Anthraquinone derivatives induce G2/M cell cycle arrest and apoptosis in NTUB1 cells

Article abstract of DOI:10.1016/j.bmc.2011.07.021

Thirteen anthraquinone derivatives 5-17 including two 3-(3- alkylaminopropoxy)-9,10-anthraquinone (NHA) derivatives 5 and 6, and 11 1-hydroxy-3-(3-alkylaminopropoxy)-9,10-anthraquinone (MHA) derivatives 7-17 were synthesized, evaluated for cytotoxicities

Full text of DOI:10.1016/j.bmc.2011.07.021

Bioorganic & Medicinal Chemistry 19 (2011) 5670–5678
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Anthraquinone derivatives induce G2/M cell cycle arrest and apoptosis
in NTUB1 cells
Huang-Yao Tu a, , A-Mei Huang b, , Chi-Huang Teng a, , Tzyh-Chyuan Hour b, Shyh-Chyun Yang a,
,
⇑
Yeong-Shiau Pu c, Chun-Nan Lin d,e,
⇑
a Faculty of Pharmacy, School of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ROC
b Graduate Institute of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ROC
c Department of Urology, College of Medicine, National Taiwan University, Taipei 100, Taiwan, ROC
d Faculty of Fragrance and Cosmetics, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ROC
e Department of Biological Science and Technology, School of Medicine, China Medical University, Taichung 404, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirteen anthraquinone derivatives 5–17 including two 3-(3-alkylaminopropoxy)-9,10-anthraquinone
(NHA) derivatives 5 and 6, and 11 1-hydroxy-3-(3-alkylaminopropoxy)-9,10-anthraquinone (MHA)
derivatives 7–17 were synthesized, evaluated for cytotoxicities against two cancer cell lines, and assayed
the generation of reactive oxygen species (ROS) in NTUB1 cells (a human bladder carcinoma cell line).
Compound 9 bearing a pyrrolidinyl group induced the stronger cytotoxic effect than those of other syn-
thesized NHA and MHA derivatives. Exposure of NTUB1 cells to, 9, 13, and 17 for 24 h significantly
increased the production of ROS, respectively. Flow cytometric analysis exhibited that the exposure of
NTUB1 cells to the selective 9 led to the G2/M phase arrest accompanied by an increase of apoptotic cell
death after the incubation for 24 h. Compound 9 induced up-regulation of cyclinB1 and p21 expressions.
Biological results suggested that the induction of G2/M arrest, apoptosis, and cell death by 9 may asso-
ciate with increased expression of p21 and cyclin B1, elevation of Bax and p53 levels, and generation
of ROS in the cell. In conclusion, these series of compounds may be used as anticancer agents.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 30 April 2011
Revised 12 July 2011
Accepted 13 July 2011
Available online 21 July 2011
Keywords:
Synthesis
Anthraquinone
Reactive oxygen species
Anticancer
p21
CyclinB1
Bax
p53
1. Introduction
moderate increase of ROS may promote cell proliferation. The
amount of ROS in cancer cells is higher than that in normal cells.
Anthraquinone derivatives have many biological activities
including antimicrobial,1 antileukemia,2,3 and antitumor activi-
ties.4 Previously we have reported the synthesis and cytotoxic
When the increase of ROS in cancer cells reaches the toxic thresh-
old, it may overwhelm the antioxidant capacity of the cell and trig-
ger cell death. Many ROS-modulating agents such as arsenic
trioxide and elesclomol are used in cancer treatment because of
inducing the ROS accumulation in cancer cells.
In recent years, emodin, 1,3,8-trihydroxy-6-methyl-anthraqui-
none, inducing cytotoxicity through a ROS-dependent and p53
mechanism was reported.10 As part of our efforts to continually de-
velop potential anticancer agents, we further synthesized a new
series of NHA and MHA derivatives and reported their structures
and cytotoxic relationships against two cancer cell lines, induction
of the ROS accumulation, and mechanism of action in the present
paper.
effects of
a series of 1-hydroxy-3-(x-alkylaminopropoxy)-9,
10-anthraquinone,5 1,3-dihydroxy-9,10-anthraquinone (DHA),
1-hydroxy-3-(3-alkylaminopropoxy)-9,10-anthraquinone (MHA),
and 3-(3-alkylaminopropoxy)-9,10-anthraquinone (NHA) deriva-
tives.6 Doxorubicin and daunorubicin, well-known analogues of
anthraquinones, are used as anticancer drugs. It is valuable to
continuously synthesize other series of anthraquinone derivatives
and evaluate their anticancer activities.
Reactive oxygen species (ROS) are involved in multistage carci-
nogenesis. ROS may cause oxidative DNA damage and loss of DNA
repair capacity.7,8 It was reported that a radical therapeutic ap-
proach targeted cancer cells by ROS-mediated mechanism.9
A
2. Results and discussion
2.1. Chemistry
⇑
Corresponding authors. Tel.: +886 7 3121101; fax: +886 7 5562365.
E-mail addresses: scyang@cc.kmu.edu.tw (S.-C. Yang), lincna@cc.kmu.edu.tw
As shown in Scheme 1 and Table 1, a new series of MHA and
NHA derivatives were synthesized by the method described
(C.-N. Lin).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.021

H.-Y. Tu et al. / Bioorg. Med. Chem. 19 (2011) 5670–5678
5671
O
R
O
R
O
O
R
a
b
O
Br
OH
O
R'
O
O
1R=H
2R=OH
3 R=H
4 R=OH
5, 6 R=H
7-17 R=OH
Scheme 1. Reagents: (a) 1,3-dibromopropane, KOH, MeOH (b) various amines.
Table 1
Structure, yield, and cytotoxicity (ED50 values in
l
M)a of NHA and MHA derivatives
O
R
R'
O
O
5-17
Compound
R
R0
Yield (%)
NTUB1
PC3
N
5
6
7
H
15
30
35
—
—
H
24.45 0.88
22.93 2.43
HN
HN
OH
>50 (23.20 6.08)
18.87 1.02
>50 (19.20 9.08)
13.95 1.43
8
9
OH
OH
41
33
HN
N
9.72 1.21
7.64 0.68
8.89 0.09
N
10
11
OH
OH
25
28
12.30 3.05
OH
HN
OH
12.00 1.19
9.07 0.00
12
13
14
15
OH
OH
OH
OH
N
42
40
44
30
32.62 0.00
28.48 0.11
17.73 0.68
9.12 0.32
N
O
20.40 1.71
8.37 0.30
NH
N
N
N
>30 (15.54 6.73)
>50 (44.36 3.84)
OH
16
OH
OH
28
40
>50 (18.75 9.97)
>50 (18.54 5.03)
N
N
O
17
50.90 2.23
3.27 0.10
>30 (12.44 7.57)
4.56 0.76
O
N
Cisplatin
a
Cytotoxity was assessed by the MTT assay after treating with different concentrations of compounds for 72 h. Data were presented as mean SD (n = 5).Compound 1–17
or cisplatin dissolved in DMSO, 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. —, not determined. When 50% inhibition could not be reached at the highest concentration, the percentage of inhibition is
given in parentheses.
elsewhere.5 Briefly, potassium salt of 1 or 3 was allowed to react
with 1,3-dibromoalkane in appropriate solvent and then aminated
with appropriate amines to give various NHA and MHA derivatives.
14 indicated the most potent cytotoxic activity against NTUB1 cells
with an ED50 value of 8.37 0.30 M.
l
Cytotoxic activities of MHA derivatives 7–17 bearing a hydroxy
group at C-1 were stronger than those of NHA derivatives 6 except
for 7, 12, and 15–17. Most compounds with a hydroxy group at C-1
enhanced cytotoxic effects against all cancer cell lines used in this
study. Compound 8 showed stronger cytotoxic effect against cancer
cell lines used in Table 1 than those of 7. It clearly indicated that an
increase of carbon number of alkyl amine group such as 8 enhanced
the cytotoxicity against those cancer cell lines used in Table 1. Com-
pound 9 bearing a pyrrolidinyl or cyclohexylamino side chain, such
as 9 and 14, revealed potent cytotoxic activity than those of com-
pounds bearing other heterocyclic ring. The hydroxylation at C-2
of the N-alkylamino side chain of 8 such as 11 enhanced the
2.2. Biological results and discussion
Cytotoxic activities of synthesized NHA (5 and 6) and MHA
(7–17) derivatives were studied against NTUB1 (a human bladder
carcinoma cell line) and PC3 (a human prostate cancer cell line)
cancer cell lines. Results are listed in Table 1, most of these anthra-
quinone derivatives 5–17 showed cytotoxic activities against the
cancer cell line used in Table 1. Among these synthesized NHA
and MHA derivatives, 9 showed the strongest cytotoxic activity
against PC3 cells with an ED50 value of 7.64 0.68 lM. Compound

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H.-Y. Tu et al. / Bioorg. Med. Chem. 19 (2011) 5670–5678
cytotoxicity against NTUB1 and PC3 cancer cell lines. Compounds
with a piperazinyl moiety such as 15–17 did not display significant
cytotoxic activities against cancer cell lines used in Table 1.
For further evaluation of cytotoxicities on NTUB1, PC3, and SV-
HUC1 (an immortalized normal human uroepithelial cell line) cells,
cells were treated with different concentrations of selective 9 (3
As shown in Figure 4A, exposure of NTUB1 cells to 1 mM of NAC,
analyzedby (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) (MTT) method, exhibited slightly inhibitory cell growth
while exposure of NTUB1 with 9 combined with NAC significantly
increased the cell growth of NTUB1 cells. Immuno-fluorescence
staining with H2DCFDA showed that NAC attenuated the fluorescent
intensity through reducing the ROS accumulation in NTUB1 cells
(Fig. 4B). Doxorubicin could induce the ROS accumulation in cancer
cells11 used as the positive control. It also supported that the
cytotoxicity induced by 9 associated with the ROS accumulation in
NTUB1 cells.
and 10 lM), 13 (10 and 30 lM), and 17 (30 and 50 lM). The results
showed that 9 revealed less cytotoxicity against SV-HUC1 cells
than those of selective compounds against NTUB1 and PC3 cells
(Fig. 1A–C).
We further examined the effect of 9, 13, and 17 on intracellular
ROS generation in NTUB1 cells. It reported that exposure of NTUB1
cells to cisplatin, used as positive control, increased ROS accumula-
tion. N-acetyl cysteine (NAC), a thiol antioxidant, was used as the
negative control. As shown in Figure 2A–C, the exposure of NTUB1
Results of FACS analysis showed that exposure of NTUB1 cells to
20 lM 9 induced the G2/M phase arrest. The cell morphology of
NTUB1 cells was further examined by Immuno-fluorescence stain-
ing with 40,6-diamidino-2-phenylindole (DAPI) (Fig. 5). Doxorubi-
cin was used as a contrast because it was reported inducing G2/
cells to 20 lM of cisplatin and different concentrations of 9, 13, and
17 for 24 h caused an increase of the ROS accumulation in a dose-
dependent manner. Treatment of NTUB1 cells with 1 mM of NAC
attenuated the oxidation of H2DCFDA (2,7-dichlorodihydrofluores-
cein) in NTUB1 cells. It suggested that these compounds are able to
generate intracellular ROS and subsequently induced cell death.
Effects of cisplatin and 9 on the cell cycle progression were
determined by using fluorescence-activated cell sorting (FACS)
analysis in propidium iodide (PI)-stained NTUB1 cells. As shown
in Figure 3A and B, NTUB1 cells treated with 20 lM of cisplatin
for 24 h led to an accumulation of cells in G1 and S phases with
the concomitant increased the population of sub-G1 phase. NTUB1
cells was exposed to 10 and 20 lM 9, for 24 h induced G1 and G2/
M phase arrest and apoptosis.12 After treatment of 20
lM 9 for
24 h, the tetraploid DNA increased in NTUB1 cells. Most of NTUB1
cells did not exhibit the progression to the mitosis (Fig. 5). It sug-
gested that 20 lM 9 may influence the expression of protein in G2
phase of NTUB1 cells.
Progression of the cell cycle is controlled by the actions of var-
ious types of cyclins and cyclin-dependent protein kinase (Cdks).
As progression of the cell cycle moves into G2 phase, Cdk1 and
B-type cyclin (B1 abd B2) complexes play important roles in the
G2/M transition and M phase progression.13 p21 is present during
G1 phase while its levels decrease during S phase and increase dur-
ing G2 phase.14 Western blot analysis (Fig. 6) indicated that NTUB1
M phase arrest with the concomitant decrease and increase of the
cells were treated with 20
lM 9 for 24 h induced up-regulation of
population of sub-G1 phase, respectively.
cyclin B1 and p21 expressions in NTUB1 cells while NTUB1 cells
Figure 1. Cytotoxicities of 9 (A), 13 (B), and 17 (C) against NTUB1, PC3, and SV-HUC1 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.

H.-Y. Tu et al. / Bioorg. Med. Chem. 19 (2011) 5670–5678
5673
Figure 2. The effect of 9, 13, and 17 on the production of ROS in NTUB1 cells. (A) (a) control; (b) 1 mM NAC (N-acetyl cysteine, the negative control); (c) 20
lM cisplatin (the
positive control); (d) 10 lM 9; (e) 20 lM 9; (f) 20 lM 13; (g) 40 lM 13; (h) 20 lM 17; (i) 40 lM 17. Cells were treated 1 mM NAC, 20 lM cisplatin, and different
concentrations of 9, 13, and 17 for 24 h, respectively, and the amount of ROS was assayed by H2DCFDA staining. (B) Results were repeated by three independent experiments
and calculated the control as 0% increased ROS. ⁄P value <0.05 and ⁄⁄P <0.01 compared to amount of increased ROS of the control, respectively. (C) Overlapped data of control,
20 lM cisplatin, and 10 lM and 20 lM 9.
were treated with 10
lM 9 for 24 h induced down-regulation of
decreased and increased after 24 h of 10 and 20 lM 9 exposure
cyclinB1 and up-regulation of p21 expressions in NTUB1 cells.
Treatment of androgen-sensitive (LNCaP) cells with kuguacin J
resulted in significant G1-phase arrest of cell cycle progression,
along with reduction of cyclinD1, cyclinE, cdk2, and CDK4, and in-
crease of p21 and p27 at the protein level.15 The above result sug-
gested that the cell cycle arrest of NTUB1 cells at G1 phase induced
to NTUB1 cells. In contrast the GAPDH level, an internal control,
did not change. FACS analysis demonstrated that a decreased and
a marked accumulation of sub-G1 peaks induced by 10 and
20
by 20
and p53 levels detected after 24 h of 20
cell death or inhibition of cell growth induced by 10 l
l
M 9 in NTUB1 cells. It suggested that the apoptosis induced
M 9 in NTUB1 cells may correlate with elevation of Bax
M 9 treatment but the
M 9 in
l
l
by 10
lM 9 may correlate with up-regulation of p21 expression
and independent with the decrease of expression of cyclinB1.
Cell cycle progression is well regulated by the timing of the
expression of cell-specific cyclins. It was reported that treatment
with deoxypodophyllotoxin (DPPT) in Hela cells induced G2/M
phase arrest.16 In the Western blot analysis, treatment with DPPT
in Hela cells resulted in a rapid increase in cyclin B1 expression
was observed within 3 h of DPPT treatment that remained elevated
for up to 24 h. The expression of cyclin B1 returned to near control
level by 48 h.16 Based on the above results, it suggested that the
NTUB1 cells may correlate G1 phase arrest and may be indepen-
dent with down-regulation expression of P53 and Bax. It needs
to further examine the detailed molecular mechanism of cell cycle
arrest at G1 phase induced by 10 lM 9.
The level p53 is controlled by Mdm2 protein which degrades
p53 soon after its synthsis.18 When the cells are suffering certain
types of genotaxic stress, ATM or Chk2 can phosphorylate p53 at
multiple sites, thus preventing Mdm2-mediated degradation.18 It
also needs to study the levels of phosphor-ATM, phosphor-Chk2,
cell cycle arrest of NTUB1 cells at G2/M phase induced by 20
lM
and phpspho-p53 after 20 lM 9 exposure in NTUB1 cells.
9 may correlate with up-regulation of cyclin B1 and p21 expres-
sions in NTUB1 cells. It needs to examine the regulation of cyclin
B1 by the timing of the expression of cyclinB1.
3. Conclusion
The tumor suppressor p53 may activate mitochondrial-
mediated apoptosis by inducing the expression of proapoptotic
genes, including Bax, PUMA, and Noxa.17 Western blot analysis
(Fig. 6), demonstrated that the expression of p53 and Bax
It was reported that some NHA derivatives did not exert stron-
ger cytotoxicities than those of MHA derivatives.19 We further syn-
thesized a series of MHA and NHA derivatives and studied their

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H.-Y. Tu et al. / Bioorg. Med. Chem. 19 (2011) 5670–5678
Figure 3. Flow cytometry analysis of cisplatin and 9-treated NTUB1 cells. (A) NTUB1 cells (3 ꢁ 105 cells/ 6 cm dish) was treated with no compound (control) (a), cisplatin
20 lM (b), 10 lM 9 (c), and 20 lM 9 (d) for 24 h. At the time indicated, cells were stained with propidium iodide (PI), DNA contents were analyzed by flow cytometry, and
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. (B) The bars showed the distribution of 9-treated NTUB1 cells in the phases of the cell cycle. Values shown were means SD (n = 4) of the percentage of cells in
individual phases of the cell cycle from four independent experiments. ⁄P <0.05 and ⁄⁄P <0.01 compared to the control value, respectively.
cytotoxicities against two kinds of cancer cell lines. Among these
synthesized MHA and NHA derivatives, compound 9 revealed the
most potent cytotoxicity against cancer cell lines used in Table 1.
4. Experimental section
4.1. General experimental procedures
Exposure of 10 lM of 9, 10 and 30 lM of 13, and 30 and 50 lM
of 17 to SV-HUC1 cells, respectively, showed less cytotoxic effects
than those of NTUB1 and PC3 cells treated with corresponding con-
centrations of 9, 13, and 17. In the ROS assay, exposure of NTUB1
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 spectrometer, and masses
were obtained on a JMX-HX 100 mass spectrometer. Elemental
analyses were within 0.4% of the theoretical values, unless other-
wise noted. Chromatography was performed using a flash-column
technique on Silica Gel 60 supplied by E. Merck.
cells to 20
lM of 9 induced the increase of generation of ROS than
those of 20
lM of 13 and 17. NAC, an antioxidant, antagonized the
cytotoxicity of 9 in NTUB1 cells. It showed that 9 exhibited the
cytotoxicity in NTUB1 cells associated with the ROS accumulation.
Compound 9 induced G2/M arrest and apoptosis may correlate
with the increased expression p21 and cyclin B1 in 20
treated NTUB1 cells, and up-regulation of Bax and p53 expressions
in 20 M 9-treated NTUB1 cells. The induction of cell death
through G2/M phase arrest and apoptosis by 20 M 9 may corre-
late with accumulation of ROS and up-regulation of p21, cyclin
B1, p53, and Bax expression. Our study may not elucidate the
detailed mechanism of this series of anthraquinone derivatives in-
duced inhibition of tumor cell growth, but it may encourage the
development of additional series of anthraquinone derivatives as
anticancer agent.
lM 9-
4.2. 3-[3-(Dimethylamino)propoxy]-9,10-anthraquinone (5)
l
Compound 3 (60 mg, 0.17 mmol) in EtOH (40 mL) was added
dimethylamine (130 mg, 1.7 mmol) and then refluxed for 1 h. The
product was purified by column chromatography (silica gel and
MeOH) to give 5 (7 mg, 0.03 mmol, 15%). IR (KBr) 3384, 1676,
l
1654, 1590 cmꢀ1 1H NMR (CD3OD): d 2.29 (2H, t, –OCH2CH2–),
.
2.98 (6H, s, Me), 3.41 (2H, t, J = 8.0 Hz, –CH2N(CH3)2), 4.34 (2H, t,
J = 6.0 Hz, –OCH2–), 7.41 (1H, dd, J = 8.8, 2.8 Hz, H-2), 7.77 (1H, d, J
=2.8 Hz, H-4), 7.87 (2H, m, H-6, and H-7), 8.26 (3H, m, H-1, H-5,

H.-Y. Tu et al. / Bioorg. Med. Chem. 19 (2011) 5670–5678
5675
Figure 4. (A) Cytotoxicity of 9 and 9 combined with NAC. Cell viability was assessed by the MTT assay after treating with no compound (control), NAC, 9, and the combination
of NAC and 9 for 72 h. The data shown represent mean SD (n = 3) for one experiment performed in triplicate. ⁄P value <0.05 compared to the control value. (B) Immuno-
fluorescence staining with H2DCFDA validated NAC antagonizing the ROS accumulation in 9-treated NTUB1 cells. NTUB1 cells were incubated with no compound, NAC,
doxorubicin, 9, and the combination of NAC and 9 for 24 h, stained with H2DCFDA and microscoped.
Figure 5. Immuno-fluorescent staining of NTUB1 cells with DAPI after the treatment with no compound (control), 10 lM doxorubicin, and 10 lM and 20 lM 9 for 24 h.
and H-8). 13C NMR (CD3OD): d 25.6 (–OCH2CH2–), 43.7 (2 ꢁ Me),
56.5 (–CH2N(CH3)2), 66.8 (–OCH2–), 112.1 (C-4), 122.1 (C-2), 128.0
(C-5 and C-8), 128.6 (C-9a), 130.8 (C-1), 134.1 (C-8a), 134.9 (C-
10a), 135.1 (C-6), 135.6 (C-7), 135.9 (C-4a), 164.8 (C-3), 183.3 (C-
9), 184.2 (C-10). EIMS (70 eV) m/z (%rel. int.): 309(12) [M]+. HREIMS
m/z [M]+ 309.1367 (calcd for C19H19NO3, 309.1364).

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H.-Y. Tu et al. / Bioorg. Med. Chem. 19 (2011) 5670–5678
(–NHCH2–), 48.9 (–CH2NH–), 66.5 (–OCH2–), 107.2 (C-2), 108.1
(C-4), 110.7 (C-9a), 126.9 (C-8), 127.4 (C-5), 133.3 (C-8a and
C-10a), 135.1 (C-6), 135.2 (C-7), 135.3 (C-4a), 165.0 (C-1), 165.4
(C-3), 182.1 (C-10), 186.7 (C-9). EIMS (70 eV) m/z (%rel. int.): 339
(7) [M]+. HREIMS m/z [M+1]+ 340.1550 (calcd for C20H22NO4,
340.1549).
4.6. 1-Hydroxy-3-[3-(pyrrolidin-1-yl)propoxy]-9,10-
anthraquinoneꢂHCl (9)
Compound4 (80 mg, 0.22 mmol) in EtOH (40 mL) was added pyr-
rolidine (150 mg, 2.2 mmol). The mixture was treated and purified
as 5 to yield 9 (25 mg, 0.07 mmol, 33%) IR (KBr) 3421, 1670, 1637,
1589 cmꢀ1. 1H NMR (CDCl3): d 2.15 (2H, m, –OCH2CH2–), 2.29 (2H,
m, –CH2CH2–), 2.55 (2H, m, –CH2CH2–), 2.86 (2H, m, –NCH2–), 3.33
(2H, m, –NCH2–), 3.90 (2H, m, –CH2N–), 4.23 (2H, t, J = 6.4 Hz,
–OCH2–), 6.69 (1H, d, J = 2.4 Hz, H-2), 7.32 (1H, d, J = 2.4, H-4),
Figure 6. Regulations of p53, Bax, p21, and cyclinB1 in 9-treated NTUB1 cells were
analyzed. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the
internal control.
4.3. 3-[3-(Ethylamino)propoxy]-9,10-anthraquinoneꢂHCl (6)
Compound 3 (60 mg, 0.17 mmol) in EtOH (40 mL) was added
ethylamine (130 mg, 1.7 mmol). The mixture was treated and puri-
fied as 5 to yield 6 (15 mg, 0.05 mmol, 30%). IR (KBr) 3447, 1677,
7.81 (2H, m, H-6 and H-7), 8.29 (2H, m, H-5 and H-8), 12.10 (1H,
ꢃ
br s, NH), 12.86 (1H, s, OH-1). 13C NMR (CDCl3): d 23.5 (–CH2CH2–),
25.5 (–OCH2CH2–), 53.1 (–CH2NH–), 54.0 (–N(CH2CH2)2), 65.5
(–OCH2–), 107.0 (C-2), 107.6 (C-4), 110.6 (C-9a), 126.9 (C-8), 127.4
(C-5), 133.4 (C-8a and C-10a), 134.3 (C-6), 134.4 (C-7), 135.1
(C-4a), 164.6 (C-1), 165.4 (C-3), 182.3 (C-10), 186.9 (C-9). EIMS
(70 eV) m/z (%rel. int.): 351 (8) [M]+. HREIMS m/z [M]+ 351.1467
(calcd for C21H21NO4, 351.1470).
1655, 1590 cmꢀ1 1H NMR (DMSO-d6): d 1.21 (3H, t, J = 7.3 Hz,
.
Me), 2.10 (2H, m, –OCH2CH2–), 3.01 (2H, q, J = 7.2 Hz, –CH2CH3),
3.12 (2H, t, J = 7.6 Hz, –CH2NH–), 4.31 (2H, t, J = 6.0 Hz, –OCH2–),
7.45 (1H, dd, J = 8.8, 2.8 Hz, H-2), 7.62 (1H, d, J = 2.8 Hz, H-4),
7.92 (2H, m, H-6 and H-7), 8.19 (3H, m, H-1, H-5, and H-8), 8.45
(2H, br s, NH2þ). 13C NMR (DMSO-d6):
d 11.0 (Me), 25.4
(–OCH2CH2–), 42.1 (–CH2CH3), 43.5 (–CH2NH–), 65.6 (–OCH2–),
110.7 (C-4), 121.1 (C-2), 126.5 (C-5), 126.7 (C-8), 126.7 (C-9a),
129.6 (C-1), 133.0 (C-8a), 133.1 (C-10a), 134.3 (C-6), 134.7 (C-7),
135.0 (C-4a), 163.0 (C-3), 181.3 (C-9), 182.4 (C-10). EIMS (70 eV)
m/z (%rel. int.): 309 (11) [M]+. HREIMS m/z [M+1]+ 310.1441 (calcd
for C19H20NO3, 310.1443).
4.7. 1-Hydroxy-3-{3-[(2-hydroxyethyl)methylamino]propoxy}-
9,10-anthraquinoneꢂHCl (10)
Compound 4 (80 mg, 0.22 mmol) in EtOH (40 mL) was added
N-methylethanolamine (170 mg, 2.2 mmol). The mixture was
treated and purified as 5 to yield 10 (20 mg, 0.06 mmol, 25%).
IR (KBr) 3421, 1670, 1637, 1589 cmꢀ1 1H NMR (DMSO-d6): d
.
4.4. 1-Hydroxy-3-[3-(ethylamino)propoxy]-9,10-anthraquinone
ꢂHCl (7)
2.22 (2H, m, –OCH2CH2–), 2.82 (3H, s, –NCH3), 3.33 (4H, br s,
–CH2NCH2–), 3.77 (2H, m, –NCH2CH2OH), 4.25 (2H, t, J = 6.4 Hz,
–OCH2–), 5.37 (1H, t, J = 5.0 Hz, –CH2OH), 6.90 (1H, d, J = 2.4 Hz,
Compound 4 (80 mg, 0.22 mmol) in EtOH (40 mL) was added
ethylamine (90 mg, 2.2 mmol). The mixture was treated and puri-
fied as 5 to yield 7 (25 mg, 0.08 mmol, 35%). IR (KBr) 3447, 1675,
H-2), 7.21 (1H, d, J = 2.4 Hz, H-4), 7.92 (2H, m, H-6 and H-
ꢃ
7), 8.19 (2H, m, H-5 and H-8), 10.06 (1H, s, ffl NH), 12.76
(1H, s, OH-1). 13C NMR (DMSO-d6): d 23.1 (–OCH2CH2–),
52.6 (ꢀNCH2—), 55.2 (ꢀNCH2—), 56.9 (ꢀNCH3), 66.1 (–OCH2–),
106.7 (C-2), 107.7 (C-4), 110.6 (C-9a), 126.5 (C-8), 126.9
(C-5), 132.9 (C-8a and C-10a), 134.7 (C-6), 134.8 (C7), 134.9
(C-4a), 164.6 (C-1), 164.9 (C-3), 181.6 (C-10), 186.3 (C-9).
EIMS (70 eV) m/z (%rel. int.): 355 (2) [M]+. HREIMS m/z
[M+1]+ 356.1500 (calcd for C20H22NO5, 356.1498).
1635, 1591 cmꢀ1 1H NMR (DMSO-d6): d 1.22 (3H, t, J = 6.8 Hz,
.
Me), 2.14 (2H, m, –OCH2CH2–), 2.97 (2H, m, –CH2CH3), 3.07 (2H,
m, –CH2NH–), 4.28 (2H, t, J = 6.4 Hz, –OCH2–), 6.90 (1H, d,
J = 2.4 Hz, H-2), 7.22 (1H, d, J = 2.4 Hz, H-4), 7.94 (2H, m, H-6 and
H-7), 8.20 (2H, m, H-5 and H-8), 8.87 (2H, br s, NH2þ), 12.76 (1H,
s, OH-1). 13C NMR (DMSO-d6): d 11.1 (Me), 25.3 (–OCH2CH2–),
42.0 (–CH2CH3), 43.4 (–CH2NH–), 66.0 (–OCH2–), 106.8 (C-2),
107.7 (C-4), 110.3 (C-9a), 126.5 (C-8), 127.0 (C-5), 132.9 (C-8a
and C-10a), 134.7 (C-6), 134.8 (C-7), 134.9 (C-4a), 164.6 (C-1),
165.0 (C-3), 181.7 (C-10), 186.3 (C-9). EIMS (70 eV) m/z (% rel.
int.): 325 (6) [M]+. HREIMS m/z [M+1]+ 326.1391 (calcd for
4.8. 1-Hydroxy-3-[3-(2-hydroxypropylamino)propoxy]-9,10-
anthraquinoneꢂHCl (11)
C
19H20NO4, 326.1392).
Compound 4 (80 mg, 0.22 mmol) in EtOH (40 mL) was added 1-
amino-2-propanol (170 mg, 2.2 mmol). The mixture was treated
and purified as 5 to yield 11 (21 mg, 0.06 mmol, 28%). IR (KBr)
4.5. 1-Hydroxy-3-[3-(propylamino)propoxy]-9,10-
anthraquinoneꢂHCl (8)
3420, 1670, 1630, 1590 cmꢀ1 1H NMR (DMSO-d6): d 1.13 (3H, d,
.
J = 6 Hz, Me), 2.17 (2H, m, –OCH2CH2–), 2.80 (1H, m, –NHCHH–),
3.00 (1H, m, –NHCHH–), 3.10 (2H, m, –CH2NH–), 3.99 (1H,
m,–CH2CH(OH)–), 4.27 (2H, t, J = 6.4 Hz, –OCH2–), 5.40 (1H, br s,
ꢀCHOH), 6.89 (1H, d, J = 2.4 Hz, H-2), 7.20 (1H, d, J = 2.4 Hz, H-4),
Compound 4 (80 mg, 0.22 mmol) in EtOH (40 mL) was added
propylamine (130 mg, 2.2 mmol). The mixture was treated and
purified as 5 to yield 8 (31 mg, 0.09 mmol, 41%). IR (KBr) 3447,
1675, 1634, 1590 cmꢀ1 1H NMR (DMSO-d6):
. d 0.92 (3H, t,
7.94ꢃ(2H, m, H-6 and H-7), 8.17 (2H, m, H-5 and H-8), 8.71 (H, br
ꢃ
s, ꢀ NHH), 8.94 (H, br s, ꢀ NHH), 12.76 (1H, s, OH-1). 13C NMR
(DMSO-d6): d 21.1 (Me), 25.0 (–OCH2CH2–), 44.3 (–NHCH2–), 53.5
(–CH2NH–), 62.2 (ꢀCHOH), 66.1 (–OCH2–), 106.8 (C-2), 107.7
(C-4), 110.3 (C-9a), 126.5 (C-8), 126.9 (C-5), 132.9 (C-8a), 132.9
(C-10a), 134.7 (C-6), 134.8 (C-7), 134.9 (C-4a), 164.6 (C-1), 165.0
(C-3), 181.6 (C-10), 186.3 (C-9). EIMS (70 eV) m/z (% rel. int.): 355
J = 7.2 Hz, Me), 1.67 (2H, m, –CH2CH3), 2.18 (2H, m, –OCH2CH2–),
2.86 (2H, m, –NHCH2–), 3.06 (2H, m, –CH2NH–), 4.27 (2H, t,
J = 6.4 Hz, –OCH2–), 6.86 (1H, d, J = 2.4 Hz, H-2), 7.17 (1H, d,
J = 2.4 Hz, H-4), 7.92 (2H, m, H-6 and H-7), 8.17 (2H, m, H-5 and
H-8), 9.11 (2H, br s, NH2þ), 12.67 (1H, s, OH-1). 13C NMR (DMSO-
d6):
d 11.5 (Me), 19.4 (–CH2CH3), 25.6 (–OCH2CH2–), 44.3

H.-Y. Tu et al. / Bioorg. Med. Chem. 19 (2011) 5670–5678
5677
(1) [M]+. HREIMS m/z [M+1]+ 356.1500 (calcd for C20H22NO5,
356.1498).
and purified as 5 to yield 15 (25 mg, 0.07 mmol, 30%). IR (KBr)
3420, 1670, 1636, 1589 cmꢀ1 1H NMR (DMSO-d6): d 1.60 (2H,
.
m, –OCH2CH2–), 2.20 (3H, br s, –CH3), 2.74 (4H, br s, 2 ꢁ
–NCH2CH2–), 2.94 (4H, br s, 2 ꢁ –NCH2CH2–), 3.40 (2H, m,
–CH2N–), 4.18 (2H, t, J = 6.0 Hz, –OCH2–), 6.17 (1H, d, J = 2.4 Hz,
H-2), 7.37 (1H, d, J = 2.4 Hz, H-4), 7.80 (2H, m, H-6 and H-7), 8.27
(2H, m, H-5 and H-8), 12.86 (1H, s, OH-1). 13C NMR (DMSO-d6): d
43.6 (Me), 49.7 (2 ꢁ –NCH2CH2–), 53.6 (2 ꢁ –NCH2CH2– and
–CH2N–), 66.5 (–OCH2–), 107.3 (C-2), 107.7 (C-4), 126.8 (C-5),
127.4 (C-8), 133.4 (C-10a), 133.5 (C-8a), 134.2 (C-6), 134.4 (C-7),
135.1 (C-4a), 165.3 (C-1 and C-3), 182.5 (C-10), 186.8 (C-9). EIMS
(70 eV) m/z (% rel. int.): 380 (3) [M]+. HREIMS m/z [M]+ 380.1739
(calcd for C22H24N2O4, 380.1736).
4.9. 1-Hydroxy-3-[3-(piperidin-1-yl)propoxy]-9,10-
anthraquinoneꢂHCl (12)
Compound 4 (80 mg, 0.22 mmol) in EtOH (40 mL) was added
piperidine (190 mg, 2.2 mmol). The mixture was treated and purified
as 5 to yield 12 (34 mg, 0.09 mmol, 42%). IR (KBr) 3447, 1670, 1654,
1589 cmꢀ1 1H NMR (DMSO-d6): d 1.40 (H, m, –NCH2CH2CHH–),
.
1.77 (5H, m, –NCH2CH2CHH–), 2.22 (2H, m, –OCH2CH2–), 2.90 (2H,
m, –NCH2–), 3.22 (2H, m, –NCH2–), 3.48 (2H, m, —CH2Nꢁ), 4.27 (2H,
t, J = 6.4 Hz, –OCH2–), 6.91 (1H, d, J = 2.4 Hz, H-2), 7.23 (1H, d,
J = 2.4 Hz, H-4), 7.95 (2H, m, H-6 and H-7), 8.23 (2H, m, H-5 and H-
ꢃ
8), 9.80 (H, br s, NH), 12.77 (1H, s, OH-1). 13C NMR (DMSO-d6): d
21.8 (–OCH2CH2–), 22.9 (–N(CH2CH2)2–), 23.8 (–NCH2CH2CH2–),
52.5 (–N(CH2CH2)2–), 53.5 (—CH2Nꢁ), 66.6 (–OCH2–), 107.2 (C-2),
108.2 (C-4), 110.8 (C-9a), 127.0 (C-8), 127.4 (C-5), 133.4 (C-8a and
C-10a), 135.2 (C-6), 135.3 (C-7), 135.3 (C-4a), 165.1 (C-1), 165.4 (C-
3), 182.1 (C-10), 186.8 (C-9). EIMS (70 eV) m/z (% rel. int.): 365 (5)
[M]+. HREIMS m/z [M+1]+ 366.1706 (calcd for C22H24NO4, 366.1705).
4.13. 1-Hydroxy-3-{3-[4-(2-hydroxyethyl)piperazin-1-yl]pro-
poxy}-9,10-anthraquinone (16)
Compound 4 (80 mg, 0.22 mmol) in EtOH (40 mL) was added
ethanolpiperazine (290 mg, 2.2 mmol). The mixture was treated
and purified as 5 to yield 16 (25 mg, 0.06 mmol, 28%). IR (KBr)
3446, 1670, 1637, 1577 cmꢀ1 1H NMR (DMSO-d6): d 2.10 (2H, br
.
s, –OCH2CH2–), 3.20 (2H, br s, –CH2NH–), 3.48 (10H, br s, 10 ꢁ
–NCH2CH2–), 3.78 (2H, br s, –CH2OH–), 4.28 (2H, t, J = 6.0 Hz,
–OCH2–), 6.93 (1H, d, J = 2.4 Hz, H-2), 7.24 (1H, d, J = 2.4 Hz, H-4),
7.95 (2H, m, H-6 and H-7), 8.24 (2H, m, H-5 and H-8), 12.78 (1H,
4.10. 1-Hydroxy-3-[3-(morpholin-4-yl)propoxy]-9,10-
anthraquinoneꢂHCl (13)
Compound 4 (80 mg, 0.22 mmol) in EtOH (40 mL) was added
morpholine (190 mg, 2.2 mmol). The mixture was treated and puri-
fied as 5 to yield 13 (32 mg, 0.09 mmol, 40%). IR (KBr) 3420, 1670,
1637, 1588 cmꢀ1. 1H NMR (DMSO-d6): d 2.24 (2H, m, –OCH2CH2–),
3.09 (2H, m, –NCH2–), 3.30 (2H, br s, –NCH2–), 3.47 (2H, m,
–CH2N–), 3.80 (2H, m, –CH2CH2O–), 3.98 (2H, m, –CH2CH2O–), 4.28
(2H, t, J = 6.0 Hz, –OCH2–), 6.91 (1H, d, J = 2.4 Hz, H-2), 7.22 (1H, d,
s, OH-1). 13C NMR (DMSO-d6):
d 23.1 (–OCH2CH2–), 48.2
(–NCH2CH2–), 48.7 (–NCH2CH2–), 52.9 (–CH2N–), 55.2 (2 ꢁ
–NCH2CH2–), 58.0 (–CH2OH–), 66.0 (–OCH2–), 106.8 (C-2), 107.8
(C-4), 110.3 (C-9a), 126.4 (C-5), 127.0 (C-8), 132.9 (C-8a), 132.9
(C-10a), 134.7 (C-4a), 134.8 (C-6), 134.9 (C-7), 164.6 (C-1), 164.9
(C-3), 182.7 (C-10), 186.3 (C-9). EIMS (70 eV) m/z (% rel. int.): 410
(5) [M]+. HREIMS m/z [M+1]+ 411.1918 (calcd for C23H27N2O5,
411.1920).
J = 2.4 Hz, H-4), 7.94 (2H, m, H-6 and H-7), 8.20 (2H, m, H-5 and H-
ꢃ
8), 10.95 (1H, br s, N H), 12.77 (1H, s, OH-1). 13C NMR (DMSO-d6):
d 22.8 (–OCH2CH2–), 51.1 (N(CH2CH2)2O), 53.2 (–CH2NH–), 63.2
(N(CH2CH2)2O), 66.1 (–OCH2–), 106.7 (C-2), 107.7 (C-4), 110.3
(C-9a), 126.5 (C-8), 127.0 (C-5), 132.9 (C-8a and C-10a), 134.7
(C-6), 134.8 (C-7), 134.9 (C-4a), 164.6 (C-1), 164.9 (C-3), 181.7
(C-10), 186.3 (C-9). EIMS (70 eV) m/z (% rel. int.): 367 (3) [M]+.
HREIMS m/z [M+1]+ 368.1499 (calcd for C21H22NO5, 368.1498).
4.14. 4-[3-(4-Hydroxy-9,10-dioxo-9,10-dihydro-anthracen-2-
yloxy)-propyl]-piperazine-1-carboxylic acid ethyl esterꢂ2HCl
(17)
Compound 4 (80 mg, 0.22 mmol) in EtOH (40 mL) was added 1-
piperzine-carboxylic acid ethyl ester (350 mg, 2.2 mmol). The mix-
ture was treated and purified as 5 to yield 17 (39 mg, 0.09 mmol,
4.11. 1-Hydroxy-3-[3-(cyclohexylamino)propoxy]-9,10-
anthraquinoneꢂHCl (14)
40%). IR (KBr) 3446, 1670, 1637, 1588 cmꢀ1 1H NMR (DMSO-d6):
.
d 1.18 (3H, t, J = 6.0 Hz, Me), 2.23 (2H, m, –OCH2CH2–), 3.07 (4H,
m, 2 ꢁ –NCH2CH2–), 3.26 (2H, m, –CH2N–), 3.57 (4H, m, 2 ꢁ
–NCH2CH2–), 4.07 (4H, m, –OCH2CH3 and ꢀNCH2—), 4.28 (2H, t,
J = 6.0 Hz, –OCH2–), 6.91 (1H, d, J = 2.4 Hz, H-2), 7.22 (1H, d,
J = 2.4 Hz, H-4), 7.94 (2H, m, H-6 and H-7), 8.18 (2H, m, H-5 and
Compound 4 (80 mg, 0.22 mmol) in EtOH (40 mL) was added cyclo-
hexylamine (220 mg, 2.2 mmol). The mixture was treated and purified
as 5 to yield 14 (37 mg, 0.10 mmol, 44%). IR (KBr) 3446, 1670, 1637,
1577 cmꢀ1. 1H NMR (DMSO-d6): d 1.11 (1H, m, –CH2CH2CHH–), 1.26
(4H, m, 2 ꢁ –CH2CH2–), 1.60 (1H, m, –CH2CH2CHH–), 1.78 (2H, m,
–CH2CH2–), 2.08 (2H, m, –CH2CH2–), 2.12 (2H, m, –OCH2CH2–), 3.04
(1H, m, –NHCH–), 3.12 (2H, m, –CH2NH–), 4.29 (2H, t, J = 6.0 Hz,
–OCH2–), 6.92 (1H, d, J = 2.4 Hz, H-2), 7.25 (1H, d, J = 2.4 Hz, H-4),
ꢃ
ꢃ
H-8), 9.11 (1H, br s, ffl NH), 10.83 (1H, br s, ffl NH), 12.77 (1H, s,
OH-1). 13C NMR (DMSO-d6): d 14.5 (Me), 23.0 (–OCH2CH2–), 42.3
(2 ꢁ –NCH2CH2–), 50.5 (2 ꢁ –NCH2CH2–), 52.9 (–CH2N–), 61.4
(–OCH2CH3), 66.0 (–OCH2–), 106.7 (C-2), 107.8 (C-4), 110.4
(C-9a), 126.5 (C-5), 127.0 (C-8), 132.9 (C-8a), 132.9 (C-10a), 134.7
(C-4a), 134.8 (C-6), 134.9 (C-7a), 154.3 (C@O), 164.5 (C-1), 164.9
(C-3), 181.7 (C-10), 186.3 (C-9). EIMS (70 eV) m/z (% rel. int.): 439
(22) [M+1]+. HREIMS m/z [M+1]+ 439.1872 (calcd for C24H27N2O6,
439.1869).
7.95 (2H, m, H-6 and H-7), 8.22 (2H, m, H-5 and H-8), 8.49 (2H, br s,
ꢃ
ꢀ NHH), 12.80 (1H, s, OH-1). 13C NMR (DMSO-d6): d 23.8 (–CH2–),
24.7 (–OCH2CH2–), 25.4 (–CH2–), 28.6 (2 ꢁ –CH2CH2–), 40.9
(–NHCH–), 56.0 (–CH2NH–), 65.9 (–OCH2–), 106.8 (C-2), 107.6 (C-4),
110.4 (C-9a), 126.5 (C-5), 127.0 (C-8), 132.9 (C-8a and C-10a), 134.8
(C-4a and C-6), 134.9 (C-7), 164.6 (C-1), 164.9 (C-3), 181.7 (C-10),
186.4 (C-9). EIMS (70 eV) m/z (% rel. int.): 379 (10) [M]+. HREIMS m/z
[M+1]+ 380.1860 (calcd for C23H26NO4, 380.1862).
4.15. Assays of NTUB1, PC3, and SV-HUC1 cell lines
NTUB1, an immortalized human urothelial carcinoma cell line,
was established from a high-grade bladder cancer.20 PC3 (a human
prostate cancer cell line), and SV-HUC1 (a SV-40 immortalized
human uroepithelial cell line) were obtained from ATCC. The cells
were maintained in RPMI 1640 (for NTUB1 and PC3 cells) or F12
medium (for SV-HUC1) supplemented with 10% FBS, 100 unit/mL
4.12. 1-Hydroxy-3-[3-(4-methylpiperazin-1-yl)propoxy]-9,10-
anthraquinone (15)
Compound 4 (80 mg, 0.22 mmol) in EtOH (40 mL) was added
methylpiperazine (220 mg, 2.2 mmol). The mixture was treated

5678
H.-Y. Tu et al. / Bioorg. Med. Chem. 19 (2011) 5670–5678
penicillin-G, 100
lg/mL streptomycin and 2 mM
L
-glutamine. The
4.19. Western blot analysis
cells were cultured at 37 °C in a humidified atmosphere containing
5% CO2.
Cells were harvested by trypsinization and resuspended with
suitable amount of PBS adjusted with the cell numbers. The cells
were mixed with equal volume of 2 ꢁ sample buffer and boiled
for 10 min twice to denature the proteins. Cell extracts were sepa-
rated 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 and 0.1%
Tween-20). The membranes were incubated with specific antibod-
ies at 4 °C overnight. The membranes were washed with TBST buf-
fer and incubated with secondary antibody at room temperature
for another 1 h. Signals were detected by chemiluminescence ECL
reagent after TBST wash and visualized on Fuji SuperRX film.
For evaluating the cytotoxic effect of tested compounds and cis-
platin, a modified MTT assay was performed.21–23 Briefly, the cells
were plated at a density of 1000 (for NTUB1 and PC3 cells) and
5000 (for SV-HUC1 cells) cells/well in 96-well plates and incubated
at 37 °C overnight before drug exposure. Cells were then cultured
in the presence of graded concentrations of tested compounds at
37 °C for 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 1000 ꢁ g for 10 min,
media were removed and 150 lL DMSO were added to each well.
The proportions of surviving 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 IC50 values of each group were
calculated by the median-effect analysis and presented as
mean standard deviation (SD).
4.20. Statistical analysis
Data were expressed as mean SD. Statistical analysis were per-
formed using the Bonferroni t-test method after ANOVA for multi-
group comparison and the student’s t-test method for two group
comparison, with p<0.05 was considered to be statistically significant.
4.16. Quantitative analysis of intracellular reactive oxygen
species (ROS)
Acknowledgment
Production of ROS was analyzed by flow cytometry as described
previously.22 Briefly, cells were plated and treated as indicated
conditions. 10 lM H2DCFDA (Molecular Probes, Eugene, OR) was
added to the treated cells 30 min prior harvest. The cells were col-
lected by trypsinization and washed with PBS. The green fluores-
cence of intracellular DCF (20,70-dichlorofluorescein) was then
analyzed immediately by FACScan flow cytometer with a 525-nm
band pass filter (Becton Dickinson).
This work was partially supported by a research grant from the
National Science Council of the Republic of China (NSC97-2320-B-
037-014 and NSC97-2320-B-037-018-MY3).
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Lin, C.-N. Bioorg. Med. Chem. 2009, 17, 7265.
23. Tu, H.-Y.; Huang, A.-M.; Hour, T.-C.; Yang, S.-C.; Pu, Y.-S.; Lin, C.-N. Bioorg. Med.
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4.18. Immunofluorescence microscopy
NTUB1 cells (3 ꢁ 105 cells) plated on the 6-cm dish were treated
no compound as control and indicated compounds for 24 h. After
treatment, cells were fixed with 2% formaldehyde/PBS for 20 min,
washed with PBS, and cold methanol (ꢀ20 °C) for 3 min. After
washing with PBS, cells were added H2DCFDA in PBS and incubated
for 30 min at 37 °C. Then cells were washed with PBS and stained
with DAPI (Sigma, Co) in dark room for 1 h at room temperature.
After being washed with PBS, cells were examined with Axioskop
2 plus fluorescence microscope.
24. Huang, A.-M.; Montagna, C.; Sharan, S.; Ni, Y.; Ried, T.; Sterneck, E. Oncogene
2004, 23, 1549.