Structure-dependent inhibition of bladder and pancreatic cancer cell growth by 2-substituted glycyrrhetinic and ursolic acid derivatives

Article abstract of DOI:10.1016/j.bmcl.2008.03.031

Derivatives of oleanolic acid, ursolic acid and glycyrrhetinic acid substituted with electron-withdrawing groups at the 2-position in the A-ring which also contains a 1-en-3-one structure are potent inhibitors of cancer cell growth. In this study, we have compared the effects of several 2-substituted analogs of triterpenoid acid methyl esters derived from ursolic and glycyrrhetinic acid on proliferation of KU7 and 253JB-V bladder and Panc-1 and Panc-28 pancreatic cancer cells. The results show that the 2-cyano and 2-trifluoromethyl derivatives were the most active compounds. The glycyrrhetinic acid derivatives with the rearranged C-ring containing the 9(11)-en-12-one structure were generally more active than the corresponding 12-en-11-one isomers. However, differences in growth inhibitory IC₅₀ values were highly variable and dependent on the 2-substituent (CN vs CF₃) and cancer cell context.

Full text of DOI:10.1016/j.bmcl.2008.03.031

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2633–2639
Structure-dependent inhibition of bladder and pancreatic cancer
cell growth by 2-substituted glycyrrhetinic
and ursolic acid derivatives
Gayathri Chadalapaka,a, Indira Jutooru,a, Alan McAlees,b
Tom Stefanacb and Stephen Safea,c,
*
aDepartment of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843-4466, USA
bWellington Laboratories, 345 Southgate Dr., Guelph, Ont., Canada N1G 3M5
cInstitute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX 77030-3303, USA
Received 31 January 2008; revised 6 March 2008; accepted 7 March 2008
Available online 14 March 2008
Abstract—Derivatives of oleanolic acid, ursolic acid and glycyrrhetinic acid substituted with electron-withdrawing groups at the 2-
position in the A-ring which also contains a 1-en-3-one structure are potent inhibitors of cancer cell growth. In this study, we have
compared the effects of several 2-substituted analogs of triterpenoid acid methyl esters derived from ursolic and glycyrrhetinic acid
on proliferation of KU7 and 253JB-V bladder and Panc-1 and Panc-28 pancreatic cancer cells. The results show that the 2-cyano
and 2-trifluoromethyl derivatives were the most active compounds. The glycyrrhetinic acid derivatives with the rearranged C-ring
containing the 9(11)-en-12-one structure were generally more active than the corresponding 12-en-11-one isomers. However, differ-
ences in growth inhibitory IC50 values were highly variable and dependent on the 2-substituent (CN vs CF3) and cancer cell context.
ꢀ 2008 Elsevier Ltd. All rights reserved.
Pentacyclic triterpenoid acids such as betulinic acid, ole-
anolic acid, ursolic acid, and glycyrrhetinic acid are phy-
tochemicals that have been extensively used in
traditional medicines for treatment of a wide variety of
human ailments.1–4 Most of these compounds exhibit
anti-inflammatory and anticarcinogenic activities as well
as a large number of compound-specific effects. For
example, the major bioactive component of licorice ex-
tracts is glycyrrhizic acid glycoside which is readily
hydrolyzed to glycyrrhetinic acid and these extracts/
compounds possess anti-inflammatory, antiviral and
endocrine activities.4 All of these triterpenoid acids have
been used as building blocks for the synthesis of more
active analogs. Oleanolic acid has been converted into
2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO),
the corresponding methyl ester (CDDO-Me) and other
structurally related analogs, and these compounds are
potent anticancer agents.5–9 These synthetic triterpe-
noids activate the nuclear receptor peroxisome prolifer-
ator-activated receptor c (PPARc)9 and also induce
several other responses including apoptosis in various
cancer cell lines. The cytotoxicity and anti-inflammatory
activity of CDDO-Me and related compounds are due
to the 2-cyano group and the 1-en-3-one and 9-en-12-
one functionalities in the A- and C-rings, respectively.7,8
We also showed that the glycyrrhetinic acid analog
methyl 2-cyano-3,11-dioxo-18b-olean-1,12-dien-30-oate
(b-CDODA-Me) and the corresponding 18a derivative
(a-CDODA-Me) which contain 2-cyano-1-en-3-one
and 12-en-11-one functionalities in their A- and C-rings,
respectively, activated PPARc and were also highly
cytotoxic in colon cancer cells.10 Similar results were ob-
tained for the betulinic acid derivatives containing a
2-cyano-1 en-3-one function.11 In this study, we have
further investigated the structure-dependent growth
inhibitory effects of b-CDODA-Me and analogs
containing different electronegative 2-substituents and
a selected number of the corresponding ursolic acid
2-substituted-1-ene-3-one derivatives.12 In addition, we
also show that for the glycyrrhetinic acid derivatives
growth inhibitory differences of the 9-en-12-one and
12-en-11-one isomers are lower than previously reported
Keywords: Glycyrrhetinate analogs; Growth inhibition; Bladder can-
cer; Pancreatic cancer.
*
Corresponding author. Tel.: +1 979 845 5988; fax: +1 979 862
4929; e-mail: ssafe@cvm.tamu.edu
These authors contributed equally to this study.
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.031

2634
G. Chadalapaka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2633–2639
for anti-inflammatory activity of CDDO-Me and the 12-
en-11-one isomers.8 Growth inhibition by the 2-substi-
tuted glycyrrhetinic acid and ursolic acid methyl ester
analogs is dependent on the nature of the 2-substituent
and cancer cell context among two bladder (KU7 and
253JB-V) and two pancreatic (Panc-1 and Panc-28) can-
cer cell lines.13
Previous studies with CDDO-Me and related com-
pounds showed that the 1-en-3-one structure containing
electronegative 2-substituent groups such as cyano were
highly cytotoxic5–9 and similar results were observed for
b-CDODA-Me, the corresponding 2-cyano analog of
glycyrrhetinic acid.10 In this study, we used 253JB-V
and KU7 bladder and Panc-1 and Panc-28 pancreatic
cancer cells to investigate the growth inhibitory effects14
of CDODA-Me analogs containing several electronega-
tive 2-substituents including iodo, cyano, trifluoro-
methyl, dimethylphosphonyl and methanesulfonyl
groups (Table 1). b-CDODA, the parent-free acid deriv-
ative of b-CDODA-Me is used as a control for this
group of compounds, and IC50 values for inhibition of
bladder and pancreatic cancer cell growth varied from
5.9 to 7.3 lM. The corresponding range of IC50 values
for b-CDODA-Me was 0.25–1.80 lM indicating that
in cell culture studies the methyl ester derivatives were
more potent than the free acid (b-CDODA) as previ-
ously reported for growth inhibition of colon cancer
cells.10 The effects of compounds unsubstituted at C-2
were >7-fold less active than the 2-cyano derivatives
(data not shown) and these results were similar to those
observed for these compounds in colon cancer cells.10
Based on the synthetic scheme which used the 2-iodo
derivative (4) as a precursor, we synthesized the 2-tri-
fluoromethyl, dimethylphosphonyl and methanesulfonyl
derivatives and determined the effects of different elec-
tronegative substituents on their inhibition of bladder
and pancreatic cancer cell growth (Table 1). The IC50
values were lowest for 2-cyano and 2-trifluoromethyl
(b-CF3DODA-Me) derivatives; however, their relative
potencies were dependent on cell context; b-CF3DO-
DA-Me was more active than b-CDODA-Me in KU7
(IC50: 0.38 vs 1.59 lM), Panc-1 (IC50: 0.82 vs 1.22 lM)
and Panc-28 (IC50: 1.14 vs 1.80 lM) cells, whereas b-
CDODA-Me was more active in 253JB-V cells (IC50:
0.25 vs 0.67 lM). Both the 2-dimethylphosphonyl and
2-methanesulfonyl analogs were relatively inactive with
IC50 values ranging from 3.34 to 12.0 lM over the four
cell lines.
The synthesis of a series of 2-substituted glycyrrhetinic
acid derivatives is summarized in Scheme 1. The free
acid (1) was esterified at 0 ꢁC with ethereal diazometh-
ane to give methyl glycyrrhetinate (2) which was then
treated with 4 equiv of 2-iodoxybenzoic acid (IBX) in
DMSO at 85 ꢁC for 21 h. The resulting methyl 3,11-di-
oxo-18b-oleana-1,12-dien-30-oate product (3) was then
converted into the 2-iodo derivative (4) by treating with
2 equiv of iodine and 3 equiv of pyridine in tetrahydro-
furan (refluxing). The methyl 2-iodo-3,11-dioxo-18b-ole-
ana-1,12-dien-30-oate derivative (4) was used as the
precursor for the synthesis of the 2-cyano (5), 2-meth-
anesulfonyl (6), 2-trifluoromethyl (7) and 2-dimethyl-
phosphonyl (8) analogs (Scheme 1). The 2-cyano
derivative was prepared by treating 4 with 2 equiv of cu-
prous cyanide in N-methylpyrrolidinone (NMP) for 2 h
at 130 ꢁC. The 2-methanesulfonyl derivative was ob-
tained by treating 4 with sodium methanesulfinate and
cuprous iodide in DMSO at 120–125 ꢁC for 20 h. The
2-dimethylphosphonyl analog was synthesized by treat-
ing 4 with dimethylphosphite, cesium carbonate, N,N-
dimethylethylenediamine in toluene at 95–100 ꢁC for
26 h. Finally the 2-trifluoromethyl derivative was ob-
tained by treating 4 with cuprous iodide and methyl
2,2-difluoro-2-(fluorosulfonyl) acetate in dimethylform-
amide/HMPT at 70 ꢁC for 20 h. The synthetic scheme
used for conversion of ursolic acid (9) into the 2-iodo
derivative (12) and the corresponding 2-cyano (13) and
2-trifluoromethyl (14) derivatives (Scheme 2) involved
intermediates (10) and (11) and was identical to that car-
ried out for conversion of methyl glycyrrhetinate into
the analogous 2-substituted compound as illustrated in
Scheme 1. Previous studies with oleanolic acid deriva-
tives reported the synthesis of several analogs including
CDDO which contain the 1-en-3-one A-ring and a 9-en-
12-one C-ring,8 and Scheme 3 outlines an analogous
route for conversion of methyl glycyrrhetinate into the
rearranged C-ring analogs of CDODA-Me. Methyl gly-
cyrrhetinate was reduced with H2/platinum oxide cata-
lyst in acetic acid, then acetylated with acetic
anhydride/pyridine in dimethylaminopyridine (DMAP)
to give the acetylated ester (15) in which the 9-oxo group
had been reduced. Treatment with m-chloroperbenzoic
acid (m-CPBA) in methylene chloride gave the 12,13-
epoxide which was converted into the 12-oxo derivative
by boron trifluoride etherate in methylene chloride. Bro-
mination of the 12-oxo derivative followed by dehydro-
bromination in acetic anhydride followed by basic
hydrolysis of the acetate group gave the rearranged C-
ring derivative of methyl glycyrrhetinate (16). Subse-
quent treatment with 2-iodoxybenzoic acid and iodine/
pyridine in tetrahydrofuran gave methyl 2-iodo-3,12-di-
oxo-oleana-1,9-dien-30-oate (17) which is converted into
the C-ring rearranged cyano (18) and trifluoromethyl
(19) derivatives as outlined in Schemes 1 and 2.
Since the 2-cyano and 2-trifluoromethyl derivatives
were the most active of the glycyrrhetinic acid group
of 2-substituted 1-en-3-one compounds, we synthesized
a similar series of analogs from methyl ursolate and
compared their growth inhibitory IC50 values to the
2-iodo analog and methyl ursolate (Table 2). IC50 val-
ues for methyl ursolate (9) and the 2-iodo-1-en-3-one
analog (12) varied from 6.13 to 11.75 and 4.90 to
13.50 lM, respectively, in the bladder and pancreatic
cancer cell lines and there was less than a 2-fold differ-
ence in their IC50 values. IC50 values for the 2-cyano
(13) and 2-trifluoromethyl (14) analogs varied from
0.17 to 0.97 and 0.17 to 1.13 lM, respectively, and
were at least an order of magnitude more potent in
the growth inhibition assay than the 2-iodo compound
or methyl ursolate. These data confirm that 2-cyano
and 2-trifluoromethyl groups coupled with introduction
of a 1-en-3-one functionality into the A-ring of methyl
ursolate resulted in enhanced growth inhibition with
comparable IC50 values in KU7, 253JB-V, Panc-1
and Panc-28 cancer cells.

G. Chadalapaka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2633–2639
2635
CO2H
O
CH2N2
CO2CH3
Et2O, 0°C
IBX (4 equiv)
CO2CH3
OH
O
DMSO, 80-85°C
1
Iodine/Pyridine
O
CO2CH3
Tetrahydrofuran
OH
O
2
O
I
3
O
4
CO2CH3
O
CO2CH3
CuCN (20 equiv)
NaO2SCH3DMSO
NC
4
NMP, 130°C, 2 hr
Cul, 120-125°C, 20 hr
O
O
O
O
S
5
H3C
CO2CH3
O
6
O
F3C
CO2CH3
O
O
O
7
(H3CO)2P
O
8
Scheme 1. Synthesis of 2-substituted-1-en-3-one derivative of methyl glycyrrhetinate.
Previous studies with CDDO-Me and related com-
pounds showed that the functionality of the C-ring
markedly influenced the activity of oleanolic acid deriv-
atives with 2-cyano-1-en-3-one functionality.9 For
example, CDDO-Me which contains a 9(11)-en-12-one
C-ring structure is 200 times more potent as an inhibitor
of nitric oxide production in mouse macrophages than
the corresponding 12-en-11-one isomer.9 We therefore
synthesized a series of 2-iodo, 2-cyano, and 2-trifluoro-
methyl-1-en-3-one analogs of glycyrrhetinic acid which
also contain a 9(11)-en-12-one C-ring functionality (Ta-
ble 3) and compared their growth inhibitory effects to
the 12-en-11-one isomers (Table 1). The effects of the
C-ring en-one functionality on the activity of these iso-
mers were dependent not only on the 2-substituent but
also on cell context. IC50 values were similar for both
2-iodo isomers in 253JB-V, KU7 and Panc-1 cells,
whereas IC50values for the 12-en-11-one and 9(11)-en-
12-one isomers in Panc-28 cells were 12.75 and
3.66 lM, respectively. Thus, for the 2-iodo derivative,
the C-ring structure had minimal effects on growth
inhibitory activity, except for one cell line where IC50
differences were <4-fold.
functionalities were more dramatic and varied from a
2.2- (253JB-V cells) to a 39.1-fold (Panc-28) lower IC50
values for isomers containing the former C-ring struc-
ture. These differences for the 2-cyano derivatives of
methyl glycyrrhetinate were significantly lower than dif-
ferences observed for CDDO-Me and the corresponding
12-en-11-one isomer where IC50 values for inhibition of
nitric oxide production in mouse macrophages were 0.1
and 20 nM, respectively, a 200-fold difference.9 We also
directly compared the effects of the rearranged 2-cyano
methyl glycyrrhetinate derivative (18) with CDDO-Me
in the four cell lines; the former compound (18) was
more active than CDDO-Me in Panc-1 and Panc-28
cells, whereas IC50 values were similar in KU7 cells
and CDDO-Me (IC50 = 0.03 lM) was more active in
253JB-V cells. Thus, a direct comparison of the 2-cyano
derivative of methyl glycyrrhetinate (18) and CDDO-Me
(derived from methyl oleanolate) on their inhibition of
cancer cell proliferation indicates that both synthetic
triterpenoids are highly potent and differences in their
IC50 values in four cell lines were less than an order of
magnitude.
The antiproliferative activity of the trifluoromethyl
derivative (6) (Table 1) was <2-fold lower than the
9(11)-en-12-one isomer in 253JB-V and Panc-1 cells,
Differences in IC50 values for the 2-cyano methyl glycyr-
rhetinate isomers with 12-en-11-one and 9(11)-en-12-one

2636
G. Chadalapaka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2633–2639
CH2N2
IBX (4 equiv)
Et2O, 0°C
DMSO, 80-85°C
CO2H
CO2CH3
CO2CH3
HO
O
HO
9
10
11
I2 (3 equiv), pyridine
(5 equiv)
THF reflux. 3d
CO2CH3
NC
O
CO2CH3
I
13
O
12
CO2CH3
F3C
O
14
Scheme 2. Synthesis of 2-substituted-1-en-3-one derivatives of methyl ursolate.
CO2CH3
CO2CH3
O
a, b, c, d
e, f
O
AcO
15
HO
2
CO2CH3
CO2CH3
O
O
(i) g, h
17: X = I (i) g, h
18: X = CN (ii) i
19: X = CF3 (iii) j
(ii) i
(iii) j
X
O
HO
16
a: H2/PtO2 in acetic acid,
g: iodoxybenzoic acid/DMSO,
h: iodine/pyridine in tetrahydrofuran,
i: cuprous cyanide in NMP,
b: acetic anhydride/pyridine in DMAP,
c: m-CBPA in methylene chloride,
d: boron trifluoride etherate in methylene
chloride
j: methyl 2,2-difluoro-2-
(fluorosulfonyl)acetate, cuprous
iodide in dimethyl formamide/HMPT
e: bromine/HBr in acetic acid
f: potassium hydroxide in methanol
Scheme 3. Synthesis of C-ring rearranged analogs of CDODA-Me.

G. Chadalapaka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2633–2639
Table 1. Cytotoxicity of 2-substituted compounds derived from methyl glycyrrhetinate
2637
CO2R1
O
R2
IC50 (lM)
253JB-V
KU7
Panc-1
Panc-28
R1 = H:R2 = CN
6.10
2.67
0.25
0.67
11.97
7.90
5.88
3.04
1.59
0.38
3.34
3.73
3.81
4.08
1.22
0.82
7.69
6.11
7.32
12.75
1.80
1.14
9.75
8.14
R1 = CH3:R2 = I (4)
R1 = CH3:R2 = CN (5)
R1 = CH3:R2 = CF3 (6)
R1 = CH3:R2 = CH3SO2 (7)
R1 = CH3:R2 = (CH3O)2PO (8)
Table 2. Cytotoxicity of 2-substituted compounds derived from methyl ursolate
CO2 R1
R2
IC50 (lM)
253JB-V
KU7
Panc-1
Panc-28
Methyl ursolate (a)
6.13
4.90
0.17
0.17
8.95
6.02
0.30
0.47
11.75
6.91
0.53
0.65
10.58
13.49
0.97
R1 = CH3:R2 = I (12)
R1 = CH3:R2 = CN (13)
R1 = CH3:R2 = CF3 (14)
1.13
Table 3. Cytotoxicity of 2-substituted compounds derived from methyl glycyrrhetinate with C-ring rearrangement
CO2R1
O
R2
IC50 (lM)
253JB-V
KU7
Panc-1
Panc-28
CDDO-Me
0.03
3.62
0.11
0.36
0.12
2.61
0.12
1.37
0.27
4.45
0.07
0.68
0.29
3.66
0.05
1.13
R1 = CH3:R2 = I (17)
R1 = CH3:R2 = CN (17)
R1 = CH3:R2 = CF3 (18)

2638
G. Chadalapaka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2633–2639
fluoromethyl analog was more active (IC50 = 0.38 lM)
KU7 CELLS
A
in KU7 cells than the corresponding 9(11)-en-12-one
isomer (IC50 = 1.37 lM). Figure 1 illustrates the struc-
ture-dependent differences in growth inhibitory activities
in KU7 cells of the 2-cyano and 2-trifluoromethyl ana-
logs containing the unrearranged 12-en-11-one (5 and
7) and rearranged 9(11)-en-12-one (18 and 19) C-ring
in the methyl glycyrrhetinate series of isomers.
#5 (2-CN: 12-en-11-one)
3
2.5
2
DMSO
0.5 μM
1.0 μM
2.5 μM
1.5
1
In summary, the results of this study show that the anti-
proliferative activities of several 2-substituted glycyrrh-
etinic acid and ursolic acid derivatives containing the
1-en-3-one functionality in their A-rings were maximal
for both the cyano and trifluoromethyl derivatives.
The corresponding 2-iodo substituted analogs were less
active (Tables 1 and 2). The 2-dimethylphosphonyl
and 2-methanesulfonyl derivatives of glycyrrhetinic acid
(Table 1) also exhibited higher IC50 values than the cor-
responding 2-cyano and 2-trifluoromethyl derivatives.
The rationale for the observed differences in the activity
of the different 2-substituted analogs containing electro-
negative substituents is unclear and is currently being
investigated using other assay systems. Compared to
previous studies with oleanolic acid derivatives,5–9 the
effects of the C-ring functionality [9(11)-en-12-one vs
12-en-11-one] on inhibition of cancer cell growth were
minimal (Tables 1 and 3) and dependent on the 2-substi-
tuent (cyano vs trifluoromethyl) (Fig. 1) and cancer cell
context. This suggests that for the glycyrrhetinic acid
derivatives it may not be necessary to carry out the addi-
tional synthetic steps to convert the 12-en-11-one to the
9(11)-en-12-one functionality in the C-ring. This is con-
firmed, in part, by ongoing in vivo studies in mouse
xenograft experiments where significant inhibition of tu-
mor growth by b-CDODA-Me is observed at doses of
10–15 mg/kg/day (data not shown). Ongoing work in-
cludes further investigation of structure-dependent anti-
carcinogenic potencies of the cyano and trifluoromethyl
glycyrrhetinic acid analogs, and studies of their underly-
ing mechanism of action and clinical applications.
0.5
0
0
2
4
6
Time (Days)
KU7 CELLS
B 2.5
#18 (2-CN: 9(11)-en-12-one)
DMSO
0.01 μM
0.1 μM
0.5 μM
2
1.5
1
0.5
0
0
2
4
6
Time (Days)
KU7 CELLS
#7 (2-CF3: 12-en-11-one)
C
1.6
1.4
1.2
1
DMSO
0.01 μM
0.1 μM
0.5 μM
0.8
0.6
0.4
0.2
0
0
2
4
6
Time (Days)
Acknowledgments
KU7 CELLS
D
2.5
2
#19 (2-CF3: 9(11)-en-12-one)
The financial assistance of the National Institutes of
Health (ES09106 and CA108718) and the Texas Agri-
cultural Experiment Station is gratefully acknowledged.
DMSO
0.01 μM
0.1 μM
0.5 μM
1.5
1
References and notes
0.5
0
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0
2
4
6
Time (Days)
Figure 1. Effects of 2-CN- and 2-CF3-1-en-3-one analogs containing
12-en-11-one or 9(11)-en-12-one functionality in the C-ring.
whereas similar IC50 values were observed in Panc-28
cells. In contrast the 12-en-11-one (unrearranged) tri-

G. Chadalapaka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2633–2639
2639
7. Honda, T.; Gribble, G. W.; Suh, N.; Finlay, H. J.;
Rounds, B. V.; Bore, L.; Favaloro, F. G., Jr.; Wang, Y.;
Sporn, M. B. J. Med. Chem. 2000, 43, 1866.
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Favaloro, F. G., Jr.; Suh, N.; Wang, Y.; Sporn, M. B.;
Gribble, G. W. J. Med. Chem. 2000, 43, 4233.
9. Wang, Y.; Porter, W. W.; Suh, N.; Honda, T.; Gribble, G.
W.; Leesnitzer, L. M.; Plunket, K. D.; Mangelsdorf, D. J.;
Blanchard, S. G.; Willson, T. M.; Sporn, M. B. Mol.
Endocrinol. 2000, 14, 1550.
10. Chintharlapalli, S.; Papineni, S.; Jutooru, I.; McAlees, A.;
Safe, S. Mol. Cancer Ther. 2007, 6, 1588.
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12. Chemicals: The ursolic acid and glycyrrhetinic acid starting
materials were purchased from Sigma–Aldrich (St. Louis,
MO) and all chemical reagents used for synthesis were
purchased from Sigma–Aldrich. Dulbecco’s modified/Ham’s
F-12 and RPMI media and the antibiotic/antimycotic
solution were also obtained from Sigma–Aldrich.
13. Cell lines, chemicals and other materials: KU7 human
bladder cancer and Panc-1 pancreatic cancer cells were
obtained from the American Type Culture Collection
(Manassas, VA) and 253JB-V cells were provided by Dr.
A. Kamat, M.D Anderson Cancer Center, Houston, TX.
The Panc-28 cell line was a generous gift from Dr. Paul
Chiao, The University of Texas M.D. Anderson Cancer
Center (Houston, TX). Both 253JB-V and KU7 cell lines
were maintained in RPMI 1640 supplemented with 10%
fetal bovine serum (FBS), 0.15% sodium bicarbonate,
0.011% sodium pyruvate, 0.24% Hepes and 10 ml/L of
antibiotic/antimycotic cocktail solution. The pancreatic
cancer cells were maintained in Dulbecco’s modified/
Ham’s F-12 with phenol red supplemented with 0.22%
sodium bicarbonate, 0.011% sodium pyruvate, 5% fetal
bovine serum and 10 ml/L 100· antibiotic/antimycotic
solution. The cells were grown in 150 cm2 culture plates in
an air/CO2 (95:5) atmosphere at 37 ꢁC and passaged
approximately every 3 days.
14. Cell proliferation assays: All four cancer cell lines (3 · 104
cells per well) were plated using DMEM:Ham’s F-12
medium containing 2.5% charcoal-stripped FBS in 12-well
plates and left to attach for 24 h. Cells were then treated
with either vehicle (DMSO) or the indicated concentra-
tions of different compounds. Fresh medium and test
compounds were added every 48 h and cells were then
counted at the indicated times using a Coulter Z1 particle
counter. Each experiment was done in triplicate, and
results are expressed as means SE for each determina-
tion. The IC50 values were calculated using linear regres-
sion method and are expressed in lM.