DifferentialRetentionof DifferentialRetentionofa a-VitaminEIsCorrelated
Jing Ni,1See-Too Pang,2and Shuyuan Yeh1*
1DepartmentofUrologyandPathology ,UniversityofRochester,Rochester ,NewYork
2DepartmentofSurgery ,DivisionofUrology ,ChangGungMemorialHospital,TaoYuan,Taiwan
cancer (PCa).Interestingly, differentprostatecancer cellshavedifferentsensitivity to a-Vit Eor
VES treatment. The goal of this study is to determine whether cellular Vit E bioavailability and
its transport proteins are important contributing factors.
METHODS. a-Vit E and its ester form, VES, were used to treat prostate cancer LNCaP, PC3,
andDU145cells,andtheirgrowthratesweredeterminedbyMTT assay.Cellularlevels ofVitE
were quantified using HPLC as the index of bioavailability. The expression levels of Vit E
transport proteins were determined by real-time PCR.
while both LNCaP and PC3 cells were sensitive to 20 mM VES treatment. Coordinately, cellular
levels of a-Vit E and VES positively correlated to their inhibitory effects. Further study found
expression levels of Vit E transport proteins, including tocopherol associated protein (TAP),
scavenger receptor class B type I (SR-BI), a-tocopherol transfer protein (TTP), and ATP binding
cassette transporter A1 (ABCA1), were different in various PCa cells, which may contribute to
cellular Vit E bioavailability. This notion is further supported by the findings that over-
expression or knockdown of TTP could coordinately alter cellular a-Vit E levels in PCa cells.
CONCLUSION. Antiproliferative efficacy of a-Vit E is correlated with its cellular bioavail-
the growth inhibition efficacy of Vit E in prostate cancer cells. Prostate
# 2007 Wiley-Liss, Inc.
KEY WORDS: vitamin E succinate; prostate cancer; TAP; TTP; ABCA1; SR-BI
Prostate cancer is the second leading fatal cancer in
American men. While initially androgen dependent
and responsive to androgen ablation therapy, prostate
cancer will eventually relapse and become androgen-
independent. There is no cure for patients with
androgen-independent prostate cancer .
Epidemiological studies show that daily supple-
mentation of Vit E may reduce the incidence and
mortality of prostate cancer [3–5]. The Vit E family
contains eight members, a-, b-, g-, d-tocopherol and a-,
b-, g-, d-tocotrienol. Among them, a-tocopherol (a-Vit
E) is the most bioactive form in the body, making up
more than 90% of Vit E in mammalian tissues .
Because Vit E is structurally unstable and easily
to increase stability, including VES. VES is one of the
cells in vitro. Moreover, VES selectively inhibits the
growth of cancer cells, but not non-malignant cells .
We previously showed that VES suppresses prostate
Abbreviations: PCa, prostate cancer; a-Vit E, a-tocopherol; VES,
a-vitamin E succinate, a-tocopherol succinate; TAP, a-tocopherol
associated protein; ABCA1, ATP binding cassette transporter A1;
TTP, a-tocopherol transfer protein; SR-BI, scavenger receptor class B
type I; HPLC, high performance liquid chromatography.
Grant sponsor: NIH; Grant number: DK60912.
*Correspondence to: Dr. Shuyuan Yeh, 601 Elmwood Ave., Box 656,
Rochester, NY 14642. E-mail: Shuyuan_yeh@urmc.rochester.edu
Received 18 July 2006; Accepted 9 August 2006
Published online in Wiley InterScience
? 2007 Wiley-Liss,Inc.
cancer LNCaP cells, but not primary-cultured prostate
fibroblasts . VES suppresses prostate cancer cell
growth via multiple mechanisms, including down-
regulation of the AR/PSA signaling pathway ,
blocking cell cycle progression [9,10], suppressing
invasion , and inducing apoptosis [12,13].
Interestingly, a-Vit E analog suppresses different
prostate cancer cells with different efficacy. We
hypothesize this may be, at least partly, due to
differential accumulation of Vit E in prostate cancer
AlthoughVitEisfat-soluble,its uptakeintothe cells
is not just through passive transport. Instead, several
Vit E-binding proteins/transport proteins mediate Vit
E uptake and efflux in vivo and in vitro . For
example, a-tocopherol transfer protein (TTP) can
selectively bind and release a-Vit E from the liver into
the serum. Deficiency of TTP will increase Vit E
accumulation in the liver and deplete a-Vit E from
peripheral tissue in mice . Functional loss of TTP
can result in ataxia with Vit E deficiency (AVED) in
humans . a-Tocopherol associated protein (TAP),
and VES uptake into the prostate cancer cells to
facilitate Vit E anti-proliferation function .
In this study, we mainly focused on comparing the
retention of a-Vit E and VES in prostate cancer cells.
Our results suggest that VES had much better anti-
proliferation effects on prostate cancer cells than a-Vit
a greater amount of cellular Vit E is correlated with Vit
of Vit E transported genes in prostate cancer cells may
contribute to their different cellular Vit E amounts.
Therefore, targeting on the expression of Vit E-binding
protein is another strategy for facilitating the chemo-
preventive and chemotherapeutic effects of a-Vit E
analog on prostate cancer.
a-, d- tocopherol, VES, and ascorbic acid (vitamin C)
were purchased from Sigma.
The LNCaP, PC3, and DU145 cells were obtained
VA). The LNCaP and PC3 cells were maintained in
RPMI 1640 medium supplemented with 8% fetal
bovine serum (FBS) and 100 mg/ml penicillin-strepto-
mycin. The DU145 cells were maintained in DMEM
medium supplemented with 8% fetal bovine serum
(FBS) and 100 mg/ml penicillin-streptomycin. All cells
were incubated in a humidified atmosphere of 5% CO2
in air at 378C.
The cells were seeded into 12- or 24-well plates.
or VES. At the indicated time, 0.5 ml of MTT (0.5 mg/
ml) was added. After 3 hr incubation at 378C, 1 ml
Absorbency was read at a wavelength of 595 nm. The
experiment was performed in quadruplicate.
After a-Vit E or VES treatment, the cells were
harvested and counted. Cell pellets were lysed with
1 nmol d-tocopherol, followed by 0.4 ml ethanol, then
frozen, and thawed four times. Vit E isoforms were
extracted with 0.8 ml hexane. The hexane extract
was taken to dryness under N2in a TurboVap1LV
concentration workstation (Zymark, MA). The residue
was dissolved in 2.5% ascorbated in methanol (1 ml)
and analyzed (50 ml) by HPLC.
Cellular VES concentrations were determined by
measuring a-Vit E concentrations before and after base
hydrolysis. The cell extractions were divided into two
equal portions. The second portion was hydrolyzed
using 0.2 ml of 4 M KOH overnight, then neutralized
with 0.2 ml 4 M HCl, and extracted with 0.8 ml hexane.
The hexane extract was taken to dryness under N2, the
residue dissolved in 2.5% ascorbate in methanol and
analyzed (50 ml) by high-performance liquid chroma-
tography (HPLC) .
eluted with MeOH: H2O 96:4 (V/V) at a flow rate of
by Empower software (Waters).
According to the manufacturer’s instructions, total
RNA was isolated using Trizol (Invitrogen, Carlsbad,
CA), 2 mg RNA was then subjected to reverse tran-
scription using Superscript III (Invitrogen). The real-
Mix (Biorad, Hercules, CA). Specific primers (Table I)
were designed according to Beacon Designer software.
PCR was performed at 948C for 3 min, and 40 cycles
on an iCycler iQ Multi-color real-time PCR detection
system (Biorad). Each sample was run in triplicate.
The Prostate DOI 10.1002/pros
Full length TTP was isolated from cDNA of prostate
HisMax (Invitrogen). TTP siRNA was constructed
using a DNA-based vector pSuperior-retro-puro (Oli-
goEngine, WA) that contained the puromycin resis-
tance marker. The oligonucleotides containing the
siRNA sequences targeting hTTP, GTGGCATCCA-
TTTGATAAA, or GGAACGGATTCACATGCAT, was
subcloned into the BglII–HindIII site of pSuperior-
retro-puro vector to generate TTPsi593 and TTPsi675,
respectively. All constructions were verified by
Full length hTTP plasmid was transfected into
LNCaP cells using an efficient electroporation system
that yieldedmore than
TTPsiRNA were transduced into DU145 cells by
a-Vit EandVESInhibit PCaCellGrowth
As an initial attempt to compare the effect of a-Vit E
on prostate cancer cells with VES, we treated prostate
cancer LNCaP, PC3, and DU145 cells with a-Vit E or
in human serum . Treatment with 20 mM a-Vit E
yielded growth inhibition of AR-positive LNCaP cells
by 14% within 6 days, but no inhibition of AR-negative
PC3 and DU145 cells. In contrast, 20 mM VES showed
significant anti-proliferative activity on LNCaP cells
and PC3 cells up to 55%, but no inhibition on DU145
cells. These data suggested that VES has better anti-
proliferation effects than a-Vit E. DU145 cells are
insensitive to 20 mM a-Vit E and VES treatment.
is due to its greater bioavailability, we assessed the
and DU145 cells. As shown in Figure 2A, within 48 hr,
a-Vit E can be accumulated in LNCaP cells at least 50%
more than that in PC3 and DU145 cells. These data
indicate that the higher cellular retention ability of
a-Vit E is associated with increased a-Vit E-mediated
growth inhibition in LNCaP cells.
Next, we examined cellular VES levels after the cells
were exposed to 20 mM VES for 12, 24, and 48 hr. As
shown in Figure 2B, within 48 hr, cellular VES
concentrations were approximately 10 nmol/106cells
in LNCaP and PC3 cells, as opposed to ?4 nmol/106
observation that 20 mM VES significantly suppressed
(Fig. 1), suggesting higher cellular retention ability of
VES might be associated with the increased VES
sensitivity in LNCaP and PC3 cells.
VES can be hydrolyzed to a-Vit E by esterase, which
could reduce VES’s growth inhibitionory activity
(Fig. 1). However, the efficiency of VES hydrolysis in
PCa cells remains unknown. Here, we also measured
the total Vit E (VESþa-Vit E) amount. As shown in
Figure 2C, after VES treatment for 48 hr, total Vit E
amount was ?10 nmol/106cells in both LNCaP and
PC3 cells, whereas it was only ?5 nmol/106cells in
DU145 cells, suggesting that DU145 cells retain much
less total Vit E. We then calculated the ratio between
In LNCaP and PC3 cells, 7–10% of VES can be con-
verted to a-Vit E, while in DU145 cells, the rate was
15–23% (Fig. 3D). Uptake, efflux, and metabolism of
VES will determine the final cellular VES levels. The
high VES hydrolysis rate in DU145 cells can contribute
to the low cellular VES levels. Furthermore, amount of
totalVitE(VESþa-Vit E)inDU145cellsismuch lower
than that in LNCaP and PC3 cells, suggesting that low
uptake or/and high efflux process may be the main
factors for the low cellular VES amount in DU145 cells.
Taken together, low accumulation of cellular VES in
DU145 cells may be one of the reasons that DU145 cells
are insensitive or less sensitive to 20 mM VES.
To determine whether DU145 cells are sensitive to
higher dose ofVES treatment,we evaluatedthe IC50 of
VES in prostate cancer cells. As shown in Figure 3, VES
The Prostate DOI 10.1002/pros
TABLE I. ThePrimersofVitET ransportProteinsforReal-TimePCR
Gene symbol GenBank numberForward primer (50-30)Reverse primer (50-30)
inhibited prostate cancer cell growth with IC50
20 mM for both LNCaP cells and PC3 cells, and 50 mM
for DU145 cells, indicating a 2.5-fold difference in the
dose of IC50. Further study found cellular VES levels
reached 17 nmol/106cells inside the DU145 cells after
50 mM VES treatment (Fig. 3D), but 4 mmol/106cells
after 20 mM VES treatment. There is 4-fold increase
of the cellular VES levels when using 2.5-fold higher
dose to treat cells. It appears that the intracellular VES
levels is a better indicator for VES anti-proliferatative
Taken together, the efficacy of a-Vit E or VES among
different prostate cancer cells may be, at least partly,
dependent on cellular a-Vit E or VES bioavailability.
The Prostate DOI 10.1002/pros
Fig. 1. Thegrowtheffectof20mMa-VitEand20mMVESonPCa
4 NiandY eh
Vit E is a fat-soluble nutrient, however, its transport
into the cells is not solely via a passive process. One of
the potential mechanisms for the differential retention
of Vit E in various prostate cancer cells may be due to
different expression levels of Vit E transporters with
that TTP and lipid efflux gene ATP binding cassette
transporter A1 (ABCA1) can transfer the a-Vit E out
of the cells [15,21,22], while TAP and high density
which mainly mediates selective cholesterol uptake,
may facilitate a-Vit E uptake inside the cells [17,23,24].
can be mediated by TAP . Thus, we first compared
these genes’ mRNA expression in different PCa cell
lines. As shown in Figure 4A, TAP had the highest
expression in PC3 cells. The expression of SR-BI was
similar in LNCaP, PC3, and DU145 cells (Fig. 4B). On
expression in DU145 cells, modest expression in PC3
cells, and least expression in LNCaP cells (Fig. 4C).
ABCA1 expression levels in DU145 and PC3 cells were
similar, and much higher than that in LNCaP cells
(Fig. 4D). These data suggested that high ability to
effluxwith low ability touptakeVitEmay bethemajor
reason why DU145 cells have such low retention of Vit
of Vit E in PC3 cells and low efflux with low uptake of
Vit E in LNCaP cells may balance the retention of
vitamin E which contributes to their relatively high
accumulations of Vit E.
The Prostate DOI 10.1002/pros
with 0,10, 20, 30,40, 50, 60, 70, 80,90,100 mMVES for 6 days.Cell
growth was determined by MTTassay.The diagram represented
one setofexperiments.The IC50 wererepresentedasmeanvalue
Fig. 2. The cellular concentrations of Vit E/VES in PCa cells.
PC3, and DU145 cells were treatedwith 20 mM a-Vit E.Cells were
was extractedwithhexane anddetectedby HPLC.The datawere
representedas themeanvalues ?SEM of atleastfiveindependent
experiments. Statistics P value is compared to the data of LNCaP
cells at the same timepoint.*,P<0.05.The concentrations ofVES
LNCaP,PC3, and DU145 were treatedwith 20 mMVES.Cellswere
were determined by measuring the amount of a-Vit E in cellular
extracts before and after base hydrolysis.Cellular VES hydrolysis
rateiscalculatedby theratiobetweencellulara-VitE to totalVitE
of at least four independent experiments. a, b are the statistical
P valueby whichthedataofDU145cellsiscomparedwiththedata
ofLNCaPcells at the same timepoint.a<0.05;b<0.01.c,dis the
We further asked if TTP affects Vit E accumulation
in PCa cells by knockdown of TTP in DU145 cells.
Two DNA-based siRNAs constructs targeting TTP
(TTPsi573 and TTPsi675) had been generated. As
shown inFigure5A, thepooledTTPsiRNA-transfected
reduced TTP expression by around 40%, while the
empty vector control (DU-V) did not significantly
change TTP mRNA levels, compared to parental
DU145 cells. We then treated these cells with 20 mM
a-Vit E or VES for 24 h, and examined the total
cellular Vit E concentration. As shown in Figure 5B,C,
DU-TTPsi573 and DU-TTPsi675 cells can increase
cellular a-Vit E concentration about 1.8-fold compared
to the parental DU145 cells. Interestingly, there is no
significant difference on VES concentration among
DU-TTPsi573, DU-TTPsi675, DU-V, and DU145 cells
(Fig. 5C). To further characterize TTP function in PCa
cells, we ectopically restored TTP expression back in
(Fig. 5D). The cellular Vit E and VES concentrations
were then examined after 24 h of treatment. As shown
in Figure 5E and F, the concentration of a-Vit E, but not
VES, was significantly reduced in TTP-transfected
LNCaP cells (LN-TTP) compared to vector control
cells (LN-V). Therefore, our data indicated that TTP
mediates the efflux of a-Vit E but not VES in PCa cells.
Taken together, our data suggested that the expres-
sion levels of TTP and probably other Vit E transport
proteins might influence cellular Vit E concentrations
in different PCa cells.
The androgen receptor (AR) is required for normal
prostate development and prostate cancer cell prolif-
eration and survival [25–27]. We have identified that
downregulationofARsignaling contributestothe VES
LNCaP cells and AR-negative PC3 cells are equally
only genetic difference responsible for VES-mediated
inhibition of prostate cancer cells growth.Inthis study,
we found that cellular Vit E bioavailability may
represent another factor for Vit E inhibition effects on
prostate cancer cells. This notion was supported by the
observation that the cellular Vit E bioavailability is
The Prostate DOI 10.1002/pros
Fig. 4. ThedifferentialexpressionlevelsofVitEtransportgenesinPCacells.ThemRNAexpressionlevelsofVitEabsorptiongenesTAP(A),
6 NiandY eh
positively correlated with Vit E anti-proliferactive
activity in prostate cancer cells.
TTP has high expression in liver and low expression
information is available to show the expression status
of TTP in prostate. Using RT-PCR, we isolated TTP full
length cDNA and did not identify any mutation in
benign prostate and prostate cancer cells. TTP function
in prostate is not well known. With the help of TTP,
a-Vit E may be supplied from the prostate epithelial
The Prostate DOI 10.1002/pros
Fig. 5. TTPmodulates thecellularVitEcontentinprostatecancercells.A:TTPexpressionswereknockeddowninDU145cellsbysiRNA
threeindependentexperiments. Statistics P valueiscomparedto the data ofDU145cells.D:TTPwas ectopicallyexpressedinLNCaPcells.
treatment.LN-TTPor empty vector cells LN-V were treatedwith 20 mM a-Vit E or VES for1day.Cells were harvested and counted at the
cells to the seminal fluid to nourish sperm. Interest-
ingly, the mRNA expression level of TTP is much
higher in prostate cancer cell line DU145 compared to
other prostate cancer cells. We used knockdown stra-
tegy to study the function of TTP in prostate cancer
cells, however, our siRNA only knocked down 40% of
TTP mRNA levels, still higher than that in prostate
cancer LNCaP and PC3 cells. Moreover, since the
cultured medium contains excess amount of Vit E, the
function of TTP to efflux a-Vit E may be inhibited.
Nevertheless, our data suggested that TTP mediated
the transport of a-Vit E, but not VES in prostate cancer
in PCa cells after the same dose treatment. TTP selec-
tively transports a-Vit E from the liver into the serum
after Vit E absorption in the intestine [29,30]. Our
current studies suggested that TTP can mediate the
transport of a-Vit E, but not the a-Vit E ester analog,
studies show that TAP not only mediates a-Vit E, but
also mediates VES uptake . The specificity of TTP
may contribute to higher VES amount than a-Vit E
levels after same dose treatment. However, we do not
exclude that other genes may regulate Vit E/Vit E
to the final cellular Vit E amount. In this study, we also
found DU145 cells have a higher rate of hydrolysis of
VES to a-Vit E (Fig. 2D), therefore increased resistance
to VES treatment. It is likely esterase expression levels
are higher in DU145 cells. Supportively, Kline et al.
cancer cells might cause them to be insensitive to VES
the rate of a-Vit E metabolism (converting a-Vit E into
other metabolites) is faster in DU145 cells than that in
LNCaP and PC3 cells. Thus, the total cellular concen-
tration of a-Vit E and VES is lower in the DU145 cells.
Further studies are needed to verify this possibilities in
Different prostate cancers have different sensitiv-
ities to a-Vit E treatment. This difference was, at least
partly, associated with the cellular accumulation or
suggested that Vit E-binding proteins and other
transport proteins might contribute to the retention of
a-Vit E in PCa cells.
We thank Karen Wolf and Susan R. Schoen for
manuscript preparation. This work is partly supported
by NIH grant DK60912.
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