Rewiring of chemically modified MAPK pathway and its

downstream targets by rosmarinic acid in mice dermal cancer

R. SHARMILA1, K. GHOSH2, S. MANOHARAN1* 1 Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar

608002, Tamilnadu, India. 2 Unit of Toxicology, Department of Zoology, School of Life Sciences, Bharathiar University,

Coimbatore 641046, Tamilnadu, India.

*Corresponding author: Dr. S. Manoharan,

Associate Professor

Department of Biochemistry and Biotechnology, Annamalai University,

Annamalainagar 608002, Tamilnadu, India,

Telephone: + 91-4144-239141 (Extn. 230), Fax: +04144 238 080,

E-mail: sakshiman@rediffmail.com

Abstract

Rewiring of mitogen activated protein kinase (MAPK) signaling pathway and its downstream

targets by the natural compounds is renowned as a valuable approach to hinder the cancer

development. In the present study, we aimed to investigate the mechanisms of rosmarinic acid (RA)

on the expression pattern of MAPK signaling proteins and its downstream targets in mice dermal

cancer and docking interaction of RA with ERK-2 protein, which have not been elucidated

previously. Dermal cancer was induced at the depilated back of mice by applying DMBA (20µg in

0.1 ml acetone) twice a week for 8 weeks. Histopathology was used to examine the deformities in

dermal layers. Immunohistochemical analysis, ELISA and RT-PCR were utilized to observe the

expression profile of p-ERK1/2, p-JNK1/2, p-p38, c-fos, PCNA, NF-κB p65, COX-2 and iNOS. The

interaction of RA with ERK-2 protein was performed by molecular docking study. Massive

histological deformities were observed in DMBA treated dermal tissues from different weeks (8th,

14th, 17th, 21st & 25th weeks) and the intense immuno expression of p-ERK1/2 protein was

observed in these periods. RA (100 mg/kg bw) could significantly reduce the DMBA induced

expression of p-ERK1/2, p-JNK1/2, p-p38, c-fos, NF-κB p65, COX-2 and iNOS in dermal tissues.

Furthermore, the inhibitory effect of RA on ERK-2 protein was confirmed by molecular docking

analysis. From the results of this study, we could confer that RA inhibits ERK-2 activation, thereby

rewiring the DMBA-induced MAPK signaling cascade in mice dermal cancer.

Key words: Dermal cancer, MAPK signaling, Proliferation, Rosmarinic acid.

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Introduction

Dermal cancer is the most common cancer diagnosed with one in every three cancers.

Globally, the incidence of non-melanoma and melanoma dermal cancer has been rising up to 2 to

3 million and 132 thousand per year, respectively. [1] Every day, the skin is exposed to

accumulation of stresses includes exposure to UV (ultra violet) induced tissue damage, noxious

agents and microorganisms, which might lead to dermal cancer. [2] The physiological alterations

of melanocytes by the chronic exposure of UV enhance the secretion of paracrine acting factors,

which stimulate the activation of ERK/ MAPK (extracellular-signal regulating kinase/ mitogen

activated protein kinase) pathway. [3,4] This pathway plays a pivotal role in balancing the

differentiation and proliferation of melanocytes and its unregulation was found in patients with

90% of melanomas. [5] The aberrant activation of the MAPK pathway targeting the AP-1

(activator protein-1) transcription factor (c-jun and c-fos), where it targets the downstream of

ERK, which plays a major role in the protection of melanoma cells. [6]

The persistent ERK activation enhances the expression of various cytokines, which are

the activators of nuclear factor-κB (NF-κB). Upregulation of NF-κB modulate cell proliferation

and cell death, which was linked with melanoma tumor progression; [7] further, this drives the

constitutive expression of melanoma promoting genes, such as cyclooxygenase-2 (COX-2),

inducible nitric oxide synthase (iNOS) and proliferative cell nuclear antigen (PCNA). [8,9]

These overall alterations by the MAPK pathway favors such modifications in DNA and nuclear

proteins, thereby protect tumor cells from apoptosis and allows cancer cell growth and survival.

[10]

In the majority of cancer therapies, the drug-resistance involving inability to rewiring the

MAPK pathway, which leads to disease progression. [5] Moreover, the treatment with some

therapeutic agents and small molecule inhibitors to target MAPK pathway has ended in

secondary malignancies production. [11] So, there is an urgent need to target and rewiring the

MAPK pathway by utilizing the natural compound might be a great opportunity to inhibit dermal

cancer. Rosmarinic acid (RA, C18H16O8) is one of the high constituents of Orthosiphon

stamineus, [12] has been widely used as a food preservative and is shown to be safe due to its

various pharmacological properties. [13] Lee et al., (2008) tested the usefulness of RA in the

treatment of atopic dermatitis. [14] In our previous dermal cancer experiment, RA favorably

normalized the DMBA-induced changes in the antioxidant status, tumor suppressor gene and

apoptotic markers. [15] These findings are quite intriguing, and the present study has

investigated that RA inhibits the tumor promotion via an abrogation of the MAPK signaling

pathway in the DMBA induced mice dermal cancer model.

Materials and Methods

Chemicals

Rosmarinic acid, DMBA, Trizol reagent, primers for GAPDH, iNOS and COX-2, Red

Taqman Amplification Kit and 3,3′-diaminobenzidine were purchased from Sigma-Aldrich

Chemical Pvt. Ltd., USA. Antibodies for NF-κB p65, PCNA, p-p38, p-JNK1/2 and p-ERK1/2,

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were purchased from Santa Cruz Biotechnology, USA. Cayman’s COX activity assay kit was

procured from Cayman Chemical (Ann Arbor, MI, USA) and Mouse Proto-oncogene c-fos ELISA

kit was obtained from My BioSource, Inc (San Diego, CA, USA). Power BlockTM reagent and

goat anti-mouse IgG-HRP polyclonal antibody were obtained from BioGenex (San Ramon, CA,

USA).

Experimental Animals

Healthy male Swiss albino mice (4–6 weeks old; weighing 15-20 g) were purchased from

the National Institute of Nutrition, Hyderabad, India and maintained in the Central Animal

House, Annamalai University. The animals were housed in polypropylene cages and were

provided with a standard pellet diet and water ad libitum and maintained under controlled

conditions of temperature (22+ 2ºC) and humidity (65-70%), with a 12 h light/dark cycle. The

animal treatment and protocol employed was approved by the Institutional Animal Ethics

Committee (Registration number 160/1999/CPCSEA; Proposal No. 698), Annamalai University.

The animals were kept in compliance with the “Guide for the care and use of laboratory animals”

and committee for the purpose of control and supervision on experimental animals.

Experimental Design

A total number of 24 male Swiss albino mice were randomized into 4 groups of 6 mice

each. The depilatory cream was applied from the back of each mouse to remove hair, and the

experimental study was started after two days. The dermal carcinogenesis was induced by the

method of Azuine and Bhide (1992). [16] The depilated back of groups I and II mice were painted

with DMBA (25 µg in 0.1 ml acetone/mouse, twice a week for 8 weeks). In addition to DMBA,

group II mice were orally administered with RA (100 mg/kg bw in 1 ml distilled water, thrice a

week for 25 weeks) starting 1 week before the DMBA treatment. Group III mice were orally

administered with RA alone throughout the experimental period. The depilated back of group IV

mice were painted with acetone (0.1 ml/mouse, twice a week for 8 weeks) and served as the

vehicle control. At the end of 25 weeks, all the animals were sacrificed by cervical dislocation.

Histopathology

Tumor tissues were fixed in 10% formalin and embedded in paraffin. 2-3µm sections were

cut using a rotary microtome and stained with hematoxylin and eosin.

Enzyme Linked Immuno Sorbent Assay (ELISA)

Activities of COX-2 and c-fos in the dermal tissues were measured by using Cayman’s COX

activity assay kit and USCN c-fos activity assay kit respectively according to the manufacturer’s

instructions.

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Immunohistochemistry

Dermal tissues from control and experimental animals in each group were fixed and

embedded in paraffin wax from buffered formalin (10%). Sections (2–3 µm) of tissues were

made using a rotary microtome and placed on polylysine coated clean glass slides for drying at

37°C and then it was used for immunohistochemical studies. Paraffin-embedded tissue sections

were deparaffinised and rehydrated through graded ethanol with distilled water. Endogenous

peroxidase was blocked by incubation with hydrogen peroxide (3%) in methanol for 10 min. The

antigen retrieval was achieved by microwaving in citrate buffer solution (pH 6.0) for 10 min,

followed by washing step with Tris-buffered saline (pH 7.6). The tissue sections were then

incubated with power BlockTM reagent, a universal proteinaceous blocking reagent, for 15 min at

room temperature to block the non-specific bindings. These tissue sections were then incubated

with the specific primary antibody (p-p38, p-ERK1/2, p-JNK1/2, PCNA & NF-κB p65)

overnight at 4°C. The bound primary antibody was then incubated with the secondary antibody

that is conjugated with horseradish peroxidase at room temperature for 30 min. After rinsing

with the Tris-buffered saline, the antigen-antibody complex was detected using 3, 3´-

diaminobenzidine, which is the substrate of the enzyme horseradish peroxidase. When the

acceptable color intensity was reached, the slides were washed with counter-stain hematoxylin,

and was then washed, dried and covered with a mounting medium, and viewed under a

microscope.

RT-PCR

The total cellular RNA was extracted from the dermal tissue using Trizol reagent. The

concentration and purity of RNA preparation were checked by using a Nano spectrophotometer

(Implen, Germany) measuring the absorbed at 260 and 280nm. Total RNA (2.0 µg) was reverse

transcribed to cDNA in a reaction mixture containing 1µl of oligo (dT) primer (0.2 µg / ml), 1 µl of

RNase inhibitor (10 U / ml), 1µl of 0.1 M DTT, 4µl of 5x reaction buffer, 2 µl of 30 mMdNTP mix

(7.5 mM each), 0.5 µl of M-MuLV reverse transcriptase (50 U/µl) and made up to 20 µl with DEPC

water and kept at 37°C for 1h and then heated at 95°C for 2 min. The primer sequences for iNOS,

COX-2 and GAPDH were as follows: iNOS: 5'-GTGTTCCACCAGGAGATGTTG-3' as forward

and 5'-CTCCTGCCCACTGAGTTCGTC-3' as reverse, COX-2: 5'-

TTCAAAAGAAGTGCTGGAAAAGGT-3' as forward and 5'-

GATCATCTCTACCTGAGTGTCTTT-3' as reverse and GAPDH:

5'-AATGCATCCTGCACCACCAA-3' as forward and 5'-GTAGCCATATTCATTGTCATA-3'

as reverse primer. Reactions were run in a total volume of 50 µl, including 25 µl PCR reaction

mix (RED Taq-Ready Mix), 2 µl of each primer at 10 µM concentrations, 5 µl of cDNA and 16

µl of DEPC water. The PCR conditions used were as follows: 95°C for 5 min, 40 cycles of 30

sec at 95°C, 30 sec at 52 to 60°C (based on the target), and 60 sec at 72°C.

All PCR products were run on 1.5% agarose stained with ethidium bromide and

visualized by UV transilluminator. Semi-quantitative determination of PCR products was

performed using the gel documentation system (UV Tech, Genie). The relative expression of

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each studied gene (R) was calculated using the following formula, R = densitometrical units of

each studied gene/densitometrical units of GAPDH.

Molecular Docking Analysis

Grid-Based Ligand Docking With Energetics (Glide) was used for docking studies, which

is a part of Schrodinger (version9.3.518) suites, with a system configuration of Intel (R) Xeon

(R) Quad-Core CPU E3-1225 V2 @3.20 GHz with Linux Operating system. For ligand and

protein preparation, Maestrov9.0 Graphical User Interface (GUI) workspace was used. The

ligand structure used for the docking calculation was retrieved as Mol file from the ChemSpider

Database that contains information on small molecules.

LigPrep module of Schrodinger suites was used for developing various tautomers of the

ligand molecule. The protein structure for ERK2 (PDB ID: 1TVO) was retrieved from Protein Data

Bank. The PDB structure was edited by removing water molecules and by adding polar hydrogen

atoms for satisfying their apt valancies. The edited protein structure was then prepared using the

Protein Preparation Wizard in the Schrodinger suites, which utilizes the Optimized Potential for

Liquid Simulations-All Atoms (OPLS-AA) force fields for energy minimization. Following

protein preparation, Grid preparation was carried out for assigning the appropriate area of

interest for ligand-protein docking. The ligand docking was carried out using extra-precision

(XP) mode. The docking results were visualized using Maestro 10.3 visualizer and Discovery

studio 4.1 visualizer (DassaultSystèmes BIOVIA, Discovery Studio Modeling Environment,

Release 4.1, San Diego: DassaultSystèmes, 2013).

Statistical Analysis

All the statistical analysis were performed using one-way analysis of variance (ANOVA),

that was followed by Duncan's multiple range test (DMRT) in SPSS version 17.0 for Windows

(SPSS, Tokyo, Japan). All values are represented as mean ± SD (n=6) and p < 0.05 were

considered statistically significant.

Results

Dermal cancer study

Histopathological observations

DMBA treated mice from different weeks and histopathological manifestations in dermal

tissues were depicted in Fig. 1 (a-r). Histopathological observations revealed that DMBA treated

animals from different weeks (8th, 14th, 17th, 21st & 25th weeks) exhibited step-by-step massive

histological deformities in dermal layers, characterized by dysplasia, hyperplasia, infiltration of

inflammatory cells, hyperchromasia, loss of polarity, nest of well-differentiated squamous cells,

increase mitotic activity, keratin pearl formation, exophytic papillomatous projections, keratin

plugs formation within the tumor crypts and invasion of the perineural space.

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Immunohistochemical analysis of p-ERK1/2

The immunohistochemical patterns of p-ERK1/2 in the dermal tissues of tumor induced

mice were shown in Fig. 1 (s-v). Intense staining of p-ERK1/2 expression was observed in

DMBA painted dermal tissues from different weeks (8th, 14th, 17th, 21st & 25th weeks), which

suggested that the constant activation of ERK1/2 protein was participated in the different stages

of dermal tumor, ranging from initiation to progression. So, this work further analysing the effect

of RA on ERK protein by using immunohistochemical and molecular docking studies, thereby

evaluating the anti-cancer potential of RA.

After DMBA Application

8th week

14th week

17th week

21st week

j a

s k c b l

t n m e d

u p o g

f

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25th week

Fig. 1 a-i DMBA treated mice from different weeks (8th, 14th, 17th, 21st & 25th weeks). j-r

histopathological manifestations in DMBA treated dermal tissues from different weeks (8th, 14th,

17th, 21st & 25th weeks) shows step-by-step massive histological deformities. s-v Representative

photomicrographs of immunohistochemical pattern of p-ERK1/2 in the dermal tissues of tumor

induced mice from different weeks (8th, 14th, 17th, 21st & 25th weeks).

DMBA alone

(25µg in 0.1 ml

acetone/mouse)

DMBA+RA

(100 mg/kg bw)

RA alone

(100 mg/kg bw)

Control (0.1 ml

acetone/mouse)

Immuno

Expression

p-p38

p-ERK1/2

p-JNK1/2

COX-2

v r q i h

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Chemoprevention study

Effect of RA on immunoexpression of phosphorylated MAPKinases, PCNA and NF-κBp65,

Immunohistochemical evaluation (Figs. 2a & 2b) in the dermal tissues showed increased

expression of p-p38, p-JNK1/2, p-ERK1/2, PCNA and NF-κB p65 in mice painted with DMBA

(Group I) as compared to control mice (Group IV). Oral administration of 100 mg/kg bw of RA

pretreatment to DMBA treated mice (Group II) resulted in a significant (p < 0.05) decrease in the

immunoreactivity of these proteins compared to DMBA-treated mice. No apparent differences

were observed between the control (Group IV) and RA alone treated (Group III) mice.

Fig. 2 a Representative photomicrographs of immunohistochemical staining of NF-κB p65,

PCNA, p-JNK1/2, p-ERK1/2 and p-p38 in mice dermal tissues (20x). b Bar diagram depicts the

intensity of immunoreaction. The immunoreaction was measured by the image analyzing system

and recorded with the range from 0 to 10. Data shown are mean ± SD of six independent

experiments. #Values differ significantly (p < 0.05) from control. *Values differ significantly (p

< 0.05) from DMBA treated.

Fig. 2 a Representative photomicrographs of immunohistochemical staining of NF-κB p65,

PCNA, p-JNK1/2, p-ERK1/2 and p-p38 in mice dermal tissues (20x). b Bar diagram depicts the

intensity of immunoreaction. The immunoreaction was measured by the image analyzing system

# #

#

##

*

*

*

*

*

0

1

2

3

4

5

6

7

8

9

10

NF-κB p65 PCNA p-p38 p-ERK1/2 p-JNK 1/2

Imm

nu

no

reacti

on

DMBA DMBA + RA RA Control

NF-κB p65

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and recorded with the range from 0 to 10. Data shown are mean ± SD of six independent

experiments. #Values differ significantly (p < 0.05) from control. *Values differ significantly (p

< 0.05) from DMBA treated.

Effect of RA on COX-2 and c-fos

The levels of COX-2 and c-fos in the dermal tissues of control and experimental mice

were evaluated by ELISA method (Figs. 3a & 3b). DMBA painted mice (Group I) showed a

significant (p < 0.05) increase in the levels of COX-2 and c-fos as compared to control mice

(Group IV). Pretreatment with RA has significantly (p < 0.05) decreased the levels of COX-2

and c-fos (Group II). There was no significant difference between the control (Group IV) and RA

alone treated (Group III) mice.

Fig. 3a Fig. 3b

Fig. 3 a & b Effect of RA on levels of COX-2 and c-fos in the dermal tissues of control and

experimental animals respectively (ELISA method). Data shown are mean ± SD of six

independent experiments. #Values differ significantly (p < 0.05) from control. *Values differ

significantly (p < 0.05) from DMBA treated.

Effect of RA on mRNA expression of COX-2 and iNOS

The mRNA expression pattern of COX-2 and iNOS in the control and experimental mice

was depicted in Figs. 4a & 4b. DMBA treatment (Group I) induced a significant increase in the

mRNA expression of COX-2 and iNOS when compared to control mice (p < 0.05). RA (100

mg/kg bw) pretreatment significantly reduced COX-2 and iNOS mRNA expression in Group II.

Similar patterns of mRNA expressions were observed in the control and RA alone treated

groups.

#

*

0

2

4

6

8

10

12

14

16

18

20

DMBA DMBA +RA

RA Control

nm

ol/

min

/ m

l

COX-2

#

*

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

DMBA DMBA +RA

RA Control

ng

/ m

l

c-fos

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Fig. 4 a Effect of RA on COX-2 and iNOS mRNA expressions in the dermal tissues of control

and experimental animals. b COX-2 and iNOS mRNA expression was normalized to the

expression level of the GAPDH mRNA expression. The changes relative to control are

represented in the bar diagram. Data shown are mean ± SD of six independent experiments. #

Values differ significantly (p < 0.05) from control. *Values differ significantly (p < 0.05) from

DMBA treated.

Molecular interaction of RA with ERK-2

Fig. 5 (a-d) shows the docking analysis of RA with ERK-2 protein. The interaction

between RA and ERK-2 involved two H-Bond with SER 153 (Bond length: 1.70, 1.74) and ASP

111 (Bond length: 1.82, 1.89), one H-Bond with LYS 54 (Bond length: 1.76) and GLN 105

(Bond length: 2.16), along with one salt bridge with LYS 54 (Bond length: 4.56), one pi-alkyl

interaction with LEU 156, ILE 31, VAL 39 (Bond length: 4.88, 5.29,5.43) and pi-sulfur

interaction with CYS 166 (Bond length: 4.24). The binding free energy obtained for RA for the

target protein was found to be -8.745. Moreover, this docking analysis revealed the interactions

##

* *

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

COX-2 iNOS

fold

ch

an

ge

DMBA DMBA + RA RA Control

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of RA with three amino acid sequences (GLN 105, LEU 156, CYS 166) of ATP pocket in the

ERK-2 protein. Thus, RA can inhibit the activation of ERK-2 protein by inhibiting the ATP binding

site.

Fig. 5a

Fig. 5b

Fig. 5c

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Fig. 5d

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Discussion

There is a growing research evidences that proved the inhibition of MAPK pathway are

valuable for preventing cancer. [5,6] The activation of ERK1/2 simultaneously participates in the

migration and the speed of invasion in B‑cell chronic lymphocytic leukemia and dermal cancer

as well. [10,17,18] Increased ERK activity in tumor cells might be responsible for the malignant

transformation, which suggested that the ERK protein was frequently participated in the different

stages of tumor, ranging from initiation to progression. In the present study, massive histological

deformities were observed in DMBA treated dermal tissues from different weeks (8th, 14th, 17th,

21st & 25th weeks). The strong immuno expression of p-ERK1/2 protein was observed in these

periods suggests that phosphorylation and activation of ERK1/2 participated in all deformities of

dermal cancer development and progression; this includes proliferation, survival, hypoxia,

invasion and angiogenesis. So, this work further analysing the anti-tumor effect of RA on

DMBA induced phosphorylated form of ERK1/2, JNK1/2 and p38, c-fos, PCNA, NF-κB p65,

COX-2 and iNOS expression.

Screening the agents from natural sources that interfere with the intracellular signaling

mechanisms governing the transcription of MAPK represents a useful surrogate biomarker and

considered to be a potential anti-cancer agent. Scheckel and their coworkers (2008) demonstrated

that RA antagonized the activator protein-1-dependent activation of COX-2 expression in human

cancer. [19] Both in colon cancer HT-29 cells and breast cancer MCF-7 cells, RA suppressing

the activation of ERK1/2 by repressed the binding of c-jun and c-fos. [20,21] In human

leukemia U937 cells, RA reportedly inhibited the tumor necrosis factor-alpha induced NF-κB

activation by suppressing the NF-κB phosphorylation and NF-κB-dependent anti-apoptotic

proteins. [22] Our data is paralleling those of these investigations, reporting that the treatment of

DMBA induced dermal carcinogenesis with 100 mg/ kg bw of RA suppressed the NF-κB p65,

COX-2, iNOS and PCNA expression by blocking the activation of MAPK family proteins and

activator protein c-fos.

Lee et al. (2016) have investigated in HaCaT cells indicated that ERK1/2 pathway was

needed to activate the p38 and the AP-1, which was required for COX-2 up-regulation, which

disrupt skin barrier function. [23] Scientific evidences revealed an association between the ERK

activity and increased nuclear translocation of NF-κB and subsequent DNA binding in various

cell types. [19] Moreover, the activation of ERK-1/2 influences VEGF (vascular endothelial

growth factor) expression, which is responsible for angiogenesis of tumor cells. Thus, inhibition

of ERK1/2 results in the diminution of microvessel formation. [24] The enhanced expression of

PCNA, an epidermal proliferative marker has downstream effects on the signaling activation of

MAPK/ERK1/2 and thus strongly associated to the circumstance of cellular proliferation. [25]

Both in in vitro and in vivo, RA modulated the ERK signaling pathway, which inturn inhibited

tumor metastasis in colon carcinoma cells. [26,27] However, in the present study, rosmarinic

acid pretreatment markedly inhibited the MAPK family protein phosphorylation and PCNA

expression, which further inhibited the tumor growth and regression by stimulating apoptotic cell

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death. The apoptotic potential of rosmarinic acid was proved by our previous study. [15] In

addition, the docking analysis of ERK-2 protein with RA in the present study revealed the

presence of conventional hydrogen bonds (H-Bond), salt bridge, pi-alkyl and pi-sulfur interactions.

Furthermore, this study shows the interaction of RA with the ATP binding pocket (GLN 105,

LEU 156, CYS 166) of ERK-2 also validates the efficiency of RA to control cancer. In-silico

approach from the previous study supports the effect of RA on COX-2 inhibition through

hydrogen bonding with COX-2 active site residues (ARG 120 and SER 353). [28] In the light of

previous findings, the present study suggests that RA might exert its anticarcinogenic effect

through its anti-inflammatory and anti-proliferative effects.

Marnett et al. (2000) studied that a targeted deletion of the iNOS gene resulted in a

significant reduction in peroxynitrite and decrease in prostaglandin production. [29] Further, it

was strongly proved in mouse skin model that the topical application of the peroxynitrite

releasing compound SIN-1 significantly increased the COX-2 expression through NF-kB

activation [30] and was associated with increased metastatic and invasive behavior of cancer

cells. [31] Therefore, these findings suggested that peroxynitrite increased the COX2 induced

skin inflammation. Our findings further demonstrate that the rosmarinic acid inhibits DMBA-

induced expressions of COX-2 and iNOS in mouse skin through suppression of the

phosphorylated form of upstream signaling enzymes such as ERK, p38 and JNK, c-fos, PCNA

and NF-κB activation, which might represent the molecular mechanisms underlying the anti-

tumor promoting effect of rosmarinic acid. Furthermore, the docking evidence strongly suggested

that RA has the potential to rewiring the DMBA altered MAPK pathway, thereby inhibit mice

dermal cancer (Fig. 6).

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Fig. 6

Conclusions

The results of the present study thus reveal that RA at a dose of 100 mg/kg b.w inhibited

oncogenic MAPK signaling to abrogate dysregulated inflammation and abnormal cell proliferation,

which in turn prevented DMBA-induced mice dermal carcinogenesis. These promising results

might unveil a potential research field for rosmarinic acid and its clinical applications. Further

studies are warranted to elucidate the anticancer effect of RA on the mechanism of angiogenesis

and metastasis of dermal tumor cells.

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Fig. 5 a Photographs shows the docking analysis of ERK-2 (protein) with RA (ligand). b

Molecular docking analysis represents RA form hydrogen bond with SER 153, ASP 111, LYS 54

and GLN 105 of ERK-2. c & d Shows the number of bonds, bond length and aminoacids involved

in the docking analysis.

Fig. 6 Schematic representation for rewiring of chemically modified mitogenic signals in mice

dermal tissues by rosmarinic acid.

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