Suppression of cell cycle progression by flavonoids: dependence on the aryl hydrocarbon receptor
John J. Reiners, Jr1,
Russell Clift and
Patricia Mathieu
Institute of Chemical Toxicology, Wayne State University, 2727 Second Avenue, Detroit, MI 48201, USA
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Abstract
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Some flavonoids are ligands of the aryl hydrocarbon receptor (AHR) and cause cell cycle arrest. The dependency of the cytostatic effects of five flavonoids (flavone,
-naphthoflavone, apigenin, 3'-methoxy-4'-nitroflavone and 2'-amino-3'-methoxyflavone) on a functional AHR was examined in AHR-containing rat hepatoma 5L cells and an AHR-deficient cell line (BP8) derived from the 5L line. The potent AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was cytostatic to the 5L line due to the induction of a G1 arrest and dramatically elevated steady-state levels of CYP1A1 mRNA. TCDD affected neither the proliferation nor CYP1A1 mRNA contents of BP8 cells. With the exception of apigenin, the flavonoids under study induced G1 arrest in both 5L and BP8 cells when used at concentrations at which they functioned as AHR agonists, but not antagonists. Apigenin-treated 5L and BP8 cultures primarily arrested in G2/M. The AHR-containing murine hepatoma cell line 1c1c7 arrested following exposure to AHR agonist concentrations of flavone and
-naphthoflavone, but not TCDD. Unlike the G1 arrest observed in 5L cultures, the latter two flavonoids caused principally G2/M arrest in 1c1c7 cells. These studies demonstrate that the cytostatic activities of flavonoids do not require the AHR and the site of checkpoint arrest with a specific flavonoid can vary with cell type.
Abbreviations:
-NF,
-naphthoflavone or 7,8-benzoflavone; AHR, aryl hydrocarbon receptor; DMSO, dimethylsulfoxide; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase kinase; PBS, phosphate-buffered saline; PD98059, 2'-amino-3'-methoxyflavone; PD168641, 3'-methoxy-4'-nitroflavone; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin
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Introduction
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The flavonoids are a diverse family of chemicals commonly found in fruits and vegetables. Numerous members of this family have cytostatic (1), apoptotic (2,3), anti-inflammatory (4,5), anti-angiogenic (1) and estrogenic (6) activities. In addition, several flavonoids are potent modulators of both the expression and activities of specific cytochrome P450 genes/proteins (79). Because of these activities, flavonoids have attracted attention as possible chemoprotective or chemotherapeutic agents.
The cytostatic and apoptotic activities of the flavonoids have been attributed to their modulation of several biological processes. Flavonoids are potent inhibitors of processes involved in mitogen signaling or DNA synthesis, including mitogen-activated receptor tyrosine kinase activities (2,10,11), protein kinase C (1012), mitogen-activated protein kinase kinase (MEK) (13) and topoisomerase II (1). A select group of flavonoids also up-regulate the expression of the cdk inhibitor p21WAF1 (14) and/or inhibit cdk2/4 or cdc2 activities (1416). These properties seemingly provide an explanation for the G1/S and/or G2/M arrests seen in some cell types following flavonoid exposure. However, other factors must influence the checkpoint at which cells arrest. For example, apigenin induces exclusively G2/M arrest in human HL60 cells (14) but G1/S and G2/M arrest in human diploid fibroblasts (15).
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor (17,18). Numerous xenobiotics, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are ligands of the AHR. A variety of biological activities have been attributed to TCDD. One such activity is the induction of G1 arrest in some cell types (19,20). Recent studies, involving analyses of cells of the rat hepatoma 5L lineage expressing different amounts of the AHR, demonstrated that TCDD induction of G1 arrest requires a functional AHR (20).
Many flavonoids are AHR ligands (7,8,21). However, the biological activities invoked by their binding to the AHR are concentration dependent. At the lower end of the concentration range at which they bind to the AHR many flavonoids function as AHR antagonists (8,21,22). As antagonists they bind to the receptor and can compete with TCDD for binding, but do not transform the AHR into an active transcription factor. However, at higher concentrations the same flavonoids function as AHR agonists and duplicate many of the activities of TCDD, including transcriptional activation of the CYP1A1 gene (8,21,22). We are unaware of any published comparison of concentrations at which selected flavonoids are cytostatic and function as AHR ligands. However, the concentrations at which numerous flavonoids have been reported to be cytostatic are well within the range defining flavonoidAHR binding (1,7,8,21).
The current study was designed to determine whether the cytostatic effects of flavonoids could be attributed to their functioning as AHR ligands. As a test system we employed rat hepatoma 5L and BP8 cells, the latter being an AHR null variant of 5L cells which does not G1 arrest following exposure to TCDD (20). As an ancillary system we also employed murine hepatoma 1c1c7 cells, a cell line that is well characterized with respect to its AHR content and responsiveness to TCDD and several flavonoids (2224). The flavonoids examined (Figure 1
) were chosen on the basis of existing literature documenting the concentrations at which the agents are cytostatic, function as AHR antagonists/agonists and/or inhibitors of MEK. Collectively, our studies demonstrate that the cytostatic properties of flavonoids are not dependent upon the presence of a functional AHR.
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Materials and methods
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Chemicals
Flavone was purchased from Aldrich Chemical Co. (Milwaukee, WI). Apigenin and
-naphthoflavone (
-NF) were obtained from Sigma Chemical Co. (St Louis, MO). TCDD and 2'-amino-3'-methoxyflavone (PD98059) were purchased from ChemSyn (Lenexa, KS) and New England Biolabs (Beverley, MA), respectively. 3'-Methoxy-4'-nitroflavone (PD168641) was the gift of the Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co. (Ann Arbor, MI).
Cell culture and treatments
The rat hepatoma 5L and BP8 cell lines were gifts of Dr Martin Gottlicher (Research Center, Karlsruhe, Germany). Both lines were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin and grown at 37°C in a humidified atmosphere containing 5% CO2. For cell proliferation studies exponentially growing cultures were trypsinized and plated at low density in 60 mm dishes. Cultures were initially washed and refed 2024 h after plating and then exposed to chemical agents ~24 h later. In a few studies the washing step was skipped and cultures were treated ~24 h after plating. The two protocols yielded similar results when examined in a side by side comparison.
Wild-type Hepa 1c1c7 cells (obtained from Dr J.Whitlock, Stanford University, CA) were cultured at 32°C as described previously (24).
Cultures were treated with varying concentrations of chemicals dissolved in dimethylsulfoxide (DMSO) (absolute volume of solvent <0.1% of medium volume). Details of treatment are provided in the text. Cells were harvested by exposure to solutions of 0.25% trypsin, 0.1 mM EDTA, counted and assessed for ability to exclude trypan blue.
RNA preparation and northern blot analyses
Total cellular RNA was isolated according to the acidic phenol extraction method of Chomeczynski and Sacchi (25). RNA was resolved on 1.2% agaroseformaldehyde gels and transferred to nylon membranes as described previously (13). The probes used for detection of 7S and rat and murine CYP1A1 RNAs and the conditions used for hybridization have been described in detail (24,26).
Cell cycle analyses
Cells were plated in 100 mm culture dishes at densities that ensured that they would still be in exponential growth at the time of harvest. Cultures were treated and harvested as described above. After trypsinization cells were washed once in phosphate-buffered saline (PBS), pelleted, and resuspended in PBS, which was followed by addition of an equal volume of ethanol. The resulting cell suspension was stored for a minimum of 3 days at 4°C before being processed for DNA analyses. For DNA analyses suspensions of ~1x106 cells were pelleted and resuspended in PBS containing glucose, 100 U/ml RNase A and 50 µg/ml propidium iodide. After a 2 h incubation in the dark the cells were analyzed with a Becton Dickinson FACScalibur instrument and the percentages of cells in the G1, S and G2/M stages of the cell cycle were determined with a DNA histogram-fitting program (MODFIT; Verity Software, Topsham, ME). A minimum of 104 events/sample was collected for subsequent analyses.
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Results
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Effects of TCDD and flavonoids on CYP1A1 mRNA steady-state content in 5L and BP8 cells
Exposure of 5L cultures to 1 nM TCDD resulted in a pronounced accumulation of CYP1A1 mRNA (Figure 2
) within 6 h of exposure. Exposure to flavone,
-NF and PD98059 also caused concentration-dependent elevations of steady-state CYP1A1 mRNA contents in 5L cells (Figure 2
). All of these flavonoids markedly increased CYP1A1 mRNA contents when used at concentrations
20 µM, but had no detectable effects at 1 µM. CYP1A1 mRNA contents were also elevated in 5L cultures following exposure to apigenin, but to a lesser extent than that observed with flavone,
-NF and PD98059 (Figure 2
).

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Fig. 2. CYP1A1 mRNA contents of 5L cells treated with flavonoids. Exponentially growing cultures of 5L cells were exposed to DMSO, TCDD (2 nM) or varying concentrations of flavonoids for 6 h prior to being harvested for isolation of RNA and analyses of CYP1A1 and 7S RNAs.
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The flavonoid PD168641 is a high affinity ligand of the AHR which functions exclusively as an AHR antagonist (7,21). Exposure of 5L cultures to concentrations of PD168641 as high as 1 µM had no effect on CYP1A1 mRNA content (Figure 2
). This concentration is at least 50-fold greater than that needed to inhibit TCDD binding to the AHR and activation of CYP1A1 (7,21).
Analyses identical to those presented in Figure 2
were also performed with the BP8 cell line. We were unable to detect either constitutive levels of CYP1A1 mRNA in this cell line or an accumulation of CYP1A1 mRNA following exposure to TCDD or any of the five flavonoids under study by northern blot analysis (J.J.Reiners, unpublished observations).
Cytostatic effects of TCDD and flavonoids in 5L and BP8 cells
Exposure of 5L cultures to concentrations of TCDD
1 nM resulted in a transient suppression of proliferation (Figure 3
). In contrast, 1 or 10 nM TCDD had no effect on BP8 proliferation (Figure 3
). The AHR ligand and antagonist PD168641 had no effect on the proliferation of either 5L or BP8 cultures at even the highest concentration tested (e.g. 1 µM; Figure 3
). A sub-agonist concentration (1 µM) of flavone,
-NF, apigenin or PD98059 did not affect the proliferation of either 5L or BP8 cultures (Figure 3
). In contrast, all four flavonoids were cytostatic to both cell lines when used at concentrations (
20 µM) at which they functioned as AHR agonists (Figure 3
). In general, 5L cells were as sensitive or more sensitive than BP8 cells to the anti-proliferative effects of the flavonoids. Indeed, flavone concentrations
20 µM completely suppressed the proliferation of 5L cells, but were only modestly cytostatic to BP8 cells.

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Fig. 3. Cytostatic effects of flavonoids on 5L and BP8 cells. Exponentially growing cultures of 5L and BP8 cells were exposed to DMSO, varied concentrations of TCDD or flavonoids prior to being harvested for determination of cell number. One set of untreated culture dishes was harvested at the time of treatment to serve as the 0 time control. Data represent means ± SD of determinations made on three to four culture dishes. All analyses were reproduced in a minimum of one additional study. Symbols are defined in the figure.
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In almost all cases examined, the anti-proliferative effects of the various flavonoids did not reflect agent cytotoxicity (J.J.Reiners, unpublished observations). There were only two notable exceptions. Exposure of 5L cultures to 50 µM flavone or 40 µM apigenin led to a time-dependent accumulation of trypan blue permeable cells (~40% after 72 h treatment). In contrast, 50 µM flavone was not cytotoxic to BP8 cells and 40 µM apigenin was only weakly cytotoxic (~15% killing after 72 h treatment).
Cell cycle analyses of flavonoid-treated 5L and BP8 cells
Flow cytometric analyses of cellular DNA content were used to determine if the cytostatic effects of TCDD and the flavonoids reflected an arrest at a specific cell cycle checkpoint. TCDD treatment of 5L cells resulted in a G1 arrest, as indicated by a dramatic increase in the percentage of cells having diploid DNA contents (Figure 4A
), and concomitant reductions in the percentages of S and G2/M phase cells (Figure 4B and C
, respectively). In agreement with the proliferation data presented in Figure 3
, TCDD had no effect on the cell cycle distribution of BP8 cells (Figure 4DF
). Flavone,
-NF and PD98059, like TCDD, also induced a pronounced G1 arrest in 5L cultures when used at AHR agonist concentrations (4050 µM; Figure 4A
). A G1 arrest also occurred in BP8 cultures treated with 50 µM flavone, 50 µM
-NF and 40 µM PD98059 (Figure 4D
). However, in accord with the cell proliferation data reported in Figure 3
, the percentage of BP8 cells in G1 was less than that seen in 5L cultures treated with comparable concentrations of the flavonoids. Apigenin induced a dramatic accumulation of G2/M cells in both 5L and BP8 cultures (Figure 4C and F
).

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Fig. 4. Cell cycle distribution analyses of flavonoid-treated 5L and BP8 cells. Exponentially growing cultures of 5L and BP8 cells were exposed to DMSO, TCDD or 1, 40 or 50 µM concentrations of flavonoids prior to being harvested for per cell DNA analyses by FACs. Data represent analyses of 104 cells. Results similar to those depicted in the figure were obtained in a second independent study. Symbols are defined in the figure.
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Cell cycle analyses were also performed on 5L and BP8 cultures following exposure to a non-AHR agonist concentration (1 µM) of flavonoids. In no instance did 1 µM flavone,
-NF, apigenin or PD98059 cause a perturbation of the cell cycle (1 µM flavone data are presented in Figure 4
; other results are unpublished observations).
Effects of TCDD and flavonoids on CYP1A1 mRNA steady-state content in 1c1c7 cells
The murine hepatoma cell line 1c1c7 expresses relatively high levels of the AHR (27) and is very responsive to TCDD, as indicated by monitoring the transcriptional activation of Cyp1a1 (Figure 5
). CYP1A1 mRNA contents were also elevated in 1c1c7 cultures within 6 h of exposure to either 50 µM flavone or
-NF (Figure 5
). A 1 µM concentration of either flavonoid had no effect on CYP1A1 mRNA steady-state content (Figure 5
).

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Fig. 5. CYP1A1 mRNA contents of 1c1c7 cells treated with flavonoids. Exponentially growing 1c1c7 cultures were exposed to DMSO, TCDD (5 nM) or varying concentrations of flavonoids for 6 h prior to being harvested for isolation of RNA and analyses of CYP1A1 and 7S RNAs.
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Cytostatic effects of TCDD and flavonoids in 1c1c7 hepatoma cells
Exposure of 1c1c7 cultures to a concentration of TCDD capable of transcriptionally activating Cyp1a1 had no effect on cell proliferation (Figure 6A
), viability (Figure 6D
) or cell cycle distribution (Figure 6GI
). In contrast, an AHR agonist concentration (50 µM) of flavone or
-NF totally suppressed the proliferation of 1c1c7 cells (Figure 6B and C
). This suppression occurred in the absence of significant cytotoxicity (Figure 6E and F
). In flavone-treated cultures the cells in G1 initially arrested in G1, whereas cells in S phase progressed into G2/M and subsequently arrested (Figure 6GI
). However, within 72 h of treatment the flavone-induced G1 arrest was lost and the cells progressed into the S and G2/M phases of the cell cycle. In marked contrast to what was observed with flavone, no G1 arrest occurred in 1c1c7 cultures following exposure to
-NF (Figure 6G
). Instead,
-NF-treated cells underwent a sustained G2/M arrest (Figure 6I
).

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Fig. 6. Cytostatic and cytotoxic effects of flavonoids on 1c1c7 cells. Exponentially growing cultures were exposed to DMSO, TCDD (5 nM) or varied concentrations of flavonoids prior to being harvested for determination of cell number (AC), viability (DF) and DNA content (GI). One set of untreated culture dishes was harvested at the time of treatment to serve as the 0 time control. Data represent means ± SD of determinations made on three to four culture dishes. All analyses were reproduced in a minimum of one additional study. Symbols are defined in the figure.
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Discussion
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One goal of the current study was to determine if the cytostatic activities of flavonoids were related to their functioning as AHR ligands. Flavone,
-NF and TCDD were all capable of transforming the AHR of 1c1c7 cells into a functional transcription factor, as indicated by the accumulation of CYP1A1 mRNA. However, only the two flavonoids inhibited 1c1c7 cell proliferation. Since TCDD could transform the AHR into a functional transcription factor but did not duplicate the cytostatic effects of the flavonoids, it seems unlikely that the cytostatic effects of the flavonoids in this cell line reflected consequences of their interaction with the AHR. Similarly, although three of the flavonoids under study were cytostatic to 5L cells when used at concentrations at which they functioned as AHR agonists, and induced a G1 arrest similar to the known AHR agonist TCDD, the same compounds also induced a G1 arrest in AHR null BP8 cells. Furthermore, we recently found that pretreatment of 5L cultures with the AHR antagonist PD168641 suppressed the cytostatic activity of TCDD (J.J.Reiners, unpublished observation). However, a similar pretreatment of 5L cultures with PD168641 did not suppress the cytostatic effects of either apigenin or flavone. This latter study suggests that these two flavonoids suppress 5L proliferation by a process that does not involve the AHR. Collectively, these results demonstrate that the cytostatic activity of flavonoids is not dependent upon their interaction with the AHR.
Apigenin is cytostatic towards many cell types. However, the checkpoint at which cell cycle arrest occurs varies markedly amongst cell types. It induces predominantly a G2/M arrest in human HL60 cells (15), B104 rat neuronal cells (28) and the immortal murine keratinocyte C50 cell line (15), but a mixed G1 and G2/M arrest in human diploid foreskin fibroblasts (14). HL60 cells express low levels of AHR mRNA and are not responsive to TCDD, as assessed by measurement of CYP1A1 mRNA (29). Similarly, the conditions used for the culturing of the C50 line suppress activation of Ah battery genes in primary cultures of murine keratinocytes by TCDD (30). Conversely, it has been reported that established fibroblast cell lines and primary fibroblast cultures constitutively express functional AHR (31,32). Although these studies may be interpreted as suggesting a correlation between AHR content/function and the checkpoint at which apigenin causes cell cycle arrest, our studies do not support such a correlation. Specifically, apigenin-induced a G2/M arrest in both AHR null BP8 cells and the parental AHR-containing 5L cell line. Hence, the checkpoint at which apigenin arrests cells is not AHR-dependent.
PD98059 is a commonly used reagent in the signal transduction field because of the specificity with which it inhibits MEK (33,34). Studies employing PD98059 suggest that a functional MEK/extracellular signal-regulated kinase (ERK) pathway is required for mitogen-induced cell proliferation in several cell types (3538). Reports by several investigators, including ourselves, have demonstrated that the phosphorylated, active forms of ERK1/2 are absent in a variety of cell types treated with concentrations of PD98059
20 µM (13,33,34). Such concentrations suppressed the proliferation of both 5L and BP8 cells in the current study. Insufficient data are available to address the issue of whether flavonoids in general are MEK inhibitors. However, the current investigation, when coupled with our recent report (13), clearly demonstrates that the cytostatic activity of at least one flavonoid is unrelated to its ability to inhibit MEK/ERKs. Specifically, the concentration (20 µM) at which flavone was very cytostatic to 5L cultures is minimally 10-fold less than the ID50 required for MEK inhibition (13).
The cytostatic activity of TCDD has been documented in several cell types besides 5L cells. TCDD suppresses the proliferation of estrogen receptor-negative MDA-MD-486 human breast cancer cells (39) and estrogen receptor-positive MCF-7 human breast cancer cells (19,40). In the former case suppression is mediated by the production of a cytokine (i.e. transforming growth factor
), which inhibits proliferation in an autocrine fashion (39). In the latter case, the cytostatic effects of TCDD reflect its abilities to function as an anti-estrogen and down-regulate a battery of proliferative processes normally activated by 17ß-estradiol (19). TCDD has also been reported to inhibit the proliferation of T47D human breast cancer cells (41) and sparsely seeded primary rat hepatocytes in response to epidermal growth factor (42). In this latter example, parallel studies performed with hepatocytes having a low affinity AHR implicated the AHR as being the mediator of the effects of TCDD. In contrast to these reports, our studies with 1c1c7 cells clearly demonstrate that TCDD is not necessarily cytostatic to cells having a functional AHR. We have also observed that concentrations of TCDD capable of transcriptionally activating several members of the Ah battery in the human breast epithelial cell line MCF10A do not affect the proliferation of such cells (43; unpublished observation). Hence, TCDD-dependent transformation of the AHR does not ensure the induction of a cytostatic process. Other factors, in concert with the AHR, must contribute to the susceptibility of a cell to the cytostatic effects of TCDD.
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Acknowledgments
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This work was supported by National Institutes of Health grant CA34469 and utilized the services of the Cell Culture and the Imaging and Cytometry Facility Cores, which are supported by National Institutes of Environmental Health Sciences grant P30ES06639.
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Notes
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1 To whom correspondence should be addressedEmail: john.reiners.jr{at}wayne.edu 
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Received November 19, 1998;
revised March 12, 1999;
accepted April 12, 1999.