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Published OnlineFirst June 1, 2011; DOI: 10.1158/1535-7163.MCT-10-1062
Molecular
Cancer
Therapeutics
Preclinical Development
Sodium Butyrate Inhibits the Self-Renewal Capacity of
Endometrial Tumor Side-Population Cells by
Inducing a DNA Damage Response
Kiyoko Kato1, Aya Kuhara2, Tomoko Yoneda2, Takafumi Inoue2, Tomoka Takao2, Tatsuhiro Ohgami2,
Li Dan2, Ayumi Kuboyama2, Soshi Kusunoki1, Satoru Takeda1, and Norio Wake2
Abstract
We previously isolated side-population (SP) cells from a human endometrial cancer cell line, Hec1, and
determined that Hec1-SP cells have cancer stem–like cell features. In this study, we isolated SP cells and nonSP (NSP) cells derived from a rat endometrial cell line expressing human [12Val] KRAS (RK12V cells) and
determined the SP phenotype. RK12V-SP cells showed self-renewal capacity, the potential to develop into
stromal cells, reduced expression levels of differentiation markers, long-term proliferating capacity in
cultures, and enhanced tumorigenicity, indicating that RK12V-SP cells have cancer stem–like cell features.
RK12V-SP cells also display higher resistance to conventional chemotherapeutic drugs. In contrast, treatment
with a histone deacetylases (HDAC) inhibitor, sodium butyrate (NaB), reduced self-renewal capacity and
completely suppressed colony formation of RK12V-SP cells in a soft agar. The levels of intracellular reactive
oxygen species (ROS) and the number of gH2AX foci were increased by NaB treatment of both RK12V-SP cells
and RK12V-NSP cells. The expression levels of gH2AX, p21, p27, and phospho-p38 mitogen-activated protein
kinase were enhanced in RK12V-SP cells compared with RK12V-NSP cells. These results imply that treatment
with NaB induced production of intracellular ROS and DNA damage in both RK12V-SP and RK12V-NSP cells.
Following NaB treatment, DNA damage response signals were enhanced more in RK12V-SP cells than in
RK12V-NSP cells. This is the first article on an inhibitory effect of NaB on proliferation of endometrial cancer
stem–like cells. HDAC inhibitors may represent an attractive antitumor therapy based upon their inhibitory
effects on cancer stem–like cells. Mol Cancer Ther; 10(8); 1430–9. 2011 AACR.
Introduction
Endometrial cancer is the most common gynecologic malignancy in the industrialized world and can
be classified into 2 different clinicopathologic types,
estrogen-related endometrial cancers (type I) and nonestrogen-related endometrial cancers (type II). The most
frequent genetic alteration in type I endometrial cancers
is PTEN inactivation, followed by microsatellite instability and mutations of the KRAS and b-catenin. In type II
endometrial cancers, TP53 mutation is the most frequent
genetic alteration, followed by amplification of ERBB2
(1). Some of these pathways are important determinants of stem cell activity (Wnt-, b-catenin, and PTEN;
Authors' Affiliations: 1Department of Obstetrics and Gynecology, Faculty
of Medicine, Juntendo University, Hongo 2-1-1, Bunkyo-ku, Tokyo; and
2
Department of Obstetrics and Gynecology, School of Medicine, Kyushu
University Maidashi 3-1-1, Higashi-ku, Fukuoka, Japan
Corresponding Author: Kiyoko Kato, Department of Obstetrics and
Gynecology, Faculty of Medicine, Juntendo University, Hongo 2-1-1,
Bunkyo-ku, Tokyo 113-8431, Japan. Phone: 81-3-5802-1100; Fax: 813-5689-7460; E-mail: [email protected]
doi: 10.1158/1535-7163.MCT-10-1062
2011 American Association for Cancer Research.
1430
refs. 2–4). These findings suggest a stem cell contribution
to endometrial carcinoma development.
Recent evidence suggests that cancer stem–like cells
exist in several malignant tumors, such as leukemia (5, 6),
breast cancer (7), and brain tumors (8), and that these
stem cells express surface markers similar to those
expressed by normal stem cells in each tissue (5, 9). Stem
cell subpopulations [side-population (SP) cells] have
been identified in many mammals, including humans,
on the basis of the ability of these cells to efflux the
fluorescent dye Hoechst 33342 (10). The SP phenotype
is associated with a high expression level of the ATPbinding cassette transporter protein ABCG2/Bcrp1 (11).
Established malignant cell lines, which have been maintained for many years in culture, have also been shown
to contain SP cells as a minor subpopulation (12). Friel
and colleagues showed that SP cells derived from the
endometrial cancer cell lines (An3CA) had features of
cancer stem–like cells including low proliferative activity
during 9 days of cultivation, chemoresistance, and
enhanced tumorigenicity (13). Hubbard and colleagues
showed that a small population of clonogenic cells from
endometrial cancer tissues showed self-renewing, differentiating, and tumorigenic properties (14). Gotte and
colleagues showed that the adult stem cell marker
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An Effect of HDAC Inhibitor on Cancer SP Cell Proliferation
Musashi-1 was coexpressed with Notch-1 in a subpopulation of endometrial cells (15). Furthermore, they
showed that telomerase and Musashi-1 expressing cells
were significantly increased in proliferative endometrium, endometriosis, and endometrial carcinoma tissue,
compared with secretary endometrium, suggesting the
concept of a stem cell origin of endometriosis and endometrial carcinoma. Most recently, they showed that short
interfering RNA depletion of Musashi-1, an adult stem
cell marker enriched in the SP, in the endometrial carcinoma cell line Ishikawa leads to interference with the
Notch signaling pathway and p21 expression, resulting in
an antiproliferative effect and induction of apoptosis (16).
The regulation of histone acetylation is a major mechanism controlling cellular differentiation and the biological
phenotype of cancer cells (17). Histone deacetylases
(HDAC) and histone acetyl transferases are enzymes that
ensure the homeostatic levels of histone acetylation.
Deregulated HDAC activity has been found in certain
human cancers (18–22). Several studies have shown the
antiproliferative or the proapoptotic effects of HDAC
inhibitors (HDACi) on endometrial cancer cells (23–25).
Thus, HDACs are important therapeutic targets for cancer
and several HDACi’s are in various stages of clinical
development (26, 27). However, the effect of HDACi’s
on proliferation of cancer stem cells is unknown.
We have isolated SP cells from normal human endometrium and from a human endometrial cancer cell line,
Hec1, and characterized their properties (28, 29). We have
shown that Hec1-SP cells have cancer stem–like cell
features. In this study, SP cells and non-SP (NSP) cells
derived from a rat endometrial cell line expressing
human [12Val] KRAS (RK12V cells) were isolated. We
analyzed the biological characteristics and assessed the
antiproliferative effect of a HDAC inhibitor (NaB).
Materials and Methods
Plasmid
pZIP-Neo SV(X)1 containing [12Val] human KRAS 4B
cDNA was a gift from Dr. Channing Der (University of
North Carolina; refs. 30, 31). The pZeo-vector was purchased from Invitrogen. We cut the 1.1-kb fragment
containing human [12Val] KRAS 4B cDNA from the
pZIP-Neo SV(X)1 construct with BamHI and legated it
to the BamHI site of the pZeo vector.
Cell culture
A rat endometrial cell line (RENT4) was used in this
study. RENT4 cells were established by Wiehle and
colleagues (32) and obtained from the European Collection of Cell Cultures. No authentication for the cell line
was done by the authors.
RENT4 cells harboring mutant [12Val] versions of
KRAS4B (RK12V cells) were established by transfecting
RENT4 cells with pZeo constructs, containing cDNA
sequences encoding [12Val] KRAS by using Lipofectamine (Invitrogen). Stably transfected cells were selected
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and isolated in growth medium containing 400 mg/mL of
Zeocin (Invitrogen) to establish cell lines expressing
KRAS protein, previously described elsewhere (33).
Pooled populations were used for the assay. Cells were
cultured with Dulbecco’s Modified Eagle’s Medium
(DMEM; Nissui Seika) supplemented with 20 mg/mL
Gly-His-Lys, 2 mmol/L glutamine, 80 IU insulin (Sigma),
and 10% FBS (Hyclone; ref. 32). Cells used were always
less than 20 passages.
Isolation of SP cells
To identify and isolate Hec1 and RK12 V SP cells, the
cells were dislodged from the culture dishes with trypsin
and EDTA, washed, and suspended at a concentration of
106 cells per milliliter in DMEM containing 2% FBS. The
cells were then labeled in the same medium at 37 C for 90
minutes with 2.5 mg/mL Hoechst 33342 dye (Molecular
Probes), either alone or in combination with 50 mmol/L
verapamil (Sigma). Finally, the cells were counterstained
with 1 mg/mL propidium iodide (PI) to label dead cells.
The cells were then analyzed in the EPICS ALTRA
HyPerSort (Beckman Coulter) by using dual-wavelength
analysis (blue, 424–444 nm; red, 675 nm) after excitation
with 350 nm UV light. PI-positive cells were excluded
from the analysis.
The SP cells were separated by flow cytometry (the
EPICS ALTRA HyPerSort) from the NSP cells and both
fractions were seeded in a mesenchymal stem cell maintenance medium (MF medium; TOYOBO) and 10% FBS
on a collagen-coated 24-well plates (2 cm2; Iwaki). The
cells were cultured for 2 to 4 weeks. The cells were then
transferred to collagen-coated 60-mm plates.
Growth rate assay
Cells were plated in a MF medium in the presence or
absence of NaB (Sigma-Aldrich). Cell viability was determined by using trypan blue exclusion assay. Floating
cells were washed away and the cells were detached
from dishes by 0.25% trypsin. Collected cells were stained
with 0.4% trypan blue and were counted by using
hematocytometer.
Self-renewal assays
SP cells or NSP cells were plated in 24-well collagencoated dishes (10 cells/cm2). SP cells, but not NSP cells,
formed colonies. Cells from individual colonies of SP cells
were reseeded at 10 cells/cm2 in triplicate in 60-mm
collagen-coated plates to generate colonies. Colonies
were monitored to ensure they were derived from single
cells. The secondary colonies were reseeded in a similar
manner to generate tertiary colonies. The cloning plates
were stained with the crystal violet solution (Sigma).
Soft agar assays
For the anchorage-independent growth assays in soft
agar, 1 104 cells were seeded in 60-mm dishes containing growth medium, supplemented with 10% FBS and
0.3% Bactoagar over a hardened 0.5% agar base layer in
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Kato et al.
the presence or absence of 2 or 5 mmol/L NaB. Cells were
incubated for 3 weeks and the number of colonies per 4
cm2 was counted under a microscope.
In vivo tumor formation assays
We inoculated 1 104 cells in Matrigel (BD Matrigel
Basement Membrane Matrix High Concentration; BD
Bioscience) into the subcutaneous connective tissue of
5-weeks-old nude mice (Balb nu/nu). After 6 weeks, mice
were killed and the tumors excised. All mouse experiments were approved by the animal ethics committee of
Kyushu University.
In vitro sensitivity to chemotherapeutic agents
RK12V-SP cells and RK12V-NSP cells were cultured for
96 hours in the presence or absence of 1 mmol/L cisplatin,
1 mmol/L doxorubicin, or 10 nmol/L paclitaxel. Viable
cells determined by the trypan blue exclusion assay were
counted by using a hemocytometer.
Cell-cycle analysis
The DNA content of cells was measured by flow cytometric analysis (EPICSXL; Beckman Coulter) by using the
PI staining method. Cells (2 105) in 6-cm plates were
treated with different concentrations of NaB for 24 hours.
After treatment, the attached cells were washed twice
with ice-cold PBS and suspended in NP-40 lysis buffer
(3.4 mmol/L sodium citrate, 10 mmol/L NaCl, and 0.1%
NP-40) containing 0.5% PI. The proportion of cells in G1,
S, and G2–M phases was determined from DNA histograms by using CellQuest software (Beckton Dickinson).
Antibodies
Primary antibodies used in this study were as follows:
CD13 monoclonal antibody (3D8), vimentin monoclonal
antibody (V9), p21 polyclonal antibody (C-19), p27
monoclonal antibody (F-8), b-actin antibody (C4), histone H3(FL-136), and Ac-histone H3(Lys 9/14), all
obtained from Santa Cruz Biotechnology, Inc. a-Smooth
muscle actin monoclonal antibody (1A4) was purchased
from MBL. Phospho-histone 2AX (Ser139), p38 mitogenactivated protein kinase (MAPK) antibody, and phospho-p38 MAPK (Thr180/Tyr182) were obtained from
Cell Signaling Technology, Inc.
Immunohistochemistry
Formalin-fixed histologic tumor sections from nude mice
or cultured cells were used. Cultured cells were incubated
on glass chamber slides (LAB-TEK; Nalge Nunc International Corp.) and fixed by treatment with 10% formalin.
Sections were rinsed twice in PBS (pH 7.4) for 5 minutes
each. Samples were then incubated with 4% blocking horse
serum (Vector Laboratories) for 1 hour at room temperature in a humidified chamber, followed by incubation with
the primary antibody (200 mg/mL, 1:100 diluted). We also
used nonimmune mouse or rabbit IgG as a control for the
primary antibody. Staining with the primary or control
antibody was conducted overnight at 4 C. Bound antibo-
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Mol Cancer Ther; 10(8) August 2011
dies were detected with a biotinylated anti-rabbit IgG
secondary antibody (1.5 mg/mL) and an avidin–biotin
complex linked to horseradish peroxidase (Vectastain,
Vector Laboratories), followed by incubation with diaminobenzidine tetrahydrochloride as the substrate.
Analysis of the levels of intracellular reactive
oxygen species
The levels of intracellular reactive oxygen species
(ROS) were evaluated by flow cytometry after staining
RK12V-SP cells and RK12V-NSP cells with the CMH2DCFDA probe (Invitrogen). Both cells were cultured
with MF medium in the presence or absence of 5 mmol/L
NaB for 72 hours and incubated with phenol red-free
Opti-MEM containing 1 mmol/L CM-H2DCFDA for
30 minutes. Cells were washed in PBS and collected in
0.5 mL PBS. Fluorescently stained cells were transferred
to polystyrene tubes (Falcon) and were subjected to flow
cytometric analysis (FACScan, Becton Dickinson) by
using CellQuest software for data acquisition and analysis. The levels of intracellular ROS were shown as mean
fluorescence values.
Analysis of gH2AX foci
Cells were incubated on glass chamber slides (LABTEK; Nalge Nunc International Corp.) in the presence or
absence of 5 mmol/L NaB for 24 hours and cells were
fixed by treatment with 10% formalin. gH2AX foci were
analyzed by immunohistochemistry by using phosphohistone 2AX (Ser139) antibody. gH2AX were counted
visually by microscopy, examining 10 cells in 3 different
areas of the slide per each condition. Three independent
experiments were conducted.
Western blotting
To detect each protein expression, subconfluent cells
were lysed with ice-cold lysis buffer (20 mmol/L TrisHCl, pH 8.0, 1% Triton X-100, 10% glycerol, 137 mmol/L
NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 50 mmol/L
NaF, and 1 mmol/L Na3VO4) containing freshly added
protease inhibitors (1 mmol/L phenyl methyl sulphonylfluoride, 1 mg/mL leupeptin, and 10 mg/mL aprotinin;
Sigma). After centrifugation at 13,000 g for 10 minutes
to remove debris, 10 mg of the proteins were subjected to
SDS-PAGE and transferred onto a nitrocellulose membrane in a semi-dry transfer cell (Bio Rad Laboratories).
The blots were incubated with diluted primary antibodies overnight at 4 C. After incubation with each primary
antibody (1:1,000 diluted), the blots were incubated with
horseradish peroxidase-linked anti-rabbit antibodies and
analyzed with an ECL system (Amersham Bioscience).
The levels of protein expression were quantitated by
using ImageJ software.
Microdissection and DNA extraction
Glass slides with an overlay of 4 mm of thin LM Film
(PALM Microlaser Technologies GmbH) were prepared.
Formalin-fixed, paraffin-embedded tumor tissue was cut
Molecular Cancer Therapeutics
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An Effect of HDAC Inhibitor on Cancer SP Cell Proliferation
by using an ABI PRISM Big Dye Termination Ver3.1
Cycle Sequencing Kit according to the manufacturer’s
instructions and the ABI PRISM 3100 (Applied Biosystems). The primer used for sequencing was 50 -TTGAAACCCAAGGTACATTTCA-30 (antisense) for KRAS DNA.
into 7-mm sections and placed on the slides. The sections
were then deparaffinized and stained with hematoxylin
and eosin. By using a laser microdissection system (Leica
Microsystems), tumor cells or stromal cells were isolated
into the cap of a 0.5-mL microtube. After retrieval of the
cells, 50 mL proteinase K solution (Pico Pure DNA Extraction kit; ARCTURUS) was added into the cap. The DNA
was extracted by overnight incubation at 65 C. The solution was then boiled for 10 minutes to inactivate the
proteinase K.
Data analysis
Data are represented with the means SEM and were
analyzed with Student’s t test. A P value of less than 0.05
was considered statistically significant.
PCR of the KRAS gene
To amplify the KRAS gene (cDNA for RK12V-SP
tumor), PCR by using a T3000 thermal cycler was performed (Biometra). The primers used for PCR were as
follows: 50 -GACTGAATATAAACTT-30 (sense); 50 -CATAATTACACACTTTGTCTT-30 for human KRAS cDNA.
The PCR cycling conditions were as follows: (i) preheating for 2 minutes at 94 C, 39 cycles of denaturation for 1
minute at 94 C, annealing for 30 seconds at 59.3 C, and
extension for 1 minute at 72 C, (ii) preheating for 2
minutes at 94 C, 39 cycles of denaturation for 1 minute
at 94 C, annealing for 30 seconds at 59.3 C, and extension
for 1 minute at 72 C. After the last cycle, a final extension
of 5 minutes at 72 C was added.
Results
RK12V-SP cells show features of cancer
stem–like cells
First of all, we analyzed self-renewal capacity of K12VSP cells and potential to develop into stromal cells. When
RK12V-SP cells were plated in collagen-coated dishes (10
cells/cm2), they proliferated and formed large (>2 mm)
colonies. In contrast, RK12V-NSP cells formed only small
colonies (Fig. 1A). We tested their self-renewal capacity
by evaluating serial colony forming potential. Primary
colonies of RK12V-SP cells or RK12V-NSP cells were
dissociated into single cells and then cultured these cells
in 60-mm collagen-coated plates (10 cells/cm2). A single
cell from the secondary colony of RK12V-SP cells generated tertiary colonies. RK12V-NSP cells did not form the
secondary colonies (Fig. 1B).
Next, we investigated whether RK12V-SP cells had the
ability to develop into stromal cells. RK12V-SP cells formed
large, invasive tumors with extracellular matrix–enriched
stroma-like tissues in nude mice as previously shown (29).
Determination of the KRAS sequence
PCR products were electrophoresed on a 2% agarose
gel and the band corresponding to the desired target
cut from the gel. DNA was extracted by using the GFX
PCR DNA Gel Band Purification Kit (GE Healthcare).
Direct sequencing of the PCR product was then done
A
Colony passage
1
B
2
3
20.0
CFU (%)
SP
19.2
SP
NSP
15.0
10.0
5.0
NSP
22.8
25.0
0.0
5.7
1.1
0.0
1
2
3
Colony passage
bars = 10 mm
Figure 1. RK12V-SP cells possess self-renewal capacity. A, RK12V-SP cells and RK12V-NSP cells were plated in collagen-coated dishes (10 cells/cm2)
and cultured with MF medium for 7 days. RK12V-SP cells proliferated and formed large (>2 mm) colonies. In contrast, RK12V-NSP cells formed only
small colonies (colony passage 1). The primary colonies of RK12V-SP cells or RK12V-NSP cells were dissociated into single cells and then cultured in 60-mm
collagen-coated plates (10 cells/cm2). A single cell from the secondary colony of RK12V-SP cells generated tertiary colonies. RK12V-NSP cells did not
form the secondary colonies (colony passage 2 or 3). The cloning plates were stained with crystal violet solution. Scale bar, 10 mm. B, colony numbers
were counted in triplicate. Colony-forming units (CFU) in each passage are shown. Error bar represents SEM from CFU in 3 independent experiments.
CFU, the number of colonies (>2 mm) per total plating cells.
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Kato et al.
chemotherapy) on the proliferation of RK12V-SP cells
and RK12V-NSP cells (Fig. 3). Ninety-six hours of incubation of RK12V-NSP cells with medium containing these
chemotherapeutic drugs inhibited proliferation compared with control. Relative to control, the extent of
inhibition was 61% in 1 mmol/L cisplatin, 51% in10
nmol/L paclitaxel, and 56% in 1 mmol/L doxorubicin.
All of these drugs inhibited the proliferation of RK12VNSP cells significantly compared with control (P < 0.001).
In contrast, none of these drugs had an inhibitory effect
on the growth of RK12V-SP cells.
We microdissected CD13-positive stroma-like cells and
CD13-negative tumor cells, respectively, and sequenced
exon 1 (codons 27–35) in the KRAS gene (Fig. 2A). Several
bases in this region differ between the human and mouse,
enabling the origin of the cells to be determined. As
expected, both tumor cells (data not shown) and the surrounding CD13-positive stroma-like cells contained the
human KRAS gene sequences (Fig. 2B). Three different
regions containing stroma-like cells in RK12V-SP tumors
were microdissected. Human KRAS gene sequences were
detected in all of them. As RK12V cells were transfected
with human mutant [12Val] KRAS 4B cDNA, these results
clearly show that the surrounding stroma-like cells, at least
in part, originated from the inoculated RK12V-SP cells.
Treatment of NaB inhibits self-renewal capacity of
RK12V-SP cells
We previously showed that 2 mmol/L NaB, a shortchain fatty acid HDAC inhibitor, induced senescence and
apoptosis in several types of gynecologic cancer (34).
Takai and colleagues showed that the effective dose of
NaB that inhibited 50% clonal growth of the endometrial
RK12V-SP cells display higher resistance to
conventional chemotherapeutic drugs
Next, we investigated the effect of cisplatin, paclitaxel,
and doxorubicin (clinically used for endometrial cancer
Microdissection
A
CD13
After
Before
a
b
B
< human >
Primer 2 (for RK12V-SP tumor)
5′
3′
5′
3′
5′
Sequence of KRAS gene
17
27
30 31 32 33 34 35
AGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAG
TCACGGAACTGCTATGTCGATTAAGTCTTAGTAAAACACCTGCTTATACTAGGTTGTTATCTC
< mouse >
27
30 31 32 33 34 35
AGTGCCTTGACGATACAGCTAATTCAGATCACTTTGTGGATGAGTACGACCCTACGATACAG
TCACGGAACTGCTATGTCGATTAAGTCTTAGTGAAACACCTACTCATGCTGGGATGCTATCTC
T
C A
T T A
A
a: RK12V-SP CD13 positive cells
GATTCC
intron
5′
3′
3′
5′
A G T G C C T T G A C G ATA C A G C TA AT T C A G A AT C AT T T T G T G G A C G A ATAT G AT C C A A C A ATA G A G G AT T C C
19 21 23 25 27 21 31 35 35 37 39 41 43 45 47 49 51 63 55 57 59 62 63 65 61 71 73 75 77 79 81 83 85
3′
Figure 2. RK12V-SP cells differentiate to stromal-like cells. A, CD13-positive stroma-like cells (a) and CD13-negative tumor cells (b) in tumor-derived
RK12V-SP cells were microdissected, respectively (magnification 50 in left panel, 100 in right panel). B, DNA sequence of exon 1 (codons 17–39)
in the KRAS gene is shown. The third base of codon 27 and codons 30–35 (underlined) differ between human and mouse. An intron is inserted between
codons 37 and 38. DNA sequences of microdissected tumor cells and stroma-like cells in the K12V-SP tumor were analyzed. Both the tumor cells
(data not shown) and stroma-like cells contained human KRAS DNA. Arrowhead, third base of codon 27, codons 31–35. Three different regions containing
stromal-like cells in RK12V-SP tumors were microdissected. The human KRAS gene sequences were detected in all of them (data not shown).
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An Effect of HDAC Inhibitor on Cancer SP Cell Proliferation
RK12V-SP
RK12V-NSP
Relative ratio (%)
Relative ratio (%)
100
100
*
*
50
0
* P < 0.001
*
*
*
*
*
50
None
CDDP
PTX
DOX
(1 μmol/L) (10 nmol/L) (1 μmol/L)
0
None
CDDP
PTX
DOX
(1 μmol/L) (10 nmol/L) (1 μmol/L)
Day 4 (n = 10)
Figure 3. RK12V-SP cells display higher resistance to conventional chemotherapeutic drugs. RK12V-SP cells and RK12V-NSP cells (2 104) were plated and
cultured with DMEM containing 10% FBS in the presence or absence of 1 mmol/L cisplatin, 10 nmol/L paclitaxel, and 1 mmol/L doxorubicin for 96 hours. The
proportion of viable cells relative to control after incubation is shown. Inhibition (relative to control) was 61% in 1 mmol/L cisplatin, 51% in10 nmol/L
paclitaxel, and 56% in 1 mmol/L doxorubicin. All of these drugs inhibited the proliferation of RK12V-NSP cells significantly compared with control (P < 0.001).
In contrast, none of these drugs had an inhibitory effect on growth of RK12V-SP cells. Error bar represents SEM in 10 independent experiments.
cancer cell lines ranged between 8.3 104 and 4.1 103
mol/L (23). On the basis of these previous data, we
examined the effect of 2 or 5 mmol/L NaB on cell proliferation of both RK12V-SP cells and RK12V-NSP cells.
We first confirmed by Western blot that the levels of
acetylated H3 were enhanced in both RK12V-SP cells and
RK12V-NSP cells treated with 2 mmol/L NaB for 72
hours compared with that in untreated cells (Fig. 4A).
Treatment with 2 or 5 mmol/L NaB for 96 hours
significantly inhibited cell proliferation of RK12V-SP cells
as well as RK12V-NSP cells (Fig. 4B; P < 0.01). RK12V-SP
cells have a potential to regenerate SP cells after incubation, which is an important characteristics of stem-like
cells. Treatment with NaB for 24 hours significantly
inhibited the proportion of reproduced SP cells (control,
15%; 2 mmol/L NaB, 1.5% P < 0.02; 5 mmol/L NaB,
0.023% P < 0.01; n ¼ 4, Fig. 4C a). The primary colonyforming potential of RK12V-SP cells was completely
suppressed by treatment by 2 mmol/L NaB (Fig. 4C b).
Next, we analyzed the cell-cycle alteration in response
to NaB by using flow cytometry with PI staining. Treatment with NaB for 24 hours resulted in a significant
decrease in the fraction of RK12V-SP cells in S phase in
a dose-dependent manner (control, 22.5%; 2 mmol/L
NaB, 14%; 5 mmol/L NaB, 10%; P < 0.05; Fig. 4D). Conversely, the percentages of cells in a sub-G1 population
increased, but it was not statistically significant. In
RK12V-NSP cells, there was no significant change in
the cell-cycle phase distribution.
NaB treatment suppresses colony formation of
RK12V-SP cells in soft agar cultures
Next, we investigated the effect of NaB treatment on
tumorigenicity of RK12V-SP cells and RK12V-NSP cells
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by evaluating colony forming efficiency in soft agar
cultures in the presence or absence of 2 or 5 mmol/L
NaB. After 3 weeks of culture, RK12V-SP cells yielded
many large colonies in the control culture lacking NaB
(Fig. 5). In contrast, no colony formed in soft agar
cultures in RK12V-NSP cells. Treatment with NaB (both
2 and 5 mmol/L) suppressed colony formation of
RK12V-SP cells in soft agar cultures. As anticipated,
RK12V-NSP cells did not form colonies in soft agar
cultures.
RK12V-SP cells show higher susceptibility to
NaB-induced DNA damage
Finally, we investigated the molecular mechanism
associated with the inhibitory effect of NaB treatment
of RK12V-SP cells. Recently, we reported that treatment
with NaB induced cell death in several cancer cell lines
mediated by enhanced ROS levels, DNA damage
response signals, and upregulation of p21 (35). Thus,
we examined the change of these signal levels in
RK12V-SP cells and RK12V-NSP cells.
The levels of intracellular ROS were enhanced by 5
mmol/L NaB treatment in both RK12V-SP cells and
RK12V-NSP cells (Fig. 6A). Phosphorylated H2AX
(gH2AX) foci, which are indicators of DNA damage, were
assessed by immunohistochemistry. The numbers of
gH2AX foci were increased by 5 mmol/L NaB treatment
in both RK12V-SP cells and RK12V-NSP cells (Fig. 6B).
The levels of gH2AX proteins were markedly increased in
RK12V-SP cells (44-fold) compared with that in RK12VNSP cells (2-fold; Fig. 6C). The p21, p27, and phospho-p38
MAPK expression levels were also enhanced more in
RK12V-SP cells than in RK12V-NSP cells. These results
imply that treatment with NaB induced production of
Mol Cancer Ther; 10(8) August 2011
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Kato et al.
A
RK12V-SP
0
NaB
RK12V-NSP
C
2 mmol/L 0 2 mmol/L
Acetylated-H3
Relative ratio
1.0
1.73
1.0
1.46
NaB
H3
B
1.2
1
0.8
0.6
0.4
0.2
0
NaB
SP
P < 0.01
L
ol/
m
0m
m
2m
ol/
L
NSP
1.2
1
0.8
0.6
0.4
P < 0.01 0.2
0
m
5m
ol/
L
SP
20
15
10
5
0
a
P < 0.02
P < 0.01
Control 2 mmol/L 5 mmol/L
b
P < 0.01 P < 0.01
m
0m
L
ol/
L
ol/
m
2m
L
ol/
m
5m
NaB
–
2 nmol/L
D
SP
45 %
40
35
30
25
20
15
10
5
0
sub-G1
NSP
Control
NaB 2 mmol/L
NaB 5 mmol/L
* P < 0.05
*
G1
S
*
G2/M
45 %
40
35
30
25
20
15
10
5
0
Control
NaB 2 mmol/L
NaB 5 mmol/L
sub-G1
G1
S
G2/M
Figure 4. Treatment of NaB inhibits self-renewal capacity of RK12V-SP cells. A, RK12V-SP cells and RK12V-NSP cells were cultured with MF
medium in the presence or absence of 2 mmol/L NaB for 72 hours. The level of acetylated H3 or nonacetylated H3 was analyzed by Western blot. The
relative ratio of acetylated H3 levels by NaB treatment to control is shown. Representative data are shown. Similar results were obtained 3 times.
B, RK12V-SP cells and RK12V-NSP cells (2 104) were plated and cultured with DMEM containing 10% FBS in the presence or absence of 2 or 5 mmol/L
NaB for 96 hours. The proportion of viable cells relative to control after incubation is shown. Treatment with 2 or 5 mmol/L NaB for 96 hours
significantly inhibited cell proliferation of RK12V-SP cells as well as RK12V-NSP cells (P < 0.01). Error bar represents SEM in 3 independent experiments.
C, a, RK12V-SP cells were reanalyzed by flow cytometry 2 weeks after culture in the presence or absence of NaB. Treatment of NaB for 24 hours
significantly inhibited the proportion of reproduced SP cells (control, 15%; 2 mmol/L NaB, 1.5%, P < 0.02; 5 mmol/L NaB, 0.023%, P < 0.01). Error
bar represents SEM in 4 independent experiments. In b, RK12V-SP cells were plated in collagen-coated dishes (10 cells/cm2) and cultured with MF
medium in the presence or absence of 2 mmol/L NaB for 7 days. Treatment with 2 mmol/L NaB completely suppressed primary colony-forming
potential of RK12V-SP cells. D, flow cytometric analysis of cell-cycle phase distribution following treatment with NaB. In RK12V-SP cells, treatment
with 2 or 5 mmol/L NaB for 24 hours resulted in a significant decrease in the fraction of cells in S phase in a dose-dependent manner (control
22.5%, 2 mmol/L NaB, 14%; 5 mmol/L NaB, 10%; P < 0.05). In RK12V-NSP cells, there were no significant changes in the cell-cycle phase distribution.
Error bar represents SEM in 3 independent experiments.
intracellular ROS and DNA damage in both RK12V-SP
and RK12V-NSP cells.
Signals involved in DNA damage responses were
enhanced more in RK12V-SP cells (cancer stem–like cells)
than in RK12V-NSP cells by NaB treatment.
Discussion
In this study, we analyzed the biological characteristics
of the SP fraction derived from rat endometrial cells
harboring human mutant [12Val] KRAS 4B genes
1436
Mol Cancer Ther; 10(8) August 2011
(RK12V cells). We determined that the phenotype of
RK12V-SP cells resembled that of cancer stem–like cells.
We found that the HDAC inhibitor, NaB inhibited proliferation of RK12V-SP and RK12V-NSP cells. Although
these findings may not be representative of all endometrial carcinomas, this is the first report of an inhibitory
effect of NaB on proliferation of endometrial cancer stem–
like cells.
As previously shown, RK12V-SP cells have reduced
expression levels of certain differentiation markers (CD9
and CD13), reduced long-term proliferating capacity in
Molecular Cancer Therapeutics
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Published OnlineFirst June 1, 2011; DOI: 10.1158/1535-7163.MCT-10-1062
An Effect of HDAC Inhibitor on Cancer SP Cell Proliferation
NaB
Figure 5. Treatment with NaB
completely suppressed colony
formation of RK12V-SP cells in
soft agar cultures. RK12V-SP cells
and RK12V-NSP cells (1 104
cells) were seeded in 60-mm
plates containing growth medium,
supplemented with 10% FBS and
0.3% Bactoagar over a hardened
0.5% agar base layer in the
presence or absence of 2 or 5
mmol/L NaB. After 3 weeks of
culture, control cells in the
absence of NaB formed many
large RK12V-SP colonies.
Treatment with NaB suppressed
RK12V-SP colony formation. In
contrast, no colony formed in soft
agar cultures of RK12V-NSP cells.
Error bar represents SEM in 3
independent experiments.
0 mmol/L
5 mmol/L
SP
NSP
(×60)
Number of colonies/4 cm2
180.0 166.8
160.0
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.1
0.0
NaB 0 nmol/L
culture, and enhanced tumorigenicity (29). We found in
this study that RK12V-SP cells possessed self-renewal
capacity and the potential to develop into stromal cells.
These results indicate that RK12V-SP cells have cancer
stem–like cell features as that of SP cells derived from a
human endometrial cancer cell line, Hec1 cells.
It has been known that SP cells (but not NSP cells)
express MDR transporter proteins, such as ABCG2/
Brcp1 (11, 36). As expected, RK12V-SP cells displayed
higher resistance to conventional chemotherapy (Fig. 3),
indicating a requirement for new targets for the treatment
of cancer stem–like cells. To develop new approaches to
molecular cancer therapy, we did the microarray assays
to identify the overexpressed genes in RK12V-SP cells
compared with those in RK12V-NSP cells (data not
shown). The expression level of a number of genes,
including cytokines and growth factors, was enhanced
in RK12V-SP cells (Kato and colleagues, manuscript in
preparation), suggesting that multiple signal pathways
exist to maintain the phenotype of SP cells. It would be
difficult to identify a single, selective molecular target to
SP cells.
HDACi’s have multiple biological effects, including
growth arrest, apoptosis, senescence, ROS facilitated
cell death, mitotic cell death, and antiangiogenesis
(17). HDACi’s include short-chain fatty acids (e.g.,
butyrates and valporic acid), organic hydroxamic acids
[trichostatin A and suberoyl anilide bishydroxamine
(SAHA)], cyclic tetra peptides (e.g., trapoxin), and bezamides (e.g., MS-275). Trichostatin A, NaB, valporic acid,
and SAHA can inhibit malignant cells in vitro and in vivo
www.aacrjournals.org
2 mmol/L
SP
NSP
0.0 0.0
0.0 0.0
2 nmol/L
5 nmol/L
(37–39). We have previously shown that NaB induced
p21 expression, resulting in growth arrest and cell death
(34). Recently, we have also shown that DNA damage
signals were involved in NaB-induced cell death (35). In
this study, NaB inhibited primary colony-forming
potential and regeneration of SP cells, indicating suppression of self-renewal capacity. Most recently, Conti
and colleagues showed that SAHA treatment of cancer
cells slows down replication forks, activates dormant
origins, and induces DNA damage (40). They also found
gH2AX could be used as a convenient pharmacodynamic biomarker for HDACi-induced DNA damage
(41). We showed that gH2AX was markedly enhanced
in RK12V-SP cells compared with that in RK12V-NSP
cells, implying that RK12V-SP cells were highly sensitive to NaB-induced DNA damage (Fig. 6). These results
indicated that cancer stem–like cells, which are resistant
to conventional chemotherapy, were sensitive to treatment with HDACi’s. It is believed that stem cells generally proliferate slowly. Burgess and colleagues
showed that HDACi’s can induce death of transformed
cells in both proliferative and nonproliferative phases of
the cell cycle (42).
Clinical trials of several HDACi’s are currently
underway, as monotherapies or in combination with
other anticancer drugs and radiation. A total of 140
studies for cancer are found in website of U.S. NIH
(www.clinicaltrials.gov). SAHA and depsipeptide have
been approved by the FDA for cancer therapy (26, 27).
HDACi’s emerge as promising drugs for targeting of
cancer stem–like cells in clinical setting.
Mol Cancer Ther; 10(8) August 2011
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Published OnlineFirst June 1, 2011; DOI: 10.1158/1535-7163.MCT-10-1062
Kato et al.
A
B
Average number of
γH2AX foci per cells
5
Mean fluorescence value
100
4
3
50
2
1
0
C
0
DMSO
NaB
5 nmol/L
RK12V-SP
DMSO
DMSO
NaB
5 nmol/L
RK12V-NSP
RK12V-SP
NaB 5 mmol/L
0h
6h
12 h
0h
6h
12 h
0h
6h
NSP
12 h
0h
6h
12 h
γH2AX
9.2
10.1
1.0
2.0
2.1
Relative ratio 1.0
19.9
44.5
1.0
1.3
2.6
2.9
2.6
1.0
1.0
1.0
Phosphop38MAPK
p27
Relative ratio 1.0
RK12V-NSP
SP
p21
Relative ratio 1.0
NaB
5 nmol/L
NaB 5 mmol/L
NSP
SP
DMSO
NaB
5 nmol/L
2.6
2.7
1.0
1.7
1.5
β-Actin
Relative ratio 1.0
p38MAPK
Figure 6. RK12V-SP cells show high responsiveness to NaB-induced DNA damage. A, the levels of intracellular ROS were evaluated by flow cytometric
analysis after staining RK12V-SP cells and RK12V-NSP cells with the CM-H2DCFDA probe. Both types of cells were cultured with MF medium in the
presence or absence of 5 mmol/L NaB for 72 hours and incubated with phenol red free-Opti-MEM containing 1 mmol/L CM-H2DCFDA for 30 minutes.
The levels of intracellular ROS are shown as mean fluorescence value. Error bar represents SEM in 3 independent experiments. The ROS levels were enhanced
by 5 mmol/L NaB treatment in of RK12V-SP cells and RK12V-NSP cells. B, phosphorylated H2AX (gH2AX) foci were assessed by immunohistochemistry
by using phospho-histone 2AX (Ser139) antibody. gH2AX were counted visually by microscopy, examining 10 cells in 3 different areas of the slide
per each condition. Error bar represents SEM in 3 independent experiments. The number of gH2AX foci was increased by 5 mmol/L NaB in the treatment of
both RK12V-SP cells and RK12V-NSP cells. DMSO, dimethyl sulfoxide. C, RK12V-SP cells and RK12V-NSP cells were cultured with MF medium in the
presence or absence of 5 mmol/L NaB for 6 or 12 hours. The level of each protein was analyzed by Western blot and the relative ratio to control is shown.
The levels of gH2AX proteins were markedly increased in RK12V-SP cells (44-fold) compared with that in RK12V-NSP cells (2-fold). Expression levels
of p21, p27, and phospho-p38 MAPK were also enhanced more in RK12V-SP cells than in RK12V-NSP cells. b-Actin was the internal control. Representative
data are shown. Similar results were obtained 3 times.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
The authors thank Dr. Channing Der (University of North Carolina)
for generously donating pZIP neo SV (X)1-K-Ras4B(12V) and Miwako
Ando for technical assistance. The authors also thank Drs. Ryota
Souzaki, Tatsuro Tajiri, and Tomoaki Taguchi (Department of Pediatric
Surgery, Kyushu University) for technical advice on a laser microdissection.
Grant Support
This work was supported by grants-in-aid 20659259, 22659302, and
22591869 from the Ministry of Education, Culture, Sports, Science and
Technology, Japan and the Environment Technology Development Fund
of the Ministry of the Environment, Japan for K. Kato.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received November 29, 2010; revised April 26, 2011; accepted May 24,
2011; published OnlineFirst June 1, 2011.
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