PHYTOTHERAPY RESEARCH Phytother. Res. (2014) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5232
Modulating Potential of L-Sulforaphane in the Expression of Cytochrome P450 to Identify Potential Targets for Breast Cancer Chemoprevention and Therapy Using Breast Cell Lines Barbara Licznerska,1 Hanna Szaefer,1 Iwona Matuszak,1 Marek Murias2 and Wanda Baer-Dubowska1* 1
Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences, Poznań, Poland Department of Toxicology, Poznan University of Medical Sciences, Poznań, Poland
2
The L-sulforaphane (SFN) component of broccoli sprout showed anticancer activity in several preclinical studies including breast cancer. Estrogens are considered major risk factors in breast carcinogenesis. The aim of this study was to evaluate the effect of SFN on the expression of cytochrome P450 involved in the estrogen metabolism in breast cancer cell lines MCF7 and MDA-MB-231 and in non-tumorigenic MCF10A cell line. The expression of CYP19, CYP1A1, 1A2, 1B1 was determined at the transcript and protein levels. There were found some remarkable differences in the effect of SFN at a dose of 5 μmol/L on CYP19 expression: in ER(+) MCF7 significant reduction, in ER( ) MDA-MB-231 an increased expression and unchanged expression in MCF10A cell line. The effect of SFN on CYPs (1A1, 1A2, 1B1) involved in estrogen catabolism was to a lesser extent dependent on breast cell line. The slightly reduced CYP1A1 protein level was observed in all cell lines tested. An increased level of CYP1A2 and decreased level of CYP1B1 expression were found in MCF10A. These results indicate that the naturally occurring L isomer of SFN may affect the expression of P450s involved in estrogen metabolism. This effect may contribute to the anticancer activity of SFN in breast tissue. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: L-Sulforaphane; CYP19; CYP1A1; CYP1A2; CYP1B1; breast cancer chemoprevention. Abbreviations: AhR, hydrocarbon receptor; ER, estrogen receptor; GST, glutathione S-transferase; NQO1, NAD(P)H:quinone oxidoreductase; PBGD, porphobilinogen deaminase; SFN, L-sulforaphane; TBP, TATA box binding protein.
INTRODUCTION Cruciferous vegetables are a rich source of bioactive isothiocyanates. Among them, sulforaphane [1-isothiocyanato4-methylsulfinyl-butane (SFN)] in some cruciferous vegetables, especially in broccoli sprouts, is one of the most abundant [Cornblatt et al., 2007; Zhang et al., 1992]. This compound has been proved to possess anticancer activity in several preclinical studies including breast cancer. A preliminary clinical trial involving healthy women undergoing reduction mammoplasty further supported the SFN potential in chemoprevention of breast cancer [Cornblatt et al., 2007]. SFN is known as a potent activator of Nrf2 pathway and inducer of detoxification enzymes such as NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase (GST) [Jeong et al., 2005, Keum et al., 2009, Munday and Munday, 2004, Zhang et al., 2006]. These enzymes might be responsible for the decreased DNA-adduct formation in normal mammary cells exposed to benzo[a]pyrene and 1,6-dinitropyrene * Correspondence to: Wanda Baer-Dubowska, Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences, Poznań, Poland. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
[Singletary and MacDonald, 2000] as well as depurinating estrogen-DNA adducts [Yang et al., 2013]. These experimental findings are further supported by some epidemiological data suggesting a protective association between increased consumption of broccoli or Brussels sprouts and breast cancer [Ambrosone et al., 2004, Fowke et al., 2003, Terry et al., 2001]. Estrogens are considered major risk factors in breast carcinogenesis. Two mechanisms for estrogen carcinogenesis have been proposed: estrogen-induced proliferation [in estrogen receptor (ER) positive cells] and the formation of reactive estrogen metabolites [Watson et al., 1999]. In the metabolism of estrogens, a crucial role is played by cytochrome P450. The de novo estrogen synthesis from androgens requires aromatase activity, encoded by CYP19. The oxidative catabolism of estrogens is catalyzed mainly by CYP1A1/CYP1A2 and CYP1B1. The action of these enzymes can convert 17-β estradiol in breast epithelium into its 2-hydroxyestradiol and 4-hydroxyestradiol. While 2-hydroxyestradiol is mainly catalyzed by CYP1A2 and CYP3A4 in the liver, and by CYP1A1 in extra-hepatic tissues, CYP1B1 is highly expressed in estrogen target tissues including mammary and catalyzes the 4-hydroxylation of estradiol. Because 4-hydroxyestradiol generates free radicals from Received 17 March 2014 Revised 20 May 2014 Accepted 31 August 2014
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the reductive–oxidative cycling with the corresponding semiquinone and quinone forms, which cause cellular damage, the specific and local formation of 4-hydroxyestradiol is important for breast carcinogenesis [Tsuchiya et al., 2005]. By the induction of NQO1 and GSTs, compounds like SFN may detoxify estrogen metabolites protecting against DNA damage, but equally important is targeting the specific CYPs involved in their activation. In contrast to the induction of phase II enzymes by SFN, its effect on CYPs was the subject of only two studies performed in rat or murine hepatocytes and human HepG2 cells. Moreover, the results of these studies were inconsistent. While the earlier investigations indicated the inhibition of CYP1A1, the results of a more recent study showed CYP1A1 induction [Anwar-Mohamed and El-Kadi, 2009, Mahéo et al., 1997]. None of the available studies evaluated the effect of SFN on CYP19 expression. The expression of CYP1 family is controlled by the aryl hydrocarbon receptor (AhR). AhR initiates also the degradation of the ER. Thus, the effect of specific AhR ligand may depend on breast cell ER status [Okino et al., 2009]. The aim of the present study was to evaluate the effect of the naturally occurring L isomer of SFN on the expression of different cytochrome P450 isoforms (CYP19, CYP1A1, CYP1A2, CYP1B1) in human breast epithelial cell lines differing in ER status, tumorigenic (ER-positive MCF7 and ER-negative MDA-MB-231) and non-tumorigenic/immortalized (MCF10A).
MATERIALS AND METHODS Chemicals. L-sulforaphane (1-isothiocyanato-4methylsulfinyl-butane, SFN), dithiothreitol, antibiotics solution (104 U penicillin, 10 mg streptomycin, 25 μg amphotericin B), bovine serum albumin, dimethylsulfoxide (DMSO), fetal bovine serum, Dulbecco’s modified Eagle’s medium (DMEM), 1 mg/mL hydrocortisone, 10 mg/mL insulin, 100 μg/mL epidermal growth factor, horse serum, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), radioimmunoprecipitation assay (RIPA) buffer, trypsin, Tris, and tRNA from Escherichia coli were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Primary antibodies against CYP1A1, CYP1A2 and β-actin and secondary antibodies were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary and secondary antibodies against CYP1B1, purified standard CYP1B1 were obtained from BD Biosciences (Woburn, MA, USA). Protease inhibitor tablets were obtained from Roche Diagnostics GmbH (Penzberg, Germany). SFN was dissolved in DMSO at a concentration of 100 mmol/L and stored at 20°C.
Cell culture and treatment. MCF7 (ECACC 86012803) and MDA-MB-231 (ECACC 92020424) cell lines were purchased from the European Collection of Cell Cultures (Salisbury, Wiltshire, UK). MCF10A (ATTC CRL-10317) cell line was a gift of Dr Blażej Rubiś (Dept Clinical Chem Molecular Diagnostics, PUMS). MCF7 and MDA-MB-231 cell lines were cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotics solution. To assess the effects of SFN, the cells Copyright © 2014 John Wiley & Sons, Ltd.
were grown in the presence of 5% fetal bovine serum. MCF10A cell line were cultured in DMEM supplemented with 0.02 μg/mL of epidermal growth factor, 0.5 μg/mL of hydrocortisone, 1 μg/mL insulin solution, 5% horse serum, and 1% antibiotics solution. Experiments were conducted at a cell density of 70% confluence at standard conditions (5% CO2/95% air). After a 24 h preincubation, the cells were treated with SFN at the doses selected based on cytotoxicity assay: 5, 10, or 20 μmol/L. The incubation was continued for subsequent 72 h. Control cells were treated with vehicle (DMSO). The concentration of DMSO did not exceed 0.1%.
Viability assay. The effect of SFN on cell viability was assessed with the MTT assay, according to standard protocols. The cells were seeded in a 96-well culture plate. After a 24 h preincubation in the culture medium, 1 to 80 μmol/L of SFN was added and the cells were incubated for 72 h. Subsequently, the cells were washed with PBS buffer, and fresh medium containing MTT salt (0.5 mg/mL) was added to the wells. After a 4 h incubation, the formazan crystals were dissolved in acidic isopropanol and the absorbance was measured at 570 and 690 nm.
Apoptosis detection. The cell apoptosis ELISA detection kit (Roche Diagnostics, Penzberg, Germany) was used to detect apoptosis in breast cancer cells treated for 72 h with SFN according to manufacturer’s protocol as described previously [Licznerska et al., 2013].
Measurements of CYP450 mRNA transcripts (quantitative real-time PCR). Total RNA was isolated, using the GenElute Mammalian Total RNA Miniprep Kit (Sigma, St. Louis, MO, USA), and the first-strand complementary DNA (cDNA) was generated from total RNA using the RevertAid First Strand cDNA Synthesis Kit (Fermantas, St. Leon-Rot, Germany) according to manufacturers’ recommendations. Primer pairs capable of hybridization with unique regions of the appropriate gene sequence were designed in Beacon Designer (PREMIER Biosoft Intern.) as mentioned previously [Licznerska et al., 2013, Szaefer et al., 2012]. The quantitative real-time PCR was performed using SyberGreen on the Chromo4 (Bio-Rad Laboratories, CA, USA). All reactions were run in triplicate. The protocol started with a 5 min enzyme activation at 95°C, followed by 40 cycles of 95°C for 15 s; 56°C for 20 s; 72°C for 20 s and the final elongation at 72°C for 5 min. The melting curve analysis was used for product size verification. Experiments were normalized for the expression of porphobilinogen deaminase (PBGD) and TATA box binding protein (TBP). The Pfaffl relative method was used for fold change quantification.
Measurements of cytochrome protein levels (Western blot). Whole cell lysates were prepared using the RIPA buffer. Proteins were separated on 10% sodium dodecyl sulfate-PAGE gels and transferred to nitrocellulose membranes [Szaefer et al., 2012]. After blocking with 10% skimmed milk, the proteins were probed with goat Phytother. Res. (2014)
L-SULFORAPHANE AFFECTS CYTOCHROME P450 IN BREAST CELLS
polyclonal CYP19, goat polyclonal CYP1A1, goat polyclonal CYP1A2, rabbit anti-rat CYP1B1, and rabbit anti-mouse β-actin antibodies. As the secondary antibodies, the alkaline phosphatase-labeled anti-goat IgG or anti-rabbit IgG were used. Protein contents were measured using albumin as a standard and the β-actin protein as an internal control. The amount of immunereactive product in each lane was determined by densitometric scanning using a BioRad GS710 Image Densitometer (BioRad Laboratories, Hercules, CA, USA). The values were calculated as relative absorbance units (RQ) per mg protein.
Statistical analysis. Statistical analysis was performed by one-way ANOVA. The statistical significance between the experimental groups and their respective controls was assessed by Tukey’s post hoc test, at P < 0.05.
MDA-MB-231 cell line exhibited intermediate sensitivity to SFN after a 72 h treatment. Based on these findings, all the subsequent studies were conducted at concentrations of SFN ranging from 5 to 20 μmol/L assuring viability above 70%. Effects of SFN on apoptosis The results of ELISA test detecting histone/DNA fragments are shown in Fig. 1b. SFN in a dose 5 and 20 μmol/L induced apoptosis in estrogen-independent breast cancer MDA-MB-231 cell line (1.45 to 4.49-fold change respectively) and in non-tumorigenic MCF10A cell line treated with 5 μmol/L dose of this compound (1.85-fold change), while in estrogen-dependent breast cancer MCF7 cell line antiapoptotic effect was observed, particularly at 5 μmol/L dose (0.59-fold change). Effects of SFN on the expression of cytochrome P450
RESULTS Effect of SFN on cell viability The treatment with SFN reduced the viability of all tested cell lines, basically in a dose-dependent manner (Fig. 1a). The MCF7 cell line was the most sensitive [50% inhibitory concentration (IC50) 40 μmol/L], while non-tumorigenic MCF10A cells were least prone to the cytotoxic effect of SFN (IC50 > 80 μmol/L), while
The expression of CYP19, CYP1A1, 1A2, 1B1 was determined at the transcript and protein levels. The constitutive expression of CYP19 and CYP1 family isoforms was detected in all tested cell lines, and no significant differences, except CYP1B1, in the expression were found between the cell lines tested. The treatment with SFN changed their expression in a dose and cell line-dependent manner. In estrogen-dependent MCF7 cell line, SFN in a lower dose (5 μmol/L) significantly reduced both CYP19 transcript (0.43-fold change, Fig. 2a/Table 1) and protein levels (0.89-fold change, Fig. 2b/Table 1). The higher dose of SFN (20 μmol/L) diminished also CYP19 protein (0.79-fold change, Fig. 2b/Table 1). In contrast, in estrogen-independent MDA-MB-231 cell line, SFN in a dose of 5 μmol/L increased both CYP19 transcript (1.56-fold change, Fig. 2a/Table 1) and protein levels (1.59-fold change, Fig. 2b/Table 1). In a higher 20 μmol/L dose of SFN, an increased aromatase protein level (1.6-fold change, Fig. 2b/Table 1) was found. The expression of CYP19 was not affected in MCF10A cell line. However, in MCF10A cell line, SFN increased the expression of CYP1A2 (Fig. 3b/Table 2, Fig. 4b/Table 3), and at 5 μmol/L dose, it slightly reduced CYP1B1 transcript (Fig. 3c/Table 2). The increase of CYP1A2 protein level, but not its transcript, was also observed in MDA-MB-231 cell line (Fig. 4b/Table 3). In this cell line, the slight decrease of CYP1B1 was also noticed (Fig. 3c/Table 2). CYP1A1 protein level was decreased in all cell lines; however, the dose-dependent effect was found only in MDA-MB-231 cell line (Fig. 4a/Table 3).
DISCUSSION
Figure 1. The effect of L-sulforaphane (SFN) on the cytotoxicity (a) and apoptosis (b) in MCF7, MDA-MB-231, and MCF10A cell lines. The mean values ± SEM from three independent experiments run in triplicate are presented. The asterisk above the bar denotes statistically significant differences of mean values from the control group (P < 0.05). Copyright © 2014 John Wiley & Sons, Ltd.
Estrogens are considered a major risk factor for breast cancer. In their synthesis and oxidative metabolism, the key role is played by cytochrome P450. In this study, the effect of L-sulforaphane on the expression of CYPs was evaluated in two breast cancer cell lines differing in ER status, ER-positive MCF7 and ER-negative MDA-MB231, along with immortalized breast non-tumorigenic MCF10A cell line. In accordance with our previous and Phytother. Res. (2014)
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Figure 2. The effect of 72 h incubation with L-sulforaphane (SFN) on the level of the CYP19 transcript (a) and protein (b) in MCF7, MDA-MB-231, and MCF10A cell lines. The values were calculated as a relative change in transcript or protein level in comparison with control cells (expression equals 1). The mean values ± SEM from three independent experiments run in duplicate (protein) or triplicate (mRNA) are presented. The asterisk above the bar denotes significant differences of mean values from the control group (P < 0.05).
Table 1. The effect of L-sulforaphane on the level of the CYP19 transcript and protein in breast cell lines Cell line MCF7 MDA-MB-231 MCF10A
SFN [μmol/L]
CYP19 mRNA
CYP19 protein
5 20 5 20 5 10
0.434 ± 0.004* 0.858 ± 0.351 1.560 ± 0.126* 1.143 ± 0.979 1.719 ± 0.412 1.767 ± 0.484
0.885 ± 0.028* 0.794 ± 0.020* 1.590 ± 0.015* 1.600 ± 0.039* 0.987 ± 0.025 1.030 ± 0.025
The values were calculated as a relative change in transcript or protein level in comparison with control cells (expression equals 1). The mean values ± SEM from three independent experiments run in duplicate (protein) or triplicate (mRNA) are presented. *Significantly different of mean values from the control group (P < 0.05).
other studies, constitutive expression of CYP19 and CYP1 family isoforms was detected in all tested cell lines [Licznerska et al., 2013, Szaefer et al., 2012]. Moreover, the higher relative level of CYP1B1 mRNA in tumorigenic Copyright © 2014 John Wiley & Sons, Ltd.
Figure 3. The effect of 72 h incubation with L-sulforaphane (SFN) on the transcript level of the CYP1A1 (a), CYP1A2 (b), CYP1B1 (c) in MCF7, MDA-MB-231, and MCF10A cell lines. The values were calculated as a relative change in transcript level in comparison to control cells (expression equals 1). The mean values ± SEM from three independent experiments run in triplicate are presented. The asterisk above the bar denotes significant differences of mean values from the control group (P < 0.05).
cell lines (MCF7 and MDA-MB-231) in comparison to non-tumorigenic (MCF10A), one may confirm the possible role of CYP1B1 in breast cancer etiology. The treatment with SFN affected the viability of these phenotypically distinct breast epithelial cells in a specific manner. While the viability of MCF7 cell line was significantly decreased, MDA-MB-231 and MCF10A cell lines were much less affected within the tested concentration range (1–80 μmol/L). Moreover, IC50 values in MCF7 and MDA-MB-231 were significantly higher than those found by other authors even after a shorter (24 h) treatment [Pawlik et al. 2013, Tseng et al. 2004]. One reason of such a discrepancy might be the fact that in these studies, D,L-SFN, a synthetic analogue of the naturally occurring L-SFN, was used, while in our study, the L-SFN isomer was applied. Thus, our results reflect better the dietary effect of this broccoli constituent. It also has to be stressed that the doses of L-SFN applied in our study were lower than the level of SFN metabolites detected in rat mammary tissue following a single oral Phytother. Res. (2014)
L-SULFORAPHANE AFFECTS CYTOCHROME P450 IN BREAST CELLS
Table 2. The effect of L-sulforaphane on the level of the CYP1A1, CYP1A2 and CYP1B1 transcripts in breast cell lines Cell line
SFN [μmol/L]
CYP1A1
CYP1A2
CYP1B1
5 20 5 20 5 10
1.063 ± 0.352 0.943 ± 0.157 0.875 ± 0.053 1.181 ± 0.137 1.033 ± 0.026 0.919 ± 0.073
0.981 ± 0.098 1.827 ± 0.355 0.401 ± 0.281 0.338 ± 0.093* 1.314 ± 0.051* 1.262 ± 0.003*
1.058 ± 0.104 1.298 ± 0.318 1.252 ± 0.201 1.146 ± 0.050* 0.870 ± 0.042* 1.767 ± 0.484
MCF7 MDA-MB-231 MCF10A
The values were calculated as a relative change in transcript level in comparison to control cells (expression equals 1). The mean values ± SEM from three independent experiments run in triplicate are presented. *Significantly different of mean values from the control group (P < 0.05).
Figure 4. The effect of 72 h incubation with L-sulforaphane (SFN) on the protein level of the CYP1A1 (a), CYP1A2 (b), CYP1B1 (c) in MCF7, MDA-MB-231, and MCF10A cell lines. The values were calculated as a relative change in protein level in comparison to control cells (expression equals 1). The mean values ± SEM from three independent experiments run in duplicate are presented. The asterisk above the bar denotes significant differences of mean values from the control group (P < 0.05).
150 μmol/L dose of SFN and in human breast tissue after a single dose of broccoli sprout preparation containing 200 μM of SFN [Cornblatt et al., 2007]. Our study also confirmed the induction of apoptosis in ER( ) MDA-MB-231 cell line. Again, this effect was achieved in a higher dose in comparison with racemic SFN used by the other authors [Pawlik et al., 2013, Sakao and Singh, 2012]. Moreover, L-SFN in the dose Copyright © 2014 John Wiley & Sons, Ltd.
as low as 5 μmol/L induced apoptosis also in nontumorigenic MCF10A cell line but exerted antiapoptotic effect in MCF7 cell line. The latter observation is in contrast to the data presented by Pledgie-Tracy et al. [2007] for racemic SFN where no differences in the sensitivity to apoptosis assessed by DNA fragmentation between MDA-MB-231 and MCF7 breast cell lines were found. However, DNA fragmentation was observed only after 96 h but not at the earlier time points such as 72 h evaluated in our study by ELISA assessment of cytoplasmic histone/DNA fragments. Moreover, the pattern of apoptotic protein expression seen in racemic SFN-treated MDA-MB-231 cell line was markedly different from changes induced by SFN in the other tree breast cancer cell lines examined including MCF7 and suggested that distinct pathways of apoptosis were induced by SFN in these cell lines. Thus, further studies are required in order to explain the cell-specific difference in sensitivity to apoptosis induced by L-SFN. Among the cytochrome P450, L-SFN in a 5 and 20 μmol/L dose affected the CYP19 expression in MCF7 cell line the most. Aromatase, encoded by this isoform, plays a crucial role in local production of estrogen in the breast tissue. The ability to decrease its level is a very good prognostic factor in breast cancer treatment and perhaps in prophylaxis. Thereby, we provided the evidence that SFN might downregulate the CYP19 expression on both transcript and protein level in ER positive cancer breast cells. In contrast, in the estrogen-independent MDA-MB-231, an increase of CYP19 mRNA and protein was observed at a dose 5 μmol/L. Such a trend indicates that the mechanism of anticancer action of SFN in breast carcinogenesis could be associated with ER status. Moreover, SFN-mediated aromatase level decrease in MCF7 cell line that may be mediated by the downregulation of the ERα expression was noticed by Ramirez and Singletary [2009]. The detection of the constitutive CYP19 expression in nontumorigenic MCF10A cell line indicates that aromatase may be a target not only for treatment but also for prevention of breast cancer. The effect of SFN on cytochrome P450 involved in estrogen catabolism was to a lesser extent dependent on epithelial breast cell line. The slightly reduced CYP1A1 protein level was observed in all cell lines tested. An increased level of CYP1A2 expression was found in MCF10A. CYP1B1 protein was unaffected despite the increased level of its mRNA, particularly in ER-negative MDA-MB-231 and non-tumorigenic MCF10A cell line. Although the positive effect of SFN on phase II drug metabolizing enzymes is well established, Phytother. Res. (2014)
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Table 3. The effect of L-sulforaphane on the level of the CYP1A1, CYP1A2 and CYP1B1 proteins in breast cell lines Cell line
SFN [μmol/L]
CYP1A1
CYP1A2
CYP1B1
5 20 5 20 5 10
0.881 ± 0.005* 0.926 ± 0.049 0.920 ± 0.008* 0.880 ± 0.020* 0.810 ± 0.028* 1.020 ± 0.029
1.005 ± 0.052 0.993 ± 0.020 1.160 ± 0.010* 1.170 ± 0.007* 1.300 ± 0.030* 1.050 ± 0.009
0.970 ± 0.028 0.984 ± 0.015 0.990 ± 0.012 0.950 ± 0.009 0.930 ± 0.072 1.010 ± 0.139
MCF7 MDA-MB-231 MCF10A
The values were calculated as a relative change in protein level in comparison to control cells (expression equals 1). The mean values ± SEM from three independent experiments run in duplicate are presented. *Significantly different of mean values from the control group (P < 0.05).
its effect on the phase I bioactivating cytochrome P450 is still a matter of debate. The early studies [Mahéo et al., 1997] showed the unchanged expression (mRNA) of CYP1A2 in human HepG2 cells but reduced activity of CYP1A1 in rat primary hepatocytes as the result of D, L-SFN treatment. Contrary, a more recent study revealed the induction of CYP1A1 mRNA in murine Hep 1c1c7 and human hepatocytes HepG2 cells [Anwar-Mohamed and El-Kadi, 2009]. This effect was observed at the doses similar to that applied in our study to breast epithelial cells. Our study may confirm this effect but only on the CYP1A1 transcript level and in to a lesser extent. Moreover, these authors demonstrated that SFN can directly induce the CYP1A1 gene expression in an AhR-dependent manner [Anwar-Mohamed and El-Kadi, 2009]. One reason of the differences in the results of our study and that mentioned in the preceding texts could be, beside the cell type, the time point of CYPs mRNA and protein measurement. We evaluated the effect of SFN after 72 h, which may better reflect the in vivo situation [Moiseeva et al., 2007], while the induction of Cyp1a1 mRNA in murine Hepa1c1c7 was observed up to 24 h with maximal induction at 6 h time point [Anwar-Mohamed and El-Kadi, 2009]. On the other hand, in breast cells, particularly MCF7 cell line, in contrast to hepatocytes, cross talk between AhR and ER has to be considered [MacPherson and Matthews, 2009]. In this regard, it was shown that resveratrol in nanomolar concentrations was able to repress AhR-mediated induction of CYP1A1 in an ERα-dependent manner without reducing the level of AhR complex at the CYP1A1 promoter. In contrast,
10 μM of resveratrol markedly reduced AhR complex on the CYP1A1 promoter and resulted in nearcomplete inhibition of CYP1A1 expression and metabolic activity [Perdew et al., 2010]. Thus, further studies including cross talk between ERα and AhR also using nanomolar SFN concentrations are necessary to explain the mechanism of the inhibition or induction of CYPs involved in estrogen catabolism. The application of the inducers of specific isoform of P450 may be also considered as it was shown that D,L-SFN may inhibit CYP1A1 and CYP1A2 protein level and activity induced by polycyclic aromatic hydrocarbons in MCF7 cell line [Skupinska et al., 2009]. To sum up, our study showed that the naturally occurring L-isomer of SFN may affect the cytochrome P450 involved in estrogen synthesis and catabolism. This effect is partly dependent on breast cell ER status. Although the exact mechanism of this SFN activity requires further studies, our results support a rationale for future clinical evaluation of sulforaphane or the extract of broccoli/broccoli sprouts for breast cancer chemoprevention.
Acknowledgement This work was supported by the Ministry of Science and Higher Education of Poland, grant N405 3390.
Conflict of Interest The authors declare that they have no conflict of interest.
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Singletary K, MacDonald C. 2000. Inhibition of benzo[a]pyrene- and 1,6-dinitropyrene-DNA adduct formation in human mammary epithelial cells by dibenzoylmethane and sulforaphane. Cancer Lett 155: 47–54. Skupinska K, Misiewicz-Krzeminska I, Lubelska K, KasprzyckaGuttman T. 2009. The effect of isothiocyanates on CYP1A1 and CYP1A2 activities induced by polycyclic aromatic hydrocarbons in Mcf7 cells. Toxicol In Vitro 23: 763–71. Szaefer H, Licznerska B, Krajka-Kuźniak V, Bartoszek A, BaerDubowska W. 2012. Modulation of CYP1A1, CYP1A2 and CYP1B1 expression by cabbage juices and indoles in human breast cell lines. Nutr Cancer 64: 879–88. Terry P, Wolk A, Persson I, Magnusson C. 2001. Brassica vegetables and breast cancer risk. JAMA 285: 2975–7. Tseng E, Scott-Ramsay EA, Morris ME. 2004. Dietary organic isothiocyanates are cytotoxic in human breast cancer MCF-7 and mammary epithelial MCF-12A cell lines. Exp Biol Med 229: 835–42. Tsuchiya Y, Nakajima M, Yokoi T. 2005. Cytochrome P450-mediated metabolism of estrogens and its regulation in human. Cancer Lett 227: 115–124. Watson CS, Gametchu B, Norfleet AM, Cambell CH, Thomas ML. 1999. Rapid, nongenomic action of estrogens. LOWOC J 1: 21–28. Yang L, Zahid M, Liao Y, Rogan EG, Cavalieri EL, Davidson NE, Yager JD, Visvanathan K, Groopman JD, Kensler TW. 2013. Reduced formation of depurinating estrogen-DNA adducts by sulforaphane or KEAP1 disruption in human mammary epithelial MCF-10A cells. Carcinogenesis 34: 2587–92. Zhang Y, Talalay P, Cho CG, Posner GH. 1992. A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. Proc Natl Acad Sci 89: 2399–2403 Zhang Y, Munday R, Jobson HE, Munday CM, Lister C, Wilson P, Fahey JW, Mhawech-Fauceglia P. 2006. Induction of GST and NQO1 in cultured bladder cells and in the urinary bladders of rats by an extract of broccoli (Brassica oleracea) sprouts. J Agric Food Chem 54: 9370–76.
Phytother. Res. (2014)
Modulating Potential of L-Sulforaphane in the Expression of Cytochrome P450 to Identify Potential Targets for Breast Cancer Chemoprevention and Therapy Using Breast Cell Lines Barbara Licznerska,1 Hanna Szaefer,1 Iwona Matuszak,1 Marek Murias2 and Wanda Baer-Dubowska1* 1
Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences, Poznań, Poland Department of Toxicology, Poznan University of Medical Sciences, Poznań, Poland
2
The L-sulforaphane (SFN) component of broccoli sprout showed anticancer activity in several preclinical studies including breast cancer. Estrogens are considered major risk factors in breast carcinogenesis. The aim of this study was to evaluate the effect of SFN on the expression of cytochrome P450 involved in the estrogen metabolism in breast cancer cell lines MCF7 and MDA-MB-231 and in non-tumorigenic MCF10A cell line. The expression of CYP19, CYP1A1, 1A2, 1B1 was determined at the transcript and protein levels. There were found some remarkable differences in the effect of SFN at a dose of 5 μmol/L on CYP19 expression: in ER(+) MCF7 significant reduction, in ER( ) MDA-MB-231 an increased expression and unchanged expression in MCF10A cell line. The effect of SFN on CYPs (1A1, 1A2, 1B1) involved in estrogen catabolism was to a lesser extent dependent on breast cell line. The slightly reduced CYP1A1 protein level was observed in all cell lines tested. An increased level of CYP1A2 and decreased level of CYP1B1 expression were found in MCF10A. These results indicate that the naturally occurring L isomer of SFN may affect the expression of P450s involved in estrogen metabolism. This effect may contribute to the anticancer activity of SFN in breast tissue. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: L-Sulforaphane; CYP19; CYP1A1; CYP1A2; CYP1B1; breast cancer chemoprevention. Abbreviations: AhR, hydrocarbon receptor; ER, estrogen receptor; GST, glutathione S-transferase; NQO1, NAD(P)H:quinone oxidoreductase; PBGD, porphobilinogen deaminase; SFN, L-sulforaphane; TBP, TATA box binding protein.
INTRODUCTION Cruciferous vegetables are a rich source of bioactive isothiocyanates. Among them, sulforaphane [1-isothiocyanato4-methylsulfinyl-butane (SFN)] in some cruciferous vegetables, especially in broccoli sprouts, is one of the most abundant [Cornblatt et al., 2007; Zhang et al., 1992]. This compound has been proved to possess anticancer activity in several preclinical studies including breast cancer. A preliminary clinical trial involving healthy women undergoing reduction mammoplasty further supported the SFN potential in chemoprevention of breast cancer [Cornblatt et al., 2007]. SFN is known as a potent activator of Nrf2 pathway and inducer of detoxification enzymes such as NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase (GST) [Jeong et al., 2005, Keum et al., 2009, Munday and Munday, 2004, Zhang et al., 2006]. These enzymes might be responsible for the decreased DNA-adduct formation in normal mammary cells exposed to benzo[a]pyrene and 1,6-dinitropyrene * Correspondence to: Wanda Baer-Dubowska, Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences, Poznań, Poland. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
[Singletary and MacDonald, 2000] as well as depurinating estrogen-DNA adducts [Yang et al., 2013]. These experimental findings are further supported by some epidemiological data suggesting a protective association between increased consumption of broccoli or Brussels sprouts and breast cancer [Ambrosone et al., 2004, Fowke et al., 2003, Terry et al., 2001]. Estrogens are considered major risk factors in breast carcinogenesis. Two mechanisms for estrogen carcinogenesis have been proposed: estrogen-induced proliferation [in estrogen receptor (ER) positive cells] and the formation of reactive estrogen metabolites [Watson et al., 1999]. In the metabolism of estrogens, a crucial role is played by cytochrome P450. The de novo estrogen synthesis from androgens requires aromatase activity, encoded by CYP19. The oxidative catabolism of estrogens is catalyzed mainly by CYP1A1/CYP1A2 and CYP1B1. The action of these enzymes can convert 17-β estradiol in breast epithelium into its 2-hydroxyestradiol and 4-hydroxyestradiol. While 2-hydroxyestradiol is mainly catalyzed by CYP1A2 and CYP3A4 in the liver, and by CYP1A1 in extra-hepatic tissues, CYP1B1 is highly expressed in estrogen target tissues including mammary and catalyzes the 4-hydroxylation of estradiol. Because 4-hydroxyestradiol generates free radicals from Received 17 March 2014 Revised 20 May 2014 Accepted 31 August 2014
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the reductive–oxidative cycling with the corresponding semiquinone and quinone forms, which cause cellular damage, the specific and local formation of 4-hydroxyestradiol is important for breast carcinogenesis [Tsuchiya et al., 2005]. By the induction of NQO1 and GSTs, compounds like SFN may detoxify estrogen metabolites protecting against DNA damage, but equally important is targeting the specific CYPs involved in their activation. In contrast to the induction of phase II enzymes by SFN, its effect on CYPs was the subject of only two studies performed in rat or murine hepatocytes and human HepG2 cells. Moreover, the results of these studies were inconsistent. While the earlier investigations indicated the inhibition of CYP1A1, the results of a more recent study showed CYP1A1 induction [Anwar-Mohamed and El-Kadi, 2009, Mahéo et al., 1997]. None of the available studies evaluated the effect of SFN on CYP19 expression. The expression of CYP1 family is controlled by the aryl hydrocarbon receptor (AhR). AhR initiates also the degradation of the ER. Thus, the effect of specific AhR ligand may depend on breast cell ER status [Okino et al., 2009]. The aim of the present study was to evaluate the effect of the naturally occurring L isomer of SFN on the expression of different cytochrome P450 isoforms (CYP19, CYP1A1, CYP1A2, CYP1B1) in human breast epithelial cell lines differing in ER status, tumorigenic (ER-positive MCF7 and ER-negative MDA-MB-231) and non-tumorigenic/immortalized (MCF10A).
MATERIALS AND METHODS Chemicals. L-sulforaphane (1-isothiocyanato-4methylsulfinyl-butane, SFN), dithiothreitol, antibiotics solution (104 U penicillin, 10 mg streptomycin, 25 μg amphotericin B), bovine serum albumin, dimethylsulfoxide (DMSO), fetal bovine serum, Dulbecco’s modified Eagle’s medium (DMEM), 1 mg/mL hydrocortisone, 10 mg/mL insulin, 100 μg/mL epidermal growth factor, horse serum, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), radioimmunoprecipitation assay (RIPA) buffer, trypsin, Tris, and tRNA from Escherichia coli were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Primary antibodies against CYP1A1, CYP1A2 and β-actin and secondary antibodies were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary and secondary antibodies against CYP1B1, purified standard CYP1B1 were obtained from BD Biosciences (Woburn, MA, USA). Protease inhibitor tablets were obtained from Roche Diagnostics GmbH (Penzberg, Germany). SFN was dissolved in DMSO at a concentration of 100 mmol/L and stored at 20°C.
Cell culture and treatment. MCF7 (ECACC 86012803) and MDA-MB-231 (ECACC 92020424) cell lines were purchased from the European Collection of Cell Cultures (Salisbury, Wiltshire, UK). MCF10A (ATTC CRL-10317) cell line was a gift of Dr Blażej Rubiś (Dept Clinical Chem Molecular Diagnostics, PUMS). MCF7 and MDA-MB-231 cell lines were cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotics solution. To assess the effects of SFN, the cells Copyright © 2014 John Wiley & Sons, Ltd.
were grown in the presence of 5% fetal bovine serum. MCF10A cell line were cultured in DMEM supplemented with 0.02 μg/mL of epidermal growth factor, 0.5 μg/mL of hydrocortisone, 1 μg/mL insulin solution, 5% horse serum, and 1% antibiotics solution. Experiments were conducted at a cell density of 70% confluence at standard conditions (5% CO2/95% air). After a 24 h preincubation, the cells were treated with SFN at the doses selected based on cytotoxicity assay: 5, 10, or 20 μmol/L. The incubation was continued for subsequent 72 h. Control cells were treated with vehicle (DMSO). The concentration of DMSO did not exceed 0.1%.
Viability assay. The effect of SFN on cell viability was assessed with the MTT assay, according to standard protocols. The cells were seeded in a 96-well culture plate. After a 24 h preincubation in the culture medium, 1 to 80 μmol/L of SFN was added and the cells were incubated for 72 h. Subsequently, the cells were washed with PBS buffer, and fresh medium containing MTT salt (0.5 mg/mL) was added to the wells. After a 4 h incubation, the formazan crystals were dissolved in acidic isopropanol and the absorbance was measured at 570 and 690 nm.
Apoptosis detection. The cell apoptosis ELISA detection kit (Roche Diagnostics, Penzberg, Germany) was used to detect apoptosis in breast cancer cells treated for 72 h with SFN according to manufacturer’s protocol as described previously [Licznerska et al., 2013].
Measurements of CYP450 mRNA transcripts (quantitative real-time PCR). Total RNA was isolated, using the GenElute Mammalian Total RNA Miniprep Kit (Sigma, St. Louis, MO, USA), and the first-strand complementary DNA (cDNA) was generated from total RNA using the RevertAid First Strand cDNA Synthesis Kit (Fermantas, St. Leon-Rot, Germany) according to manufacturers’ recommendations. Primer pairs capable of hybridization with unique regions of the appropriate gene sequence were designed in Beacon Designer (PREMIER Biosoft Intern.) as mentioned previously [Licznerska et al., 2013, Szaefer et al., 2012]. The quantitative real-time PCR was performed using SyberGreen on the Chromo4 (Bio-Rad Laboratories, CA, USA). All reactions were run in triplicate. The protocol started with a 5 min enzyme activation at 95°C, followed by 40 cycles of 95°C for 15 s; 56°C for 20 s; 72°C for 20 s and the final elongation at 72°C for 5 min. The melting curve analysis was used for product size verification. Experiments were normalized for the expression of porphobilinogen deaminase (PBGD) and TATA box binding protein (TBP). The Pfaffl relative method was used for fold change quantification.
Measurements of cytochrome protein levels (Western blot). Whole cell lysates were prepared using the RIPA buffer. Proteins were separated on 10% sodium dodecyl sulfate-PAGE gels and transferred to nitrocellulose membranes [Szaefer et al., 2012]. After blocking with 10% skimmed milk, the proteins were probed with goat Phytother. Res. (2014)
L-SULFORAPHANE AFFECTS CYTOCHROME P450 IN BREAST CELLS
polyclonal CYP19, goat polyclonal CYP1A1, goat polyclonal CYP1A2, rabbit anti-rat CYP1B1, and rabbit anti-mouse β-actin antibodies. As the secondary antibodies, the alkaline phosphatase-labeled anti-goat IgG or anti-rabbit IgG were used. Protein contents were measured using albumin as a standard and the β-actin protein as an internal control. The amount of immunereactive product in each lane was determined by densitometric scanning using a BioRad GS710 Image Densitometer (BioRad Laboratories, Hercules, CA, USA). The values were calculated as relative absorbance units (RQ) per mg protein.
Statistical analysis. Statistical analysis was performed by one-way ANOVA. The statistical significance between the experimental groups and their respective controls was assessed by Tukey’s post hoc test, at P < 0.05.
MDA-MB-231 cell line exhibited intermediate sensitivity to SFN after a 72 h treatment. Based on these findings, all the subsequent studies were conducted at concentrations of SFN ranging from 5 to 20 μmol/L assuring viability above 70%. Effects of SFN on apoptosis The results of ELISA test detecting histone/DNA fragments are shown in Fig. 1b. SFN in a dose 5 and 20 μmol/L induced apoptosis in estrogen-independent breast cancer MDA-MB-231 cell line (1.45 to 4.49-fold change respectively) and in non-tumorigenic MCF10A cell line treated with 5 μmol/L dose of this compound (1.85-fold change), while in estrogen-dependent breast cancer MCF7 cell line antiapoptotic effect was observed, particularly at 5 μmol/L dose (0.59-fold change). Effects of SFN on the expression of cytochrome P450
RESULTS Effect of SFN on cell viability The treatment with SFN reduced the viability of all tested cell lines, basically in a dose-dependent manner (Fig. 1a). The MCF7 cell line was the most sensitive [50% inhibitory concentration (IC50) 40 μmol/L], while non-tumorigenic MCF10A cells were least prone to the cytotoxic effect of SFN (IC50 > 80 μmol/L), while
The expression of CYP19, CYP1A1, 1A2, 1B1 was determined at the transcript and protein levels. The constitutive expression of CYP19 and CYP1 family isoforms was detected in all tested cell lines, and no significant differences, except CYP1B1, in the expression were found between the cell lines tested. The treatment with SFN changed their expression in a dose and cell line-dependent manner. In estrogen-dependent MCF7 cell line, SFN in a lower dose (5 μmol/L) significantly reduced both CYP19 transcript (0.43-fold change, Fig. 2a/Table 1) and protein levels (0.89-fold change, Fig. 2b/Table 1). The higher dose of SFN (20 μmol/L) diminished also CYP19 protein (0.79-fold change, Fig. 2b/Table 1). In contrast, in estrogen-independent MDA-MB-231 cell line, SFN in a dose of 5 μmol/L increased both CYP19 transcript (1.56-fold change, Fig. 2a/Table 1) and protein levels (1.59-fold change, Fig. 2b/Table 1). In a higher 20 μmol/L dose of SFN, an increased aromatase protein level (1.6-fold change, Fig. 2b/Table 1) was found. The expression of CYP19 was not affected in MCF10A cell line. However, in MCF10A cell line, SFN increased the expression of CYP1A2 (Fig. 3b/Table 2, Fig. 4b/Table 3), and at 5 μmol/L dose, it slightly reduced CYP1B1 transcript (Fig. 3c/Table 2). The increase of CYP1A2 protein level, but not its transcript, was also observed in MDA-MB-231 cell line (Fig. 4b/Table 3). In this cell line, the slight decrease of CYP1B1 was also noticed (Fig. 3c/Table 2). CYP1A1 protein level was decreased in all cell lines; however, the dose-dependent effect was found only in MDA-MB-231 cell line (Fig. 4a/Table 3).
DISCUSSION
Figure 1. The effect of L-sulforaphane (SFN) on the cytotoxicity (a) and apoptosis (b) in MCF7, MDA-MB-231, and MCF10A cell lines. The mean values ± SEM from three independent experiments run in triplicate are presented. The asterisk above the bar denotes statistically significant differences of mean values from the control group (P < 0.05). Copyright © 2014 John Wiley & Sons, Ltd.
Estrogens are considered a major risk factor for breast cancer. In their synthesis and oxidative metabolism, the key role is played by cytochrome P450. In this study, the effect of L-sulforaphane on the expression of CYPs was evaluated in two breast cancer cell lines differing in ER status, ER-positive MCF7 and ER-negative MDA-MB231, along with immortalized breast non-tumorigenic MCF10A cell line. In accordance with our previous and Phytother. Res. (2014)
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Figure 2. The effect of 72 h incubation with L-sulforaphane (SFN) on the level of the CYP19 transcript (a) and protein (b) in MCF7, MDA-MB-231, and MCF10A cell lines. The values were calculated as a relative change in transcript or protein level in comparison with control cells (expression equals 1). The mean values ± SEM from three independent experiments run in duplicate (protein) or triplicate (mRNA) are presented. The asterisk above the bar denotes significant differences of mean values from the control group (P < 0.05).
Table 1. The effect of L-sulforaphane on the level of the CYP19 transcript and protein in breast cell lines Cell line MCF7 MDA-MB-231 MCF10A
SFN [μmol/L]
CYP19 mRNA
CYP19 protein
5 20 5 20 5 10
0.434 ± 0.004* 0.858 ± 0.351 1.560 ± 0.126* 1.143 ± 0.979 1.719 ± 0.412 1.767 ± 0.484
0.885 ± 0.028* 0.794 ± 0.020* 1.590 ± 0.015* 1.600 ± 0.039* 0.987 ± 0.025 1.030 ± 0.025
The values were calculated as a relative change in transcript or protein level in comparison with control cells (expression equals 1). The mean values ± SEM from three independent experiments run in duplicate (protein) or triplicate (mRNA) are presented. *Significantly different of mean values from the control group (P < 0.05).
other studies, constitutive expression of CYP19 and CYP1 family isoforms was detected in all tested cell lines [Licznerska et al., 2013, Szaefer et al., 2012]. Moreover, the higher relative level of CYP1B1 mRNA in tumorigenic Copyright © 2014 John Wiley & Sons, Ltd.
Figure 3. The effect of 72 h incubation with L-sulforaphane (SFN) on the transcript level of the CYP1A1 (a), CYP1A2 (b), CYP1B1 (c) in MCF7, MDA-MB-231, and MCF10A cell lines. The values were calculated as a relative change in transcript level in comparison to control cells (expression equals 1). The mean values ± SEM from three independent experiments run in triplicate are presented. The asterisk above the bar denotes significant differences of mean values from the control group (P < 0.05).
cell lines (MCF7 and MDA-MB-231) in comparison to non-tumorigenic (MCF10A), one may confirm the possible role of CYP1B1 in breast cancer etiology. The treatment with SFN affected the viability of these phenotypically distinct breast epithelial cells in a specific manner. While the viability of MCF7 cell line was significantly decreased, MDA-MB-231 and MCF10A cell lines were much less affected within the tested concentration range (1–80 μmol/L). Moreover, IC50 values in MCF7 and MDA-MB-231 were significantly higher than those found by other authors even after a shorter (24 h) treatment [Pawlik et al. 2013, Tseng et al. 2004]. One reason of such a discrepancy might be the fact that in these studies, D,L-SFN, a synthetic analogue of the naturally occurring L-SFN, was used, while in our study, the L-SFN isomer was applied. Thus, our results reflect better the dietary effect of this broccoli constituent. It also has to be stressed that the doses of L-SFN applied in our study were lower than the level of SFN metabolites detected in rat mammary tissue following a single oral Phytother. Res. (2014)
L-SULFORAPHANE AFFECTS CYTOCHROME P450 IN BREAST CELLS
Table 2. The effect of L-sulforaphane on the level of the CYP1A1, CYP1A2 and CYP1B1 transcripts in breast cell lines Cell line
SFN [μmol/L]
CYP1A1
CYP1A2
CYP1B1
5 20 5 20 5 10
1.063 ± 0.352 0.943 ± 0.157 0.875 ± 0.053 1.181 ± 0.137 1.033 ± 0.026 0.919 ± 0.073
0.981 ± 0.098 1.827 ± 0.355 0.401 ± 0.281 0.338 ± 0.093* 1.314 ± 0.051* 1.262 ± 0.003*
1.058 ± 0.104 1.298 ± 0.318 1.252 ± 0.201 1.146 ± 0.050* 0.870 ± 0.042* 1.767 ± 0.484
MCF7 MDA-MB-231 MCF10A
The values were calculated as a relative change in transcript level in comparison to control cells (expression equals 1). The mean values ± SEM from three independent experiments run in triplicate are presented. *Significantly different of mean values from the control group (P < 0.05).
Figure 4. The effect of 72 h incubation with L-sulforaphane (SFN) on the protein level of the CYP1A1 (a), CYP1A2 (b), CYP1B1 (c) in MCF7, MDA-MB-231, and MCF10A cell lines. The values were calculated as a relative change in protein level in comparison to control cells (expression equals 1). The mean values ± SEM from three independent experiments run in duplicate are presented. The asterisk above the bar denotes significant differences of mean values from the control group (P < 0.05).
150 μmol/L dose of SFN and in human breast tissue after a single dose of broccoli sprout preparation containing 200 μM of SFN [Cornblatt et al., 2007]. Our study also confirmed the induction of apoptosis in ER( ) MDA-MB-231 cell line. Again, this effect was achieved in a higher dose in comparison with racemic SFN used by the other authors [Pawlik et al., 2013, Sakao and Singh, 2012]. Moreover, L-SFN in the dose Copyright © 2014 John Wiley & Sons, Ltd.
as low as 5 μmol/L induced apoptosis also in nontumorigenic MCF10A cell line but exerted antiapoptotic effect in MCF7 cell line. The latter observation is in contrast to the data presented by Pledgie-Tracy et al. [2007] for racemic SFN where no differences in the sensitivity to apoptosis assessed by DNA fragmentation between MDA-MB-231 and MCF7 breast cell lines were found. However, DNA fragmentation was observed only after 96 h but not at the earlier time points such as 72 h evaluated in our study by ELISA assessment of cytoplasmic histone/DNA fragments. Moreover, the pattern of apoptotic protein expression seen in racemic SFN-treated MDA-MB-231 cell line was markedly different from changes induced by SFN in the other tree breast cancer cell lines examined including MCF7 and suggested that distinct pathways of apoptosis were induced by SFN in these cell lines. Thus, further studies are required in order to explain the cell-specific difference in sensitivity to apoptosis induced by L-SFN. Among the cytochrome P450, L-SFN in a 5 and 20 μmol/L dose affected the CYP19 expression in MCF7 cell line the most. Aromatase, encoded by this isoform, plays a crucial role in local production of estrogen in the breast tissue. The ability to decrease its level is a very good prognostic factor in breast cancer treatment and perhaps in prophylaxis. Thereby, we provided the evidence that SFN might downregulate the CYP19 expression on both transcript and protein level in ER positive cancer breast cells. In contrast, in the estrogen-independent MDA-MB-231, an increase of CYP19 mRNA and protein was observed at a dose 5 μmol/L. Such a trend indicates that the mechanism of anticancer action of SFN in breast carcinogenesis could be associated with ER status. Moreover, SFN-mediated aromatase level decrease in MCF7 cell line that may be mediated by the downregulation of the ERα expression was noticed by Ramirez and Singletary [2009]. The detection of the constitutive CYP19 expression in nontumorigenic MCF10A cell line indicates that aromatase may be a target not only for treatment but also for prevention of breast cancer. The effect of SFN on cytochrome P450 involved in estrogen catabolism was to a lesser extent dependent on epithelial breast cell line. The slightly reduced CYP1A1 protein level was observed in all cell lines tested. An increased level of CYP1A2 expression was found in MCF10A. CYP1B1 protein was unaffected despite the increased level of its mRNA, particularly in ER-negative MDA-MB-231 and non-tumorigenic MCF10A cell line. Although the positive effect of SFN on phase II drug metabolizing enzymes is well established, Phytother. Res. (2014)
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Table 3. The effect of L-sulforaphane on the level of the CYP1A1, CYP1A2 and CYP1B1 proteins in breast cell lines Cell line
SFN [μmol/L]
CYP1A1
CYP1A2
CYP1B1
5 20 5 20 5 10
0.881 ± 0.005* 0.926 ± 0.049 0.920 ± 0.008* 0.880 ± 0.020* 0.810 ± 0.028* 1.020 ± 0.029
1.005 ± 0.052 0.993 ± 0.020 1.160 ± 0.010* 1.170 ± 0.007* 1.300 ± 0.030* 1.050 ± 0.009
0.970 ± 0.028 0.984 ± 0.015 0.990 ± 0.012 0.950 ± 0.009 0.930 ± 0.072 1.010 ± 0.139
MCF7 MDA-MB-231 MCF10A
The values were calculated as a relative change in protein level in comparison to control cells (expression equals 1). The mean values ± SEM from three independent experiments run in duplicate are presented. *Significantly different of mean values from the control group (P < 0.05).
its effect on the phase I bioactivating cytochrome P450 is still a matter of debate. The early studies [Mahéo et al., 1997] showed the unchanged expression (mRNA) of CYP1A2 in human HepG2 cells but reduced activity of CYP1A1 in rat primary hepatocytes as the result of D, L-SFN treatment. Contrary, a more recent study revealed the induction of CYP1A1 mRNA in murine Hep 1c1c7 and human hepatocytes HepG2 cells [Anwar-Mohamed and El-Kadi, 2009]. This effect was observed at the doses similar to that applied in our study to breast epithelial cells. Our study may confirm this effect but only on the CYP1A1 transcript level and in to a lesser extent. Moreover, these authors demonstrated that SFN can directly induce the CYP1A1 gene expression in an AhR-dependent manner [Anwar-Mohamed and El-Kadi, 2009]. One reason of the differences in the results of our study and that mentioned in the preceding texts could be, beside the cell type, the time point of CYPs mRNA and protein measurement. We evaluated the effect of SFN after 72 h, which may better reflect the in vivo situation [Moiseeva et al., 2007], while the induction of Cyp1a1 mRNA in murine Hepa1c1c7 was observed up to 24 h with maximal induction at 6 h time point [Anwar-Mohamed and El-Kadi, 2009]. On the other hand, in breast cells, particularly MCF7 cell line, in contrast to hepatocytes, cross talk between AhR and ER has to be considered [MacPherson and Matthews, 2009]. In this regard, it was shown that resveratrol in nanomolar concentrations was able to repress AhR-mediated induction of CYP1A1 in an ERα-dependent manner without reducing the level of AhR complex at the CYP1A1 promoter. In contrast,
10 μM of resveratrol markedly reduced AhR complex on the CYP1A1 promoter and resulted in nearcomplete inhibition of CYP1A1 expression and metabolic activity [Perdew et al., 2010]. Thus, further studies including cross talk between ERα and AhR also using nanomolar SFN concentrations are necessary to explain the mechanism of the inhibition or induction of CYPs involved in estrogen catabolism. The application of the inducers of specific isoform of P450 may be also considered as it was shown that D,L-SFN may inhibit CYP1A1 and CYP1A2 protein level and activity induced by polycyclic aromatic hydrocarbons in MCF7 cell line [Skupinska et al., 2009]. To sum up, our study showed that the naturally occurring L-isomer of SFN may affect the cytochrome P450 involved in estrogen synthesis and catabolism. This effect is partly dependent on breast cell ER status. Although the exact mechanism of this SFN activity requires further studies, our results support a rationale for future clinical evaluation of sulforaphane or the extract of broccoli/broccoli sprouts for breast cancer chemoprevention.
Acknowledgement This work was supported by the Ministry of Science and Higher Education of Poland, grant N405 3390.
Conflict of Interest The authors declare that they have no conflict of interest.
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