Impact of methotrexate on oxidative stress

and apoptosis markers in psoriatic patients

Tamilselvi Elango, Haripriya Dayalan,

Pushpa Gnanaraj, Hemamalini
Malligarjunan & Swapna Subramanian
Clinical and Experimental Medicine
ISSN 1591-8890
Clin Exp Med
DOI 10.1007/s10238-013-0252-7

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Clin Exp Med
DOI 10.1007/s10238-013-0252-7

ORIGINAL ARTICLE

Impact of methotrexate on oxidative stress and apoptosis

markers in psoriatic patients
Tamilselvi Elango Haripriya Dayalan
Pushpa Gnanaraj Hemamalini Malligarjunan
Swapna Subramanian

Received: 11 February 2013 / Accepted: 24 July 2013

 Springer-Verlag Italia 2013

Abstract Methotrexate (MTX), a cytotoxic chemotherapeutic agent, is considered an effective drug in the treatment of psoriasis. The aim of this study was to find out
whether the effect of MTX treatment in psoriasis is due to
oxidative stress-induced apoptosis. Psoriasis vulgaris
patients (58 in number) were recruited for this study.
Healthy volunteers (45 in number) served as control.
Samples of psoriatic patients were collected and analyzed
for total reactive oxygen species (ROS), malondialdehyde
(MDA) levels, nitrite, nitrate levels and the activities of
antioxidants like superoxide dismutase (SOD), catalase
(CAT) and total antioxidant status (TAS) and also the
protein expression of caspase-3, before (Day 0) and after
(at the end of 6 and 12 weeks) MTX treatment. Our results
show a significant increase in tissue ROS and plasma MDA
after MTX treatment when compared with before MTX
treatment in psoriasis patients (p \ 0.001). The levels of
serum nitrite and nitrate were decreased significantly after
MTX treatment (p \ 0.001). The activities of plasma SOD,
TAS and serum CAT levels were decreased, but not significantly after 12 weeks of treatment. The expression of
caspase-3 was increased after MTX treatment. In conclusion, MTX induce apoptosis through oxidative stress by
reducing NO and increasing caspase-3 levels. MTXinduced apoptosis may account for the beneficial effect of

T. Elango
H. Dayalan (&)
H. Malligarjunan

S. Subramanian
Department of Medical Research, SRM Medical College,
Hospital and Research Centre, Kattankulathur 603203,
Tamilnadu, India
e-mail: dhpsrm@yahoo.in
P. Gnanaraj
Department of Dermatology, SRM Medical College, Hospital
and Research Centre, Kattankulathur 603203, Tamilnadu, India

MTX treatment in psoriasis patients, which is characterized

by acanthosis.
Keywords Reactive oxygen species

Methotrexate
Apoptosis
Psoriasis

Introduction
Skin is a major target of oxidative stress due to reactive
oxygen species (ROS) that originate in the environment
and in the skin itself. Reactive oxygen species (ROS)mediated oxidative stress is involved in a vast number of
biological responses causing DNA modification, lipid
peroxidation and production of inflammatory cytokines [1].
This may contribute to the pathogenesis of many inflammatory skin diseases, including psoriasis [13].
In inflammation, apoptosis is frequently delayed, a
mechanism that contributes to accumulation of inflammatory cells. The generation of ROS by inflammatory cells
contributes to host defense mechanisms and tissue damage.
On the other hand, ROS are important in signal transduction events required for essential cell functions. In respect
of apoptosis, ROS is in general associated with induction of
death. However, under certain circumstances, ROS can
also be antiapoptotic [4].
The principal histological features of psoriasis are epidermal hyperplasia or abnormal differentiation, dilated
blood vessels in dermis and predominantly infiltration of
leukocytes into dermis causing inflammation [5]. It is
considered a T-helper 1 (Th1) disease based on the increase
in cytokines of the Th1 pathway such as interferon gamma,
interleukin (IL) 2 and 12, as found in psoriatic plaques [6].
Keratinocytes play a major role in this chronic inflammatory disease [7]. Decrease in apoptosis is also suggested as

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a specific pathogenic phenomenon in psoriasis [8, 9].

Acanthosis in psoriatic skin is resulted from diminished
epidermal apoptosis [10], while induction of apoptosis is
involved in the regression of psoriatic hyperplasia [8, 9].
In psoriasis, ROS also play a central role which stimulate
cell proliferation and trigger undifferentiated keratinocytes
to form a defective stratum corneum [11]. Abnormal
keratinocyte growth in psoriasis, therefore, may be partially
due to the elevated O2- generated by PMNs and psoriatic
dermal fibroblasts [12]. Nitric oxide (NO) is also considered
as a potent regulator of keratinocyte growth and differentiation and seems to be a strong candidate in the pathogenesis of psoriasis [1315]. NO is a highly reactive free
radical with a very short half-life. The labile nature of NO
makes it impossible to analyze serum and tissue levels. The
nonfunctional metabolites of NO, nitrite and nitrate, are
helpful to search the amount of NO. Nitrite levels are
considered the marker of NO in tissue and other body fluids.
It is important to study nitrate levels as the nitrite is reduced
into nitrate by oxyhemoglobin, in blood [1619].
In 2000, study by Laporte et al. [9] showed that PUVA
therapy in psoriasis patients induces apoptosis, which causes
regression of psoriatic hyperplasia. Another study in 2006 by
Kruger et al. [20] also showed that the apoptosis of keratinocytes explain the rapid and sustained therapeutic effect of
infliximab in psoriasis. Therapies based on the regulation of
keratinocyte proliferation are potentially useful in treatment
of psoriasis because the restored homeostatic control of
keratinocyte growth and differentiation is crucial for
recovery from psoriatic to normal epidermis [21].
Methotrexate (MTX) is considered as a gold standard
therapy for moderate to severe psoriasis [22]. Although the
effectiveness of MTX in the treatment of psoriasis is very
well established, the mechanism of action is poorly
understood [23, 24]. But MTX is suggested to act primarily
as an anti-inflammatory and immunosuppressant drug [25].
ROS generation by MTX is important for cytostasis in
monocytes and cytotoxicity T-cells. Furthermore, MTX
caused a reduction in monocyte adhesion to endothelial
cells. These showed the generation of ROS is an important
mechanism in the immunosuppressive effects of MTX.
Therefore, its clinical efficacy can be attributed to multiple
targets [26]. Hence, the present study was carried out to
study the role of MTX in oxidative stress-induced apoptosis of proliferating keratinocytes in psoriasis patients.

were recruited for this study. The age of all patients ranged
from 18 to 70 years (mean SD, 46.4 14.1 years), and
there were 27 men and 31 women. Healthy volunteers (45
in number) consisting of 21 men and 24 women, aged from
21 to 70 years (mean SD, 44.6 15.5 years) served as
control. Psoriasis patients with [18 years of age, who had
more than 20 % body surface area involvement and had not
received any topical or systemic therapy for at least a
month, were included in this study. Patients with unstable
psoriasis, liver and renal impairment, infertility, anemia,
excessive alcohol intake or any other systemic diseases,
children (\18 years old), as well as pregnancy and lactating women were excluded from this study. The study
protocol was approved by the Institutional Ethical Committee. Informed consent documents in regional language
were signed by all the patients.
Psoriatic patients were treated with 7.5 mg of methotrexate per week for 12 weeks. Folic acid was given at
5 mg once daily except on the day of MTX for 12 weeks.
During systemic treatment, no concomitant antipsoriatic
therapy was permitted, with the exception of emollients.
Collection of samples
Blood samples of psoriatic patients were collected and
analyzed before (Day 0) and after (6 and 12 weeks) treatment with MTX. In control subjects, blood samples were
collected only once. Blood was collected in blood collection tube, allowed to clot for 30 min and centrifuged at
2,500 rpm for 10 min, and the serum was separated.
Freshly isolated heparinized peripheral blood samples were
immediately centrifuged for 10 min at 500 g, and the
plasma were separated.
In each patient, lesional and nonlesional skin biopsies
(10 mM) were taken after local anesthesia lidocaine
hydrochloride and adrenaline bitartrate. I.P. lesional biopsies were taken at before (Day 0) and after (6 and 12 weeks)
treatment with MTX. Nonlesional skin biopsy served as a
control, which was collected only once. Biopsies of psoriatic lesional skin were taken within a lesion, 1 cm from the
edge of the plaque border. Biopsies of nonlesional skin were
taken 2 cm beyond the plaque border. All tissue specimens
were immediately immersed in protease inhibitor cocktail
and finally stored at -20 C until further use.
Estimation of oxidative markers and antioxidants

Materials and methods

Patients details and treatment regimen
Psoriasis vulgaris patients (58 in number) who visited
Dermatology Department, SRM Hospital, Kattankulathur,

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Plasma lipid peroxidation was estimated colorimetrically

by measuring malondialdehyde (MDA) level in psoriasis
patients by double heating method of Draper and Hadley
1990 [27]. Serum nitrite (NO2) and nitrate (NO3) were
measured by using a Griess reaction [19] and using the
enzymatic one-step assay with nitrate reductase [28].

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Keratinocytes were isolated from all the skin biopsies as

described by Piskin et al. 2003 [29]. The total reactive
oxygen species (ROS) in isolated keratinocytes were
assayed spectrofluorometrically as described by Davina
et al. [30].
Plasma SOD was determined by the method of Das et al.
[31]. Serum catalase (CAT) activity was assayed according
to the method of Goth [32]. Plasma total antioxidant
capacity (TAC) was determined by the method of Miller
and Rice-Evans et al. [33].
Western blot analysis
Total cell extracts were prepared from skin biopsy as
described by [34]. The caspase-3 protein expression was
determined by Western blot analysis [35]. Band intensity
was analyzed by using ImageJ software (http://rsb.info.nih.
gov/ij/).
Statistical analysis
The results were expressed as mean SD. Data were
analyzed using Spss 16.0 software. Control and psoriatic
patients (Day 0) were compared using independent sample
t test. Nonlesional and lesional skin biopsy samples were
compared by paired t test. Before (Day 0) and after (6 and
12 weeks), MTX treatment psoriatic samples were compared by repeated measures ANOVA. p \ 0.05 was considered to be significant.

Results
Six patients were dropped out of this study: two patients
due to transient increase in alanine aminotransferase (ALT)
activity and four patients were dropped out due to difficulty
in follow-up.
Figure 1 indicates tissue total ROS levels before and
after treatment with MTX in psoriatic patients. Tissue ROS

Fig. 1 Effect of methotrexate on total reactive oxygen species in skin

biopsy of psoriasis patients. All values were expressed as
mean SD, a denotes comparison of lesional with nonlesional skin
biopsy, b denotes comparison of 6 and 12 weeks methotrexate
treatment with lesional skin biopsy, $ denotes p \ 0.001

was significantly (p \ 0.001) higher in lesional skin biopsy

compared to nonlesional skin biopsy (0.29 0.06 to
0.60 0.06). MTX treatment further significantly
(p \ 0.001) elevated the oxidative stress markers in psoriatic patients (0.60 0.06 to 1.22 0.14).
Table 1 shows the effect of MTX on lipid peroxidation
and antioxidant levels in psoriasis patients. Our results
show that the plasma MDA levels in psoriatic patients were
higher significantly (p \ 0.001) with mean difference
1.997 compared to the plasma of healthy controls. MTX
treatment for 6 and 12 weeks showed significant increase
(p \ 0.001) in plasma MDA levels with mean difference of
1.465 & 2.459 compared to Day 0.
Plasma SOD, TAS and serum catalase were significantly
decreased in (p \ 0.001) psoriasis patients, as compared to
healthy controls. After 6 and 12 months treatment with
methotrexate, the plasma SOD, TAC and serum catalase
levels were decreased, but not significantly in psoriasis
patients.
Figure 2 shows the effect of MTX on nitratenitrite
levels in psoriasis patients. Our results show that serum

Table 1 Effect of MTX on lipid peroxidation and antioxidant levels in psoriasis patients
S. no.

Parameters

Control

Psoriasis (Day 0)

After MTX treatment

6th week

Plasma MDA (nmol/ml)

12th week

1.20 0.23

3.20 0.49a$

4.66 0.95b$

5.66 0.83b$

a$

4.72 0.48

4.85 0.51

Plasma SOD (U/mL)

5.68 0.43

4.68 0.41

Serum catalase (KU/L)

61.6 2.31

48.6 6.41a$

49.5 8.12

50.5 7.28

Plasma TAS (lmol/L)

1,014.2 59.3

899.37 55.5a$

897.04 62.3

893.63 61.8

$ denotes p \ 0.001
All values were expressed as mean SD
a

Comparison of Day 0 with control

Comparison of 6 and 12 weeks methotrexate treatment with Day 0

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Discussion

Fig. 2 Levels of serum nitrite and nitrate levels in psoriatic patients

after methotrexate treatment. All values were expressed as
mean SD, a denotes comparison of Day 0 with control,
b denotes comparison of 6 and 12 weeks methotrexate treatment
with Day 0, $ denotes p \ 0.001

levels of nitritenitrate were significantly higher in psoriasis patients with mean difference of 14.6, 44.4 compared
to controls and their levels were decreased significantly
(p \ 0.001) after MTX treatment with mean difference of
9.85, 26.97, respectively.
The expression of caspase-3 levels was analyzed
using Western blot analysis. A significant difference in
the expression of the caspase-3 protein was detected in
all the samples (Fig. 3a). Downregulation (p \ 0.001) of
caspase-3 was observed in lesional skin biopsy compared
to nonlesional skin biopsy. After MTX treatment, the
expression of caspase-3 was increased significantly
(p \ 0.001) as shown by densitometric analysis
(Fig. 3b).

Fig. 3 a Expression of caspase3 analyzed by Western blotting.

The protein extracts (50 lg of
protein) were separated by SDSPAGE on a 1020 % gradient
gel. After electro-blotting, the
separated proteins were probed
with an antibody recognizing
caspase-3 and b-actin from 10
psoriatic patients.
b Densitometric analysis of the
Western blot bands for caspase3 normalized to b-actin,
a denotes comparison of Day 0
with nonlesional skin biopsy,
b denotes comparison of 6 and
12 weeks methotrexate
treatment with Day 0, $ denotes
p \ 0.001

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Increased ROS production in patients of psoriasis and

decreased concentration of antioxidants leads to oxidative
stress which is a feature of psoriasis [36].
Our results showed increased oxidative stress markers
like MDA and total ROS in psoriasis patients compared to
healthy controls. Similarly, other reports also showed that
patients with psoriasis exhibit increased concentrations of
MDA [3740]. ROS-induced oxidation of polyunsaturated
fatty acids in biological systems results in the formation of
lipid peroxidation products such as MDA [4042].
Increased MDA levels may be due to the intraepidermal
penetration of activated polymorphonuclear leukocytes that
leads to increased ROS production provided by NADPH
oxidase and proteolytic enzymes in psoriasis [43]. Even
though there is increased ROS level in psoriasis, increased
NO level [16] opposes the apoptosis-triggering effect of
ROS by inducing heat-shock proteins [4].
The elevated oxidative stress biomarkers in psoriasis
patients causes activation of phospholipase A2, production
of many mediators by arachidonate, deactivation of adenylate cyclase and activation of guanylate cyclase leading to
decrease in the cAMP/cGMP ratio responsible for epidermal proliferation in patients of psoriasis [44].
Further induction of oxidative stress in psoriasis patients
by MTX may be due to its inhibition of dihydrobiopterin to
tetrahydrobiopterin reduction, thus causes the elevated
ROS levels which in turn activates JNK, that leads to
increased sensitivity of apoptosis [45] in proliferating

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Clin Exp Med

keratinocytes. Studies by Darren C Phillips et al. 2003 [26]

showed that the elevation in peroxide is responsible to the
cellular responses and functional consequence of MTX
treatment. The increase in MDA and tissue total ROS in
psoriasis patients by MTX proves that MTX stimulate
ROS-induced apoptosis in proliferating keratinocytes.
Our results showed increased serum nitrite and nitrate
levels in psoriasis patients compared to healthy controls.
Orem et al. [16] and Gokhale et al. [46] also observed
increased NO production in psoriasis patients and also
showed a significant correlation with PASI. This may be
due to the overexpression of iNOS mRNA in psoriatic
lesions [47]. In 2009, Cals-Grierson and Ormerod [48]
suggested that NO stimulates the epithelial cells and
releases chemokines and growth mediators which are
important for the proliferation and angiogenesis of
keratinocytes.
We observed that after MTX treatment, the levels of
serum nitrite and nitrate were reduced in psoriasis patients.
MTX inhibits NO by means of constitutive and/or inducible NO synthases inhibition [4952]. MTX decreased NO
levels, thereby causing ROS-induced apoptosis of proliferating keratinocytes in psoriasis.
Our results showed that plasma SOD, TAC and serum
catalase activities were significantly decreased in psoriasis
patients, as compared to healthy controls. Decreased SOD
activity would lead to buildup of H2O2. H2O2 generated
through other processes have also been reported to induce
CAT inactivation [53]. These might be related to epidermal hyperproliferation, because the ROS are thought to
induce cell proliferation in keratinocytes. Also decreased
SOD activity observed in the present study might develop
inflammatory and immune processes [54, 55]. MTX
therapy did not have an impact on the decreased levels of
plasma SOD, TAC and serum catalase in psoriasis
patients.
We observed that the protein expression of caspase-3
was downregulated in lesional compared to nonlesional
skin biopsy. This may be due to increase in NO levels; NO
inhibit apoptosis by nitrosylating essential components of
the apoptosis machinery such as caspases [56]. TNF alpha
increases production of nitric oxide, which causes partial
inactivation of caspase-3 [57].
Our results showed that MTX treatment causes upregulation of caspase-3 in psoriasis patients compared to lesional skin biopsy. This may be due to decrease in NO
level; thereby, it decreases nitrosylation of caspases and
thereby facilitates apoptosis. Methotrexate also decreases
TNF alpha [58] and NFKB levels [59] thereby activates
JNK through transcriptional factor GADD45beta. JNK
directly activates Bid causing cytochrome c release [60]
and caspase-3 activation in proliferating keratinocytes. To
conclude, MTX induce apoptosis through oxidative stress

by reducing NO and increasing caspase-3 levels. MTXinduced apoptosis may account for the beneficial effect of
MTX treatment in psoriasis patients, which is characterized
by acanthosis.
Acknowledgments The authors greatly acknowledge SRM University for their financial support.
Conflict of interest
interest.

The authors state that there is no conflict of

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