Cancer Letters 253 (2007) 124–130

www.elsevier.com/locate/canlet

Resveratrol sensitization of DU145 prostate cancer cells

to ionizing radiation is associated to ceramide increase
Francesca Scarlatti a, Giusy Sala a, Clara Ricci a, Claudio Maioli b,
Franco Milani b, Marco Minella c, Marco Botturi d, Riccardo Ghidoni a,*

a
Laboratory of Biochemistry & Mol Biology, San Paolo Medical School, University of Milan, Italy
b
Institute of Radiological Sciences, University of Milan, Italy
c
Servizio di Fisica Sanitaria, Niguarda Hospital, Milan, Italy
d
Radiotherapy Service, Niguarda Hospital, Milan, Italy

Received 29 November 2006; received in revised form 18 January 2007; accepted 19 January 2007

Abstract

Radiotherapy is an established therapeutic modality for prostate cancer. Since it is well known that radiotherapy is lim-
ited due to its severe toxicity towards normal cells at high dose and minimal effect at low dose, the search for biological
compounds that increase the sensitivity of tumors cells to radiation may improve the efficacy of therapy. Resveratrol, a
natural antioxidant, was shown to inhibit carcinogenesis in animal models, and to block the process of tumor initiation
and progression. The purpose of this study was to examine whether or not resveratrol can sensitize DU145, an andro-
gen-independent human prostate cancer cell line, to ionizing radiation. We report here that DU145 cells are resistant to
ionizing radiation-induced cell death, but pretreatment with resveratrol significantly enhances cell death. Resveratrol acts
synergistically with ionizing radiation to inhibit cell survival in vitro. Resveratrol also potentiates ionizing radiation-
induced ceramide accumulation, by promoting its de novo biosynthesis. This confirms ceramide as an effective mediator
of the anticancer potential induced by resveratrol.
Ó 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Radioresistance; Sphingolipid signaling; Synergism

1. Introduction impairs the efficacy of tumor radiotherapy. Prostate

Ionizing radiation is used as a primary treatment
for all stages of locally confined prostate cancer and
is frequently used as a treatment for the palliation
of distant disease [1]. Radioresistance markedly
*
Corresponding author. Tel.: +39 0250323250; fax: +39
0250323245.
E-mail address: riccardo.ghidoni@unimi.it (R. Ghidoni).
cancer is regarded as relatively resistant to radia-
tions [2,3]. Consistent with this notion, androgen-
independent DU145 human prostate cancer cells
manifest resistance to radiation-induced apoptotic
death [4–6].
Resveratrol (3,5,4 0 -trans-trihydroxystilbene), a
natural product from grapes and present in red
wine, is known to affect a broad range of intracellu-
lar mediators involved in the initiation, promotion

0304-3835/$ - see front matter Ó 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.canlet.2007.01.014
 F. Scarlatti et al. / Cancer Letters 253 (2007) 124–130
and progression of cancer [7–9]. Exposure of
DU145 cells to resveratrol resulted in a dose-depen-
dent growth inhibition [10] and apoptosis [11–13]
caused by down-regulation of cyclin E and cyclin-
dependent kinase 2 kinase activities [14], and inhibi-
tion of DNA synthesis [15].
In some forms of radiation-induced cell death,
primary lesions to DNA trigger the apoptotic
response [16,17]. Nevertheless exposure to ionizing
radiation leads to damage of several cellular targets,
but how signals from different targets are integrated
to determine the cell fate is not clear. Ceramide, has
been proposed as a mediator of apoptosis and the
cellular response to stress [18], including exposure
to ionizing radiation [19–26].
We have previously demonstrated that resvera-
trol has a ceramide-mediated antiproliferative and
proapoptotic effect in highly metastatic breast [27]
and prostate cancer cells [28]. We reported that
apoptotic cell death was dependent upon raised cel-
lular ceramide levels caused by its increased de novo
synthesis. For this reason, we hypothesize that res-
veratrol reverses radioresistance of DU145 cells by
increasing the production of de novo ceramide
thereby causing cell death. Thus, the purpose of this
study was to investigate whether or not resveratrol
can potentiate the response of DU145 prostate can-
cer cells to radiation and whether or not this poten-
tiation is associated with an increase in cellular
ceramide levels.
2. Materials and methods
2.1. Reagents
Resveratrol from Cayman Chemical Company, Ann
Arbor, Michigan, was dissolved in ethanol before use.
Trypan blue and myriocin were from Sigma–Aldrich, St.
Louis, Missouri, reference lipids from Avanti Polar Lip-
ids, Alabaster, AL. [c32-P]ATP (3 Ci/lmol) was from Per-
kin–Elmer Life and Analytical Sciences, Wellesley, MA;
cell culture media and fetal calf serum were from Euro-
Clone Life Science Division, Milan, Italy; penicillin and
streptomycin were from Invitrogen, Carlsbad, California;
Giemsa was from Merck S.p.A. Darmstadt, Germany;
diacylglycerol kinase (2.69 U/mg protein) was from Cal-
biochem, San Diego, CA.
2.2. Cell lines and treatment
The human prostate cancer cell line DU145 from
American Type Culture Collection (ATCC), Manassas,
VA was maintained at 37 °C in 5% CO2 in Dulbecco’s
125
modified Eagle’s medium, supplemented with 5% fetal
bovine serum and 100 ng/ml each of penicillin and
streptomycin.
DU145 cells were seeded in 24-well tissue culture at
104 cells/well in triplicate and allowed to adhere for
24 h. Then cells were treated with increasing doses of res-
veratrol or 0.05% of ethanol (as vehicle) for 72 h. The cul-
ture medium was replaced with fresh medium and cells
were irradiated as described below. Following ionizing
radiation, cells were returned to the incubator overnight
and cell viability and ceramide levels were evaluated.
2.3. Irradiation protocol
Cells were irradiated (0.5–2.0 Gy/day) for three con-
secutive days with a Pantak Therapax DXT 300RX
machine operating at 300 kV, 10 mA, 3.6 mm Cu half-
value-layer (HVL) and at dose rate of 0.63 Gy/min.
Dosimetry was performed on a regular basis with a
0.3 cm3 ionization chamber (PTW 23332), which was con-
nected to an electrometer (PTW Unidos). The chamber
was placed in parallel to the irradiated cell flasks. Dose
homogeneity was evaluated as being within 2%. Control
cultures were not irradiated but otherwise treated like
the irradiated cultures.
2.4. Cell viability assay
Cell viability was determined using a trypan blue
exclusion test after the final dose of radiation [29]. Cells
were harvested using trypsin, stained with 0.4% trypan
blue and trypan blue-negative cells were counted using a
phase contrast microscope.
2.5. Clonogenicity assay
Cell survival was determined by clonogenic assay after
the final radiation dose [29]. Cells were trypsinized under
sterile conditions and plated into 6-well tissue culture
plates at 103 cells/well in triplicate for each condition.
Cells were allowed to grow for an additional 7–10 days
and then were fixed with absolute methanol, air-dried
for 2 h, stained with Giemsa (1:10 in water by vol), and
counted by Gel-Doc Quantity-One software, Bio-Rad
Laboratories, Hercules, CA. Groups of 50 or more cells
were counted as colonies.
2.6. Analysis of the multiple drug effects
The combination effects of resveratrol and radiation
were analyzed according to the computerized method of
Chou and Talalay [30] using CalcuSyn software (Biosoft,
Inc., Cambridge, United Kingdom). This analysis of drug
interactions is based on the median effect principle (31).
126 F. Scarlatti et al. / Cancer Letters 253 (2007) 124–130
The analysis involves plotting dose–effect curves for each
agent and for multiple diluted, fixed ratio combinations of
agents using the median effect equation:
fa =fu ¼ ðD=Dm Þm
where D is the dose, Dm is the dose required to reach a
50% effect (e.g. 50% inhibition of cell growth), fa is the
fraction affected by D (e.g. 0.9 if cell growth is inhibited
by 90%), fu is the unaffected fraction (1  fa), and m is
the coefficient of sigmoidicity of the dose–effect curve.
2.7. Endogenous ceramide assay
Cells were harvested in methanol and lipids were
extracted according to Bligh and Dyer [31]. Total endog-
enous ceramide levels were measured using the diacylglyc-
erol kinase assay as described previously [32]. Briefly,
30 nmol of extracted lipids, determined as inorganic phos-
phate [33] were incubated at room temperature for 45 min
in the presence of 20 ll b-octylglucoside/ dioleoylphos-
phatidylglycerol, 4/1, by vol mixed micelles, 2 mM dithio-
threitol, 6 lg of diacylglycerol kinase containing
membranes, 1 mM ATP and 1.3 lCi of [32P]ATP in a final
volume of 100 ll. At the end of reaction, lipids were
extracted and separated by TLC, with chloroform/ace-
tone/methanol/acetic acid/water (10/4/3/2/1, by vol).
Radioactive ceramide phosphate spots were visualized
by autoradiography, scraped and counted in a scintillator
counter. Ceramide levels were compared to the level of
total phospholipids.
2.8. Statistical analysis
Data for cell viability, clonogenicity assays, and cera-
mide are presented as means of three experiments ± SD.
Differences between groups were analyzed using the Stu-
dent’s t-test and a value of p < 0.05 was considered
significant.
3. Results
3.1. Resveratrol acts synergistically with ionizing radiations
to induce cell death in DU145 cells
To investigate whether DU145 cells are resistant to
ionizing radiation treatment we tested cell survival and
cell death in response to increasing doses of radiation.
The treatment with multifractionated radiation was
unable to markedly reduce the clonogenic activity of
DU145 cells assessed by the colony formation assay.
The percent of colonies remaining with ionizing radia-
tion was only 20% less compared to untreated control
cells (Fig. 1, upper left panel). After 3 days exposure
of DU145 cells to multifractionated radiation, cell via-
bility was affected significantly only at the maximal dose
(2 Gy per day) (Fig. 1, upper right panel). These results
confirm the radioresistance of DU145 cells. To evaluate
whether or not DU145 cells are sensitive to the natural
chemotherapeutic resveratrol we tested cell survival and
cell death after treatment with increasing doses of this
agent. Exposure of DU145 cells to resveratrol for 72 h
affected cell survival in a dose-dependent manner
(Fig. 1, middle left panel) and induced a striking
increase in cell death even at a concentration of 2 lM
(Fig. 1, middle right panel). The IC50 values of ionizing
radiation and resveratrol in DU145 cells were estimated
to be 2.17 Gy and 2.15 lM, respectively (Table 1). To
investigate whether resveratrol sensitized the response
of DU145 cells to radiation, we evaluated the cell death
and clonogenic survival after pretreatment of cells with
resveratrol and subsequent treatment with ionizing radi-
ation. The combined treatment with resveratrol and
radiation dramatically reduced cell survival and number
of viable cells compared to ionizing radiation treatment
alone (Fig. 1, lower panel). When the resveratrol and
radiation were used in combination, the IC50 value for
ionizing radiation was reduced to 1.0 Gy (Table 1).
These data indicate that pretreatment with resveratrol
results in greater susceptibility of DU145 cells to ioniz-
ing radiation-induced cytotoxicity. In order to quantita-
tively evaluate the effect of the combination of
resveratrol with ionizing radiation, the dose reduction
index (DRI) and combination index (CI) were calcu-
lated from the data in Table 1 using a Calcusyn soft-
ware programme at 50%, 60%, 75%, 90%, and 95%
inhibition levels of cell viability [34,35]. DRI values
indicate that the synergic combination caused 2.2–7.8-
fold reduction of the ionizing radiation dose and 4.0–
40.1-fold reduction of resveratrol dose (Table 2). The
values of the CI suggest that the combination of ioniz-
ing radiation and resveratrol was additive at 50% and
60% inhibition, synergistic at 75% and 90%, and
strongly synergistic at 95% (Table 2). Accordingly, res-
veratrol acted synergistically with radiation at when cell
inhibition was greater than 60%.
3.2. Endogenous ceramide increases after combined
treatment in DU145 cells
To investigate whether an increase of intracellular
ceramide is associated with increased radio-sensitivity
of DU145 prostate cancer cells acquired after pretreat-
ment with resveratrol, we evaluated the level of this
lipid using the diacylglycerol kinase assay (Fig. 2, upper
panel) and the corresponding cell viability (Fig. 2, lower
panel). Endogenous ceramide increased 2.3-fold after
combined treatment compared to untreated cells and
1.6- and 2.0-fold, respectively, during separate radiation
and resveratrol treatments. Concurrently, cell viability
was 10% after combined treatment compared to
untreated cells, and 83% and 22%, respectively, during
separate radiation and resveratrol treatments. Thus,
 F. Scarlatti et al. / Cancer Letters 253 (2007) 124–130 127

Fig. 1. Biological effects of ionizing radiation or resveratrol on DU145 prostate cancer cells. Upper panel: Cells were treated with
increasing doses of radiation (0.5–2.0 Gy every day) for three consecutive days and the colonies (left) and cell viability (right) were
evaluated as described in Section 2. Values are the means ± SD of three independent experiments. Middle panel: Cells were treated with
increasing doses of resveratrol (0.5–32 lM) for 72 h and the colonies (left) and cell viability (right) were evaluated as described in Section 2.
Vehicle treatment was identical to untreated cells. Values are the means ± SD of three independent experiments. Data are expressed as
percentage of cell colonies or viability vs untreated cells. *p < 0.05 (Student’s t-test). Lower panel: Cells were pretreated with increasing
doses of resveratrol (0.5–2 lM) or 0.05% ethanol (as vehicle) for 72 h and then exposed to multifractionated ionizing radiation (0.5–2.0 Gy
every day) for three consecutive days and then cell survival (left) and cell viability (right) were estimated as described in Section 2. Values
are normalized to untreated cells.

we suggest that the increase of ceramide observed in 4. Discussion

cells treated with the both agents is correlated to resve-
ratrol-induced sensitivity of DU145 cells. To clarify the
mechanism of ceramide generation, we incubated cells
with myriocin, an inhibitor of serine palmitoyl transfer-
ase, the rate-limiting enzyme of de novo ceramide bio-
synthesis. Myriocin was able to reverses ceramide
generation after both resveratrol alone treatment and
combined treatments (Fig. 2, upper panel), concurrent
with a dramatic decrease in cell death (Fig. 2, lower
panel).
Most prostate cancers are androgen-dependent
at their early stages; nevertheless, they will
ultimately become androgen-independent and
refractory to most available therapies. Although
multiple reasons cause cancer disease recurrence, a
major factor may be the existence of tumor cell pop-
ulations that are unable to undergo apoptotic cell
death and thus become resistant to chemotherapy
128 F. Scarlatti et al. / Cancer Letters 253 (2007) 124–130

Table 1
Dose–effect relationship parameters of single and combined
treatments on growth of DU145 cellsa
Drug dose Fa b Dmb
Radiation (Gy/day) Res (lM)
0.5 0.05 2.17 (Gy)
1 0.41
1.5 0.18
2 0.50
0.5 0.26 2.15 (lM)
2 0.55
4 0.58
16 0.76
Radiation (Gy/day)+ Res (lM)
0.5 0.5 0.16 1.0 (Gy)
1 2 0.55
1.5 4 0.79
2 16 0.97
Radiobiological parameters calculated from cell viability assay of
Fig. 1 (lower right panel), based on linear-quadratic model.
a
Method of cytotoxicity assay is described in Section 2.
b
The parameters Fa and Dm, are the fractional inhibition and
dose at 50% inhibition (equivalent to IC50 value).

Table 2
Computer simulated CI and DRI values for ionizing radiation
and resveratrola
Inhibition CIb DRIc DRIc Fig. 2. Stimulatory effect of resveratrol and ionizing radiation on
Radiation Resveratrol cellular ceramide production in DU145 cells and reversion by
(%) myriocin. DU145 cells were treated with vehicles (0.05% ethanol
50 1.05 (additive) 2.47 1.55 and/or methanol), 16 lM resveratrol, 50 nM myriocin and
60 1.05 (additive) 5.66 1.15 multifractionated ionizing radiation 1.0 Gy every day for three
75 0.71 (synergism) 2.18 3.99 days as described in Section 2. After treatments, ceramide levels
90 0.42 (synergism) 4.91 4.55 (upper panel) and cell viability (lower panel) were evaluated.
95 0.15 (strong synergism) 7.88 40.11 Vehicle treatments were identical to untreated cells. Data are
normalized to control = 100% for cell viability and control = 1
The synergism analysis of resveratrol with radiation was per-
for ceramide analysis. Values are the means ± SD of three
formed with Chou–Talalay’s combination index and multiple
independent experiments. **p < 0.01; *p < 0.05 (Student’s t-test).
drug-dose effect analysis method as described in Section 2.
a
The values were determined using computer program from
the data presented in Table 1. obstacle to the curative potential of radiation ther-
b
Combination index (CI, 95% confidence intervals). CI < 1,
=1, and >1 indicates synergism, additive effect, and antagonism,
apy. Thus, any agent that may expand the effects
respectively. of ionizing radiation and overcome cell resistance
c
Dose reduction index: fold of dose reduction for each drug in would be very attractive. Among these agents we
combination, for a given degree of inhibition, when compared observed that resveratrol, at different doses,
with the dose of each drug alone for the same degree of strongly decreased both cell survival and viable cells
inhibition.
in DU145 cells. The use of a combined treatment
with both agents (resveratrol and ionizing radiation)
or radiotherapy. Generally those cells that survive decreased the number of colonies and cell viability
the initial ionizing radiation are those that are com- in DU145 cells compared to radiation treatment
petent to repopulate the irradiated area and to alone. Resveratrol is a radiosensitizing agent in dif-
metastasize. ferent cancer cell lines, and at high concentrations it
In agreement with a previous study [5], we con- has been shown to enhance radiation toxicity in
firm here that DU145 cells are notably resistant to HeLa, K-562, IM-9 [36], and EOL-1 [37] tumor cell
treatment with increasing doses of multifractioned lines by preventing repopulation, as well as by
ionizing radiation. Since radioresistance is still an repairing radiation damage. Moreover, in HeLa
 F. Scarlatti et al. / Cancer Letters 253 (2007) 124–130
cells, resveratrol, enhanced the response to ionizing
radiation by inducing a cell cycle checkpoint at S
phase and COX-1 inhibition [38].
We also quantified resveratrol and radiation
interaction by median-effect analysis software (Cal-
cuSyn). For therapeutic improvement against can-
cer a synergistic combination must pursue a dose
reduction factor for the component drugs [34].
The combination index (CI) can be considered as
an algebric expression of the isobologram [39] and
determines the degree and the nature of the interac-
tion of drugs, e.g. CI < 1, CI = 1, and CI > 1 are
indicative of synergistic, additive, and antagonistic
effects, respectively [35,39]. The dose reduction
index (DRI), at a given degree of effect, serves as
an important index for the determination of dose
reduction, which may consequently lead to less tox-
icity and improvement of therapeutic efficacy. In
our experiments dose reduction values indicated
that the combination of the two treatments (resvera-
trol and radiation) caused 2.2–7.8-fold reduction in
the dose of ionizing radiation and 4.0–40.1-fold in
the dose of resveratrol. Since irradiation is toxic,
any reduction in the dose at a particular degree of
effect should reduce toxicity and enhance the thera-
peutic value. Our data show that resveratrol exhibits
significant levels of synergy with ionizing radiation.
Notably, synergy occurs at low doses of ionizing
radiation, which fall well within the range of clini-
cally relevant doses (0.5–2.0 Gy).
The synergistic effect occurs at the ionizing radi-
ation doses of 1.5 Gy. The CI and DRI values also
suggest that it may be possible to decrease the dose
of ionizing radiation using combined treatments,
without sacrificing efficacy. This notion has impor-
tant clinical implications because the incidence of
morbidity caused by ionizing radiation increases
with radiation dose [40].
Ultimately, we demonstrated that addition of
resveratrol enhanced the generation of ionizing
radiation-induced ceramide in DU145 cells. Cera-
mide generation was due to an increased of its de
novo biosynthesis, since myriocin was able to par-
tially abrogate the effects of resveratrol alone and
combined treatments. We showed that combined
treatment with resveratrol and radiation induced
more ceramide accumulation than radiation or res-
veratrol alone.
Previously, we demonstrated that resveratrol was
able to stimulate accumulation of endogenous cera-
mide as a lipid mediator of apoptotic and cell death
in breast [27], and prostate cancer cells [28].
129
The addition of resveratrol might lead to sus-
tained ceramide accumulation in cancer cells, sensi-
tizing them to radiation-induced cell death and
growth inhibition. Therefore we speculate that irra-
diated-DU145 cells become sensitive to cell death
and growth inhibition as a result of an increased
production of resveratrol-induced apoptotic de novo
ceramide. In conclusion, pretreatment with resvera-
trol enhanced tumor cell killing and inhibited the
clonogenic survival in resistant irradiated-DU145
cells. Interaction analysis of the combined treatment
showed that resveratrol affected synergistically the
cellular response to ionizing radiation and that this
event may be mediated by an increase in cellular
de novo ceramide levels.
Acknowledgments
This study was supported by intramural grants
from of University of Milan. We are indebted to
Dr. Stuart Moore for English style and grammar
revision used in this paper.
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