Influence OF anticancer drugs on DNA methylation in liver of female mice

doi:10.4236/ajmb.2011.12008

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American Journal of Molecular Biology, 2011, 1, 62-69

doi:10.4236/ajmb.2011.12008 Published Online July 2011 (http://www.SciRP.org/journal/ajmb/

AJMB

Published Online July 2011 in SciRes. http://www.scirp.org/journal/AJMB

Influence of anticancer drugs on DNA methylation in liver of

female mice

Shaden Muawia Hanafy1, Tarek Abd El-Raouf Salem1, Amal Ahmed Abd El-Aziz1,

Bahgat Abd El-Ghafar El Fiky2, Mahmoud Abd El-Azeim Shokair1

1Molecular Biology Department, Genetic Engineering and Biotechnology Institute, Minufia University, Shibin El Kom, Egypt;

2Animal Biotechnology Department, Genetic Engineering and Biotechnology Institute, Minufia University, Shibin El Kom, Egypt.

E-mail: [email protected]

Received 18 March 2011; revised 16 May 2011; accepted 29 May 2011.

ABSTRACT

Epigenetic changes such as DNA methylation regu-

late gene expression in normal development. Meth-

otrexate and Adriamycine are antineoplastic drugs

that target DNA and enzymes acting on DNA. We

aimed to evaluate their cytotoxic effect on cell lines

and on female mice to investigate the in vivo influ-

ence of both drugs on the DNA methylation and sub-

sequently the protein expression. The total level of

DNA methylation showed a significant reduction

from 62.2% to 36.7%, 36.6% as compared to control

group, when using different doses of MTX and ADR.

Hepatic protein pattern revealed five bands with low

MW (16 - 6.1 KDa) in acute and LD50 doses. In con-

clusion DNA methylation is influenced by anticancer

drugs in a dose—dependent manner. Some specific

protein fragments may be considered as a potential

markers associated with high dose of anticancer

drugs.

Keywords: Epigenetic; DNA Methylation; Methotrexate

(MTX); Adriamycine (ADR)

1. INTRODUCTION

Cancer is uncontrolled growth of cells coupled with ma-

lignant behavior; invasion and metastasis. It is thought to

be caused by the interaction between genetic susceptibil-

ity and environmental toxins. Most of chemotherapeutic

drugs work by impairing mitosis and/or inducing apop-

tosis. Several anticancer drugs target DNA or enzyme

acting on the DNA [1]. The resistance of tumor cells to

different antineoplastic agent is an obstacle for cancer

chemotherapy. The main mechanism in drug resistance

is the multi-drug resistance (MDR) phenomenon, which

constitutes the reduction of intracellular drug level due

to the P-glycoprotein pump function [2]. Drug develop-

ment programs for identification of new cancer chemo-

therapeutic agents involve extensive preclinical evalua-

tion of vast numbers of chemicals for detection of anti-

neoplastic activity. Animal models have always played

an important role, and also cell culture systems have

figured largely in the field of cancer chemotherapy,

where the potential value of such systems for cytotoxic-

ity and viability testing is now widely accepted [3].

Methotrexate (MTX)—is an antimetabolite and anti-

folate drug which is used in treatment for many neoplas-

tic disorders and some autoimmune diseases. It inhibits

the synthesis of nucleic acids and subsequently proteins

[4]. However, MTX, at certain dose, exhibited a toxic

side effect to normal cells and organs in the body. Also,

using of MTX for long period can increase the risk of

toxicity [5]. Many studies proved the ability of MTX to

inhibit dihydrofolate reductase enzyme (DHFR) which

converts dihydrofolate to the active tetrahydrofolate

compound which is essential for DNA methylation. [6].

Adryamycin (ADR) is an anthracycline isolated from

streptomycin peucetius. It is commonly used in the treat-

ment of a wide range of cancer including haematological

malignancies, carcinomas, sarcomas and lymphomas. It

prevents DNA replication by acting as topoisomerase

inhibitor [7].

DNA methylation is a major biochemical modification,

typically occurs at 5’-CpG (Cytosine-phosphate-guanine

sites). They are regions have a higher GC content than

the genome average and they may repress transcription

[8]. In mammals, almost 60% of all promoters localize

within CpG region. These regions are commonly devoid

of methylation, while the rest have a methylation pattern

and base composition indistinguishable from bulk DNA

[9]. There is an inverse relationship between CpG me-

thylation and transcriptional activity. Evidence that has

S. M. Hanafy et al. / American Journal of Molecular Biology 1 (2011) 62-69 63

accumulated in the past 10 years suggests that cancer

cells usurp this physiologic mechanism and use it to

their benefit by inactivating tumor suppressor genes

leading to cancer progression. Hypomethylating agents

or DNA methylation inhibitors could be used for the

reversal of aberrant DNA methylation and therefore re-

store the function of silenced genes in cancer causing

growth arrest in tumor cells [10,11]. Upon these obser-

vations, we designed this work to investigate the poten-

tial activity of two common anticancer drugs, MTX and

ADR as DNA hypomethylating agents that may lead to

hyper expression of some genes associated with tumor

suppression.

2. MATERIALS AND METHODES

2.1. Cell Lines and Cytotoxic Drugs

Three different types of cell lines were used for in vitro

study; human larynex carcinoma cell line (Hep2, ATCC

No. CCL-23), human hepatocyte carcinoma cell line

(HepG2, ATCC No. HB-8065) and monkey kidney cell

line (Vero, ECACC No. 84113001). Cell lines were

maintained and grown in RPMI culture medium supple-

mented with 15% fetal bovine serum (FBS), 10 mmol/L

HEPES, 1 mmol/L sodium pyruvate, 4.5 g/L glucose, 1.5

g/L sodium bicarbonate, and 5% penicillin/streptomycin

(Reagents and chemicals were obtained from Sigma/Al-

drich, USA). Two different chemotherapeutic drugs were

used, methotrexate (10 mg/1ml; Ebewe Co. Italy) and

adriamycin (2 mg/1ml; Pharmacia Co. Italy). Drugs were

diluted in 0.9% physiological saline for 1X concentra-

tion.

2.2. In-Vitro Cytotoxic Study

Cells were seeded in 96-well flat-bottomed microtiter

plates (100 µl/well) under complete aseptic condition

and incubated in 5% CO2 incubator at 37˚C for 24 hr to

reach complete monolayer. Serial dilutions of tested

drugs were titrated in triplicate to the cells; in addition to

the control wells that left without drugs. The plate was

then incubated in 5% CO2 incubator at 37˚C for 24 hr

and 72 hr to investigate the LD50 and cytopathic effect

of the tested drugs. For recovery period bioassay, plates

were incubated for 7 days in which growth medium was

renewed every 2 days.

2.3. In-Vivo Cytotoxic Study

The toxicity study was carried out using 70 female

Balb/c mice weighing 20 - 25 g each. They were main-

tained on animal cubes (Feeds Nigeria Ltd), provided

with water ad libitum and were allowed to acclimatize to

the laboratory conditions for seven days before the ex-

periment. Three doses were selected for each drug ac-

cording to LD50 determinaion on cell lines: For adria-

mycine; 0.4, 0.04 and 0.004 mg/100g, BW for acute,

LD50 and therapeutic dose, respectively. While, For

metotrexate; 2, 0.2 and 0.02 mg/100g, BW for acute,

LD50 and therapeutic dose, respectively. Animals were

divided randomly into 7 groups (10 mice each), six

groups of animals were injected subcutaneously with

these dosese three times a week for 1 month. In addition,

ten normal non-injected mice served as control.

2.4. Biochemical and Hematological

Investingtions

The serum activity of liver enzymes, ALT and AST, were

determined according to the method of Reitman and

Frankel [12]. Also, serum albumin [13], urea [14] and

creatinine [15] were estimated. On the other hand, level

of haemoglobin (Hb) and total leucocytic count [16]

were determined.

2.5. Histopathological Examination

At the end of experiment, animals were sacrificed by

cervical dislocation. Liver, kidney and spleen were im-

mediately excised and processed for histopathological

examination. Briefly, paraffin sections of fixed tissues

were cut in 5 µm thickness; stained with hematoxylin

and eosin (H&E) and then examined microscopically.

Histopathological changes were graded according to

Portmann, et al. [17].

2.6. SDS-PAGE for Hepatic Proteins

Half gram of liver tissue was placed in ice-cold PBS,

minced and homogenized by using Teflon-glass ho-

mogenizer. The homogenates were spun and the clear

supernatants were transferred into clean tube, the protein

content was determined according to the method of

Bradford [18]. Analysis of hepatic proteins was carried

out by using SDS-polyacrylamide gel electrophoresis

(SDS-PAGE) as described by Laemmli [19]. Briefly,

total protein liver extracts (20 μg) from different groups

of animals were loaded onto 15% polyacrylamide gel

and subjected to 80 V for 30 minutes. At the end of mi-

gration, gel was stained with Coomassie blue for 2 hr

and then the excess of stain was removed by using gla-

cial acetic acid for 4 hr. The gel was visualized by using

white light and photographed.

2.7. Determination of Hepatic DNA Methylation

Analysis

Isolation of the mouse hepatic DNA was done by using

DNeasy Tissue Kit (Qiagen, Germany). For restriction

analysis, we used two enzymes—Msp I and Hpa II

(Moraxella species and Haemophilus parainfluenzae,

respectively, MBI, Fermentas, Lithuania). Both enzymes

cut DNA in the sequence:

S. M. Hanafy et al. / American Journal of Molecular Biology 1 (2011) 62-69

Figure 1. Percentage of cytopathic effects 24 hr after incuba-

tion with drugs and recovery percentage after 72 hr from drugs

elimination.

…..5’…..C↓CGG…..3’

…..3’…..GG C↑C…..5’

MspI and HpaII differ in sensitivity to DNA methyla-

tion. MSPI cleaves outer and inner methylated cytosines

(mCCGG or CmCGG). While Hpa II cleaves outer cyto-

sines in this DNA sequence (mCCGG). On this base, we

could to determine the percentage of methylated frage-

ments of genomic DNA. The samples of DNA were

analyzed by using 1% agarose gel in TAE buffer con-

taining ethidium bromide (at final concentration of 1

mg/ml) [20]. 1kb DNA (ladder 250 - 10000 bp-Promega,

USA) was used as standard DNA. The electrophoresis

was performed at 100 mA for 3 hours. Individual frag-

ments of DNA were detected by UV-trans-illuminator

and photographed. For densitometrical scanning of DNA

preparations and data evaluation, we used the Gel do-

cumentation system, the software Microsoft Photo Editor,

Ingenius Syncene Bioimaging, Canada, software version

2.8. Statistical Analysis

Statistical analysis was done using the statistical package

SPSS version 10. Comparison of mean values of studied

variables among different groups was done using

ANOVA test. p < 0.05 was considered to be significant.

3. RESULTS

3.1. In-Vitro Cytotoxicity Study on Hep2,

HepG2 and Vero Cell Lines

He percentage of cytopathic effect of MTX and ADR

were calculated and presented in Figure 1. Results

showed that MTX exhibited higher cytopathic effect

against HepG2, Hep2 and Vero cell lines as compared to

that of ADR after 24 hr exposure. Furthermore, HepG2

cells were more susceptible to the toxicity of MTX and

ADR as it exhibited a significant percentage of growth

Figure 2. Biochemical parameters among different injected

groups with ADR compared to control.

Figure 3. Biochemical parameters among different injected

groups with MTX compared to control.

Figure 4. A photomicrograph of normal liver of female mice (a)

and injected with therapeutic MTX (b) showing dilated central

vein congested with blood cells, fatty changes and vacuolar

degeneration of hepatocytes.

S. M. Hanafy et al. / American Journal of Molecular Biology 1 (2011) 62-69 65

Figure 5. A photomicrograph of normal kidney of female mice

(a) and treated with therapeutic ADR (b) showing normal pat-

tern of glomeruli with normal subcapsular space, dilated corti-

cal tubules and peritubular inflammatory cells.

inhibition (79.1% and 71.5%, respectively); while the

percentage of recovered cells after 72 hr were 23.6% and

25.7%, respectively. In contrast, the Hep2 and Vero cell

lines exposure time 24 hr were more resistant to the cy-

totoxic effect of both MTX and ADR (54.6% and 43.1%

for Hep2, 58.2% and 41.6% for Vero cell line); while the

recovery cells after 72 hr proved the differential cyto-

toxicity for MTX and ADR.

3.2. Biochemical and Hematological Investiga-

tions

Treatment of ADR group of mice with different doses,

revealed a significant difference between the sub groups

when compared with control. The difference was very

highly significant in case of AST, blood urea, serum

creatinine and haemoglobin. (p = 0.001) as shown in

Figure 2. While, in case of MTX group. The difference

was very highly significant between the subgroups

compared to control in AST, ALT, blood urea and hae-

moglobin (p = 0.000) as shown in Figure 3.

3.3. Histopathology

Liver, kidney and spleen biopsies of female mice in-

jected with therapeutic doses of MTX and ADR for one

month showed some histopathological changes when

Figure 6. A photomicrograph of normal spleen of female mice

(a) and treated with therapeutic MTX showing apoptosis and

bleeding of splenic cells.

Figure 7. SDS-PAGE of hepatic proteins of studied groups:

M: marker, Lane1: control, Lane2: Acute ADR, Lane3: LD50

ADR, Lane4: Therapeutic ADR, Lane5: Acute MTX, Lane 6:

LD50 MTX, Lane7: Therapeutic MTX.

compared to normal control group as shown in Figures

4, 5, 6(a) and (b)). While, in case of toxic doses, it

showed marked histopathological changes especially in

kidney.

3.4. Protein analysis

Figure 7 illustrates the pattern of hepatic protein elec-

S. M. Hanafy et al. / American Journal of Molecular Biology 1 (2011) 62-69

Figure 8. Msp I digestion of hepatic DNA isolated from the

groups of study.

trophoresis of the studied groups of animals. Regarding

the band with MW 130 KDa was presented in normal

and therapeutic ADR dose. The band which had MW

136 KDa was presented in normal only and disappeared

in all groups. The bands which had MW 134.06, 130.44,

120.30, 100.31 and 97.45 KDa were presented in normal

and therapeutic ADR and therapeutic MTX. These bands

were not observed in animals injected with acute and

LD50 doses of ADR or MTX. This band can be consid-

ered as a potential marker associated with therapeutic

dose of anticancer drugs. They showed appearance of

two bands (18.1 and 16.7 KDa) in animals treated with

acute, LD50 and therapeutic doses of ADR and MTX,

while not shown in control groups. Five bands had been

shown with low molecular weight (range size 16 - 6.1

KDa) in animals exposed to acute and LD50 doses of

ADR and MTX. These bands were not observed in the

controls, and in animals injected with therapeutic dose of

ADR and MTX. These bands can be considered as a

potential marker associated with high dose of anticancer

drugs.

3.5. DNA Methylation Study

Representative electrophoreograms of hepatocyte DNA

of control and injected groups digested with Msp I or

HpaII were demonstrated in Figures 8 and 9. The elec-

trophoreograms were scanned densitometrically. The

scans were divided into molecular weight intervals calcu-

lated from the migration of the standard DNA. On densito-

metrical scans, curves of non-digested DNA and DNA

treated by the restriction enzymes had different shapes.

The shape of curves was influenced also by the MTX

and ADR (Figures 9, 11(a) and (b)). On the basis of

densitometrical scans, molecular weight distribution of

products of DNA cleavage with restriction enzymes Msp

I and Hpa II was calculated. The results of analyses of

DNA fragments obtained by treatment with the restric-

tion enzymes Msp I and Hpa II, summarized in Table 1.

Figure 9. HpaII digestion of hepatic DNA isolated from the

groups of study.

Figure 10. Densitometer scans of Msp I digested-hepatic DNA:

(a) control and MTX groups (b) control and ADR groups.

Where: Black line represents control dose, Green line repre-

sents LD50 dose, Red line represents acute dose,Blue line

represents therapeutic dose.

Figure 11. Densitometer scans of HpaII digested-hepatic DNA:

(a) control and MTX groups (b) control and ADR groups.

Where: Black line represents control dose, Green line repre-

sents LD50 dose, Red line represents acute dose,Blue line

represents therapeutic dose.

It showed that the total level of DNA methylation was

influenced not only by anticancer drugs but also by their

doses. Treatments related alterations liver DNA methyla-

tion in the typical methylation sequence C-C-G-G were

expressed as a total methylation percentage in different

MTX and ADR subgroups. Total methylation percentage

was markedly reduced from 62.2% (control) to 36.7% by

the action of the hypomethylating agents MTX and ADR.

S. M. Hanafy et al. / American Journal of Molecular Biology 1 (2011) 62-69 67

Table 1. Percentage of hepatic DNA methylation among dif-

ferent groups of the study.

Sample N MW

(Kb) Mn

(Kb) R %

Methylation P

Control

Msp I 12 2.5 0.86 2.90

Hpa II 12 3.0 0.53 5.66

62.2 0.001124

Therapeutic MTX

Msp I 12 3.1 0.67 4.62

Hpa II 12 2.9 0.49 5.91

36.7 0.079478

Acute MTX

Msp I 12 1.75 0.69 2.53

Hpa II 12 3.21 0.50 6.42

38.0 0.001124

LD50 MTX

Msp I 12 3.01 0.78 3.85

Hpa II 12 2.89 0.56 5.16

39.3 0.001124

Therapeutic ADR

Msp I 12 3.7 0.97 3.81

Hpa II 12 3.25 0.71 4.57

36.6 0.003341

Acute ADR

Msp I 12 2.25 0.74 3.04

Hpa II 12 3.01 0.54 5.57

37.0 0.079478

LD50 ADR

Msp I 12 3.32 0.81 4.09

Hpa II 12 2.99 0.58 5.15

36.9 0.003341

The mass average MW = , where Wi is the mass fraction and Mi

the average weight for interval i. Individual intervals were obtained from

gel photographs, which were scanned; scans were divided into MW inter-

vals calculated from the migration of standard DNA molecules. The number

average MW: Mn = , where Xi is number fraction for interval i.

For the number average distribution, the relative number of molecules

under each interval was summed and the number in each interval of mole-

cules was taken as a fraction of the total DNA. R is the ratio (MW/Mn), a

change in the value of r indicated a change in the shape of the distribution.

Percentage of methylation = 1 – (Mn Msp I)/(Mn HpaII) × 100.

Xi Mi

The reduction was in a dose-dependent manner. There

was a highly significant reduction in MTX therapeutic,

LD50 and ADR therapeutic as well as LD50 in com-

parison to control group (p = 0.0011, 0.0033, respec-

tively). Significant difference was found especially in

products of cleavage with enzyme MspI, where a de-

crease in DNA fragments of medium molecular weight

(1 - 7 kb) occurred between the therapeutic MTX dose

and therapeutic ADR dose than control. MTX and ADR

of acute and LD50 did not induce significant changes in

molecular weight distribution of restriction fragments of

DNA isolated from acute and LD50 treatment with MTX

or ADR.

4. DISCUSSION

Epigenetic changes such as DNA methylation act to re-

gulate gene expression in normal mammals. In the pre-

sent study, the higher level of DNA methylation ob-

served in control than treated female mice was probably

connected with alterations in the gene expression of the

hepatocytes. It was obviously recognized in the SDS-

PAGE for total soluble hepatic protein patterns. As it

showed absence or presence of some protein bands after

treatment with anti-cancer drugs comparing to untreated

group. This finding is in agreement with the report of

David [21] and this observation relies on the type of

drug used and in a dose-dependent manner. Five bands

had been shown with low molecular weight (16 - 6.1

KDa) after treatment with acute or LD50 dose of ADR,

and meanwhile with acute and LD50 dose of MTX.

These bands were not observed either in the control or

therapeutic ADR or therapeutic MTX. These bands can

be considered as a potential marker associated with high

dose of anticancer drugs. The changes in band intensity

or density could be explained on the basis of cytoge-

netical abnormalities produced by these drugs. Donna et

al., [22] concluded that the increase in band intensities

or densities could be due to the gene duplication pro-

duced by induction of bridges, breaks, laggards, and

micronuclei. The disappearance of some bands could be

attributed to the loss of some genetic material. It seems

possible that interaction of diet and contaminants or

drugs by inducing changes in DNA methylation and ab-

errant gene expression. Specific methylation alterations

are associated with changes in gene expression and this

association is described by the simple hypothesis that

methylation turns some genes off and others on. Our

data and data from several studies indicated that DNA

methylation changes are much complicated and its pat-

tern is generally discontinuous. This can be observed by

comparing Msp I partial digested DNA with comparable

Hpa II digests. Since differential methylation clearly

exists in DNA, it is likely that gene expression has

evolved to utilize these differences (up and down of

regulation different genes) [23]. This was strongly sup-

ported by our data and reflected by different protein

banding pattern for the different treated groups of animal

comparing to control. Inhibition of DNA methylation

(hypomethylation) as a result of anti cancer drugs or

post-radiation therapy had been reported by Igor, et al.

[24]. Hypomethylation showed the loss of long inter-

spersed nucleotide element-1 (LINE-1) CpG methylation

in spleen.

Recent study of Basak, et al. [25] reported that the

cytotoxic effect of MTX is associated with apoptosis

enhancement, as it may be related to hyperhomocys-

teinemia and deoxyribonucleotide pool imbalances. Con-

clud- ing that there was an altered expression of MTHFR

enhanced MTX—induced myelosuppression in mice,

after evaluating that in the major hemolytic organ spleen.

DNA methylation-related anticancer drugs had gained

increasing attention over the past decades due to the ab-

errant DNA methylation to development of drug resis-

tant tumors cells. Hence the acquired drug resistance

represents a frequent obstacle which hampers efficient

chemotherapy of cancers [26]. Recently, Boettcher et al.

[27] characterized DNA methylation change which aris-

S. M. Hanafy et al. / American Journal of Molecular Biology 1 (2011) 62-69

es from treatment of tumor cells with the adriamycine.

DNA methylation level from CpG islands linked to

twenty eight genes whose expression levels had been

shown to contribute with the resistance against DNA

double strand break induced drugs. These data were

supported in some way to our data in documenting DNA

methylation by different doses of doxorubicin drug in

non-tumor female mice [28] assessed the CpG methyla-

tion aberrations induced by pixantrone and doxorubicin.

A characteristic that may determine the most cancer

types to specific drug treatments and is a marker of drug

sensitivity. Moreover, Winter-Vann, et al. [29] suggested

that MTX has an additional mechanism of action besides

it is a potent product inhibitor of cellular methyltrans-

ferases. It is also having an inhibitiory effect on Ras sig-

naling that regulates cell growth and differentiation. Be-

cause carboxyl methylation of Ras is important for

proper plasma membrane localization and function, they

reported that after MTX treatment of DKOB8 cells, Ras

methylation is decreased by almost 90% and subse-

quently inhibition of carboxyl methylation.

5. CONCLUSIONS

DNA methylation is influenced by anticancer drugs

(MTX and ADR) and this influence was in a dose-

dependent manner; as they exhibited reduction in DNA

methylation with varying degrees in liver genomic DNA.

Some specific protein bands may be considered as a po-

tential markers associated with high doses of anticancer

drugs. Treatment with DNA methylation inhibitors may

reactivate epigenetically silenced genes and has been

shown to restore normal gene function. Further studies

are recommended to characterize the protein fragments

associated with anticancer drugs treatment.

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