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INT ERNATIONAL JOUR NAL OF ONCOLOGY
Abstract. Natural components continue to be an important
source for the discovery and development of novel anticancer
agents. Polymethoxyavones are a class of avonoids found in
citrus fruits and medicinal plants used in traditional medicine.
In the present study, the anticancer activity of the well-known
nobiletin (5,6,7,8,3',4'-hexamethoxyflavone) was compared
against its less studied structural isomer 5,6,7,3',4',5'-hexa-
methoxyflavone. These compounds were evaluated on the
Hs578T triple-negative breast cancer cell line and its more
migratory subclone Hs578Ts(i)8. 5,6,7,3',4',5'-hexamethoxya-
vone was found to be less toxic than nobiletin, while a similar
growth inhibitory effect was observed after 72 h. Additionally,
5,6,7,3',4',5'-hexamethoxyavone arrested the cell cycle in the
G2/M phase, while no effect was observed on apoptosis or the
migratory behavior of these cells. Furthermore, mechanistic
studies revealed that the growth inhibition was concomitant
with reduced phosphorylation levels of signaling molecules
in the MAPK and Akt pathways as well as cell cycle regula-
tors, involved in regulating cell proliferation, survival and cell
cycle. In summary, the present study is the rst to report on
the anticancer activities of 5,6,7,3',4',5'-hexamethoxyavone
and to provide evidence that this avone could have a greater
potential than nobiletin for prevention or treatment of triple-
negative breast cancer.
Introduction
Compounds derived from natural products play an important
role in the discovery of clinically suitable therapeutic agents.
This is particularly true for anticancer medicines, with almost
70% of the novel anticancer drugs approved, over the past six
decades, coming from natural products or based on the knowl-
edge gained from natural products (1).
Recently, a renewed interest for avonoids as anticancer
agents has been catalyzed by avopiridol (Alvocidib), a potent
cyclin-dependent kinase (CDK) inhibitor, which has been
granted orphan drug designation by the FDA in 2014 to treat
patients with acute myeloid leukemia (2). Flavonoids are found
throughout the plant kingdom and several epidemiological
studies suggest that dietary intake of avonoids is responsible
for chemoprevention (3). Of particular interest are the polyme-
thoxyavones (PMFs), avones substituted with two or more
methoxy groups. This subclass of avonoids is thought to be
superior to polyhydroxylated avonoids due to their increased
metabolic stability, oral bioavailability and consequently
improved cancer chemopreventive activities (4).
PMFs can be found in high concentrations in the peel of
several Citrus species and in medicinal plants used in tradi-
tional medicine (5-7). Studies on the a nticancer activity of PMFs
have mostly been focused on nobiletin. This 5,6,7,8,3',4'-hexa-
methoxyavone has been shown to be effective in vitro and
in vivo by affecting several cellular activities, including inhi-
bition of cell proliferation, invasion and migration, inducing
cell cycle arrest as well as reducing angiogenesis, signaling
pathways and bioactivation by CYP1 (8-11). Notable also, is
its predominant anticancer activity in MDA-MB-468 cells
which indicates a potential role of nobiletin for the prevention
of triple-negative breast cancer (TNBC) (12), an aggressive
and highly metastatic subtype with poor prognosis for which
hormonal therapy is not benecial and chemotherapy remains
the only treatment (13).
Studies with different Citrus species and medicinal plants
indicate a high structural variability in PMF content, including
5,6,7,3',4',5'-Hexamethoxyavone inhibits growth of triple-
negative breast cancer cells via suppression of MAPK
and Akt signaling pathways and arresting cell cycle
NATASHA BORAH1*, SHIMARA GUNAWARDANA1*, HAYDEE TORRES1,
SUSAN McDONNELL2 and SEVERINE VAN SLAMBROUCK1
1Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA;
2UCD School of Chemical and Bioprocess Engineering, University College Dublin, Beleld Dublin 4, Ireland
Received July 27, 2017; Accepted September 25, 2017
DOI: 10.3892/ijo.2017.4157
Correspondence to: Dr Séverine Van Slambrouck, Department
of Chemistry and Biochemistry, South Dakota State University,
Chemistry-box 2202, Brookings, SD 57007, USA
E-mail: severine.vanslambrouck@sdstate.edu
Abbreviations: TNBC, triple-negative breast cancer; PMF,
polymethoxyflavones; HMF, hexamethoxyflavones; FDA, Food and
Drug Administration; MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated kinase; SAPK, stress-activated
protein kinase; JNK, c-Jun N-terminal kinase; HRP, horseradish
peroxidase; CDK, cyclin-dependent kinase; Chk, checkpoint kinase,
Cdc, cell division cycle
Key words: hexamethoxyflavones, cytotoxicity, growth, signaling,
phosphokinase array, cell cycle, triple-negative breast cancer
BORAH et al: HEXAMETHOXYFLAVONES AND TNBC
2
the presence of smaller methoxyflavones and structural
isomers. While several reports suggest that the anticancer
activity from avonoids is profoundly affected by their compo-
sition and structure, limited studies are published on the effect
of these less known congeners (4), such as 5,6,7,3',4',5'-hexa-
methoxyavone. This avone has the same structural formula
as nobiletin and has been isolated from Citrus reticulate and
Ageratum conyzoides (Fig. 1). The compound was found to
be cytotoxic against P-388 mouse leukemia cells, but not
against the HT-29 human colon adenocarcinoma cell line
and to suppress the degranulation from antigen-stimulated rat
basophil RBL-2H3 cells through its effect on signaling as Syk/
PLCγ's/PKC and mitogen-activated protein kinase (MAPK)
pathways and Ca2+ inux (14,15).
The present study aimed at investigating the possible
anticancer effects of 5,6,7,3',4',5'-hexamethoxyflavone and
comparison against the well-studied nobiletin in the Hs578T
progression model of TNBC. This in vitro cell system
comprises the Hs578T TNBC cell line and its more metastatic
and isogenic variant Hs578Ts(i)8 and embodies an elegant
experimental model for studying the anticancer activity of
both hexamethoxyavones in TNBC and on TNBC progres-
sion (16).
Materials and methods
Antibodies and other reagents. Antibodies directed against
p-ERK (D13.14.4E), p-JNK/SAPK (81E11), p-Akt (D9E), p-p38
MAPK (D3F9), p-Chk2 (C13C1), p-Chk1 (133D3), p-Cdc2
(10A11) and anti-β-actin (D6A8) or β-tubulin (9F3) antibodies
as well as camptothecin were from Cell Signaling Technology
(Danvers, MA, USA). Anti-mouse and anti-rabbit alkaline phos-
phatase-labeled secondary antibodies, the BCA protein assay
reagent kit and trypan blue solution were from Thermo Fisher
Scientic (Waltham, MA, USA). Drug toxicity was evaluated
through measurement of mitochondrial dehydrogenase activi-
ties with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) reagent (Sigma-Aldrich, St. Louis, MO, USA).
Nobiletin and 5,6,7,3',4',5'-hexamethoxyavone were obtained
from Alkemist Labs (Costa Mesa, CA, USA).
Cell culture. The human mesenchymal breast cancer
Hs578T cells and the derivative cell line Hs578Ts(i)8 were a
kind gift from Dr S. McDonnell (UCD School of Chemical
and Bioprocess Engineering, University College Dublin,
Ireland) (16) and were grown in Dulbecco's modied Eagle's
medium (DMEM) supplemented with 10% (v/v) fetal bovine
serum (FBS), 100 IU/ml penicillin, 100 µg/ml streptomycin
and 0.01 mg/ml bovine insulin (Thermo Fisher Scientic) at
37˚C equilibrated with 5% (v/v) CO2 in humidied air. The
TNBC cells used in the present study were frozen in liquid
nitrogen when not in use and were not passaged in our labora-
tory for >15 weeks.
Assay for cell viability. The effect of nobiletin and
5,6,7,3',4',5'-hexamethoxyavone on cell viability was tested in
accordance with Romijn et al (17). Briey, mitochondrial dehy-
drogenase activities were measured by an MTT reagent. Cells
were seeded in 96-well plates at an initial density of 1.5x104
cells in 100 µl culture medium. After overnight incubation,
cells were treated with nobiletin and 5,6,7,3',4',5'-hexamethoxy-
avone at a nal concentration of 100, 50 and 10 µM. After 24
and 72 h of incubation, 100 µl medium was removed prior to
the addition of MTT reagent, and formed formazan crystals
were dissolved in 200 µl dimethyl sulfoxide (DMSO). Four
independent experiments were completed to determine the
mean absorbance referring to cell viability, using a Cytation™
3 Cell Imaging Multi-mode reader with Gen5 software (BioTek
Instruments, Inc., Winooski, VT, USA) and were expressed in
percentage as compared to DMSO-treated control cells. In each
experiment, eight wells were used per condition.
Cell counting. Cells were seeded in 25-cm2 culture asks at an
initial density of ~1.5x105 cells in 5 ml cultu re medium and were
treated with nobiletin and 5,6,7,3',4',5'-hexamethoxyavone at
a nal concentration of 100 µM after overnight incubation. The
cells were allowed to grow for 24, 48 and 72 h, harvested using
trypsin/EDTA and counted with a TC20™ automated cell
counter (Bio-Rad Laboratories, Hercules, CA, USA). At least
three independent experiments were performed to determine
the mean value, which is presented as a percentage compared
to the DMSO-treated controls.
Wound healing assay. Cells were grown in 24-well plates
until conuency and washed twice with phosphate-buffered
saline (PBS). A scratch was made using a P200 pipette tip
and 1 ml of medium in the presence of DMSO, nobiletin or
5,6,7,3',4',5'-hexamethoxyavone at 100 µM, was added. Cell
migration was monitored and images were collected after
17 h, with an EVOS® XL Core Cell Imaging (Thermo Fisher
Scientic). ImageJ software was used to estimate the cell free
area of the wounds (18). The distances over which the cells
migrated were measured in three independent experiments
and expressed as percentage compared to DMSO-treated
Hs578T and Hs578Ts(i)8 cells.
Proteome Proler Human Phospho-kinase Array. Cells at
70% confluency were washed three times, serum-starved
overnight, washed again and stimulated for 2 h with complete
culture medium, followed by a treatment of 10 min with
100 µM nobiletin and 5,6,7,3',4',5'-hexamethoxyavone prior
to cell lysis. Lysates were made using kit components and the
array experiments were performed following the manufactur-
er's instructions (R&D Systems, Inc., Minneapolis, MN, USA).
Briey, aliquots of cell lysates, containing 250 µg of protein,
were incubated overnight with a human phospho-kinase array
membrane containing capture antibodies against 43 different
Figure 1. Structures of hexamethoxyavones: 5,6,7,3',4',5'-hexamethoxya-
vone and nobiletin.
INT ERNATIONAL JOUR NAL OF ONCOLOGY 3
kinase phosphorylation sites. After washing, the membranes
were incubated with biotinylated antibodies, streptavidin-HRP
and chemiluminescent reagent for detection of phosphorylated
protein at each capture spot on the membranes. Images were
taken with a LI-COR® Odyssey Fc and analyzed with Image
Studio 5.0 (LI-COR Biosciences, Lincoln, NE, USA) for deter-
mination of mean pixel density and further analyzed using
Excel.
Western blotting. Conuent cell cultures (70%) were washed
three times and for MAPK and Akt signaling experiments
serum-starved overnight, washed again and stimulated for
2 h with complete culture medium prior to treatment with
100 µM nobiletin and 5,6,7,3',4',5'-hexamethoxyavone for
indicated times. For investigation of cell cycle regulators, 70%
conuent cultures were not stimulated and washed three times.
Subsequently, cells were lysed using lysis buffer containing
1% Triton X-100 and 1% Halt™ Protease Inhibitor Cocktail
(Thermo Fisher Scientific). Aliquots of lysates containing
25-30 µg of proteins were boiled for 5 min in SDS-PAGE
sample buffer supplemented with 5% β-mercaptoethanol,
electrophoresed on 10% or 4-15% gradient Mini-PROTEAN®
TGX™ gels and transferred to PVDF membranes (Bio-Rad
Laboratories). After transfer, membranes were incubated
with relevant antibodies against p-ERK, p-JNK/SAPK, p-p38
MAPK, p-Akt, p-Chk2 and Chk1, p-Cdc2 and β-tubulin or
β-actin, as loading controls, followed by incubation with a
secondary alkaline-phosphatase anti-rabbit antibody and
stained with NBT/BCIP (1:50 in 0.1 M Tris-HCl, 0.05 M
MgCl2 and 0.1 M NaCl at pH 9.5).
Flow cytometric analysis. Cells were treated for 72 h with
100 µM nobiletin and 5,6,7,3',4',5'-hexamethoxyflavone.
Floating cells and trypsinized adherent cells were combined
and washed with cold PBS or cell culture medium. For detection
of apoptosis, cells were stained with an Annexin V/7-AAD kit
(Beckman Coulter, Miami, FL, USA) using the manufacturer's
protocol. In brief, cells were incubated with Annexin V and
7-AAD in ice-cold binding buffer in the dark. After 15 min the
samples were mixed with more binding buffer and analyzed
within 30 min. Positive controls for apoptosis induction
included Hs578T and Hs578Ts(i)8 cells treated with 10 µM
camptothecin for 72 h. Cell cycle analysis was investigated
by adding Vybrant® DyeCycle™ Green Stain (Thermo Fisher
Scientific) to 1 ml cell suspension at a final concentration
of 250 nM. After 30-min incubation at 37˚C, the samples
were analyzed by flow cytometry and compared against
DMSO-treated cells. All these experiments were performed
on a CytoFLEX flow cytometer (Beckman Coulter) using
CytExpert 2.0 software.
Statistics. All treatments were matched and carried out at least
3 times. Data were analyzed using Excel, for determination
of mean, standard deviation (SD) and Student's t-test (95%).
The intensity of the immunoblotted bands was quantied by
densitometry, using statistical software Scion Image (Scion
Corp., Frederick, MD, USA).
Results
Effect of nobiletin and 5,6,7,3',4',5'-hexamethoxyavone on
cell viability. Hs578T and the more invasive Hs578Ts(i)8 cells
were treated for 24 and 72 h with nobiletin and 5,6,7, 3',4',5'-hexa-
methoxyavone at concentrations of 10, 50 and 100 µM and
cytotoxicity was determined by the MTT-test. The effects of
these compounds on cell viability are shown in Table I. Overall,
the compounds did not have a major toxic effect toward the cell
viability of the tested cell lines at 100 µM, after 24 and 72 h
and consequently no IC50 values could be determined. Instead,
nobiletin generally reduced the cell viability of the Hs578T
and Hs578Ts(i)8 cell lines with ~30% after 24 h and 30-50%
after 72 h at its highest concentration, while no noteworthy
effect was observed for 5,6,7,3',4',5'-hexamethoxyavone after
24 h (upper panel), and a small drop after 72 h (lower panel).
Notably, we found that both nobiletin and 5,6,7,3',4',5'-hexame-
thoxyavone at 100 µM showed a greater toxicity toward the
more invasive Hs578Ts(i)8 variant cell line, decreasing the cell
viability to 51.6 and 68.1%, respectively, after 72 h.
Nobiletin and 5,6,7,3',4',5'-hexamethoxyavone inhibit cell
growth. The growth inhibitory effects of the compounds
at 100 µM were evaluated after 24, 48 and 72 h by cell
counting. Nobiletin and 5,6,7,3',4',5'-hexamethoxyflavone
markedly inhibited the growth of Hs578T (Fig. 2A) and
Table I. Percentage of cell viability of Hs578T and Hs578Ts(i)8 after 24 and 72 h nobiletin and 5,6,7,3',4',5'-hexamethoxyavone
treatment.
Nobiletin Hexamethoxyavone
------------------------------------------------------------------------------------------ -----------------------------------------------------------------------------------------
100 µM 50 µM 10 µM 100 µM 50 µM 10 µM
(24 h)
Hs578T 71.4±6.1 98.4±2.7 99.0±0.9 92.7±4.4 97.5±1.3 95.9±4.3
Hs578Ts(i)8 70.0±9.0 88.5±2.0 93.0±3.9 85.9±7.2 88.4±6.7 92.6±7.6
(72 h)
Hs578T 73.2±13.4 98.9±12.1 99.7±2.8 81.9±1.6 84.5±3.6 90.2±13.0
Hs578Ts(i)8 51.6±7.7 84.7±4.6 103.7±3.6 68.1±7.7 80.9±5.3 93.7±11.1
Results are expressed as mean % cell viability vs. DMSO-treated cells, of four independent experiments with n=8 per condition in each
experiment, determined by MTT assays.
BORAH et al: HEXAMETHOXYFLAVONES AND TNBC
4
Hs 578Ts (i)8 cells (Fig. 2B) in a time-dependent manner.
After 24 h, limited effects were observed. After 48 and 72 h,
however, nobiletin signicantly reduced the amount of cells
by roughly 40 and 50%, respectively. The growth inhibition
following 5,6,7,3',4',5'-hexamethoxyflavone treatment was
most pronounced after 72 h, and reached a level (~50%) almost
similar to nobiletin. It should be noted, that the reduction in
number of cells grown in the presence of nobiletin is relatively
comparable to the data obtained in the MTT-assays (Table I),
which may suggest a confounding effect due to its effect on
cell viability.
Nobiletin and cell migration. In our previous study, we
published that the Hs578Ts(i)8 cells migrate twice as fast
compared to the parenta l Hs578T cells in a wound heali ng assay
under normal growth conditions (19), which was in line with
the earlier published increased migratory capacity of the more
invasive Hs578Ts(i)8 cell line using the BD Matrigel™Invasion
Chamber assay system (16). Herein, we tested the potential of
nobiletin and 5,6,7,3',4',5'-hexamethoxyavone to inhibit the
migratory behavior, and this particularly of the Hs578Ts(i)8
cells. Results shown in Fig. 3 reveal that nobiletin at a concen-
tration of 100 µM signicantly reduced the migration by 40%,
and that there was no selectivity toward the more migratory
and invasive Hs578Ts(i)8 cells. In contrast, no effect was seen
for 5,6,7,3',4',5'-hexamethoxyavone and the wound was closed
after 17 h, similar to the DMSO-control conditions. Yet again,
the effect of nobiletin should be viewed with caution, as the
growth inhibitory activity may confound the effect of nobiletin
on cellular migration.
Effect of nobiletin and 5,6,7,3',4',5'-hexamethoxyavone on
apoptosis. Annexin V-FITC/7-AAD double staining was used
to examine the effect of the two hexamethoxyavones on the
induction of cellular apoptosis. There are several compounds
available to induce apoptosis in cancer cells in studies,
in vitro. In vivo, taxanes have been shown to be benecial in
Figure 2. Growth inhibitory effects on Hs578T (A) and Hs578Ts(i)8 cells (B)
after treatment with 100 µM nobiletin (closed bars) and 5,6,7,3',4',5'-hexame-
thoxyavone (open bars). The number of cells was determined by counting
and expressed as mean % ± SD of three independent experiments with
n=3 per condition in each experiment. *P<0.05, statistical difference from
DMSO-treated control cells.
Figure 3. Effects on wound healing after 17-h treatment with 100 µM
nobiletin (closed bars) and 5,6,7,3',4',5'-hexamethoxyavone (open bars).
Percentage of closure of wound, resulting from measuring the difference in
width at t=0 and t=17, are expressed as mean % ± SD of three independent
experiments, with n=6 per condition in each experiment. *P<0.05, statistical
difference f rom DMSO-treated control cells.
Figure 4. Effects of 100 µM nobiletin and 5,6,7,3',4',5'-hexamethoxyavone,
as well as 10 µM camptothecin treatment for 72 h on apoptosis, in Hs578T
(upper panel) and Hs578Ts(i)8 cells (lower panel). (A) Cells were trypsinized,
combined with oating cells prior to staining with Annexin V-FITC/7-AAD.
Profiles a re representative examples of four independent experiments.
(B) Percentage of live (open bars), early apoptotic (light grey bars), late apop-
totic (grey bars) and necrotic (closed bars) cells, expressed as mean % ± SD
of four independent experiments.
INT ERNATIONAL JOUR NAL OF ONCOLOGY 5
neoadjuvant, adjuvant, and metastatic settings in TNBC (20),
and this specically toward the basal-like (BL) 1 and 2 TNBC
subtypes as compared to the mesenchymal-like and luminal
androgen receptor subtypes (21). Since the Hs578T/ Hs578Ts(i)8
breast cancer model represents mesenchymal-like TNBC,
the well-known and universally used inducer of apoptosis,
camptothecin, was used. While the addition of camptothecin
in a nal concentration of 5-10 µM and incubation in a time
range of 4-24 h usually allows the evaluation of apoptosis in
many cell lines, Hs578T and Hs58Ts(i)8 cells did not respond
to the treatment within that time frame or after 48 h (data
not shown). Instead, camptothecin-induced apoptosis was
observed after 72 h at a concentration of 10 µM. Consequently,
Hs578T and Hs578Ts(i)8 cells were exposed to nobiletin and
5,6,7,3',4',5'-hexamethoxyavone for 72 h, and oating cells
and adherent cells were combined prior to staining. Fig. 4A
and the quantification in Fig. 4B indicate no significant
differences in the Annexin V-FITC positive and 7-AAD
positive or negative cells after 72-h treatment with nobiletin
and 5,6,7,3',4',5'-hexamethoxyavone at a nal concentration
of 100 µM as compared to DMSO and this in both cell lines.
These results suggest that Hs578T and Hs578Ts(i)8 cells: i) are
quite resistant toward camptothecin-induced apoptosis; and
ii) do not undergo apoptosis after exposure to nobiletin and
5,6,7,3',4',5'-hexamethoxyavone (Fig. 4).
Nobiletin and 5,6,7,3',4',5'-hexamethoxyflavone inhibit
activation of distinct signaling molecules. With increasing
knowledge on and complexity of signaling pathways as well
as the fact that these pathways are highly cell-type specic,
phospho-kinase arrays were performed alongside western
blotting, to take an unbiased approach for analysis of affected
Figure 5. (A) Effect of a 10-min treatment with 100 µM nobiletin and 5,6,7,3',4',5'-hexamethoxyavone, after overnight serum-starvation and stimulation for
2 h with complete cultu re media, on kinase phosphorylation sites using phospho-kinase arrays in Hs578T (upper panel) and Hs578Ts(i)8 cells (lower panel). (B)
Quantication by mean pixel density, expressed as means of % inhibition of nobiletin (closed bars) and 5,6,7,3',4', 5'-hexamethoxyavone (open bars) in Hs578T
an d H s578Ts (i)8 cells vs DMSO-t reated cells. Results were obtained from 2 independent experiments. Lower panel: western blot analysis of phosphorylated
proteins in Hs578T (C) and Hs578Ts(i)8 cells (D). Cells were serum-sta rved overnight, stimulated with complete culture medium for 2 h and treated for
indicated times with 100 µM nobiletin and 5,6,7,3',4',5'-hexamethoxyavone. Cell lysates were analyzed by TGXTM gels, transferred to PVDF membranes and
use of the corresponding primary antibodies against k inase phosphorylation sites and tubulin as loading controls. Blots are representative examples of at least
three independent experiments. Scion Image densitometry analysis of bands comparing the relative levels of phosphorylation after 10 min 100 µM nobiletin
and 5,6,7,3',4',5'-hexamethoxyavone as compared to DMSO-treatment. Bar graphs are means ± SD from at least three independent experiments. *P<0.05,
statistical difference from DMSO-treated cells. ND, not detected.
BORAH et al: HEXAMETHOXYFLAVONES AND TNBC
6
signaling molecules and pathways upon treatment with the
two hexamethoxyavones. This was particularly important,
given the uniqueness of the cell model system and the limited
information available. Initially, western blotting was performed
to determine an approximate optimal time-point in the cell
lines used, for nobiletin and 5,6,7,3',4',5'-hexamethoxyavone-
mediated inhibition of phosphorylated ERK and Akt. The
phosphorylation of these two signaling molecules is often
found to be elevated after hormone or growth factor stimulation
and inhibited following exposure to PMFs, such as nobiletin
and tangeretin (8,22). Limiting factors to use this approach
include the fact that these cell lines are triple-negative and
the poor comprehension of the importance of growth factors
in the behavior of the Hs578T and more migratory and
invasive Hs578Ts(i)8 cells. The MAPK and Akt pathways are
known to be activated by a variety of factors, among them
receptor tyrosine kinases, integrin, cytokine and G-protein
coupled receptors. Therefore, the method of overnight serum-
starvation and serum stimulation was applied to achieve
phosphorylation and demonstrate inhibition by nobiletin and
5,6,7,3',4',5'-hexamethoxyavone. Responsiveness to serum
stimulation, after overnight serum-starvation, with complete
culture media was tested over time and 2 h proved to be an
optimal stimulus, while a significant inhibition of the two
hexamethoxyavones could be observed as early as ~5-10 min
after 100 µM treatment, with 10 min used in the subsequent
array experiments.
The phospho-kinase array revealed a considerable amount
of information, with most notably the decrease of p-ERK
phosphorylation by nobiletin and 5,6,7,3',4',5'-hexamethoxy-
avone as compared to DMSO-treated cells. A similar trend
was observed for p-JNK1/2/3, although the effect was less
pronounced. Moreover, both hexamethoxyavones inhibited
the phosphorylation levels of Akt, while the phosphorylation
of checkpoint kinase 2 (Chk2) seemed to be solely reduced
upon 10 min of nobiletin treatment. Additionally, phos-
phorylated p38 MAPK was only detected in the Hs578T
cell line, and was inhibited by the two hexamethoxyavones
(HMFs) (Fig. 5A and B), while it was below the detection limit
of the array for the Hs578Ts(i)8 cells. It must be mentioned
Figure 6. Effects on the cell cycle after treatment with 100 µM nobiletin and 5,6,7,3',4',5'-hexamethoxyavone for 72 h in Hs578T (upper panel) and Hs578Ts(i)8
cells (lower panel). (A) Cells were trypsinized, combined with oating cells prior to staining with Vybrant® DyeCycle™ Green. Proles are representative
examples of four independent experiments. (B) Percentage of population in sub G0 (open bars), G 0/G1 (light grey bars), S (grey bars) and G2/M (closed bars)
cells, expressed as mean % ± SD of three independent experiments. *P<0.05, statistical difference from DMSO-treated cells. Lower panel: western blot analysis
of phosphorylated Chk2 and Cdc2 in Hs578T (C) as well as Chk1 in Hs578Ts(i)8 cells (D). Cells were treated for indicated times with 100 µM nobiletin and
5,6,7,3',4',5'-hexamethoxyavone. Cell lysates were analyzed by TGX™ gels, transferred to PVDF membranes and use of the corresponding primary antibodies
against kinase phosphorylation sites and β-actin as loading controls. Blots are representative examples of at least three to ve independent experiments. Scion
Image densitometry analysis of bands comparing the relative levels of phosphorylated p-Ch k2 (solid line) and p-Cdc2 (dashed line) in Hs578T cells as well as
p-Chk1 (dotted line) in Hs578Ts(i)8 cells, after 100 µM nobiletin (closed circles) and 5,6,7,3',4',5'-hexamethoxyavone (open circles) as compared to DMSO-
treatment. Line graphs are means ± SD from three to ve independent experiments.
INT ERNATIONAL JOUR NAL OF ONCOLOGY 7
that only two arrays per cell line and per component were
performed to obtain initial data. Even though these arrays
would allow for the quantication of the relative phosphory-
lation levels, the obtained results were further conrmed by
western blotting in kinetics experiments in Hs578T (Fig. 5C)
and Hs578Ts(i)8 (Fig. 5D) cells. Serum stimulation induced the
phosphorylation of all kinases as compared to unstimulated
cells, except for Chk2. Chk2 was not only found highly phos-
phorylated in these cell lines after serum-starvation but also
under normal growth conditions (data not shown). Nobiletin
and 5,6,7,3',4',5'-hexamethoxyavone were able to inhibit the
serum-induced phosphorylation of ERK, SAPK/JNK and Akt
in a time range of 5-20 min in Hs578T and Hs578Ts(i)8 cells.
Additionally, signicant Chk2 phosphorylation inhibition was
evident for nobiletin after 5 min, whereas 5,6,7,3',4',5'-hexa-
methoxyflavone significantly reduced the phosphorylation
levels after treatments of 20 min and longer. Also in the
western blotting experiments, phosphorylated p38 MAPK
could only be clearly detected in the Hs578T cells under the
experimental conditions used, and was reduced by nobiletin
and 5,6,7,3',4',5'-hexamethoxyflavone over the investigated
time range (Fig. 5C and D).
Nobiletin and 5,6,7,3',4',5'-hexamethoxyflavone affect
the cell cycle. As a result of the significant nobiletin- and
5,6,7,3',4',5'-hexamethoxyavone-mediated inhibition of Chk2
phosphorylation in the previous phosphokinase arrays and
western blot experiments, the effect of both hexamethoxya-
vones on the cell cycle was evaluated by performing a Vybrant®
DyeCycle™ green staining. Comparison with DMSO-treated
cells indicated that both hexamethoxyavones upon treatment
with 100 µM for 72 h, but not 48 h (data not shown), were
able to cause a slight increase in G2/M arrest (Fig. 6A and B).
While 5,6,7,3',4',5'-hexamethoxyavone elevated the subpopu-
lation of Hs578T and Hs578Ts(i)8 cells by 16.1 and 13.4%,
respectively, nobiletin only signicantly enhanced the G2/M
arrest in Hs578T cells by 14.3%. Western blot results in
Fig. 6C and D revealed that Chk2 phosphorylation at T68
decreased after 10 min in the presence of nobiletin (P<0.05)
and 30 min with 5,6,7,3',4',5'-hexamethoxyavone (P<0.05),
without prior serum-starvation and stimulation, in both cell
lines. Additionally, Chk1 phosphorylation levels were below or
near the detection limit in the Hs578T cell lines, whereas weak
levels were detected in the more invasive variant, which were
not notably altered in the presence of the HMFs. Furthermore,
phosphorylation levels of the cell division cycle 2 (Cdc2),
known as CDK1, which acts downstream from Chk1 and regu-
lates G2/M arrests, were found signicantly decreased after
30-min exposure to nobiletin and 5,6,7,3',4',5'-hexamethoxy-
avone in the Hs578T and after 30 to 60 min in Hs578Ts(i)8
cells (P<0.05).
Discussion
The present study reports for the first time on the anti-
cancer activity of 5,6,7,3',4',5'-hexamethoxyflavone. This
polymethoxyavone is a structural isomer of 5,6,7,8,3',4'-hexa-
methoxyavone, known as nobiletin. They differ in the position
of one methoxy group either on ring A or B (Fig. 1). While
nobiletin is widely studied and known for its benecial effects
on cancer cells, 5,6,7,3',4',5'-hexamethoxyavone is not, despite
being isolated from the same sources and the recognized
concept of isomerism affecting biological activity (4,13,15,23).
In the present study, we used the Hs578T/Hs578Ts(i)8
breast cancer progression model (16,19) for several reasons,
including the potential role of nobiletin against TNBC (12) and
in prevention of metastasis (8-10). Our results indicated that
5,6,7,3',4',5'-hexamethoxyavone is less toxic than nobiletin
against Hs578T and Hs578Ts(i)8 cells, while exerting a nearly
similar effect as nobiletin in the cell counting experiments
after 72-h treatments. Additionally, only nobiletin reduced
the migratory behavior with almost 40% within 17 h in both
cell lines. The effects of nobiletin on cell viability are in the
same range as previously published studies using MCF-7,
T47D, MDA-MB-231 and SKBR3 breast cancer cells. Notably,
these studies also reported that nobiletin reduced the migra-
tory behavior of MCF-7 cells by 40% in wound-healing
experiments and invasion by 30% using the Matrigel inva-
sion assay, while a 60% reduction in invasion was observed
in MDA-MB-231 cells (24,25). These ndings as well as the
observations presented in this study should be interpreted
and used with caution, given that nobiletin decreased the cell
viability and number of cells by 20-30% within 24 h which
may confound the effect on migration and invasion. This idea
is conrmed by the fact that 5,6,7,3',4',5'-hexamethoxyavone
did not inuence migration, while showing limited to no effect
on cell viability and cell counts after 24 h. Other structural
congeners with methoxy groups at position 3',4',5' of the B-ring
include sinensetin and 5,6,7,8,3',4',5'-heptamethoxyflavone.
Unfortunately, few studies are published on the potential
anticancer activity of the structurally related penta- and hepta-
methoxyavone. A recent study suggested that sinensetin has
an antiproliferative effect and the ability to induce apoptosis
at concentrations of 50 µM and higher (26). These results are
quite remarkable given the poor solubility in water and the
typical solvents as DMSO, ethanol or methanol and conse-
quently questions the validity of the results.
Further investigations on the possible mechanisms of
action of 5,6,7,3',4',5'-hexamethoxyflavone and nobiletin
resulting in a decreased number of cells suggested that 72 h
treatments with either compounds were not able to induce
apoptosis in the Hs578T and Hs578Ts(i)8 cells as compared
to camptothecin. It must be mentioned though that nobiletin
studies are controversial and inconclusive when it comes to its
effect on apoptosis. One reasoning could be that certain cell
lines, such as the Hs578T cell line and its more invasive variant
Hs578Ts(i)8 as well as glioma cell lines (8) are more resistant
to apoptosis, and that nobiletin or other PMFs are not potent
enough to induce an apoptotic effect similar to camptothecin.
The use of Proteome Profiler Human phospho-kinase
arrays allowed for an objective screening of altered phos-
phorylation levels of 43 different kinases. The array data as
well as the conrming western blotting results, using pathway
activation and potential inhibition as described by Burkhard
and Shapiro (27), revealed that 5,6,7,3',4',5'-hexamethoxya-
vone treatment mainly inhibited MAPK and Akt signaling
pathways, recognized for inuencing cell proliferation, cell
differentiation and cell death. These ndings were compa-
rable to nobiletin and were also obtained without pathway
stimulation for which a signicant inhibition was detected
BORAH et al: HEXAMETHOXYFLAVONES AND TNBC
8
after 30-min exposure to both hexamethoxyavones (data
not shown). Numerous studies link the effect of nobiletin
to the inhibition of MAPK and Akt signaling pathways and
the small non-receptor tyrosine kinases involved. This study
contributes to this knowledge and adds TNBC cell lines to
the list, while the effect of 5,6,7,3',4',5'-hexamethoxyavone
on signaling is novel and puts this avone in a similar group
as nobiletin and tangeretin, as potential chemopreventive or
therapeutic agent.
Ad dit iona l ly, both hexame thoxy avone s redu ced the phos -
phorylation levels of cell cycle checkpoint kinase 2 (Chk2)
and induced a subtle arrest at G2/M in the TNBC cell lines.
Usually, Chk2 is phosphorylated at T68 after DNA damage,
such as ionizing radiation and UV irradiation, however in the
Hs578T/Hs578Ts(i)8 cell model high phosphorylation levels
were seen in untreated or solvent treated conditions, which
suggests that repair mechanisms are constitutively active in
these cells. Furthermore, several reports indicate that Chk2
induces a cell cycle arrest at G1/S and G2/M (28). Our results
do not show a G1/S arrest, instead the hexamethoxyfla-
vones cause a G2/M arrest. This outcome is in contrast to
the G1/S arrest of nobiletin on MDA-MB-468 TNBC cells
reported by Chen et al (12). However, this study included
not only the effect on G2/M but also Chk2 phosphorylation
and is supported by previous studies in which particularly
Chk2 was proven to be required for the G2/M arrests trig-
gered by naturally-occurring chemopreventive agents (29).
On the other hand, G2/M arrests typically dependent on
a Chk1-associated signaling pathway leading to the inhibi-
tion of cyclin B1/Cdc2 activity, with Cdc2 also known as
cyclin dependent kinase 1 (CDK1). Chk1 is activated by
phosphorylation on S345 and subsequently inhibits Cdc25C
phosphatase by phosphorylating S216. This Cdc25C plays
an important role in the dephosphorylation and activation of
CDK1/Cdc2 on T14/Y15 needed for G2/M transition (30,31).
In this study, Chk1 phosphorylation was difcult to detect
in the Hs578T, whereas very weak levels were observed in
Hs578Ts(i)8 cells. Nobiletin and 5,6,7,3',4',5'-hexamethoxya-
vone did not seem to signicantly affect the phosphorylation
levels of Chk1, while the phosphorylation of Cdc2 was found
to be suppressed after 30 min and more, which suggests that
both hexamethoxyflavones induce the activation of Cdc2
needed for G2/M transition. These results are not in accor-
dance with the observed G2/M arrest, thus, it seems that
Chk1 and Chk2 may have additional roles in the nobiletin
and 5,6,7,3',4',5'-hexamethoxyavone-mediated G2/M arrest.
Interestingly, these observations are not uncommon, several
groups have demonstrated a G2/M cell cycle arrest following
the use of herbal derivatives. These studies mentioned that the
mechanism of G2/M arrest may be secondary to antimitotic
effects in contrast to checkpoint modulation late in G2 (32).
Furthermore, research using flavopiridol or silibinin with
other chemotherapeutic agents showed increased cytotox-
icity associated with G1 and G2 arrests, while avopiridol is
known to mediate its effect via inhibition of cdks, silibinin
in combination with doxorubicin was found to decrease the
expression of Cdc25C and Cdc2 (32). These correlations hint
that downregulation of G2/M cell cycle regulators resulting
in increased G2/M arrest could be a mechanism as well.
Further investigations will be needed to clarify the mecha-
nisms of G2/M arrest and determine whether nobiletin and
5,6,7,3',4',5'-hexamethoxyavone have an antimitotic effect
on the Hs578T and Hs578Ts(i)8 cells or suppress cell cycle
regulators.
In conclusion, our results indicate that 5,6,7,3',4',5'-hexa-
methoxyavone has several anticancer properties and could be
a more valuable component than nobiletin. This initial study
showed that 5,6,7,3',4',5'-hexamethoxyavone is less toxic than
nobiletin, and shows comparable growth inhibition of TNBC
cells. This effect could be ascribed in part to a suppression of
MAPK and Akt signaling pathways and to the induction of
a G2/M cell cycle arrest. Taken together, the present results
suggest that 5,6,7,3',4',5'-hexamethoxyflavone may possess
potential as a novel preventive or therapeutic agent in the treat-
ment of TNBC.
Acknowledgements
The present study was supported by the National Science
Foundation/EPSCoR Cooperative Agreement #IIA-1355423,
the South Dakota Research and Innovation Center, BioSNTR,
and by the State of South Dakota.
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