A monocarbonyl analogue of curcumin, 1,5-bis(3-hydroxyphenyl)-1,4- pentadiene-3-one (Ca 37), exhibits potent growth suppressive activity and enhances the inhibitory effect of curcumin on human prostate cancer cells

Article abstract of DOI:10.1007/s10495-013-0947-y

Prostate carcinoma is one of the leading causes of cancer-related morbidity and mortality in males in western countries. Curcumin exhibits growth-suppressive activity against several cancers, including prostate cancer, but it has poor bioavailability. The purpose of this study was to evaluate the anticancer potency and mechanism of a curcumin analogue, 1,5-bis(3- hydroxyphenyl)-1,4-pentadiene-3-one (Ca 37), in human prostate cancer. Studies were performed in established human prostate cancer cell lines (PC-3 and DU145) as well as in a murine xenograft tumor (PC-3) model. Ca 37 presented a preferential suppression capacity against growth and migration toward prostate cancer cells compared with curcumin. Ca 37 impaired the bioenergetics system, promoted cell cycle arrest and apoptosis activation in PC-3 cells. In addition, 0.5 μmol (6.65 mg/kg body weight) of Ca 37 significantly inhibited the growth of the prostate xenografted tumors, whereas 6 μmol (110 mg/kg body weight) of curcumin had little effect. Furthermore, a combination of Ca 37 and curcumin resulted in enhanced antitumor activity in prostate cancer cells. N-Acetylcysteine abrogated both reactive oxygen species (ROS) production and viability loss induced by Ca 37 but partially prevented growth inhibition in PC-3 cells treated with curcumin alone, or a combination with Ca 37. The data indicate that induction of ROS plays a vital role in the growth inhibitory effect of Ca 37 in PC-3 cells. This study suggests that Ca 37, alone or in combination with curcumin, may be a promising anticancer agent for prostate cancer therapy.

Full text of DOI:10.1007/s10495-013-0947-y

Apoptosis (2014) 19:542–553
DOI 10.1007/s10495-013-0947-y
ORIGINAL PAPER
A monocarbonyl analogue of curcumin, 1,5-bis(3-hydroxyphenyl)-
1,4-pentadiene-3-one (Ca 37), exhibits potent growth suppressive
activity and enhances the inhibitory effect of curcumin on human
prostate cancer cells
Cheng Luo
Hua Li Zhihui Feng
Jiankang Liu
•
Yan Li
•
Bo Zhou
Yuan Li
•
Liang Yang
Jiangang Long
•
•
•
•
•
Published online: 3 December 2013
Ó Springer Science+Business Media New York 2013
Abstract Prostate carcinoma is one of the leading causes
of cancer-related morbidity and mortality in males in
western countries. Curcumin exhibits growth-suppressive
activity against several cancers, including prostate cancer,
but it has poor bioavailability. The purpose of this study
was to evaluate the anticancer potency and mechanism of a
curcumin analogue, 1,5-bis(3-hydroxyphenyl)-1,4-pentadi-
ene-3-one (Ca 37), in human prostate cancer. Studies were
performed in established human prostate cancer cell lines
6 lmol (110 mg/kg body weight) of curcumin had little
effect. Furthermore, a combination of Ca 37 and curcumin
resulted in enhanced antitumor activity in prostate cancer
cells. N-Acetylcysteine abrogated both reactive oxygen
species (ROS) production and viability loss induced by Ca
37 but partially prevented growth inhibition in PC-3 cells
treated with curcumin alone, or a combination with Ca 37.
The data indicate that induction of ROS plays a vital role in
the growth inhibitory effect of Ca 37 in PC-3 cells. This
study suggests that Ca 37, alone or in combination with
curcumin, may be a promising anticancer agent for prostate
cancer therapy.
(
(
PC-3 and DU145) as well as in a murine xenograft tumor
PC-3) model. Ca 37 presented a preferential suppression
capacity against growth and migration toward prostate
cancer cells compared with curcumin. Ca 37 impaired the
bioenergetics system, promoted cell cycle arrest and
apoptosis activation in PC-3 cells. In addition, 0.5 lmol
Keywords Curcumin analogue Ca 37 ꢀ Prostate
cancer ꢀ Reactive oxygen species ꢀ Tumor xenograft
(
6.65 mg/kg body weight) of Ca 37 significantly inhibited
the growth of the prostate xenografted tumors, whereas
Abbreviations
CI
Combination index
Complex I
Complex II
NADH–CoQ oxidoreductase
Succinate–CoQ oxidoreductase
C. Luo ꢀ L. Yang ꢀ H. Li ꢀ Z. Feng ꢀ Y. Li ꢀ
J. Long ꢀ J. Liu (&)
Center for Mitochondrial Biology and Medicine, The Key
Laboratory of Biomedical Information Engineering of Ministry
of Education, School of Life Science and Technology and
Frontier Institute of Life Science, FIST, Xi’an Jiaotong
University, Xi’an 710049, China
Complex III CoQ–cytochrome c reductase
Complex IV Cytochrome c oxidase
DCIP
DMSO
FBS
2,6-Dichlorobenzenone–indophenol
Dimethyl sulfoxide
e-mail: j.liu@mail.xjtu.edu.cn
Fetal bovine serum
ROS
Reactive oxygen species
Reduced glutathione
C. Luo
e-mail: luoke123@gmail.com
GSH
0
0
H DCFDA
2
2 ,7 -Dichlorodihydrofluorescein diacetate
2-(p-iodophenyl)-3(p-nitrophenyl)-5-
phenyl tetrazolium chloride
a-Ketoglutarate dehydrogenase complex
Lactic acid
Y. Li
INT
Center for Bioinformatics, The Key Laboratory of Biomedical
Information Engineering of Ministry of Education, School of
Life Science and Technology, Xi’an Jiaotong University,
Xi’an 710049, China
a-KGDH
LA
LDH
Lactate dehydrogenase
B. Zhou
State Key Laboratory of Applied Organic Chemistry,
Lanzhou University, Lanzhou 730000, China
MDH
NAC
Malate dehydrogenase
N-Acetylcysteine
1
23

Apoptosis (2014) 19:542–553
543
However, the poor water solubility and bioavailability of
curcumin severely curtails its anti-cancer capacity [7]. Many
structural analogues of curcumin have been synthesized to
improve its bioavailability [8]. However, many analogues are
very expensive for researches or clinic trials, i.e. EF-24
NBT
Nitroblue tetrazolium
PARP
PI
Poly(ADP-ribose) polymerase
Propidium iodide
PMS
Phenazine methosulfate
Ribonuclease A
RNase A
(E8409-5 mg, US$ 69.30, sigma) and FLLL31 (F9057-5 mg,
US$ 88.50, sigma). Derivatives with low costs and favorable
anti-cancer effects should be developed to benefit the general
public. 1,5-bis(3-hydroxyphenyl)-1,4-pentadiene-3-one, a
monocarbonyl analogue of curcumin, named Ca 37 herein,
was chosen to evaluate its anticancer potency and mechanism
against human prostate cancer for its low-cost synthesis. We
found that Ca 37 exerts a preferential toxicity toward prostate
cancer cells which is dependent on ROS induction, similar to
that of other curcumin analogues [9–11].
Introduction
Prostate cancer is the most frequently diagnosed cancer and
the second-leading cause of cancer death among men in the
United States. According to a cancer statistical report from
the American Cancer Society, there will be approximately
2
41,740 new cases and 28,170 deaths from prostate cancer
occurring in the United States in 2012 [1]. Curcumin, a
polyphenolic molecule extracted from turmeric, exhibits
promising chemopreventive activity against prostate cancer
through anti-proliferation and pro-apoptosis activity, pre-
vention of cell motility and metastasis, regulation of the
inflammatory response and down-regulation of the andro-
gen receptors and co-factors [2].
Materials and methods
Materials
N-Acetyl-cysteine (NAC), CoQ1, crystal violet, decyl-
ubiquinone, curcumin, 2-(p-iodophenyl)-3(p-nitrophenyl)-
Many cancer cells are resistant to chemotherapy due to
deregulated apoptosis. The evaluation of the ability to
overcome the resistance of tumor cells towards apoptosis,
i.e., apoptosis activation, is a routine way to screen chemo-
therapy drugs. Apoptosis can be divided into two pathways:
the intrinsic pathway (signaling involving the mitochondria)
and the extrinsic pathway (signaling through death receptors
on the cell surface). Pro-apoptosis proteins of the Bcl-2
family, sharing homology in the Bcl-2 homology regions
5
-phenyl tetrazolium chloride (INT), dimethyl sulfoxide
(DMSO), cisplatin, doxorubicin, 2,6-dichlorobenzenone-
indophenol (DCIP), 3-hydroxy-benzaldehyde, glucose-6-
phosphate dehydrogenase, CoASH, thiamine pyrophos-
phate, propidium iodide (PI), phenazine methosulfate
(PMS), and an anti-actin primary antibody were obtained
from Sigma (St. Louis, MO). RPMI-1640 medium, peni-
0
0
cillin, streptomycin, 2 ,7 -dichlorodihydrofluorescein diac-
etate (H DCFDA) and trypsin were purchased from
2
(
BH1–BH3) with conserved sequence motifs, initiate the
Invitrogen (Carlsbad, CA). Primary antibodies for Bax,
Bak, Bad, Bcl-xL, Bcl-2, and poly(ADP-ribose) polymer-
ase (PARP) were obtained from Cell Signaling Technol-
ogy, Inc. (Beverly, MA). Cyclin A, B1, D1, and E were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Secondary HRP conjugated anti-mouse/rabbit antibodies
were purchased from Jackson ImmunoResearch Laborato-
ries, Inc. (West Grove, PA). Ribonuclease A (RNase A)
was obtained from Takara Biotechnology (Dalian) Co.,
Ltd. (Dalian, Liaoning, China). Fetal bovine serum (FBS)
was obtained from PAA Laboratories GmbH (Linz, Aus-
tria). Hoechst 33342 and cell lysis buffer were obtained
from Beyotime Biotechnology (Haimen, Jiangsu, China).
Lactate dehydrogenase (LDH) activity and lactic acid (LA)
content kits were obtained from the Nanjing Jiancheng
Bioengineering Institute (Nanjing, Jiangsu, China).
mitochondrial pathway, leading to cytochrome c release and
then the activation of caspases [3]. The mitochondria are
intracellular power houses and a major source of reactive
oxygen species (ROS), including superoxide anion radicals,
hydroxyl radicals, and hydrogen peroxide (H O ). ROS are
2
2
active mediators in the regulation of cell dysfunction and
death at different levels via induction of protein carbonyl-
ation, lipid peroxidation and DNA damage [4]. In curcumin-
treated prostate cancer cells, there is a decrease in anti-
apoptosis proteins Bcl-2 and Bcl-xL expression, an increase
in the pro-apoptosis proteins Bax and Bak, and a transloca-
tion of Bax to the mitochondria. ROS generation, cyto-
chrome c release and caspase-3 activation are involved in the
induction of apoptosis by curcumin [5]. However, a study by
Hilchie et al. [6] suggested that the cytotoxic effect of cur-
cumin did not involve ROS generation in PC-3 cells. The
researchers compared apoptosis suppression and the ROS
scavenging effects of the exogenous antioxidants, including
reduced glutathione (GSH), N-Acetylcysteine (NAC),
ascorbic acid, and ebselen.
Synthesis of curcumin analogue Ca 37
Ca 37 was synthesized based on previously described
methods [12, 13]. Briefly, aqueous NaOH (20 % (wt),
123

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Apoptosis (2014) 19:542–553
1
5 mL, 75 mmol) was added dropwise to a vigorously
stirred solution of 3-hydroxy-benzaldehyde (51 mmol) and
ketone (25 mmol) in absolute ethanol (8 mL). The reaction
proceeded under stirring at room temperature for 48 h,
distilled water (40 mL) was added, and the solution was
neutralized by HCl. The yellow precipitate was filtered off,
washed with distilled water and dried under a vacuum. The
resulting product was directly charged onto a silica gel
column and eluted with a mixture of ethyl acetate/petro-
leum to obtain the pure product: a yellow solid; yield:
1
1
3.7 %; mp 202–204 °C; H NMR (400 MHz, (CD ) CO),
3
2
Fig. 1 Chemical structure diagram of curcumin and its analogue Ca
37
d 8.57 (s, 2 H, –OH), 7.72 (d, J = 16.0 Hz, 2 H), 7.20–7.30
(
1
m, 8 H), 6.93 (ddd, J = 7.6, 2.4, 1.2 Hz, 2 H); C NMR
3
(
100 MHz, (CD ) CO), d 189.0, 158.8, 143.6, 137.5,
3
2
?
30.9, 126.6, 120.9, 118.5, 115.7; EIMS, m/z 266 [M] .
1
the cells were lysed with buffer (10 mmol/L Tris,
The purity of Ca 37 was determined to be 99.87 % by
150 mmol/L NaCl, 0.1 mmol/L EDTA, 0.5 % Triton
HPLC. HPLC analysis was performed using a Waters 600
X-100, pH 7.5), centrifuged at 13,000g for 10 min, and
instrument with photodiode array detector, and a Symmetry
TM
then the supernatant was collected. The fluorescence
Shield
RP18 (3.9 9 150 mm, 5 lm particle size,
Waters) with a mixture of isopropanol/n-hexane (5:95, v/v)
intensity of the supernatant was recorded with a Thermo
TM
Scientific Fluoroskan
Ascent FL at 485 nm excitation
as an eluent. The flow rate was set at 0.5 mL/min.
and 535 nm emission and normalized by cellular protein
content.
Cell culture
Chromatin condensation
Human androgen-independent prostate cancer PC-3 (from
Chinese Academy of Sciences Committee Type Culture
Collection Cell Bank/CAS Shanghai Institutes for Biologic
Sciences Cell Resource Center) and DU145 (from the cell
bank of the Kunming Institute of Zoology, Chinese
Academy of Sciences) cells were maintained at 37 °C with
The cells were fixed with 4 % paraformaldehyde, followed
by staining with Hoechst 33342 (Beyotime Biotechnology,
Jiangsu, China). The fluorescent photographs of chromatin
condensation were taken with a fluorescence microscope
(Olympus IX71).
5
% CO in RPMI 1640 medium containing 10 % FBS,
2
100 units/mL penicillin and 100 lg/mL streptomycin in a
humidified incubator (Thermo Fisher Scientific, Inc.,
Cell cycle analysis
Waltham, MA).
The cells were fixed with 75 % ethanol in -20 °C, fol-
lowed by staining with PI solution (0.1 % Triton-100,
Cell viability/cytotoxicity assay
2
00 lg/mL RNase A, 20 lg/mL PI). The analysis of cell
TM
cycle phases was performed using a BD FACSCanto
Flow Cytometer according to manufacturer’s instructions.
A crystal violet-based staining assay was used to assess the
cytotoxicity of curcumin and Ca 37 [14]. After treatment
with the compounds, the cells were fixed with 4 % para-
formaldehyde and stained with 0.1 % crystal violet at room
temperature, and then excess stain was washed away. The
crystal violet in stained cells was dissolved in 30 % acetic
Mitochondrial respiratory chain complex and citric acid
cycle enzyme activity
acid, and the absorbance was recorded with a microplate
TM
The mitochondria in PC-3 cells were isolated with a differ-
ential centrifugation method at 4 °C as follows, similar to our
previous work [16]. After swelling in ice-cold RSB (a
hypotonic buffer that causes tissue culture cells to swell,
spectrophotometer (Thermo Scientific Multiskan
trum) at 570 nm.
Spec-
Determination of intracellular reactive oxygen species
ROS)
10 mmol/L NaCl, 1.5 mmol/L MgCl , 10 mmol/L Tris–
2
(
HCl, pH 7.5) for 15 min, the cells were homogenized with
several strokes of a small-clearance pestle in media
(210 mmol/L mannitol, 70 mmol/L sucrose, 5 mmol/L
Tris–HCl, 1 mmol/L EDTA, pH 7.5) in a 2 mL Dounce
homogenizer. The homogenate was centrifuged at 1,300g for
0
0
Intracellular ROS were measured by 2 ,7 -dichlorodihy-
drofluorescein diacetate (H DCFDA), which is non-fluo-
2
rescent unless oxidized within the cell [15]. After staining,
1
23

Apoptosis (2014) 19:542–553
545
5
1
min, the supernatant was collected and centrifuged at
200 lmol/L NADH), followed by scanning at 600 nm for
2 min at 30 °C [17].
7,000g for 15 min to obtain mitochondrial particles. The
mitochondrial protein concentrations were determined using
TM
a BCA Protein Assay kit (Thermo Scientific). The mito-
Succinate–CoQ oxidoreductase (Complex II) was asses-
sed in a reaction mixture (50 mmol/L potassium phosphate
buffer pH 7.8, 0.05 mmol/L DCIP, 2 mmol/L EDTA, 0.1 %
BSA, 3 lmol/L rotenone, 1 lmol/L antimycin A, 0.2 mmol/
chondria were stored at -80 °C before assayed.
NADH–CoQ oxidoreductase (Complex I) activity was
assessed in a reaction mixture (50 mmol/L Tris–HCl, pH
L NaN , 0.2 mmol/L ATP, 0.05 mmol/L CoQ1, 10 mmol/L
3
8.1, 0.05 mmol/L DCIP, 0.35 % BSA, 1 lmol/L antimycin
A, 0.2 mmol/L NaN3, 0.05 mmol/L coenzyme Q1,
succinate), followed by scanning at 600 nm for 2 min at
30 °C [18].
Fig. 2 Ca 37 exhibits potential
inhibitory effect against prostate
cancer cells. The viabilities of
PC-3 cells (a) and DU145 cells
(
b) treated with the indicated
concentrations of curcumin (a1,
b1) or Ca 37 (a2, b2); c wound
healing assay of PC-3 cells and
DU145 cells; d1 the activities of
respiration chain complex
enzymes (Complex I to
Complex IV) and citric acid
cycle enzymes (MDH and a-
KGDH) in Ca 37-treated PC-3
cells; d2 the LDH activity and
LA content in Ca 37-treated PC-
3
cells; e1 representative images
and e2 quantitative data of cell
cycle arrest in 10 lmol/L Ca
3
7-treated PC-3 cells; f1
western blot of Bcl-2 family
proteins and poly(ADP-ribose)
polymerase (PARP) in 10 lmol/
L Ca 37-treated PC-3 cells; f2
the semi-quantified statistics for
the western blot normalized
with b-actin; g chromatin
condensation in Ca 37-treated
PC-3 cells. *p \ 0.05,
*
*p \ 0.01 vs. non-treated
control group (vehicle group).
All data were from at least 3
independent repeat experiments
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Apoptosis (2014) 19:542–553
Fig. 2 continued
CoQ–cytochrome c reductase (Complex III) was assessed in
Lactate dehydrogenase (LDH) activity and lactic acid
(LA) content assessment
a reaction mixture (50 mmol/L Tris–HCl pH 7.8, 0.2 mmol/L
NaN , 0.05 % Tween-20, 0.01 % BSA, 0.05 mmol/L cyto-
3
chrome c, 0.05 mmol/L decylubiquinol), followed by scanning
at 550 nm for 2 min at 30 °C [19].
Cytochrome c oxidase (Complex IV) was measured in a
reaction mixture (50 mmol/L potassium phosphate buffer
pH 7.0, 0.1 % BSA, 0.2 % Tween-20, 0.05 mmol/L
reduced cytochrome c), followed by scanning at 550 nm
for 2 min at 30 °C [20].
Lactate dehydrogenase activity was assessed with a
colorimetric method using a commercial kit (Nanjing
Jiancheng Bioengineering Institute, Nanjing, Jiangsu,
China). According to the manufacturer’s instructions, the
resulting pyruvate was obtained from lactic acid via
lactate dehydrogenase-mediated conversion of 2,4-dini-
trophenylhydrazine into a visible brownish red hydra-
zone in alkaline solutions, and the absorbance of the
hydrazone was recorded with a microplate spectropho-
tometer (Thermo Scientific Multiskan Spectrum) at
440 nm.
a-Ketoglutarate dehydrogenase complex (a-KGDH)
was assayed in a reaction mixture (35 mmol/L potassium
phosphate buffer pH 7.25, 2 mmol/L NaN , 0.5 mmol/L
3
EDTA, 2.5 lmol/L rotenone, 5 mmol/L MgCl , 0.5 mmol/
2
?
L NAD , 0.2 mmol/L thiamine pyrophosphate, 2 mmol/L
Lactic acid content was measured by following NADH
production by coupling it to the reduction of nitroblue
tetrazolium (NBT) via the intermediate electron carrier
phenazine methosulfate (PMS) using a commercial kit
(Nanjing Jiancheng Bioengineering Institute, Nanjing,
Jiangsu, China). The absorbance of NBT formazan was
recorded with a microplate spectrophotometer (Thermo
Scientific Multiskan Spectrum) at 530 nm.
a-ketoglutarate, 0.04 mmol/L CoASH), followed by scan-
ning at 340 nm for 2 min at 30 °C [21].
Malate dehydrogenase (MDH) activity was assayed in a
reaction mixture (15 mmol/L malate, 3 lmol/L rotenone,
?
.1 %BSA, 0.5 mmol/LNAD , 0.6 mmol/LINT, 0.2 mmol/
0
L PMS, 50 mmol/L potassium phosphate buffer pH 9), fol-
lowed by scanning at 500 nm for 2 min at 30 °C [16].
1
23

Apoptosis (2014) 19:542–553
547
Fig. 3 Ca 37 suppresses
prostate cancer tumor xenograft
growth in vivo. The tumor
volumes (a) and mice body
weights (b) of Ca 37-treated
nude mice; the photographs (c1)
and weights (c2) of the tumors
from the Ca 37-treated nude
mice; the tumor volumes
(
(
d) and mice body weights
e) of curcumin-treated nude
mice; f1 western blot of cell
cycle regulation proteins from
protein extracts of tumors from
the Ca 37-treated nude mice; f2
the semi-quantified statistics for
the western blot (f1) normalized
with b-actin. *p \ 0.05,
*
*p \ 0.01 vs. vehicle group.
All data were from at least three
independent repeat experiments
Wound healing migration assay
of Medicine Laboratory Animal Center (Xi’an, Shannxi,
China). The animal care and treatments were performed in
accordance with the animal experimentation guidelines of
Xi’an Jiaotong University. All mice were maintained with
ad libitum water and sterilized standard mouse chow in
sterile filter-capped microisolator cages on a 12 h light/
dark cycle in the same animal center. Prostate cancer PC-3
The wound healing assay is a simple method to study direc-
tional cell migration in vitro. Cells were plated in a 12-well
plate and grown to confluence. A wound was created by
dragging a 200 lL pipette tip across the surface of a mono-
layerculture, andthecellulardebriswaswashedoutwithPBS.
The cells were treated with curcumin or Ca 37 for 2 days,
during which time vehicle treatment cells always grew to
convergence across the wound, and then images were taken.
7
cells were suspended in RPMI 1640 (1 9 10 /100 lL/
mouse) and injected s.c. into the right flank of the mice.
When the injected tumor cells grew to a size of approxi-
3
mately 100 mm , the tumor-bearing mice were then divi-
In vivo PC-3 tumor xenograft model
ded randomly into treatment groups (five mice per group).
The mice received an i.p. injection of DMSO vehicle,
0.25 lmol Ca 37 (3.33 mg/kg body weight), 0.5 lmol Ca
37 (6.65 mg/kg body weight), or 6 lmol curcumin
Five-week-old male athymic nude mice (BALB/c-nu/nu)
were obtained from the Xi’an Jiaotong University College
123

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Apoptosis (2014) 19:542–553
Fig. 4 Reactive oxygen species (ROS) is involved in Ca 37 induced
growth inhibition in prostate cancer PC-3 cells. a Increased ROS
production in PC-3 cells with Ca 37 treatment; b NAC abrogated
time-dependent ROS production in 10 lmol/L Ca 37-treated PC-3
cells; c NAC abrogated Bad expression in PC-3 cells treated with
normalized with b-actin; NAC prevented growth inhibition of PC-3
cells induced by Ca 37 (d) and curcumin (e). *p \ 0.05, **p \ 0.01
&
&
vs. vehicle group;
#
p \ 0.01 vs. 5 lmol/L Ca 37 treatment group;
#
@@
p \ 0.01 vs. 10 lmol/L Ca 37 treatment group;
p \ 0.01 vs.
!!
40 lmol/L curcumin treatment group; p \ 0.01 vs. 60 lmol/L
curcumin treatment group. All data were from at least three
independent repeat experiments
10 lmol/L Ca 37 for 4 h: the lower panel presents a representative
blot and the upper panel presents the semi-quantified statistics
(
110 mg/kg body weight) every other day for 16 days.
incubated with appropriate secondary antibodies for 1 h at
room temperature. The western blots were developed using
an Enhanced Chemiluminescence Kit (Millipore).
Tumor growth was determined by caliper measurement of
the length (L) and width (W) every 4 days, and tumor
volume was calculated on the basis of the following for-
2
mula: volume = 0.52 9 L 9 W . The body weights of the
Statistical analysis
mice were measured every 4 days during the 16-day
treatment period. At the end of study, the tumors were
excised, weighed and then lysed for western blot analysis.
All assays were conducted with at least three independent
experiments. Data are presented as the mean ± SEM. The
statistical significance was evaluated by one-way analysis
of variance (ANOVA), followed by Fisher’s LSD test. All
analyses were performed using the IBM SPSS Statistics
software. Significant differences are indicated as follows:
*p \ 0.05, **p \ 0.01.
Western blot analysis
The cells or tumors were treated with extraction buffer
from Beyotime Biotechnology (Haimen, Jiangsu, China)
and centrifugated at 17,000g for 15 min at 4 °C. The
supernatants were collected, and the protein concentrations
TM
were determined using a BCA
Protein Assay Kit. Ali-
Results
quots containing approximately 20 lg of protein were
subjected to SDS polyacrylamide gel electrophoresis and
then electroblotted onto nitrocellulose membranes (Milli-
pore). After being blocked with 5 % nonfat milk in TBST,
the membranes were incubated with primary antibodies at
The curcumin analogue Ca 37 exhibits potential
inhibitory effects in human androgen-independent
prostate cancer cells
4 °C overnight. After washing away the un-combined pri-
mary antibodies with TBST, the membranes were
Compared with curcumin, Ca 37 significantly suppresses
the proliferation of human androgen-independent prostate
1
23

Apoptosis (2014) 19:542–553
549
cancer PC-3 and DU145 cells in a dose-dependent manner
within a 24 h period. The IC50 value of Ca 37 was 4.7 or
The curcumin analogue Ca 37 suppresses prostate
cancer tumor xenograft growth in vivo
1
0.5 lmol/L, a value seven or threefold lower than that of
curcumin (IC , 33.8 or 34.3 lmol/L), in PC-3 cells
To determine whether the growth inhibition potential of Ca
37 in vitro could be achieved in vivo, we examined the
ability of Ca 37 to suppress the growth of human prostate
cancer PC-3 cell xenografts in nude mice. There was a
significant inhibition of the growth of the tumor xenografts
when mice were treated with 0.5 lmol Ca 37 (Fig. 3a), but
not when treated with 6 lmol curcumin (Fig. 3d), which is
similar to previous results obtained by Khor et al. [22]. The
weights of the excised tumors from the Ca 37-treated mice
ranged from 140 to 300 mg, whereas those from the
vehicle group ranged from 220 to 480 mg (Fig. 3c). The
effects of the compounds on body weight are shown in
Figs. 3b, e. There was an increase in the expression of
cyclins (Cyclin E and D1) regulating G1/S transition but a
decrease in Cyclin A and Cyclin B1, which regulate S/G2
transition and G2/M phase transition, respectively
(Fig. 3f). The aberrant expression of cyclins indicates that
Ca 37 promotes cancer cells entrance into the cell cycle
5
0
(
Fig. 2a1, a2) or DU145 cells (Fig. 2b1, b2), respectively.
Ca 37 also exhibited greater inhibitory capacity against
prostate cancer PC-3 or DU145 cell migration in scratch-
wound assays (Fig. 2c). The bioenergetics system, includ-
ing the mitochondrial fractions (mitochondrial respiration
chain complex enzymes (Complex I to Complex IV) and
citric acid cycle enzymes (MDH and a-KGDH activities))
(
Fig. 2d1) and cytoplasmic fractions (LDH activity, LA
content) (Fig. 2d2), was reduced in Ca 37-treated PC-3
cells. After 8 h treatment with Ca 37, the cell percentage in
the G2 phase increased with corresponding decrease in the
G1 phase of PC-3 cells (Fig. 2e), indicating that the cells
were arrested in G2 phase. There were significant increases
in the pro-apoptotic proteins Bad, Bak, activated PARP
(
(
89-kD fragment) based on statistical analysis results
Fig. 2f), and condensed chromatin in the nuclei (Fig. 2g)
in Ca 37-treated PC-3 cells, indicating apoptosis involved.
@
@
!!
Fig. 5 Ca 37 enhances curcumin induced growth inhibition of
prostate cancer cell. The viabilities of cells treated with combinations
of curcumin and Ca 37 (a1, b1), cisplatin and Ca 37 (a2, b2),
doxorubicin and Ca 37 (a3, b3) for 24 h in a PC-3 cells and b DU145
p \ 0.01 vs. 1.25 lmol/L doxorubicin group, p \ 0.01 vs.
2.5 lmol/L doxorubicin group; b1 *p \ 0.05 **p \ 0.01 vs. vehicle
#
#
$$
group, p \ 0.01 vs. Ca 37 group, p \ 0.01 vs. 5 lmol/L curcumin
group, p \ 0.01 vs. 10 lmol/L curcumin group,
^^
&&
p \ 0.01 vs.
#
#
cells. a1 *p \ 0.05, **p \ 0.01 vs. vehicle group; p \ 0.01 vs. Ca
20 lmol/L curcumin group; b2 **p \ 0.01 vs. vehicle group,
@
@
!!
##
3
7 group;
p \ 0.01 vs. 1.25 lmol/L curcumin group; p \ 0.01
p \ 0.01 vs. Ca 37 group; b3 *p \ 0.05, **p \ 0.01 vs. vehicle
$
$$
##
vs. 2.5 lmol/L curcumin group; p \ 0.05, p \ 0.01 vs. 5 lmol/L
curcumin group;
&
p \ 0.01 vs. 20 lmol/L curcumin group; a2 **p \ 0.01 vs.
group, p \ 0.01 vs. Ca 37 group; c Ca 37 enhanced curcumin-
^^
p \ 0.01 vs. 10 lmol/L curcumin group;
induced Bad expression and cleavage of PARP in PC-3 and DU145
cells; d NAC partially prevented growth inhibition of PC-3 cells
induced by a combination of 10 lmol/L curcumin and 2.5 lmol/L Ca
37 following 24 h of treatment: **p \ 0.01 vs. vehicle group,
&
##
@@
vehicle group,
2
p \ 0.01 vs. Ca 37 group,
p \ 0.01 vs.
!
!
.5 lmol/L cisplatin group, p \ 0.01 vs. 5 lmol/L cisplatin group,
$$
^^
##
p \ 0.01 vs. 10 lmol/L cisplatin group, p \ 0.01 vs. 20 lmol/L
p \ 0.01 vs. the combination treatment group. All data were from at
&&
cisplatin group,
*
p \ 0.01 vs. 40 lmol/L cisplatin group. a3
least three independent repeat experiments
#
#
*p \ 0.01 vs. vehicle group,
p \ 0.01 vs. Ca 37 group,
123

5
50
Apoptosis (2014) 19:542–553
Fig. 5 continued
arrest, which is in accordance with the in vitro result
induced by Ca 37 but only partially protected cells from the
reduced viability induced by curcumin (Fig. 4d, e). These
results indicate a different cell death-promoting mechanism
induced by curcumin and Ca 37 in PC-3 cells.
(
Fig. 2e).
Reactive oxygen species (ROS) is involved in Ca
7-induced growth inhibition in prostate cancer PC-3
3
cells
Ca 37 enhances curcumin-induced growth inhibition
of prostate cancer cells
The results of the present study indicated that exposure of
PC-3 cells to Ca 37 for 12 h resulted in the generation of
ROS (Fig. 4a) and that N-Acetyl-cysteine (NAC) abrogated
time-dependent ROS production (Fig. 4b). Moreover, NAC
prevented Ca 37-induced Bad increase (Fig. 4c). NAC
significantly prevented growth inhibition of PC-3 cells
The treatment of PC-3 cells with Ca 37 (2.5 or 3.5 lmol/L)
and increasing doses of curcumin (1.25–20 lmol/L) for
24 h resulted in a dose-dependent inhibition of cell growth
(Fig. 5a1). Moreover, the treatment of DU145 cells with
Ca 37 (5 lmol/L) and increasing doses of curcumin
1
23

Apoptosis (2014) 19:542–553
551
Fig. 5 continued
(
1.25–20 lmol/L) for 24 h also resulted in a dose-depen-
countries than in eastern developing countries due to the
differences in diet and lifestyle between developed and
developing countries [23–25]. Curcumin, a major yellow
pigment in turmeric, can suppress prostate cancer cell
growth in vitro and in vivo by regulating multiple signaling
pathways, including inflammation, cell proliferation, cell
motility, and apoptosis [2]. However, studies reveal that
serum and tissue levels of curcumin are very low due to
poor absorption, rapid metabolism and elimination in the
body, which curtails the bioavailability of curcumin [7]. To
improve the bioavailability of curcumin, structural ana-
logues of curcumin have been synthesized and reported to
exhibit anti-cancer capacities both in vitro and in vivo [9–
11, 26–28]. 1,5-Bis(3-hydroxyphenyl)-1,4-pentadiene-3-
one, Ca 37, a novel monocarbonyl analogue of curcumin
(Fig. 1), exhibits superior potential inhibitory capacities
against prostate cancer cell growth and migration (Fig. 2),
suppressive effects against prostate cancer tumor xenograft
growth (Fig. 3) comparing with curcumin.
dent inhibition of cell growth (Fig. 5b1). Those combina-
tion treatments resulted in a more effective inhibition than
that of single-compound treatment. The additive effects of
the combination of Ca 37 and curcumin suggest a possible
synergism (combination index (CI) = 0.93 \ 1 for
2
.5 lmol/L Ca 37 and 20 lmol/L curcumin in PC-3 cells,
CI = 0.96 \ 1 for 5 lmol/L Ca 37 and 20 lmol/L curcu-
min in DU145 cells). However, only a small enhancement
of the inhibitory effect was found in the combinations of
Ca 37 and high doses of cisplatin (20, 40 lmol/L)
(
Fig. 5a2) but not with the combination of Ca 37 and
doxorubicin (Fig. 5a3) in PC-3 cells. Furthermore, there
were no additive effects associated with combinations of
Ca 37 and cisplatin (Fig. 5b2) or doxorubicin (Fig. 5b3) in
DU145 cells.
We evaluated several factors affecting cell growth to
understand the molecular basis of the synergism or additive
effects of combination. The combination of Ca 37 and
curcumin induced an increase in the expression of the pro-
apoptotic protein Bad and cleavage of PARP both in PC-3
and DU145 cells (Fig. 5c). However, NAC partially pre-
vented the growth inhibition of PC-3 cells induced by a
combination of 2.5 lmol/L Ca 37 and 10 lmol/L curcu-
min, which suggests that ROS production was partially
involved in this process (Fig. 5d).
Some monocarbonyl or monoketone analogues of cur-
cumin have been synthesized to improve the anti-cancer
capacity of curcumin, such as FLLL11 and FLLL12 [29,
30], B19 [31], and B63 [32]. Similar to Ca 37, a b-diketone
moiety deletion exists in those analogues. They possess
only one carbonyl or ketone and a central methylene car-
bon, in addition to a 5-carbon tether length between the
aromatic rings (curcumin has a 7-carbon tether). Due to the
b-diketone moiety deletion in the analogues, they are more
stable in a pH 7.4 solution than curcumin. After oral
administration, the analogues exhibits decreased degree
and speed of metabolism, and much higher peak concen-
tration and a decrease of clearance in the plasma of healthy
rats [33]. These traits may explain why Ca 37 is more
effective as an anti-prostate cancer agent than curcumin.
ROS (superoxide, hydroxyl radicals, and hydrogen per-
oxide) promote oxidative damage, such as protein carbon-
ylation, lipid peroxidation and DNA damage, in
biomacromolecules. Meanwhile, a small amount of ROS
Discussion
The present study indicates that the novel curcumin ana-
logue Ca 37 possesses great potential as a promising anti-
prostate cancer therapeutic agent comparing with curcu-
min. Prostate cancer occurs mainly above the age of 50 and
is a common cause of cancer death in men. As the aging
population is increasing, the number of prostate cancer
patients is growing accordingly. Generally, the incidence
rates of prostate cancer are higher in western developed
123

5
52
Apoptosis (2014) 19:542–553
act as secondary messengers and mediate various signaling
cascades related to cellular proliferation, mutation and
genetic instability in cancer cells. ROS might function as
double-edged swords in cancer therapeutics through an
imbalance between ROS generation and elimination. Cells
can resist ROS under a cell-death threshold. Once exceed-
ing the threshold, the cells will die [34]. The ROS levels are
higher in prostate cancer cell lines than in normal cell lines,
implying that cancer cells may be more vulnerable to ROS
than normal cells [35]. The generation of ROS increased in
Ca 37-treated PC-3 cells and could be abrogated by the ROS
scavenger NAC. Moreover, the viability loss prevented by
NAC revealed the involvement of ROS in Ca 37-induced
growth inhibition. In comparison, curcumin-induced via-
bility loss was only partially involved in ROS (Fig. 4).
It is an interesting phenomenon that Ca 37 enhances the
growth inhibitory effect of curcumin, but not doxorubicin
or cisplatin, to prostate cancer cells. In present research,
ROS plays a vital role in Ca 37 treated prostate cancers.
Doxorubicin and cisplatin are also reported to induce
cancer cell death through ROS generation [36, 37]. How-
ever, curcumin exhibits therapeutic promise for prostate
cancer through various pathways [2], whereas ROS is only
partially involved. The combination of two or more dif-
ferent pathways (ROS and others) may explain why Ca 37
enhances the growth inhibitory effect of curcumin. It is a
hint for us to pay attention to the synergy between struc-
tural analogues with different mechanisms but similar
biological effects.
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5. Shankar S, Srivastava RK (2007) Involvement of Bcl-2 family
0
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1
1
In conclusion, the curcumin analogue Ca 37 suppresses
both the proliferation of prostate cancer cells in vitro and
prostate cancer tumor xenograft growth. In addition, Ca 37
enhances the inhibitory growth effects of curcumin. ROS
induction is involved in preferential toxicity of Ca 37
toward prostate cancer cells. Moreover, cell cycle arrest
mediated Ca 37-induced tumor cell growth inhibition
in vitro and in vivo. Therefore, we believe that Ca 37 has
the potential to be used as an anti-cancer candidate for
prostate cancer therapy.
1
1
6
8(2):101–111
Acknowledgments This study was partially supported by the
National Natural Science Foundation of China, Key Program
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3
0930105, Foundation of Xi’an Jiaotong University, New Century
Excellent Talents in University, the National Natural Science Foun-
dation of China (Grant No. 31070740), and the 985 and 211 Projects
of Xi’an Jiaotong University.
1
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method for a more specific and accurate assay of mitochondrial
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