phenothiazines induce abortive autophagy leading to cancer cell death

207 Pages·2017·6.96 MB·English

by  Catherine T. Charles

TThhee TTeexxaass MMeeddiiccaall CCeenntteerr LLiibbrraarryy
DDiiggiittaallCCoommmmoonnss@@TTMMCC
The University of Texas MD Anderson Cancer
The University of Texas MD Anderson Cancer
Center UTHealth Graduate School of
Center UTHealth Graduate School of
Biomedical Sciences Dissertations and Theses
Biomedical Sciences
(Open Access)
8-2012
PPHHEENNOOTTHHIIAAZZIINNEESS IINNDDUUCCEE AABBOORRTTIIVVEE AAUUTTOOPPHHAAGGYY LLEEAADDIINNGG TTOO
CCAANNCCEERR CCEELLLL DDEEAATTHH
Catherine T. Charles
Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations
Part of the Medicine and Health Sciences Commons
RReeccoommmmeennddeedd CCiittaattiioonn
Charles, Catherine T., "PHENOTHIAZINES INDUCE ABORTIVE AUTOPHAGY LEADING TO CANCER CELL
DEATH" (2012). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of
Biomedical Sciences Dissertations and Theses (Open Access). 294.
https://digitalcommons.library.tmc.edu/utgsbs_dissertations/294
This Dissertation (PhD) is brought to you for free and
open access by the The University of Texas MD Anderson
Cancer Center UTHealth Graduate School of Biomedical
Sciences at DigitalCommons@TMC. It has been
accepted for inclusion in The University of Texas MD
Anderson Cancer Center UTHealth Graduate School of
Biomedical Sciences Dissertations and Theses (Open
Access) by an authorized administrator of
DigitalCommons@TMC. For more information, please
contact [email protected].
PHENOTHIAZINES INDUCE ABORTIVE AUTOPHAGY
LEADING TO CANCER CELL DEATH
by
Catherine Nguyen Charles, B.A., B.S.

APPROVED:

______________________________
Gordon B. Mills, M.D., Ph.D.
Supervisory Professor

______________________________
Peter J. Davies, M.D., Ph.D.

____________________________
Honami Naora, Ph.D.

______________________________
David McConkey, Ph.D.

______________________________
Huang Peng, M.D., Ph.D.

_______________________________

Dean, Graduate School of Biomedical Sciences
The University of Texas Health Science Center at Houston
PHENOTHIAZINES INDUCE ABORTIVE AUTOPHAGY
LEADING TO CANCER CELL DEATH

A
DISSERTATION
Presented to the Faculty of
The University of Texas
Health Science Center at Houston
and
The University of Texas
M. D. Anderson Cancer Center
Graduate School of Biomedical Sciences
in Partial Fulfillment
of the Requirements
for the Degree of

DOCTOR OF PHILOSOPHY

by

Catherine Nguyen Charles, B.A., B.S.
Houston, Texas
August, 2012
ACKNOWLEDGMENTS

I am grateful for the privilege and opportunity to train in Dr. Gordon Mills’
laboratory. I thank Dr. Mills for the countless hours of discussion, guidance, and for
providing me with amazing opportunities to collaborate with experts in various
aspects of cancer cell signaling. My experiences have built my knowledge of
systems biology and prepared me for my future endeavors. I am also thankful for
the support and efforts of my current committee members: Drs. Peter Davies,
Honami Naora, David McConkey, Huang Peng, and past committee members Drs.
Joseph Alcorn, Garth Powis, Jeffrey Myers, and Wei Zhang. I am especially
grateful for Drs. Davies and Naora, who have been guiding me from the beginning
of my PhD studies. I would like to also thank the current and former members of
Dr. Mills’ lab for their instructions on various techniques and their guidance
throughout my studies: Yiling Lu, Qinghua Yu, Shuangxing Yu and Nancy Shih. I’d
like to thank Zhang Dong for teaching me mouse study techniques and helping me
in my xenograft models. I’d like to thank Jane Li for her help in preparing my
lysates from my animal studies for RPPA and Nancy for pushing hard to get the
sample processed in the core on time to complete my studies. I’m grateful to Jae-
Ho Cheong, Jiyong Liang and Sofie Claerhout their discussions of various
experimental designs for my studies. I’ve greatly benefited from Sofie’s expertise
and willingness to discuss and share with me the key aspects of autophagy
signaling studies and for the countless hours of image collections on the confocal
microscopy. I am indebted to Sofie for in her valuable inputs throughout my project.
iii
I’ve received countless input and support from the amazing post-docs in the Mills’
research group: Sofie Claerhout, Shreya Mitra, and Jennifer Molina. Doris Siwak
worked with me for months in the data analysis and the generation of heatmaps for
my RPPA studies and for cheering me on in the end. I thank Zhenlin Ju, Wenbin
Lu and Fan Zhang for their help in the statistical analysis of my data. I would like to
thank everyone in the Department of Systems Biology for being such a wonderful
group of people to work with. I’ve had the opportunity to interact with several
faculties who’ve given me great help with input on my project. I’d like to thank the
students in Dr. Mills’ lab and in the Department of Systems Biology for their
companionship. I also would like to thank the administrative team of the
Department of Systems Biology for their support throughout my studies.
Additionally, I would like to thank Drs. William Bornmann and David Maxwell,
who were my co-mentors in my pharmacoinformatics training. I have benefited from
the generosity of the Dunn family through their foundation, and I had the privilege of
meeting them and thank them for the Prestwick Compound Library which I used at
the earlier stage of my project. Finally, I would like to acknowledge the Keck Center
Pharmacoinformatics Training Program of the Gulf Coast Consortia for providing
partial funding of my studies through the Pharmacoinformatics Predoctoral
Fellowship.

iv
DEDICATION

This dissertation is dedicated to my family whose love and support helped
me through my long journey of my Ph.D. education.

v
PHENOTHIAZINES INDUCE ABORTIVE AUTOPHAGY
LEADING TO CANCER CELL DEATH
Publication No.________

Catherine Nguyen Charles, B.A, B.S
Supervisory Professor: Gordon B. Mills, M.D., Ph.D.

Autophagy is an evolutionarily conserved process that functions to maintain
homeostasis and provides energy during nutrient deprivation and environmental
stresses for the survival of cells by delivering cytoplasmic contents to the lysosomes
for recycling and energy generation. Dysregulation of this process has been linked
to human diseases including immune disorders, neurodegenerative muscular
diseases and cancer.
Autophagy is a double edged sword in that it has both pro-survival and pro-
death roles in cancer cells. Its cancer suppressive roles include the clearance of
damaged organelles, which could otherwise lead to inflammation and therefore
promote tumorigenesis. In its pro-survival role, autophagy allows cancer cells to
overcome cytotoxic stresses generated the cancer environment or cancer
treatments such as chemotherapy and evade cell death. A better understanding of
how drugs that perturb autophagy affect cancer cell signaling is of critical
importance to improve the cancer treatment arsenal.
vi
In order to gain insights in the relationship between autophagy and drug
treatments, we conducted a high-throughput drug screen to identify autophagy
modulators. Our high-throughput screen utilized image based fluorescent
microscopy for single cell analysis to identify chemical perturbants of the autophagic
process. Phenothiazines emerged as the largest family of drugs that alter the
autophagic process by increasing LC3-II punctae levels in different cancer cell lines.
In addition, we observed multiple biological effects in cancer cells treated with
phenothiazines. Those antitumorigenic effects include decreased cell migration,
cell viability, and ATP production along with abortive autophagy. Our studies
highlight the potential role of phenothiazines as agents for combinational therapy
with other chemotherapeutic agents in the treatment of different cancers.

vii
TABLE OF CONTENTS

ACKNOWLEDGMENTS…………………………………………………………………. iii
DEDICATION……………………………………………………………………………….v
ABSTRACT…………………………………………………………………………………vi
TABLE OF CONTENTS………………………………………………………………….viii
LIST OF FIGURES………………………………………………………………………..xii
LIST OF TABLES………………………………………………………………………….xv
LIST OF ABBREVIATIONS……………………………………………………………...xvi
CHAPTER 1: INTRODUCTION
Autophagy………………………………………………………………………….. 2
Physiological functions of autophagy…………………………………………… 2
Autophagy functions as quality control of proteins and organelles……………3
Autophagy is a multi-step process………………………………………………..5
The core regulators of the autophagosome machinery……………………….. 6
Monitoring autophagy………………………………………………………………7
Autophagy and cancer……………………………………………………………11
Targeting autophagy differences in cancer versus normal cells…………….13
Phenothiazines……………………………………………………………………16
Expression of dopamine receptors outside of central nervous system……..18
Phenothiazines and cancer…………………………………………….………..20
Dissertation Overview……………………….……………………………………22
viii
CHAPTER 2: MATERIALS AND METHODS
Compounds and reagents………………………………………………………..24
Cell lines…………………………………………………………………………...24
Fluorescent image based high-throughput drug screen for autophagy……..26
Modulators
siRNA transfection………………………………………………………………..27
SDS-PAGE and western blot analysis …………………………………………27
Antibodies………………………………………………………………………….28
Transmission electron microscopy (TEM)……………………………………...29
Crystal violet cell viability assay…………………………………………………30
Cell viability and proliferation assay ……………………………………………30
Caspase-7 activity assay…………………………………………………………30
ATP assay…………………………………………………………………………31
DiOC apoptosis Assay….…………………. …………………………………..31
6
Clonogenic viability assay………………………………………………………..32
Matrigel invasion assay…………………………………………………………..32
Confocal microscopy……………………………………………………………..33
Mice studies……………………………………………………………………….34
Tumor implantation and treatment………………………………………………34
Reverse phase protein array (RPPA)…………………………………………..35

ix