(hepg2) cells anil amichund chu

146 Pages·2015·3.38 MB·English

AN INVESTIGATION INTO THE BIOCHEMICAL,
MOLECULAR AND EPIGENETIC EFFECTS OF FUMONISIN B
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IN LIVER (HEPG2) CELLS

By

ANIL AMICHUND CHUTURGOON

Submitted in fulfillment for the degree of Doctor of Philosophy (Medical
Biochemistry), School of Laboratory Medicine and Medical Sciences, College of
Health Sciences, University of KwaZulu-Natal

December 2014
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Dedication

I am deeply indebted to my Spritual Master and Guru– Shree Ramakrishna for always
guiding me on the path of Righteousness and Truth.
“Our duty is to encourage every one in his struggle to live up to his highest idea, and
strive at the same time to make the ideal as near as possible to the Truth.”
Swami Vivekananda

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Acknowledgements
Dr Devan Moodley (Harvard Medical School, Boston, USA)
Thank you for your support, friendship and advice. You have an amazing and
intelligent mind and your unselfish nature knows no bounds. Your enthusiasm and
ability for hard work is very inspiring. May you long continue to inspire young
minds.
Dr Alisa Phulukdaree (Medical School, University of Pretoria)
Thank you for your dedication and compassion for science. You are an incredible
individual with an insatiable thirst for new discoveries. Your friendship and
assistance will always be greatly valued.
My family
Thanks to my wife Reena for her unconditional support and encouragement – and for
being strong for my family during this time; Your passion and drive sets an example
for us all to follow. I am grateful for your unselfish sacrifices you made and still
make for us. My two dear children, Shekhar and Sanam, for “technology” assistance
and being the “best kids a parent can wish for”.
My parents
Although you did not have an opportunity to educate yourself – you instilled in me
the value of good education and I appreciate all the sacrifices you made to allow me
to pursue my dreams.

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PUBLICATIONS

1. Chuturgoon, A.A., Phulukdaree, A., and Moodley, D. (2014). Fumonisin
B induces global DNA hypomethylation in HepG2 cells – an alternative
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mechanism of action. Toxicology 315, 65-69.
(DOI: 10.1016/j.tox.2013.11.004)

2. Chuturgoon, A.A., Phulukdaree, A., and Moodley, D. (2014). Fumonisin
B modulates expression of human cytochrome P 1B1 in human
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hepatoma (HepG2) cells by repressing mir-27b. Toxicology Letters
227(1), 50-55.
(DOI: 10.1016/j.toxlet.2014.02.026)

3. Chuturgoon, A.A., Phulukdaree, A., and Moodley, D. (2014).
Fumonisin B inhibits apoptosis in HepG2 cells by inducing Birc-8/ILP-2.
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Toxicology Letters (In press, February 2015)
(Manuscript number: TOXLET-D-14-01036)

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PRESENTATIONS

International
1. Chuturgoon AA, Phulukdaree A and Moodley D. Fumonisin B induces
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global DNA hypomethylation and modulates cytochrome P 1B1 by
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repressing miR-27b in HepG2 cells. Eurotox 2014 – Edinburgh, Scotland.
Toxicology Letters; 2014 P3-8; 229S, P146.

2. Chuturgoon AA, Phulukdaree A and Moodley D. Fumonisin B inhibits
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apoptosis in HepG2 cells by inducing the IAP protein – BIRC8/ILP-2.
Accepted Abstract. SOT 2015: 54th Annual Meeting and ToxExpo; San
Diego, California (USA): 22- 26 March 2015.

National
1. Chuturgoon, AA, Phulukdaree A and Moodley D. UNIPATH 2014: Unifying
Pathology and Laboratory Medicine in the era of P4 Medicine: (19-21
September 2014). The epigenetic properties of Fumonisin B . Pretoria, South
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Africa.

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ABSTRACT

Fumonisins are carcinogenic mycotoxins that occur world wide in maize and maize-
based products intended for human consumption. Consumption of fumonisin-
contaminated maize as a staple diet has been associated with oesophageal and liver
cancer in South Africa and China. Fumonisin B (FB ) inhibits sphingolipid
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biosynthesis and has been implicated in cancer promoting activity in animals and
humans. FB disrupts DNA methylation and induces chromatin modifications in
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human hepatoma (HepG2) cells. In this study FB (IC =200µM) altered liver enzyme
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expression of DNA methyltransferases and demethylases. DNA methyltransferase
activities of DNMT1, 3a and 3b were significantly decreased, whilst both DNA
methylase (MBD2) activity and expression was significantly up-regulated resulting in
global DNA hypomethylation. In addition the histone demethylases, KDM5B and
KDM5C, expression was increased. FACS data confirmed FB significantly increased
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global DNA hypomethylation – a process that causes chromatin instability. Next the
effect of FB on miRNA expression was evaluated; FB significantly down-regulated
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(11 fold) expression of miR-27b. MiR-27b modulates expression of human
cytochrome P (CYP1B1) that catalyzes the metabolic activation of many
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procarcinogens. In order to directly assess the effect of miR-27b on CYP1B1 mRNA
levels, liver cells were transfected with the mimic to miR-27b. CYP1B1 mRNA and
protein expression was significantly up-regulated by 1.8- fold and 2.6- fold
respectively. CYP1B1 is post-transcriptionally regulated by miR-27b suggesting that
FB - induced modulation of miR-27b in hepatic cells may be an additional mode of
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hepatic neoplastic transformation. Finally, the effect of FB on the apoptotic pathway
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in HepG2 cells was investigated using an mRNA expression array panel of pro- and
anti- apoptotic molecules. FB significantly increased an AIP family member - BIRC-
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8/ILP-2 (8-fold) in an apoptosis array. In addition, ILP2 protein expression was
increased (2.3-fold) with a corresponding decrease in Smac/DIABLO protein levels
(1.7-fold). Further analysis showed an FB (0µM, 50µM, 100µM, 200µM) dose-
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dependent increase in BIRC-8/ILP-2 mRNA and protein expression in HepG2 cells.
This data suggests that FB modulates apoptosis in a complex dose-dependent
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regulation of pro- and anti-apoptotic molecules – and it is not a matter of simply
switching on or off.
In conclusion, the data shows that FB possess epigenetic properties by inducing
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global DNA hypomethylation, modulating miRNA expression, and increasing
expression of the AIP protein family (BIRC8/ILP-2) that may lead to liver
tumourigenesis.

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LIST OF FIGURES

Figure 2.1: Chemical structure of (A) Fumonisin B and its similarities
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to sphingoid bases (B) Sphinganine ad (C) Sphingosine……………...8

Figure 2.2: The de novo synthesis of sphingolipids in animal cells;
the points at which Fumonisin B disrupts this process……………...12
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Figure 2.3: The geographical representation of oesophageal cancer
incidence between males and females in 2009……………………….17

Figure 2.4: The process of methylation and demethylation of cytosine
by DNA methyl transferases (DNMT) and MDB2…………………..20

Figure 2.5: The process of miRNA biogenesis, maturation and
regulation……………………………………………………………..24

Figure 2.6: MicroRNAs function in A: normal tissue, B: as a tumour
suppressor and C: as an oncogene……………………………………25

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Figure 2.7: The activation of the extrinsic and intrinsic pathways of
apoptosis……………………………………………………………...28

Figure 2.8: The inhibitors of apoptosis (IAP) protein functions
as caspase inhibitors halting the execution phase of apoptosis………32

Figure 3.1: Fumonisin B induced changes in expression levels of DNA
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methyltransferases and DNA demethylases in HepG2 cells………...60

Figure 3.2: Fumonisin B decreased expression of DNA methyltransferases
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and increased MBD2 protein expression…………………………….61

Figure 3.3: Fumonisin B induced global DNA hypomethylation in HepG2 cells
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as assessed using flow cytometry…………………………………….62

Figure 3.4: Fumonisin B increased DNA comet tails in HepG2 cells…………...62
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Figure 4.1: MiRNA profiles in Fumonisin B treated HepG2 cells………………81
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Figure 4.2: Hierarchical cluster analysis of miRNA expression in
Fumonisin B treated HepG2 cells…………………………………...82
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