S-Alk(en)ylmercaptocysteine: Chemical synthesis, biological activities, and redox-related mechanism

Article abstract of DOI:10.1021/jf305486q

S-Alk(en)ylmercaptocysteine (CySSR, R = methyl, ethyl, propyl, 1-propenyl, and allyl), which are the putative metabolites of Allium thiosulfinates, were chemically synthesized. CySSR, but not the corresponding monosulfide species S-alk(en)yl cysteine (CyS

Full text of DOI:10.1021/jf305486q

Article
pubs.acs.org/JAFC
S‑Alk(en)ylmercaptocysteine: Chemical Synthesis, Biological
Activities, and Redox-Related Mechanism
Guodong Zhang*,† and Kirk L. Parkin*
Department of Food Science, University of WisconsinMadison, Babcock Hall, 1605 Linden Drive, Madison, Wisconsin 53706,
United States
ABSTRACT: S-Alk(en)ylmercaptocysteine (CySSR, R = methyl, ethyl, propyl, 1-propenyl, and allyl), which are the putative
metabolites of Allium thiosulfinates, were chemically synthesized. CySSR, but not the corresponding monosulfide species S-
alk(en)yl cysteine (CySR), were able to induce quinone reductase (QR, a representative phase II enzyme) in Hepa 1c1c7 cells
and inhibit nitric oxide (NO, an inflammatory biomarker) formation in lipopolysaccharide (LPS)-activated RAW 264.7 cells.
These results indicate the importance of the disulfide bond for the biological activities of CySSR. Glutathione (GSH) and N-
acetylcysteine (NAC), but not other types of cellular antioxidants, suppressed multiple biological activities of CySSR in vitro. The
inhibitory effects of GSH and NAC on the biological activities of CySSR were correlated with a glutaredoxin (Grx)-dependent
intracellular reduction of CySSR to generate cysteine and RSH, which were secreted into the extracellular medium.
KEYWORDS: allium, thiosulfinates, S-alk(en)ylmercaptocysteine, S-1-propenylmercaptocysteine, phase II enzyme, inflammation,
glutaredoxin
including antioxidant,10 phase II enzyme inducing and anti-
INTRODUCTION
■
inflammatory,11 apoptosis inducing,12 antiproliferative,13 and
Epidemiological studies have consistently shown that con-
antimetastatic14 effects. CySSA has been shown to covalently
sumption of Allium vegetables (such as onions, garlic, chives,
modify protein thiols.15,16 A recent study shows that it
and leeks) is associated with reduced incidence of many human
disrupted microtubule polymerization via covalent modifica-
diseases.1−4 The health benefits of Allium vegetables are widely
tions of cysteine residues of microtubule.12 Therefore, CySSA
attributed to the enzyme-evolved organosulfur compounds
could exert its biological activities via a redox-related
called thiosulfinates (TS).5 TS and related organosulfur
mechanism.
compounds are highly unstable in vivo and are rapidly
For the preparation of CySSR, we have reported the
metabolized to mixed disulfide conjugates with cellular cysteine
and glutathione, named S-alk(en)ylmercaptocysteine (CySSR)
chemical synthesis of non-1-propenyl CySSR (R = methyl,
ethyl, propyl, and allyl). However, this method cannot be used
to prepare the cysteine conjugate with 1-propenyl TS, S-1-
and S-alk(en)ylmercaptoglutathione (GSSR) respectively (Fig-
ure 1).5−9 CySSR and GSSR are proposed to be the active
species which contribute to the health-promoting effects of
Allium vegetables. Indeed, S-allylmercaptocysteine (CySSA, an
active ingredient of aged garlic extract) has been marketed as a
dietary supplement for decades and is a promising cancer
chemoprotective agent with multiple biological effects,
propenylmercaptocysteine (CySSPe).11 1-Propenyl TS are the
dominate TS species of some Allium tissues such as onions, and
are the most challenging TS to be chemically synthesized.5,17 1-
Propenyl TS haven been shown to have the most potent phase
II enzyme inducing activities among major Allium TS.18
Therefore the biological activities of CySSPe could be critical
to understand the health-promoting effects of onions. Here we
report a novel chemical synthesis strategy to prepare CySSPe.
The biological activities and redox-related mechanisms of
CySSR (R = methyl, ethyl, propyl, allyl, and 1-propenyl) were
also studied in cell cultures.
MATERIALS AND METHODS
■
Materials and Chemicals. All chemicals were obtained from
Sigma-Aldrich (St. Louis, MO). All solvents used for extraction or
chromatography were purchased from Fisher Scientific (Hampton,
NH). Hepa 1c1c7 and RW 264.7 cells were purchased from ATCC
(Manassas, VA). Cell culture medium and supplements were from
Invitrogen (Carlsbad, CA). NMR data were collected on Varian Unity-
Figure 1. Chemical structures of CySSR in this study. Abbreviations:
CySSM, S-methylmercaptocysteine; CySSE, S-ethylmercaptocysteine;
CySSP, S-propylmercaptocysteine; CySSA, S-allylmercaptocysteine;
CySSPe, S-1-propenylmercaptocysteine.
Received: December 24, 2012
Revised: February 1, 2013
Accepted: February 4, 2013
Published: February 4, 2013
© 2013 American Chemical Society
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a
Scheme 1. Chemical Synthesis of CySSR
a
(A) Chemical synthesis of non-1-propenyl S-alk(en)ylmercaptocysteine (CySSR, R = methyl, ethyl, propyl, and allyl). (B) Chemical synthesis of S-
1-propenylmercaptocysteine (CySSPe). (C) Proposed mechanism for the reaction of N-Boc-cysteine sulfenyl bromide and TrSPe to generate Boc-
CySSPe.
Inova 400 and 500 MHz NMR spectrometers (Analytical
Instrumentation Center, School of Pharmacy, UW-Madison). High-
resolution ESI-MS data were collected on an Agilent ESI-TOF mass
spectrometer (Mass Spectrometry/Proteomics Facility, Biotechnology
Center, UW-Madison).
Chemical Synthesis of Non-1-propenyl CySSR (R = Methyl,
Ethyl, Propyl, and Allyl). The synthesis of CySSR (R = methyl,
ethyl, propyl, and allyl) was carried out as described earlier.11 As
shown in Scheme 1, dithiophosphoric-alk(en)yl disulfides were
prepared by the reaction of (5,5-dimethyl-2-thiono-1,3,2-
dioxophosphorinanyl)sulfenyl bromide and RSH (R = Me, Et, Pr,
and All, for R = Me, MeSNa was used) in CH2Cl2. CySSR were
synthesized by the reaction of the corresponding dithiophosphoric-
alk(en)yl disulfides with L-cysteine using Et3N as a base in a mixed
solution of ethanol and H2O (50:50, vol:vol).
Chemical Synthesis of S-1-Propenylmercaptocysteine (Cy-
SSPe). Sodium (2 g) was added to 200 mL of methanol in a reflux
apparatus, stirred until the sodium was totally dissolved, and 25 g of
triphenylmethanethiol (TrSH) was added. The reaction mixture was
refluxed for 1 h, and 10 mL of allyl bromide was added and refluxed for
another 2 h. The solution was allowed to cool, and 19 g of particulate
material, trityl allyl sulfide (TrSAll), was obtained by filtration using
filter papers and was used directly for the next step without any
purification. tert-BuOK (11 g) was added to a solution of 19 g of
TrSAll in 100 mL of anhydrous tetrahydrofuran (THF) and 200 mL of
tert-BuOH, stirred at 20−22 °C overnight. The solvent in the reaction
mixture was removed by rotary evaporation, water and CH2Cl2 were
added to the residue, and the product was extracted into the CH2Cl2
phase. Trityl 1-propenyl sulfide (TrSPe) was obtained as a white color
powder by purification of the CH2Cl2 extract on a silica gel column
eluted with 10% CH2Cl2 in hexane.
reaction mixture was purified on a silica gel flash column eluted with
5% methanol in CH2Cl2 to provide 0.32 g (yield 20%) of Boc-CySSPe
as a yellow oil. To remove the Boc group, 0.84 mL of trifluoroacetic
acid (TFA) was added to a solution of 0.32 g of Boc-CySSPe in 0.84
mL of CH2Cl2 at 0 °C, stirred at 0 °C for 1.5 h. Solvent was removed
from reaction mixture under vacuum, and the residue was washed with
CH2Cl2, allowing CySSPe to be obtained as a white solid. The
structure of CySSPe was confirmed by comparing NMR data with a
published result.19 1H NMR (D2O, 500 MHz): δ 4.35 (dd, J = 4.0, 8.0
Hz, 1H, H-2), 3.37 (dd, J = 4.0, 15.5 Hz, 1H, H-3α), 3.21 (dd, J = 8.0,
15.0 Hz, 1H, H-3β), 6.17 (m, 2H, H-4 and H-5), 1.81 (m, 3H, H-6).
HR-ESI-MS: m/z [M + H]+ at 194.0308 (calcd 194.0309), [M − H]−
at 192.0166 (calcd 192.0153).
DTNB Assay of Extracellular Thiols in Cell Culture Medium.
Extracellular thiols were assayed by a slightly modified method
described by Biaglow et al.20 RAW 264.7 cells (ATCC, 5 × 104 cells
per well) were cultured in 200 μL of Dulbecco’s modified Eagle’s
medium (DMEM) in a 96-well plate for 24 h. The medium was
decanted and washed with Dulbecco’s phosphate buffered saline
(DPBS, Invitrogen, fortified with 10 mM glucose), then a 100 μL
sample serially diluted in DPBS buffer was added to each well, and an
equal volume of 1 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in
DPBS buffer was added. Absorbance at 412 nm was semicontinuously
measured to record kinetics of extracellular thiol formation (via
measuring 5-thio-2-nitrobenzoic acid (TNB) anion formation) over 3
h. In another experiment, cells were treated with compounds in DPBS
buffer, and at predetermined incubation times, 100 μL of DPBS
medium was removed and mixed with an equal volume of 1 mM
DTNB solution in DPBS buffer to measure thiol concentration. A
series of cysteine solutions were prepared to establish a standard curve
for calculation of thiol concentration in the samples.
Bromine (0.13 mL) was added to a suspension of 1.2 g of Boc-
cystine in 50 mL of CH2Cl2 at −80 °C, and stirred at −80 °C for 10−
15 min. Then 1.7 g of TrSPe dissolved in 10 mL of CH2Cl2 was added,
and the mixture was stirred for another 30 min at 20−22 °C. The
HPLC Analysis of Extracellular Thiols by DTNB Derivatiza-
tion. To identify the extracellular thiols produced by treated cells, at
different time intervals, 100 μL of DPBS medium was removed and
mixed with an equal volume of 1 mM DTNB solution in DPBS and
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incubated for 5 min. After centrifuging, the clarified sample was
subjected to HPLC analysis on a Jupiter 4 μm Proteo 90 Å, 250 × 10
mm HPLC column (Phenomenex, CA) to resolve the TNB-SS-thiol
disulfide conjugates using a gradient of solvent A (water with 0.1%
trifluoroacetic acid) in solvent B (80% acetonitrile in water with
0.085% trifluoroacetic acid). Solvent B was started at 5%, then
increased to 30% in 9 min, increased further to 80% in 7 min, and then
kept at 80% for 5 min before decreasing to 5% in 4 min, and held there
for 10 min. Flow rate was 0.25 mL/min, and detection at 326 nm.21
Glutaredoxin (Grx) Assay. The enzymatic reduction of CySSR by
glutaredoxin (human glutaredoxin, American Diagnostica, Stamford,
CT) was measured by a spectrophotometric assay. An assay mixture
containing 0.15 mM CySSR, 0.5 mM GSH, 0.2 mM NADPH, and 2
U/mL glutathione reductase (GR) was incubated in the absence or
presence of 40 μM Grx in 100 mM Tris-HCl, pH = 7.5. The kinetics of
the reaction was semicontinuously monitored by NADPH oxidation at
340 nm.
(Boc-CySSPe) with an isolable yield of ∼20%. Removing Boc
group by TFA generated the final product S-1-propenylmer-
capto-L-cysteine (CySSPe). Scheme 1C shows a proposed
reaction mechanism for the formation of Boc-CySSPe,
involving the formation of a sulfonium bromide intermediate
of which selective C−S bond cleavage is mediated by the trityl
group because trityl carbocation is stable whereas 1-propenyl
carbocation is unstable.24,25 For all synthesized CySSR
conjugates (R = methyl, ethyl, propyl, allyl, and 1-propenyl),
1
> 98% purity was obtained based on H NMR analysis.
CySSR Induces Phase II Enzyme Induction and
Inhibits LPS-Induced NO Formation in Vitro. Figure 2A
Quinone Reductase (QR) Induction Assay. A bioassay based on
cultured murine hepatoma cells (Hepa 1c1c7) was used to assess QR
induction essentially as described earlier.22 Hepa 1c1c7 cells were
grown for 24 h in 96-well plates (5 × 103 cells per well) in 200 μL of
minimal essential medium (MEM) supplemented with 10% fetal
bovine serum (FBS) and antibiotics at 37 °C in 5% CO2 in air. The
medium was replaced with fresh MEM medium containing test
compounds, and the cells were incubated for 48 h. A standard assay
cocktail was prepared, and QR activity was determined by measuring
absorbance of the reduced tetrazolium dye over a 10 min period at 490
nm. Cell viability was determined by crystal violet assay at 610 nm.
The degree of QR induction was calculated as the ratio of QR activity
in the treated (induced) sample relative to the untreated control
sample. The concentration required for doubling the specific activity of
QR relative to nontreated control cells was used as an indicator of
inducer potency, expressed as CD values.
LPS-Induced Nitric Oxide (NO) Assay. Mouse macrophage cells
(RAW 264.7) were cultured in Dulbecco’s modified Eagle’s medium
(DMEM, Invitrogen) supplemented with 15% FBS at 37 °C in 5%
CO2 in a 96-well plate (5 × 104 cells per well) for 24 h. The medium
was then replaced with fresh medium containing test compounds and
1 μg/mL lipopolysaccharide (LPS). The plate was incubated for
another 24 h. A 100 μL sample was removed from the culture
supernatant of each well and mixed with an equal amount of freshly
prepared Griess reagent and incubated for 10 min, and then the
absorbance was measured at 542 nm. Cell viability was measured by
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay at 550 nm.23
Figure 2. CySSR induce QR activity and inhibit LPS-induced NO
production in vitro. (A) QR inducing assay of CySSR (R = methyl,
ethyl, propyl, 1-propenyl, and allyl) in Hepa 1c1c7 cells. (B) LPS-
induced NO production assay of CySSR in RAW 264.7 cells. Results
are expressed as mean SD from three independent experiments.
shows the QR inducing activities of CySSR (R = methyl, ethyl,
propyl, 1-propenyl, and allyl) in Hepa 1c1c7 cells, with an order
of potency CySSPe > CySSA > CySSP ≈ CySSE ≈ CySSM.
CySSPe was the most potent QR inducer with a CD value ∼25
μM, which was more potent than other CySSR species
including the most intensively studied water-soluble Allium
organosulfur compound CySSA (CD value of CySSA ≈ 125
μM), as well as CySSM, CySSE, CySSP with CD values 246−
439 μM.
The structures of CySSR compounds differ in the existence
and position of a double bond in the R group. CySSPe, which
has a double bond adjacent to the disulfide bond, showed the
most potent QR inducing activity. CySSA, which contains a
double bond one carbon atom away from the disulfide bond,
was the second most potent. CySSM, CySSE and CySSP, which
are devoid of double bond in the R group, were the least potent
compounds and exhibited similar potency for QR induction.
The existence of an electron-rich double bond and the distance
of the double bond to the disulfide bond in the structure of
CySSR might be correlated with the chemical reactivity of the
disulfide bond (such as thiol−disulfide exchange reaction) due
to the inductive effect. These structure−activity relationships
suggest a model where the CySSR may exert their phase II
enzyme inducing effects through a direct interaction with
RESULTS AND DISCUSSION
■
Chemical Synthesis of CySSR. Non-1-propenyl cysteine
mixed disulfide conjugates CySSR (R = methyl, ethyl, propyl,
and allyl) were synthesized by the reaction of dithiophosphoric-
alk(en)yl disulfide with cysteine (Scheme 1A).11 However this
method cannot be used to prepare the 1-propenyl conjugate
CySSPe, because dithiophosphoric-1-propenyl disulfide failed
to react with cysteine to generate the corresponding 1-propenyl
conjugate CySSPe. This result indicated a different synthetic
strategy was required for the preparation of 1-propenyl disulfide
conjugate.
In this study, the reaction of a sulfenyl halide with trityl 1-
propenyl sulfide (TrSPe) was deployed to prepare CySSPe
(Scheme 1B). This strategy was simpler than the previously
reported method for CySSPe synthesis.19 Trityl (E/Z)-1-
1
propenyl sulfide (TrSPe, E:Z ≈ 6:1 estimated from H NMR
analysis) was synthesized by isomerization of trityl allyl sulfide
(TrSAll) using tert-BuOK in a mixed solvent of tert-BuOH and
THF.19 Boc-L-cystine reacts with bromine in CH2Cl2 to form
N-Boc-L-cysteine sulfenyl bromide, which quickly reacts with
(E/Z)-TrSPe to form S-1-propenylmercapto-N-Boc-L-cysteine
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cysteine residues of Keap1 protein.26 Previous research has
shown that a common mechanistic feature of phase II enzyme
inducers is that they directly modify active cysteine residues of
Keap1 protein, leading to dissociation of Nrf2 protein from
Keap1 and activation of Nrf2 signaling.26 In support of this
hypothesis, CySSA has been shown to covalently modify
protein thiols.12,15,16 Further studies are required to investigate
whether CySSR activates Nrf2 signaling through a direct
interaction with cysteine residues of Keap1 protein. The effects
of CySSR on phase I enzymes (P450 enzymes) are not studied
here, however, previous studies have shown that CySSA inhibits
P450 2E1 in animals.27,28
For LPS-induced NO production, CySSPe was a weaker NO
inhibitory agent (IC50 ≈ 120 μM) compared with CySSA (IC50
≈ 36 μM) in LPS-activated RAW 264.7 cells. The potency of
CySSPe was similar to the other CySSR species with saturated
R groups (R = methyl, ethyl and allyl, IC50 ≈ 130−260 μM).11
These results indicate that CySSR compounds may induce QR
and inhibit LPS-induced NO via different mechanisms; more
studies are required to characterize the potential anti-
inflammatory effects of CySSR compounds.
An order of potency for Allium TS of 1-Pe TS > allicin >
PrSS(O)Pr > EtSS(O)Et > MeSS(O)Me was observed for QR
induction in Hepa 1c1c7 cells (ref 18 and unpublished results
of Qin Ren and Kirk Parkin), which was similar to the order of
potency of CySSR for QR induction. 1-Propenyl TS (PeSS-
(O)Pr, PrSS(O)Pe, PeSS(O)Me) showed the most potent QR
inducing activity with CD values ∼10 μM,18 which was more
potent than allicin (AllSS(O)All, CD = 28 μM) and other TS
species including PrSS(O)Pr (CD = 38 μM), EtSS(O)Et (CD
= 60 μM), and MeSS(O)Me (CD = 100 μM) (unpublished
results, Qin Ren and Kirk Parkin). For NO inhibition, allicin is
the only TS species which has been studied (IC50 = 15−20
μM), which was more potent than CySSA (IC50 = 36 μM).29
Since 1 molar TS can react with 2 molar cysteine to generate 2
molar CySSR, thus the potency of CySSR and TS for phase II
enzyme induction and anti-inflammation are well correlated.
These data support that the CySSR conjugates may be the
bioactive species responsible for the health benefits of Allium
TS.
Disulfide Bond Is Essential for the Biological Activities
of CySSR. The biological activities of CySSR and CySR were
compared to investigate the roles of the disulfide (−SS−) bond.
Figure 3 shows that CySR (R = methyl, ethyl, and allyl) was
inactive to induce QR or inhibit LPS-induced NO production
in vitro. In the tested dose range, CySSR dose-dependently
induced QR activity in Hepa 1c1c7 cells (Figure 3A) and
inhibited NO formation in LPS-activated RAW 264.7 cells
(Figure 3B). In contrast, CySR showed no such effects at doses
as high as 1 mM, which is consistent with a previous study.30
CySSA has been shown to have more potent antiproliferative
activity than CySA.13 These results demonstrate the importance
of the disulfide bond for the biological activities of CySSR. The
disulfide bond may not be the only required structural moiety
because the CySSR analogue cystine (Cy-SS-Cy) or disulfides
RSSR (diallyl disulfide, dipropyl disulfide) does not have QR
inducing or NO inhibitory effects in vitro (data not shown, see
ref 30). Diallyl disulfide has been shown to have potent phase II
enzyme inducing activities in animals,31 suggesting a potential
metabolic activation of this compound in vivo.
Figure 3. Compared with CySSR, CySR is inactive to induce QR or
inhibit LPS-induced NO production. QR (A) and NO (B) assay of
CySSR and CySR (R = methyl, ethyl, and allyl). Results are expressed
as mean SD from three independent experiments.
of oxidative stress via generation of ROS or depletion of
intracellular GSH, leading to modulation of redox sensitive
signaling pathways.32 For the organosulfur compounds, the
diallyl trisulfide (DATS)-induced Nrf2 activation has been
shown to be suppressed by cellular antioxidants such as GSH,
N-acetylcysteine (NAC, a GSH boosting agent), and micro-
somal epoxide hydrolase,33 supporting a potential redox-related
mechanism involved in the phase II enzyme induction of
DATS.
Here we investigate whether the biological activities of
CySSR are mediated by a redox-related mechanism, using
cellular antioxidants GSH, NAC, and ascorbic acid (ASC).
Using CySSA and CySSPe as examples, we found that GSH
and NAC dose-dependently suppressed multiple biological
effects of CySSA and CySSPe (Figure 4). Treatments of 162
μM CySSA or 32.4 μM CySSPe alone caused ∼2-fold increase
of QR activity after 48 h treatment in Hepa 1c1c7 cells, while
coaddition of GSH dose-dependently inhibited CySSA- or
CySSPe-induced QR induction. With coaddition of 1 mM
GSH, the CySSA- or CySSPe-induced QR induction was
almost abolished (Figure 4A,B). For LPS-induced NO
production in RAW 264.7 cells, treatment with 32.4 μM
CySSA or 162 μM CySSPe inhibited 50−60% of NO
production, and the inhibitory effects were also dose-depend-
ently reversed by coaddition of GSH (Figure 4C,D). For cell
proliferation, treatment with 647.7 μM CySSA or 259 μM
CySSPe caused severe cell death after 48 h treatment in Hepa
1c1c7 cells, while coaddition of GSH dose-dependently
“rescued” the cells from the cytotoxicity of CySSA and CySSPe
(Figure 4E,F). NAC also showed a similar inhibitory effect on
the biological activities of CySSA and CySSPe (data not
shown). GSH or NAC treatments alone had no effect on QR
induction, NO inhibition, or cellular proliferation in the tested
dose range (data not shown). Incubation of GSH or NAC with
CySSA or CySSPe showed no chemical reaction (or
degradation of CySSA or CySSPe, data not shown). In contrast
to GSH and NAC, coaddition of other cellular antioxidants
Coaddition of GSH and NAC, Not ASC, Suppresses the
Biological Effects of CySSR. Many cancer chemopreventive
agents have been shown to exert their effects through induction
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Figure 4. Coaddition of GSH suppressed multiple biological activities of CySSA and CySSPe, including QR induction, inhibition of LPS-induced
NO production, and inhibition of cell proliferation. (A, B) Hepa 1c1c7 cells were treated with 162 μM CySSA (A) or 32.4 μM CySSPe (B), with or
without varied doses of GSH. After 48 h treatment, QR activity and cell viability were measured. (C, D) LPS-stimulated RAW 264.7 cells were
treated with 32.4 μM CySSA (C) or 162 μM CySSPe (D), with or without coaddition of varied doses of GSH. After 24 h treatment, NO production
in the medium and cell viability were measured. (E, F) Hepa 1c1c7 cells were treated with 647.7 μM CySSA (E) or 259 μM CySSPe (F), with or
without coaddition of varied doses of GSH. After 48 h treatment, cell viability was measured. Results are expressed as mean SD from three
independent experiments.
such as ASC had no effect to suppress the biological activities of
CySSA and CySSPe (data not shown).
and allyl) in DPBS buffer led to a dose- and time-dependent
increase of extracellular thiols, as measured by a semi-
continuous DTNB assay (Figure 5A). The control cells treated
with DTNB alone showed almost no thiol increase, indicating
the produced thiols are induced by and/or sourced from
CySSR compounds. Glucose was found to be required for this
process, as the production of extracellular thiols was not
observed without addition of glucose in DPBS buffer (data not
shown). The experiment was done in DPBS buffer instead of
complete cell culture medium for easy manipulation of the
extracellular environment.
HPLC was employed to identify the produced thiol species
after DTNB derivatization by analyzing the formed TNB-SS-
thiol conjugates. For each CySSR compound, four peaks were
observed on HPLC: besides DTNB (peak 3) and TNB (peak
2), the other two peaks were found to be TNB-SS-cysteine
(peak 1) and TNB-SS-R (peaks 4−8) that were confirmed by
coelution with standards (Figure 5B). These results indicate
that the evolved extracellular thiols from CySSR species include
cysteine and RSH (other thiol species could also be in the
extracellular medium), which are reducing products of CySSR.
This result supports a model where CySSR undergoes a cellular
reduction to generate cysteine and RSH which remain an end-
up in the extracellular culture medium. A similar phenomenon
of cellular disulfide reduction to form extracellular thiol species
has been described in a previous study.20
Together, these results support a model that CySSR exerts its
biological activities via induction of cellular redox stress, which
activates redox signaling pathways and causes beneficial effects
such as phase II enzyme induction and anti-inflammation.
Induction of redox stress has been demonstrated to be a general
mechanistic feature of many chemopreventive compounds.32 In
addition, our results seem to suggest a specific thiol-dependent
redox mechanism of CySSR, rather than a general redox
mechanism since only GSH and NAC instead of other cellular
antioxidants such as ASC suppressed the effects of CySSR. ASC
decreases ROS level and has been shown to inhibit the
biological activity of ROS-generating compound such as 3,3′-
diindolylmethane.34 Both GSH and NAC boost the level of
intracellular GSH, thus it is likely that the selective inhibitory
effects of GSH and NAC on the biological activities of CySSR
were due to an intracellular GSH-dependent “detoxification”
process. Addition of GSH or NAC to cells increased the level of
intracellular GSH, favoring the GSH-dependent metabolism of
CySSR to generate less-active metabolites. Therefore next we
studied the metabolism of CySSR in vitro, and tested whether
GSH is involved in the metabolism of CySSR in cell cultures.
CySSR Is Metabolized To Form Cysteine and RSH in
Extracellular Medium in Vitro. RAW 264.7 macrophage
cells exposed to CySSR (R = methyl, ethyl, propyl, 1-propenyl,
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Figure 5. CySSR was metabolized to form cysteine and RSH in the extracellular medium, via a likely mechanism involving the metabolism by
glutaredoxin (Grx). (A) Treatment with CySSA in DPBS buffer (fortified with 10 mM glucose) caused a time- and dose-dependent increase of thiols
in medium of RAW 264.7 cells, measured by a DTNB assay. (B) HPLC analysis of cell medium of CySSR-treated RAW 264.7 cells after DTNB
assay, demonstrating the formed thiols are cysteine and RSH derived from CySSR. The RAW 264.7 cells were treated with 62.5 μg/mL CySSR and
0.5 mM DTNB in 10 mM glucose-fortified DPBS buffer for 3 h at room temperature; then 100 μL of medium was taken out, centrifuged, and
subjected to HPLC analysis, detection at 326 nm. Panel from top to bottom: (A) CySSM, (B) CySSE, (C) CySSP, (D) CySSA, and (E) CySSPe.
Peak 1: TNB-SS-cysteine. Peak 2: TNB. Peak 3: DTNB. Peak 4: TNB-SS-Me. Peak 5: TNB-SS-Et. Peak 6: TNB-SS-Pr. Peak 7: TNB-SS-All. Peak 8:
TNB-SS-1-Pe. (C) Glutaredoxin (Grx) enzymatic assay of CySSA in 100 mM Tris-HCl (pH = 7.5). (D) Proposed mechanism for the redox-related
cellular responses of CySSR.
CySSR could be reduced to form cysteine and RSH via
multiple mechanisms. In the present study, we found that
CySSR is a substrate of glutaredoxin (Grx). Figure 5C shows
the enzymatic assay of Grx, using a standard Grx assay system
which contains Grx, GSH reductase (GR), GSH, NADPH, and
CySSR.35 Addition of Grx caused a ∼5-fold increase in initial
reaction velocity of CySSR reduction compared with the non-
Grx control, supporting that CySSR is a substrate of Grx. To
further study whether Grx was involved in cellular reduction of
CySSR in RAW 264.7 cells as shown in Figure 5A,B, cells were
treated with CySSR together with CdCl2, a chemical inhibitor
of Grx.36 Coaddition of 2.5−10 μM CdCl2 inhibited 30−40%
of CySSA-induced extracellular thiol production after 1 h
treatment in RAW 264.7 cells (data not shown). MTT assay of
the cells after the treatment showed no decrease of cell viability,
indicating the observed inhibitory effects were not due to loss
in cell number (data not shown). Together, these results imply
that CySSR could be reduced by a Grx-dependent process to
form cysteine and RSH in vitro, and the produced cysteine and
RSH are then secreted to extracellular medium, most likely via
multiple drug resistance protein (MRP) (proposed model
shown in Figure 5D). Other cellular reducing enzymes, such as
thioredoxin reductase (TRxR), thioredoxin (Trx), and GR,
could also be involved in the cellular reduction of CySSR. The
Grx-mediated disulfide bond reduction requires NADPH,
which might be derived from glucose metabolism through
glucose-6-phosphate dehydrogenase (G6PDH). This would
explain the requirement of glucose in the DPBS medium for
extracellular thiol production of CySSR. The Grx-dependent
reduction of CySSR may at least partially explain the selective
inhibitory effects of GSH and NAC on the biological activities
of CySSR. Coaddition of GSH or NAC, but not other types of
cellular antioxidants such as ASC, increases the level of
intracellular GSH, favoring the Grx-dependent metabolism of
CySSR since Grx has a high Km value for GSH.37 This process
leads to removal of CySSR from intracellular space, and the
formed cysteine and RSH are inactive for QR induction and
NO inhibition (data not shown), leading to decreased
biological effects of CySSR species.
Interestingly, S-alk(en)ylmercaptoglutathione (GSSR, R =
methyl, ethyl, propyl, 1-propenyl, and allyl), which showed
similar effects of CySSR to induce QR and inhibit LPS-induced
NO formation,11 did not induce extracellular thiol production
(data not shown). This result indicates GSSR might not enter
intracellular space, because if GSSR pass cell membrane, GSSR
would be intracellularly reduced to form RSH, which would be
secreted into extracellular medium. Multidrug resistance
protein transporters (MRP) function to export glutathione S-
conjugates, GSH, and GSSG from cells;38 importation of these
chemotypes and the similarly structured GSSR species into cells
would pose a futile cycle. It would be expected that GSSR
would remain in extracellular space until further metabolized.
Together, in the present study we report the chemical
synthesis, biological activities and redox-related mechanisms of
CySSR compounds. The 1-propenyl conjugate CySSPe showed
the most potent phase II enzyme inducing activities, which is
well correlated with the potent effects of the Allium 1-propenyl
TS. CySSR compounds were metabolized to form cysteine and
RSH, which were secreted into extracellular medium, via a
process which could involve Grx-dependent enzymatic activity.
This GSH/Grx-dependent metabolism could explain the
selective inhibitory effects of GSH and NAC on the biological
activities of CySSR.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: klparkin@wisc.edu (K.L.P.) and gdzhang@ucdavis.
edu (G.Z.).
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Journal of Agricultural and Food Chemistry
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†Department of Entomology and Comprehensive Cancer
Center, University of California, Davis, One Shield Ave,
Davis, CA, 95616.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by the University of Wisconsin,
College of Agricultural and Life Sciences, in part through an
Agricultural Research Station Hatch grant (WIS01184)and the
American Institute for Cancer Research (project 10A050).
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