Novel mechanisms of quinone toxicity

165 Pages·2015·6 MB·English

NOVEL MECHANISMS OF QUINONE TOXICITY
by
RUI XIONG
B.S, China Pharmaceutical University, 2009

A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
Toxicology Program
2015
This thesis for the Doctor of Philosophy degree by
Rui Xiong
has been approved for the
Toxicology Program
by

Manisha Patel, Chair
David Ross, Advisor
David Siegel
Jared Brown
Wenbo Zhou

Date : 12/18/2015

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Rui Xiong (Ph.D., Toxicology)
Novel Mechanisms of Quinone Toxicity
Thesis directed by Professor David Ross.
ABSTRACT
This thesis investigated the role of xenobiotic and endogenous quinones in modulating
major intracellular protein handling systems including the 20/26S proteasome, autophagy,
the ER stress response, heat shock proteins (HSPs) and aggresome formation.
The activation sequence of diverse protein handling systems was first defined using
the proteasome inhibitor MG132 in rat dopaminergic N27 cells. The data suggested that
after proteasomal inhibition, the ER stress response was most rapidly triggered and
subsequent induction of autophagy and Hsp70 expression was important for protection
against accumulation of intracellular protein aggregates. Three model quinones including
the environmentally relevant 1,4-benzoquinone (BQ), the vitamin K analog menadione
(MD) and aminochrome (AC) a reactive dopamine oxidation product, were utilized to
determine quinone-induced protein handling changes in N27 cells. The results
demonstrated that the nature and extent of protein handling changes were quinone-specific.
BQ and AC could inhibit proteasomal activity and trigger a rapid activation of the ER
stress response and autophagy in N27 cells. AC induced aggresome formation and caused
the most pronounced protein handling changes, while MD had little effect on any protein
handling system. The effect of NAD(P)H:quinone oxidoreductase 1 (NQO1) was also
quinone-dependent. NQO1 inhibited the ER stress response and protected against BQ
toxicity but potentiated both the ER stress response and toxicity of AC and MD.
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Finally, the ability of 19-substituted benzoquinone ansamycins (19BQAs) novel
antitumor Hsp90 inhibitors, to modulate protein handling systems was defined using
human dopaminergic SH-SY5Y (5Y) cells. The results demonstrated that 19BQAs had
decreased toxicity in 5Y cells relative to their parent quinones geldanamycin (GA), 17-
AAG and 17-DMAG while retaining the ability to induce HSP expression and autophagy.
Whether 19BQAs could protect against protein aggregation and toxicity was further
examined using an A53T α-synuclein Parkinson’s disease (PD) cellular model. 19-Phenyl-
GA (19-Ph-GA), a lead compound in the GA series significantly decreased the oligomer
formation and toxicity of A53T α-synuclein in 5Y cells. Mechanistic studies demonstrated
that 19-Ph-GA activated Hsp70, Hsp27 and autophagy and attenuated mTOR activation in
A53T α-Syn overexpressing cells, all of which might contribute to protection against
A53T α-Syn oligomer formation and toxicity.

The form and content of this abstract are approved. I recommend its publication.
Approved: David Ross

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DEDICATION
This thesis is dedicated to my loving parents, Rong Zhou and Wenxin Xiong, for their
caring, support and encouragement during the challenges of graduate school and life. My
Mom is a senior procurator and my Dad is an experienced surgeon. They are always
passionate, dedicated to their work and helped many people in need and made positive
contributions to society. They are role models for me and have influenced my personal
and academic goals.

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ACKNOWLEDGEMENTS
First and foremost, I would like to express the deepest gratitude to my advisor Dr.
David Ross. I would never have been able to complete this thesis without his excellent
guidance, support and encouragement. During the past five years, he patiently helped me
to develop my interest in toxicology, build my confidence in conducting scientific
research and improve my writing skills. Dr. Ross is incredibly insightful, organized and
also a great problem solver, all of these qualities were extremely helpful in moving
forward my research project. I would also like to thank Dr. David Siegel who trained me
and taught me many useful techniques. From him, I learned many invaluable attributes
including passion and motivation for science that will be extremely helpful as my career
moves forward.
I would like to thank Dr. Dennis Petersen and Dr. Manisha Patel for their scientific
guidance and suggestions in my work. A special thanks to Dr. Wenbo Zhou, he kindly
provided α-synuclein expressing adenoviruses to me and helped with my cell culture
problems. I would also like to thank current and former Ross lab members, including Dr.
Jadwiga Kepa who helped with generating isogenic cell lines and Dr. Chao Yan who
always made time to discuss scientific problems with me.
I would also like to acknowledge my best friends including Lingzi Cai, Xiang Zhai,
Yingzi Jin and Hong Li. They have always been there for me whenever I am in need.
Finally, I would like to express my appreciation to the incredible family I have. My
cousin, Ying Zhang brings her optimistic attitude to my life and always cheers me up
when I face challenges. My Mom in China received numerous calls from me and patiently
listened to my problems and provided encouragement and perspective. I would also like to
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thank my fiancé, Yue Wu. He has been patient with me and provided me with his constant
support and encouragement.

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TABLE OF CONTENTS
CHAPTER
I. REVIEW OF THE LITERATURE AND STATEMENT OF PURPOSE 1
General mechanism of quinone toxicity 1
Major protein handling systems and the capability of quinones to modulate protein
handling activities in cells 4
The ubiquitin-proteasome system (UPS) 4
Autophagy 8
The Endoplasmic Reticulum (ER) stress response/Unfolded Protein Response (UPR)
11
Heat Shock Proteins (HSPs) 13
Aggresome Formation 15
Implications of AC induced protein handling changes in the pathogenesis of
Parkinson’s disease 17
The role of NQO1 in quinone mediated toxicity 20
Purpose and significance of the study 22
II. THE ACTIVATION SEQUENCE OF CELLULAR PROTEIN HANDLING
SYSTEMS AFTER PROTEASOMAL INHIBITION IN DOPAMINERGIC
CELLS 25
Introduction 25
Materials and methods 26
Results 30
Exposure to MG132 induced caspase 3 cleavage and toxicity in N27 cells 30
MG132 inhibited proteasomal activity and induced the accumulation of
polyubiquitinated proteins in a time-dependent manner in N27 cells 32
Induction of the ER stress response was tightly associated with early
proteasomal inhibition in N27 cells 34
Proteasomal inhibition induced autophagy 35
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Prolonged proteasomal inhibition resulted in formation of aggresome-like
inclusion bodies and induction of Hsp70 in N27 cells 37
Involvement of autophagy, but not the ER stress response or HSP response, in
the clearance of aggresomes during recovery from proteasomal inhibition 40
Discussion 43
III. QUINONE-INDUCED PROTEIN HANDLING CHANGES: IMPLICATIONS
FOR MAJOR PROTEIN HANDLING SYSTEMS IN QUINONE-MEDIATED
TOXICITY 47
Introduction 47
Materials and Methods 48
Results 56
Inhibition of proteasomal activity by quinones 56
BQ induced proteasomal inhibition was tightly associated with induction of the
ER stress response and autophagy, but had little effect on other major protein
handling systems (HSPs and formation of aggresome-like inclusion bodies) 60
Dopamine derived AC triggered the ER stress response, induction of
autophagy and formation of aggresome-like inclusion bodies in N27 cells 63
MD exerted little influence on the ER stress response and autophagy 68
Overexpression of NQO1 in N27 cells 69
Modulation of quinone-induced ER stress response by NQO1 and its
relationship to toxicity 70
NQO1 increased quinone induced oxidative stress that may contribute to the
activation of the ER stress response and toxicity 73
Discussion 76
IV. A NOVEL HSP90 INHIBITOR ACTIVATES COMPENSATORY HEAT
SHOCK PROTEIN RESPONSES AND AUTOPHAGY AND ALLEVIATES
MUTANT A53T α-SYNUCLEIN TOXICITY 81
Introduction 81
Materials and Methods 83
Results 88
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Decreased toxicity of 19BQAs relative to their parent quinone geldanamycin
(GA) in SH-SY5Y cells 88
19BQAs are potent inducers of heat shock proteins 91
Overexpression of A53T α-synuclein (α-Syn) resulted in protein handling
changes and toxicity 92
19-Ph-GA alleviated A53T α-Syn induced toxicity 96
19-Ph-GA activated HSP and autophagy that may contribute to the decreased
oligomer formation and toxicity of A53T α-Syn 98
19-Ph-GA significantly down regulated mTOR/p70S6K signaling in A53T
α-Syn overexpressing cells 102
Discussion 105
V. SUMMARY AND FUTURE DIRECTIONS 110
REFERENCES 117
APPENDIX 146
A. 19BQAS IN THE 17-AAG AND 17-DMAG SERIES STUDIED IN CHAPTER IV
146

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