Hesperetin Induced G1-Phase Cell Cycle Arrest in Human Breast Cancer MCF-7 Cells: Involvement of CDK4 and p21

NUTRITION AND CANCER, 59(1), 115–119
          C 2007, Lawrence Erlbaum Associates, Inc.

        Hesperetin Induced G1-Phase Cell Cycle Arrest in Human Breast
             Cancer MCF-7 Cells: Involvement of CDK4 and p21

                                                                Eun Jeong Choi

Abstract: This study was to investigate the effects of hes-                      nature. Dietary hesperidin is deglycosylated to hesperetin
peretin on cell proliferation and cell cycle arrest and ex-                      by intestinal bacteria prior to absorption (12), and hesperidin
plored the mechanism for these effects in breast carcinoma                       may be considered a pro-drug, which is metabolized to hes-
MCF-7 cells. Hesperetin significantly inhibited cell prolifer-                   peretin (13). In several animal studies of the absorption,
ation in a dose-dependent manner after treatment for 48 and                      bioavailability, and pharmacokinetics of hesperidin, hes-
72 h and resulted in significant cell cycle arrest in the G1                     peridin has not been observed in plasma, bile, or urine (14–
phase. In the G1-phase related proteins, hesperetin down-                        16). Several reports have described the beneficial biological
regulates the cyclin-dependent kinases (CDKs) and cyclins                        effects of hesperidin. However, these studies may have in-
and upregulates p21Cip1 and p27Kip1 in cells treated with                        advertently investigated the biological effects of hesperidin
hesperetin for 48 h and 72 h. After 72 h treatment, these phe-                   because hesperidin is not found in the body. To understand
nomenons were more pronounced. Hesperetin treatment at                           the biological activities of hesperidin, it is necessary to study
high concentration for 72 h resulted in a decrease in CDK2                       the molecule’s biologically active form. Moreover, relatively
and CDK4 together with cyclin D. In addition, hesperetin                         little has been reported about hesperetin in comparison with
increases the binding of CDK4 with p21Cip1 but not p27Kip1                       hesperidin, and only a few in vitro studies have assessed it.
or p57Kip2 . Taken together, our data suggest for the first time                 Hesperetin is reported to be a powerful radical scavenger
that the regulation of CDK4 and p21Cip1 may participate                          and to promote cellular antioxidant defense-related enzyme
in the anticancer activity pathway of hesperetin in MCF-7                        activity (17–18). Hesperetin has also shown potential anti-
cells.                                                                           carcinogenic activity as an antiangiogenic agent in mES cells
                                                                                 (19). Thus, hesperetin may be useful in nutritional therapy
                                                                                 and chemotherapy. We investigated the possible anticarcino-
                                                                                 genic effects of hesperetin through the inhibition of cell pro-
                                                                                 liferation and cell cycle arrest in human breast carcinoma
                                                                                 MCF-7 cells, which are regarded as an in vitro breast cancer
   Flavonoids are the most abundant natural antioxidants
in food. Several epidemiological studies have supported the
hypothesis that the antioxidant action of flavonoids may re-
duce the risk of developing cancer and cardiovascular disease
                                                                                                      Materials and Methods
(1–4). Reports based on the in vitro action of flavonoids on
cancer cells have found various anticancer effects such as
                                                                                 The Cells Culture and Hesperetin Treatment
the inhibition of cell proliferation and kinase activity and the
induction of apoptosis (4,5–7).                                                      MCF-7 cells were purchased from the Korean Cell Line
   Flavonoids have been shown to inhibit the proliferation                       Bank (Seoul, Korea). Cells were routinely maintained in
of cultured human cancer cell lines (8–9). The observed an-                      RPMI 1640 (Gibco BRL, MD), supplemented with 10%
tiproliferative property of flavonoids suggests that these com-                  fetal bovine serum and antibiotics (50 U/ml of penicillin
pounds may inhibit the cell cycle or induce apoptosis (10).                      and 50 µg/ml streptomycin; Gibco) at 37◦ C in a humid-
However, many of the mechanisms underlying the potential                         ified atmosphere containing 5% CO2 . Cells were treated
anticancer activity of flavonoids are not fully understood.                      with hesperetin ranging from 1 to 100 µM for 24 h, 48 h,
   Hesperetin (3 ,5,7-trihydroxy-4-methoxyflavanone), a                         and 72 h. Hesperetin was purchased from Sigma and dis-
member of the flavanone subclass of flavonoids, is found                         solved in dimethyl sulfoxide (DMSO; final concentration
in fruit sources including various citrus species (11).                          0.1% in medium). All experiments were done in triplicate or
Hesperetin occurs as hesperidin (its glycoside form) in                          quadruple.

   E. J. Choi is affiliated with the Plant Resources Research Institute, Duksung Women’s University, Seoul, South Korea.
Cell Proliferation and Cell Death Assay
   Cell proliferation was determined using the methyl thia-
zolyl tetrazolium (MTT) assay. At 24 h, 48 h, and 72 h point,
the cells exposed to hesperetin were added to MTT. DMSO
was added 4 h later to each well to dissolve the resulting for-
mazan crystals, and then absorbance was recorded at 490 nm
in a microplate reader SpectraMax Plus; Molecular Devices,

Cell Cycle Distribution
    Cells were then harvested, washed with cold phosphate
buffered solution (PBS), and processed for cell cycle analy-
sis. Briefly, the cells were fixed in absolute ethanol and stored   Figure 1. Effect of hesperetin on cell proliferation of MCF-7 cells. Cells
at –20◦ C for later analysis. The fixed cells were centrifuged      were exposed to either vehicle (0.1% dimethyl sulfoxide in medium) or
                                                                    hesperetin (1–100 µM) and incubated for 24 h, 48 h, and 72 h. All data
at 1,000 rpm and washed with cold PBS twice. Ribonucle-
                                                                    are reported as the percentage change in comparison with the vehicle-only
ase A (20 µg/ml final concentration) and propidium iodide           group, which were arbitrarily assigned 100% viability. ∗ , P < 0.001, signif-
staining solution (50 µg/ml final concentration) was added          icantly different from the vehicle-only group (hesperetin concentration =
to the cells and incubated for 30 min at 37◦ C in the dark.         “0”).
The cells were analyzed a FACS Calibur instrument (BD
Biosciences, San Jose, CA) equipped with CellQuest version
                                                                    tured in sample buffer, and then analyzed by immunoblotting
3.3 software. ModFit LT version 3.1 trial cell cycle analysis
                                                                    using p21Cip1 , p27Kip1 , and p57Kip2 .
software was used to determine the percentage of cells in the
different phases of the cell cycle.

                                                                    Statistical Analyses
Immunoblotting and Immunoprecipitation Assay
                                                                       All data were expressed as percent compared with vehicle-
    Cells were lysed in radio-immunoprecipitation assay             treated control cells, which were arbitrarily assigned 100%.
buffer (1% NP-40, 150 mM NaCl, 0.05% 4-chloro-2,5-                  Data were analyzed by one-way analysis of variance fol-
dimethoxyamphetamine, 1% sodium dodecyl sulfate (SDS),              lowed by Dunnett’s multiple comparison test (Sigma Stat,
50mM Tris, pH 7.5) containing protease inhibitor for 1 h            Jandel, San Rafael, CA). For all comparisons, differences
at 4◦ C. The supernatant was separated by centrifugation,           were considered statistically significant at P < 0.05.
and protein concentration was determined by Bradford
protein assay kit II (Bio-rad Laboratories, CA). Proteins
(25 µg/well) denatured with sample buffer were separated
by 10% SDS-polyacrylamide gel. Proteins were transferred
onto nitrocellulose membranes (0.45 µm). The membranes
                                                                    Inhibition of Cell Proliferation by Hesperetin
were blocked with a 1% bovine serum albumin solution
for 3 h, washed twice with PBS containing 0.2% Tween-                  The effects of hesperetin on proliferation of human breast
20, and incubated with the primary antibody overnight at            cancer MCF-7 cells were measured using an MTT assay. The
4◦ C. Antibodies against cyclin-dependent kinases (CDKs)            treatment of MCF-7 cells with hesperetin for 24 h did not
CDK2, CDK4, CDK6, cyclin D, cyclin E, p16INK4a , p18INK4c ,         affect cell proliferation (Fig. 1). Hesperetin significantly de-
p21Cip1 , p27Kip1 , p57Kip2 , and β-actin were purchased from       creased cell proliferation after 48 h and 72 h of treatment in
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and used to         a dose- and time-dependent manner (P < 0.05). Cell prolif-
probe the separate membranes. The next day, the immunore-           eration was decreased up to 21.07% with 100 µM hesperetin
action was continued with the secondary goat antirabbit             for 72 h compared to the control.
horseradish-peroxidase–conjugated antibody after washing
for 2 h at room temperature. The specific protein bands were
detected by Opti-4CN Substrate kit (Bio-rad Laboratories).
                                                                    Hesperetin-Induced Cell Cycle Arrest
    For CDK4-CDK inhibitors binding assay, cells were
treated with vehicle or 100 µM of hesperetin for 48 h and              Hesperetin-induced cell cycle arrest in MCF-7 cells was
72 h. CDK4 from cell lysates (250 µg protein) was immuno-           investigated (Fig. 2A). Dose-dependent, hesperetin-induced
precipitated using anti-CDK4 antibody (4 µg) and precleared         cell cycle arrest in the G1 phase was observed when MCF-7
protein A/G-plus agarose beads (Santa Cruz) for overnight           cells were treated with hesperetin for 48 h or 72 h but not
at 4◦ C. The precipitates were washed with lysis buffer, dena-      for 24 h. A significant increase in the number of G1-phase

116                                                                                                         Nutrition and Cancer 2007
Figure 2. Effect of hesperetin on cell cycle distribution of MCF-7 cells. Values are expressed as percentage of the cell population in the G1, S, and G1/M
phase of cell cycle. Cells were exposed to either vehicle (0.1% dimethyl sulfoxide in medium) or hesperetin (1–100 µM) and incubated for 48 h and 72 h.
∗ , P < 0.05, significantly different from the vehicle-only group (hesperetin concentration = “0”).

cells was seen with hesperetin treatment at concentrations
of 50 and 100 µM (61.6% and 67.2% of the cell population
compared with control value 49.2%, respectively, P < 0.05).
After exposure to 10, 50, and 100 µM hesperetin for 72 h,
we observed that the proportion of MCF-7 cells in the G1
phase increased from 50.9% to 62.1%, 50.9% to 65.3%, and
50.9% to 77.0%, respectively, and the proportion of S-phase
cells decreased from 39.8% to 26.8%, 39.8% to 25.8%, and
39.8% to 15.4%, respectively.
   The effect of hesperetin on the expression of G1-involved
cell cycle regulatory proteins are shown in Fig. 3A. Hes-
peretin treatment resulted in a marked reduction in the ex-
pression of CDK4 in a dose-dependent manner after 48 and
72 h of treatment. Similarly, the expression of cyclin D was
inhibited after 48 h of 50 µM hesperetin. A reduction in
the expression of CDK2 and cyclin E was observed at the
maximum concentration of 100 µM hesperetin for 72 h. The
inhibitors of CDK 4 (INK4), p16INK4a ,and p18INK4c did not
differ compared with vehicle-treated MCF-7 cells. The ex-
pression levels of the p21Cip1 and p27Kip1 were increased
in response to hesperetin treatment compared with vehicle-
treated MCF-7 cells. When cells were treated with hesperetin
for 24 h, expression of cell cycle regulatory proteins were not
affected (data not shown).
   Also, the formation of CDK4–p21Cip1 in response to hes-
peretin treatment was increased in MCF-7 cells, although
CDK4–p27Kip1 and CDK4–p57Kip2 complex levels did not
change (Fig. 3B).
                                                                                Figure 3. Effect of hesperetin on genes related G1 phase of MCF-7 cells.
                                                                                Protein expression (A) and binding assay of cyclin-dependent kinase (CDK)
                                                                                4 and CDK inhibitory subunits (CKIs) (B) [immunoprecipitation (IP)]. Cells
                                                                                were exposed to either vehicle (0.1% dimethyl sulfoxide in medium) or
Hesperetin-Induced Apoptosis                                                    hesperetin (1–100 µM) and incubated for 48 h and 72 h.

   To investigate whether apoptosis was induced by hes-
peretin in MCF-7 cells, we measured the expression of Bcl-2                                                   Discussion
and Bcl-2 associated x protein (Bax) as markers of apop-
tosis (Fig. 4). Hesperetin decreased the expression of Bcl-2                       It is well known that flavonoids act as general cell growth
and increased that of Bax in a dose-dependent manner at                         inhibitors (20). This biological capacity of flavonoids sug-
48 h. More pronounced changes were observed in cells                            gests their potential use in cancer chemotherapy. Hesperetin
treated with hesperetin for 72 h.                                               inhibits vessel structure formation (an antiangiogenic effect)

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and p21Cip1 , indicating that the inhibition of cell prolifera-
                                                                              tion and the contribution to the apoptotic process is a possible
                                                                              mechanism by which hesperetin acts on cancer cells.
                                                                                  Moreover, the G1-phase cell cycle arrest induced by hes-
                                                                              peretin resulted in apoptosis in MCF-7 cells. Hesperetin
                                                                              induced a dose- and time-dependent decrease in Bcl-2 ex-
                                                                              pression and increase in Bax expression in MCF-7 cells.
                                                                              Apoptosis is a multistep process important in controlling cell
Figure 4. Effect of hesperetin on Bcl-2 and Bcl-2 associated x protein        number and proliferation as part of normal development;
(Bax) expression of MCF-7 cells. Cells were exposed to either vehicle (0.1%   the Bax genes and members of the Bcl-2 family such as
dimethyl sulfoxide in medium) or hesperetin (1–100 µM) and incubated for      bcl-2 are involved in the control of apoptotic pathways. De-
48 h.
                                                                              creased expression of Bcl-2 and increased expression of Bax
                                                                              are correlated with the response to anticancer compounds
in mouse embryonic stem cells, suggesting that its inhibitory                 for chemotherapy in cancer cell lines. In addition, the loss
effects may be associated with its prooxidant activity (19).                  of Bcl-2 expression can promote the induction of apoptosis
Thus, hesperetin is a potential anticancer agent. Moreover,                   (27–29). An anticancer drug that initiates apoptosis may be
unlike other flavonoids that have prooxidant activity, no                     useful as a new category of chemotherapeutic agent.
hesperetin-induced cytotoxicity was observed, even at high                        In conclusion, our results indicate that hesperetin treat-
concentrations (100 µM). Thus, we investigated the anti-                      ment leads to the inhibition of cell proliferation, the induction
cancer effects of hesperetin on breast carcinoma MCF-7 cells                  of cell cycle arrest at the G1 phase, and apoptosis. Addition-
in which hesperetin inhibits cell proliferation by causing cell               ally, our data suggest for the first time that the regulation of
cycle arrest.                                                                 CDK4 and p21Cip1 may participate in the anticancer activ-
   To assess whether the hesperetin-induced inhibition of cell                ity of hesperetin in MCF-7 cells. Therefore, we suggest that
growth was mediated via alterations in cell cycle progression,                hesperetin is worthy of further study to assess its potential
cells were exposed to either vehicle (0.1% DMSO) or vari-                     as an anticancer drug; it remains to be determined whether
ous concentrations of hesperetin (1, 5, 10, 50, 100 µM). Hes-                 hesperetin has anticarcinogenic activity in vivo.
peretin significantly inhibited cell proliferation in a dose- and
time-dependent manner. Hesperetin can inhibit the prolifer-
ation of both estrogen receptor-negative MDA-MB-435 and                                         Acknowledgments and Notes
estrogen receptor-positive MCF-7 human breast cancer cells
in vitroin vitro (21–22). Our results supported these obser-                     This work was supported by the Korea Research Foundation Grant funded
vations; the exposure of MCF-7 cells to hesperetin resulted                   by the Korean Government (MOEHRD, KRF–2006–311–F00127). Ad-
in a significant inhibition of cell proliferation.                            dress correspondence to EJ Choi, Ph.D., Plant Resource Research Institute,
                                                                              Duksung Women’s University, 419 Ssangmun-dong, Dobong-ku, 132–714,
   We evaluated the mechanism by which hesperetin inhib-
                                                                              Seoul, South Korea. Phone: +82-2-901-8663. FAX: 82-2-901-8661. E-mail:
ited cell proliferation using a cell cycle analysis. The eu-                  ejchoi@duksung.ac.kr.
karyotic cell cycle is controlled by catalytic complexes of
CDK/cyclin that coordinate internal and external signals at                      Submitted 14 January 2007; accepted in final form 27 March 2007.
several key checkpoints (23). G1 arrest leads cells to undergo
repair or to follow the apoptosis pathway (24). Activated                                                   References
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