CARDIOLIPIN PROFILING IN METASTATIC BREAST CANCER CELLSthesis.honors.olemiss.edu/1104/1/thesis final...

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CARDIOLIPIN PROFILING IN METASTATIC BREAST CANCER CELLS

By: Meghan Houston McNeely

A thesis submitted to the faculty of The University of Mississippi in partial fulfillment of the requirements of the Sally McDonnell Barksdale Honors College.

Oxford May 2018

Approved by:

_________________________________ Advisor: Dr. Yu-Dong Zhou

_________________________________ Co-Advisor/Reader: Dr. Dale G. Nagle

_________________________________ Reader: Dr. Kristopher Harrell

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© 2018

Meghan Houston McNeely

ALL RIGHTS RESERVED

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ACKNOWLEGMENTS

First, I would like to thank Drs. Zhou and Nagle for guiding me throughout the

production of this thesis. Without Dr. Zhou’s insight, I am not sure I would have found a

research topic that interests me as much as this one has. I would also like to thank Dr.

Nagle’s graduate student, Jawaher Alkhamisy, for assisting me in the various

experiments involved in this research. I have learned countless valuable experimental

techniques through Jawaher and Dr. Zhou’s leadership and direction. Last, I would like to

thank my family and friends for being such an incredible support system throughout the

stress of these past few semesters.

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ABSTRACT

MEGHAN HOUSTON MCNEELY:

Cardiolipin Profiling in Metastatic Breast Cancer Cells

Six human breast cancer cell lines (MDA-MB-231, LM, BoM, T47D, MCF7, and

MCF7-BoM) were treated with three differentially targeted phospholipase inhibitors

(bromoenol lactone, tricyclodecan-9-yl-xanthogenate, and halopemide) as well as various

dietary oils (canola oil, avocado oil, coconut oil, olive oil, sesame oil, and soybean oil).

Cell viability assays were performed to examine the effects of each inhibitor and oil in

varying concentrations on the proliferation of each breast cancer cell line. The goal was

to detect changes in cardiolipin concentration within each cell line after exposure to a

series of various phospholipase inhibitors and oils. Liquid chromatography-mass

spectrometry (LC-MS) was performed on a cardiolipin standard as well as lipid extract

samples from each cell line in order to detect changes in cardiolipin composition before

and after the addition of test compounds. Because the MS results for the cell lines did not

display a peak for the cardiolipin standard, we could not use these results as a valid

means for determining the changes in cardiolipin composition between the samples.

However, BEL, halopemide, D609, canola oil, avocado oil, coconut oil, and olive oil did

prove to cause a statistically significant difference in the cell viability of various breast

cancer cell lines.

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TABLE OF CONTENTS LIST OF FIGURES.............................................................................................................vi

CHAPTER I: Introduction...................................................................................................1

1.1 Breast Cancer Background................................................................................1

1.2 Metastasis...........................................................................................................2

1.3 Mitochondria and Cardiolipin Background.......................................................3

1.4 Cardiolipin Remodeling.....................................................................................5

1.5 Conclusions......................................................................................................10

CHAPTER II: Cardiolipin Profile in Metastatic Breast Cancer Cells...............................11

2.1 Introduction......................................................................................................11

2.2 Results..............................................................................................................16

2.3 Discussion........................................................................................................26

2.4 Materials and Methods.....................................................................................27

2.4.1 Cell viability assay............................................................................27

2.4.2 Mitochondrial isolation.....................................................................30

2.4.3 Cardiolipin extraction.......................................................................31

2.4.4 Mass spectrometry............................................................................32

2.4.5 Data...................................................................................................32

2.4.6 Statistic analysis................................................................................32

REFERENCES..................................................................................................................33

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LIST OF FIGURES Figure 1. Chemical Structure of Cardiolipin........................................................................5

Figure 2. Cardiolipin Biosynthesis and Remodeling...........................................................8

Figure 3.1 Chemical Structures of Selected Phospholipase Inhibitors...............................12

Figure 3.2 Hydrolyzation of Phosphatidylinositol 4,5-bisphosphate by PLC...................13

Table 1. Source and Pathological Features of Tumors Used to Derive Selected Breast

Cancer Cell Lines...............................................................................................................16

Figure 4. Impact of Bromoenol Lactone (BEL) on Breast Tumor Cell Viability..............17

Figure 5. Impact of The Tricyclodecan-9-yl-xanthogenate (D609) on Breast Tumor Cell

Viability.............................................................................................................................18

Figure 6. Impact of Halopemide on Breast Tumor Cell Viability......................................19

Figure 7. Impact of Canola Oil on Breast Tumor Cell Viability........................................20

Figure 8. Impact of Avocado Oil on Breast Tumor Cell Viability.....................................21

Figure 9. Impact of Coconut Oil on Breast Tumor Cell Viability.....................................22

Figure 10. Impact of Olive Oil on Breast Tumor Cell Viability........................................23

Figure 11. Impact of Soybean Oil on Breast Tumor Cell Viability...................................24

Figure 12. Impact of Sesame Oil on Breast Tumor Cell Viability.....................................25

Figure 13. Mass Spectrometry Analysis Cardiolipin Standard and Cell Extracts.............26

1

CHAPTER I

Introduction

Meghan Houston McNeely

1.1 Breast Cancer Background

Breast cancer is the leading cause of cancer-related death in women in Western

countries, with roughly 41,000 deaths resulting from breast cancer each year within the

United States1. Approximately 250,000 cases of breast cancer are diagnosed annually in

the United States, and breast cancer accounts for about one in every three cancers

diagnosed in American women1,2. Despite improvements in early breast cancer screening,

the prevalence of breast cancer diagnoses has nonetheless remained relatively stable since

the 1970’s3. In fact, many predict that in the coming years, the rate of breast cancer

diagnoses will only continue to rise, presenting an enormous need for the discovery of

effective treatment options1.

As they are widely available and relatively inexpensive, mammograms have become

the preferred method for screening patients for early indications of breast cancer2.

Mammograms are based on X-ray photographs of the patient’s breast, and they can be

used to detect early abnormalities in the breast before they are able to be felt as lumps by

a woman or her physician2. Along with their role in screening for signs of breast cancer,

mammograms are also crucial in following the progression of cancer in patients whom

have already been diagnosed4. While the rate of breast cancer diagnoses has not

2

decreased since the 1970’s, the rate of breast cancer mortalities has significantly

decreased4. This suggests that improvements in screening methods, such as

mammography, have allowed breast cancer cases to be diagnosed and treated more

efficiently, leading to a concurrent decrease in these patients’ mortality.

Radiation therapy is widely used in the treatment of many cancers such as primary

and secondary breast cancer1. However, treatment options such as cytotoxic

chemotherapy, are often inadequate when it comes to treating malignant tumors which

have spread to distant areas within the body1. Many improvements have been made in

breast cancer therapies, leading to longer survival rates for patients diagnosed with

metastatic breast cancer3. However, approximately 90% of deaths associated with breast

cancer still result from metastasis of the tumor cells1. Non-traditional treatments are

being studied in the hope of discovering alternative treatments for cancer patients,

especially for those fighting metastatic cancers.

1.2 Metastasis

Cancer is defined by eight hallmarks: “growth factor independence or self-

sufficiency, insensitivity to anti-growth signals, avoidance of programmed cell death,

ability to recruit a dedicated blood supply, immortalization by reactivation of telomerase,

ability to invade adjacent normal tissues and metastasize to distant sites, reprogrammed

energy metabolism, and evading immune destruction”5. Approximately 6% of patients

whom have been diagnosed with early-stage breast cancer experience such metastatic

potential, in which their cancer has already spread to distant sites within the body3. The

metastatic ability of many cancer cells often contributes largely to the mortality

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associated with a cancer diagnosis, due partly to an overall lack of understanding when it

comes to the cellular and molecular mechanisms of cancer metastasis6,7. The metastatic

process is comprised of multiple elements, such as: tumor cell intravasation (or the

entering of the tumor cell into the bloodstream), survival in bodily circulation,

extravasation into an organ distantly located within the body, and a resulting unrestricted

growth within an organ6,8. Breast cancer metastasis frequently occurs in the lungs, brain,

liver, and bones6.

Evidence indicates that breast cancer metastasis to the brain can typically occur years

after a tumor in the breast has been removed9. This, therefore, suggests that the cancer

cells must acquire specialized functions to successfully allow metastasis to the brain9. It

is conventionally recognized that metastasis involves an interaction between the tumor

cell and its host microenvironment7,10. However, the specific alterations that provide a

cancer cell with the necessary attributes to establish metastatic lesions within its

environment are not widely understood. One explanation of such cancer cell alterations

lies within a modification of its mitochondrial function. Studies have shown that an

impairment to the apoptotic mechanisms present within the mitochondria of a cell can

ultimately facilitate the conversion of a normal cell into a cancerous cell11.

1.3 Mitochondria and Cardiolipin Background

In addition to being the cellular ‘powerhouse’ for ATP production, mitochondria are

crucial to anabolic metabolism, as well as serving a vital role in integrating both pro-

death and pro-survival signals. The role of the mitochondria in regulating cell death

signals allows it to definitively control apoptosis. Due to its role in programmed cell

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death, it is said that the mitochondria “hide(s) the weapons of cellular suicide”12. When

tumor cells suppress apoptotic signals from the mitochondria, the cell is able to avoid cell

death and therefore promote the proliferation of a tumor within the body11. This

contributes to one of the previously mentioned major hallmarks of cancer, specifically the

insensitivity of cancer cells to anti-proliferative signals.

Cardiolipins (CLs) are an essential component in mitochondrial structure and

function. These bridged phospholipid dimers, which are located almost exclusively

within the inner mitochondrial membrane, are the only phospholipid found primarily in a

single location within the cell. Cardiolipin molecules are dispersed asymmetrically

among the inner mitochondrial membrane, contributing to their ability to perform various

signaling functions13. Aside from contributing to cellular signaling, cardiolipin molecules

are also crucial in maintaining structural integrity within the inner mitochondrial

membrane14.

Cardiolipin structure has been widely preserved in eukaryotic cells. Their structure

contains a glycerol backbone that bridges two phosphatidylglycerols in order to form a

dimeric molecule14. The tails of the structure are comprised of four acyl chains, and the

polar head carries two negative charges due to the associated phosphate groups13,15. In

comparison to prokaryotic cardiolipin, eukaryotic cardiolipins possess longer acyl chains

that are less saturated. The polyunsaturated nature of the eukaryotic cardiolipin tails

allows the molecule to undergo oxidation by cytochrome c oxidase13. This

polyunsaturation and oxidation is crucial in regulating the release of pro-apoptotic signals

from the cardiolipin in the mitochondrial membrane into the cytosol11. Disturbances

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within the cardiolipin structure and function, therefore, can result in various defects in the

signaling functions of the mitochondria, such as those that regulate cell death13.

Figure 1. Chemical Structure of Cardiolipin16. The four fatty acyl chains of the CL

represented by R1, R1¢, R2 and R2¢, can exist in varying lengths and degrees of

unsaturation. This provides an opportunity for the creation of many diverse CL

molecules. The polyunsaturated nature of the tails results in high susceptibility of the

molecule to cytochrome c oxidase-mediated oxidation16.

1.4 Cardiolipin Remodeling

Unlike most membrane lipids, which are synthesized in the endoplasmic reticulum,

cardiolipin is synthesized primarily in the inner mitochondrial membrane and is involved

in various, diverse functions of the mitochondria17. This diversity of functions is due

primarily to the unique structure of this phospholipid. The four fatty acid tails of the

cardiolipin along with the negative charges associated with the two phosphate heads

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allow numerous interactions between the phospholipid and various cellular proteins17.

Regulation of the synthesis and remodeling of a cell’s cardiolipin, as well as acyl

degradation and oxidation, largely contributes to the unique cardiolipin profile within a

particular cell17.

The process of synthesizing cardiolipin de novo is highly conserved between

yeast and mammals. The first reaction involved in synthesizing CL requires the

conversion of mitochondrial phosphatidic acid (PA) to CDP-diacylglycerol (CDP-DAG).

This is executed via Tam41, the mitochondrial CDP-DAG synthase. Pgs1 (previously

known as the PEL1 gene) then catalyzes the transfer of a phosphatidyl group from CDP-

DAG to glycerol-3-phosphate. This forms phosphatidylglycerol phosphate, or PGP, that

is, in turn, dephosphorylated to form PG, or phosphatidylglycerol. In yeast, this step is

catalyzed by Gep4 (as seen in Figure 2A). In mammals, PTPMT1 (protein tyrosine

phosphatase, mitochondrial 1) serves as the catalyst of this dephosphorylation. Finally,

Crd1 (cardiolipin synthase) catalyzes the final reaction in the de novo synthesis pathway

by adding a phosphatidyl group from CDP-DAG to PG to form the CL. The acyl chains

of the newly synthesized CL are primarily saturated (CLSAT). However, CLSAT is

deacylated by Cld1 (cardiolipin-specific deacylase 1) to form a monolysocardiolipin

(MLCL). This MLCL can be reacylated by tafazzin, a Taz1 gene product located in the

outer face of the inner mitochondrial membrane as well as the inner face of the outer

mitochondrial membrane17.

The enzymes involved in cardiolipin synthesis do not exhibit acyl specificity,

allowing CL remodeling to occur in which acyl chains are removed from the

phospholipid and replaced17. This remodeling occurs either through a two-step process or

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a one-step process. The two-step process, or the deacylation-reacylation Lands cycle,

involves a phospholipid being deacylated (at which point it is referred to as a

lysophospholipid). Then, acyl groups from nearby phospholipids are added to the

lysophospholipid in a reacylation step. In the one-step process, acyl chains are exchanged

between neighboring phospholipids in a process known as transacylation17. Tafazzin

(Taz1) can serve as a transacylase and transfer an acyl group between phospholipid and

lysophospholipid, contributing to the one-step CL remodeling mechanism. Likewise,

tafazzin can also contribute to the two-step method of remodeling. Tafazzin’s crucial role

in the two-step method of CL remodeling is further proven through the fact that a tafazzin

deficiency ends the acylation step of the two-step method17.

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Figure 2. Cardiolipin Biosynthesis and Remodeling17. The synthesis of cardiolipin in

the inner mitochondrial membrane is highly conserved between yeast to mammals.

Cardiolipin synthesis is shown in (A) as it occurs in the yeast Saccharomyces cerevisiae.

The synthesis of CLSAT represents the end cardiolipin product, which typically possesses

saturated acyl chains. The conversion of CLSAT to CLUNSAT by tafazzin (Taz1)

summarizes the process of CL remodeling. Cardiolipin remodeling, shown in more detail

in (B) can occur via two mechanisms: a two-step deacylation-reacylation cycle or a one-

step transacylation. Taffazin can transacylate CL in one step, or it can reacylate MLCL in

the two-step process. (Permission to use figures received from publisher).

(A)

(B)

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It has been demonstrated that specific alterations to lipids contribute to multiple

cellular functions and disease states. Even slight alterations to fatty acid chains, such as

those within the cardiolipin structure, can lead to very contrasting properties between

molecular phospholipid species16. Various analytical methods such as chromatography

and mass spectrometry have been used in order to gain insight into such alterations and

their resulting effects, contributing largely to the field of lipidomics. These analyses are

useful in acquiring information on lipid distribution and content within cells16. Mass

spectrometry serves as the primary method for analyzing lipids, and it functions by “the

detection of ions (as charged lipid molecules) after their separation within an

electromagnetic field”16. Specifically, the field of lipidomics has been used in studies by

Kagan and coworkers in order to analyze cardiolipin content within the brain following

traumatic brain injuries16. These studies demonstrate that peroxidation of cardiolipin is

necessary for the release of pro-apoptotic signals from the mitochondria16.

Kagan and coworkers stimulated proapoptotic signals in the mitochondria of

tumor cells as a potential target in anticancer treatment11. Specifically, this therapy aims

to target an early stage of mitochondria-dependent apoptosis, which involves the

oxidation of cardiolipin by cytochrome c oxidase11. Such efforts aim to suppress

proapoptotic signals in the normal cells which surround the cancerous cells11. Further

evidence of the role of cardiolipin abnormalities in cancer is provided by animal-based

studies, such as the discovery by Peyta and coworkers of an increase in cardiolipin

content in cancer rat models and in a murine brain tumors13.

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1.5 Conclusions

The overall impact and deadly prevalence of breast cancer in women warrants a need

for an exploration of new therapeutic options. Modern treatments are relatively

ineffective in treating highly metastatic forms of breast cancer. Further, the metastasis of

breast tumors contributes largely to the disease mortality. Efforts have been made to

determine which alterations within a cancer cell allow for its metastatic potential, and the

answer may lie within the mitochondrial membrane of the tumor cell. Cardiolipins,

unique to this membrane, regulate pro/anti-apoptotic signals which can contribute largely

to the proliferation (or lack thereof) of cells such as breast cancer cells. The field of

lipidomics uses analytical methods such as mass spectrometry to examine phospholipid

content (such as cardiolipin content) within cells. Alterations in cardiolipin composition

may play a crucial role in regulating breast cancer cell survival and metastasis.

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CHAPTER II

Cardiolipin Profiling in Metastatic Breast Cancer Cells

Jawaher Alkhamisy, Meghan McNeely, Dale G. Nagle, and Yu-Dong Zhou

*MM performed cell viability experiments, mitochondrial isolation, cardiolipin

extraction, and statistical analyses

2.1 Introduction

In an attempt to study the effect of cardiolipin profile on the metastatic potential of

breast cancer cells, the effects of three differentially targeted phospholipase inhibitors

were evaluated in various breast cancer cell lines. Cell viability assays ware performed in

order to examine the effects of each inhibitor on the proliferation/viability of each cell

line. Bromoenol lactone (BEL), the first inhibitor used, serves as an inhibitor of calcium-

independent phospholipase A2 (iPLA2)18. The iPLA2 family of phospholipases functions

in membrane phospholipid remodeling19. The second inhibitor used was tricyclodecan-9-

yl-xanthogenate (D609), which is a competitive inhibitor of phosphatidylcholine-specific

phospholipase C (PC-PLC)20. Phospholipase C functions in converting

phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3)

diacylglycerol (DAG)21. The DAG and IP3 are both crucial second messengers which are

important in the regulation of various cellular metabolic pathways21. Lastly, halopemide

was used as an inhibitor of phospholipase D (PLD)22. Phospholipase D plays a vital role

12

in the production of phosphatidic acid (PA) and choline by the hydrolysis of

phosphatidylcholine (PC), and it also functions in altering cell growth and

morphology22,23. This production of PA and choline is important in beginning many lipid

signaling pathways mediated by PA as well as other cellular messengers such as DAG22.

Figure 3.1 Chemical Structures of Selected Phospholipase Inhibitors. The three

selected inhibitors target specific phospholipases involved in cardiolipin biosynthesis and

remodeling. The molecular structures of (A) Bromoenol lactone (BEL), an inhibitor of

calcium-independent phospholipase A2 (iPLA2)18, (B) Tricyclodecan-9-yl-xanthogenate

(D609), an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC)20, and

(C) Halopemide, an inhibitor of phospholipase D (PLD)22 are shown above.

(A)

(C)

(B)

13

Figure 3.2 Hydrolyzation of Phosphatidylinositol 4,5-bisphosphate (PIP2) by

Phospholipase C21. Phospholipase C (PLC) hydrolyzes phosphatidylinositol 4,5-

bisphosphate (PIP2) to yield the second messengers inositol 1,4,5-trisphosphate (IP3) and

diacylglycerol (DAG). This pathway is shown in red. IP3 and DAG are second

messengers, which are substrates for other molecules such as phosphatidic acid (PA) and

inositol polyphosphates. Related regulatory targets are shown in green, while other

singling molecules are represented in blue21.

In addition to the phospholipase inhibitors, the cell lines were also treated with

various dietary oils in order to determine the effect of these oil types and concentrations

on breast tumor cell proliferation. Evidence has established a link between dietary lipid

consumption and the species of cardiolipin that is produced during CL synthesis24.

Additionally, the consumption of dietary oils, specifically canola oil, has been linked to

decreased tumor incidence in patients with colon cancer25. These studies indicate that a

canola oil-rich diet significantly decreased both colon cancer incidence and multiplicity

PIP2

PLC

DAG PA

IP3 PIP3

PKC TRPC Chimerin (RhoGAP) Munc13-1

Channels PLD Endocytosis Anchors

Kinases Scaffolds Motility Transcription

Kinases

ICa2+

IPn

14

in rats when compared to the effects of corn oil25. Canola oil contains a higher ratio of ω-

3 fatty acids to ω-6 fatty acids (a 1:2 ratio) as compared to corn oil25. The ideal ratio of ω-

3 fatty acids to ω-6 fatty acids is about 1:1 to 1:4, whereas the average American diet

consist of the oils in a ratio between 1:10 and 1:2525. Studies have shown that the

chemopreventative mechanism of ω-3 fatty acids in canola oil has to do with the

inhibition of cyclooxygenase (COX) as well as the synthesis of arachidonic acid (AA)25.

Additionally, ω-3 fatty acids are proven to reduce prostaglandin production and

inflammation26. High levels of ω-6 fatty acids, on the other hand, are linked to increased

prostaglandin production, which increases the cytokine activity associated with

inflammation26. Research has shown that diets high in ω-6 fatty acids may also increase

the growth rate of cancers other than colon cancers, including breast cancer25.

Six dietary oils were selected (canola oil, avocado oil, coconut oil, olive oil,

sesame oil, and soybean oil). Whereas canola oil has a relatively low ratio of ω-3 fatty

acids to ω-6 fatty acids (1:2), olive oil has a much higher ω-6 fatty acid content, with a

ratio of 1:10. Soybean oil also has a very high ω-6 fatty acid content27. However, coconut

oil, like canola oil, is also relatively low in ω-6 fatty acid content27.

The saturated vs. unsaturated nature of these six dietary oils is also relevant to the

degree of breast cancer cell proliferation. In a study by Serge Hardy and coworkers, the

effect of saturated and unsaturated free fatty acids (FFAs) on the proliferation and

apoptosis of the breast cancer cell line MDA-MB-231 was examined28. The study found

that unsaturated fatty acids increase breast cancer cell proliferation. Saturated fatty acids,

on the other hand, inhibit proliferation and cause apoptosis28. The saturated fatty acids

decrease the levels of mitochondrial CL, which is necessary for cytochrome c retention.

15

The release of cytochrome c, a mitochondrial proapoptotic protein, results in the

activation of caspases which cause cell death28. The six oils used in this study possess

varying degrees of unsaturation. Soybean oil is the least saturated of the six and is about

60% polyunsaturated29. Coconut oil, on the other hand, is about 75% saturated29. Olive

oil, avocado oil, and canola oil, are all monounsaturated (77%, 68%, and 60%

monounsaturated relatively)29.

After performing cell viability experiments for each breast cancer cell line with

the various phospholipase inhibitors and oils, mitochondria were isolated from the

various cell cultures and liquid chromatography-mass spectrometry was performed in

order to analyze their cardiolipin profiles. The goal was to study the working hypothesis

for this experiment: that cardiolipin composition plays a major role in the mitochondrial

functioning of metastatic breast cancer cells.

The cell lines selected were MDA-MB-231, LM, BoM, T47D, MCF7, and MCF7-

BoM. MDA-MB-231 is a widely studied triple-negative breast cancer (TNBC) cell line30.

The BoM and LM cell lines are bone metastatic and lung metastatic subclones derived

from the MDA-MB-231 cell line6,7. The T47D is an estrogen-dependent cell line which

exhibits many low-level genetic abnormalities30. The MCF7, on the other hand, possesses

many genetic abnormalities with high amplification30. The MCF7-BoM is a metastatic

variant of the MCF7 breast cancer cell line31.

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CELL LINE GENE

CLUSTER ER PR SOURCE TUMOR

TYPE AGE MDA-MB-231 BaB - [-] PE AC 51

T47D Lu + [+] PE IDC 54

MCF7 Lu + [+] PE IDC 69

Table 1. Source and Pathological Features of Tumors Used to Derive Selected Breast

Cancer Cell Lines30. MDA-MB-23, T47D, and MCF7 were all derived from the pleural

effusion of a metastatic breast cancer patient. The T47D and MCF7 cell lines were

derived from a luminal gene cluster, whereas MDA-MB-231 was derived from a basal

gene cluster. (BaB= basal. Lu= luminal. PE= pleural effusion. AC=adenocarcinoma.

IDC= invasive ductal carcinoma)30.

2.2 Results

Data comparison was performed on cell viability data with one-way ANOVA

followed by Bonferroni post hoc analyses using GraphPad Prism 7. Differences between

data sets were considered statistically significant when p < 0.05.

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Figure 4. Impact of Bromoenol Lactone (BEL) on Breast Tumor Cell Viability. (A)

Structure of BEL. (B) Percent control of BEL inhibitor (B1552) on breast cancer cell

lines. Data are shown as average ± standard deviation (n=4). (*represents statistical

significance).

0 1 3 10 300

50

100

150

BoM

BEL concentration (µM)

% C

ontro

l

0 1 3 10 300

50

100

150

LM


% C

ontro

l

*

0 .1 .3 1 3 10 300

50

100

150

200

MCF7


% C

ontro

l

0 1 3 10 300

50

100

150

T47D


% C

ontro

l

0 .1 .3 1 3 10 300

50

100

150

MDA-MB-231


% C

ontro

l

*

0 .1 .3 1 3 10 300

50

100

150

MCF7-BoM


% C

ontro

l

*

*

(A)

(B)

18

Figure 5. Impact of The Tricyclodecan-9-yl-xanthogenate (D609) on Breast Tumor

Cell Viability. (A) Structure of D609. (B) Percent control of D609 inhibitor (T8543) on

breast cancer cell lines. Data are shown as average ± standard deviation (n=4).

(*represents statistical significance).

0 1 3 10 30100

0

50

100

150

BoM

D609 concentration (µM)

% C

ontro

l *

0 1 3 10 30100

0

50

100

150

LM


% C

ontro

l

0 1 3 10 30100

0

50

100

150

MCF7


% C

ontro

l

0 10 30100

0

50

100

150

T47D


% C

ontro

l

*

0 1 3 10 30100

0

50

100

150

MDA-MB-231


% C

ontro

l *

0 1 3 10 30100

0

50

100

150

MCF7-BoM


% C

ontro

l

**

(A)

(B)

19

Figure 6. Impact of Halopemide on Breast Tumor Cell Viability. (A) Structure of

halopemide. (B) Percent control of halopemide inhibitor (H3041) on breast cancer cell

lines. Data are shown as average ± standard deviation (n=4). (*represents statistical

significance).

0 .03 .1 .3 1 3 100

50

100

150

BoM

Halopemide concentration (µM)

% Co

ntro

l

0 .03 .1 .3 1 3 100

50

100

150

LM


% Co

ntro

l

0 .03 .1 .3 1 3 100

50

100

150

200

MCF7


% Co

ntro

l

* *

0 .03 .1 .3 1 3 100

50

100

150

T47D


% Co

ntro

l *

0 .03 .1 .3 1 3 100

50

100

150

MDA-MB-231


% Co

ntro

l

0 .03 .1 .3 1 3 100

50

100

150

MCF7-BoM


% Co

ntro

l

(A)

(B)

20

Figure 7. Impact of Canola Oil on Breast Tumor Cell Viability. Percent control of

canola oil on breast cancer cell lines. Data are shown as average ± standard deviation

(n=2). (*represents statistical significance).

0% .3% 1% 3%0

50

100

150

BoM

Canola Oil concentration (µM)

% C

ontro

l

0% .3% 1% 3%0

50

100

150

MDA-MB-231


% C

ontro

l

**

0% .3% 1% 3%0

50

100

150

MCF7-BoM


% C

ontro

l

***

0% .3% 1% 3%0

50

100

150

LM


% C

ontro

l

0% .3% 1% 3%0

50

100

150

MCF7


% C

ontro

l

21

Figure 8. Impact of Avocado Oil on Breast Tumor Cell Viability. Percent control of

avocado oil on breast cancer cell lines. Data are shown as average ± standard deviation


0% .3% 1% 3%0

50

100

150

BoM

Avocado Oil concentration (µM)

% C

ontro

l

0% .3% 1% 3%0

50

100

150

MDA-MB-231


% C

ontro

l

*

0% .3% 1% 3%0

50

100

150

MCF7-BoM


% C

ontro

l

*

0% .3% 1% 3%0

50

100

150

LM


% C

ontro

l

0% .3% 1% 3%0

50

100

150

MCF7


% C

ontro

l

*

22

Figure 9. Impact of Coconut Oil on Breast Tumor Cell Viability. Percent control of

coconut oil on breast cancer cell lines. Data are shown as average ± standard deviation


0% .3% 1% 3%0

50

100

150

BoM

Coconut Oil concentration (µM)

% C

ontro

l

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MDA-MB-231


% C

ontro

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% C

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* **

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LM


% C

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Figure 10. Impact of Olive Oil on Breast Tumor Cell Viability. Percent control of olive

oil on breast cancer cell lines. Data are shown as average ± standard deviation (n=2).

(*represents statistical significance).

0% .3% 1% 3%0

50

100

150

200

BoM

Olive Oil concentration (µM)

% C

ontro

l *

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% C

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% C

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LM


% C

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MCF7


% C

ontro

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Figure 11. Impact of Soybean Oil on Breast Tumor Cell Viability. Percent control of

soybean oil on breast cancer cell lines. Data are shown as average ± standard deviation

(n=2).

0% .3% 1% 3%0

50

100

150

BoM

Soybean Oil concentration (µM)

% C

ontro

l

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MDA-MB-231


% C

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% C

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% C

ontro

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Figure 12. Impact of Sesame Oil on Breast Tumor Cell Viability. Percent control of

sesame oil on breast cancer cell lines. Data are shown as average ± standard deviation

(n=2).

0% .3% 1% 3%0

50

100

150

200

BoM

Sesame Oil concentration (µM)

% C

ontr

ol

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% C

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ol

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% C

ontr

ol

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Figure 13. Mass Spectrometry Analysis of Cardiolipin Standard and Cell Extracts.

Liquid chromatography-mass spectrometry was performed on the breast cancer cells prior

to the addition of the three inhibitors. The cardiolipin standard peak can be seen at 3.32

minutes on the MS of the cardiolipin-only sample. The cardiolipin peak did not appear on

the MS results for the cell lines, which does not allow for comparison of the cell

cardiolipin compositions before and after the addition of the phospholipase inhibitors.

2.3 Discussion

The goal of these experiments was to detect changes in cardiolipin composition

within breast cancer cell lines by their exposure to three differentially targeted

phospholipase inhibitors. The first step of our experiment was to set up a method for

detecting changes in cardiolipin composition within the cell lines. Liquid

chromatography-mass spectrometry (LC-MS) was performed on each cell line as well as

a cardiolipin standard. The next step was to treat the cells with the inhibitors and assess

27

for changes in cardiolipin composition by comparing the mass spectrometry results

before and after the addition of the compounds. The cardiolipin standard peak can be

definitively observed in the MS results for the cardiolipin-only sample. However, the MS

results for the breast cancer cell lines did not display the peak for the cardiolipin standard.

Therefore, we cannot use the MS data we gathered as a valid means for comparing

cardiolipin composition before and after the addition of the inhibitory compounds.

However, statistically significant differences in cell viability results were observed in the

breast tumor cell lines as a result of treatment with each of the three phospholipase

inhibitors (BEL, halopemide, D609), as well as canola oil, avocado oil, coconut oil, and

olive oil. (See figures 4-10). Soybean oil and sesame oil did not provide statistically

significant results.

2.4 Materials and Methods

2.4.1 Cell viability assay

Cell culture was performed for each cell line before the cell viability assay was

executed. Cells were maintained in medium with RPMI-1640 with glutamine (Corning),

with 10% (v/v) fetal calf serum (FCS, Hyclone), and 50 units mL-1 penicillin and 50 µg

mL-1 streptomycin (Sigma). Before adding the cells to each culture, 9 mL of the prepared

media was placed in the incubator for 10 to 15 minutes. The cells were then taken from

the liquid nitrogen and defrosted in the incubator for about five minutes. Cells (1 mL) of

each respective cell line was added to the media in the 100 mm plate. Each plate was

incubated at 37°C overnight (95% Air/5% CO2). The next morning, the cells were

checked under the microscope to ensure they were healthy. The media was aspirated from

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each culture, and regular media (without FCS or antibiotic) was used to wash the cells.

Once the cells were washed, 10 mL of the prepared media was again added to each

culture.

Next, the media was again aspirated and 1 mL of Trypsin-EDTA (0.25%, Gibco) was

used in order to detach the cells. The cultures were put in the incubator for five minutes.

Once the cells were floating, 10 mL of the prepared media was added to inactive the

trypsin. The cells and media were gathered using a pipette and transferred to a 15 mL

centrifuge tube. The tubes were centrifuged at 600 rpm for 5 minutes (Jouan C412) and

the media was aspirated. To detach the cells, 5 µL of the prepared media was added.

When performing the cell viability assay, the basic rule followed was to gather 3x106

cells per 10 mL solution. A 12 mL solution was therefore prepared with 3.6x106 cells.

Using the cell density from the cultures performed (0.68x106 cells/mL), the volume of

cells to be gathered was found to be 5.15 mL by dividing the cell number (3.6x106) by

the cell density. The solution was completed to 12 mL using 6.85 mL of prepared media.

The solution containing 5.15 mL cells and 6.85 mL media was placed in a reservoir.

Using a pipette, 100 µL of the solution was added to each well on a 96-well plate. The

plate was shaken on a vortex on low speed for 1 minute. The stock solutions for the

inhibitors were then prepared.

Because BEL is stable at a maximum concentration of 25 g/L, the stock solution was

prepared to be 20 g/L. This concentration along with the inhibitor’s molecular weight

(317.18 g/mole) was used to calculate the molarity as 63 x 104 µM. To simplify the

calculations, we used 60 x 104 mM. This yielded 5 mg dissolved in 262.7 µL of DMSO

for the stock solution. A 1:10 dilution was performed in order to provide samples with the

29

desired concentrations (30, 10, 3, and 1 µM) from the stock solution. First, 1.5 µL of the

compound was added to 1,498.5 µL media to yield the 30 µM sample. 400 µL of this mix

was added to a new tube along with 800 µL media in order to yield the 10 µM sample.

Another 100 µL from the 30 µM sample was added to a third tube along with 900 µL

media in order to yield a 3 µM sample. Lastly, 100 µL from the 10 µM sample was

transferred to a fourth tube with 900 µL media. This yielded the last concentration needed

for BEL: 1 µM.

The stock solution for halopemide was prepared next. Halopemide is stable at a

maximum concentration of 10 g/L. This concentration along with the MW of halopemide

(416.88 g/mole) was used to calculate the molarity as 20 M. This yielded 5 mg

halopemide dissolved in 599.7 µL of DMSO for the stock solution. A 1:10 dilution was

again performed, this time to create samples with concentrations of 10, 3, 1, 0.3, 0.1, and

0.03 µM.

The D609 solution was then prepared. D609 is stable at a maximum concentration of

20 g/L. This concentration along with the MW of D609 (266.48 g/mole) was used to

calculate the molarity as 75 mM. The stock solution was prepared using by adding the 5

mg of D609 to 250 µL of DMSO. 3.99 µL of the stock solution was added to 1498.5 µL

media to form a 100 µM solution. Next, 300 µL of this mix was added to 700 µL of

media in a separate tube to produce a 30 µM solution. A 1:10 dilution was used to make a

10 µM solution and a 1µM solution from the original 100 µM solution. A 1:10 dilution

was, again, used to make a 3 µM solution from the 30 µM solution.

The inhibitors in various concentrations were added to the cells which have been

seeded onto 96-well plates. Various oils in differing concentrations were also added to

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the cells in order to examine the effect of the oils on tumor cell proliferation. Canola oil,

avocado oil, coconut oil, olive oil, sesame oil, and soybean oil were used at

concentrations of 3%, 1%, and 0.3%. Additionally, a media control was included in the

96-well plate as well as 10 µM cycloheximide (CHX). In order to fix the cells, 100 µL of

media was removed from each well and 100 µL of 20% TCA was added to the wells. The

cells were incubated at 4°C for one hour. The plates were then washed by placing the

plates into a bucket of tap water four times. Each plate was left to air dry at room

temperature. Next, in order to stain the cells, 80 µL of SRB was added to the cells at

room temperature for 10 minutes. After rinsing with 1% acetic acid three times, 100 µL

of Tris base solution was added to the cells. The plates were placed on a shaker and the

absorbance of each plate was taken using the microarray at 510 nm.

2.4.2 Mitochondrial isolation32

The PIERCE Mitochondria Isolation Kit for Cultured Cells was used in order to

extract intact mitochondria from the cell cultures. Approximately 20 x106 cells were

gathered from each of the five cell lines in order to create the five samples. First, the

protease inhibitor was added to Reagants A and C. The protease inhibitor (240 mL) was

added to 4.8 mL of Reagant A, and 390 µL of the protease inhibitor was added to 7.8 mL

of Reagant C. Cells from each cell line were suspended in 2,000 µL of 1x concentrated

Dulbecco's Phosphate-Buffered Saline (DPBS) and transferred to separate 2 mL

centrifuge tubes. The samples were centrifuged for two minutes and the supernatant was

removed. Next, 800 µL of Reagant A was added to each sample, and the samples were

put in a medium speed vortex for five seconds. Each sample was then incubated on ice

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for two minutes. Next, 10 µL of Reagant B was added and the samples were again

vortexed for five seconds, this time on maximum speed. The tubes were incubated on ice

for an additional five minutes, and vortexed at maximum speed for five seconds at each

one-minute interval. Each sample was centrifuged at 600 x g at 4°C for 10 minutes. Next,

the supernatant from each tube was transferred to a new 2.0 mL tube and centrifuged at

12,000 x g at 4°C for 15 minutes. The supernatant was again transferred to a new tube,

with the pellet containing the isolated mitochondria. Reagant C (500 µL) was added to

the pellet and the samples were centrifuged at 12,000 x g for 5 minutes. The supernatant

was discarded. The isolated mitochondria in the pellet was kept at -80°C before

processing.

2.4.3 Cardiolipin extraction

A cardiolipin extraction was performed from both the whole cell as well as the

isolated mitochondria. First, 0.05 µL (or 50 µg) of tetramyristyl-CL (1,1',2,2'-

tetratetradecanoyl cardiolipin, Avanti) was added to the sample. Next, 4.2 µL

chloroform/methanol solution (2:1 v/v) was added. Then, 7.5 µL 0.05% butylated

hydroxytoluene (BHT) was added and the sample was vortexed for two minutes. The

liquid and aqueous phases were separated by adding 800 µL of 0.01 M HCl. The sample

was vortexed at high speed for two minutes and then centrifuged at 1,000 rpm and 25°C

for 5 minutes. The lipid phase (at bottom) was collected and dried under Argon.

32

2.4.4 Mass spectrometry

Liquid chromatography-mass spectrometry (LC-MS) was performed on the

cardiolipin sample as well as the breast cancer cell lines in order to detect a cardiolipin

standard within the cells. The instrument used to perform the mass spectrometry analysis

was Waters Xevo G2-S QTOF. Cardiolipin (1 µg/mL) in isopropyl alcohol (i-PrOH) served

as the standard for the mass spectrometry analysis. The cardiolipin standard was then

meant to be used as a means to compare cardiolipin alterations in each sample after the

addition of the phospholipase inhibitors.

2.4.5 Data

The percent control that each inhibitor had on the proliferation of each cell line was

found using measures of absorbance found with a microarray. Each 96-well plate was

stained with Sulforhodamine B (SRB) and the absorbance of each well was measured at

510 nm. This data was then inserted into an excel sheet. The average absorbance for each

condition as well as the control was found. These values were then used to calculate the

percent control for each condition. This was done by dividing the average absorbance of

the particular condition by the average absorbance for the control samples in each plate.

2.4.6 Statistic analysis

Data comparison was performed with one-way ANOVA followed by Bonferroni post

hoc analyses using GraphPad Prism 7. Differences between data sets were considered

statistically significant when p < 0.05.

33

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