent phosphorylation and activation of receptor-regulated Smad2/3, thereby blocking TGF-b signaling [12]. Epidemiological data have concluded that diet may be the most important environmental factor involved in the etiology of various cancers [13]. In vivo studies also demonstrated that natural products have been a good source of novel chemotherapy and prevention for the progression of carcinogenesis. Consumption of soy has been found to reduce colon cancer risk in some human populations and animal studies [14]. Soy products are high in phytochemicals and the most abundant isoflavone in soy is genistein. GenisteinÕs effects on cells cancer include inhibition of proliferation, induction of differentiation [15], apoptosis [16], and arrest of cells at cell cycle checkpoints [17,18]. Despite genistein and TGF-b1 have similar inhibitory effects on cell growth, little information is known regarding the actions of one might involve or even require the other. We herein reported that genistein positively regulated TGF-b1/Smad pathway in mouse colon MC-26 cells, which confirmed the hypothesis that inhibitory effect of genistein on colon cancer cells involved activation of Smad signaling that could aid in its evaluation for colon tumor prevention.
Materials and methods Cell culture. Mouse colon cancer MC-26 cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 100 lg/ml streptomycin, and 100 U/ml penicillin, and grown in 5% CO2 atmosphere at 37 C with 100% humidity. Real-time quantitative PCR. Total RNA was isolated from genistein-treated MC-26 cells using Trizol (Life Technologies). RNA was further purified by using the RNeasy kit (Qiagen). The concentration of extracted RNA from each group was adjusted to 200 ng/ll based on the absorbance value measured at 260 nm. A 25 ll reaction mixture containing 2 lg of total RNA was reverse transcribed to cDNA using oligo(dT) primers and SuperScript II RT-polymerase (Life Technologies). Real-time quantitative PCR was performed using the ABI PRISM 7700 sequence detector (Applied Biosystems) in combination with SYBRgreen dye. The reaction was performed at 50 C for 2 min, 95 C for 10 min, followed by 40 cycles at 95 C for 15 s and 60 C for 1 min in triplicate and analyzed using the comparative Ct method according to the TaqMan manual. The following primers were used: for TGF-b1, 5 0 -GGTAACCGGCTGCTGACC-3 0 and 5 0 -GCCCT GTATTCCGTCTCCTCCTTG-3 0 with a product of 102 bp; for GADPH, 5 0 -GGCCTTCCGTGTTCCTAC-3 0 and 5 0 -TGTCATCAT ACTTGGCAGGTT-3 0 with a product of 87 bp. Trypan blue exclusion assay. To neutralize TGF-b1 growth factor activity, MC-26 cells were seeded at a density of 1 · 104 per well in 12well plates and treated with 60 lM genistein alone or in co-treatment
with a mouse monoclonal anti-TGF-b1 antibody (5–15 lg/ml) (R&D Systems) for 24 h. Cells were then trypsinized, stained with 0.4% trypan blue (Sigma), and counted using a hemocytometer. Only the unstained live cells were counted. Transient transfection. A FLAG-tagged murine Smad7 cDNA was isolated from pcDNA3-FLAG-Smad7 and then inserted into the pBabepuro retroviral vector [19,20]. High-titer retroviral supernatants were obtained through transient transfection of retroviral plasmid DNA into the Ecopak packaging cell line (Clontech). Virus was purified over two CsCl gradients and dialyzed against a buffer containing Tris–HCl (pH 7.5), 10% glycerol, and 1 mM MgCl2 [21]. MC-26 Cells were seeded at a density of 1 · 104 per well in 6-well plates. Cells were infected with either Smad7 or pBabepuro retrovirus on day 3 using Fugene6 (Roche), followed by selection with 2 lg/ml of puromycin medium for 3 days. The infected and selected MC-26 cells were cultured for further biochemical analysis. MTT colorimetric assay. The MTT test, based on the enzymatic reduction of the tetrazolium salt MTT in viable/metabolically active cells, was used to monitor cell growth. The Smad7-infected cells were plated at a density of 5 · 103 cells/well, using 96-well microtiter plates (Costar), and allowed to adhere overnight prior to 60 lM genistein treatment for 24 h. Colorimetric changes were measured using a Microtiter plate reader with a 570 nm filter. Apoptosis analysis. The formation of DNA fragments, an indicator of apoptosis, was measured using the Cell Death Detection ELISA (Roche, Indiana) according to the protocols provided by the manufacturer. Briefly, the Smad7-infected cells were manipulated in the same way as MTT assay. The cells were then lysed to release cytoplasmic histone-associated DNA fragments. Cell lysates were prepared and placed into streptavidin-coated microplates. These were incubated for 2 h at room temperature with anti-histone–biotin and anti-DNA–peroxidase antibodies. The 96-well plates were read at 405 nm to quantitate the amount of nucleosomes bound to the plate. Relative apoptosis was determined by a ratio of the average absorbance of the treatment wells to the average absorbance of the control wells. Western blot. Total lysates from infected and non-infected MC-26 cells were prepared with lysis buffer (1% NP-40, 10% glycerol, 20 mM Tris–Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaVO4, and 10 mM NaPO4) in the presence of a protease inhibitor solution consisting of 1 mM phenylmethyl sulfonylfluoride (PMSF), 1 lg/ml aprotinin, 1 lg/ml leupeptin, and 1 lg/ml pepstatin. Lysates were then subjected to 12% SDS–PAGE, transferred to nitrocellulose membranes (50 lg/lane), and incubated with primary antibodies and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) according to manufacturerÕs protocol. Bound antibody was visualized using enhanced chemiluminescence (Amersham Biosciences). Assay for caspase-3 activity. After Smad7-infected cells were treated by genistein for 24 h, the cells were washed twice with cold phosphate-buffered saline (PBS) and resuspended in the same lysis buffer as Western blot, left on ice for 40 min, then centrifuged at 8000g for 5 min. Caspase-3 activity was measured using fluorogenic substrate peptides 7-amine-4methylcoumarine (Pharmingen). The supernatant containing 100 lg of whole cell lysates was incubated with 100 lM substrate peptide in 100 ll lysis buffer at 37 C for 1 h. Absorbance of the samples was read at 405 nm in a microtiter plate reader using a sample without substrate peptides as a blank. Caspase3 activity of each sample was calculated according to the formula given below: caspase-3 activity ¼
OD of test sample OD of blank . OD of control OD of blank
Electrophoretic mobility shift assay (EMSA). MC-26 cells were plated at a density of 5 · 104 per well in 6-well plates and grown to 70% confluency. The cells were treated with various concentrations of genistein for 24 h. The cells were washed with PBS, scraped, pelleted by
Z. Yu et al. / Biochemical and Biophysical Research Communications 333 (2005) 827–832 centrifugation at 400g for 2 min at 4 C, and then frozen at 80 C. At the time of analysis, the cells were suspended in ice-cold PBS. Nuclear extracts were isolated by using the CelLytic NuCLEAR Extraction Kit (Sigma). Five microgram nuclear extract was incubated for 20 min on ice in binding reaction buffer (10 mM Hepes–KOH, pH 7.9, 4% glycerol, 40 mM KCl, 0.4 mM EDTA, and 0.4 mM dithiothreitol) in the presence of 1 lg poly(dI–dC), prior to the addition of 32P-labeled oligonucleotide probe (5 · 104 cpm) (Santa Cruz) for another 20 min of incubation at room temperature. Protein–DNA complexes were resolved in 5% polyacrylamide gels containing 0.5· TBE. Gels were dried and visualized by autoradiography. For competition experiments, nuclear extracts were incubated with a 50- and 100-fold molar excess of an unlabeled oligonucleotide probe containing the consensus SBE (CAGA Smad-binding elements, SBE). Statistical analysis. All data are presented as mean values ± SD (standard deviation). The data were evaluated by a one-way ANOVA followed by least significant difference (LSD) test as a post hoc test or DunnettÕs T3 test. Statistical significance was at p < 0.05.
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Antibody for TGF-b1 partially reversed the growth inhibition by genistein In this set experiment, we evaluated the effect of a mouse monoclonal anti-TGF-b1 neutralizing antibody (5–15 lg/ml) on cell growth in the presence of 60 lM genistein. Trypan blue exclusion assay showed that addition of a neutralizing TGF-b1 antibody reversed genistein-mediated growth inhibition. Co-incubation with 15 lg/ml anti-TGF-b1 antibody significantly attenuated the inhibitory effect of genistein, whereas the addition of the same concentration of an irrelevant IgG had no significant effect, convincingly suggesting that TGFb1 plays a critical role in growth inhibition mediated by genistein (Fig. 2). Smad7 overexpression blocked genistein-mediated growth inhibition
Results Genistein increased TGF-b1 mRNA expression in MC-26 cells Since genistein and TGF-b1 have similar inhibitory effects on cell growth, including regulating cell proliferation, apoptosis, differentiation, etc., it was reasonable to speculate that the actions of one might involve or even require the other. Therefore, the effect of genistein on TGF-b1 expression was examined. Real-time RTPCR analyses revealed that genistein dose-dependently stimulated TGF-b1 mRNA level in mouse colon cancer MC-26 cells (Fig. 1).
Smad7 is an inhibitory Smad that blocks TGF-b1 signaling. To further determine whether TGF-b1 signaling was involved in genistein-mediated inhibitory effects on colon cancer cells, we infected MC-26 cells with a FLAG-tagged mouse Smad7 retrovirus. Smad7 overexpression attenuated growth inhibition and apoptosis mediated by genistein (Figs. 3A and B) through inhibiting the upregulation of p21 and caspase-3 activity by genistein (Figs. 3C and D). Genistein induced formation of Smad–DNA complexes and phosphorylation of Smad2/3 To further confirm genistein-mediated growth inhibition involving TGF-b1/Smad signaling, we performed
Fig. 1. Genistein enhanced TGF-b1 mRNA expression in mouse colon cancer MC-26 cells. Real-time quantitative PCR was used to quantify the TGF-b1 mRNA in MC-26 cell treated with given concentrations of genistein for 24 h. Levels of TGF-b1 and G3PDH expression in each sample were determined by using the relative standard curve method. A relative amount of DNA of TGF-b1 was expressed as a ratio to GADPH DNA, and the TGF-b1 level in vehicle control (0 lM) was set to 1. This experiment was run twice. In each experiment, samples were run in triplicate. Data are expressed as means ± SD. a, p < 0.05; b, p < 0.01. Con, ethanol vehicle control; GS, genistein.
Fig. 2. Anti-TGF-b1 antibody neutralized genistein-mediated growth inhibition in MC-26 cells. MC-26 cells were cultured with genistein alone (60 lM), or combinations of genistein and anti-TGF-b1 neutralizing antibody (5, 10, and 15 lg/ml) or genistein and an irrelevant IgG (15 lg/ ml), as indicated for 24 h. Control cells only received vehicle. Trypan blue exclusion assay was employed to determined cell growth. Only the unstained live cells were counted. Values are means ± SD (n = 5). a, p < 0.05 versus vehicle control; b, p < 0.05 versus geinstein. Con, ethanol vehicle control; GS, genistein; Ab, anti-transforming growth factor-b1.
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Fig. 3. Expression of retrovirally transduced Smad7 blocks inhibitory effect of genistein. Mouse colon cancer MC-26 cells were infected with either the control pBabepuro (pBabe) or pBabeSmad7 (Smad7) and selected with puromycin. After the infected and selected cells were treated with 60 lM genistein (GS) for 24 h, the cells were collected and used for biochemical analysis. The data here were obtained from triplicate experiments and expressed as means ± SD. a, p < 0.05 versus control pBabepuro; b, p < 0.05 versus pBabepuro + genistein. (A) MTT assay was used to determine cell growth. (B) Cell Death Detection ELISA kit was employed to determine formation of NDA fragments, an indictor of apoptosis. (C) Western blot analysis was used to determine cell cycle inhibitor gene p21 protein expression. (D) Fluorogenic substrate peptide 7-amine-4-methylcoumarine was used to probe capase-3 activity.
EMSA and Western blot to explore critical molecular changes of this pathway. As data indicated, after 24 h of treatment, genistein strongly induced Smad protein binding to probes containing SBE sequences (CAGA Smad-binding elements, SBE) (Fig. 4A). The competition reaction proved that Smad–DNA complex is specific. Genistein also produced a slight increase in phosphorylation of Smad2/3 (Fig. 4B). These data suggested that genistein activates Smad proteins to translocate to the nucleus and bind to SBE in MC-26 cells.
Discussion Chemically, isoflavones belong to the group of polyphenols. The most important food source is soy and the most abundant isoflavone is genistein. Soy isoflavones are felt to protect against different cancers [22], cardiovascular disease [23], and bone loss [24]. Many studies have demonstrated the effect of soy isoflavones on specific target molecules and signaling pathways, including
but not limited to, cell proliferation and differentiation, cell cycle regulation, apoptosis, angiogenesis, cell adhesion and migration, metastasis, and activity of different enzymes [25–28]. TGF-b1 was a potent inhibitor of cell proliferation, and a rapid inducer of growth arrest and apoptosis in epithelial tissue [29]. It inhibits the cell cycle through a partial block in the cell transition form G1 to S phase by downregulating components of the cell cycle and upregulating cell cycle inhibitors [30]. An apoptotic effect of TGF-b1 manifests with morphological features, like cell shrinkage, chromatin condensation, nuclear pycnosis, fragmentation of the nucleus, and formation of apoptotic bodies. Execution of TGF-b1-induced apoptosis occurs by the action of caspase-3 and is regulated via a mitochondrial pathway [31]. Mouse colon cancer cell line MC-26 was derived from a chemically induced transplantable colon cancer (CT26) in the mouse [32]. MC-26 cells express Smad2 and Smad3, which are important direct downstream targets of TGF-b type I receptor and also express Smad4, which
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forms heteromeric complexes with Smad2 and Smad3 to enter the nucleus as transcription regulators [33]. Unlike most human colorectal cancer cells, MC-26 cells are sensitive to the tumor suppressor effects of TGF-b1, as demonstrated by the ability of TGF-b1 to inhibit cell growth and induce apoptosis. Thus, MC-26 cells have a functional TGF-b1 signaling pathway and are a useful model for examining the tumor suppressor effects of TGF-b1 in colorectal cancers [33,34]. Since genistein and TGF-b1 have similar inhibitory effects on epithelial cancer cell growth, it was reasonable to speculate that the actions of one might involve or even require the other. The results described here demonstrated that, indeed, the actions of genistein on colon cancer cells in part not completely involved TGF-b1 signaling. Genistein treatment increased TGF-b1 mRNA
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expression and 15 lg/ml mouse monoclonal neutralizing anti-TGF-b1 antibody significantly blocked inhibitory effect of genistein in colon cancer MC-26 cells, suggesting that TGF-b1 plays a critical role in growth inhibition mediated by genistein. Smads are a novel class of proteins that function as transcription regulators and either bind directly to specific cis regulatory elements of the promoters or interact with other transcription regulators [35,36]. There are several classes of Smads. The receptor-activated Smads (Smad2 and Smad3) are phosphorylated at their carboxyl-terminal serines by the type I receptors. The co-Smads (Smad4) form heteromeric complexes with receptor-activator Smads to enter the nucleus. Smad7 is an inhibitor of TGF-b1 signaling by binding to the TbRI receptor and preventing phosphorylation of Smad2 and Smad3 [37]. To examine the role of elevated Smad7 in growth-inhibitory effect of genistein, we have used retroviruses to introduce Smad7 into MC-26 cells by using adenoviral vector. As expected, Smad7 overexpression reversed genistein-induced growth inhibition and apoptosis as determined by MTT assay and cell death ELISA kit. Smad7 overexpression also decreased genistein-induced upregulation of cell cycle inhibitor gene p21 protein and apoptosis executor caspase-3 activity. In the final test set, our data indicated that genistein induced an evident formation of Smad–DNA complexes and phosphorylation of Smad2/3. This further confirmed that TGF-b1/Smad signaling pathway was involved inhibitory effects of genistein on colon cancer cells. Taken together, these results first demonstrated that TGF-b1/Smad pathway has an important role in the prevention of colon tumor by genistein.
References
Fig. 4. Genistein induced formation of Smad–DNA complexes and phosphorylation of Smad2/3. MC-26 cells were treated with given concentrations of genistein for 24 h. The cells were then collected and used for biochemical analysis. (A) Whole cell lysates were subjected to Western blot analysis using antibodies against phosphoserine Smad2 (p-Smad2), p-Smad2 or total Smad2/3. Immunoblots representative of triplicate independent experiments are shown. Lane 1, vehicle control; lane 2, 20 lM; lane 3, 40 lM; lane 4, 60 lM. Con, ethanol vehicle control; GS, genistein. (B) EMSA was performed using nuclear extracts from genistein-treated MC-26 cells. Competition reaction was performed with 50- and 100-fold molar excess of an unlabelled oligonucleotide probe and proved the specificity of Smad–DNA complex. Lane 1, 50· competition reaction; lane 2, 100· competition reaction; lane 2, vehicle control; lane 4, 20 lM genistein; lane 5, 40 lM genistein; lane 6, 60 lM genistein.
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Inhibitory effect of genistein on mouse colon cancer MC-26 cells involved TGF-b1/Smad pathway Zengli Yu a,*, Yunan Tang b, Dongsheng Hu a, Juan Li a a
b
School of Public Health, Zhengzhou University, Zhengzhou 450052, China Laboratory of Neuroimmunology, Department of Neurology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7025, USA Received 24 May 2005 Available online 13 June 2005
Abstract TGF-b1/signaling has been shown to be associated with proapoptotic and antimitotic activities in epithelial tissues. Genistein, a major component of soybean isoflavone, has multiple functions resulting in anticancer proliferation. We herein showed that genistein dose-dependently increased TGF-b1 mRNA expression in mouse colon cancer MC-26 cells. A mouse monoclonal anti-TGF-b1 neutralizing antibody partially, but not completely, blocked the growth inhibition by genistein. By using adenoviral vector, we demonstrated that Smad7 overexpression attenuated genistein-induced growth inhibition and apoptosis as determined by MTT and apoptosis ELISA. Smad7 overexpression also inhibited upregulation of p21 and caspase-3 activity by geinistein. To further confirm inhibitory effect of genistein in MC-26 cells require TGF-b1/Smad signaling, we employed Western blot and electrophoretic mobility shift assay to detect formation of Smad–DNA complexes and phosphorylation of Smad2 and Smad3, respectively. Data revealed that genistein induced an evident formation of Smad–DNA complexes and phosphorylation of Smad2 and Smad3, indicating increased TGF-b1 signaling. Taken together, these findings first provided insights into possible molecular mechanisms of growth inhibition by genistein that required Smad signaling, which could aid in its evaluation for colon tumor prevention. 2005 Published by Elsevier Inc. Keywords: Colon cancer; MC-26 cells; Genistein; TGF-b1; Smad proteins
Colon cancer is a major cause of morbidity and mortality in the Western world. The process of colon carcinogenesis involves a sequential transformation of ‘‘normal’’ colonic epithelial cells into precancerous lesions identified as ACF, which then progress into adenomas, carcinomas, and, finally, metastatic tumors [1]. Transforming growth factor-b1 (TGF-b1) belongs to a family of multifunctional cytokines that regulate a variety of biological responses such as proliferation, differentiation, apoptosis, and development [2,3]. Recent studies demonstrated that TGF-b1 signaling might play
*
Corresponding author. E-mail address: [email protected] (Z. Yu).
0006-291X/$ - see front matter 2005 Published by Elsevier Inc. doi:10.1016/j.bbrc.2005.05.177
a critical role in early and late stages of colon carcinogenesis [1]. TGF-b1 exerts its biological effects by interacting with two transmembrane receptors, type I and type II, that have serine/threonine kinase domains in the intracellular region. Binding of ligand to TGF-b receptors induces formation of a heteromeric complex of TbRI and TbRII receptors [4]. Formation of this heteromeric complex enables the TbRII to phosphorylate TbRI, resulting in activation of TbRI kinase [5]. TbRI phosphorylates and thereby activates transcription factors Smad2 and Smad3 [6,7]. Phosphorylated Smad2 and/ or Smad3 then bind their common partner, Smad4, to form a heteromeric complex, which then translocates to and accumulates in the nucleus, where it acts as a
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transcription factor [8,6]. The DNA-binding ability of Smad proteins is achieved mainly by their MH1 and MH2 (Mad homologous domain, MH) domains [9]. For example, Smad3 and Smad4 have been shown to associate with the palindromic sequence GTCTAGAC [10], as well as with GTCT or AGAC motifs in many promoters [11]. The actions of TGF-b1 are antagonized by Smad7, which interacts stably with TbRI to prevent phosphorylation and activation of receptor-regulated Smad2/3, thereby blocking TGF-b signaling [12]. Epidemiological data have concluded that diet may be the most important environmental factor involved in the etiology of various cancers [13]. In vivo studies also demonstrated that natural products have been a good source of novel chemotherapy and prevention for the progression of carcinogenesis. Consumption of soy has been found to reduce colon cancer risk in some human populations and animal studies [14]. Soy products are high in phytochemicals and the most abundant isoflavone in soy is genistein. GenisteinÕs effects on cells cancer include inhibition of proliferation, induction of differentiation [15], apoptosis [16], and arrest of cells at cell cycle checkpoints [17,18]. Despite genistein and TGF-b1 have similar inhibitory effects on cell growth, little information is known regarding the actions of one might involve or even require the other. We herein reported that genistein positively regulated TGF-b1/Smad pathway in mouse colon MC-26 cells, which confirmed the hypothesis that inhibitory effect of genistein on colon cancer cells involved activation of Smad signaling that could aid in its evaluation for colon tumor prevention.
Materials and methods Cell culture. Mouse colon cancer MC-26 cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 100 lg/ml streptomycin, and 100 U/ml penicillin, and grown in 5% CO2 atmosphere at 37 C with 100% humidity. Real-time quantitative PCR. Total RNA was isolated from genistein-treated MC-26 cells using Trizol (Life Technologies). RNA was further purified by using the RNeasy kit (Qiagen). The concentration of extracted RNA from each group was adjusted to 200 ng/ll based on the absorbance value measured at 260 nm. A 25 ll reaction mixture containing 2 lg of total RNA was reverse transcribed to cDNA using oligo(dT) primers and SuperScript II RT-polymerase (Life Technologies). Real-time quantitative PCR was performed using the ABI PRISM 7700 sequence detector (Applied Biosystems) in combination with SYBRgreen dye. The reaction was performed at 50 C for 2 min, 95 C for 10 min, followed by 40 cycles at 95 C for 15 s and 60 C for 1 min in triplicate and analyzed using the comparative Ct method according to the TaqMan manual. The following primers were used: for TGF-b1, 5 0 -GGTAACCGGCTGCTGACC-3 0 and 5 0 -GCCCT GTATTCCGTCTCCTCCTTG-3 0 with a product of 102 bp; for GADPH, 5 0 -GGCCTTCCGTGTTCCTAC-3 0 and 5 0 -TGTCATCAT ACTTGGCAGGTT-3 0 with a product of 87 bp. Trypan blue exclusion assay. To neutralize TGF-b1 growth factor activity, MC-26 cells were seeded at a density of 1 · 104 per well in 12well plates and treated with 60 lM genistein alone or in co-treatment
with a mouse monoclonal anti-TGF-b1 antibody (5–15 lg/ml) (R&D Systems) for 24 h. Cells were then trypsinized, stained with 0.4% trypan blue (Sigma), and counted using a hemocytometer. Only the unstained live cells were counted. Transient transfection. A FLAG-tagged murine Smad7 cDNA was isolated from pcDNA3-FLAG-Smad7 and then inserted into the pBabepuro retroviral vector [19,20]. High-titer retroviral supernatants were obtained through transient transfection of retroviral plasmid DNA into the Ecopak packaging cell line (Clontech). Virus was purified over two CsCl gradients and dialyzed against a buffer containing Tris–HCl (pH 7.5), 10% glycerol, and 1 mM MgCl2 [21]. MC-26 Cells were seeded at a density of 1 · 104 per well in 6-well plates. Cells were infected with either Smad7 or pBabepuro retrovirus on day 3 using Fugene6 (Roche), followed by selection with 2 lg/ml of puromycin medium for 3 days. The infected and selected MC-26 cells were cultured for further biochemical analysis. MTT colorimetric assay. The MTT test, based on the enzymatic reduction of the tetrazolium salt MTT in viable/metabolically active cells, was used to monitor cell growth. The Smad7-infected cells were plated at a density of 5 · 103 cells/well, using 96-well microtiter plates (Costar), and allowed to adhere overnight prior to 60 lM genistein treatment for 24 h. Colorimetric changes were measured using a Microtiter plate reader with a 570 nm filter. Apoptosis analysis. The formation of DNA fragments, an indicator of apoptosis, was measured using the Cell Death Detection ELISA (Roche, Indiana) according to the protocols provided by the manufacturer. Briefly, the Smad7-infected cells were manipulated in the same way as MTT assay. The cells were then lysed to release cytoplasmic histone-associated DNA fragments. Cell lysates were prepared and placed into streptavidin-coated microplates. These were incubated for 2 h at room temperature with anti-histone–biotin and anti-DNA–peroxidase antibodies. The 96-well plates were read at 405 nm to quantitate the amount of nucleosomes bound to the plate. Relative apoptosis was determined by a ratio of the average absorbance of the treatment wells to the average absorbance of the control wells. Western blot. Total lysates from infected and non-infected MC-26 cells were prepared with lysis buffer (1% NP-40, 10% glycerol, 20 mM Tris–Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaVO4, and 10 mM NaPO4) in the presence of a protease inhibitor solution consisting of 1 mM phenylmethyl sulfonylfluoride (PMSF), 1 lg/ml aprotinin, 1 lg/ml leupeptin, and 1 lg/ml pepstatin. Lysates were then subjected to 12% SDS–PAGE, transferred to nitrocellulose membranes (50 lg/lane), and incubated with primary antibodies and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) according to manufacturerÕs protocol. Bound antibody was visualized using enhanced chemiluminescence (Amersham Biosciences). Assay for caspase-3 activity. After Smad7-infected cells were treated by genistein for 24 h, the cells were washed twice with cold phosphate-buffered saline (PBS) and resuspended in the same lysis buffer as Western blot, left on ice for 40 min, then centrifuged at 8000g for 5 min. Caspase-3 activity was measured using fluorogenic substrate peptides 7-amine-4methylcoumarine (Pharmingen). The supernatant containing 100 lg of whole cell lysates was incubated with 100 lM substrate peptide in 100 ll lysis buffer at 37 C for 1 h. Absorbance of the samples was read at 405 nm in a microtiter plate reader using a sample without substrate peptides as a blank. Caspase3 activity of each sample was calculated according to the formula given below: caspase-3 activity ¼
OD of test sample OD of blank . OD of control OD of blank
Electrophoretic mobility shift assay (EMSA). MC-26 cells were plated at a density of 5 · 104 per well in 6-well plates and grown to 70% confluency. The cells were treated with various concentrations of genistein for 24 h. The cells were washed with PBS, scraped, pelleted by
Z. Yu et al. / Biochemical and Biophysical Research Communications 333 (2005) 827–832 centrifugation at 400g for 2 min at 4 C, and then frozen at 80 C. At the time of analysis, the cells were suspended in ice-cold PBS. Nuclear extracts were isolated by using the CelLytic NuCLEAR Extraction Kit (Sigma). Five microgram nuclear extract was incubated for 20 min on ice in binding reaction buffer (10 mM Hepes–KOH, pH 7.9, 4% glycerol, 40 mM KCl, 0.4 mM EDTA, and 0.4 mM dithiothreitol) in the presence of 1 lg poly(dI–dC), prior to the addition of 32P-labeled oligonucleotide probe (5 · 104 cpm) (Santa Cruz) for another 20 min of incubation at room temperature. Protein–DNA complexes were resolved in 5% polyacrylamide gels containing 0.5· TBE. Gels were dried and visualized by autoradiography. For competition experiments, nuclear extracts were incubated with a 50- and 100-fold molar excess of an unlabeled oligonucleotide probe containing the consensus SBE (CAGA Smad-binding elements, SBE). Statistical analysis. All data are presented as mean values ± SD (standard deviation). The data were evaluated by a one-way ANOVA followed by least significant difference (LSD) test as a post hoc test or DunnettÕs T3 test. Statistical significance was at p < 0.05.
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Antibody for TGF-b1 partially reversed the growth inhibition by genistein In this set experiment, we evaluated the effect of a mouse monoclonal anti-TGF-b1 neutralizing antibody (5–15 lg/ml) on cell growth in the presence of 60 lM genistein. Trypan blue exclusion assay showed that addition of a neutralizing TGF-b1 antibody reversed genistein-mediated growth inhibition. Co-incubation with 15 lg/ml anti-TGF-b1 antibody significantly attenuated the inhibitory effect of genistein, whereas the addition of the same concentration of an irrelevant IgG had no significant effect, convincingly suggesting that TGFb1 plays a critical role in growth inhibition mediated by genistein (Fig. 2). Smad7 overexpression blocked genistein-mediated growth inhibition
Results Genistein increased TGF-b1 mRNA expression in MC-26 cells Since genistein and TGF-b1 have similar inhibitory effects on cell growth, including regulating cell proliferation, apoptosis, differentiation, etc., it was reasonable to speculate that the actions of one might involve or even require the other. Therefore, the effect of genistein on TGF-b1 expression was examined. Real-time RTPCR analyses revealed that genistein dose-dependently stimulated TGF-b1 mRNA level in mouse colon cancer MC-26 cells (Fig. 1).
Smad7 is an inhibitory Smad that blocks TGF-b1 signaling. To further determine whether TGF-b1 signaling was involved in genistein-mediated inhibitory effects on colon cancer cells, we infected MC-26 cells with a FLAG-tagged mouse Smad7 retrovirus. Smad7 overexpression attenuated growth inhibition and apoptosis mediated by genistein (Figs. 3A and B) through inhibiting the upregulation of p21 and caspase-3 activity by genistein (Figs. 3C and D). Genistein induced formation of Smad–DNA complexes and phosphorylation of Smad2/3 To further confirm genistein-mediated growth inhibition involving TGF-b1/Smad signaling, we performed
Fig. 1. Genistein enhanced TGF-b1 mRNA expression in mouse colon cancer MC-26 cells. Real-time quantitative PCR was used to quantify the TGF-b1 mRNA in MC-26 cell treated with given concentrations of genistein for 24 h. Levels of TGF-b1 and G3PDH expression in each sample were determined by using the relative standard curve method. A relative amount of DNA of TGF-b1 was expressed as a ratio to GADPH DNA, and the TGF-b1 level in vehicle control (0 lM) was set to 1. This experiment was run twice. In each experiment, samples were run in triplicate. Data are expressed as means ± SD. a, p < 0.05; b, p < 0.01. Con, ethanol vehicle control; GS, genistein.
Fig. 2. Anti-TGF-b1 antibody neutralized genistein-mediated growth inhibition in MC-26 cells. MC-26 cells were cultured with genistein alone (60 lM), or combinations of genistein and anti-TGF-b1 neutralizing antibody (5, 10, and 15 lg/ml) or genistein and an irrelevant IgG (15 lg/ ml), as indicated for 24 h. Control cells only received vehicle. Trypan blue exclusion assay was employed to determined cell growth. Only the unstained live cells were counted. Values are means ± SD (n = 5). a, p < 0.05 versus vehicle control; b, p < 0.05 versus geinstein. Con, ethanol vehicle control; GS, genistein; Ab, anti-transforming growth factor-b1.
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Fig. 3. Expression of retrovirally transduced Smad7 blocks inhibitory effect of genistein. Mouse colon cancer MC-26 cells were infected with either the control pBabepuro (pBabe) or pBabeSmad7 (Smad7) and selected with puromycin. After the infected and selected cells were treated with 60 lM genistein (GS) for 24 h, the cells were collected and used for biochemical analysis. The data here were obtained from triplicate experiments and expressed as means ± SD. a, p < 0.05 versus control pBabepuro; b, p < 0.05 versus pBabepuro + genistein. (A) MTT assay was used to determine cell growth. (B) Cell Death Detection ELISA kit was employed to determine formation of NDA fragments, an indictor of apoptosis. (C) Western blot analysis was used to determine cell cycle inhibitor gene p21 protein expression. (D) Fluorogenic substrate peptide 7-amine-4-methylcoumarine was used to probe capase-3 activity.
EMSA and Western blot to explore critical molecular changes of this pathway. As data indicated, after 24 h of treatment, genistein strongly induced Smad protein binding to probes containing SBE sequences (CAGA Smad-binding elements, SBE) (Fig. 4A). The competition reaction proved that Smad–DNA complex is specific. Genistein also produced a slight increase in phosphorylation of Smad2/3 (Fig. 4B). These data suggested that genistein activates Smad proteins to translocate to the nucleus and bind to SBE in MC-26 cells.
Discussion Chemically, isoflavones belong to the group of polyphenols. The most important food source is soy and the most abundant isoflavone is genistein. Soy isoflavones are felt to protect against different cancers [22], cardiovascular disease [23], and bone loss [24]. Many studies have demonstrated the effect of soy isoflavones on specific target molecules and signaling pathways, including
but not limited to, cell proliferation and differentiation, cell cycle regulation, apoptosis, angiogenesis, cell adhesion and migration, metastasis, and activity of different enzymes [25–28]. TGF-b1 was a potent inhibitor of cell proliferation, and a rapid inducer of growth arrest and apoptosis in epithelial tissue [29]. It inhibits the cell cycle through a partial block in the cell transition form G1 to S phase by downregulating components of the cell cycle and upregulating cell cycle inhibitors [30]. An apoptotic effect of TGF-b1 manifests with morphological features, like cell shrinkage, chromatin condensation, nuclear pycnosis, fragmentation of the nucleus, and formation of apoptotic bodies. Execution of TGF-b1-induced apoptosis occurs by the action of caspase-3 and is regulated via a mitochondrial pathway [31]. Mouse colon cancer cell line MC-26 was derived from a chemically induced transplantable colon cancer (CT26) in the mouse [32]. MC-26 cells express Smad2 and Smad3, which are important direct downstream targets of TGF-b type I receptor and also express Smad4, which
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forms heteromeric complexes with Smad2 and Smad3 to enter the nucleus as transcription regulators [33]. Unlike most human colorectal cancer cells, MC-26 cells are sensitive to the tumor suppressor effects of TGF-b1, as demonstrated by the ability of TGF-b1 to inhibit cell growth and induce apoptosis. Thus, MC-26 cells have a functional TGF-b1 signaling pathway and are a useful model for examining the tumor suppressor effects of TGF-b1 in colorectal cancers [33,34]. Since genistein and TGF-b1 have similar inhibitory effects on epithelial cancer cell growth, it was reasonable to speculate that the actions of one might involve or even require the other. The results described here demonstrated that, indeed, the actions of genistein on colon cancer cells in part not completely involved TGF-b1 signaling. Genistein treatment increased TGF-b1 mRNA
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expression and 15 lg/ml mouse monoclonal neutralizing anti-TGF-b1 antibody significantly blocked inhibitory effect of genistein in colon cancer MC-26 cells, suggesting that TGF-b1 plays a critical role in growth inhibition mediated by genistein. Smads are a novel class of proteins that function as transcription regulators and either bind directly to specific cis regulatory elements of the promoters or interact with other transcription regulators [35,36]. There are several classes of Smads. The receptor-activated Smads (Smad2 and Smad3) are phosphorylated at their carboxyl-terminal serines by the type I receptors. The co-Smads (Smad4) form heteromeric complexes with receptor-activator Smads to enter the nucleus. Smad7 is an inhibitor of TGF-b1 signaling by binding to the TbRI receptor and preventing phosphorylation of Smad2 and Smad3 [37]. To examine the role of elevated Smad7 in growth-inhibitory effect of genistein, we have used retroviruses to introduce Smad7 into MC-26 cells by using adenoviral vector. As expected, Smad7 overexpression reversed genistein-induced growth inhibition and apoptosis as determined by MTT assay and cell death ELISA kit. Smad7 overexpression also decreased genistein-induced upregulation of cell cycle inhibitor gene p21 protein and apoptosis executor caspase-3 activity. In the final test set, our data indicated that genistein induced an evident formation of Smad–DNA complexes and phosphorylation of Smad2/3. This further confirmed that TGF-b1/Smad signaling pathway was involved inhibitory effects of genistein on colon cancer cells. Taken together, these results first demonstrated that TGF-b1/Smad pathway has an important role in the prevention of colon tumor by genistein.
References
Fig. 4. Genistein induced formation of Smad–DNA complexes and phosphorylation of Smad2/3. MC-26 cells were treated with given concentrations of genistein for 24 h. The cells were then collected and used for biochemical analysis. (A) Whole cell lysates were subjected to Western blot analysis using antibodies against phosphoserine Smad2 (p-Smad2), p-Smad2 or total Smad2/3. Immunoblots representative of triplicate independent experiments are shown. Lane 1, vehicle control; lane 2, 20 lM; lane 3, 40 lM; lane 4, 60 lM. Con, ethanol vehicle control; GS, genistein. (B) EMSA was performed using nuclear extracts from genistein-treated MC-26 cells. Competition reaction was performed with 50- and 100-fold molar excess of an unlabelled oligonucleotide probe and proved the specificity of Smad–DNA complex. Lane 1, 50· competition reaction; lane 2, 100· competition reaction; lane 2, vehicle control; lane 4, 20 lM genistein; lane 5, 40 lM genistein; lane 6, 60 lM genistein.
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