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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