Faizaa Ali University of East Anglia University of Suffolk

241 Pages·2017·4.88 MB·English

The role of phytomolecules in the prevention of
bone cell apoptosis–potential role in bone health

Faizaa Ali
A thesis submitted for PhD degree in Medical Sciences

University of East Anglia
Norwich Medical School

University of Suffolk
Department of Science
and Technology
March 2018
ACKNOWLEDGEMENTS

I would like to express my extreme gratitude to my primary supervisor Dr. Val
Mann and my previous primary supervisor Prof. Brendon Noble for the sup-
port I have received during the undertaking of this study. I would also like to
thank my secondary supervisor Prof. Ian Clark at University of East Anglia
and Dr. Chris Turner at University of Suffolk and to all the staff at the School
of Bioscience at University of Suffolk. Finally, big thanks to my family for
their support during my study.

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DECLARATION

The work in this thesis is the result of my own investigations and the views
expressed are those of myself, except the modification of the two phytoestro-
gens Genistein and Daidzein which was carried out by Dr. Sinan Battah, visit-
ing fellow at Essex University, School of Biological Science.

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ABSTRACT

Estrogen withdrawal after the menopause leads to an increased rate of bone
remodeling, excessive osteoclast activity and a greater fracture risk. Hor-
mone replacement therapy (HRT) was prescribed as a treatment for post-
menopausal women to prevent bone loss; however, HRT is associated with
an elevated incidence of cardiovascular disease, stroke and cancer. These
side effects led to an interest in naturally occurring compounds with estrogen-
ic action such as phytoestrogens (PEs), which are non-steroidal-plant derived
compounds. The aim of this study describes investigation of the protective
effect of phytoestrogen molecules (Genistein, Daidzein, Resveratrol, 6-
Gingerol and Fucosterol) in the prevention of reactive oxygen species in-
duced by H O cell death in MLOY4 osteocyte like cell line. All compounds
2 2
tested significantly (p<0.05) reduced osteocyte apoptosis and decreased re-
active oxygen species activity. PEs have multiple effects on different bone
cells, reducing osteoblast apoptosis and inhibiting RANKL inducing osteo-
clast formation. The protective effect of Genistein, Daidzein and Resveratrol
was not prevented by estrogen receptor inhibitor ICI 182780, suggesting a
potential direct antioxidant effect. Additionally, this study offers evidence for
bone protection conferred by PEs through demonstrating their potent inhibito-
ry effect on cytokine release and activating antioxidant enzymes such as
Catalase (CAT). The antioxidant effect of PEs was thought to be due to their
similar phenolic structure to estrogen; therefore Genistein and Daidzein were
modified by masking their hydroxyl groups and tested for their protective ef-
fects on osteocyte apoptosis. Not all modified compounds protected osteo-
cytes from oxidative stress induced by hydrogen peroxide (H O ). This study
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provides novel evidence that PEs have multiple effects on bone cell activity.
Thus, PEs may not be the only treatment but possible supplementation of
pharmacological or alternative to pharmacological treatment of post-
menopausal osteoporosis.

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS ................................................................................ i
DECLARATION .............................................................................................. ii
ABSTRACT .................................................................................................... iii
TABLE OF CONTENTS .................................................................................. v
ABBREVIATIONS GLOSSARY ..................................................................... ix
LIST OF FIGURES ....................................................................................... xii
LIST OF TABLES ......................................................................................... xvi
CHAPTER ONE: INTRODUCTIO......................................…………………….1
1.1 Bone biology ......................................................................................... 2
1.2 Bone Cells ............................................................................................ 7
1.3 The osteoblast ...................................................................................... 8
1.4 The osteoclast .................................................................................... 11
1.4.1 Factors that affect osteoclastogenesis ......................................... 11
1.4.1.1 Receptor activator of NF-kB (RANKL) .................................... 11
1.4.1.2 Tumor Necrosis Factor -  (TNF-) ........................................ 15
1.4.1.3 Interleukin-6 (IL-6) .................................................................. 17
1.5 Osteocytes .......................................................................................... 19
1.6 Osteocyte function .............................................................................. 20
1.6.1 Blood-calcium/phosphate homeostasis ........................................ 20
1.6.2 The osteocyte as a mechanosensor ............................................. 22
1.6.3 Bone remodeling .......................................................................... 24
1.7 Hormone receptors on osteocytes ...................................................... 27
1.7.1 Estrogen ....................................................................................... 29
1.8 Apoptosis ............................................................................................ 32
1.9 Bcl-2 family and mitochondrial role in apoptosis ................................. 33
1.10 Caspases (Cysteine Aspartate Protease) ......................................... 34
1.11 Death receptors ................................................................................ 35
1.12 Osteocyte Apoptosis ......................................................................... 37
1.12.1 The role of osteocytes in bone disease ...................................... 40
1.13 Osteoclast apoptosis ........................................................................ 41
1.14 Osteoblast apoptosis ........................................................................ 42
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1.15 Oxidative stress and its role in promotion of osteoporosis ................ 43
1.16 Osteoporosis .................................................................................... 48
1.16.1 Osteoporosis and estrogen ........................................................ 50
1.17 Current treatments for osteoporosis ................................................. 51
1.17.1 Calcium & Vitamin D ................................................................... 51
1.17.2 Bisphosphonates ........................................................................ 52
1.17.3 Hormone replacement therapy (HRT) ........................................ 53
1.17.4 Selective estrogen receptor modulators (SERMs) ...................... 54
1.17.5 Parathyroid Hormone ................................................................. 55
1.17.6 Calcitonin .................................................................................... 56
1.18 Phytoestrogens ................................................................................. 56
1.19 Genistein (GEN) ............................................................................... 60
1.20 Daidzein (DZ).................................................................................... 62
1.21 Resveratrol (Resv) ............................................................................ 63
1.22 Fucosterol ......................................................................................... 65
1.23 6-Gingerol ......................................................................................... 66
Hypothesis ................................................................................................ 67
Aim of study .............................................................................................. 67
CHAPTER TWO: MATERIALS AND METHODS ......................................... 68
2.1 Materials: ............................................................................................ 69
2.2 Methods: ............................................................................................. 69
2.2.1 Cell culture: .................................................................................. 69
2.2.2 MC3T3-E1 cell culture .................................................................. 70
2.2.3 Cell culture for RAW264.7 ............................................................ 70
2.2.4 Osteoclast differentiation .............................................................. 70
2.2.5 Assesment of Tartrate-Resistant Acid Phosphatase activity ....... 71
2.2.6 Fixation ......................................................................................... 71
2.2.7 DAPI staining ................................................................................ 72
2.2.8 Induction of apoptotic bodies using H O ..................................... 72
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2.2.9 Characterization of apoptotic bodies by Annexin V FITC binding
and PI staining. ...................................................................................... 72
2.2.10 Treatment of MLO-Y4 cells with 17β-Estradiol or phytoestrogens
(Genistein, Diadzein and Resveratrol): .................................................. 73
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2.2.11 Treatment of MLO-Y4 cells with Estrogen Receptor Inhibitor prior
to treatment with 17β-Estradiol or phytoestrogen (Genistein, Diadzein
and Resveratrol): ................................................................................... 74
2.2.12 Detection of Caspase 3 and 7 activities ...................................... 74
2.2.13 Detection of intracellular reactive oxygen species ...................... 75
2.2.14 Enzyme-linked immunosorbent assay (ELISA) for cytokines
(RANKL, IL-6 and TNF-α) ...................................................................... 75
2.2.15 Modification of Genistein and Daidzein ...................................... 77
2.2.16 Catalase Activity Assay .............................................................. 78
Statistical analysis ........................................................................................ 78
CHAPTER THREE: INVESTIGATION OF THE EFFECT OF
PHYTOESTROGENS IN THE PREVENSION OF OXIDANT INDUCED
APOPTOSIS IN MLO-Y4 CELLS ................................................................. 79
3.1 Introduction ......................................................................................... 80
3.2 Characterisation of osteocyte apoptotic bodies .................................. 83
3.3 The anti-apoptotic effect of E2 on H O induced apoptosis in MLOY4 86
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3.4 The anti-apoptotic effect of Genistein, Daidazein and Resveratrol on
osteocyte apoptosis induced by H O ...................................................... 87
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3.5. Effect of Fucosterol on oxidative stress induced by Hydrogen Peroxide
in MLO-Y4 cells ........................................................................................ 90
3.6 Effect of 6-Gingerol on protecting MLO-Y4 cells from apoptosis ........ 91
3.7 Detection of intracellular ROS ............................................................ 92
3.7.1 Effect of Fucosterol on intracellular ROS generation .................... 96
3.7.2 Effect of 6-Gingerol on intracellular ROS generation .................... 97
3.8 Investigation of role of E2 and Genistein, Daidzein and Resveratrol on
caspase3, 7 activation .............................................................................. 98
3.9 Effect of 6-Gingerol on caspase activity ............................................ 103
3.10 Discussion ...................................................................................... 104
CHAPTER FOUR: THE EFFECT OF PHYTOESTROGEN ON HYDROGEN
PEROXIDE INDUCED APOPTOSIS IN MC3T3-E1 AND RAW 264.7 CELLS
DIFFERENTIATION ................................................................................... 111
4.1 Introduction ....................................................................................... 112
4.2 Saving effect of PEs on MC3T3-E1 cells .......................................... 113
4.3 Measurement of intracellular ROS .................................................... 115
4.4 Effects of PEs on differentiation of RAW 264.7 cells and Tartrate-
resistant Acid Phosphatase .................................................................... 118
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4.5 Discussion ........................................................................................ 120
CHAPTER FIVE: INVESTIGATION OF POTENTIAL SAVING MECHANISM
OF PHYTOESTROGEN ............................................................................ 125
5.1 Introduction ....................................................................................... 126
5.2 Investigation of Estrogen Receptor Inhibitor effect on anti-apoptotic
effect of estrogen and phytoestrogen ..................................................... 127
5.3 Effect of Genistein, Daidzein and Resveratrol on catalase (CAT)
enzyme activity ....................................................................................... 133
5.4 Modification and the structural rational strategy ............................... 135
5.5 Fluorometric assay of MLO-Y4 cells treated with modified Genistein
and Daidzein via H2DCF-DA .................................................................. 139
5.6 The anti-apoptotic effect of modified Genistein and Daidazein on
osteocyte apoptosis induced by H O . .................................................... 141
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5.7 Discussion ........................................................................................ 143
CHAPTER SIX: THE EFFECT OF PHYTOESTROGENS ON CYTOKINE
PRODUCTION IN MLO-Y4 IN RESPONSE TO HYDROGEN PEROXIDE 149
6.1 INTRODUCTION .............................................................................. 150
6.2 Measurement of RANKL release ..................................................... 152
6.3 Measurement of TNF-α release ........................................................ 155
6.4 Effect of PEs on IL-6 released in MLO-Y4 cells ................................ 159
6.5 Discussion ........................................................................................ 163
CHAPTER SEVEN: GENERAL DISCUSSION .......................................... 168
7.1 Discussion ........................................................................................ 169
7.2 Future work ....................................................................................... 176
REFFERENCES ........................................................................................ 178

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ABBREVIATIONS GLOSSARY

1,25(OH)3 VitaminD3
ALP Alkaline phosphatase
ATP Adenosine triphosphate
BMU Basic multicellular unit
BSUs Basic structure units
BH3 BCL-2 homology 3
Bcl-2 B cell leukemia/lymphoma 2-like proteins
BMSCs Bone marrow stromal cells
BLCs Bone lining cells
BMD Bone mineral density
BRU Bone remodelling unit
BMP Bone morphogenic protein
Caspase Cysteine Aspartate Protease
CFU-O Colony- forming unit-osteoprogenitor
CX43 Connexin43
DC-STAMP Dendritic specific transmembrane protein
ERα Estrogen receptor alpha
ERβ Estrogen receptor Beta
ERK1/2 Extracellular signal-regulated kinase 1/2
FADD FAS-associated death domain
FGF Fibroblast growth factor
FOXO Forkhead box-O
FPPs Farnesyl diphosphate synthase
GLa Carboxyglutamic acid
Gpx1 Glutathione peroxidase 1
GM-CSF Granulocyte macrophage colony stimulating factor
HRT Hormone replacement therapy
H O Hydrogen peroxide
2 2
IFN-γ Interferone-gamma
IGF-1 Insulin-like growth factor-1
IL-1α Interlukin 1 alpha
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