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CHEM. RES.

CHINESE UNIVERSITIES 2012, 28(6), 10171021



*Corresponding author. E-mail: congxl888@hotmail.com
Received September 21, 2012; accepted October 12, 2012.
Supported by the National Natural Science Foundation of China(Nos.81001298, 81241093), the Project of China Postdoc-
toral Science Foundation(No.20100481058), the High-tech Industrial Development Project of Jilin Province, China
(No.20090633), the Specialized Research Fund for the Doctoral Program of Higher Education of China(No.20100061120028) and
the Scientific Research Foundation of Jilin Province, China(Nos.20080739, 200905169).

Procaine Inhibiting Human Bladder Cancer Cell Proliferation by
Inducing Apoptosis and Demethylating APAF1
CpG Island Hypermethylated
SUN Ran
1
, CHANG Li-ping
2
, WANG Kai-chen
3
,
SUN Hong-yan
1
and CONG Xian-ling
1,4*

1. Tissue Bank, China-Japan Union Hospital, Jilin University, Changchun 130033, P. R. China;
2. Department of Cardiopathy, the Affiliated Hospital of Changchun University of Chinese Medicine,
Changchun 130021, P. R. China; 3. Department of Urinary Surgery,
4. Department of Dermatology, China-Japan Union Hospital, Jilin University,
Changchun 130033, P. R. China


Abstract Studies have shown that aberrant DNA methylation of apoptotic protease activating factor-1(APAF1) is an
important epigenetic mechanism of gene regulation in the progression of bladder cancer. In this article, we have
proved that procaine, an inhibitor of DNA methyltransferases, could inhibit the proliferation of T24 and 5637 human
bladder cancer cells by inducing their apoptosis. The mechanism studies reveal that procaine could induce demethy-
lation of APAF1 gene in T24 or 5637 cells, subsequently activating caspase-3/9. It was also shown that the serum so-
luble fas ligand(sFasL) was activated, and the expression of matrix metallopeptidase 9(MMP-9) was down-regulated.
Procaine seems to induce cell death by different pathways, and it might be used as a potential agent for bladder cancer
treatment.
Keywords Bladder cancer cell; Epigenetics; DNA methylation; Apoptotic protease activating factor-1(APAF1);
Procaine
Article ID 1005-9040(2012)-06-1017-05

1 Introduction
Bladder cancer is the second leading cause of cancer death
in the patients with urinary tract malignancies in the world and
is increasingly being diagnosed in China
[1]
. Cancer is characte-
rized by global hypomethylation, with the hypermethylation of
a subset of gene promoters contained within CpG islands
leading to gene silencing. Hypermethylation of those genes
involving cell-cycle control, cell invasion and cell architecture,
DNA damage repair and apoptosis is an important epigenetic
mechanism of gene regulation and plays essential roles in
bladder cancer initiation and progression
[27]
.
Apoptotic pathways are targets for epigenetic silencing
and several apoptosis-related genes, such as B-cell CLL/
lymphoma 2(BCL2), death-associated protein kinase(DAPK),
apoptotic peptidase activating factor 1(APAF1), telomerase
reverse transcriptase(TERT), are directly or indirectly regulated
by methylation
[811]
. APAF1 plays essential roles in the intrin-
sic pathway of apoptosis and is important for cellular responses
to DNA damage
[12]
. When DNA is damaged by chemothera-
peutic agents, oncogenic stimuli, UV and ionizing radiation,
cytochrome C will be released from the mitochondria. It binds
to APAF1 in the cytosol and facilitates a conformational change
of APAF1 to expose its CARD domain. Subsequently, APAF1
oligomerizes through the exposed CARD domain and catalyzes
autoactivation of caspase-9, leading to the serial activation of
downstream effector caspases such as caspase-3, caspase-6, and
caspase-7, which results in apoptotic cell death
[1315]
.
We will report herein that Procaine hydrochloride, a DNA
methyltransferase inhibitor approved by the United States Food
and Drug Administration for the treatment of cardiac arrhyth-
mias, could inhibit the proliferation of bladder cancer cells via
demethylating the profile of promoter-CpG Island, the common
epigenetic change in human bladder cancer
[16]
.
2 Materials and Methods
2.1 Cell Culture
T24 and 5637 cells(Human bladder cancer cell lines) were
obtained from the American Type Culture Collection(ATCC),
and grown in RPMI 1640-medium supplemented with 10%
(volume fraction) fetal bovine serum(Hyclone) and 2 mmol/L
1018 CHEM. RES. CHINESE UNIVERSITIES Vol.28

glutamine, and antibiotics(100 U/mL penicillin and 100 g/mL
streptomycin) at 37 C in a humidified 5% CO
2
atmosphere.
2.2 Cell Proliferation Assay
T24 or 5637 cells(110
4
cells per well) were plated in a
24-well plate, and the cells were treated with procaine(04
mmol/L) for 48 h at 37 C. The cell proliferation rate was
determined by the assay with Cell Counting Kit-8(Keygen
Biotech) and expressed as mean A
570
value[(absorbance value)
standard deviations(SD)] of triplicate wells.
2.3 Apoptosis Detection
The cells were treated with procaine(4 mmol/L) for 48 h
and stained with 5 mg/L Hoechst 33258(Sigma) for 30 min at
37 C, then visualized under a fluorescence microscope with
standard excitation filters. Apoptotic cells were defined as cells
showing nuclear and cytoplasmic shrinkage, chromatin con-
densation and the presence of apoptotic bodies. At least 200
cells were counted and the percentage of apoptotic cells(apop-
totic index) was calculated at 600 magnification.
2.4 DNA Bisulfite Conversion
After 2000 ng of genomic DNA was extracted from
bladder cancer cells via Wizard Genomic DNA Purification
Kit(Promega), bisulfite conversion reactions were conducted
with the help of EZ DNA Methylation-Gold Kit(ZYMO)
according to manufacturers instructions.
2.5 RNA Extraction and Real-time Polymerase
Chain Reaction(RT-PCR) Analysis
Total RNA from bladder cells was isolated with the SV
Total RNA Isolation Kit(Promega) according to the manufac-
turers protocol. RNA was reversely transcribed via the Prime-
Script RT Master Reverse Transcription Kit(Takara); and RT-
PCR was then performed with Epidermal growth factor recep-
tor(EGFR), sFasL or MMP-9 specific primers via the SYBR
Premix Ex Taq GC(Takara). The sequences of the primers were
as follows: EGFR 5-GGACGACGTGGTGGATGCCG-3
(Forward), EGFR 5-GGCGCCTGTGGGGTCTGAGC-3
(Reverse); MMP-9 5-TTCGACGGGAAGGACGGGCT-3
(Forward), MMP-9 5-TGGAACCACGACGCCCTTGC-3
(Reverse); sFasL 5-ACAGGG TCCCGT CCTTGACACC-3
(Forward); sFasL 5-CAGGCCAGCCCCAGCAAACG-3
(Reverse). GAPDH was used as a housekeeping gene for nor-
malization. The expression of each mRNA was defined from
the threshold cycle(Ct), and relative expression levels were
calculated by the 2
Ct
method after normalization.
2.6 Real-time Methylation-specific PCR(MSP)
Real-time MSP analysis of bisulphite-treated DNA was
performed in triplicate. The levels of unmethylated and methy-
lated species were determined by a standard curve constructed
by the serial dilutions of SssI-methylated healthy donor DNA.
GAPDH was used as a control for normalization. The primers
of GAPDH were the same as those described in RT-PCR. The
data on CpG methylation in APAF1 gene were used to design
methylation-specific PCR primers for the APAF1 promoter
region. The primers to detect the methylated form were 5-
GTTTAGTAGTGGGGTTTATTGG-3(Forward) and 5-TTC-
GGGTAAAAGGGATAGAATTAGA-3(Reverse), and the pri-
mers to detect the unmethylated form were 5-AAAAACCTC-
CAAAAATTAACAAA-3(Forward) and 5-TATAACGCCC-
TTCCCCCGACGACG-3(Reverse). The methylated primer set
generated 169-base-pair(bp) product(M) and unmethylated
primer set generated 119 bp product(U). Methylation-specific
PCR cycling conditions consisted of one incubation of 15 min
at 95 C, followed by 40 cycles of a denaturation at 95 C for
30 s, an annealing at 61 C for 30 s, an extension at 72 C for
30 s, and a final extension at 72 C for 5 min. The methylation
index(MI) was calculated as previously described
[17]
.
2.7 Caspase-3 and Caspase-9 Assays
Caspase-3 and caspase-9 assays were performed according
to the manufacturers instructions(Keygen Biotech).
2.8 Statistical Analysis
Statistical analyses were performed with the aid of SPSS
Software, P values less than 0.05 were taken as significant.
3 Results
3.1 Inhibitory Effects of Procaine on 5637 and
T24 Cells
To investigate the potential inhibitory effects of procaine
on 5637 and T24 cells, we tested the inhibitory effect of
procaine on human bladder cells. Procaine hydrochloride was
diluted in phosphate buffer solution(PBS). After the treatment
of them with procaine for 48 h, microscopic observation of the
cells showed a significant reduction in cell numbers, as com-
pared to that of the PBS control(Fig.1). The result shows that
procaine was capable of inhibiting the proliferation of 5637 or
T24 cells significantly in a dose-dependent manner. IC
50
value
was 3.39 mmol/L for 5637 cells, and 3.20 mmol/L for T24
cells(Fig.2).












Fig.1 Photographs(200) of 5637(A, C) and T24(B, D)
cells incubated with PBS(A, B) or procaine(C, D) for
48 h
No.6 SUN Ran et al. 1019







Fig.2 Dose-effect curves of procaine on T24(A) and
5637(B) bladder cancer cell lines
Each bar represents the meanSD.
3.2 Procaine Demethylated DNA of APAF1 in
T24 and 5637 Bladder Cancer Cells
To determine whether procaine could demethylate the
DNA of APAF1, we incubated 5637 and T24 cells with PBS or
procaine. After incubation for 48 h, 5637 and T24 cells were
harvested for APAF1 methylation, real-time MSP assay was
performed. The methylation index was calculated, results are
representative of three independent experiments.
The results in Fig.3 show that the methylation index of
APAF1 gene in the bladder cells treated with procaine was
obviously lower than that in the bladder cells treated with
PBS(P<0.05). Presumably, we speculated that procaine might
inhibit DNA methylation process.








Fig.3 Comparison of methylation indexes of APAF1
gene after cell line treated with procaine and
PBS
(A) T24 cells; (B) 5637 cells. Columns are expressed as meanSD of
three independent experiments.
3.3 Procaine Induced Apoptosis via Caspase
Pathway, Up-regulation of sFasL, and Inhibition of
MMP-9 Expression in T24 and 5637 Cell Lines
The morphological changes of the apoptosis cells were
assayed under a fluorescence microscope, followed by staining
with Hoechst 33258[Fig.4(AD)]. When viewed with a
phase-contrast microscope, 5637 and T24 cells treated with
PBS remained flat with a uniform polygonal shape and exhi-
bited typical growth patterns. Compared with cells treated with
PBS, 5637 or T24 cells treated with procaine for 24 h exhibited
much less cyto- plasmic organelles. Most of the cells treated
with procaine for at least 48 h resulted in morphological
changes distinguished by their characteristic patterns of apop-
tosis, including nuclear condensation, cytoplasmic rounding.
The apoptotic cell number increased 25 fold after treatment
of the cells with procaine[Fig.5(A)].











Fig.4 Fluorescence microscopy images(600) of T24 and
5637 cells followed by staining with Hoechst 33258
(A) 5637 cells treated with PBS showing intact nuclear morphology; (B)
5637 cells treated with procaine for 48 h showing typical punctuated nuclear
morphology of apoptotic cells; (C) T24 cells treated with PBS showing
intact nuclear morphology; (D) T24 cells treated with procaine for 48 h
showing typical punctuated nuclear morphology of apoptotic cells.
















Fig.5 Percentages of apoptosis T24 and 5637 cells
treated with procaine(A) and expressions of
caspase-3(B, C) and caspase-9(D, E) protein
in T24(B, D) and 5637(C, E) cells treated with
procaine for 48 h
Results are the representative of three independent experiments. Columns
are expressed as meanSD of three independent experiments.
Caspases have been shown to be activated during apopto-
sis of many cells and play critical roles in both the initiation
and progression of apoptosis. In order to clarify the mechanism
of procaine induced apoptosis of the cells, we examined the
expression of caspase-3/9 in procaine-induced T24 and 5637
bladder cancer cells. The results in Fig.5(BE) show that pro-
caine could induce activation of caspase-3 and caspase-9 in
T24 cells, but could not in 5637 cells.
To determine whether procaine could inhibit the prolife-
ration via other pathways, T24 or 5637 cells were treated with
or without procaine. After 48 h, the cells were harvested
for EGFR, MMP-9, sFasL mRNA expression by RT-PCR. The
1020 CHEM. RES. CHINESE UNIVERSITIES Vol.28

results in Fig.6 show that procaine could up-regulate the
expression of sFasL, and inhibit the expression of MMP-9 in
T24 and 5637 cells.

















Fig.6 Expressions of EGFR(A,D), MMP-9(B,E), sFasL(C,F) in T24(AC) and 5637(DF) cells
treated with or without procaine for 48 h
Columns are expressed as meanSD of three independent experiments.
4 Discussion
DNA hypermethylation is the most common and best cha-
racterized epigenetic abnormality in human malignancies
[1823]
.
In this study, we focused on APAF1, an apoptosis-related gene.
APAF1 gene encodes a cytoplasmic protein that activates cas-
pase-9 to stimulate the subsequent caspase cascade that com-
mits the cells to apoptosis, and activates caspase-3 to regulate
tumor cells repopulation
[24]
. Our results show that APAF1 gene
was hypermethylated in the bladder cancer tissue at statistically
significantly higher frequency, while it was methylated in the
noncancerous controls(unpublished results). Analysis by MSP,
100 of 112 patients with bladder cancer exhibited APAF1
hypermethylation. This indicates that APAF1, served as an
apoptosis-related gene, might play an important role in the
pathogenesis of bladder cancer.
Somatic CpG island DNA methylation changes have been
reported to occur in the early stage of pathogenesis in many
human cancers, suggesting that DNA methyltransferase inhibi-
tors might be considered as a drug candidate for cancer chemo-
prevention and treatment
[2527]
. Procainamide, a widely used
anti-arrythmic, causes DNA hypomethylation in the human cell
line
[2832]
. The anti-tumor mechanism of procaine can be di-
vided into two parts: one is procaine closely integrated through
the densely populated areas of CpG island hypermethylation,
and induces demethylation of the tumor suppressor genes, the
other is that procaine also inhibits the growth of tumor cells by
preventing mitosis
[33]
. Currently, procaine has entered Phase II
clinical trials as a new antitumor reagent
[34]
. However, there has
not been a clear report whether procaine could induce the de-
methylation pattern of tumor suppressor gene promoter in uri-
nary tract tumors. In this study, we found that procaine dis-
played dramatic effect on inhibiting the proliferation of human
bladder cells by demethylating APAF1 CpG Island hyperme-
thylated followed by triggering caspase-induced apoptosis
pathways.
As mentioned above, similar phenomenon was also ob-
served in our study, that is, procaine could up-regulate sFasL,
and inhibit the expression of MMP-9 in T24 and 5637 cells.
Based on these results, procaine seemed to induce cell death by
different pathways.
The initiation and progression of cancer is controlled by
both genetic and epigenetic events. Unlike genetic alterations,
which are almost impossible to reverse, epigenetic aberrations
are potentially reversible, allowing the malignant cell popula-
tion to revert to a more normal state. Taken together, our data
demonstrate that procaine may inhibit bladder cancer cell
proliferation by inducing APAF1 demethylation. Our results
suggest that procaine could be used for the control of bladder
cancer. However, further studies are needed to validate our
findings in appropriate animal models. The exact mechanism of
procaine induced cell death needs more detailed and in-depth
fundamental studies.

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