Inhibitory effects of cancer cell proliferation by novel histone deacetylase inhibitors involve p21/WAF1 induction and G2/M arrest

Article abstract of DOI:10.1248/bpb.28.849

Two compounds were synthesized which have a structural component other than those of our new series histone deacetylase (HDAC) inhibitors to determine the structure-activity relationship. It was also examined whether the inhibitory effects on cancer cell proliferation by HDAC inhibitors involve p21/WAF1 induction and G₁ or G₂/M arrest in p53-mutated MG63 human osteosarcoma cells as do other HDAC inhibitors. It was demonstrated that inhibitors with the 2-naphthylcarbonyl group and hydroxamic acid at both termimal sides as well as the phenylene component at the center of molecule markedly induce the p21/WAF1 protein by stimulating p21/WAF1 gene promoter activity. Furthermore, cell cycle analysis revealed that these compounds arrest MG63 cells in the G₂/M phase.

Full text of DOI:10.1248/bpb.28.849

May 2005
Biol. Pharm. Bull. 28(5) 849—853 (2005)
849
Inhibitory Effects of Cancer Cell Proliferation by Novel Histone
Deacetylase Inhibitors Involve p21/WAF1 Induction and G2/M Arrest
Taishi MAEDA,a Yasuo NAGAOKA,a,b Yuki KAWAI,a Nobumasa TAKAGAKI,c Chikako YASUDA,c
Shingo YOGOSAWA,c Yoshihiro SOWA,c Toshiyuki SAKAI,c and Shinichi UESATO*,a,b
a Department of Biotechnology, Faculty of Engineering, Kansai University; b High Technology Researach Center, Kansai
University; Suita, Osaka 564–8680, Japan: and c Department of Molecular-Targeting Cancer Prevention, Kyoto
Prefectural University of Medicine, Graduate School of Medical Science; Kyoto 602–8566, Japan.
Received November 24, 2004; accepted February 2, 2005
Two compounds were synthesized which have a structural component other than those of our new series his-
tone deacetylase (HDAC) inhibitors to determine the structure–activity relationship. It was also examined
whether the inhibitory effects on cancer cell proliferation by HDAC inhibitors involve p21/WAF1 induction and
G1 or G2/M arrest in p53-mutated MG63 human osteosarcoma cells as do other HDAC inhibitors. It was demon-
strated that inhibitors with the 2-naphthylcarbonyl group and hydroxamic acid at both termimal sides as well as
the phenylene component at the center of molecule markedly induce the p21/WAF1 protein by stimulating
p21/WAF1 gene promoter activity. Furthermore, cell cycle analysis revealed that these compounds arrest MG63
cells in the G2/M phase.
Key words histone deacetylase inhibitor; p21/WAF1; MG63 cell; HCT116 cell; G2/M phase; cell cycle arrest
Histone acetyltransferases (HATs) and histone deacety- group) other than those of the HDAC inhibitors 1—4 to de-
lases (HDACs) play a crucial role in gene expression through termine whether these groups can be substitutes for pheny-
reversible acetylation and deacetylation of histones. HAT-me- lene and hydroxamic acid groups in 1—4, making contacts
diated hyperacetylation of lysine residues in the N-terminal with the walls and zinc ion of the HDAC pocket, respec-
tails of core histones loosens the histone–DNA binding and tively. We also examined whether the inhibitory effects on
activates gene transcription. In contrast, HDAC-catalyzed cancer cell proliferation by the novel histone deacetylase in-
deacetylation of acetylated lysine residues leads to tight his- hibitors involve p21/WAF1 induction and G1 or G2/M arrest
tone–DNA binding, which restricts the access of transcrip- as do other HDAC inhibitors through assessment of Western
tional factors.1—3) These enzymes are known to correlate blot, luciferase, and cell cycle progression assays.
with cell cycle progression, and their deregulation is associ-
Chemistry Compounds 5 and 6 were synthesized in the
ated with tumorigenesis.4—6) HDAC inhibitors appear to be following way (Chart 1): Condensation of methyl 6-amino-2-
attractive new antitumor candidates because they are ex- naphthoate hydrochloride and phenylacetyl chloride in the
pected to suppress the cell cycle progression of human tumor presence of Et3N gave 5a, which, after conversion to car-
cells and cause apoptosis by inducing the expression of cell boxylic acid 5b, was reacted with BOP-Cl and O-benzylhy-
cycle-arresting genes such as p21/WAF17) and GADD45.8) droxyamine hydrochloride to give 5c. It was then hydro-
Recently, X-ray crystallographic analysis has disclosed the genated over 10% Pd-C, yielding the desired 5. Condensation
interaction mode between the HDAC-like protein (HDLP)
from the bacterium Aquifex aeolicus and the natural HDAC
inhibitor TSA from actinomyces; TSA binds inside the
pocket of HDLP, making tight contacts with residues at the
rim, walls, and bottom of the pocket. The zinc ion near the
bottom is coordinated by the hydroxamic acid of TSA.9) For
the design of a new series HDAC inhibitors, we chose a ben-
zyl component for interaction with the walls of a HDAC
pocket, a bicyclic arylcarbonyl component for capping the
pocket, and the hydroxamic acid for capture of the zinc ion at
the bottom. Among the synthesized HDAC inhibitors, com-
pound 1 showed promising inhibitory efficacies against the
proliferation of HCT116 human colorectal carcinoma cells
(IC50 0.7 mM) and HDACs activities (IC50 0.044 mM) (Table
1).10) Further improvement of the pharmacokinetic profile af-
forded the nitrogen-introduced compounds 2—4. These com-
pounds were improved in water solubility. Compound 2 ex-
hibited inhibitory activities stronger than those of suberoy-
lanilide hydroxamic acid (SAHA) against a panel of cancer
cells as well as the maximal 185% T/C of survival rate in the
experiments with P388 cell-inoculated mice.11)
In the present study, we synthesized two derivatives 5 and
6 with a structural component (2,6-naphthylene or thiol Chart 1. Synthetic Pathways of 5 and 6
∗ To whom correspondence should be addressed. e-mail: uesato@ipcku.kansai-u.ac.jp
© 2005 Pharmaceutical Society of Japan

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Vol. 28, No. 5
of [4-(aminomethyl)phenyl]methanol and phenylacetyl chlo- residue was recrystallized from EtOAc to give the pure com-
ride in the presence of Et3N gave 6a, which was treated with pound 5c (0.15 g, 0.37 mmol, 23% yield). mp 229.6—
HBr to give benzyl brimide 6b. Replacement reaction of 232.1 °C. IR (KBr) cmꢁ1: 3292, 1643, 1537, 1313, 1228,
1
bromine (6b) with thio-ureido (6c) followed by alkaline 1028, 812, 696. H-NMR (DMSO-d6) d: 3.31 (2H, d, Jꢂ5.9
treatment yielded the desired 6.
Hz, PhCH2), 4.95 (2H, s, OCH2), 7.22—8.36 (16H, m, arom.
H16), 10.55 (1H, t, Jꢂ5.9 Hz, NHOH), 11.85 (1H, s,
CH2CONH). EI-MS m/z 410 (Mꢃ), 395, 304, 186, 91. HR-
EI-MS m/z: 410.1640 (Calcd for C25H22N2O3: 410.1630).
N-(Hydroxy)-6-[(phenylacetyl)amino]-2-naphthamide
MATERIAL AND METHODS
Melting points were determined on a Yanagimoto MP-32
micromelting point apparatus and are uncorrected. IR spectra (5) Ten percent Pd-C (77 mg) was added to a solution of 5c
were recorded on Shimadzu FTIR-8400 infrared spectropho- (0.10 g, 0.24 mmol) in MeOH (30 ml), and the mixture was
tometer. Low-resolution (LR)-FAB-MS spectra were mea- stirred under a H2 atmosphere overnight. The catalyst was re-
sured on a JEOL JMS-HX 100 instrument, whereas high-res- moved by filtration and washed successively with MeOH and
olution (HR)- and LR-electron impact (EI)-MS spectra were CHCl3. The combined filtrate was evaporated to give pure
1
measured on a JEOL The Tandem MStation JMS-700. H- compound 5 (67.4 mg, 0.21 mmol, 87.5% yield). mp 230.0—
NMR spectra were recorded on JEOL EX-270 (270 MHz) 230.5 °C (dec.). IR (KBr) cmꢁ1: 3030, 1651, 1554, 1023,
1
and JEOL EX-400 (400 MHz) instruments using tetramethyl- 898, 771. H-NMR (DMSO-d6) d: 3.69 (2H, d, Jꢂ5.9 Hz,
silane as an internal standard. Analytical TLC and PLC were CH2), 7.22—8.34 (11H, m, arom. H11), 9.00 (1H, t, Jꢂ5.9
performed using Silica gel 60 F254 (Merck, 0.25 and 0.5 mm, Hz, NHOH), 10.46 (1H, s, CH2CONH). FAB-MS m/z: 321
respectively) glass plates. Column chromatography was per- (MꢃHꢃ), 277, 185, 93, 75; HR-FAB-MS m/z: 321.1245
formed using Silica Gel 60 (70—230 mesh ASTM). TSA (Calcd for C19H17N2O3: 321.1239).
was purchased from Sigma-Aldrich Co.
N-[4-(Hydroxymethyl)benzyl]-2-naphthamide (6a) 2-
Methyl 6-[(Phenylacetyl)amino]-2-naphthoate (5a) To Naphthoyl chloride (4.73 g, 21.8 mmol) was reacted with [4-
a suspension of methyl 6-amino-2-naphthoate hydrochloride (aminomethyl)phenyl]methanol (3.02 g, 22.0 mmol) in the
(3.0 g, 14.9 mmol) in absolute DMF (60 ml) were added Et3N presence of Et3N (9.0 ml, 88.3 mmol) in the same way as for
(8.3 ml, 60.0 mmol) and phenylacetyl chloride (2.16 ml, 5a, yielding 6a (3.86 g, 13.2 mmol, 60.2% yield). mp
16.4 mmol). The mixture was stirred at room temperature 163.0—164.0 °C. IR (KBr) cmꢁ1: 2886, 1544, 954, 837, 781,
1
overnight. After being evaporated, the residue was dissolved 482. H-NMR (CD3OD) d: 4.72 (2H, s, NHCH2Phe), 4.80
in CHCl3 (200 ml) and washed successively with 1 N HCl (2H, s, PheCH2OH), 6.54 (2H, m, arom. H2), 7.36—7.39
(3ꢀ50 ml), saturated NaHCO3 (3ꢀ50 ml), and brine (3ꢀ (3H, m, arom. H3), 7.50—7.64 (2H, m, arom. H2), 7.82—
50 ml). The organic layer was dried and concentrated to dry- 7.86 (2H, m, arom. H2), 7.88—7.92 (2H, m, arom. H2). FAB-
ness. The resulting residue was recrystallized from CHCl3–n- MS m/z: 292 (MꢃHꢃ), 277, 185. HR-FAB-MS m/z:
hexane to give the pure compound 5a (1.3 g, 4.3 mmol, 292.1340 (Calcd for C19H18NO2: 292.1338).
30.0% yield). mp 177.5—180.9 °C. IR (KBr) cmꢁ1: 3031,
N-[4-(Bromomethyl)benzyl]-2-naphthamide (6b)
A
1
1714, 1656, 1584, 1238, 1097, 775, 545. H-NMR (CDCl3) mixture of 6a in 49% aqueous HBr was heated at 90 °C for
d: 3.38 (2H, d, Jꢂ5.9 Hz, PhCH2), 3.95 (3H, s, CO2CH3), 10 min. After cooling, the resulting precipitate was filtered
7.33—8.20 (11H, m, arom. H11), 8.50 (1H, s, CH2CONH); off and washed with water to give pure compound 6b (1.72 g,
EI-MS m/z: 319 (Mꢃ), 201, 170, 91; HR-EI-MS m/z: 4.9 mmol, 90.6% yield). mp 171.2—177.0 °C (dec.). IR
1
319.1207 (Calcd for C20H17NO3: 319.1208).
(KBr) cmꢁ1: 1633, 1548, 1510, 1232, 871, 838, 742. H-
6-[(Phenylacetyl)amino]-2-naphthoic Acid (5b) LiOH NMR (DMSO-d6) d: 4.46 (2H, s, NHCH2), 4.56 (2H, s,
1 M (7.8 ml, 7.8 mmol) was added to a solution of 5a (1.1 g, CH2Br), 6.90 (4H, m, arom. H4), 7.3—8.4 (7H, m, arom. H7).
3.5 mmol) in a mixture of water (0.5 ml) and THF (15.1 ml), FAB-MS m/z: 354 (MꢃHꢃ), 297, 185, 171, 157, 155. HR-
and the mixture was stirred at room temperature overnight. FAB-MS m/z: 354.0521 (Calcd for C19H17BrNO: 354.0494).
After evaporation of THF, the residual water solution was ad-
4-[(2-Naphthoylamino)methyl]benzyl Imidothiocarba-
justed to pH 3 with 1 N HCl. The resulting precipitate was fil- mate (6c) To a suspension of 6b in DMSO (10 ml) was
tered off and washed with water to give the pure compound added thiourea (123.8 g, 1.63 mmol), and the mixture was
5b (1.0 g, 3.3 mmol, 94% yield). mp 361.1—363.5 °C. IR stirred at room temperature for 18 h and then lyophilized to
(KBr) cmꢁ1: 3031, 1651, 1611, 1546, 1392, 1261, 925, 786. remove the solvent. The resulting residue was recrystallized
1H-NMR (CD3OD) d: 4.45 (2H, s, PhCH2), 7.94—8.98 from CHCl3 to give pure compound 6c (1.3 g, 4.3 mmol,
(11H, m, arom. H11). EI-MS m/z: 305 (Mꢃ), 187, 91, 44; HR- 30.0% yield). mp 217.3—218.7 °C. IR (KBr) cmꢁ1: 3855,
EI-MS m/z: 305.1025 (Calcd for C19H15NO3: 305.1052).
3845, 3726, 3622, 3008, 1537, 783. 1H-NMR (DMSO-d6) d:
N-(Benzyloxy)-6-[(phenylacetyl)amino]-2-naphthamide 3.51 (2H, s, CH2-S), 4.53 (2H, d, Jꢂ5.6, CH2Br), 7.3—9.2
(5c) To a suspension of O-benzylhydroxylamine hy- (11H, m, arom. H11). FAB-MS m/z: 350 (MꢃHꢃ). HR-FAB-
drochloride (0.31 g, 1.97 mmol) in absolute DMF (8.2 ml) MS m/z: 350.1342 (Calcd for C20H20N3OS: 350.1327).
were added 5b (0.50 g, 1.6 mmol), BOP-Cl (0.50 g,
N-[4-(Sulfanylmethyl)benzyl]-2-naphthamide (6)
A
1.96 mmol), and Et3N (0.67 ml, 4.8 mmol). The mixture was suspension of 6c in aqueous 6% NaOH was refluxed for 8 h.
stirred at room temperature overnight. After being evapo- After cooling, the solution was adjusted to pH 3 with HCl
rated, the residue was dissolved in CHCl3 (100 ml) and 6 M. The resulting precipitate was filtered off and washed
washed successively with 1 N HCl (3ꢀ20 ml), saturated with water to give pure compound 6 (51 mg, 0.17 mmol,
NaHCO3 (3ꢀ20 ml), and brine (3ꢀ20 ml). The organic layer 63.4% yield). mp 213.4—216.3 °C. IR (KBr) cmꢁ1: 3303,
was dried over Na2SO4 and concentrated. The resulting 1695, 1639, 1546, 1508, 1417, 1355, 1313, 831, 781, 738.

May 2005
851
1H-NMR (DMSO-d6) d: 3.76 (2H, s, CH2SH), 4.51 (2H, d,
Jꢂ6.0 Hz, NHCH2), 7.2—9.3 (11H, m, arom. H11). FAB-MS
m/z: 308 (MꢃHꢃ); HR-FAB-MS m/z: 308.1090 (Calcd for
C19H18NOS: 308.1109).
Evaluation of Histone Deacetylase Inhibitory Activities
Partial purification of HDACs and measurement of inhibitory
activities of test compounds against HDACs using [3H]-
acetylated histones were performed according to the proce-
dure by Mori et al.12) IC50 values in Table 1 represent the
molar concentrations (mM) required to inhibit the HDACs by
50%.
Cell Culture HCT116 cells (purchased from the ATCC)
were cultured in McCoy’s 5a medium (Invitrogen) supple-
mented with 10% fetal bovine serum (FBS), streptomycin
2.5 mg/ml, and penicillin 2.5 units/ml at 37 °C in a humidi-
fied atmosphere of 5% CO2. Cells were passaged every 2 d
into 60-mm dishes by washing twice with phosphate-
buffered saline (PBS) and incubated with 0.25% trypsin and
0.03% EDTA for 5 min at 37 °C prior to resuspension in
growth medium. MG63 human osteosarcoma cells (kindly
provided by Dr. Y. Yanase, Wakayama Medical College)
were cultured in Dulbecco’s modified Eagle’s medium (Invit-
rogen) supplemented with 10% FBS, streptomycin 10 mg/ml,
and penicillin 10 units/ml at 37 °C in a humidified atmos-
phere of 5% CO2. Cells were passaged every 3 d into 100-
mm dishes by washing twice with PBS and incubated with
0.05% trypsin and 0.02% EDTA for 3 min at 37 °C prior to
resuspension in growth medium.
Luc, was a kind gift from Dr. B. Vogelstein. Transient trans-
fection was performed at 60% confluency in 12-well titer
plates with the calcium phosphate technique using Cellphect
transfecting agent (Amersham-Pharmacia). Luciferase activi-
ties were measured using a luminometer, and were normal-
ized for the amount of the protein in cell lysates. All experi-
ments were carried out in triplicate.
Western Blot Analysis MG63 cells in 100-mm plates
were treated with 1—6 at the various concentrations indi-
cated in DMEM for 24 h. After the medium was aspirated,
the cells were washed with cold PBS and ice-cold radioim-
munoprecipitation assay (RIPA) buffer. Western blot analysis
was performed using mouse monoclonal antibody recogniz-
ing p21 (PM15091A from Pharmingen) as the primary anti-
body. The signal was detected with a chemiluminescence
system (ECL, Amersham-Pharmacia). Protein concentration
was determined using the Bio-Rad Protein Assay Kit.
RESULTS AND DISCUSSION
Table 1 shows the inhibitory activities of 1—6 against the
proliferation of HCT116 cells as well as HDACs. Com-
pounds 1—4 had comparatively low IC50 values (HCT116,
0.7—3.9 mM; HDAC, 0.026—0.044 mM). However, replace-
Table 1. Inhibition against HCT116 Cell Growth and HDACs
HCT116
HDACs
Compd.
Structure
IC50 (mM)
IC50 (mM)
Evaluation of Growth Inhibition against HCT116 Cells
Cells were plated in 96-well plates at densities of
1.0ꢀ105/ml. On the same day, compounds were added, and
the cells were cultured for 2 d. After addition of WST-1 and
1-methoxy-5-methyl-phenazinium methylsulfate, the OD450
and OD630 were measured in the conventional way.11)
Analysis of Cell Cycle Progression Cells were removed
from culture dishes by trypsinization and collected by cen-
trifugation. After washing with PBS, cells were suspended in
PBS containing 0.1% Triton X-100 to prepare nuclei. After
the suspension was filtered through 50-nm nylon mesh, 0.1%
RNase and 50 mg/ml propidium iodide were added. DNA
contents in stained nuclei were analyzed with FACSCalibur
(Becton Dickinson). The suspension of cells was analyzed
for each DNA histogram. The number of stained nuclei in
each phase was measured according to the Mod FitLT pro-
gram in the FACSCalibur system.
1
2
0.7
3.9
0.044
0.039
3
4
5
3.6
3.8
0.043
0.026
7.7
80.4
6
85.8
0.5
>10
Plasmid Transfection an Assessment of Luciferase
Assay The human wild-type p21/WAF1 promoter, pWWP-
TSA
0.003
Fig. 1. Compound 1, 2, 3, or 4 Stimulates Endogenous p21/WAF1 Protein Expression
Lysates (30 mg) of MG63 cells exposed to various concentrations of 1, 2, 3, 4, 5, or 6 as indicated or 0.1% DMSO for 24 h were examined using SDS-polyacrylamide gel elec-
trophoresis and analyzed with immunoblotting using antibodies for p21/WAF1 and a-tubulin.

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Vol. 28, No. 5
DNA replication and cell proliferation.15)
ment of the 1,4-phenylene and 2-naphthylcarbonyl group in 1
with the 2,6-naphthylene and phenylcarbonyl group in 5 as
well as of hydroxamic acid in 1 with thiol in 6 resulted in
much greater IC50 values.
The observed increase in the p21/WAF1 level as well as
the cell cycle arrest in the G2/M phase strongly suggest that
1—4 are potent inhibitors of cell cycle progression. Al-
though the p21/WAF1 gene is a p53-regulated one,16) it was
upregulated by 1, 2, 3, or 4 in the p53-mutated MG63 cells.
Since the p53 gene is frequently mutated,17) clarification of
the p53-independent activating pathway of the p21/WAF1
gene might contribute to cancer therapy.
We analyzed the effects of 1—6 and TSA on p21/WAF1
protein expression in MG63 cells. As shown in Fig. 1, treat-
ment with 1—6 induced p21/WAF1 protein expression in a
dose-dependent manner in Western blot analysis, although
the protein levels induced by 5 and 6 were much lower than
those by 1—4, in consonance with the low HDAC and
HCT116 inhibition of 5 and 6. In contrast, the level of alpha-
tubulin was unaltered by each treatment (Fig. 1). We thus in-
vestigated whether these compounds could stimulate the ac-
tivity of the p21/WAF1 gene promoter in cells transiently
transfected with the 2.4-kb wild-type p21/WAF1 promoter–
luciferase fusion plasmid pWWP-Luc. The p21/WAF1 gene
promoter activity was markedly stimulated by 1—4, in line
with the enhancement of the protein expression, as illustrated
in Fig. 2. Further, the effects of these compounds on cell
cycle progression of MG63 cells were investigated by mea-
suring the DNA content of nuclei of MG63 cells with flow
cytometry. Exposure to 1 mM of 4 or to 10 mM each of 1, 2,
and 3 for 24 h apparently decreased S-phase cells, whereas it
caused accumulation of cells in the G2/M phase (Fig. 3). In-
terestingly, the levels of p21/WAF1 gene expression and
G2/M phase accumulation were mutually correlated. We have
so far shown that p21/WAF1 is upregulated by HDAC in-
hibitors including TSA and sodium butyrate in MG63 cells;
this was associated with G2/M arrest7) It is also known that
enhancement of p21/WAF1 causes G1 arrest.13) p21/WAF1
inhibits cyclin-dependent kinase (CDK) activities such as
CDK2, CDK4, or CDK6 promoting G1/S-phase transition.13)
In addition to its role in G1 transition, p21/WAF1 reaccumu-
lates in nuclei near the G2/M boundary and causes a transient
block in the late G2 phase.14) In addition to CDK regulation,
p21/WAF1 binds directly to PCNA, which is associated with
Fig. 2. Compoud 1, 2, 3, or 4 Activates p21/WAF1 Promoter Activity
MG63 cells were transiently transfected with the pWWP-Luc reporter plasmid, and
luciferase activity was measured after incubation with or without various concentra-
tions of 1, 2, 3, 4 or 5. Relative luciferase activity is shown as raw light units (RLU) in
cell lysates/mg of protein. All results are the meanꢄS.E. of at least three independent
experiments, and the significant differences from the control (0.1% DMSO) were deter-
mined by Student’s test and are indicated by an asterisk (pꢅ0.05).
Fig. 3. Treatment with 1, 2, 3, or 4 Causes G2/M Arrest
MG63 cells were treated with or without various concentrations of 1, 2, 3, 4, or 5 as indicated for 24 h, and their DNA content was analyzed using fluorescence flow cytometry.
The percentages of the population in the G0/G1, G2/M, and S phase are indicated. The experiment was repeated three times and representative histograms are shown.

May 2005
853
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Acknowledgments Part of this research was financially
supported by a Grant-in-Aid from the Japan Society for the
Promotion of Science, Kansai University Special Aid for
Promotion of Research and Education, 2002, and a Grant-in-
Aid from the Ministry of Education, Culture, Sports, Science
and Technology, Japan. We are grateful to Mr. H. Mori and
Ms. T. Yamada for the HDAC inhibitory assay done at the
Medical Biology Research Laboratories of Fujisawa Pharma-
ceutical Co., Ltd.
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