Resveratrol and diallyl disulfide enhance curcumin-induced sarcoma cell apoptosis.

Abstract

Malignant tumors of mesenchimal origin such as rhabdomyosarcoma and osteosarcoma are highly aggressive pedriatic malignancies with a poor prognosis. Indeed, the initial response to chemotherapy is followed by chemoresistance. Diallyl disulfide (DADS), resveratrol (RES) and curcumin (CUR) are dietary chemopreventive phytochemicals which have been reported to have antineoplastic activity on rhabdomyosarcoma and osteosarcoma cells as single drugs. In this study we evaluated whether, as compared to the single compounds, the combination of DADS+RES, DADS+CUR and RES+CUR resulted in an enhancement of their antitumor potential on malignant rhabdoid (SJ-RH4, RD/18) or osteosarcoma (Saos-2) cell lines. Through FACS analysis and activated caspase-3 labeling we demonstrate that CUR induces apoptosis of rabdomyosarcoma and osteosarcoma cells and that this effect is potentiated when CUR is combined with RES or DADS. Further, we explored the effects of the compounds, alone or in combination, on signal transduction pathways involved in apoptosis and growth of cancer cells and show that in rhabdomyosarcoma cells the apoptotic effect of CUR, either alone or in combination, is independent of p53 activity. Our findings suggest that CUR and CUR-based combinations may have relevance for the treatment of p53-deficient cancers, which are often unaffected by conventional chemotherapies or radiotherapy.

Full text

Page 1

[Frontiers in Bioscience 17, 498-508, January 1, 2012]
498
Resveratrol and diallyl disulfide enhance curcumin-induced sarcoma cell apoptosis

Laura Masuelli1, Laura Marzocchella2, Chiara Focaccetti3, Ilaria Tresoldi2, Camilla Palumbo2, Valerio Izzi2, Monica
Benvenuto2, Massimo Fantini2, Florigio Lista4, Umberto Tarantino5, Andrea Modesti2, Fabio Galvano6, Roberto Bei2

1Department of Experimental Medicine, University of Rome, Sapienza, Rome, Italy, 2Department of Experimental Medicine and
Biochemical Sciences, University of Rome, Tor Vergata, Rome, Italy, 3Department of Biology, STA, Rome, Italy, 4Army Medical
and Veterinary Research Center, Rome, Italy, 5Department of Surgery, University of Rome, Tor Vergata, Rome, Italy,
6Department of Biological Chemistry, Medical Chemistry and Molecular Biology, University of Catania, Catania, Italy

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Reagents
3.2. Cell lines and treatments
3.3. Sulforhodamine B (SRB) assay
3.4. Ultrastructural analysis
3.5. FACS analysis
3.6. In situ detection of apoptosis
3.7. Preparation of cell lysates and Western Blotting
3.8. Statistical analysis
4. Results
4.1. Inhibition of malignant rhabdoid (SJ-RH4, RD/18) and osteosarcoma (Saos-2) cell survival by DADS, RES and
CUR alone or in combination
4.2. Morphological features of SJ-RH4 cells after treatment with DADS, RES and CUR alone or in combination
4.3. RES and DADS potentiate the apoptotic effect of CUR on SJ-RH4 and Saos-2 cell lines
4.4. Effect of DADS, RES and CUR alone or in combination on apoptosis and pro-survival signaling proteins
5. Discussion
6. Acknowledgements
7. References

1.ABSTRACT

Malignant tumors of mesenchimal origin such as
rhabdomyosarcoma and osteosarcoma are highly
aggressive pedriatic malignancies with a poor prognosis.
Indeed, the initial response to chemotherapy is followed by
chemoresistance. Diallyl disulfide (DADS), resveratrol
(RES) and curcumin (CUR) are dietary chemopreventive
phytochemicals which have been reported to have
antineoplastic activity on rhabdomyosarcoma and
osteosarcoma cells as single drugs. In this study we
evaluated whether, as compared to the single compounds,
the combination of DADS+RES, DADS+CUR and
RES+CUR resulted in an enhancement of their antitumor
potential on malignant rhabdoid (SJ-RH4, RD/18) or
osteosarcoma (Saos-2) cell lines. Through FACS analysis
and activated caspase-3 labeling we demonstrate that CUR
induces apoptosis of rabdomyosarcoma and osteosarcoma
cells and that this effect is potentiated when CUR is
combined with RES or DADS. Further, we explored the
effects of the compounds, alone or in combination, on
signal transduction pathways involved in apoptosis and
growth of cancer cells and show that in rhabdomyosarcoma
cells the apoptotic effect of CUR, either alone or in
combination, is independent of p53 activity. Our findings
suggest that CUR and CUR-based combinations may have
relevance for the treatment of p53-deficient cancers, which
are often unaffected by conventional chemotherapies or
radiotherapy.
2. INTRODUCTION

Malignant tumors of mesenchimal origin are in
most of the cases highly aggressive pedriatic malignancies
and although the prognosis of these tumors has improved, it
still remains poor (1-3). Current treatment modalities are
based on the combination of chemotherapy and
radiotherapy (1-3). However, the initial response to
chemotherapy is followed by chemoresistance (1-3). Thus,
discovery and development of innovative drugs can
supplement the pharmaceutical armamentarium presently
used for the treatment of rhabdomyosarcoma and
osteosarcoma (4-5). Diallyl disulfide (DADS), an organo-
sulfur compound derived from garlic (Allium sativum) has
been demonstrated to inhibit proliferation of various types
of tumors (6-9). Resveratrol (3,4’,5-trihydroxy-trans-
stilbene) (RES), a polyphenol compound isolated from grapes,
berries, plums, peanuts and pines, has several biological
properties, including antioxidant, anti-inflammatory, anticancer
and anti-aging activities (10-12). Curcumin [1,7-bis-(4-
hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] (CUR),
a polyphenol compound found in the spice turmeric, a product
of the plant Curcuma longa, has been revealed to have anti-
tumor, anti-inflammatory, antioxidant, immunomodulatory and
antimicrobial activities both in rodents and in humans (13-15).

It has been reported that DADS, RES and CUR
inhibit the growth of rhabdomyosarcoma and osteosarcoma
cell lines when employed as single drugs (16-23). In

Page 2

Resveratrol and diallyl disulfide and curcumin-induced apoptosis

499
particular, RES has been found to inhibit the growth of
osteosarcoma cells through the activation of the
ERKs/p53 signaling pathway (17) and to induce
apoptosis with minor effects on normal osteoblasts (16).
Further, it exerts a
rhabdomyosarcoma cell proliferation (18). CUR has
been demonstrated to induce cell cycle arrest and
apoptosis in human osteosarcoma cells (19-20) and to
inhibit growth of Rh1 and Rh30 rhabdomyosarcoma
cells by inhibiting phosphorylation of the mammalian
target of rapamycin (mTOR) (21). In addition, CUR has
been reported to induce apoptosis and to regulate
radiosensitivity in Ewing's sarcoma cells (22, 23).
Finally, garlic extract and Diallyl trisulfide inhibited the
growth of rat sarcoma cells and the osteosarcoma cell
line Saos-2, respectively (24, 25).

The aim of this study was to determine whether
the combination of two of these dietary phytochemicals
(DADS+RES, DADS+CUR and RES+CUR) resulted in
an enhancement of their antitumor activities on
malignant rhabdoid (SJ-RH4, RD/18) or osteosarcoma
(Saos-2) cell lines, as compared to the single compounds.
In addition we explored their effect and interaction on
signal transduction pathways involved in apoptosis and
growth of cancer cells.

3. MATERIALS AND METHODS

3.1. Reagents
DMSO, diallyl disulphide (DADS), trans-
resveratrol (RES), curcumin from Curcuma Longa
(CUR), Sulforhodamine B (SRB), staurosporine and
Hoechst 33342 were purchased from Sigma-Aldrich
(Milan, Italy). Rabbit polyclonal anti-Bax and mouse
monoclonal anti-Bcl-2 antibodies were obtained from
BD Pharmingen (BD Biosciences). Antibodies against
ERK1/2 (C-14), phospho-ERK (E-4), AKT and p53
(DO-1) were obtained from Santa Cruz Biotechnology
(CA, USA). The anti-activated caspase 3 antibody was
purchased from Cell Signaling Technology (MA, USA).
The goat anti-rabbit IgG Alexa fluor-594-conjugated
secondary antibody was from Invitrogen (Milan, Italy)
and the goat anti-mouse or -rabbit IgG peroxidase-
conjugated secondary antibodies were from Sigma-
Aldrich.

3.2. Cell lines and treatments
Malignant rhabdoid (SJ-RH4, RD/18) and
osteosarcoma (Saos-2) cell lines were obtained from
Prof. P.L. Lollini (University of Bologna, Italy) and
Prof. C. Dominici (Sapienza University, Rome, Italy)
and maintained in RPMI containing 10% fetal bovine
serum, 100 u/ml penicillin, and 100 µg/ml streptomycin
(complete medium). Cells were grown at 37°C in a
humidified incubator with an atmosphere of 5% CO2.
SJ-RH4 and RD/18 cells are of alveolar and embryonal
histotype respectively (26). For treatments, cells were
incubated for the indicated times in the presence of
DADS, RES and CUR alone or in combination of two
compounds (dose range 6-50 µM) or vehicle control
(DMSO ≤0.1%).
strong inhibition of
3.3. Sulforhodamine B (SRB) assay
Cells were seeded at 4 x 103/well in 96-well plates
and incubated at 37°C to allow cell attachment. After 24
hours, the medium was changed and the cells were treated
with DADS, RES and CUR alone or in combination or with
DMSO and incubated for 48 hours. Cells were then fixed
with cold trichloroacetic acid (final concentration 10%) for
1 hour at 4°C. After 4 washes with distilled water, the
plates were air-dried and stained for 30 min with 0.4%
(wt/vol) SRB in 1% acetic acid. After 4 washes with 1%
acetic acid to remove the unbound dye, the plates were air-
dried and cell-bound SRB was dissolved with 200 µl/well
of 10 mM unbuffered Tris base solution, pH 10. The optical
density (O.D.) of the samples was determined at 540 nm
using a spectrophotometric plate-reader. The percentage
survival of the cultures treated with the compounds or
DMSO was calculated by normalization of their O.D.
values to those of untreated control cultures (27, 28). The
experiments were performed in triplicate and repeated three
times.

3.4. Ultrastructural analysis
Ultrastructural analyses were performed on SJ-
RH4 cells untreated or treated with DADS, RES and CUR,
alone or in combination. Cells were fixed in 2.5%
glutaraldehyde in PBS pH 7.4 and the samples were
processed for transmission electron microscopy following
routine procedures (29, 30).

3.5. FACS analysis
Asynchronized log-phase growing cells (60%
confluent, about 2.5 x 105/well in 6-well plates) were
treated with DADS, RES and CUR alone or in combination
or with DMSO in complete culture medium. After 48
hours, adherent as well as suspended cells were harvested,
centrifuged at 1,500 rpm for 10 min and washed twice with
cold phosphate buffered saline (PBS) as previously
described (28, 31). Cell pellets were resuspended in 70%
ethanol for 1 hour at −20°C. Cells were then washed twice
with cold PBS, centrifuged at 1,500 rpm for 10 min,
incubated for 1 hour in the dark with propidium iodide (25
µg/ml final concentration in 0.1% citrate and 0.1% Triton
X-100) and analyzed by flow cytometry using a
FACSCalibur cytometer running CellQuest software.

3.6. In situ detection of apoptosis
For in situ detection of programmed cell death,
SJ-RH4 and Saos-2 cells were seeded at 5 × 103 cells/well
in 8-well chamber-slides and, after 24 hours, treated with
25 µM of DADS, RES and CUR alone or in combination of
two compounds, or with vehicle control. After 48 hours,
cells were fixed in 4% formaldehyde for 15 min, washed
and incubated with an anti-activated caspase-3 polyclonal
antibody for 1 hour. After additional washings the cells
were labeled with a goat anti-rabbit IgG Alexa fluor-594-
conjugated antibody for 30 min (32). After a third washing
the cells were incubated with 0.1 µg/ml Hoechst 33342 and
mounted under a coverslip with glycerol. Cells treated for
24 hours with staurosporine 1 µM were used as positive
control. The percentage of apoptotic cells was calculated by
determining the ratio between the cells positive for
activated caspase-3 and the total number of cells present in

Page 3

Resveratrol and diallyl disulfide and curcumin-induced apoptosis

500
five randomly chosen microscopic fields. Cell counts were
done in a blinded fashion.

3.7. Preparation of cell lysates and Western Blotting
About 1 x 106 cells were seeded in 100 mm tissue
culture dishes 24 hours prior to the addition of 25 µM of
compounds alone or in combination or vehicle control. For
determining Erk phosphorylation levels upon serum
stimulation, SJ-RH4 cells were treated for 32 hours with 25
µM of compounds or DMSO, starved for 16 hours in
serum-free medium containing 0.2% bovine serum albumin
in the presence of compounds or DMSO and then either left
unstimulated or stimulated for 5 minutes with 20% of
serum. At the endpoint of the experiments, the cells were
harvested, washed twice with cold PBS and lysed in RIPA
buffer (Triton X-100 1%, SDS 0.1%, NaCl 200 mM, Tris
HCl 50 mM pH 7.5, PMSF 1 mM, NaOV 1 mM). After 30
minutes at 4°C, the mixtures were centrifuged at 12,000 g
for 15 minutes and the supernatants were analyzed by
western blotting (32, 33).

For immunoblot analysis, 50 µg of cell lysates
were resolved in 10% SDS-PAGE and then transferred to
nitrocellulose membranes. Equal loading and transfer of
proteins was verified by Ponceau red staining of the filters.
After blocking, the membranes were incubated with
specific primary antibodies at the concentration of 1-2
µg/ml overnight at 4˚C. After washing, the filters were
incubated with goat anti-mouse or -rabbit IgG peroxidase-
conjugated antibodies
chemiluminescence as previously described (32-34).

3.8. Statistical analysis
Data distribution of cell survival and FACS
analyses were preliminarily verified by the Kolmogorov-
Smirnov test, and data sets were analyzed by one-way
analysis of variance (ANOVA) followed by Newman-
Keuls test. Differences were regarded as significant when p
value was less than 0.05. Differences in number of
apoptotic cells were evaluated by a two-tailed t-test.

4. RESULTS

4.1. Inhibition of malignant rhabdoid (SJ-RH4, RD/18)
and osteosarcoma (Saos-2) cell survival by DADS, RES
and CUR alone or in combination
Survival of malignant rhabdoid (SJ-RH4, RD/18)
or osteosarcoma (Saos-2) cells was evaluated by the SRB
assay after exposure to increasing doses of DADS, RES
and CUR (6 to 50 µM), alone or in combination of two
compounds (DADS+RES, DADS+CUR, RES+CUR) or
vehicle control for 48 hours. CUR was the most effective
compound inhibiting cell survival (Figure 1). The effect of
CUR was dose-dependent
significance at all doses tested as compared to vehicle
control treatments. RES significantly decreased cell
survival in all three cell lines at high doses (25-50 µM),
whereas at 12 µM it
rhabdomyosarcoma cells. On the other hand, DADS
significantly inhibited cell growth of the SJ-RH4 cell line
only and only at the highest concentration (Figure 1).
Besides, the effects obtained with equimolar combinations
and developed by
and gained statistical
was effective only in
of
RES+CUR) were not significantly different from those
obtained using the more potent compound present in each
combination (Figure 1).

4.2. Morphological features of SJ-RH4 cells after
treatment with DADS, RES and CUR alone or in
combination
Morphological features of SJ-RH4 cells after
treatment with DADS, RES and CUR, alone or in
combination at the concentration of 25 µM were examined
by transmission electron microscopy. Untreated and
DMSO-treated cells were used for comparison. Treatment
of cells with DMSO did not cause significant changes as
compared to untreated cells (not shown). DMSO-treated
SJ-RH4 cells (Figure 2, a) showed heterogeneous forms
with a predominance of stretched over round cells. The
nuclei appeared large, mainly formed of euchromatin with
low dense heterochromatin in the periphery. In the
cytoplasm numerous mitochondria in the condensed phase,
cisterns of rough endoplasmic reticulum, glycogen, rare
vacuoles, and not organized filaments were detected.
Conversely, treatment with DADS induced the presence of
numerous cytoplasmic vacuoles, surrounded by single or
double membrane, the latter being likely of autophagic
origin (Figure 2, b). In addition, some mitochondria were
swollen and the rough endoplasmic reticulum decreased.
Treatment with RES induced the presence of cracks in the
cytoplasm, while major alterations were found in the
mitochondria which appeared greatly shrunken and
condensed (Figure 2, c). Among all the treatments, CUR
was confirmed to be the most harmful to the cells. In fact,
most of the cells showed alterations related to apoptosis,
with condensation of cytoplasm, presence of vacuoles of
various sizes, intact mitochondria and nuclear pyknosis.
Rare features of necrosis were also observed (Figure 2, d).
Cells simultaneously treated with the combination
DADS+RES, appeared swollen with rarefaction of the
cytoplasm in which mitochondria appeared swollen as well
and with abnormal cristae. In addition several vacuoles
were observed (Figure 2, e). DADS+CUR and RES+CUR
combined treatments produced morphological effects
similar to those observed in the samples treated with CUR
only. In fact, apoptotic features such as cell shrinkage,
nuclear condensation and
cytoplasmic vacuoles were prevalent (Figure 2, f-g).

4.3. RES and DADS potentiate the apoptotic effect of
CUR on SJ-RH4 and Saos-2 cell lines
In order to determine the effect of the compounds
alone or in combination on apoptosis and cell cycle
distribution of malignant rhabdoid (SJ-RH4, RD/18) or
osteosarcoma (Saos-2) cells, a FACS analysis of DNA
content was performed. Figure 3 shows a representative
experiment in which the effects of increasing doses of
compounds administered alone or in combination are
compared with each other as well as with those obtained
with DMSO vehicle only. As compared to DMSO, DADS
treatment scarcely affected cell cycle in the different cell
lines, except that RD/18 cells treated with 6-50 µM DADS
accumulated in the G2/M phase (p<0.05). Treatment with
RES resulted in a dose-dependent decrease in the
two compounds (DADS+RES, DADS+CUR,
presence of numerous

Page 4

Resveratrol and diallyl disulfide and curcumin-induced apoptosis

501


Figure 1. Effect of DADS, RES and CUR alone or in combination on rhabdomyosacoma or osteosarcoma cell survival. Survival
of rhabdomyosacoma (SJ-RH4, RD/18) and osteosarcoma (Saos-2) cells was assessed by the SRB assay after 48 hours of
treatment with DMSO or DADS, RES and CUR alone or in equimolar combinations of two compounds (DADS+RES,
DADS+CUR, RES+CUR). The percentage survival of the cultures treated with compounds or DMSO as vehicle control was
calculated by normalization of their O.D. values to those of untreated control cultures. Results reported are mean ± SD values
from three experiments performed in triplicates. #: p≤0.0001; §: p≤0.001; *: p≤0.05 vs. cultures treated with DMSO.




Figure 2. Morphological features of SJ-RH4 cells after treatment with DADS, RES and CUR alone or in combination.
Ultrastructural analysis performed on SJ-RH4 cells treated for 48 hours with (a) DMSO as vehicle control (original magnification
(o.m.) x3700), (b) DADS (o.m. x6500), (c) RES (o.m. x6500), (d) CUR (o.m. x3700), (e) DADS+RES (o.m. x6500), (f)
DADS+CUR (o.m. x6500), and (g) RES+CUR (o.m. x5900), at 25 µM. The arrow in (b) indicates a double membrane vacuole
(high magnification in the square); the arrow in (c) indicates mitochondrial alterations (high magnification in the square).

percentage of cells in G0/G1 and G2/M phases (p<0.05)
and an increased percentage of cells in the S phase
(p<0.05). In addition, RES treatment induced a modest to
moderate increase in the apoptotic sub-G0 population at
high doses (Figure 3 and Table 1). On the other hand, in all
cell lines treatment with CUR resulted in a marked, dose-
dependent increase of the percentage cells in the sub-G0
phase and a decrease in the number of cells in G0/G1,

Page 5

Resveratrol and diallyl disulfide and curcumin-induced apoptosis

502

Table 1. Effects of DADS, RES and CUR alone or in combination on the percentage of rhabdomyosarcoma and osteosarcoma
cells in the sub G0 phase

SJ-RH4

mean1 p
DMS0
1,09
DADS 6.2
0,82
DADS 12.5
1,18
DADS 25
1,06
DADS 50
1,88
RES 6.2
1,04
RES 12.5
1,21
RES 25
2,46
RES 50
3,29 2
CUR 6.2
0,78
CUR 12.5
7,69 2
CUR 25
54,26 2
CUR 50
72,18 2
DADS+RES 6.2
0,68
DADS+RES 12.5
1,24
DADS+RES 25
2,36

DADS+RES 50
5,33
DADS+CUR 6.2
0,88
DADS+CUR 12.5
10,42
DADS+CUR 25
64,82 3
DADS+CUR 50
81,98 3
RES+CUR 6.2
1,19
RES+CUR 12.5
18,70 3
RES+CUR 25
71,30 3
RES+CUR 50
84,60 3
1Percentage of cells in the sub G0 phase were calculated using CellQuest software. The data are representative of three
experiments. DADS, RES and CUR were used in the range 6.2-50 µM. Statistical significance of the effects obtained with single-
compound treatments was calculated vs. those obtained with DMSO (2p<0.05 vs. DMSO); statistical significance of the effects
obtained with combined treatments was calculated vs. those obtained with the more potent compound present in each
combination (3p<0.001 vs. CUR; 4p<0.001 vs. RES)

(p<0.001), S (p<0.001) and G2/M (p<0.001) (Figure 3 and
Table 1).

As for the effects of combined treatments, the
DADS+RES combination induced a dose-dependent
decrease in the percentage of SJ-RH4 and RD/18 cells in
G0/G1 (p<0.05) and G2/M (p<0.01) phases and an increase
of those in S phase (p<0.01), similar to those obtained with
RES alone. Thus RES counteracted the increase of cells in
G2/M induced by DADS in the RD/18 cell line. In addition,
in RD/18 cells this combination treatment at the higher
dose induced an increase in the percentage of cells in sub-
G0 as compared to treatment with RES alone (p<0.001)
(Figure 3 and Table 1). In particular, the apoptotic rate
obtained with the combined treatment was 1.4 times higher
than that obtained with RES alone, i.e. the more potent
compound present in the combination. Conversely, in the
Saos-2 cell line the treatment with DADS+RES
significantly decreased the percentage of cells in G0/G1 as
compared to DADS and RES alone (p<0.05), while it did
not affect the G2/M phase. Based on these results, DADS
and RES appeared to interact in a cell-context dependent
manner.

Although the effects of DADS+CUR were on the
whole similar to those obtained with CUR alone, at high
concentrations this combination induced a significant
increase in the percentage of SJ-RH4 cells in sub-G0 as
compared to DADS and CUR alone (p<0.001) (Figure 3
and Table 1). Similarly, treatment with RES+CUR induced
in SJ-RH4 and Saos-2 a significant, dose-dependent
RD/18
mean
0,98
0,63
0,58
0,49
0,89
0,99
1,49
8,21
19,84
1,18
2,70
73,37
80,37
2,96
1,34
7,16
28,54
0,82
2,74
75,13
83,55
1,35
6,67
81,43
90,44

Saos-2
mean
1,10
0,82
1,10
1,43
1,34
1,80
1,81
3,97
6,46
1,03
1,53
17,6
66,3
1,60
3,10
10,42
14,48
1,10
1,57
25,40
66,80
2,29
6,72
39,70
82,23

p







2
2


2
2



4








p







2
2


2
2









3
3
3
increase in the percentage of apoptotic, sub-G0 cells as
compared to either compound administered alone
(p<0.001), accompanied by a decrease in G2/M, G0/G1 and
S phases (p<0.001) (Figure 3 and Table 1). From the
comparison of the apoptotic rates obtained with the
RES+CUR combination and those obtained with CUR
alone, it emerged that in SJ-RH4 cells the combined
treatment allowed to reduce the dose of CUR required to
achieve an apoptotic rate of 30%, 50% or 70% by 1.2, 1.2
and 1.9 times, respectively. Similarly, in Saos-2 cells the
dose of CUR required to achieve an apoptotic rate of 30%
or 50% could be reduced 1.5 and 1.3 times, respectively,
through the combination of CUR with RES.

It is of note that, according to the results of the
SRB assays, the reduction of cell survival obtained with
DADS+CUR and RES+CUR was not significantly
different from that obtained using CUR alone, the more
potent compound present in the combinations, while the
results of FACS analysis indicate that CUR-induced
apoptosis was potentiated by the combination with DADS
in SJ-RH4 cells and by the combination with RES in SJ-
RH4 and Saos-2 cells. However, this apparent discrepancy
is reconciled when considering that SRB has been
demonstrated to stain live as well as recently died cells
(35). Still, to further corroborate the data on apoptotic cell
death obtained by FACS analysis, SJ-RH4 and Saos-2 cells
were labeled with an anti-activated caspase-3 polyclonal
antibody after treatment with DADS, RES and CUR alone
or in combination at 25 µM for 48 hours. Cells treated with
DMSO vehicle were used as negative control, while cells

Page 6

Resveratrol and diallyl disulfide and curcumin-induced apoptosis

503

Figure 3. Effects of DADS, RES and CUR alone or in combination on apoptosis and cell cycle distribution of rhabdomyosacoma
and osteosarcoma cells. FACS analysis of DNA content performed on SJ-RH4, RD/18 and Saos-2 cells treated for 48 hours with
DMSO or DADS, RES and CUR alone or in combination. In all cell lines CUR induced an increase of the percentage of cells in
sub G0 (M1) and a decrease of the percentage of cells in G0/G1 (M2), S (M3) and G2/M (M4) in a dose-dependent manner.
DADS increased the sub G0 peak induced by CUR in SJ-RH4 cells when the compounds were used at the highest concentration.
RES increased the sub G0 peaks induced by CUR in SJ-RH4 and Saos-2 cells in a dose-dependent manner.

Page 7

Resveratrol and diallyl disulfide and curcumin-induced apoptosis

504




Figure 4. In situ detection of apoptosis. Induction of apoptosis in SJ-RH4 cells by DADS, RES and CUR alone or in
combination, DMSO as vehicle control and staurosporine as positive control, as assessed by immunolabeling with an anti-
activated-caspase 3 polyclonal antibody. DADS, RES and CUR alone or in combination were used at 25 µM for 48 hours;
staurosporine was used at 1 µM for 24 hours. Original magnification x200. Nuclei were counterstained with Hoechst.

treated with staurosporine at 1 µM for 24 hours were used
as positive control. Representative pictures of immunolabeled
SJ-RH4 cells are reported in Figure 4. According to activated
caspase 3 positivity, the treatment with staurosporine resulted
in apoptotic rates of about 95-96% in either cell line, whereas
treatment with DMSO, DADS and RES had no relevant effect
on the induction of apoptosis in SJ-RH4 and Saos-2. On the
other hand, CUR induced a significant increase of apoptosis in
both cell lines. Furthermore, the apoptotic rate of SJ-RH4 cells
treated with DADS+CUR was significantly higher than that
obtained with CUR alone (46.4% vs. 36.4%, p<0.001).
Similarly, the apoptotic rates of SJ-RH4 and Saos-2 cells
treated with the RES+CUR combination were higher than
those observed after treatment with CUR alone (50.8% vs.
36.4% for SJ-RH4 cells, p<0.001; 30% vs. 18.4% for Saos-2
cells, p<0.001), consistent with the results obtained by FACS
analysis.

4.4. Effect of DADS, RES and CUR alone or in
combination on apoptosis and pro-survival signaling
proteins
The expression levels of apoptosis and pro-
survival signaling proteins were investigated by western
blotting in SJ-RH4 and RD/18 cells treated for 48 hours with
the three compounds alone or in combination at 25 µM. A
representative experiment is illustrated in Figure 5, upper
panels. CUR either alone or in combination with DADS or
RES induced in both cell lines the appearance of the 18 kDa
Bax isoform (p18 Bax), which is known as a more potent
inducer of apoptotic cell death than the full-length p21 Bax
(36). The appearance of p18 Bax was concomitant with an
overall decrease of Bcl-2 expression in both cell lines.
Conversely, p18 Bax was not observed nor the ratio between
Bax and Bcl-2 significantly modified after treatment with
DADS and RES alone or in combination. In addition, DADS
and RES alone or in combination did not change or slightly
decreased the expression of the pro-survival signaling
protein AKT. Conversely, CUR and CUR combinations
strongly reduced AKT expression levels. Collectively,
these results are in agreement with the reported apoptotic
activity of the compounds at the employed concentration.

Next, the expression of p53 was analyzed in order to
determine whether apoptosis induced by CUR in SJ-RH4 and
RD/18 cells was p53-dependent. Unlike RD/18 cells, where
p53 expression was decreased by CUR treatment, SJ-RH4
cells did not express p53, indicating that CUR can trigger p53-
independent apoptosis in rhabdomyosarcoma cells.

Finally, it was investigated the effect of the
compounds on Erk phosphorylation (Figure 5, upper panels).
In both SJ-RH4 and RD/18 cells DADS had no notable effects
on phospho-Erk levels, RES increased Erk phosphorylation
and a further increase was observed after treatment with
DADS+RES. On the other hand, while CUR reduced
phospho-Erk levels in SJ-RH4 cells, the same compound
increased phospho-Erk in the RD/18 cell line. Of note, when
CUR was administered with RES it was able to counteract the
increased Erk phosphorylation induced by RES in SJ-RH4
cells. In order to substantiate the observed cell context-
dependent modulation of Erk signaling, both cell lines were
treated for 32 hours, starved for 16 hours in serum-free
medium and then stimulated for 5 minutes with 20% of serum
in the continuous presence of the three compounds alone or in
combination (Figure 5, lower panels). In SJ-RH4 cells all
compounds inhibited serum-mediated Erk phosphorylation as
compared to DMSO, the maximal inhibitory activity being
obtained with CUR either alone or in combination with DADS
or RES. Conversely, in RD/18 cells CUR, either alone or in
combination with DADS or RES, increased Erk
posphorylation upon serum stimulation as compared to
DMSO vehicle treatment, while no major effects were
observed with DADS and RES.

Page 8

Resveratrol and diallyl disulfide and curcumin-induced apoptosis

505


Figure 5. Effect of DADS, RES and CUR alone or in combination on apoptosis and pro-survival signaling proteins. Upper
panels: assessment of Bax, Bcl-2, AKT, p53, phosphorylated Erk (p-Erk) and Erk levels by western blotting in SJ-RH4 and
RD/18 cells treated for 48 hours with DADS, RES and CUR alone or in combination at 25 µM or with DMSO as vehicle.
Lower panels: assessment of p-Erk levels upon serum stimulation of SJ-RH4 cells treated with DMSO or with the three
compounds alone or in combination: after 32 hours of treatment, the cells were starved for 16 hours and then stimulated for 5
minutes with 20% of serum in the continuous presence of compounds or DMSO. Shown are representative experiments.

5. DISCUSSION

Chemoprevention involves the use of natural or synthetic
substances to prevent certain diseases such as cancer.
Indeed, chemoprevention has been proven to be relatively
successful in preventing cancer (37-42) and several
chemopreventive agents are now being tested for their anti-
cancer therapeutic activity in tumor bearing hosts (37, 41-
43). Therapeutic approaches based on drug combinations
aim at increasing clinical responses while lowering toxicity
and the incidence of drug resistance. The advantage of
combining multiple agents stems from the fact that each
agent can have a single target or mechanism of action or
different agents may share the same target or mechanism of
action against cancer cells (44, 45). Therefore the
combination treatment could either increase the number of
targets and/or mechanisms of action or potentiate the
effects on the same target, thus lowering the drug
concentrations needed to exert a biological effect against
cancer cells.

CUR, RES and DADS have been demonstrated to
have anti-tumor activity both in vitro and in vivo using
animal models (6-25). Furthermore, some clinical trials
have highlighted the potential role of CUR as an
antineoplastic agent (37, 46). However, pharmacokinetic
studies have shown that this compound is poorly absorbed
and rapidly eliminated from the human body (37, 46). In
the first report of the joint FAO/WHO expert committee on
food additives it was established that an acceptable daily
intake (ADI) for CUR is of 0–0.1 mg/kg body weight (47).
Thus it may be beneficial to find other chemopreventive
agents that can potentiate the anti-cancer activity of CUR.

In this study we provide evidence that CUR
induces apoptosis of rhabdomyosarcoma and osteosarcoma

Page 9

Resveratrol and diallyl disulfide and curcumin-induced apoptosis

506
cell lines and that the combination of RES or DADS plus
CUR potentiates the apoptotic effect of CUR. In particular,
in SJ-RH4 cells the RES+CUR combined treatment
allowed to reduce the dose of CUR required to achieve an
apoptotic rate of 30%, 50% or 70% by 1.2, 1.2 and 1.9
times, respectively. Similarly, in Saos-2 cells the dose of
CUR required to achieve an apoptotic rate of 30% or 50%
could be reduced 1.5 and 1.3 times, respectively, through
the combination of CUR with RES.

We also demonstrate that CUR induced the
expression of p18 Bax in both SJ-RH4 and RD/18
rhabdomyosarcoma cell lines. This truncated isoform of
Bax is known as a more potent inducer of apoptotic cell
death than the full-lenght p21 Bax (36). Conversely, CUR
down-regulated the expression of the anti-apoptotic protein
Bcl-2 and strongly reduced the expression of the pro-
survival kinase AKT. Indeed, AKT promotes cell survival
and resistance to apoptosis by sequestering different protein
targets, including the FOXO family of forkhead
transcription factor and the pro-apoptotic protein Bad, as
well as by activating the pro-survival transcription factor
NF-kB (48). Most notably, we showed that CUR alone or
in combination induced apoptosis of rhabdomyosarcoma
cells independently of p53 activity. This finding indicates
that CUR may have relevance for the treatment of p53-
deficient cancers, which are often unaffected by
conventional chemotherapies or irradiation therapy.

Interestingly, the effects of CUR on the
modulation of basal and serum-induced Erk activity
appeared different in SJ-RH4 and RD/18 cells. Indeed,
while CUR reduced phospho-Erk levels in both
unstimulated and serum-stimulated SJ-RH4 cells, the
opposite effect was observed in the RD/18 cell line. In this
respect it has been demonstrated that, depending on the
specific context, Erk activation can either protect or
contribute to drug-induced cell death (49, 50). Indeed,
despite the opposite and cell type-specific modulation of
Erk activity induced by CUR in SJ-RH4 (alveolar
histotype) and RD/18
rhabdomyosarcoma cells, this compound was able to
produce a marked increase of the apoptotic rate of both cell
lines.

In conclusion, our study provides evidence that
the treatment of rhabdomyosarcoma or osteosarcoma cells
with combinations of CUR plus RES or DADS can be more
effective in inducing apoptosis than the treatment with
CUR as a single compound. Still, additional studies
performed both in vitro and in vivo will be needed to fully
define the therapeutic potential of these compounds.

6. ACKNOWLEDGEMENTS

The authors thank Prof. P.L. Lollini (University
of Bologna, Italy) and Prof. C. Dominici (Sapienza
University, Rome, Italy) for providing SJ-RH4, RD/18 and
Saos-2 cell lines and Barbara Bulgarini for editorial
assistance in the preparation of the manuscript. This study
was supported by grant MIUR-PRIN 2007-2009 and RSA-
University of Rome, Tor Vergata.
(embryonal histotype)
7. REFERENCES

1. D. Walterhouse and A. Watson: Optimal management
strategies for rhabdomyosarcoma in children. Paediatr
Drugs 9, 391-400 (2007)

2. R. S. Benjamin and S. R. Patel: Pediatric and adult
osteosarcoma: comparisons and contrasts in presentation
and therapy. Cancer Treat Res 152, 355-363 (2009)

3. H. Lünenbürger, C. Lanvers-Kaminsky, B. Lechtape
and M. C. Frühwald: Systematic analysis of the
antiproliferative effects of novel and standard anticancer
agents in rhabdoid tumor cell lines. Anticancer Drugs
21, 514-522 (2010)

4. M. Wachtel and B. W. Schäfer: Targets for cancer
therapy in childhood sarcomas. Cancer Treat Rev 36,
318-327 (2010)

5. S. Y. Kim and L.J. Helman: Strategies to explore new
approaches in the investigation and treatment of
osteosarcoma. Cancer Treat Res 152, 517-528 (2009)

6. F. Khanum, K. R. Anilakumar and K. R.
Viswanathan: Anticarcinogenic properties of garlic: a
review. Crit Rev Food Sci Nutr 44, 479-488 (2004)

7. A. Herman-Antosiewicz and S. V. Singh: Signal
transduction pathways leading to cell cycle arrest and
apoptosis induction in cancer cells by Allium vegetable-
derived organosulfur compounds: a review. Mutat Res 555,
121-131 (2004)

8. Y. Shukla and N. Kalra: Cancer chemoprevention with
garlic and its constituents. Cancer Lett 247, 167-181 (2007)

9. A. A. Powolny and S. V. Singh: Multitargeted prevention
and therapy of cancer by diallyl trisulfide and related Allium
vegetable-derived organosulfur compounds. Cancer Lett 269,
305-314 (2008)

10. A. Bishayee: Cancer prevention and treatment with
resveratrol: from rodent studies to clinical trials. Cancer Prev
Res (Phila) 2, 409-418 (2009)

11. S. K. Goswami and D. K. Das: Resveratrol and
chemoprevention. Cancer Lett 284, 1-6 (2009)

12. J. K. Kundu and Y. J. Surh: Cancer chemopreventive and
therapeutic potential of
perspectives. Cancer Lett 269, 243-261 (2008)

13. G. Bar-Sela, R. Epelbaum and M. Schaffer: Curcumin
as an anti-cancer agent: review of the gap between basic
and clinical applications. Curr Med Chem 17, 190-197
(2010)

14. J. Epstein, I. R. Sanderson and T. T. Macdonald:
Curcumin as a therapeutic agent: the evidence from in
vitro, animal and human studies. Br J Nutr 103, 1545-1557
(2010)
resveratrol: mechanistic

Page 10

Resveratrol and diallyl disulfide and curcumin-induced apoptosis

507
15. P. Anand, C. Sundaram, S. Jhurani, A. B.
Kunnumakkara and B. B. Aggarwal: Curcumin and cancer:
an "old-age" disease with an "age-old" solution. Cancer
Lett 267, 133-164 (2008)

16. Y. Li, C. M. Bäckesjö, L. A. Haldosén and U.
Lindgren: Resveratrol inhibits proliferation and promotes
apoptosis of osteosarcoma cells. Eur J Pharmacol 609, 13-
18 (2009)

17. M. Alkhalaf and S. Jaffal: Potent antiproliferative
effects of resveratrol on human osteosarcoma SJSA1 cells:
novel cellular mechanisms involving the ERKs/p53
cascade. Free Radic Biol Med 41, 318-325 (2006)

18. A. W. Chow, G. Murillo, C. Yu, R. B. van Breemen, A. W.
Boddie, J. M. Pezzuto, T. K. Das Gupta and R. G. Mehta:
Resveratrol inhibits rhabdomyosarcoma cell proliferation. Eur
J Cancer Prev 14, 351-356 (2005)

19. D. S. Lee, M. K. Lee and J. H. Kim: Curcumin induces cell
cycle arrest and apoptosis in human osteosarcoma (HOS) cells.
Anticancer Res 29, 5039-5044 (2009)

20. D. K. Walters, R. Muff, B. Langsam, W. Born and B.
Fuchs: Cytotoxic effects of curcumin on osteosarcoma cell
lines. Invest New Drugs 26, 289-297 (2008)

21. C. S. Beevers, F. Li, L. Liu and S. Huang: Curcumin
inhibits the mammalian target of rapamycin-mediated
signaling pathways in cancer cells. Int J Cancer 119, 757-764
(2006)

22. M. Singh, A. Pandey, C. A. Karikari, G. Singh and D.
Rakheja: Cell cycle inhibition and apoptosis induced by
curcumin in Ewing sarcoma cell line SK-NEP-1. Med Oncol
27, 1096-1101 (2010)

23. J. Veeraraghavan, M. Natarajan, T. S. Herman and N.
Aravindan: Curcumin-altered p53-response genes regulate
radiosensitivity in p53-mutant Ewing's sarcoma cells.
Anticancer Res 30, 4007-4015 (2010)

24. X. Hu, B. N. Cao, G. Hu, J. He, D. Q. Yang and Y. S.
Wan: Attenuation of cell migration and induction of cell death
by aged garlic extract in rat sarcoma cells. Int J Mol Med 9,
641-643 (2002)

25. Y. K. Zhang, X. H. Zhang, J. M. Li, S. Sun de, Q. Yang
and D. M. Diao: A proteomic study on a human osteosarcoma
cell line Saos-2 treated with diallyl trisulfide. Anticancer Drugs
20, 702-712 (2009)

26. S. Croci, L. Landuzzi, A. Astolfi, G. Nicoletti, A. Rosolen,
F. Sartori, M. Y. Follo, N. Oliver, C. De Giovanni, P. Nanni
and P. L. Lollini: Inhibition of connective tissue growth factor
(CTGF/CCN2) expression decreases the survival and
myogenic differentiation of human rhabdomyosarcoma cells.
Cancer Res 64, 1730-1736 (2004)

27. L. Masuelli, C. Focaccetti, V. Cereda, F. Lista, D.
Vitolo, P. Trono, P. Gallo, A. Amici, P. Monaci, M. Mattei,
M. Modesti, G. Forni, M. H. Kraus, R. Muraro, A. Modesti
and R. Bei: Gene-specific inhibition of breast carcinoma in
BALB-neuT mice by active immunization with rat Neu or
human ErbB receptors. Int J Oncol 30, 381-392 (2007)

28. L. Masuelli, L. Marzocchella, A. Quaranta, C.
Palumbo, G. Pompa, V. Izzi, A. Canini, A. Modesti, F.
Galvano and R. Bei: Apigenin induces apoptosis and
impairs head and neck carcinomas EGFR/ErbB2 signaling.
Front Biosci 16, 1060-1068 (2011)

29. L. Masuelli, P. Trono, L. Marzocchella, M. A. Mrozek,
C. Palumbo, M. Minieri, F. Carotenuto, R. Fiaccavento, A.
Nardi, F. Galvano, P. Di Nardo, A. Modesti and R. Bei:
Intercalated disk remodeling in delta-sarcoglycan-deficient
hamsters fed with an alpha-linolenic acid-enriched diet. Int
J Mol Med 21, 41-48 (2008)

30. G. Tumino, L. Masuelli, R. Bei, L. Simonelli, A.
Santoro and S. Francipane: Topical treatment of chronic
venous ulcers with sucralfate: a placebo controlled
randomized study. Int J Mol Med 22, 17-23 (2008)

31. R. Bei, L. Masuelli, P. Trono, P. L. Orvietani, S. Losito,
L. Marzocchella, D. Vitolo, L. Albonici, M. A. Mrozek, E.
Di Gennaro, F. Lista, G. Faggioni, F. Ionna, L. Binaglia, V.
Manzari, A. Budillon and A. Modesti: The ribosomal P0
protein induces a spontaneous immune response in patients
with head and neck advanced stage carcinoma that is not
dependent on its overexpression in carcinomas. Int J Oncol
31, 1301-1308 (2007)

32. L. Masuelli, L. Marzocchella, C. Focaccetti, F. Lista, A.
Nardi, A. Scardino, M. Mattei, M. Turriziani, M. Modesti,
G. Forni, J. Schlom, A. Modesti and R. Bei: Local delivery
of recombinant vaccinia virus encoding for neu counteracts
growth of mammary tumors more efficiently than systemic
delivery in neu transgenic mice. Cancer Immunol
Immunother 59, 1247-1258 (2010)

33. R. Bei, L. Masuelli, E. Moriconi, V. Visco, A. Moretti,
M. H. Kraus and R. Muraro: Immune responses to all ErbB
family receptors detectable in serum of cancer patients.
Oncogene 18, 1267-1275 (1999)

34. R. Bei, A. Budillon, L. Masuelli, V. Cereda, D. Vitolo,
E. Di Gennaro, V. Ripavecchia, C. Palumbo, F. Ionna, S.
Losito, A. Modesti, M. H. Kraus and R. Muraro: Frequent
overexpression of multiple ErbB receptors by head and
neck squamous cell carcinoma contrasts with rare antibody
immunity in patients. J Pathol 204, 317-325 (2004)

35. Y. P. Keepers, P. E. Pizao, G. J. Peters, J. van Ark-Otte,
B. Winograd and H. M. Pinedo: Comparison of the
sulforhodamine B protein and tetrazolium (MTT) assays
for in vitro chemosensitivity testing. Eur J Cancer 27, 897-
900 (1991)

36. H. Toyota, N. Yanase, T. Yoshimoto, M. Moriyama, T.
Sudo and J. Mizuguchi: Calpain-induced Bax-cleavage
product is a more potent inducer of apoptotic cell death
than wild-type Bax. Cancer Lett 189, 221-230 (2003)

Page 11

Resveratrol and diallyl disulfide and curcumin-induced apoptosis

508
37. A. Shehzad, F. Wahid and Y. S. Lee: Curcumin in
cancer chemoprevention:
pharmacokinetics, bioavailability, and clinical trials. Arch
Pharm (Weinheim) 343, 489-499 (2010)

38. R. G. Mehta, G. Murillo, R. Naithani and X. Peng:
Cancer chemoprevention by natural products: how far have
we come? Pharm Res 27, 950-961 (2010)

39. P. H. Brown: Chemoprevention clinical trials: it is time
to turn success into progress. Cancer Epidemiol
Biomarkers Prev 16, 1531-1532 (2007)

40. E. N. Scott, A. J. Gescher, W. P. Steward and K.
Brown: Development of
chemopreventive agents: biomarkers and choice of dose for
early clinical trials. Cancer Prev Res (Phila) 2, 525-530
(2009)

41. J. J. Johnson and H. Mukhtar: Curcumin for
chemoprevention of colon cancer. Cancer Lett 255, 170-
181 (2007)

42. S. C. Thomasset, D. P. Berry, G. Garcea, T. Marczylo,
W. P. Steward and A. J. Gescher: Dietary polyphenolic
phytochemicals--promising cancer chemopreventive agents
in humans? A review of their clinical properties. Int J
Cancer 120, 451-458 (2007)

43. K. R. Patel, V. A. Brown, D. J. Jones, R. G. Britton, D.
Hemingway, A. S. Miller, K. P. West, T. D. Booth, M.
Perloff, J. A. Crowell, D. E. Brenner, W. P. Steward, A. J.
Gescher and K. Brown: Clinical pharmacology of
resveratrol and its metabolites in colorectal cancer patients.
Cancer Res 70, 7392-7399 (2010)

44. A. R. Amin, D. Wang, H. Zhang, S. Peng, H. J. Shin, J.
C. Brandes, M. Tighiouart, F. R. Khuri, Z. G. Chen and D.
M. Shin: Enhanced anti-tumor activity by the combination
of the natural compounds (-)-epigallocatechin-3-gallate and
luteolin: potential role of p53. J Biol Chem 285, 34557-
34565 (2010)

45. H. Jiang, X. Shang, H. Wu, G. Huang, Y. Wang, S. Al-
Holou, S. C. Gautam and M. Chopp: Combination
treatment with resveratrol and sulforaphane induces
apoptosis in human U251 glioma cells. Neurochem Res 35,
152-161 (2010)

46. A. S. Strimpakos and R. A. Sharma: Curcumin:
preventive and therapeutic properties in laboratory studies
and clinical trials. Antioxid Redox Signal 10, 511-545
(2008)

47. Evaluation of certain food additives. Fifty-first report of
the Joint/WHO expert Committee on food additives. World
Health Organ Tech Rep Ser 891( i-viii), 1-168 (2000)

48. P. O-charoenrat, P. H. Rhys-Evans, H. Modjtahedi and
S. A. Eccles: The role of c-erbB receptors and ligands in
head and neck squamous cell carcinoma. Oral Oncol 38,
627-640 (2002)
molecular targets,
dietary phytochemical
49. S. Cagnol and J. C. Chambard: ERK and cell death:
mechanisms of ERK-induced
autophagy and senescence. FEBS J 277, 2-21 (2010)

50. J. A. McCubrey, L. S. Steelman, W. H. Chappell, S. L.
Abrams, E. W. Wong, F. Chang, B. Lehmann, D. M.
Terrian, M. Milella, A. Tafuri, F. Stivala, M. Libra, J.
Basecke, C. Evangelisti, A. M. Martelli and R. A.
Franklin: Roles of the Raf/MEK/ERK pathway in cell
growth, malignant transformation and drug resistance.
Biochim Biophys Acta 1773, 1263-1284 (2007)

Key Words: Curcumin, Combined Treatments, Sarcoma,
Apoptosis

Send correspondence to: Roberto Bei, Department of
Experimental Medicine and Biochemical Sciences,
University of Rome, Tor Vergata, Via Montpellier 1, 00133
Rome, Tel: 39-06-72596514, Fax: 39-06-72596506, E-
mail: bei@med.uniroma2.it

http://www.bioscience.org/current/vol17.htm





cell death-apoptosis,