Lactam-based HDAC inhibitors for anticancer chemotherapy: Restoration of RUNX3 by posttranslational modification and epigenetic control

Article abstract of DOI:10.1002/cmdc.201300393

Expression and stability of the tumor suppressor runt-related transcription factora 3 (RUNX3) are regulated by histone deacetylase (HDAC). HDAC inhibition alters epigenetic and posttranslational stability of RUNX3, leading to tumor suppression. However, HDAC inhibitors can nonselectively alter global gene expression through chromatin remodeling. Thus, lactam-based HDAC inhibitors were screened to identify potent protein stabilizers that maintain RUNX3 stability by acetylation. RUNX activity and HDAC inhibition were determined for 111 lactam-based analogues through a cell-based RUNX activation and HDAC inhibition assay. 3-[1-(4-Bromobenzyl)-2-oxo-2,5-dihydro-1H-pyrrol-3-yl]-N- hydroxypropanamide (11-8) significantly increased RUNX3 acetylation and stability with relatively low RUNX3 mRNA expression and HDAC inhibitory activity. This compound showed significant antitumor effects, which were stronger than SAHA, in an MKN28 xenograft model. Thus, we propose a novel strategy, in which HDAC inhibitors serve as antitumor chemotherapeutic agents that selectively target epigenetic regulation and protein stability of RUNX3.

Full text of DOI:10.1002/cmdc.201300393

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DOI: 10.1002/cmdc.201300393
Lactam-Based HDAC Inhibitors for Anticancer
Chemotherapy: Restoration of RUNX3 by Posttranslational
Modification and Epigenetic Control
Misun Cho,[a] Eunhyun Choi,[c] Jae Hyun Kim,[a] Hwan Kim,[a] Hwan Mook Kim,[d] Jang Ik Lee,[e]
Ki-Chul Hwang,[f] Hyun-Jung Kim,*[b] and Gyoonhee Han*[a]
Expression and stability of the tumor suppressor runt-related
transcription factor 3 (RUNX3) are regulated by histone deace-
tylase (HDAC). HDAC inhibition alters epigenetic and posttrans-
lational stability of RUNX3, leading to tumor suppression. How-
ever, HDAC inhibitors can nonselectively alter global gene ex-
pression through chromatin remodeling. Thus, lactam-based
HDAC inhibitors were screened to identify potent protein sta-
bilizers that maintain RUNX3 stability by acetylation. RUNX ac-
tivity and HDAC inhibition were determined for 111 lactam-
based analogues through a cell-based RUNX activation and
HDAC inhibition assay. 3-[1-(4-Bromobenzyl)-2-oxo-2,5-dihydro-
1H-pyrrol-3-yl]-N-hydroxypropanamide (11-8) significantly in-
creased RUNX3 acetylation and stability with relatively low
RUNX3 mRNA expression and HDAC inhibitory activity. This
compound showed significant antitumor effects, which were
stronger than SAHA, in an MKN28 xenograft model. Thus, we
propose a novel strategy, in which HDAC inhibitors serve as
antitumor chemotherapeutic agents that selectively target epi-
genetic regulation and protein stability of RUNX3.
Introduction
Histone deacetylases (HDACs) are a family of enzymes that cat-
alyze histone deacetylation and subsequent epigenetic regula-
tion of gene transcription. HDACs are associated with the
pathogenesis of several diseases, including cancers.[1] Recent
research indicates that HDACs also regulate deacetylation of
nonhistone proteins, such as, transcription factors, chaperones,
structural proteins, and tumor suppressor proteins.[2] Therefore,
pharmacological inhibitors of HDACs are considered to be po-
tential therapeutic agents for treating cancers.[3] Two HDAC in-
hibitors, vorinostat (SAHA, 1) and romidepsin (FK228), have
been approved by the US Food and Drug Administration (FDA)
for treatment of cutaneous T-cell lymphoma (CTCL).[4]
In gastric cancer, the functional inactivation of runt-related
transcription factor 3 (RUNX3) is related to tumor develop-
ment. Thus, RUNX3 can function as a tumor suppressor.[5]
RUNX3 activity is regulated by epigenetic silencing and post-
translational modification.[5b,6] Hypermethylation of the CpG
island, histone H3K9 methylation, H3 deacetylation, and
H3K27 methylation in the RUNX3 promoter repress transcrip-
tion of RUNX3.[5b] Nuclear translocation and stability of RUNX3
are regulated by transforming growth factor (TGF)-b signaling.
Activation of TGF-b stimulates nuclear translocation of endoge-
nous RUNX3, which inhibits cancer cell growth. In addition,
TGF-b activation enhances acetylation of RUNX3 by p300 his-
tone acetyltransferase (HAT) and stabilizes RUNX3 due to inhib-
ition of ubiquitin-mediated degradation.[6c] Recent studies
demonstrate that HDAC inhibitors, such as trichostatin A (TSA,
2), contribute to reactivation and stabilization of RUNX3 and
subsequent inhibition of cancer cell growth.[6b,c,7]
[a] Dr. M. Cho, J. H. Kim, Dr. H. Kim, Prof. G. Han
Translational Research Center for Protein Function Control (TRCP)
Department of Biotechnology and
Department of Biomedical Sciences (WCU Program)
Yonsei University, Seodaemun-gu, Seoul 120-752 (Republic of Korea)
E-mail: gyoonhee@yonsei.ac.kr
[b] Dr. H.-J. Kim
BioRunx Co. Ltd., 12 Gaesin-dong, Huengduk-gu
Cheongju, Chungbuk 361-763 (Republic of Korea)
E-mail: hjkim7@yuhs.ac
[c] Dr. E. Choi
Severance Integrative Research Institute for
Cerebral and Cardiovascular Disease
Yonsei University Health System
Seodaemun-Gu, Seoul 120-752 (Republic of Korea)
[d] Prof. H. M. Kim
College of Pharmacy, Gachon University of Medicine and Science
Incheon 406-799 (Republic of Korea)
[e] Prof. J. I. Lee
HDAC inhibitors have emerged as putative epigenetic drugs
for anticancer treatment. However, these agents have substan-
tial limitations, including epigenetic non-specificity (pleiotropic
effects) and drug resistance.[8] The traditional rationale of epi-
genetic cancer therapy, which focuses on histone proteins,
needs to be refined to selectively modify tumor-specific nonhi-
stone proteins.[9] Restoration and stabilization of RUNX3 by
HDAC inhibition is a novel approach for anticancer chemother-
College of Pharmacy, Yonsei University
85 Songdogwahak-ro, Yeonsu-gu, Incheon 406-840 (Republic of Korea)
[f] Prof. K.-C. Hwang
Severance Biomedical Science Institute
Cardiovascular Research Institute
Yonsei University College of Medicine
Seodaemun-gu, Seoul 120-752 (Republic of Korea)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300393.
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apy. We developed lactam-based HDAC inhibitors and selected
those inhibitors that restored RUNX3 transcriptional expression
by histone acetylation and stabilization of RUNX3 protein by
acetylation at the posttranslational level.
In the present study, we investigated the effects of our in-
house chemical library of lactam-based HDAC inhibitors on the
restoration of RUNX3 gene expression and RUNX3 acetylation
and stabilization. Cell-based mechanistic studies provided a ra-
tionale for in vivo analysis. Our results suggest that epigenetic
and posttranslational regulation of RUNX3 by lactam-based
HDAC inhibitors might contribute to attenuation of gastric
cancer pathogenesis. These novel data suggest that HDAC in-
hibitors can be developed as target-specific anticancer agents.
Results and Discussion
Chemical library of lactam-based HDAC inhibitors
In our previous studies, we reported novel lactam-based HDAC
inhibitors (10, 11) that consist of three parts. The inhibitors
have diverse cap groups with substituted aromatic rings, d- or
g-lactam cores, and hydroxamate moieties that function as
zinc binders (Figure 1).[10] Several compounds potently inhibit-
ed HDAC activities and reduced cancer cell growth.[10,11]
Through docking simulation study and quantitative structure–
activity relationship (QSAR), we indicated that our active
lactam compounds bound to the active pocket of HDAC; hy-
Scheme 1. General synthetic procedures of lactam-based HDAC inhibitors
(10, m=2; 11, m=1; n=1–4). Reagents and conditions: a) EDC, DMAP,
CH2Cl2; b) Grubbs catalyst I, CH2Cl2; c) Grubbs catalyst II, CH2Cl2; d) KONH2
(1.7m in MeOH), MeOH, 08C.
Grubbs’ catalyst II (2–3 mol%) produced the d-lactam
rings (8) and Grubbs’ catalyst I generated the g-
lactam rings (9) in good yields. The final compounds
(10, 11), which were transformed from methyl esters
into hydroxamic acids, were obtained by subsequent
reaction with potassium hydroxylamine in methanol
at 08C.[11a,b]
A total of 111 lactam-based HDAC inhibitors were
prepared to study their abilities to restore and stabi-
lize RUNX3. There were 50 d-lactam- and 62 g-
lactam-based analogues with a wide spectrum of
HDAC inhibitory activities ranging from IC50 values of
0.01 mm to more than 10 mm (see table S1).
Figure 1. HDAC inhibitors.
Screening: RUNX activity and HDAC inhibitory activity
droxamate moieties were chelated with a zinc ion in the active
site similar to TSA and SAHA, and the aromatic cap groups had
hydrophobic interactions with the enzyme surface.[12] The
lactam core interacted with the hydrophobic aromatic side
chain, and g-lactam especially gave stabilization by p–p inter-
action with phenylalanine residues in the narrow active pock-
et.[10b,11b,c,12]
We needed an assay system that could evaluate whether our
lactam-based HDAC inhibitors affected RUNX proteins. We
used an effective luciferase assay system in 6xOSE2-C2C12 cells
to monitor RUNX transcriptional activity (Figure 2 and
table S1).[13] RUNX activity is presented as percentage of activa-
tion compared to a positive control, fibroblast growth factor
(FGF)-2, which is known to simulate RUNX2 expression and
function and highly stimulates 6xOSE2-luciferase activity.[14] All
three RUNX proteins share structural similarity and recognize
the same consensus sequences in the promoter regions of
target genes.[14] Overexpression of RUNX3 also stimulated
RUNX activity (data not shown). In addition, RUNX3 is ex-
The synthetic procedures of d- and g-lactam based HDAC in-
hibitors (10, 11) are presented in Scheme 1. The secondary
amines (3, 4), which were obtained by N-alkylation or reductive
amination, were coupled with monoacid (5) using a 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide (EDC)-mediated coupling
reaction to afford amines 6 and 7. Metathesis reaction with
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Figure 2. Percentage of RUNX transcriptional activation relative to FGF-2. Data represent the meanÆstandard deviation (SD) of at least two independent ex-
periments, and the order of compound names are provided in table S1. Compounds that show >100% RUNX activity are highlighted with black arrows.
pressed in this cell line.[13] Based on these studies, the 6xOSE2-
C2C12 cell-based assay is useful for screening compounds that
regulate the activity of RUNX3 and other RUNX family mem-
bers. Lactam analogues were compared to SAHA, which was
the first epigenetic anticancer drug approved by the US
FDA.[15]
of 0.03–0.1 mm (10-18: 0.05 mm, 11-58: 0.03 mm, 10-29: 0.1 mm).
Group III compounds displayed a wide spectrum of HDAC in-
hibitory activities from 0.3 mm to 0.01 mm (see table S1). SAHA
(1) showed moderate RUNX activity [(80.2Æ0.41)%] and HDAC
inhibitory activity [IC50 =(0.11Æ0.021) mm] as much as group III
compounds (see table S1). Based on these results and classifi-
cation, we confirmed that all compounds in Table 1 show
potent HDAC inhibition with IC50 values <0.3 mm (see
table S1). Although potency of HDAC inhibition does not
always correlate with potency of RUNX activation, it is definite-
ly involved in regulation of RUNX transcriptional activity.
We screened the effects of 111 compounds on RUNX tran-
scriptional activity in 6xOSE2-C2C12 cells (Figure 2). Eighteen
lactam analogues induced RUNX activity to greater than 100%
and were selected for further analysis (Table 1). Three activa-
tors, 11-36, 11-48 and 11-46, which induced RUNX activation
by >200%, were categorized as group I. Three good activa-
tors, 10-18, 11-58 and 10-29, which induced RUNX activation
from 150% to 200%, were classified as group II, and twelve ac-
tivators, 11-28, 11-37, 11-27, 10-47, 11-57, 11-53, 11-60, 11-
55, 11-8, 10-5, 10-22 and 10-30, induced RUNX activation
from 100% to 150% (Table 1).
Regulation of RUNX3 acetylation
RUNX3 acetylation by p300 plays a key role in the mainte-
nance of RUNX3 stability and transcriptional activity. Deacetyla-
tion of acetylated RUNX3 by HDACs promotes proteasome-
mediated degradation of RUNX3.[6c] We examined RUNX3 ace-
tylation and protein levels after treating cells with the HDAC
inhibitors. Myc-tagged RUNX3 expression vectors were trans-
fected into HEK293 cells fol-
Compounds of group I showed HDAC inhibitory activities
from 0.05 mm to 0.1 mm (11-36: 0.05 mm, 11-48: 0.07 mm, 11-46:
0.1 mm) and group II compounds had IC50 values in the range
lowed by treatment with select-
ed HDAC inhibitors.[6c] All of the
Table 1. Grouping of lactam-based HDAC inhibitors by RUNX transcriptional activation.
group I and II compounds (11-
36, 11-48, 11-46, 10-18, 11-58,
and 10-29) and three com-
pounds (10-47, 11-37, and 11-8)
in group III were examined in
this experiment (Figure 3A). TSA
(2) is a positive control, because
it is known to increase expres-
Relative RUNX transcriptional activation to FGF-2 [%][a]
Group I: >200%
Compd RUNX [%]
Group II: 150–200%
Compd RUNX [%]
Group III: 100–150%
Compd RUNX [%] Compd RUNX [%]
Compd RUNX [%]
11-36 215.4Æ1.56
11-48 266.9Æ7.95
11-46 206.1Æ2.15
10-18 170.8Æ1.52
11-58 155.4Æ4.95
10-29 185.5Æ1.70
11-28 115.2Æ7.67 11-57 115.3Æ7.12 11-8
11-37 145.5Æ0.87 11-53 111.4Æ0.84 10-5
102.6Æ3.94
102.5Æ0.59
11-27 110.3Æ2.99 11-60 111.0Æ4.60 10-22 105.4Æ0.80
10-47 132.1Æ0.53 11-55 108.2Æ5.87 10-30 115.4Æ2.74
[a] Data represent the meanÆSD of at least two independent experiments. Compounds are listed in the order
sion
and
acetylation
of
of HDAC inhibitory activity (see table S1).
RUNX3.[6c] The ratio of acetylated
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Figure 3. RUNX3 acetylation of selected lactam-based HDAC inhibitors. A) Acetylated RUNX3 levels were analyzed by immunoprecipitation in HEK293 cells at
1 mm test compound; B) Relative densitometry ratio of RUNX3 acetylation based on the results of the western blot shown in Figure 3A (at 1 mm test com-
pound); C) Selected lactam-based HDAC inhibitors based on RUNX3 acetylation assay.
RUNX3 mRNA expression and RUNX3 stability
RUNX3 following treatment with each of the above com-
pounds relative to levels following treatment with TSA is
shown in Figure 3B.
RUNX3 has been reported to be reactivated by an HDAC inhib-
itor in the MKN28 human gastric cancer cell line, in which the
RUNX3 gene has a hemizygous deletion.[5c] Reverse transcrip-
tase polymerase chain reaction (RT-PCR) was performed to de-
termine if 11-8, 11-58, and 11-48 regulate RUNX3 at the tran-
scriptional level. SNU16 cells were used as a positive control
for detecting RUNX3 mRNA, because RUNX3 is highly ex-
pressed in this cell line despite the presence of a mutation in
RUNX3 (Figure 4).[5c] Compound 11-48 strongly induced RUNX3
mRNA expression. Induction of RUNX3 gene expression by
1 mm 11-48 was much higher than that achieved by TSA (2,
Figure 4A). Compound 11-8 weakly induced RUNX3 mRNA ex-
pression. At the same concentration (1 mm), 11-58 and SAHA
(1) poorly induced RUNX3 mRNA expression.
Compounds 11-8 and 11-58 were tested in concentration
dependent manner to confirm this result (Figure 4B). Com-
pound 11-8 showed very low levels of RUNX3 mRNA expres-
sion, whereas 11-58 moderately expressed RUNX3 mRNA at
0.1 mm and 1 mm. At 10 mm, both compounds remarkably in-
creased RUNX3 mRNA expression, comparable to the positive
control SNU16 (Figure 4B). Despite slight discrepancies in the
levels of induction of RUNX3 mRNA in Figure 4A and 4B, 11-8
and 11-58 showed weak effects on RUNX3 mRNA expression
at less than 1 mm in comparison with 11-48.
Most of the compounds, including SAHA (1), 11-8, 11-36,
11-37, 10-18, 10-29, 11-48, 11-46, and 11-58, significantly in-
creased the levels of acetylated RUNX3, resulting in RUNX3 sta-
bilization compared to control (Figure 3A). In particular, 11-8,
11-37, 10-18, 11-48, 11-58, and SAHA (1) dramatically in-
creased acetylation of RUNX3 compared to TSA (Figure 3). The
highest levels of acetylated RUNX3 and protein stabilization
were achieved by 11-27, 10-18, 11-48, 11-58, and 11-8 in
groups I, II, and III, respectively (Figure 3A,B). Among these, the
HDAC inhibitory activities of 11-37 and 10-18 [IC50 =(0.02Æ
0.0134) mm and (0.03Æ0.0234) mm, respectively] were signifi-
cantly more potent. Thus, 11-37 and 10-18 might promote
nonselective chromatin remodeling and associated changes in
global gene expression. In comparison to TSA (2), 10-18 ach-
ieved weaker stabilization of RUNX3 protein, while it signifi-
cantly promoted RUNX expression (Table 1). Therefore, we ex-
cluded these compounds from the subsequent studies. Thus,
11-8, 11-48, and 11-58, which had g-lactam cores and bromo-
substituents on the aromatic rings, were selected as the lead
compounds for further experiments (Figure 3C).
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levels and altered gene expression profiles in cells, indicating
global chromatin remodeling, 11-48 might cause nonspecific
gene expression at the transcription level. Based on these
result, RUNX3 restoration by 11-8 and 11-58 was relatively
more regulated by RUNX3 acetylation by posttranslational
modification rather than controlling RUNX3 gene expression at
the transcriptional level. Therefore, 11-8 and 11-58 might be
more suitable HDAC inhibitors for our strategy on RUNX3-tar-
geted drug discovery.
Reduced tumor growth in xenograft models
In vivo xenograft experiments were performed to evaluate an-
titumor activities of 11-8, 11-58, and 11-48. MKN28 cells were
subcutaneously implanted into nude mice. Compounds were
administered intravenously at 20 mgkgÀ1 daily for two weeks
when tumor volumes reached 50–80 mm3. Intravenous admin-
istration was performed to minimize the effects of ADME (ab-
sorption, distribution, metabolism and elimination). Tumor
volume was measured every two or three days for 14 days.
Compounds 11-8, 11-48, and 11-58 significantly inhibited
tumor growth compared to vehicle control (Figure 6A,B). The
positive control, SAHA, also showed significant tumor growth
inhibition. There were no significant reductions in body weight
in the treatment groups (Fig-
Figure 4. RUNX3 mRNA expression in MKN28 cells after treatment with 11–
8, 11–48, and 11–58 (1 mm each). A) The effects of the three compounds
and SAHA on RUNX mRNA expression; B) Dose-dependent effects of 11–8
and 11–58 on RUNX3 mRNA expression. All of the compounds were treated
at micromolar concentration (mm); 2, TSA; 1, SAHA, Cont., control.
The effects of compounds 11-8, 11-58, and 11-48 on RUNX3
stability were evaluated in MKN28-RUNX3-Lac cells (Figure 5).
Because this stable cell line expresses RUNX3 only after treat-
ment with isopropyl-b-d-thiogalactopyranoside (IPTG),[15] it is
a useful cell-based system to monitor RUNX3 stability together
ure 6A,B). The tumor growth in-
hibitory activities of compounds
11-8, 11-48, and 11-58 were sim-
ilar to SAHA at tumor volume
and tumor weight (Table 2).
Compound 11-8 and 11-48 in-
hibited tumor growth at tumor
volume (57.5% and 35.1%, re-
Figure 5. Stability of RUNX3 and acetylated histone H3 (Ac-H3) in MKN28-Lac-RUNX3.
spectively) and weight (58.1%
and 40.6%, respectively) similar
with a RUNX3 immunoprecipitation assay. Cells were pretreat-
ed with IPTG for 24 h to induce maximum RUNX3 expression,
at which time the RUNX3 expression level was the same in all
groups. Cells were then treated with test compounds for an
Table 2. Tumor growth inhibition in xenograft models.[a]
Compd
Volume [%]
Weight [%]
Expt I
SAHA
11-48
11-8
44.6
35.1
57.5
45.6
40.6
58.1
additional 24 h without IPTG treatment to monitor RUNX3 sta-
bility. Acetylation of histone H3 was also examined to monitor
the effects of compounds on histone proteins. All compounds,
11-8, 11-58, 11-48 and SAHA, showed similar levels of RUNX3
stability and histone H3 acetylation at each concentration;
their RUNX3 stability and histone acetylation were slightly in-
creased at 1 mm, and they showed excessive increases of
RUNX3 stability and histone H3 acetylation at 10 mm (Figure 5).
RUNX3 stability at 10 mm was caused by effects of both tran-
scriptional expression of RUNX3 and posttranslational acetyla-
tion of RUNX3.
Expt II
SAHA
11-58
56.3
45.2
51.0
46.7
[a] Tumor inhibition compared to vehicle. For details, see Figure 6.
to SAHA (44.6% for tumor volume and 45.6% for weight), and
11-58 (45.2% for tumor volume and 46.7 for weight) also
showed tumor growth inhibition like SAHA (56.3% for tumor
volume and 51.0% for weight).
Taken together, although RUNX3 stability in the groups
treated with 11-8 or 11-58 was similar to those of the group
treated with 11-48 at 1 mm, they induced relatively low levels
of RUNX3 mRNA expression as compared with 11-48, which
showed the strongest induction of RUNX3 mRNA expression.
Because HDAC inhibitors cause strong induction of mRNA
All three compounds showed similar HDAC inhibitory poten-
cy and RUNX3 stability as well as in vivo tumor growth inhibi-
tory activity. Among them, 11-48 showed a remarkably high
level of RUNX3 mRNA expression, which could be an indicator
for a nonselective epigenetic activator. Therefore, 11-58 and
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a
novel anticancer epigenetic
approach with HDAC inhibitors
for anticancer drug develop-
ment.
Experimental Section
Chemistry
Chemical data for 18 hit and lead
compounds were published in pre-
vious papers: 10-5 and 10-22 are
presented in ref. [16] and [10a], re-
spectively; 10-18, 10-29, 10-30
and 10-47 are published in
ref. [11a]; 11-27, 11-28, 11-36, 11-
37 and 11-48 are shown in
ref. [10b]; 11-8, 11-46, 11-53, 11-
55, 11-57, 11-58, 11-60 are report-
ed in ref. [11c]. For convenience,
synthetic procedures and charac-
terization data of these com-
pounds are presented in the Sup-
porting Information together with
data for intermediate compounds
3-47, 4-48, 5, 6-47, 7-48, 8-47 and
9-48, which are not reported else-
where.
Figure 6. In vivo antitumor activity of 11–8, 11–48, and 11–58 in a xenograft model. A) Experiment I: average
tumor volume and body weight changes of vehicle (control), SAHA, 11–8, and 11–48. B) Experiment II: average
tumor volume and body weight changes of vehicle (control), SAHA, and 11–58. Experiment I and II were per-
formed with xenografts of the human stomach cancer cell line MKN28 in nude mice; experiment I, n=5; experi-
ment II, n=6; standard errors are provided in the Supporting Information (table S2 and S3); *p<0.05, **p<0.01,
***p<0.001, Student t-test; DVt (tumor volume)=Vt (measurement of the tumor volume)ÀV0 (initial tumor
volume).
Biology
HDAC inhibition assay: HDAC inhib-
ition assays were performed as de-
scribed in previous papers.[17]
Cell cultures: All tissue culture media and antibiotics were pur-
chased from Hyclone (Logan, UT, USA). HEK293 and C2C12-6xOSE
cells were maintained in DMEM with 10% fetal bovine serum
(FBS) and antibiotics [penicillin (100 IUmLÀ1), streptomycin
(100 mgmLÀ1)] (Invitrogen, Grand Island, NY, USA ) at 378C in a 5%
CO2 atmosphere. MKN28, MKN28-Lac-RX3, and SNU16 cells were
maintained in RPMI1640 media with 10% FBS and antibiotics. The
MKN28-Lac-RX3 stable cell line was kindly provided by Prof. Sul-
Chul Bae (Chungbuk National University, Cheongju, South
Korea).[15] Treatment with 2 mm isopropyl-b-d-thiogalactopyrano-
side (IPTG, Sigma–Aldrich, St. Louis, MO, USA) was performed for
24 h to induce RUNX3. Drugs were then added to cell cultures for
an additional 24 h to assess effects on RUNX3 stability.
11-8 were selected as potent RUNX3 stabilizers for gastric
cancer therapy.
Conclusions
Epigenetic activation often leads to nonselective gene expres-
sion and side effects, which are considered to be major limita-
tions of HDAC inhibitors.[15] The approach of stabilizing pro-
teins with HDAC inhibitors is an alternative strategy for restor-
ing expression of tumor suppressors through target-selective
acetylation. Specifically, RUNX3 can be stabilized by HDAC in-
hibitors, resulting in restoration of its tumor suppressor func-
tions. In this study, 11-8, 11-58, and 11-48 were selected by
measuring acetylation and stabilization of RUNX3. These inhibi-
tors also showed good in vivo anticancer efficacy. However,
11-48 was not selected as a candidate because of very strong
epigenetic effects at the transcription level. In previous reports,
we already demonstrated that compound 11-8 is orally avail-
able with good pharmacokinetic properties,[11c] whereas 11-58
is not (unpublished data). Therefore, 11-8 should be the better
candidate to regulate RUNX3 stabilization through epigenetic
and target-specific posttranslational modification. Further stud-
ies towards the identification of the underlying mechanisms by
which these novel HDAC inhibitors stabilize RUNX3 are in
progress. The current study provides important insights into
Screening for RUNX activity: The C2C12-6xOSE cell line was kindly
provided by Prof. Hyun-Mo Ryoo (Seoul National University, Seoul,
South Korea). Cells were plated at 1ꢁ104 cells per well in 96-well
plates. FGF-2 and drugs (1 mm each) were added to cells the next
day. After 24 h, cells were harvested and analyzed by luciferase
assay with the Bright-Glo Luciferase Assay System according to the
manufacturer’s protocol (Promega, Madison, WI, USA). Lysates
were analyzed with the GloMax-multi Detection System (Promega).
Fibroblast growth factor (FGF)-2 was used as a positive control for
RUNX activation.
Western blot: After cells were lysed with radio-immunoprecipitation
assay (RIPA) buffer (25 mm Tris-HCl, pH 7.6, 150 mm NaCl, 1% NP-
40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, SDS),
protein concentrations were determined with a bicinchoninic acid
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assay (BCA) protein assay kit (Pierce, Rockford, IL, USA). Equal
amounts of protein were fractionated by SDS-PAGE and transferred
onto a nitrocellulose membrane (BioRad, Richmond, CA, USA).
After blocking with 5% skim milk in phosphate buffered saline
(PBS) containing 0.1% Tween 20, membranes were incubated with
the appropriate primary antibodies at 48C overnight. Proteins were
software package (SigmaStat, SPSS Science, Chicago, IL, USA). Stu-
dent’s t-test was performed for individual comparisons. Multiple
comparisons were assessed by one-way ANOVA or regression anal-
ysis.
detected with horseradish peroxidase (HRP)-conjugated secondary Acknowledgements
antibodies and enhanced chemiluminescence (ECL) kit (Amersham
Pharmacia Biotech, Buckinghamshire, UK). Antibodies against myc
This research was supported by a grant of the Translational Re-
(9E10), b-actin, and tubulin were from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). Anti-acetylated lysine and anti-acetylated
histone 3 antibodies were purchased from Cell Signaling (Danvers,
MA, USA).
search Center for Protein Function Control (2009-0083522), the
Korea Health 21 R&D Project, and Ministry of Health and Welfare
(A120478), Republic of Korea. This work was also supported by
the National Research Foundation of Korea (NRF), grant funded
by the Korean government (MEST) (NRF-2008-2005600 to H.J.K).
Immunoprecipitation: The myc-RUNX3 expression vector was kindly
provided by Prof. Sul-Chul Bae (Chungbuk National University,
Cheongju, South Korea). The myc-RUNX3 expression vector was
transfected into HEK293 cells with Lipofectamine 2000 (Invitrogen).
Transfected cells were treated with HDAC inhibitors at 1 mm the
next day for an additional 24 h. Cells were lysed in ice-cold lysis
buffer (25 mm HEPES, pH 7.5, 150 mm NaCl, 1% Nonidet P-40,
0.25% sodium deoxycholate, 10% glycerol, 25 mm NaF, 1 mm
EDTA, 1 mm Na3VO4) and cleared by centrifugation. For immuno-
precipitation experiments, 500 mg of protein was incubated with
anti-myc antibody and precipitated with protein G beads (Pierce,
Rockford, IL, USA) at 48C. The beads were washed three times with
cold lysis buffer, and the immunoprecipitates were analyzed by
western blot with an antibody against acetylated lysine.
Keywords: drug discovery · histone deacetylase · inhibitors ·
lactam · runt-related transcription factor 3
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Received: October 7, 2013
Published online on December 2, 2013
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ChemMedChem 2014, 9, 649 – 656 656