Lantabetulic acid derivatives induce G1 arrest and apoptosis in human prostate cancer cells

Article abstract of DOI:10.1002/ardp.201300224

Ten new lantabetulic acid (1) derivatives 2-11 were synthesized and their cytotoxicities against human prostate cancer cells were evaluated. PC3 cells treated with 10 μM 8 exhibited the most potent G1 phase arrest. In addition, 10 μM 8 markedly decreased the levels of cyclin E and cdk2 and caused an increase in the p21 and p27 levels, while 20 μM 8 mainly led to cell death through the apoptotic pathway, which correlated with an increase in reactive oxygen species levels, decreased expression levels of Bcl-2 and caspase-8, the induction of mitochondrial changes, and decreased levels of cytochrome c in mitochondria. The dual action of 8 could provide a new approach for the development of chemotherapeutic drugs. Ten new lantabetulic acid (1) derivatives 2-11 were synthesized and evaluated for their cytotoxic activities against human prostate cancer cells (PC3). Compound 8 led to G1 phase arrest (10 μM) as well as to apoptosis induction (20 μM). The dual action of 8 could provide a new approach for the development of chemotherapeutic drugs.

Full text of DOI:10.1002/ardp.201300224

42
Arch. Pharm. Chem. Life Sci. 2014, 347, 42–53
Full Paper
Lantabetulic Acid Derivatives Induce G1 Arrest and Apoptosis
in Human Prostate Cancer Cells
Kai-Wei Lin1, Zih-You Lin1, A-Mei Huang2, Jing-Ru Weng3, Ming-Hong Yen4, Shyh-Chyun Yang1*,
and Chun-Nan Lin1,3
1
Faculty of Fragrance and Cosmetics, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan
Institute of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
Department of Biological Science and Technology, School of Medicine, China Medical University, Taichung,
2
3
Taiwan
4
Institute of Natural Product, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan
Ten new lantabetulic acid (1) derivatives 2–11 were synthesized and their cytotoxicities against human
prostate cancer cells were evaluated. PC3 cells treated with 10 mM 8 exhibited the most potent G1 phase
arrest. In addition, 10 mM 8 markedly decreased the levels of cyclin E and cdk2 and caused an increase
in the p21 and p27 levels, while 20 mM 8 mainly led to cell death through the apoptotic pathway, which
correlated with an increase in reactive oxygen species levels, decreased expression levels of Bcl-2
and caspase-8, the induction of mitochondrial changes, and decreased levels of cytochrome c in
mitochondria. The dual action of 8 could provide a new approach for the development of
chemotherapeutic drugs.
Keywords: Apoptosis / Cytotoxicity / G1 arrest / Lantabetulic acid / Synthesis
Received: June 13, 2013; Revised: August 30, 2013; Accepted: September 5, 2013
DOI 10.1002/ardp.201300224
Introduction
tive, antioxidant, analgesic, cytotoxic, and antidepressive
effects [4]. Recently, a large number of synthetic derivatives of
natural triterpenoids have contributed to the pharmacologi-
cal characterization of these compounds [4].
Prostate cancer is the most often diagnosed cancer and the
most common malignant tumor in men in the Western
countries [1]. Current therapies, including radical prostatec-
tomy, local radiotherapy, or brachytherapy, have limited
efficacy against the metastatic disease [2]. Consumption of
plant-derived foods such as polyphenolic phytochemicals
with potent antioxidant activity may reduce the incidence of
prostate cancer [3]. Indeed, there has been an increase in the
use of dietary plant-derived food to prevent or treat cancer.
Hence, the development of plant-derived natural products
with strong antioxidant activity could appreciably reduce the
disease-related mortality.
Recently, we have reported several series of natural
triterpenoid derivatives and evaluated them as potent
anticancer agents [5, 6]. In addition, it has been reported
that the natural triterpenoid lantabetulic acid (1), which is
structurally different from other types of triterpenoids, causes
NUGC-3 and HONE-1 cells to show survival rates of 10%
and 38%, respectively [7]. However, a report on a series of
structures of lantabetulic acid derivatives in relationship to
their cytotoxic activities has so far not appeared in the
literature. Therefore, we synthesized a series of lantabetulic
acid derivatives, evaluated their cytotoxic effects against the
human prostate cancer cell line PC3, and discussed their
structure–cytotoxicity relationships. In this study, we also
reported the possible mechanism of induced cell death of the
selective compound 8.
Natural or semisynthetic triterpenoids have attracted
much interest because of their structural diversity and their
broad spectrum of pharmacological activities, such as anti-
inflammatory, gastroprotective, antimicrobial, antinocicep-
Correspondence: Chun-Nan Lin, Faculty of Fragrance and Cosmetics,
College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708,
Taiwan.
E-mail: lincna@cc.kmu.edu.tw
Fax: þ886 7 5562365
ꢀAdditional correspondence: Shyh-Chyun Yang,
E-mail: scyang@kmu.edu.tw
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43
Results and discussion
13C NMR spectrum of 1 (Table 2) was assigned by 1D and 2D NMR
techniques and is reported in this study.
Chemistry
The 1H NMR spectrum of 2 exhibited proton signals for five
tertiary methyls at d 0.83 (3H, s, Me-26), 0.94 (6H, s, Me-24 and
Me-27), 1.01 (3H, s, Me-23), and 1.67 (3H, s, Me-30), a methine
proton signal at d 2.97 (1H, dt, J ¼ 10.8, 4.4 Hz, Hb-19), a methyl
ester group at d 3.65 (3H, s, COOCH3), and two methylene
proton signals at d 3.71 (1H, d, J ¼ 8.8 Hz, Ha-25), 4.22 (1H, d,
J ¼ 8.8 Hz, Hb-25), 4.60 (1H, brs, Ha-29), and 4.72 (1H, brs, Hb-
29). The above 1H and 13C NMR spectra of 2 were almost
identical to the corresponding proton and carbon signals of 1
(Table 2), except for the appearance of an additional methyl
ester of proton and carbon signals at d 3.65 and 51.3 in the
spectrum of 2, respectively. In addition, the chemical shift of
C-28 of 2 was downfielded from d 180.1 to 176.6 compared to
that of 1. The above result supports that the structure of 2 was
characterized as shown in Scheme 1. The NMR spectra of 3–8
were similar to the corresponding proton and carbon signals
of 2, except for signals of an ester group substituted at C-17
and the chemical shift of Me-26 of 8 upfielded from d 0.83 to
0.65 compared to that of 2 (Table 2 and Experimental section).
The result also supports that the structures of 3–8 were
characterized as shown in Scheme 1.
To increase the anticancer activity of 1 isolated from the roots of
Rhus javanica L. var. roxburghiana, various 1 derivatives were
synthesized through chemical modification at the carboxylic
acid group at C-17 and the alcoholic group at C-3 (Table 1 and
Scheme 1). The C-17-COOH of 1 in anhydrous CHCl3 was
esterified with trimethylsilyldiazomethane (TMSCHN2) at room
temperature to yield 2, while the C-17-COOH of 1 in acetone was
treated with K2CO3, different alkyl halides or benzyl halide, or
2-chloroethanol or a-monochlorohydrin, and refluxed for 24 h
at 80°C, to yield compounds 3–8. In turn, the C-3-OH of 1 in
anhydrous pyridine was reacted with different anhydrides
of carboxylic acid or different carboxylic acids and refluxed at
80°C for 24 h, to yield compounds 9–11. The characterization of
1
the known compound 1 by H NMR has been reported in the
literature [7, 8]. For further identification of its structure, the
Table 1. Cytotoxicity (ED50 values in mM)a) of lantabetulic acid
derivatives.
In turn, the 1H NMR spectrum of 9 indicated proton signals
for five tertiary methyls at d 0.91 (3H, s, Me-26), 0.93
(3H, s, Me-27), 1.02 (3H, s, Me-24), 1.07 (3H, s, Me-23), and
1.69 (3H, s, Me-30), and an acetyl group at d 1.94 (3H, s, OAc).
The above 1H and 13C NMR spectra were similar to the
corresponding proton and carbon signals of 1 (Table 2 and
Experimental section), except for the carbon signals at C-3 and
C-23, and presenting an additional acetyl group at d 1.94 and
two carbon signals at d 20.9 and 171.1. In addition, the
chemical shift of C-3 was downfielded from d 98.4 to 04.7
compared to that of 1 (Table 2). This suggests that the
structure of 9 was characterized as shown in Scheme 1. The
NMR spectra of 10 and 11 were similar to the corresponding
data of 9, except for the carbon signals of C-3 and C-23, and
presenting additional signals of acyl groups substituted at C-3
of 10 and 11 (Table 2 and Experimental section), respectively.
The result also supports the characterization of the structures
of 10 and 11 as shown in Scheme 1. The IR, EIMS, and HREIMS
results also supported the characterization of this series of
compounds as shown in Scheme 1 (see Experimental section).
R2
O
O
R1O
_
1 11
Compd.
R1
R2
PC3
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
OH
OCH3
22.09 ꢁ 1.38
16.21 ꢁ 1.21
12.86 ꢁ 2.31
21.36 ꢁ 4.29
OCH2CH3
O(CH2)2OH
OCH2CHOHCH2OH 19.34 ꢁ 1.86
O(CH2)2CH3
OCH(CH3)2
OCH2C6H5
OH
7.02 ꢁ 2.23
10.36 ꢁ 1.07
6.80 ꢁ 2.97
21.93 ꢁ 2.96
14.44 ꢁ 1.51
9.51 ꢁ 2.41
4.56 ꢁ 0.76
H
9
10
11
Cisplatin
COCH3
COCH2CH3
COCH CHCH
OH
OH
–
–
3
Biology
PC3 cells were treated with various concentrations of 1 and
its derivatives 2–11 for 72 h and their effects on cell growth
were assessed by MTT assay (Table 1). Growth of the PC3 cells
was inhibited by all tested compounds in a concentration-
dependent manner, as shown in Table 1.
a)
Cytotoxicity was assayed by the MTT assay after treating with
various concentrations of compounds for 72 h. Data were
presented as means ꢁ SD (n ¼ 5). Compounds 1ꢂ11 or cisplatin
dissolved in DMSO were diluted with culture medium containing
0.1% DMSO, respectively. The cultured cells were treated with
culture medium containing 0.1% DMSO. Cisplatin was used as a
positive control.
The alkanoylation at C-3 of 1 with acetic anhydride resulted
in stronger cytotoxicity against the PC3 cells than that of 1,
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K.-W. Lin et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 42–53
2: R1 = H
R2 = OMe
3: R1 = H
R2 = OC2H5
a
11: R1 = COCH=CHCH3
R2 = OH
b
j
4: R1 = H
29
19
R2 = O(CH2)2OH
30
20
18
21
c
12
26
22
13
14
R2
11
25
i
17
16
28
1
10: R1 = COCH2CH3
9
O
2
3
R2 = OH
8
7
15
10
d
O
4
5: R1 = H
R2 = OCH2CHOHCH2OH
27
5
R1O
6
23
24
1: R1 = H
R2 = OH
h
e
6: R1 = H
R2 = O(CH3)2CH3
9: R1 = COCH3
R2 = OH
g
f
7: R1 = H
R2 = OCH(CH3)2
8: R1 = H
R2 = OCH2C6H5
Scheme 1. Synthesis of lantabetulic acid (1) derivatives. Reagents and conditions: (a) CHCl3, TMSCHN2, rt, 1 h; (b) acetone, K2CO3,
CH3CH2I, reflux, 80°C, 24 h; (c) acetone, K2CO3, CH2ClCH2OH, reflux, 80°C, 24 h; (d) acetone, K2CO3, a-monochlorohydrine, reflux, 80°C,
24 h; (e) acetone, K2CO3, CH3CH2CH2I, reflux, 80°C, 24 h; (f) acetone, K2CO3, (CH3)2CHI, reflux, 80°C, 24 h; (g) acetone, K2CO3,
benzylbromide, reflux, 80°C, 24 h; (h) pyridine, acetic anhydride, stirring, 60°C, 24 h; (i) pyridine, propionic acid, stirring, 60°C, 24 h; and (j) N,
N0-dicyclohexylcarbodiimide (in pyridine), crotonic anhydride, stirring, 60°C, 24 h.
while an increase in the carbon number of the alkyl chain of
aliphatic acids, such as in 10, appreciably enhanced the
cytotoxic effect against PC3 cells, as shown in Table 1. The
alkanoylation at C-3 with an unsaturated alkyl chain of
aliphatic acids, such as in 11, significantly enhanced
the cytotoxicity against the same cancer cell line (Table 1).
In turn, the esterification at C-17 of 1 with TMSCHN2 or
alkyl halides resulted in stronger cytotoxicity against PC3
cells than that of 1, while an increase in the carbon number
of the alkyl chain, such as in 3 and 6, significantly enhanced
the cytotoxic effect compared to that of 1 (Table 1). The
esterification at C-17 of 1 with a branched alkyl halide, such
as in 7, revealed weaker cell growth inhibition of PC3
cells than after the esterification at C-17 of 1 with the
corresponding straight-chain alkyl halide, such as in 6
(Table 1). The esterification at C-17 of 1 with benzyl halide,
such as in 8, revealed the strongest cytotoxicity against
PC3 cells in this series of compounds (Table 1). The
esterification at C-17 of 1 with 1-chloroethanol or a-mono-
chlorohydrin decreased the cytotoxic effect against PC3 cells
compared to the esterification at C-17 with the corresponding
alkyl halide, such as in 4 and 5.
The above results and discussion obtained from the
structure–cytotoxicity relationships suggest that this series
of compounds shows cytotoxicity against PC3 cells and that
the 3-hydroxy-17-benzyl ester of 1, such as 8, exhibited
the most potent cytotoxic effects against PC3 cells. This
compound could be used as a lead compound for the further
design and synthesis of novel anticancer agents.
To further evaluate the cytotoxic effects of derivatives of 1,
the mechanisms of cell death induced by the selective
compound 8 were studied in vitro in PC3 cells. Treatment of
PC3 cells with various concentrations of 8 for different
incubation times induced cell death in a concentration-
dependent manner, except for PC3 cells treated with 1 mM 8
for 72 h. Among them, exposure of PC3 cells to 8 for 72 h
exhibited the most potent cytotoxic effects (Fig. 1).
The generation of reactive oxygen species (ROS) is part of
the mechanism by which most chemotherapeutic agents kill
tumor cells [2]. We investigated whether the generation of
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Arch. Pharm. Chem. Life Sci. 2014, 347, 42–53
Anticancer Triterpenoids Induce G1 Arrest and Apoptosis
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Table 2. 13C NMR spectral data for compounds 1–11 (in CDCl3).
Position
1
2
3
4
5
6
7
8
9
10
11
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
C-30
35.1
29.5
98.4
40.4
49.6
19.7
32.0
39.6
45.2
35.6
22.2
25.7
38.4
42.1
29.8
32.0
56.3
49.0
46.7
150.3
30.5
36.9
26.8
19.5
67.7
16.5
14.5
180.1
109.9
19.5
35.6
29.7
98.3
40.3
49.7
19.6
32.0
39.6
45.2
35.2
22.2
25.8
38.4
42.1
29.6
32.1
56.5
49.2
46.8
150.4
30.6
36.9
26.9
19.4
68.0
16.2
14.3
176.6
109.6
18.4
51.3
35.6
29.6
98.3
40.3
49.7
19.7
32.0
39.7
45.2
35.2
22.2
25.8
38.4
42.1
29.5
32.0
56.4
49.1
46.8
150.5
30.6
36.9
26.8
19.4
68.0
16.2
14.3
176.0
109.5
18.4
35.5
29.7
98.3
40.3
49.5
19.7
31.9
39.6
45.2
35.1
22.2
25.7
38.4
42.1
29.5
32.0
56.4
49.1
46.7
150.5
30.5
36.9
26.8
19.4
67.9
16.1
14.3
176.5
109.7
18.4
35.5
29.7
98.3
40.3
49.5
19.7
31.9
39.6
45.2
35.1
22.2
25.8
38.4
42.1
29.5
32.0
56.7
49.1
46.7
150.5
30.5
36.9
26.8
19.4
68.0
16.2
14.3
176.5
109.7
18.4
35.5
29.6
98.3
40.3
49.7
19.7
32.0
39.6
45.2
35.2
22.2
25.8
38.4
42.1
29.5
32.0
56.5
49.1
46.8
150.5
30.6
37.0
26.8
19.4
68.0
16.2
14.3
176.1
109.5
18.4
35.5
29.6
98.3
40.3
49.7
19.7
31.7
39.7
45.2
35.2
22.2
25.8
38.4
42.1
29.5
32.0
56.3
49.0
46.9
150.6
30.6
37.0
26.8
19.4
68.0
16.2
14.3
176.5
109.5
18.4
35.5
29.6
98.3
40.3
49.7
19.7
31.9
39.6
45.2
35.5
22.1
25.8
38.3
42.1
29.5
32.0
56.5
49.1
46.7
150.4
30.5
36.8
26.8
19.4
67.5
16.0
14.3
175.7
109.6
18.4
36.9
30.5
104.7
40.4
51.5
19.6
33.2
38.6
46.8
34.4
22.2
25.7
38.8
42.5
29.9
32.0
56.3
49.1
45.8
150.2
30.5
36.9
29.3a)
19.4
64.5
16.0
14.9
181.6
109.8
19.4
36.9
30.5
104.7
40.4
51.5
19.6
33.2
40.4
46.8
34.4
22.3
25.7
38.8
42.5
29.9
32.0
56.3
49.1
45.8
150.2
30.5
36.9
29.3a)
19.5
64.7
16.0
14.9
182.0
109.8
19.4
37.0
30.5
104.7
40.4
51.5
19.6
33.1
40.4
46.8
34.4
22.1
25.7
38.8
42.5
29.9
32.0
56.3
49.1
45.8
150.2
30.5
37.0
29.3a)
19.5
64.6
16.0
14.9
181.8
109.8
19.4
COOCH3
COOCH2CH3
COOCH2CH3
COOCH2CH2OH
COOCH2CH2OH
59.8
14.3
61.5
65.5
Position
5
6
7
8
9
10
11
COOCH2CHOHCH2OH
COOCH2CHOHCH2OH
COOCH2CHOHCH2OH
COOCH2CH2CH3
COOCH2CH2CH3
COOCH2CH2CH3
COOCH(CH3)2
63.5
70.5
64.7
65.5
1.64
0.95
66.8
21.7
21.7
COOCH(CH3)2
COOCH2C6H5
COOCH2C6H5
65.7
121.8 ꢃ 2
128.2 ꢃ 2
128.5
136.4
CH3COO
CH3COO
CH3CH2COO
CH3CH2COO
CH3CH2COO
–
20.9
171.1
8.9
27.4
174.6
CH CH CHCOO
38.9
130.1
118.7
171.1
–
3
–
CH CH CHCOO
–
3
–
CH CH CHCOO
–
3
–
CH CH CHCOO
–
3
a)
Signals may be revised.
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intracellular ROS is part of the mechanism by which 8 induces
apoptosis in PC3 cells. The generation of ROS by 8 was assessed
by the fluorescent dye, H2DCFDA, which preferentially detects
intracellular ROS (Fig. 2). As shown in Fig. 2, exposure of cells
to 20 mM cisplatin and 5, 10, or 20 mM 8 caused a significant
increase in the level of intracellular generation of ROS, while
exposure of the cells to 1 mM N-acetylcysteine (NAC) caused a
decrease in the level of intracellular ROS. These results
indicate that various concentrations of 8 are able to generate
intracellular ROS and subsequently induce cell death. This
further suggests that the generation of ROS induced by 20 mM
8 may be correlated with the mechanism by which 8 induces
cell death.
Cell cycle arrest and apoptosis are the most common causes
of cell growth inhibition. To explore the underlying
mechanism of growth suppression induced by 8, cell cycle
analysis was performed by cell sorting of propidium iodide
(PI)-stained PC3 cells. As shown in Fig. 3, cells treated with 5
and 10 mM 8 for 24 h appeared to appreciably accumulate in
the G1 phase of the cell cycle, but cells treated with 20 mM 8
for 24 h showed a significantly increased population of sub-G1
Figure 1. Antiproliferative effects of various concentrations of 8 on
PC3 cells. Cells were incubated with various concentrations of 8 and
cell growth was determined after 24, 48, and 72 h of treatment. The
data shown represent the means ꢁ SD (n ¼ 5) for one experiment;
p < 0.05 (a), p < 0.01 (b), and p < 0.001 (c) compared to the control
values.
Figure 2. Effects of 8 and cisplatin on the production of ROS in PC3 cells. PC3 cells were treated with (A) control, (B) 20 mM cisplatin,
(C) 1 mM NAC, (D) 5 mM 8, (E) 10 mM 8, and (F) 20 mM 8 for 24 h, and the amount of ROS was assayed by H2DCFDA staining. Each sampling
measured the mean fluorescence intensity (MFI) of 3 ꢃ 105 cells corrected for autofluorescence. The control cells were treated with vehicle.
Similar results were obtained in three repeated experiments. (G) Each bar represents the means ꢁ SD of the MFI of 3 ꢃ 105 cells corrected
for autofluorescence from three independent experiments; p < 0.01 (a) compared to the respective control value.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 42–53
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Figure 3. Flow cytometry analysis of PC3 cells treated with cisplatin and 8. PC3 cells (3 ꢃ 105 cells/6-cm dish) were treated with vehicle
(control) (A), 20 mM cisplatin (B), 5 mM 8 (C), 10 mM 8 (D), or 20 mM 8 (E) for 24 h. At the indicated time, cells were stained with PI, and
the DNA contents were analyzed by flow cytometry; apoptosis was measured by the accumulation of sub-G1 DNA contents in the cells. The
control cells were treated with vehicle. The results are representative of three independent experiments. The bars (F) show the distribution
of PC3 cells treated with cisplatin and 8 in the phases of the cell cycle. The values show the percentages of cells in the individual phases
of the cell cycle from three independent experiments; p < 0.05 (a) and p < 0.01 (b) compared to the respective control value.
phase cells, which are apoptotic cells. These results suggest
that the induction of cell death by 5 and 10 mM 8 is mainly due
to arresting cells in the G1 phase of the cell cycle, while the
induction of cell death by 20 mM 8 is mainly due to apoptosis.
Induction of cell apoptosis was further confirmed by using
a dual staining approach with PI and annexin V-FITC staining.
Annexin V-positive/PI-negative (early apoptotic) and annexin
V-positive/PI-positive (late apoptotic) cells were increased in
PC3 cells treated with 8 for 24 h. The results from the
apoptosis assays revealed that the cytotoxicity induced by
20 mM 8 was due to apoptosis and necrosis (Fig. 4). The number
of both early apoptotic and late apoptotic cells was increased
about sixfold at 20 mM 8, compared to the untreated cells.
We also validated that the apoptotic effect of treatment with
20 mM 8 for 24 h was inhibited by pretreatment with NAC
(Fig. 4). This result indicates that ROS generation is required
for apoptosis induced by 20 mM 8.
It has been suggested that ROS overproduction induced a
reduction in the mitochondrial membrane potential (MMP)
(Dcm) as well as mitochondrial dysfunction [9]. Thus, to detect
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K.-W. Lin et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 42–53
Figure 4. The graphs depict apoptotic and necrotic populations of PC3 cells double stained with PI- and FITC-labeled annexin V. PC3 cells
were treated with vehicle (control), 5, 10, or 20 mM 8, and pre-incubated with 1 mM NAC for 0.4 h prior to exposure to 20 mM 8 for 24 h.
The results are expressed as percent of total cells.
changes in the MMP, the specific fluorescent probe JC-1
(5,50,6,60-tetrachloro-1,10,3,30-tetraethyl benzimidazolyl carbo-
cyanine iodide) was used. As shown in Fig. 5, the positive
control carbonyl cyanide 3-chlorophenylhydrazone (CCCP)
and 20 mM 8 significantly increased the number of JC-1
monomers (R2) and decreased that of JC-1 aggregates (R1). This
suggests that 20 mM 8 dramatically decreased the Dcm in PC3
cells. The marked collapse of the MMP suggests that a
dysfunction of mitochondria is involved in the oxidative burst
and apoptosis induced by 20 mM 8. Hence, we demonstrated
that 20 mM 8 induced cell death in PC3 cells mainly by
apoptosis through the mitochondria-mediated pathway.
Regulation of cell cycle progression in cancer cells is
considered to be a potentially effective mechanism for growth
control [10]. Since treatment of cells with 10 mM 8 for 24 h
exhibited the most potent G1 phase arrest, we next
determined the expression of cell cycle-related protein levels
by 10 mM 8. The expression of the cyclin-dependent kinase 2
(cdk2) inhibitors, p21 and p27, which regulate the progres-
sion of cells in the G1 phase of the cell cycle, was assessed. The
protein levels of p21 and p27 were increased following
treatment with 10 mM 8 for 24 h (Fig. 6A). In addition,
the expression levels of the G1 positive regulators, cdk2 and
cyclin E, were down-regulated following treatment with
10 mM 8 for 24 h (Fig. 6A). This indicates that the G1 arrest
induced by 10 mM 8 might be mediated through the
up-regulation of the p21 and p27 proteins, which enhance
the formation of heterotrimeric complexes with G1-S cyclin-
dependent kinases and cyclins, thereby inhibiting their
activities [10]. The above results revealed that exposure of
cells to 10 mM 8 had antiproliferative effects on tumor cells,
which accumulated in the G1 phase of the cell cycle, caused by
the decreased activity of a key driver of S phase progression,
cdk2 [11]. This suggests that 10 mM 8 could arrest the
checkpoint of G1/S transition and inhibit cancer cells to
freely enter into the next S phase.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 42–53
Anticancer Triterpenoids Induce G1 Arrest and Apoptosis
49
Figure 5. Collapse of MMP induced by CCCP and 8. PC3 cells were treated without CCCP and 8 (control; A), 10 mM CCCP (B), 5 mM 8 (C),
10 mM 8 (D), or 20 mM 8 (E) for 24 h. Appropriate gates were made to define JC-1 aggregates (R1) and JC-1 monomers (R2). The bars
(F) show the percentages of depolarized populations and the results are shown as the means ꢁ SD of triplicate experiments: p < 0.05
(a) compared to the respective control value.
Anti-apoptotic proteins (e.g., Bcl-2 and Bcl-xL) are essential
for the regulation of apoptosis through caspase signaling [12].
Previous studies have exhibited that caspase-8 cleaves Bid to
form tBid, which through direct correlation with an anti-
apoptotic member of the Bcl-2 family releases pro-apoptotic
Bax or Bak to generate pores in the mitochondrial membrane,
leading to the cytochrome c-dependent caspase-3 pathway [13,
14]. Since Western blot analysis displayed that the expression
levels of Bcl-2 and caspase-3 and -8 were decreased in response
to treatment with 20 mM 8 (Fig. 6B), this may suggest that
caspase-3 activation elicited by 20 mM 8 might result from
upstream caspases such as caspase-8. Thus, caspase-8 might
play an initiating role.
Since an increase in the permeability of the outer
mitochondrial membrane leads to the release of several
apoptotic factors, such as cytochrome c, we investigated
whether the apoptosis induced by 20 mM 8 in PC3 cells
involved the release of cytochrome c. Western blot analysis
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50
K.-W. Lin et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 42–53
a Varian UNITY-400 spectrometer. Low-resolution mass spectra
and high-resolution mass spectra data were obtained on a JMX-HX
100 mass spectrometer. Chromatography was performed using
the flash-column technique on Silica Gel 60 supplied by E. Merck.
Chemistry
Extraction of lantabetulic acid (1)
Compound 1 was isolated from the roots of R. javanica L. var.
roxburghiana. The structure was determined by various spectro-
scopic methods and compared to data reported in the
literature [7].
Esterification at the C-17 carboxylic acid of 1
Methyl 3,25-epoxy-3a-hydroxylup-20(29)-en-28-oate (2): To
compound 1 (50 mg, 0.11 mmol) in CHCl3 two drops of TMSCHN2
were added . The reaction mixture was stirred at room temperature
for 1h. The mixture was concentrated to dryness under reduced
pressure. The crude product was purified by chromatography
using n-hexane/acetone (5:0.5) to give a colorless powder (21mg,
Figure 6. Compound 8 alters the expressions of proteins involved
in the cell cycle and apoptosis. The levels of (A) cell cycle-related
proteins (p21, p27, cdk2, and cyclin E) and (B) apoptosis-related
proteins (Bcl-2, caspase-3, caspase-8, and cytochrome c) were
determined in PC3 cells treated with vehicle (control) or 10 or 20 mM
8, for 24 h. The Western blot analysis shown here is representative
of three independent experiments with similar results. b-Tubulin
and b-actin were employed as loading controls, respectively.
25
0.04mmol, 41%), ½aꢄD 59 (c 0.1, CHCl3), IR (KBr) cmꢂ1 3390, 1724,
1639. 1H NMR (CDCl3) d 0.83 (3H, s, Me-26), 0.94 (3H, s, Me-27), 0.95
(3H, s, Me-24), 1.01 (3H, s, Me-23), 1.67 (3H, s, Me-30), 2.97 (1H, dt,
J ¼ 10.8, 4.4 Hz, Hb-19), 3.65 (3H, s, COOCH3), 3.71 (1H, d, J ¼ 8.8 Hz,
Ha-25), 4.22 (1H, d, J ¼ 8.8Hz, Hb-25), 4.60 (1H, brs, Ha-29), 4.72 (1H,
brs, Hb-29). 13C NMR (CDCl3) see Table 2. EIMS (70 eV) m/z (% rel. int.),
484 [M]þ (24). HREIMS m/z calcd. for C31H48O4, 484.3552; found:
484.3534.
indicated that the level of cytochrome c in the mitochondria
decreased upon treatment with 20 mM 8 and correlated with a
decrease in the MMP (Figs. 5E, F and 6B).
General procedure for the esterification at the C-17
carboxylic acid of 1
Conclusion
To compound 1 (30 mg, 0.06 mmol) in acetone K2CO3 (6.39 mg,
0.05 mmol) and different alkyl halides or benzyl halide or 2-
chloroethanol or a-monochlorohydrin (each at 0.3 mmol) was
added. The reaction mixture was refluxed for 24 h. The mixture
was concentrated to dryness under reduced pressure, diluted
with 10% HCl solution, and extracted with EtOAc. The organic
phase was dried over anhydrous Na2SO4, filtered, and concen-
trated in vacuo to give the crude product. The crude product was
purified by chromatography using n-hexane/acetone as eluate to
afford the purified compounds 3–8.
In this study, we report on the design, synthesis, and
biological evaluation of lantabetulic acid (1) and its
derivatives. Most of the compounds displayed significant
cytotoxic effects against human prostate cancer cells. The
selective compound 8, at concentrations of 5 and 10 mM,
mainly induced cell death through the induction of G1 phase
arrest in PC3 cells, while 20 mM 8 mainly induced cell death
through the apoptotic pathway (which correlated with
increased intracellular ROS generation), decreased the
expression of Bcl-2 and caspase-8, induced mitochondrial
Ethyl 3,25-epoxy-3a-hydroxylup-20(29)-en-28-oate (3)
Compound 3 was prepared from 1 (30 mg, 0.06mmol) following
the general procedure described for the esterification at the C-17
carboxylic acid of 1, using ethyl iodide as alkyl halide moiety.
changes and decreased the level of cytochrome
c in
mitochondria. This may also correlate with the decreased
expression of caspase-3. The dual action of 8 could provide a
new approach for the development of chemotherapeutic
drugs.
Compound
3 was obtained as colorless powder (29.9mg,
25
0.06mmol, 94%), ½aꢄD 52 (c 0.1, CH2Cl2), IR (KBr) cmꢂ1 3392,
1719, 1640. 1H NMR (CDCl3) d 0.83 (3H, s, Me-26), 0.93 (3H, s, Me-27),
0.95 (3H, s, Me-24), 1.00 (3H, s, Me-23), 1.24 (3H, t, J ¼ 6.8 Hz,
COOCH2CH3), 1.67 (3H, s, Me-30), 2.99 (1H, dt, J ¼ 10.8, 4.4 Hz, Hb-
19), 3.71 (1H, d, J ¼ 8.8 Hz, Ha-25), 4.21 (1H, d, J ¼ 8.8 Hz, Hb-25), 4.12
(2H, m, COOCH2CH3), 4.59 (1H, brs, Ha-29), 4.71 (1H, brs, Hb-29).
13C NMR (CDCl3) see Table 2. EIMS (70 eV) m/z (% rel. int.), 498 [M]þ
(90). HREIMS m/z calcd. for C32H50O4, 498.3708; found: 498.3710.
Experimental
Reagents, starting material, and solvents were purchased from
commercial suppliers. Cisplatin was obtained from Pharmacia
Upjohn (Milan, Italy). All culture reagents were obtained from
Gibco BRL. Optical rotations were recorded with a JASCO-370
polarimeter using CH2Cl2 as solvent. IR spectra were determined
20-Hydroxyethyl 3,25-epoxy-3a-hydroxylup-20(29)-en-28-
oate (4)
Compound 4 was prepared from 1 (30 mg, 0.06 mmol) following
the general procedure described for the esterification at the C-17
with
a Perkin-Elmer system 2000 FTIR spectrophotometer.
1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on
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Arch. Pharm. Chem. Life Sci. 2014, 347, 42–53
Anticancer Triterpenoids Induce G1 Arrest and Apoptosis
51
carboxylic acid of 1 using 2-chloroethanol to substitute for the
alkyl halide moiety. Compound 4 was obtained as colorless
Benzyl 3,25-epoxy-3a-hydroxylup-20(29)-en-28-oate (8)
Compound 8 was prepared from 1 (30 mg, 0.06 mmol) following
the general procedure described for the esterification at the C-17
carboxylic acid of 1 using benzyl bromide as alkyl halide moiety.
25
powder (25.9 mg, 0.05 mmol, 79%), ½aꢄD 28 (c 0.1, CHCl3), IR (KBr)
cmꢂ1 3398, 1720, 1645. 1H NMR (CDCl3) d 0.83 (3H, s, Me-26), 0.93
(3H, s, Me-27), 0.94 (3H, s, Me-24), 1.00 (3H, s, Me-23), 1.68 (3H, s,
Me-30), 2.97 (1H, dt, J ¼ 12.2, 6.8 Hz, Hb-19), 3.70 (1H, d, J ¼ 8.8 Hz,
Ha-25), 3.80 (2H, t, J ¼ 4.8 Hz, COOCH2CH2OH), 4.20 (1H, d,
J ¼ 8.8 Hz, Hb-25), 4.21 (2H, m, COOCH2CH2OH), 4.59 (1H, brs,
Ha-29), 4.71 (1H, brs, Hb-29). 13C NMR (CDCl3) see Table 2. EIMS
(70 eV) m/z (% rel. int.) 514 [M]þ (39). HREIMS m/z calcd. for
C32H50O5, 514.3658; found: 514.3646.
Compound
8 was obtained as colorless powder (25.9 mg,
25
0.05 mmol, 77%), ½aꢄD 51 (c 0.1, CHCl3), IR (KBr) cmꢂ1 3393,
1721, 1646. 1H NMR (CDCl3) d 0.65 (3H, s, Me-26), 0.91 (3H, s, Me-
27), 0.95 (3H, s, Me-24), 1.00 (3H, s, Me-23), 1.67 (3H, s, Me-30), 3.00
(1H, dt, J ¼ 10.8, 4.4 Hz, Hb-19), 3.69 (1H, d, J ¼ 8.8 Hz, Ha-25), 4.19
(1H, d, J ¼ 8.8 Hz, Hb-25), 4.59 (1H, brs, Ha-29), 4.71 (1H, brs, Hb-29),
5.11 (2H, d, J ¼ 12.4 Hz, COOCH2C6H5). 7.35 (5H, m, COOCH2C6H5).
13C NMR (CDCl3) see Table 2. EIMS (70 eV) m/z (% rel. int.) 583
[MþNa]þ (100). HREIMS m/z calcd. for C37H52O4, 560.3865; found:
560.3870.
20,30-Dihydroxypropyl 3,25-epoxy-3a-hydroxylup-20(29)-
en-28-oate (5)
Compound 5 was prepared from 1 (30 mg, 0.06 mmol) following
the general procedure described for the esterification at the C-17
carboxylic acid of 1 using a-monochlorohydrin to substitute
for the alkyl halide moiety. Compound 5 was obtained as
General procedure for the alkanoylation at the C-3 hydroxyl
of 1
To compound 1 (30 mg, 0.06 mmol) in 2 mL pyridine different
anhydrides of carboxylic acid (0.3 mL, 3.17 mmol) or different
carboxylic acids (0.3 mL, 4.01 mmol) were added. The mixture was
refluxed at 80°C for 24 h, neutralized with saturated sodium
carbonate solution, and extracted with EtOAc. The organic phase
was concentrated in vacuo to give the crude product. The crude
product was purified by chromatography using n-hexane/acetone
as eluate to afford the purified compounds 9–13.
25
colorless powder (24.2 mg, 0.04 mmol, 74%), ½aꢄD 35 (c 0.1,
1
CHCl3), IR (KBr) cmꢂ1 3391, 1722, 1641. H NMR (CDCl3) d 0.83
(3H, s, Me-26), 0.94 (3H, s, Me-27), 0.95 (3H, s, Me-24), 1.00
(3H, s, Me-23), 1.67 (3H, s, Me-30), 2.96 (1H, dt, J ¼ 11.6, 5.2 Hz,
Hb-19), 3.60 (1H, m, COOCH2CHOHCHHOH), 3.68 (1H, m,
COOCH2CHOHCHHOH), 3.72 (1H, d, J ¼ 8.8 Hz, Ha-25), 3.90
(1H, m, COOCH2CHOHCH2OH), 4.11 (1H, d, J ¼ 6.4 Hz, COOCHH-
CHOHCH2OH), 4.21 (1H, d, J ¼ 8.8 Hz, Hb-25), 4.23 (1H, d,
J ¼ 6.4 Hz, COOCHHCHOHCH2OH), 4.60 (1H, brs, Ha-29), 4.71 (1H,
brs, Hb-29). 13C NMR (CDCl3) see Table 2. EIMS (70 eV) m/z (% rel.
int.) 544 [M]þ (43). HREIMS m/z calcd. for C33H52O6, 544.3764;
found: 544.3768.
3a-Acetoxy-3,25-epoxylup-20(29)-en-28-oic acid (9)
Compound 9 was prepared from 1 (30 mg, 0.06 mmol) following
the general procedure described for the alkanoylation at the C-3
hydroxyl of 1 using acetic anhydride as anhydride moiety.
Compound
9 was obtained as colorless powder (24.6 mg,
25
0.05 mmol, 80%), ½aꢄD 57 (c 0.1, CH2Cl2), IR (KBr) cmꢂ1 1736,
Propyl 3,25-epoxy-3a-hydroxylup-20(29)-en-28-oate (6)
Compound 6 was prepared from 1 (30 mg, 0.06 mmol) following
the general procedure described for the esterification at the C-17
carboxylic acid of 1 using propyl iodide as alkyl halide moiety.
1701, 1646. 1H NMR (CDCl3)
d 0.91 (3H, s, Me-26), 0.93
(3H, s, Me-27), 1.02 (3H, s, Me-24), 1.07 (3H, s, Me-23), 1.69
(3H, s, Me-30), 1.94 (3H, s, OCOCH3), 3.00 (1H, dt, J ¼ 10.8, 6.0 Hz,
Hb-19), 4.07 (1H, d, J ¼ 11.6 Hz, Ha-25), 4.21 (1H, d, J ¼ 11.6 Hz,
Hb-25), 4.61 (1H, brs, Ha-29), 4.73 (1H, brs, Hb-29). 13C NMR (CDCl3)
see Table 2. EIMS (70 eV) m/z (% rel. int.) 512 [M]þ (3). HREIMS m/z
calcd. for C32H48O5, 512.3502; found: 512.3511.
Compound
6 was obtained as colorless powder (27.6 mg,
25
0.05 mmol, 90%), ½aꢄD 57 (c 0.1, CHCl3), IR (KBr) cmꢂ1 3393,
1720, 1639. 1H NMR (CDCl3) d 0.82 (3H, s, Me-26), 0.93 (3H, s,
Me-27), 0.95 (3H, s, Me-24), 0.95 (3H, t, J ¼ 7.2 Hz, COOCH2CH2CH3),
1.00 (3H, s, Me-23), 1.64 (2H, m, COOCH2CH2CH3), 1.68 (3H, s,
Me-30), 2.99 (1H, dt, J ¼ 10.8, 4.4 Hz, Hb-19), 3.70 (1H, d, J ¼ 8.8 Hz,
Ha-25), 4.02 (2H, m, COOCH2CH2CH3), 4.21 (1H, d, J ¼ 8.8 Hz,
Hb-25), 4.59 (1H, brs, Ha-29), 4.71 (1H, brs, Hb-29). 13C NMR (CDCl3)
see Table 2. EIMS (70 eV) m/z (% rel. int.) 512 [M]þ (99). HREIMS m/z
calcd. for C33H52O4, 512.3865; found: 512.3871.
3,25-Epoxy-3a-propionoxylup-20(29)-en-28-oic acid (10)
Compound 10 was prepared from 1 (30 mg, 0.06 mmol) following
the general procedure described for the alkanoylation at the C-3
hydroxyl of 1 using propionic acid as carboxylic acid moiety.
Compound 10 was obtained as colorless powder (22.4 mg,
25
0.04 mmol, 87.1%), ½aꢄD 36 (c 0.1, CH2Cl2), IR (KBr) cmꢂ1 1730,
1701, 1650. 1H NMR (CDCl3) d 0.91 (3H, s, Me-26), 0.93 (3H, s, Me-
27), 1.01 (3H, s, Me-24), 1.07 (3H, s, Me-23), 1.07 (3H, t, J ¼ 4.8 Hz,
OCOCH2CH3), 1.69 (3H, s, Me-30), 1.98 (2H, q, J ¼ 7.2 Hz,
OCOCH2CH3), 3.00 (1H, dt, J ¼ 10.8, 4.4 Hz, Hb-19), 4.06 (1H,
d, J ¼ 11.6 Hz, Ha-25), 4.22 (1H, d, J ¼ 11.6 Hz, Hb-25), 4.61 (1H, brs,
Ha-29), 4.73 (1H, brs, Hb-29). 13C NMR (CDCl3) see Table 2. EIMS
(70 eV) m/z (% rel. int.) 526 [M]þ (4). HREIMS m/z calcd. for C33H50O5,
526.3658; found: 526.3666.
Isoropyl 3,25-epoxy-3a-hydroxylup-20(29)-en-28-oate (7)
Compound 7 was prepared from 1 (30 mg, 0.06 mmol) following
the general procedure described for the esterification at the C-17
carboxylic acid of 1 using isopropyl iodide as alkyl halide moiety.
Compound
7 was obtained as colorless powder (30.4 mg,
25
0.06 mmol, 99%), ½aꢄD 19 (c 0.1, CHCl3), IR (KBr) cmꢂ1 3396,
1716, 1650. 1H NMR (CDCl3) d 0.83 (3H, s, Me-26), 0.93 (3H, s, Me-
27), 0.94 (3H, s, Me-24), 1.00 (3H, s, Me-23), 1.21, 1.23 (6H, d,
J ¼ 3.2 Hz, COOCH(CH3)2), 1.67 (3H, s, Me-30), 3.00 (1H, dt, J ¼ 6.8,
3.6 Hz, Hb-19), 3.70 (1H, d, J ¼ 8.8 Hz, Ha-25), 4.21 (1H, d, J ¼ 8.8 Hz,
Hb-25), 4.58 (1H, brs, Ha-29), 4.71 (1H, brs, Hb-29), 4.99
(1H, m, COOCH(CH3)2). 13C NMR (CDCl3) see Table 2. EIMS
(70 eV) m/z (% rel. int.) 512 [M]þ (43). HREIMS m/z calcd. for
C33H52O4, 512.3865; found: 512.3864.
3a-Crotonoxy-3,25-epoxylup-20(29)-en-28-oic acid (11)
Compound 11 was prepared from 1 (30 mg, 0.06 mmol) following
the general procedure described for the alkanoylation at the C-3
hydroxyl of 1 using N,N0-dicyclohexylcarbodiimide (in pyridine) as
substitute for pyridine and crotonic anhydride as anhydride
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52
K.-W. Lin et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 42–53
moiety. Compound 11 was obtained as colorless powder (22.3 mg,
Flow cytometry analysis
25
0.04 mmol, 69%), ½aꢄD 49 (c 0.1, CH2Cl2), IR (KBr) cmꢂ1 1731, 1698,
The DNA content was determined following PI staining of the
cells as previously described [17]. Briefly, 8 ꢃ 105 cells were plated
and treated with 20 mM cisplatin and various concentrations of 8
for 24 h. These cells were harvested by trypsinization, washed
with 1ꢃ PBS, and fixed in ice-cold MeOH at ꢂ20°C. After
overnight incubation, the cells were washed with PBS and
incubated with 50 mg/mL PI (Sigma) and 50 mg/mL RNase A
(Sigma) in PBS at room temperature for 30 min. The fractions of
cells in each phase of the cell cycle were analyzed using a
FACScan flow cytometer and CellQuest software (Becton
Dickinson).
1639. 1H NMR (CDCl3) d 0.90 (3H, s, Me-26), 0.93 (3H, s, Me-27), 1.01
(3H, s, Me-24), 1.06 (3H, s, Me-23), 1.68 (3H, s, Me-30), 1.98
–
(3H, m, OCOCH CHCH ), 2.99 (1H, dt, J ¼ 10.8, 4.4 Hz, H -19), 4.05
–
3
b
(1H, d, J ¼ 11.6 Hz, Ha-25), 4.22 (1H, d, J ¼ 11.6 Hz, Hb-25), 4.61
(1H, brs, Ha-29), 4.73 (1H, brs, Hb-29), 5.14 (1H, d, J ¼ 10.4 Hz,
–
–
–
OCOCH CHCH ), 5.19 (1H, m, J ¼ 10.4 Hz, OCOCH CHCH ).
–
3
3
13C NMR (CDCl3) see Table 2. EIMS (70 eV) (% rel. int.) 538 [M]þ
(7). HREIMS m/z calcd. for C34H50O5, 538.3658; found: 538.3644.
Biological assays
Cell culture and MTT assay for cell viability
Measurement of the mitochondrial membrane potential
PC3 cells (3 ꢃ 105) treated with 10 mM CCCP and different
concentrations of 8 were incubated for 30 min with 1 mM of
5,50,6,60-tetrachloro-1,10,3,30-tetraethyl benzimidazolyl carbocya-
nine iodide (JC-1) (Molecular Probes) in culture medium. The
resulting cells were washed three times with PBS and dislodged
with trypsin–EDTA. Cells were collected in PBS/2% BSA, washed
twice by centrifugation (800g, 5 min), and resuspended in 0.3 mL
PBS/2% BSA for analysis by a FACSCalibur flow cytometer (Becton
Dickinson). The cytometer settings were optimized for green (R1)
and red (R2) fluorescence and the ratio of JC-1 aggregates (red
fluorescence) to JC-1 monomers (green fluorescence) was
calculated to represent the MMP [18].
Human PC3 cells were maintained in RPMI 1640 medium
supplemented with 10% fetal bovine serum (FBS), 100 U/mL
penicillin-G, 100 mg/mL streptomycin, and 2 mM L-glutamine [15].
The cells were cultured at 37°C in a humidified atmosphere
containing 5% CO2. For evaluating the cytotoxic effects of the
tested compounds and the positive control cisplatin, a modified 3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT;
Sigma Chemical Co.) assay was performed [15]. Briefly, the cells
were plated at a density of 1800 cells/well in 96-well plates and
incubated at 37°C overnight before drug exposure. The cells were
then cultured in the presence of graded concentrations of the
tested compounds 1–11 and cisplatin (Pharmacia & Upjohn), at
37°C for 72 h, and various concentrations of 8 at 37°C for 24, 48,
and 72 h. At the end of the culture period, 50 mL MTT (2 mg/mL
in PB) was added to each well and allowed to react for 3 h.
Following centrifugation of the plates at 1000g for 10 min, the
medium was removed and 150 mL of DMSO was added to each
well. The proportions of surviving cells were determined by
absorbance spectrometry at 540 nm using an MRX (DYNEXCO)
microplate reader. The cell viability was expressed as the
percentage of the viable cells under control culture conditions.
The IC50 values of each group were calculated by the median-
effect analysis and presented as means ꢁ standard deviation (SD).
Western blot analysis
Cells were harvested by trypsinization and resuspended in a
suitable amount of PBS to adjust the cell numbers. The cells were
mixed with an equal volume of 2ꢃ sample buffer and boiled twice
for 10 min to denature the proteins. Cell extracts were separated
by SDS–PAGE. The proteins were transferred to nitrocellulose
membranes (Millipore, Billerica, MA, USA) using a semi-dry
blotter. The blotted membranes were treated with 5% w/v
skimmed milk in TBST buffer (100 mM tris–HCl pH 7.5, 150 mM
NaCl, 0.1% Tween-20). The membranes were incubated with
specific antibodies at 4°C overnight. The membranes were
washed with TBST buffer and incubated with the secondary
antibody at room temperature for another 1 h. Signals were
detected by chemiluminescence ECL reagent after TBST washing
and visualized on Fuji SuperRX film.
Quantitative analysis of intracellular ROS
Production of ROS was analyzed by flow cytometry as described
previously [16]. Briefly, cells were plated and treated as indicated.
Ten micromolar of 2,7-dichlorodihydrofluorescein diacetate
(H2DCFDA; Molecular Probes, Eugene, OR, USA) was added to
the treated cells 30 min prior to harvesting. The cells were
collected by trypsinization and washed with PBS. The green
fluorescence of intracellular 20,70-dichlorofluorescein (DCF) was
then analyzed immediately by a FACScan flow cytometer with a
525-nm band pass filter (Becton Dickinson, Germany).
Monoclonal antibodies specific for b-actin and cytochrome c
and antibodies against cyclin E, cdk2, cdk4, p21, p27, and b-
tubulin were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). Bcl-2, caspase-3, and caspase-8 were purchased
from Cell Signaling (Beverly, MA, USA).
Preparation of mitochondrial and cytosolic lysates
PC3 cells were washed with PBS, resuspended in ice-cold lysis
buffer (250 mM sucrose, 20 mM N-(2-hydroxyethyl)piperazine-N0-
(2-ethanesulfonic acid) (HEPES) pH 7.5, 10 mM KCl, 1.5 mM
MgCl2, 1 mM each of EGTA, EDTA, DTT, and PMSF, and 10 mg/mL
each of leupepten, aprotinin, and pepstatin A), and incubated
for 20 min. The cells were centrifuged at 750g for 10 min at 4°C,
and the supernatants were further centrifuged at 10,000g for
25 min at 4°C in order to prepare the cytosolic fraction. The
remaining pellets were resuspended in lysis buffer and used as
the mitochondrial fraction after centrifugation at 10,000g for
25 min [19].
Apoptosis assays
Apoptosis was measured using an annexin V-FITC apoptosis
detection kit (BD PharMingen, San Jose, CA, USA). Cells cultured
in 6-cm dishes were trypsinized and collected by centrifugation.
The cell pellet was washed, resuspended in 1ꢃ binding buffer,
and stained with annexin V-FITC, as recommended by the
manufacturer. Cells were also stained with PI to detect necrosis or
late apoptosis. The distribution of viable (FITC/PI-negative), early
apoptotic (FITC-positive/PI-negative), late apoptotic (FITC/PI-dou-
ble positive), and necrotic (PI-positive, FITC-negative) cells was
determined using
Dickinson). The results are expressed as percent of total cells.
a BD FACSAria flow cytometer (Becton
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 42–53
Anticancer Triterpenoids Induce G1 Arrest and Apoptosis
53
Data analysis
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2005, 52, 833–841.
Data were expressed as means ꢁ SD. Statistical analyses were
performed using the Bonferroni t-test method after ANOVA for
multigroup comparison and Student’s t-test method for two-
group comparison; p < 0.05 was considered to be statistically
significant.
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This work was supported by a grant from the National Science Council
of the Republic of China (NSC 101-2320-B-037-038).
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The authors have declared no conflict of interest.
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ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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