Download Functional and physical interaction between the

Carcinogenesis vol.31 no.8 pp.1360–1366, 2010
doi:10.1093/carcin/bgq114
Advance Access publication June 7, 2010
Functional and physical interaction between the selenium-binding protein 1 (SBP1) and
the glutathione peroxidase 1 selenoprotein
Wenfeng Fang1,2,3, Marci L.Goldberg1, Nicole M.Pohl1,
Xiuli Bi1, Chang Tong1, Bin Xiong3, Timothy J.Koh4,
Alan M.Diamond1,5 and Wancai Yang1,5,
1
Department of Pathology, University of Illinois at Chicago, 840 South Wood
Street, CSN 130, Chicago, IL 60612, USA, 2The Open Laboratory for
Overseas Scientists and 3Department of Oncology, Zhongnan Hospital, Wuhan
University, Wuhan 430071, China and 4Department of Kinesiology and
Nutrition and 5Cancer Center, University of Illinois at Chicago, Chicago, IL
60612, USA
To whom correspondence should be addressed. Tel: þ1 312 355 4154;
Fax: þ1 312 996 7586;
Email: [email protected]
Selenium-binding protein (SBP) 1 is present in reduced levels in
several cancer types as compared with normal tissues, and lower
levels are associated with poor clinical prognosis. Another
selenium-containing protein, glutathione peroxidase 1 (GPX1),
has been associated with cancer risk and development. The interaction between these representatives of different classes of selenoproteins was investigated. Increasing SBP1 levels in either
human colorectal or breast cancer cells by transfection of an
expression construct resulted in the reduction of GPX1 enzyme
activity. Increased expression of GPX1 in the same cell types resulted in the transcriptional and translational repression of SBP1,
as evidenced by the reduction of SBP1 messenger RNA and protein and the inhibition of transcription measured using an SBP1
reporter construct. The opposing effects of SBP1 and GPX1 on
each other were also observed when GPX1 was increased by supplementing the media of these tissue culture cells with selenium,
and the effect of selenium on SBP1 was shown to be GPX1
dependent. Decreasing or increasing GPX1 levels in colonic
epithelial cells of mice fed a selenium-deficient, -adequate or
-supplemented diet resulted in the opposing effect on SBP1 levels.
These data are explained in part by the demonstration that SBP1
and GPX1 form a physical association, as determined by coimmunoprecipitation and fluorescence resonance energy transfer assay.
The results presented establish an interaction between two distinct selenium-containing proteins that may enhance the understanding of the mechanisms by which selenium and selenoproteins
affect carcinogenesis in humans.
Introduction
Mammalian selenium-containing proteins fall into three distinct categories (1,2). In one, selenium is erroneously substituted for sulfur in
sulfur-containing amino acids due to the similarity in structure between these two elements. The second class consists of proteins in
which selenium is a constituent of the amino acid selenocysteine,
which is inserted cotranslationally into selenoproteins in response to
a UGA codon in the corresponding messenger RNA (mRNA) (3). The
third class is composed of selenium-binding proteins (SBPs), which
bind selenium by a mechanism that has yet to be clarified. Given the
wealth of data indicating that low non-toxic levels of selenium can
prevent cancer in animal models, and human epidemiology establishing an inverse association between dietary selenium intake and the
risk of several cancer types (4–10), there is considerable interest in
investigating the role of selenium-containing proteins in cancer etiology, although the Selenium and Vitamin E Cancer Prevention Trial
Abbreviations: cDNA, complementary DNA; GFP, green fluorescent protein;
GPX1, glutathione peroxidase 1; HA, hemagglutinin; mRNA, messenger RNA;
PCR, polymerase chain reaction; SBP, selenium-binding protein 1.
showed lack of efficacy of selenium (11) and other studies have indicated that higher selenium intake may be associated with increased
risk of diabetes (11–13).
The human SBP gene (SBP1, SELENBP1 or hSP56) (14) is located
on chromosome 1 at q21–22 and is the homologue of the mouse SP56
gene that was originally reported as a 56 kDa mouse protein that
stably bound 75selenium (15,16). The human complementary DNA
(cDNA) contains a 472 amino acid-encoding open reading frame (14)
and protein resides both in the nucleus and in the cytoplasm (17). It is
expressed in a variety of tissue types, including the heart, lung, kidney
and tissues of the digestive tract. The function of SBP1 is unknown
although it may be involved in intra-golgi transport (18). SBP1 was
recently reported to interact with the von Hippel–Lindau protein
(pVHL)-interacting deubiquitinating enzyme 1 (VDU1) and might
play a role in ubiquitination/deubiquitination-mediated protein degradation pathways in a selenium-dependent manner (19). We and
others have recently reported that the levels of SBP1 are significantly
decreased in various epithelial cancers, including those of the prostate
(20), stomach (21), ovary (22), lung (17) and colon (23,24). More
recently, we found that SBP1 becomes silenced by methylation in
colon cancers and can influence sensitivity to oxidative stress, cell
migration and tumorigenesis (25). Decreased expression of SBP1 in
lung and colorectal cancer is also associated with poor prognosis
(17,23,24).
Among the 25 human selenocysteine-containing proteins, there is
considerable evidence that the cytosolic form of glutathione peroxidase 1 (GPX1) is associated with cancer risk. GPX1 is a ubiquitously
expressed enzyme that detoxifies hydroperoxides using reducing
equivalents from glutathione. Human studies have demonstrated that
a functional GPX1 gene polymorphism at codon 198 resulting in
either a proline (Pro) or a leucine (Leu) at that position is associated
with increased risk for several types of cancer, including those of the
head and neck, breast, colon, lung and prostate (26,27). In addition,
allelic loss at the GPX1 locus is a common event in cancer development (28,29). This, as well as in vitro data indicating that reduced
GPX1 expression can sensitize mammalian cells to DNA damage (30)
and that overexpression can have the opposite effect (31), support
a role for GPX1 levels in determining cancer risk. Given the cumulative data indicating possible roles of both SBP1 and GPX1 in cancer
development and/or outcome, the interaction of these two seleniumassociated proteins was investigated in several model systems in the
present study.
Materials and methods
Cell lines and cell culture
The human colon cancer cell line HCT116 and breast cancer cell line MCF-7
were obtained from the American Type Culture Collection (Manassas, VA).
Derivative MCF-7 cell lines obtained by transfection of a GPX1 expression
construct (designed MCF-7-GPX1) and a control vector-only (pLNCX)transfected cell line MCF-7 (designed MCF-7-vector) were previously generated
(28). HCT116 cells were cultured in McCoy’s 5A medium with 10% fetal bovine
serum and antibiotics (10 000 U/ml penicillin and 10 lg/ml streptomycin).
The selenium concentration of the fetal bovine serum was determined to be
253 nM by the Analytical Services laboratory at South Dakota State
University. MCF-7 cells were cultured in modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics (10 000 U/ml penicillin
and 10 lg/ml streptomycin). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. Sodium selenite (Sigma, St Louis, MO) was
dissolved in phosphate-buffered saline and stored at 20°C until used.
Plasmids construction and establishment of SBP1 stable transfection cell line
A human SBP1 expression plasmid (pIRES2-SBP1) was generated using normal colon mucosa DNA as template for polymerase chain reaction (PCR)
amplification and subcloning into the pIRES2 vector (Clontech Laboratory,
Ó The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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GPX1 and SBP1 interaction and regulation
Mountain View, CA) using the following primers: forward 5#-ATCTCGAGCTATGGCTACGAAATGTGGGAAT-3# and reverse 5#-ATGGATCCTCAAATCCAGATGTCAGAGCTAC-3#. The PCR product was digested with XhoI
and HindIII and the digestion products were ligated into the vector. Another
SBP1 expression plasmid was generated by inserting human SBP1 cDNA into
pcDNA3-hemagglutinin (HA) vector (Clontech Laboratory) using the forward
primer 5#-ATCTCGAGATGGCTACGAAATGTGGGAATT-3# and reverse
primer 5#- ATTCTAGATCAAATCCAGATGTCAGAGCTAC-3# with XhoI
and XbaI digestion. Similarly, a human GPX1 cDNA was amplified using
the forward primer 5#-TCAAGCTTGTGCTGCTCGGCTAGCGGCG-3# and
reverse primer 5#-ATGGATCCCTGCTACACCCGGCACTTTAT-3#, respectively, and inserted into pECFP-C1 at the HindIII and BamH1 sites. A mutant
GPX1 plasmid (pECFP-GPX1-Cys) was constructed by changing the selenocysteine (UGA) into a cysteine (UGC) using two-step PCR and ligation. Two
sets of primers were used for the PCR amplification: set 1 forward primer
5#-TCAAGCTTGTGCTGCTCGGCTAGCGGCG-3# and GPX1-Cys reverse
primer 5#-.
CGCTGCAGCTCGTTCATCTGGGTGTAGTCCCGGACCGTGGTGCCGCA-3#; set 2 GPX1-Cys forward primer 5#-AGCTGCAGCGGCGCCTCGGA3# and reverse primer 5#-ATGGATCCCTGCTACACCCGGCACTTTAT-3#.
These two products were digested with PtsI and ligated and then were inserted
into the pECFP-C1 vector at the HindIII and BamH1 sites. A human SBP1
promoter (2572 to þ1) reporter construct (designed pGL4-SBP1) was generated by PCR amplification of the human SBP1 promoter and insertion into
the pGL4 luciferase reporter at the XhoI and HindIII sites using a forward
primer 5#-ATCTCGAGCCCATACATACCAAGCAA-3# and reverse primer
5#-TCAAGCTTCGGGTTTGCTGTGCTGGTGTC-3#. The sequences of all
generated recombinant constructs were confirmed by sequencing. Transfection
of plasmids into the human colon cancer cells HCT116 was accomplished
using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Clones were selected
using 500 lg/ml of G418.
Quantitative real-time PCR
To quantify expression, total RNA was isolated and cDNA was synthesized
using TaqMan MultiScribe Reverse Transcriptase (Applied Biosystems, Foster
City, CA) as described recently (24). Quantitative real-time PCR analysis was
performed using an ABI Prism 7900-HT Sequence Detection System (96-well,
Applied Biosystems) using the forward primer 5#-CCAAGCTCATCACCTGGTCT-3# and reverse primer 5#-TCGATGTCAATGGTCTGGAA3# for GPX1. Primers used for quantification of mRNA levels for SBP1 and
b-actin were previously reported (24).
Measurement of GPX activity
GPX activity was measured using a standard coupled spectrophotometric
method as described by Samuels et al. (32). Briefly, cells were washed with
ice-cold phosphate-buffered saline twice, scraped and transferred to a 1.5 ml
tube. Cells were resuspended in sodium phosphate buffer (0.1 M, pH7.0) and
lysed by sonication. Determination of GPX1 activity was calculated based on
the consumption of reduced nicotinamide adenine dinucleotide phosphate in
a reaction containing reduced glutathione, glutathione reductase, cell lysate
and hydrogen peroxide. Units of GPX1 activity are expressed as nanomoles of
reduced nicotinamide adenine dinucleotide phosphate oxidized per minute per
milligram protein. Assays were conducted in triplicate by using three independently generated lysates from separate cultures.
Immunoblotting
Cells were harvested and suspended in RIPA lysis buffer (Upstate Biotechnology, Lake Placid, NY) containing a protease inhibitor cocktail (Sigma–Aldrich
Corporation, St Louis, MO). After incubation on ice for 15 min, cell lysates
were centrifuged at 12 000 r.p.m. for 15 min at 4°C. The protein content of the
supernatant was determined using the Bio-Rad Protein Assay Reagent
(Bio-Rad Laboratories, Hercules, CA). Proteins were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to
polyvinylidene difluoride membranes (Bio-Rad Laboratories). Blots were
probed with antibodies specific for SBP1, GPX1 (Medical and Biological
Laboratories Co., Nagoya, Japan) and b-actin (Sigma–Aldrich Corporation).
Signals were detected by enhanced chemiluminescence Plus reagents (Amersham Pharmacia, Piscataway, NJ). The signals were quantified by densitometry
using Quantity One software (Bio-Rad) and normalized to b-actin.
SBP1 promoter activity
Transfected cells were plated in 24-well plates and cotransfected with either
the pGL4-SBP1 or the pGL4 control luciferase reporter plasmid, along with
a Renilla luciferase plasmid used for transfection efficiency normalization
using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s
instructions. At 48 h after transfection, the cells were harvested and the firefly
and Renilla luciferase activities were measured using a dual luciferase kit
(Promega, Madison, WI). The firefly luciferase data for each sample was
normalized based on transfection efficiency using Renilla luciferase activity.
Each experiment was repeated at least three times.
Mouse diets and tissue preparation
Fifteen C57Bl/6 mice were purchased from Harlan Laboratories (Indianapolis,
IN) and provided a Torula yeast-based diet containing 0, 0.1 or 0.4 p.p.m.
selenium in the form of sodium selenite for 10 weeks, based on the feeding
scheme described by Novoselov et al. (33). The 0.1 p.p.m. diet would approximate the 55 mg/day intake in humans that is the Recommended Daily Allowance and 0.4 p.p.m would approximate the 200 mg/day commonly used in
human chemoprevention trials with selenium. Mouse colonic and duodenal
epithelial cells were isolated by incubating with 15 mM ethylenediaminetetraacetic acid buffer for 30 min at 37°C, and cell pellets were collected by
centrifuging at 4°C as described recently (34). Cell lysates were used for
immunoblotting analysis using Anti-SBP1 (Medical and Biological Laboratories Co.) and anti-GPX1 (Abcam, Cambridge, MA) antibodies.
Coimmunoprecipitation analysis
HCT116 cells were cotransfected with the SBP1 expression plasmid pcDNA3HA-SBP1 and GPX1 expression plasmid pECFP-GPX1, GPX1 mutation
plasmid pECFP-GPX1-Cys or empty vector pECFP. Twenty-four hours
posttransfection, the cells were harvested and lysed in RIPA lysis buffer
(20 mM Tris–HCL, pH 8.0, 150 mM NaCl, 1% Triton X-100 and protease
inhibitor cocktail) and then the cell lysates were immunoprecipitated with antigreen fluorescent protein (GFP) antibody (recognition of pECFP-GPX1), the
latter precipitate was probed with anti-HA antibody (for SBP1). The immunocomplex was visualized using enhance chemiluminescence reagent plus
(Amersham Biosciences, Piscataway, NJ).
Results
SBP1 negatively regulates GPX1 activity
SBP1 and GPX1 represent distinct classes of proteins with the selenium moiety of GPX1 present as the amino acid selenocysteine. In
order to determine whether the levels of SBP1 could influence GPX
activity, we used a human colon cancer cell line (HCT116) that has
undetectable levels of SBP1 but significant levels of GPX1 protein
and enzyme activity (Figure 1). Increasing SBP1 levels in these cells
by transfection of an expression construct resulted in an 50% reduction in GPX1 activity as compared with cells transfected with
parental pIRES2 vector alone (Figure 1A). The reduction in GPX1
activity observed in SBP1-transfected cells was considerably more
than the amount seen when cells were transfected with the pIRES2
vector alone. Changes in GPX enzyme activity as a consequence of
the transfection process of the magnitude shown have been observed
previously (Yang, et al, unpublished results), indicating the necessity
for this particular control. As seen in Figure 1, the observed reduction
in GPX enzyme activity was posttranslational, not being associated
with lowering either GPX1 protein (Figure 1B) or mRNA levels
(Figure 1C).
Although colon-derived cells typically express both the GPX1 and
the gastrointestinal-GPX, GPX2, HCT116 cells contain relatively little GPX2 (Yang, et al, unpublished results). To confirm that SBP1
overexpression could specifically inhibit GPX1 activity and extend
these observations to a different cell type, SBP1 expression was increased in MCF-7 human breast carcinoma cells by transfection of the
SBP1 expression construct. MCF-7 cells do not express detectable
levels of GPX1 mRNA and display low or undetectable total GPX1
enzyme activity (28). Using a previously generated derivative MCF-7
cell line engineered such that all of its detectable GPX activity is
exclusively due to GPX1 (28) as the recipient of the SBP1 expression
construct, it was shown that overexpression of SBP1 caused a dramatic
reduction, 90%, of GPX activity in these cells (Figure 1D).
GPX1 negatively regulates SBP1
The results shown in Figure 1 establish that SBP1 could negatively
regulate GPX1 activity. To determine whether GPX1 could affect
SBP1 abundance, parental and GPX1-expressing MCF-7 cells were
examined for SBP1 expression and protein levels (Figure 2). Increased expression of GPX1 resulted in a significant decline in
SBP1 protein levels as compared with either the parental MCF-7 or
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W.Fang et al.
Fig. 2. GPX1 negatively regulated SBP1 expression. (A) SBP1 protein was
inhibited by GPX1. MCF-7 parental, MCF-7-vector and MCF-7-GPX1
stable transfected cells were lysed and subjected to immunoblotting for SBP1
and GPX1. SBP1 and GPX1 signals were quantified and normalized to
b-actin, a loading control. Relative intensities were indicated under lanes.
(B) SBP1 mRNA was significantly reduced in MCF-7-GPX1 cells compared
with the MCF-7-vector cells ( P , 0.01) (n 5 3). (C) GPX1 inhibited SBP1
promoter activity. MCF-7-vector and MCF-7-GPX1 cells were cotransfected
with Renilla and the pGL4 control or pGL4-SBP1 promoter. Twenty-four
hours after transfection, cells were harvested and luciferase activity
determined ( P , 0.01). (n 5 3). Each bar represents the mean ± SD of
samples obtained in triplicate.
Fig. 1. SBP1 inhibited GPX1 activity in HCT116 cells and MCF-7 cells but
did not affect GPX1 protein and mRNA levels. (A) Endogenous GPX1
activity was inhibited by SBP1 in HCT116 cells. HCT116 parental (mock),
pIRES2 empty vector control (pIRES2) and SBP1 overexpressing HCT116
cells (SBP1) were lysed and GPX1 activity determined ( P , 0.001) (n 5 3,
indicating the experiments were repeated three times). (B) GPX1 protein was
not affected by SBP1 overexpression. The HCT116 parental, pIRES2 empty
vector control (pIRES2) and SBP1 overexpressing HCT116 cells (SBP1)
were lysed and immunoblotted to determine GPX1 and SBP1 protein levels.
GPX1 signals were quantified by densitometry and normalized to b-actin,
a loading control. (C) GPX1 mRNA levels were not affected by SBP1
overexpression. Quantitative real-time PCR was used to quantify GPX1
mRNA in HCT116 parental, pIRES2 control and SBP1 overexpressing
HCT116 cells. The data were normalized to HCT116 parental cells (n 5 3).
(D) GPX1 activity derived from an expression construct was inhibited by
SBP1 in MCF-7-GPX1 cells. MCF-7-GPX1 cells were treated with
transfection reagents without plasmid (mock), pIRES2 control vector or
pIRES2-SBP1 plasmid. Forty-eight hours after transfection, cells were
harvested and GPX1 activity was determined ( P , 0.001) (n 5 3).
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the MCF-7-vector cells (Figure 2A). This reduction in SBP1 protein
levels was associated with 50% reduction in SBP1 mRNA levels as
determined by Quantitative real-time PCR (Figure 2B). To determine
whether the observed reduction in SBP1 RNA by GPX1 was due to
either an effect on transcription or RNA stability, a reporter construct
was generated using human SBP1 promoter sequences and luciferase
as the reporter protein. As seen in Figure 2C, overexpression of GPX1
inhibited transcription from the SBP1 promoter driving the reporter
gene.
Contrasting effects of selenium supplementation on SBP1 and GPX1
The data presented above indicated that increasing GPX1 by transfection resulted in reduction of SBP1 and increasing SBP1 by transfection resulted in the reduction of GPX1 activity. An alternative
approach to enhance GPX1 is by the addition of sodium selenite.
GPX1 activity in cell lines is generally inducible by low physiologically relevant doses of selenium (27). The media of HCT116-SBP1
and MCF-7-GPX1 cells were each supplemented with 25, 50, 100 or
GPX1 and SBP1 interaction and regulation
tained by western blotting (data not shown). One possible explanation
for these results is that SBP1 and GPX1 are competing for the available cellular selenium pool. A selenium titration study was performed
on HCT116-SBP1 cells using an expanded range of selenium supplementation, from 0 to 5 lM (Figure 4B). GPX1 levels increased and
SBP1 declined with supplementation, reaching a maximum at 250 nM
and then the trends reversed with increasing supplementation with
GPX1 declining and SBP1 increasing at the higher selenium concentrations. Similar results were obtained with MCF-7-GPX1 transfectants (data not shown).
Effects of selenium status on SBP1 and GPX1 in mouse intestinal
epithelial cells
The inverse association between SBP1 and GPX1 observed in tissue
culture cells was examined in intestinal epithelial cells obtained from
C57Bl/6 mice fed a selenium-deficient (0 p.p.m.), -adequate (0.1
p.p.m.) or -enriched (0.4 p.p.m.) diet. The levels of selenium in the
diet were selected as the lowest will result in the a dramatic reduction
in GPX1 in most tissues, the mid range corresponds to the amount of
selenium recommended for human intake and the highest dose is one
used for chemoprevention studies and would correspond to the level
used in human chemoprevention trials (35). Animals were fed the
respective diets for 10 weeks and colon and duodenal epithelial cells
were isolated for western blotting analysis using anti-SBP1 and antiGPX1 antibodies. As shown in Figure 5, there were very low levels of
GPX1 in cells derived from the animals maintained on the seleniumdeficient diet, much higher levels in those fed the ‘adequate’ diet
and GPX1 protein was the highest in cells obtained from mice on
selenium-supplemented diet. Consistent with the cell culture studies,
there was an inverse association between GPX1 levels and SBP1
levels in intestinal epithelial cells derived from both colon and
duodenum.
Fig. 3. Contrasting effects of sodium selenite on SBP1 and GPX1.
(A) Sodium selenite increased GPX1 expression and reduced SBP1 expression
in the MCF-7-GPX1 cells with addition of 0, 25, 100 or 250 nM sodium
selenite to the basal media, which contained at least 30 nM Se from the 10%
fetal bovine serum for 48 h. Cells were harvested and immunoblotted to detect
SBP1 and GPX1. SBP1 and GPX1 signals were quantified by densitometry
and normalized to b-actin, a loading control, with the relative intensities
indicated under the lanes. Similar experiments were performed in a HCT116SBP1 stably transfected cell line. (B) SBP1 was not affected by sodium
selenite in the MCF-7 cells that did not express GPX1. (C) All experiments
were repeated at least three times and representative blots shown.
250 nM of selenium in the form of sodium selenite for 48 h and levels
of GPX1 and SBP1 were determined by western blotting (Figure 3A
and B). As is typically observed, there was a dose–response with GPX
enzyme activity increasing with selenium supplementation (data not
shown). Consistent with what was observed in the transfection studies, there was an inverse association between the increase of GPX1
that occurred in a dose-dependent manner and the reduction of SBP1.
Since MCF-7 is null for GPX1 (28), use of these cells allows one to
determine whether the reduction in SBP1 that occurred with increasing selenium concentration was GPX1 dependent. MCF-7 cells transfected only with an empty vector were incubated with increasing
selenium concentration under the same conditions described above
for the GPX1 expressing lines and GPX1 and SBP1 were quantified.
As expected, no GPX1 signal was detectable in the western blot
(Figure 3C). In contrast to the results obtained with GPX1-expressing
cells, the MCF-7 cells did not exhibit a selenium-dependent decline in
SBP1, indicating that the selenium-mediated increment of GPX1 was
responsible for the corresponding decline of SBP1.
The relationship between GPX1 and SBP1 was further examined
by quantifying GPX1 enzyme activity in HCT116 transfected with
either SBP1 or vector and incubating the cells with increasing
amounts of selenium (Figure 4A). It is apparent that baseline GPX1
activity was lower in the SBP1-expressing cells and the seleniummediated increase in activity was attenuated at the tested doses. The
obtained enzyme activity corresponded to the levels of protein ob-
Direct interaction between SBP1 and GPX1
The ability of SBP1 and GPX1 to influence each others abundance
and/or activity raised the possibility that there were direct interactions
between these two proteins. To investigate this possibility, a plasmid
that expressed an HA-tagged SBP1 (pcDNA3-HA-SBP) was cotransfected with an expression construct containing a GFP-GPX1 fusion
protein expression construct (pECFP-GPX1), mutant GPX1 plasmid
(pECFP-GPX1-CYS) or the empty vector (pECFP) into HCT116
cells. Extracts from transfectants were analyzed by western blotting
(Figure 6A). Using antibodies directed against GFP, it is apparent that
the vector-only transfectant expressed GFP, whereas the cells expressing the GFP-GPX1 fusion protein revealed a larger protein whose
molecular weight was consistent with that expected from the fusion
(49 KD). As seen with the earlier studies, cells expressing the GFP–
GPX1 fusion also expressed less SBP1 as indicated by the reduced
signal seen with anti-HA antibodies. In order to determine whether
SBP1 and GPX1 physically bound each other, extracts from the three
transfectants were immunoprecipitated using anti-GFP antibodies and
probed with anti-HA to probe for the SBP1-HA-tagged protein in the
precipitates. As seen in Figure 6B, HA-tagged SBP1 was only present
in immunoprecipitates from cells that were also expressing the GFP–
GPX1 fusion proteins, including a derivative GPX1 in which the
selenocysteine coding UGA triplet was changed to the codon of
cysteine (GPX1-Cys), indicating the binding of SBP1 to GPX1. This
also excluded the possibility that the interaction occurs by the binding
of SBP1 to the selenium moiety of GPX1. The interaction between
SBP1 and GPX1 was further supported by fluorescence resonance
energy transfer assay (supplementary Figure S1 is available at Carcinogenesis Online).
Discussion
The goal of the work presented in this manuscript was to investigate
whether two proteins representing different classes of seleniumcontaining molecules functionally interacted. The first of these,
SBP1, binds selenium and low levels of SBP1 are associated with
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W.Fang et al.
Fig. 4. (A) SBP1 attenuated sodium selenite-induced increase of GPX1 activity in HCT116 cells. HCT116-pIRES2 control and HCT116-SBP1 stable cells were
treated with addition of 0, 25, 100 or 250 nM sodium selenite to the basal media, which contained at least 30 nM Se from the 10% fetal bovine serum. Forty-eight
hours after treatment, the cells were lysed and GPX1 activity was determined. (B) GPX1 levels increased and SBP1 declined with supplementation of sodium
selenite, reaching a maximum at 250 nM and then the trends reversed with increasing supplementation with GPX1 declining and SBP1 increasing at the higher
selenium concentrations. SBP1 and GPX1 signals were quantified by densitometry and normalized to b-actin, a loading control, with the relative intensities
indicated under the lanes. All experiments were repeated at least three times and representative blots shown.
cancer and poor clinical outcome (17,23,24). The second, GPX1,
contains selenium as the amino acid selenocysteine (2), and increasing or decreasing GPX1 levels has an inverse effect of susceptibility
of cells to DNA damage (31,36–39). Human data have indicated that
functional polymorphisms in GPX1 are associated with increased
cancer risk and allelic loss of GPX1 frequently occurs during cancer
development (26,28,29). SBP1 and/or GPX1 are therefore potential
mediators of the chemopreventive effects of selenium identified by
human epidemiology and animal supplementation studies.
Increasing the levels of SBP1 in either colon-derived HCT116 cells
or MCF-7 breast carcinoma cells resulted in a consequential decline in
GPX1 enzyme activity. How SBP1 affected GPX1 activity remains to
be determined, but neither GPX1 RNA nor protein levels were
changed (Figure 1). One possible mechanism to explain these results
would involve a direct physical interaction between the two proteins
resulting in reduced GPX activity. Support for this notion can be seen
in the coprecipitation data presented in Figure 6 showing that SBP1
and GPX1 do form a complex. How these proteins interact remains to
be determined, but it is unlikely that this interaction occurs by the
binding of SBP1 to the selenium atom at the GPX1 active site as the
selenium is probably to be inaccessible to a molecule the size of SBP1
and SBP1 is able to bind mutant GPX1 (Figure 6B).
As seen in Figure 2, overexpression of GPX1 resulted in the reduction of SBP1 in the same cell lines used to investigate the effects of
SBP1 overexpression. This effect is due, at least in part, to the inhibition of SBP1 transcription, as determined by the GPX1-mediated
reduction in both SBP1 mRNA (Figure 2B) and expression from an
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SBP1 promoter-driven reporter gene (Figure 2C). Little is known
about the regulation of SBP1 transcription, but it is possible that
SBP1 expression is dependent on reactive oxygen-responsive transcriptional elements, and the reduction in peroxides that is expected
to be achieved with increased GPX1 activity results in the attenuation
of transcription. A search for an antioxidant response element (40) in
the DNA region upstream of the SBP1 transcription start site revealed
two sequences (GTGACCCTGGC and TGACACCAGC) that are very
similar to the consensus antioxidant response element recognition
motif (40), located just 3# of the transcription start site, although
the functionality of this sequence as an antioxidant response element
remains to be established.
Also consistent with the studies using ectopic expression of either
SBP1 or GPX1, selenium titration experiments indicated an inverse
association in the expression of these proteins. GPX1 protein levels
rose with selenium supplementation of HCT116- and MCF-7-derived
cell lines with a corresponding decline in SBP1. This effect was dependent on the presence of GPX1 and not some other consequence or
function of selenium. This was established by showing the seleniumassociated decline in SBP1 did not occur in GPX1 null MCF-7
cells (Figure 3C). The relationship between SBP1 and GPX1
was also apparent in mice fed a selenium-deficient, -adequate
or -supplemented diet. Selenium deficiency resulted in a dramatic
reduction of GPX1 in colonic and duodenal epithelial cells, whereas
selenium supplementation resulted in elevated GPX1 as compared
with those animals fed a selenium-adequate diet. For each level of
dietary intake, there was a corresponding decline in SBP1 levels,
GPX1 and SBP1 interaction and regulation
Fig. 5. Dietary selenium status affected GPX1 and SBP1 expression in mouse
colonic (A) and duodenal epithelial cells (B). Mice were fed with seleniumadequate diet (0.1 p.p.m.), selenium-deficient diet (0 p.p.m., Se-deficiency) or
selenium-enriched diet (0.4 p.p.m. Se-supplemental) for 10 weeks. Intestinal
epithelial cells were isolated for western blotting analysis for GPX1 and SBP1.
b-actin was used as loading control. Each lane represented one individual
mouse and three mice from each group were studied.
extending the results obtained in cell culture to animals. It is particularly noteworthy that supra nutritional amounts of selenium in the
diet that have been associated with cancer prevention in animals
studies, including those investigating colon cancer prevention (41),
resulted in changes in the levels of the two investigated seleniumcontaining proteins.
Collectively, the work presented herein establishes an interaction
among selenium availability, the amount of a protein that binds
selenium—SBP1 and another protein in which selenium is present
as the amino acid selenocysteine. These data raise questions as to
whether higher levels of GPX1 in tumors promote carcinogenesis
by suppressing SBP1 and whether SBP1 has biological functions
beyond its interaction with GPX1. Furthermore, there may be biological consequences of the regulatory interaction between GPX1 and
SBP1 that reflect on the potential health consequences of selenium
dietary intake. An inverse association between dietary selenium intake and cancer risk has been reported for several organ systems (42).
Encouraged by the results that were initially presented for the Nutritional Prevention of Cancer selenium supplementation trial (43,44),
the large cancer prevention study selenium and vitamin E cancer
prevention trial that investigated the efficacy of selenium, both in
concert with vitamin E and alone, in the prevention of prostate cancer
was initiated (45). This study was terminated early, in part due to the
lack of efficacy of selenium (11), although the analysis of the cohort
following stratification by selenium status and genetic polymorphisms
has not yet been reported. Data from selenium and vitamin E cancer
prevention trial, as well as an additional recent studies, have indicated
that higher selenium intake may also be associated with increased risk
of diabetes (11–13). Whether any of the possible benefits or risks of
selenium intake are due to consequential effects on GPX1 and SBP1
remains to be determined.
Supplementary material
Supplementary FigureS1 can be found at http://carcin.oxfordjournals
.org/
Funding
National Cancer Institute (CA112081 to W.Y. and CA127943 to
A.M.D.); American Institute for Cancer Research (05A121 to
W.Y.); State Scholarship Fund of China (2007-3020) to F.W.
Acknowledgements
We would like to thank Dr Frank J.M.van Kuppeveld (Radboud University
Nijmegen Medical Centre, Netherlands) for providing the pECFP and pEYFPECFP plasmids and thank Dr Dong Hu (University of Illinois at Chicago, IL)
for technical assistance.
Conflict of Interest Statement: None declared.
References
Fig. 6. SBP1 interacts with GPX1 as determined by immunoprecipitation
(IP). HCT116 cells were cotransfected with SBP1 expression plasmid
pcDNA-HA-SBP1 and GPX1 expression plasmid pECFP-GPX1 or empty
vector pECFP or Sec-Cys GPX1 plasmid (pECFP-GPX1-Cys). (A) Wholecell lysates were analyzed by immunoblotting (IB) to confirm transfection
efficiency and downregulation of SBP1 by GPX1. (B) The cell lysates were
incubated with anti-GFP antibody to immunoprecipitate pECFP-GPX1 and
pECFP-GPX1-Cys and then probed with anti-HA antibody to detect the HAtagged SBP1. Non-specific peptide (NSP) in the same blot showed the equal
immunoprecipitation of pECFP, pECFP-GPX1 and pECFP-GPX1-Cys. Lane
1, cell lysate from cells cotransfected with HA-SBP1 and pECFP; lane 2, cell
lysate from cells cotransfected with HA-SBP1 and pECFP-GPX1 and lane 3,
cell lysate from cells cotransfected with HA-SBP1 and pECFP-GPX1-Cys.
Equal amount of protein were added to the gel and all experiments were
repeated at least three times with representative blots shown.
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Received September 23, 2009; revised May 25, 2010; accepted May 29, 2010