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UNIVERSITI PUTRA MALAYSIA
CHARACTERISATION OF INDUCED RAT MAMMARY GLAND TUMOR AND THE ANTITUMOR EFFECT OF RECOMBINANT
HUMAN ERYTHROPOIETIN AND TAMOXIFEN
SAIRAH ABDUL KARIM
IB 2009 12
CHARACTERISATION OF INDUCED RAT MAMMARY GLAND TUMOR AND THE
ANTITUMOR EFFECT OF RECOMBINANT HUMAN ERYTHROPOIETIN AND TAMOXIFEN
SAIRAH ABDUL KARIM
DOCTOR OF PHILOSOPHY UNIVERSITI PUTRA MALAYSIA
2009
CHARACTERISATION OF INDUCED RAT MAMMARY GLAND TUMOR AND THE ANTITUMOR EFFECT OF RECOMBINANT HUMAN
ERYTHROPOIETIN AND TAMOXIFEN
By
SAIRAH ABDUL KARIM
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in fulfillment of the Requirements for the Degree of Doctor of
Philosophy
2009
ii
DEDICATION
To my parents, Mr. Abdul Karim Jabar and Mrs. Noli
Othman…………………………..
To my beloved husband Mohd. Fadhil Ahmad and my lovely children,
Faiz Isqandar and Aleya Yasmin who bring me great happiness……….
iii
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Doctor of Philosophy
CHARACTERISATION OF INDUCED RAT MAMMARY GLAND TUMOR AND THE ANTITUMOR EFFECT OF RECOMBINANT HUMAN ERYHTROPOIETIN
AND TAMOXIFEN
By
SAIRAH ABDUL KARIM
2008
Chairman : Professor Rasedee Abdullah, PhD Faculty : Institute of Bioscience Breast cancer is the most common cancer and the incidence and mortality rate
had remained high. In Malaysia alone, breast cancers accounted for 31% of all
new cancer cases and are among the most fatal cancers. Since breast cancers
are complex diseases, there is no single marker that is both sensitive and
specific for early detection of the disease. The present study was undertaken to
characterize rat mammary gland tumors as a model for breast cancers and to
determine parameters that could be used as early tumor markers. The study
also undertook to determine the effect of recombinant human erythropoietin
(rHuEPO) and Tamoxifen on the rat mammary gland tumor.
In the first part of the study serum biochemical parameters, angiogenic factors
and tumor markers, tumor histopathology and ultrastructure and expression of
estrogen (ER) and erythropoietin receptors (EPOR) were determined. Twenty
female Sprague-Dawley rats, aged six to seven weeks were divided into two
iv
groups of 10 rats per group. The rats were treated intragastrically, the first
group with 20 mg 7,12-dimethylbenz(a)anthracene (DMBA) per rat to induce
mammary tumor development and the second group with 1 mL 0.9% normal
saline and served as the control. The animals were palpated weekly for tumor
mass and sacrificed two weeks after tumor occurrence. Blood was withdrawn
through cardiac puncture before tumor induction and weekly thereafter. Serum
biochemical parameters analysed were alkaline phosphatase (ALP), aspartate
aminotransferase (AST), alanine transferase (ALT), lactate dehydrogenase
(LDH), creatinine kinase (CK), glucose, blood urea nitrogen (BUN) and
creatinine by a chemistry analyser using standard diagnostic kits. Serum tumor
markers,namely α-fetoprotein (AFP) and CA15-3 were analysed using
automated immunoassay analyser, while the angiogenic factors, matrix
metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF)
were determined by the ELISA technique. Tumor tissues excised from
sacrificed animals were subjected to histopathological analysis and ER-α and
EPOR determination through immunohistochemistry (IHC). Tumor ultrastructure
was examined by transmission electron microscopy (TEM).
The results showed higher ALT, ALP and AST concentrations in the DMBA-
treated than the control group reflecting abnormal liver function. Pronounced
increases in serum LDH were also observed in the DMBA-treated group.
Angiogenic factor estimations showed that serum MMP-2 levels remained high
throughout the study and seemed to have played a greater role than VEGF in
v
early stage tumorigenesis. Serum tumor markers, AFP and CA 15.3 were not
detected in either the treated or control rats. Histopathological analysis showed
features typical of neoplastic cells which were enlarged nuclei, conspicuous
nucleoli nuclear pleomorphism, high nuclear to cytoplasma ratio,
hyperchromasia, and epithelial cell and stroma hyperplasia. These features are
similar to that found in breast cancers. Immunohistochemical analysis showed
that ER-α and EPOR were present in the DMBA-induced rat mammary tumor,
which also resemble human breast cancers. Transmission electron microscopy
analysis of the tumor demonstrated the co-existence of apoptosis, necrosis and
aponecrosis which may be used in the determination of mammary gland tumor
and breast cancer development in the early stages.
In conclusion, the combination of serum liver-related enzymes, serum MMP-2
and histological changes, ER-α and EPOR expression, evidences of apoptosis,
necrosis and aponecrosis may form the panel for screening and determination
of early mammary gland tumors in high risk cancer patients.
The second phase of the study involved the development of a xenograft-
induced mammary gland tumor model in rats as a substitute for the
conventional drug-induced method. The xenograft-induced mammary gland
tumor seems to be reliable and can produce tumors within a short period. The
xenograft model was then used to evaluate the effects of rHuEPO, Tamoxifen
and Tamoxifen-rHuEPO combination on mammary gland tumor growth and
vi
angiogenesis. In this study, 24 rats were divided into four groups of six rats
each. Each rat was treated orally: Group 1 with 60 IU rHuEPO; Group 2 with 20
mg Tamoxifen; Group 3 with a combination of 20 mg Tamoxifen and 60 IU
rHuEPO; Group 4 with 1 mL 0.9% normal saline and served as the control. The
results showed that rHuEPO did not promote mammary tumor growth and in
fact may enhance the cytotoxicity of Tamoxifen through the stimulation of
proapoptotic and antiproliferative effects. This study suggests that rHuEPO
treatment in cancer patients may not only be beneficial for the alleviation of
anemia due to the disease, but also augments the effect of certain
chemotherapeutic drugs used in the treatment of cancers.
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk Ijazah Doktor Falsafah
PENCIRIAN TUMOR KELENJAR MAMA TIKUS TERARUH DAN KESAN ANTITUMOR ERITROPOIETIN MANUSIA REKOMBINAN DAN TAMOXIFEN
Oleh
SAIRAH ABDUL KARIM
2008
Pengerusi : Profesor Rasedee Abdullah, PhD
vii
Fakulti : Institut Biosains Kanser payudara merupakan kanser yang sangat biasa berlaku dan kadar
insidens dan kematiannya kekal tinggi. Di Malaysia sahaja, kanser payudara
menyumbang sebanyak 31% kepada kes baru kanser dan merupakan kanser
yang paling banyak menyebabkan kematian. Memandangkan kanser payudara
adalah penyakit yang kompleks, tiada satu petanda tunggal yang peka dan
khusus untuk pengesanan awal penyakit ini. Kajian ini telah diambil untuk
mencirikan tumor kelenjar mama tikus sebagai model untuk kanser payudara
and menentukan parameter yang boleh digunakan sebagai petanda awal
tumor. Kajian ini juga telah dijalankan bagi menentukan kesan eritropoietin
manusia rekombinan (rHuEPO) dan Tamoxifen terhadap tumor kelenjar mama
tikus.
Di dalam bahagian pertama kajian, parameter biokimia serum, faktor
angiogenesis dan petanda tumor, histopatologi tumor dan ultrastruktur dan
pernyataan reseptor estrogen (ER) dan eritropoietin (EPOR) telah ditentukan.
Di dalam kajian ini, 20 ekor tikus betina Sprague-Dawley, berumur 6 hingga 7
minggu telah dibahagikan kepada dua kumpulan terdiri daripada 10 ekor tikus
setiap kumpulan. Tikus-tikus ini diperlakukan secara intragaster, kumpulan
pertama dengan 20 mg 7,12-dimetilbenz(a)antrasin (DMBA) setiap ekor untuk
mengaruhkan pembentukan tumor dan kumpulan kedua diberi 1 mL 0.9%
larutan salina normal dan bertindak sebagai kumpulan kawalan. Haiwan
dipalpatkan setiap minggu untuk pengesanan tumor dan dimusnahkan dua
viii
minggu selepas kejadian tumor. Darah diambil secara tusukan jantung sebelum
pembentukan tumor diaruhkan dan setiap minggu seterusnya. Parameter
biokimia serum yang dianalisis ialah alkalin fosfatase (ALP), aspartat
aminotransefase (AST), alanin transferase (ALT), laktat dehidrogenase (LDH),
glukosa, urea nitrogen darah (BUN) dan kreatinin secara penganalisis kimia
dan kit diagnostik standard. Petanda tumor serum, α-fetoprotein (AFP) dan CA
15.3 telah dianalisis menggunakan penganalisis immunoassai automatik,
sementara faktor angiogenesis, matrik metalloproteinase-2 (MMP-2) dan faktor
pertumbuhan endotelium vesel (VEGF) telah ditentukan menggunakan teknik
ELISA. Pada tisu tumor yang diambil dari haiwan yang dimusnahkan, dilakukan
analisis histopatologi dan penentuan reseptor ER-α dan EPOR secara
immunohistokimia (IHC). Ultrastruktur tumor telah diperiksa menggunakan
mikroskop elektron transmisi (TEM).
Keputusan kajian menunjukkan kepekatan ALT, ALP dan AST lebih tinggi
dalam tikus kumpulan DMBA-diperlaku berbanding kumpulan kawalan,
mencerminkan keabnormalan fungsi hati. Peningkatan nyata LDH serum
diperhatikan dalam kumpulan DMBA-diperlaku. Penganggaran faktor
angiogenesis menunjukkan bahawa paras MMP-2 serum kekal tinggi
sepanjang kajian dan nampaknya memain peranan lebih besar daripada VEGF
pada peringkat awal tumorigenesis. Petanda tumor serum, AFP dan CA15.3
tidak dikesan samada dalam tikus tumor kelenjar mama DMBA-teraruh atau
kawalan. Analisis histopatologi menunjukkan sifat yang tipikal untuk sel
neoplasia seperti nukleus besar, pleomorfisma nukleus ketara, nisbah nuklear
ix
kepada sitoplasma tinggi, hiperkromasia dan hiperplasia sel epithelium dan
stroma. Semua sifat ini menyerupai apa yang terdapat pada kanser payudara.
Analisis immunohistokimia menunjukkan kehadiran ER-α dan EPOR pada
tumor kelenjar mama DMBA-teraruh yang juga menyerupai kanser payudara
manusia. Analisis elektron mikroskop transmisi terhadap tumor menunjukkan
pewujudan bersama apoptosis, nekrosis dan aponekrosis yang mungkin boleh
diguna dalam penentuan perkembangan tumor kelenjar mama dan kanser
payudara peringkat awal.
Sebagai kesimpulan, gabungan enzim berkaitan hati, MMP-2 serum dan
perubahan histologi, pernyataan ER-α dan EPOR, bukti apoptosis, nekrosis dan
aponekrosis mungkin boleh jadi panel bagi penyaringan dan penentuan untuk
peringkat awal tumor kelenjar mama bagi pesakit-pesakit berisiko tinggi untuk
kanser.
Fasa kedua kajian ini melibatkan pembentukan tumor kelenjar mama xenograf-
teraruh pada tikus sebagai ganti kepada kaedah drug-teraruh biasa. Tumor
kelenjar mama xenograf-teraruh adalah stabil dan penghasilan tumor berlaku
dalam masa yang singkat. Model xenograf ini seterusnya digunakan untuk
menilai kesan rHuEPO, Tamoxifen dan gabungan Tamoxifen-rHuEPO terhadap
pertumbuhan tumor kelenjar mama dan angiogenesis. Dalam kajian ini, 24 ekor
tikus dibahagikan kepada empat kumpulan terdiri daripada enam ekor setiap
kumpulan. Setiap ekor tikus diperlakukan secara oral: Kumpulan 1 diberi 60 IU
x
rHuEPO; Kumpulan 2 diberi 20 mg Tamoxifen; Kumpulan 3 diberi gabungan 20
mg Tamoxifen dan 60 IU rHuEPO; Kumpulan 4 diberi 1 mL 0.9% larutan salina
normal dan bertindak sebagai kawalan. Keputusan kajian menunjukkan
rHuEPO tidak menggalakan pertumbuhan tumor kelenjar mama malah mungkin
meningkatkan lagi kesitotoksikan Tamoxifen melalui perangsangan kesan
proapoptosis dan antiproliferatif. Kajian ini mencadangkan rawatan rHuEPO
bagi pesakit kanser bukan hanya berfaedah untuk meringankan anemia
disebabkan penyakit tersebut, tetapi juga memperkuatkan kesan kesitotoksikan
beberapa drug kemoterapi tertentu yang diguna dalam rawatan kanser.
xi
ACKNOWLEDGEMENTS
I would sincerely like to extend my greatest and deepest appreciation to my
main supervisor, Prof. Dr. Rasedee Abdullah for his guidance, advice and
encouragement which he unselfishly gave throughout the course of this study. I
would also like to thank to my co-supervisors, Prof. Dato Dr. Sheikh Omar
Abdul Rahman and Assoc. Prof. Dr. Rozita Rosli for their suggestions and
support.
Sincere gratitude and appreciation are forwarded to Dr. Hazilawati Hamzah,
Mrs. Fauziah Nordin, Mr. Halmi Othman, Mr. Abdullah Misron, staff in
Hematology Laboratory of Faculty of Veterinary Medicine for their technical
assistance during this study. Thanks also forwarded to staff in Electron
Microscopy and Imaging Unit, Institute of Bioscience (IBS) and to all my friends
in UPM.
Last but not least, I would like to extend my extreme gratitude to my sporting
and understanding loving husband, Mr. Mohd. Fadhil Ahmad, my inspired
children, Faiz Isqandar and Aleya Yasmin, my supportive parents and family
members for their constant support and encouragement.
xii
I certify that an Examination Committee has met on 13 August 2009 to conduct the final examination of Sairah Abdul Karim on her Doctor of Philosophy thesis entitled Characterisation of Induced Rat Mammary Gland Tumor and the Antitumor Effect of Recombinant Human Erythropoietin and Tamoxifen in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the Doctor of Philosophy. Members of the Examination Committee are as follows: Mohd Hair Mohd Bejo, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman) Abdul Rani Bahaman, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Examiner 1) Mohd Ali Rajion, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Examiner 2) Yasmin Hanum, PhD Associate Professor, Universiti Kebangsaan Malaysia External Examiner, _____________________ Professor/Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date :
xiii
This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows: Rasedee Abdullah, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman) Dato’ Sheikh Omar Abdul Rahman Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Rozita Rosli, PhD Associate Professor Faculty of Medicine and Health Science Universiti Putra Malaysia (Member) ______________________ AINI IDERIS, PhD Professor/Dean School of Graduate Studies Universiti Putra Malaysia Date:
xiv
DECLARATION I declare that the thesis is my original work except for quotations and citations which have been duly acknowledge. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institutions. ______________________________ SAIRAH ABDUL KARIM Date: 25 November 2008
xv
TABLE OF CONTENTS
Page DEDICATION ii ABSTRACT iii ABSTRAK vii ACKNOWLEDGEMENTS xi APPROVAL xii DECLARATION xiv LIST OF FIGURES xvii LIST OF TABLES xx LIST OF ABBREVIATIONS xxi CHAPTER
I INTRODUCTION 1
II LITERATURE REVIEW 3 Breast Cancer Overview 3 Introduction 3 Human Mammary Gland Tumors 5 Predisposition
Hormones Genes
6 6 7
Screening and Early Detection Imaging Tumor Markers Current Techniques in Breast Cancer Detection Enzymes Linked Immunosorbent Assay (ELISA) and Polymerase Chain Reaction (PCR) Immunohistochemistry Chromosome Analysis Comparative Genomic Analysis Fluorescence In Situ Hybridization (FISH) Proteomics Serum Biochemistry
9 10 11 14 14
14 15 15 16 17 18
Management of Breast Cancer Surgery Chemotherapy Drug Delivery Targeted Therapy
19 19 20 22 22
Tumor Angiogenesis Vascular Endothelial Growth Factor Matrix Metalloproteinases
24 26 27
Prognosis and Predictive Factors of Breast Cancer 28
xvi
Cell Death In Cancer Apoptosis Differences between Apoptosis and Necrosis
30 30 34
Erythropoietin Therapy in Cancer-Related Anemia 35 Relevant of Animal Models for Cancer Research 40 Animal Models for Human Diseases
Transgenic Animal Models Animal Ethics in Research Rodent Mammary Gland Tumor
40 42 43 44
III SERUM ENZYME AND METABOLITE
CONCENTRATIONS DURING DMBA-INDUCED MAMMARY TUMORGENESIS IN FEMALE SPRAGUE-DAWLEY RATS
45
Introduction 45 Materials and Methods 46 Animals and Management 46 Tumor Induction 46 Body Weight and Tumor Occurrence 47 Blood Collection and Serum Samples 47 Serum Enzymes and Metabolites Analysis 47 Statistical Analysis 48 Results 49 Discussion 60
IV SERUM ANGIOGENIC FACTOR AND TUMOR MARKER CONCENTRATIONS IN FEMALE SPRAGUE-DAWLEY RATS DURING TUMORGENESIS
67
Introduction 67 Materials and Methods 68 Animals and Management 68 Blood Collection and Serum Samples 68 Serum Angiogenic Factor Analysis 68 Serum Tumor Markers Analysis 69 Results 70 Discussion 73
V HISTOPATHOLOGY AND EXPRESSION OF ESTROGEN AND ERYTHROPOIETIN RECEPTORS ON DMBA-INDUCED MAMMARY TUMOR IN FEMALE SPRAGUE- DAWLEY RATS
77
Introduction 77 Materials and Methods 78 Animals and Management 78 Tumour Measurement and Histopathological
xvii
Analysis 78 Immunohistochemical Staining for ER-α 81 Immunohistochemical Staining for EPOR 81 Results 83 Discussion 85
VI ULTRASTRUCTURE OF EARLY STAGE MAMMARY TUMOR IN FEMALE SPRAGUE-DAWLEY RAT
104 Introduction 104 Materials and Methods 105 Animals and Management 105 Transmission Electron Microscopy Procedures 105 Morphometric Analysis 105 Results 107 Discussion 116
VII EFFECTS OF rHuEPO TOWARDS THE TUMOR GROWTH AND ANGIOGENESIS IN MAMMARY TUMOR XENOGRAFT MODELS
122 Introduction 122 Materials and Methods 123 Animals Management 123 Xenograft Technique for Mammary Tumor
Induction
123 Preparation of rHuEPO 123 Preparation of Tamoxifen
Experimental Designs Tumor Mass Measurement Blood Collection VEGF and MMP-2 Analysis Histopathological Analysis Results
124 124 124 125 125 125 127
Discussion 142
VIII GENERAL DISCUSSION AND CONCLUSION 142
REFERENCES 148 APPENDICES 176 BIODATA OF THE AUTHOR 184
xix
LIST OF FIGURES
Figure Page
1 Weekly changes in animal body weight in DMBA-treated and control rats.
54
2 Weekly changes in serum ALT of DMBA-treated and
control rats. 56
3 Weekly changes in serum ALP of DMBA-treated and
control rats. 57
4 Weekly changes in serum AST of DMBA-treated and
control rats. 58
5 Weekly changes in serum LDH of DMBA-treated and
control rats. 59
6 Weekly changes in serum glucose in DMBA-treated
and control rats. 61
7 Weekly changes in BUN of DMBA-treated and control
rats. 62
8 Weekly changes of serum creatinine of DMBA-treated
and control rats. 63
9 Mammary tumor tissues dissected from female
Sprague-Dawley rat 2 week after detection. The tumor showed a nodule appearance.
10 Mammary tumor section from a female Sprague-
Dawley rat with grade 1 mitotic activity (arrows) (H&E,x1000)
11 Mammary tumor section from a female Sprague-
Dawley rat with moderate nuclear pleomorphism (grade II) (H&E, x1000).
12 Mammary tumor section from a female Sprague-
Dawley rat with grade II tubule formation (10-75%) (H&E, x400)
xx
13 Mammary tumour section from a female Sprague-Dawley rat with mild hyperplasia (H&E, x400)
14 Mammary tumour section from a female Sprague-
Dawley rat with focal necrosis (H&E, x200)
15 Normal mammary gland tissue of Sprague-Dawley rat
showing single layer of epithelial cells surrounding each tubules (H&E, x200)
16 Immunohistochemical staining of ER-α in normal
mammary gland tissue shows negative immunostaining in the epithelial cells of the tubules (DAB)
17 Immunohistochemical staining of ER-α in the
mammary tumor from a female Sprague-Dawley rat showing strong intensity immunostaining (brown) (DAB)
18 Immunohistochemical staining of ER-α in the
mammary tumor from a female Sprague-Dawley rat showing medium intensity immunostaining (brown) (DAB)
19 Immunohistochemical staining of EPOR in the normal
mammary gland tissue from a female Sprague-Dawley rat showing nuclear staining in several cells (arrows) and classified as level 1 (DAB, x400)
20 Immunohistochemical staining of EPOR in the
mammary tumor from a female Sprague-Dawley rat showing homogenous nuclear staining (DAB)
21 Immunohistochemical staining of EPOR in the
mammary tumor from a female Sprague-Dawley rat showing strong nuclear staining of tumor cells with granular appearance (arrow) (DAB)
22 Immunohistochemical staining of EPOR in the
mammary tumor from a female Sprague-Dawley rat showing cell with cytoplasmic EPOR staining (arrow) and granular or non-homogenous appearance in most of the tumor cells (DAB)
xxi
23 Mammary tumor tissue from female rat showing cells pleomorphism with perinuclear chromatin condensation suggesting the cell undergoing apoptosis and the presence of multiple nucleoli. The amorphous dense masses (arrows) are necrotic cells.
24 Mammary tumor tissue from female rat showing
aponecrotic degeneration (red arrow). The cytoplasm is scanty and contained very few organelles, variable-sized vacuoles (yellow arrow) and disrupted mitochondria. The nucleus still intact and the nuclear envelope convoluted and irregular. Heterochromatin is prominent at the perinuclear region (green arrow).
25 Mammary tumor tissue section from female rat shows
swelling cell with relatively large nucleus and a prominent nucleolus (N). The cytoplasm contains numerous variable sized of vacuoles and defective cristae of mitochondria are noted (arrows).
26 Mammary tumor tissue from female rat showing
bizarre nuclear shape with an indentation and the cytoplasm contains numerous cisternae rough endoplasmic reticulum (rER) (arrows).
27 Mammary tumor tissue from female rat showing a cell
with very distinctive multiple nucleoli (N) and indented nuclear membrane (arrows).
28 Mammary tumor tissue from female rat showing
transverse section of eosinophil which can be recognized by the presence electron dense-stripe crystals with cytoplasmic projection (arrows) or pseudopodias.
29 Mammary tumor tissue from female rat showing a
plasma cell (centre) with eccentrically-positioned nucleus and very extensive rough endoplasmic reticulum (rER) in the cytoplasm. Mast cell (left) is characterised by the presence of large dense granules.
30 A female Sprague-Dawley rat with mammary tumor
induced by xenograft technique. Note the tumor is localized at the thoracic region (arrow)
xxii
31 Rate of mammary tumor progression in control rats and rats treated with Tamoxifen, rHuEPO and Tamoxifen-rHuEPO combination.
32 Serum MMP-2 concentration in control rats and rats
treated with Tamoxifen, rHuEPO and Tamoxifen-rHuEPO combination.
33 Mammary tumor section from control group showing
a mitotic figure (arrow) (H&E, x400)
34 Mammary tumor section from the rHuEPO group
showing several mitotic figures (arrow) (H&E, x600)
xx
LIST OF PLATES
Plate Page
1 Mammary tumor tissues dissected from female Sprague-Dawley rat 2 week after detection. The tumor showed a nodule appearance.
88
2 Mammary tumor section from a female Sprague-
Dawley rat with grade 1 mitotic activity (arrows) (H&E,x1000)
90
3 Mammary tumor section from a female Sprague-
Dawley rat with moderate nuclear pleomorphism (grade II) (H&E, x1000).
90
4 Mammary tumor section from a female Sprague-
Dawley rat with grade II tubule formation (10-75%) (H&E, x400)
91
5 Mammary tumour section from a female Sprague-
Dawley rat with mild hyperplasia (H&E, x400) 91
6 Mammary tumour section from a female Sprague-
Dawley rat with focal necrosis (H&E, x200) 92
7 Normal mammary gland tissue of Sprague-Dawley rat
showing single layer of epithelial cells surrounding each tubules (H&E, x200)
92
8 Immunohistochemical staining of ER-α in normal
mammary gland tissue shows negative immunostaining in the epithelial cells of the tubules (DAB)
95
9 Immunohistochemical staining of ER-α in the mammary
tumor from a female Sprague-Dawley rat showing strong intensity immunostaining (brown) (DAB)
95
10 Immunohistochemical staining of ER-α in the mammary
tumor from a female Sprague-Dawley rat showing medium intensity immunostaining (brown) (DAB)
96
11 Immunohistochemical staining of EPOR in the normal
mammary gland tissue from a female Sprague-Dawley rat showing nuclear staining in several cells (arrows) and classified as level 1 (DAB, x400)
98
xxi
12 Immunohistochemical staining of EPOR in the
mammary tumor from a female Sprague-Dawley rat showing homogenous nuclear staining (DAB)
98
13 Immunohistochemical staining of EPOR in the
mammary tumor from a female Sprague-Dawley rat showing strong nuclear staining of tumor cells with granular appearance (arrow) (DAB)
99
14 Immunohistochemical staining of EPOR in the
mammary tumor from a female Sprague-Dawley rat showing cell with cytoplasmic EPOR staining (arrow) and granular or non-homogenous appearance in most of the tumor cells (DAB)
99
15 Mammary tumor tissue from female rat showing cells
pleomorphism with perinuclear chromatin condensation suggesting the cell undergoing apoptosis and the presence of multiple nucleoli. The amorphous dense masses (arrows) are necrotic cells.
114
16 Mammary tumor tissue from female rat showing
aponecrotic degeneration (red arrow). The cytoplasm is scanty and contained very few organelles, variable-sized vacuoles (yellow arrow) and disrupted mitochondria. The nucleus still intact and the nuclear envelope convoluted and irregular. Heterochromatin is prominent at the perinuclear region (green arrow).
115
17 Mammary tumor tissue section from female rat shows
swelling cell with relatively large nucleus and a prominent nucleolus (N). The cytoplasm contains numerous variable sized of vacuoles and defective cristae of mitochondria are noted (arrows).
116
18 Mammary tumor tissue from female rat showing bizarre
nuclear shape with an indentation and the cytoplasm contains numerous cisternae rough endoplasmic reticulum (rER) (arrows).
117
19 Mammary tumor tissue from female rat showing a cell
with very distinctive multiple nucleoli (N) and indented nuclear membrane (arrows).
118
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