Ursolic acid derivatives induce cell cycle arrest and apoptosis in NTUB1 cells associated with reactive oxygen species

Article abstract of DOI:10.1016/j.bmc.2009.08.046

Twenty-three ursolic acid (1) derivatives 2-24 including nine new 1 derivatives 5, 7-11, 20-22 were synthesized and evaluated for cytotoxicities against NTUB1 cells (human bladder cancer cell line). Compounds 5 and 17 with an isopropyl ester moiety at C-17-COOH and a succinyl moiety at C-3-OH showed potent inhibitory effect on growth of NTUB1 cells. Compounds 23 and 24 with seco-structures prepared from 1 also showed the increase of the cytotoxicity against NTUB1 cells. Exposure of NTUB1 to 5 (40 μM) and 23 (20 and 50 μM) for 24 h significantly increased the production of reactive oxygen species (ROS) while exposure of NTUB1 to 5 (20 and 40 μM) and 23 (20 and 50 μM) for 48 h also significantly increased the production of ROS while exposure of cells to 17 did not increase the amount of ROS. Flow cytometric analysis exhibited that treatment of NTUB1 with 5 or 17 or 23 led to the cell cycle arrest accompanied by an increase in apoptotic cell death after 24 or 48 h. These data suggest that the presentation of G1 phase arrest and apoptosis in 5- and 23-treated NTUB1 for 24 h mediated through increased amount of ROS in cells exposed with 5 and 23, respectively, while the presence of G2/M arrest before accumulation of cells in sub-G1 phase in 5-treated cells for 48 h also due to increased amount of ROS in cells exposed with 5. The inhibition of tubulin polymerization and cell cycle arrest at G2/M following by apoptosis presented in the cell cycle of 23 also mediates through the increase amount of ROS induced by treating NTUB1 with 23 for 48 h.

Full text of DOI:10.1016/j.bmc.2009.08.046

Bioorganic & Medicinal Chemistry 17 (2009) 7265–7274
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Ursolic acid derivatives induce cell cycle arrest and apoptosis in NTUB1
cells associated with reactive oxygen species
Huang-Yao Tu a, , A-Mei Huang b, , Bai-Luh Wei c, , Kim-Hong Gan a, Tzyh-Chyuan Hour b,
d
a,c,
*
Shyh-Chyun Yang a, , Yeong-Shiau Pu , Chun-Nan Lin
*
a Faculty of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan
b Institute of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
c Department of Life Science, National Taitung University, Taitung 950, Taiwan
d Department of Urology, College of Medicine, National Taiwan University, Taipei 100, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty-three ursolic acid (1) derivatives 2–24 including nine new 1 derivatives 5, 7–11, 20–22 were syn-
thesized and evaluated for cytotoxicities against NTUB1 cells (human bladder cancer cell line). Com-
pounds 5 and 17 with an isopropyl ester moiety at C-17-COOH and a succinyl moiety at C-3-OH
showed potent inhibitory effect on growth of NTUB1 cells. Compounds 23 and 24 with seco-structures
prepared from 1 also showed the increase of the cytotoxicity against NTUB1 cells. Exposure of NTUB1
Received 6 May 2009
Revised 21 August 2009
Accepted 22 August 2009
Available online 27 August 2009
to 5 (40
l
M) and 23 (20 and 50
l
M) for 24 h significantly increased the production of reactive oxygen
M) and 23 (20 and 50 M) for 48 h also signif-
Keywords:
Synthesis
species (ROS) while exposure of NTUB1 to 5 (20 and 40
l
l
icantly increased the production of ROS while exposure of cells to 17 did not increase the amount of ROS.
Flow cytometric analysis exhibited that treatment of NTUB1 with 5 or 17 or 23 led to the cell cycle arrest
accompanied by an increase in apoptotic cell death after 24 or 48 h. These data suggest that the presen-
tation of G1 phase arrest and apoptosis in 5- and 23-treated NTUB1 for 24 h mediated through increased
amount of ROS in cells exposed with 5 and 23, respectively, while the presence of G2/M arrest before
accumulation of cells in sub-G1 phase in 5-treated cells for 48 h also due to increased amount of ROS
in cells exposed with 5. The inhibition of tubulin polymerization and cell cycle arrest at G2/M following
by apoptosis presented in the cell cycle of 23 also mediates through the increase amount of ROS induced
by treating NTUB1 with 23 for 48 h.
Ursolic acid derivatives
Cytotoxicity
NTUB1
ROS
Cell cycle
Anticancer
Seco-compound
SAR
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
as signaling molecules and as such they may have a role in cell cy-
cle progression.7
Triterpenoids abundantly exist in plant kingdom. The triterpe-
noids, ursolic acid (1) and 1 derivatives, have been reported to have
antitumor,1,2 antiinflammatory,3 antiviral,4 and antioxidant
activities.5,6
Reactive oxygen species (ROS) are resulting in oxidation of var-
ious cell constituents as DNA, lipid, and proteins and consequently
these oxidations may cause damage to the cellular substance lead-
ing to cell death as the ultimate consequence. ROS have been
implicated in a number of disease including various forms of
non-hormone dependent cancers, atherosclerosis, ischemic reper-
fusion injury, neurodegenerative diseases, chronic inflammatory
disease, such as rheumatoid and psoriatic arthritis, and some fac-
tors underlying the aging process itself. ROS might also play a role
Several anticancer agents, such as arsenic trioxide, doxorubicin,
bleomycin, cisplatin, 5-Fu, and paclitaxel have been shown to in-
duce ROS generation in cancer cells. Various mechanisms have
been described, including respiratory chain disruption, redox cy-
cling, or p53-mediated mitochondrial oxidase activation.8
Recently, several structures and cytotoxic relationships have
been reported.9 However, a series of structures and cytotoxic rela-
tionships of 1 derivatives did not appear in literature. Based on the
above reason, we synthesized a series of 1 derivatives, evaluated
their cytotoxicities against human NTUB1 cells, and discussed their
structures and cytotoxic relationships and mechanisms of action.
2. Results and discussion
2.1. Chemistry
* Corresponding authors. Tel.: +886 7 3121101; fax: +886 7 3210638 (S.-C.Y.);
tel.: +886 7 3121101; fax: +886 7 5562365 (C.-N.L.).
E-mail addresses: scyang@kmu.edu.tw (S.-C. Yang), lincna@cc.kmu.edu.tw (C.-N.
Lin).
Leaves of loquat were extracted with NaOH solution. The NaOH
solution was separated by partition with organic solvent. The
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.046

7266
H.-Y. Tu et al. / Bioorg. Med. Chem. 17 (2009) 7265–7274
aqueous layer was neutralized with HCl solution to yield a precip-
itate. The precipitate was separated and purified by silica gel chro-
matography to yield 1. Compound 1 in acetone reacted with K2CO3
and various alkyl halides to afford different esters (2–11 and 19)
(Scheme 1). The hydroxyl group at C-3 in the ring A was treated
with various anhydrides to form different esters (12–17) (Scheme
2). Compound 1 was oxidized to the 3-keto compound 18 with
CrO3 in DMF and the treatment of 3-oxo-derivative 18 with m-
chloroperbenzoic acid (m-CPBA) afford lactone 20. Compound 18
in anhydrous MeOH/benzene was esterified with trimethylsilyldia-
zomethane (TMSCHN2) at room temperature to yield 19 (Scheme
3). Lactone 20 was cleavaged in the appropriate amount of MeOH
and H2SO4 as the catalyst to yield seco-compound 21 and cleavaged
with KOH in MeOH to yield seco-compound 22 (Scheme 3).
seco-Compound 22 in MeOH reacted with H2SO4 to yield seco-com-
pound 23. Compound 23 in anhydrous MeOH/benzene was esteri-
fied with trimethylsilyldiazomethane (TMSCHN2) at room
temperature to yield 24 (Scheme 3). The known 1 derivatives, 2–
4, 6, 12–19, 23, and 24, were identified by spectroscopic data and
compared with those data reported in literatures while the new
1 derivatives were also characterized by various spectroscopic
methods and compared with those data reported in literatures.
a
COOH
COOH
HO
RO
1
12: R = -COCH3
13: R = -COCH2CH3
14: R = -COCH2CH2CH3
15: R = -COCH=CHCH3
16: R = -COCH2CH2(CH3)2
17: R = -COCH2CH2COOH
Scheme 2. Reagents and conditions: (a) anhydrous acetic acid or various carboxylic
acid, DCC, DMAP, pyridine.
compound obtained from 22, such as 23, indicated the potently
cytotoxic activity and its dimethyl ester further enhanced the
inhibitory effect on the cell growth. It clearly indicated that the
3,4-seco-compound with dimethyl ester moieties substituted as
C-3 and C-17 obtained from cleavage of ring A displayed stronger
and concentration-dependent effect against NTUB1 cell growth.
For further evaluated the cytotoxic effect of 1 derivatives and
mechanisms of induced cancer cell death in vitro, cytotoxicities
of selective compounds 5, 17, and 23 against PC3 and A549 were
studied and compared with those of cytotoxicities against
NTUB1 cells (Fig. 1). As shown in Fig. 1, 5, 17, and 23 did not
exhibited stronger cytotoxic effects against PC3 and A549 than
2.2. Biological results and discussion
Cytotoxicities of 1 and its derivatives against NTUB1 cells were
studied, and cisplatin was used as the positive control. As shown in
Tables 1 and 2, compound 1 and most of its derivative showed sig-
nificant cytotoxic activities against NTUB1 cells. The esterification
of C-17-COOH of 1 and 18 with methyl halide such as 2 and 19 de-
creased the cytotoxic activity against NTUB1 cells, but the esterifi-
cation of 21 such as 24 enhanced the cytotoxicity against NTUB1
cells. The C-17-COOH of 1 esterified with the increased alkyl chain
of halides such as 3–8, 10 or prenyl halide such as 9 indicated that
all these compounds revealed stronger cytotoxicities than that of 2
against NTUB1 cells. The hydroxylation of C-17 ethyl ester of 3
such as 10 attenuated the cytotoxicity against NTUB1 cells. The
acetylation of C-3-OH of 1 enhanced the cytotoxic activity while
the C-3-OH of 1 modified to succinic ester, such as 17, potently en-
hanced the cytotoxicity activity against NTUB1 cells. Increasing
carbon chain of ester moieties attenuated the cytotoxic effect while
increasing the unsaturation of the same carbon chain of ester such
as 15 enhanced cytotoxic effect. The oxidation of C-3-OH (1 and 2)
to keto group (18 and 19) increased cytotoxicities while lactone
derivative 20 obtained from 18 and 3,4-seco-compounds 21 and
22 obtained from 20 weakened cytotoxic activities. The 3,4-seco-
those of cytotoxicities against NTUB1 cells while 50 lM 5, 17,
and 23 indicated same cytotoxic effect against these three hu-
man cancer lines.
ROS induce programed cell death or necrosis, induce or sup-
press the expression of many genes, and activate cell signaling cas-
cades.10 We first examined the effect of 5 and 23 on intracellular
ROS levels in NTUB1 cells. Exposure of cells to 10
40 M 5, and 20 and 50 M 23 for 24 h caused a significant in-
crease in intracellular ROS while this effect was inhibited by N-
acetyl-cysteine [NAC, a thiol antioxidant agent (Fig. 2)]. Exposure
of cells to 10 lM cisplatin, 20 and 40 lM 5, and 20 and 50 lM 23
for 48 h also caused a significant increase in intracellular ROS while
lM cisplatin,
l
l
this effect did not inhibit by NAC (Fig. 3).
ROS cause a wide range of adaptive cellular responses ranging
from transient growth arrest to permanent growth arrest, to apop-
tosis or to necrosis, dependent on the level of ROS. These responses
to allow organism to remove damage caused by ROS or allow
organisms to remove damage cells.7
29
30
20
21
22
19
12
26
18
17
11
25
a
13
14
COOH
1
4
COOR
9
28
2
16
10
8
15
3
27
7
5
HO
HO
6
23
24
2: R = -CH3
3: R = -CH2CH3
1
4: R = -CH2CH2CH3
5: R = -CH(CH3)2
6: R = -CH2CH2CH2CH3
7: R = -CH2CH2CH2CH2CH3
8: R = -CH2CH2CH2CH2CH2CH3
9: R =
10: R = -CH2CH2OH
11: R = -CH2COOC(CH3)3
Scheme 1. Reagents and conditions: (a) alkyl halide, K2CO3, (CH3)2CO.

H.-Y. Tu et al. / Bioorg. Med. Chem. 17 (2009) 7265–7274
7267
a
c
d
COOH
COOR
COOH
COOH
MeOOC
O
O
HO
O
18: R = -H
19: R = -CH3
1
20
21
b
e
f
COOH
COOR2
HOOC
HO
R1OOC
23: R1 = -H, R2 = -H
24: R1 = -CH3, R2 = -CH3
22
b
Scheme 3. Reagents and conditions: (a) CrO3, H2SO4, DMF; (b) TMSCHN2, MeOH, and benzene; (c) m-CPBA, CHCl3; (d) MeOH, H2SO4; (e) KOH, MeOH; (f) H2SO4, MeOH.
Table 1
Table 2
Cytotoxicities of 1–17 against NTUB1 cellsa (IC50 values in
l
M)
Cytotoxicities of 18–24 against NTUB1 cellsa (IC50 values in
lM)
COOH
COOH
COOR1
COOR3
O
O
R2O
HO
O
20
18, 19
1-11
12-17
Compound R1
R2
Cisplatin
3.27 0.10
29.44 1.90
1
2
3
4
5
6
7
8
H
CH3
CH2CH3
CH2CH2CH3
CH(CH3)2
CH2CH2CH2CH3
CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH3
37.13
13.45
18.28
0
0
0
COOH
COOR
5 HOOC
R4OOC
7.97 0.48
15.64 1.31
27.79 1.63
26.16 2.51
HO
22
21, 23, 24
9
10.93 2.01
Compound
R3
R4
R5
H
10
11
12
13
14
15
16
17
CH2CH2OH
CH2COOC(CH3)3
19.53
>30
14.27 2.14
0
18
19
20
21
22
23
24
H
CH3
21.44 15.50
29.57 4.65
l
M (64.85%)
COCH3
COCH2CH3
COCH2CH2CH3
COCH@CHCH3
>30
>30
>30
lM (63.91%)
lM (62.77%)
lM (56.04%)
>30
>30
l
l
M (72.53%)
M (76.47%)
0
CH3
11.94
H
CH3
H
CH3
25.49 1.46
15.63 1.82
COCH2CH2(CH3)2 30.98 4.14
COCH2CH2COOH 8.65 2.89
a
Data are presented as mean SD (n = 5). Compounds 18–24 dissolved in DMSO,
a
Data are presented as mean SD (n = 5). Compounds 1–17 or cisplatin dissolved
were diluted with culture medium containing 0.1% DMSO, respectively. The control
cells were treated with culture medium containing 0.1% DMSO. Cisplatin was used
as a positive control. When 50% inhibition could not reached at the highest con-
centration, then % of viability is given in parentheses.
in DMSO, were diluted with culture medium containing 0.1% DMSO, respectively.
The control cells were treated with culture medium containing 0.1% DMSO. Cis-
platin was used as a positive control. When 50% inhibition could not reached at the
highest concentration, then % of viability is given in parentheses.
ment with 10 and 20 lM cisplatin for 24 h led to a dose-
Whereas cell proliferation and differentiation are specifically in
the G1 phase and the G1–S transition in the cell cycle, the onco-
genic progress exerts its greatest effect by targeting particular reg-
ulators of G1 phase progression.11 The effect of positive control
cisplatin, 5, 17, and 23 on cell cycle progression was determined
by using fluorescence-activated cell sorting (FACS) analysis in pro-
pidium iodide-stained NTUB1 cells. As shown in Figure 4, treat-
dependent accumulation of cells in the G1 phase with a concomi-
tant increase in the population of sub-G1 phase. Treatment with 20
and 40
in a dose-dependent manner, accompanied by an increase in the
apoptotic cell death while treatment with 20–40 M 17 for 24 h in-
duced G2/M arrest before accumulation of cells in sub-G1 phase.
As shown in Figure 5, treatment with 10 M cisplatin for 48 h re-
lM 5 and 20 and 50 lM 23 for 24 h induced G1 phase arrest
l
l

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H.-Y. Tu et al. / Bioorg. Med. Chem. 17 (2009) 7265–7274
Figure 1. Cytotoxicities of 5 (A), 17 (B), and 23 (C) against NTUB1, PC3, and A549 cells. Cell viability was assessed by the MTT assay after treating with different
concentrations of compounds for 72 h. The data shown represent mean SD (n = 3) for one experiment performed in triplicate. *p <0.05, **p <0.01, and ***p <0.001 compared
to the control value, respectively.
sulted in S phase arrest while treatment with 20–40
50 M 23 for 48 h arrest G2/M phase before accumulation of cells
in sub-G1 phase.
l
M 5 and 20–
nology, Santa Cruz, CA, USA). Untreated NTUB1 cells (control)
showed diffuse staining throughout the cytoplasm and dense per-
inuclear staining. Treatment with taxol resulted in a distinctive
microtubule bundle that was likely due to stabilization of the rigid
microtubule network. Treatment with Compound 23, however, re-
sulted supported that cells treated with 23 for 48 h prevents
microtubule assembly in vivo by inhibiting tubulin polymerization.
l
It has been known that cellular ROS are essential to cell survival,
but the effect of ROS on cell is complex. Experimentally, a low con-
centration of H2O2 causes a moderate increase in proliferation of
many tumor cell lines, whereas a higher level results in slowed
growth, cell cycle arrest, and apoptosis or even necrosis.12 Treat-
ment of cells with 5 for 24 and 48 h exhibited induced G1 phase
and G2/M arrest, respectively, and increased the amount of ROS
in cells. It indicated that the cell cycle arrest and apoptosis induced
by 5 were correlated with ROS. Future investigation should address
the detailed mechanism of action inducing the cell cycle arrests at
G1 or G2/M phase and apoptosis. Treatment of cells with 20 and
3. Conclusion
Twenty-three ursolic acid (1) derivatives were synthesized and
a several of synthetic compounds indicated significant cytotoxic ef-
fects against NTUB1 cells. Compounds 5 and 23 revealed a partial
mechanism by which 5 and 23 mediated through generation of
ROS in NTUB1 cells induce inhibition of tubulin polymerization,
G2/M and G1 cell cycle arrest, and apoptosis. Our study may not
elucidate the detailed mechanism of synthetic ursolic acid deriva-
tives induced inhibition of tumor cell growth, but may encourage
the development of novel, efficient, and less toxic anticancer
agents targeting microtubules.
40 lM 17 for 24 h induces G2/M arrest before accumulation of cells
in sub-G1 phase. Further investigation should also address the de-
tailed mechanism of action of cell cycle arrest at G2/M phase and
apoptosis. The induction of increased amount of ROS and G1 phase
arrest by treatment of cells with 20 and 50 lM 23 for 24 h. It also
showed that the induction of G1 phase arrest by treatment of cells
with 23 for 24 h is due to the increased amount of ROS induced by
23 in cells. Treatment of cells with 20 lM 23 for 48 h resulted in
G2/M phase arrest.
4. Experimental section
All inhibition of tubulin polymerization has been implicated in
G2/M phase cell cycle arrest in various cancer cell lines.13 To iden-
tify these findings, we investigated whether treatment of cells with
4.1. General experimental procedures
20 lM 23 for 48 h affects in vivo microtubular architecture (Fig. 6).
Microtubule assembly was analyzed by indirect immunofluores-
cence staining using an anti-tubulin antibody (Santa Cruz Biotech-
IR spectra were determined with
a Perkin–Elmer system
2000 FTIR spectrophotometer. 1H (400 MHz) and 13C
(100 MHz) NMR were recorded on a Varian UNITY-400 spec-

H.-Y. Tu et al. / Bioorg. Med. Chem. 17 (2009) 7265–7274
7269
Figure 2. The effect of 5 and 23 on the production of ROS in NTUB1 cells. (A) Control; (B) 1 mM NAC; (C) 10
lM cisplatin; (D) 10 lM 5; (E) 20 lM 5; (F) 40 lM 5; (G) 20 lM
23; (H) 50 M 23. Cells were treated 1 mM NAC, 10 M cisplatin, and different concentrations of 5 and 23 for 24 h, respectively, and the amount of ROS was assayed by DCFH-
l
l
DA staining. Results were repeated by three independent experiments.
trometer, and mass were obtained on a JMX-HX 100 mass spec-
trometer. Mass analyses were within 0.4% of the theoretical
values, unless otherwise noted. Chromatography was performed
using a flash-column technique on Silica Gel 60 supplied by E.
Merck.
by silica gel chromatography and recrystallized for several times to
give ursolic acid (1) (21.9 g).
4.3. General procedure for esterification at C-17 carboxylic acid
of 1
4.2. Extraction of ursolic acid (1)
To a solution of 1 (30 mg, 0.07 mmol) in acetone was added
K2CO3 (20 mg, 0.14 mmol) and different alkyl halide (0.14 mmol).
The reaction mixture was stirred at the room temperature over-
night. The mixture was concentrated to dryness under reduced
pressure, diluted with water (30 mL), and extracted with DCM
(30 mL ꢀ 3). The organic phase was dried over Na2SO4, filtered,
and concentrated in vacuo to give the crude product. The crude
product was purified by chromatography using EtOAc/n-hexane
to afford purified compounds 2–11.
The method for extracting ursolic acid from leaves of loquat
(Eriobotrya japonica) has been reported. We used modified method
to isolate large amount of ursolic acid (1). Leaves of loquat (10 kg)
were extracted by methanol. The MeOH extract was shaken under
ultrasonic wave for 90 min at 50 °C. The extract was added 1%
NaOH solution to form salt and filtered. The solution was neutral-
ized with c-HCl to afford light yellow solid. The solid was purified

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H.-Y. Tu et al. / Bioorg. Med. Chem. 17 (2009) 7265–7274
Figure 3. The effect of 5 and 23 on the production of ROS in NTUB1 cells. (A) Control; (B) 1 mM NAC; (C) 10
lM cisplatin; (D) 10 lM 5; (E) 20 lM 5; (F) 40 lM 5; (G) 20 lM
23; (H) 50 M 23. Cells were treated 1 mM NAC, 10 M cisplatin, and different concentrations of 5 and 23 for 48 h, respectively, and the amount of ROS was assayed by DCFH-
l
l
DA staining. Results were repeated by three independent experiments.
4.3.1. Isopropyl 3b-hydroxyurs-12-en-28-oate (5)
21.8 (–CH(CH3)2), 23.3 (C-27), 23.4 (C-30), 24.1 (C-16), 27.2 (C-2),
28.0 (C-15), 28.1 (C-23), 30.7 (C-21), 33.2 (C-7), 36.6 (C-22), 36.9
(C-10), 38.6 (C-4), 38.7 (C-1), 38.9 (C-20), 39.1 (C-19), 39.6 (C-8),
42.1 (C-14), 47.6 (C-9), 47.7 (C-17), 52.8 (C-18), 55.2 (C-5), 66.9
(–COOCH(CH3)2), 79.0 (C-3), 125.5 (C-12), 138.1 (C-13), 176.9 (C-
28). EIMS (70 eV) m/z (% rel. int.): 498 [M]+ (3). HREIMS: calcd
for C33H54O3: 498.4073, found: 498.4071.
Compound 5 was prepared from 1 (50 mg, 0.11 mmol) follow-
ing the general procedure described for esterification at C-17 car-
boxylic acid using isopropyl iodide as alkyl halide moiety.
Compound 5 was obtained as a white solid (49.2 mg, 0.10 mmol,
90%), ½a 2D5
ꢁ
= +50. IR (KBr): 3447, 1715 cmꢂ1 1H NMR (CDCl3): d
.
0.77 (3H, m, H-24), 0.82 (3H, s, H-25), 0.85 (3H, d, J = 6.4 Hz, H-
30), 0.91 (3H, s, H-26), 0.93 (3H, d, J = 6.4 Hz, H-29), 0.98 (3H, s,
H-27), 1.07 (3H, s, H-23), 1.16 (3H, d, J = 6.0 Hz, –CH(CH3)2), 1.19
(3H, d, J = 6.0 Hz, –CH(CH3)2), 2.21 (1H, d, J = 11.2 Hz, H-18), 3.21
4.3.2. Pentyl 3b-hydroxyurs-12-en-28-oate (7)
Compound 7 was prepared from 1 (50 mg, 0.11 mmol) follow-
ing the general procedure described for esterification at C-17
carboxylic acid using pentyl iodide as alkyl halide moiety. Com-
pound 7 was obtained as a white solid (48.5 mg, 0.09 mmol,
(1H dd, J = 10.8, 5.2 Hz, H-3a), 4.91 (1H, m, –CH(CH3)2), 5.24 (1H,
t, J = 4.0 Hz, H-12).13C NMR (CDCl3): d 15.5 (C-24), 15.6 (C-25),
17.0 (C-11), 17.3 (C-26), 18.3 (C-6), 21.2 (C-29), 21.7 (–CH(CH3)2),

H.-Y. Tu et al. / Bioorg. Med. Chem. 17 (2009) 7265–7274
7271
Figure 4. Flow cytometry analysis of cisplatin, 5-, 17-, and 23-treated NTUB1 cells. NTUB1 cells (8 ꢀ 105 cells/10 cm dish) were treated with absence of cisplatin or
compounds (control, A), 10 lM cisplatin (B), 20 lM cisplatin (C), 10 lM 5 (D), 20 lM 5 (E), 40 lM 5 (F), 20 lM 17 (G), 40 lM 17 (H), 20 lM 23 (I), or 50 lM 23 (J) for 24 h. At
the time indicated, cells were stained with propidium iodide (PI), and DNA contents were analyzed by flow cytometry, apoptosis was measured by the accumulation of sub-
G1 DNA contents in cells. The control cells were treated with medium. Results were representative of three independent experiments.
84%), ½a 2D5
ꢁ
= +38. IR (KBr): 3447, 1718 cmꢂ1
0.75 (3H, s, H-25), 0.77 (3H, s, H-24), 0.85 (3H, d, J = 6.4 Hz, H-
30), 0.91 (3H, s, H-26), 0.91 (3H, t, J = 7.2 Hz, –CH2CH3), 0.93 (3H,
.
1H NMR (CDCl3): d
d, J = 6.4 Hz, H-29), 0.98 (3H, s, H-27), 1.07 (3H, s, H-23), 2.22
(1H, d, J = 10.8 Hz, H-18), 3.21 (1H, dd, J = 10.8, 4.8 Hz, H-3 ),
3.97 (2H, m, –COOCH2–), 5.23 (1H, t, J = 3.6 Hz, H-12). 13C NMR
a

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H.-Y. Tu et al. / Bioorg. Med. Chem. 17 (2009) 7265–7274
Figure 5. Flow cytometry analysis of cisplatin, 5-, 17-, and 23-treated NTUB1 cells. NTUB1 cells (8 ꢀ 105 cells/10 cm dish) were treated with absence of cisplatin or
compounds (control, A), 10 lM cisplatin (B), 20 lM 5 (C), 40 lM 5 (D), 20 lM 23 (E), or 50 lM 23 (F) for 48 h. At the time indicated, cells were stained with propidium iodide
(PI), and DNA contents were analyzed by flow cytometry, apoptosis was measured by the accumulation of sub-G1 DNA contents in cells. The control cells were treated with
medium. Results were representative of three independent experiments.
Figure 6. Effect of taxol and 23 on microtubule assembly. NTUB1 cells were treated with 10 nM taxol and 50
lM 23 for 24 h, and stained with anti-a-tubulin antibody and
Rodamine-conjugated secondary antibody. The cellular microtubules were observed by Axioskop 2 plus fluorescence microscope.
(CDCl3): d 14.0 (–CH2CH3), 15.4 (C-24), 15.6 (C-25), 17.0 (C-11),
17.1 (C-26), 18.3 (C-6), 21.2 (C-29), 22.3 (–CH2CH3), 23.3 (C-27),
23.5 (C-30), 24.2 (C-16), 27.2 (C-2), 28.0 (C-15), 28.1
(–CH2CH2CH3), 28.2 (C-23), 28.3 (–OCH2CH2–), 30.7 (C-21), 33.0
(C-7), 36.7 (C-22), 36.9 (C-10), 38.6 (C-1), 38.7 (C-4), 38.9 (C-20),
39.1 (C-19), 39.5 (C-8), 42.0 (C-14), 47.5 (C-9), 48.0 (C-17), 52.8
(C-18), 55.2 (C-5), 64.3 (–COOCH2–), 79.0 (C-3), 125.5 (C-12),
138.2 (C-13), 177.6 (C-28). EIMS (70 eV) m/z (% rel. int.): 526
[M]+ (3). HREIMS: calcd for C35H58O3: 526.4385, found: 526.3910.
boxylic acid using hexyl iodide as alkyl halide moiety. Compound
8 was obtained as a white solid (26.5 mg, 0.05 mmol, 45%),
½
a 2D5 = +34. IR (KBr): 3446, 1718 cmꢂ1 1H NMR (CDCl3): d 0.75
.
(3H, s, H-25), 0.77 (3H, s, H-24), 0.86 (3H, t, J = 7.6 Hz, –CH2CH3),
0.88 (3H, d, J = 6.4 Hz, H-30), 0.91 (3H, s, H-26), 0.93 (3H, d,
J = 6.4 Hz, H-29), 0.98 (3H, s, H-27), 1.07 (3H, s, H-23), 2.22 (1H,
ꢁ
d, J = 11.2 Hz, H-18), 3.21 (1H, dd, J = 9.6, 4.8 Hz, H-3a), 3.97 (2H,
m, –COOCH2–), 5.23 (1H, t, J = 3.6 Hz, H-12). 13C NMR (CDCl3): d
14.0 (–CH2CH3), 15.4 (C-24), 15.6 (C-25), 17.0 (C-11), 17.1 (C-26),
18.3 (C-6), 21.2 (C-29), 22.6 (–CH2CH3), 23.3 (C-27), 23.5 (C-30),
24.2 (C-16), 25.7 (OCH2CH2CH2–), 27.2 (C-2), 28.0 (C-15), 28.1 (C-
23), 28.5 (–CH2CH2CH3), 30.7 (C-21), 31.4 (–OCH2CH2–), 33.0 (C-
7), 36.7 (C-22), 36.9 (C-10), 38.6 (C-1), 38.7 (C-4), 38.9 (C-20),
4.3.3. Hexyl 3b-hydroxyurs-12-en-28-oate (8)
Compound 8 was prepared from 1 (50 mg, 0.11 mmol) follow-
ing the general procedure described for esterification at C-17 car-

H.-Y. Tu et al. / Bioorg. Med. Chem. 17 (2009) 7265–7274
7273
39.1 (C-19), 39.5 (C-8), 42.0 (C-14), 47.5 (C-9), 48.0 (C-17), 52.8 (C-
18), 55.2 (C-5), 64.3 (–COOCH2–), 79.0 (C-3), 125.5 (C-12), 138.2 (C-
13), 177.6 (C-28). EIMS (70 eV) m/z (% rel. int.): 540 [M]+ (5). HRE-
IMS: calcd for C36H60O3: 540.4542, found: 540.4538.
(C-14), 47.6 (C-9), 48.0 (C-17), 52.7 (C-18), 55.2 (C-5), 60.9
(–COOCH2–), 79.0 (C-3), 125.7 (C-12), 138.0 (C-13), 167.1
(–CH2CO–), 176.7 (C-28). EIMS (70 eV) m/z (% rel. int.): 570 [M]+
(6). HREIMS: calcd for C36H58O5: 570.4283, found: 570.4291.
4.3.4. 30-Methyl-20-butenyl 3b-hydroxyurs-12-en-28-oate (9)
Compound 9 was prepared from 1 (50 mg, 0.11 mmol) follow-
ing the general procedure described for esterification at C-17 car-
boxylic acid using 1-bromo-3-methyl-2-butene as alkyl halide
moiety. Compound 9 was obtained as a white solid (17.5 mg,
4.4. General procedure for esterification at C-3 hydroxy group
of 1
Compound 1 (50 mg, 0.11 mmol) was dissolved in different
anhydride (1 mL) and pyridine (1 mL), and the solution was stirred
at room temperature for 6 h. The reaction mixture was added
water (10 mL) and partitioned with DCM (15 mL ꢀ 3). The organic
solution was dried over Na2SO4 and concentrated in vacuo to give
crude product. The crude product was purified by chromatography
on a column of silica gel eluted with EtOAc/n-hexane to obtain
compounds 12–17.
0.03 mmol, 30%), ½a D25
ꢁ
= +36. IR (KBr): 3447, 1720 cmꢂ1 1H NMR
.
(CDCl3): d 0.75 (3H, s, H-25), 0.77 (3H, s, H-24), 0.85 (3H, d,
J = 6.4 Hz, H-30), 0.92 (3H, s, H-26), 0.94 (3H, d, J = 6.4 Hz, H-29),
0.99 (3H, s, H-27), 1.07 (3H, s, H-23), 1.68 (3H, s, –CH@C(CH3)2),
1.74 (3H, s, –CH@C(CH3)2), 2.23 (1H, d, J = 11.2 Hz, H-18), 3.23
(1H, dd, J = 10.8, 4.8 Hz, H-3a), 4.48 (1H, dd, J = 10.4, 7.2 Hz, –
COOCHH–), 4.51 (1H, dd, J = 10.4, 7.6 Hz, –COOCHH–), 5.24 (1H, t,
J = 4.0 Hz, H-12), 5.30 (1H, t, J = 7.6 Hz, –CH@C(CH3)2). 13C NMR
(CDCl3): d 15.4 (C-24), 15.6 (C-25), 17.0 (C-11), 17.1 (C-26), 18.0
(–CH@C(CH3)2), 18.3 (C-6), 21.2 (C-29), 23.3 (C-27), 23.5 (C-30),
24.2 (C-16), 25.8 (–CH@C(CH3)2), 27.2 (C-2), 28.0 (C-15), 28.1 (C-
23), 30.7 (C-21), 33.1 (C-7), 36.6 (C-22), 37.0 (C-10), 38.6 (C-1),
38.7 (C-4), 38.8 (C-20), 39.1 (C-19), 39.5 (C-8), 42.1 (C-14), 47.6
(C-9), 48.0 (C-17), 52.9 (C-18), 55.2 (C-5), 61.0 (–COOCH2–), 79.0
(C-3), 119.1 (–CH@(CH3)2), 125.5 (C-12), 138.1 (–CH@C(CH3)2),
138.2 (C-13), 177.5 (C-28). ESIMS (70 eV) m/z (% rel. int.): 547.
HRESIMS: calcd for C35H56O3Na: 547.4127, found: 547.4123.
4.5. 4-Hydroxy-3,4-seco-ursan-12-en-28-oic acid 3,4 lactone
(20)
A mixture of 18 (150 mg, 0.3 mmol) and 70–75% 3-chloroper-
oxybenzoic acid (200 mg, 0.8–0.9 mmol) in CHCl3 (10 mL) was stir-
red at room temperature for 72 h. More chloroform (20 mL) was
added and the organic layer was washed with aq KI (5%), aq
Na2SO3, water, aq NaHCO3 solutions. The solution was extracted
with CHCl3 (30 mL ꢀ 3), dried over Na2SO4, and concentrated in va-
cuo to give the crude product. The crude product was purified by
chromatography on a column of silica gel eluted with EtOAc/
DCM (1:6) to obtain 20 (91 mg, 0.19 mmol, 59%) as a white pow-
4.3.5. 20-Hydroxyethyl 3b-hydroxyurs-12-en-28-oate (10)
Compound 10 was prepared from 1 (50 mg, 0.11 mmol) follow-
ing the general procedure described for esterification at C-17 car-
boxylic acid using 2-chloroethanol as alkyl halide moiety.
Compound 10 was obtained as a white solid (28.5 mg, 0.06 mmol,
der, ½a 2D5
ꢁ
= +49. IR (KBr): 3448, 1756, 1714 cmꢂ1 1H NMR (CDCl3):
.
d 0.80 (3H, s, H-25), 0.85 (3H, d, J = 6.4 Hz, H-30), 0.95 (3H, d,
J = 6.0 Hz, H-29), 1.01 (3H, s, H-26), 1.07 (3H, s, H-27), 1.26 (3H,
s, H-24), 1.28 (3H, s, H-23), 2.20 (1H, d, J = 11.2 Hz, H-18), 5.25
(1H, t, J = 3.6 Hz, H-12). 13C NMR (CDCl3): d 13.6 (C-25), 16.8 (C-
26), 17.0 (C-11), 19.7 (C-24), 20.8 (C-27), 21.1 (C-30), 23.3 (C-29),
23.9 (C-6), 24.0 (C-16), 27.9 (C-15), 29.6 (C-7), 30.5 (C-21), 31.8
(C-2), 36.6 (C-22), 37.0 (C-9), 38.8 (C-20), 39.0 (C-19), 42.1 (C-
14), 45.3 (C-8), 47.9 (C-17), 52.5 (C-5), 55.2 (C-18), 74.9 (C-4),
125.3 (C-12), 138.1 (C-13), 175.8 (C-3), 182.8 (C-28). EIMS
(70 eV) m/z (% rel. int.): 470 [M]+ (2.3). HREIMS: calcd for
C30H46O4: 470.3395, found: 470.2825.
52%), ½a 2D5
ꢁ
= +35. IR (KBr): 3433, 1718 cmꢂ1 1H NMR (CDCl3): d
.
0.76 (3H, s, H-25), 0.77 (3H, s, H-24), 0.86 (3H, d, J = 6.4 Hz, H-
30), 0.91 (3H, s, H-26), 0.94 (3H, d, J = 6.4 Hz, H-29), 1.01 (3H, s,
H-27), 1.09 (3H, s, H-23), 2.24 (1H, d, J = 10.8 Hz, H-18), 3.21 (1H,
dd, J = 11.2, 4.8 Hz, H-3a), 3.79 (2H, m, –COOCH2–), 4.08 (1H,
ddd, J = 11.6, 5.6, 3.2 Hz, –CHHOH), 4.20 (1H, ddd, J = 11.6, 6.0,
3.6 Hz, –CHHOH), 5.26 (1H, t, J = 3.6 Hz, H-12). 13C NMR (CDCl3):
d 15.4 (C-24), 15.6 (C-25), 17.0 (C-11), 17.1 (C-26), 18.3 (C-6),
21.1 (C-29), 23.3 (C-27), 23.5 (C-30), 24.2 (C-16), 27.2 (C-2), 27.9
(C-15), 28.1 (C-23), 30.6 (C-21), 33.0 (C-7), 36.7 (C-22), 36.9 (C-
10), 38.6 (C-1), 38.7 (C-4), 38.8 (C-20), 39.1 (C-19), 39.5 (C-8),
42.2 (C-14), 47.5 (C-9), 48.3 (C-17), 53.0 (C-18), 55.2 (C-5), 61.4
(–CH2OH), 66.0 (–COOCH2–), 79.0 (C-3), 125.3 (C-12), 138.9 (C-
13), 177.9 (C-28). EIMS (70 eV) m/z (% rel. int.): 500 [M]+ (3). HRE-
IMS: calcd for C32H52O4: 500.3865, found: 500.3863.
4.6. Methyl 3,4-seco-ursan-4(23),12-dien-28-oic 3-oat (21)
A mixture of 18 (100 mg, 0.2 mmol) and 70–75% 3-chloroper-
oxybenzoic acid (100 mg, 0.4–0.5 mmol) in CHCl3 (10 mL) was stir-
red at room temperature for 72 h. The mixture was concentrated to
dryness under reduced pressure, added MeOH (10 mL) and c-
H2SO4 (three drops), and stirred at room temperature for 24 h.
The mixture was concentrated to dryness under reduced pressure
again, washed with aq NaHCO3 solution, extracted with CHCl3
(20 mL ꢀ 3), dried over Na2SO4, and concentrated in vacuo to give
the crude product. The crude product was purified by chromatog-
raphy on a column of silica gel eluted with EtOAc/n-hexane (1:3) to
4.3.6. tert-Butoxycarbonylmethyl 3b-hydroxyurs-12-en-28-
oate (11)
Compound 11 was prepared from 1 (50 mg, 0.11 mmol) follow-
ing the general procedure described for esterification at C-17 car-
boxylic acid using chloroacetic acid tert-butyl ester as alkyl
halide moiety. Compound 11 was obtained as a white solid
obtain 21 (22.0 mg, 0.05 mmol, 21%) as a white powder, ½a D25
ꢁ
= +8.
(38.9 mg, 0.07 mmol, 62%), ½a D25
ꢁ
= +20. IR (KBr): 3447, 1733 cmꢂ1
.
IR (KBr): 3461, 1735, 1693 cmꢂ1. 1H NMR (CDCl3): d 0.82 (3H, s, H-
27), 0.86 (3H, d, J = 6.4 Hz, H-30), 0.92 (3H, s, H-25), 0.94 (3H, d,
J = 6.4 Hz, H-29), 1.09 (3H, s, H-26), 1.72 (3H, s, H-24), 2.25 (1H,
d, J = 11.2 Hz, H-18), 3.65 (3H, s, –COOCH3), 4.64 (1H, s, –C@CHH),
4.86 (1H, s, –C@CHH), 5.26 (1H, t, J = 3.2 Hz, H-12). 13C NMR
(CDCl3): d 17.0 (C-11), 17.2 (C-26), 19.5 (C-25), 21.1 (C-29), 23.4
(C-27), 23.4 (C-24), 23.5 (C-30), 24.0 (C-16), 24.3 (C-6), 28.0 (C-
15), 28.5 (C-2), 30.6 (C-21), 31.6 (C-1), 33.9 (C-7), 36.7 (C-22),
37.7 (C-9), 38.8 (C-20), 39.1 (C-19), 39.2 (C-8), 39.2 (C-10), 42.4
(C-14), 48.0 (C-17), 50.3 (C-5), 51.6 (–COOCH3), 52.6 (C-18),
1H NMR (CDCl3): d 0.72 (3H, s, H-25), 0.78 (3H, s, H-24), 0.86
(3H, d, J = 6.4 Hz, H-30), 0.91 (3H, s, H-26), 0.94 (3H, d, J = 6.4 Hz,
H-29), 0.98 (3H, s, H-27), 1.08 (3H, s, H-23), 1.45 (9H, s, –
C(CH3)3), 2.25 (1H, d, J = 11.2 Hz, H-18), 3.21 (1H, dd, J = 9.6,
3.6 Hz, H-3a), 4.42 (2H, m, –COOCH2–), 5.25 (1H, t, J = 3.6 Hz, H-
12). 13C NMR (CDCl3): d 15.4 (C-24), 15.6 (C-25), 16.9 (C-26), 17.0
(C-11), 18.3 (C-6), 21.2 (C-29), 23.3 (C-27), 23.5 (C-30), 24.2 (C-
16), 27.2 (C-2), 28.0 (–OC(CH3)3), 28.0 (–OC(CH3)3), 28.1 (C-23),
29.7(–OC(CH3)3), 30.6 (C-21), 33.0 (C-7), 36.5 (C-22), 36.9 (C-10),
38.6 (C-1), 38.7 (C-4), 38.8 (C-20), 39.1 (C-19), 39.5 (C-8), 42.0

7274
H.-Y. Tu et al. / Bioorg. Med. Chem. 17 (2009) 7265–7274
113.6 (C-23), 125.7 (C-12), 137.9 (C-13), 147.3 (C-4), 174.6 (C-28),
183.4 (C-3). EIMS (70 eV) m/z (% rel. int.): 484 [M]+ (23). HREIMS:
calcd for C31H48O4: 484.3552, found: 484.3549.
4.10. Quantitative analysis of intracellular reactive oxygen
species (ROS)
Production of ROS was analyzed by flow cytometry as described
previously.17 Briefly, cells were plated and treated as indicated
conditions. Ten-micromolar dichlorofluorescein diacetate (DCFH-
DA; Molecular Probes, Eugene, OR) was added to the treated cells
30 min prior harvest. The cells were collected by trypsinization
and washed with PBS. The green fluorescence of intracellular DCF
(20,70-dichlorofluorescein) was then analyzed immediately by FAC-
Scan flow cytometer with a 525-nm band pass filter (Becton
Dickinson).
4.7. 3,4-seco-Ursan-4-hydroxy-12-en-3,28-dioic acid (22)
Compound 20 (30 mg, 0.06 mmol) in 5% methanolic KOH was
kept at room temperature for 48 h. The organic layer was removed
under pressure. The mixture was added water (10 mL) and ex-
tracted with EtOAc (10 mL ꢀ 3) to give the crude product. The
crude product was purified by column chromatography eluted
with acetone:DCM (1:3) and MeOH to obtain 22 (15 mg,
0.03 mmol, 48%) as a white powder, ½a D25
ꢁ
= +35. IR (KBr): 3447,
1690 cmꢂ1
.
1H NMR (CD3OD): d 0.89 (3H, d, J = 6.4, H-30), 0.91
4.11. Immunofluorescence microscopy
(3H, d, J = 6.4 Hz, H-29), 0.97 (3H, s, H-25), 1.10 (3H, s, H-26),
1.14 (3H, s, H-27), 1.25 (3H, s, H-24), 1.27 (3H, s, H-23), 2.22 (1H,
NTUB1 cells plated on coverslips were treated with no com-
d, J = 11.6, H-18), 2.30 (1H, m, H -2), 2.49 (1H, m, Hb-2), 5.27
pound as control, 10 nM taxol, 50 lM compound 23 for 24 h. After
a
(1H, t, J = 3.6 Hz, H-12). 13C NMR (CD3OD): d 17.7 (C-26), 17.8 (C-
11), 18.1 (C-25), 20.6 (C-29), 21.6 (C-6), 23.4 (C-27), 23.8 (C-30),
24.1 (C-16), 28.3 (C-24), 29.2 (C-15), 30.4 (C-2), 31.8 (C-21), 32.7
(C-7), 33.7 (C-23), 35.7 (C-22), 38.1 (C-20), 40.1 (C-19), 40.6 (C-
10), 42.1 (C-14), 43.8 (C-8), 49.0 (C-17), 52.9 (C-18), 53.0 (C-5),
54.4 (C-9), 76.2 (C-4), 127.2 (C-12), 139.4 (C-13), 181.7 (C-28 and
C-3). ESIMS (70 eV) m/z: 511 [M+Na]+. HRESIMS: calcd for
C30H48O5Na: 511.3399, found: 511.3402.
treatment, cells were fixed with 2% formaldehyde/PBS for 20 min,
washed with PBS, and cold methanol (ꢂ20 °C) for 3 min. After
washing with PBS, cells were added anti-a-tubulin monoclonal
antibody (Sigma, Co) in PBS and incubated for 3 h at room temper-
ature. Then cells were washed with PBS and reincubated with Rod-
amine-conjugated secondary antibody (Sigma, Co) in dark room for
1 h at room temperature. After being washed with PBS, coverslips
were mounted with 80% glycerol in PBS and examined with Axio-
skop 2 plus fluorescence microscope.18
4.8. Cell culture and MTT assay for cell viability/proliferation
4.12. Statistical analysis
NTUB1, a human bladder cancer cell line, was established from
a high-grade bladder cancer,14 PC3, and A549 cells were main-
tained in RPMI 1640 medium supplemented with 10% fetal bovine
Data were expressed as means SD. Statistical analysis were
performed using the Bonferroni t-test method after ANOVA for
multigroup comparison and the student’s t-test method for two
group comparison, with p <0.05 was considered to be statistically
significant.
serum (FBS), 100 unit/mL penicillin-G, 100
lg/mL streptomycin,
and 2 mM -glutamine. The cells were cultured at 37 °C in a humid-
L
ified atmosphere containing 5% CO2.
For evaluating the cytotoxic effect of the tested compound with
cisplatin, a modified 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltet-
razolium bromide (MTT, Sigma Chemical Co.) assay was per-
formed.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. Cells were then cultured in the presence of graded con-
centrations of the test compound with or without various concen-
trations of cisplatin (Pharmacia & Upjohn, Milan, Italy) at 37 °C for
Acknowledgment
This work was supported by a research grant from the National
Science Council of the Republic of China (NSC95-2320-B-037-037).
References and notes
72 h. At the end of the culture period, 50 lL of MTT (2 mg/mL in
PBS) was added to each well and allowed to react for 3 h. Following
centrifugation of plates at 1000g for 10 min, media were removed
and 150 lL DMSO were added to each well. The proportions of sur-
viving cells were determined by absorbance spectrometry at
540 nm using MRX (DYNEXCO) microplate reader. The cell viability
was expressed as a percentage to the viable cells of control culture
condition. The IC50s of each group were calculated by the median-
effect analysis and presented as mean standard deviation (SD).
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4.9. Flow cytometry analysis
DNA content was determined following propidium iodide (PI)
staining of cells as previously described.16 Briefly, 8 ꢀ 105 cells
were plated and treated with various concentrations of cisplatin
and various concentrations of 5, 17, and 23 for 24 or 48 h, respec-
tively. These cells were harvested by trypsinization, washed with
1ꢀ PBS, and fixed in ice-cold MeOH at ꢂ20 °C. After overnight incu-
bation, the cells were washed with PBS and incubated with 50
lg/
mL propidium iodide (Sigma, Co) and 50 g/mL RNase A (Sigma,
l
17. Pu, Y.-S.; Hour, T.-C.; Chen, J.; Huang, C.-Y.; Guan, J.-Y.; Lu, S.-H. Anticancer
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Co) in PBS at room temperature for 30 min. The fractions of cells
in each phase of cell cycle were analyzed using FACScan flow
cytometer and Cell Quest software (Becton Dickinson).