Tangeretin inhibits the proliferation of human breast cancer cells via CYP1A1/CYP1B1 enzyme induction and CYP1A1/CYP1B1–mediated metabolism to the product 4′ hydroxy tangeretin

Article abstract of DOI:10.1016/j.tiv.2018.04.001

Tangeretin is a polymethoxylated flavone with multifaceted anticancer activity. In the present study, the metabolism of tangeretin was evaluated in the CYP1 expressing human breast cancer cell lines MCF7 and MDA–MB–468 and in the normal breast cell line M

Full text of DOI:10.1016/j.tiv.2018.04.001

Accepted Manuscript
Tangeretin inhibits the proliferation of human breast cancer cells
via CYP1A1/CYP1B1 enzyme induction and CYP1A1/
CYP1B1–mediated metabolism to the product 4′ hydroxy
tangeretin
Somchaiya Surichan, Randolph R. Arroo, Aristidis M. Tsatsakis,
Vasilis P. Androutsopoulos
PII:
S0887-2333(18)30125-5
doi:10.1016/j.tiv.2018.04.001
TIV 4263
DOI:
Reference:
To appear in:
Toxicology in Vitro
Received date:
Revised date:
Accepted date:
20 December 2017
5 March 2018
1 April 2018
Please cite this article as: Somchaiya Surichan, Randolph R. Arroo, Aristidis M. Tsatsakis,
Vasilis P. Androutsopoulos , Tangeretin inhibits the proliferation of human breast cancer
cells via CYP1A1/CYP1B1 enzyme induction and CYP1A1/CYP1B1–mediated
metabolism to the product 4′ hydroxy tangeretin. The address for the corresponding
author was captured as affiliation for all authors. Please check if appropriate. Tiv(2018),
doi:10.1016/j.tiv.2018.04.001
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TANGERETIN INHIBITS THE PROLIFERATION OF
HUMAN BREAST CANCER CELLS VIA CYP1A1/CYP1B1
ENZYME INDUCTION AND CYP1A1/CYP1B1–MEDIATED
METABOLISM TO THE PRODUCT 4ʹ HYDROXY
TANGERETIN
1
1
2
Somchaiya Surichan , Randolph R. Arroo , Aristidis M. Tsatsakis , Vasilis P.
2
Androutsopoulos *
1
De Montfort University, Leicester School of Pharmacy, The Gateway, Leicester, LE1
9
BH, UK
2
Laboratory of Toxicology, University of Crete, Medical School, Voutes, Heraklion,
7
1409, Crete, Greece
*
Correspondence: Dr Vasilis P. Androutsopoulos, Laboratory of Toxicology,
University of Crete, Voutes, Heraklion, 71409, Crete, Greece
Telephone: 00 30 2810 394633
E-mail: androuts@uoc.gr

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Abstract
Tangeretin is a polymethoxylated flavone with multifaceted anticancer activity. In the
present study, the metabolism of tangeretin was evaluated in the CYP1 expressing
human breast cancer cell lines MCF7 and MDA–MB–468 and in the normal breast
cell line MCF10A. Tangeretin was converted to 4ʹ OH tangeretin by recombinant
CYP1 enzymes and by CYP1 enzymes expressed in MCF7 and MDA–MB–468 cells.
This metabolite was absent in MCF10A cells that did not express CYP1 enzymes.
Tangeretin exhibited submicromolar IC50 (0.25±0.15 μM) in MDA–MB–468 cells,
whereas it was less active in MCF7 cells (39.3±1.5 μM) and completely inactive in
MCF10A cells (>100 μM). In MDA–MB–468 cells that were coincubated with the
CYP1 inhibitor acacetin, an approximately 70–fold increase was noted in the IC50
(
18±1.6 μM) of tangeretin. In the presence of the CYP1 inhibitor acacetin, the
conversion of tangeretin to 4ʹ OH tangeretin was significantly reduced in MDA–MB–
68 cells (2.55±0.19 μM vs. 6.33±0.12 μM). The mechanism of antiproliferative
4
action involved cell cycle arrest at the G1 phase for MCF7 and MDA–MB–468 cells.
Tangeretin was further shown to induce CYP1 enzyme activity and
CYP1A1/CYP1B1 protein expression in MCF7 and MDA–MB–468 cells. These
results suggest that tangeretin inhibits the proliferation of breast cancer cells via
CYP1A1/CYP1B1–mediated metabolism to the product 4ʹ hydroxy tangeretin.
Keywords
Flavonoids, tangeretin, cytochrome P450 CYP1 enzymes, antiproliferation, breast
cancer

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Abbreviations
CYP1, cytochrome P450 CYP1B1, CYP1A1 and CYP1A2; HPLC, high pressure
liquid chromatography; IC50, 50% inhibitory concentration; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;

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1
. Introduction
Flavonoids are polyphenolic molecules with multiple modes of biological action.
Their cancer preventative and/or anticancer activity has been attributed to various
different mechanisms including the induction of apoptosis, the modulation of cell
signaling, the inhibition of the cell cycle and notably the process of mitosis, the
induction and/or modulation of autophagy, the inhibition of invasion and metastasis
and the interaction with Reactive Oxygen Species (ROS) and ROS signaling (Johnson
and de Mejia, 2013; Qi et al., 2015; Hsieh et al., 2016; Cheng et al., 2015; Salmela et
al., 2012; Sosa et al., 2013; Kim et al., 2015; Jo et al., 2015; Li et al., 2013;
Doleckova et al., 2012). In addition to the aforementioned biological activities, the
interactions of natural dietary flavonoids with xenobiotic phase I metabolizing
enzymes, notably the cytochrome P450 CYP1 enzymes, have been investigated by
recent studies (Androutsopoulos et al., 2008; Androutsopoulos et al., 2009a;
Androutsopoulos et al., 2009b; Androutsopoulos et al., 2009c; Androutsopoulos et al.,
2
009d; Androutsopoulos et al., 2010; Androutsopoulos and Spandidos 2013;
Androutsopoulos and Tsatsakis 2014). CYP1 enzymes participate in the metabolic
activation of pro–carcinogens to their ultimate carcinogenic derivatives, whereas
recently an anti–inflammatory role of these enzymes was demonstrated in
Cyp1a1/1a2/1b1 (⁻/⁻) C57BL/6J triple knockout mice compared with C57BL/6J wild-
type mice, via the regulation of the metabolism of arachidonic acid lipid mediators of
inflammation (Nebert and Dalton 2006; Divanovic et al., 2013). With regard to
dietary flavonoids, a cancer therapeutic effect of these compounds has been
demonstrated in breast and liver cancer cells via their intracellular metabolism by
CYP1 enzymes (Androutsopoulos et al., 2008; Androutsopoulos et al., 2009a;
Androutsopoulos et al., 2009b; Androutsopoulos et al., 2009d; Androutsopoulos and

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Spandidos, 2013). These compounds are oxidized to their corresponding hydroxylated
derivatives via aromatic hydroxylation and/or demethylation reactions occurring at
the B and/or A rings of the polyphenolic moiety (Androutsopoulos et al., 2008;
Androutsopoulos et al., 2009a; Androutsopoulos et al., 2009b; Androutsopoulos et al.,
2
009d; Androutsopoulos and Spandidos 2013; Androutsopoulos and Tsatsakis 2014).
The resulting metabolites inhibit cancer cell growth at equivalent and/or higher levels
compared with the corresponding parent compounds (Androutsopoulos et al., 2008;
Androutsopoulos et al., 2009a; Androutsopoulos et al., 2009b; Androutsopoulos et al.,
2
009d). It is important to note that synergistic effects resulting from the combined
action of the parent flavonoid compound and the CYP1–metabolite have been
documented, thus accounting for the overall antiproliferative activity
(Androutsopoulos and Spandidos 2013).
Tangeretin is a fully methoxylated flavone that is present in the peel of citrus fruits
and has demonstrated anticancer activity via distinct mechanisms of action.
Tangeretin causes G2/M arrest and induces apoptosis in glioma cells by modulating
Phosphatase and tensin homolog (PTEN) protein, and the cell cycle regulating genes,
namely D1 and cdc2, whereas in human meningioma cells this compound induces
apoptosis by enhanced phosphorylation of glycogen synthase 3 β (GSK3β) and
inhibition of the Wnt5/β–catenin pathway (Ma et al., 2016; Das et al., 2015).
Tangeretin was further shown to inhibit breast cancer formation in a rat model of
7
,12-dimethyl benz[a]anthracene (DMBA)–induced mammary carcinogenesis via
p53/p21 upregulation and inhibition of metastasis by downregulation of MMP2,
MMP9 and VEGF expression (Arivazhagan and Sorimuthu Pillai 2014; Lakshmi and
Subramanian 2014). Despite these promising studies, the metabolism of tangeretin by
CYP1 enzymes remains poorly defined. With the exception of a previous study by

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Walle and Walle that examined tangeretin metabolism by recombinant CYP enzymes
and the hepatic liver S9 fraction, there is no evidence in the literature regarding the
association of CYP1 enzymes and the cancer therapeutic effect of tangeretin (Walle
and Walle, 2007). In the present study, the metabolism of tangeretin was investigated
in the CYP1 expressing breast cancer cell lines MCF7 and MDA–MB–468 and the
normal breast cell line MCF10A. The data suggest that tangeretin is metabolized to
one major conversion product by a demethylation reaction at the B ring that in turn
inhibits cancer cell growth.
2
2
3
. Materials and Methods
.1 Chemicals and antibodies
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium
iodide (PI), 7-ethoxyresorufin, resorufin, α-napthoflavone, acacetin, tissue culture
reagents and media, Western blotting lysis buffer and DTT were purchased from
Sigma Aldrich (St Louis, MO, USA). Tangeretin and 4ʹ OH tangeretin were
purchased from Apin chemicals (Abingdon, UK). Western blotting reagents were
from Bio-Rad (Berkeley, CA, USA). The polyclonal antibody for CYP1A1 was from
Daiichi Pure Chemicals (Gentest corporation, MA, USA), whereas the monoclonal
antibodies for CYP1B1 from Auvation Limited (Glasgow, Scotland, UK), and for β-
actin from Cell signaling (Leiden, Netherlands). Secondary antibodies for western
blotting were from Santa Cruz Biotechnology (Dallas, TX, USA).
2
.2 Cell culture
MCF7 and MDA–MB–468 cells were maintained in RPMI with glutamine (2 mM)
containing 10% heat-inactivated FBS and penicillin/streptomycin. MDA–MB–468

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cells were grown in RPMI without phenol red, whereas MCF10A cells were grown in
DMEM:F12 with insulin (10 μg/ml), hydrocortisone (500 ng/ml), EGF (20 ng/ml),
1
0% FBS and 2 mM glutamine. The cells were grown in a humidified incubator at 37

C in 5% CO /95% air and passaged every 3 to 4 days using trypsin EDTA (0.25%
2
v/v).
2
.3 Tangeretin metabolism
Recombinant CYP1 enzymes and control microsomes were incubated with 10 μM of
tangeretin at 37 °C in the presence of NADPH (0.5 mM), MgCl (0.5 mM) and
2
phosphate buffer (20 mM). The recombinant enzymes and the control microsomes
were purchased from BD Biosciences (CYP1A1+reductase supersomes catalogue
number 456211, CYP1B1+reductase supersomes catalogue number 456220,
CYP1A2+reductase supersomes catalogue number 456203 and insect cell control
microsomes catalogue number 456200). Time points were obtained at 0, 5, 10, 15, 20
and 25 min intervals. The reactions were terminated by addition of equal volumes of
methanol and acetic acid at a 100:1 ratio. The samples were centrifuged at 3,500 g for
2
0 min at 4C and the supernatants were analyzed by reversed phase HPLC.
2
.4 HPLC analysis
The methodology has been described in detail in previous publications
Androutsopoulos et al., 2009a, Androutsopoulos et al., 2008). A Luna C18 4.6 x 150
(
mm 5 µ column was used and the mobile phase consisted of solvents A and B.
Solvent A comprised 1% acetonitrile and 0.5% acetic acid in H O and solvent B 4%
2
acetonitrile and 0.5% acetic acid in CH OH. The following gradient was used: 60%
3
solvent A and 40% solvent B at time=0 min and 10% solvent A and 90% solvent B at
time=10 min. The final conditions were maintained for 1 min and the composition of

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the solvents was adjusted to the initial conditions with 8 min remaining for column
equilibration after each run. The detection of tangeretin concentration was monitored
using a Waters Series 200 UV detector (Waters, Hertfordshire, UK) at 327 nm. The
concentration of tangeretin was estimated by a calibration curve covering the range of
0
.05–10 M. The assay was carried out at 37C and the flow rate was 1 ml/min. The
average recovery for tangeretin was 93% and the retention time using the
aforementioned parameters was 17.9 min. The limit of detection of both compounds
was estimated at 0.05 μM and the accuracy and precisions were in the range of 95-
9
9% and 1.7-5% for tangeretin and 93-98% and 1.5-4.9% for TP1, respectively.
2
.5 LC-MS analysis
Mass spectrometry analysis of tangeretin was conducted on an Agilent 1100 Series
LC/MSD Trap XCT systems with a photodiode array detector. The separation of
tangeretin was conducted using a Phenomenex Luna 5μ C18 (250×4.6mm) column
and the following solvents: (A) water, (B) methanol and (C) acetonitrile. The initial
conditions were as follows: 59% solvent A, 40% solvent B and 1% solvent C. The
analytical column temperature was 40˚C and the flow rate was 0.8 ml/min. A total of
5
0 μL of sample solution was analyzed. A linear gradient was initiated immediately
on the start of the analysis with solvent B rising to 90% and solvent C rising to 4%
over 15 min. These solvent compositions were held for 2 min prior to the initial
condition reset (using a flow rate of 0.8 ml/min). A re-equilibration time of 8 min was
allowed between each sample analysis. The photodiode array detector was set at 330
nm. The LC flow was controlled from the photodiode array detector into the mass
spectrometer without stream splitting. The MS determination was acquired in
electrospray ionisation (ESI) positive mode. The molecular ions ([M+H]+) were

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detected in the range of 200–500 using an auto–MS mode. Nitrogen was used as the
nebulizing gas at 60 psi and as drying gas with the flow rate of 10 L/min at 350˚C.
The MS data were analyzed using LC/MSD Trap software 5.3. The Retention time
(Rt) of tangeretin under these conditions was 17.9 min.
2
.6 MTT assay
4
MCF7, MDA–MB–468 and MCF10A cells (1 x 10 cells/ml) were seeded in 96-well
plates and the antiproliferative effect of tangeretin was examined as described
previously (Androutsopoulos et al., 2008). The inhibition experiments were conducted
in the presence of 0.5 μM of the CYP1A1 inhibitor acacetin. Similar results were
noted using higher concentrations of 1 μM of acacetin as well as for the CYP1A1
inhibitor α-napthoflavone.
2
.7 FACS analysis
MDA–MB–468, MCF7 and MCF10A were grown in T25 flasks at approximately
0% confluence. The cells were treated with tangeretin at concentrations of 10 μM in
3
the presence and/or absence of CYP1 inhibitors (0.5–1 μM) for 24 and 48 h. The cells
were subsequently washed with PBS, removed by trypsin/EDTA and centrifuged at
3
,500 rpm for 5 min. The cells were fixed in 70% ethanol for at least 24 h at –20°C.
The ethanol was removed and PBS containing PI (50 μg/ml) and RNAse (100 μg/ml)
was added to the cells that were incubated further for 30 min at 37°C. Flow cytometry
was conducted using a Beckman Coulter flow cytometer at PMT4 and at least 10,000
events were acquired for analysis. The Multicycle analysis 2.0 Software was used for
the determination of the cell cycle corresponding to the samples.
2
.8 Western blotting

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MCF7, MDA–MB–468 and MCF10A cells were cultured in T25 flasks at a density of
5
5
×10 cells/ml. The cells were washed once with PBS and lysed with 100 μl of lysis
buffer that contained protease inhibitor cocktail and DL-dithiothreitol (DTT, 1 mM).
The cells were subsequently sonicated on ice for 5 min and centrifuged at 13,000 rpm
at 4°C for 15 min. The protein concentration required for the experiment was adjusted
to 0.7 mg/ml for each sample, and the protein extract was mixed with sample buffer
that contained 5% mercaptoethanol at a 1:1 ratio. The samples were loaded on an
acrylamide gel containing 10% acrylamide for the resolving gel and 5% acrylamide
for the stacking gel and electrophoresis was carried out for 1 h at 120 V. Following
electrophoresis, the proteins were transferred by wet blotting to a PVDF membrane.
The membrane was incubated in 10% milk/0.05% TBST at room temperature for 1 h
by continuous shaking in order to block the non specific binding sites. The primary
antibodies against CYP1A1, CYP1B1 and β-actin were added to the membrane at
1
:800, 1:500 and 1: 3,000 dilutions, respectively at 4 °C overnight. The membrane
was subsequently washed three times with 0.05% TBST and incubated with the
secondary antibody against HRP (1:2,000) diluted in 1% milk/0.05% TBST at room
temperature for 1.5 h. The membrane was finally exposed to ECL reagents, and the
expression profile of the proteins was developed on film.
2
.9 EROD assay
MCF7 and/or MDA–MB–468 cells (2×10 cells/ml) were plated in 96–well plates. The
cells were incubated at 37˚C, 5% CO for 24 h and subsequently tangeretin was added at a
5
2
concentration range of 3, 10 and 30 μM, respectively. The induction of EROD activity
was assayed at the 24 h time point. Following the end of the treatment period, the medium
was aspirated and the cells were washed with PBS thrice. A total of 200 μl of medium
containing 5 μM 7–ethoxyresorufin and 1.5mM salicylamide were added to each well,

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and the plates were further incubated for 1 h. The reactions were terminated by addition
of 150 μl of ice–cold methanol to the wells, followed by centrifugation at 3,500 rpm at
4˚C for 15 min. Subsequently, 200 μl were transferred to a clear bottom black 96–well
plate and the amount of resorufin produced was measured using a fluorescence plate
reader with an excitation and emission wavelength of 530 nm and 590 nm, respectively.
The data were analyzed, following subtraction of the blank fluorescence that contained
resorufin alone from each sample reading. The concentration of resorufin was measured
using a calibration curve. The units of enzyme activity were expressed as concentration of
resorufin formed per time, per amount of cells.
Although 7-ethoxyresorufin O-deethylase is a marker of cytochrome P450 CYP1A1
activity (Burke and Mayer, 1974), the same substrate has affinity for CYP1A2 and
CYP1B1 enzymes, yet to lesser extents. The concentrations of 3, 10 and 30 μM of
tangeretin were selected based on previous studies conducted on flavonoids published
by our group and others. The early studies of Ciolino have used a concentration range
of 1-5 μM (Ciolino et al., 1998, Ciolino et al., 1999). Subsequent studies from our
group have used the same or higher concentrations with those used by the study of
Ciolino et al. for the structurally similar flavonoids cirsiliol, eupatorin and eupatorin-
5
-methyl ether (Androutsopoulos et al., 2009c, Androutsopoulos and Tsatsakis 2014).
2
.10 Statistical analysis
The data were presented as the mean of at least three independent measurements and
were analyzed by the paired t–test, the unpaired t–test and the One-Way analysis of
variance using GraphPadPrism. The error bars represent mean ± STDEV for at least
n=3 independent experiments.
3
. Results

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3
.1 Recombinant CYP1 enzymes metabolize tangeretin
The metabolism of tangeretin by recombinant CYP1 enzymes was examined by LC–
MS analysis. A main metabolite was formed, which was assigned as TP1. The mass
spectrum of TP1 indicated the presence of an ion at 359.1, whereas the ion that
corresponded to the parent compound tangeretin was 373.1 (Fig. 1B). The difference
in the molecular weights of tangeretin and TP1 (373–359=14) was determined by
their molecular ions, and suggested that a de–methoxylation occurred in the initial
structure of tangeretin (Fig. 1A). The comparison with an authentic standard of 4ʹ OH
tangeretin revealed that this compound had the same retention time as TP1, thus
confirming the identity of this metabolite as 4ʹ OH tangeretin (Fig. 1A).
3
.2 CYP1 enzymes metabolize tangeretin in MCF7 and MDA–MB–468 human breast
cancer cells
The human breast cancer cell lines MCF7 and MDA–MB–468 have been employed in
previous studies for the investigation of the CYP1–mediated metabolism and
bioactivation of dietary flavonoids (Androutsopoulos et al., 2008). In the present
study, the metabolism of tangeretin was examined in the aforementioned cell lines. A
main metabolite that matched the retention time of TP1 was identified following
incubation of tangeretin with MDA–MB–468 and/or MCF7 cells for 24 h (Fig. 2A).
This metabolite was absent in MCF10A cell extracts (Fig. 2A,B). The reduction of the
parent compound was higher in MDA–MB–468 compared with MCF7 cells following
2
4 h of incubation (Fig. 2A, B). The metabolism of tangeretin to TP1 was enhanced in
MCF7 cells that were pretreated with the potent CYP1 inducer Tetrachloro-dibenzo-
p-dioxin (TCDD, 10 nM) for 24 h, whereas the metabolism of this compound in
MDA–MB–468 cells was evident from 1 h of incubation and gradually increased at

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the 3, 6 and 9 h time points, respectively (Fig. 2C,D). LC–MS was further used in
order to add insight in the identification of the primary metabolite of tangeretin. The
LC chromatogram of the assay matched that noted from the HPLC experiments with
regard to the retention time of the compounds, whereas the MS analysis revealed that
TP1 exhibited a molecular mass of 359 (Fig. 3A,B). TP1 was identified as 4ʹ OH
tangeretin by comparison with an authentic standard. Thus, the main metabolite of
tangeretin in the CYP1 expressing cell lines MDA–MB–468 and MCF7 was 4ʹ OH
tangeretin. It is important to note that the analysis of the intracellular metabolism
revealed that approximately 35% of the metabolite and parent compound were present
in the intracellular compartment and the remaining fraction was present in the
extracellular part. A similar finding has been published previously for the flavonoids
diosmetin and luteolin in MCF7 cells (Androutsopoulos et al., 2009a).
3
.3 Tangeretin is bioactivated in the CYP1–expressing cell lines MDA–MB–468 and
MCF7
Previous studies have demonstrated that MDA–MB–468 cell constitutively express
CYP1 enzymes compared with MCF10A cells that express little or no CYP1 protein,
whereas MCF7 cells express minimal amounts of CYP1 enzyme levels that are
increased following pretreatment with a CYP1 inducer (Androutsopoulos et al., 2008;
Androutsopoulos et al., 2009a). The present study utilized the aforementioned cell
lines to investigate the antiproliferative activity of tangeretin. This compound was
considerably active in MDA–MB–468 cells and indicated a submicromolar IC50
(0.25±0.15 μM), whereas it demonstrated minimal antiproliferative activity in
MCF10A cells (>100 μM) (Fig. 4A). In MCF7 cells the IC50 of tangeretin (39.3±1.5
μM, Fig. 4B) was considerably higher compared with MDA–MB–468 cells, whereas

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pretreatment of the cells with TCDD (10 nM) for 24 h decreased the overall IC50
(9.7± 0.5 μM, Fig. 4B). The results were significantly different between the two
different types of treatments (MCF7 +– TCDD)(Fig. 4B).
The cytotoxicty of tangeretin in MDA–MB–468 cells was investigated in the presence
of the CYP1 inhibitors acacetin and α–napthoflavone (Fig. 4C). The IC50 of
tangeretin was considerably increased from 0.25±0.15 μM to 18±1.6 μM μM in the
presence of the CYP1 inhibitor acacetin (Fig. 4C). Similar results were noted for the
CYP1 inhibitor α–napthoflavone. The cytotoxicity of the metabolite TP1 was
evaluated in the 3 breast cell lines and it was found that this compound was more
active in MDA–MB–468 compared with MCF7 and MCF10A cells (Fig. 4D). The
IC50 values of TP1 for the three cell lines were as follows: MCF7 (18.4±0.7 μM),
MDA-MB-468 (2.6±0.3 μM) and MCF10A (68.6±2.7 μM) (Fig. 4D).
In addition, metabolic studies revealed that in the presence of the CYP1 inhibitors the
conversion of 4ʹ OH tangeretin from tangeretin was substantially reduced both in
MDA–MB–468 and MCF7 cells (Fig. 4E, F). Specifically, an approximately 3–fold
decrease (2.55±0.19 μM vs. 6.33±0.12 μM) was noted for the conversion of TP1 by
tangeretin, in the presence of the CYP1 inhibitors compared with the incubation of the
parent compound with MDA–MB–468 cells alone (Fig. 4F).
The antiproliferative activity of tangeretin in the 2 human breast cancer cell lines and
the normal breast cell line was further investigated by flow cytometry analysis. The
cells
were
treated
with
10
μM
of
tangeretin.
This concentration (10 μM) has been used in our previous studies conducted on
structurally similar flavonoids, such as diosmetin and eupatorin (Androutsopoulos et
al., 2008, Androutsopoulos et al., 2009b, Androutsopoulos and Spandidos, 2013).
This is usually an approximation based on the evidence that a compound with

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submicromolar IC50 (e.g. 0.5 μM) at the 96 h exposure period will require a
substantially higher concentration for a shorter time of exposure with the cells, such
as 24 h. In addition, 10 μM is the standard concentration used in the metabolic
studies. The concentration of 0.5 μM for acacetin has also been previously employed
in the study of eupatorin (Androutsopoulos et al., 2008).
Treatment of MDA–MB–468 cells with tangeretin (10 μM) for 24 and 48 h resulted in
a significant increase of the population of cells in the G1 phase (Fig. 5A–D, K,
P<0.05). This increase was accompanied by a concomitant significant decrease in the
percentage of cells in the S phase in the tangeretin–treated (Fig. 5A–D, K, P<0.05)
samples. The effect of tangeretin in MDA–MB–468 cells was partially reversible as in
the presence of the CYP1 inhibitor acacetin, a significant decrease in the population
of the cells at G1 phase was noted compared with the single treatment of tangeretin
(Fig. 5A,B,D,K, P<0.05).
MCF7 cells appeared less sensitive to tangeretin treatment and although significant
differences were noted between the control and the treated samples, these were
somewhat lower in magnitude compared to those noted in MDA–MB–468 cells (Fig.
5
E–G, L). In contrast to the breast cancer cell lines, MCF10A cells were unaffected
by tangeretin treatment (10 μM) at either 24 and/or 48 h (Fig. 5H–J, M).
.4 Tangeretin induces CYP1 enzyme expression in MCF7 and MDA–MB–468 cells
3
The initial experiments in MCF7 cells revealed metabolism of tangeretin in the
absence of TCDD pretreatment to the conversion product 4ʹ OH tangeretin (Fig. 2A).
In addition, the previous study on nobiletin by Surichan and colleagues demonstrated
that this structurally similar polymethoxyflavone was capable of inducing its own
metabolism in MCF7 cells via induction of CYP1 enzyme expression (Surichan et al.,
2
012). Based on this evidence, the induction of CYP1 enzyme expression by

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tangeretin was investigated in MCF7 and MDA–MB–468 cells following treatment
with this compound for 24 h. CYP1 enzyme activity was significantly increased in
MCF7 and MDA–MB–468 cells following treatment with 3, 10 and 30 μM of
tangeretin (Fig. 6A,B). In MCF7 cells, CYP1 enzyme activity indicated a substantial
increase at 3 μM that peaked at 10 and 30 μM of compound treatment compared with
control cells (Fig. 6B). In contrast to MCF7, MDA–MB–468 cells exhibited a basal
expression of CYP1 enzyme activity that was increased considerably following
treatment with 3, 10 and 30 μM of tangeretin (Fig. 6A). This increase was similar
among all treatments, as opposed to MCF7 cells where a dose response was noted for
the concentration range 0-10 μM (Fig. 6B). The maximum activity in MDA–MB–468
cells was nearly half of that noted for 10 nM of TCDD alone (152±3.7 vs. 225±12.1
6
pmol/min/10 cells, Fig. 6A). A similar trend was noted for MCF7 cells, with the
exception that the induction caused by TCDD was considerably higher compared with
6
that caused by 30 μM of tangeretin treatment (53±1.4 vs. 21±1.1 pmol/min/10 cells,
Fig. 6B). At the highest concentration of 30 μM of tangeretin, an approximately 3–
fold and 20–fold increase in CYP1 enzyme activity was observed compared with
control cells, for MDA–MB–468 and MCF7 cells, respectively (152±3.7 vs. 55±5.4
6
6
pmol/min/10 cells, 21±1.1 vs. 0.9±0.05 vs. pmol/min/10 cells Fig. 6A,B).
The induction of CYP1 enzyme expression by tangeretin was further monitored by
western immunoblotting. Tangeretin induced CYP1A1 protein in a dose dependent
manner in MDA–MB–468 and MCF7 cells (3–30 μM) (Fig. 6C,D). The induction of
CYP1A1 protein at 3 μM of tangeretin treatment was lesser in MCF7 compared with
MDA–MB–468 cells, whereas the induction of CYP1A1 in MDA–MB–468 cells was
approximately half of that noted for TCDD, as opposed to MCF7 cells (Fig. 6C,D).
With regard to MCF7 cells, the induction of CYP1A1 by tangeretin was lower than

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half of that noted for TCDD (Fig. 6D). In contrast to CYP1A1, tangeretin induced
CYP1B1 protein expression at minimal levels in MDA–MB–468 cells compared with
DMSO control samples (Fig. 6C).
4
. Discussion
Cytochrome P450s are enzymes that can activate a wide diversity of xenobiotics
Jiang et al., 2017; Vogel et al., 2017; Russo et al., 2017). In the present study, the
(
interactions of cytochrome P450 CYP1 enzymes with tangeretin were investigated.
CYP1 enzymes metabolized tangeretin to the conversion product TP1 that was
identified as 4ʹOH tangeretin. In addition, tangeretin was metabolized to 4ʹOH
tangeretin in the human breast cancer cell lines MDA–MB–468 and MCF7, whereas
this conversion was absent in the normal breast cell line MCF10A. 4ʹOH tangeretin
exhibited higher antiproliferative activity compared with tangeretin that resulted in the
bioactivation of tangeretin in the CYP1 expressing breast cancer cell lines, as
demonstrated by metabolic, cell cycle and cytotoxicity studies in the presence of the
CYP1 inhibitors acacetin and α-napthoflavone. Furthermore, tangeretin was capable
of inducing CYP1 enzyme activity and CYP1A1/CYP1B1 enzyme expression in
MDA–MB–468 and MCF7 cells. The data suggest that CYP1 enzymes can activate
tangeretin to the more potent antiproliferative agent 4ʹOH tangeretin in human breast
cancer cells.
The metabolism of tangeretin by CYP1 enzymes was investigated concisely in an
early study conducted by Walle and Walle in 2007 (Walle and Walle, 2007). The
authors examined the metabolism of a range of methoxylated flavones, including
mono–, di–, tri– and fully methoxylated compounds, by human liver microsomes and
recombinant CYP3A4, CYP2C9, CYP2D6 and CYP1A1/CYP1A2 enzymes (Walle

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and Walle, 2007). Oxidative demethylation was demonstrated as the rate–limiting
metabolic reaction for fully methylated flavones, which is in agreement with the
present study and our earlier report that investigated the metabolism of the fully
methylated flavone nobiletin by CYP1 enzymes (Walle and Walle 2007; Surichan et
al., 2012). However, Walle and Walle did not examine the metabolism of tangeretin
by CYP1B1 and did not identify the corresponding conversion products as opposed to
the present study. In an earlier study conducted by Breinholt and colleagues, the
identity of the CYP1A2, CYP3A4, CYP2D6 and CYP2C9–catalyzed metabolites of
tangeretin was verified by LC/MS (Breinholt et al., 2003). The major metabolic routes
involved one demethylation step at the 4ʹ position of the B ring and 2 demethylation
steps at the 5 and 6 position of the A ring that yielded the corresponding metabolites
4
ʹ hydroxy tangeretin and 5,6 dihydroxy tangeretin, respectively (Breinholt et al.,
2
003). In addition, the demethylation at the 4ʹ position of the B ring has been
previously reported as the main CYP1–mediated metabolic reaction for the
structurally similar flavones nobiletin and eupatorin (Surichan et al., 2012;
Androutsopoulos et al., 2008). Although the present study demonstrated that the
major conversion product of CYP1A1 and CYP1B1 metabolism of tangeretin was 4ʹ
hydroxy tangeretin, the presence of additional metabolites was not evident notably in
the human breast cancer cell lines MCF7 and MDA–MB–468. The possible
explanations for this outcome include the reduced levels of CYP1A1/CYP1B1
enzyme activity compared with the recombinant CYP1 enzymes.
The metabolism of tangeretin to 4ʹ hydroxy tangeretin enhanced the antiproliferative
activity of the parent compound, as determined by cell viability and cell metabolism
studies. Furthermore, it is important to note that the mechanism of action involved G1
arrest of MDA–MB–468 cells that was reversed in the presence of the CYP1

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inhibitors acacetin and α-napthoflavone. The data are in agreement with previous
reports that demonstrated cytostatic effects of tangeretin in colon cancer, breast cancer
and yeast cells (Pan et al., 2002; Morley et al., 2007; Chong et al., 2013; Arivazhagan
and Sorimuthu Pillai 2014). Induction of G1 arrest in combination with upregulation
of p53/p21 and downregulation of p–Rb and cyclin D1 were caused by tangeretin
treatment in rat breast cancer cells and human colon cancer cells, respectively
(Arivazhagan and Sorimuthu Pillai 2014; Pan et al., 2002). Moreover, induction of
apoptosis, inhibition of VEGF activity and downregulation of MMP–2 and MMP–9
are additional modes of action that have been reported for tangeretin (He et al., 2015;
Charoensinphon et al., 2013; Arivazhagan and Sorimuthu Pillai 2014). The present
study provides additional evidence regarding the antiproliferative activity of the
CYP1–metabolite 4ʹ hydroxy tangeretin. This novel mechanism of action that
includes CYP1–mediated bioactivation of tangeretin to 4ʹ hydroxy tangeretin may
apply for other types of cancer cells, such as colon cancer cells that express active
CYP1 enzymes.
With regard to the potency of tangeretin in inhibiting the proliferation of human
breast cancer cells, this compound exhibited high activity in MDA–MB–468
compared with MCF7 cells. By contrast, MCF10A cells were notably unaffected as
the IC50 of tangeretin noted in this cell line was higher than 100 μM. Tangeretin
exhibited an activation factor of approximately 72–fold in MDA–MB–468 cells
compared with MDA–MB–468 cells that were treated with tangeretin and CYP1
inhibitors (18 μM/0.25 μM). A similar trend has been previously demonstrated for the
flavonoids sinensetin, genkwanin and eupatorin in our earlier reports
(Androutsopoulos et al., 2009d; Androutsopoulos et al., 2008). These compounds
exhibited IC50 values for MDA–MB–468 cells of 0.2, 1.6 and 0.5 μM, respectively,

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whereas in MCF10A cells they were considerably less active (65, 75 and 50 μM,
respectively). In the presence of the CYP1 inhibitor acacetin, the IC50 values of
sinensetin, eupatorin and genkwanin were considerably increased (from 0.2 to 13.5
μM for sinensetin, from 0.5 to 15 μM for eupatorin and from 1.6 to 12.5 μM for
genkwanin) (Androutsopoulos et al., 2009d; Androutsopoulos et al., 2008). The data
reported in the present study are in agreement with the aforementioned studies and
suggest that the degree of methoxylation in the flavone moiety enhances the
antiproliferative activity of these compounds in MDA–MB–468 cells due to
cytochrome P450 CYP1–metabolism. This is verified by the observation that
sinensetin and tangeretin that are both penta–methoxylated flavones exhibited
submicromolar IC50 values with similar activation factors in the presence of CYP1
inhibitors (68–fold vs. 72–fold). In addition, MCF7 cells are less sensitive to the
CYP1-activating effect of tangeretin and dietary flavonoids, as CYP1 activity is
reduced following removal of TCDD, whereas basal activity of CYP1 enzymes in
MCF7 cells is a lot lower than that in MDA-MB-468 cells (Androutsopoulos et al.,
2
008; Androutsopoulos et al., 2009a). Furthermore, MCF7 cells are less sensitive
compared with MDA-MB-468 cells to the antiproliferative action of flavonoids and
their metabolites (Androutsopoulos et al., 2009a; Androutsopoulos et al., 2009b;
Wilsher et al. 2017).
In addition, the data indicated that tangeretin induced CYP1 enzyme activity and
CYP1 enzyme expression in MCF7 and MDA–MB–468 human breast cancer cells.
CYP1A and CYP2B enzyme activities and CYP1A and CYP2B enzyme expression
were increased by tangeretin treatment in rat liver tissues, as demonstrated by an early
study by Canivenc–Lavier et al. (Canivenc–Lavier et al. 1996). Additional studies
have documented CYP1 inductive effects by the structurally similar flavones

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eupatorin, cirsiliol, eupatorin–5–methyl ether and nobiletin in human breast cancer
cells at similar concentrations to those used for tangeretin in the current report
(Androutsopoulos et al., 2009c, Androutsopoulos and Tsatsakis 2014, Surichan et al.,
2
012). Hydroxylated and monomethoxylated flavones such as quercetin and
diosmetin, respectively, have also been shown to induce CYP1A1 mRNA and
CYP1A enzyme activity via direct binding to the AhR in MCF7 breast cancer cells
(Ciolino et al., 1999; Ciolino et al., 1998). It was suggested that the lipophilicity of
these natural compounds that is determined by their methoxy substitutions affected
their binding to the AhR and consequently the induction of CYP1 enzyme activity. It
is important to note that the induction of CYP1B1 protein by tangeretin is shown for
the first time as previous studies were mainly focused on the interactions of CYP1A1
and CYP1A enzymes with dietary flavones (Canivenc–Lavier et al. 1996; Ciolino et
al., 1999; Ciolino et al., 1998).
In conclusion, the present study investigated the anticancer effects of tangeretin with
regard to CYP1–mediated metabolism, CYP1–enzyme induction and inhibition of
cellular proliferation of human breast cancer cells. The data demonstrate that
tangeretin is activated in human breast cancer cells due to CYP1–mediated conversion
to the metabolite TP1 that in turn results in G1 arrest of the cells (Fig. 7). These
effects were absent in normal breast cells that do not express CYP1 enzymes. The
findings suggest that tangeretin could possess therapeutic implications in breast
cancer cells that express active CYP1 enzymes (Fig. 7). The clinical response of a
patient to tangeretin and similar CYP1-activated prodrugs will depend on his/her
endogenous levels of CYP1 expression. This is a prerequisite regarding the successful
application of this type of targeted therapy and previous studies conducted by our
group have addressed the enzymatic levels of CYP1 enzymes in human tumors

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(Androutsopoulos et al., 2013, Spyrou et al., 2014). However, larger studies are
required to profile the pattern of active CYP1 expression in a larger panel of tumor
samples.
In conclusion, tangeretin may be considered as a selective CYP1–activated natural
product that can be further evaluated for its cancer therapeutic efficacy in vivo. It is
important to note that tangeretin can be used in vivo at concentrations as high as 50
mg/kg (Avirazhagan and Sorimuthu Pillai 2014). Tangeretin has been shown to be
effective in inhibiting TNBS-induced colitis and TNBS-induced body loss at a dose of
2
0 mg/kg in C57BL/6 mice (Eun et al. 2017), whereas it exerted no significant
toxicity in vivo at a concentration of 30 mg/kg following administration to nude mice
for 28 days (Zhang et al. 2015). Consequently, high doses of tangeretin may possess
therapeutic activity against breast cancer.
Conflict of interest
None declared.
Acknowledgments
This research was funded by DeMontfort University and by the University of Crete
ELKE KA2590 grant.

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