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Biphasic effect of daidzein on cell growth of human colon cancer cells
Food and Chemical Toxicology 42 (2004) 1641–1646 www.elsevier.com/locate/foodchemtox
Biphasic effect of daidzein on cell growth of human colon cancer cells J.M. Guo
a,*
, B.X. Xiao a, D.H. Liu b, M. Grant c, S. Zhang b, Y.F. Lai a, Y.B. Guo c, Q. Liu a a
c
School of Medicine, Ningbo University, Ningbo 315211, China b Ningbo No. 2 Hospital, Ningbo 315010, China Royal Victoria Hospital, McGill University Health Center, Montreal, Canada H3A1A1 Received 21 October 2003; accepted 2 June 2004
Abstract Colorectal cancer is one of the most common cancers in the world, poorly responding to available chemotherapeutic agents. To investigate whether natural molecules can inhibit colon cancer progression, we investigated a principle phytoestrogen found in soybean known as daidzein, and determined its effects on the human colon cancer cell line LoVo. LoVo cells were treated with 0.1, 1, 5, 10, 50 and 100 lM daidzein for 2, 3, 4 or 5 d. The results indicated that daidzein stimulated the growth of LoVo cells at 0.1 and 1 lM whereas at higher concentrations (10, 50 and 100 lM) cell growth was inhibited in a dose-dependent manner. Treatment of daidzein at 10, 50 and 100 lM resulted in cell cycle arrest at G0/G1 phase, DNA fragmentation and increases in caspase-3 activity. There were no changes in alkaline phosphatase activity (ALP), an indicator of cell differentiation, upon treatment with daidzein when compared to controls. These results indicate that daidzein has a biphasic effect on LoVo cell growth and its tumor suppressive effect is by means of cell cycle arrest and apoptosis but not through cell differentiation. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Phytoestrogens; Daidzein; Human colon cancer cell line; Cell growth; Cell cycle
1. Introduction The global variation in cancer incidence reflect a relation between population dietary habits and the relative risk of developing cancer, thus the role of diet on the control of cancer risk has drawn widespread attention (Horn-Ross et al., 2000; Lee et al., 1991). Epidemiological studies have shown that high soy consumption as found in Asian populations is associated with a reduced risk of breast, colon, and prostate cancer (Messina and Barnes, 1991). Initially these studies largely began due to the results from a report by Barnes
Abbreviations: A, absorbance; AFC, 7-amino-4-trifluoromethyl coumarin; ALP, alkaline phosphatase; ANOVA, analysis of variance; DEVD, Asp–Glu–Val–Asp; DMSO, dimethylsulfoxide; EDTA, ethylenediaminotetraacetic acid; MNU, N-methyl-N-nitrosourea; MTT, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PBS, phosphate buffered saline; SPSS, statistical package for social science * Corresponding author. Tel.: +86-57487600758; fax: +8657487608638. E-mail address: [email protected] (J.M. Guo). 0278-6915/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2004.06.001
and coworkers who demonstrated that, administration of soy protein to a typical laboratory diet significantly decreased chemically induced rat mammary cancer (Barnes et al., 1990). Since then many reports have followed studying the effects of soy as an anticancer agent particularly against breast cancer. There are several known bioactive components found in soybean however, one class that has attracted much attention is the class of phytoestrogens known as isoflavones. In total, there are 12 known soybean isoflavone isomers. The most abundant isoflavones include the glucosides, genistin and daidzin and their respective aglycones, genistein and daidzein (Bingham et al., 1998). Genistein is believed to be the primary bioactive isomer in soybean and is the most studied in its class. Its biological effects include the inhibition of tyrosine kinases, DNA topoisomerases, angiogenesis and it has been shown to act as an antioxidant (Fotsis et al., 1993; Robinson et al., 1993; Spinozzi et al., 1994; Wei et al., 1993). Recently, daidzein was reported to have other biological properties including estrogen-like and estrogenindependent effects (Alexandersen et al., 2001; Gao
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et al., 2001; Guo et al., 2004; Messina and Loprinzi, 2001). However, the biological effects of daidzein are not as well understood compared to that of genistein. In order to determine whether daidzein has effects on other cell systems and could be of therapeutic potential for other types of cancers, we studied its effects on the human colon cancer cell line LoVo, and investigated its mechanisms of action.
2. Materials and methods 2.1. Materials Daidzein and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO). RPMI-1640 Medium was from Life Technologies (Grand Island, NY). The human colon cancer cell line LoVo was obtained from the Cell Bank of the Chinese Academy of Sciences (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences). 2.2. Cell culture LoVo cells were cultured in plastic flasks or multiwell plates at 37 °C in a humidified atmosphere of 5% CO2 with RPMI-1640 medium containing 10% fetal calf serum, 50 U/ml penicillin and 50 lg/ml streptomycin. The medium was changed every other day. Exponentially growing cells were used in experiments. For quantitative assays of proliferation, 104 cells/ml were seeded in 96-well plates in regular growth medium and incubated for 24 h. Cells were then incubated in medium with various concentrations of daidzein dissolved in dimethylsulfoxide (DMSO) or 0.1% DMSO (as a control; Lyn-Cook et al., 1999). The concentrations of daidzein used were 0.1, 1, 5, 10, 50 and 100 lM, respectively. Cells were then treated for 2, 3, 4 or 5 d and monitored for cell growth using the MTT assay. 2.3. MTT assay Four independent sets of 12 wells were used for each dose along with controls in this assay (Plumb et al., 1989). 30 ll of MTT solution (2 mg/ml in phosphate buffered saline, PBS) was added into each of the 96 wells. After the cells were incubated at 37 °C for 4 h, the medium was removed and 150 ll of DMSO was added to solubilize the formazan. The microplate was shaken on a rotary platform for 10 min. Finally, the absorbance (A) was measured at 570 and 630 nm using a Wellscan (Labsystems, USA). The mean A from the 12 wells was calculated. Growth inhibition was calculated in percent as follows: ½ðAcontrol Aexperiment Þ=Acontrol 100 (Guo et al., 2004). The mean of four independent assays was
determined to analyze the effect of daidzein on LoVo cell growth. 2.4. Cell cycle analysis and apoptosis measurement 106 LoVo cells/ml were treated with 5, 10, 50 and 100 lM daidzein for 2, 3, 4 or 5 d. Cells were harvested with 0.25% trypsin/0.05% EDTA and sedimented by centrifugation at 3000 rpm for 5 min at room temperature. After the supernatant was removed, ice-cold 70% ethanol was added. DNA was stained with DNA-Prep Coulter Reagents Kit (Beckman Coulter, Inc., Miami, FL) according to the manufacturer’s instructions. The cell cycle distribution was estimated by flow cytometric DNA analysis according to standard procedures (Liu et al., 1998). The cell cycle was analyzed with Coulter Flow Cytometry (Beckman Coulter, Inc., Miami, FL). The percentage of cells in different cell cycle phases (G0/ G1, S, G2/M phase) was calculated using Coulter Epicx XL-MCL DNA Analysis Software. The sub-G1 peak was considered as a measure of apoptosis (Patel et al., 1998; Su et al., 2000). Three wells of a 6-well plate were used for each dose and timed treatment. Four independent experiments were performed in this analysis. 2.5. DNA fragmentation A concentration 106 LoVo cells/ml was treated with 5, 10, 50 and 100 lM daidzein for 2, 3, 4 or 5 d before being harvested. DNA was isolated using a DNA isolation kit (Qiagen GmbH, Germany), and then separated on a 1.5% agarose gel stained with ethidium bromide at 4 V/ cm for 3 h. DNA fragments were observed under ultraviolet light (Liu et al., 1998). Four independent experiments were performed in this analysis. 2.6. Caspase-3 activity assay The caspase-3 activity was measured using a commercially available kit (BD Biosciences Clontech, San Francisco, CA) according to the manufacturer’s instructions. Enzymatic cleavage of substrate by caspase-3 results in the release of free 7-amino-4-trifluoromethyl coumarin (AFC), which emits a yellow–green fluorescence and is measured fluorometrically. LoVo cells seeded at a density of 106 cells/ml were treated with 5, 10, 50 and 100 lM daidzein for 2, 3, 4 or 5 d before being harvested. Cells were homogenized in 500 ll of lysis buffer. Samples were centrifuged at 16 000g for 10 min at 4 °C and normalized for protein amount. The fluorescent substrate, DEVD (Asp–Glu–Val–Asp)– AFC, was incubated with 50 ll of supernatant at 37 °C for 60 min. Fluorescence of the cleaved substrate was measured using a fluorometer at an excitation wavelength of 400 nm and an emission wavelength of 505 nm (Stefanis et al., 1996). An AFC calibration curve was
J.M. Guo et al. / Food and Chemical Toxicology 42 (2004) 1641–1646
established by serial dilution of a pure AFC solution provided in the kit. The caspase-3 activity was expressed as pmol AFC/h/mg protein. Four independent experiments were performed in this assay.
50
2.7. Determination of relative alkaline phosphatase activities
20
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0.1 µM 1 µM 5 µM 10 µM 50 µM 100 µM
40 30
#
4
LoVo cells were seeded at a density of 10 cells/ml and treated with 1, 5, 10, and 100 lM daidzein for 2, 3, 4 or 5 d before being assayed for alkaline phosphatase (ALP, EC 3 1 3 1) activity. Cells were then dissolved with 0.25% sodium deoxycholate. Finally, the ALP activities were measured by dynamics assay with an Automatic Biochemistry Analyser (Beckman Coulter, Inc., Miami, FL). The relative enzyme activity was expressed as ALP/total protein (U/g) (Herz and Halwer, 1990; Honma et al., 1980). Six wells of a 12-well plate were used for each dose and treated time. Four independent experiments were performed in this analysis.
Inhibitive rate (%)
* *
10
*
0 -10 *
-20
*
-30 2
3
4
5
Days
Fig. 1. Effects of daidzein on LoVo cell growth. LoVo cells treated with 0.1, 1, 5, 10, 50 and 100 lM daidzein for 2, 3, 4 or 5 d. DMSO (0.1%) was used as a control. Cell growth was measured using the MTT assay. The inhibition rate was calculated as described in Section 2. Means ± SEM represent four independent experiments; P < 0:05; # P < 0:01 compared to controls.
2.8. Statistics
3. Results 3.1. MTT assay results Growth curves of LoVo cells treated with the indicated concentrations of daidzein for 2, 3, 4 or 5 d, were found to be biphasic (Fig. 1). Daidzein stimulated the cell growth at concentrations of 0.1 and 1 lM ðP < 0:05Þ. In contrast, cells treated with higher doses of daidzein, 5–100 lM, cell growth was inhibited in a dosedependent manner (10 and 50 lM, P < 0:05; 100 lM, P < 0:01).
100 #
Percentage of G0/ G1 phase
Statistical analysis was performed using SPSS version 10.0. ANOVA was used to analyze statistical comparisons between groups (Lyn-Cook et al., 1999). The level of significance was set at P < 0:05.
80
2 days 3 days 4 days 5 days
# # #
60
40
20
0 0
5
10
50
100
Concentration
3.2. Flow cytometric analysis
Fig. 2. G0/G1 phase arrest of daidzein on LoVo cells. LoVo cells were incubated with 5, 10, 50 and 100 lM daidzein for 2, 3, 4 or 5 d. DMSO (0.1%) was used as a control. Cell cycle distributions were analyzed by flow cytometry. The percentage of cells in G0/G1 phase was calculated. Each point represents the mean ± SEM of four independent assays. # , compared with control, P < 0:01.
In order to decipher the suppressive mechanisms of daidzein on LoVo cells, we monitored changes in cell cycle distribution by flow cytometry. Treatment of daidzein resulted in a dose-dependent increase in the distribution of cells at G0/G1 phase (Fig. 2). At concentrations of 50 and 100 lM, a sub-G1 peak was observed.
ular-weight genomic DNA was separated on a 1.5% agarose gel. As shown in Fig. 3, DNA fragmentation was observed upon treatment in a dose-dependent manner. To verify that DNA fragmentation induced by daidzein treatment was indeed due to apoptosis through caspase activation, we measured caspase-3 activity.
3.3. DNA fragmentation analysis
3.4. Caspase-3 activity
To determine whether daidzein induced apoptosis we began by looking at DNA fragmentation. Low-molec-
Cleavage of the peptide substrate DEVD–AFC was used as an indicator of caspase-3 protease activity. The
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3.5. Determination of alkaline phosphatase activity In order to determine whether daidzein was capable of inducing the differentiation of LoVo cells we measured alkaline phosphatase activity (ALP), a commonly used indicator of cell differentiation. Using the dynamics assay to measure for ALP activity, we were unable to observe any statistical significance between the ALP of control and treated cells ðP > 0:05Þ.
4. Discussion Fig. 3. Agarose gel electrophoresis of DNA fragmentation. LoVo cells were treated with 5, 10, 50 and 100 lM daidzein for 3 d before being harvested. The DNA was isolated and subjected to electrophoresis on a 1.5% agarose gel (4 V/cm for 3 h) and stained with ethidium bromide. DNA fragments were observed under ultraviolet light. M, DNA marker; Lanes 1–4: 100, 50, 10 and 5 lM daidzein; Lane 5, control.
Caspase 3 activity (pmol AFC/hr/mg protein)
levels of caspase-3 activity in cells treated with 10, 50 and 100 lM daidzein were significantly higher than controls (10, 50 and 100 lM, P < 0:01; Fig. 4). This suggests that daidzein is capable of inducing apoptosis in LoVo cells in a caspase-dependent manner. The effect was dose-dependent however; longer incubation times did not result in increases in maximal activity. Therefore, caspase activation occurs relatively early but remains constant and does not accumulate throughout the duration of treatment.
350 Control 5 µM 10 µM 50 µM 100 µM
300
#
#
250
200 #
150
100 2
3
4
5
Days
Fig. 4. Changes of caspase-3 activity after treatment with daidzein. LoVo cells treated with 5, 10, 50 and 100 lM daidzein for 2, 3, 4 or 5 d were harvested. Samples were processed followed by incubation with the caspase-3 cleavage substrate DEVD–AFC. The fluorescence of cleaved substrate was measured using a fluorometer at an excitation wavelength of 400 nm and an emission wavelength of 505 nm. The caspase-3 activity is expressed as pmol AFC/h/mg protein. Each point represents the mean ± SEM of four independent assays. # , compared with control, P < 0:01.
There has been an increasing popularity towards the use of natural compounds to improve human health. For that reason consumption of soyfoods has been on an increase in the US with projected sales of $6 billion for 2005 (Messina and Loprinzi, 2001). Isoflavones, often referred to as phytoestrogens, are natural compounds derived from plants and exhibit estrogen-like activity (Tham et al., 1998). Studies have shown that injection of the isoflavone daidzein reduced N-methylN-nitrosourea (MNU)-induced mammary carcinogenesis by approximately 20% (Constantinou et al., 1996). Furthermore, soybean extract used to treat three colon cancer cell lines Caco-2, SW620 and H-29 cells suggested that exposure to soybean extract or isoflavones affected morphology and survival of colon cancer cells (Zhu et al., 2002). In this study, we found that daidzein inhibited the growth of LoVo cells in a dose-dependent manner at concentrations from 5 to 100 lM. At the higher concentrations, cells were arrested at G0/G1 phase and underwent apoptosis. We demonstrate that the antitumorigenic effects of daidzein may be derived from G0/G1 phase arrest and/or through apoptosis. Several studies have shown that daidzein could arrest the human melanoma cell line OCM-1 and the human gastric cancer cell line HGC-27 at G2/M phase, however, treatment with genistein resulted in a G0/G1 phase arrest (Casagrande and Darbon, 2001; Matsukawa et al., 1993). Other studies have demonstrated that daidzein induced apoptosis in human bladder cancer cells without altering cell cycle progression (Su et al., 2000). This suggests that multiple mechanisms may be responsible for the anticancer effects of isoflavones on different types of cancers. A volunteer study found that subjects who ingested 60 g of backed soybean powder (containing 28.5 mg daidzein) had about 1.56 ± 0.34 lM diadzein in their plasma (Watanabe et al., 1998). Our results suggest that in order to benefit from the antitumorigenic effects of daidzein on colon cancer, individuals would have to eat approximately 600 g of soybean per day. However, this amount may vary due to the other known bioactive compounds found in soybean (Bingham et al., 1998;
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Fotsis et al., 1993; Robinson et al., 1993; Spinozzi et al., 1994; Wei et al., 1993). The effect of daidzein on LoVo cell growth was found to be biphasic. Concentrations of 0.1 and 1 lM of daidzein stimulated cell growth. These results are similar to that of genistein on the estrogen-receptor-positive breast cancer cell line MCF-7 (Maggiolini et al., 2001). The proliferative effects of genistein on human breast cancer cells were mediated by estrogen receptors but its cytotoxic effects were independent of its estrogen-like activity (Maggiolini et al., 2001). There have been some reports linking colon cancer to estrogen (Fiorelli et al., 1999; Singh et al., 1993). One study found that estrogen stimulated the growth of LoVo cells suggesting that estrogen receptor inhibitors could block their growth (Arai et al., 2000). As a result, the effects of daidzein on LoVo cells may be through estrogen receptors. At low concentrations, daidzein appears to have estrogen-like effects by stimulating the growth of cancer cells however, at higher concentrations, daidzein appears to have an anti-estrogen effect resulting in inhibition of cell growth. Aside from cell cycle alteration, another important mechanism in inducing an anticancer effect is cell differentiation. Alkaline phosphatase (ALP) activity is a useful marker for the determination of differentiation in colon cancer cells (Stich et al., 1989). We measured the ALP activity in colon cancer cells treated with daidzein and found no difference between treated and controls. This suggests that daidzein has no effect on the differentiation of LoVo cells. In conclusion, we have shown that daidzein affects the growth and cell cycle distributions of LoVo cells. Furthermore, the suppressive effects of daidzein may be through cell cycle arrest and apoptosis but not through cell differentiation. These results highlight the importance of daidzein as an anticancer agent and may offer therapeutic potential against colon cancer. Acknowledgements This work was supported by Scientific Research Fund of Zhejiang Provincial Education Department (No. 20010217). References Alexandersen, P., Haarbo, J., Breinholt, V., Christiansen, C., 2001. Dietary phytoestrogens and estrogen inhibit experimental atherosclerosis. Climacteric. 4, 151–159. Arai, N., Strom, A., Rafter, J.J., Gustafsson, J.A., 2000. Estrogen receptor beta mRNA in colon cancer cells: growth effects of estrogen and genistein. Biochem. Biophys. Res. Commun. 270, 425–431. Barnes, S., Grubbs, C., Setchell, K.D., Carlson, J., 1990. Soybeans inhibit mammary tumors in models of breast cancer. Prog. Clin. Biol. Res. 347, 239–253.
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Biphasic effect of daidzein on cell growth of human colon cancer cells J.M. Guo
a,*
, B.X. Xiao a, D.H. Liu b, M. Grant c, S. Zhang b, Y.F. Lai a, Y.B. Guo c, Q. Liu a a
c
School of Medicine, Ningbo University, Ningbo 315211, China b Ningbo No. 2 Hospital, Ningbo 315010, China Royal Victoria Hospital, McGill University Health Center, Montreal, Canada H3A1A1 Received 21 October 2003; accepted 2 June 2004
Abstract Colorectal cancer is one of the most common cancers in the world, poorly responding to available chemotherapeutic agents. To investigate whether natural molecules can inhibit colon cancer progression, we investigated a principle phytoestrogen found in soybean known as daidzein, and determined its effects on the human colon cancer cell line LoVo. LoVo cells were treated with 0.1, 1, 5, 10, 50 and 100 lM daidzein for 2, 3, 4 or 5 d. The results indicated that daidzein stimulated the growth of LoVo cells at 0.1 and 1 lM whereas at higher concentrations (10, 50 and 100 lM) cell growth was inhibited in a dose-dependent manner. Treatment of daidzein at 10, 50 and 100 lM resulted in cell cycle arrest at G0/G1 phase, DNA fragmentation and increases in caspase-3 activity. There were no changes in alkaline phosphatase activity (ALP), an indicator of cell differentiation, upon treatment with daidzein when compared to controls. These results indicate that daidzein has a biphasic effect on LoVo cell growth and its tumor suppressive effect is by means of cell cycle arrest and apoptosis but not through cell differentiation. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Phytoestrogens; Daidzein; Human colon cancer cell line; Cell growth; Cell cycle
1. Introduction The global variation in cancer incidence reflect a relation between population dietary habits and the relative risk of developing cancer, thus the role of diet on the control of cancer risk has drawn widespread attention (Horn-Ross et al., 2000; Lee et al., 1991). Epidemiological studies have shown that high soy consumption as found in Asian populations is associated with a reduced risk of breast, colon, and prostate cancer (Messina and Barnes, 1991). Initially these studies largely began due to the results from a report by Barnes
Abbreviations: A, absorbance; AFC, 7-amino-4-trifluoromethyl coumarin; ALP, alkaline phosphatase; ANOVA, analysis of variance; DEVD, Asp–Glu–Val–Asp; DMSO, dimethylsulfoxide; EDTA, ethylenediaminotetraacetic acid; MNU, N-methyl-N-nitrosourea; MTT, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PBS, phosphate buffered saline; SPSS, statistical package for social science * Corresponding author. Tel.: +86-57487600758; fax: +8657487608638. E-mail address: [email protected] (J.M. Guo). 0278-6915/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2004.06.001
and coworkers who demonstrated that, administration of soy protein to a typical laboratory diet significantly decreased chemically induced rat mammary cancer (Barnes et al., 1990). Since then many reports have followed studying the effects of soy as an anticancer agent particularly against breast cancer. There are several known bioactive components found in soybean however, one class that has attracted much attention is the class of phytoestrogens known as isoflavones. In total, there are 12 known soybean isoflavone isomers. The most abundant isoflavones include the glucosides, genistin and daidzin and their respective aglycones, genistein and daidzein (Bingham et al., 1998). Genistein is believed to be the primary bioactive isomer in soybean and is the most studied in its class. Its biological effects include the inhibition of tyrosine kinases, DNA topoisomerases, angiogenesis and it has been shown to act as an antioxidant (Fotsis et al., 1993; Robinson et al., 1993; Spinozzi et al., 1994; Wei et al., 1993). Recently, daidzein was reported to have other biological properties including estrogen-like and estrogenindependent effects (Alexandersen et al., 2001; Gao
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et al., 2001; Guo et al., 2004; Messina and Loprinzi, 2001). However, the biological effects of daidzein are not as well understood compared to that of genistein. In order to determine whether daidzein has effects on other cell systems and could be of therapeutic potential for other types of cancers, we studied its effects on the human colon cancer cell line LoVo, and investigated its mechanisms of action.
2. Materials and methods 2.1. Materials Daidzein and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO). RPMI-1640 Medium was from Life Technologies (Grand Island, NY). The human colon cancer cell line LoVo was obtained from the Cell Bank of the Chinese Academy of Sciences (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences). 2.2. Cell culture LoVo cells were cultured in plastic flasks or multiwell plates at 37 °C in a humidified atmosphere of 5% CO2 with RPMI-1640 medium containing 10% fetal calf serum, 50 U/ml penicillin and 50 lg/ml streptomycin. The medium was changed every other day. Exponentially growing cells were used in experiments. For quantitative assays of proliferation, 104 cells/ml were seeded in 96-well plates in regular growth medium and incubated for 24 h. Cells were then incubated in medium with various concentrations of daidzein dissolved in dimethylsulfoxide (DMSO) or 0.1% DMSO (as a control; Lyn-Cook et al., 1999). The concentrations of daidzein used were 0.1, 1, 5, 10, 50 and 100 lM, respectively. Cells were then treated for 2, 3, 4 or 5 d and monitored for cell growth using the MTT assay. 2.3. MTT assay Four independent sets of 12 wells were used for each dose along with controls in this assay (Plumb et al., 1989). 30 ll of MTT solution (2 mg/ml in phosphate buffered saline, PBS) was added into each of the 96 wells. After the cells were incubated at 37 °C for 4 h, the medium was removed and 150 ll of DMSO was added to solubilize the formazan. The microplate was shaken on a rotary platform for 10 min. Finally, the absorbance (A) was measured at 570 and 630 nm using a Wellscan (Labsystems, USA). The mean A from the 12 wells was calculated. Growth inhibition was calculated in percent as follows: ½ðAcontrol Aexperiment Þ=Acontrol 100 (Guo et al., 2004). The mean of four independent assays was
determined to analyze the effect of daidzein on LoVo cell growth. 2.4. Cell cycle analysis and apoptosis measurement 106 LoVo cells/ml were treated with 5, 10, 50 and 100 lM daidzein for 2, 3, 4 or 5 d. Cells were harvested with 0.25% trypsin/0.05% EDTA and sedimented by centrifugation at 3000 rpm for 5 min at room temperature. After the supernatant was removed, ice-cold 70% ethanol was added. DNA was stained with DNA-Prep Coulter Reagents Kit (Beckman Coulter, Inc., Miami, FL) according to the manufacturer’s instructions. The cell cycle distribution was estimated by flow cytometric DNA analysis according to standard procedures (Liu et al., 1998). The cell cycle was analyzed with Coulter Flow Cytometry (Beckman Coulter, Inc., Miami, FL). The percentage of cells in different cell cycle phases (G0/ G1, S, G2/M phase) was calculated using Coulter Epicx XL-MCL DNA Analysis Software. The sub-G1 peak was considered as a measure of apoptosis (Patel et al., 1998; Su et al., 2000). Three wells of a 6-well plate were used for each dose and timed treatment. Four independent experiments were performed in this analysis. 2.5. DNA fragmentation A concentration 106 LoVo cells/ml was treated with 5, 10, 50 and 100 lM daidzein for 2, 3, 4 or 5 d before being harvested. DNA was isolated using a DNA isolation kit (Qiagen GmbH, Germany), and then separated on a 1.5% agarose gel stained with ethidium bromide at 4 V/ cm for 3 h. DNA fragments were observed under ultraviolet light (Liu et al., 1998). Four independent experiments were performed in this analysis. 2.6. Caspase-3 activity assay The caspase-3 activity was measured using a commercially available kit (BD Biosciences Clontech, San Francisco, CA) according to the manufacturer’s instructions. Enzymatic cleavage of substrate by caspase-3 results in the release of free 7-amino-4-trifluoromethyl coumarin (AFC), which emits a yellow–green fluorescence and is measured fluorometrically. LoVo cells seeded at a density of 106 cells/ml were treated with 5, 10, 50 and 100 lM daidzein for 2, 3, 4 or 5 d before being harvested. Cells were homogenized in 500 ll of lysis buffer. Samples were centrifuged at 16 000g for 10 min at 4 °C and normalized for protein amount. The fluorescent substrate, DEVD (Asp–Glu–Val–Asp)– AFC, was incubated with 50 ll of supernatant at 37 °C for 60 min. Fluorescence of the cleaved substrate was measured using a fluorometer at an excitation wavelength of 400 nm and an emission wavelength of 505 nm (Stefanis et al., 1996). An AFC calibration curve was
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established by serial dilution of a pure AFC solution provided in the kit. The caspase-3 activity was expressed as pmol AFC/h/mg protein. Four independent experiments were performed in this assay.
50
2.7. Determination of relative alkaline phosphatase activities
20
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0.1 µM 1 µM 5 µM 10 µM 50 µM 100 µM
40 30
#
4
LoVo cells were seeded at a density of 10 cells/ml and treated with 1, 5, 10, and 100 lM daidzein for 2, 3, 4 or 5 d before being assayed for alkaline phosphatase (ALP, EC 3 1 3 1) activity. Cells were then dissolved with 0.25% sodium deoxycholate. Finally, the ALP activities were measured by dynamics assay with an Automatic Biochemistry Analyser (Beckman Coulter, Inc., Miami, FL). The relative enzyme activity was expressed as ALP/total protein (U/g) (Herz and Halwer, 1990; Honma et al., 1980). Six wells of a 12-well plate were used for each dose and treated time. Four independent experiments were performed in this analysis.
Inhibitive rate (%)
* *
10
*
0 -10 *
-20
*
-30 2
3
4
5
Days
Fig. 1. Effects of daidzein on LoVo cell growth. LoVo cells treated with 0.1, 1, 5, 10, 50 and 100 lM daidzein for 2, 3, 4 or 5 d. DMSO (0.1%) was used as a control. Cell growth was measured using the MTT assay. The inhibition rate was calculated as described in Section 2. Means ± SEM represent four independent experiments; P < 0:05; # P < 0:01 compared to controls.
2.8. Statistics
3. Results 3.1. MTT assay results Growth curves of LoVo cells treated with the indicated concentrations of daidzein for 2, 3, 4 or 5 d, were found to be biphasic (Fig. 1). Daidzein stimulated the cell growth at concentrations of 0.1 and 1 lM ðP < 0:05Þ. In contrast, cells treated with higher doses of daidzein, 5–100 lM, cell growth was inhibited in a dosedependent manner (10 and 50 lM, P < 0:05; 100 lM, P < 0:01).
100 #
Percentage of G0/ G1 phase
Statistical analysis was performed using SPSS version 10.0. ANOVA was used to analyze statistical comparisons between groups (Lyn-Cook et al., 1999). The level of significance was set at P < 0:05.
80
2 days 3 days 4 days 5 days
# # #
60
40
20
0 0
5
10
50
100
Concentration
3.2. Flow cytometric analysis
Fig. 2. G0/G1 phase arrest of daidzein on LoVo cells. LoVo cells were incubated with 5, 10, 50 and 100 lM daidzein for 2, 3, 4 or 5 d. DMSO (0.1%) was used as a control. Cell cycle distributions were analyzed by flow cytometry. The percentage of cells in G0/G1 phase was calculated. Each point represents the mean ± SEM of four independent assays. # , compared with control, P < 0:01.
In order to decipher the suppressive mechanisms of daidzein on LoVo cells, we monitored changes in cell cycle distribution by flow cytometry. Treatment of daidzein resulted in a dose-dependent increase in the distribution of cells at G0/G1 phase (Fig. 2). At concentrations of 50 and 100 lM, a sub-G1 peak was observed.
ular-weight genomic DNA was separated on a 1.5% agarose gel. As shown in Fig. 3, DNA fragmentation was observed upon treatment in a dose-dependent manner. To verify that DNA fragmentation induced by daidzein treatment was indeed due to apoptosis through caspase activation, we measured caspase-3 activity.
3.3. DNA fragmentation analysis
3.4. Caspase-3 activity
To determine whether daidzein induced apoptosis we began by looking at DNA fragmentation. Low-molec-
Cleavage of the peptide substrate DEVD–AFC was used as an indicator of caspase-3 protease activity. The
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3.5. Determination of alkaline phosphatase activity In order to determine whether daidzein was capable of inducing the differentiation of LoVo cells we measured alkaline phosphatase activity (ALP), a commonly used indicator of cell differentiation. Using the dynamics assay to measure for ALP activity, we were unable to observe any statistical significance between the ALP of control and treated cells ðP > 0:05Þ.
4. Discussion Fig. 3. Agarose gel electrophoresis of DNA fragmentation. LoVo cells were treated with 5, 10, 50 and 100 lM daidzein for 3 d before being harvested. The DNA was isolated and subjected to electrophoresis on a 1.5% agarose gel (4 V/cm for 3 h) and stained with ethidium bromide. DNA fragments were observed under ultraviolet light. M, DNA marker; Lanes 1–4: 100, 50, 10 and 5 lM daidzein; Lane 5, control.
Caspase 3 activity (pmol AFC/hr/mg protein)
levels of caspase-3 activity in cells treated with 10, 50 and 100 lM daidzein were significantly higher than controls (10, 50 and 100 lM, P < 0:01; Fig. 4). This suggests that daidzein is capable of inducing apoptosis in LoVo cells in a caspase-dependent manner. The effect was dose-dependent however; longer incubation times did not result in increases in maximal activity. Therefore, caspase activation occurs relatively early but remains constant and does not accumulate throughout the duration of treatment.
350 Control 5 µM 10 µM 50 µM 100 µM
300
#
#
250
200 #
150
100 2
3
4
5
Days
Fig. 4. Changes of caspase-3 activity after treatment with daidzein. LoVo cells treated with 5, 10, 50 and 100 lM daidzein for 2, 3, 4 or 5 d were harvested. Samples were processed followed by incubation with the caspase-3 cleavage substrate DEVD–AFC. The fluorescence of cleaved substrate was measured using a fluorometer at an excitation wavelength of 400 nm and an emission wavelength of 505 nm. The caspase-3 activity is expressed as pmol AFC/h/mg protein. Each point represents the mean ± SEM of four independent assays. # , compared with control, P < 0:01.
There has been an increasing popularity towards the use of natural compounds to improve human health. For that reason consumption of soyfoods has been on an increase in the US with projected sales of $6 billion for 2005 (Messina and Loprinzi, 2001). Isoflavones, often referred to as phytoestrogens, are natural compounds derived from plants and exhibit estrogen-like activity (Tham et al., 1998). Studies have shown that injection of the isoflavone daidzein reduced N-methylN-nitrosourea (MNU)-induced mammary carcinogenesis by approximately 20% (Constantinou et al., 1996). Furthermore, soybean extract used to treat three colon cancer cell lines Caco-2, SW620 and H-29 cells suggested that exposure to soybean extract or isoflavones affected morphology and survival of colon cancer cells (Zhu et al., 2002). In this study, we found that daidzein inhibited the growth of LoVo cells in a dose-dependent manner at concentrations from 5 to 100 lM. At the higher concentrations, cells were arrested at G0/G1 phase and underwent apoptosis. We demonstrate that the antitumorigenic effects of daidzein may be derived from G0/G1 phase arrest and/or through apoptosis. Several studies have shown that daidzein could arrest the human melanoma cell line OCM-1 and the human gastric cancer cell line HGC-27 at G2/M phase, however, treatment with genistein resulted in a G0/G1 phase arrest (Casagrande and Darbon, 2001; Matsukawa et al., 1993). Other studies have demonstrated that daidzein induced apoptosis in human bladder cancer cells without altering cell cycle progression (Su et al., 2000). This suggests that multiple mechanisms may be responsible for the anticancer effects of isoflavones on different types of cancers. A volunteer study found that subjects who ingested 60 g of backed soybean powder (containing 28.5 mg daidzein) had about 1.56 ± 0.34 lM diadzein in their plasma (Watanabe et al., 1998). Our results suggest that in order to benefit from the antitumorigenic effects of daidzein on colon cancer, individuals would have to eat approximately 600 g of soybean per day. However, this amount may vary due to the other known bioactive compounds found in soybean (Bingham et al., 1998;
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Fotsis et al., 1993; Robinson et al., 1993; Spinozzi et al., 1994; Wei et al., 1993). The effect of daidzein on LoVo cell growth was found to be biphasic. Concentrations of 0.1 and 1 lM of daidzein stimulated cell growth. These results are similar to that of genistein on the estrogen-receptor-positive breast cancer cell line MCF-7 (Maggiolini et al., 2001). The proliferative effects of genistein on human breast cancer cells were mediated by estrogen receptors but its cytotoxic effects were independent of its estrogen-like activity (Maggiolini et al., 2001). There have been some reports linking colon cancer to estrogen (Fiorelli et al., 1999; Singh et al., 1993). One study found that estrogen stimulated the growth of LoVo cells suggesting that estrogen receptor inhibitors could block their growth (Arai et al., 2000). As a result, the effects of daidzein on LoVo cells may be through estrogen receptors. At low concentrations, daidzein appears to have estrogen-like effects by stimulating the growth of cancer cells however, at higher concentrations, daidzein appears to have an anti-estrogen effect resulting in inhibition of cell growth. Aside from cell cycle alteration, another important mechanism in inducing an anticancer effect is cell differentiation. Alkaline phosphatase (ALP) activity is a useful marker for the determination of differentiation in colon cancer cells (Stich et al., 1989). We measured the ALP activity in colon cancer cells treated with daidzein and found no difference between treated and controls. This suggests that daidzein has no effect on the differentiation of LoVo cells. In conclusion, we have shown that daidzein affects the growth and cell cycle distributions of LoVo cells. Furthermore, the suppressive effects of daidzein may be through cell cycle arrest and apoptosis but not through cell differentiation. These results highlight the importance of daidzein as an anticancer agent and may offer therapeutic potential against colon cancer. Acknowledgements This work was supported by Scientific Research Fund of Zhejiang Provincial Education Department (No. 20010217). References Alexandersen, P., Haarbo, J., Breinholt, V., Christiansen, C., 2001. Dietary phytoestrogens and estrogen inhibit experimental atherosclerosis. Climacteric. 4, 151–159. Arai, N., Strom, A., Rafter, J.J., Gustafsson, J.A., 2000. Estrogen receptor beta mRNA in colon cancer cells: growth effects of estrogen and genistein. Biochem. Biophys. Res. Commun. 270, 425–431. Barnes, S., Grubbs, C., Setchell, K.D., Carlson, J., 1990. Soybeans inhibit mammary tumors in models of breast cancer. Prog. Clin. Biol. Res. 347, 239–253.
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