Role of Acetylsalicylic acid on Cyclooxygenase-2 expression in UVB-irradiated mouse skin utilizing immunohistochemistry
A thesis Article Submitted to the College of Veterinary Medicine, University of Slemani in Partial Fulfillment of the Requirements for the Degree of Master science in pathology By Snur Mohammad Amen Hassan BVSc
Under Supervision of Assist Professor Dr. Nabil Salmo M.B., Ch. B., Msc (histopath) 2011
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Supervisor’s Certification I hereby certify that this thesis has been prepared under my supervision at the Department of pathology, College of Veterinary Medicine, University of Sulaimani, in partial fulfillment of the requirements for degree of MSc. in veterinary Pathology.
Supervised by Assist. Prof. Dr. Nabil Salmo Head –Department of Pathology and Forensic Pathology- School of Medicine-Faculty of Medical Sciences/Sulaimani University 25/ 11/2011
Department report In the view of the available recommendation, I forward this thesis for debate by the examining committee.
Dr. Nawzad Rasheed Head of pathology Department 27 / 10/2011 iii
Committee certification The examining committee, certify that after reading this thesis, have examined the student (Snur Mohammad Amen Hassan) in its contents and that, in our opinion, it meets the standard of a thesis for the degree of Msc in pathology.
Instructor Dr.
Professor
Dr. Ali H. Hassan
Dr. Entisar R. Al-kennany
Member
Member
8-12-2011
8-12-2011
Assist. Professor
Professor
Dr. Nabil A. Salmo
Dr. Hiwa B. Banna
Supervisor
Chairman
8-12-2011
8-12-2011
Resolution of the College Council Approved by the college committee graduate studies Professor. Dr. Aumaid Othman Dean of the College of Veterinary Medicin University of Sulaimani
29/ 2/2011
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Dedication To my parents, who are my sun and moon To my brothers To my sisters
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Abstract Ultraviolet type B (UVB) is a primary cause of skin cancer. UVB is a predominant cancer-causing agent; being able to initiate, promote and progress the development of skin cancer. During initiation, UVB can cause chromosomal alterations and mutations via direct DNA damage and/or production of reactive oxygen species. Tumor promotions occur via epigenetic effects, such as altered gene expression. Chronic exposure to UVB results in the induction of high levels of COX-2 expression in the skin. COX-2 has been determined to contribute to tumorigenesis and the malignant phenotype of tumor cells via increase production of prostaglandins, inhibition of apoptosis, increased angiogenesis,
invasiveness,
modulation
of
inflammation
and
immunosuppression. It is well established that Acetylsalicylic acid reduces the risk of skin cancer via inhibition of cyclooxygenase activity, the key enzyme in prostaglandin biosynthesis, inhibits angiogenesis, inhibits cell proliferation and induces apoptosis, which are considered to be an important mechanisms for their anti-tumor activity and prevention of carcinogenesis. To induce skin tumor in mice subjected to UVB exposure, to see the effect of Acetylsalicylic acid by reducing skin tumor and COX-2 expression and to demonstrate the applicability of IHC in evaluating the COX-2 protein expression in mouse skin and to see its up-regulation upon UVB exposure. A prospective study was conducted from October 10, 2010 through March 10, 2011 in the Veterinary Medicine Teaching Hospital and histopathology laboratory of Shorsh Hospital in Sulaimani Governorate. The experimental animal of this prospective study were an albino mice of the Mus musculus species, BALB/c strain. Fifty mice underwent this study and were divided into 3 groups; Group (1); 10 mice were considered as control group; Group (2); 20 mice were considered as exposure group (exposed to UVB light) and Group (3); 20 mice were considered as treatment group (exposed to UVB vi
light and treated with Acetylsalicylic acid). Mice were treated 4 days/week with Acetyl salicylic acid one week before UVB exposure. After this, the mice were treated with Acetylsalicylic acid and exposed to UVB light 4 days/ week throughout the study. Both groups (exposure and treatment) of mice were subjected to UVB irradiation 4 days/week for 20 minutes/day and for a period of 5 months. At the end of each month, incisional biopsies were taken from each group, for detection of epidermal changes or tumor develpoment in irradiated shaved area in control, exposure and treatment groups. All animals were anesthetized using general anesthetic drug (Xylazine-Ketamine). Biopsies were taken and then the tissue samples were fixed in 10% formalin, processed and embedded in paraffin blocks. Three sections of 5µm thickness were taken from each paraffin embedded tissue block. The first section was mounted on an ordinary slide for H&E staining for detection of any histological lesions. The second section was for Giemsa stain for mast cells counting while the third section was mounted on positively charged slide, then proceeding with the process of immunohistochemistry staining for COX-2. The COX-2 expression was scored by two observers and recognized as brown cytoplasm. The scores of COX-2 expression in exposure and treatment groups, as indicated by using the immunohistochemical stain, showed that the highest percentage of COX-2 expression in exposure group was 1+ with a frequency of 10(50%) and lowest percentage of COX-2 expression
was
3+ with a
frequency 3(15%), while the highest percentage of COX-2 expression
in
treatment group was 0 with a frequency of 9(45%) and lowest percentage of COX-2 expression was 3+ with a frequency of 1(5%). The effect of UVB on decreasing numbers of apoptotic bodies was highly significant in exposure group. While apoptotic bodies were increased in treatment group and there was a highly significant. The effect of UVB on increasing numbers of mast cells was vii
highly significant in exposure group. While mast cells were decreased in treatment group and there was a highly significant. It was concluded from this study that UVB-is one of the causative agents which induced or developed SK in mice, COX-2 expression is increased in SK, oral administration of Acetylsalicylic acid effectively prevents UVB-induced SK, Acetylsalicylic acid reduced epidermal changes and tumor development due to its capacity to increase apoptosis in proliferating UVB-damaged keratinocytes and UVB triggers or increased dermal mast cells.
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Aknowledgement ALLAH, the Almighty, embraced me with His blessings and guided me to the path to complete this work. I express my deep sense of gratitude and humble regards to my supervisor, Assit. Professor Dr. Nabil Salmo, pathology department, college of Medicine, University of Suialnmani, who I have been fortunate to work with. His timely guidance, suggestions and constant inspiration enabled me to complete this thesis. I am honored to express my gratitude and indebtedness to the Dean, Professor Dr. Aumaid Othman for permitting me to carry out this study. I express my special appreciation and attitude to Dr. Rzgar Sulaiman, former manager of Veterinary Teaching Hospital and all the staff of Veterinary Teaching Hospital for their support and help in carrying out my work. I express my profound sense of gratitude to my teacher Dr Sheelan, for her support and encouragement to carry out this study. My sincere thanks to my graduate college Dr Azad K. Saed for his help and kind cooporation during this study. I express my special appreciation and attitude to all staffs and technicians in the Histopathology lab in Shoresh Hospital for their support and particulary Dr. Michael Hughson, and Miss Wasan.
My heartful thanks to my parents, my sisters and my brothers and friends for their inspiration, goodwill and the support they have given me. Last, bu not the least, I am thankful to all thoser who helped me directly and indirectly in carring out my stud
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Subject
Page No.
Chapter One: Introduction Introduction Aims of the study Chapter two: Literature Review 2.1: Histology of Normal Skin 2.1.1: Introduction 2.1.2: Skin structure in human 2.1.3: Skin histology in albino mice 2.2: Light 2.2.1: Ultraviolet light (UV light) 2.2.2: Biological effects of exposure to UVR 2.2.3: Effects of UVB light on DNA 2.3: COX-2 2.3.1: Introduction 2.3.2: Structure of COX-2 2.3.3: Function of COX-2 2.3.4: Regulation of COX-2 expression 2.3.5: COX-2 and tumorigenesis 2.4: UVR induced skin tumors 2.4.1: Definition 2.4.2: Mechanisms of induction of skin tumor by UVB radiation and COX-2 expression 2.4.3: Types of epidermal tumors – induced by UVR 2.5: Acetylsalicylic acid 2.5.1: Introduction 2.5.2: Mechanisms of action and therapeutic uses 2.5.3: Role of Acetylsalicylic acid in skin tumor 2.6: Immunohistochemistry 2.6.1: Introduction 2.6.2: Immunohistochemistry methods 2.6.3: Applications of immunohistochemistry Chapter three: Materials and Methods 3.1: Animal model 3.2: Treatment group with Acetylsalicylic acid 3.3: UVB irradiation
1 3 4 4 4 5 5 6 9 10 11 11 12 13 17 19 23 23 23 29 33 33 34 35 43 43 44 46 47 47 48 x
3.4: Collection of samples 3.5: Materials 3.5.1: Equipments 3.5.2: Reagents and Solutions 3.6: Methods 3.6.1: Samples preparation 3.6.2: Immunohistochemistry staining Procedure 3.6.3: Giemsa staining procedure 3.7: Slide interpretation 3.7.1: H & E slide interpretation 3.7.2: Immunohistochemical scoring 3.8: Statistical analysis Chapter four: Results 4.1: Gross and microscopic finding 4.1.1: Control group 4.1.2: Exposed group 4.1.3: Treatment group (exposed to UVB and treated with Acetylsalicylic acid administration) 4.2: Mean number of apoptotic bodies/10HPF 4.2.1: Mean number of apoptotic bodies/10HPF in exposure Group 4.2.2: Mean number of apoptotic bodies/10HPF in treatment Group 4.2.3: Mean number of apoptotic bodies/10HPF in exposure and treatment groups 4.3: Mean number of mast cells/1HPF 4.3.1: Mean number of mast cells/1HPF in exposure group 4.3.2: Mean number of mast cells/1HPF in treatment group 4.3.3: Mean number of mast cells/1HPF in exposure and treatment groups 4.4: Correlation between apoptotic bodies and mast cells 4.4.1: Correlation between total number of apoptotic bodies/10HPF and mean number of mast cells/1HPF in exposure group 4.4.2: Correlation between total the number of apoptotic bodies/10HPF and mean number of mast cells/1HPF treatment group
48 48 48 49 50 50 50 52 53 53 53 54 55 55 55 56 71 71 73 75 77 77 79 81 84 84
85
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4.5: Immunohistochemical scoring of COX-2 expression 4.5.1: Effect of UVB on frequency of COX-2 expression scores and their percentages in exposure group 4.5.2: Effect of Acetylsalicylic acid on the frequency of COX2 expression scores and their percentages in treatment Group 4.5.3:- COX-2 expression scores and their percentages in exposure and treatment group Chapter five: Discussions, Conclusions and Recommendations Discussions Conclusions Recommendations References
86 86 89
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93 97 98 99
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Figure No. Titles Page No. 2-1 The ultraviolet (UV) component of the electromagnetic 8 spectrum. 2-2 Schematic representation of the COX-2 structure. 13 2-3 Cyclo-oxygenase enzymes in prostanoid synthesis 15 2-4 Schematic diagram illustrating the proposed mechanisms 18 by which C2 enhances LPS-inducible COX-2 expression. 2-5 NF-IL6-β is a bifunctional protein in COX-2 transcription. 19 2-6 COX-2 induced angiogenesis. 21 2-7 Proposed pathways for the anti-apoptotic effect of 22 PGE2/EP4. 2-8 A model for induction of skin cancer by UV. 25 2-9 A scheme of the NF-κB pathway. 27 2-10 Potential mechanisms of Aspirin -induced apoptosis. 37 2-11 Diagram illustrating how mitochondrial pathway proteins 39 Bax, Bid, and Smac integrate into the caspase cascade in Aspirin-induced apoptosis. 2-12 COX-2 inhibition by NSAIDs results in decreased VEGF 42 production, suppressed mitogenic response and vascular permeability in response to VEGF and inhibited integrinαVβ3 dependent Rac activation and endothelial cell migration. 4-1 Normal skin appearance in a mouse of control group. 59 4-2 Microscopic view of a skin section obtained from a mouse 59 of the control group. It shows normal histological appearance. H & E stain, (X 100). 4-3 Appearance of skin in mice of the different experimental 60 groups. A: Normal appearance of skin in a mouse of the control group. B: Thickening of the skin in a mouse of treatment group.C: Brown and tan keratotic plugs (scales or crusts) in a mouse of the exposure group. 4-4 Brown or tan colored, irregular lesions were seen on the 60 skin in a mouse of the exposure group (arrows) 4-5 A velvety to granular, light brown skin in a mouse of the 61 exposure group (arrow). 4-6 Microscopic view of skin section obtained from a mouse 61 of exposure group. It shows acanthotic SK as indicated by xiii
4-7
4-8
4-9
4-10
4-11
4-12
4-13
4-14
4-15 4-16
the marked acanthosis, papillomatosis, horn cyst and pseudo horn cyst. H & E stain, (X40). This is a higher magnification view of the tissue section illustrated in figure (4-6). It shows acanthotic SK as indicated by papillomatosis(fibrovascular cord in its center), horn cyst and pseudo horn cyst. H & E stain, (X100). Microscopical feature of acanthotic SK. It shows squamous eddies indicated by whorling aggregates of eosinophilic squamous cells in a mouse of exposure group (arrow). H & E stain, (X 400). Microscopic view of skin section obtained from mouse of exposure group. It shows clonal SK as indicated by welldefined multiple nests of basaloid cells. H & E stain, (X 100). This is a higher magnification view of the tissue section illustrated in figure (4-9). It shows the nests of basaloid cells are separated by strand of cells with small dark nuclei. H & E stain, (X 400). Sun burn cells were seen within epidermis in skin section obtained from a mouse of the exposure group (arrow). H & E stain, (X 400). Microscopic view of dermis in exposure group. It shows infiltration by inflammatory cells (arrows). H & E stain, (X400). Microscopic view of a skin section obtained from a mouse of the treatment group. It shows the presence of numerous mast cells (arrows) within dermal layer. (A) H & E stain. (B) Giemsa stain, (X 400). Microscopic view of a skin section obtained from a mouse of the exposure group. It shows the presence of numerous mast cells (arrows) within dermal layer. Giemsa stain, (100X). Presence of flat, rounded or slightly elevated skin lesions in a mouse of treatment group (arrow). Microscopic view of skin section obtained from a mouse of treatment group. It shows acanthotic SK as indicated by the marked acanthosis, horn cyst and pseudo horn cyst
62
62
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65
66 66
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4-17
4-18 4-19 4-20 4-21 4-22
4-23
4-24
4-25
4-26 4-27 4-28 4-29
(arrow). H & E stain, (X 100). Microscopic view of skin section obtained from a mouse of treatment group. It shows clonal SK as indicated by well-defined multiple nests of basaloid cells. H & E stain, (X 100). Thickening of the skin in a mouse of treatment group. Mild epidermal hyperplasia in skin of a mouse of treatment group. H & E stain, (X 400). Moderate epidermal hyperplasia in skin of a mouse of treatment group. H & E stain, (X400).
67
67 68 68
Severe epidermal hyperplasia in skin of a mouse of treatment group. H & E stain, (X400). Histological appearance of various degrees of hyperplasia in treatment group compared to skin of control group. A: Control group showed normal skin appearance, B: Mild hyperplasia, C: Moderate hyperplasia and D: Severe hyperplasia in mice of treatment group. H & E stain, (X100). Few sun burn cells were seen within epidermis in skin section obtained from a mouse of the treatment group (arrows). H & E stain, (X 400). Microscopic view of a skin section obtained from a mouse of the treatment group. It shows the presence of numerous mast cells (arrow) within dermal layer. Giemsa stain, (400X). Mouse had blindness in both eyes due to photoconjuctivits in exposure group.
69
Column chart showing mean number of apoptotic bodies/10HPF in mice of exposure group. Column chart showing mean number of apoptotic bodies/10HPF in mice of treatment group. Column chart showing mean number of apoptotic bodies/10HPF in exposure and treatment groups. Sun burn cells were seen within epidermis in skin section obtained from a mouse: (A). It shows one apoptotic bodies in exposure group. (B) It shows 5 apoptotic bodies in
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69
70
70
71
75 76 77
xv
4-30
4-31 4-32 4-33
4-34
4-35 4-36 4-37 4-38
4-39 4-40
4-41
4-42
treatment group (arrows). H & E stain, (400X). Chart column showing mean number of mast cells/1HPF in Clonal and Acanthotic SK cases in mice exposure group. Column chart showing mean number of mast cells/1HPF in treatment group. Column chart showing mean number of mast cells /1HPF in exposure and treatment groups. Microscopic view of a dermis of skin section obtained from a mouse stained by H & E: (A) It shows few number of mast cells in treatment group. (B) It shows huge number of mast cells in exposure group (arrows). (X100). Microscopic view of a dermis of skin section obtained from a mouse stained by Giemsa: A) It shows few number of mast cells in treatment group. (B) It shows huge number of mast cells in Exposure group (arrows). (X400). Score 1+ COX-2 expression in exposure group (X 400). Score 2+ COX-2 expression in exposure group (X400). Score 3+ COX-2 expression in exposure group (X 400). Pie chart showing the effect of UVB on the scores of COX-2 expression and their percentages in exposure group. Score 0 COX-2 expression in treatment group (X 400). Different immunohistochemical scores appearance of COX-2 expresssion in treatment group: (A) Score 0 COX2 expression (X 400). (B) Score 1+ COX-2 expression (X 400). (C) Score 2+ COX-2 expression (X 400). (D) Score 3+ COX-2 expression (X 100). Pie chart showing the effect of Acetylsalicylic acid on the scores of COX-2 expression and their percentages in treatment group. Column chart showing COX-2 expression scores and their percentages in exposure and treatment groups.
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81 82 83
83
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90 90
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List of Tables Table No.
Titles
Page No.
4-1
Number and percentages of benign seborrheic keratosis in exposure and treatment group.
58
4-2
Number and percentages of different degrees of hyperplasia in treatment group.
58
4-3
Histological feature of both types of SK in exposure group.
58
4-4
Mean number of apoptotic Bodies/10HPF in Clonal and Acanthotic SK cases in mice in exposure group Mean number of apoptotic bodies/10HPF in treatment group. Mean number of apoptotic bodies /10HPF in exposure and treatment groups. Mean number of mast cells /1HPF in Clonal and Acanthotic SK cases in mice of exposure group Mean number of mast cells /1HPF in treatment group.
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Mean number of mast cells /1HPF in exposure and treatment groups. Inverse correlation between total number of apoptotic bodies/10HPF and mean number of mast cells/1HPF in exposure groups. Inverse correlation between total number of apoptotic bodies /10HPF and mean number of mast cells/1HPF in treatment group.
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4-5 4-6 4-7 4-8 4-9 4-10
4-11
74 76 78 80
84
85
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6-4 PP
6-4 photoproduct
AA
Arachidonic acid
Ab
Antibody
ABC
Avidin–biotin complex
AK
Actinic keratosis
AKT
Protein kinase B
Akt
V- akt murine thymoma viral oncogen homolog
Apaf
Apoptosis protease-activating factor
APC
Antigen presenting cell
B7-2
(CD86) costimulation to T cells for proliferation
B7-I
(CD80) costimulation to T cells for proliferation
Bad
Bcl- XL /Bcl-2-associated death promoter
Bax
Bcl-2 -associated x protein
BCC
Basal cell carcinoma
Bcl-2
B-cell lymphoma 2 protein
C
Cytosin
C/EBP
CAAT/enhancer box binding protein
cAMP
Cyclic adenosine monophosphate
Cdc42
Interactive binding domain
CHS
Contact hypersensitivity reactions
CIE
International Commission on Illumination
C-JNK
C-Jun N-terminal kinase
COX
Cyclooxygenase
COX-2
Cyclooxygenase-2
CPD
Cyclobutane pyrimidine dimmer
CRE
cAMP response element
CREB
cAMP responsive element binding protein
DNA
Deoxyribonucleic acid
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ECM
Extracellular matrix
EGFR
Epidermal growth factor receptor
Egr
Early growth response
EMR
Electromagnetic radiation
EP
E-Prostanoid
ERK
Extracellular signal regulated kinases
FADD
Fas-associated via death domain protein
FGF
Fibroblast growth factor
GDP
Guanosine diphosphate
GSK
Glycogen synthase kinase
GTP
Guanosine triphosphate
H&E
Haematoxylin and Eosin
HPF
High power field
HPV
Human Papilloma virus
HRP
Horse radish peroxidase
IAP
Inhibitor of apoptosis protein
IFN-γ
Interferon-gamma
IgG
Immunoglobulin gamma
IHC
Immunohistochemistry
IkB
Inhibitors of kappa B
IKK
I kappa B kinase
IL
Interleukin
IR
Infrared
JAK
Janus-associated kinases
LAB
Labeled avidin–biotin
LC
Langerhans cell
LPS
Lipopolysaccharide
LSAB
Labeled streptavidin-biotin
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MAPK
Mitogen activated protein kinases
MBD
Membrane binding domain
MCV
Merkel cell polyomavirus
MM
Malignant melanoma
MMP
Matrix-metalloproteinases
NBCCS
Nevoid basal cell carcinoma syndrome
NFAT
Nuclear factor of activated T cells
NF-IL-6
Nuclear factor-interleukin -6
NF-κB
Nuclear factor κB
nm
Nano meters
NMSC
Non –Melanoma skin cancer
NSAID
Non steroidal anti inflammatory drug
p300
Coactivator p300 promoter gene
P38 kinase
Exhibit protein kinase activity
P53
Tumor suppressor gene
PAP
Peroxidase–antiperoxidase
PDGF
Platletes derived growth factor
PGD2
Prostaglandin D2
PGE2
Prostaglandin E2
PGF2
Prostaglandin F2
PGG2
Prostaglandin G2
PGH2
Prostaglandin endoperoxide H2
PGHS-2
Prostaglandin synthase-2
PGI2
Prostacyclin
PGs
Prostaglandins
PI3K
Phosphatidylinositol 3-kinase
PKA
Protein kinase A
PKC
Protein kinase C
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PPARγ
Peroxisome proliferator-activated receptor gamma
Ptc
Tumor suppressor gene
Rac
Interactive binding domain
Ras
Proto-oncogen Rat sarcoma
ROS
Reactive oxygen species
SBC
Sun burn cell
SCC
Squamous cell carcinoma
Ser
Serine
SIS
Skin immune system
SK
Seborrheic keratosis
T
Thymine
TAF
Activator transcription factor
TNF
Tumor necrosis factor
TNF-α
Tumor necrosis factor –alpha
TPA
Tumor promotor activator
TRAIL
Tumor necrosis factor-related apoptosis-inducing ligand
TXA2
Thromboxane A2
Tyr
Tyrosine
UCA
Urocanic acid
UPS
Ubiquitin proteasome system
UVA
Ultraviolet A
UVB
Ultraviolet B
UVC
Ultraviolet C
UVR
Ultra violet radiation
VEGF
Vascular endothelial growth factor
v-Ras
Oncogene protein involving in human neoplasms
v-Src
V- Src sarcoma (Schmidt- Ruppin- A2 ) viral oncogen homolog
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Chapter One Introduction
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INTRODUTION 1.1: Introduction Nonmelanoma skin cancer (NMSC) is the most common malignancy in fair-skinned people. In the United States alone, it is estimated that 1.2 million new cases of NMSC are diagnosed each year. The vast majority of NMSC are comprised of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), which arise primarily on sun-exposed regions of the body implicating ultraviolet (UV) radiation as the main etiological agent (Buckman et al., 1998; Wilgus et al., 2003). Skin cancer incidence has been increasing in recent years due to aging and higher levels of UV radiation reaching the surface of the earth as a result of ozone depletion. Even minor changes in the ozone are thought to have a substantial impact on UV carcinogensis as a 1% decrease in coloumn ozone has been calculated to result in a 1.56% increase in carcinogenic UV radiation peneterance and a concomitant increase in NMSC by 2.7% (Bachelor and Owens, 2009). UVB is a complete carcinogen, being able to initiate, promote
and
progress the development of skin cancer. However, numerous additional factors including gender, immunosuppressive status and vitamin D levels all contribute to the development of this most common cancer type (Bair et al., 2002; Bachelor et al., 2005; Oberyszn, 2008). Exposure to UV induces a number of pathological changes initiated in mammalian skin, including erythema, edema, hyperplasia, sunburn cell formation, immune suppression and changes in expression of numerous genes 1
associated with proliferation, differentiation and skin cancer (Ley and Reeve, 1997; Fischer et al., 2003). Chronic exposure to UV irradiation leads to the constitutive upregulation of cyclo-oxygenase-2(COX-2) expression in the skin.The involvement of COX2 in the carcinogenesis process is mediated by its increased production of its enzymatic primary product, Prostaglandin E2 (PG E2). PGE2 has been shown to have a variety of activities that can contribute to tumor development and growth (Lee et al., 2005; Tober et al., 2006; Rundhaug and Fischer, 2008a). COX-2 has been determined to contribute to tumorigenesis and malignant phenotype of tumor cells via the inhibition of apoptosis, increased angiogenesis, invasiveness, modulation of inflammation and immune suppression (Tjiu et al., 2006). Nonsteroidal anti-inflammatory drugs (NSAIDs) block the cyclooxygnase enzyme which are widely used as therapeutic agents for the treatment of pain and inflammation (Dannhardt and Kiefer, 2001; Limongelli et al., 2010).The effect of NSAIDs on chemoprevention and tumor regression has been shown in animal models, epidimologic studies and in treatment of patients (Moore and Simmons, 2000).
2
Immunohistochemistry or IHC refers to the process of localizing proteins in cells of a tissue section exploiting the principle of antibodies binding specifically to antigens in biological tissues (Ramos-Vara, 2005). Immunohistochemistry has surpassed other techniques in its effectiveness in the in-situ preservation and detection of antigens. Immunopathology has become a valuable or even an essential adjunct to diagnostic pathology. It is affirmed that diagnostic IHC is indispensable in surgical pathology for diagnosis, therapy and prognosis (Marcol et al., 2000).
Aims of the study:To induce skin tumor in mouse upon UVB exposure, to see the effect of Acetylsalicylic acid by reducing skin tumor and COX-2 expression in mice, to evaluate the COX-2 protein expression in mouse skin after UVB exposure and to demonstrate the applicability of IHC in evaluating the COX-2 protein expression in mouse skin and to see its up-regulation upon UVB exposure.
3
LITRATURE REVIEW 2.1: Histology of Normal Skin 2.1.1: Introduction The skin is the heaviest organ of the body, accounting for about 16% of total body weight and in adults representing 1.2-1.3m2 of
surface to the
external environment (Junqueira and Carneiro, 2003). Skin is a highly organized structure consisting of three main layers called the epidermis, the dermis and the hypodermis. Skin has several functions including; protection, sensation, thermoregulation,communication, vitamin D metabolism and also self repairing after injury (Benbow, 1995; Varani, 1998; Kessel,1996).
2.1.2: Skin structure in human (Junqueira and Carneiro, 2003; Topping et al., 2006).
A-Epidermis The epidermis consists mainly of a stratified squamous keratinized epithelium, but also contains three less abundant cell types; melanocytes, Langerhans cell, Merkels‟cells and inflammatory cells. From the dermis outward, the epidermis consists of five layers of keratin producing cells (keratinocytes); stratum basale (stratum germinativum), stratum spinosum (spinous or prickle cell layer), stratum granulosum, stratum lucidum and stratum corneum (horny layer).
B-Dermis The dermis is the connective tissue that supports the epidermis and binds it to the subcutaneous tissue (hypodermis). The major fibers of dermis are collagen fibers and elastin.The dermis contains two layers, the thin papillary layer is composed of loose connective tissue; fibroblasts and other connective 4
tissue cells and the reticular layer which is thicker and composed of irregular dense connective tissue layer.
C-Hypodermis (subcutis) The subcutis is the fat layer immediately below the dermis and epidermis. It is also called subcutaneous tissue, hypodermis or panniculus. The subcutis mainly consists of fat cells (adipocytes), nerves and blood vessels.
2.1.3: Skin histology in albino mice The microanatomy of mouse skin is similar to the skin of other furred animals but differs from the human skin. The mouse skin consists of an external epithelium (epidermis which consist of 2-3 layers), a thick layer of connective tissue (dermis) and a layer of adipose tissue (hypodermis or panniculus adiposus). A thin layer of striated muscle, known as panniculus carnosus, separates the skin from other structures. The mouse skin has no eccrine sweat glands; these are located in the footpad only. Melanin pigment is absent and the mouse does not have normal rete ridges where the lower aspects of epidermis form ridges of cells that extend into the dermis (Bloom and Fawcett, 1994; Hedrich et al., 2004).
2.2: Light Light is the narrow part of the wide electromagnetic spectrum that our eyes can perceive and produce visual sensation. The energy of the sun reaching the earth is known as electromagnetic radiation. Electromagnetic radiation (EMR) moves through space (not just “outer space”) but the
atmosphere,
buildings, eye lens, etc). EMR has a very wide spectrum reaching from ultra
5
short wavelength
(<10-14 m) for cosmic radiation to radio frequency where
waves can be kilometers long (Hill, 2009). The electromagnetic spectrum is divided into six major types of radiation, theses include radiowaves (including microwaves), light (including ultraviolet, visible and infrared), heat radiation, X-rays, gamma rays and cosmic rays. The spectrum of visible light started from deep purple; close to UV radiation to warm red; close to infra-red (IR) radiation (Halliday et al., 2001;Gibson, 2003). Radiation in general is everywhere, but is invisible and penetrating.The radiation deposits energy in the environment and so creates reactive chemical species known as free radicals (Reynolds and Schecker, 1995).
2.2.1: Ultraviolet light (UV light) UV radiation comprises the wavelengths from 200-400 nm which are shorter than that of visible light, but longer than X- rays; this unique portion accounts for 3% of all solar radiation reaching the earth. It is so named because the spectrum consists of electromagnetic waves with frequencies higher than those that human identify as the color violet (Soehnge et al., 1997; Lucas et al., 2006). Most UV light on earth comes from the sun. When UV light reaches the atmosphere, it reacts with oxygen molecules to create ozone. This reaction is what causes the ozone layer to form around the earth. Almost all of the short wave UV light is absorbed by the ozone layer before it can reach the earth‟s surface. It is classified by International Commission on Illumination (CIE) into three groups: (Soehnge et al., 1997; Pentland et al., 1999) (Figure 2-1). a- UVA (Long wave)
320-400nms
b- UVB (Medium wave)
290-320nms
c- UVC
200-290nms
(Short wave)
6
A. Ultraviolet A (UVA):UVA rays account for up to 95% of the UV radiation reaching the earth‟s surface nearly 100 times more than the amount of UVB reaching the earth (Tripp et al., 2003; Jung et al., 2008). UVA is not filtered out in the atmosphere. It is easily transmitted through air and glass. Its level remains constant throughout the day; 50% of UVA radiation respectively is absorbed into the stratum corneum and epidermis, but the rest penetrates deeply into the dermis (Parrish et al., 1982; Bruls et al., 1984). UVA which penetrates the skin more deeply than UVB has long been known to play a major part in skin-ageing and wrinkling (photo-ageing), it is capable of causing damage to collagen fibers and destroying vitamins A and D in skin. UVA tans and penetrates ocular media. 50% of solar UVA can reach the depth of melanocytes, whereas only 9-14% of UVB reaches that level. UVA produces oxidative DNA damage more efficiently than UVB as reactive oxygen species (ROS) and it radiation is said to be involved in the development of melanoma in fish (Soehnge et al., 1997; Clydesdale et al., 2001).
B. Ultra violet B (UVB):UVB is only partially blocked by the ozone layer and compromises approximately 1-10% of the UVR reaching the earth. Most of the UVB radiation is absorbed in the stratum corneum and epidermis and only 5-10% can reach basal keratinocytes and dermis. UVB does not pass through the glass and has highest intensity during noontime (Kaidbey et al., 1979; Bruls et al., 1984). Wavelengths in the UVB region of the solar spectrum are absorbed into the skin superficially, producing erythema, burns and eventually skin cancer. UVB erythema threshold is 1,000 times lower than the erythema threshold of the UVA and it is much more effective in causing damage to living tissue than UVA (Zigman et al., 1976; Javeri et al., 2008; Gao et al., 2010). 7
UVB is also known to upregulate genes expression through intracellular signal transduction pathways, which may contribute to developing skin cancer at the tumor promotion stage (Rünger, 2007; Rundhaug and Fischer, 2008a).
Ultraviolet C (UVC):UVC radiation however does not reache the surface of earth, since virtually all is absorbed by atmospheric ozone. UVC radiation emitted from artificial light sources can cause DNA damage in cells to the level of spinosum layer, but it does not penetrate the basale layer. UVC is efficiently absorbed by cellular and mitochondrial DNA and produce pyrimidine dimmers, such as (CPD) and (6-4 PP). UVC kills bacteria and is used in germicidal lamp (Potten et al., 1993; McKenzie, 1995).
Figure 2-1: The ultraviolet (UV) component of the electromagnetic spectrum (Soehnge et al., 1997).
8
2.2.2: Biological effects of exposure to UVR The biological effects of UVR depend on the wavelengths concerned. Sources emitting radiation with wavelengths longer than 200 nm are serious health hazards and since UVR has such low penetrating power, the effects are confined mainly to the eyes and the skin (Beitollahi and Farvadin, 2007).
I. Effect of UVR on the skin Exposure to UV rays can have both, positive and negative effects. The ultimate effects depend primarily on the level of exposure to UV rays the individual receives.
A- Positive effects: (Black and Gavin, 2006; Beitollahi and Farvadin, 2007; Engelsen, 2010). 1-The formation of vitamin D3 is the most significant of the positive effects which is used above all to prevent rickets. 2- UV light is also used for therapeutic purposes, such as in treating skin disease like psoriasis. 3- UV is also used for cosmetic purposes.
B- Negative effects: The negative effect on skin is of two types, acute and chronic. Acute effects appear within a few hours of exposure while chronic effects are longer lasting. Acute effects are sunburn and photoallergy; chronic effects are ageing of skin and skin cancer (DeFabo, 2004; Burford et al., 2005; Maier, 2008). 1- Sunburn (erythema): Inflammation of the skin that disappears after several days. This results in tanning (pigmentation) and thickening of the top-most epidermal (stratum corneum) skin layer which in turn increases the body‟s resistance against a renewed burn.
9
2- Photoxic reactions (photoallergy): The combination of UVR and certain chemicals (e.g; particular cosmetics and medication) can cause toxic reaction and trigger allergic reaction. 3- Skin ageing: Long term exposure to UVR can make the skin dry, leathery, rough, slack and wrinkly. 4- Skin cancer: Excessive and long term UV rays exposure can lead to skin cancer.
II.
Effects of UVR on eye (Young, 2006). 1- Inflammation of the cornea (keratitis) and photo- conjunctivitis: UV rays destroy the outer most cells of the cornea and /or the conjunctiva. This phenomena is known to mountain climbers as “snow blindness” and to welders as “flashing”. 2- Clouding of the lens (cataract): Long term exposure to UVR can lead to an irreversible clouding of the lens tissue.
2.2.3: Effects of UVB light on DNA DNA is one of the key targets for UV-induced damage in a variety of organisms ranging from bacteria to humans (Van der Leun and De Gruijl, 1993; Kumari et al., 2008). Among UVR, UVB is the most deleterious that induces two of the most abundant mutagenic and cytotoxic DNA lesions. UVB is absorbed directly by DNA to create mutagenic photoproducts or lesions in DNA between adjacent pyrimidines in the form of dimmers between adjacent thymine or cytosine residues, such as cyclobutane –pyrimidine dimmers (CPDs), 6-4 photoproducts (6-4 PPs) (Griffiths and Ling, 1993; Britt, 1995; Clydesdale et al., 2001 and Ichihashi et al., 2003). Dimer formation DNA lesion has been shown to occur in the epidermal basal and supra basal layers. Pyrimidine dimer formation is proposed as
an initiation step in mutagenesis and tumors 10
formation, because it was found to be closely linked to the generation of mutation in tumor suppressor genes expressed
in UV-induced skin cancer
(Stege et al., 2000; Carol and Trow, 2008).
2.3: COX-2 2.3.1: Introduction Cyclooxygenase-2 (COX-2) is induced as an immediate-early gene in most cells. Various extracellular stimuli, including growth factors, cytokines, tumor promoters, peroxisomal proliferators and carcinogens, induce COX-2 expression. Although transcriptional regulation of COX-2 has been studied extensively, post-transcriptional mechanisms are also important for COX-2 expression (liu et al., 2001). COX-2 is induced physiologically during the mitogenic response occurring in wound healing and, very significantly, has been found to be overexpressed in many types of pre-malignant and malignant neoplasms in humans and other organisms (Morre and Simmons, 2000). COX-2 has been determined to contribute to tumorigenesis and the malignant phenotype of tumor cells via increase production of prostaglandins, inhibition of apoptosis, increased angiogenesis,
invasiveness,
modulation
of
the
inflammation
and
immunosuppression (Dempke et al., 2001; Xu and Chun, 2002; Tjiu et al., 2006 ). Human genes for COX-2 map is located in chromosome 1(1q25.2–q25.3) (Taketo, 1998; Fosslien, 2001). Simillar to humans, mouse COX-2 map is also located on chromosome 1(Taketo, 1998). The cyclooxygenase isoforms, COX-1 and COX-2, are involved in the biosynthesis of prostaglandin E2, a major prostaglandin involved in epidermal homeostasis and repair (Hsi et al., 1999; Zhang and Bowden, 2008; Tripp et al., 2003). 11
COX-2 and prostaglandin E2 (PGE2) expressions in human skin are increased after UVB irradiation, especially in aged skin (Seo et al., 2003). Cultured human keratinocytes display increased COX-2 protein level and PG E2 excretion after UVB irradiation (Muller-Decker et al., 1995; Buckman et al., 1998). Experimentally, increases in COX-2 expression and proliferation of basal layer keratinocytes have been demonstrated in Skh-1 hairless mice after UVB irradiation (Fischer et al., 2003). PGE2 plays a key role in normal skin homeostasis, but it can also act as a tumor promoter, controlling many of the behaviors typical of cancer cells (Lupulescu, 1978). PGE2 can stimulate increased proliferation, altered adherence, increased migration and enhanced invasiveness of cancer cells (Vanderveen et al., 1986; Tsujii and DuBois, 1995; Buchanan et al., 2003 and Kawamori et al., 2003). The proposition that COX-2 is causally linked to cancer offers a new approach to extending our knowledge of neoplasia and improving treatment of the disease. This identification of an enzyme catalyzing fatty acid oxidation as a rate limiting step in the progress from normal cell growth through hyperplasia on to neoplasia has opened up a whole new field in cancer research (Ghosh et al., 2010).
2.3.2: Structure of COX-2 COX-2 proteins contain 587 amino acids, its 3D structure of COX-2 has been determined by X-ray crystallography. COX-2 enzyme is a homodimer. Each monomer comprises three domains; an N-terminal EGF-like domain, a membrane-binding domain and a C-terminus catalytic domain that comprises approximately 480 amino acids (~80% of the protein) and contains the two distinct catalytic sites – the COX and the peroxidase active sites. The EGF-like domain appears to be the dimerization domain holding the monomers together through hydrophobic interactions, hydrogen bonding and salt bridges (Smith et 12
al., 2000, Knights et al., 2011). Two important amino acids implicated in COX2 function tyr371 and ser516, are crucial for activity as in figure (2-2). COX-2 proteins are inactive until the tyrosyl radical is generated and hence this reaction is the rate-determining step in the initiation of the COX-2 catalytic cycle. During the course of the oxygenation of arachidonic acid, the tyrosyl radical is initially reduced to tyrosine, but is regenerated in the last step of the catalytic cycle by the peroxy radical precursor to PGG2. Regeneration of the tyrosyl radical is essential for continued oxygenation of arachidonic acid. The serine at the 516 is the site of Acetylsalicylic acid acetylation (Harris, 2003).
Figure 2-2: Schematic representation of the COX-2 structure (Harris, 2003).
2.3.3: Function of COX-2 At the beginning of the 1980s, many research laboratories speculated about the existence of more than one COX enzyme. Habenicht and colleagues reported in 1985 about two-peak induction in prostaglandin synthesis (Habenicht et al., 1985). This study was indicative of the existence of constitutive and inducible forms of COX enzyme. Subsequently, COX-2 was discovered in 1991, as a primary response gene (Kujubu et al., 1991; Xie et al., 1991).
13
The COX enzyme exists in two main isoforms, namely COX-1 and COX2. The two cyclooxygenase (COX) enzymes catalyze the conversion of arachidonic acid and production of PGs, prostacyclin and TXA2 via three steps: (i) Liberation of arachidonic acid (AA) from membrane phospholipids by the action of phospholipase enzymes A2; (ii) Arachidonic acid is then available to metabolism by cycloxygenase to form the unstable intermediate cyclic endoperoxides prostaglandin G2 and PGH2; (iii) PGH2 is then transformed enzymatically by Thromboxane synthase in platelets to Thromboxane A2 (TXA2) and by Prostacyclin synthase in endothelial cells to Prostacyclin I2 (PGI2). Other prostaglandins (PGE2, PGD2 and PGF2α) are formed in both cell types and leukocytes by PG synthase Figure (2-3) (Dogne et al., 2005; Grosser, 2006).
14
Figure 2-3: Cyclo-oxygenase enzymes in prostanoid synthesis (Legan, 2003). The COX-1 enzyme (the housekeeping enzyme) is found in most tissues and serves regular physiological function of prostanoids, but could be upregulated in particular cell types (Rouzer and Marnett , 2008 ; Mitchell, 2010). COX-3 is the third and most recently discovered cyclooxygenase COX isozyme and is actively being studied for its similarities and differences to the COX-1 and COX-2 enzymes. The sequence difference may decrease the enzymatic potential of COX-3 to generate PGE2. Currently, one hypothesis suggests that COX-3 exhibits the same role in prostaglandin synthesis,
15
ultimately regulating pain and fever (Chandrasekharan et al., 2002; Botting, 2003). COX-2 is found in the vascular endothelium, brain, kidney, skin, bone and female reproductive system and is also involved in certain physiological processes. However, it is induced by inflammatory stimuli such as bacterial endotoxin and cytokines (Tanabe and Tohnai, 2002; Mitchell, 2010). PGs play critical roles in normal physiological processes including platelet aggregation, maintenance of the gastric mucosa integrity and reproduction in addition to these physiological effects. PGs regulate inflammation, fever and pain and play an important role in the pathogenesis of cancer. PGE2 manifests its biological activities via four known G protein coupled membrane receptors: EP1 to EP4. These receptors differ in their PGE2 binding affinities and their downstream signal-transduction pathways (Tober et al., 2006). Prostacyclin is considered an independent mediator. PGI2 (prostaglandin I2) in eicosanoid nomenclature and is a member of the prostanoids (together with the prostaglandins and thromboxane). Prostacyclin is a potent endogenous anticoagulant for platelets and a strong vasodilator, it plays an essential role in the maintenance of vascular homeostasis, whereas TXA2 is a potent vasoconstrictor and a promoter of platelet aggregation. In addition, TXA2 regulates renal hemodynamics and sodium handling (Remuzzi et al., 1992, Welch and Wilcox, 1992). As a consequence of their opposing roles, an imbalance in PGI2 or TXA2 production has been implicated in the pathophysiology of many thrombotic and cardiovascular disease (Cotran et al., 1994; Caugh et al., 2001; Hermenegildo et al., 2005). The prostacyclin plays an important role in the atheroprotection that is associated with female gender. Estrogens can upregulate the expression of COX-2 dependent prostacyclin in vascular smooth muscle cells (Dannenberg and DuBois, 2003; Egan et al., 2004). 16
2.3.4: Regulation of COX-2 expression COX-2 appears to play an important role in skin carcinogenesis caused by solar ultraviolet radiation (UVR): UVR induces COX-2 expression in human skin. COX-2 expression is undetectable in most normal tissues. Important exceptions to this rule are the brain and renal cortex where constitutive COX-2 expression occurs (Mifflin and Powell, 2001) and a variety of factors have been reported to stimulate COX-2 expression. LPS was the first inducer of COX-2 expression that was identified
in
macrophage (Lee
et al.,
1992).
Proinflammatory factors (e.g. IL-1, TNF- α, IFN-γ, LPS, TPA), hormones (e.g. Follicle-stimulating hormone, luteinizing hormone, estrogen), growth factors (e.g. EGF, PDGF, FGF) and oncogenes (e.g. v-Src, v-Ras) have been reported to induce COX-2 expression (Tanabe and Tohnai, 2002). Sequence analysis of the 5′-flanking region of COX-2 gene reveals that there are many consensus cis-elements that regulate the transcription of COX-2. However, in all species studied, only a limited number of elements are known to be involved in the regulation of COX-2 gene expression, i.e., the C/EBP-NFIL6 (CAAT/enhancer binding protein), generation of cAMP, activation of protein kinase C isoforms, generation of inositol trisphosphates, generation of ceramide, activation of mitogen activated protein kinases (MAPKs) such as cJun N-terminal kinase (JNK), P38 kinase and extracellular signal regulated kinases (ERKs), as well as Janus-associated kinases (JAKs)
(Mifflin and
Powell , 2001; Klein et al., 2007) . The ceramide activates MAP kinases, including p38 kinase, JNK and ERK1/2. The pathways of the MAP kinases, in particular p38 kinase and ERK1/2, regulate the expression of COX-2. The signaling pathway of ceramide is likely to produce cross-talk with that of COX-2 expression. MAP kinases control the expression of COX-2 by LPS. p38 kinase play a critical role in LPSinducible or C2-potentiated COX-2 induction, as evidenced by complete 17
blockage of COX-2 induction by p38 kinase inhibition (Cho et al., 2002) (Figure 2-4). Epidermal growth factor (EGF) and UV light stimulate expression of COX-2 in primary keratinocytes in vitro and in the epidermis in vivo. The EGFregulated COX-2 expression is mediated through p38 MAPK signaling pathway and positively correlates with NF-IL6-β expression. NF-IL6-β coordinates with c-Jun on COX-2 transcriptional activation by receptor could directly bind to CCAAT/enhancer-binding protein (C/EBP) and cyclic AMP-response element (CRE) sites of the COX-2 promoter. CRE site was a more specific response to EGF inducibility of COX-2 gene. NF-IL6-β was also acetylated by p300 and acetylation of NF-IL6-β enhanced the COX-2 promoter activity stimulated by NF-IL6-β itself. In vivo-DNA binding assay demonstrated that EGF stimulated the recruitment of p300 and NF-IL6-β to the COX-2 promoter (Wang et al., 2006) (Figure 2-5).
Figure 2-4: Schematic diagram illustrate the proposed mechanisms by which C2 enhances LPS-inducible COX-2 expression (Cho et al., 2002).
18
Figure 2-5: NF-IL6-β is a bifunctional protein in COX-2 transcription (Wang et al., 2006).
2.3.5: COX-2 and tumorigenesis COX-2 expression is aberrantly increased in (various) human epithelial cancers in colorectum, esophagus, stomach, lung and bladder. These findings suggest that upregulation of COX-2 may be a common mechanism in epithelial carcinogenesis (Li et al., 2003). COX-2 is localized in neoplastic (epithelial) cells, microvascular endothelial cells and stromal fibroblasts. Through the released PG it enhances carcinogenesis with increasing angiogenesis, inhibiting apoptosis, activating matrix metalloproteinases, suppressing cell mediated antitumor immune response and protection against damage by cytotoxic agents (Legan, 2003).
A- The role of COX-2 in angiogenesis Small tumors are able to grow because they can obtain nutrients and oxygen by diffusion. For tumors to enlarge further they need to develop new collateral blood vessels to provide the essential nutrients for invasion, growth and subsequent metastasis. The formation of new vessels is termed neovascularisation. This is driven by the process of angiogenesis, the sprouting of 19
new vessels from existing vasculature. With tumor-associated angiogenesis, the cancer cell releases various pro-angiogenic factors (including angiogenin, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and transforms the growth factor (TGF-β). These stimulate endothelial cell proliferation, migration and invasion. The receptor for PDGF is also important in angiogenesis as it is central to the recruitment of pericytes, the cells that surround and support capillaries (Clarke and Sharma, 2006). Recent studies have demonstrated that COX-2 could affect tumorigenesis via several different mechanisms. COX-2 has also been reported to induce angiogenesis (Dempke et al., 2001; Rozic et al., 2001; Şahin et al., 2009) (Figure 2-6). The inducible COX-2 is an important mediator of angiogenesis and tumor growth. The proangiogenic effects of COX-2 are mediated primarily by three products of arachidonic metabolism: TXA2, PGE2 and PGI2. Downstream proangiogenic actions of these eicosanoid products include: (1) Production of VEGF, (2) Promotion of vascular sprouting, migration and tube formation, (3) Enhanced endothelial cell survival via Bcl-2 expression and Akt signaling, (4) Induction of matrix metalloproteinases, (5) Activation of epidermal growth factor receptor-mediated angiogenesis and (6) Suppression of interleukin-12 production (Sawaoka et al., 1999; Gately , 2000; Zha et al., 2004 and Chu et al., 2004).
20
Figure 2-6: COX-2 induced angiogenesis (Zha et al., 2004).
B- COX -2 mediated resistance to apoptosis Apoptosis or programmed cell death is a normal component of the development and health of multicellular organisms. Cells die in response to a variety of stimuli, during apoptosis they do so in a controlled and regulated manner (Cohen, 1997; Potten and Wilson, 2004). Decreased sensitivity to apoptosis was associated with increased levels of PGE2 and Bcl-2 protein induced by COX-2 overexpression (Chu et al., 2004). Increasing resistance to apoptosis has been proposed as another major mechanism for the effect of COX-2 in tumorigenesis; mediated by increased expression of the anti-apoptotic factor Bcl-2 and TGF-β (Zha et al., 2004). PGE2 is also known to regulate antiapoptotic gene products such as Bcl-2 and IAP. PKA also inactivates glycogen synthase kinase (GSK) and proapoptotic Bad by phosphorylation. EP4 has been reported to activate a phosphoinositol-3kinase (PI3K)–dependent pathway leading to the phosphorylation of Akt. Akt 21
can inactivate several proapoptotic proteins; including Bad, Bax and caspase-9 but can also activate antiapoptotic proteins including NF-κB and CREB (Datta et al., 1999; Lizcano et al., 2000; Regan, 2003) (Figure 2-7).
Figure 2-7: Proposed pathways for the anti-apoptotic effect of PGE2/EP4 (May, 2011).
22
C- COX-2 role in tumor growth and metastasis The ability of malignant cells to break loose from their own tissue is dependent on the invasiveness of the tumor and the ability to digest biological membranes
by
matrix-metalloproteinases
(MMPs),
which
reduce
the
intercellular anchorage; COX-2 enhanced expression of MMP-2 play important role in invasiveness and metastasis (Zahner et al., 1997; Chu et al., 2004).
2.4: UVR induced skin tumors 2.4.1: Definition Skin tumor is growths with multiple phenotypes and differing degrees of malignancy. The three most common malignant skin cancers are basal cell carcinoma, squamous cell carcinoma and melanoma. Skin tumors generally develop in the epidermis (the outermost layer of skin), so a tumor is usually clearly visible. This makes most skin cancers detectable in the early stages. Unlike many other cancers, including those originating in the lung, pancreas and stomach. Skin cancer is rarely fatal, with the exception of melanoma (Cotran et al., 1994 and Alam and Ratner, 2001; Gerdes and Yuspa, 2005).
2.4.2: Mechanisms of induction of skin tumor by UVB radiation and COX-2 expression The epidermal keratinocytes of the skin are the most susceptible to damage from UV exposure, due to their localization relative to the skin surface. Therefore, most skin cancers in humans arise from the epidermis. The cellular and molecular events that contribute to the development of UV-induced skin cancer is a complex process involving at least two distinct pathways that interact or converge to cause skin tumor.
23
One pathway involves the action of UV on target cells (keratinocytes) for neoplastic transformation both through genetic and epigenetic changes. The effects of UV on the host‟s immune system (Ouhtit and Ananthaswamy, 2001; Melnikova and Ananthaswamy, 2005). The alterations are classically defined as occurring in stages; initiation involves DNA damage leading to mutation; this is followed by promotion, which involves enhanced proliferation and altered cell behavior and finally progression results from subsequent genetic changes such as loss of heterozygosity and gene amplification (Rundhaug and Fischer, 2010b). Initiation occurs by exposure of the skin to the UV light which causes a genetic mutation (either an activating mutation in a proto-oncogene such as Hras or an inactivating mutation in a tumor suppressor gene such as p53) in the stem cell or progenitor cell compartment (Sarasin, 1999; Rundhaug and Fischer, 2010b). Mutations in the p53 gene, especially C → T or CC → TT transitions, considered as UV-molecular signature. The p53 mutation in keratinocytes is probably an initiating event in UV skin carcinogenesis, because cells containing p53 mutations are relatively more resistant to UV-induced apoptosis and they can acquire a growth advantage (Ouhtit and Ananthaswamy, 2001). The proto-oncogenes H-ras, K-ras and N-ras encode 21-kDa proteins share about 70% sequence homology. Which are located in the inner cell surface. Ras proteins participate in signal transduction by binding to GTP. Signals involved in growth control, such as binding of activators to cell surface receptors, are transferred from the cell surface to the nucleus. Mutations that activate ras genes occur less frequently in human skin cancers than mutations in the p53 gene. ras mutations have been reported to occur at 10-40% frequency in human skin cancers (Soehnge et al., 1997; Caulin et al., 2007). Another tumor suppressor gene known as patched (ptc) has been implicated in the development of basal cell carcinoma (BCC). The human ptc 24
gene was found localized with the map location of nevoid basal cell carcinoma syndrome (NBCCS) on chromosome 9 at 9q22.3 (Hahn et al., 1996; Johnson et al., 1996; Gailani and Bale, 1997) (Figure 2-8).
Figure 2-8: A model for induction of skin cancer by UV (Soehnge et al., 1997). One of the cellular processes that are crucial for skin tumor promotion is the induction of cell proliferation and maintenance of a sustained hyperplasia. Regenerative proliferation, such as that due to repetitive UV light exposure, can promote tumorigenesis which induces cell proliferation (Tharappel et al., 2002). In general, the effects of tumor promoters are reversible for a limited number of applications. However, their prolonged epigenetic effects result in irreversible genetic events in the later stages of tumor promotion. Tumor promotion leads to altered gene expression and identification of these critical events offer targets for chemoprevention and/or therapy (Rundhaug and Fischer, 2010b). 25
COX-2 serves as excellent biomarker to observe inflammatory responses after UVB irradiation. PPARγ directly or indirectly regulates COX-2 expression. Therefore, PPARγ may be a regulatory molecule of the UVBinduced inflammatory response in the skin. UVB irradiation induces gene expression of growth factors that regulate cellular proliferation. For example, UVB activates mitogenic signals such as mitogen activated protein kinase (MAPK) through signal transduction cascades. Additionally, epidermal growth factor receptor (EGFR) activation is an elicited UVB response which stimulates expression of COX-2. If UVB irradiation mutates a cell at the time of proliferation and the damaged DNA is not repaired, this mutation may be passed on to the daughter cells; resulting in unregulated cellular proliferation and possible subsequent tumor formation. This increase in cellular proliferation due to UVB irradiation disrupts the balance of the cell cycle progression and plays an important role in tumor promotion and development in epidermal tumor (Martel, 2008). Chronic UVB exposure causes epidermal cell damage that can lead to skin cancer. P53 has been reported to regulate the expression of COX-2 and prostaglandins produced by COX-2 have been shown to covalently bind wildtype (WT) p53 and prevent its nuclear accumulation. Thus, alterations in p53 activity represent an additional mechanism by which COX-2 could influence epidermal responses to UV exposure (Subbaramaiah et al., 1999; Marwaha et al., 2005; Fritsche et al., 2007). Chronic UVR-induced inflammatory cytokines mediated by NF-κB reportedly play important roles in photoaging and cancer. NF-κB is activated upon UV irradiation and induces the various genes including COX-2 (Kim et al., 2005). The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor kappa-B (NF-κB) pathway positively affects COX-2 expression. PI3K
26
directly activates AKT, which in turn phosphorylates and activates the I-B kinase (IKK) leading to NF-κB activation (Adams et al., 2006) (Figure 2-9).
Figure 2-9: A scheme of the NF-κB pathway (Nakanishi and Toi, 2005). UV
effects
are
studied
by
two
models
of
UV-induced
immunosuppression: local and systemic. In the local immunosuppression the site of hapten application corresponds to the site of UV irradiation, resulting in an impaired Contact Hypersensitivity Reactions (CHS) induction and formation of antigen-specific T-suppressor cells. In the “systemic immunosuppression” model the site of hapten application is far from the irradiation site (Damian et al., 2008; Beissert and Schwarz, 2009 and Amerio et al., 2009).
27
In the local immunosuppression model the mechanism of immune disregulation is mediated directly through the UV induced alteration in Antigen Presenting Cell (APC) function. UV irradiation reduces the number of Langerhans cell (LC) in the skin and alters their morphology. The drastic decrease in APC number seems important for UV-induced immunosuppression only in the afferent phase of the cellular immune response since the depletion of LC during the elicitation phase causes an enhancement of CHS response in mice (Elmets et al., 1983; Aubin, 2003; Amerio et al., 2009). Several in vitro experiments which have demonstrated the reduction of the expression of B7-1, B7-2 and CD 3 after irradiation, have confirmed that this could be one of the major mechanism of APC function impairment in animal models as well as in humans (Amerio et al., 2009) . In the “systemic” immunosuppression, circulating cytokines or factors are involved since there is no direct contact between UV irradiated APC and hapten. Cytokines implicated include: Interleukin-1, 10, 12 (IL-1, IL-10, IL-12), tumor necrosis factor α (TNF- α) and tumor growth factor-β (TGF-β) (Schwarz, 2005; Ullrich, 2007; Amerio et al., 2009). Urocanic acid (UCA) is one of the major UV absorbing components in the stratum corneum. It is produced during the process of keratinization and undergoes a trans to cis isomerization after interaction with UVB. Some studies have suggested that the amount of cis-UCA produced after UV irradiation is insufficient to suppress CHS, however there is a broad body of evidence that cis-UCA has a significant effect on CHS immunosuppression both locally and systemically in mice as well as in human after irradiation (Noonan and Defabo, 1992; Kondo et al., 1995; Van Strien and Korstange, 1995). Cis-UCA mechanism of action is still unclear it is thought that this molecule plays a role in UVB mediated Immunosuppression through its effects on cells and on cytokines production (Van Strien et al., 1995; Amerio et al., 2009). 28
2.4.3: Types of epidermal tumors – induced by UVR A- Papilloma Papilloma or „wart‟, as it is commonly referred to, is a benign neoplasm of stratified squamous epithelium. Papilloma refers to a benign epithelial tumor growing exophytically (projected outward) in finger-like fronds. Grossly, papilloma appears as single or more often multiple, raised, flat or cauliflower growths with either a smooth or more often an irregular surface. The face, neck and oral mucosa are the most common sites, but they can arise any where including the esophagus, rumen and external genital tract (Jones et al., 1997). Histopathologicaly, a papilloma consists of an elevated mass composed of multiple papillary projections of fibrovascular connective tissue covered by a well differentiated layer of heavily keratinized or cornified stratified squamous epithelium. Most spontaneously occurring papillomas of humans and animals are induced by DNA –containing papilloma virus (Jones et al., 1997). In experimental animals, prolonged exposure to devices emitting primarily UVB was a cause of papilloma in mice and rats (IARC, 1992).
B-Basal cell carcinoma (BCC) (IARC, 1992; Livolsi et al., 1993; Rubin and Farber, 1995 and Kumar et al., 2003). BCC derived from the basal cells of the epidermis. This tumor occurs predominantly on the hair-bearing surfaces of an adult.The face and the scalp areas are the regions most commonly affected. Mucous membranes, the palms and the soles are never involved. BCC very rarely metastasizes. This lesion is related to chronic sun exposure (particularly UVA and UVB irradiation) or large doses of X-ray radiation which are important predisposing factors. BCC can occur as single or multiple lesions, which measure a few centimeters in diameter and has a raised, rolled borders, with a central area of depression that may be ulcerated. 29
Histopathologically, tumor cells resemble those in the normal basal cell layer of the epidermis. They arise from epidermis follicular epithelium and do not occur on mucosal surfaces. Two pattern are seen, either multifocal growths originating from epidermis and extending over several square centimeters or more of skin surface (multifocal superficial type), or nodular lesions growing downward deeply in to the dermis as cords and islands of variably basophilic cells with hyperchromatic nuclei, embedded in a mucinous matrix and often surrounded by many fibroblasts and lymphocytes. The cells forming the periphery of the tumor cell islands tend to be arranged radially with their long axes in approximately paralled alignment (palisading).
C- Squamaous cell carcinoma (SCC) SCC can occur any where on the skin or on the mucous membranes. In 1994 in the US, the life time risk of squamous -cell carcinoma was 9%-14% among men and 4%-5% among women (Alam and Ratner, 2001). This lesion commonly formed on sun-damaged skin (particularly by 3 types of UVR), but it can also arise in association with ulcers, scars and foci of chronic osteomyelitis. The lesion consists of a shallow ulcer surrounded by wide, elevated and indurate borders. The ulcer may be covered by a crust with a red granular base. Occasionally, a raised, verrucous lesion occurs without evidence of ulceration
(IARC, 1992; Cotran et al., 1994; Kumar et al., 2003 and Goljan , 2004) . Histpathologically, the tumor consists of irregular masses of epidermal cells, which proliferate and invade the dermis. The squamous cells may show different degree of anaplasia, with prominent hyperchromatic nuclei. Intercellular bridges may be absent. Individual cells will undergo keratinization and pearl formation and mitoses are present.
30
D- Malignat melanoma (MM) (Cotran et al., 1994; Kumar et al., 2003 and Kumur et al., 2005). The most malignant of the cutaneous neoplasms, arises from the epidermal melanocytes. MM is rare before puberty, but fatal cases have been reported in children. Exposure to ultraviolet radiation (UVA and UVB) is one of the major contributors to the development of melanoma. The four types of MM are superficial spreading, nodular, lentigomaligna and acral lentiginous. The superficial spreading and nodular types are the most common. Histpathologically, the tumor originates at the dermo-epidermal junction where irregular activity occurs, with streaming of atypical and malignant nevus cells invasion down towards the dermis. The tumor cells may vary in size and shape, but most have large nuclei with prominent nucleoli and abundant granular eosinophilic cytoplasm. Multinucleate, bizarre giant cells are present and mitoses are common in Lentigo maligna melanoma, an increased number of melanocytes are present in the basal layer of the epidermis, some of which show atypia.
E- Merkle cell carcinoma (MCC) (Cotran et al., 1994; Kumar et al., 2005; Gupta et al., 2006; Elder et al., 2009). This rare neoplasm is derived from the merkle cell of the epidermis. Ultraviolet light (sun) exposure probably contributes to MCC development in a large number of cases and MCC can occur together with other sun exposurerelated skin cancers that are not infected with Merkel cell polyomavirus (MCV). Lesions may clinically present as ulcerated nodules and thus resemble eroded BCC or relatively nonpigmented forms of MM. These tumors are capable of metastasis and are potentially lethal. They are composed of small, round malignant cells containing nuerosecretory-type cytoplasmic granules.The tumor
31
cells also share some features with epithelial cells, expressing a type of keratin within their cytoplasm.
F- Keratoachanthoma Keratoachanthoma is a rapidly developing neoplasm that histologically may mimic well differentiated SCC. Often it will heal spontaneously, without treatment. Men are often more affected than women and lesions most frequently affect sun-exposed skin of white skinned people of 50 years. Keratoachanthoma appears clinically as flesh-colored, dome shaped nodules with a central keratinfilled plug (Underwood, 2004; Goljan, 2004; Elder et al., 2009). Histopathology, keratoachanthoma are characterized histologicaly by a central, keratin-filled crater surrounded by proliferating epithelial cells that extend upward in liplike fashion over the sides of the crater and downward into the dermis as irregular tongues. This epithelium is composed of enlarged cells showing evidence of reactive cytologic atypia. These cells have a characteristically „glass‟ eosinophilic cytoplasm and produce keratin abruptly (Kumar et al., 2003; Kumar et al., 2005).
G- Actinic keratosis (AK): Actinic keratos is also called „solar keratosis‟ or „senile keratosis‟ and „Bowen‟s disease‟. AK is a premalignant condition of thick, scaly, or crusty patches of skin. The growths start out as flat scaly patches and later grow into a tough, wart-like area, appearing skin-colored, pink, brown, pigmented or hyperkeratotic (Alam, 2006; Turkington and Dover, 2007). It is more common in fair-skinned people. It is associated with those who are frequently exposed to the sun, as it is usually accompanied by solar damage (particularly by the 3 types of UVR). Since some of these pre-cancers progress to squamous cell carcinoma, they should be treated. Untreated lesions have up to 20% risk of 32
progression to squamous cell carcinoma (IARC, 1992; Barton et al., 2003; Quaedvlieg et al., 2006; Prajapati and Barankin, 2008) Histopathologically, actinic keratoses share features with squamous cell carcinoma. Actinic keratosis is an epidermal lesion characterized by aggregates of atypical, pleomorphic keratinocytes at the basal layer that may extend upwards to involve the granular and cornified layers. The epidermis itself shows an abnormal architecture, with acanthosis, parakeratosis and dyskeratosis. Cellular atypia is present and the keratinocytes vary in size and shape. Mitotic figures are present. This presentation may resemble Bowen‟s disease or carcinoma in situ (Cockerell, 2000; Smoller, 2006).
2.5: Acetylsalicylic acid 2.5.1: Introduction Acetylsalicylic acid was discovered as byproduct of coal tar in the fifth century BC. A physician who wrote about a bitter powder that came from the bark of the willow tree and eased pains and reduced fever was German chemist Charles Gerhardt in 1853 and later prepared by another German chemist, Hoffman. The therapeutic effectiveness of Acetylsalicylic acid as an antiinflammatory–analgesic–antipyretic was described by Heinrich Dreser in 1899. Acetylsalicylic acid is believed to be derived from the German word for Acetylsalicylic acid, acetyl spirsaure (from spirea, a plant from which salicylic acid had been prepared for years and saure the German word for acid ) (Smith and Reynard, 1992; Koester,1993; Schrör, 2009). Acetylsalicylic acid, arguably the world‟s favourite drug, has been around since the late nineteenth century, but it was not until the late 1970s that its ability to inhibit prostaglandin production by the cyclooxygenase enzyme was identified as the basis of its therapeutic action (Flower, 2003). 33
Acetylsalicylic acid and other NSAIDs that inhibit COX-2 induce tumor cell apoptosis and reduce COX-2-tumor induced angiogenesis which is considered to be an important mechanism for their anti-tumor activity and prevention of tumorigenesis (Williams et al., 2000; Toyota et al., 2000; Fosslien, 2001). The chemopreventive effect of NSAIDs, particularly Acetylsalicylic acid, on NMSC has been repeatedly shown in animal and in vitro studies in humans. Some experimental studies have reported that topical or oral NSAIDs may lead to regression of skin neoplasms (Al-Waili, 1989; Al-Saleem, 1993; Grau et al., 2006 and Asgari et al., 2010).
2.5.2: Mechanism of action and therapeutic use A-Anti inflammatory effects: Acetylsalicylic acid is non-selective inhibitor of both COX isoforms (COX-1 & COX-2), it irreversibly inhibits COX and inhibits platelet aggregation
(Vane and Botting, 2003; Katzung, 2004). NSAID may be
categorized according to their COX specificity as: COX-2 selective compounds, whose selectivity for inhibiting COX-2 is at least 5 times that for COX-1. The group includes rofecoxib, celecoxib, melocoxib, etodolac and nabumetone. Non-COX-2 selective compounds, which comprise all other NSAIDs. These drugs inhibit COX-1 as much as, or even more than, COX-2, like Acetylsalicylic acid (Bennett and Brown, 2003).
B-Analgesic effects Acetylsalicylic acid is more effective in reducing pain of mild to moderate intensity through its effects on inflammation and because it probably inhibits pain stimuli. It has been shown that Acetylsalicylic acid-like drugs reduce the enhanced nociceptor activity in damaged tissue, probably as a result 34
of prostaglandin synthesis inhibition (Brune et al., 1992).The anti-inflammatory effects of Acetylsalicylic acid act synergistically with the opioids to enhance analgesia, used for the treatment of painful joint conditions like osteoarthritis, rheumatoid arthritis, arthritis of systemic lupus erythematosus, psoriasis and spondyloarthropathies (Kuhnert, 2000; Dannhardt and Kiefer, 2001; Dhikav et al., 2002 and Dugowson and Gnanashanmugam, 2006) .
C-Antipyretic effects: Acetylsalicylic acid‟s antipyretic effect is probably mediated by both COX inhibition in the central nervous system and inhibition of IL-1 (which is released from macrophages during episodes of inflammation) (Vane and Botting, 2003; Katzung, 2004).
D-Antiplatelet effects: Acetylsalicylic acid leads to irreversible platelet inhibition. Inhibition of platelet aggregation is attributable to the inhibition of platelet synthesis of TXA2, a potent vasoconstrictor and inducer of platelet aggregation. Therefore, single low dose of Acetylsalicylic acid produces a slightly prolonged bleeding time, which doubles if it is continued for a week (Vane and Botting, 2003; Katzung, 2004). Acetylsalicylic acid decreases the incidence of transient ischemic attacks, unstable angina, coronary artery thrombosis with myocardial infarction and thrombosis after coronary artery by -pass grafting (Verstraete, 1994; Hennekens, 1997; Das, 2005; Bennett et al., 2003).
2.5.3: Role of Acetylsalicylic acid in skin tumor It has been demonstrated by epidemiological studies, a large number of in-vitro and animal experiments and several clinical studies that non-selective NSAIDs can restrain the development and growth of different types of cancer. 35
Long-term use of Acetylsalicylic acid and other NSAIDs has been shown to reduce the risk of colon cancer and cancer of gastrointestinal organs as well as of cancer of the breast, prostate, lung and skin (Zhang et al., 1999; Fosslien, 2001). Understanding the effect of Acetylsalicylic acid provides substantial insights into the mechanisms by which these unique agents inhibit neoplastic proliferation by inducing apoptosis and inhibiting angiogenesis and enable better strategies for its prevention and treatment (Rao and Reddy, 2004).
I. Acetylsalicylic acid: Stimulation of Apoptosis Programmed cell death or apoptosis, is needed to maintain homeostasis in continuously replicating tissues such as that of the skin. It is important to note that non-selective NSAIDs COX-2 inhibitors (Acetylsalicylic acid) stimulate apoptosis and suppresses cell proliferation (Shiff et al., 1996; Malisetty et al., 2003; Rao and Reddy, 2004). Several mechanisms have been proposed for Acetylsalicylic acid induced
apoptosis,
which
are
COX-2-independent.
These
include
downregulation of nuclear factor-kappa B (NF-κB) activity, alteration in the levels of pro and anti-apoptotic proteins, activation of extrinsic and intrinsic pathways of apoptosis, inhibition of proteasome function, cell cycle arrest and generation of stress response and activation of stress kinases. Some of the above-mentioned apoptosis-induced mechanisms could also be regulated through COX-2-dependent pathways (Andrews et al., 2002; Takada et al., 2004; Jana, 2008) (Figure 2-10).
36
Figure (2-10): Potential mechanisms of Acetylsalicylic acid -induced apoptosis (Jana, 2008). A-Downregulation of NF-κB pathway NF-κB is a ubiquitous factor that regulates the transcription of many genes involved in immune and inflammatory responses as well as cell survival and cell death. NF-κB is localized in the cytoplasm in an inactive form in association with a family of inhibitory proteins called inhibitors of kappa B (IκBs). In response to multiple activating signals, IκB is phosphorylated and subsequently degraded by the proteasome. The rapid degradation of IκB proteins unmasks the nuclear localization signals of NF-κB, which then translocate to the nucleus and activate the transcription of multiple genes. Activation of NF-κB seems to stimulate some pathways that promote cell death, while other pathways promote cell survival. Several reports have suggested that Acetylsalicylic acid could promote apoptosis through the inhibition of NF-κB 37
activity. They also inhibit tumor necrosis factor-α (TNF- α)-mediated NF-κB activation (Kopp and Ghosh, 1994; Wong et al., 2003; Jana, 2008). The inhibition of NF-κB enhanced apoptosis in certain tumors and blockade of NFκB predisposed murine skin to squamous cell carcinoma. They showed that in normal human epidermal cells, NF-κB triggered cell-cycle arrests. Thus, these reports suggest that suppression of NF-κB could be tumorigenic under some conditions (Aggarwal, 2004). B-Activation of intrinsic and extrinsic apoptotic pathways Acetylsalicylic acid has been found to induce apoptosis through mitochondrial pathways by cytochrome -C release and activation of caspase-9 and extrinsic pathways by activation of caspase-8. Release of cytochrome-C from mitochondria is acentral event in apoptosis. It has also been demonstrated that cytochrome-C release from mitochondria is an early event in Acetylsalicylic acid -induced apoptosis. Cytochrome-C released into the cytosol can bind with the apoptotic protease activating factor- 1 (apaf-1) and form the apoptosome complex, which in turn leads to the sequential activation of caspase-9 and caspase-3. Activation of caspase-8 could also play an important role in Acetylsalicylic acid-induced apoptosis (Redlak et al., 2005; Jana, 2008) (Figure 2-11).
38
Figure (2-11): Diagram illustrating how mitochondrial pathway proteins Bax, Bid and Smac integrate into the caspase cascade in Acetylsalicylic acid-induced apoptosis (Redlak et al., 2005). C- Alteration in the levels of pro- and anti-apoptotic proteins Upregulation of pro-apoptotic proteins and downregulation of antiapoptotic proteins is also a possible target for Acetylsalicylic acid mediated apoptosis.
Acetylsalicylic acid has been shown to downregulate Bcl-2-
expression and induce the expression of Bax, Bad and p53.
Bax could
translocate from the cytosol to the outer mitochondrial membrane and make pores in the membrane to release cytochrome-C. Downregulation of Bcl-2 could further promote the release of cytochrome-C. Cytochrome- C release from mitochondria can also be induced by arachidonic acid and ceremide, which are 39
increased during COX-2 inhibition (Rshikesh and Sadhana, 2003; Jana, 2008; Suzuki et al., 2010).
D-Inhibition of proteasome function and cell cycle arrest Acetylsalicylic acid also could induce cell cycle arrest and apoptosis through inhibition of proteasome function. The Ubiquitin Proteasome System (UPS) is the cell‟s major extralysosomal pathway responsible for intracellular protein degradation in eukaryotes. This pathway is involved in the degradation of several critical regulatory proteins associated with regulation of the cell cycle and differentiation. Proteasome inhibitors also inhibit NF-κB activity, induce oxidative and endoplasmic (ER) stress and activate various stress kinases. Treatment of Acetylsalicylic acid in various cell lines has been found to decrease proteasome activity and increase accumulation of ubiquitylated proteins, which correlates with its effect on cell death. Acetylsalicylic acid and other NSAIDs exposure also increases the intracellular accumulation of various proteasomal substrates which are pro-apoptotic, like Bax, IκB-a, p53, p21waf1/Cip1
and
p27kip1.
Increased
accumulation
of
p27kip1
or
p21waf1/Cip1 would result in cell cycle arrest at the G1/S phase and leads to apoptosis (Redondo et al., 2003; Dikshit et al., 2006).
II. Acetylsalicylic acid: Modulation of Angiogenesis COX-2 expression is widely induced in the angiogenic vasculature of colorectal adenomatous polyps and in carcinomas of the colon, lung, breast, esophagus, prostate and skin. Acetylsalicylic acid is required to block vascular endothelial tube formation. Many molecular targets may be achieved by directed anti-angiogenesis. The most important target is VEGF, as blocking its synthesis, immunoneutralisation or impairing transduction of the signal from surface receptor to the inside of cell nuclei inhibiting not only neoangiogenesis
40
but also development of the neoplasm itself and spreading of metastasis (Ste˛pien, et al., 2002). COX-2 inhibitors inhibit angiogenesis through a combined inhibition of angiogenic growth factors production, response to angiogenic factor and impairment of endothelial cell survival and migration. Inhibition of COX-2 resulted in a diminished integrin -αVβ3dependent activation Cdc42 and Rac, two members of the Rho family of GTPases that regulate cytoskeletal organization and cell migration (Dormond et al., 2001; Compare et al., 2010) Mechanisms by which Acetylsalicylic acid inhibit angiogenesis appear to be multifactorial. Some of these mechanisms include; inhibition of mitogenactivated protein (Erk2) kinase activity, suppression of cell cycle proteins, inhibition of early growth response (Egr-1) gene activation, interference with hypoxia inducible factor 1 and VEGF gene activation, increased production of the
angiogenesis
inhibitor-endostatin,
inhibition
of
endothelial
cell
proliferation, migration, spreading and induction of endothelial apoptosis (Tarnawski and Jones, 2003; Mehta and Mohandas, 2010) (Figure 2-13).
41
Figure 2-12: COX-2 inhibition by NSAIDs results in decreased VEGF production, suppressed mitogenic response and vascular permeability in response to VEGF and inhibited integrin-αVβ3 dependent Rac activation and endothelial cell migration (Tarnawski and Jones, 2003).
42
2.6: Immunohistochemistry 2.6.1: Introduction Immunohistochemistry (IHC) refers to the process of localizing proteins in the cells of a tissue section exploiting the principle of antibodies binding specifically to antigens in biological tissues. It takes its name from „immune‟, in reference to antibodies used in the procedure and „histo‟ meaning tissues (Ramos-Vara, 2005). IHC is a key tool for the analysis of localization of target molecules within tissues. It is used routinely for almost every aspect of modern biomedical research. Technical ease of use, rapidity and reliability usually determine the techniques utilized in academic or medical settings (Levin, 2004; Goldstein et al., 2007). Immunohistochemical technique has equipped the histopathologist with the tools needed to tackle the most common diagnostic problems in tumor pathology especially the characterization of the undifferentiated or poorly differentiated malignant tumors, whether primary or metastatic. No other method during the past fifty years has had such a major impact on histopathology (Delellis and Dayal, 1987; Chan, 2000; Coindre, 2003). The publication of a paper by Coons et al. in 1941 describing an immunofluorescence technique for detecting cellular antigens in tissue sections marked the beginning of immunohistochemistry. Since then IHC has become a valuable tool in both diagnosis and research of infectious and neoplastic diseases in a variety of animals (Ramos-Vara, 2005). Simply defined, immunohistochemistry is the study of antigen to antibody interactions and how these reactions are visualized in tissues. The primary antibody is applied to the tissue, where the antigen is „suspected‟ to be present. The antigen is made up of a combination of several proteins in a specific sequence and conformation. The site on the antigen where the antibody binds is referred to as the „epitope‟ and is made of 5 to 16 amino acids, which 43
may represent a small percentage of the length of the total antigen. One antigen may have multiple „epitopes‟ (antibody binding sites). Each binding site is given a different name, in reference to the antibody clone name. The antibody will bind to the epitope and the detection system used will allow for visualization
of
this
antibody-antigen
reaction
(Elias,
2003).
Immunocytochemistry provides the most direct method for identifying both the cellular and subcellular distribution of proteins and can provide a relatively rapid indication of gene expression or protein distribution (Mello and Fire, 1995).
2.6.2: Immunohistochemistry methods An antigen-antibody interaction can be visualized using the following methods: 1-Direct method is a one-step staining method and involves a labeled antibody (Different labels have been used, including; fluorochromes, enzymes, colloidal gold and biotin) reacting directly with the antigen in tissue sections. While this technique utilizes only one antibody and therefore is simple and rapid, the sensitivity is lower due to little signal amplification, in comparison with different indirect methods and is also less commonly used. 2- Indirect methods involve an unlabeled primary antibody (first layer) that binds to the target antigen and a labeled secondary antibody (second layer) that reacts with the primary antibody. The secondary antibody must be raised against the IgG of the animal species in which the primary antibody has been raised. This method is more sensitive than direct detection strategies because of signal amplification due to the binding of several secondary antibodies to each primary antibody, if the secondary antibody is conjugated to the fluorescent or enzyme receptor (Carson, 1997; Ramos- Vara, 2005; Mashhood, 2008). 3-Avidin–biotin complex (ABC) method is standard IHC method and widely used technique for immunhistochemical staining. Avidin, a large glycoprotein, 44
can be labeled with peroxidase or fluorescent and has a very high affinity for biotin. The technique involves three layers. The first layer is unlabeled primary antibody. The second layer is biotinylated secondary antibody. The third layer is a complex of avidin-biotin peroxidase linked with appropriate label. The peroxidase is then developed by the DAB or other substrates to produce different colorimetric end products. 4- Labeled avidin-biotin (LAB) or labeled streptavidin-biotin (LSAB) method. Streptavidin, derived from Streptococcus avidini, is a recent innovation for substitution of avidin. LSAB is technically similar to the standard ABC method. The first layer is unlabeled primary antibody. The second layer is biotinylated secondary antibody. The third layer is Enzyme-Streptavidin conjugates (HRPStreptavidin or AP-Streptavidin) to replace the complex of avidin-biotin peroxidase. A recent report suggests that LSAB method is about 5 to 10 times more sensitive than standard ABC method. 5-Peroxidase–antiperoxidase (PAP) method is a further development of the indirect technique and it involves a third layer which is a rabbit antibody to peroxidase, coupled with peroxidase to make a very stable peroxidase antiperoxidase complex. The sensitivity is about 100 to 1000 times higher, since the peroxidase molecule is not chemically conjugated to the anti IgG but immunologically bound and loses none of its enzyme activity. 6-Polymeric Methods are based on dextran polymer technology and new methods of polymerizing enzymes and attaching these polymers to antibody (Boenisch, 2001; Ramos- Vara, 2005; Chen et al., 2010).
45
2.6.3: Applications of immunohistochemistry 1-
Diagnosis
of
tumors
of
uncertain
histogenesis
(undifferentiated
malignancies). 2-To identify abnormal protein deposits within cells. 3- IHC is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue. 4- IHC is a highly sensitive and specific method, especially advantageous as a diagnostic tool for infectious diseases . 5- Categorization of leukemias and lymphomas. 6- Identifying the origin and type of secondary deposits. 7- Identification of hormone receptors which are of prognostic value as estrogen and progestron in breast cancer to determine the mode of treatment (Levin, 2004; Tuffaha and Muin, 2008).
46
MATERIALS AND METHODS 3.1: Animal model A prospective study was conducted from October 10, 2010 through to March 10, 2011in two different locations. The first part of the study which dealt with housing, irradiation and treatment of mice was performed in Veterinary Medicine Teaching Hospital and the second part which included convential Haematoxylin and Eosin (H & E) staining and immunohistochemistry technique was performed in histopathology laboratory of Shorsh Hospital in Sulaimani Governorate. The experiment animal of this prospective study were an albino mice of Mus musculus species, BALB/c strain, of both sexes with approximately the same age (3-4 weeks) and weight (20-25gm). The albino mice were housed in climate quarters (25°C), with a 12/12 hours dark/light cycle under a white fluorescent light. Fifty mice underwent this experiment and were divided into 3 groups; group 1was kept as control group and included 10 mice (not exposed and not treated with Acetyl salicylic acid); group 2 which included 20 mice was considered as exposure group (exposed to UVB light) and group 3 which included 20 mice was considered as treatment group (exposed to UVB light and treated with Acetylsalicylic acid).
3.2: Treatment group with Acetylsalicylic acid The COX- 2 inhibitor used in this study was, a non selective one (Acetyl salicylic acid). Mice were treated 4 days/week with Acetylsalicylic acid one week before UVB exposure. After that the mice were treated with Acetylsalicylic acid and exposed to UVB light 4 days/week throughout the whole experimental period for about 5 months. Acetylsalicylic acid was a white powder; 20mg of this powder were weighed by a sensitive balance and dissolved into 25ml of warm distilled water in a sterile flask, shaked well until 47
powder dissolved completely. Each mouse was treated with 1ml (0.8mg / day in a single dose) of Acetyl salicylic acid orally by a sterile syringe.
3.3: UVB irradiation group The mice were subjected to UVB irradiation with a calculated power of 53 mj/sec using a lamp of 312 nm wavelength, 15 watts; Vilber-LourmatFrance. The mice were exposed to UVB light for 4days/week for 20 minutes repeated for 5 months. This was done after making a window by shaving the mice‟s back (5X2 cm).
3.4: Collection of samples At the end of each month and for each group incisional biopsies were taken from the irradiated shaved area of exposure and treatment group for detection of any pathologic abnormalities. All animals were anesthetized using general anesthetic drug (XylazineKetamine: 0.1ml/10g of body weight) as recommended dose intraperitonially
(In a sterile 10 ml tube with a rubber stopper, mix 1ml of ketamine (100mg/ml) + 0.1ml of xylazine (100mg/ml) + 8.9ml of sterile water for injection, shacked well before use). Then tissue samples were fixed in 10% formalin, processed and embedded in paraffin blocks.
3.5: Materials 3.5.1: Equipments 1- Ultraviolet lamp (53 mj/sec, 312 nm wavelength, 15 watts; Vilber-Lourmat France) 2- Sakura rotary microtome (Acuu-Cut SRM200-Japan) 3- Water bath 4- Oven 5- Pressure cooker 48
6- Light microscope (Olympus 6V20 WHAL-Japan) 7- Incubator and humid chamber 8- Sensitive balance (Mettler Toledo- Switzerland) 9- Positively charged slides (Fisherbrand -U.S.A) 10- Ordinary glass slides 11- Glass staining jars and Coplin jars 12- Slide holder 13- Cover slips 14- Wash bottles 15- Cylinders and flasks 16- Filter paper 17- Pap pen -Dako 18- Absorbent wipes 19- Syringes 20- Timer (stop-watch) 21- Surgical gloves 22- Manual camera (Sony- Japan)
3.5.2: Reagents and Solutions 1- Acetylsalicylic acid (Hoz Company Ltd) 2- Distilled water 3- Xylazine (Ceva sante animale- France) 4- Ketamine (Holden medica- India) 5- Ethanol (Absolute, 90% and 70%) 6- Xylene 7- Counter stain (Mayer‟s Hematoxylin) 8- Mounting medium (DPX) 9- Monoclonal mouse anti-Human COX-2 10- Dako antibody diluents 49
®
11- Visualization System {Dako Cytomation EnVision + Dual Link SystemHRP (DAB+)} 12- Dako wash buffer (concentrated Tris –buffered saline solution, PH 7.6) 13- Target retrieval solution (PH 9.0) 14- Endogenous peroxidase blocking agent 15- Protein blocking agent (Human serum) 16- Giemsa stain (R1”Giemsa stock solution” and R2 buffer solution “).
3.6: Methods 3.6.1: Samples preparation Three sections of 5µm thickness were taken from each paraffin embedded tissue block. The first section was mounted on an ordinary slide for H&E staining for detection of any histological lesions. The second section was for Giemsa stain for mast cells counting while the third section was mounted on positively
charged
slide,
then
proceeding
with
the
process
of
immunohistochemistry staining for COX-2 following the protocol that was ®
supplied with the kit of COX-2 using Dako Cytomation En Vision + Dual Link System- HRP (DAB+).
3.6.2: Immunohistochemistry staining Procedure 1- Formalin fixed-paraffin embedded sections were cut into 5µm thickness, then placed on positively charged slides to be stained. 2- The sections were backed in the oven (over night at 56°C) and then dewaxed in xylene for 5-10 minutes. 3- The sections were then rehydrated using graded alcohol (ethanol) in descending concentrations to water:a- Absolute ethanol for 10 minutes. b- 90% ethanol for 5 minutes. 50
c- 70% ethanol for 5 minutes. 4- The sections were washed in running tap water for 5 minutes and then placed in 3 changes of wash buffer, 2 minutes each time. 5-The pressure cooker was filled with 1L of distilled water then covered and switched on until its temperature reached 95-98°C. 6- A coplin jar was filled with target retrieval solution with slides, placed in pressure cooker with an open lid for about 10 minutes, then the lid was closed tightly and the coplin jar with the all slides were left for 15 minutes. 7- The coplin jar with slides removed from the pressure cooker and allowed to cool down for 20 minutes at room temperature. 8- The slides were washed 3 times, 2 minutes each time with a wash buffer. 9- The slides were tapped off and the area around the specimen wiped to remove any remaining liquid and the section encircled with pap pen. 10- Few drops of endogenous peroxidase blocking agent were applied to cover the specimen and incubated in humid chamber for about 10 minutes at room temperature, then rinsed with wash buffer 3 times, 2 minutes each time. 11- Few drops of protein blocking agent (human serum) were placed on each specimen and incubated in humid chamber for about 10 minutes at room temperature. The slides were then blotted with out washing. 12- Few drops of previously prepared
primary antibody (1µl of monoclonal
mouse anti-human COX-2+ 100 µl of Dako Antibody diluents) were applied to cover the specimens and incubate for 1 hour in humid chamber at room temperature, then rinsed in wash buffer 2 times, 2 minutes each time. 13- Sections were then covered by few drops of labeled polymer (secondary antibody) and incubated for one hour in humid chamber at room temperature, then rinsed with wash buffer 2 times, 2 minutes each time. 14- Few drops of previously prepared DAB + substrate-chromogen solution (1 drop of chromogen + 1ml of substrate) were applied to cover the specimens and
51
incubated for 3-5 minutes in humid chamber at room temperature, then washed under running tap water for 5 minutes. 15-The slides were immersed in a bath of aqueous Haematoxylin for less than one minute, then rinsed gently under running tap water. 16-The slides were dehydrated consecutively by dipping in staining jars containing the following:a- 70% ethanol for 5 minutes. b- 90% ethanol for 5 minutes. c- Absolute ethanol for 5 minutes. d- Xylene for 5-10 minutes. 17- The slides were then mounted using mounting medium (DPX) and covered with cover slips and left to dry.
Results: COX-2 staining
brown
Back ground
blue
3.6.3: Giemsa staining procedure Following the protocol that was supplied with the kit of using Giemsa Stain Kit, Artisan™ 1-Formalin fixed-paraffin embedded sections were cut into 5µm thickness and then placed on ordinary slides to be stained. 2- The sections were backed in the oven (over night at 56°C) and then dewaxed in xylene for 5-10 minutes 3-The sections were then rehydrated using graded alcohol (ethanol) in descending concentrations to water:a- Absolute ethanol for 10 minutes. b- 90% ethanol for 5 minutes. c- 70% ethanol for 5 minutes. 52
4- The sections were washed under running tap water for 2 minutes. 5- Sections were covered by 15 drops of Giemsa stain solution for 1 minute. 6- Sections were then covered by 30 drops of R2 Wright‟s stain buffer solution until the metallic shine color appeared. 7- Wash sections under running tap water for 2 minutes, then dehydrated by putting in a jar containing xylene for about 2 minutes. 8- The slides were then mounted using mounting medium (DPX) and covered with cover slips and left to dry. Results: Mast cells Background
violet blue
3.7: Slide interpretation 3.7.1: H & E slide interpretation The H & E stained slides prepared from control, irradiated and treated groups biopsies were examined thoroughly for any histological lesion or changes in epidermis and dermis. This was done by two independent observers.
3.7.2: Immunohistochemical scoring COX-2 immunostaining was evaluated by two independent pathologists. The staining was categorized as either positive or negative based on two parameters: (1) The percentage of positive cells scored on a scale of 0-3; 0 = 0%, 1= <25%, 2 = 26-50%, 3>50% positive cells and (2) The strength of staining intensity scored on a scale of 0-3; 0 = negative, 1 = weak or light staining, 2 = moderate staining, 3 = intense or strong staining. COX-2 protein expression was classified in to a 0-3 point scale: 1+; <25% positive cells for weak staining , 2+; 26-50% positive cells for moderate staining, 3+; >50% positive cells for strong staining (Hong et al., 2004). 53
3.8: Statistical analysis The data obtained from our observations were analyzed using ANOVA, Duncan‟s test and Pearson‟s correlation.
54
RESULTS 4.1: Gross and microscopic finding: 4.1.1: Control group The mice of control group had normal appearance of skin grossly (Figure 4-1) and microscopically (Figure 4-2) compared to exposure and treatment group (Figure 4-3).
4.1.2: Exposed group Gross skin lesions were detected in all mice of this group was benign tumor of seborrheic keratosis. These lesions appeared slightly raised (flat or round); measured only few mms in diameter, occasionally reaching 1cm in variable colors, most were tan or brown with friable consistency. Some had smooth surface but characteristically showed keratotic plugs (Figures 4-4 and 45). Microscopic finding of acanthotic SK was diagnosed in 12 (60%) mice as indicated by: 1- Thickening of epidermal layer that resulted from basaloid cell proliferation, with variable degree of hyperkeratosis and papillamatosis. 2- Presence of epidermal cysts filled with keratin (horn cyst) which is a common feature of SK. Some of these cysts resulted from infolding of epidermis and called pseudohorn cysts (Figures 4-6 and 4-7). 3- Presence of squmaous eddies (whorling aggregates of eosinophilic squamous cells) (Figure 4-8 and tables 4-2 and 4-3). Microscopic finding of clonal SK was diagnosed in 8 (40%) mice as indicated by the presence proliferation of sharply demarcated intraepithelial nests of basaloid or pale cells; in some cases the nests are composed of larger cells with conspicuous intercellular bridges, with nests separated by strand of cells with small dark nuclei (Figures 4-9 and 4-10).
55
The epidermis also contained small shrinked cells with pyknotic nuclei and deep eosinophilic cytoplasm especially located in supra basal layer of the skin and regarded as sunburn cells (SBC) or called apoptotic keratinocytes (Figure 4-11). The dermis showed infiltration of inflammatory cells including; neutrophills, macrophages and lymphocytes (Figure 4-12). In addition, dermis showed a number of mast cells which had cytoplasmic granules located in lower and upper dermis but especially in upper dermis surrounding blood capillaries (Figures 4-13 and 4-14).
4.1.3: Treatment group (exposed to UVB and treated with Acetylsalicylic acid administration) Mice irradiated with UVB and orally administrated mouse with Acetylsalicylic acid showed development of seborrheic keratosis 2 mice out of 20. Gross pathologic examination of skin biopsies obtained from these 2 mice showed the appearance of flat, rounded or slightly elevated lesions of variable size with light (pale) color (Figure 4-15). Microscopically, these lesions in the first mouse was diagnosed as acanthotic seborrheic keratosis (SK) as indicated by marked acanthosis, prominant papillomatosis, hyperkeratosis, characteristics horn cyst & pseudo horn cyst (Table 4-1 and figure 4-16). Where as the lesions of the second mouse was diagnosed as clonal SK as indicated by well-defined multiple nests of basaloid cells which are located within the epidermis; some of these nests contain keratin (Figure 4-17). The gross pathological examination of the remaining 18 mice showed thickening of their skin was diagnosed by touching (palpation) (Figure 4-18). Microscopically, this thickening was diagnosed to variable degrees of epidermal hyperplasia which was classified according to number of epidermal layers regarded as mild = (4-6 layers) (Figure 4-19), moderate= (7-9 layers) (Figure 456
20) and severe= (≥10 layers) (Figure 4-21). The numbers and percentages and the comparison between the different degrees of hyperplasia in treatment group (Table 4-2 and figure 4-22). The epidermis also contained small shrinked cells with pyknotic nuclei and deep eosinophilic cytoplasm especially located in supra basal layer of skin. These cells known as sunburn cells (SBC) or called apoptotic keratinocytes (Figure 4-23). The dermis showed a number of mast cells with cytoplasmic granules located in lower and upper dermis but especially in upper dermis surrounding blood capillaries (Figure 4-24).
In the exposure and treatment groups, mice suffered from keratitis and conjunctivitis some of them became blind, due to the UVB irradiation for long time period (Figure 4-25).
57
Table 4-1: Number and percentage of benign seborrheic keratosis in exposure and treatment group. Groups
No. & percentage of Acanthotic SK
Exposure group ( 20 cases) Treatment group (20 cases)
No. & percentage of Clonal SK
12
(60%)
8
(40%)
1
(5%)
1
(5%)
Table 4-2: Number and percentage of different degrees of hyperplasia in mice of treatment group. Degree of hyperplasia
Number
Percentage %
Mild hyperplasia
14
70%
Moderate hyperplasia
2
10%
Severe hyperplasia
2
10%
Table 4-3: Histological feature of both types of SK in exposure group. Types of SK
Squamous eddies
Papillomatosis Clonality Acanthosis
Acanthotic SK
Present
Present
Absent
Present
Clonal SK
Present
Absent
Present
Absent
SK: Seborrheic keratosis
58
Figure 4-1: Normal skin appearance in a mouse of control group.
Figure 4-2: Microscopic view of a skin section obtained from a mouse of the control group. It shows normal histological appearance. H & E stain, (X 100). 59
Figure 4-3: Appearance of skin in mice of the different experimental groups. A: Normal appearance of skin in a mouse of the control group. B: Thickening of the skin in a mouse of treatment group.C: Brown and tan keratotic plugs (scales or crusts) in a mouse of the exposure group.
Figure 4-4: Brown or tan colored, irregular lesions were seen on the skin in a mouse of the exposure group (arrows). 60
Figure 4-5: A velvety to granular, light brown skin in a mouse of the exposure group (arrow).
Figure 4-6: Microscopic view of skin section obtained from a mouse of exposure group. It shows acanthotic SK as indicated by the marked acanthosis, papillomatosis, horn cyst and pseudo horn cyst. H & E stain, (X40). 61
Figure 4-7: This is a higher magnification view of the tissue section illustrated in figure (4-6).
It shows acanthotic SK as indicated by papillomatosis(fibrovascular cord in its center), horn cyst and pseudo horn cyst. H & E stain, (X100).
Figure 4-8: Microscopical feature of acanthotic SK. It shows squamous eddies indicated by whorling aggregates of eosinophilic squamous cells in a mouse of exposure group (arrow). H & E stain, (X 400). 62
Figure 4-9: Microscopic view of skin section obtained from mouse of exposure group. It shows clonal SK as indicated by well-defined multiple nests of basaloid cells. H & E stain, (X 100).
Figure 4-10: This is a higher magnification view of the tissue section illustrated in figure (49). It shows the nests of basaloid cells are separated by strand of cells with small dark nuclei. H & E stain, (X 400). 63
Figure 4-11: Sun burn cells were seen within epidermis in skin section obtained from a mouse of the exposure group (arrow). H & E stain, (X 400).
Figure 4-12: Microscopic view of dermis in exposure group. It shows infiltration by inflammatory cells (arrows). H & E stain, (X400). 64
Figure 4-13: Microscopic view of a skin section obtained from a mouse of the treatment group. It shows the presence of numerous mast cells (arrows) within dermal layer. (A) H & E stain. (B) Giemsa stain, (X 400).
Figure 4-14: Microscopic view of a skin section obtained from a mouse of the exposure group. It shows the presence of numerous mast cells (arrows) within dermal layer. Giemsa stain, (100X). 65
Figure 4-15: Presence of flat, rounded or slightly elevated skin lesions in a mouse of treatment group (arrow).
Figure 4-16: Microscopic view of skin section obtained from a mouse of treatment group. It shows acanthotic SK as indicated by the marked acanthosis, horn cyst and pseudo horn cyst (arrow). H & E stain, (X 100). 66
Figure 4-17: Microscopic view of skin section obtained from a mouse of treatment group. It shows clonal SK as indicated by well-defined multiple nests of basaloid cells. H & E stain, (X 100).
Figure 4-18: Thickening of the skin in a mouse of treatment group. 67
Figure 4-19:- Mild epidermal hyperplasia in skin of a mouse of treatment group. H & E stain, (X 400).
Figure 4-20: Moderate epidermal hyperplasia in skin of a mouse of treatment group. H & E stain, (X400). 68
Figure 4-21: Severe epidermal hyperplasia in skin of a mouse of treatment group. H & E stain, (X400).
Figure 4-22: Histological appearance of various degrees of hyperplasia in treatment group compared to skin of control group. A: Control group showed normal skin appearance, B: Mild hyperplasia, C: Moderate hyperplasia and D: Severe hyperplasia in mice of treatment group. H & E stain, (X100). 69
Figure 4-23: Few sun burn cells were seen within epidermis in skin section obtained from a mouse of the treatment group (arrows). H & E stain, (X 400).
Figure 4-24: Microscopic view of a skin section obtained from a mouse of the treatment group. It shows the presence of numerous mast cells (arrow) within dermal layer. Giemsa stain, (400X). 70
Figure 4-25: Mouse had blindness in both eyes due to photoconjuctivits in exposure group.
4.2: Mean number of apoptotic bodies/10HPF: 4.2.1: Mean number of apoptotic bodies/10HPF in exposure group: Results of this study revealed strong effects of chronic UVB irradiation on apoptotic bodies in seborrheic keratosis i.e. the mean number of apoptotic bodies were decreased remarkably in chronically irradiated mice. For example, apoptotic bodies were decreased in number in acanthotic SK with a range of 12 and a mean number of 1.000/10HPF, while mildly decreased in clonal types of SK with a range of 2-4 and mean number of 3.125/10HPF. The P-value was 0.0001 with A-B symbols (according to F test and Duncan’s test). This indicated highly significant effects of chronic UVB exposure on apoptotic bodies in two types of SK (Table 4-4 and figure 4-26).
71
Table 4-4: Mean number of apoptotic Bodies/10HPF in Clonal and Acanthotic SK cases in mice in exposure group
Types of SK
No. of apoptotic
Mean/10HPF
Bodies Clonal SK
2-4
3.125 A
Acanthotic SK
1-2
1.000 B
SK: Seborrheic keratosis HPF: High power field With in the last column, the mean values that do not have common capital letters (A-B) vary from each other (P<0.05)
Mean number of apoptotic bodies in 10 high power field
Mean number of apoptotic bodies in 10 high power field in exposure group
Figure 4-26: Column chart showing mean number of apoptotic bodies/10HPF in mice of exposure group. 72
4.2.2: Mean number of apoptotic bodies/10HPF in treatment group: According to results of this study there was very strong effect of Acetylsalicylic acid on increasing number of apoptotic bodies in treatment group (with exception of the two tumor cases) by chronic UVB irradiation. For example, there was mildly increased number of apoptotic bodies in mice that suffered severe hyperplasia with a range of 4-6 and mean number of 5.000/10HPF, moderately increased number of apoptotic bodies
showed a
range of 4-9 with mean number of 6.500/10HPF in moderate hyperplasia. Markedly increased in mild hyperplasia demonstrated a range of 6-14 and mean number of 8.8576/10HPF. The P-value of 0.0001 with a (A-B-C-D) symbols (according to F test and Duncan‟s test). This indicated highly significant effect of Acetylsalicylic acid in increasing number of apoptotic bodies in different degrees of hyperplasia in treatment group (Table 4-5 and figure 4-27).
73
Table 4-5: Mean number of apoptotic bodies/10HPF in treatment group.
Epidermal lesion
No. of apoptotic Mean/10HPF Bodies
Mild hyperplasia
6-14
8.857
A
Moderate hyperplasia
4-9
6.500
B
Severe hyperplasia
4-6
5.000
C
Clonal SK
4
4.000
C
Acanthotic SK
2
2.000
D
SK: Seborrheic keratosis HPF: High power field With in the last column, the mean values that do not have common capital letters (A-D) vary from each other (P<0.05)
74
Mean number of apoptotic bodies in 10 high power field
Mean number of apoptotic bodies in 10 high power field in treatment group
Figure 4-27: Column chart showing mean number of apoptotic bodies/10HPF in mice of treatment group.
4.2.3: Mean number of apoptotic bodies/10HPF in exposure and treatment groups: Results revealed that there was a strong effect of Acetylsalicylic acid in reducing effect of chronic UVB irradiation on tumor development in exposure group by effects of UVB irradiation in reducing the number of apoptotic bodies and in treatment group by increasing number of apoptotic bodies. For example, apoptotic bodies were decreased or reduced in exposure group with a range of 1-4 and mean number of 1.850/10HPF, while apoptotic bodies were increased in treatment group with a range of 4-14 and mean number of 7.650/10HPF. Pvalue was 0.0001 with A-B symbols (according to F test and Duncan‟s test). This indicated a highly significant relation of both groups in numbering of apoptotic bodies and its role in reducing tumor development through 75
Acetylsalicylic acid administration in treatment group (Table 4-6 and figures 428 and 4-29). Table 4-6: Mean number of apoptotic bodies/1HPF in exposure and treatment groups.
Groups
No. of apoptotic
Mean/10HPF
Bodies Treatment group
4-14
7.650 A
Exposure group
1- 4
1.850
B
SK: Seborrheic keratosis HPF: High power field With in the last column, the mean values that do not have common capital letters (A-B) vary from each other (P<0.05)
Mean number of apoptotic bodies in 10 high power field
Mean number of apoptotic bodies in 10 high power field in exposure and treatment group
Figure 4-28: Column chart showing mean number of apoptotic bodies/10HPF in exposure and treatment groups. 76
Figure 4-29: Sun burn cells were seen within epidermis in skin section obtained from a mouse: (A). It shows one apoptotic bodies in exposure group. (B) It shows 5 apoptotic bodies in treatment group (arrows). H & E stain, (400X).
4.3: Mean number of mast cells/1HPF: 4.3.1: Mean number of mast cells/1HPF in exposure group: Results showed that there was a strong effect of UVB on increasing number of mast cells in Mus musculus species, BALB/c strain mice i.e. chronic UVB irradiation increased number of mast cells. For example, moderately increased number of mast cells in acanthotic SK with a range of 6-15 and mean number of 9.375/1HPF, while markedly increased number of mast cells in clonal SK with a range of 24-28 and with a mean number of 25.438/1HPF. The P- value was 0.001with A-B symbols (according to F test and Duncan‟s test). This indicated a highly significant effect of chronic UVB irradiation on the increasing number of mast cells in Musmusculus species, BALB/c strain (Table 4-7 and figure 4-30). 77
Table 4-7: Mean number of mast cells /1HPF in Clonal and Acanthotic SK cases in mice of exposure group.
Types of SK
No. of mast cells
Mean/1HPF
Clonal SK
24-28
25.438
A
Acanthotic SK
6-15
9.375
B
SK: Seborrheic keratosis HPF: High power field With in the last column, the mean values that do not have common capital letters (A-B) vary from each other (P<0.05)
Mean number of mast cells in 1 high power field
Mean number of mast cells in 1 high power field in exposure group
Figure 4-30: Chart column showing mean number of mast cells/1HPF in Clonal and Acanthotic SK cases in mice exposure group. 78
4.3.2: Mean number of mast cells/1HPF in treatment group: Results showed
there was a strong effect of Acetylsalicylic acid in
reducing the number of mast cells despite chronic UVB irradiation. For example, Mast cells were absent or reduced in the mouse that showed mild hyperplasia with a range of 0.5-1 and mean number of 0.829/1HPF. The same was noted for mouse that showed moderate hyperplasia with a range of 1.2-1.4 and mean number of 1.300/1HPF and the same was also observed in mouse that showed severe hyperplasia with a range of 1.4-2 and mean number of 1.700/1HPF. In the mouse that revealed acanthotic SK, 2 mast cells were observed with mean number of 2.00 /1HPF and in clonal SK the range of 8 and mean number of 8.000/1HPF. The P- value was 0.0001 with A-B-C symbols (according to F test and Duncan‟s test). This indicates a highly significant effect of Acetylsalicylic acid in reducing the number of mast cell with different degrees of hyperplasia in treatment group (Table 4-8 and figure 4-31).
79
Table 4-8: Mean number of mast cells/1HPF in treatment group.
Epidermal lesion
No. of mast cells
Mean/1HPF
Clonal SK
8
8.000
A
Acanthotic SK
2
2.000
B
Severe hyperplasia
1.4-2
1.700
B
Moderate hyperplasia
1.2-1.4
1.300
C
Mild hyperplasia
0.4-1
0.829
C
SK: Seborrheic keratosis HPF: High power field With in the last column, the mean values that do not have common capital letters (A-C) vary from each other (P<0.05)
80
Mean number of mast cells in 1 high power field
Mean number of mast cells in 1 high power field in treatment group
Figure 4-31: Column chart showing mean number of mast cells/1HPF in treatment group.
4.3.3: Mean number of mast cells/1HPF in exposure and treatment groups: Results revealed there was a strong effect of Acetylsalicylic acid in reducing the effect of chronic UVB irradiation in treatment group by decreasing number of mast cells and increasing number of mast cells in exposure group. For example, mast cell numbers were decreased in the treatment group with a range of 0.4-2 and mean number of 1.380/1HPF, while mast cells increased in exposure group with a range of 6-28 and mean number of 15.8 /1HPF. The Pvalue was 0.0001 with A-B symbols (according to F test and Duncan‟s test) (Table 4-9 and figures 4-32, 4-33 and 4-34).
81
Table 4-9: Mean number of mast cells/1HPF in exposure and treatment groups.
Groups
No. of mast cells
Mean/1HPF
Exposure group
6-28
15.800
A
Treatment group
0.4-2
1.380
B
SK: Seborrheic keratosis HPF: High power field With in the last column, the mean values that do not have common capital letters (A-B) vary from each other (P<0.05)
Mean number of mast cells in 1 high power field
Mean number of mast cells in 1 high powerfield in treatment and exposure groups
Figure 4-32: Column chart showing mean number of mast cells /1HPF in exposure and treatment groups.
82
Figure 4-33: Microscopic view of a dermis of skin section obtained from a mouse stained by H & E: (A) It shows few number of mast cells in treatment group. (B) It shows huge number of mast cells in exposure group (arrows). (X100).
Figure 4-34: Microscopic view of a dermis of skin section obtained from a mouse stained by Giemsa: A) It shows few number of mast cells in treatment group. (B) It shows huge number of mast cells in Exposure group (arrows). (X400). 83
4.4: Correlation between apoptotic bodies and mast cells: 4.4.1:- Correlation between total number of apoptotic bodies/10HPF and mean number of mast cells/1HPF in exposure group: The results of this study showed a strong inverse correlation between total number of apoptotic bodies/10HPF and mean number of mast cells/1HPF in chronic UVB irradiation in exposure group i.e. Such that, the decreasing numbers of apoptotic bodies were related inversely to the increasing numbers of mast cells with a P- value of 0.01 according to Pearson's correlation coefficient test. This indicated a highly significant inverse correlation between the increasing numbers of mast cells with the decreasing numbers of apoptotic bodies related to the effect of UVB irradiation (Table 4-10).
Table 4-10: Inverse correlation between total number of apoptotic bodies/10HPF and mean number of mast cells/1HPF in exposure group. Observed value
0.78**
2 tailed P-value
0.01
Alpha
0.05
**
Conclusion: At the level of significance Alpha=0.050 the decision is to reject the null hypothesis of absence of correlation; in other words, the correlation is significant.
84
4.4.2:
Correlation
between
total
the
number
of
apoptotic
bodies/10HPF and mean number of mast cells/1HPF treatment group: Results showed a strong inverse correlation between apoptotic bodies and mast cells i.e., decreasing number of apoptotic bodies related to the increasing number of mast cells in Acetylsalicylic acid administed group with chronic UVB irradiation, with a P-value of 0.01which indicated a highly significant inverse correlation between the number of mast cells and number of apoptotic bodies due to the effect of Acetylsalicylic acid in treatment group (Table 4-11).
Table 4-11: Correlation between total number of apoptotic bodies/10HPF and mean number of mast cells/1HPF in treatment group. Observed value
0.87**
2 tailed P-value
0.01
Alpha
0.05
**
Conclusion: At the level of significance Alpha=0.050 the decision is to reject the null hypothesis of absence of correlation; in other words, the correlation is significant.
85
4.5: Immunohistochemical scoring of COX-2 expression: Immunohistochemical staining of COX-2 in irradiated and treatment groups demonstrated cytoplasmic accumulation of COX-2 protein and were recognized as brown discoloration of the keratinocytes cytoplasm (Figure 4-3540) The COX-2 protein expression was classified into 0-3 point-scale: 0, 0%, 1+, <25% positive cells for weak staining, 2+, 26-50% positive cells for moderate staining, 3+, >50% positive cells for strong staining.
4.5.1: Effect of UVB on frequency of COX-2 expression scores and their percentages in exposure group: The results of COX-2 expression in mice of the exposure group were as follows: 1- Score 1+ was observed in 10 mice with a percentage of 50 (Figure 4-35). 2- Score 2+ was observed in 7 mice with a percentage of 35 (Figure 4-36). 3- Score 3+ was observed in 3 mice with a percentage of 15 (Figure4-37). This indicated that chronic UVB irradiation has a direct effect on COX-2 expression compared to control group which showed score 0 (Figure 4-38 and 4-39).
86
Figure 4-35: Score 1+ COX-2 expression in exposure group (X 400)
Figure 4-36: Score 2+ COX-2 expression in exposure group (X400). 87
Figure 4-37: Score 3+ COX-2 expression in exposure group (X 400).
88
Scores of COX-2 expression in exposure group. Score 0 Score 1+ Score 2+ 0 10 7
Score 3+ 3
Scores for COX-2 expression in exposure group
Score 2+ 35%
Series1, Score 0, 0, 0%
Score 1+ 50%
Score 3+ 15%
Score 0
Score 1+
Score 2+
Score 3+
Figure 4-38: Pie chart showing the effect of UVB on the scores of COX-2 their percentages in exposure group.
expression and
4.5.2:- Effect of Acetylsalicylic acid on the frequency of COX-2 expression scores and their percentages in treatment group: The results of COX-2 expression in mice of the treatment group were as follows: 1- Score 0 was observed in 9 mice with a percentage of 45 (Figure 4-39) 2- Score 1+ was observed in 8 mice with a percentage of 40 3- Score 2+ was observed in 2 mice with a percentage of 10 4- Score 3+ was observed in 1 mouse with a percentage of 5 This indicates the effect of Acetylsalicylic acid by highly reducing COX-2 expression in treatment group by chronic UVB irradiation (Figure 4-40 and 441).
89
Figure 4-39: Score 0 COX-2 expression in treatment group (X 400).
Figure 4-40: Different immunohistochemical scores appearance of COX-2 expresssion in treatment group: (A) Score 0 COX-2 expression (X 400). (B) Score 1+ COX-2 expression (X 400). (C) Score 2+ COX-2 expression (X 400). (D) Score 3+ COX-2 expression (X 100). 90
Scores of COX-2 expression in treatment group Score 0 9
Score 1+ 8
Score 2+ 2
Score 3+ 1
Scores for COX-2 expression in treatment group
Score 1+ 40%
Score 2+Score 3+ 5% 10%
Score 0
Score 1+
Score 0 45%
Score 2+
Score 3+
Figure 4-41: Pie chart showing the effect of Acetylsalicylic acid on the scores of COX-2 expression and their percentages in treatment group.
4.5.3:- COX-2 expression scores and their percentages in exposure and treatment group: The results of COX-2 expression revealed a strong effect of UVB in exposure group in comparison with treatment group as indicated by the following: 1- The highest COX-2 expression score in exposure group was 1+ with a frequency of 10 compared to 0 score with a frequency of 9 in treatment group. 2- The lowest COX-2 expression score in exposure group was 3+ with a frequency of 3 compared to same score with a frequency of 1 in treatment group. This indicated a marked effect of Acetylsalicylic acid in reducing COX-2 91
expression and reducing the effect of chronic UVB irradiation, while in exposure group the COX-2 expression was increased by UVB irradiation (Figure 4-42).
Scores of COX-2 expression Groups
Score o
Score 1
Score 2
Score 3
Exposure group
0
10
7
3
Treatment group
9
8
2
1
Scores for COX-2 expression 10 9 8
groups
7
3 2 1
0
exposure group
treatment group
Figure 4-42: Column chart showing COX-2 expression scores and their percentages in exposure and treatment groups.
92
Discussion UVR is a very common carcinogen. Chronic exposure to sunlight is the most important environmental risk factor for skin cancer. This is predominantly due to the damaging effect of UVB on the DNA. UVB also induces also sunburn cells, i.e. apoptotic keratinocytes, which are crucial protective mechanism against the carcinogenic effects of UVB irradiation (Claerhout et al., 2006; Svobodova et al., 2006). Skin cancer is an important model system for the study of cancer chemoprevention. The ease with which clinical and histological changes may be followed in skin cancer is a great advantage over malignancies of other organ systems. The majority of human malignancies are developed through a sequence of distinct pathophysiologic processes in which multiple genetic alterations or defects are accumulated (Horn and Gordon, 2001; Marks and Furstenberger, 2000; Chun et al., 2004). Seborrheic keratosis (SK) is thickened (acanthotic) epidermis caused by a proliferation of benign, basaloid epidermal cells associated with varying amounts of hyperkeratosis, with horn or keratin cysts and characterized by slow growth without tendency to spontaneous regression and it may be familiar with autosomal dominant inheritance. Usually it is of unknown etiology and not related to sun exposure or viral etiology (Kumar et al., 2005; Elder et al., 2009). Seborrheic keratoses are the most common benign tumors in older people. Seborrheic keratoses have a variety of clinical appearances and they develop from the proliferation of epidermal cells. Although no specific etiologic factors have been identified, they occur more frequently in sunlight-exposed areas (Kwon et al., 2003; Hafner et al., 2007; Naruke et al., 2008 and Balin, 2009). A mutation of a gene coding for a growth factor receptor, (FGFR3), has 93
been associated with seborrheic keratosis (Logie et al., 2005; Hafner et al., 2007; Nakai et al., 2010) Two studies evaluated the expression of COX-2 in seborrheic keratosis. Immunohistochemical staining applied to detect COX-2 expression showed that COX-2 was slightly expressed in SK other than genes like p63 and BCL-2 (Yue-ping et al., 2006; Wu et al., 2007). In this study the animal models were mice as used by Chun et al., (2004), who documented that the mice model properly reflects the concept of multistage carcinogenesis in skin tumor formation. Our study showed a highly significant association between chronic UVB irradiation and development of seborrheic keratosis in exposure group and indicated that UVB with a long duration initiated and promoted seborreic keratosis development which is in disagreement with the studies of Kwon et al., (2003), Kumar et al., (2005) and Elder et al., (2009), who showed that the sun may or may not be related to the development of seborreic keratosis, but this is in agreement with that of Haw et al., (2009). In fact, this article was only a research on UVB as causative agent that induced SK. In this study, COX-2 expression was increased in cases of SK induced by chronic UVB irradiation in exposure group. This means that COX-2 has it an important role in promoting SK development after initiation by UVBR which is in disagreement with Yue-Ping et al., (2006) and Wu et al., (2007) who showed that COX-2 was slightly expressed in SK. To my knowledge no study has been published on over expression of COX-2 in SK. A moderate increase is observed in the rates of apoptosis in all varieties of seborrheic keratoses compared to normal skin (Balin, 2009). Rate of apoptosis in SK is not significantly different from normal skin (Bowen et al., 94
2004). Our results revealed that the decrease of apoptotic cells differed according to the types of SK that showed a mildly decreased apoptosis in clonal type of SK while for acanthotic SK apoptotic bodies were absent or decreased in number and this finding was in disagreement with Bowen et al., (2004) and Balin, (2009) who showed that apoptosis moderately increased or its rate did not differ compared to normal skin. Mast cells have been found to play a critical role in the suppression of immune reactions and not only through production of inhibitory cytokine IL-10. Thus, mast cell infiltration into tumor may possibly remodel tumor microenvironment and profoundly influence tumor behavior by participating and regulating inflammatory and immune reactions. However, although some studies have shown that mast cells promote tumor angiogenesis and tumor growth because of their properties as inflammatory cells, the roles of mast cells in tumor progression has not thus far been understood completely (Huang et al., 2008). Interleukin (IL)-10 and tumor necrosis factor (TNF)-α are the two cytokines most strongly implicated in signaling the immunosuppressive effects of UVB. Furthermore, IL-10 and TNF-α may be produced by additional cells. TNF-α production has been reported from mast cells and fibroblasts after UVB irradiation (Walsh, 1995 and De Kossodo et al., 1995). In the present study there was an increase of mast cells in exposure group due to the effect of chronic UVBR, this was in agreement with Kligman and Murphy, (2008); Chacón-Salinas et al., (2011) who documented that Chronic UVB irradiation increases the number of mast cells in the hairless mouse. UVBenhanced increased the number of mast cells in our animal model (Mus musculus species, BALB/c strain). This finding indicates that this strain was susceptible to UVB irradiation for increasing the number of mast cells and it 95
also indicates development of SK in all mice in exposure group and indicated a strong relation between mast cells, chronic UVB irradiation the increase in number of mast cells is a possible indicator of immunosupression a; as reported by (Huang et al., 2008) who stated that mast cell infiltration into tumor may possibly remodel tumor microenvironment and profoundly influence tumor behavior by participating and regulating inflammatory and immune reactions. To my knowledge no work has ever mentioned an increased number of mast cells in SK and the results of this study was the first of its kind to be reported. In this current study it found that Acetylsalicylic acid was effective in reducing tumor development in chronically exposed mice in treatment group (5 month-irradiation) and showed only 2 cases out of 20 cases of mice which had SK due to irreversible inhibition of COX-2 by Acetylsalicylic acid, enhanced apoptosis and reduced mast cell infiltration which
played a role in
tumorigenesis. To my knowledge no study has ever been published on the effects of Acetylsalicylic acid on SK tumorigenesis and thus our results were the first to show the role of Acetylsalicylic acid in reducing SK development in mice. Our result showed down regulation of COX-2 expression in treatment group by Acetylsalicylic acid administration when compared with exposure group. This indicated that Acetylsalicylic acid was effective in blocking COX-2 enzyme and its production of prostaglandins especially those related to the reduction of SK. Our results showed highly significant relation between Acetylsalicylic acid use and COX-2 expression. To my knowledge no studies have ever been published on the role of Acetylsalicylic acid in reducing COX-2 expression in SK in mice by chronic UVB irradiation. Our study was the first to prove this. In this study the increased number of apoptotic bodies in treatment group was due to the action of Acetylsalicylic acid which led to the increase of apoptosis when compared to exposure group and showed a highly significant 96
relation and eventually the number of tumor development also decreased. There was also an increase in the number of apoptotic bodies which was one of Acetylsalicylic acid‟s effects and a factor to reduce tumorgenesis in treatment group despite chronic UVB irradiation. To my knowledge no study has ever been reported about the effect of Acetylsalicylic acid on apoptosis in SK. In this study we detected a rather significant effect of Acetylsalicylic acid in reducing the number of inflammatory mast cells in chronic UVB irradiation in SK when compared to exposure group and this was due to the anti inflammatory effect of Acetylsalicylic acid on mast cells and decreased role in immunosuppressant in SK development. The present study was first to show the role of Acetylsalicylic acid on decreasing mast cells in SK in mice by chronic UVB irradiation. This study showed a very crucial correlation between mast cells and apoptotic bodies. This correlation reversed in direction; so in exposure group the apoptotic bodies decreased while mast cells increased but in treatment group there was a decrease in mast cells and increase in apoptosis. This was related to the effect of Acetylsalicylic acid in treatment group. To our knowledge no study has ever been published on this correlation and so this study was the first to prove this.
Conclusions: 1- UVB-is one of the causative agents which induced or developed SK in mice. 2- COX-2 expression is increased in SK in mice. 3- Oral administration of Acetylsalicylic acid effectively reduced UVB-induced SK in mice.
97
4- Acetylsalicylic acid reduced epidermal changes and tumor development due to its capacity to increase apoptosis in proliferating UVB-damaged keratinocytes in mice. 5- UVB triggers or increased dermal mast cells.
Recommendations: 1- The feasibility of using chemo-preventive medicine to inhibit COX-2induced tumor in future research studies. 2- Administration of low dose of Acetylsalicylic acid to reduce several types of tumors and further studies will be needed to document this. 3- Further studies on the relation between SK with COX-2 and other genes like P38, ERK1/2,
EGFR are required to further understand their role in
determining other unkown causative agents for SK development. 4- Increase knowledge and applications of diagnostic molecular pathology like FISH and recombinant DNA technology for diagnosing the inherited gene disorders in SK pathology.
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الخالصة رؼذ األشؼخ فىق اٌجٕفغغُخ ِٓ ٔىع ة ) (UVBأحذ األ عجبة اٌشئُغُخ ٌغشغبْ اٌغٍذ .فهٍ لبدسح ػًٍ ثذء ورحفُض و رطىس عشغبْ اٌغٍذ .فً ِشحٍخ اٌجذي َّ ،ىٓ أْ رغجت هزٖ االشؼخ رغُُشاد فٍ اٌىشوِىعىِبد وغفشاد فٍ اٌحّط إٌىوٌ اٌذَىوغً ِٓ DNAخالي اٌزٍف اٌّجبششو أو إٔزبط أٔىاع األوغغُٓ اٌفؼبٌخَ .حذس اٌىسَ ػٓ غشَك رؼضَض اٌزأصُشاد فىق اٌغُُٕخ ِ ،epigeneticضً رغُششفشح اٌغُٕبد .إْ اٌزؼشض اٌّضِٓ إًٌ األشؼخ فىق اٌجٕفغغُخ َؤدي إًٌ اٌزؼجُش ػٓ ِغزىَبد ػبٌُخ ِٓ COX-2فٍ اٌغٍذ .رُ إصجبد دوس COX-2فٍ رىىْ األوساَ اٌخجُضخ وإٌّػ اٌظبهشٌ ِٓ اٌخالَب اٌغشغبُٔخ ِٓ خالي دوسٖ فً صَبدح إٔزبط اٌجشوعزبعالٔذَٓ ورضجُػ اٌخالَب وصَبدح األوػُخ اٌذِىَخ، اٌغضو و رغُش فٍ اٌحبالد االٌزهبثُخ وإٌّبػخ ِٓ .اٌضبثذ أْ األعجشَٓ َمًٍ ِٓ ِخبغش االصبثخ ثغشغبْ اٌغٍذ ػٓ غشَك رضجُػ ٔشبغ أٔضَّبد األوغذح اٌحٍمُخ و هٍ ِٓ اإلٔضَّبد اٌحُىَخ اٌشئُغُخ وزىىْ اٌجشوعزبعالٔذَٓ ،وَحىي دوْ رىىَٓ األوػُخ اٌذِىَخ وَحىي أَعب دوْ رىبصش اٌخالَب وَؤدٌ إًٌ ِىد اٌخالَب اٌّجشِظ ،واٌزٍ رؼزجش ِٓ اٌُِبد اٌهبِخ ٌٍٕشبغ اٌّعبد ٌٍىسَ و اٌىلبَخ ِٓ اٌغشغبْ رهذف هزٖ اٌذساعخ إًٌ ِحبوٌخ أحذاس وسَ فً اٌغٍذ ِٓ خالي رؼشَط فئشاْ إًٌ األشؼخ فىق اٌجٕفغغُخ ِٓ ٔىع ة وإظهبس ِذي ِالئّخ اٌىُُّب إٌغُغُخ إٌّبػجخ IHCفٍ رمُُُ رؼجُشثشورُٓ COX-2فٍ عٍذ اٌفئشاْ اٌّؼشظخ ًٌ UVBو رأصُش حبِط أعُزًُ اٌغبٌُغٍُُه فً اٌحذ ِٓ اٌىسَ اٌغٍذٌ و رأصُشٖ فً رغُش رؼجُش .COX-2 أعشَذ هزٖ اٌذساعخ االعزطالػُخ ٌٍفزشح ِٓ 10أوزىثش 0202و حزً ِ10بسط 0200فٍ ِغزشفً اٌطت اٌجُطشٌ اٌزؼٍٍُّ وِخزجشاألِشاض إٌغُغً فٍ ِغزشفً شىسػ فٍ ِحبفظخ اٌغٍُّبُٔخ. إٌّىرط اٌحُىأٍ اٌّغزخذَ فٍ هزٖ اٌذساعخ االعزطالػُخ وبْ اٌفئشاْ اٌجُعبء اٌزٍ وبٔذ ِٓ ٔىع عالٌخ .BALB/cخعغ ٌهزٖ اٌذساعخ خّغىْ فأسح ولغّذ إًٌ صالس ِغبِغ ،اٌّغّىػخ ) (1ورعّذ 10 فئشاْ وّغّىػخ عُطشح ،اٌّغّىػخ ) 2 (2وأػزجشد فُهب اٌفئشاْ فُهب وّغّىػخ اٌزؼشض ) رزؼشض ألشؼخ فىق اٌجٕفغغُخ( واٌّغّىػخ ) 20 (3وأػزجشد اٌفئشاْ وّغّىػخ اٌؼالط ) رزؼشض ألشؼخ فىق اٌجٕفغغُخ ورؼبًِ ِغ حّط األعُزًُ عبٌُغٍُه ( .ػىٌغذ اٌفئشاْ ِغ حبِط أعُزًُ اٌغبٌُغٍُُه ٌّذح أعجىع واحذ لجً اٌزؼشض ٌألشؼخ وٌّذح 4أَبَ /أعجىع .ثؼذهب رؼشظذ اٌفئشاْ (فً وً ِغّىػخ) ٌألشؼخ فىق اٌجٕفغغُخ ٌّذح أسثؼخ اَبَ فٍ االعجىع و ٌّذح 02دلُمخ و ٌفزشح اِزذد إًٌ خّغخ اشهش.
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فٍ ٔهبَخ وً شهش و ِٓ وً ِغّىػخ أخزد خضػبد ٔغُغُخ ٌٍىشف ػٓ اٌزغُشاد إٌغُغُخ فً اٌغٍذ أو ػٓ ظهىس وسَ فٍ إٌّطمخ اٌّؼشظخ ٌالشؼخ و اٌزٍ أصًَ ِٕهب اٌشؼش ِغجمب فٍ ِغّىػزٍ اٌزؼشض واٌؼالط .ووبٔذ عُّغ اٌحُىأبد لذ خذسد ثبعزخذاَ ِخذس ػبَ (صَالصَٓ -اٌىُزبُِٓ) .وأخزد خضػبد ٔغُغُخ و ِٓ ثؼذهب صجزذ فٍ ِحٍىي اٌفىسِبٌُٓ ) )٪02و ػىٍِذ ثّىاد وبٌىحىي واٌضآٍَُ وأٔزهذ ثبٌحصىي ػًٍ اٌىزٍخ اٌشّؼُخ ولذ رُ اعزحصبي صالصخ ششائح ٔغغُخ و ثغّه ِٓ μm5وً وزٍخ ثبسافُُٕخٌ .ىْ اٌّمطغ األوي ثصجغخ االَىصَٓ و اٌهُّبرىوغٍُُٓ E & Hورٌه ٌٍجحش ػٓ اٌ رغُشاد ٔغُغُُخ ِحزٍّخ وػٓ أٌ شزور رشخُصٍ ِشظٌٍ ،ىْ اٌّمطغ اٌضبٍٔ ثصجغخ ا اٌىُُّب ٌٕغُغُخ إٌّبػُخ ًٌ COX-2ودلمذ ٔزبئظ اٌصجغخ ػٓ غشَك اصُٕٓ ِٓ اخزصبصً ػٍُ االِشاض وثشىً ِغزمً ووشفذ ػٓ وعىد ٌىْ ثٍٕ فٍ اٌغبَزىثالصَ و ثؼذ ِشاعؼخ اٌششائح اٌضعبعُخ اٌٍّىٔخ ثصجغخ اٌهُّبرىوغٍُٓ واالَىعُٓ ٌ ,ىحع وعىد خالَب فً ِٕطمخ ِبرحذ األدِخ وشخصذ هزٖ اٌخالَب ػًٍ أٔهب خالَخ اٌجذَٕخ Mast cellsوألعً رأوُذ رٌه ٌىٔذ اٌششَحخ اٌجبسافُُٕخ اٌضبٌضخ ثصجغخ اٌىُّضا. أظهشد ٔزبئظ اٌىُُّب إٌغُغُخ إٌّبػُخ فً ٌٍىشف ػٓ رؼجُش اي COX-2فٍ ِغّىػزً اٌزؼشض و اٌؼالط ,إْ أػًٍ ٔغجخ ٌٍزؼجُش ػٓ اي ِ COX-2غّىػخ اٌزؼشض وبٔذ )٪50( 0+وإْ ألً ٔغجخ ٌٍزؼجُشػٓ اي COX-2وبٔذ )٪15( 3+فٍ حُٓ اْ أػًٍ ٔغجخ ٌٍزؼجُشػٓ اي COX-2ظّٓ ِغّىػخ اٌؼالط وبٔذ (صفش) ( )٪45و الً ٔغجخ ٌٍزؼجُش فٍ ٔفظ اٌّغّىػخ وبٔذ ٌ . )٪5( 3+ىحع وعىد ػاللخ احصبئُخ لىَخ ثُٓ رأصُش اشؼخ UVBػًٍ رمًٍُ ػذد خالَب حشق اٌشّظ ))sun burn ظّٓ ِغّىػخ اٌزؼشض و وزٌه ٌىحع وعىد صَبدح فٍ ػذد خالَب حشوق اٌشّظ ظّٓ ِغّىػخ اٌؼالط و وبٔذ هٕبن ػاللخ احصبئُخ ِهّخ ٌزأصش اي UVBػًٍ ػذد خالَب اٌجذَٕخ ( )mast cellsفٍ ِغّىػخ اٌزؼشض ثبٌّمبسٔخ ػذد هزٖ اٌخالَب فٍ ِغّىػخ اٌؼالط. دٌذ هزٖ اٌذساعخ ػًٍ إْ االشؼخ فىق اٌجٕفغغُخ ِٓ ٔىع (ة) ، UVBهٍ أحذ ِٓ اٌؼىاًِ اٌّغججخ ٌظهىس و رط َىس Seborrheic keratosisفٍ اٌفئشاْ وإْ هٕبٌه ػاللخ غشدَخ ثُٓ رؼجُش ثشورُٓ اي COX-2واٌزؼشض ٌألشؼخ فىق اٌجٕفغغُخ (ة) ظّٓ ِغّىػزٍ اٌزؼشض واٌؼالط واْ اػطبء ِحٍىي حبِط األعُزًُ عبٌُغٍُه ػٓ غشَك اٌفُ لذ لًٍ ِٓ ظهىس اٌىسَ اٌحُّذ Seborrheic keratosisو وزٌه لًٍ ِٓ رؼجُش ثشورُٓ COX-2فً اٌغٍذ.
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ثوختة
.شيَسثةجنة ى ثيَشت تًشكى شه روو وه نه وشه يى له جؤرى (ب) هؤكاريَكى شه ره كًه له دروشت بىونى تًشكى شه روو وه نه وشه يى طسنكرتيو هؤكارى دروشت بىونى شيَسثةجنة ى ثيَشتة كه ده تىانًت ببًتة هؤى ده شت له كاتى (قؤناغى ) ده .يانداى (ثة ره شه ندى) وه ثيَشخشنت له طة شه ثيَدانى شيَسثةجنة ى ثيَشت ,ثيَكسدن ) به زِيَطاى تيَكضونى زِاشته وخؤ DNAستثيَكسدندا ئه تىانيَت ببيَتة هؤى طؤزِاى له كرؤمؤسؤمه كاندا وتيَكضوى له ( قؤناغى يانداى رووئه دات له زِيَطاى reactive oxygen species).ياى وه ثة يدابىونى مادده ى كًنًايى ( به ركه وتهى به رده وامى تًشكى شه روو وه نه ).وه كى طؤزِاى له ده ركه وتهى جًهداepigeneticكار يطة رى ( COXثسؤتًهى ( ).به راده يه كى به رز له ثيَشتدا ( COX-2وشه يى ده بيَتة هؤى به رزبىنه وه ى ده ركه وتهى) ده شتهًشانكراوه به وه ى كه به شدارى ده كات له دروشت بىونى شيَسثةجنة داو دياريكردنى شًىه شيَسثةجنه ى 2 ترشها ك (زيانبه خش) بؤخانه كانى شيَسثةجنه له زيَطاى زيادبىونى به ريه م يًهانى (دروشت بىونى) زيادبىونى دروشت بىونه وه apoptosis),زِيَطةطستو (نههيَشتنى) مردنى ثسؤطسامى خانه كاى (,ثسؤشتاطالنديو ) و طؤزِيهى حالَةته كانى يه وكردى invasivenessته خش بىوى (angiogenesis),ى مىولىوله كانى خىيَن ( شه مليَنساوه كه ده رمانى ئه سثسيو ده بيَتة هؤى كه م بىونه وه ى مه ترشى تىوش بىوى به شيَسثة .و به رطسيكردى كه ئه نسميًكى شه ره كًه له به ريه م يًهانى COX).جنه ى ثيَشت له زيَكاى زِيَطة طستو له جاالكى ئه نسميى ( زِيَطة كرتو له زيادبىونى خانه كاى و ,زِيَطة طستو له دروشت بىونه وه ى مىولىوله كانى خىيَن ,ثسؤشتاطالنديو ) كه داده نريت به طسنطرتيو شيؤه كانى به ئه جنام طة ياندى له apoptosisزيادبىونى مردنى ثسؤطسامى خانه ( .دذى جاالكى شيَسثةجنه و خؤثاراشنت له شيَسثة جنه
يه ولَداى بى دروشت بىونى شيَسثة جنه ى ثيَشت له مشكدا به به ركه وتهى تًشكى شه روو وه نه وشه يى ده شتهًشانكردنى تىاناى زِيَطاى كًنًا شانه به رطسى له يهلَشة نطاندنى ده ركه وتهى UVB),له جىرى (ب) ( 129
) به )COX-2له ثيَشتى مشكدا و بؤ بًهًهى به رزبىنه وه ى راده ى ده ركه وتو له ثسؤتًهى (COX-2ثسؤتًهى ( به ركه وتهى تًشكى شه روو وه نه وشه يى له جؤرى (ب) وه كار يطة رى ده رمانى ئه سثسيو له كه م بىونه وه ى COX-2).شيَسثة جنه ى ثيَشت و ده ركه وتهى (
ئه م تىيَر يهه وه يه م ده شت ثيَكسد له 01ى ما نطى زِيَبة نداى يه تا 01ى ما نطى خه رماناى شاىل تىيَريهه وه كه م به جٌ هيَنا له نه خىشخانه ى فيَسكارى ثزيشكى فيَتيَسنه رى و تاقيطة ى 1100,ز ئه و ئاذةله ى به كارم هيَنا له تىيَريهه وه كه مدا .هشتؤثاسؤلؤجى له نهخؤشخانه ى شؤزِش له ثاريَزَطاى سليَمانى ثة جنا ( )41مشك به Mus musculus species, BALB/c strain).بريتى بىو له مشكى سثى له جؤرى ( ; 01مشك بىوى دانراى وه كى كؤنرتؤل; كارهيَنساى له م تىيَريهه وه يه دا و دابه ش كراى بى 2طسوب طسوثى ()0 11مشك بىوى دانراى وه كى طسوثى به ركه وتو به تًشكى شه روو وه نه وشه يى له جؤرى (ب) و ;طسوثى ()1 11مشك بىوى دانراى وه كى طسوثى ضازةسةز (به تًشكى شه روو وه نه وشه يى له جؤرى (ب) و ;طسوثى ()2 مشكه كاى له (طسوثى ضازةسةز) و ضازةسةزكراى به ده رمانى ئه سثسيو .ضازةسةزكردنًاى به ده رمانى ئه سثسيو) يه ك يه فته ثيَش ده شت ثيَكسدنًاى به به ركه وتو به تًشكى شه روو وه نه وشه يى له جؤرى (ب) بى ماوه ى 3زِؤذ له يه فته يه كدا ثاشاى مشكه كاى به رئه كه وتو به تًشكى شه روو وه نه وشه يى له جؤرى (ب) ضازةسةزئه كراى يه ريه كيَك له وطسوثانه ( له يه ردوو طسوثى .بؤ ماوه ى 3زؤذ له يه فته يه كدا به دريَراى ماوه ى تىيَريهه وه كه مشكه كاى) به رئه كه وتو به تًشكى شه روو وه نه وشه يى له جؤرى (ب) بؤماوه ى 3زِؤذ له يه فته يه كدا بؤ11 .ده قًقه زِؤذانه بؤ ماوه ى 4مانط ) وه رطريا بؤ دياريكردنى يه biopsiesدواى كىتاى ياتهى يه ر مانطيَك بؤ يه ر طسوثيَك 1منىنه ى ( ) ياى ده ركه وتهى شيَسثةجنه له ناوجه بٌ مىوه تًشك به ركه epidermisرطؤزِانكاريًه ك له ضينى شانه ى ( يه مىو ئاذه له كاى به نج ده كراى به به كار هيَنانى ده رمانى به جنى طشتى .وتىوه كاى له يه ر 1طسوثةكه دا ) جيَ )epidermisوه رده طريا ثاشاى منىونه كاى (شانه ى ضينى (Xylazine-Ketamin).( biopsies 130
طريده كرا له طرياوةى فؤرمالني بهزِيَرة ى ( )%01ثاشاى به يه نكاوه كانى ئاماده كردنى شانه دا ده براى يه تا وه رده طريا 5µmثاشاى 2ثار ضة به ئه شتىورىparaffin embedded tissue block).ثةيدابىونى ( ) بؤ دياريكردنى H & Eيه كه مًاى زِةنط ده كرا به بؤياخى (paraffin embedded tissue block).له ( دووه مًاى زِةنط ده كرا بهزِيَطاى كًنًا شانه ى .يه ر طؤزِانكاريًه كى نائاشايى تايبه ت به ليكؤلًهه وه ى نه خؤشى شاره زايًانى بىارى نه ) 2ثؤليَن كرا له اليه ى COX-2ده ركه وتهى (COX-2).به رطسى تايبه ت به ( خؤشًسانى به شيَوةى جًا و ناشرايه وه به بىونى شايتىثالزمى قاوه ى وه شًًه مًاى زِةنط ده كرا به بؤياخى Giemsa)).
له م تىيَريهه وه دا كه به به كار هيَنانى بؤ ياخى كًنًا شانه به رطسى بؤ زانًهى ثؤليَنى ده ركه وتهى ) له طسوثى به ركه COX-2به رزتريو زِيَرةى ده ركه وتهى ( ).له طسوثى به ركه وتىو و طسوبى ده رماى(COX-2 ) له طسوثى به ركه )٪COX-2ونسمرتيو زِيَرةى ده ركه وتهى (50وتىو به ثؤىل 0بىو كه به بسِى ذما ره ى 01كه ( ) له COX-2له كاتًكدا به رزتريو زِيَرةى ده ركه وتهى ( 15)٪,كه ( 23بىو كه به بسِى ذما ره ى وتىو به ثؤىل ) له )٪COX-2ونسمرتيو زِيَرةى ده ركه وتهى (45كه ( 9طسوثى ده رماى به ثؤىل صفر بىو كه به برى زما ره ى له رووى شتاشًتكه وه به يىه نديه كى بههيَز 25)٪.بىو كه به برى ذما ره ى 0كه ( طسوثى به ركه وتىو به ثؤىل بىو له نًىاى كا ريكه رى تًشكى شه روو وه نه وشه يى له جؤرى (ب) له شه ركه م بىونى خانه كانى ثسؤطسامى له كاتيَكدا خا نه كانى ثسوطسامى مردى له P1110 =.1,له طسوثى به ركه وتىو كه نرخى )(apoptosisمردى له رووى شتاشًتكه وه به يىه نديه P1110 =.1.طسوثى ده رماى زيادى كرد ثة يىه نديه كى به هيَزبىوكه نرخى كى به هيَز يه بىو له نيواى كا ريكه رى تًشكى شه روو وه نه وشه يى له جؤرى (ب) له شه رزيادبىونى خا نه كانى ) له 1mast cellsله كاتيَكدا خا نه كانى ( )P1110 =.له طسوثى به ركه وتىو كه نرخى (mast cells P1110 =.1.طسوثى ده رماى كه مى كرد ثة يىه نديه كى به هيَز بىوكه نرخى
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) يهكيَكة له UVBئه م تىيَر يهه وه يه ئه يصه ملًهًت كه تًشكى شه روو وه نه وشه يى له جؤرى (ب) ( Seborrheicهؤكاره كانى تىوشبىوى كه ده بيَتة هؤى دروشت بىونى ياى طة شه كردنى شيَسثةجنه ى جؤرى ( ) ثة يىه ندى يه يه به به ركه وتهى تًشكى شه روو وه نه وشه يى COX-2ده ركه وتهى ثسؤتًهى (keratosis) , له جؤرى (ب) له مشكه كاندا و ثيَدانى ده رمانى ئه سثسيو له زِيَطاى ده مه وه كاريطة رى يه يه له كه م بىونه وه ى ) و كه م بىونه وه )UVBله مشكى به ركه وتىو به – تًشكى ( Seborrheic keratosisشيَسثةجنه ى جؤرى ( COX-2).ى ده ركه وتهى ثسؤتًهى (
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