Post on 07-Feb-2023
Demethylating agent 5-aza-2-deoxycytidine enhances
susceptibility of breast cancer cells to anticancer agents
Sameer Mirza • Gayatri Sharma • Pranav Pandya •
Ranju Ralhan
Received: 7 October 2009 / Accepted: 17 April 2010
Ó Springer Science+Business Media, LLC. 2010
Abstract DNA methylation plays an important role in
regulation of gene expression and is increasingly being
recognized as a determinant of chemosensitivity of human
cancers. With the aim of improving the chemotherapeutic
efficacy of breast carcinoma, the effect of DNA methyl-
transferase inhibitor, 5-Aza-20-deoxycytidine (5-aza-CdR),
on the chemosensitivity of anticancer drugs was investi-
gated. The cytotoxicity of paclitaxel (PTX), adriamycin
(ADR), and 5-fluorouracil (5-FU) was analyzed against
human breast cancer cell lines, MDA MB 231 and MCF 7
cell lines using the MTT assay, and the synergy of 5-aza-
CdR and these agents was determined by Drewinko’s
fraction method. The effects of each single agent or the
combined treatment on cell cycle arrest were analyzed by
flow cytometric analysis. We also investigated the effect of
each single agent or the combined treatment of anticancer
drugs with 5-aza-CdR on the methylation status of the
selected genes by methylation specific PCR. In MDA MB
231 cells, a synergistic antiproliferative effect was
observed with a combination of 10 lM 5-aza-CdR and
these three anticancer drugs, while in MCF 7 cells, a
semiadditive effect was observed. Treatment with 5-aza-
CdR and anticancer drug resulted in partial demethylation
of a panel of genes including RARb2, Slit2, GSTP1, and
MGMT. Based on these findings, we propose that 5-aza-
CdR enhances the chemosensitivity of anticancer drugs in
breast cancer cells and may be a promising approach for
increasing the chemotherapeutic potential of these anti-
cancer agents for more effective management of breast
carcinomas.
Keywords Demethylating agent �
5-Aza-20-deoxycytidine � Breast carcinoma �
Anticancer agents � Chemosensitivity
Introduction
Aberrant DNA methylation plays an important role in car-
cinogenesis and tumor apoptosis. In particular, tumor sup-
pressors are often rendered non-functional and, often this
loss of function is associated with epigenetic modifications,
primarily DNA methylation. DNA hypermethylation is
carried out by DNA methyltransferases, which catalyze the
covalent addition of a methyl group from a donor S-adeno-
sylmethionine to the 5th position of cytosine, predominantly
within the CpG dinucleotide [1]. 5-Aza-20-deoxycytidine
(5-aza-CdR), a DNA methyltransferase inhibitor, has been
used to reverse methylation and reactivate the expression of
silenced genes. 5-aza-CdR suppressed the growth of several
tumors in vitro, and showed clinical utility against hema-
topoietic malignancies, metastatic lung carcinoma, and
Sameer Mirza and Gayatri Sharma have contributed equally to this
manuscript.
S. Mirza � G. Sharma � R. Ralhan
Department of Biochemistry, All India Institute of Medical
Sciences, Ansari Nagar, New Delhi 110029, India
G. Sharma � P. Pandya
Dev Sanskriti Vishwa Vidhyalaya, Haridwar, India
R. Ralhan
Department of Otolaryngology-Head and Neck Surgery, Mount
Sinai Hospital, University of Toronto, Toronto, ON, Canada
R. Ralhan (&)
Joseph and Mildred Sonshine Family Centre for Head & Neck
Diseases, Mount Sinai Hospital, Joseph & Wolf Lebovic Health
Complex, 600 University Avenue, Room 6-500,
Toronto, ON M5G 1X5, Canada
e-mail: ralhanr@gmail.com
123
Mol Cell Biochem
DOI 10.1007/s11010-010-0473-y
gastric cancer [2–4]. Moreover, 5-aza-CdR also increased
the effect of chemotherapeutic agents and sensitized tumor
cells to chemotherapy regimens [4, 5].
Breast cancer represents a major health problem; with
over 1 million new cases per annum, and is the second
leading cause of death from cancer in women [6]. In spite
of increasing incidence, breast cancer mortality has
declined in majority of developed countries, partly attrib-
utable to benefits of adjuvant and neoadjuvant chemo-
therapy [7]. DNA methylation has been shown to affect
silencing of chemosensitivity-related genes; hence inhibi-
tors of methylation may enhance the sensitivity of cancer
cells to anti-cancer drugs [8]. Indeed, these epigenetic
inhibitors have their broadest activity in chemosensitizing
refractory cancers in combination with conventional anti-
cancer drugs.
With the aim of improving the chemotherapeutic effi-
cacy of breast carcinoma, we investigated a novel combi-
nation strategy with DNMT inhibitor (5-aza-CdR) and
anticancer agents—PTX, ADR, and 5-FU—widely used in
breast cancer therapy. In the present study, we determined
the antiproliferative effects of 5-aza-CdR in combination
with these three chemotherapeutic agents in breast cancer
cells. The increase in susceptibility of breast cancer cells to
PTX, 5-FU, and ADR by addition of 5-aza-CdR suggested
that combination chemotherapy with these agents might be
clinically useful for breast cancer management.
Materials and methods
Cell lines and chemotherapeutic agents
Breast cancer cell lines: MCF 7 (ERa-positive) and MDA
MB 231 (ERa-negative) were used in this study as models
for ER-positive and ER-negative breast cancers. MDA MB
231 cells were grown in L-15 medium supplemented with
10% FBS and 10 lg/ml ciprofloxacin. MCF 7 cells were
grown in DMEM supplemented with 10% FBS and 10 lg/ml
ciprofloxacin. Both cell lines were cultured as monolayers in
complete medium and incubated in a humidified atmosphere
of 5% CO2 at 37°C. 5-aza-CdR, PTX, 5-FU, and ADR were
obtained from Sigma Chemical Co. (Bangalore, India).
Measurement of cell growth in vitro
MTT assay
MCF 7 and MDA MB 231 cells were harvested by tryp-
sinization, resuspended in DMEM supplemented with 10%
FBS and plated (2 9 103 cells/100 ll) in 96-well tissue
culture plates (Corning BV, Schipol-Rijk, The Netherlands).
The cells were treated with 5-aza-CdR (10 lMinMCF 7 and
12 lM in MDA MB 231) for 72 h. Thereafter, cells were
treated with/without different concentrations of anticancer
drugs (PTX, ADR, and 5-FU) using appropriate controls.
Cell viability was measured by MTT [3-(4, 5-dimethylthi-
azol-2-yl)-2, 5-diphenyltetrazolium bromide] assay. Briefly,
after 24 h of treatment, 10 ll of 1 mg/mlMTT solutionwere
added to each well, and the plate was incubated for 4 h,
allowing viable cells to reduce the yellow MTT to dark-blue
formazan crystals, which were dissolved in 100 ll of
DMSO. The absorbance was measured at 595 nm using a
microplate reader (Micro Scan, MS5608A, ECIL, Hydera-
bad, India). The IC50was determined as a drug concentration
showing 50% cell growth inhibition as compared to the
untreated control cells. The potential synergy between the
drugs and 5-aza-CdR was evaluated using Drewinko’s
fraction method [9]. The synergistic, semiadditive, and
antagonistic interactions were determined when the calcu-
lated value was less than the expected value, more than the
expected value but less than the drugs’ value, and more than
the drugs’ value, respectively. The expected value was cal-
culated by the combined effects (%) = the effects of the
anticancer drug/control * the effects of the 5-aza-CdR/
control * 100.
Flow cytometry
In order to determine the effect of 5-aza-CdR, PTX, ADR,
and 5-FU on the cell cycle, MCF 7 and MDA MB 231
(2 9 106 cells) were treated with 5-aza-CdR, PTX, ADR,
and 5-FU at their respective IC50 doses and fixed with 70%
ethanol. After overnight incubation at -20°C, cells were
washed with PBS prior to staining with propidium iodide
(PI) (10 mg/ml PI; 0.5% Tween-20; 0.1% RNase in 0.01 M
phosphate buffered saline pH 7.2 (PBS)). The cells were
analyzed using BD FACS CantoTM (BD, Singapore) and
data were analyzed using the BD FACSDiva software (BD
Biosciences, CA).
Methylation specific PCR (MSP) and RT-PCR
MDA MB 231 and MCF 7 (1.5 9 106 cells) were plated in
10 cm culture dishes and incubated overnight prior to
treatment with 5-aza-CdR, either alone or in combination
with PTX, ADR, and 5-FU. The cells were harvested and
divided into two aliquots, one of which was extracted with
TRI Reagent (Sigma, Banglore, India) to isolate RNA for
RT-PCR and the other was used for DNA isolation using a
DNeasy Tissue kit according to the manufacturer’s
instruction (DNeasy Tissue Kit, Qiagen, Hilden, Germany).
DNA samples were treated with sodium bisulfite and
subsequently DNA was extracted using a Wizard DNA
cleanup kit followed by ethanol precipitation and
Mol Cell Biochem
123
resuspension in double distilled water. MSP analyses were
performed for RARb2, Slit2, GSTP1, and MGMT using
primers specific for methylated and unmethylated DNA
products as described in our previous reports [10–14]. The
MSP products were resolved on 2% agarose gels and
visualized by ethidium bromide staining.
Cell lysis and western blot
Cells were lysed on ice using radioimmunoprecipitation
buffer (0.05 mol/l Tris–HCl, pH 7.4, 0.15 mol/l NaCl,
0.25% deoxycholic acid, 1% NP-40, 1 mmol/l ethylene-
diaminetetraacetic acid, 0.5 mmol/l dithiothreitol, 1 mmol/l
phenylmethylsulfonyl fluoride, 5 mg/ml leupeptin, and
10 mg/ml aprotinin). Lysates were then centrifuged at
13,000 rpm at 4°C for 10 min. Protein extracts were solu-
bilized in sodium dodecyl sulfate (SDS) gel loading buffer
(60 mmol/l Tris base, 2% SDS, 10% glycerol, and 5% b
mercaptoethanol). Samples containing equal amounts of
protein (50 lg) were separated on an 8% SDS-polyacryl-
amide gel electrophoresis and electroblotted onto Immobi-
lon-P membranes (Millipore, Bedford, Massachusetts,
USA) in a transfer buffer. Immunoblotting was performed
using antibodies against p53 (1: 1000, Santa Cruz, sc-263),
p21 (1: 500, Santa Cruz, sc-71811), Bcl2 (1: 500 Santa
Cruz, sc-7382), BAX (1: 200 Santa Cruz, sc-70406), cas-
pase 9 (1: 200 Santa Cruz, sc-56076), and anti-b-actin
antibodies (1: 500 Santa Cruz, sc-69879); as an internal
control. The signal was developed with enhanced chemi-
luminescence (Pierce) after incubation with appropriate
secondary antibodies.
Results
5-Aza-CdR increased the efficiency of anticancer drugs
5-Aza-CdR marginally suppressed cell proliferation in both
MDA MB 231 (12 lM) and MCF 7 (10 lM) cells (Fig. 1);
the cell viability in 5-aza-CdR treated cells was 83 and
75% in MDA MB 231 and MCF 7 cells, respectively. Anti-
cancer drugs were added to these cells at their IC50 for each
cell line (Table 1). The proliferation rates in response to
combined exposure were evaluated by comparison with the
expected additive effect. The proliferation rates for MDA
MB 231 cells treated with PTX, ADR, and 5-FU alone
were reduced to 59, 55, and 55%, respectively. The pro-
liferation rates for MDA MB 231 cells treated with a
combination of 5-aza-CdR and PTX or ADR or 5-FU were
43, 40, and 42%, respectively. The proliferation effects on
MDA MB 231 cells treated with the combination of 5-aza-
CdR and anticancer drugs were lower than the expected
additive effects, suggesting that the combinations of
5-aza-CdR with PTX, ADR, or 5-FU showed synergistic
effects (Fig. 1). The proliferation rates for MCF 7 cells in
response to PTX, ADR, or 5-FU were 55, 54, and 51%,
respectively, while 5-aza-CdR in combination with PTX,
ADR, or 5-FU, these rates were reduced to 47, 44, and
40%, respectively. In MCF 7 cells, the proliferation effect
of combination of 5-aza-CdR and drugs was more than the
expected additive effect, but lesser than each drugs’ value
alone, suggesting that the combination of 5-aza-CdR with
PTX, ADR, or 5-FU showed a semi-additive effect. The
semi-additive effect of combination of 5-aza-CdR with
5-FU only reached statistical significance (Fig. 1).
Effect of 5-aza-CdR, PTX, ADR, and 5-FU on cell
cycle in breast cancer cell lines
The DNA content was analyzed by flow cytometry. The
percentages of cell populations in each phase of cell cycle
are indicated in the histograms (Fig. 2). In MDA MB 231
cells, 5-aza-CdR treatment showed an increase in sub-G0
(12%) as compared with the untreated control (7%).
Anticancer drugs, PTX, ADR, and 5-FU alone also showed
an increase in sub-G0 (25, 25, and 31%, respectively)
compared with the untreated control (7%). In addition,
increase in sub-G0 induced by the combined exposure to
5-aza-CdR and anticancer drugs, PTX, ADR, and 5-FU
were 35, 40, and 38%, respectively. In MCF-7 cells, 5-aza-
CdR treatment did not show increase in sub-G0 (6%)
compared with the control (4%). The treatment of PTX,
ADR, and 5-FU alone showed an increase in sub-G0 of 10,
18, and 25%, respectively, and the increase induced by
combined exposure of 5-aza-CdR with PTX, ADR, and
5-FU were 19, 23, and 35%, respectively.
In MDA MB 231 cells, 5-aza-CdR treatment showed an
increase in G2/M phase (25%) as compared with 16% in
the control untreated cells. Anticancer drugs, PTX and
5-FU alone also showed an increase in G2/M phase—25
and 28%, respectively, whereas ADR alone showed
decrease in G2/M phase. In addition, increase in G2/M
phase induced by combined exposure of 5-aza-CdR with
anticancer drugs, PTX, ADR and 5-FU was 35, 29, and
26% respectively. In MCF 7 cells, 5-aza-CdR treatment
showed increase in G2/M phase (25%) compared with 16%
in the control. Treatment with PTX, ADR, and 5-FU alone
showed an increase in G2/M phase—28, 23, and 18%,
respectively, and the increase induced by combined expo-
sure of 5-aza-CdR with PTX, ADR, and 5-FU was 36, 30,
and 20%, respectively. This clearly suggests that 5-aza-
CdR inhibits proliferation by inducing G2/M arrest and
slight increase in sub-G0 phase. The combined exposure of
5-aza-CdR with PTX, ADR, and 5-FU showed an increase
in sub-G0 and G2/M arrest of the cells.
Mol Cell Biochem
123
Effect of 5-aza-CdR, PTX, ADR, and 5-FU
on hypermethylated genes
Methylation specific PCR of the panel of genes—RARb2,
Slit2, GSTP1, and MGMT—was carried out after treatment
of MCF 7 and MDA MB 231 with 5-aza-CdR at the above-
mentioned dose and time. Partial demethylation of our
panel of genes was observed at 12 lM (72 h) in MDA MB
231 and at 10 lM (72 h) in MCF 7. Promoter methylation
of genes RARb2, Slit2, GSTP1, and MGMT was partially
reversed in all the cell lines, when treated with 5-aza-CdR,
but there was no change in the methylation status of these
genes when treated with PTX, ADR, and 5-FU alone.
Promoter methylation status of a panel of genes—RARb2,
Slit2, GSTP1, and MGMT—was also determined in cells
treated with 5-aza-CdR for 72 h followed by 24 h exposure
with PTX, ADR, and 5-FU. There was partial demethyla-
tion of all panels of genes when PTX, ADR, and 5-FU were
combined with 5-aza-CdR. Figure 3a–d shows the effect of
PTX, ADR, and 5-FU combination with 5-aza-CdR, on
methylation status of a panel of genes RARb2, Slit2,
GSTP1, and MGMT.
Effect of 5-aza-CdR, PTX, ADR, and 5-FU on p21WAF1
transcript level
The effect of treatment of 5-aza-CdR, PTX, ADR, and 5-
FU alone or combined treatment of PTX, ADR, and 5-FU
with 5-aza-CdR on the transcript levels of p21WAF1 in MCF
7 and MDA MB 231 cell lines was analyzed by RT PCR.
p21WAF1 transcript levels were altered by 5-aza-CdR
treatment in all the cell lines. The treatment with anticancer
drugs alone, PTX, ADR, and 5-FU, resulted in twofolds
increase in p21WAF1 transcripts (Fig. 4). In addition, the
0
20
40
60
80
100
Control
5-aza-CdR
PTX
Expected value
PTX+5-aza-CdR
ADR
Expected value
ADR+5-aza-CdR
5-FU
Expected value
5-FU+5-aza-CdR
0
20
40
60
80
100
Con
trol
5-az
a-CdR PTX
Expec
ted
valu
e
PTX+5-az
a-CdR AD
R
Expec
ted
valu
e
ADR+5-
aza-
CdR
5-FU
Expec
ted
valu
e
5-FU
+5-az
a-CdR
MD
A M
B 2
31
MC
F 7
* * *
*NSNS
Fig. 1 Synergistic or
semiadditive effects of 5-aza-
CdR with anticancer drugs in
breast cancer cell lines.
A synergistic antiproliferative
effect in response to a
combination of 5-aza-CdR with
PTX, ADR, and 5-FU was
observed in MDA MB 231.
A semiadditive effect was
observed in response to
combination of 5-aza-CdR with
5-FU in MCF 7. Each anticancer
drug was added at the IC50 for
each cell line. The synergistic
and semiadditive interactions
were determined when the value
was less than the expected value
and more than the expected
value but less than the drugs’
value. The expected value of the
combined effects (%) = effects
of anticancer drug/
control * effects of 5-aza-CdR/
control * 100. The results are
presented as the mean of three
independent experiments, and
bars indicate the standard
deviation. * P\ 0.05 compared
with anticancer drug alone
Table 1 IC50 of MDA MB 231 and MCF 7 cells of anticancer drugs
Drugs MDA MB 231 MCF 7
Paclitaxel 65 nM 75 nM
Adiramycin 5.5 lM 5.5 lM
5-Fluorouracil 12 nM 10 nM
Mol Cell Biochem
123
combined treatment with 5-aza-CdR and PTX increased
the p21WAF1 transcript levels from threefolds to fivefolds,
whereas combined treatment of 5-aza-CdR with ADR and
5-FU increased the p21WAF1 transcript levels from two-
folds to threefolds in MCF 7 and MDA MB 231 (Fig. 4).
Effect of 5-aza-CdR, PTX, ADR, and 5-FU on p21WAF1
and p53
The effect of treatment of 5-aza-CdR, PTX, ADR, and
5-FU alone or combined treatment of PTX, ADR, and 5-FU
with 5-aza-CdR on p21WAF1 and p53 protein expression in
MCF 7 and MDA MB 231 cell lines was analyzed by
western blotting. A elevated level of p53 was observed
after the treatment of 5-aza-CdR, PTX, ADR, and 5-FU or
with the combination in MCF 7 while p21WAF1 expression
were altered in both the cell lines. The treatment with
anticancer drugs alone, PTX, ADR, and 5-FU, resulted in
twofolds increase in p21WAF1 (Fig. 5). In addition, the
combined treatment with 5-aza-CdR and PTX increased
the p21WAF1 transcript levels from threefolds to fivefolds,
whereas combined treatment of 5-aza-CdR with ADR and
5-FU increased the p21WAF1 and p53 protein expression
from twofolds to threefolds in MCF 7 and MDA MB 231
(Fig. 5).In addition to these proteins, the combined treat-
ment also increases the level of proapoptotic protein BAX
in both the cell lines while there is a decrease in the level of
antiapoptotic protein Bcl2. However, there was slight
increase in caspase 9 in both the cell lines (Fig. 5).
Discussion
5-aza-CdR has been shown to reverse methylation and
reactivate the expression of silenced genes and thus
sparked interest as an antitumor agent [8]. 5-aza-CdR
ctr
ADR
PTX
5-FU
5-aza-CdR
+ 5-FU
+ PTX
+ ADR
0%
20%
40%
60%
80%
100%
CTR
5-az
a-CdR PTX
5-az
a-CdR
5-Fu
5-az
a-CdR
+5-
FUAD
R
5-az
a-CdR
+AD
R
G2-M S G1 SUB G0
+ ADR
ctr
ADR
PTX
5-FU
+ 5 FU
+ PTX
0%
20%
40%
60%
80%
100%
CTR
5-az
a-CdR
PTX
5-az
a-CdR
5-Fu
5-az
a-CdR
+5-
FUADR
5-az
a-CdR
+ADR
G2-M S G1 Sub G0
A B
5-aza-CdR
5-aza-CdR
5-aza-CdR
5-aza-CdR
5-aza-CdR
5-aza-CdR
5-aza-CdR
Fig. 2 Effect of 5-aza-CdR with anticancer drugs on cell cycle
distribution in breast cancer cell lines. Cell cycle distribution analysis
was carried out by FACS for control and 5-aza-CdR and PTX, ADR,
and 5-FU treated MDA MB 231 (a) and MCF 7 (b). Data are
presented as mean of % events from three independent experiments
by 100% stacked column graph for control and experimental groups
Mol Cell Biochem
123
augmented the effects of chemotherapeutic agents against
tumor cells, including lung cancer [15], melanoma [16],
and breast cancer [17]. 5-aza-CdR was also reported to
sensitize hepatoma and pancreatic cancer cells to 5-FU
[18]. In the present study, diverse patterns of synergy were
observed for different combinations of chemotherapy
agents with 5-aza-CdR. In MDA MB 231 cells, synergistic
antiproliferative effects were observed in response to
U:146 bpM:145bp
RAR 2
L U M U M U M U M U M U M U M U M U M
SLIT2
U:150 bpM:150 bp
L U M U M U M U M U M U M U M U M U M
A
B
U:93 bpM:122 bp
GSTP1
U:97 bpM:91 bp
MGMT
L U M U M U M U M U M U M U M U M U M
L U M U M U M U M U M U M U M U M U M
C
D
Fig. 3 MSP analysis of RARb2, SLIT2, GSTP1, and MGMT genes in
breast cell lines. a RARb2 panel viewed from left to right shows a
50-bp ladder as molecular weight marker, a water control for
contamination in the PCR reaction, MDA MB 231 shows presence of
methylated DNA in untreated cells, 5-aza-CdR treated cells showed
complete demethylation, MDA MB 231 treated with ADR shows
presence of methylated DNA while combined treatment with 5-aza-
CdR and PTX shows both methylated and unmethylated DNA. MDA
MB 231 treated with ADR shows presence of methylated DNA, while
for combined treatment with 5-aza-CdR and PTX shows both
methylated and unmethylated DNA. MDA MB 231 treated with
5-FU shows presence of methylated DNA, while for combined
treatment with 5-aza-CdR and 5-FU shows both methylated and
unmethylated DNA. b Slit2 panel viewed from left to right shows a
50-bp ladder as molecular weight marker, a water control for
contamination in the PCR reaction, MDA MB 231 shows presence of
methylated DNA in untreated cells, 5-aza-CdR treated cells showed
complete demethylation, MDA MB 231 treated with ADR shows
presence of methylated DNA, while combined treatment with 5-aza-
CdR and PTX shows both methylated and unmethylated DNA. MDA
MB 231 treated with ADR shows presence of methylated DNA, while
for combined treatment with 5-aza-CdR and PTX shows both
methylated and unmethylated DNA. MDA MB 231 treated with
5-FU shows presence of methylated DNA while for combined
treatment with 5-aza-CdR and 5-FU, shows both methylated and
unmethylated DNA. c GSTP1 panel viewed from left to right shows a
50-bp ladder as molecular weight marker, a water control for
contamination in the PCR reaction, MCF 7 shows presence of
methylated DNA in untreated cells, 5-aza-CdR treated cells showed
complete demethylation, MCF 7 treated with ADR shows presence of
methylated DNA, while combined treatment with 5-aza-CdR and
PTX shows both methylated and unmethylated DNA. MCF 7 treated
with ADR shows presence of methylated DNA, while for combined
treatment with 5-aza-CdR and PTX, shows both methylated and
unmethylated DNA. MCF 7 treated with 5-FU shows presence of
methylated DNA while for combined treatment with 5-aza-CdR and
5-FU, shows both methylated and unmethylated DNA. d MGMT
panel viewed from left to right shows a 50-bp ladder as molecular
weight marker, a water control for contamination in the PCR reaction,
MDA MB 231 shows presence of methylated DNA in untreated cells,
5-aza-CdR treated cells showed complete demethylation, MDA MB
231 treated with ADR shows presence of methylated DNA while
combined treatment with 5-aza-CdR and PTX shows both methylated
and unmethylated DNA. MDA MB 231 treated with ADR shows
presence of methylated DNA, while for combined treatment with
5-aza-CdR and PTX shows both methylated and unmethylated DNA.
MDA MB 231 treated with 5-FU shows presence of methylated DNA,
while for combined treatment with 5-aza-CdR and 5-FU, shows both
methylated and unmethylated DNA
Mol Cell Biochem
123
5-aza-CdR in combination with PTX, ADR, and 5-FU,
while in MCF 7, semiadditive antiproliferative effects were
observed, though only the effect with 5-aza-CdR and 5-FU
reached statistical significance. Our findings suggest that
5-aza-CdR could have varied chemotherapeutic efficacy
with different types of anticancer drugs in breast cancer.
Although 5-aza-CdR as a single agent has demonstrated
little activity in solid tumors, the present findings suggested
that combination of 5-aza-CdR with chemotherapeutic
drugs may be clinically useful and might improve the
response rates in patients with breast cancer. In the present
study, diverse patterns of synergy were observed for dif-
ferent combinations of chemotherapy agents with 5-aza-
CdR. In MDA MB 231 cells, synergistic antiproliferative
effects were observed in response to 5-aza-CdR in com-
bination with PTX, ADR, and 5-FU, while in MCF 7,
semiadditive antiproliferative effects were observed,
though only the effect with 5-aza-CdR and 5-FU reached
statistical significance. Our findings suggest that 5-aza-
CdR could have varied chemotherapeutic efficacy with
different types of anticancer drugs in breast cancer.
Although 5-aza-CdR as a single agent has demonstrated
0
1
2
3
4
MDA MB 231 MCF 7
Control
5-aza-CdR
PTX
ADR
5-FU
PTX + 5-aza-CdR
ADR+ 5-aza-CdR
5-FU +5-aza-CdR
MCF 7
FO
LD
CH
AN
GE
200 bp
MDA MB 231
200 bp
461 bp 461 bp
Fig. 4 RT-PCR analysis of p21WAF/CIP in breast cancer cell lines.
Panel a from left to right shows Lane 1, no band in negative control,
untreated cells, 5-aza-CdR, ADR, ADR ? 5-aza-CdR, PTX, PTX ?
5-aza-CdR, 5-FU, and 5-FU ? 5-aza-CdR treated cells showed a
200 bp transcript in MDA MB 231 cell lines; Lane 2 shows a 461 bp
product of b-actin in the respective untreated and treatedMDAMB231
cells. Panel b Lane 1, no band in negative control, untreated cells,
5-aza-CdR, ADR, ADR ? 5-aza-CdR, PTX, PTX ? 5-aza-CdR,
5-FU, and 5-FU ? 5-aza-CdR treated cells showed a 200 bp transcript
in MCF 7 cell lines; Lane 2 showed a 461 bp product of b-actin in the
respective untreated and treated MCF 7 cells
p53
p21
p21
BAX
Bcl2
ACTIN
ACTIN
BAX
Bcl2
MDA MB 231MCF 7
Caspase 9
Caspase 9
Fig. 5 WB analysis of p21WAF/CIP, p53, BAX, Bcl2, and caspase 9 in
breast cancer cell lines. Western blot analysis on the expression of
proapoptotic and antiapoptotic elements in MCF 7 (a) and MDA MB
231 (b) cells treated with 5-aza-CdR, ADR, ADR ?5-aza-CdR, PTX,
PTX ? 5-aza-CdR, 5-FU, and 5-FU ? 5-aza-CdR
Mol Cell Biochem
123
little activity in solid tumors, the present findings suggested
that combination of 5-aza-CdR with chemotherapeutic
drugs may be clinically useful and might improve the
response rates in patients with breast cancer.
Furthermore, we investigated the mechanism of synergy
of 5-aza-CdR with chemotherapeutic drugs. 5-aza-CdR
inhibited cell growth by induction of G2/M cell cycle
arrest. In contrast, the effect of chemotherapeutic drugs
depended on both apoptosis induction and G2/M arrest.
MDA MB 231 and MCF 7 cells treated with combination
of 5-aza-CdR and PTX, ADR, or 5-FU resulted in a greater
percentage of cells in the sub-G0 and G2/M phases, com-
pared with the cells treated with 5-aza-CdR or PTX, ADR,
or 5-FU alone. These findings provided insight into the
plausible mechanism underlying the augmentation of the
effect of 5-aza-CdR and PTX, ADR, or 5-FU in breast
cancer cells. Cell cycle progression is regulated by the
interaction between cyclins and cyclin-dependent kinases
(CDKs). p21WAF1 is a member of the CIP/KIP protein
family, which inhibits CDKs activity. Increased expression
of p21WAF1 may play an important role in the growth arrest
induced in transformed cells [19]. The p53 and p21WAF1
tumor suppressor genes are known to be involved in
apoptosis. Functional p53 can also induce p21WAF1, and an
increased level of p21WAF1 can decrease the activity of
cyclin-dependent kinases (CDKs), resulting in growth
arrest [20]. Hence, functional p53 is important in p53-
dependent pathway leading to apoptosis.
The treatment of these cells with 5-aza-CdR, PTX,
ADR, and 5-FU alone or combined treatment of PTX,
ADR, and 5-FU up-regulated the expression of p53 after
treatment in MCF 7, while p21WAF1 was induced in both
the cell lines. An increase in p21WAF1 induced by 5-aza-
CdR, PTX, ADR, and 5-FU alone or combined treatment of
PTX, ADR, and 5-FU results in G2 or early M phase arrest,
cells fail to progress to mitosis and are destined to apop-
tosis. Thus, the up-regulation of p21WAF1 may be another
molecular mechanism through which 5-aza-CdR, PTX,
ADR, and 5-FU alone or combined treatment of PTX,
ADR, and 5-FU inhibits cancer cell growth and induces
apoptosis. We have similar results with MDA MB 231 cell,
which is ER-negative and possesses mutant p53. Therefore,
the effects of 5-aza-CdR, PTX, ADR, and 5-FU alone or
combined treatment of PTX, ADR, and 5-FU on cell
growth inhibition and induction of apoptosis are indepen-
dent with ER and p53. In our preliminary assay, 5-aza-CdR
(10–12 lM) treatment for 96 h showed complete demeth-
ylation of our panel of genes, but did not show synergistic
and additive effect with anticancer drugs, whereas these
doses when combined with anticancer drugs for 72 h
increased the effect of these drugs. Our study is limited in
aspect that we have not carried out an in depth analysis of
the mechanism of synergy between 5-aza-CdR and PTX,
ADR, and 5-FU, though we showed that the synergistic and
semiadditive effect of 5-aza-CdR on chemotherapeutical
agents is not likely to be related to our panel of genes. The
particular molecular mechanisms or pathways involved in
the interaction and synergy of these two agents in vivo
should be clarified in the future.
Bax and Bcl-2 have been reported to play a major role in
determining whether cells will undergo apoptosis under
experimental conditions that promote cell death. Increased
expression of Bax can induce apoptosis [21], while Bcl-2
protects cells from apoptosis [21]. Our data showed only a
slight decrease of Bcl-2 expression in breast cancer cells
after treatment with 5-aza-CdR, PTX, ADR, and 5-FU
alone or combined treatment of PTX, ADR and 5-FU. The
expressions of Bax, however, were significantly up-regu-
lated in breast cancer cells after treatment. Our results of
Bax and bcl2 expression directly corroborate flow cytom-
etry results that there is increase apoptosis. Bax harbors a
high homologous sequence with Bcl-2 and forms hetero-
dimers with Bcl-2. Thus, Bax is a protein that antagonizes
the anti-apoptotic function of Bcl-2. Our results suggest
that up-regulation of Bax and down-regulation of Bcl-2
may be one of the molecular mechanisms through which
5-aza-CdR, PTX, ADR, and 5-FU alone or combined
treatment of PTX, ADR, and 5-FU induces apoptosis.
However, further the effect on caspase 9 is also found to be
up-regulated. Therefore, with these markers, we can
hypothesize the apoptosis is the main phenomena involved
in it. In conclusion, our data suggest that 5-aza-CdR can
increase the chemosensitivity of PTX, ADR, and 5-FU in
breast cancer cells by altering cell cycle kinetics. Our
results might provide a rationale for combination chemo-
therapy of DNA methyltransferase inhibitors with tradi-
tional anticancer drugs in breast cancers.
Acknowledgments S.M. is thankful to UGC for providing him SRF
Fellowship.
Conflicts of Interest None.
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