Regular paper

The influence of an antiestrogen, indole-3-carbinol (I3C) on the expression of CYP1A1, CYP1B1 and AhR genes was investigated in an attempt to establish whether I3C could increase the expression of genes involved in estrone metabolism. Another purpose was to examine the proliferation of an estrogen-dependent breast cancer cell (MCF-7 line) under the influence of I3C and both I3C and DDT. In MCF-7 cells incubated with I3C or I3C and DDT combined, quantitative RT-PCR analysis revealed a significant increase in the level of CYP1A1, AhR, and CYP1B1 transcripts. The proliferation rate of MCF-7 cells was increased by treatment with DDT or estradiol (E2), whereas I3C did not affect the proliferation of MCF-7 cells but greatly reduced the stimulatory effect of DDT, and abolished the effect of E2. The level of p21 transcript, encoding p21 protein involved in the cell cycle, was increased several-fold by I3C comparing to its level in cells incubated with estradiol or DDT. The results suggest that the proliferation of MCF-7 cells is accompanied not only by expression of genes encoding cytochromes involved in estrogen metabolism, but also by changes in the expression of other genes including that encoding p21 protein involved in the cell cycle.


INTRODUCTION
Cytochromes P450, the protein products of CYP genes are components of estrone hydroxylase enzyme system.Cytochromes P450 1A1 and P450 1A2 are responsible for 2-hydroxylation, which is the main route of estrogen elimination and provides a mechanism protecting the cells against cancer (Jefcoate et al., 2000).On the other hand, the action of cytochrome P450 1B1 involved in 4-hydroxylation results in the synthesis of potentially carcinogenic catechol estrogens.The expression of CYP1A and CYP1B gene families is mediated by aryl hydrocarbon receptor (AhR) (Safe, 2001).
Indole-3-carbinol (I3C) is a natural anti-estrogen isolated from cruciferous species (Michnovicz & Bradlow, 1990).Its affinity for estrogen receptor is weak and the anti-estrogenic properties are due to an increase of CYP1A1 transcription and a decrease of the expression of proteins activating the cell cycle (Tiwari et al., 1994).
The main purpose of the present study was to examine the expression of CYP1A and CYP1B1, as well as AhR, in order to determine whether low doses of the natural anti-estrogen, I3C influence the expression of these genes in estrogen-dependent breast cancer cells (MCF-7 line).Studies on CYP1A1 and CYP1B1 expression have been reported (Brockdorff et al., 2000;Coumoul et al., 2001).However, the doses of xenoestrogens used in those experiments were very high and exceeded those found in the polluted environment.Another purpose of this study was to examine the effect of I3C, shown to protect cells against cancer (Bradlow et al., 1999), on estradiol (E2)-induced proliferation of MCF-7 cells, as well as the effect of this compound on cell proliferation in-

MATERIALS AND METHODS
Cell culture and incubation with test substances.The estrogen-dependent MCF-7 cell line was kindly donated by Dr. S. Szala (Institute of Oncology, Gliwice, Poland).Cells were cultured at 37 o C in 5% CO 2 /95% air in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotic-antimycotic (Sigma, UK) to reach approx.80% confluency.
Test substances.I3C, DDT and estradiol (Sigma, UK) were dissolved in ethanol and kept at -20 o C. Working solutions were diluted with the medium just before experiments to a final concentrations of: 10 -8 M I3C, 10 -6 M DDT and 10 -12 M estradiol (E2).The final concentration of ethanol in the incubation medium did not exceed 0.01%.
Expression assay.MCF-7 cells were incubated with the test substances in Dulbecco's modified Eagle's medium (DMEM) without phenol red, supplemented with ITS 100 × Supplement Medium (1.0 mg/ml of insulin, 0.55 mg/ml of transferrin and 0.5 μg/ml of sodium selenite) (Sigma, UK).After 2 to 48 h of incubation the cells were counted and total RNA was isolated (Chomczynski, 1993) using TRI Reagent (Sigma, UK).Poly A + RNA was reversetranscribed with the use of Superscript II RNase H Reverse Transcriptase (Invitrogen, USA) according to the producer's instructions, and cDNA fragments were amplified with the use of specific primers (Table 1).In order to quantify the concentration of specific mRNAs, real-time PCR was conducted using the LightCycler System (Roche Scientific, USA) and QuantiTect SybrGreen PCR kit (Qiagen, USA).The number of transcript copies was normalized to the number of cells (Bustin, 2002).Amplification of p21 cDNA was conducted in a total volume of 20 μl containing: 1 μl of template, 5 pmol of each primer, 200 μM of each dNTP, 2 μl of reaction buffer and 1 unit of RedTAQ Polymerase (Sigma, UK).Twentyfive PCR cycles, each consisting of: 5 s denaturation at 94 o C, 20 s annealing at 64 o C followed by 20 s elongation at 72 o C. The amplified fragments were separated by electrophoresis in 2.5% agarose gel, analyzed by densitometry and quantified with the use of BandLeader software.
Proliferation assay.Cells were cultured for 48 h in DMEM without phenol red (Sigma, UK) supplemented with 10% fetal bovine serum devoid of steroids (Sonnenschein et al., 1995).When 30% confluence was reached, test substances were added to the final concentrations of: 10 -8 M I3C, 10 -6 M DDT and 10 -12 M E2 and the cell proliferation monitored.At different time intervals, 5% MTT [1-(4,5dimethylthiazol-2-yl)-3,5-diphenylformazan] (Sigma, UK) was added.After 2 h of incubation the medium was removed and the cells were treated with DMSO (dimethylsulfoxide) (Sigma, UK) in order to visualize the metabolized MTT (Carmichael et al., 1987).Proliferation rate was expressed as the number of cells after 48 h of incubation with xenoestrogens compared to the untreated control.
Statistics.The results of expression and proliferation studies (each experiment was repeated three times) were subjected to ANOVA, while the results of p21 expression were analyzed by Student's t-test.The significance of the differences was tested at the level of P < 0.05.

RESULTS
In MCF-7 cells incubated with I3C, the levels of CYP1A1 and CYP1B1 mRNA were significantly increased the latter being over 3-times higher than the former.The highest level of CYP1A1 transcripts was detected after 4 h, while that of the CYP1B1 transcripts after 2 h of treatment.A significant increase in AhR transcript appeared as early as 2 h of incubation with I3C and was maintained until 12 h.There was no change in the transcript levels upon incubation with estradiol, while DDT caused an expected increase in the expression of the three genes, with the highest levels being detected after 4 h (Fig. 1).
As expected, estradiol and DDT significantly enhanced proliferation of MCF-7 cells, I3C alone did not have any effect, but abolished the stimulatory effect of estradiol, and slightly, but significantly reduced the effect of DDT on cell proliferation (Fig. 2).
Semi-quantitative RT-PCR analysis revealed that the level of mRNA encoding an estrogen-dependent cell cycle protein, p21 was increased severalfold by I3C, while the effects of DDT and estradiol were negligible (Fig. 3).

The influence of I3C on the expression of CYP1A1, CYP1B1 and AhR
An increased expression of CYP1A1 under the influence of I3C in breast cancer cell lines and in mammary gland was described before (Horn et al., 2002), and an increase of CYP1A1 protein was observed after 12 h (Tiwari et al., 1994).Our results revealed the highest CYP1A1 transcript level after 4 h of incubation with I3C, indicating that it pre-I3C affects CYP expression ceded CYP1A1 protein accumulation.The increase in CYP1A1 transcript level, in turn, was preceded by accumulation of AhR transcripts.This suggested that AhR mediated the effect of I3C on CYP1A1 expression.Our results confirmed earlier observations that I3C and its metabolites in µM concentration induced the accumulation of AhR mRNA, followed by increased CYP1A1 expression (Jellinck et al., 1993;Chen et al., 1998).However, the concentration of I3C used in those studies was five orders of magnitude higher than in the present report, and no kinetic data were presented.Therefore our observation on a rapid accumulation of AhR transcripts under low doses (10 nM) of I3C should be considered as being novel and of physiological significance.
CYP1B1 expression has been detected in both mammary gland and breast cancer cells (Hellmold et al., 1998;Iscan et al., 2001).CYP1B1 expression was also demonstrated in human carcinoma cells treated with the I3C metabolite diindolemethane (Sanderson et al., 2001).However, in mammary gland of the rat (Horn et al., 2002), as well as in human prostate cells (Leibelt et al., 2003;Li et al., 2003), no I3C-induced CYP1B1 expression could be demonstrated.On the other hand, in MCF-7 cells CYP1B1 expression was induced by a typical agonist of AhR, TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), which exhibits a much higher affinity for AhR than I3C (Spink et al., 1998;Angus et al., 1999;Coumoul    MCF-7 cells were grown to 50% confluency and were subsequently incubated for 24 h with I3C (10 -8 M), and/ or DDT (10 -6 M) in DMEM containing 10% FBS, devoid of phenol red and steroids.After incubation total RNA was extracted and reverse transcribed.A fragment of p21 cDNA (499 bp) was amplified by polymerase chain reaction (PCR) and the PCR product was analyzed by electrophoresis on 1.5% agarose gel.The experiment was repeated three times and typical gel is shown.(A) Ethidium bromide staining of agarose gel.(B) Densitometric analysis of the gel.

2007
M. Ociepa-Zawal and others et al., 2001).Our results demonstrate that in MCF-7 cells the CYP1B1 expression induced by I3C was preceded by increased expression of AhR.Therefore it seems that I3C, thought to enhance the expression of the protective CYP1A1, also increases the expression of CYP1B1.In MCF-7 cells constitutive and induced expression of CYP1B1 and only induced expression of CYP1A1 was observed.
Steroid hydroxylases, related to cytochromes P450 1A1 and P450 1B1, catalyze 2-OH-E2 and 4-OH-E2 synthesis, respectively (Lee et al., 2003).While stimulation of the formation of the 2-OH derivative formation protects the cells against potent estrogen accumulation, the 4-OH derivative accumulation is considered a breast cancer risk factor (Jefcoate et al., 2000).Our results indicate that the protective effect of I3C is due to an enhanced CYP1A1/CYP1B1 expression ratio rather than being caused by an increase in CYP1A1 expression alone.

The influence of indole-3-carbinol on proliferation of MCF-7 cells
Our results were consistent with earlier reports (Liu et al., 1994;Tiwari et al., 1994;Diel et al., 2002) and showed that both I3C and DDT affected the proliferation of the estrogen-dependent MCF-7 cells, but the inhibition by I3C of DDT-induced proliferation was never shown before.This effect of I3C might be due to changes in CYP1A1/CYP1B1 expression ratio, and was AhR-mediated.DDT in concentrations used in our experiments was 100-times less effective in binding to the estrogen receptor (ER) than estradiol in a physiological concentration (Zava et al., 1997).Consequently, I3C affects the metabolism of estrogens by changing the CYP1A1/CYP1B1 expression ratio rather than by blocking the ER (Chen et al., 1998).
Our results were supported by the examination of p21 gene expression in MCF-7 cells.p21 (p21 CIP1 ) is a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, suppressed by estrogens in a mechanism involving proteolysis and protein sequestration (Foster et al., 2001).Similar to earlier studies, the concentration of p21 gene transcript in MCF-7 cells under the influence of estradiol was low (Cariou et al., 2000;Lai et al., 2001).Sanders (1998) showed that I3C affected the expression of cell cycle proteins, but rather at the level of cyclin-dependent kinase 6 (CDK6) than kinase inhibitors.In our experiment I3C markedly enhanced the expression of p21, which was earlier demonstrated by Firestone and Bjeldanes (2003) and Cover et al. (1998) and indicated a protective role of I3C.The concentration of p21 gene transcripts under the influence of DDT was as low as under the influence of estradiol, suggesting that the effects of xenoestro-gens and natural estrogens on cell cycle protein expression in MCF-7 cells are similar.
The results of our study showed that I3C influences the expression of CYP1A1 as well as CYP1B1 and inhibits the proliferation of breast cancer MCF-7 cells induced by E2 and DDT.The protective affect of I3C might therefore be due not only to changes of the expression of CYP genes involved in estrogen metabolism, but also other factors including cell cycle proteins.

Figure 1 .
Figure 1.Effects of I3C, DDT and estradiol on CYP1A1, CYP1B1 and AhR expression.MCF-7 cells were incubated for the time indicated with 10 -8 M I3C, 10 -6 M DDT or 10 -12 M estradiol in a defined medium (DMEM) devoid of phenol red.Following incubation, cells were counted, total RNA was extracted, poly A + RNA reverse transcribed and P450 1A1, P450 1B1 as well as AhR cDNAs were amplified and quantified by realtime PCR as described in Materials and Methods.Results were normalized to the cell number.Open bars, untreated control; hatched bars, E2; filled bars, I3C; double hatched bars, DDT.*P < 0.05,**P < 0.001 (Newman-Keul test).

Figure 3 .
Figure 3.Effect of xenoestrogens on p21 mRNA content in MCF-7 cells.MCF-7 cells were grown to 50% confluency and were subsequently incubated for 24 h with I3C (10 -8 M), and/ or DDT (10 -6 M) in DMEM containing 10% FBS, devoid of phenol red and steroids.After incubation total RNA was extracted and reverse transcribed.A fragment of p21 cDNA (499 bp) was amplified by polymerase chain reaction (PCR) and the PCR product was analyzed by electrophoresis on 1.5% agarose gel.The experiment was repeated three times and typical gel is shown.(A) Ethidium bromide staining of agarose gel.(B) Densitometric analysis of the gel.