The Journal of Nutrition
Biochemical, Molecular, and Genetic Mechanisms
A g-Tocopherol-Rich Mixture of Tocopherols
Maintains Nrf2 Expression in Prostate Tumors
of TRAMP Mice via Epigenetic Inhibition of
Ying Huang,3,5Tin Oo Khor,3,5Limin Shu,3Constance Lay-Lay Saw,3Tien-Yuan Wu,3Nanjoo Suh,4
Chung S. Yang,4and Ah-Ng Tony Kong3*
3Department of Pharmaceutics, and4Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology,
Center for Cancer Prevention Research, Earnest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ
Nuclear factor-erythroid 2-related factor 2 (Nrf2) plays a pivotal role in maintaining cellular redox homeostasis and
eliminating reactive toxic species. Nrf2 is epigenetically suppressed due to CpG hypermethylation in prostate tumors from
the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. We previously showed that dietary feeding of a g-
tocopherol–rich mixture of tocopherols (g-TmT) suppressed prostate tumorigenesis in TRAMP mice associated with
higher Nrf2 protein expression. We hypothesized that g-TmT may maintain Nrf2 through epigenetic inhibition of promoter
CpG methylation. In this study, 8-wk-old male TRAMP mice were fed 0.1% g-TmT or a control diet for 16 wk. The
methylation in the Nrf2 promoter was inhibited in the prostate of the g-TmT group compared with the control group.
Protein expressions of DNA methyltransferase (DNMT), including DNMT1, DNMT3A, and DNMT3B, were lower in the
prostate of the g-TmT group than in the controls. TRAMP-C1 cells were treated with 30 mmol/L of g-TmT or blank medium
for 5 d. The methylation in the Nrf2 promoterwas inhibited in the g-TmT–treated cells compared withthe untreated cells at
d 5, and mRNA and protein expressions of Nrf2 and NAD(P)H:quinone oxidoreductase 1 were higher. Interestingly, only
DNMT3B was inhibited in the g-TmT–treated cells compared with the untreated cells. In the aggregate, our findings
demonstrate that g-TmT could inhibit CpG methylationin the Nrf2 promoterin theprostate of TRAMP miceand in TRAMP-
C1 cells, which might lead to higher Nrf2 expressionand potentially contribute to the prevention of prostate tumorigenesis
in this TRAMP model.J. Nutr. 142: 818–823, 2012.
Nrf2 (nuclear factor-erythroid 2-related factor 2) is a trans-
cription factor that plays pivotal role in maintaining cellular
redox homeostasis and elimination of carcinogens and reactive
intermediates (1,2). Accumulating evidence has demonstrated
that Nrf2-decient mice are more susceptible to carcinogenic,
inflammatory, and oxidative insults (3,4). Furthermore, it has
been found that Nrf2 and its downstream target GST (glutathi-
one-S-transferase) are suppressed in human and TRAMP (the
transgenic adenocarcinoma of the mouse prostate) prostate
cancer associated with excessive reactive oxygen species (5).
Higher reactive oxygen species levels could cause genetic and
epigenetic instability and transduce a variety of signals for tumor
cell survival, proliferation,andinvasion(5,6). Although the direct
relationship between the loss of Nrf2 and prostate carcinogenesis
isyettobe established,maintainingNrf2expression appearstobe
critical in retaining cellular adaptability to environmental and
endogenous stresses and to delay or prevent the development of
The suppression of Nrf2 in prostate tumors of TRAMP mice
and TRAMP-C1 cells was found to be caused by CpG
hypermethylation in the promoter, especially at the first 5 CpG
(7). These CpG are hypermethylated in tumorigenic TRAMP-C1
cells but not in nontumorigenic TRAMP-C3 cells (8). Treatment
with DNMT (DNA methyltransferase) inhibitor 5-aza-29-deox-
ycytidine and HDAC (histone deacetylase) inhibitor trichostatin
A could restore Nrf2 expression in TRAMP-C1 cells (7).
However, it may not be feasible to use 5-aza-29-deoxycytidine
as a cancer chemopreventive agent chronically due to its toxicity,
and therefore great effort has been made in looking for effective
epigenetic interventions through the use of relatively nontoxic
natural compounds (9).
1Supported by NIH grant R01-CA152826.
2Author disclosures: Y. Huang, T. O. Khor, L. Shu, C. L. Saw, T. Y. Wu, N. Suh,
C. S. Yang, and A. N. Kong, no conflicts of interest.
5Y.H. and T.O.K. contributed equally to the project.
* To whom correspondence should be addressed. E-mail: KongT@pharmacy.
ã 2012 American Society for Nutrition.
Manuscript received October 12, 2011. Initial review completed November 16, 2011. Revision accepted February 22, 2012.
First published online March 28, 2012; doi:10.3945/jn.111.153114.
Vitamin E refers to a group of lipid-soluble compounds
consisting of 8 structurally related tocopherols (a-, b-, g-, and
d-) and tocotrienols (a-, b-, g-, and d-). They are well-known
natural antioxidants and are abundant in a variety of foods,
including vegetable oils, nuts, and whole grains (10). Epidemi-
ological studies revealed that a higher serum g-tocopherol level
is associated with a reduced risk of prostate cancer (11), but
large-scale clinical trials with a-tocopherol supplementation
demonstrated inconsistent efficacy against prostate cancer
(12,13). g-TmT (g-tocopherol-rich mixture of tocopherols) is a
by-product of the refining of soybean oil and typically contains
57% g-tocopherol, 24% d-tocopherol, 13% a-tocopherol, and
1.5% b-tocopherol. g-TmT has been shown to inhibit carcino-
genesis in different types of cancer, including prostate, colon,
lung, and mammary (14–17).
We reported that dietary feeding of 0.1% g-TmT could
inhibit prostate tumorigenesis in TRAMP mice along with
higher Nrf2 expression, but the potential mechanisms remain
unknown (17). Hence, the present study was undertaken to
investigate whether g-TmT would maintain Nrf2 expression by
inhibiting CpG methylation in TRAMP mice and TRAMP-C1
Methods and Materials
Mice. Female heterozygous C57BL/TGN TRAMP mice, line PB Tag
8247NG, and male C57BL/6 mice were purchased from Jackson
Laboratory. TRAMP females were crossed with C57BL/6 males and
the first or second generation of transgenic males was chosen for the
study. The genotype of the offspring was determined by a PCR-based
method (18). Mice were housed in cages with wood chip bedding in a
temperature-controlled room (20–228C) with a 12-h-light/-dark cycle,
with a relative humidityof 45–55%in Rutgers Animal Care Facility. The
study was carried out using an IACUC-approved protocol at Rutgers
Mouse study design. To test whether higher Nrf2 expression in
TRAMP prostates after g-TmT treatment was associated with decreased
promoter methylation, we repeated the treatment of 0.1% g-TmT
diet in 8-wk-old TRAMP mice for 16 wk, as previously performed (17).
Eight-week-old male TRAMP mice were randomlyassigned to treatment
(n = 7) and control (n = 6) groups. Mice in the treatment group were
fed 0.1% mixed tocopherols in an AIN-93M diet (19). g-TmT
was purchased from Cognis and contained 130.0 mg of a-tocopherol,
15.0 mg of b-tocopherol, 243.0 mg of d-tocopherol, and 568.0 mg of g-
tocopherol/g. At wk 24, mice were killed by CO2asphyxiation and the
genitourinary apparatus including the prostate, the seminal vesicles, and
the bladder were collected, snap-frozen in liquid nitrogen, and stored in
280oC for further analysis.
Archived prostate tissues of TRAMP mice at the age of 12 wk (n = 4),
18 wk (n = 3), and 24 wk (n = 5) and nontransgenic C57BL/6 mice at the
age of 12 wk (n = 2) and 24 wk (n = 2) were used for DNA extraction to
determine the methylation status of the Nrf2 promoter at different ages.
,1 y before DNA collection. Mice were fed Purina Mouse Chow 5015.
Cell culture and treatment. TRAMP-C1 cells were cultured in DMEM
containing 10% FBS and antibiotics. Cells were grown at 37oC in a
humidified 5% CO2atmosphere. g-TmT was dissolved in DMSO to
make a stock solution containing 100 mmol/L total tocopherols
consisting of 13.0 mmol/L a-tocopherol, 1.5 mmol/L b-tocopherol,
25.0 mmol/L d-tocopherol, and 60.5 mmol/L g-tocopherol. TRAMP-C1
cells were treated with 30 mmol/L of g-TmT in DMEM containing 1%
FBS for 5 d and harvested.
Bisulfite sequencing. Genomic DNA was isolated from TRAMP
prostate tissues and TRAMP-C1 cells using a QIAamp mini kit (Qiagen).
DNA (800 ng) was bisulfite converted using an EZ DNA Methylation-
Gold kit (Zymo Research). TA cloning was performed as previously
described (7). For each sample, 5–10 clones were chosen for sequenc-
ing. Plasmid DNA was sequenced using T7 primer (Genewiz) at the
Rutgers Sequencing Core facility. The methylation percentage was
calculated as the number of methylated CpG over the total number of
Western-blot analyses. DNMT are key enzymes catalyzing the
addition of the methyl group to cytosine and play a critical role in
establishing DNA methylation patterns (20). To investigate whether
inhibition of methylation in the Nrf2 promoter was related to down-
regulation of any of DNMT, we determined the protein expression of
DNMT in the prostate tissues of TRAMP mice and TRAMP-C1 cells.
Two prostate specimens in the same group were combined for protein
extraction. The detailed procedure of Western blotting was previously
described (7). Protein bands were visualized by Supersignal West Femto
(Pierce) and documented by Gel Documentation 2000 system (Bio-Rad).
Protein expressions were semiquantitated by densitometry using ImageJ
program. Antibodies against DNMT1, DNMT3A, and DNMT3B were
purchased from Imgenex. Antibodies against Nrf2 (sc-722), NQO1
[NAD(P)H:quinone oxidoreductase 1; sc-16464], and b-actin (sc-1616)
were purchased from Santa Cruz Biotechnology.
RNA extraction and RT-PCR. RNA was extracted using a Qiagen
RNeasy mini kit and converted to cDNA (TaqMan). Conditions for
qPCR were described (18). Relative expression was analyzed by aDDCt
method using RQ Manager 1.2 and GAPDH expression was used as
internal control. The forward and reverse primers for Nrf2 amplification
were 59-TCACACGAGATGAGCTTAGGGCAA-39 and 59-TACAGTT-
CTGGGCGGCGACTTTAT-39. Primers for Nqo1 were 59-AAGAGCT-
TTAGGGTCGTCTTGGCA-39 and 59-AGCCTCCTTCATGGCGTAG-
TTGAA-39. Primers for GAPDH were 59-TCAACAGCAACTCCCACT-
CTTCCA-39 and 59-ACCCTGTTGCTGTAGCCGTATTCA-39.
Statistical analyses. Data are mean 6 SEM. Palpable tumor incidence
was evaluated using the Fisher exact test. The methylation percentages
of the Nrf2 promoter in the archived prostate samples were compared
using 1-way ANOVA followed by Tukey’s Studentized range test. For
all other determinations, Student’s t-test or Welch’s t-test was used.
SAS, version 9.2, was used for all statistical analyses. All P values
correspond to 2-sided hypothesis tests and P , 0.05 was regarded as
CpG methylation in the Nrf2 promoter increases during
prostate tumorigenesis in TRAMP mice. In the present study,
we examined the methylation pattern of the first 5 CpG in the
prostate of TRAMP and nontransgenic mice at different ages. In
the prostate of TRAMP mice, the methylation of these CpG
significantly increased from 12 to 24 wk (Fig. 1A,C), whereas in
the prostate of nontransgenic mice, the methylation remained
unchanged (Fig. 1B,C).
Dietary 0.1% g-TmT inhibits CpG hypermethylation in the
Nrf2 promoter in the prostate of TRAMP mice. The palpable
tumor incidence (Table 1) was significantly lower in the g-TmT
group than in the control group, which is consistent with the
previous study (17). The methylation of the first 5 CpG in the
Nrf2 promoter was significantly lower in the g-TmT group than
in the control group (Fig. 2A,B). Nrf2 mRNA expression tended
to be higher in the g-TmT group than in the control group (P =
0.069) (Fig. 2C).
promoter in TRAMP-C1 cells. From the above in vivo study,
reverses CpG hypermethylation inthe Nrf2
g-TmT maintains Nrf2 epigenetically819
which demonstrated that g-TmT inhibited CpG methylation in
the Nrf2 promoter, we next investigated whether g-TmT could
reverse CpG hypermethylation in TRAMP-C1 cells. The meth-
ylation of the first 5 CpG in the Nrf2 promoter was inhibited in
the g-TmT–treated cells compared with the untreated cells at d 5
(Fig. 3). Cell viability was not affected by the treatment (data not
g-TmT induces mRNA and protein expressions of Nrf2
and Nqo1 in TRAMP-C1 cells. We examined the expression of
Nrf2 to see whether reduced promoter methylation could
reactivate gene expression. The mRNA and protein expressions
of Nrf2 and NQO1 were induced in TRAMP-C1 cells treated
with 30 mmol/L of g-TmT compared with the control cells on d
5 (Fig. 4).
g-TmT suppresses the expression of DNMT in the prostate
of TRAMP mice and TRAMP-C1 cells. In the prostate of
TRAMP mice, the protein levels of DNMT, including DNMT1,
DNMT3A, and DNMT3B, were all lower in the g-TmT group
than in the control group (Fig. 5A). Interestingly, only DNMT3B
was suppressed when TRAMP-C1 cells were treated with 30
mmol/L of g-TmT for 5 d (Fig. 5B). These results suggest the
potential role of DNMT in CpG methylation and demethylation
regulated by g-TmT.
g-TmT has been shown to inhibit prostate tumorigenesis in
TRAMP mice associated with higher Nrf2 expression (17);
however, the molecular mechanism remains unclear. In the
present study, we show that g-TmT treatment prevented CpG
hypermethylation in the Nrf2 promoter in vivo and reversed its
hypermethylation in vitro, which might contribute to higher
Nrf2 expression. Tocopherols are extensively studied with
respect to their antioxidative, antiinflammatory, and antiproli-
ferative effects (21), yet to the best of our knowledge, their
effects on epigenetic modification have not been reported.
The progression of prostate tumorigenesis in the TRAMP
model is associated with abnormal DNA methylation events
with both locus-specific hypermethylation and global genomic
hypomethylation (22). The present study demonstrated that
methylation of the first 5 CpG in the Nrf2 promoter increased
during prostate cancer development in TRAMP mice, especially
at the late stage (Fig. 1A,C). These CpG are critical in regulating
the expression of Nrf2, and the increased methylation may
promoter in archived prostate samples from TRAMP (A) and non-
transgenic (B) mice and the methylation percentage (C) at various
ages. In C, data are mean 6 SEM, n = 3–5 (TRAMP) or 2
(nontransgenic). In TRAMP mice, means without a common letter
differ, P , 0.05. TRAMP, transgenic adenocarcinoma of the mouse
Methylation patterns of the first 5 CpG in the Nrf2
Palpable tumor incidence in TRAMP mice fed control
or 0.1% g-TmT diet for 16 wk1
Current studyPrevious study2
Groupn Palpable tumor incidencen Palpable tumor incidence
1*Differs from the control group, P , 0.05. TRAMP, transgenic adenocarcinoma of
the mouse prostate; g-TmT, g-tocopherol-rich mixture of tocopherols.
2Adapted from (17).
promoter (A), the overall methylation percentage (B), and the mRNA
expression of Nrf2 (C) in the prostate of TRAMP mice fed a control or
0.1% g-TmT diet. Data are mean 6 SEM, n = 4 (control) or 7 (g-TmT).
*Different from control, P , 0.05. TRAMP, transgenic adenocarci-
noma of the mouse prostate; g-TmT, g-tocopherol-rich mixture of
Methylation patterns of the first 5 CpG in the Nrf2
percentage (B) of the first 5 CpG in the Nrf2 promoter in TRAMP-C1
cells following treatment with 30 mmol/L of g-TmT for 5 d. Data are
mean 6 SEM, n = 3. *Different from control, P , 0.05. TRAMP,
transgenic adenocarcinoma of the mouse prostate; g-TmT, g-tocopherol-
rich mixture of tocopherols.
The methylation pattern (A) and the overall methylation
820Huang et al.
contribute to the lower Nrf2 expression in prostate tumors of
TRAMP mice, as previously reported (7). NRF2 is also found to
be repressed in human prostate cancer (5); however, future
studies are warranted to investigate whether NRF2 inactivation
in human prostate cancer is caused by CpG hypermethylation.
Prostate cancer in TRAMP mice appears to involve excessive
oxidative stress, accompanied by increased damage to DNA,
protein, and lipid (6). Because Nrf2 plays a central role in
adapting the cells to environmental and endogenous stresses (1),
the loss of Nrf2 expression would potentially make the prostate
of TRAMP mice more vulnerable to insults, because Nrf2-
targeted enzymes such as the SOD, UGT1A1, NQO1, and GST
family are also lost during tumorigenesis (5,17,23). g-TmT
inhibited CpG methylation (Figs. 2A,B and 3) and elevated Nrf2
and its downstream antioxidant enzyme NQO1 in TRAMP-C1
cells (Fig. 4), which could potentially contribute to the preven-
tion against prostate cancer.
Some natural phytochemicals have been shown to reactivate
the expression of silenced genes in tumor cells through epige-
netic modifications (9,24). Possible mechanisms could be related
to inhibition of DNMT and/or HDAC. For instance, green tea
polyphenols, sulforaphane, and curcumin have been reported to
inhibit both DNMT and HDAC (24–26). In the present study,
we showed that dietary g-TmT feeding suppressed the expres-
sions of all 3 DNMT in TRAMP mice (Fig. 5A). Lower
expression of DNMT could prevent promoter CpG hyper-
methylation in the prostate of TRAMP mice, including the Nrf2
promoter during the early stages of tumorigenesis. Furthermore,
metabolites of tocopherols are hypothetical HDAC inhibitors as
predicted by molecular modeling (24). We speculate that histone
modifications might also contribute to the lower CpG methyl-
ation and higher Nrf2 expression after g-TmT treatment in vivo.
Further study is needed to explore the effect of g-TmTon HDAC
and histone modifications.
The human body preferentially retains a-tocopherol despite
the high g-tocopherol intake from the typical American diet
(21). This is achieved in part by the selectivity of the hepatic a-
tocopherol transfer protein, which facilitates the entrance of a-
tocopherol into the circulatory system, while the non-a-tocoph-
erols undergo fast metabolism mediated by the cytochrome
P450 (27). In immunodeficient mice fed 0.1% g-TmT, a-
tocopherol remained the most abundant form in the prostate,
though its concentration was not greater than in the control
group (28). The concentrations of g- and d-tocopherol in the
prostate increased by 2- to 3-fold following 0.1% g-TmT
treatment. Another study showed that the urinary excretions of
tocopherol metabolites such as g- and d-carboxymethyl hydrox-
ychromans dramatically increased in immunodeficient mice
following 0.17–0.3% of g-TmT feeding (29). These tocopherol
levels reported in mice suggest that the observed epigenetic effect
in the prostate of TRAMP mice in the present study may be
attributed to the single or combined effects of g- and d-
tocopherol and their metabolites.
As a proof-of-concept, we demonstrated that g-TmT could
reverse hypermethylation of the Nrf2 promoter using TRAMP-
C1 cells (Fig. 3). However, DNMT3B, but not DNMT1 or
DNMT3A, was suppressed in the g-TmT–treated cells at d 5
(Fig. 5B). There are several possible explanations for the
sion of Nrf2 and NQO1 in TRAMP-C1 cells following
the treatment with 30 mmol/L of g-TmT for 5 d.
Three independent experiments were carried out.
Data are mean 6 SEM, n = 3. *Different from
control, P , 0.05. C, control; TRAMP, the trans-
genic adenocarcinoma of mouse prostate; g-TmT,
g-tocopherol-rich mixture of tocopherols; T, g-TmT
The mRNA (A) and protein (B) expres-
DNMT3A, and DNMT3B in the prostate of TRAMP
mice fed a control or 0.1% g-TmT diet (A) and in
TRAMP-C1 cells (B) treated with 30 mmol/L of g-
TmT for 5 d. Values are mean 6 SEM, n = 4–7
(TRAMP mice) or 3 (TRAMP-C1 cells). *Different
from control, P , 0.05. C, control; g-TmT, g-
tocopherol-rich mixture of tocopherols; T, g-TmT
Protein expressions of DNMT1,
g-TmT maintains Nrf2 epigenetically821
differences between the in vivo and in vitro results. First, the
TRAMP study revealed primary prevention, in which g-TmT
blocked the expression of DNMT proteins and the methylation
of the Nrf2 promoter during prostate tumorigenesis. The in vitro
study was carried out in a prostate cancer cell line, in which the
Nrf2 promoter is already hypermethylated (7), and the g-TmT
treatment reversed the hypermethylation. Second, prostate
tumors from TRAMP mice are a heterogeneous population (8)
compared with the TRAMP-C1 cells, which are relatively
homogenous and therefore could result in different outcomes
upon g-TmT treatment. Third, the concentrations of different
tocopherols and their metabolites in prostate tissues and
TRAMP-C1 cells might be different. It has been reported that
only a small portion of tocopherols in cell culture medium can be
metabolized by human prostate cancer cells (30). Hence, it is
likely that TRAMP-C1 cells are mostly exposed to the parent
tocopherols as they are supplemented in the medium. In con-
trast, mice can metabolize tocopherols extensively in vivo, gen-
erating high concentrations of carboxychromanol metabolites
(29), some of which have been shown to possess superior
biological activity compared with the parent tocopherols (31–33).
In summary, in our present study, we showed that g-TmT
prevents CpG methylation in the Nrf2 promoter in vivo in the
prostate of TRAMP mice and reverses hypermethylation of the
Nrf2promoter invitroinTRAMP-C1 cells,associated with lower
contribute to higher Nrf2 expression, which potentially plays a
role in the prevention of prostate tumorigenesis in TRAMP mice.
Y.H. discussed, designed, conducted the research, analyzed the
data, and wrote the manuscript; T.O.K. discussed, designed,
conducted the research, and wrote the manuscript; N.S. and
C.S.Y. discussed and designed the research; A.N.K. discussed
and designed the research, wrote the manuscript and had
primary responsibility for the final content; and L.S., C.L-L.S.,
and T-Y.W. conducted the research. All authors read and
approved the final manuscript.
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