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.
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:
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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
5.
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.
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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-
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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.
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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
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-
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S. M. Hanafy et al. / American Journal of Molecular Biology 1 (2011) 62-69 68
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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