Cysteine-Derived Antitumor Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 2993
mycin, 3 mM HEPES, and incubated at 5% CO
2
, 99% humidity
Refer en ces
at 37 °C for 16 h before treatment. Cells were treated with 10
µg/mL of drug and RNA harvested at the indicated times
following treatment. Total RNA was extracted from cells using
the Qiagen RNeasy Kit as per manufacturer’s instructions.
RNA was analyzed for sufficient quality by formamide agarose
gel electrophoresis and quantified by spectrophotometry.
(
1) Marks, P. A.; Richon, V. M.; Kiyokawa, H.; Rifkind, R. A.
Inducing differentiation of transformed cells with hybrid polar
compounds: a cell cycle-dependent process. Proc. Natl. Acad.
Sci. U.S.A. 1994, 91, 10251-10254.
(
2) Rifkind, R. A.; Richon, V. M.; Marks, P. A. Induced differentia-
tion, the cell cycle and the treatment of cancer. Pharmacol. Ther.
1
996, 69, 97-102.
p 21 Exp r ession . The semiquantitative analysis of mRNA
(3) Leszczyniecka, M.; Roberts, T.; Dent, P.; Grant, S.; Fisher P. B.
Differentiation therapy of human cancer: basic science and
clinical implications. Pharmacol. Ther. 2001, 90, 105-156.
WAF1/Cip1
expression of p21
was carried out by RT-PCR. First
strand synthesis was performed using 2 µg of total RNA with
.5 µg of oligo (dT)15 and 200 U SuperScript II (Invitrogen,
(
4) Vigushin, D. M.; Coombes, R. C. Histone deacetylase inhibitors
in cancer treatment. Anti-Cancer Drugs. 2002, 13, 1-13.
5) Marks, P. A.; Rifkind, R. A.; Richon, V. M.; Breslow, R.; Miller,
T.; Kelly, W. K. Histone deacetylases and cancer: causes and
therapies. Nature 2001, 1, 194-202.
6) Richon, V. M.; Emiliani, S.; Verdin, S.; Webb, Y.; Breslow, R.;
Rifkind, R. A.; Marks, P. A. A class of hybrid polar inducers of
transformed cell differentiation inhibits histone deacetylases.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3003-3007.
0
Carlsbad, CA), at 42 °C for 50 min in a final volume of 20 µL.
Polymerase chain reaction was performed using 10 µL of a 1
in 10 dilution of the first strand cDNA, under standard
conditions with the polymerase DyNAzyme (Finnzymes, Mel-
(
(
bourne, Australia). Oligonucleotide primers and conditions
WAF1/Cip1
used in the PCR were as follows: p21
F 5′-ATT AGC
WAF1/Cip1
AGC GGA ACA AGG AGT CAG ACA T-3′, p21
R 5′-
(7) Parsons, P. G.; Hansen, C.; Fairlie, D. P.; West, M. L.; Danoy,
P. A. C.; Sturm, R. A.; Dunn, I. S.; Pedley, J .; Ablett, E. M. Tumor
Selectivity and Transcriptional Activation by Azalaic Bishydrox-
amic Acid in Human Melanocytic Cells. Biochem. Pharmacol.
CTG TGA AAG ACA CAG AAC AGT ACA GGG T-3′ with
initial denaturation at 94 °C for 7 min, 27 cycles of 94 °C for
4
5 s, 60 °C for 40 s and 72 °C for 60 s, with the final extension
1
997, 53, 1719-1724.
for 5 min; GAPDH F 5′-GGC TCT CCA GAA CAT CAT CCC
TGC-3′, GAPDH R 5′-GGG TGT CGC TGT TGA AGT CAG
AGG-3′ with initial denaturation at 94 °C for 7 min, 25 cycles
of 94 °C for 45 s, 62 °C for 40 s and 72 °C for 60 s, with the
final extension for 5 min. Products were analyzed by agarose
gel electrophoresis and visualized on a UV light box. Product
intensity was determined to increase linearly with number of
cycles and amount of mRNA used, by densiometric analysis
(
8) Su, G. H.; Sohn, T. A.; Ryu, B.; Kern, S, E. A Novel Histone
Deacetylase Inhibitor Identified by High-Troughput Transcrip-
tional Screening of a Compound Library. Cancer Res. 2000, 60,
3
137-3142.
(
9) Butler, L. M.; Agus, D. B.; Sher, H. I.; Higgins, B.; Rose, A.;
Cordon-Cardo, C.; Thaler, H. T.; Rifkind, R. A.; Marks, P. A.;
Richon, V. M.. Suberoylanilide Hydroxamic Acid, an inhibitor
of Histone Deacetylase, Suppresses the Growth of Prostate
Cancer Cells in Vitro and in Vivo. Cancer Res. 2000, 60, 5165-
5
170.
using ImageQuaNT 4.2 software (Molecular Dynamics, Sunny-
(
10) Tsuji, N.; Kobayashi, M.; Nagashima, K.; Wakisaka, Y.; Koizumi,
WAF1/Cip1
vale, CA). Quantitation of p21
induction was also
K. A new antifungal antibiotic, trichostatin. J . Antibiot. 1976,
performed by densitometric analysis using ImageQuaNT 4.2
software following normalization to GAPDH product intensity.
2
9, 1-6.
(11) Yoshida, M.; Kijima, M.; Akita, M.; Beppu, T. Potent and specifik
Inhibition of Mammalian Histone Deacetylase Both in Vivo and
in Vitro by Trichostatin A. J . Biol. Chem.1990, 265, 17174-
Mor p h ologica l Rever sion . Cells were plated into 96-well
3
microtiter plates at 5 × 10 cells/well and allowed to adhere
1
7179.
overnight. Compounds were added to culture medium at the
indicated concentrations and plates incubated in the above
conditions for 24 h. Cells were then washed once with Hank’s
Balanced Salt Solution (HBSS; Gibco/Invitrogen, Grand Island,
NY), and fixed in 4% buffered formalin for 1 h at room
temperature. The fixed cells were then washed once further
with HBSS and stained with 1% Crystal Violet in methanol
for 5 min. Excess stain was removed by washing with tap
water, before the microtiter plate being air-dried at 37 °C.
Photographs were taken using a Leica DMIRB inverted
microscope.
(
12) Kijima, M.; Yoshida, M.; Sugita, K.; Horinouchi, S.; Beppu, T.
Trapoxin, an Antitumor Cyclic Tetrapeptide, Is an Irreverible
Inhibitor of Mammalian Histone Deacetylase. J . Biol. Chem.1993,
2
68, 22429-22435.
(
13) Darkin-Rattray, S. J .; Gurnett, A. M.; Myers, R. W.; Dulski, P.
M.; Crumley, T. M.; Allocco, J . J .; Cannova, C.; Meinke, P. T.;
Colletti, S. L.; et al. Apicidin: a novel antiprotozoal agent that
inhibits parasite histone deacetylase. Proc. Nat. Acad. Sci. U.S.A.
1
996, 93, 13143-13147.
(
(
(
14) Yoshida, M.; Furumai, R.; Nishiyama, M.; Komatsu, Y.; Nishino,
N.; Horinouchi, S. Histone deacetylase as a new target for cancer
chemotherapy. Cancer Chemother. Pharmacol. 2001, 48, S20-
S26.
15) Furumai, R.; Komatsu, Y.; Nishino, N.; Khochbin, S.: Yoshida,
M.; Horinouchi, S. Potent histone deacetylase inhibitors built
from trichostatin A and cyclic tetrapeptide antibiotics including
trapoxin. Proc. Nat. Acad. Sci. U.S.A.. 2001, 98, 87-92.
16) Murray, P. J .; Kranz, M.; Ladlow, M.; Taylor, S.; Berst, F.;
Holmes, A. B.; Keavey, K. N.; J axa-Chamiec, A.; Seale, P. W.;
Stead, P.; Upton, R. J .; Croft, S. L.; Clegg, W.; Elsegood, M. R.
J . The synthesis of cyclic tetrapeptiod analogues of the antipro-
tozoal natural product Apicidin. Bioorg. Med. Chem. Lett. 2001,
Ack n ow led gm en t. We thank the Australian Re-
search Council and The Hans Werth e´ n Foundation for
fellowship support to D.F. and P.K. and NHMRC for
support to P.P.
Ap p en d ix
1
1, 773-776.
(
17) Taunton, J .; Collins, J . L.; Schreiber, S. L. Synthesis of Natural
and Modified Trapoxins, Useful Reagents for Exploring Histone
Deacetylase Function. J . Am. Chem. Soc. 1996, 118, 10412-
Abbr eviation s. Ac ) acetyl; DCM ) dichloromethane;
DIPEA ) diisopropylamine; DMAP ) 4-(dimethylami-
no)pyridine; DMBA ) 1,3-dimethylbarbituric acid; DMF
1
0422.
(
18) Komatsu, Y.; Tomizaki, K.; Tsukamoto, M.; Kato, T.; Nishino,
N.; Sato, S.; Yamori, T.; Tsuruo, T.; Furumai, R.; Yoshida, M.;
Horinouchi, S.; Hayashi, H. Cyclic Hydroxamic-acid-containing
Peptide 31, a Potent Synthetic Histone Deacetylase Inhibitor
with Antitumor Activity. Cancer Res. 2001, 61, 4459-4460.
)
dimethylformamide; EtOAc ) ethyl acetate; Fmoc-
OSu ) 9-fluorenylmethyloxycarbonyl-N-hydroxysuccin-
imide; HATU ) O-(7-azabenzotriazol-1-yl)-1,1,3,3-tet-
ramethyluronium hexafluorophosphate; HBTU ) [(benzo-
triazolyl)oxy]-N′,N′,N′,N′-tetramethyluronium hexafluo-
rophosphate; rpHPLC ) reverse phase high perfor-
mance liquid chromatography; LRMS ) low resolution
mass spectroscopy; TFA ) trifluoroacetic acid; THF )
tetrahydrofuran; TSA ) trichostatin A.
(19) Cairns B. R. Emerging roles for chromatin remodelling in cancer
biology. Trends Cell Biol. 2001, 11, S15-S21.
(
20) Cress, W. D.; Seto, E. Histone Deacetylase, Transcriptional
Control, and Cancer. J . Cell. Physiol. 2000, 184, 1-16.
(21) Schlake, T.; Klehr-Wirth, D.; Yoshida, M.; Beppu, T.; Bode, J .
Gene expression within a chromatin domain: the role of core
histone hyperacetylation. Biochemistry 1994, 33, 4197-4206.
(22) Marks, P. A.; Richon, V. M.; Breslow, R.; Rifkind, R. A. Histone
deacetylase inhibitors as new cancer drugs. Curr. Opin. Oncol.
2
001, 13, 477-483.
Su p p or tin g In for m a tion Ava ila ble: Mass spectral and
HPLC data. This material is available free of charge via the
Internet at http://pubs.acs.org.
(
23) Grozinger, C. M.; Schreiber, S. L. Deacetylase Enzymes: Biologi-
cal Functions and the Use of Small-Molecule Inhibitors. Chem.
Biol. 2002, 9, 3-16.