Download Tangeretin induces cell-cycle G1 arrest through inhibiting cyclin

Carcinogenesis vol.23 no.10 pp.1677–1684, 2002
Tangeretin induces cell-cycle G1 arrest through inhibiting
cyclin-dependent kinases 2 and 4 activities as well as elevating
Cdk inhibitors p21 and p27 in human colorectal carcinoma cells
Min-Hsiung Pan1,4, Wei-Jen Chen1, Shoei-Yn Lin-Shiau2,
Chi-Tang Ho3 and Jen-Kun Lin1*
Institutes of 1Biochemistry and 2Toxicology, College of Medicine,
National Taiwan University, Taipei, Taiwan, 3Department of Food Science,
Cook College, Rutgers University, New Brunswick, NJ 08901, USA and
4Department of Marine Food Science, National Kaohsiung Institute of
Marine Technology, Kaohsiung, Taiwan
Tangeretin (5,6,7,8,4’-pentamethoxyflavone) is concentrated
in the peel of citrus fruits. DNA flow cytometric analysis
indicated that tangeretin blocked cell cycle progression at
G1 phase in colorectal carcinoma COLO 205 cells. Over a
24 h exposure to tangeretin, the degree of phosphorylation
of Rb was decreased after 12 h and G1 arrest developed. The
protein expression of cyclins A, D1, and E reduced slightly
under the same conditions. Immunocomplex kinase experiments showed that tangeretin inhibited the activities of
cyclin-dependent kinases 2 (Cdk2) and 4 (Cdk4) in a dosedependent manner in the cell-free system. As the cells were
exposed to tangeretin (50 µM) over 48 h a gradual loss of
both Cdk2 and 4 kinase activities occurred. Tangeretin also
increased the content of the Cdk inhibitor p21 protein and
this effect correlated with the elevation in p53 levels. In
addition, tangeretin also increased the level of the Cdk
inhibitor p27 protein within 18 h. These results suggest that
tangeretin either exerts its growth-inhibitory effects through
modulation of the activities of several key G1 regulatory
proteins, such as Cdk2 and Cdk4, or mediates the increase
of Cdk inhibitors p21 and p27.
Introduction
Flavonoids are widespread in fruit and vegetables. They are
intensively studied for their role in human health, including
cancer prevention. Citrus flavonoids have a broad spectrum of
biological activity, including anticarcinogenic and antitumor
activities (1). It is suggested that cancer induction can be
prevented by ingestion of certain food ingredients (2), and
flavonoids, in citrus fruits and juices are among the most
prominent cancer-preventing agents (3–5). The biological
effects of flavonoids seem to occur mainly through their
interaction with protein tyrosine kinases (6) and cyclooxygenase (7). Polymethoxylated flavonoids, such as tangeretin
and nobiletin, are more potent inhibitors of tumor cell growth
than free hydroxylated flavonoids. They also possess potent
anti-invasive and anti-metastatic activities (8,9).
Tangeretin is a polymethoxylated flavone, 5,6,7,8,4’-pentamethoxyflavone, which is concentrated in the peel of citrus
Abbreviations: CAK, Cdk-activating kinase; Cdk, cyclin-dependent kinase;
CKI, Cdk inhibitor; EGCG, (-)-epigallocatechin-3-gallate; IP, immunoprecipitation; PCNA, proliferating cell nuclear antigen.
© Oxford University Press
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*To whom correspondence should be sent at Institute of Biochemistry,
College of Medicine, National Taiwan University, No 1, Section 1,
Jen-Ai Road, Taipei, Taiwan
Email: [email protected]
fruits (10) (Figure 1), and probably acts as a natural resistance
factor against pathogenic fungi (11). Grapefruit and orange
juice have been shown to inhibit human breast cancer cell
proliferation (12) and to interact with drug administration (13).
Several biological activities have been shown for tangeretin
itself, including the ability to enhance gap junctional intercellular communication (14), to counteract tumor promoterinduced inhibition of intercellular communication (15) and to
inhibit cancer cell proliferation (16).
Previous studies have shown that many flavonoids exhibit
potent antitumor activity against several rodent and human
cancer cell lines (17,18). The antitumor properties of some
flavonoids have been studied with respect to apoptosis and
cell cycle arrest. Quercetin has been shown to impair the G1
to the S phase transition in a human gastric cancer cell line
and also to cause apoptosis in several cell lines (19). The
molecular mechanisms of cell cycle arrest by flavonoids remain
largely unclear, but appear to involve modulation of multiple
cell cycle regulatory proteins. The eukaryotic cell cycle is
regulated through the sequential activation and inactivation of
cyclin-dependent kinases (Cdks) that drive cell cycle progression through the phosphorylation (20) and dephosphorylation
of several regulatory proteins.
In normal cells, Cdks exist predominantly in quaternary
complexes consisting of a Cdk, a cyclin, a proliferating cell
nuclear antigen (PCNA) and a 21 kDa protein (p21) (21). Cdk
activation requires cyclin binding and phosphorylation of
conserved threonine residue by Cdk-activating kinase (CAK).
The activated Cdk–cyclin complexes can be changed to an
inactive state by phosphorylation of a conserved threonine–
tyrosine pair or binding to Cdk inhibitory subunits (CKIs).
Progression from G1 to S phase in mammalian cells is regulated
by the accumulation of cyclins D, E and A, which bind to and
activate different Cdk catalytic subunits. The activation of
Cdk4–cyclin D and/or Cdk6–cyclin D complex is necessary
for transition from early to mid G1 phase. Transition through
mid G1 to S phase is regulated by activation of the Cdk2–
cyclin E complex. Progression through late G1 to S phase also
requires the presence of Cdk2–cyclin A complex (22). The
retinoblastoma tumor suppressor protein (Rb) is a critical target
protein that is phosphorylated via these Cdk–cyclin complexes
(23). Rb controled gene expression is mediated by a family of
heterodimeric transcriptional regulators, collectively termed
the E2F, which can transactivate genes whose products are
important for transition from G1 to S phase (24). Phosphorylation of Rb frees these regulators, enabling them to transactivate
the target genes. Therefore, the hypophosphorylated forms
of Rb are predominantly found in G0–G1 phase, but the
hyperphosphorylated forms of Rb are required during S and
G2–M phase (23).
Recent studies show that Cdk regulation involves a diverse
family of proteins, termed the CKIs (Cdk inhibitors), that bind
and inactivate Cdk–cyclin complexes. In mammalian cells
CKIs fall into two classes: (i) p21 (Cip1/Waf1/Cap20/Sdi1/
M.-H. Pan et al.
system and in cultured cells. The effects of these flavonoids
on the p53 protein levels were also investigated.
Materials and methods
Cell culture
The cell line COLO 205 (CCL-222; American Type Culture Collection) was
developed from a poorly differentiated human colon adenocarcinoma. Cell
lines were maintained in RPMI-1640 supplemented with 10% FCS (Gibco
BRL, Grand Island, NY), 1% penicillin/streptomycin–1% glutamate and kept
at 37°C in a humidified atmosphere of 5% CO2 in air.
Determination of cell growth curve
Human colon cancer (5⫻104) cells were plated in 35 mm petri dishes. The
next day, the medium was changed and various flavonoids were added. Control
cells were treated with DMSO to a final concentration of 0.05% (v/v). At the end
of incubation, cells were harvested for cell count using a hemocytometer (17).
Flow cytometric cell analysis
Cell cycle distribution was analyzed by flow cytometry as described previously
(35). Briefly, cells were trypsinized, washed once with PBS, and fixed in
100% ethanol for 1 h at –20°C. Fixed cells were washed with PBS, incubated
with 0.5 ml PBS containing 0.05% RNase and 0.5% Triton X-100 for 30 min
at 37°C and stained with propidium iodide. The stained cells were analyzed
using a FAScan laser flow cytometer (Becton Dickinson, San Jose, CA).
Fig. 1. Structures of the different flavonoids.
Pic1), p27 (Kip1), and p57 (Kip2) are related proteins with
a preference for Cdk2– and Cdk4–cyclin complexes; (ii)
p16INK4A, p15INK4B, p18INK4C and p19INK4D are closely
related CKIs specific for Cdk4– and Cdk6–cyclin complexes
(25). The majority of in vivo studies suggest that the inhibitory
effect of p21 is largely exerted during the G1 phase of the cell
cycle, with preferential binding to Cdk4- and Cdk2- containing
complexes, and that it either inhibits their kinase activities
or prevents their activation by CAK (26). In addition, the
regulation of p21 is largely dependent on the presence of
functional p53, a transcriptional regulator that mediates cell
cycle arrest following DNA damage (27) and in senescence
(28). However, the expression of p21 in a variety of tissues
from p53 null mice suggests that it is also regulated by a
p53-independent mechanism (29). These effects probably
play a role in the ability of p53 to act as a tumor suppressor.
In addition, a number of chemopreventive agents have
been shown to exert their anti-tumorigenic activity through
p53-dependent mechanisms (30–33).
In this study, we examined the effects of tangeretin and
other flavonoids on the growth of colorectal carcinoma COLO
205 cells, expression of G1 cyclins, phosphorylation state of
Rb, and kinase activities of Cdk2 and Cdk4 in a cell-free
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Western blot analysis
Equal amounts of total cellular proteins (50 µg) were resolved by SDS–
polyacrylamide gel electrophoresis (PAGE), transferred onto polyvinylidene
difluoride (PVDF) membranes (Amersham, Arlington, IL), and then probed
with primary antibody followed by secondary antibody conjugated with
horseradish peroxidase or alkaline phosphatase. The immunocomplexes were
visualized using enhanced chemiluminescence kits (α-tubulin, Rb, cyclin A,
cyclin E, Cdk2, Cdk4, p21, p27, p53 and cyclin D1).
In vitro and cell culture Cdks kinase assay
For in vitro Cdks kinase assay, exponentially growing COLO 205 cells were
washed with cold PBS and lysed with Gold lysis buffer [10% glycerol, 1%
Triton X-100, 1 mM sodium orthovanadate, 1 mM EGTA, 5 mM EDTA,
10 mM NaF, 1 mM sodium pyrophosphate, 20 mM Tris–HCl pH 7.9, 100 µM
β-glycerophosphate, 137 mM NaCl, 1 mM PMSF, 10 µg/ml aprotinin and
10 µg/ml leupeptin] for 30 min at 4°C. The cell lysate was clarified by
centrifugation at 12 000 g for 10 min at 4°C. 4 mg of protein were incubated
with anti-Cdk2 or anti-Cdk4 antibody and protein A/G plus agarose (Santa
Cruz Biotechnology, Santa Cruz, CA) for 18 h at 4°C. The immunoprecipitate
was washed three times with immunoprecipitate buffer [1% Triton X-100,
150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM
sodium vanadate, 0.2 mM PMSF, 0.5% NP-40] and three times with kinase
buffer (50 mM Hepes, pH 7.4, 10 mM MgCl2, 2.5 mM EDTA, 10 mM
β-glycerophosphate, 1 mM NaF, 1 mM DTT for Cdk4; 50 mM Hepes,
pH 7.4, 10 mM MgCl2, 2.5 mM EDTA, 1 mM DTT for Cdk2) another three
times and separated into 6–8 tubes. The kinase reactions were carried out in
a final volume of 40 µl containing 2 µg histone H1 (Calbiochem, CA) or
1 µg Gst-Rb (Santa Cruz), 20 µM cold ATP, 5 µCi [γ-32P]ATP (5000 Ci/mmol,
Amersham) and incubated for 20 min at 25°C. Each sample was mixed with
10 µl of 5⫻Laemmli’s loading buffer to stop the reaction, heated for 10 min
at 100°C, and subjected to SDS–PAGE. The gels were dried, visualized by
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Materials
Apigenin, kaempferol, myricetin, quercetin, rutin, luteolin and all protease
inhibitors were purchased from Sigma (St Louis, MO). Tangeretin and nobiletin
were isolated from orange peel extract rich in polymethoxyflavones which
was provided by Florida Flavors Inc. (Lakeland, FL). The extract (5 g) was
subjected to silica gel column chromatography. The column was eluted with
chloroform containing increasing amounts of ethyl acetate followed by ethyl
acetate and methanol. Tangeretin and nobiletin were isolated as two major
products at 72 mg and 52 mg, respectively. The purity of these two compounds
were ⬎⬎98% as judged by HPLC. Tangeretin and nobiletin were identified
by comparison of 1H and 13C NMR spectra with literature values (34). The
antibodies of p27, cyclins A, D1, E and Cdk2 were obtained from the Santa
Cruz Biotechnology (Santa Cruz, CA); anti-p21 monoclonal antibody was
obtained from Transduction Laboratory (Lexington, KY); anti-α-tubulin monoclonal antibody from Oncogene Science (Cambridge, UK); anti-Rb monoclonal
antibody and Cdk4 from Upstate Biotechnology (Lake Placid, NY); p53 from
Oncogene Research Products (Cambridge, MA); anti-pRb antibody from Cell
Signaling (Beverly, MA). It is important to be precise about which antibodies
are used, especially with respect to anti-phospho-Rb.
Tangeretin induces G1 arrest in human colorectal carcinoma cells
Table I. Effect of different flavonoids on the growth of COLO 205 cells
Compounds
Apigenin
Kaemperol
Myricetin
Qucercetin
Rutin
Luteolin
Tangeretin
Nobiletin
IC50 (µM)
⬎100
⬎100
⬎100
⬎100
⬎100
47.6 ⫾ 0.15
37.5 ⫾ 0.12
66.2 ⫾ 1.25
Cells were treated with various concentrations of flavonoids for 24 h. The
numbers of viable cells were determined by counting the trypan blueexcluding cells in a hemocytometer. Each experiment was independently
performed three times and expressed as mean ⫾ SE.
Results
Several flavonoids inhibit cell proliferation
Previous studies have shown that flavonoids are potent antiproliferation (17–18) and anticancer agents (36). Here we
investigated the anti-proliferation of eight structurally related
flavonoids: apigenin, luteolin, quercetin, rutin, tangeretin,
nobiletin, myricetin and kaempferol. The structures of these
flavonoids are illustrated in Figure. 1. To assess the inhibitory
effects of selected flavonoids on the growth of colon cancer
cells, we first determined the growth rates of COLO 205 colon
cancer cells. Exponentially growing cultures of COLO 205
cells were continuously cultured in the absence or presence of
different concentrations of flavonoids. After 48 h of treatment,
the cell growth rates were determined by trypan blue exclusion.
As shown in Table I, luteolin and nobiletin induced a dosedependent inhibited cell proliferation, assuming an IC50 value
of ~47.6 µM and 66.2 µM, but the potencies of their inhibition were lower than that by tangeretin (IC50, 37.5 µM).
Tangeretin strongly inhibited COLO 205 cell growth (Table I).
Exponentially growing COLO 205 cultures rapidly underwent
growth inhibition with the addition of various concentrations
of tangeretin, as evidenced by a decrease of cell proliferation
over the experimental period (Figure 2).
Tangeretin induces G0/G1 cell cycle arrest in COLO 205 cells
In this study, we demonstrated that tangeretin induced
significant growth inhibition of human colon cancer cells
(Figure 2). In order to determine whether tangeretin has a cell
cycle arrest effect in human COLO 205 colon cancer cells,
the cells treated with DMSO or tangeretin for 12 and 24 h
were subjected to flow cytometric analysis after staining their
DNA. Histograms of flow cytometric data are shown in Figure
3. Control cells (DMSO) progressed through the cell cycle
well. In contrast, tangeretin-treated COLO 205 cells were
blocked in the G0/G1 phase (over 90%) after 24 h treatment.
Effects of tangeretin on pRb phosphorylation, cyclins and Cdks
2 and 4 protein expression
The phosphorylation mediated by both cyclin D/Cdk4 and
cyclin E/Cdk2 of the Rb protein is required for the cells to
Fig. 2. Effects of tangeretin on the growth of COLO 205 cells. Cells were
treated without (d) or with various concentrations of tangeretin and cell
number was determined every 12 h thereafter, the numbers of viable cells
were determined by counting the trypan blue-excluding cells in a
hemocytometer. Data represent means ⫾ SE.
Fig. 3. Tangeretin caused cell cycle arrest at G0/G1 phase. Cells were
treated with DMSO (0.05%) or tangeretin (50 µM) for indicated time, and
cell cycle analysis was measured as described in Materials and methods.
Data shown are representative of at least three independent experiments.
progress from G1 into S phase in those cells possessing a
functional pRb. To elucidate the arrest point of tangeretintreated COLO 205 cells in the G1 phase we analyzed the
phosphorylation state of pRb and the expression of G1 cyclin
protein and Cdks 2 and 4. As shown in Figure 4A, the degree
of phosphorylation of Rb was decreased after 12 h of 50 µM
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autoradiography, and quantitated by densitometry (IS-1000 Digital Imaging
System).
For cell culture Cdks kinase assay, cells (2⫻105/dish) were precultured in
100 mm plastic dishes for 2 days and treated for various periods. Cell lysis
and immunoprecipitation were performed as described above. Kinase assay
was carried out in 50 µl of kinase buffer with Cdk2 or Cdk4-immunoprecipitate
from 250 µg of protein of the lysate. The kinase reaction was performed as
described above.
M.-H. Pan et al.
tangeretin treatment compared with total Rb protein. Exposure
to 50 µM tangeretin, the levels of cyclins A, D1 and E were
analyzed by immunoblotting over a 24 h period. As shown in
Figure 4B and C, cyclin A, D1 and E levels did not change with
increasing time of 0.05% DMSO exposure, while tangeretin
appeared to have a suppressing effect on the levels of cyclin
A, cyclin D1 and E. By contrast, there was no change in the
protein expression of Cdks 2 and 4, which are associated
with Rb phosphorylation. However, small changes in the levels
of cyclins were not sufficient to explain the reduction of the
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phosphorylation of Rb. Therefore, we subsequently investigated the effects of tangeretin on the activities of Cdks 2 and
4 in a cell-free system and in cultured cells, as well as its
effects on the expression of Cdk inhibitory subunits (CKIs).
Effects of tangeretin on the activities of Cdks 2 and 4 in a
cell-free system and in cultured cells
To examine whether tangeretin would directly inhibit Cdks in
COLO 205 cells, we first evaluated the effect of tangeretin on
the kinase activities of Cdks 2 and 4 in cell-free systems.
The kinase activities of Cdks 2 and 4 were measured after
immunoprecipitation from exponentially growing COLO 205
cells using anti-Cdk2 or anti-Cdk4 antibodies, then tangeretin
was added and histone H1 (for Cdk2) or Gst–Rb fusion protein
(for Cdk4) as substrates. As shown in Figures 5A and 6A,
the in vitro inhibition of Cdks 2 and 4 by tangeretin was
concentration-dependent, assuming an IC50 of ~26.5 µM and
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Fig. 4. Effects of tangeretin on the Rb protein phosphorylation and the
protein expression of G1 cyclins as well as Cdk2 and Cdk4. (A) COLO 205
cells were treated with 50 µM of tangeretin for the time indicated (top). The
phosphorylated Rb and total Rb protein were detected with specific
antibodies. (B) COLO 205 cells were treated with 50 µM of tangeretin or
(C) DMSO [final concentration of 0.05% (v/v)] for different time periods.
The cell lysis and Western blotting were performed as Materials and
methods indicate. Western blot data presented are representative of those
obtained in at least three separate experiments.
Fig. 5. Effects of tangeretin on the activities of Cdk2 kinase in cell-free
system and cultured cell. (A) Cdk2 immuno complex was prepared from
growing COLO 205 cells and reacted with [γ-32P]ATP, various
concentrations of tangeretin and substrates (histone H1) for 20 min at room
temperature as described under Materials and methods. Quantification of the
phosphorylated-histone H1 was performed by densitometric analysis
(IS-1000 Digital System). Data represent the means ⫾ SE of three samples.
(B) COLO 205 cells were treated with 50 µM of tangeretin for increasing
periods. (C) Cells were treated with different concentrations of tangeretin
for 24 h. Total cell lysates were used for immunoprecipitation, the kinase
activities were assayed with histone H1 as substrates. The experiments were
performed as described under Materials and methods. Data shown are
representative of at least three independent experiments.
Tangeretin induces G1 arrest in human colorectal carcinoma cells
19.2 µM for Cdk2 and Cdk4 respectively. We next examined
the activities of Cdks 2 and 4 from tangeretin-treated COLO
205 cells. The cells were treated with 50 µM tangeretin for
increasing periods, and the kinase activity was then determined
by the immunoprecipitation method described above. As shown
in Figure 5B and 6B, the kinase activities of both Cdks 2 and
4 were inhibited in a time-dependent manner, and ⬎90% of
the activities were inhibited compared with untreated control
cells (100%) after 12 h treatment. On the other hand, the
inhibition of Cdks 2 and 4 activities by tangeretin were
also concentration-dependent in cultured COLO 205 cells
(Figure 5C and 6C). These findings were consistent with the
lack of Cdks 2 and 4 activities in tangeretin-treated cells,
the underphosphorylated form of Rb, and the failure of these
cells to progress from G1 to S phase.
Effects of tangeretin on protein expression of Cdk inhibitors
p27Kip1, p21Cip1and p53 in colon cancer cells
To examine further whether tangeretin could induce other
members of the Cdk inhibitor protein family, we investigated
the effect of tangeretin on the expression of p27 and p21
proteins COLO 205 cells. The expression of p27 protein was
significantly increased within 18 h after exposure to 50 µM
tangeretin (Figure 7A). As with p27 protein, tangeretin treatment
induced a significant increase of p21 protein. We studied further
the effect of tangeretin on the levels of p27 and p21 proteins. As
shown in Figure 7B, tangeretin markedly up-regulated the level
of p27 and p21 in a concentration-dependent manner. The
increase of p21 is reported to be regulated by either a p53dependent or p53-independent mechanism. To determine
whether the growth-inhibitory response to tangeretin was
dependent on the p53 status, additional experiments were
performed as described below, and it was found that p53
protein was also induced by tangeretin in COLO 205 cells.
Effects of tangeretin and related compounds on the expression
of p53 in COLO 205 colon cancer cell line
The p53 tumor suppressor has been shown to be a key regulator
in interpreting the extrinsic signals that induce cell cycle arrest
(37). Flavonoids such as apigenin have been reported to induce
the expression of p53 protein in mouse fibroblasts (38). To
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Fig. 6. Effects of tangeretin on the activities of Cdk4 kinase in cell-free
system and cultured cell. (A) Cdk4 immuno complex was prepared from
growing COLO 205 cells and reacted with [γ-32P]ATP, various
concentrations of tangeretin and substrates (GST-Rb) for 20 min at room
temperature as described under Materials and methods. Quantification of the
phosphorylated-GST-Rb was performed by densitometetric analysis (IS-1000
Digital System). Data represent the means ⫾ SE of three samples. (B)
COLO 205 cells were treated with 50 µM of tangeretin for increasing
periods. (C) Cells were treated with different concentrations of tangeretin
for 24 h. Total cell lysates were used for immunoprecipitation, the kinase
activities were assayed with GST-Rb as substrates. The experiments were
performed as described under Materials and methods. Data shown are
representative of at least three independent experiments.
Fig. 7. Effects of tangeretin on protein expression of p27, p21 and p53 in
COLO 205 cells. (A) COLO 205 cells were treated with 50 µM of
tangeretin for the time indicated. (B) Cells were treated with various
concentrations of tangeretin for 24 h. Cell lysis and western blotting were
performed as described under Materials and methods. Data shown are
representative of at least three independent experiments.
M.-H. Pan et al.
investigate the structure–activity relations the effects of selected
flavonoids on the expression of p53 were examined by western
blotting. Among these compounds, tangeretin and nobiletin
exhibited rather strong induced activities in the COLO 205 cells
(Figure 8A). However, tangeretin was the most potent inducer
of p53 and induced p53 protein expression in a dose-dependent
manner (Figure 8B). These results suggest that p53 protein plays
a critical role in the tangeretin-induced G0/G1 cell cycle arrest.
Discussion
Flavonoids are naturally occurring plant polyphenols found in
abundance in diets rich in fruit, vegetable and plant-derived
beverages such as tea. Epidemiological studies have shown
that the intake of certain vegetables, fruits and tea in the daily
diet provides effective cancer prevention (39). Polymethoxyflavonoids, including tangeretin, occur exclusively in citrus
fruits, and the Dancy tangerine has the highest total, containing
approximately five times the amount found in the peel of
sweet orange varieties (40). Citrus flavonoids have a broad
spectrum of biological activity including anticarcinogenic and
antitumor activities. Polymethoxylated flavonoids, such as
tangeretin and nobiletin, are more potent inhibitors of tumor
cell growth than hydroxylated flavonoids (1). Tangeretin was
reported to inhibit tumor invasion and metastasis (8) and to
induce apoptosis in HL-60 cells (9). In addition, nobiletin was
reported to induce differentiation of mouse myeloid leukemia
cells (41), to exhibit anti-proliferative activity towards a human
squamous cell carcinoma cell line (42), to exert antimutagenic
activity (43), to suppress the induction of matrix metalloproteinase-9 (44), and to inhibit phorbol ester-induced skin
inflammation, oxidative stress, and tumor promotion in mice
(5). Our previous report demonstrated that (-)-epigallocatechin3-gallate (EGCG) could block cell cycle progression in the
G1 phase in human breast carcinoma MCF-7 cells (34). In
the present study, we showed that tangeretin also arrested
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Fig. 8. Effects of tangeretin and related compounds on protein expression of
p53 in COLO 205 cells. (A) COLO 205 cells were treated with 50 µM of
various compounds for 24 h (C, control; A, apigenin; K, kaempferol; M,
myricetin; R, rutin; Q, quercetin; T, tangeretin; N, nobiletin; L, luteolin).
(B) COLO 205 cells treated with increasing doses of tangeretin for 24 h.
Cell lysis and western blotting were performed as described under Materials
and methods. Data shown are representative of at least three independent
experiments.
cell cycle progression in the G1 phase in human colorectal
carcinoma COLO 205 cells (Figure 3). As shown in Figure 2,
among selected flavonoids, tangeretin was more potent to
inhibit COLO 205 cell proliferation. Previous studies showed
that many flavonoids, such as apigenin, genistein and quercetin,
which have a different structure than tangeretin, induce G2/M
arrest in several cell lines (45–47). On the other hand, many
flavonoids also induce G1 arrest (47). These results suggest
that arrest of the cell cycle by flavonoids or tangeretin is
an effect which is dependent on the structural groups of
these agents.
Our previous studies have shown that EGCG exerts
inhibitory activity against Cdk2 in cell-free systems (35).
Tangeretin also exhibited a concentration-dependent inhibitory
effect, not only on Cdk2 but also on Cdk4 obtained from
cycling COLO 205 cells, resulting in an IC50 of ~26.5 µM
and 19.2 µM, respectively (Figure 5A and 6A). The kinase
activities of Cdks 2 and 4 were inhibited by 50 µM tangeretin
in a time-dependent (Figure 5B and 6B) and a dose-dependent
(Figure 5C and 6C) manner in cultured cells. The Cdks 2 and
4 inhibition due to these direct and indirect actions inhibited
phosphorylation of Rb by tangeretin (Figure 4A). However,
Cdk activation and inhibition also requires phosphorylation or
dephosphorylation at some conserved amino acid residues
(20). It is therefore possible that tangeretin inhibits the Cdkactivating kinases (CAKs) or activates the Cdk-inactivating
phosphatases, which are regulators for Cdks. Additional studies
are needed to determine whether the inhibition of CAKs or
activation of Cdk-inactivating phosphatases contributes to
the inhibition of Cdks 2 and 4. In addition, Cdk6 might
phosphorylate pRb in cells. Issues of whether or not tangeretin
directly inhibits Cdk6 activity remain to be addressed.
There was little change in the levels of cyclin D1 and E
after 24 h of tangeretin exposure (Figure 4B). But, it seems
that the protein amount of cyclins was not the main causative
effect on the activities of Cdks in tangeretin-treated COLO
205 cells. However, we cannot rule out the possibility that
tangeretin might block cyclin binding to Cdk, thereby inhibiting
the kinase complex activities of Cdks.
The tumor suppressor, p53, has been implicated in a variety
of cellular processes (48). However, the undisputed roles of
p53 are the induction of cell growth arrest and apoptosis (27).
Among the transcriptional targets of p53, the Cdk inhibitor
p21Cip1 plays a key role in mediating G1 arrest (49). Another
CKI is p27Kip1, which mediates growth arrest and is thought
to play a critical role in negative regulation of cell division
in vivo (22,50). The outcome of CKIs induction in most cells
is cessation of cell proliferation, differentiation, or even cell
death. Since an inhibition of CKIs activity is one of the factors
causing uncontrolled proliferation of tumor cells, one possible
strategy to control cancer cell proliferation is to induce CKIs
expression, which would lead to G0/G1 arrest and stop tumor
cell growth. In this study, we observed that tangeretin can
cease cell proliferation in COLO 205 cells which possess
functional p53 and induce the increase of p53 protein in a
time-dependent manner. G1 arrest produced by tangeretin
occurred with an accompanying p21Cip1 and p27Kip1 accumulation (Figure 7). The p21Cip1 continued to show a markedly
up-regulation from 18 h to 24 h after exposure to tangeretin.
Inhibition of Cdk activity might also have occurred through
up-regulation of p21Cip1 by tangeretin. These effects might be
triggered by an increased expression of p53 by tangeretin. In
this study, tangeretin also increased the p27 protein level in
Tangeretin induces G1 arrest in human colorectal carcinoma cells
Acknowledgments:
This study was supported by the National Science Council NSC 90-2320-B002-163, NSC 90-2320-B-002-164, NSC 90-2313-B-022-004; by the National
Health Research Institute, NHRI-EX918913BL; by the National Research
Institute for Chinese Medicine, NRICM 90102; and by the Ministry of
Education, ME 89-B-FA01-1.
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