Studies on the metabolism and biological activity of the epimers of sulindac

Article abstract of DOI:10.1124/dmd.110.037663

Sulindac is a nonsteroidal, anti-inflammatory drug (NSAID) that has also been studied for its anticancer activity. Recent studies suggest that sulindac and its metabolites act by sensitizing cancer cells to oxidizing agents and drugs that affect mitochondrial function, resulting in the production of reactive oxygen species and death by apoptosis. In contrast, normal cells are not killed under these conditions and, in some instances, are protected against oxidative stress. Sulindac has a methyl sulfoxide moiety with a chiral center and was used in all of the previous studies as a mixture of the R- and S-epimers. Because epimers of a compound can have very different chemical and biological properties, we have separated the R- and S-epimers of sulindac, studied their individual metabolism, and performed preliminary experiments on their effect on normal and lung cancer cells exposed to oxidative stress. Previous results had indicated that the reduction of (S)-sulindac to sulindac sulfide, the active NSAID, was catalyzed by methionine sulfoxide reductase (Msr) A. In the present study, we purified an enzyme that reduces (R)-sulindac and resembles MsrB in its substrate specificity. The oxidation of both epimers to sulindac sulfone is catalyzed primarily by the microsomal cytochrome P450 (P450) system, and the individual enzymes responsible have been identified. (S)-Sulindac increases the activity of the P450 system better than (R)-sulindac, but both epimers increase primarily the enzymes that oxidize (R)-sulindac. Both epimers can protect normal lung cells against oxidative damage and enhance the killing of lung cancer cells exposed to oxidative stress. Copyright

Full text of DOI:10.1124/dmd.110.037663

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090-9556/11/3906-1014–1021$25.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
DMD 39:1014–1021, 2011
Vol. 39, No. 6
37663/3688349
Printed in U.S.A.
Studies on the Metabolism and Biological Activity of the
Epimers of Sulindac
David Brunell, Daphna Sagher, Shailaja Kesaraju, Nathan Brot, and Herbert Weissbach
Center for Molecular Biology and Biotechnology, Florida Atlantic University, John D. MacArthur Campus, Jupiter, Florida
Received December 10, 2010; accepted March 7, 2011
ABSTRACT:
Sulindac is a nonsteroidal, anti-inflammatory drug (NSAID) that has their effect on normal and lung cancer cells exposed to oxidative
also been studied for its anticancer activity. Recent studies sug- stress. Previous results had indicated that the reduction of (S)-
gest that sulindac and its metabolites act by sensitizing cancer sulindac to sulindac sulfide, the active NSAID, was catalyzed by
cells to oxidizing agents and drugs that affect mitochondrial func- methionine sulfoxide reductase (Msr) A. In the present study, we
tion, resulting in the production of reactive oxygen species and
death by apoptosis. In contrast, normal cells are not killed under
purified an enzyme that reduces (R)-sulindac and resembles MsrB
in its substrate specificity. The oxidation of both epimers to sulin-
these conditions and, in some instances, are protected against dac sulfone is catalyzed primarily by the microsomal cytochrome
oxidative stress. Sulindac has a methyl sulfoxide moiety with a P450 (P450) system, and the individual enzymes responsible have
chiral center and was used in all of the previous studies as a
been identified. (S)-Sulindac increases the activity of the P450
mixture of the R- and S-epimers. Because epimers of a compound system better than (R)-sulindac, but both epimers increase primar-
can have very different chemical and biological properties, we ily the enzymes that oxidize (R)-sulindac. Both epimers can protect
have separated the R- and S-epimers of sulindac, studied their normal lung cells against oxidative damage and enhance the killing
individual metabolism, and performed preliminary experiments on of lung cancer cells exposed to oxidative stress.
Introduction
against oxidative damage and aging (for review, see Weissbach et al.,
005). This enzyme family includes two major classes, MsrA and
MsrB, that specifically reduce the S- and R-epimers of methionine
sulfoxide, Met-S-(o) and Met-R-(o), respectively (Sharov et al., 1999;
Grimaud et al., 2001). In mammalian cells there is one MsrA gene and
three MsrB genes, referred to as MsrB1, MsrB2, and MsrB3 (Kim and
2
Sulindac was originally developed as a nonsteroidal, anti-inflam-
matory drug (NSAID) (Van Arman et al., 1976; Huskisson and Scott,
978). Sulindac is a prodrug with a sulfoxide moiety that requires in
vivo reduction to sulindac sulfide, the pharmacologically active me-
tabolite (Duggan et al., 1977). Because sulindac has a chiral sulfur
1
center, it can exist as either the R- or S-epimer. Earlier in vivo studies Gladyshev, 2004). In addition to protein-bound Met-S-(o), MsrA can
on the metabolism of sulindac, containing a mixture of both epimers, efficiently reduce the S-epimer of free methionine sulfoxide and other
showed that sulindac could be reduced to sulindac sulfide and oxi- methyl sulfoxide compounds (Moskovitz et al., 1996). That the S-
dized to sulindac sulfone (Fig. 1) (Duggan et al., 1977; Ratnayake et epimer of sulindac was specifically reduced by MsrA was verified in
al., 1981).
later studies (Etienne et al., 2003). In contrast, MsrB from E. coli and
PilB from Neisseria gonorrhoeae appear to be relatively specific for
Met-R-(o) in proteins with very weak activity toward free Met-R-(o)
Little is known about the enzymes that are involved in the reduction
of sulindac to sulindac sulfide or the oxidation of sulindac to sulindac
sulfone. Our initial interest in sulindac arose from the finding that this
drug, which contains a methyl sulfoxide moiety, is a substrate for
methionine sulfoxide reductase (Msr) A (Moskovitz et al., 1996;
Etienne et al., 2003). The Msr family of enzymes are primarily
responsible for the reduction of protein-bound methionine sulfoxide to
methionine and appear to play an important role in protecting cells
(
Grimaud et al., 2001; Lowther et al., 2002). However, a partially
purified preparation of MsrB1 from mouse liver has been reported to
reduce free Met-R-(o) (Moskovitz et al., 2002).
With regard to the oxidation of sulindac to sulindac sulfone, sulin-
dac oxidation in liver microsomes was reported (Kitamura and Tat-
sumi, 1982) and found to be enhanced by 3-methylcholanthrene
treatment. It has been reported (Ciolino et al., 2006, 2008) that
sulindac induces some members of the microsomal P450 system via a
mechanism involving the aryl hydrocarbon receptor (AHR), but the
specific enzymes responsible for sulindac oxidation were not identi-
fied. No studies on the oxidation of the individual epimers of sulindac
have been reported.
The research was funded in part by the National Institutes of Health National
Cancer Institute [Grant R15-CA12200-01 A1] (to H.W.); and Florida State Univer-
sity Research Commercialization Assistance [Grant R94007] (to H.W.).
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.037663.
ABBREVIATIONS: NSAID, nonsteroidal anti-inflammatory; Msr, methionine sulfoxide reductase; Met-S-(o), S-epimer of methionine sulfoxide;
Met-R-(o), R-epimer of methionine sulfoxide; AHR, aryl hydrocarbon receptor; TBHP, tert-butyl hydroperoxide; DABS, 4-N,N-dimethylaminoazo-
benzene-4-sulfonyl; DTT, dithiothreitol; TrxA, thioredoxin; TrxB, thioredoxin reductase; HPLC, high-performance liquid chromatography.
1014

METABOLISM AND BIOLOGICAL ACTIVITY OF SULINDAC EPIMERS
1015
FIG. 1. Structure of sulindac and its reduced and oxidized metabolites. The methyl sulfoxide moiety of sulindac can be reduced to sulindac sulfide or oxidized to sulindac
sulfone.
In addition to its anti-inflammatory effects, sulindac and its metab- prepared them by separation of the epimers using a modification of a procedure
described previously (Hamman et al., 2000). The sulindac epimers were
dissolved in 1.0 M Tris buffer, pH 8.0. DABS-S-Met-(o) and DABS-R-Met-(o)
were prepared as described previously (Minetti et al., 1994). Clones containing
the genes for E. coli TrxA and TrxB, E. coli and bovine MsrA, and human
MsrB2 and MsrB3 were overexpressed in E. coli, and the respective proteins
were purified as described previously (Rahman et al., 1992; Moskovitz et al.,
olites have been shown to have anticancer activity in a number of
cancer cell lines and have also been tested clinically (Hixson et al.,
1
994; Taketo, 1998a,b; Gwyn and Sinicrope, 2002). There has been
renewed interest in sulindac because of several reports that sulindac
can selectively kill cancer cells in culture when combined with com-
pounds that affect mitochondrial respiration (Park et al., 2008; Seo et
al., 2008; Marchetti et al., 2009). As an example, recent results from
our laboratory, using sulindac with either hydrogen peroxide or tert-
1
996; Sagher et al., 2006a,b). Human and rat recombinant cytochrome P450
enzymes (Supersomes) were obtained from BD Gentest (Woburn, MA). The
individual P450 enzymes contained various amounts of NADPH cytochrome
butyl hydroperoxide (TBHP), showed that mitochondrial dysfunction, P450 reductase, and rat 2B1 and 3A2 and human 3A4 and 2C8 enzymes also
leading to ROS production, was responsible for the selective, en- contained cytochrome b . Sprague-Dawley rats were supplied by Charles River
5
hanced killing of colon, lung, and skin cancer cells in culture, In Laboratories, Inc. (Wilmington, MA). A GST fusion protein (36.5 kDa)
containing the first 95 amino acids of mammalian MsrB1 (10.5 kDa) and the
addition, there have been two promising clinical studies using sulin-
anti-SepX1 antibody were purchased from Abnova (Taipei City, Taiwan). The
dac drug combinations. A limited, proof-of-concept clinical study,
GST tag was removed with PreScission protease (GE Healthcare, Little Chal-
using sulindac and hydrogen peroxide for the treatment of actinic
font, Buckinghamshire, UK).
keratoses, showed a 50% cure rate (Resnick et al., 2009). In a second,
In Vivo Experiments. The sulindac epimers (2 mg/animal) were adminis-
tered by intraperitoneal injection to 20 rats, 10 receiving the R-epimer and 10
receiving the S-epimer. Animals were euthanized 0.5 to 4 h postinjection.
Heparinized blood was then collected, and the plasma was separated by
larger, 3-year clinical study, sulindac was used in combination with
difluoromethylornithine, an inhibitor of ornithine decarboxylase, to
determine its effect on the recurrence of colon polyps. The patients
receiving the drug combination showed an 80% decrease in recur- centrifugation at 18,000g for 20 min. Liver, skin, and brain tissues were
rence of colon polyps and a 90% drop in the appearance of adeno- minced and then homogenized using 5 strokes in a Potter-Elvehjem homoge-
carcinomas, compared with the control group of patients (Meyskens et nizer with 3 volumes of 50 mM Tris-Cl, pH 7.4 (buffer A). The homogenates
were centrifuged for 20 min at 16,000g, and the supernatant was assayed.
HPLC Assay for Msr Activity and Sulindac Reduction. The Msr assay
was based on the reduction of DABS-Met(o) to DABS-Met, adapted to HPLC
al., 2008). However, there is no information on the activity of the
individual sulindac epimers in any of the anticancer studies.
In contrast to the results in studies with cancer cells, pretreatment
analysis (Minetti et al., 1994; Moskovitz et al., 1997). Reaction mixtures
of normal lung cells with sulindac followed by exposure to TBHP
contained 100 mM Tris, pH 7.4, 20 nmol of the indicated DABS-Met(o)
protected these cells against oxidative stress (Marchetti et al., 2009).
These studies have recently been extended to cardiac tissue using a
Langendorf model, under conditions of ischemia and reoxygenation
known to cause oxidative damage to cardiac tissue. It was shown that
epimer, and partially purified rat liver extract in a final volume of 100 ␮l. The
reducing system consisted of either 15 mM DTT or the Trx system [1 mM
NADPH, 1.2 ␮g of TrxB, and 5 ␮g of TrxA]. Incubations were performed at
3
7°C for the time specified. Under these conditions, the enzymatic reduction
under these conditions, sulindac protected cardiac tissue from oxida- was proportional to the enzyme concentration and time until 75% (15 nmol) of
tive damage by a chemical preconditioning mechanism (Moench et the substrate was reduced. The reactions were terminated by adding 300 ␮l of
al., 2009).
acetonitrile. After centrifugation, 20 ␮l of the supernatant was injected onto a
4
.6 ϫ 75 mm C18 column (3.5-␮m particle size; Waters, Milford, MA) and
Because it is well established that epimers of a given compound can
have distinct biological properties, we were interested in studying the
R- and S-epimers of sulindac because of the diverse biological activ-
ities of sulindac. In the present report we have separated the R- and
S-epimers of sulindac with two goals. The first was to elucidate the
enzymatic systems involved in their metabolism, and the second was
to examine their effect on normal lung and lung cancer cells exposed
to oxidative stress.
eluted with a buffer consisting of 55% 50 mM sodium acetate buffer, pH 4.73,
and 45% acetonitrile. DABS-Met(o) and DABS-Met eluted at 0.98 and 1.7
min, respectively. Detection was by absorbance at 436 nm.
The enzymatic reduction of sulindac was assayed under the same reaction
conditions but using 20 nmol of sulindac or its epimers as substrate. The
amount of sulindac sulfide formed was assayed by HPLC using the same C18
column, and detection was at 330 nm. In these experiments, the eluting
solution was 25% 50 mM sodium acetate buffer, pH 4.73, and 75% acetoni-
trile, and sulindac and sulindac sulfide eluted at 0.88 and 1.37 min, respec-
tively. For sulindac sulfone detection, the eluting solution contained 50% 50
mM sodium acetate buffer, pH 4.73, and 50% acetonitrile, and the elution
Materials and Methods
Materials. Methionine sulfoxide, dabsyl chloride (4-N,N-dimethylamino-
azobenzene-4-sulfonyl chloride) (DABS), sulindac [(Z)-5-fluoro-2-methyl- times for sulindac, sulindac sulfone, and sulindac sulfide, were 1.1, 1.5, and 7.1
1
6
-[p-(methylsulfinyl)benzylidene]indene-3-acetic acid], NADPH, glucose min, respectively.
-phosphate, and dithiothreitol (DTT) were purchased from Sigma-Aldrich (St.
Isolation of Hepatic Microsomes and Oxidation of the Sulindac
Louis, MO), unless specified otherwise. Sulindac R- and S-epimers were Epimers. Fresh rat liver was minced and then homogenized using 5 strokes in
purchased from Custom Synthesis, Inc. (Delray Beach, FL), and we also a Potter-Elvehjem homogenizer with 3 volumes of 50 mM Tris-Cl, pH 7.4. The

1
016
BRUNELL ET AL.
homogenate was centrifuged for 15 min at 10,000g, the pellet was discarded,
and the supernatant was spun at 100,000g for 2 h to obtain crude microsomes.
The microsomes were washed by resuspension in 3 volumes of the above
buffer and centrifugation at 100,000g for 1 h. The microsomal pellet was
finally resuspended in 3 volumes of the above buffer, aliquoted, and frozen at
Ϫ80°C. The protein concentration was 25 mg/ml. In a total volume of 100 ␮l,
of the sulindac S-epimer with the P450 enzymes because the activity was quite
low. The lower substrate concentration made it possible to detect the low levels
of sulindac sulfone in the presence of the large excess of sulindac coming off
the HPLC column. Positive controls for enzyme activity were performed with
various substrates according to the manufacturer’s (BD Gentest) recommen-
dations. Two separate batches of the most-active P450 enzymes were pur-
chased from the manufacturer, and they gave similar results.
HepG2 Cells Exposed to Sulindac Show Increased Activity of P450
Enzymes. HepG2 human hepatocarcinoma cells were obtained from American
Type Culture Collection (Manassas, VA) and cultured in modified Eagle’s
medium containing 10% fetal bovine serum. Cells were grown in 24-well
plates to 80% confluence. For induction, 1 ml of fresh medium containing a
2
0 nmol of each sulindac epimer were incubated with 20 to 80 ␮g of
microsomes and a NADPH-regenerating system (1.5 mM NADPH, 5 mM
glucose 6-phosphate, 150 ng of glucose-6-phosphate dehydrogenase, and 5
mM MgCl ) in potassium phosphate buffer (pH 7.4, 100 mM) for 60 min
2
at 37°C. The reaction was then stopped with 3 volumes of acetonitrile and
centrifuged. The supernatant was fractionated by HPLC as described
above.
1
25 ␮M concentration of either the R- or S-epimer of sulindac was added per
Oxidation of the Sulindac Epimers by Recombinant P450 Enzymes.
Recombinant rat or human P450 enzymes (Supersomes) were incubated with
each sulindac epimer and a NADPH-regenerating system and analyzed via
HPLC as described above. Protein concentration curves and kinetic experi-
ments were initially performed with the individual isoforms that showed the
highest activities with the sulindac R- and S-epimers to ensure that the
enzymatic reactions were linear with respect to protein concentration and for
time periods up to 90 min. Once the enzymatic parameters were established,
well, and the cells were incubated in 5% CO at 37°C for the times indicated.
2
To make certain that the number of cells was similar in each experiment, all
wells were seeded simultaneously, and the sulindac epimers were added at the
appropriate times relative to the end of the induction period. The cells were
washed with phosphate-buffered saline and 100 ␮l/well of fresh medium
containing 200 ␮M (R)-sulindac or (S)-sulindac were added for the assay. This
second incubation was carried out for 1 h at 37°C. After centrifugation, the
each P450 enzyme was assayed at least three times. In addition, it was medium was analyzed by HPLC for sulindac and its metabolites after addition
determined that the concentration of substrate (sulindac epimer) was saturating
of 3 volumes of acetonitrile as described above. Control experiments showed
between 200 and 500 ␮M and routinely either 200 or 500 ␮M substrate was that the cell pellets contained only trace amounts of sulindac sulfone; therefore,
used. The lower concentration of substrate was routinely used for the oxidation only the medium was analyzed in these studies.
FIG. 2. Separation of sulindac and its metabolites from the plasma
of rats 4 h after intraperitoneal injection of the R- and S-epimers of
sulindac (see Materials and Methods). Plasma samples were run on
a C18 column and separated as described under Materials and
Methods, using conditions suitable for detection of sulindac sulfone.
Sulindac appears at 1.1 min, sulindac sulfone at 1.5 min, and sulindac
sulfide at 7.1 min. A, (S)-sulindac metabolites. B, (R)-sulindac metab-
olites. The ratio of sulindac sulfone to sulindac sulfide was 2.35 Ϯ 0.19
(mean Ϯ S.E.M.) in the animals treated with the R-epimer and 1.25 Ϯ
0.49 in the animals treated with the S-epimer (n ϭ 10, p Ͻ 0.05).

METABOLISM AND BIOLOGICAL ACTIVITY OF SULINDAC EPIMERS
1017
Purification of (R)-Sulindac Reductase from Rat Liver. Rat livers were
TABLE 1
homogenized in a Waring blender at high speed for 60 s in 3 volumes of 50
mM Tris buffer, pH 7.4, containing 1 mM DTT and 1 mM EDTA. The
homogenate was centrifuged for 20 min at 15,000g, and the supernatant was
further centrifuged for 2 h at 100,000g (S-100). The supernatant was subjected
to ammonium sulfate precipitation, and the proteins precipitating between 30
and 70% saturation were resuspended in 20 mM Tris-Cl, pH 8, 1 mM DTT,
and 1 mM EDTA and dialyzed against this buffer. Three grams of protein were
applied to a 100-ml DEAE column and eluted using 20 mM Tris-Cl, pH 8, 1
mM DTT, and 1 mM EDTA with a salt gradient from 0 to 200 mM KCl. The
major activity peak eluted at a salt concentration of approximately 100 mM.
The active fractions from several runs were pooled, concentrated, and applied
to a G50 superfine gel filtration column equilibrated with 50 mM Tris, pH 7.4,
containing 1 mM DTT, 1 mM EDTA, and 0.15 M NaCl. The active fractions
Sulindac oxidation by cytochrome P450 enzymes
Each enzyme was tested at least three times with each epimer. The substrate
concentrations used were 500 ␮M for the R-epimer and 200 ␮M for the S-epimer; further
details are described under Materials and Methods. Not included in the table are enzymes
with very low activity for both epimers, including rat 3A2 and 2B1 and human 2C8, 2C9,
2
C19, and 2D6. Values in the table are given as mean Ϯ S.E.
P450 Enzyme
(R)-Sulindac
(S)-Sulindac
pmol sulindac/pmol enzyme/h
Rat 1A1
Rat 1A2
380 Ϯ 22
460 Ϯ 15
130 Ϯ 5.1
73 Ϯ 0.8
47 Ϯ 1.2
22 Ϯ 1.0
24 Ϯ 2.8
34 Ϯ 3.5
0 Ϯ 0
Human 1A2
Human 1B1
Human 3A4
29 Ϯ 3.5
(
21–26) were used for gel analyses, Western blots, and other assays.
SDS-Polyacrylamide Gel Electrophoresis and Western Blot of Purifi-
result from plasma is shown in Fig. 2. The ratio of sulindac sulfone to
sulindac sulfide in plasma was greater by a factor of more than 2 when
rats were treated with the R-epimer compared with the S-epimer (see
legend to Fig. 2). Sulindac, sulindac sulfone, and sulindac sulfide
cation Fractions. Proteins from various purification steps were separated on 4
to 12% NuPAGE gels (Invitrogen, Carlsbad, CA), with 2 ␮g of protein being
loaded into each lane. After vertical electrophoretic separation, the proteins
were blotted to a polyvinylidene difluoride membrane and probed with rabbit
polyclonal antibody (Abnova) against SepX1 (MsrB1). A truncated, recombi- were also found in liver, skin, and brain tissues but at lower concen-
nant MsrB1 protein was used as a positive control, with and without cleavage trations than that in plasma (data not shown).
of the GST moiety by PreScission protease (see Materials).
Oxidation of Sulindac Epimers by Rat Liver Microsomes and
Purified P450 Enzymes. Because many drugs are metabolized by the
P450 system, the ability of rat liver microsomes to oxidize both the R-
Results
Sulindac Metabolites Detected in Normal Rat Tissues. It is and S-epimers was examined. As seen in Fig. 3, rat liver microsomes,
known that sulindac, which is an equal mixture of the R- and S- under uninduced conditions, catalyzed the oxidation of both R- and
epimers, can be reduced to sulindac sulfide and oxidized to sulindac S-epimers. The reaction was dependent on microsome concentration,
sulfone in vivo (Duggan et al., 1977). As a first step in establishing the and no activity was seen in the absence of the NADPH-regenerating
in vivo metabolic pathways for the sulindac epimers, rats received system (data not shown). Furthermore, no activity was detected in the
intraperitoneal injections of the individual epimers (see Materials and liver cytosolic fraction (data not shown). Under these in vitro condi-
Methods). The sulindac metabolites found in plasma, liver, skin, and tions with crude microsomes, the S-epimer is oxidized to the sulfone
brain were then analyzed by HPLC, and, regardless of which epimer at a rate roughly twice that of the R-epimer. This is in contrast to what
was given, sulindac and its two metabolites, sulindac sulfone and was seen in the plasma of rats injected with the sulindac R- and
sulindac sulfide, were found at detectable levels. A typical HPLC S-epimers (Fig. 2), in which the R-epimer was converted to the
sulfone more efficiently than the S-epimer. The lower production of
sulfone from the R-epimer in the isolated microsomes is probably due
to the fact that the microsomes in these experiments were from
animals not exposed to sulindac, so there was no induction of the P450
system (see below).
To determine which specific P450 enzymes might be responsible
for metabolism of the sulindac epimers, 11 rat or human recombinant
P450 enzymes were incubated with each epimer and a NADPH-
regenerating system (see Materials and Methods), and the metabolites
were analyzed by HPLC. Table 1 lists the activity of the five most
active enzymes. The other P450 enzymes having low activities with
both epimers are listed in the explanation to the table. The primary
P450 enzymes responsible for R-epimer oxidation were rat and human
1
A2, rat 1A1, and human 1B1, which are under control of the AHR.
For oxidation of the S-epimer, the enzymes with the highest activity
are human 1A2 and 3A4. It should be stressed that because the P450
Supersomes used are from two different species and are supplied by
the manufacturer with varying but saturating amounts of NADPH
cytochrome P450 reductase and some contain cytochrome b5 (see
Materials and Methods), the activity values obtained are only meant
to provide an overview of which purified enzymes showed activity
with each sulindac epimer under the conditions used and may not
correspond to what occurs in vivo.
FIG. 3. Effect of rat liver microsome concentration on sulindac sulfone formation.
Reaction mixtures and HPLC analysis are as described under Materials and Meth-
ods. The results shown, representing a microsome preparation from one of six rats,
Induction of P450 Enzyme Activity by the Sulindac Epimers.
Previous studies showed that sulindac could induce several of the
demonstrate a concentration dependence on the amount of microsomes added and P450 enzymes that were regulated by the AHR (Ciolino et al., 2006,
greater amount of sulfone formation from the S-epimer than from the R-epimer. For
the six microsome preparations that were tested, the ratio of the amount of sulindac
sulfone formed from the S-epimer to that formed from the R-epimer was 2.1 Ϯ 0.31
2
008). These authors used ethoxyresorufin as a substrate to measure
induction of the P450 system but did not measure the induction of the
P450 enzymes that oxidize sulindac or the effect of the sulindac
(
mean Ϯ S.E.M.) when 40 ␮g of microsomal protein was used.

1
018
BRUNELL ET AL.
FIG. 4. Induction of sulindac oxidation in HepG2 human hepatoma cells. A, induction by pretreatment of cells with (R)-sulindac. B, induction by pretreatment of cells with
S)-sulindac. Cells in 24-well plates were incubated for the indicated time with a 125 ␮M concentration of the inducing epimer, followed by a 200 ␮M concentration of
(
either (R)-sulindac or (S)-sulindac, as described under Materials and Methods. The results are expressed as picomoles per 100-␮l incubation reaction. This experiment was
repeated twice with a similar pattern seen in both experiments.
epimers in their P450 induction experiments. To see whether sulindac human MsrB2 and B3, which are known to catalyze the reduction of
increased the activity of P450 enzymes involved in its own metabo- protein-bound methionine-(R)-sulfoxide, were tested and found to
lism, human HepG2 cells were treated for periods ranging from 2 to have no activity in reducing the (R)-sulindac epimer (data not shown).
2
4 h with either (R)- or (S)-sulindac. After the pretreatment period, the Recombinant mammalian MsrB1 was not available, but its partial
medium containing sulindac was removed and replaced with fresh purification from mouse liver has been described previously (Mosko-
medium containing either the R- or S-epimer for a 1-h incubation vitz et al., 2002), and it was shown that the purified enzyme reduced
period (see Materials and Methods). After this second incubation, both free Met-R-(o) as well as DABS-R-Met(o), a substrate that
metabolites in the medium (see Materials and Methods) were ana- mimics Met(o) in peptide linkage. Thus, it was possible that MsrB1
lyzed by HPLC. As shown in Fig. 4A, pretreatment with the R-epimer might also reduce the R-epimer of sulindac. We therefore set out to
increases the activity of enzymes that can oxidize the R-epimer but not purify both the (R)-sulindac reductase activity and DABS-R-Met(o)
the S-epimer to the sulfone. Pretreatment of the cells with the S- activity from a rat liver S-100 fraction to see whether the two activ-
epimer results in a much higher level of increase in the activity of ities copurified (see details of purification under Materials and Meth-
enzymes that can oxidize both the R- and S-epimers (Fig. 4B). It ods). A summary of a typical purification of (R)-sulindac reducing
should be noted that the R-epimer is oxidized at a faster rate than the activity is shown in Table 2. A 270-fold purification was obtained in
S-epimer, regardless of which epimer is used during the preincubation this run, and in several other runs the purification varied between 250-
(
induction) period. We assume that the sulindac epimers are inducing and 350-fold. (S)-Sulindac reducing activity was also tested on these
the P450 enzymes in these experiments because Ciolino et al. (2006, fractions (data not shown). This activity, presumably mostly due to
008) have clearly shown that sulindac and its metabolites can induce MsrA, was twice the (R)-sulindac reductase activity in the original
2
several P450 enzymes. However, we should stress that we have not S-100, but the final G50 fraction had only a trace of (S)-sulindac
directly shown that the P450 enzymes are induced, only that there is reductase activity (Ͻ6%), which was not further identified. In
an increase in P450 enzymatic activity after the cells are exposed to Table 3, the purification of both (R)-sulindac and DABS-R-Met(o)
sulindac over a 24-h period.
reductase activities from a typical purification are compared. It can be
Partial Purification of an (R)-Sulindac Reductase with MsrB- seen that the purification of both activities is very similar at each step,
Like Activity. The reduction of (S)-sulindac has been shown to be suggesting that the same enzyme may be responsible for both activ-
catalyzed by MsrA (Moskovitz et al., 1996; Etienne et al., 2003). The ities. In addition, the reducing requirement for both activities at the
reductase for (R)-sulindac was of particular interest because there is final purification stage was similar, because DTT, but not reduced
presently no known enzyme that catalyzes this reaction. Several Trx, could serve as the reducing agent (data not shown). Mammalian
different MsrB enzymes, including recombinant E. coli MsrB and MsrB1 has a molecular mass of 12.8 kDa, and, as shown in Fig. 5A,
TABLE 3
TABLE 2
Copurification of (R)-sulindac reductase and DABS-R-Met(o) reductase
Purification of (R)-sulindac reductase from rat liver
The purification factor at the different steps of purification was measured using either 200
M (R)-sulindac or 200 ␮M DABS-R-Met(o), as described under Materials and Methods.
Aliquots from each step of purification were incubated with 200 ␮M (R)-sulindac as
described under Materials and Methods.
␮
Purification Factor
Step Specific Activity
Purification Factor Recovery
Step
(R)-Sulindac Reductase
DABS-R-Met(o) Reductase
nmol product formed/mg protein/h
%
S-100
NH4) SO , 30–70%
DEAE
G50
4.2
5.6
32.6
1
1.3
7.8
100
98
43
S-100
(NH4) SO , 30–70%
DEAE
G50 peak fraction
1
1.3
7.7
1
1.5
7.5
(
2
4
2
4
1160
276
40
340
256

METABOLISM AND BIOLOGICAL ACTIVITY OF SULINDAC EPIMERS
1019
shown in Fig. 6B, the two sulindac epimers showed similar enhanced
killing of lung cancer cells under the conditions used. In all these
experiments, the cells were preincubated with sulindac for 48 h before
being exposed to TBHP (see Materials and Methods and Marchetti et
al., 2009). It is concluded that the S- and R-epimers of sulindac have
similar activities toward both normal lung cells, which sulindac pro-
tects against oxidative stress, and lung cancer cells, which sulindac
enhances the killing of when exposed to oxidative stress. Studies on
the protection of normal cells against oxidative stress by sulindac
(
mixture of R- and S-epimers) have been shown to involve a precon-
ditioning mechanism (Moench et al., 2009). The selective, enhanced
killing of cancer cells exposed to sulindac and oxidative stress has
also been described elsewhere in detail and shown to involve mito-
chondrial dysfunction, leading to loss of mitochondrial membrane
potential, excess production of reactive oxygen species, and apoptosis
(
Marchetti et al., 2009). There is no reason to believe that the indi-
vidual sulindac epimers are not functioning in a similar way.
Discussion
In prior studies on sulindac, the metabolism and biological effects
were investigated primarily with sulindac preparations that contained
equal amounts of both epimers. Because individual epimers of a
compound often have different metabolic pathways and biological
effects, a complete understanding of a drug’s metabolism requires
study of the individual epimers. This may be especially true for
sulindac, which has been shown recently to have unique anticancer
FIG. 5. Gel analysis of purified (R)-sulindac reductase and Western blot analysis
for MsrB1. Proteins from the indicated purification steps (see Materials and
Methods) were separated on 4 to 12% NuPAGE gels. Two micrograms of protein
were applied in each lane. A, Coomassie-stained fractions: lane 1, S-100; lane 2,
(
NH ) SO , 30 to 70%; lane 3, DEAE fraction; lane 4, G50 peak activity fraction
4 2 4
(
no. 25). B, Western blot analysis of the G50 fraction 25 as in A, along with a
standard for MsrB1. Lane 1, G50 (no. 25); lane 2, incomplete digest of MsrB1-GST
fusion protein standard (36.5 kDa), with the truncated MsrB1 10.5 kDa fragment
clearly visible (see Materials and Methods).
there is a prominent Coomassie-stained band of this size in the
most-purified fraction. Western blot analysis, using the SepX1 anti-
body (see Materials) against a GST fusion protein containing the
N-terminal 95 amino acids of MsrB1, also provided evidence of
MsrB1 at this position (Fig. 5B, lane 1). Figure 5B, lane 2, is a
positive antibody control using the GST-MsrB1 fusion protein. This
protein (36.5 kDa) was partially digested with PreScission protease,
resulting in a second, lower band corresponding to the truncated
MsrB1 (10.5 kDa). Taken together, these results strongly indicate that
MsrB1 is present in the most-active fractions and may be responsible
for the reduction of (R)-sulindac in the rat liver extracts.
Effect of Sulindac on Normal Lung and Lung Cancer Cells
Exposed to Oxidative Stress. Our previous results (Marchetti et al.,
2
009) showed that normal lung cells, when pretreated with sulindac,
were protected against oxidative stress. In Fig. 6A, we tested the
individual sulindac epimers to determine whether they both had a
protective effect using normal lung cells exposed to TBHP (see
Materials and Methods). Under the conditions used, normal lung cells
are quite sensitive to TBHP as indicated by the loss of viability in the
absence of any drug. It can be seen that both epimers are able to
protect the normal lung cells against TBHP oxidation. There is an
indication that the (R)-sulindac epimer is slightly more protective than
the S-epimer, but only at higher concentrations of TBHP, and we do
not think that this difference has physiological or therapeutic
significance.
FIG. 6. Effect of sulindac epimers on the viability of lung cells exposed to oxidative
stress. Cells were incubated in the presence of 500 ␮M (R)-sulindac (F), 500 ␮M
(S)-sulindac (E), or no drug (f) for 48 h before exposure to TBHP, as described
The lung cancer cells are more resistant to TBHP, as shown in Fig. under Materials and Methods and in Marchetti et al. (2009). Viability is expressed
as percentage viable cells not exposed to TBHP. A, normal lung cells. B, lung
cancer cells. Data points are graphed as mean and S.E. bars from a set of five
repeats. ء, treatment conditions under which sulindac-treated cells showed a statis-
been shown to selectively kill these cells (Marchetti et al., 2009). As tically significant (p Ͻ 0.05) departure from controls (no drug).
6
B. In contrast to what is seen in normal cells, pretreatment of lung
cancer cells with sulindac followed by exposure to oxidative stress has

1
020
BRUNELL ET AL.
activity in combination with other agents (Meyskens et al., 2008; Park induction of enzyme activity in the cell culture experiments is due
et al., 2008; Seo et al., 2008; Marchetti et al., 2009; Resnick et al., primarily to sulindac and not to any of its metabolites, because only
2
009; Pangburn et al., 2010) and to protect normal cells against a small fraction of the sulindac was metabolized in these experiments.
oxidative stress (Marchetti et al., 2009). Both of these activities of However, preliminary experiments have shown that sulindac sulfide
sulindac are unrelated to its NSAID activity. can induce P450 enzyme activity similar to what was seen with the
In the present studies using rats, we have demonstrated that both the sulindac R-epimer, and thus we cannot rule out the possibility that
sulfone and sulfide metabolites are present in plasma liver, skin, and some of the increase in P450 activity seen with the sulindac epimers
brain after injection with either sulindac epimer. The oxidation of each is due to the sulindac metabolites.
epimer with microsomes and commercially obtained, purified cyto-
Regarding the reduction of sulindac to sulindac sulfide, our earlier
chrome P450 enzymes has allowed us to identify some of the P450 studies showed that MsrA reduced the S-epimer of sulindac (Etienne
enzymes that may be involved in the oxidation of the two epimers in et al., 2003). However, MsrB2 and MsrB3, which are capable of
vivo. Of those tested, the major purified P450 enzymes responsible for reducing the R-epimer of peptide-bound methionine sulfoxide, cannot
oxidation of the R-epimer include rat and human 1A2, rat 1A1, and reduce (R)-sulindac to sulindac sulfide (data not shown). This finding
human 1B1, whereas human 1A2 appeared to be most active with the prompted us to search for the enzyme(s) that could reduce the R-
S-epimer. From previously published rat feeding experiments, it epimer of sulindac, especially because sulindac sulfide was present in
seems that sulindac (a mixture of both epimers) did induce hepatic the plasma of rats fed the R-epimer. A 250- to 350-fold purified
mRNA expression of CYP1A1, CYP1A2, and CYP1B1 (Ciolino et enzyme fraction that can reduce both the R-epimer of sulindac and
al., 2008), although sulindac oxidation was not tested in these studies. DABS-Met-R-(o), the latter being a known substrate for the MsrB
In fact, CYP1A1 showed the highest level of mRNA induction by the enzymes, has been obtained. Preliminary data suggest that the enzyme
mixed epimers in these previous studies and showed high activity for may be MsrB1, a selenoprotein, because the activities that reduce both
oxidation of the R-epimer in our studies. It should also be mentioned substrates copurify, and gel analyses of the most-purified preparation
that there is one other report showing that sulindac sulfide can be show a band, recognized by the anti-MsrB1 antibody, at the expected
oxidized to sulindac in vitro by a flavin-containing monooxygenase, molecular weight. Matrix-assisted laser desorption ionization analysis
although the physiological significance of this reaction in vivo is not showed that this purified preparation was grossly contaminated with
known (Hamman et al., 2000). Induction of P450 enzyme activity in fatty acid binding protein (data not shown), which prevented us from
HepG2 cells using the individual epimers showed more induced obtaining selenium measurements that could support the presence of
activity toward the R-epimer than toward the S-epimer. It was sur- MsrB1 in the preparation. However, we must be cautious at this time
prising that the induction of enzyme activity with the R-epimer in concluding that MsrB1 is the active factor that reduces (R)-sulin-
showed little or no increase in P450 activity toward the S-epimer. We dac, because a recombinant MsrB1 (a generous gift from V. N.
have not found any other report using chiral drugs for which this is the Gladyshev, Harvard Medical School, Boston, MA), with cysteine at
case. When the S-epimer was used to induce the P450 activity, once position 95 instead of selenocysteine, did not have detectable activity
again the induced activity was primarily toward the R-epimer, al- with (R)-sulindac even at high enzyme concentrations, under condi-
though in these experiments there was a low level of induction of tions in which DABS-R-Met(o), a known substrate for MsrB, was
P450 activity toward the S-epimer. It was also of interest that the readily reduced by this enzyme. This recombinant enzyme also could
S-epimer has a greater capacity to induce the P450 enzyme system use thioredoxin as a reducing system, whereas the rat liver fraction
than the R-epimer. It is possible that the S-epimer has a higher affinity that we purified could use DTT, but not thioredoxin. More studies are
for the AHR or other P450-inducing receptors, but that the induced needed to clarify this issue. One possibility is to look at (R)-sulindac
P450 enzyme activity oxidizes the R-epimer at a greater rate than the reductase activity in MsrB1 knockout mice or use MsrB1 small
S-epimer in whole cells. This finding is in agreement with the in vivo interfering RNA. Such studies will not provide a clear answer because
plasma levels of the metabolites. In summary, our results indicate that the bulk of the (R)-sulindac reductase activity in rat liver was not in
after induction of P450 enzyme activity with either epimer, the R- the protein fraction we purified. The DEAE fraction that contained the
epimer of sulindac is more readily converted to the sulfone than the highest (R)-sulindac reductase specific activity as well as the MsrB1
S-epimer in cells in culture and in vivo. We have assumed that the activity represented approximately 30% of the total (R)-sulindac re-
FIG. 7. Summary of P450 enzymes involved in
the metabolism of the sulindac epimers. See
text for details.

METABOLISM AND BIOLOGICAL ACTIVITY OF SULINDAC EPIMERS
1021
tive sulfoxidation of sulindac sulfide by flavin-containing monooxygenases. Comparison of
human liver and kidney microsomes and mammalian enzymes. Biochem Pharmacol 60:7–17.
Hixson LJ, Alberts DS, Krutzsch M, Einsphar J, Brendel K, Gross PH, Paranka NS, Baier M,
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ductase activity in the DEAE fractions. Thus, knocking out MsrB1
would give only a partial loss of (R)-sulindac reductase activity in the
crude cell extracts and would require more extensive purification.
Figure 7 summarizes our present knowledge of the metabolism of the
R- and S-epimers of sulindac.
Kim HY and Gladyshev VN (2004) Methionine sulfoxide reduction in mammals: characteriza-
Finally, because of the recent interest in the role of sulindac in
protecting normal cells against oxidative stress while sensitizing can-
cer cells to agents that affect mitochondrial function, we have checked
the effect of both epimers on the protection of normal lung cells
against oxidative stress as well as the ability of the sulindac epimers
to selectively enhance the killing of lung cancer cells exposed to
oxidative stress. Our purpose here was not to investigate specific
mechanisms of normal cell protection or cancer killing by the sulindac
epimers and oxidative stress, because mechanism studies with sulin-
dac (mixture of epimers) have been described elsewhere (Marchetti et
al., 2009; Moench et al., 2009). As mentioned, sulindac can protect
cells against oxidative damage by a preconditioning mechanism (Mo-
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exposed to oxidative stress involves mitochondrial dysfunction and
death as a result of reactive oxygen species production (Marchetti et
al., 2009). The goal of the present studies was to compare the relative
efficacy of the two epimers. Both epimers showed similar protection
of lung normal cells to oxidative stress and enhanced killing of cancer
cells exposed to oxidative stress. However, differences in the metab-
olism of the sulindac epimers could have therapeutic significance. The
R-epimer may have a better safety profile owing to its more efficient
conversion to the sulindac sulfone, which is not a cyclooxygenase
inhibitor.
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Kitamura S and Tatsumi K (1982) In vitro metabolism of sulindac and sulindac sulfide:
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Marchetti M, Resnick L, Gamliel E, Kesaraju S, Weissbach H, and Binninger D (2009) Sulindac
enhances the killing of cancer cells exposed to oxidative stress. PloS ONE 4:e5804.
Meyskens FL Jr, McLaren CE, Pelot D, Fujikawa-Brooks S, Carpenter PM, Hawk E, Kelloff G,
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Acknowledgments
We thank Dr. V. N. Gladyshev for his generous gift of recombinant MsrB1
with a Cys residue in place of the selenocysteine and Diana Navarro for her
help in preparing Western blots.
8
:29–32.
Authorship Contributions
Sagher D, Brunell D, Brot N, Vallee BL, and Weissbach H (2006a) Selenocompounds can serve
Participated in research design: Brunell, Brot, and Weissbach.
Conducted experiments: Brunell, Sagher, and Kesaraju.
Performed data analysis: Brunell and Sagher.
Wrote or contributed to the writing of the manuscript: Brunell, Sagher,
Brot, and Weissbach.
as oxidoreductants with the methionine sulfoxide reductase enzymes. J Biol Chem 281:
3
1184–31187.
Sagher D, Brunell D, Hejtmancik JF, Kantorow M, Brot N, and Weissbach H (2006b) Thionein
can serve as a reducing agent for the methionine sulfoxide reductases. Proc Natl Acad Sci USA
103:8656–8661.
Seo SK, Jin HO, Lee HC, Woo SH, Kim ES, Yoo DH, Lee SJ, An S, Rhee CH, Hong SI, et al.
(2008) Combined effects of sulindac and suberoylanilide hydroxamic acid on apoptosis
Other: Weissbach initiated the project.
induction in human lung cancer cells. Mol Pharmacol 73:1005–1012.
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protein-bound methionine sulfoxide by methionine sulfoxide reductase. FEBS Lett 455:247–
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