Isoflavone genistein protects high glucose-induced human aortic endothelial cell apoptosis through estrogen receptor-mediated pathway

86
Journal of Geriatric Cardiology June 2008 Vol 5 No 2
Laboratory Research
Isoflavone genistein protects high glucose-induced human
aortic endothelial cell apoptosis through estrogen
receptor-mediated pathway
Wenwen Zhong, Yang Liu, Guang Yang , Hui Tian
Department of Geriatric Endocrinology, Chinese PLA General Hospital, Beijing, 100853, China
Objective The aim of this study was to determine if isoflavone genistien has protective effects against high glucose-induced cell
apoptosis in human aortic endlthelial cells, and investigate the possible mechanism for this protection. Methods Human aortic
endothelial cells subjected to normal (5mmol/L) or high glucose (25mmol/L) were treated with genistein at 0, 50, 100nmol/L. Parallel
experiments were performed with 100nM 17b-estradiol, and also in the presence and absence of the pure anti-estrogen ICI-182,780
(100nmol/L). The effects on cell apoptotic DNA fragmentation were determined using cell death ELISA, and the effects on cellular
proliferation were determined using tritiated thymidine incorporation assay. Estrogen receptor expression was detected by Taqman
quantitative PCR. Results Genistein at 100nmol/L significantly reduced high glucose-induced DNA fragmentation, and reversed cell
DNA synthesis inhibition (P <0.001) after 24 hours’ incubation. The effect of genistein was completely blocked by ICI-182,780
administration. Estrogen receptor beta, but not alpha was found to be expressed in these cells. Conclusion Isoflavone genistein
shows protection against high glucose-induced cell damage through estrogen receptor beta, reducing apoptotic DNA damage and
protecting from the inhibition of cell proliferation.(J Geriatr Cardiol 2008; 5:86-90.)
Key Words phytoestrogen; endothelial cell; high glucose; estrogen receptor; apoptosis
Introduction
dant levels and preventing oxidative DNA damage.11
The studies performed here were to determine the
Phytoestrogens are a group of biologically active plant
protection from high glucose induced oxidative stress by
substances with chemical structures similar to the endog-
the isoflavone genistein on human aortic endothelial cells
enous estrogen, allowing them to bind to estrogen recep-
(HAoECs), and to determine if these effects were through
tors (ER), though with lower affinity.1,4 Epidemiologically,
the estrogen receptor or via an alternative pathway.
there is a lower incidence of cardiovascular disease in Asian
countries that have a high soy phytoestrogen consump-
Materials and methods
tion in their diet.5 The anti-oxidative properties of
phytoestrogens may be responsible in part for their poten-
Reagents
tial protective effects. Genistein at physiological concen-
All reagents and chemicals were obtained from Sigma,
trations reduced free radicals damage to MC3T3-E1 cells
Poole, UK unless otherwise stated. All reagents for tissue
more effectively than either vitamin E or C,6 glucose-trig-
culture were obtained from Invitrogen, UK. The cell death
gered oxidation of LDL can be effectively prevented by
ELISA was obtained from Roche Diagnostics Ltd.
genistein;7 genistein and equol were shown to protect
against H O -induced DNA damage in human lymphocytes,
Cell culture
2
2
more than anti-oxidant vitamins and estradiol.8 Genistein
HAoECs were purchased from PromCel Ltd, and cul-
also plays a role in the scavenging of reactive oxygen spe-
tured in endothelial cell growth media (phenol red free) that
cies and lipid peroxidation.9 Daidzein has been shown to
was supplemented with 2% fetal calf serum, 5.0 ng/ml epi-
increase catalase mRNA expression and activate the cata-
dermal growth factor, 0.5 ng/ml vascular endothelial growth
lase promoter region,10 and phytoestrogens can increase
factor, 10 ng/ml basic fibroblast factor, 20 ng/ml R3 IGF-1,
total glutathione levels, thus decreasing intracellular oxi-
22.5ì g/ml heparin, 50 ng/ml amphotericin B and 50 ì g/ml
gentamicin. This medium kit initially contained supplements
of ascorbic acid and hydrocortisone, which were omitted
Corresponding author: Dr. Zhong Wenwen, Department of Geriat-
for this study, and the concentration of D-glucose is 5 mmol/L.
ric Endocrinology, The Chinese PLA General Hospital, 28 Fu Xing
Road, Beijing 100853, P.R. China.
Cells were seeded into culture plates at the density of
Tel:13031105535; Email: chinazhongww@yahoo.com.cn
2.5×105/ml and incubated at 37°C in an atmosphere of 5%
CO . Control cells received normal 5mmol/L glucose medium;
2

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Journal of Geriatric Cardiology June 2008 Vol 5 No 2 87
high D-glucose treated cells had this medium supplemented
sodium hydroxide. The final lysates were transferred to a
with D-glucose to achieve 11.3 or 25mM glucose in total.
scintillation vial with 10ml of Ecosint H (National
Cells with or without the pure anti-estrogen ICI182,780
Diagnostics, Hull, UK) for counting.
(100nM) received genistein or 17-estradiol (1nM) for 24
hours. All experiments were performed in triplicates.
Real-time quantitative PCR
Cell RNA was extracted using Trizol reagent and
Cell death detection ELISA
treated with DNAse to remove genomic DNA. RNA was
To detect the DNA fragmentation of apoptotic cells,
then reverse-transcribed to cDNA using M-MLV Reserve
the Cell Death Detection ELISA assay was used according
Transcriptase. The GeneAmp 5700 sequence detection sys-
to the manufacturer’s protocol. Briefly, cells were lysed with
tem (Applied Biosystems, UK) for real-time PCR was used
200ì l lysis buffer and centrifuged at 4,000rpm for 10min,
t o m e a s u r e t h e r e l a t i v e l e v e l o f g e n e e x p r e s s i o n
and 20ì l supernatant of each sample was transferred into a
quantitatively. Each sample was amplified with primers and
streptavidin-coated microtitre plate (MTP). 80ì l of the
probed for the target gene as well as the housekeeping gene,
immunoreagent mixture was added, washed and 100ì l ABTs
human â-glucuronidase (hGUS). The sequences for target
substrate solution added. A MTP reader (Anthos 2010,
gene were obtained from NCBI, and the primers and probes
Anthos Labtech Instruments) was used to measure the ab-
were designed using Primer Express software (Applied
sorbance at the wavelength of 405nm with 492nm as a
Biosystems), and shown in Table 1. The results were
reference.
analysed using GeneAmp 5700 software, and the baseline
and threshold were set manually.
Cytotoxicity detection assay of LDH
To determine if glucose levels may affect the expres-
Necrosis was determined through the activity of lac-
sion of the ERs, the effect of 5 and 25mM glucose on the ER
tate dehydrogenase (LDH) released from the cytosol; the
mRNA expression was undertaken over 30min, 2hours, 16
Cytotoxicity Detection Kit (Roche Diagnostics Ltd, UK) was
hours, 24 hours, 48 hours and 72 hours, and determined by
used according to the manufacturer’s protocol. Briefly,
quantitative PCR.
100 ì l culture supernatant of each sample was transferred
into a 96-well plate, followed by adding 100 ì l reaction mix-
Statistical analysis
ture to each well. The samples were incubated for 20 min-
Statistical analysis was performed on at least three
utes in the dark at room temperature and the absorbance
replicates using ANOVA with Tukeys post hoc analysis
was measured by the MTP reader at the wavelength of 492
(SPSS version 11, SPSS UK Ltd, Surrey, UK). Data are ex-
nm with 602 nm as a reference.
pressed as mean ± standard deviation (SD). Values of
P <0.05 were considered statistically significant.
Tritiated thymidine incorporation assay
Each sample received 0.25uCi of [3H]-TdR (Amersham
Results
International, Amersham, UK) and was incubated for 3 hours
at 37°C. After incubation the media was removed and cells
High glucose induced cell apoptsis and cell proliferation
washed with phosphate buffered saline. 0.5ml of 10% trichlo-
inhibition in HAoECs
roacetic acid was then added to each sample for 1 hour to
HAoECs were found to react differently to different
precipitate the DNA. Unbound thymidine was removed by
glucose concentrations. Compared to 5 mM D-glucose
a gentle wash with 4°C phosphate buffered saline. The DNA
group, 25 mM and 40 mM D-glucose significantly increased
was then dissolved in 500µl of 0.1% Triton ¡Á100 in 10%
cell apoptotic level and reduced proliferation level (*P<0.05),
Table 1 Sequences of primers/probes for target genes
Target gene
Primer/Probe
Sequence (5’-3’)
ERá
Forward
CAA GGG AAG TAT GGC TAT GGA ATC
Reverse
CTC ACA GGA CCA GAC TCC ATA ATG
Probe
CCT GAA GCA TAG TCA TTC CAC ACT GCA GAC
ERá46
Forward
AAG AGG GTG CCA GGC TTT G
Reverse
GTC CAA GAG CAA GTT AGG AGC AA
Probe
CTA GAG ATC CTG ATG ATT GGT CTC GTC TGGC
ERâ
Forward
CGA CAA GGA GTT GGT ACA CAT GA
Reverse
AAC AGG CTG AGC TCC ACA AAG
Probe
CAG CTG GGC CAA GAA GAT TCC CG

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88
Journal of Geriatric Cardiology June 2008 Vol 5 No 2
but this was not observed with 11.3 mM glucose-treated
elevated apoptotic level from 0.597±0.005 to 0.430±0.004,
group. The treatment of 40 mM D-glucose caused further
reducing 33.5% of induced apoptosis. The apoptotic level
cell damages than 25 mM D-glucose (P<0.05). Detailed data
in 100 nM genistein-treated cells was even lower than that
were shown in Figure 1A&B.
in 10 nM genistein group. The ÄOD value of this group was
only 0.297±0.003, and about 60.1% of apoptosis was
A
suppressed. 1 nM genistein did not reduce the apoptotic
level caused by high glucose. Similar results were found
with cell proliferation detected by [3H]-TdR incorporation
assay. 17â -estradiol was also found to have protective ef-
fects against high glucose-induced cell damages in HAoECs.
Estrogen receptor expression in HAoECs
Quantitative PCR showed that only ER â and not ER
á or the soluble type of ER á, ER á 46, was expressed in this
cell model (Figure 2A). The time course of 5mM and 25mM
glucose showed no effect on the expression of the ER â
m RNA over 30min to 72 hours (Figure 2B).
B
Anti-estrogen ICI182,780 completely blocked the protec-
tive effects of genistein against high glucose in HAoECs
Table 3 showed that cells treated with genistein or
17â -estradiol had significantly higher proliferation level
compared to the high glucose group (P<0.05), as also shown
in Table 2. The co-treatment of ICI182, 780 significantly re-
duced the cell proliferation to the level comparable to high
glucose-treated group. Similar results were obtained from
apoptosis detection that genistein and 17â-estradiol reduced
apoptotic level, but with the co-treatment of ICI182, 780, the
apoptotic levels were elevated back to the level similar to
high glucose group (Table 3). The anti-oestrogen ICI182,
Figure 1 High glucose altered cell proliferation and cell
death in HAoECs
780 alone did not cause reduced cell proliferation or in-
creased apoptosis (data not shown).
Genistein reversed high glucose-induced cell apoptosis
and proliferation inhibition in HAoECs
Discussion
Table 2 showed that genistein at physiological con-
centrations was able to reduce high glucose-triggered
Data here demonstrated the deleterious effect of high
apoptosis and proliferation inhibition in HAoECs. The
glucose on endothelial proliferation and survival. Clinically,
apoptotic DNA fragmentation level in 25mM D-glucose
hyperglycaemia and oxidative stress are well-recognized risk
treated group was significantly higher than the normal con-
factors for atherosclerotic diseases, especially the cardio-
trol (P<0.05). Genistein at 50 nM significantly reduced the
vascular complications in diabetes mellitus. Due to the lo-
Table 2 Effects of genistein and 17b-estradiol on high glucose-induced HAoECs apoptosis and proliferation inhibition
ELISA assay
[3H]-TdR assay
OD value
P value
â-reading
P value
Contro
l0.098±0.003
3450.71±30.31
25mM D-glucose
0.597±0.005
<0.05*
2146.63±27.38 <0.05*
1nM genistein
0.589±0.006
>0.05*
2152.47±29.44 >0.05*
50nM genistein
0.430±0.003
<0.05*
2493.52±30.24 <0.05*
100nM genistein
0.297±0.003
<0.05*
2792.15±30.17 <0.05*
1nM 17?-estradiol
0.306±0.004
<0.05*
2812.36±25.74 <0.05*
*compared with the control detected by the same method;
*compared with the value of 25mM D-glucose group detected by the same method; compared to 50nM genistein group detected by the same method

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Journal of Geriatric Cardiology June 2008 Vol 5 No 2 89
muscle cell proliferation. It was also demonstrated that oxi-
dative stress- or high glucose-induced endothelial damages
i n c l u d e d r e d u c e d p r o d u c t o f n i t r i c o x i d e 12,13 a n d
overexpression of adhesion molecules14,15 which facilitated
leukocyte/monocyte- endothelium interaction and migration
of leukocytes/monocytes into the sub-endothelial layer of
blood vessels. These, together with the impaired lipid pro-
file in blood formed the base of atherosclerotic diseases in
diabetes.
The soy isoflavones, genistein (5,7,4'-trihydroxyisoflavone)
is the representative phytoestrogen that functions as a pu-
tative protective agent against cancer, cardiovascular
disease, and osteoporosis. The beneficial effects of
phytoestrogen on cardiovascular protection have been in-
dicated by the relatively lower incidence of cardiovascular
diseases in people with higher phytoestrogen consumption5.
In this study, genistein significantly inhibited high glucose-
induced cell apoptosis, indicating that they had protective
effects against high glucose induced oxidative cell dam-
ages in cultured human aortic endothelial cells. This is in
accord with reports in literature showing the protective ef-
fect of genistein on free radical-induced oxidative damage
in other cell types like MC3T3-E1 osteoblast-like cells or
A. Estrogen receptor b was found to be expressed in HAoECs
human lymphocytes.8 The results from proliferation assay
- ¡ñ- hGUS; - ¡ø-ERb
mirrored the effects on apoptosis, with genistein protecting
B. 25mM D-glucose did not change the expression level of ER? over
against the reduction in proliferation from high glucose-
72 hours incubation
induced oxidative stress. 17â-estradiol also showed protec-
Figure 2 Estrogen receptor b expression in HAoECs
tive effect in our system, but at much lower doses, the rea-
son for which might be attributed to the different binding
cation among all the constituents in the vessel wall, endot-
affinities among these agents.
helial cells might be the first to be affected by high glucose
The protective effects of genistein and 17â-estradiol
concentrations or oxidative stress, thus, endothelial dys-
were reversed by the pure antiestrogen ICI182,780 indicat-
function in diabetes might play a vital role in the progress of
ing their protective effects being mediated through the es-
diabetic complications. The inhibited proliferation and
trogen receptor alone, and that no additional mechanism of
apoptotic cell death induced by high glucose exposure and/
action through an alternative pathway was occurring. The
or oxidative stress might result in an in vivo loss of integrity
anti-estrogen ICI182,780 is commonly used to block the ac-
in the endothelial monolayer of blood vessels, leaving the
tivation of ER.16 Our findings are consistent with the
underlined basement membrane exposed to blood ingredi-
literature, and it has been reported that the anti-apoptotic
ents including platelets which aggregated to form thrombi
effect of genistein in neurons was ER dependent and was
and secreted various growth factors to trigger smooth
Table 3 Anti-estrogen ICI182,780 blocked the effects of genistein and 17b-estradiol on high
glucose-induced cell damage in HAoECs
ELISA assay
[3H]-TdR assay
OD Value
P Value
â-reading
P Value
Normal Control
0.098±0.003
3450.71±30.31
25mM D-glucose
0.597±0.005
<0.05
2146.63±27.38 <0.05
100nM genistein
0.297±0.003
<0.05*
2792.15±30.17 <0.05*
+100nM AE
0. 589±0.002
>0.05*
2152.47±29.44 >0.05*
1nM 17?-estradiol
0.306±0.004
<0.05*
2812.36±25.74 <0.05*
+100nM AE
0.592±0.004
>0.05*
2212.76±31.33 >0.05*
*compared with 25mM D-glucose group detected by the same method;
AE: anti-estrogen ICI182,780

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90
Journal of Geriatric Cardiology June 2008 Vol 5 No 2
reversed by anti-estrogen co-treatment.16 The mRNA of ERâ
monal and biochemical mechanisms and associations. Scand J
but not
Clin Lab Invest Suppl 1990; 201: 3-23.
á was found to be expressed in HAoECs in our
6. Ju YH, Allred CD, Allred KF, et al. Physiological concentrations
system. Whether this is due to the techniques employed or
of dietary genistein dose-dependently stimulate growth of es-
the culture conditions used it is unclear why ERs have been
trogen-dependent human breast cancer (MCF-7) tumors im-
found by some but not by others. There was not direct
planted in athymic nude mice. J Nutr 2001 Nov;131(11):2957-62.
effect of glucose on the expression of ER mRNA, which
7. Exner M, Hermann M, Hofbauer R, et al. Genistein prevents the
suggested that high glucose is having an effect downstream
glucose autoxidation mediated atherogenic modification of low
of transcription, perhaps at the level of translation or ER
density lipoprotein. Free Radic Res 2001;34: 101-12.
cofactors.
8. Sierens J, Hartley JA, Campbell MJ, et al. Effect of phytoestrogen
In conclusion, isoflavone genistein protected against
and antioxidant supplementation on oxidative DNA damage
assessed using the comet assay. Mutat Res 2001;485: 169-76.
high glucose-induced cell apoptosis and proliferation inhi-
9. Patel RP, Boersma BJ, Crawford JH, et al. Antioxidant mecha-
bition in human aortic endothelial cells that express ERâ, by
nisms of isoflavones in lipid systems: paradoxical effects of
an estrogen dependent mechanism, and these effects are
peroxyl radical scavenging. Free Radic Biol Med 2001;15:31
completely reversed by the antiestrogen ICI182,780. High
(12):1570-81.
glucose was likely to have an effect downstream of ER
10. Rohrdanz E, Ohler S, Tran-Thi QH & Kahl R. The phytoestrogen
transcription, which needs to be investigated in the future
daidzein affects the antioxidant enzyme system of rat hepatoma
H4IIE cells. J Nutr 2002; 132: 370-5.
work. Hypothetically, the anti-oxidative properties against
11. Mizutani K, Ikeda K, Nishikata T & Yamori Y. Phytoestrogens
high glucose of isoflavone genistein may give clinical arte-
attenuate oxidative DNA damage in vascular smooth muscle
rial endothelial cell protection through the activation of es-
cells from stroke-prone spontaneously hypertensive rats. J
trogen receptor, by which the proliferative activity, viability
hypertens 2000;18: 1833-40.
and even some active substances-secreting ability may be
12. Thomas SR, Chen K, Keaney JF, et al. Hydrogen peroxide
restored at the condition of diabetes or hyperglycemia, and
activates endothelial nitric-oxide synthase through coordinated
therefore, help delay the onset and development of cardio-
phosphorylation and dephosphorylation via a phosphoinositide
vascular complications.
3-kinase-dependent signaling pathway. J Biol Chem 2002; 277:
6017-24.
13. Booth G, Stalker TJ, Lefer AM, et al. Elevated ambient glucose
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