Table Of ContentFLAXSEED OIL INDUCES APOPTOSIS IN THE AGGRESSIVE MURINE
MELANOMA CELL LINE B16-BL6
by
Alison Buckner
A thesis submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy (PhD.) in Biomolecular Sciences
The Faculty of Graduate Studies
Laurentian University
Sudbury, Ontario, Canada
© Alison Buckner, 2014
THESIS DEFENCE COMMITTEE/COMITÉ DE SOUTENANCE DE THÈSE
Laurentian Université/Université Laurentienne
Faculty of Graduate Studies/Faculté des études supérieures
Title of Thesis
Titre de la thèse FLAXSEED OIL INDUCES APOPTOSIS IN THE AGGRESSIVE MURINE
MELANOMA CELL LINE B16-BL6
Name of Candidate
Nom du candidat Buckner, Alison
Degree
Diplôme Doctor of Philosophy
Department/Program Date of Defence August 22, 2014
Département/Programme Biomolecular Sciences Date de la soutenance
APPROVED/APPROUVÉ
Thesis Examiners/Examinateurs de thèse:
Dr. Robert Lafrenie
(Supervisor/Directeur(trice) de thèse)
Dr. Sabine Montaut
(Committee member/Membre du comité)
Dr. Carita Lanner
(Committee member/Membre du comité)
Approved for the Faculty of Graduate Studies
Dr. Francisco Diaz-Mitoma Approuvé pour la Faculté des études supérieures
(Committee member/Membre du comité) Dr. David Lesbarrères
M. David Lesbarrères
Dr. Siyaram Pandey Acting Dean, Faculty of Graduate Studies
(External Examiner/Examinateur externe) Doyen intérimaire, Faculté des études supérieures
Dr. Diane Cameron
(Internal Examiner/Examinateur interne)
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Abstract
Flaxseed is classified as a functional food and is renowned for its exceptional
nutrional value. These foods have the ability to either promote overall well-being and/or
reduce the risk of certain diseases. Flaxseed, in the form of seed-derived oil, is the
highest plant source of the omega-3 fatty acid, alpha linolenic acid. This nutraceutical
has been credited with providing protective benefits against breast, colon and prostate
cancers. Humans are unable to synthesize omega-3 fatty acids within the body and
therefore must obtain sufficient amounts of this antioxidant through diet alone.
Currently, the Western diet is grossly deficient in omega-3 fatty acids and this is
in part due to the overwhelming presence of omega-6 fatty acids used in food processing
and manufacturing techniques. As one of the highest sources of omega-3 fatty acids,
flaxseed is often taken as a supplement to help balance the ratio of omega-3 to omega-6
fatty acids in Western cultures. Although flaxseed has been beneficial in alleviating
certain symptoms in patients suffering from cardiovascular disease, diabetes, and cancer,
the underlying mechanisms remain unclear. Previous animal studies have shown that a
diet supplemented with 10% flaxseed, significantly decreased tumour growth in rodent
cancer models. Our studies include the treatment of malignant cells with flaxseed oil in
an in vitro model. We have shown that flaxseed oil has the ability to reduce cell growth
in B16-BL6 cells, an aggressive murine melanoma. Furthermore, a total of seven
different oils containing high concentrations of omega fatty acids, including flaxseed oil,
olive, sunflower, canola, sesame, peanut and grapeseed, were characterized by HPLC and
GC/MS analysis for fatty acid profiles, and these oils were also used to treat B16-BL6
cells. Although all seven oils contain various amounts of omega-3, -6 or -9 fatty acids,
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only treatment with flaxseed oil decreased the growth of the aggressive murine melanoma
cell line B16-BL6. DNA laddering, acridine-orange staining, TUNEL staining, and
FACS analysis using Annexin V and propidium iodide, showed that the flaxseed-treated
cells were undergoing apoptosis, a type of cell suicide. Therefore, due to its ability to
selectively inhibit malignant cell proliferation, flaxseed oil has significant potential as an
anti-cancer therapeutic.
Keywords
Flaxseed, omega-fatty acids, cancer, natural products, anti-cancer therapeutics
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Acknowledgements
I would like to first and foremost thank my supervisor Dr. Lafrenie for providing me with
the continued support and encouragement to complete this thesis. It has been a difficult
journey and I could not have succeeded without you.
Thank you to my family for your words of encouragement throughout the years. I cannot
express how much it means to me that all of you stood by me and never stopped
believing that I could one day complete this chapter of my life.
To Dave, I cannot thank you enough for helping me through the most stressful times in
my life and for always making me see the actual important things in my life. I look
forward to all the adventures we will have with our newest little bundle of joy.
And finally, I dedicate this chapter of my life to my beautiful baby girl Abby. You are
the reason I decided to push myself to complete this thesis. Your early arrival gave me
the confidence and courage to stand in front of my peers and defend this thesis. I promise
to support you in all that you do and I will try my best to make all your wishes come true.
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Table of Contents
Thesis Defence Committee………………………………………………………………..
Abstract…………………………………………………………………………………...iii
Acknowledgments…………………………………………………………………….......v
Table of Contents………………………………………………………………………....vi
List of Figures…………………………………………………………….…………........ix
List of Tables …………………………………...…………………………………...…...xi
List of Appendices……………………………………………………...…….….….…...xii
Abbreviations………………………………………………………………………...….xiii
1.0 Introduction……….………………………………………………………….…….….1
1.1 Traditional Use of Natural Products in Medicine..………………………………..2
1.2 Flaxseed Oil Consumption………………………………………………………...4
1.3 Functional Foods, Flaxseed and Fatty Acids…….…………………………….….6
1.4 Omega-3 and Omega-6 Structures, Classification and Desaturation...………......7
1.5 Omega-3 versus Omega-6 Functions.……...………………………………........11
1.6 Natural Products, Cell Growth and Cell Death….……...…..……………….......13
1.7 Natural Products and Chemotherapy...…..………………...…………………….17
1.8 Proposal…..…………………………………………………...……………….…20
2.0 Materials and Methods……..……..……………..…………………………………..22
2.1 Tissue Culture…..…..……………………………………………………..……..22
2.2 GC/MS of Plant Oils……………………………………………………………..22
2.3 Trypan Blue Survival Analysis…..…..…………………………………………..23
2.4 Cell Viability by Methyl Tetrazolium Blue (MTT).……....…………….….…...24
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2.5 FACS Analysis: Annexin V and Propidium Iodide Stain………………………..25
2.6 Fluorescence Microscopy: Acridine-orange, Ethidium Bromide, TUNEL,
Lysotracker and Mitotracker……..…..…………….…………………………….…..25
2.7 Metabolism Experiment………..…..………………………………………….…26
2.8 Gas Chromatography/Mass Spectrometry Analysis of Cellular Membranes...…27
3.0 Results………..……………..……………………………….…………………….…28
3.1 GC/MS FAME Profiles of Plant Oils …………………………………………...28
3.2 The effects of Plant Oils on Cell Proliferation in B16-BL6 cells………….…….35
3.3 Flaxseed oil Treatment Induces Apoptosis in B16-BL6 cells…...……….……...38
3.4 Cell Cycle and Inhibition of Cell Proliferation .…………………………………47
3.5 Lysosome Formation in Flaxseed Oil Treated B16-BL6 cells………………..…50
3.6 Mitochondrial Changes in Flaxseed Oil Treated B16-BL6 cells………………..53
3.7 Solvent Extraction of Anti-Cancer Components of Flaxseed Oil……………….58
3.8 Flaxseed Oil Metabolism in B16-BL6 Cells…………………………………….63
3.9 Flaxseed Oil Enhance Taxol-Mediated Cell Death in B16-BL6 Cells.………….69
4.0 Discussion……………..………………………………...………...………………....74
4.1 Flaxseed oil and Effects on B16-BL6 Cells…………………….………………..74
4.2 Apoptosis and Cell Morphology………………………….……………………...76
4.3 TUNEL and DNA Damage……………………………………………………....78
4.4 Annexin V-FITC and Apoptosis……………....……………………………..…..79
4.5 PI Stain and the Cell Cycle……………..……………………………..….……...81
4.6 Mitochondrial and Lysosomal Changes in Flaxseed Oil Treated Cells…………82
4.7 Omega-3 Fatty Acid Metabolism…………….……...………………..….………83
4.8 Flaxseed Oil and Chemotherapeutic Agents……………………………………..85
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4.9 Future Directions- Cell Signaling………………..…………………….………...86
5.0 Conclusion……………..………………………..…………………..………….....…88
References………………..……………………..………………………………………..90
Appendix…………………..……………………………………………………………..97
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List of Figures
Figure 1 Chemical structures of the omega-3 and omega-6 fatty acids…………………..9
Figure 2 Competing desaturation and elongation pathway of the omega-3 and omega-6
fatty acids………………………………………………………………………………...10
Figure 3 The intrinsic and extrinsic apoptotic pathways………………………………. 16
Figure 4 Chemical structures of the four major classes of chemotherapeutic agents…...18
Figure 5 GC/MS Chromatograms of oils tested and analysis of the FAME standards....29
Figure 6 GC/MS Chromatograms of three brands of flaxseed oils……….…..…….…..33
Figure 7: Effect of plant oils on cell viability of the aggressive murine melanoma B16-
BL6 cells using MTT assays……………………………………………………...…......36
Figure 8 Apoptosis analysis by acridine orange and ethidium bromide staining of flaxseed
oil treated B16-BL6 cells…………………...…………………………………..39
Figure 9 Early stage apoptosis detection by TUNEL stain in B16-BL6 cells following
flaxseed oil treatment………………………………………………………...……..……43
Figure 10 Cell death in B16-BL6 cells determined by annexin V-FITC staining following
flaxseed oil treatment…………………………………………………………….…...…45
Figure 11 Cell cycle analysis of B16-BL6 cells treated with flaxseed oil……..…...….48
Figure 12 Lysosome formation analysis in flaxseed oil-treated B16-BL6 cells by
lysotracker stain………………………………………………………………………….51
Figure 13 Mitochondrial analysis of flaxseed oil-treated B16-BL6 cells using mitotracker
red…………………………..………………………………………………………...….55
Figure 14 Media extraction of the anti-cancer components of flaxseed oil.…………....59
Figure 15 Solvent extraction of the anti-cancer components of flaxseed oil.…...…..….61
Figure 16 Omega fatty acid metabolism of flaxseed oil by B16-BL6 cells…..………...64
Figure 17 GC/MS determination of fatty acid composition of cell membranes of B16-
BL6 cells …..……………………………………………………………..……………..66
Figure 18 Flaxseed oil enhances taxol-mediated cell death……………………..……...70
ix
Figure 19 Flaxseed oil enhances 10-8M taxol-mediated cell death………………….......72
Figure 20 Proposed hypothesis of how flaxseed oil is metabolized within the cellular
membrane resulting in the activation of the apoptotic pathway…………………………88
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Description:I, Alison Buckner, hereby grant to Laurentian University and/or its agents the I further agree that permission for copying of this thesis in any manner,
