1521-0103/351/2/270–277$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/jpet.114.216754
J Pharmacol Exp Ther 351:270–277, November 2014
Coadministrating Luteolin Minimizes the Side Effects of the
Aromatase Inhibitor Letrozole
Fengjuan Li, Tsz Yan Wong, Shu-mei Lin, Simon Chow, Wing-hoi Cheung, Franky L. Chan,
Shiuan Chen, and Lai K. Leung
Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, China (F.L.); Biochemistry
Programme, School of Life Sciences, Faculty of Science (F.L), Food and Nutritional Sciences Programme , School of Life
Sciences, Faculty of Science (T.Y.W., L.K.L.), Department of Orthopaedics and Traumatology, Faculty of Medicine (Si.C., W.C.),
and School of Biomedical Sciences, Faculty of Medicine (F.L.C.), The Chinese University of Hong Kong, Shatin, Hong Kong;
Department of Food Science, National Chiayi University, Chiayi City, Taiwan, Republic of China (S.L.); and Department of Cancer
Biology, Beckman Research Institute of the City of Hope, Duarte, California (Sh.C.)
ABSTRACT
Aromatase inhibitors (AIs) have been used as adjuvant therapeutic
agents for breast cancer. Their adverse side effect on blood lipid is
well documented. Some natural compounds have been shown to
be potential AIs. In the present study, we compared the efficacy of
the flavonoid luteolin to the clinically approved AI letrozole
(Femara; Novartis Pharmaceuticals, East Hanover, NJ) in a cell
and a mouse model. In the in vitro experimental results for
aromatase inhibition, the Ki values of luteolin and letrozole were
estimated to be 2.44 mM and 0.41 nM, respectively. Both letrozole
and luteolin appeared to be competitive inhibitors. Subsequently,
an animal model was used for the comparison. Aromataseexpressing MCF-7 cells were transplanted into ovariectomized
Introduction
Since estrogen is critical in the initiation and progression of
breast cancer, estrogen receptor (ER) antagonism has been a
common strategy for treating this disease. Tamoxifen is a
widely used ER antagonist in breast cancer therapy. However, it may cause endometrial hyperplasia and elevate the
cancer risk at this tissue (Lindahl et al., 2008). Moreover, it
also increases the risk of stroke after long-term treatment
(Hernandez et al., 2008).
Another type of drug for breast cancer therapy is aromatase
or CYP19 inhibitor (AI). Estrogen is converted from cholesterol, and aromatase catalyzes the rate-limiting reaction. AIs,
which can be steroidal or nonsteroidal compounds, block the
formation of estrogen and reduce the stimulus for growth in
ER-positive tumors. Steroidal AIs bind to the active site of
aromatase, whereas their nonsteroidal counterparts reversibly
This study was supported by the Chinese University of Hong Kong Direct
Grant Project [Grant 4053047].
F.L. and T.Y.W. contributed equally to this work.
dx.doi.org/10.1124/jpet.114.216754.
athymic mice. Luteolin was given by mouth at 5, 20, and 50 mg/kg,
whereas letrozole was administered by intravenous injection. Similar to letrozole, luteolin administration reduced plasma estrogen
concentrations and suppressed the xenograft proliferation. The
regulation of cell cycle and apoptotic proteins—such as a decrease
in the expression of Bcl-xL, cyclin-A/D1/E, CDK2/4, and increase
in that of Bax—was about the same in both treatments. The most
significant disparity was on blood lipids. In contrast to letrozole,
luteolin increased fasting plasma high-density lipoprotein concentrations and produced a desirable blood lipid profile. These results
suggested that the flavonoid could be a coadjuvant therapeutic
agent without impairing the action of AIs.
interact with the enzyme (Campos, 2004). AIs are superior to
tamoxifen in treating advanced breast cancer with fewer side
effects (Bonneterre et al., 2001; Cuzick, 2003). Unlike tamoxifen,
AIs have no agonistic activity toward the ER in any tissues.
Letrozole (Femara; Novartis Pharmaceuticals, East Hanover,
NJ), or 4,49-((1H-1,2,4-triazol-1-yl)methylene)dibenzonitrile, is
an approved AI for treating estrogen-sensitive breast cancer
after surgery. Because the action of AIs is not tissue-specific,
side effects may result from prolonged estrogen deprivation.
Increased bone turnover has been observed in women treated
with letrozole (McCaig et al., 2010). Postmenopausal women
on letrozole therapy could also develop an undesirable blood
lipid profile, such as increased levels of total cholesterol, lowdensity lipoprotein cholesterol, and apolipoprotein B (Elisaf
et al., 2001).
Luteolin, or 39,49,59,79-tetrahydroxyflavone, is a common
flavonoid which can be isolated from vegetables such as celery,
broccoli, carrots, thyme, green peppers, etc. It is one of the most
potent aromatase inhibitors in the flavonoid family in vitro
(Jeong et al., 1999; van Meeuwen et al., 2008). Furthermore, it is
capable of containing the transcriptional activity of aromatase
ABBREVIATIONS: AD, mice treated with androstenedione; AD12mg LET, mice treated with androstenedione plus 2 mg/day letrozole; AD+LUT 50,
mice treated with 50 mg/kg luteolin; AI, aromatase inhibitor; ANOVA, analysis of variance; ER, estrogen receptor; HDL, high-density lipoprotein;
LDL, low-density lipoprotein; MCF-7aro, MCF-7 cells stably transfected with human CYP19; PBS, phosphate-buffered saline; TC, total cholesterol;
ThN, trabecular number.
270
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Received May 18, 2014; accepted August 15, 2014
Luteolin Inhibits Aromatase and Raises HDL In Vivo
271
TABLE 1
Treatment of mice
Treatment
Regimen
1
2
3
4
5
6
7
8
Group
Androstenedione
Luteolin
Letrozole
mg/day
mg/kg body wt/day
mg/day
0
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0
0
5
20
50
20
20
—
0
0
0
0
0
0.04
2
2
Control
AD
AD+LUT 5
AD+LUT 20
AD+LUT 50
AD+LUT 20 + 0.04mg LET
AD+LUT 20 + 2mg LET
AD+2mg LET
Materials and Methods
Chemicals. Luteolin was obtained from Indofine Chemical Co.,
Inc. (Hillsborough, NJ). Letrozole was purchased from International
Laboratory USA (San Bruno, CA). Other chemicals, if not stated, were
purchased from Sigma-Aldrich (St. Louis, MO).
Cell Culture. The breast cancer cell line MCF-7 was purchased
from American Type Culture Collection (Rockville, MD). MCF-7 cells
stably transfected with human CYP19 (MCF-7aro) were prepared as
previously described (Zhou et al., 1990). The stably transfected MCF-7
cells were maintained in minimum Eagle’s medium (Invitrogen, Grand
Island, NY) supplemented with 10% fetal bovine serum (Invitrogen/
Life Technologies, Rockville, MD) and the selection antibiotic G418
(Geneticin, 500 mg/ml; USB, Cleveland, OH). Cells were incubated at
37°C in 5% carbon dioxide and routinely subcultured when 80% confluency was reached.
Enzyme Kinetic Assay. To illustrate the aromatase-inhibitory
property of luteolin, an enzyme kinetic assay was performed using a
previously described method (Wang et al., 2008). MCF-7aro cells were
seeded in six-well plates at a density of 5–105 per well for 24 hours.
The reaction was initiated by replacing with serum-free medium
containing [1b-3H]4-androstene-3,17-dione and luteolin or letrozole.
After incubating for 1/2 hour at 37°C, the medium was removed and
mixed with an equal volume of chloroform. The mixture was then
centrifuged at 10,000g at 4°C for 10 minutes. The aqueous phase was
transferred into a tube containing 5% activated charcoal suspension.
After 30-minute incubation, the supernatant was aliquoted out for
scintillation counting. A bicinchoninic acid kit (Sigma-Aldrich) was
used to determine protein content of cells.
Animal Experimentation. This mouse model for postmenopausal
breast carcinogenesis was adopted from Yue et al. (1994), where the
tumor growth was dependent on the aromatase activity. The model was
used for testing the effect of luteolin on aromatase, blood lipid profile,
and the bone. Six-week-old female athymic mice were acquired from
the Animal Facility of Chinese University of Hong Kong. The mice were
ovariectomized, and were fed the purified and phytoestrogen-free AIN93G diet (Reeves et al., 1993). After 2 weeks of recovery, they were
transplanted with MCF-7aro cells and randomly assigned into regimens as stated in Table 1. The treatment lasted for 84 days after cell
inoculation.
Luteolin was dissolved in ethanol and diluted to 100–200 ml by
phosphate-buffered saline (PBS) for gavage, and the final ethanol
concentration was 1%. Androstenedione and letrozole were dissolved
in 0.1 ml of 0.3% hydroxyl propyl cellulose and given as subcutaneous
injections. Control mice received the carrier solvent injection; mice
treated with androstenedione (AD), androstenedione plus 2 mg/day
letrozole (AD12mg LET), and control mice were also given PBS with
1% ethanol by mouth. Before transplantation, MCF-7aro cells were
cultured as described earlier. The cells were trypsinized and suspended
in Matrigel matrix (10 mg/ml; BD Biosciences, San Jose, CA). Then
0.1 ml of cells (3 107 cells/ml) was injected into the two flanks of the
animal. Body weight, tumor size, and food intake were monitored
weekly throughout the study. Tumor volume was measured by an
electronic caliper and estimated according to the following formula:
p/(6 length width height), where length, width, and height were
the three orthogonal diameters of the tumors. The average volume of
two xenografts from the same mice was treated as one data point. At
the end of the study, the mice were fasted overnight and euthanized by
cervical dislocation. Livers and uteri were excised and weighed. These
Fig. 1. Enzyme kinetic analysis of luteolin and letrozole on aromatase
inhibition. MCF-7aro cells were cultured and assayed for aromatase activity. Concentrations of luteolin (0, 1, 2.5, 5, and 10 mM) and letrozole
(0, 2.5, 5, and 250 nM) were administered to the cells with different
substrate concentrations for 30 minutes. Aromatase enzyme activities were
plotted against substrate concentrations for luteolin (A) and letrozole (B).
The chemical structures of luteolin (A) and letrozole (B) are also displayed.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
in MCF-7 cells (Li et al., 2011). In the present study, luteolin’s
potency of aromatase inhibition and its side effects were investigated in cell and mouse models. We hypothesized that
luteolin could be a therapeutic agent with beneficial effects on
the bone and blood lipid.
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Li et al.
Immunoblot of Proteins Extracted from Tumor. Western
blot analysis was carried out for comparing the cell cycle and death of
proteins. The frozen tumors were pulverized in a Dounce homogenizer
with added liquid nitrogen. The pulverized samples were then
sonicated in lysis buffer (PBS, 1% NP40, 0.5% sodium deoxycholate,
0.1% SDS, 40 mg/l phenylmethyl-sulfonyl fluoride, 0.5 mg/l aprotinin,
0.5 mg/l leupeptin, 1.1 mM EDTA, and 0.7 mg/l pepstatin) with a cell
disruptor (Branson Ultrasonics Corp., Danbury, CT) on ice for
Fig. 2. Both luteolin and letrozole administration inhibited the growth of
MCF-7aro xenograft. Mice were inoculated with MCF-7aro cells at two
sites per mouse after ovariectomy, and were treated with letrozole and/or
luteolin. (A) Tumor volumes were estimated once a week starting from day
7 after inoculation. (B) The tumor weights were measured on the day of
sacrifice. The data were analyzed by one-way ANOVA, followed by Tukey’s
multiple comparison test. Values are means 6 S.E.M., n = 6–8 (the two
grafts from one animal were combined as one data point). Means labeled
with various letters are significantly different (order: a . b . c; P , 0.05).
organs were the respective indicators for hepatotoxicity and estrogen
status. Tumors and serum were collected and stored at 280°C until
assayed. Femurs were removed and soaked in ethanol for scanning.
The procedure was approved by the Animal Experimentation Ethics
Committee, Chinese University of Hong Kong.
Serum Estradiol Determination. Aromatase is the key enzyme
for estrogen synthesis. Serum estrogen levels in the model mice
reflected the activity of the enzyme. Serum estradiol concentrations
were measured by enzyme-linked immunosorbent assay (Cayman
Chemical Company, Ann Arbor, MI). The samples were added into
a 96-well plate coated with antibody raised against estradiol. After
incubation and development at room temperature, the absorbance at
410 nm was quantified using a microplate reader (FLUOstar; BMG
Labtechnologies GmBH, Offenburg, Germany).
Fig. 4. Luteolin counteracted uterine weight reduction under letrozole
treatment. Uteruses of the experimental animals were dissected and
weighed at sacrifice. The data were analyzed by one-way ANOVA,
followed by Tukey’s multiple comparison test. Values are means 6 S.E.M.,
n = 6–8. Means labeled with different letters are significantly different
(order: a . b . c; P , 0.05).
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 3. Reduced plasma estradiol concentration (Conc.) in luteolin-treated
mice. Blood was drawn from the animals at euthanasia. Serum estradiol
concentration was quantified by enzyme-linked immunosorbent assay.
The data were analyzed by one-way ANOVA, followed by Tukey’s multiple
comparison test. Values are means 6 S.E.M., n = 6–8. Means labeled with
different letters are significantly different (order: a . b . c; P , 0.05).
Luteolin Inhibits Aromatase and Raises HDL In Vivo
273
30 seconds for protein extraction. Thirty micrograms of protein
extract was separated on 10% SDS-PAGE gel and transferred onto an
Immobilon polyvinylidene membrane (Millipore, Bedford, MA). AntiCDK2 and 4, cyclin A, B1, E, P57, P21 (Santa Cruz Biotechnology,
Santa Cruz, CA), anti–cyclin D1 (Abcam, Cambridge, UK), anti–BclxL, Bcl-2, Bax, Bak (Santa Cruz Biotechnology), and anti–b-actin
primary (Sigma-Aldrich) and secondary antibodies conjugated
with horseradish peroxidase (Santa Cruz Biotechnology) were
used for protein detection. The targeted proteins were visualized by
autochemiluminography. The optical density readings of the images
were analyzed using the computer software ImageJ (NIH, Bethesda,
MD), and optical density of the corresponding b-actin was used for
normalization.
Bone Microarchitecture Analysis. Bone integrity is a major
concern for administering AI, so the status of bone was examined under
luteolin and letrozole treatments. High-resolution microcomputed
tomography was used for bone microarchitecture evaluation. The
femora were placed and affixed in a specimen holder submerged in 70%
ethanol. The bone volume fraction, trabecular thickness, and trabecular number were analyzed as previously described (Siu et al., 2004;
Shi et al., 2010; Chow et al., 2011).
Analysis of Serum Lipoproteins. Another side effect of AI is in
the induction of an undesirable plasma lipid profile. This part of the
experiment was to investigate the effect of luteolin on plasma lipids.
Plasma total cholesterol (TC) and total triglycerides were measured
using the respective commercial kits from Infinity (Waltham, MA)
and Stanbio Laboratories (Boerne, TX). For measuring plasma highdensity lipoprotein cholesterol (HDL), low-density lipoprotein (LDL)
and very-low-density lipoprotein were precipitated by phosphotungstic
acid and magnesium chloride (Stanbio Laboratories). HDL in the
supernatant was determined using the Infinity kit. The value of LDL
cholesterol was estimated by subtracting HDL from TC, i.e., TC – HDL 5
LDL.
Statistical Analysis. The data were analyzed by the software
package GraphPad Prism 5 for Windows, version 5.04 (GraphPad
Software, Inc., San Diego, CA). One-way analysis of variance (ANOVA)
tests were used for group comparisons; the post-hoc ranking test
(Tukey’s multiple comparison) was performed to differentiate the
treatments when P , 0.05. For the tumor volume data, two-way
ANOVA was used for analysis. As ANOVA alone would not indicate
differences among individual treatments, post-hoc ranking comparison
was needed for such differentiation (Figs. 2–7). For instance, means
labeled with a were significantly different from those labeled with b but
were not different from those labeled with a,b in the present labeling
system. The order of magnitude was shown at the end of figure legend
in parentheses.
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Fig. 5. Effect of luteolin on cell cycle protein expression. Protein expression of genes controlling the cell cycle was determined by western blot analysis.
The corresponding optical density readings are shown in the graphs on the right. The images are representations of four independent experiments with
similar results. The data were analyzed by one-way ANOVA, followed by Tukey’s multiple comparison test. Means labeled with different letters are
significantly different (order: a . b . b for cyclin A, a . b for cyclin D1 and E, a . b . c for CDK4 and 2, a . b for P57, and no differences among samples
for P21; P , 0.05).
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Li et al.
For the enzyme kinetic studies, the data were processed by nonlinear
regression and fitted into the built-in mixed model of inhibition in
GraphPad Prism 5 with the default setting. No data weight adjustments in the enzyme activity were made. Ki and a values were obtained
from the program printouts.
Results
Cell Study
Enzyme Kinetic Results. Five concentrations, i.e., 0, 1,
2.5, 5, and 10 mM, were selected for kinetic analysis for luteolin,
whereas 0, 0.0025, 0.005, and 0.25 mM were used for letrozole.
Aromatase enzyme activities were plotted against substrate
concentrations for luteolin and letrozole in Fig. 1, A and B,
respectively. Mixed-model analysis for luteolin indicated that
a competitive type of inhibition on aromatase was apparent
with a calculated Ki value of 2.44 mM (S.E. 5 1.29 mM) and
a value of 26.65 (S.E. 5 34.05). Letrozole was also shown to be
a competitive inhibitor with an estimated Ki value of 0.41 nM
(S.E. 5 0.22 nM) and a value of 26.21 (S.E. 5 17.52) in this
system. This result indicated that luteolin was a weak AI
compared with letrozole.
Animal Study
Treatments of Luteolin and Letrozole Did Not Affect
Body or Liver Weight. Mouse body weights were monitored
weekly. There were no significant differences in body weights
or liver weights (data not shown). Treatments of luteolin,
letrozole, and the combination at the given concentrations
appeared to be tolerable to the mice.
Luteolin or Letrozole Deterred Xenograft Proliferation. The tumor size of AD was significantly (P , 0.05) larger
than that of control mice starting from day 14 to the end of the
experiment. Starting from day 35, tumor volumes in mice
receiving luteolin or letrozole were smaller than that in AD mice
(Fig. 2A). Among the luteolin treatment groups, a dose-response
relationship was observed. The tumor weights measured at
sacrifice were consistent with the tumor volume results (Fig.
2B). High doses of luteolin administration could achieve a
tumor-suppressing effect comparable to that of letrozole.
Luteolin Reduced Plasma Estradiol Concentration.
Increased serum estradiol concentration was seen in AD mice
as compared with that in control mice. The concentration was
suppressed by luteolin, letrozole, or coadministration of the
two (Fig. 3). The estradiol concentration in the sham-operated
group was the highest among all groups, but was not different
(P , 0.05) from the AD group (data not shown). The aromataseexpressing xenografts synthesized a significant amount of
estradiol from the substrate, and both letrozole and luteolin
could prevent the production of estradiol.
Luteolin and Letrozole Counteracted Androgen-Induced
Uterine Growth. The uterine wet weight of androstenedioneinjected mice displayed a 2-fold increase as compared with that
of control mice. Treatment with 2 mg of letrozole or 50 mg/kg
luteolin prevented the androstenedione-induced uterine growth
(Fig. 4). No significant differences were seen with the other
treatments. The uterotropic effect of luteolin appeared to be
coming from aromatase inhibition.
Cell Cycle and Apoptotic Protein Expression in Tumors.
Luteolin at 50 mg/kg per day and letrozole at 2 mg/day altered the
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Fig. 6. Effect of luteolin on apoptotic protein expression. Protein expression of genes controlling apoptosis was determined by western blot analysis. The
corresponding optical density readings are shown in the graphs on the right. The images are representations of four independent experiments with
similar results. The data were analyzed by one-way ANOVA, followed by Tukey’s multiple comparison test. Means labeled with different letters are
significantly different (order: a . b . c . d for Bcl-xl, a . b for Bcl-2, a . b for Bax, and no differences among samples for Bak; P , 0.05).
Luteolin Inhibits Aromatase and Raises HDL In Vivo
275
proteins of the cell cycle (Fig. 5) and apoptosis (Fig. 6) in a
comparative pattern. Both drugs increased Bax and prevented the
androstenedione-induced cyclin A and E, Cdk2 and 4, and Bcl-xL
expression in the tumor samples. However, the suppression of
letrozole on Cdk4 and Bcl-xL was significantly stronger than that
curbed by luteolin. In addition, reduced expression of cyclin D1
was only observed in letrozole-treated samples. Although the
xenograft growth was suppressed by both letrozole and luteolin,
the mechanisms could be different.
Luteolin Did Not Change the Bone Status. Compared
with the ovariectomized control mice (control), AD mice
exhibited a 10% increase in trabecular number (ThN). The
estrogen status was consistent with the ThN results. Letrozole
reduced ThN, and luteolin administered at a high dosage
apparently induced a similar reduction, although the decrease
was not significant (data not shown). The result of bone volume
fraction (data not shown) showed a similar pattern as that
of ThN. The bone status would not benefit from luteolin
administration.
Luteolin Increased HDL Levels in Mice. Luteolin did
not change plasma TC or total triglycerides (data not shown),
but increased HDL levels in a dose-dependent manner (Fig.
7A). The levels in mice treated with 50 mg/kg luteolin (AD1
LUT 50) were higher than those treated with androstenedione
or AD12mg LET (Fig. 6A). When expressed as the ratio of
HDL to TC, the values of groups of AD1LUT 20 and AD1
LUT 50 were significantly higher than that of AD12mg
LET (Fig. 7B). On the other hand, the ratio of LDL/HDL in
AD1LUT 20 and AD1LUT 50 was significantly lower than
that in AD12mg LET (Fig. 7C). No matter whether administered alone or together with letrozole, luteolin could favorably
change the plasma lipid profile.
Discussion
AI is an adjuvant therapeutic drug for estrogen-responsive
breast cancer. However, it may induce adverse effects on blood
lipids (Confavreux et al., 2007; Rabaglio et al., 2009). Some
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Fig. 7. Luteolin increased HDL level and reduced the ratio of LDL/HDL. Mice were inoculated with MCF-7aro cells after ovariectomy, and were treated
with luteolin and androstenedione and letrozole. Blood was drawn from the animals at sacrifice. Serum HDL (A) and LDL (B) were measured using the
commercial enzymatic kits. Ratios of HDL/TC (C) and LDL/HDL (D) are shown. The data were analyzed by one-way ANOVA, followed by Tukey’s
multiple comparison test. Values are means 6 S.E.M., n = 6–8. Means labeled with different letters are significantly different (order: a . b . c for HDL
concentrations, a . b for LDL concentrations, a . b for HDL/TC ratio, and a . b for LDL/HDL ratio; P , 0.05). Conc., concentration.
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Li et al.
Although luteolin is widely distributed in plant foods, the
dosages used in the current study were above the normal
levels that humans consume. Nevertheless, this study could
provide the scientific basis for nutraceutical or pharmacological development of this flavone.
In conclusion, our results demonstrated that luteolin
suppressed MCF-7aro xenograft growth while raising HDL.
As a high LDL/HDL ratio is usually associated with long-term
use of AI, the administration of luteolin can be a potential
compensation mechanism without compromising on aromatase inhibition.
Acknowledgments
The authors thank Professor Howard Glauert of the Graduate
Center for Nutritional Sciences, University of Kentucky, for
proofreading this manuscript.
Authorship Contributions
Participated in research design: Leung, Li, Wong, Lin.
Conducted experiments: Li, Wong, Lin, Chow.
Contributed new reagents or analytic tools: Leung, Chan, Chen,
Cheung.
Performed data analysis: Leung, Li, Lin, Chow, Wong.
Wrote or contributed to the writing of the manuscript: Leung, Li,
Wong.
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flavonoids are able to improve plasma lipid profiles. However,
the isoflavone genistein may also compromise the action of
letrozole (Ju et al., 2008). By comparing the estimated Ki
values in the present study, the aromatase-inhibitory effect of
luteolin was about 5900-fold weaker than that of letrozole.
Unlike the antagonistic effect seen in the aforementioned study
of genistein, luteolin would not weaken the activity of AI.
Estrogen is known to influence the blood lipid profile, such as
increasing plasma HDL and decreasing LDL (Espeland et al.,
1998; Nasr and Breckwoldt, 1998). Increases in total serum
cholesterol, LDL, and serum lipid have been observed in breast
cancer patients receiving AI (Nicolaides et al., 2000; Elisaf
et al., 2001). Another study has found that exemestane and
letrozole may differentially affect HDL and LDL; nevertheless,
the ultimate LDL/HDL ratios are significantly increased in
patients treated with the two AIs (Bell et al., 2012).
The effect of luteolin on lipid metabolism has been reported
previously. Luteolin or its conjugate reduced plasma LDL and
TC in diabetic rats (Wang et al., 2012) and normal adult rats
(Azevedo et al., 2010), and elevated HDL levels in the study by
Wang et al. (2012). The flavone also prevents lipid accumulation in cultured hepatic cells (Liu et al., 2011). These
properties may reduce the risk of cardiovascular disease. In
the present study, luteolin administration could increase
HDL levels and suppress the ratio of LDL/HDL in the blood.
Moreover, the coadministration of luteolin and letrozole
reduced the LDL/HDL ratio without elevating serum estradiol and the xenograft growth rate. This suggested that
luteolin could be a cotherapeutic agent to AIs.
Bone homeostasis is regulated by the activities of osteoclasts and osteoblasts. Estrogen can directly or indirectly
prevent bone resorption by suppressing the formation and
survival of osteoclasts. AI therapy would induce bone loss by
depriving the hormone (Pfeilschifter and Diel, 2000). Luteolin
has been previously shown to increase collagen synthesis,
alkaline phosphatase activity, and osteocalcin secretion in
osteoblastic MC3T3-E1 cells (Choi, 2007). Furthermore,
luteolin could dampen osteoclast differentiation and function.
Oral administration of luteolin to ovariectomized mice causes
a significant increase in bone mineral density (Kim et al.,
2011). However, luteolin administered with letrozole or by
itself did not improve the bone status as shown by the
trabecular number in the present study.
Cyclins in the G1 phase of the cell cycle are overexpressed in
some primary breast cancer tissues and in tumor-derived cell
lines (Keyomarsi and Pardee, 1993; Keyomarsi et al., 1994;
Husdal et al., 2006), and transition from G1 into the S phase is
regulated by the activities of cyclin-associated kinases (Morgan,
1995). Luteolin has previously been shown to induce G1 arrest
in different types of cancer cells (Casagrande and Darbon, 2001;
Kobayashi et al., 2002; Lim et al., 2007). In the present study,
increased expression levels of cyclins A, D1, and E, and the
related CDK2 and 4, were observed in androstenedione-treated
mice. Treatment of luteolin or letrozole could reverse the
expression levels of these genes.
Our results also show that these agents may influence
apoptosis. Estrogen may alter Bcl-2 family gene expression in
favor of cell survival (Leung and Wang, 1999). In the current
study, androstenedione induced Bcl-xL and Bcl-2 and suppressed Bax in the tumor samples. Letrozole or luteolin
reversed the expression of Bcl-xL and Bax with no effect on
Bcl-2.
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Address correspondence to: Dr. Lai K. Leung, Food and Nutritional
Sciences Programme, School of Life Sciences, The Chinese University of Hong
Kong, Rm. 507C MMW Bldg., Shatin, N.T., Hong Kong. E-mail: laikleung@
cuhk.edu.hk
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