is induced by genistein through the expression ... - Wiley Online Library

Publication of the International Union Against Cancer

Int. J. Cancer: 105, 747–753 (2003) © 2003 Wiley-Liss, Inc.

NONSTEROIDAL ANTI-INFLAMMATORY DRUG-ACTIVATED GENE (NAG-1) IS INDUCED BY GENISTEIN THROUGH THE EXPRESSION OF P53 IN COLORECTAL CANCER CELLS Leigh C. WILSON, Seung Joon BAEK, Allison CALL and Thomas E. ELING* Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Genistein is an isoflavenoid found in soy that has antitumorigenic activities. Treatment of colorectal carcinoma HCT-116 cells with 50 ␮M genistein results in a 50% reduction in cell proliferation and a 6-fold increase in apoptosis. Genistein induces nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1), a protein with antitumorigenic activities, in a time- and concentration-dependent manner in HCT-116 cells. In addition, p53 and p21 are induced in HCT116 cells. The induction of p53 (3 hr) precedes the induction of NAG-1 (12 hr), suggesting that genistein-induced NAG-1 expression is mediated by p53. In contrast, NAG-1 is not induced by genistein in the p53-negative colorectal carcinoma cell line HCT-15. Luciferase reporter constructs of the NAG-1 promoter containing 2 p53 sites showed that the p53 sites within the NAG-1 promoter are critical to genisteininduced NAG-1 expression in p53-positive U2OS cells. The expression of p53 was critical for NAG-1 promoter activity since no promoter activity was observed with genistein treatment in HCT-15 cells. However, genistein-induced promoter activity was restored in HCT-15 cells by transfection with wild-type p53. Together our data suggest a relationship between genistein, p53 and NAG-1 forming a novel pathway responsible for the antitumorigenic activity of genistein. © 2003 Wiley-Liss, Inc.

induce apoptosis and the expression of nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1). NAG-1 is a TFG-␤ superfamily member that has antitumorigenic activity and stimulates apoptosis in colon cancer and other cell lines.23 NAG-1 is regulated at the basal level by Sp1, Sp3 and COUP-TF1 transcription factors24 and by activators of the p53 tumor suppressor gene25,26 in a prostaglandin-independent manner.27 NAG-1 is induced by other dietary compounds including resveratrol, a compound in red wine,28 and diallyl disulfide, a lipid-soluble compound from garlic,29 in a p53-dependent manner. Therefore, we decided to study the relationship between genistein, p53 and NAG-1. In our study we investigated the effect of genistein on NAG-1 expression and apoptosis in colon cancer and other cell lines and determined if the effect was p53 dependent or independent. We report here that genistein induces NAG-1 expression, induces apoptosis and suppresses cell growth. Genistein also induces p53, which regulates NAG-1 at 2 known p53 sites within the promoter.25,26 We propose that NAG-1 may play a role in the antitumorigenic activity of genistein.

Key words: genistein; colon cancer; anti-tumorigenesis; NAG-1; NSAIDs; cyclooxygenase

Cell lines and reagents Cell lines were purchased from ATCC (Rockville, MD). Human colorectal carcinoma cells, HCT-116, HCT-15, and human osteosarcoma cells, U2OS, were grown in McCoy’s 5A media (Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum and gentamicin (10 ␮g/ml). Genistein was purchased from Sigma (St. Louis, MO) and dissolved in dimethylsulfoxide (DMSO) from Sigma.

Epidemiologic studies show that a diet high in soy-containing products reduces the risk of certain cancers including breast, prostate and colon.1–3 In vivo studies also show rats maintained on a soy-based diet were significantly protected against mammary tumor induction by dimethylbenz[a]anthracene and N-methylnitrosourea.4 – 6 Soy products contain high levels of genistein, an isoflavone and phytoestrogen that is a potent inhibitor of cell proliferation and angiogenesis.7 In addition to reduced risk of cancer, genistein also improves plasma lipids, resulting in lowered LDL cholesterol in premenopausal women,8 and lowers blood pressure in men and women with mild to moderate hypertension.9 Furthermore, genistein may attenuate bone loss in lumbar spine of postmenopausal women,10 indicating multiple mechanisms are likely involved. Although the exact mechanism by which genistein exerts its antitumorigenic activity is not known, several biologic processes important in tumor development are affected by genistein. Genistein is best known as a tyrosine kinase inhibitor.11 Other mechanisms include inhibition of growth-promoting steroid hormones,12 antioxidant activity,13,14 DNA topoisomerase inhibition15,16 and inhibition of angiogenesis.7,17 Genistein also downregulates cyclooxygenase-2 (COX-2) promoter activity in colon cancer cells stably transfected with a COX-2 reporter gene system.18 COX-2 is the inducible form of COX and its activation is believed to be related to tumorigenesis as it is upregulated in tumors.19 The antitumorigenic activity of genistein may be related to the inhibition of COX expression but further examination is required. Recently, genistein was reported to trigger apoptosis in cell culture.20 –22 Our laboratory has investigated the stimulation of apoptosis in cultured cells with COX inhibitors to study antitumorigenic effects on colon cancer cells. These COX inhibitors

MATERIAL AND METHODS

Western blot analysis Cells were grown to 60 –70% confluence and treated with genistein or vehicle (DMSO) for 48 hr in complete media. Cells were rinsed 2 times with phosphate-buffered saline (PBS) and then lysed in RIPA buffer [1⫻ PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS] with a protease inhibitor cocktail added. Proteins were normalized, NuPage sample buffer (Invitrogen, Carlsbad, CA) was added and 30 ␮g of protein were electrophoresed on 4 –12% Bis/Tris NuPage gradient gels (Invitrogen). ProAbbreviations: COX, cyclooxygenase; FACS, fluorescence-activated cell sorter; MTS/PMS, Owen’s reagent-phenazine methosulphate; NAG-1, NSAID-activated gene-1; NSAIDs, nonsteroidal anti-inflammatory drugs; RIPA, radioimmunoprecipitation; TGF-␤, transforming growth factorbeta. *Correspondence to: P.O. Box 12233, Research Triangle Park, NC 27709, USA. Fax: ⫹919-541-0146. E-mail: [email protected] Received 14 August 2002; Revised 16 December 2002; Accepted 18 December 2003 DOI 10.1002/ijc.11173

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teins were transferred onto nitrocellulose membrane (Invitrogen). The membranes were blocked overnight in 10% Tris-buffered saline/0.05% Tween-20 (TBS-T) at 4°C and then probed with the appropriate antibody in 2% milk in TBS-T. The antibodies used were NAG-1,23 p53 (Santa Cruz, Santa Cruz, CA), p21 (Santa Cruz) and actin (Santa Cruz). After washing, the blots were treated with the appropriate secondary horseradish peroxidase-conjugated antibody and washed. Proteins were detected using enhanced chemiluminescence system (Amersham, Arlington Heights, IL) and exposed to Hyperfilm-MP (Amersham). Caell proliferation assay The cell proliferation assay was performed using the CellTiter 96 Aqueous Non-Radioactive cell proliferation assay kit (Promega, Madison, WI). The assay was carried out according to the manufacturer’s protocol. In 96-well plates, cells were plated at 500 cells/well in 100 ␮l of media. After 16 hr, the cells were treated with vehicle or genistein (50 ␮M) and incubated for different time points. MTS/PMS solution (20 ␮l/well) was added and incubated for 1 hr at 37°C/5% CO2. Absorbance was read at 490 nm using an ELISA plate reader (Molecular Dynamics, Menlo Park, CA). The quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture (approximately OD490 ⫽ 1 is 1 ⫻ 105 cells). Transfection using the luciferase reporter system Cells were plated in 6- or 12-well plates and grown to 40 –50% confluence in the appropriate medium. Plasmid mixtures containing 0.5–1 ␮g of NAG-1 promoter linked to luciferase and 0.05– 0.1 ␮g of pRL-null (Promega, Madison, WI) were transfected using LipofectAMINE (Life Technologies) according to the manufacturer’s protocol. After 12 hr, the medium was changed to serum free and genistein was added. Cells were treated for 48 hr and lysed in 1⫻ passive lysis buffer (Promega). Luciferase activity was determined and normalized to pRL-null luciferase activity using the Dual Luciferase Assay Kit (Promega). Apoptosis assay Apoptosis was measured using the TACS™ Annexin V-FITC kit from Trevigen (Gaithersburg, MD) according to the manufacturer’s instructions. Briefly, HCT-116 and HCT-15 cells were plated in 6-well plates and grown until they reached 50% confluence. The cells were treated with 50 ␮M genistein in complete media for 48 hr. The cells were collected and washed with PBS, incubated with the Annexin V conjugate and measured by fluorescence-activated cell sorter (FACS). A total of 7,500 cells were measured by flow cytometry using Becton Dickinson (Franklin Lakes, NJ) FACsort equipped with CellQuest software by gating on an area vs. width dot plot to exclude cell debris and cell aggregates. Cell Death Detection (nuclear matrix protein) ELISA (Oncogene, San Diego, CA) was used as a second method to measure apoptosis. The assay was carried out according to the manufacturer’s instructions by first treating HCT-15 and HCT-116 cells with genistein (50 ␮M) at various time points. The medium was then collected and used to detect soluble nuclear matrix protein by ELISA using the provided antibodies. Absorbance at 490/550 nm was measured using an ELISA plate reader (Molecular Dynamics, Menlo Park, CA). Cloning of NAG-1 promoter The NAG-1 constructs pNAG966/⫹70 (containing 2 p53 sites, p53-A and -B) and pNAG1739/⫹41 (containing 1 p53 site, p53-A) were generated as previously described.28 The pNAG1739/⫹70 construct was generated by digesting pNAG966/⫹70 and pNAG1739/⫹41 with XbaI. The fragment released from pNAG966/⫹70 containing the ⫹70 region (p53-B site) of the promoter was ligated with the fragment released from pNAG1739/ ⫹41 (p53-A site) containing the upstream region of the promoter.

The resulting plasmid was verified by sequencing using the GLprimer2 (Promega). Densitometry measurements Western blots were scanned using an Umax™Powerlook III™ scanner equipped with a transparency adapter and scanning software. Bands were quantified using Scion Image™ Beta version 4.0.2. Values were normalized using the corresponding actin bands. Numbers shown are fold increase compared to vehicletreated cells. RESULTS

Effects of genistein on growth and apoptosis in HCT-116 cells To investigate the effects of genistein on the growth of colorectal cancer cells in culture, genistein was added to the culture medium for varying lengths of time. In the presence of 50 ␮M genistein, a significant reduction on cell growth was seen: approximately 30% after 48 hr and approximately 50% after 72 hr, respectively (Fig. 1a). To determine if genistein induces apoptosis in HCT-116 (p53-positive) and HCT-15 (p53-negative) cells, the Annexin V-FITC method was used to measure apoptosis. Figure 1b shows a 6-fold increase in apoptotic cells compared to vehicletreated cells in HCT-116 cells treated with genistein for 48 hr. In contrast, HCT-15 cells showed much less apoptotic cells compared to HCT-116 cells when treated with genistein. To confirm these results, a second method was employed to measure apoptosis using cell death detection ELISA as described in Material and Method. Soluble nuclear matrix protein (NMP) that is released into culture supernatants of dead or dying cells was measured from genisteintreated HCT-116 and HCT-15 cells. Figure 1c shows a 2-fold increase in NMP in HCT-116 cells compared to untreated cells. No increase in NMP was detected in HCT-15 cells, suggesting that the presence of wild-type p53 is required for genistein-induced apoptosis in colon cancer cells. Genistein induces NAG-1 in a concentration- and time-dependent manner Since genistein induces apoptosis in HCT-116 cells and NAG-1 is a pro-apoptotic protein, we measured NAG-1 protein levels after treatment with genistein. HCT-116 cells were treated with varying concentrations of genistein for 48 hr and protein levels were analyzed by western. Genistein induced NAG-1 at concentrations as low as 25 ␮M, showed the highest induction at 50 ␮M, followed by a decrease in expression at 100 ␮M (Fig. 2a). Because 50 ␮M genistein induced NAG-1 by the greatest amount, it was used to treat HCT-116 cells for varying amounts of time. NAG-1 protein levels increased at 48 hr with vehicle treatment as seen previously in our laboratory. However, NAG-1 protein levels increased at 6 hr and continued to increase up to 48 hr after treatment (Fig. 2b). Thus, genistein induced NAG-1 in a concentration- and timedependent manner. Genistein induces p53 in a dose-dependent manner Because genistein induces apoptosis in a p53-dependent manner30 and NAG-1 is regulated by p53,25–27 we examined the expression pattern of NAG-1 and p53 in the presence of genistein to determine a possible link between NAG-1, genistein and p53. HCT-116 cells were treated with varying amounts of genistein for 48 hr and analyzed by western blot for p53 protein (Fig. 3a). Like NAG-1 (Fig. 2a), p53 was induced by genistein but at all concentrations for p53. We also measured the expression of p21 CDK inhibitor, p53 target protein, and found p21 proteins were induced by genistein. p21 induction may result from p53 induction. Further, p21 induction may contribute, at least in part, to genisteininduced cell growth arrest in HCT-116 cells. Next, HCT-116 cells were treated with 50 ␮M genistein for varying time points. As seen in Figure 3a, p53 levels increase at 3 hr and continue to increase up to 48 hr after treatment. An induction of p53 was observed at earlier time points than the 6 hr time point observed for induction of NAG-1.

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FIGURE 1 – Time dependent growth retardation of HCT-116 cells and apoptosis of HCT-116 and HCT-15 cells. (a) Effect of genistein on HCT-116 cell growth. Cells were plated at 500 cells/well and incubated with vehicle (DMSO) or genistein (50 ␮M). Cell growth was measured using PMS cell proliferation kit (Promega). Values are expressed as mean ⫾ SD of 4 to 5 replicates. (b) Apoptosis of HCT-116 and HCT-15 cells treated with genistein (50 ␮M). Cells were plated in 6-well plates and treated with vehicle (DMSO) or genistein for 48 hr and analyzed for apoptosis as described in Material and Methods. The data are represented as fold increase over apoptotic percentage of vehicle-treated cells. The results show the mean ⫾ SD of 3 separate treatments. (c) Apoptosis of HCT-116 and HCT-15 cells treated with genistein (50 ␮M) at various time points. Cell were plated in 6-well plates and treated with vehicle (DMSO) or genistein for 0, 24 and 48 hr. The media were collected and nuclear matrix protein was measured by ELISA as described in Material and Methods. The data represent increase over OD (490/595) of untreated (0 hr) cells. The results are the mean ⫾ SD of 3 separate treatments. The level of significance for the different cell populations after genistein treatment has been calculated by Student’s t-test with respect to the corresponding controls in absence of genistein. *, The changes in the numbers of cells at genistein-treated cells are significant at p ⬍ 0.001.

NAG-1 expression in a p53 mutant cell line To further investigate the hypothesis that genistein induces NAG-1 through the p53-dependent pathway, the p53 mutant cell line HCT-15 was treated with genistein at varying concentrations for 48 hr (Fig. 4). NAG-1 can be induced in HCT-15 cells with treatment of nonsteroidal anti-inflammatory drugs (NSAIDs), which induce NAG-1 in a p53-independent manner.29 As shown in Figure 4, genistein induced NAG-1 in HCT-116 cells at 25 ␮M and 50 ␮M, whereas no induction of NAG-1 was seen in HCT-15 cells. Likewise, as shown in Figure 1c, genistein did not induce apoptosis in HCT-15 cells, suggesting an association between NAG-1 and apoptosis. Genistein induces NAG-1 through p53 protein To obtain further evidence in support of the hypothesis that genistein induces NAG-1 through p53, NAG-1 promoter activity was examined. We cloned the NAG-1 promoter (⫺1739 to ⫹70) as described in Material and Methods. The constructs pNAG1739/ ⫹70 containing 2 p53 binding sites (p53-A, p53-B) and deletion

clone pNAG1739/⫹41 (p53-A) were generated by PCR. All clones were linked to the luciferase gene, transiently transfected into U2OS cells (p53 wild-type) and HCT-15 cells (p53 mutant) and treated with 50 ␮M genistein for 48 hr. Etoposide was used as a positive control for p53-dependent activation throughout these experiments and typically showed a 7-fold induction (data not shown). As shown in Figure 5a, the construct lacking the p53-B site did not significantly induce NAG-1 promoter activity in U2OS cells when treated with genistein. The construct containing p53 sites A and B (pNAG1739/⫹70) showed significantly enhanced luciferase activity when treated with genistein, suggesting that the p53-B site has a more critical role for genistein-induced NAG-1 expression. These data are consistent with previous data showing that the p53-B site is more critical for etoposide- and resveratrolinduced NAG-1 expression.26,28 In the HCT-15 cells, no induction of the NAG-1 promoter was seen with either vector used when treated with genistein, which supports the hypothesis, that p53 regulates NAG-1 expression.

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FIGURE 2 – Western analysis of NAG-1 protein levels in genistein-treated HCT-116 cells. (a) Dose response of NAG-1 protein levels. HCT-116 cells were treated with various amounts of genistein for 48 hr and western analysis was performed with total cell lysate. The bands were quantified using Scion Image software (Scion) and values for NAG-1 were normalized to actin protein levels. (b) Time course of NAG-1 protein levels. HCT-116 cells were treated with genistein (50 ␮M) or vehicle (DMSO) for various time points and analyzed by western.

FIGURE 3 – Expression of p53 and p21 in the presence of genistein. (a) HCT-116 cells were treated with genistein at various concentrations for 48 hr and total cell lysate was harvested for western analysis. Bands were quantified using Scion Image software (values for p53 and p21 were normalized to actin protein levels). (b) Time course of p53 levels. HCT-116 cells were treated with 50 ␮M genistein for various time points and analyzed by western.

Finally, to examine whether the transactivation of the NAG-1 promoter by p53 depends on p53 binding sites, we performed cotransfection experiments using p53 cDNA in an expression vector in HCT-15 (p53-negative) cells. The constructs pNAG1739/ ⫹41(p53 site A) and pNAG1739/⫹70 (p53 sites A and B) were cotransfected with either empty vector or p53 expression vector. As shown in Figure 6, the ectopic expression of p53 with the pNAG1739/⫹70 vector enhanced luciferase activity 5.5-fold in p53-negative cells compared to empty vector-transfected cells. However, pNAG1739/⫹41 did not show any significant induction when transfected with p53. This suggests that the p53-B site is a more critical site for induction by p53 and confirms that wild-type p53 is necessary for induction of NAG-1 promoter activity. DISCUSSION

Genistein is a naturally occurring isoflavanoid found in soybean that has been found to have chemopreventive properties. Although the molecular mechanisms of genistein on carcinogenesis are not clear, many different actions are known, including tyrosine kinase

FIGURE 4 – NAG-1 protein levels in the presence of genistein in HCT-116 and HCT-15 cells. HCT-116 (p53 wild type) and HCT-15 (p53 mutant) were treated with various concentrations of genistein for 48 hr and total cell lysate was harvested for western analysis. NAG-1 bands from HCT-116 cells were quantified using Scion Image software and normalized to actin values.

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FIGURE 5 – Effect of genistein on NAG-1 promoter. The pNAG1739/⫹70 and pNAG1739/⫹41 constructs were linked to luciferase and transiently transfected in U2OS or HCT-15 cells. The constructs (1 ␮g) were cotransfected with pRL-null (Promega) vector, using lipofectAMINE. After transfection, the cells were treated with genistein or vehicle for 48 hr and harvested for luciferase activity. Transfection efficiency for luciferase activity was normalized to the Renilla luciferase (pRL-null) activity. The results shown are mean of 3 independent transfections. Fold increase refers to ratio of luciferase activity of genistein-treated cells compared to vehicle-treated cells for each vector. The results show mean ⫾ SE of 3 separate transfections. Statistical analysis was performed as described in Figure 1. *, The changes in the numbers of cells at genistein-treated cells are significant at p ⬍ 0.001.

FIGURE 6 – Activation of luciferase activity in p53-dependent manner. Each construct was transiently cotransfected with either empty vector or p53 expression vector into HCT-15 (p53-negative) cells. The cells were grown for 2 days and harvested for luciferase activity. The data show induction over relative luciferase activity of empty vector-transfected cells. Values are mean ⫾ SD of 3 independent transfections. Statistical analysis was performed as described in Figure 1. *, The changes in the numbers of cells at genistein-treated cells are significant at p ⬍ 0.001.

inhibition and topoisomerase II inhibition. Our present study was conducted to test the hypothesis that genistein may also act through NAG-1 to exert its antitumorigenic effects. Our results show that genistein is an effective inhibitor of cell growth and inducer of apoptosis in the colon cancer cell line, HCT-116.

Genistein also induces NAG-1 (2-fold) in a concentration- and time-dependent manner in HCT-116 cells. The tumor suppressor p53 and cdk inhibitor p21 are also induced by genistein. Only the p53 wild-type cells had induction of NAG-1 and an increase in apoptosis after treatment with genistein. When the cell line

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HCT-15 (p53-null) was treated with genistein, there was minimal increase in apoptosis and no induction of NAG-1 protein. Furthermore, our promoter assays show that when the vital p53-B site is removed, genistein-induced expression of the NAG-1 promoter decreases markedly. In addition, we performed experiments in a cell line devoid of p53 and examined the cotransfection pNAG1739/⫹70 (p53 sites A and B) with p53 and there was a 5-fold induction of luciferase activity over the pNAG1739/⫹41 (p53-A site). Thus, genistein induces NAG-1 dependent on p53 activity. According to previous studies, the NAG-1 promoter contains 2 p53 binding sites,25,26 and the second binding site (p53-B) is more critical for the activation of NAG-1. This finding is consistent with our reports that genistein-induced NAG-1 expression is mediated mainly by the second p53 binding site. Our data are concurrent with other reports that genistein induces p53.30,31 Interestingly, other dietary compounds including resveratrol,28 diallyl disulfide29 and selenium32 also act through a p53-dependent pathway. The biologic significance of our study is the implication of genistein and its linkage to p53-mediated NAG-1 expression and apoptosis. NAG-1 is the most notable p53-induced gene as determined by cDNA array technology.33 Genistein has antitumorigenic activity but also growth-promoting effects.34 –36 Some of these findings may be due to cell type. Genistein induces TGF-␤1, a member of the TGF-␤ superfamily of growth factors.37 TGF-␤1 is

highly pleitropic and can both stimulate and inhibit cell growth in a cell-type-specific manner. Another cause for different effects may be due to the concentration of genistein used. For example, Salti et al.37 showed that at lower levels (1–2 ␮M) genistein increased cell proliferation in colon cancer cells, whereas at concentrations ⬎60 ␮M genistein caused cell cycle arrest and apoptosis. Physiologic levels of genistein obtained from normal diet are 10 ␮M in serum.37 Recently, a clinical study38 delivered doses of highly purified genistein to men, at levels higher than those previously given to humans (1–16 mg/kg body wt). They found minimal clinical toxicity even at single doses that exceed normal dietary intakes several fold. In summary, we have shown that genistein is an inducer of NAG-1, a TGF-␤ superfamily protein that has pro-apoptotic and antitumorigenic properties. We have demonstrated that genisteininduced p53 expression leads to apoptosis and slowed cell growth in colon cancer cells at 50 ␮M. These effects of genistein may be the result of NAG-1 induction. Taken together, these findings suggest a novel pathway by which genistein exerts its antitumorigenic activity. ACKNOWLEDGEMENTS

We thank Dr. J. Nixon and Mr. F. Bottone of NIEHS for their helpful comments and suggestions.

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