The use of histone deacetylase inhibitor FK228 and DNA hypomethylation
agent 5-azacytidine in human bladder cancer therapy
Jose A. Karam1, Jinhai Fan1, Jennifer Stanfield1, Edmond Richer2, Elie A. Benaim1, Eugene Frenkel3, Peter Antich2,
Arthur I. Sagalowsky1, Ralph P. Mason2and Jer-Tsong Hsieh1*
1Department of Urology, University of Texas Southwestern Medical Center, Dallas, TX
2Department of Radiology, University of Texas Southwestern Medical Center, Dallas, TX
3Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX
The long-term disease-free survival in patients with metastatic
transitional cell carcinoma (TCC) is still considerably low. Novel
chemotherapeutic agents are needed to decrease the morbidity
and mortality of TCC. In this study, we have evaluated several
epigenetic modifiers for their therapeutic application in bladder
cancer. Both histone deacetylase inhibitors (FK228, TSA) and
DNA hypomethylating agent (5-Azacytidine) were tested using in
vitro assays such as cell viability, cell cycle analysis and western
blot to determine their mechanisms of action. Drug combination
experiments were also designed to study any additive or synergis-
tic effects of these agents. In addition, two bladder cancer xeno-
graft models (one subcutaneous and one orthotopic) were
employed to assess the therapeutic efficacy of these agents in vivo.
Three agents exhibited various growth inhibitory effects on 5 dif-
ferent TCC cell lines in a dose- and time-dependent manner. In
addition to G2/M cell cycle arrest, FK228 is more potent in induct-
ing apoptosis than the two other single agents, and combination of
both FK228 and 5-Aza further enhances this effect. p21 induction
is closely associated with FK228 or TSA but not 5-Aza, which is
mediated via p53-independent pathway. Consistent with in vitro
results, FK228 exhibited a significant in vivo growth inhibition of
TCC tumor in both subcutaneous and orthotopic xenograft mod-
els. FK228 is a potent chemotherapeutic agent for TCC in vivo
with minimal undesirable side effects. The elevated p21 level
mediated via p53 independent pathway is a hallmark of FK228
mechanism of action.
' 2007 Wiley-Liss, Inc.
Key words: bladder cancer; chemotherapy; histone acetylation; DNA
In the United States, transitional cell carcinoma (TCC) of the
bladder is estimated to affect 63,210 persons in 2005, resulting in
13,180 deaths.1In recent times, patients with metastatic TCC have
experienced improved prognosis; however, the long-term disease-
free survival is still low.2Conventional cytotoxic chemotherapy is
currently the main treatment for these patients.3Several single
agents and combinations have been used, but the MVAC (Metho-
trexate, Vinblastin, Adriamycin and Cisplatin) regimen has been
yielding the best results.3,4This regimen, however, is not without
serious side effects. Treatment-related deaths can occur in up to
4% of patients.5,6Still, with the best regimens available, median
survival ranges from 13 to 15 months.4,6–8The importance of find-
ing alternatives to MVAC, which are at least as effective but with
less side effects, has encouraged several investigators to try other
chemotherapeutic combinations, such as gemcitabine plus cispla-
tin8and docetaxel plus cisplatin.9However, these agents did not
prove to be superior to the traditional MVAC regimen. Novel che-
motherapeutic regimens are currently needed to decrease the mor-
bidity and mortality of TCC.
Epigenetic mechanisms result in changes in gene expression
without altering the DNA sequence per se.10These changes involve
DNA methylation and histone modifications (such as acetylation),
which are potentially reversible. This property has made modula-
tion of epigenetic gene suppression a very attractive model to treat
cancer. Several epigenetic modifiers have been noted to modulate
these mechanisms. For example, 5-Azacytidine (5-Aza) is a cyto-
sine analogue that can integrate in DNA and RNA, resulting in
tight binding to DNA methyltransferase 1 and prevention of DNA
methylation.11Trichostatin A (TSA) isolated from Streptomyces
hygroscopicus belongs to the hydroxamate class of histone de-
acetylase (HDAC) inhibitors.12FK228 (Depsipeptide, FR901228)
is an HDAC inhibitor (HDACI) that was isolated from Chromobac-
terium violaceum.13Both TSA and FK228 target class I HDACs
which are mainly present in the nucleus.14,15The inhibition of
HDACs by HDACIs results in induction of differentiation, growth
arrest and apoptosis.16In addition, methyl-CpG binding protein
(MeCP2) can bind to methylated DNA, with subsequent recruit-
ment of a HDAC complex, and therefore preventing gene expres-
sion.17The finding that DNA methylation and histone deacetyla-
tion interact to silence genes has been used to develop new thera-
peutic regimens. DNA microarray analyses18show that the use of
DNA methyltrasferase (DNMT) inhibitors and HDACIs results in a
synergistic activation of tumor suppressor genes,19–21as well as
influencing global gene expression. Low doses of DNMT inhibitors
and HDACIs can be combined in order to decrease the occurrence
of side effects and cause a synergistic inhibition of tumor growth.22
Our objective was to evaluate the effect of three epigenetic modi-
fiers on the growth of TCC cell lines in vitro and in vivo. We
hypothesized that combining these drugs would result in additive or
synergistic effects on cell growth inhibition, as well as prolonging
growth inhibition. In addition, we studied the mechanism of action
of these agents on TCC cell lines and evaluated the therapeutic effi-
cacy using two different preclinical animal models. Together, the
outcome of this study supports the rational design of chemotherapeu-
tic trials targeting the epigenetic machinery in TCC of the bladder.
Material and methods
Cells and reagents
Human TCC cell lines T24, 253J, UMUC3, WH and TCC-SUP
were purchased from the American Type Culture Collection
(ATCC, Manassas, VA). Cells were maintained in T medium
(Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine se-
rum and 1% penicillin/streptomycin in a humidified incubator at
37?C and 5% CO2.
5-Azacytidine (5-Aza, Sigma, Saint Louis, MO) was used at
concentrations of 1, 5, 10 and 25 lM. FK228 (FR901228, Depsi-
peptide, a gift from Fujisawa Pharmaceutical Co., Japan) was used
Dr. Karam and Dr. Fan have contributed equally in this paper.
Grant sponsor: NIH; Grant number: CA95730.
*Correspondence to: Department of Urology, University of Texas
Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-
9110, USA. E-mail: JT.Hsieh@UTSouthwestern.edu
Received 19 June 2006; Accepted after revision 12 September 2006
Published online 17 January 2007 in Wiley InterScience (www.interscience.
Abbreviations: 5-Aza, 5-Azacytidine; BLI, bioluminescence imaging;
HDAC, histone deacetylase; HDACI, histone deacetylase inhibitor;
DNMT, DNA methyltransferase; PARP, Poly (ADP-ribose) Polymerase;
PBS, phosphate buffered saline; TCC, transitional cell carcinoma.
Int. J. Cancer: 120, 1795–1802 (2007)
' 2007 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
at 0.25, 0.75, 1 and 5 ng/ml. TSA (Trichostatin A, Sigma) was
used at 25, 100, 200 and 1,000 nM.
Antibodies used in this study are as follows: PARP (Poly (ADP-
ribose) Polymerase; 1:2,000, Roche, Indianapolis, IN), actin
(1:5,000, Sigma), p21 (1:400, BD Pharmingen, San Diego, CA),
p53 (1:400, BD Pharmingen), cyclin B1 (1:500, Pharmingen, San
Diego, CA) and cyclin E (1:500, Pharmingen).
Cell growth assays and IC50calculations
Cells were seeded in 24-well plates (Costar, Corning, NY) at a
concentration of 2–4 3 104cells in 2 ml of medium per well. Each
treatment condition was assessed in quadruplicate. After 24 hr, the
medium was aspirated and new medium containing the drug was
added. At the indicated time, total cell number was determined
using crystal violet assay. Briefly, the medium was aspirated and
500 ll of 1% gluteraldehyde (Sigma) in phosphate buffered saline
(PBS) was added for 15-min incubation. After removing gluteral-
dehyde, 0.5% crystal violet (Sigma) was incubated for 15 min then
plates were washed 3 times in water and left to dry at room temper-
ature. Once dry, 500 ll of Sorenson’s solution (8.967 g tri-sodium
citrate in 305 ml of distilled water, 195 ml 0.1 N HCl, 500 ml 90%
ethanol) was added to elute the crystal violet. After 30 min, 200 ll
of the resulting solution was transferred into a 96-well plate and
was read at 540 nm using ELX800 microplate reader (Bio-Tek
Instruments, Winooski, VT).
IC50values for 5-Aza, FK228 and TSA on day 3 in each of T24
and 253J cell lines were calculated using Origin1version 7.5
(OriginLab, Northampton, MA) software. IC50was considered as
the drug concentration that decreases the cell count by 50%. Non-
linear regression curve fitting was performed. The data were fitted
to an exponential first-order decay function.
Western blot analysis
Cells were seeded at a concentration of 1 3 105cells in a 100-mm
plate (Costar, Corning, NY). A single treatment was applied 24 hr af-
ter cell plating. After 3 days, the supernatant was aspirated, washed
twice with ice-cold PBS and then 100 ll of protein lysis buffer
(50 mM Hepes pH 7.5, 150 nM NaCl, 10% glycerol, 1% Triton X-
100, 1.5 mM MgCl2) with protease inhibitor cocktail was added. The
attached cells were washed with PBS twice then incubated with 500
ll of protein lysis buffer. The resulting proteins were kept at 280?C
until western blotting was done. Twenty micrograms of protein were
subjected to a 10 or 16% Criterion1SDS-PAGE (Biorad, Hercules,
CA). After transferring onto a nitrocellulose membrane (Osmonics,
Minnetonka, MN), the membrane was blocked for 1 hr with PBS
containing 5% dry milk and 0.1% Tween 20, then the primary anti-
body was incubated overnight at 4?C followed by a corresponding
secondary antibody. After extensive washing, the membranes were
developed using ECL Plus (Amersham, Piscataway, NJ).
Cell cycle analysis
T24 cells were plated at a concentration of 1 3 105cells in a
100-mm plate for 24 hr then different agents were added. After
3 days of treatment, the attached cells were trypsinized and pooled
with the suspension cells. Cells were washed twice with cold PBS
and resuspended in 0.5 ml of cold PBS then mixed with 4.5 ml of
70% ethanol. The next day, ethanol was removed and cells were
incubated for 15 min at 37?C with 1 ml propidium iodide solution
(100 ml of 0.1% v/v Triton X-100 in PBS, 20 mg DNase-free RNase
A and 2 mg of propidium iodide). Cell fluorescence was measured
with flow cytometry using FACScanTM(Becton Dickinson, San
Jose, CA). Gating was implemented to remove cell doublets.
Xenograft animal models and treatment schedule
T24-t cell line (T24-tumorigenic) was derived from a subcuta-
neous T24 tumor with higher tumorigenicity that was achieved af-
FIGURE 1 – Effect of 5-Aza, TSA and FK228 on cell growth of T24 and 253J bladder cancer cell lines. Cells were seeded in 24-well plates for
24 hr prior to adding each agent. T24 and 253J cells were treated with 5-Aza (in PBS) at 1, 5, 10 and 25 lM (a and d), FK228 at 0.25, 0.75, 1
and 5 ng/ml (b and e) and TSA (in ethanol) at 25, 100, 200 and 1,000 nM (c and f). Relative cell number was assessed using the crystal violet
assay. Optical densities were read at 540 nm. Data represent mean 6 SD.
TABLE I – IC50 VALUES FOR 5-AZA, FK228 AND TSA IN T24 AND 253J
CELLS AFTER 3 DAYS OF TREATMENT
KARAM ET AL.
ter multiple cycles of tumor harvesting and reimplantation in nude
mice. T24-t-luc (T24-tumorigenic-Luciferase) was generated by
using a lentivirus expressing a luciferase gene under the control of
CMV promoter (provide by Dr. Jerry Shay). All experimental pro-
cedures have been approved by the Institutional Animal Care and
For the subcutaneous model, athymic nude mice were injected
with a 100 lL cell suspension containing 1 3 106T24-t on the
flank (2 sites/animal). When the tumors became palpable, different
treatments were given to randomized animals (4 animals/ treat-
ment group). FK228 was dissolved in 100% ethanol and further
diluted with 5% glucose solution at 1:39 ratio before injection.
FK228 (1.0 mg/kg) or 5-Aza (5.0 mg/kg) was administrated by in-
travenous tail injection twice a week for 3 weeks. Tumors were
measured using a caliper and tumor volume in mm3was calcu-
lated using the ellipsoid formula p/6 3 length 3 width 3 depth.
Mice were killed when tumors became ulcerating, in accordance
with our institutional animal care policies.
For the orthotopic animal model, the procedure was a modifica-
tion of the technique described by Watanabe.23Briefly, female athy-
mic nude mice were anesthetized and a 24-gauge angiocatheter was
inserted into the bladder through the urethra. One hundred microli-
ter of 0.2% trypsin in 0.02% EDTA was infused and retained in the
bladder for 30 min. The bladder was then recatheterized and rinsed
with phosphate-buffered saline (PBS). Subsequently, a 100 lL cell
suspension containing 106T24-t-luc cells mixed with matrigel (1:1
ratio) was instilled into the bladder. The urethra was ligated with a
4–0 nylon suture to assure that cells were retained in the bladder.
After 3 hr the suture was removed. FK228 was dissolved in 100%
ethanol and further diluted with 5% glucose solution at 1:39 ratio
before injection. Once the tumors were detectable by biolumines-
cence imaging (BLI intensity >105photons), FK228 (1.0 mg/kg)
was administrated by intravenous tail injection twice a week for
3 weeks. BLI was performed weekly by subcutaneously injecting
450 mg/kg D-luciferin substrate in PBS (Biosynth, Neperville, IL)
into anesthetized mice as described previously24using a CCD cam-
era (charge-couple device, Scientific Imaging Technologies, Inc.,
Tigard, OR (SITe) SI-032AB CCD). Light was captured for 2 min
starting 10 min after D-luciferin injection. Image signal intensity
was quantified as the sum of all detected photon counts within the
region of interest after subtraction of background luminescence
using Igor Pro software (WaveMetrics, Lake Oswego, OR).
ANOVA was used to compare the means of cell viability of sev-
eral treatment groups. Post-hoc tests were used to find the signifi-
cant difference among the treatment groups. The Student’s t-test
was used to detect any statistically significant difference between
each treatment group and control. p values < 0.05 were considered
Dose-response and time-response effects of 5-Aza
or HDACIs on different TCC cells
The effect of the DNA hypomethylating agent 5-Aza on 2 TCC
cell lines, T24 (Fig. 1a) and 253J (Fig. 1d) was first determined.
After 1 day of treatment, 5-Aza resulted in 12–19% toxicity in
253J and 0–43% toxicity in T24 (doses from 1 to 25 lM, respec-
tively). In T24 cells, on Day 3, 5-Aza resulted in a low degree of
(32%) cytotoxicity starting at 1 lM and the major growth suppres-
sion (75%) was observed starting at 5 lM doses. On Day 5 and 7,
cells partially recovered from drug effects, after being remarkably
suppressed at concentrations above 10 lM. In 253J cells, we ob-
served a higher sensitivity to 5-Aza (73–93% cytotoxicity) as com-
pared to T24 (30–88% cytotoxicity) for concentrations between 1
FIGURE 2 – Effect of combination treatments on cell growth of TCC cell lines. Cells were seeded in 24-well plates for 24 hr prior to adding
each agent, and relative cell number was determined at Day 3 using crystal violet assay. Data represent the percentage of viability of each treat-
ment after being normalized with control. * denotes statistical significance (p < 0.05) of combination treatment compared to both single treat-
FIGURE 3 – Effect of combination treatments on preventing recurrent cell growth in T24 cells. Cells were seeded in 24-well plates for 24 hr
and a single dosage of each agent was administered. Relative cell number was determined by crystal violet assay at the indicated time. Data rep-
resent the percentage of viability of each treatment after normalization with control. * denotes statistical significance (p < 0.05) of combination
treatment compared to both single treatments.
BLADDER CANCER THERAPY WITH EPIGENETIC MODIFIERS
and 25 lM, along with some recovery after 5-day treatment at
1 lM, but markedly decreased recovery was observed at 25 lM
under the same condition.
For HDACIs, FK228 starting at 0.75 ng/ml and TSA starting at
100 nM resulted in more significant growth suppression in both
T24 (Figs. 1b and 1c) and 253J (Figs. 1c and 1f) cells. Overall,
T24 cells appear to be more resistant than 253J cells. Similar to
the treatment with 5-Aza, T24 cell lines recovered at Day 5 with
lower concentration of FK228 or TSA. IC50values for each of the
3 drugs after 3 days of treatment are shown in Table I.
We then investigated 3 different combinations of 5-Aza 5 lM,
FK228 0.75 ng/ml and TSA 100 nM in 4 different TCC cell lines
after 3 days of treatment, for possible additive or synergistic
effects. Combining 5-Aza and FK228 (Fig. 2a) resulted in additive
cytotoxicity (?90%) in 253J, WH and UMUC3 cells, but not in
TCC-SUP cells. We noted that 5-Aza alone was not as effective on
TCC-SUP as on the other 3 cell lines. On the other hand, FK228
alone resulted in the highest decrease in cell number in TCC-SUP,
and therefore masking any potential additive effect. When 5-Aza
and TSA were combined (Fig. 2b), an additive effect (75–90%)
was seen in all cell lines. Of note, TSA was not effective in WH
cells at the concentration used. Using the combination of FK228
and TSA (Fig. 2c), the additive effect was observed in UMUC3
cell lines only whereas others showed no additive effect.
To evaluate whether the recurrent cell growth can be prevented
with combination treatment, we used the most resistant cell line,
T24, in this experiment. The additive effect was observed with all
three combinations starting at Day 3 (Fig. 3). Also, cell recovery
was prevented in all 3 combinations, except in the FK228 1 TSA
combination some cell recovery was noticed at Day 7.
FK228 induces apoptosis and G2/M cell cycle arrest in T24 cells
After establishing the activity of the 3 drugs in TCC cell lines, we
investigated whether apoptosis is involved in this process (Fig. 4).
At a concentration of 0.75 ng/ml, FK228 caused 37.5% apoptosis in
T24 cells measured by flow cytometry (Fig. 4c), which can be fur-
ther potentiated by combining it with 5-Aza (67.4%) (Fig. 4e). At
low concentrations, 5-Aza and TSA did not induce apoptosis (pre-
G0/G1was 2.8% and 3.5%, respectively) in T24 cells (Figs. 4b and
4d). FK228-induced apoptosis was further confirmed by the appear-
ance of the 89 kDa PARP cleavage fragment in T24 cells using
FIGURE 4 – Cell cycle analysis of T24 cells under different treatment conditions. T24 cells were seeded at a concentration of 1 3 105cells in
100 mm plates for 24 hr and a single dosage of each agent was administered. On Day 3, cells were harvested from each treatment and stained
with propidium iodide and then analyzed with flow cytometry.
KARAM ET AL.
western blot (Fig. 5a). In all studied cell lines, PARP cleavage was
seen in the supernatant phase, but not in the attached cell population
(data not shown).
Moreover, the G2/M cell population increased from baseline af-
ter treatment with the three drugs separately at low concentrations
(5-Aza 22.2%, FK228 21.2%, TSA 22.5% as compared to control
11.7%) (Figs. 4a–4d). Noticeably, a dramatic decrease in the G2/
M cell population in T24 cells treated with FK228 1 5-Aza was
seen, because of an increased rate of apoptosis with this treatment
compared to the two other combinations (Fig. 4e).
Analysis of cell cycle regulation by FK228
To understand the possible effect of these agents on modulating
cell cycle-related proteins, we have determined the steady state
levels of cyclin B1, cyclin E, p21 and p53 in T24 cells. As shown
in Figure 5b, cyclin B1levels were unchanged. On the other hand,
cyclin E levels were increased after each single agent treatment.
Interestingly, elevated p21 levels were only detected in cells
treated with HDACIs. The fact that p53 was not detected in the
T24 cell line, before or after treatment, suggests that HDACIs-
elicited p21 protein induction is mediated by p53-independent
pathways (Fig. 5b).
FK228 results in tumor growth suppression in vivo in both
xenograft and orthotopic bladder cancer models
To evaluate whether HDACIs or DNA hypomethylating agents
can be applied as a single agent or combination treatment on TCC,
FK228 and 5-Aza were injected intravenously into athymic nude
mice bearing subcutaneous T24-t tumors (the most resistant cell
line tested in our study). A time-dependent growth inhibition was
observed with FK228 in the T24-t xenograft model: tumors were
similar in size prior to the treatment (21 vs. 16 mm3) and 7 days
after treatment (46 vs. 58 mm3). On Day 14 and 21 after treatment
(Fig. 6a), tumors were significantly smaller in the FK228-treated
group (63 vs. 132 mm3and 69 vs. 240 mm3, respectively), and to a
similar extent in the Aza/FK228 combination group (64 vs.
132 mm3and 102 vs. 240 mm3, respectively). In contrast, we failed
to detect any tumor inhibition elicited by 5-Aza alone (Fig. 6a). In
this case, it appeared that the Aza/FK228 combination did not re-
sult in further tumor inhibition compared to FK228 alone, imply-
ing that the effect noted in the combination group is likely due to
FK228 activity. Tumor size was still relatively small (90 mm3) in
the FK228 group at Day 28 (Fig. 6a). Mice treated with control or
combination therapy were sacrificed when tumors began to ulcer-
ate or when more than 10% body weight loss was noted, respec-
tively, in compliance with institutional regulations, limiting long-
term comparison of treatment groups with control.
To evaluate the therapeutic effect of FK228 on bladder TCC,
we employed an orthotopic model using the bladder instillation
method. BLI data (Fig. 6b and 6c) clearly indicated that lumines-
cence intensity was significantly lower in FK228-treated mice on
days 14 and 21 after treatment consistent with the subcutaneous
data. Noticeably, BLI also revealed the appearance of lung metas-
tasis in several animals (Fig. 6c). In most cases, FK228 alone did
not cause any significant loss of body weight in all tested animals.
Taken together, these data indicate that FK228 is a potent chemo-
therapeutic agent for TCC.
It is becoming increasingly clear that gene transcription from the
tightly packed DNA is regulated by chromatin-remodeling events,
which can render DNA either more or less accessible to transcrip-
tion factors. One of the hallmarks of the regulation for gene tran-
scription is local chromatin decondensation mediated by histone
acetylation, which leads to a reduced association between chromo-
somal DNA and histones, and subsequently increases the accession
FIGURE 5 – Analysis of apoptosis-
or cell cycle-related proteins in T24
cells under different treatment condi-
tions. T24 cells were plated at a con-
centration of 1 3 105cells in 100
mm plates and the agents were
administered 24 hr after cell plating.
Total cell lysate was prepared from
either suspension cell (a) or attached
cell (b) and subjected to western blot
analysis probed with various antibod-
ies. A431 cell lysate (Upstate, Lake
Placid, NY) was used as p53 positive
control. Actin was used as an internal
control for equal protein loading.
BLADDER CANCER THERAPY WITH EPIGENETIC MODIFIERS
of high molecular weight protein complexes of the transcription ma-
chinery. Conversely, histone deacetylation can repress transcription
by increasing histone-DNA interaction. The effect of histone acetyl-
transferases (HATs) is counterbalanced by the presence of HDACs.25
Aberrant acetylation or deacetylation leads to such diverse disor-
ders as leukemia,26epithelial cancers27and Rubinstein–Taybi syn-
drome.28,29From recent reports, HDAC-containing complexes are
involved in DNA methylation-mediated transcriptional silencing of
various tumor-suppressor genes.30In addition to gene mutations
associated with cancer cells, it is becoming more apparent that
HDAC activity is upregulated in cancer cells.31Therefore, target-
ing HDAC activity has become a new strategy for cancer chemo-
therapy; several inhibitors such as FK22832and SAHA33have been
developed and tested in phase I clinical trials.
FIGURE 6 – Therapeutic efficacy of FK228 or 5-Aza on in vivo T24 tumor model. (a) Athymic nude mice were injected subcutaneously with a
100 lL cell suspension containing 1 3 106T24-t. When the tumors became palpable, animals were randomized for different treatments and
each group contained 8 tumors. Single agent or combination of FK228 (1.0 mg/kg) and 5-Aza (5.0 mg/kg) were administrated by tail vein injec-
tion twice a week for 3 weeks. Tumor volume (mm3) was measured weekly. * denotes statistical significance (p < 0.05) when each treatment
group was compared to control each week. FK, FK228. (b,c) Orthotopic model was generated by instilling T24-t-luc into the bladder. Once the
tumor mass were detectable by BLI, FK228 (1.0 mg/kg) was administrated by intravenous tail injection twice a week for 3 weeks. Animals were
imaged weekly and luminescence intensity was determined by integrating the number of photons over a 2-min imaging time. The BLI intensity
depicted in panel (b) was calculated from the low abdominal area of each animal.
KARAM ET AL.
In addition, DNA hypermethylation has been implicated in pa-
rental gene imprinting, X chromosome inactivation and endoge-
nous retrovirus silencing, as well as in the transcriptional silencing
of tumor suppressor genes.10Although, hypermethylation of CpG
islands is also found in the 30end of some genes, the density of
DNA methylation in promoter or first exon regions correlates
inversely with the level of gene transcription.34DNA hypermeth-
ylation, particularly in the GC-rich promoter region, results in
transcription repression that is often associated with a number of
tumor suppressor gene promoters, including Rb,35p1536and
p16.37DNA hypomethylating agents such as 5-Aza can inhibit
DNA methyltransferase activity and result in gene reactivation.
Interestingly, it has also been shown that transcription repression
mediated by methyl-CpG-binding proteins involves HDAC com-
plex,38indicating that there is a close relationship between DNA
methylation and histone deacetylation.
Our data demonstrate that 5-Aza and TSA can inhibit cell
growth of TCC cells in a time and dose-dependent manner (Figs.
1a, 1c, 1d and 1f), mainly by causing G2/M cell cycle arrest (Figs.
4b and 4d). On the other hand, FK228 inhibits TCC cell growth by
inducing both G2/M cell cycle arrest and apoptosis evidenced by
PARP cleavage (Fig. 5a) and flow cytometry (Fig. 4c) experi-
ments. Also, the degree of apoptosis elicited by FK228 can be fur-
ther potentiated by combining with 5-Aza, suggesting that both
agents may have different mechanisms of action in TCC.
To unveil the mechanism of action of these agents, we have
analyzed the steady-state levels of various cell cycle regulators
in T24 cells (Fig. 5b). It appears that cyclin B1levels were not
changed. Instead, cyclin E levels were induced by these agents,
suggesting that these agents may push cells into S phase and then
cause arrest in G2/M phase by other factors.39Noticeably, a sig-
nificant induction of p21 in T24 cells is associated with HDACIs,
which is independent of p53-mediated pathway, since p53 was
not detected in this cell line. This result is consistent with
published literature showing that p53 may not be important in
HDACI-induced apoptosis.40,41Although the role of elevated
p21 in FK228-elicited cell cycle arrest and apoptosis in TCC is
still unknown and p21 has multiple activities in cell cycle such
as G2 arrest42as well as G1 arrest43–45and apoptosis,46we
believe that p21 may be critical for cell apoptosis based on our
data (Fig. 4).
5-Aza alone failed to inhibit tumor growth and also the combi-
nation only achieved the same tumor inhibition as FK228 alone,
indicating that 5-Aza, in our model, is not an effective therapeutic
agent in the same tumor cell growing as xenografts in vivo. This
contrasts to cell culture data where growth inhibition was
observed (Fig. 1a and 1d, 4b). This may be due to its short half-
life in vivo.47Considering its in vitro effect, further investigation
using its oral analog, zebularine,48is warranted.
Data from the subcutaneous tumor model (Fig. 6a) clearly indi-
cate that FK228 results in time-dependent tumor suppression, with
minimal side effects (data not shown). Mice treated with FK228
tended to survive longer, partly due to lack of morbidity from tumor
ulceration and tumor burden. Furthermore, the BLI technique can
monitor tumor growth from the orthotopic site in a real-time and
continuous manner (Fig. 6b and 6c). Ultimately, orthotopic models
are thought to be preferable and BLI allowed noninvasive detection
of a deep seated developing tumor. Importantly, BLI also revealed
additional metastases, which would not normally have been
detected. In summary, these data provide a strong rationale that
FK228 can be applied in bladder cancer chemotherapy.
We thank Ms. Angelina Contero for performing BLI studies as
a service of the Simmons Cancer Center In Vivo Cellular and Mo-
lecular Imaging Resource and Dr. Jerry Shay for providing lentivi-
rus expressing luciferase.
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