Anti-proliferation effect of 5,5-diphenyl-2-thiohydantoin (DPTH) in human vascular endothelial cells

Article abstract of DOI:10.1016/j.bcp.2003.08.018

The aim of this study was to examine the anti-proliferation effect of 5,5-diphenyl-2-thiohydantoin (DPTH), an analogue of antiepileptic drug phenytoin (5,5-diphenylhydantoin), on human umbilical vein endothelial cells (HUVEC) and its possible molecular mechanism underlying. Here we demonstrated that DPTH at a range of concentrations (12.5-50 μM) dose- and time-dependently inhibited DNA synthesis and decreased cell number in cultured HUVEC, but not human fibroblasts. DPTH was not cytotoxic at these concentrations. [3H]Thymidine incorporation and flow cytometry analyses demonstrated that treatment of HUVEC with DPTH arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that the protein level of p21 increased after DPTH treated. In contrast, the protein levels of p27, p53, cyclins A, D1, D3 and E, cyclin-dependent kinase (CDK)2, and CDK4 in HUVEC were not changed significantly after DPTH treatment. Immunoprecipitation showed that the formations of the CDK2-p21 and CDK4-p21 complex, but not the CDK2-p27 and CDK4-p27 complex, were increased in the DPTH-treated HUVEC. Kinase assay further demonstrated that both CDK2 and CDK4 kinase activities were decreased in the DPTH-treated HUVEC. Pretreatment of HUVEC with a p21 antisense oligonucleotide reversed the DPTH-induced inhibition of [3H]thymidine incorporation into HUVEC. In conclusion, these data suggest that DPTH inhibits HUVEC proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 and CDK4 kinase activities, and finally interrupts the cell cycle. The findings from the present study suggest that DPTH might have the potential to inhibit the occurrence of angiogenesis.

Full text of DOI:10.1016/j.bcp.2003.08.018

Biochemical Pharmacology 67 (2004) 67–75
Anti-proliferation effect of 5,5-diphenyl-2-thiohydantoin
(DPTH) in human vascular endothelial cells
Chun-Ru Shiha, Jender Wub, Yuan Liua, Yu-Chih Liangc, Shyr-Yi Linc,
Ming-Thau Sheud, Wen-Sen Leea,e,*
aGraduate Institute of Medical Sciences, School of Medicine, Taipei Medical University, Taipei, Taiwan, ROC
bDepartment of Medicinal Chemistry, School of Pharmacy, Taipei Medical University, Taipei, Taiwan, ROC
cDepartment of Internal Medicine, Taipei Medical University, Taipei, Taiwan, ROC
dGraduate Institute of Pharmaceutical Sciences, School of Pharmacy, Taipei Medical University, Taipei, Taiwan, ROC
eDepartment of Physiology, School of Medicine, Taipei Medical University, Taipei, Taiwan, ROC
Received 16 May 2003; accepted 21 August 2003
Abstract
The aim of this study was to examine the anti-proliferation effect of 5,5-diphenyl-2-thiohydantoin (DPTH), an analogue of antiepileptic
drug phenytoin (5,5-diphenylhydantoin), on human umbilical vein endothelial cells (HUVEC) and its possible molecular mechanism
underlying. Here we demonstrated that DPTH at a range of concentrations (12.5–50 mM) dose- and time-dependently inhibited DNA
synthesis and decreased cell number in cultured HUVEC, but not human fibroblasts. DPTH was not cytotoxic at these concentrations.
[3H]Thymidine incorporation and flow cytometry analyses demonstrated that treatment of HUVEC with DPTH arrested the cell at the G0/
G1 phase of the cell cycle. Western blot analysis revealed that the protein level of p21 increased after DPTH treated. In contrast, the protein
levels of p27, p53, cyclins A, D1, D3 and E, cyclin-dependent kinase (CDK)2, and CDK4 in HUVEC were not changed significantly after
DPTH treatment. Immunoprecipitation showed that the formations of the CDK2–p21 and CDK4–p21 complex, but not the CDK2–p27
and CDK4–p27 complex, were increased in the DPTH-treated HUVEC. Kinase assay further demonstrated that both CDK2 and CDK4
kinase activities were decreased in the DPTH-treated HUVEC. Pretreatment of HUVEC with a p21 antisense oligonucleotide reversed the
DPTH-induced inhibition of [3H]thymidine incorporation into HUVEC. In conclusion, these data suggest that DPTH inhibits HUVEC
proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 and CDK4 kinase activities, and finally interrupts the cell
cycle. The findings from the present study suggest that DPTH might have the potential to inhibit the occurrence of angiogenesis.
# 2003 Elsevier Inc. All rights reserved.
Keywords: DPTH; Angiogenesis; p21; p53; Cyclin-dependent kinase; Endothelial cells
1. Introduction
stimulators and inhibitors of angiogenesis maintains it in
balance. However, persistent and unregulated angiogenesis
is often found to be a critical causal factor in many
pathological conditions [2].
Angiogenesis, the formation of new capillary blood
vessels as extensions of existing vessels, is a complex
process regulated by multiple stimulatory and inhibitory
factors [1]. In the physiological condition, the activity of
The events that are thought to be essential for angiogen-
esis include: local degradation of the basement membrane
of the parent vessel, allowing protrusion of endothelial
cells; outward migration of endothelial cells in tandem to
form a capillary sprout; proliferation of endothelial cells
within the sprout; and the formation of a lumen with
subsequent branching. Angiogenesis is essential for many
physiological processes and important in the pathogenesis
of many disorders [1]. Normally, vascular proliferation
occurs only during embryonic development, the female
reproductive cycle and wound healing. In contrast, many
* Corresponding author. Tel.: þ886-2-27361661x3103;
fax: þ886-2-27391775.
E-mail address: wslee@tmu.edu.tw (W.-S. Lee).
Abbreviations: DPTH, 5,5-diphenyl-2-thiohydantoin; DPH, 5,5-diphe-
nylhydantoin; HUVEC, human umbilical vein endothelial cells;
HDMVEC, human dermal microvascular endothelial cells; CDK, cyclin-
dependent kinase; ECGS, endothelial cell growth supplement; M199,
medium 199; FBS, fetal bovine serum; CKI, CDK-inhibitory protein;
FACS, fluorescence-activated cell sorter.
0006-2952/$ – see front matter # 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2003.08.018

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199 (M199), trypsin-EDTA, and kanamycin were pur-
chased from Life Technologies. Fetal bovine serum
(FBS) was purchased from HyClone. Antibodies specific
for cyclins, CDKs, and CKIs were purchased from Trans-
duction Laboratories. An antibody specific for G3PDH was
purchased from Biogenesis. Anti-mouse IgG conjugated
alkaline phosphatase was purchased from Jackson Immuno
Research Laboratories. 4-Nitro blue tetrazolium (NBT)
and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) were
purchased from Kirkegaard & Perry Laboratories. Protein
assay agents were purchased from Bio-Rad.
Fig. 1. Chemical structure of DPTH.
pathological conditions (e.g. atherosclerosis, cancer, and
diabetic retinopathy) are characterized by persistent, unre-
gulated angiogenesis [2]. Control of vascular development
could permit new therapeutic approaches to these disor-
ders. During the past few years, experimental and clinical
investigators continue to search for new therapeutic stra-
tegies for preventing the occurrence of angiogenesis.
One approach, as pursued in this study, seeks to identify
medicinal agents capable of retarding the cell cycle in the
vascular endothelial cells.
DPTH (Fig. 1), an analogue of antiepileptic drug phe-
nytoin (5,5-diphenylhydantoin, DPH), has been suggested
to be a potential hypolipidemic agent [3] and a potent
goitrogenic compound [4]. Structural difference is that
2-oxo functionality of DPH is replaced as 2-thio of DPTH,
but their biological activity is very different. DPTH is
inactive as an anti-convulsant, and its prominent activities
were reported as being inhibitory against thyroxine-stimu-
lated response in mitochondria and being hypolipidemic
[5]. As far as anti-angiogenic activity is concerned, DPTH
has not been assessed yet.
2.2. Cell culture
HUVEC or human dermal microvascular endothelial
cells (HDMVEC) were grown in M199 containing 10%
FBS, ECGS (0.03 mg/mL) and kanamycin (50 U/mL) in a
humidified 378 incubator. After the cells had grown to
confluence, they were disaggregated in trypsin solution,
washed with M199 containing 10% FBS, centrifuged at
125 g for 5 min, resuspended, and then subcultured accord-
ing to standard protocols. Cells from passages 5 to 9 were
used.
2.3. [3H]Thymidine incorporation
The [3H]thymidine incorporation was performed as
previously described [7,8]. Briefly, HUVEC were applied
to 24-well plates in growth medium (M199 plus 10% FBS
and ECGS). After the cells had grown to 70–80% con-
fluence, they were rendered quiescent by incubation for
24 hr in M199 containing 2% FBS. M199 supplemented
with 10% FBS and 0.05% DMSO (control) or various
concentrations of DPTH was added to the cells and the
cultures were allowed to incubate for 21 hr. During the last
2 hr of the incubation with or without DPTH, [3H]thymidine
was added at 1 mCi/mL (1 mCi ¼ 37 kBq). Incorporated
[3H]thymidine was extracted in 0.2 N NaOH and measured
in a liquid scintillation counter.
This study was designed to investigate the inhibitory
effect of DPTH in HUVEC proliferation and its molecular
mechanism underlying. Here, we demonstrate that DPTH
dose-dependently inhibited the growth of HUVEC by
interrupting the transition of cell cycle from the G1 into
S phase. The DPTH-induced cell cycle arrest in HUVEC
occurred when the CDK2 and CDK4 activities were
inhibited just as the level of p21 protein increased.
2. Materials and methods
2.4. Cell counting
2.1. Materials
As a measurement of cell proliferation, the cells were
seeded onto 6-well 1% gelatin-coated plates and grown in
M199 supplemented with 10% FBS and ECGS. Media
without (control) and with DPTH were changed daily until
cell counting. At various times of incubation, cultures were
treated with trypsin-EDTA and the released cells were
counted in a Coulter apparatus.
DPTH was synthesized as previously described [6].
Briefly, a mixture of benzil (2.1 g, 10 mmol) and thiourea
(0.8 g, 10.5 mmol) in 95% ethanol (15 mL) was added 8 M
aqueous KOH (3 mL) and the whole was heated for 2.5 hr
and then cooled. Thin layer chromatography (SiO2,
CH2Cl2) showed the disappearance of benzil. Concentrated
HCl was added to acidify the mixture at 08 and the resulted
precipitate was collected, washed with water and then
dried. Recrystallization with alcohol gave 2.53 g (94%)
yield, mp 237–2398 (Lit. 239–2408). HEPES, glycerol,
phenylmethylsulphonyl fluoride (PMSF), SDS, Nonident
P-40 (NP-40), and endothelial cell growth supplement
(ECGS) were purchased from Sigma Chem. Medium
2.5. Viability assay
Cell viability was estimated by a modified MTT [3-(4,5-
dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide]
assay as previously described [8]. Four samples were
analyzed in each experiment.

C.-R. Shih et al. / Biochemical Pharmacology 67 (2004) 67–75
69
2.6. Protein preparation and Western blotting
anti-CDK2 or anti-CDK4 antibody (2 mg/mL) and protein
A agarose beads (1/10 V). The precipitates were washed
five times with washing buffer, and then resuspended in
sample buffer (250 mM Tris–HCl, pH 6.8, 8% SDS, 40%
glycerol, 20% b-mercaptoethanol, and 0.04% bromophe-
nol blue) and incubated at 958 for 10 min before electro-
phoresis to release the proteins from the beads.
To determine the expression levels of cyclins, CDKs,
CKIs, and G3PDH in HUVEC, the total proteins were
extracted and Western blot analyses were performed as
described previously [8,9]. Briefly, HUVEC were cultured
in 10 cm petri dishes. After reaching subconfluence, the
cells were rendered quiescent and then treated with various
concentrations of DPTH for 21 hr, and then incubated in a
humidified incubator at 378. After incubation, the cells were
washed with PBS (pH 7.4), incubated with extraction buffer
(Tris 50 mM, pH 7.5, NaCl 150 mM, PMSF 1 mM, NP-40
1%, 0.1% SDS, 10 mg/mL Aprotinin and EDTA 10 mM) on
ice, and then centrifuged at 12,000 g for 30 min. The cell
extract was then boiled in a ratio of 3:1 with sample buffer
(Tris–HCl 250 mM, pH 6.8, glycerol 40%, b-mercaptoetha-
nol 20%, SDS 8% and bromophenol blue 0.04%). Electro-
phoresis was performed using 12% SDS-polyacrylamide
gel (2 hr, 110 V, 40 mA, 50 mg protein per lane). Separated
proteins were transferred to PVDF membranes (1 hr,
400 mA), treated with 5% fat-free milk powder to block
the nonspecific IgGs, and incubated for 1 hr with specific
antibody for cyclins, CDKs, CKIs, or G3PDH. The blot was
then incubated with anti-mouse or anti-rabbit IgG linked to
alkaline phosphatase (1:1000) for 1 hr. Subsequently, the
membrane was developed with NBT/BCIP as a substrate.
2.9. CDK kinase assay
As previously described [8], CDK2 or CDK4 immuno-
precipitates from DPTH-treated and control HUVEC were
washed three times with lysis buffer and twice with kinase
assay buffer [50 mM Tris–HCl, pH 7.4, 10 mM MgCl2, and
1 mM dithiothreitol (DTT)]. Phosphorylation of histone
H1 (for CDK2) and glutathione-S-transferase/retinoblas-
toma (Gst-Rb) fusion protein (for CDK4) were measured
by incubating the beads with 40 mL of ‘‘hot’’ kinase solu-
tion [0.25 mL (2.5 mg) histone H1, 0.5 mL [g-32P]ATP
(10 mCi/mL), 0.5 mL 0.1 mM ATP, and 38.75 mL kinase
buffer] at 378 for 30 min. The reaction was stopped by
boiling the sample in SDS sample buffer for 5 min. The
products were analyzed by 10% SDS–PAGE. The gel was
dried and visualized by autoradiography.
2.10. Flow cytometry
2.7. RNA extraction and RT–PCR
As previously described [10], the cells were seeded onto
10-cm petri dishes and grown in M199 supplemented with
10% FBS and ECGS. After the cells had grown to sub-
confluence, they were rendered quiescent and challenged
with 10% FBS. Then, after release using trypsin-EDTA,
they were washed twice with PBS and fixed in 70% ethanol
at 48. Nuclear DNA was stained with a reagent containing
propidium iodine (8 mg/mL) and DNase-free RNase
(100 mg/mL) and measured using a fluorescence-activated
cell sorter (FACS).
Total RNA was isolated from cultured HUVEC and
prepared by guanidinium isothiocyanate extraction and
centrifugation through cesium chloride. The mRNA levels
were detected by reverse transcription–polymerase chain
reaction (RT–PCR) technique as previously described [9].
The p21 cDNA amplication was performed by incubating
20 ng equivalents of cDNA in 100 mM Tris–HCl buffer,
pH 8.3, containing 500 mM KCl, 15 mM MgCl2, 0.1%
gelatin, 200 mM concentration of each dNTP, and 50 U/mL
Super Taq DNA polymerase with specific oligonucleotide
primers: 50-AGGAGGCCCGTGAGCGAGCGATGGAAC-
30 and 50-ACAAGTGGGGAGGAAGTAGC-30. The cDNA
sequence of GAPDH was also amplified as a control in
the same method using the following primers: 50-CCACC-
CATGGCAAATTCCATGGCA-30 and 50-TCTAGACGG-
CAGGTCAGGTCCACC-30. PCR products were analyzed
on 1.0% agarose gels. Thermal cycle conditions were as
follows: 1 cycle at 948 for 5 min, 30 cycles at 948 for 1 min
2.11. Statistical analysis
Values represent the means ꢀ SEM. Three to four sam-
ples were analyzed in each experiment. Comparisons were
subjected to one-way ANOVA followed by Fisher’s least
significant difference test. Significance was accepted at
P < 0:05.
(for p21) or 45 s (for GAPDH), 558 for 1 min (for p21Cip1
)
3. Results
or 598 (for GAPDH) for 45 s, 728 for 2 min (for p21) or
1 min (for GAPDH), and 1 cycle at 728 for 10 min. PCR
products were analyzed on 1.8% agarose gels.
3.1. Inhibition of [3H]thymidine incorporation and
cell proliferation in HUVEC by DPTH
2.8. Immunoprecipitation
To study the anti-proliferative effect of DPTH on the
vascular endothelial cells, we examined changes in
[3H]thymidine incorporation (a measurement of DNA
synthesis) in response to DPTH treatment in subcultured
As previously described [8], CDK2 or CDK4 was
immunoprecipitated from 200 mg of protein by using

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C.-R. Shih et al. / Biochemical Pharmacology 67 (2004) 67–75
HUVEC
8
6
4
6
12 HDMVEC
HUVEC
*
*
9
6
3
4
2
*
*
*
*
*
*
*
2
0
0
0
0
12.5
25
50
0
12.5
25
50
0
12.5
25
50
(a)
(b)
(c)
DPTH Concentrations ( M)
DPTH Concentrations ( M)
DPTH Concentrations ( M)
Human fibroblast
7
4
3
2
0.20
HDMVEC
HUVEC
6
5
0.16
*
0.12
0.08
4
3
2
1
0
*
*
1
0
0.04
0
0
12.5
25
50
0
12.5
25
50
0
12.5
25
50
(d)
DPTH Concentrations (µM)
DPTH Concentrations (µM)
(e)
DPTH Concentrations (µM)
(f)
Fig. 2. Effects of DPTH on [3H]thymidine incorporation and cell growth rate in subcultured HUVEC. (a) Dose-dependent inhibition of [3H]thymidine
incorporation in HUVEC by DPTH. (b and c) Dose-dependent inhibition of vascular endothelial cell growth by DPTH treatment. The HUVEC (b) or
HDMVEC (c) were treated with DMSO without or with various doses of DPTH for 3 days, and then the cells were harvested and counted. (d) Continuous
presence of DPTH is not necessary for its anti-proliferation effect in vascular endothelial cells. The HDMVEC were treated with DMSO without or with
various doses of DPTH for 1 day, and then counted the number of cells after 6 days. A dose-dependent inhibition of cell number was still observed. (e) There
was no significant difference in viability between control and DPTH-treated HUVEC. (f) Treatment with DPTH (12.5–50 mM) for 3 days did not affect cell
growth of human fibroblasts. Three to four samples were analyzed in each group, and values represent the means ꢀ SEM. Significance was accepted at
P < 0:05. (ꢁ) DPTH-treated group different from control group.
HUVEC. As illustrated in Fig. 2a, treatment of HUVEC
with DPTH (12.5–50 mM) for 21 hr induced a decrease in
[3H]thymidine incorporation in a dose-dependent manner.
We further examined the effect of DPTH on cell number of
HUVEC. In the experiment of Fig. 2b, HUVEC were
cultured for 3 days with or without DPTH (12.5–50 mM),
and then the cells were harvested and counted. These data
showed that a reduced cell count was observed in the DPTH-
treatedHUVECascomparedwithDMSO-treatedcells. This
DPTH-induced decrease in the cell growth rate was dose-
dependent, consistent with the inhibitory effect of DPTH on
[3H]thymidine incorporation. The DPTH-induced decrease
in the cell growth rate was also observed in the other
endothelial cells, HDMVEC (Fig. 2c). In contrast, treatment
of human fibroblasts with DPTH (12.5–50 mM) for 3 days
did not cause any significant growth inhibition (Fig. 2f),
suggesting the specific effect of DPTH on HUVEC growth
inhibition. However, the continuous presence of DPTH is
not necessary for its anti-proliferation effect in vascular
endothelial cells. As illustrated in Fig. 2d, the HDMVEC
were treated with DPTH for 1 day and then counted the
number of cells after 6 days. A dose-dependent inhibition of
cell number was still observed. The DPTH-induced reduc-
tion in [3H]thymidine incorporation in HUVEC can be due
to retardation of cell cycle or cell death. To confirm that the
results of our studies of DNA synthesis and cellular pro-
liferation in HUVEC were not due to cell death caused by
DPTH treatment, we conducted viability assay by treating
the cells with DPTH for 21 hr at the doses (12.5–50 mM)
used in the studies of cell growth inhibition. MTT assays
indicated that there was no significant difference in cell
viability between control and DPTH-treated HUVEC
(Fig. 2e), suggesting that there was an inhibitory effect of
DPTHonthemechanismsforcelldivisioninthesubcultured
HUVEC.
3.2. Arrest of cell cycle in G0/G1phase by
DPTH treatment
To further investigate the cellular mechanism of the
DPTH-induced growth inhibition, FACS analyses of
DNA content in both DMSO- and DPTH-treated HUVEC

C.-R. Shih et al. / Biochemical Pharmacology 67 (2004) 67–75
71
Fig. 3. Retardation of cell cycle in HUVEC by DPTH. FACS analysis of DNA content was performed after 24 hr release from quiescence by incubation in
culture media supplemented with 10% FBS and 0.05% DMSO without (a) or with 50 mM DPTH (b). Results from a representative experiment are shown.
Percentage of cells at the G0/G1, S, or G2/M phase of the cell cycle was determined using established CellFIT DNA analysis software.
Fig. 4. Effect of DPTH on the protein levels of cyclins, CDKs and CKIs. Western blot analysis was performed to examine the changes of protein levels of
cyclins, CDKs, and CKIs in the DPTH-treated HUVEC. Proteins were extracted from the cultured HUVEC at 21 hr after DPTH treatment and probed with
proper dilutions of specific antibodies. (a) DPTH (12.5–50 mM) did not induce any significant change of the protein levels of cyclin A, D1, D3, and E, CDK2,
and CDK4. Results from a representative experiment are shown. (b) Treatment of HUVEC with DPTH (12.5–50 mM) for 21 hr dose-dependently increased
the protein levels of p21, but not p27 and p53. Results from a representative experiment are shown. Three samples were analyzed in each group, and values
shown in parentheses represent the means ꢀ SEM. Membrane was probed with anti-G3PHD antibody to verify equivalent loading. (c) The time course of p21
mRNA changes in HUVEC in response to 50 mM DPTH treatment. Values shown in parentheses represent the relative intensities (ratios of p21/GAPDH
mRNA in the HUVEC treated with DPTH vs. DMSO). RT–PCR products of GAPDH were used as an internal control. CDK, cyclin-dependent kinase.

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C.-R. Shih et al. / Biochemical Pharmacology 67 (2004) 67–75
were conducted. Initially, the cells were switched to media
with 2% FBS for 24 hr to render them quiescent and to
synchronize their cell cycle activities. Then they were
returned to media with 10% FBS without or with DPTH
(50 mM). Figure 3 showed that after 24 hr treatment of
HUVEC with DPTH induced a significant accumulation of
cells at the G0/G1 phase of the cell cycle as compared with
the cell treated with vehicle, suggesting that the observed
growth inhibition effect of DPTH was due to an arrest of
DNA replication thereby inhibiting further progress in the
cell cycle.
coordinated successive activation of certain CDKs. This
CDK activation is in turn modulated by association with
a number of regulatory subunits called cyclins, and with
a group of CDK-inhibitory proteins designated CKIs.
Accordingly, we examined the changes in cyclin and
CDK protein level in the DPTH-treated HUVEC. As
shown in Fig. 4a, treatment of HUVEC with DPTH for
21 hr at a range of concentrations (12.5–50 mM), which
caused the inhibition of [3H]thymidine incorporation and
cell growth, did not induce any significant changes of the
protein levels of cyclin A, cyclin D1, cyclin D3, cyclin E,
CDK2 and CDK4, suggesting that DPTH-induced cell
cycle arrest in HUVEC is not through alternations of the
protein levels of cyclins and CDKs. Since the CDK activity
can be also controlled by a group of CKIs, we further
examined the changes of protein levels of p21 and p27, two
known CKIs, in the DPTH-treated HUVEC. Figure 4b
showed that the protein levels of p21, but not p27, were
dose-dependently increased in the DPTH-treated HUVEC
as compared with the DMSO-treated cells (control).
3.3. Alterations in cell cycle activity by
DPTH treatment
To delineate the molecular mechanism of DPTH-
induced inhibition of HUVEC cell proliferation, we further
examined the levels of cell cycle regulatory proteins in
the DPTH-treated HUVEC. It has been generally believed
that progression of cell cycle activity is regulated by
Fig. 5. Effect of DPTH on the formations of CKI–CDK complex and CDK kinase activity. (a) DPTH-induced upregulation of the formations of CDK2–p21
and CDK4–p21 complex in a dose-dependent manner. The formations of CDK2–p27 and CDK4–p27 complex were not affected significantly by DPTH
treatment. CDK2 was immunoprecipitated by anti-CDK2 antibody, and CDK2–p21 association was detected by anti-p21 antibody, whereas CDK2–p27
association was detected by anti-p27 antibody. CDK4 was immunoprecipitated by anti-CDK4 antibody, and CDK4–p21 association was detected by anti-p21
antibody, whereas CDK4–p27 association was detected by anti-p27 antibody. Results from a representative experiment are shown. Values shown in
parentheses represent the relative intensities (ratios of CDK–p21 or CDK–p27 complex in the HUVEC treated with DPTH vs. DMSO). (b) Treatment of the
HUVEC with DPTH dose-dependently decreased the CDK2 and CDK4 kinase activities. Results from a representative experiment are shown. The CDK2 and
CDK4 kinase activities were determined as described in Section 2. Values shown in parentheses represent the relative intensities (ratios of CDK2 or CDK4
kinase activity in the HUVEC treated with DPTH vs. DMSO). CDK, cyclin-dependent kinase.

C.-R. Shih et al. / Biochemical Pharmacology 67 (2004) 67–75
73
Fig. 6. Involvement of p21 in the DPTH-induced decrease of [3H]thymidine incorporation in HUVEC. Antisense p21 or p27 oligonucleotide was added to
HUVEC at a final concentration up to 10 nM at 16 hr before the cell was challenged with 10% FBS and 50 mM DPTH for additional 21 hr. The DPTH-
induced up-regulation of p21 protein was reduced by pre-treatment of the cells with p21 antisense oligonucleotide (a, right panel). Membrane was probed
with anti-G3PHD antibody to verify equivalent loading. The levels of p21 protein in each treatment after normalized with the levels of G3PDH protein were
shown in parentheses. Pretreatment of HUVEC with AS p21 (a, left panel), but not AS p27 (b), dose-dependently reversed the DPTH-induced decrease of
[3H]thymidine incorporation. Values represent the means ꢀ SEM. AS p21, antisense p21 oligonucleotide; AS p27, antisense p27 oligonucleotide.
Using RT–PCR technique, we further demonstrated that
treatment of HUVEC with 50 mM DPTH for 3 hr induced
an up-regulation of p21 mRNA (Fig. 4c), suggesting that
transcriptional regulation was involved in the DPTH-
induced increase in p21 protein levels. The CKI exerts
its inhibitory effect on the kinase activity through binding to
cyclin–CDK complex. Accordingly, we further conducted
immunoprecipitation assay to examine the effect of DPTH
on the formation of CDK–CKI complex. In the DPTH-
treated cells, the formations of the CDK2–p21 and CDK4–
p21 complex, but not CDK2–p27 and CDK4–p27 complex,
were increased (Fig. 5a). To demonstrate that the increased
p21 protein is associated with inhibition of CDK activation,
we examined the CDK kinase activity. Figure 5b showed
that the assayable CDK2 and CDK4, kinase activities
were significantly decreased in the HUVEC treated with
DPTH. To further demonstrate that the increased p21
expression observed in the DPTH-treated HUVEC corre-
lated with G0/G1 arrest, the experiment illustrated in Fig. 6
was conducted. Thus, in the sample labeled DPTH (for
50 mM DPTH-treated alone), the [3H]thymidine incorpora-
tion was decreased. Sample DPTH þ AS p21 was pre-
treated with a p21 antisense oligonucleotide (AS), which
blocked the expression of p21 protein (Fig. 6a, right panel).
Treatment of HUVEC with AS p21 or AS p27 alone did
not cause any significant change in [3H]thymidine incor-
poration into HUVEC. Consequently, pretreatment of the
HUVEC with AS p21 dose-dependently reversed the
DPTH-induced decrease in [3H]thymidine incorporation
(Fig. 6a, left panel). In contrast, pretreatment of HUVEC
with AS p27 failed in preventing the DPTH-induced
decrease in [3H]thymidine incorporation (Fig. 6b).
4. Discussion
Control of vascular development has been suggested
to be new therapeutic approaches to many angiogenesis-

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C.-R. Shih et al. / Biochemical Pharmacology 67 (2004) 67–75
related disorders. In the present study, we demonstrated
that DPTH at a range of concentrations (12.5–50 mM)
inhibited DNA synthesis and decreased cell number in
cultured HUVEC in a dose- and time-dependent manner
(Fig. 2). These results were not due to cell death, indicating
that there was an inhibitory effect of DPTH on the mechan-
isms for cell division in the subcultured HUVEC. To our
knowledge, this is the first demonstration that DPTH
inhibits the growth of human vascular endothelial cells.
By flow cytometry analyses, we demonstrated that
DPTH treatment decreased DNA synthesis and arrested
the cells at the G0/G1 phase of the cell cycle (Fig. 3). It has
been suggested that the cell cycle is regulated by the
activation of specific CDKs and coordinated successive
activation of certain CDKs occurs late in the G1 phase and
is instrumental in the transition from the G1 to the S phase
[11,12]. This CDK activation is in turn modulated posi-
tively by their assembly with a series of regulatory subunits
called cyclins, and negatively by association with a group
of CDK-inhibitory proteins designated CKIs [13]. Cyclins
have been identified as cyclins A, D1, D3 and E, whereas
the most common CDKs are designated CDK2 and CDK4.
The formations of cyclin A–CDK2 and cyclin E–CDK2
complex occur late in the G1 phase as cells prepare to
synthesize DNA [14], and formation of the cyclin E
complex is a rate-limiting step in the G1/S transition
[15]. The basic mechanisms for cell cycle regulation
appear to be universal. In the present study, we demon-
strated that DPTH at a concentration of 50 mM, which
inhibited cell cycle arrest, did not induce any significant
changes of the protein levels of cyclins A, D1, D3 and E,
CDK2, and CDK4 in the HUVEC, indicating that the
action of DPTH on the growth of endothelial cells is not
through a mechanism to reduce the cyclins or CDKs.
Examination of the expression levels of CDK inhibitory
proteins, we found that treatment of HUVEC with DPTH
resulted in an increase in the protein level of p21, but not
p27. In accord with the established notion that p21 is one
known CDK inhibitor, we found that the formations of the
CDK2–p21 and CDK4–p21 complex, but not CDK2–p27
and CDK4–p27complex, were increased and the assayable
CDK2 and CDK4 kinase activities were decreased in the
DPTH-treated HUVEC. These findings suggest that DPTH
inhibits the CDK2 and CDK4 kinase activities through an
increase in p21 expression. The important role of p21 in the
DPTH-induced anti-proliferation in the HUVEC is con-
firmed by the antisense p21 oligonucleotide experiment
showing that pretreatment with a p21 antisense oligonu-
cleotide, but not p27 antisense oligonucleotide, reversed
the DPTH-induced inhibition in [3H]thymidine incorpora-
tion. Accordingly, we concluded that DPTH induced an
increase in p21 expression, which in turn inhibited the
CDK2 and CDK4 enzyme activities and led to the impair-
ment of HUVEC in the transition from the G1 to S phase.
How does DPTH act to regulate the level of p21 protein
remains unsolved in the present study? In response to a
50 mM DPTH treatment for 18 hr, we observed that the
level of p21 protein increased significantly (Fig. 4b). Inter-
estingly, the level of p21 mRNA in HUVEC increased after
a 3 hr exposure and then declined after 6 hr (Fig. 4c).
Similarly, our previous study done in the 12-o-tetradeca-
noylphorbol-13-acetate-treated COLO-205 cell line
showed that the p21 mRNA level increased after 1 hr
treatment with 12-o-tetradecanoylphorbol-13-acetate,
peaked at 3 hr and then declined at 6 hr, whereas the level
of p21 protein increased significantly after 6 hr exposure
and peaked after 24 hr [9]. These data indicated that the
half-life of p21 protein is much longer than its mRNA, or
else a post-translational regulation might also be involved
in the DPTH-induced increase in the p21 protein.
Although we did not perform the time course experiment
of DPTH-induced increases in the protein levels of p21,
our data suggested that transcriptional regulation might be
involved in the DPTH-induced increase in p21 protein
levels. p21 is a transcriptional target of the tumor sup-
pressor gene p53 [16,17]. Expression of p53 in the cells
can induce cell growth arrest through transcriptional
activation of p21 [18]. However, treatment of HUVEC
with DPTH did not affect the expression level of p53
protein, suggesting that p53 protein is not involved in
this process. To identify the primary target molecules of
DPTH in regulating the p21 up-regulation, more experi-
ments need to be done. In conclusion, the results from the
present studies indicate that DPTH-induced cell cycle
arrest in HUVEC occurred when the cyclin-CDK system
was inhibited just as p21 protein levels increased. The
findings from the present studies suggest the potential
applications of DPTH in the treatment of angiogenesis-
related disorders.
Acknowledgments
This work was supported by research grants from
the National Science Council of the Republic of China
(NSC90-2320-B-038-032; 91-2320-B-038-045) to Dr. Lee.
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