Oxidative species and S-glutathionyl conjugates in the apoptosis induction by allyl thiosulfate

Article abstract of DOI:10.1111/j.1742-4658.2011.08407.x

Natural allyl sulfur compounds show antiproliferative effects on tumor cells, but the biochemical mechanisms underlying the antitumorigenic properties of the organ sulfur compounds are not yet fully understood. Sodium 2-propenyl-thiosulfate is a garlic water-soluble organo-sulfane sulfur compound able to promote apoptosis in cancer cells, affecting the 'managing' of the redox state in the cell. Our studies show that sodium 2-propenyl-thiosulfate reacts spontaneously with reduced glutathione at physiological pH, leading to the formation of S-allyl-mercapto-glutathione, radicals and peroxyl species, which are able to induce inhibition of enzymes with cysteine in the catalytic site, such as sulfurtransferases. S-Allyl-mercapto-glutathione was purified and characterized by NMR and MS, and its cytotoxic effect at 500 μm on HuT 78 cells was analyzed, showing activation of the p38-MAPK pathway. Many allyl sulfur compounds are also able to promote chemoprevention by induction of xenobiotic-metabolizing enzymes, inducing down-activation or detoxification of the carcinogens. Thus, the effects of the S-allyl-mercapto-glutathione on proteins involved in the cellular detoxification system, such as glutathione S-transferase, have been evaluated both in vitro and in HuT 78 cells. Although the antitumor properties of water-soluble sulfur compounds may arise from several mechanisms and it is likely that more cellular events occur simultaneously, a relevant role is played by the formation of both reduced glutathione conjugates and radical species that affect the activity of the thiol-proteins involved in fundamental cellular processes.

Full text of DOI:10.1111/j.1742-4658.2011.08407.x

Oxidative species and S-glutathionyl conjugates in
the apoptosis induction by allyl thiosulfate
Ridvan Nepravishta1, Renato Sabelli1, Egidio Iorio2, Laura Micheli1, Maurizio Paci1
and Sonia Melino1
1 Department of Sciences and Chemical Technologies, University of Rome ‘Tor Vergata’, Italy
`
2 Department of Cellular Biology and Neurosciences, Istituto Superiore di Sanita, Rome, Italy
Keywords
Natural allyl sulfur compounds show antiproliferative effects on tumor
garlic; glutathione; organ sulfur compounds;
ROS; thiyl radicals
cells, but the biochemical mechanisms underlying the antitumorigenic prop-
erties of the organ sulfur compounds are not yet fully understood. Sodium
2-propenyl-thiosulfate is a garlic water-soluble organo-sulfane sulfur com-
pound able to promote apoptosis in cancer cells, affecting the ‘managing’
of the redox state in the cell. Our studies show that sodium 2-propenyl-
thiosulfate reacts spontaneously with reduced glutathione at physiological
pH, leading to the formation of S-allyl-mercapto-glutathione, radicals and
peroxyl species, which are able to induce inhibition of enzymes with cyste-
ine in the catalytic site, such as sulfurtransferases. S-Allyl-mercapto-gluta-
thione was purified and characterized by NMR and MS, and its cytotoxic
effect at 500 lM on HuT 78 cells was analyzed, showing activation of the
p38–MAPK pathway. Many allyl sulfur compounds are also able to
promote chemoprevention by induction of xenobiotic-metabolizing
enzymes, inducing down-activation or detoxification of the carcinogens.
Thus, the effects of the S-allyl-mercapto-glutathione on proteins involved
in the cellular detoxification system, such as glutathione S-transferase, have
been evaluated both in vitro and in HuT 78 cells. Although the antitumor
properties of water-soluble sulfur compounds may arise from several mech-
anisms and it is likely that more cellular events occur simultaneously, a rel-
evant role is played by the formation of both reduced glutathione
conjugates and radical species that affect the activity of the thiol-proteins
involved in fundamental cellular processes.
Correspondence
S. Melino, Dipartimento di Scienze e
Tecnologie Chimiche, University of Rome
‘Tor Vergata’, via della Ricerca Scientifica,
00133-Rome, Italy
Fax: +39 067 259 4328
Tel: +39 067 259 4449
E-mail: melinos@uniroma2.it
Note
Ridvan Nepravishta and Renato Sabelli
contributed equally to this work
(Received 2 August 2011, revised 14
October 2011, accepted 24 October 2011)
doi:10.1111/j.1742-4658.2011.08407.x
Introduction
The medicinal uses of Allium sativum have a long his-
tory [1] and its uses as a remedy for heart disease,
tumors and headaches are documented in the Egyptian
Codex Ebers, dating from 1550 bc. Recent studies have
validated many of the medicinal properties attributed
to garlic and its potential to lower the risk of disease.
Thus, in recent years, several studies of either oil- or
water-soluble garlic compounds have been carried out
to verify their chemopreventive and anticarcinogenic
effects, and to explain the mechanism of their action.
Chemopreventive agents may exert antiproliferative
effects via induction of cell-cycle arrest or apoptosis,
induction of terminal differentiation and inhibition of
oncogene activity or DNA synthesis [2].
Allicin is the main sulfur compound formed by alliin
upon crushing of garlic cloves [3], it is an unstable
Abbreviations
2-PTS, sodium-2-propenyl-thiosulfate; GSH, reduced glutathione; GSSA, S-allyl-mercapto-glutathione; GST, glutathione S-transferase;
HRP, horseradish peroxidise; OSC, organ sulfur compound; TMB, tetramethyl benzidine; TST, thiosulfate sulfurtransferase.
154
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R. Nepravishta et al.
OSCs a natural defense against cancer
compound and is rapidly transformed into secondary
products in vivo, such as allylmercaptans and others
that are also SH-modifying reagents [4–6]. Propenyl
thiosulfinates are major species of organ sulfur com-
pounds (OSCs) in fresh onion tissue macerates [7],
thiosulfinates can freely permeate cell membranes and
are most stable at the acidic pH of the stomach and
also at duodenal pH; furthermore, the response to this
compound has been shown to be also related to the
number of sulfur atoms [3,7–9]. Recently, many of
these OSCs have been shown to suppress the prolifera-
tion of cancer cells through the induction of pro-
grammed cell death. This induction leads to relevant
questions about the metabolism of OSCs and their role
in the activity of enzymes involved in detoxification of
the cellular system. In recent years, it has been shown
that increased garlic consumption diminishes the risk
of stomach and colorectal cancer [10]. A variety of
garlic allyl sulfur compounds have been reported to
induce growth rate arrest in tumor cells, either in cul-
ture or in vivo [11–14]. Several proteins that regulate
cellular proliferation can be damaged by S-allylsulfides
and are considered to be the target proteins involved
in the anticancer activity of garlic. Previous studies
show that expression of kinase C, cyclin-dependent
kinase 2, AP-1, p21waf1, p27Kip1, and especially NF-jB
and p53, is changed most consistently by garlic treat-
ment [13,15–17]. The biochemical mechanisms underly-
ing the antitumorigenic and antiproliferative effects of
garlic-derived OSCs are not yet fully understood,
although it seems likely that the rate of clearance of
allyl sulfur groups from cells is a determinant of the
overall response. Recently, it has been proposed that
exposure of tumor cells to allylsulfides and cysteinyl
S-conjugates affects the intracellular redox status either
by production of reactive oxygen species or reactive
nitrogen species, or by altering the reduced glutathione
(GSH) content [18]. GSH represents the main intracel-
lular redox buffer, its concentration is organ depen-
dent, varying between 2 and 10 mm [19], and its levels
modulate the redox state of the cell. It is known that
thiosulfinate, as well as some selenium compounds
[20], can react instantaneously with GSH in the living
cell to form intracellular GSH mixed-disulfide conju-
gates [6,21,22]. In this study, we used HPLC, NMR
spectroscopy, MS and cyclic voltammetry to investi-
gate the effects of the water–soluble proapoptotic gar-
lic compound, 2-propenyl-thiosulfate (2-PTS) [23–25],
on the antioxidant cellular system, by analyzing the
products of the in vitro reaction with GSH. Moreover,
the effects of the reaction products on enzymes, which
are implied in the cellular detoxification system, were
evaluated. Usually, targets for radical species in the
proteins are highly conserved cysteine residues whose
redox status is crucial for their activity. Sulfydril cen-
ters are reactive sites in redox-sensitive proteins that
are essential in functional signal transduction and tran-
scription events.
The cyanide:thiosulfate sulfurtransferase (TST;
EC 2.8.1.1) enzyme, which is characterized by the
presence of a catalytic cysteine residue and by high
structural homology with Cdc25 phosphatases [26],
was used as a model to investigate the effects of the
products of the 2-PTS–GSH reaction on the activity of
the homologous classes of enzymes with sulfydril
active centers. S-allyl-mercapto-glutathione (GSSA)
was also generated with high yield by a 2-PTS–GSH
reaction in vitro, and this glutathione derivative was
characterized and tested for its anticarcinogenic prop-
erties, suggesting a possible role for GSH in the anti-
proliferating effect of 2-PTS. It has been observed that
thiosulfinates and polysulfides can modify the drug
detoxification system by upregulation of phase II
enzymes [27–29] and furthermore, changes in the activ-
ity of detoxification enzymes may be due to reaction
with thiol groups [18,25,30]. Thus the effects of GSSA
on the cell detoxification system were investigated
in vitro and in treated HuT 78 cells.
Results
2-PTS and the cell redox system: GSSA synthesis
Previously, the effects of 2-PTS on the redox system
were investigated and oxidation of thioredoxin was
observed [25]. Thioredoxin and GSH represent the two
principal molecules that regulate the cell redox state.
In this study, we analyzed the interaction of 2-PTS
with GSH in vitro.
The reaction between GSH and 2-PTS was per-
formed in 50 mm Tris ⁄HCl buffer, pH 7.4, using a
4 : 1 2-PTS ⁄GSH molar ratio and then analyzed by
RP-HPLC. Figure 1A shows the RP-HPLC profile of
the 2-PTS⁄ GSH mixture and it is possible to see, in
addition to the a and b peaks which correspond to the
mix of 2-PTS, GSH, their oxidated forms and Tris, the
presence of a stable compound eluted at a retention
time of 21.81 min (peak c), which represents the third
principal component of the mixture. Peak c was col-
lected and then analyzed by ESI MS yielding a mass
of 380.07
0.07 m ⁄ z (Fig. 1B). The molecular mass
of peak c was compatible with a glutathione-propenyl-
sulfide derivative, also named GSSA [6]. Structural
characterization of peak c was also performed by
1H NMR spectroscopy (Fig. 1C) showing the charac-
teristics resonances of the S-allyl-mercapto-glutathione
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155

OSCs a natural defense against cancer
R. Nepravishta et al.
A
B
C
Fig. 1. Characterization of the 2-PTS ⁄ GSH mixture. (A) RP-HPLC of the PTS ⁄ GSH mixture (25 mM GSH and 100 mM 2-PTS) after 1 h of the
reaction. The elution was performed with a linear gradient of 80% CH3CN, 0.1% TFA as described in Materials and Methods (B) ESI mass
spectrum of the c peak, 380.07 0.07 m ⁄ z. (C) 1H NMR spectrum of the c peak in D2O solution.
(Table 1). GSSA formation obtained in vitro also sug-
gests a possible interaction of 2-PTS with GSH in vivo,
leading to GSSA production. A 2-PTS ⁄ GSH mixture
obtained using a 2 : 1 molar ratio was also analyzed
by RP-HPLC, resulting in a GSSA amount lower than
that obtained using a molar ratio of 4 : 1. As previ-
ously observed in the case of GSSA formation by the
allicin ⁄ GSH reaction [6], also in this case, a depen-
dence of the reaction on high pH values was observed,
indicating dependence on the concentration of GS).
is more able to inhibit TST activity in the E form of the
enzyme. Previously, we have showed that 2-PTS is able
to bind the sulfur-free form (E) of TST, inhibiting its
activity by thiolation of the catalytic cysteine [25], but
inhibition of the 2-PTS ⁄ GSH mixture was higher than
2-PTS alone at the same concentrations. By contrast,
purified GSSA is not able to significantly inhibit TST
activity in vitro (Fig. 2B), although a quenching of the
intrinsic fluorescence of both forms (ES and E) of TST
changed with the increase in GSSA concentration in
solution (Fig. 3A,C). Quenching of the intrinsic fluores-
cence of both forms of the enzyme, which are character-
ized by two different fluorescence spectra [31,32], and
the absence of inhibition may indicate unspecific interac-
tion of the GSSA with the surface of the enzyme that
does not involve the active site of the enzyme or induce
relevant conformation changes in the protein.
Cysteine–enzyme inhibition and radical species
formation by 2-PTS–GSH reaction
In a previous study, we showed the ability of 2-PTS to
induce inhibition of TST. TST from Azotobacter vine-
landii was used as model to assay the effects of both a
2-PTS⁄ GSH mixture and GSSA on thiol-containing
proteins, for high 3D structural homology with Cdc25
phosphatase and for the characteristic presence of only
one cysteine residue, which is also the catalytic residue.
The effects of the 2-PTS ⁄ GSH mixture on TST activity
were analyzed in vitro (Fig. 2), showing that the mixture
TST activity was also unaffected by preincubation
of the E form in the presence of GSH, at the same
concentrations and conditions used for the mixture
(data not shown) and no changes in the intrinsic fluo-
rescence of the E form were observed after addition of
GSH (Fig. 3B).
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R. Nepravishta et al.
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Table 1. 1H NMR chemical shifts for glutathione, sodium-2-prope-
nyl-thiosulfate (2-PTS) and their disulfide conjugates in D2O. s, sin-
glet; d, doublet; t, triplet; m, other multiplets.
electrochemical measurement of the enzyme product
[TMB(Ox)].
Figure 4A shows the current values of the oxida-
tion peak of TMB as a function of the concentration
of 2-PTS alone compared with the reaction mixture
2-PTS–GSH. As expected, in the case of the 2-PTS
mixture, the current increases with increasing reagent
concentrations, indicating the formation of a larger
amount of product. The current value of the peak is
an indirect measurement of the 2-PTS–GSH reaction
products. In fact, TMB(Ox) formation, which is due
to a reaction catalyzed by HRP, indicates the pres-
ence of peroxide ⁄ peroxyl species in the solution. Oxi-
dation of thiols by peroxidase has been shown to
yield thiyl radicals [33,35], thus we have performed
an analysis of both 2-PTS (Fig. 4A) and GSH
(Fig. S1) separately. By contrast to the mixture, single
compounds do not show a concentration-dependent
increase in the positive band in the presence of HRP,
indicating the absence of peroxyl species in the solu-
tion. Figure 4B shows the voltammograms for the
mixture at different times of the 2-PTS–GSH reaction
H chemical
shift (ppm)a
Compounds
Groups
Multiplicityb
Glutathione reduced form (GSH)
Glycine moiety
Cysteine moiety
CH2
CH
3.95
4.56
2.94
3.82
2.15
2.55
s
dd
dd
t
CH2
CH
Glutamate moiety
CH2
CH2
m
m
Glutathione oxidated form (GSSG)
Glycine moiety
Cysteine moiety
CH2
CH
3.91
3.28
2.98
3.78
2.17
2.55
3.76
6.02
5.24
s
dd
dd
t
CH2
CH
Glutamate moiety
CH2
CH2
–CH2–
CH
m
m
d
2-PTS
m
dd
CH=
S-allyl-mercapto-glutathione (GSSA)
Glycine moiety
Cysteine moiety
CH2
CH
3.95
3.24
2.97
3.79
2.17
2.55
3.38
5.89
5.21
s
dd
dd
t
CH2
CH
A
Glutamate moiety
2-PTS moiety
CH2
CH2
–CH2–
CH
m
m
d
m
dd
CH=
a
Proton chemical shift are reported with reference to 3-(trimethylsi-
b
lyl)-propionic-2,2,3,3-d4 acid sodium salt at 0 ppm. The multiplicity
given here was observed in conventional 1D spectra recorded at
400 MHz.
B
Taken together, these results suggest that TST inhi-
bition in the presence of the the 2-PTS–GSH mixture
may be due to the formation of oxidative species dur-
ing the 2-PTS–GSH reaction. The reductive ability of
thiols, particularly GSH, makes them an effective anti-
oxidant in vivo and in vitro.
Thus, we investigated the species produced during
the reaction that led to the formation of GSSA. The
formation of radicals and peroxyl species during the
2-PTS–GSH reaction was analyzed by cyclic voltam-
metry after 30 min of the reaction. A horseradish per-
oxidase (HRP; EC 1.11.1.7) reaction was used in the
presence of tetramethyl benzidine (TMB) as an elec-
tron donor [33,34]. Cyclic voltammetry was carried out
using screen-printed electrodes (2 · 10)4 m) in 0.1 m
citrate–phosphate buffer, pH 5.0. The 2-PTS–GSH
mixture was analyzed after 1 min incubation with
HRP and TMB and 70 lL of the reaction mixture
was put directly onto the screen-printed electrode for
Fig. 2. 2-PTS ⁄ GSH mixture and GSSA affect the TST activity. (A)
Inhibitory effect of 2-PTS ⁄ GSH mixture on TST activity. Time-
dependence decreasing of TST activity of 5 lM TST in 50 mM
Tris ⁄ HCl buffer, pH 8.0, 0.3 M NaCl in the presence of a threefold
molar excess of KCN (15 lM) and of 200-fold molar excess of
2-PTS (1 mM) (h) or 2-PTS ⁄ GSH mixture (1 mM and 250 lM respec-
tively) (D) at 37 ꢀC. (B) Time dependence decreasing of TST activity
(8.75 lM TST) in the presence of a threefold molar excess of KCN
(h) (26.25 lM) and of 26.25 lM KCN and a 400-fold molar excess
of GSSA (3.5 mM) ( ) at 37 ꢀC. All values are expressed in percent
of TST activity value of ES form.
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R. Nepravishta et al.
A
B
C
Fig. 3. Effects of GSSA on the TST intrinsic fluorescence. Intrinsic fluorescence of 3 lM TST (E) at different molar ratios of GSSA (A) and
GSH (B), and of 3 lM TST (ES) at different molar ratios of GSSA (C). 1 ⁄ 0 (____), 1 ⁄ 1 (
TST ⁄ GSH or GSSA c ⁄ c.
), 1 ⁄ 10 (_ _ _ _), 1 ⁄ 100 (- - - -) and 1 ⁄ 150 (
)
A
at room temperature after reaction with HRP and it
is possible to observe the time-dependent formation
of the radical species.
Cyclic voltammetry of the mixture and of the indi-
vidual components in the absence of HRP was also
performed and a significant shift in the oxidation cur-
rent over time was observable only in the case of the
2-PTS–GSH mixture, indicating formation of the radi-
cal species in the reaction (Fig. S2). Moreover, analy-
sis of the mixture at different times of the 2-PTS–GSH
reaction was also performed using a Prussian Blue
screen-printed electrode, indicating the formation of
the radical species, in agreement with previous results
(data not shown).
B
Taking into account that Allium derivatives S-allyl-
l-cysteine and diallyl trisulfide are hydrogen sulfide
(H2S)-releasing, formation of H2S during the 2-PTS–
GSH reaction was also evaluated by methylene blue
formation, but no significant H2S formation was
detected (data not shown).
Inhibition of cell-cycle progression of the HuT 78
cells by GSSA
The effects of purified GSSA on cell growth in the
human T lymphoblastoid cell line, HuT 78, were
checked and a typical time- and dose-dependent inhibi-
tion of cell growth in these cells was observed. A sta-
tistically significant decrease in the number of viable
cells was evidenced at different concentrations of
GSSA (from 0 to 0.5 mm) and at different times, com-
pared with the control (Fig. 5A). Approximately 90%
and 80% of the HuT 78 cells were viable following
exposure to 0.5 mm GSSA for 24 and 48 h, respec-
tively (Fig. 5B). The growth inhibitory effects of GSSA
Fig. 4. Cyclic voltammetric investigation of the 2-PTS ⁄ GSH mixture
in 0.1 M citrate–phosphate buffer, pH 5.0, with 0.1 M KCl. (A) Cur-
rents with respect to TMB(Ox) at different concentrations of a
2-PTS ⁄ GSH mixture (40 and 10 mM respectively) and of 2-PTS solu-
tion (40 mM) alone, in 50 mM Tris ⁄ HCl, pH 8.0, buffer after 30 min
incubation at 25 ꢀC. Before analysis, the solutions were incubated
with 20 UÆmL)1 HRP for 1 min at room temperature and 0.2 mM
TMB was added to the solutions. (B) Cyclic voltammetry of a
2-PTS ⁄ GSH mixture after 30 min (b) and 60 min (c) of the reaction.
Before the measurements, the solutions were incubated with
20 UÆmL)1 HRP for 5 min at room temperature and 0.2 mM TMB
was added to the solutions. (a) Voltammograms of TMB in buffer.
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R. Nepravishta et al.
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A
B
C
Fig. 5. Effects of GSSA on cell growth of
HuT 78 cells. (A) HuT 78 cell viability after
addition in culture of different concentra-
tions of GSSA (0, 30, 60, 125, 250 and
500 lM). Trypan Blue staining was used to
differentiate viable cells. (B) Cell viability
dependence at 24 and 48 h as a function of
the GSSA concentration. Data are
D
expressed as mean SD. *P < 0.005,
**P < 0.001 (n = 4). (C) Percentage cell-
cycle distribution of HuT 78 cells after 24
and 48 h of treatment with 0.5 mM GSSA
detected using FACSCalibur flow cytometer.
Data are expressed as mean SD.
*P < 0.001, **P < 0.05 (n = 3). (D) Western
immunoblotting showing P-p38 expression
in HuT 78 cell lysates after GSSA treatment,
30 lg of proteins from the lysates of
untreated cells (CTRL) and 0.5 mM of GSSA-
treated cells after 4, 9 and 24 h incubation.
A monoclonal anti-actin IgG was used as a
control.
were assessed by Trypan Blue dye exclusion assay.
Flow cytometric analysis of HuT 78 cells after 24 h
treatment with 0.5 mm GSSA resulted in a statistically
significant increase in the fraction of subG1 over the
control and a blockage in the G2 ⁄ M phase (Fig. 5).
We also investigated regulatory proteins that might
be involved in cell-cycle progression. p38 MAP kinase
is a member of a pathway that responds to cellular
stress and is linked to the cell cycle through senescence
and the differentiation pathway [36,37]. Western blot
analysis of HuT 78 lysates after GSSA treatment
(Fig. 5C) shows that expression of the phosphorylated
form of p38 (P-p38) was markedly increased, suggest-
ing that the antiproliferative effect of GSSA is related
to activation of the p38 pathway in time-dependent
manner in HuT 78 cells.
Effects of GSSA on glutathione S-transferase
expression and activity in vitro and in HuT 78
cells
Interestingly, it has recently been demonstrated that
GSSA is a potent quinone reductase inductor at micro-
molar concentrations [38]. In this study, the effects of
GSSA on the xenobiotic export system have been
investigated, evaluating its influence on glutathione
S-transferase (GST; EC 2.5.1.18) activity either in vitro
or in a cell system. GST activity in the presence of differ-
ent concentrations of GSSA was assayed, as described
in Materials and Methods. GSSA was able to inhibit
GST activity in a concentration-dependent manner
(Fig. 6A). IC50 and Ki were measured at ꢀ 0.6 and
0.1 mm, respectively, and were comparable with
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R. Nepravishta et al.
activity was observed, although the sulfur compound
was able to inhibit GST activity in vitro.
A
Discussion
Allyl sulfur compounds are the dominant species in
both garlic (50–94%) and fresh onion tissue macerates
[7], and are widely believed to be responsible for the
health benefits of these plants [3,7]. The study of the
biological effects of their mixed-disulfide conjugate
derivatives may highlight on their health benefits. The
bioavailability of thiosulfinates approaches 100%, and
they are metabolized rapidly in human blood in vitro,
with a half-life of < 1 min [39]. Their cellular mem-
brane permeability and high reactivity with thiols
[6,21,40] may account for the short half-life of thiosulf-
inates in physiological systems. In recent years, several
studies have focused on elucidation of the mechanism
of biological activity in garlic and its allyl sulfide deriv-
atives, such as allicin, diallyl disulfide, diallyl trisulfide
and, more recently, 2-PTS [25,41–46]. Not all cells are
equally susceptible to the deleterious effects of the gar-
lic sulfur compounds and, in particular, non-neoplastic
cells tend to be less susceptible, suggesting that the
uncontrolled proliferation of the neoplastic cell might
be also related to a sulfane sulfur deficiency due to an
incorrect functionality of the enzymes involved in their
catabolism [47]. Previously, it has been suggested that
erythrocyte GSH accelerates the oxidative damage pro-
duced by 2-PTS, indicating that this compound may
react with GSH, which might convert to GSSG, result-
ing in the activation of n-propylthiosulfate as an
oxidant [48]. In this study, we have shown that
2-PTS, at physiological conditions, is able to react
spontaneously with reduced GSH, forming the S-allyl-
mercaptoglutathione. Mixed-disulfide conjugates of
thiosulfinates with biological ⁄ physiological thiols repre-
sent major derivatives that form in the gut or through
metabolic processes in vivo. GSH mixed-disulfide con-
jugates would most likely be formed intracellularly
after consumption of thiosulfinates [6] or through met-
abolic activity [49,50]. Moreover, GSH mixed-disulfide
conjugate species might remain in the extracellular
space until metabolized, thus it is likely that GSH
mixed-disulfide conjugates and cysteine-conjugates
would be accumulated and act in different extracellular
compartments, affecting redox modulation. The bio-
logical effects on thiol-containing proteins of both a
2-PTS–GSH mixture and purified GSSA have been
tested in vitro using TST from A. vinelandii, showing
the major ability of the mixture to inhibit TST activity
with respect to 2-PTS alone. By contrast, purified
GSSA was able to interact with the enzyme, but
B
C
Fig. 6. Effects of GSSA on GST activity and expression. (A) In vitro
GST activity in the presence of different concentrations of GSSA.
The inhibition of the GST activity by GSSA showed an IC50 value of
ꢀ 0.6 0.07 mM and apparent ki of 0.10 0.01 mM. (Inner) Plot of
the GST activity. (B) GST activity of HuT 78 cells after 4, 9 and
24 h treatment with 0.5 mM GSSA. (C) Western immunoblotting
showing expression in HuT 78 cell lysates after GSSA treatment,
30 lg of proteins from the lysates of untreated cells after 24 h
(CTRL) and treated cells with 0.5 mM GSSA after 4, 9 and 24 h
incubation. A monoclonal anti-actin IgG was used as control of the
concentrations.
inhibition of the S-methylglutathione inhibitor, sug-
gesting a similar mechanism of inhibition.
The effects of GSSA on GSTP1-1 enzyme expression
and total cellular GST activity in HuT 78 cells were
analyzed. Figure 5C shows the western blot of the
HuT 78 lysates after 24 and 48 h of treatment with
0.5 mm GSSA. Densitometry measurements of western
blots, corrected for actin expression, show that no sig-
nificant variation in GST expression with respect to
controls was induced by treatment with GSSA. Total
GST activity was also assayed after GSSA treatment,
as shown in Fig. 6B, and no variation in total GST
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showed very low ability to inhibit TST activity at high
concentrations. The evidence for production during the
reaction of oxidative species, such as peroxyl species
and thiyl and ⁄ or oxygen radicals, was obtained by
cyclic voltammetry analysis. In fact, the oxidizing
agent is reduced and the thiols are rapidly oxidized,
generating thiyl and thiyl-derived radicals. Thiyl radi-
cals are oxidizing radicals with a potential redox Eꢀ
involves cell arrest in the G2 ⁄ M phase of the cell cycle
in cancer cells [44,61]. An increase in the expression of
the activated form of p38MAPK after GSSA treatment
was also observed, suggesting that the antiproliferative
effect of GSSA may be due to activation of the p38
pathway. p38MAPK is a key member of the MAPK
family and it is known that p38 is rapidly activated in
response to proinflammatory cytokines and stress and
cellular damage [62–67]. Most studies are consistent
with the interpretation that p38 plays a critical role in
progression through G2 and not mitosis. It has been
shown that activated P-p38 is distributed throughout
the cytoplasm of mitotic cells [68], and also that it is
localized in the centrosomes during spindle assembly
[69,70]. Recently, it has been demonstrated that
diallyl disulfide- and diallyl trisulfide-induced apoptosis
in some tumor cell lines is related to activation of
the p38MAPK signaling pathway [71–73]. Thus
p38MAPK activation might be a common mechanism
of action of the allyl sulfur compounds due to the for-
mation of their glutathione conjugate.
(RSÆ, H+ ⁄ RSH) = 1.35
0.04 V [51]. Once formed,
thiyl radicals can react with the thiolate or a parent
thiol to form the disulfide radical anion (RSSRÆ)).
Both these reactive species can react with molecular
oxygen, and the first can form the thiyl peroxyl radical
[52–54], which may be a damaging species involved in
protein deactivation [55]. The presence of oxygen can
also lead to sulfonylperoxyl radicals (RSOOÆ, RSOÆ,
RSO2OOÆ) [55], which also exhibit oxidizing properties.
The disulfide radical anion can also react with oxygen,
generating the superoxide radical anion: GSSGÆ) + O2
GSSG + O2Æ).
Thus, TST inhibition may be related to the presence
of these species in solution, which was shown to be
able to inhibit TST activity in vitro [56].
The induction of programmed cell death by sulfane
sulfur compounds [alk(en)yl thiosulfate, selenodigluta-
thione, allyl disulfide, etc.] poses significant questions
concerning their catabolism in the cell and on the role
in cancerogenesis of proteins involved in the detoxifica-
tion process.
Reactive oxygen species formation in tumor cells
after 2-PTS treatment was also observed in our previ-
ous study [25] and was suggested as the earliest event
in the cascade of apoptosis induction by 2-PTS treat-
ment of HuT 78 cells. Recently, it has been also
shown that 2-PTS increased 4b-phorbol 12-myristate
Interestingly, glutathione mixed-disulfide conjugates
were found to be able to inhibit nitric oxide produc-
tion in macrophage cells, showing their potential
ability to prevent cancer and the inflammatory process,
and to induce quinone reductase in murine hepatoma
cells (Hepa 1c1c7) at micromolar concentrations [39].
It is noteworthy, in fact, that anticancer effects can be
mediated through the antioxidant response element to
afford upregulation of phase II enzymes [27].
)
13-acetate-induced O2 generation, although it is also
able to accelerate the reaction catalyzed by NADPH
oxidase in canine polymorphonuclear leukocytes
[57,58]. This evidence is also in agreement with recent
studies on diallyl trisulfide compound, which suggest
that diallyl trisulfide can mediate the decline in
Cdc25C protein levels in cancer cells because of an
oxidation component [44]; this is consistent with redox
regulation of Cdc25C stability by oxidation of two
specific cysteine residues [59]. Therefore, oxidative
stress, as suggested for other OSCs [60], might repre-
sent the triggering factor in induced apoptosis by
2-PTS. Reactive oxygen species formation may be also
related to an oxidative unbalance due to both the
consumption of GSH and to the TST–thioredoxin–
thioredoxin reductase system [25].
It has been shown that the antiproliferative effects
might be attributable to a multifactorial mechanism in
which the GSTs may play a role, and the detoxifica-
tion system of tumor cells might be affected by the
glutathione derivative of OSCs. Several studies using
allyl sulfides have demonstrated a significant decrease
in GST activity in hepatocytes after treatment with a
high concentration of them [74]. Our results show that,
although GSSA is able to inhibit GST activity in vitro,
similar to the GST inhibitor S-methylglutathione, both
GSTP1-1 expression and cellular GST activity do not
change after GSSA treatment of HuT 78 cells. In gen-
eral, GSH-conjugates are exported from cells by
energy-dependent GS-xenobiotic pumps; also known
as multidrug resistance proteins, these transport mech-
anisms, designated as phase III of the detoxification
process [75], are therefore an essential component of
The effects of 2-PTS glutathionyl derivative GSSA
on HuT 78 cells were analyzed, showing a typical time-
and dose-dependent inhibition in cell growth. Flow
cytometric analysis of HuT 78 cells after GSSA treat-
ment resulted in an antiproliferative effect with a
blockage in the G2 ⁄ M phase.
These results are in agreement with the antiprolifera-
tive mechanism of most garlic compounds, which
FEBS Journal 279 (2012) 154–167 ª 2011 The Authors Journal compilation ª 2011 FEBS
161

OSCs a natural defense against cancer
R. Nepravishta et al.
cellular defense mechanisms against toxic chemicals
[76]. The phase II reaction products must eventually be
transported to complete detoxification, because accu-
mulation of these products can not only cause toxicity,
but also inhibit the phase II reactions. It has been sug-
gested that the chemopreventive agents present in gar-
lic might have a significant effect on chemotherapeutic
treatments using GS-xenobiotic pump substrates [77].
It has been observed that diallyldisulfide (DAS) is a
selective and highly potent modulator of P-glycopro-
tein-mediated multidrug resistance in human K562 leu-
kemic cells and in rodent liver [78]. Thus, further
studies are required to evaluate the effects of GSSA on
the cell detoxification system and the possibility that
this glutathione mixed-disulfide conjugate may act on
the P-glycoprotein protein cannot be ruled out. Fur-
ther studies on the relationship between sulfur metabo-
lism and the p38MAPK signal transduction pathway
in cellular apoptosis will lead to better clarification of
the mechanism of apoptosis and will have important
significance for the investigation into the antitumor
mechanisms of OSCs and the design of new drugs.
Kinetic analysis
TST activity was measured using the discontinuous colori-
metric assay, as described by Sorbo [83], in which the pro-
duction of thiocyanate from thiosulfate and cyanide was
followed at 460 nm using a Perkin–Elmer spectrometer.
GST activity was measured using the GST assay kit
(CS0410; Sigma-Aldrich, Milan, Italy). The reaction is mea-
sured by observing the conjugation of 1-chloro-2,4-dinitro-
benzene with GSH, which induces an increase in
absorbance at 340 nm. One unit of enzyme will conjugate
10.0 nmol of 1-chloro-2,4-dinitrobenzene with reduced glu-
tathione per min at 25 ꢀC [84–86]. The Ki value was calcu-
lated with the Cheng–Prusoff equation [87]:
Ki ¼ IC50=ð1 þ S=KmÞ
ð1Þ
where the IC50 is the concentration of the competitive
inhibitor producing a 50% inhibition, S is the substrate
concentration under which the study is performed and Km
is the Michaelis constant of the substrate for the enzyme
and assuming a 0.19 mm Km GSH and a competitive behav-
ior against GSH. The activity measurements were per-
formed at least three times using cellular lysates from
two different cellular treatments.
Materials and methods
Fluorescence measurements
2-PTS synthesis
All fluorescence measurements were made using a LS50
Perkin–Elmer spectrofluorimeter equipped with a thermo-
stated stirrer cell holder. The temperature was always main-
tained at 23 ꢀC. The excitation and emission bandwidths
were 5 and 3 nm, respectively. The excitation wavelength
was set at 286 nm and the spectra were recorded from 300
to 400 nm. The fluorescence measurements were performed
in the presence of different concentrations of allyl com-
pound with 3 or 5 lm enzyme in 50 mm Tris ⁄ HCl buffer,
pH 7.2. E form of the enzyme for the fluorescence spectra
was obtained by treatment with dithiothreitol in a molar
ratio of 1 : 100 TST ⁄ dithiothreitol and after treatment a
gel-filtration chromatography of the sample was performed
to remove the dithiothreitol using a Napꢁ 25 column
(GE Amersham).
2-PTS was synthesized according to a method described by
Chapelet et al. [79]. The product was dried in vacuum and
extracted with methanol. The extract was then purified by
silica gel chromatography (methanol ⁄ chloroform 45 : 55
v ⁄ v). The purity and structure of the compound were evalu-
1
ated by RP-HPLC, LC-MS and H NMR [25].
Preparation of RhdA and GST proteins
The plasmid pQER1 containing the gene coding for N-ter-
minal His-tag A. vinelandii TST was expressed and purified
as previously described [80]. The protein concentration was
0:1%
280
determined using A
= 1.3 [81] and the molecular mass
of the E form was estimated at 31 063.8 kDa by ESI MS.
TST(E) form was prepared by adding a 10-fold molar
excess of cyanide to ES rhodanese or using 4 mm dith-
iothreitol, in 50 mm Tris ⁄ HCl buffer pH 7.4 and 0.3 m
NaCl followed by 30-min incubation at room temperature.
Excess cyanide, thiocyanate or dithiothreitol were removed
using gel-filtration chromatography (Napꢁ 25 column; GE
Amersham, Milan, Italy) or by 48 h dialysis at 4 ꢀC.
ES fi E form conversion was monitored by increasing the
fluorescence quantum yield, which is accompanied by
removal of the persulfide sulfur [31,32]. GST expression
and purification were performed in Escherichia coli, as
descried by Battistoni et al. [82].
S-Allyl-mercapto-glutathione analysis
S-Allyl-mercapto-glutathione synthesis was performed using
25 mm GSH and 100 mm 2-PTS (1 : 4 v ⁄ v), in Tris ⁄ HCl buf-
fer pH 7.4. GSSA formation was evaluated using RP-HPLC
(mod. LC-10AVP; Shimadzu, Milan, Italy) with an solvent B
gradient (0–5 min, 0%; 5–25 min, 15%; 25–27 min, 15% and
27–30 min 90%), using 0.1% trifluoric acid as solvent A and
80% CH3CN, 0.1% trifluoric acid as solvent B, and a Brou-
wnlee C18 column (OD300, 250 · 4.6 mm, 7 lm). The elute
was monitored at 220 nm by UV detector (Shimadzu, Milan,
162
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R. Nepravishta et al.
OSCs a natural defense against cancer
Italy). Samples were then analyzed by ESI MS (CEINGE
Advanced Biotechnologies, Naples, Italy). High-resolution
1H NMR experiments (25 ꢀC) were performed at 400 MHz
(Bruker AVANCE spectrometer, Milan, Italy). The
compounds were resuspended and 500 lL of D2O (Sigma-
Aldrich, Milan, Italy) containing 0.1 mm 3-(trimethylsilyl)-
propionic-2,2,3,3-d4 acid sodium salt was added as the
internal standard (Merck, Montreal, Canada). A quantitative
determination of the GSSA solution was performed by
incubated at 37 ꢀC for 10 min, and terminated by the addi-
tion of 20 lL of solution I (20 mm N¢,N’-dimethyl-p-pheny-
lenediamine dihydrochloride in 7.2 m HCl) and 20 lL of
solution II (30 mm FeCl3 in 1.2 m HCl). After incubation
for 30 min at room temperature, methylene blue formation
was examined spectrophotometrically at 670 nm.
Cell proliferation assay
1
1H NMR. H NMR spectra of samples were obtained using
HuT 78 human T lymphoblastoid cells were purchased from
the Istituto Superiore di Sanita (Italy). HuT 78 (0.2 · 106)
cells were preincubated for 24 h in RPMI 1640 (GIBCO,
Milan, Italy) in the presence of 1% glutamine, 10% heat-
inactivated fetal bovive serum and antibiotics (1% penicillin
and streptomycin sulfate) at 37 ꢀC in air supplemented with
5% CO2. HuT 78 cells were treated with different concentra-
tions of GSSA and then monitored for 8, 24 and 48 h. The
cells were then collected and counted after Trypan Blue
staining (0.4% Tripan Blue solution; Sigma-Aldrich, Milan,
Italy) by optical microscopy using a Thoma chamber. The
rates of growth inhibition were calculated with respect to the
control culture, which was taken as 100% growth.
RF pulses for excitation, water signal presaturation, data
processing and data analysis as described previously [25,88].
The solution was used to form a calibration curve by
RP-HPLC using the same column and conditions described
above.
Cyclic voltammetry
Electrochemical measurements were performed at room
temperature, using a computer-controlled system, AUTO-
LAB model GPSTAT-12 with gpes software (Ecochemie,
Utrecht, The Netherlands). Screen-printed electrodes were
home-produced with a 245 DEK (Weymouth, UK) screen-
printing machine. Graphite-based ink (Elettrodag 421)
from Acheson (Milan, Italy) was used to print the working
and counter electrode. The substrate was a flexible polyes-
ter film (Autostat HT5) obtained from Autotype Italia
(Milan, Italy).
Cell-cycle analysis
The cell-cycle distribution of HuT 78 cells was measured by
flow cytometry. Harvested cells (ꢀ 0.5 · 106 cells) were
stained with 50 lgÆmL)1 propidium iodide (Sigma-Aldrich)
in NaCl ⁄ Pi buffer with 0.1% Triton X-100 and 1 mgÆmL)1
sodium citrate. They were immediately analyzed using a
flow cytometer FACSCalibur (Becton-Dickinson, San Jose,
CA, USA) and the percentage of cells in each phase of cell
cycle was evaluated according to Nicoletti et al. [91].
The electrodes were produced in foils of 48 strips. Each
sensor consists of three screen-printed elements: two carbon
electrodes acting as working and counter, respectively, and
a silver electrode acting as pseudoreference. The diameter
of the working electrode was 0.3 cm. After the printing
step, the foils were stored dry, at room temperature, in the
dark. The screen-printed electrodes were modified with
Prussian Blue [potassium iron (III)hexacyanoferrate(II)]
prior to enzyme immobilization. The working electrode sur-
face-modification protocol was based on the procedure
optimized by Ricci et al. [89]. In general, 5 lL of each
Prussian Blue precursor solution, consisting of 0.1 m of
K3Fe(CN)6 in 10 mm HCl and 20 lL of 0.1 m FeCl6 in
10 mm HCl, were applied to the working electrode. The
electrodes were incubated for 10 min in the dark and then
rinsed with 10 mm HCl. The electrodes were then left for
1 h in the oven at 100 ꢀC to obtain a dry and stable active
layer of Prussian Blue. The Prussian Blue-modified elec-
trodes were stored dry at room temperature in the dark
until use. All measurements were performed at least three
times showing good reproducibility.
Protein extraction and western blot analysis
Proteins were extracted from HuT 78 cells in 200 lL of
50 mm Tris ⁄ HCl, pH 7.09, containing a protease inhibitor
cocktail (Sigma-Aldrich, Milan, Italy) and pervanadate as
the phosphatase inhibitor, and sonicated using four steps of
5 s with 1 min standby in ice. Samples were centrifuged for
10 min at 12 000 g at 4 ꢀC. Protein contents was deter-
mined by bicinconinic acid protein assay (Sigma-Aldrich),
cell extracts (30 lg of protein) were electrophoresed on
15% polyacrylamide gel, electroblotted on a poly(vinylidene
difluoride) membrane (Applied Biosystems, Monza, Italy),
expression levels of GST were analyzed using anti-(human
GSTP1-1) monoclonal rabbit Ig (Calbiochem, Milan, Italy)
and phosphorylated p38 using anti-(P-p38) monoclonal IgG
(Sigma-Aldrich). An immunoblot with anti-actin mono-
clonal IgG (Santa-Cruz D.B.A. Italia, Milan, Italy) was
also probed for controlling the protein loading. The pro-
tein complex formed upon incubation with specific second-
ary antibodies (dilution 1 : 10 000) (Sigma-Aldrich) was
H2S assay
To estimate H2S production, a methylene blue formation
assay was performed following the method of Schmidt [90]
with minor modifications. Briefly, the reaction (200 lL) was
FEBS Journal 279 (2012) 154–167 ª 2011 The Authors Journal compilation ª 2011 FEBS
163

OSCs a natural defense against cancer
R. Nepravishta et al.
identified using a Fluorchem Imaging system (Alpha Inno-
tech Corp., Analitica De Mori, Milan, Italy) after incuba-
tion with ChemiGlow chemiluminescence substrate.
Densitometry analysis of western blots was performed using
quantity one software (Bio-Rad, Milan, Italy).
8 Shen C & Parkin KL (2000) In vitro biogeneration of
pure thiosulfinates and propanethial-S-oxide. J Agric
Food Chem 48, 6254–6260.
9 Wargovich MJ (1992) Inhibition of gastrointestinal can-
cer by organo-sulfur compounds in garlic. In Cancer
Chemoprevention (Wattenberg LW, Lipkin M, Boone
CW & Kelloff GJ eds), pp. 195–203. CRC Press, Boca
Raton, FL.
Statistical analysis
10 Fleischauer AT & Arab L (2001) Garlic and cancer:
a critical review of the epidemiologic literature. J Nutr
131, 1032S–1040S.
All experiments were carried out at least three time
(n = 3), unless otherwise indicated. Data were expressed as
mean
SD. Comparisons between control and treated
11 Knowles LM & Milner JA (2001) Possible mechanism
by which allyl sulfides suppress neoplastic cell prolifera-
tion. J Nutr 131, 1061S–1066S.
cells were made using Student’s t-test. Statistical signifi-
cance was defined as P < 0.05.
12 Knowles LM & Milner JA (2000) Allyl sulfides modify
cell growth. Drug Metabol Drug Interact 17, 81–107.
13 Knowles LM & Milner JA (2003) Diallyl disulfide
induces ERK phosphorylation and alters gene expres-
sion profiles in human colon tumor cells. J Nutr 133,
2901–2906.
Acknowledgements
We thank Prof. P. Tagliatesta for his help in the
organic synthesis of the 2-PST compound; Dr G. Vitic-
chie for her help in some experiments. We are grateful
to Prof. S. Pagani for kindly giving us the plasmid
coding for the RhdA protein.
14 Pinto JT, Qiao C, Xing J, Suffoletto BP, Schubert KB,
Rivlin RS, Huryk RF, Bacich DJ & Heston WD (2000)
Alterations of prostate biomarker expression and testos-
terone utilization in human LNCaP prostatic carcinoma
cells by garlic-derived S-allylmercaptocysteine. Prostate
45, 304–314.
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