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The role of translocator protein (TSPO) in mediating the effects of mitotane in human adrenocortical carcinoma cells

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The role of translocator protein (TSPO) in mediating the effects of mitotane in human adrenocortical carcinoma cells

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The role of translocator protein (TSPO) in mediating the effects of
mitotane in human adrenocortical carcinoma cells

By

Zhihang Shen

A Thesis Presented to the
FACULTY OF THE USC School of Pharmacy
University of Southern California
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
Molecular Pharmacology & Toxicology Program
May 2020

Copyright 2020 Zhihang Shen

ii

Table of Contents
List of Tables ................................................................................................................................. iv
List of Figures ................................................................................................................................. v
List of Abbreviations...................................................................................................................... vi
Abstract ........................................................................................................................................ vii
Chapter 1 Introduction .................................................................................................................... 1
1.1 Adrenocortical Carcinoma (ACC)......................................................................................... 1
1.1.1 Overview ........................................................................................................................ 1
1.1.2 Clinical Presentation and Histopathology Evaluation .................................................... 2
1.1.3 Therapeutic Approaches ................................................................................................. 3
1.2 Mitotane ................................................................................................................................ 5
1.2.1 Properties of Mitotane .................................................................................................... 5
1.2.2 Drug Mechanism of Mitotane ........................................................................................ 7
1.3 Translocator Protein (TSPO) ............................................................................................... 10
1.3.1 Structure, Organization and Distribution ..................................................................... 10
1.3.2 Function and Physiological Role .................................................................................. 11
Chapter 2 Materials and Methods ................................................................................................. 13
2.1 Cell Culture ......................................................................................................................... 13
2.2 Mitochontrial Preparation ................................................................................................... 14
2.3 Scatchard Assay .................................................................................................................. 15
2.4 Displacement Assay ............................................................................................................ 16
2.5 BCA Protein Assay ............................................................................................................. 17
2.6 Bradford Reagent Assay ...................................................................................................... 17
2.7 Cell Proliferation Kit (MTT) ............................................................................................... 18
2.8 Immunoblotting Assay ........................................................................................................ 18

iii

2.9 ELISA Assay ....................................................................................................................... 20
2.10 Immunocytochemistry Staining ........................................................................................ 21
Chapter 3 Results .......................................................................................................................... 23
3.1 Characterization of TSPO in Isolated MA-10, H295R, and HAC15 Mitochondria ........... 23
3.2 Inhibitory Effects of Mitotane on Cell Proliferation in MA-10, H295R, and HAC15 Cells
................................................................................................................................................... 27
3.3 The Expression change of TSPO in Response to the Mitotane Treatment in MA-10, H295R,
and HAC15 Cells ....................................................................................................................... 30
3.4 Inhibitory Effects of Mitotane on Steroid Production in MA-10, H295R, and HAC15 Cells
................................................................................................................................................... 33
3.5 The effects of mitotane on the expression and distribution of TSPO in MA-10, H295R, and
HAC15 cells .............................................................................................................................. 39
Chapter 4 Discussion .................................................................................................................... 42
References ..................................................................................................................................... 47

iv

List of Tables
Table 1: Properties of Mitotane ............................................................................................................... 6

v

List of Figures
Figure 1: Scatchard analysis of [
3
H] PK11195 binding in MA-10, H295R, and HAC15 cells .... 24
Figure 2: Binding specificity of TSPO in MA-10, H295R and HAC15 cells .............................. 26
Figure 3: Mitotane showed variable inhibitory effects on different cells types ........................... 28
Figure 4: TSPO plays an important role in the inhibitory effects of mitotane on MA-10 cells ... 29
Figure 5: The Expression change of TSPO in response to the mitotane treatment in MA-10,
H295R, and HAC15 cells ............................................................................................................. 31
Figure 6: Progesterone production in mitotane treated MA-10, H295R, and HAC15 cells upon
stimulation of dbcAMP and 22(R)-hydroxycholesterol ................................................................ 35
Figure 7: Cortisol production in mitotane treated MA-10, H295R, and HAC15 cells upon the
stimulation of dbcAMP and 22(R)-hydroxycholesterol ................................................................ 37
Figure 8: Expression and distribution of TSPO in MA-10, H295R, and HAC15 cells ................ 40

vi

List of Abbreviations
ACC Adrenocortical Carcinoma
TSPO Translocator Protein
MAMs Mitochondrial-associated Membranes
DDT Dichlorodiphenyltrichloroethane
VDAC V oltage-dependent Anion Channel
ACAT1 Acyl-coenzyme A Transferase 1
PISD Phosphatidylserine Decarboxylase
ER Endoplasmic Reticulum
PS Phosphatidylserine
PE Phosphatidylethanolamine
IMM Inner mitochondrial Membrane
STAR Steroidogenesis Acute Regulatory Protein
PBR Peripheral-type Benzodiazepine Receptor
mBzR Mitochondrial Benzodiazepine Receptor

vii

Abstract
Mitotane (2,4'-(dichlorodiphenyl)-2,2-dichloroethane) is the only drug approved for the
treatment of metastatic adrenocortical carcinoma (ACC) due to its anti-tumor and antisecretory
properties. However, its mechanism of action remains unknown. In this study, we focused our
attention on the interaction between mitochondrial TSPO (Translocator protein 18-KDa) and
mitotane in the treatment of ACC. To evaluate the adrenal specificity of mitotane action, TSPO
was analyzed in two different human adrenocortical steroid-secreting cell lines H295R and
HAC15 and mouse Leydig MA-10 cells. In vitro competition binding studies revealed that
mitotane displaced radiolabeled PK11195, a TSPO ligand and inhibitor ，with an IC50 in the
nanomolar range in MA-10 and H295R cells, but not in HAC15 cells. These findings indicated
that TSPO may constitute a direct target of the drug in the former two cell lines. Moreover, MA-
10 and H295R cell viability was reduced by mitotane. The higher survival rate of a MA-10 TSPO
knock out cell line, nG1, suggested that mitotane-inhibited MA-10 cell proliferation was TSPO-
dependent. ELISA analysis showed that mitotane can affect cell steroidogenesis in a dose-
dependent manner in all three cell types. However, confocal microscopy analysis showed that
TSPO was downregulated in MA-10 and H295R but not in HAC15 cells, suggesting that their
steroidogenesis is TSPO-dependent. Further immunoblot analyses only detected reduced
expression of TSPO in H295R but not MA-10, which might be due to its limited capacity of
detecting multiplex forms of TSPO found in MA-10 cells. In conclusion, our results suggest that
mitotane binds to TSPO and acts as a TSPO functional antagonist in MA-10 and H295R cells,
whereas its effect on HAC15 cells is TSPO-independent.

1

Chapter 1 Introduction
Adrenocortical Carcinoma (ACC) is a rare cancer with high mortality rate. Mitotane, as the only
approved drug for ACC treatment, its drug mechanism still remains unknown. TSPO is expressed
in many organs, especially in tissues containing steroid-synthesizing cells, such as adrenal. In this
study, we hypothesis that there may be some interactions between mitotane and TSPO when
treating ACC. Therefore, the introductory will come from three aspects: ACC (overview, clinical
presentation and histopathology evaluation, therapeutic approaches), mitotane (molecular
properties, drug mechanism) and TSPO (structure, organization and distribution, function and
physiological Role).
1.1 Adrenocortical Carcinoma (ACC)
1.1.1 Overview
ACC is a rare cancer associated with a poor prognosis. The estimated incidence of ACC is 1
million to 2 million cases per year around the world (Fassnacht M., 2018). ACC is most commonly
diagnosed in the fifth to seventh decade of life, but it can arise at any age (Else et al., 2014).
Although most ACCs are considered sporadic and without a known cause, a minority of cases are
attributable to known hereditary predispositions (Raymond et al., 2013).
The prognosis of ACC is very poor. By the time ACC is diagnosed, most of patients have locally
or systemically advanced disease (Kerkhofs et al., 2013). Moreover, ACC can grow rapidly and
produce excess adrenal hormones that contribute to morbidity (Vaidya et al., 2019). The
combination of its rarity and generally short survival time has resulted in few prospective or
randomized trials (Vaidya et al., 2019). So far, surgery is the cornerstone of therapy and currently
the only avenue for a potential cure (Vaidya et al., 2019). Adjuvant-based systemic medical
therapies and adjuvant radiation therapy also have been used depending on the stage and grade of
the tumor (Vaidya et al., 2019).

2

1.1.2 Clinical Presentation and Histopathology Evaluation
The clinical evaluation of ACC involves several measurements to assess adrenocortical
hormone excesses and cross-sectional imaging (Vaidya et al., 2019). ACC can autonomously
secrete one or multiple adrenal cortical hormones that can substantially compound morbidity.
Therefore, ACC is commonly diagnosed either during the evaluation of hypercortisolism,
hyperandrogenism, or primary aldosteronism or during the evaluation of a suspicious adrenal mass
that was incidentally discovered on imaging for other purposes (Fassnacht et al., 2016). Less
commonly, ACC is diagnosed during the evaluation of nonspecific symptoms that raise a concern
of malignancy, such as weight loss, fevers, night sweats, back or flank pain, or abdominal fullness
(Bancos et al., 2016). A biopsy is often tempting to make a tissue diagnosis but is strongly
discouraged because the heterogeneity of ACC can result in nonmalignant or uninterpretable
results that can be falsely reassuring. Additionally, there is a theoretic risk for seeding through the
needle track (Bancos et al., 2016).
Although a clinical evaluation often strongly suggests ACC (ie, a large heterogeneous adrenal
mass with hyperandrogenism), accurate diagnosis relies on long-term clinical observation.
However, majority of patients need to be diagnosed rapidly, thus, diagnosis is made mostly
thorugh histopathologic examination. The most commonly used histopathologic tool to make the
distinction between an adrenocortical adenoma and ACC uses Weiss criteria on hematoxylin-
eosin–stained slides (Weiss et al., 1989). Three or more of the following Weiss criteria are strongly
indicative of ACC: high nuclear grade, more than 5 mitoses per 50 high-power fields, atypical
mitotic figures, less than 25% clear cells, diffuse architecture, necrosis, venous invasion,
sinusoidal invasion, and capsular invasion (Aubert et al., 2002). To complement the Weiss criteria,
several other classification systems exist and can be useful for borderline tumors (Duregon et al.,
2011) (Duregon et al., 2013) (Doghman-Bouguerra and Lalli, 2017; Pennanen et al., 2015). The
Ki-67 proliferation index is also an important prognosis factor in cancer. Although pathologists
generally grade ACC according to the Weiss criteria, treatment algorithms also rely on Ki-67

3

proliferative index. Because the Ki-67 protein is expressed in all phases of the cell cycle except
G0 and serves as a good marker for proliferation. Ki-67 proliferative index less than or equal to
10% considered low grade which is stage I & II and a Ki-67 proliferative index greater than 10%
considered high grade which is stage III&IV (Schteingart, 2007).
The overall survival rate of ACC is less than 5 years from the time of diagnosis (Vaidya et al.,
2019). Although Stage I and stage II ACC have 5-year survival rates of 60% to 80%, respectively,
because they are most amenable to complete surgical resection, detection of ACC at these stages
is not common (Weiss, 1984). ACC is most commonly detected at locoregional disease (stage III)
or advanced disease (stage IV), where the overall survival rates at 5 years are 30% to 50% and
less than 25%, respectively (Fassnacht et al., 2012). For these reasons and the fact that expertise
in ACC care is not widespread, it has been strongly recommended that patients with ACC be
treated by multidisciplinary teams at highly experienced centers (Aubert et al., 2002). A complete
surgical resection by an experienced surgical team is critical for optimal outcome, and a
multidisciplinary approach to adjuvant systemic and localized therapies by an experienced
medical team can amplify these gains (Vaidya et al., 2019).
1.1.3 Therapeutic Approaches
A. Surgical Approach and Complications
Complete surgical resection is the only known avenue for a potential cure of ACC and even
incomplete surgical resections are associated with better long-term outcomes and survival than no
resection at all (Else et al., 2014). Therefore, open radical adrenalectomy performed by surgeons
with considerable experience with adrenal or oncologic surgery is the preferred approach (Vaidya
et al., 2019).
ACC can be adherent and locally invasive, thereby increasing complication rates. The risks and
complications of adrenalectomy greatly depend on which adrenal gland is involved. The risks of
left-sided multivisceral resections include renal failure/insufficiency, pancreatic fistula,

4

postsplenectomy sepsis, and/or delayed gastric emptying (Vaidya et al., 2019). The main risk of
resection of a right-sided ACC is massive life-threatening bleeding from injury to the inferior vena
cava (Vaidya et al., 2019). Complications that are common to both left and right open adrenal
resections include wound infection, hernia, bleeding, and adrenal insufficiency in addition to
perioperative cardiovascular morbidity, such as myocardial infarction and deep venous thrombosis
or venous thromboembolism (Vaidya et al., 2019).
B. Adjuvant Mitotane Therapy
Mitotane (2,4'-(dichlorodiphenyl)-2,2-dichloroethane) is a derivative of the insecticide
dichlorodiphenyltrichloroethane (DDT) and is the only drug approved for ACC treatment
(Schteingart, 2007). Adjuvant mitotane therapy is recommended for patients after complete
surgical resection (R0 resection) who have either stage III or stage IV disease and/or high-grade
disease of any stage (Ki-67 index >10%). However, for patients with stage I or stage II disease
that is low grade (Ki-67 ≤10%), there is no evidence-based recommendation on whether adjuvant
mitotane may be beneficial after an R0 resection.
Despite its approval for use, mitotane can induce a variety of adverse effects, including
gastrointestinal, hepatic, hematological, and neurologic complications, in addition to multiple
endocrinopathies. In order to mitigate the side effects, some supplemental treatments are
combined with mitotane to treat patients. For example, adrenal insufficiency is induced by
mitotane, so patients can be treated supplemental glucocorticoids and mineralocorticoids. When
patients suffer from hypothyroidism, they need supplemental thyroid hormone treatment.
Therefore, patients on mitotane require routine monitoring for adverse effects. This includes
regular laboratory evaluations for liver injury, blood counts, thyroid and adrenal hormone status,
cholesterol, and testosterone deficiency. In addition, patients must be seen every 3 months for
examination and imaging surveillance of the abdomen and chest to check for recurrence or
progression. In the absence of recurrence or progression, mitotane therapy and monitoring

5

continues for approximately 2 years. When recurrence is confirmed, intensification of therapy
with alternative modalities have to be considered.
C. Chemotherapy
First-line therapy for de novo metastatic or recurrent adrenal cortical carcinoma depends on a
patient’s performance status and tumor characteristics. Patients with more indolent tumors may
be managed with mitotane alone. For those patients who have aggressive tumor characteristics,
EDP (etoposide, doxorubicin, and cisplatin) with mitotane (EDP-M) is the first-line regimen for
systemic chemotherapy. According to Fassnacht and colleagues, there is a statistically significant
improvement in progression-free survival with EDP-M compared to streptozocin-mitotane (5.3
months vs 2.0 months), although the median survival time (14.8 months vs 12 months) did not
reach statistical significance (Fassnacht et al., 2012). However, during the Fassnacht study, the
patients were allowed to cross over to the alternative streptozocin-mitotane treatment at the time
of progression; therefore, it is possible that the efficacy of EDP-M may have been underestimated.
Due to the lack of approved drugs to treat ACC, most of patients who have the disease in stages
III and IV have to rely on mitotane treatment. However, the serious side effects of mitotane limit
the wide application of mitotane. For this reason, firstly we need to assess the pharmacokinetics
properties of the molecule and its mechanism of action. By knowing this can we can figure out its
molecular target in ACC and redesign appropriately the mitotane structure to reduce its side effects.
1.2 Mitotane
1.2.1 Properties of Mitotane
Mitotane is an isomer of DDT (dichlorodiphenyltrichloroethane) which was found to cause
adrenal atrophy in dogs and was later developed as an antineoplastic agent for advanced or
metastatic adrenocortical carcinoma (Bethesda, 2012). Mitotane was approved for use in the
chemotherapy of advanced adrenal carcinoma in the United States in 1970 (Bethesda, 2012). It is
available as tablets of 500 mg under the brand name Lysodren (Bethesda, 2012). The typical initial

6

dose is 2 to 6 grams daily in 3 to 4 divided doses, which can be increased to achieve a blood
concentration of 14 to 20 mg/L (Bethesda, 2012). Mitotane is available as 500 mg tablets, which
should be taken with a glass of water during meals containing fat-rich foods (Paragliola RM et al.,
2018). This is because, mitotane is a hydrophobic drug with two aromatic rings and four halogen
atoms. When taking with fat-rich food, the drug can be absorbed by small intestinal due to its
lipophilic properties. The half-life of mitotane is 18-159 days which is relatively long and vary
from person to person. The reason of this is that fat tissue can act as a reservoir for mitotane due
to its lipophilic properties, causing a prolonged half-life and potential accumulation of mitotane
(Sparagana M, 1987). Therefore, in overweight patients, it is important to monitor the plasma
concentration in order to adjust drug dosage.
Table 1. Properties of Mitotane
Name Mitotane
Chemical Structure

Molecular Weight 320.041 g/mol
Chemical Formula C14H10Cl4
Metabolism Hepatic and Renal (DrugBank)
Route of Elimination 1%~17% is excreted in the bile (DrugBank)
Half Life 18~159 days (DrugBank)
Solubility 0.1 mg/L (at 25 ℃) (DrugBank)
Melting Point 77

7

1.2.2 Drug Mechanism of Mitotane
Mitochondria have been shown to be the cellular targets of mitotane where it interferes with the
respiratory chain activity, however, the exact mechanism is unknown Hescot et al. previously
showed that mitotane induced fragmentation of the mitochondrial network and demonstrated that
this was accompanied by specific inhibition of respiratory chain complexes I and IV activity and
by enhanced mitochondrial mass, as a compensatory mechanism in response to the respiratory
chain defect (Hescot et al., 2013).
Mitochondria-associated membranes (MAMs) are subcellular structures belonging to the ER
and are reversibly tethered to the mitochondria. MAMs have been recently extensively studied
(Vance, 2014) and constitute pivotal intracellular structures controlling key cellular processes
such as apoptosis, calcium homeostasis, phospholipid metabolism, mitochondrial function,
cholesterol metabolism and steroid synthesis, notably in adrenocortical cells (Doghman-
Bouguerra and Lalli, 2017). MAMs could be the targeted molecular complex of mitotane's action,
given their structural centrality to mitochondrial function. Mitotane action has been associated
with an inhibitory effect on a number of proteins anchored to the MAM: voltage-dependent anion
channel (VDAC), dynamin-1-related protein (DRP1), Acyl-coenzyme A Transferase 1 (ACAT1),
and phosphatidylserine decarboxylase (PISD). Poli et al., showed that mitotane inhibited the
expression of voltage-dependent anion channel (VDAC), a protein anchored in the outer
mitochondrial membrane (Duregon et al., 2013). In a recent study, Sbiera et al, hypothesized that
mitotane induced endoplasmic reticulum (ER) stress through Acyl-coenzyme A Transferase 1
(ACAT1) inhibition leading to cell apoptosis (Sbiera et al., 2015).
MAMs can contain metabolic enzymes such as phosphatidylethanolamine-N-methyltransferase
(Stone and Vance, 2000). These enzymes transfer phosphatidylserine (PS) from the ER to the
mitochondria leading to phosphatidylethanolamine (PE) synthesis catalyzed by
phosphatidylserine decarboxylase (PISD), an enzyme anchored in the inner mitochondrial
membrane (Shiao et al., 1995). Tasseva et al., reported that mitotane inhibited PISD, which

8

induced a selective defect in respiratory chain complexes I and IV accompanied by increased
mitochondrial fission (Tasseva et al., 2013). Taken together, these results stimulated our search
for a specific target for mitotane related to or associated with MAM functions.
Cholesterol importation into the mitochondria is also one of the functions associated with
MAMs and initiates steroidogenesis. The steroidogenic process involves a series of substrates, the
first being cholesterol, that are metabolized by enzymes distributed in the mitochondria and
endoplasmic reticulum of steroid-forming cells (Payne and Hales, 2004). However, cholesterol is
a highly lipophilic compound originating from various intracellular locations which cannot freely
diffuse to the inner mitochondrial membrane (IMM) (Papadopoulos et al., 2015). Thus, a
transduceosome structure is able to facilitate its movement to IMM. Steroidogenesis begins with
cholesterol transfer into mitochondria through the transduceosome, a complex composed of
cytosolic proteins that include steroidogenesis acute regulatory protein (STAR), 14-3-3 adaptor
proteins, and the outer mitochondrial membrane proteins TSPO and Voltage-Dependent Anion
Channel (VDAC) (Papadopoulos et al., 2015). Cholesterol is metabolized into pregnenolone,
which is then exported back out to the cytosol for further processing by a series of enzymes into
a variety of steroids depending upon the cell type.

9

Pathways of steroid synthesis. A. Pathways of steroid synthesis in the adrenal gland. B. Pathways of steroid synthesis in Leydig
cells of testis (Gupta E., 2014).
Mitotane can also down-regulate expression of steroidogenic enzymes both in and out of the
mitochondria. Its action on adrenal steroidogenesis has been associated with the inhibition of a
number of mitochondrial cytochrome P450-dependent enzymes: cholesterol side chain cleavage
(CYP11A1), 11b-hydroxylase (CYP11B1), and 18b-hydroxylase (CYP11B2), as well as P450-
independent enzymes, such as 3b-hydroxysteroid-dehydrogenase (Asp et al., 2010). Steroidogenic
acute regulatory protein (STAR) and CYP11A1, which are involved in cholesterol importation to
the mitochondria, the rate-limiting step of steroidogenesis, are most sensitive to mitotane.
Mitotane has also been shown to affect the cytoplasmic steps in steroidogenesis. Lehmann et
al. studied the effect of 24-h mitotane treatment on human adrenocortical steroid-secreting cell
line H295R cell viability, and expression of genes involved in adrenal steroidosynthesis has been
analyzed (Asp et al., 2010). It was found that mitotane markedly inhibited expression of genes
coding for enzymes involved in generation of cortisol and dehydroepiandrosterone sulfate
(CYP11A1 and CYP17A1). Moreover, mitotane reduced viability of H295R cells, inducing cell
apoptosis triggered by increased caspase 3 and caspase 7 activity. The mitotane-induced
repression of genes in the steroidogenic pathway was confirmed by another study using the same
H295R cell line (Zsippai et al., 2012). Mitotane proved to be a strong inhibitor of 5a-reductase
activity, this effect prompted use of 5a-dihydrotestosterone as an androgen substitution in
mitotane-treated men according to Terzolo M et al. Significant mitotane-induced derangement of
cortisol and testosterone metabolism was also shown in a similar study (Ghataore et al., 2012). In
H295R and SW13, another human adrenocortical steroid-secreting cell line, mitotane inhibited
cell proliferation in a dose and a time-dependent manner and suppressed cortisol and 17-
hydroxyprogesterone through inhibition of a number of genes involved in steroidogenesis (StAR,
CYP11A1, HSD3B2, CYP11B1, and CYP11B2).

10

Mitotane also induces CYP3A4 gene expression, thus enhancing metabolic clearance of cortisol
and a variety of drugs. Mitotane was found to be a strong inducer of CYP3A4 activity leading to
glucocorticoid inactivation and a sharp rise in 6b-hydroxycortisol urinary excretion. It was
calculated that mitotane is able to inactivate 50% of administered hydrocortisone and explains
why patients on mitotane have an increased dose requirement for steroid replacement.
1.3 TSPO
1.3.1 Structure, Organization and Distribution
Translocator protein (18 kDa) (TSPO) is a well-conserved ubiquitous protein that is encoded
by nuclear DNA and localized primarily in the outer mitochondrial membrane (Anholt et al., 1986).
TSPO was previously known as the peripheral-type benzodiazepine receptor (PBR) or the
mitochondrial benzodiazepine receptor (mBzR). TSPO is a five transmembrane domain protein
that consists of 169 aminoacids (Joseph-Liauzun et al., 1998). Although TSPO is expressed in
many organs, the highest levels are found in tissues containing steroid-synthesizing cells, such as
adrenal, gonad and brain (Papadopoulos et al., 2006).
The presence of specific mitochondrial proteins that interact with TSPO has suggested that
TSPO forms a complex composed of proteins residing in both the outer and inner mitochondrial
membrane (McEnery et al., 1992). These findings suggest that TSPO is a component of the
outer/inner membrane mitochondrial contact sites and thereby facilitates the passage of
cholesterol across the aqueous intermembrane space (Garnier et al., 1994). The ability of TSPO
to form homopolymers (mainly dimers and trimers) seems to increase with mitochondrial activity,
such as during cell proliferation and activated steroid synthesis (Delavoie et al., 2003). The tissue-
specific and cell-specific protein and lipid compositions of the mitochondrial membranes may
affect and even determine cell-specific TSPO function (Lacapère and Papadopoulos, 2003).
Moreover, many cytosolic proteins have been shown to interact with TSPO (Asp et al., 2010),
which suggests that TSPO serves as a mitochondrial anchor that transduces intracellular signals
to mitochondria (Rone et al., 2009).

11

1.3.2 Function and Physiological Role
TSPO plays an important role in steroidogenesis. TSPO was shown to be a high-affinity
cholesterol binding protein. The functional structure of cholesterol binding localized to the C-
terminal end of transmembrane helix 5 (TM5) at a conserved cholesterol recognition amino acid
consensus (CRAC) domain (Delavoie et al., 2003). Because when mutations happened in some
particular amino acids of the CRAC domain, TSPO’s ability to bind cholesterol were shown to be
eliminated (Lacapè re and Papadopoulos, 2003). Exposure of steroidogenic cells to TSPO ligands,
at concentrations close to their binding affinities, were found to stimulate steroid synthesis; and
compounds that blocked TSPO action blocked hormone-induced steroid formation in cells and in
vitro and in vivo (Papadopoulos et al., 2006).
TSPO is a drug- and cholesterol-binding protein found at particularly high levels in steroid
synthesizing cells. Its aberrant expression has been linked to cancer, neurodegeneration,
neuropsychiatric disorders and primary hypogonadism (Papadopoulos et al., 2006). However,
TSPO drug ligands have been proposed as therapeutic agents to regulate steroid levels in the brain
and testis. Steroids are critical actors in a number of health issues (Papadopoulos et al., 2006).
Brain steroids serve as local regulators of neural development and excitability, and reduced levels
have been linked to depression, anxiety and neurodegeneration. Reduced serum testosterone is
common among subfertile young men and aging men, and is associated with depression, metabolic
syndrome and reduced sexual function. TSPO, then, has become an increasing protein of interest.
Its interaction with mitotane is, therefore, of some importance.
At present, using mitotane to treat ACC patients is limited because of the severity of adverse
effects. Modifying the chemical structure and/or delivery system are standard methods to reduce
toxicity and improve bioavailability. However, they require detailed knowledge of target(s) and
interactions before designing new therapies. Therefore, identification of mitotane molecular
targets should be the first step to pursue. In this study, we want to find a target or targets of
mitotane in MAMs. One possible target has already been identified. According to Hescot et

12

al., mitotane has synergistic effects with PK11195, a ligand and pharmacological inhibitor of
TSPO, so we hypothesized that mitotane may also act as a mitochondrial receptor antagonist by
binding to TSPO (Hescot et al., 2017).

13

Chapter 2 Materials and Methods
2.1 Cell culture
1. MA-10 cells
MA-10 mouse tumor Leydig cell culture medium was composed of DMEM-F12 Glutamax
(Thermor Fisher Scientific, #1056-018), 2.5% heat-inactivated horse serum (Thermor Fisher
Scientific, #16140-071), 5% heat-inactivated fetal bovine serum (Thermor Fisher Scientific,
#26050088), and 1% penicillin/streptomycin (Thermor Fisher Scientific, #15140-122). MA-10
cells were a gift from Dr. Mario Ascoli, University of Iowa. MA-10 cells were cultured in 75 cm
2

culture flasks (Corning Inc., #430641U) and maintained at 37℃ and 3.5% CO2.
MA-10 cells were passed in a ratio of 1:10 every two days, when the cell reaches 70 %
confluency. To pass MA-10 cells, culture medium was removed from the flask. The cells were
washed once in PBS followed by1 mL trypsin treatment for 2 minutes (min). When the cells had
detached from the flask, 10 mL culture medium was added to the flask to neutralize the trypsin.
Then the cells were collected in a 50 ml falcon tubes (Corning Inc., #352098) and centrifuged at
800 rpm for three minutes at room temperature. The supernatant was removed from the tube and
the cell pellet resuspended in 10 ml of fresh culture medium. Next, 1 ml of the resuspended cells
was mixed with 9 ml fresh culture medium and added to a new 75 cm
2
flask.
MA-10 cells were frozen when the density of MA-10 cells reached 80-90%. Similar to passing
the cells, MA-10 cells were dissociated by trypsin and centrifuged. After that, 1× 10
6
cells were
resuspended in 1ml freezing medium composed of 90% culture medium and 10% DMSO. The
cells were then put into a CoolCell LX controlled-rate alcohol-free cell freezing container
(Biocision, #BCS-405) for 24 hours (h) and then placed in the liquid nitrogen for long-term
storage.
To thaw MA-10 cells, a vial of frozen cells was put into a 37℃ water bath until completely
thawed, no more than 2 min. After that, the thawed cells were added to a new 75 cm
2
flask with
15 ml fresh culture. Before the flask was put into the incubator, it was shaken to make cells were

14

distributed evenly in the flask. The cells were allowed to settle, and medium was changed the next
day.
2. H295R cells
H295R human adrenal cortical carcinoma cell culture medium was composed of DMEM-F12
Glutamax, 2.5% Nu-Serum Growth Medium Supplement (Corning Inc., #355100), 0.1% ITS
Premix Universal Culture Supplement (Corning Inc., #354351), and 1% penicillin/streptomycin.
H295R cells were cultured in 75 cm
2
culture flasks and maintained at 37℃ and 5% CO2. H295R
cells (CRL-2128) were obtained from ATCC (Manassas, Virginia).
The subculture and freezing methods for H295R cells were the same as MA-10 cells. However,
the freezing medium for H295R cells was composed of 87.5% culture medium, 7.5% Nu-Serum
Growth Medium Supplement, and 5% DMSO.
3. HAC15 cells
HAC15 human adrenal cortical carcinoma cells were a gift from Dr. William Rainey,
University of Michigan. Cell culture medium was composed of DMEM-F12 Glutamax, 10 %
HyClone Cosmic Calf Serum (GE Healthcare Life Sciences, #SH30087.03), 0.1% ITS Premix
Universal Culture Supplement, 1% penicillin/streptomycin, and 0.01% Gentamicin (Thermo
Fisher Scientific, #15750060). HAC15 cells were cultured in 75 cm
2
culture flasks and maintained
at 37℃ and 3.5% CO2.
The subculture and freezing methods for HAC15 cells were the same as H295R cells.

2.2 Mitochondrial Preparation
To extract mitochondria, cells were washed in PBS two times and dissociated from the flask
using scrapers. The dissociated cells were washed in ice-cold Buffer A; composed of 250 mM
Sucrose (Sigma-Aldrich, #S0389), 50 mM Tris-Hydrochloride Buffers, pH 7.5 ± 0.1 (Corning
Inc.,# 46-030-CM), and deionized H2O; and put into 50 ml falcon tubes. Cells were centrifuged
at 800 × g for 10 min at 4℃ and then resuspended in buffer A. The cells were homogenized on
ice with 20 strokes using an electric potter homogenizer with a Teflon pestle. The cell lysates were

15

then collected in a 50 ml falcon tube and centrifuged at 800 × g for 10 min at 4℃. The supernatants
were collected into a glass tube and placed on ice. The centrifuged pellet was homogenized again
on ice with 10 stokes using the dounce with a glass pestle. The lysates were again centrifuged at
800 × g for 10 min at 4℃. The supernatants were collected and combined with the supernatants
from the previous step. The combined supernatants were centrifuged at 10,000 × g for 30 min at
4℃. the supernatant was poured off and the mitochondrial fraction at the bottom was resuspended
in Buffer A. The protein concentration of mitochondria was determined using a BCA Protein
Assay Kit (Thermo Fisher Scientific, #23225). If the mitochondria were to be stored for further
use, a proteinase inhibitor was added to the suspension in a ratio of 1:4, and the mixture stored at
-80℃.

2.3 Scatchard Assay
1. Preparation of working solutions
To prepare a stock solution of [
3
H] PK11195 (PerkinElmer, #NET885250UC), 5 μL of [
3
H]
PK11195 was added to 1 mL PBS to obtain a 60 nM solution and using this stock to make working
solutions. The working solution should be three-fold higher than the final experimental
concentrations. The final experimental concentrations of [
3
H] PK11195 ranged from 0.05 nM to
12.5 nM. To prepare a working solution of cold PK11195 ligand for non-specific measurements,
10 μM PK11195 was prepared in 100% ethanol (EtOH).
2. Competitive binding assay of cold ligand and hot ligand to mitochondria
Tubes (VWR North American, #47729-570) were prepared for three different reactions:
straight radiation, total binding, and non-specific binding. For the straight radiation reactions, 100
μL of each [
3
H] PK11195 working solution (0.05-12.5 nM) were added to 5 mL scintillation
cocktail (EcoLume, #882470) in a scintillation vial. For total binding reaction, 100 μL PBS were
mixed with 100 μL of each [
3
H] PK11195 working solutions. For non-specific binding reaction,
100 μL of cold ligand were mixed with 100 μL of each [
3
H] PK11195 working solution. Then 100
μL mitochondria extract was added to total binding and non-specific binding reaction tubes. These

16

reaction tubes were incubated at 4℃ for 90 min. The reaction mixtures for total binding and non-
specific binding reactions were filtered using a Brandel binding apparatus. In brief, the reaction
mixture was pumped onto WHAMAN GF/B 47 mm filter paper (Brandel InC., #FP-100)
preincubated with ~ 0.05% polyethlenimine for 20-30 min. The reaction tube was washed with
ice cold PBS five times, and the washes filtered through the same filter paper.The filter paper was
then put into the scintillation vial (VWR, #66022-296) containing 5 mL scintillation cocktail. The
vials were vortexed completely and incubated at room temperature overnight. The deuterium
counts per minute were measured using a Hidex 300 SL scintillation counter. The counting time
was 60 seconds.

2.4 Displacement Assay
1. Preparation of the cold ligand for competitive binding assay
To prepare a stock solution of cold ligand, 1 mg of PK11195 (Sigma-Aldrich, #C0424) was
diluted in 284 μL EtOH to make a 10 mM solution. To make a working solution, 3 μL of stock
solution of PK11195 was added to 997 μL of PBS to make a 30 μM solution. Then the 30 μM
solution was serially diluted with PBS into 3, 3× 10
-1
, 3× 10
-2
, 3× 10
-3
, 3× 10
-4
, 3× 10
-5
, 3× 10
-6
, and
3× 10
-7
μM solutions. Then 100 μL of each of these solutions were mixed with 100 μL of 2.5 nM
[
3
H] PK11195 and 100 μL of mitochondria extract to make an experimental solution.
2. Preparation of mitotane for competitive binding assay
To prepare the stock solution of mitotane (Sigma-Aldrich, #SML1885), 16.5 mg of mitotane
was diluted in 171.8 μL DMSO to make a 300 μM solution. Then the stock mitotane solution was
serially diluted with PBS into 3, 3× 10
-1
, 3× 10
-2
, 3× 10
-3
, 3× 10
-4
, 3× 10
-5
, 3× 10
-6
, and 3× 10
-7
μM
working solutions. Then 100 μL of each mitotane working solution was mixed with 100 μL of 2.5
nM [
3
H] PK11195 and 100 μL of mitochondria extract to an experimental solution.
3. Displacement assay
Tubes were prepared for four different reactions: straight radiation, total binding, non-specific
binding, and competitive binding. For straight radiation reaction, 100 μL of 2.5 nM [
3
H] PK11195

17

solution were added to 5 mL scintillation cocktail (EcoLume, #882470). For total binding reaction,
100 μL PBS was mixed with 100 μL of 2.5 nM [3H] PK11195 solution. For non-specific binding
reaction, 100 μL of cold ligand was mixed with 100 μL of 2.5 nM [
3
H] PK11195 solution. For
competitive binding reactions, 90 μL of PBS was mixed with 10 μL of each mitotane working
solution (3× 10
-7
–3 μM) and 100 μL of 2.5 nM [
3
H] PK11195 solution. Then 100 μL mitochondria
extract was added to total binding, non-specific binding, and competitive binding reaction tubes.
These reaction tubes were incubated at 4℃ for 90 min. Then the reaction mixtures of total binding,
non-specific binding, and competitive binding reaction mixture were filtered using the brandel
binding apparatus. In brief, a reaction mixture was pumped onto a WHAMAN GF/B filter paper
preincubated with~0.05% polyethlenimine for 20-30 min. The reaction tube was washed by ice
cold PBS five times and filtered onto the same filter paper using the Brandel binding apparatus.
Then the filter paper was put into a scintillation vial containing 5 mL scintillation cocktail. The
straight radiation mixture was also added to a scintillation vial. The vials were vortexed
completely and incubated at room temperature overnight. The deuterium counts per minute were
measured using the scintillation counter.

2.5 BCA Protein Assay
BCA Protein Assay Kit from Thermo Fisher Scientific is used to determine protein concentrations.
Follow the protocol to diluted Albumin (BSA) Standards. Add 25 µL of those standards and 25
µL protein samples in 96 well plates. Triplicate both BSA standard and protein samples. Then
prepare the BCA working reagent by mixing 50 parts of BCA Reagent A with 1 part of BCA
Reagent B (50:1, Reagent A:B). Add 200 µL into each well. The plate was then covered and
incubated at 37° C for 30 minutes. After the plate was cooled to room temperature, using the plate
reader to measure the absorbance at 595 nm. Use Excel to do data analysis. Generate a standard
curve and use that to determine the concentration of each sample.

2.6 Bradford Reagent

18

BSA protein standards were prepared at the following concentrations: 0 mg/mL, 25 mg/mL, 50
mg/mL, 75 mg/mL, 100 mg/mL. 20 μL of each standard were pipetted into a well in a 96-well
plate. 200 μL of Bradford Reagent from Sigma was added to each well and mixed by pipetting.
The plate was allowed to stand at room temperature for 2 min. Absorbance was measured at 595
nm using a plate reader. A standard curve was generated by plotting absorbance at 595 nm versus
protein concentration. The curve was then used to determine protein concentration of samples.

2.7 Cell Proliferation Kit 1 (MTT from Roche)
1. Cells were incubated in a 96 well plate overnight, then 100 μL mitotane was added at different
concentrations (0 μM, 10 μM, 25 μM, 50 μM, 100 μM). The plates were incubated in the incubater
for 48 h (the incubation condition depends on the cell type), then 10 μL of the yellow MTT solution
was added and allowed to sit for approximate 4 h without light. The number of cells used for each
cell line were: 1 × 10
4
for MA-10 and nG1 cells and 5 × 10
5
forH295R and HAC15 cells.
2. After incubation, purple formazan salt crystals formed. Since these salt crystals are insoluble in
aqueous solution, they were solubilized by adding 100 μL solubilization solution and incubating
the plates overnight in a humidified atmosphere.
3. The solubilized formazan products were then spectrophotometrically quantified using a plate
reader. Increased numbers of living cells result in increases in the total metabolic activity in the
sample. This increase correlates directly to the amount of purple formazan crystals formed, as
monitored by the absorbance.

2.8 Immunoblotting Assay
1. Cell Preparations
MA-10, H295R, and HAC15 cells were plated into the 6-well plates and cultured for 24 h. The
plating density of MA-10 cells was 1× 10
5
cells/well. The plating density of H295R and HAC15
cells was 1× 10
6
cells/well. 24 h later, the cells were treated with different concentrations of
mitotane (0 μM, 10 μM, 25 μM, 50 μM, 100 μM) for 48 h under the same conditions.

19

2. Protein Extraction
After 48 h of mitotane treatment, the medium was discarded, and the cells were washed by PBS
one time. 1 ml trypsin was added to each well to digest the cells. When the cells began to detach,
1 ml culture medium was added to neutralize the trypsin. The cells were collected into 50 ml
falcon tubes and centrifuged at 3000 rpm for 2 min. The supernatants were discarded, and the cells
were washed in PBS again. After centrifugation, cells were lysed in RIPA buffer for 30 min on
ice. During lysis, the cell suspension was agitated every 10 min. The cell suspension was then
centrifuged at 16,000 × g for 20 min at 4 ℃. The supernatant was collected in a 1.5 ml tube and
placed on ice.
3. Concentration Determination
The BCA Protein Assay described above was used to determine the protein concentration. After
the protein concentrations were determined, RIPA buffer was added to each sample to adjust their
protein concentrations the same. 2× loading buffer with 10% 2-mercaptoethanol was added to
samples, and the mixtures incubated at 99℃ for 5 min. The heated samples were then centrifuged
and mixed well to make the homogenous mixtures before adding to the gel.
4. SDS-PAGE
20 μL of each sample were loaded onto an SDS-PAGE gel along with molecular weight markers
(Thermo Fisher Scientific, #7331500UL) and run Wet/Tank Blotting System (Bio-RAD) at 60 V
for 15 min followed by 200 V for 45 min.
5. Transfer
PVDF membranes (Sigma) were activated with methanol for 1 min and rinsed with transfer buffer
before stacking the gel on top. The proteins were transferred to the PVDF membrane at 100 V for
90 min. Then the membranes were blocked by 5% BSA in PBST for 90 min.
6. Antibody incubation and signal development
After transfer was complete, membranes were incubated with TSPO antibody in the first day
and GAPDH was used to incubate after stripping. TSPO antibody was developed in our laboratory
and GAPDH antibody was from Thermo Fisher Scientific (#AM4300). TSPO is the protein under

20

investigation and GAPDH is used as the internal control. TSPO antibody was diluted 1:5000 and
GAPDH 1:6000. Both of them were diluted in 5% BSA in PBST at 4℃. After 24 h, membranes
were washed in PBST three times for 10 min each. The membranes were then incubated with the
secondary rabbit ant-mouse antibody (1: 6000, Abcam, #ab6728) for 60 min at room temperature.
Membranes were washed in PBST three times for 10 min each. Then, antibodies were detected
using a Clarity Western ECL Substrate system (BioRad) and visualized using an Azure c600
Western blot imaging system (Azure Biosystems, c600)
7. Stripping
The membrane was stripped for 15 min using Restore™ Western Blot Stripping Buffer
(Thermo Fisher Scientific, #21059) at room temperature with an orbital shaker. The stripped
membrane was then washed in PBST for 60 min at room temperature. and used for GAPDH
detection again, as what we did for TSPO detection.

2.9 ELISA Assay
1. Cortisol
1) Cell Preparation
H295R and HAC15 cells were cultured in 48 well plates overnight with 1 × 10
5
cells and 200 μL
medium. The same concentrations of mitotane as used above were added and the cells incubated
for 48 h as before. 200 μL of 1 mM cAMP and 20 μM 22(R)-hydroxycholesterol were added for
2 h. Incubate the cells in the same condition as growing. After 2 h, collect the medium.
2) Performing the Assay
Use culture medium to dilute the standard curve and add 50 μL culture medium in the NSB
and B0 wells. Add 50 μL standard S1-S8 and 50 μL of sample per well. Add 50 μL Cortisol
AChE Tracer to each well except the TA and Blk wells. Add 50 μL Cortisol ELISA Monoclonal
Antibody to each well except the TA, the NSB, and the Blk wells according to manufacturer’s
directions. Cover each plate with plastic film and incubate overnight at 4℃. Reconstitute 250
dtn vial Ellman’s Reagent immediately into 50 mL before use into UltraPure water. Empty the

21

wells and rinse five times with Wash Buffer. Add 200 μL of Ellman’s Reagent to each well. Add
5 μL of tracer to the TA wells. Cover the plate with plastic film. Use orbital shaker to shake for
90 min and avoid light. Read the plate at a wavelength at 420 nm.

2. Progesterone
1) Cell Preparation
Culture the cell in 96 well plates and overnight. 1 × 10
4
cells for MA-10 cells and 1 × 10
5
cells
for H295R and HAC15 cells with 100 μL medium. The same concentrations of mitotane as used
above were added and the cells incubated for 48 h as before. 200 μL of 1 mM dbcAMP and 20
μM 22(R)-hydroxycholesterol were added for 2 h.
2) Performing the Assay
According the the manufacturer’s instruction, use ELISA Buffer to dilute the standard. Add 100
μL ELISA Buffer to NSB wells. Add 50 μL ELISA Buffer to B0 wells. Add 50 μL standard S1-
S8 and 50 μL of sample per well. Add 50 μL Progesterone AChE Tracer to each well except the
TA and Blk wells. Add 50 μL Progesterone ELISA Monoclonal Antibody to each well except the
TA, the NSB, and the Blk wells. Cover each plate with plastic film and incubate for 1 h at room
temperature. Reconstitute 250 dtn vial Ellman’s Reagent immediately into 50 mL before use into
UltraPure water. Empty the wells and rinse five times with Wash Buffer. Add 200 μL of Ellman’s
Reagent to each well. Add 5 μL of tracer to the TA wells. Cover the plate with plastic film. Use
orbital shakers to shake for 60 min. Read the plate at 420 nm.

2.10 Immunocytochemistry Staining
1. Cell culture
MA-10, H295R, and HAC15 cells were plated in the 6-well plates and cultured for 24 h. The
plating density of MA-10 cells was 1× 10
5
cells for each well. The plating density of H295R and
HAC15 cells was 1× 10
6
cells for each well. 24 h later, the cells were treated by different
concentrations of mitotane (0 μM, 10 μM, 25 μM, 50 μM, 100 μM) for 48 h.

22

2. Immunocychemistry
Before fixation, the cells were incubated with Mitotraker diluted into the phenol free medium
in the ratio of 1:700 at room temperature for 30 min. Then the cells were incubated by 4% (v/v)
paraformaldehyde in PBS for 10 min at room temperature. The cells were washed by PBS for
three times and incubate 0.1% (v/v) Triton X-100 in PBS for 10 min at room temperature. Next,
the fixed cells were washed by PBS for three times and incubate in a mixture of 5% Donkey serum,
0.5% BSA, and PBS for 30 min at room temperature. The blocking cells were washed by PBS for
three times and incubated by TSPO Antibody (1:300) at 4℃ overnight. Then the cells were
washed by PBS for three times and incubated by Donkey anti goat Alex488 (1:5000, Thermo
Fisher Scientific, #A11006) at room temperature for 30 min. Finally, the cells were wash by PBS
for three times and the nuclear of cells were stained by DAPI solution.
3. Confocal imaging
Zeiss LSM 880 + Airyscan Confocal Microscope was used scan the slide, within which the
whole filed was divided into 5× 5 areas. For each of the area, 20x objective was used to scan the
fluorescent signals.

23

Chapter 3 Results
3.1 Characterization of TSPO in Isolated MA-10, H295R, and HAC15 Mitochondria
As displayed in Figure 1, Scatchard analysis of MA-10, H295R, and HAC15 isolated
mitochondria using [
3
H] PK11195 demonstrated a single class of binding sites with a dissociation
constant of 4.37 nM, 6.669 nM and 0.8406 nM, respectively, and a binding capacity of 16691
fmol/mg, 152.9 fmol/mg and 86.15 fmol/mg, respectively. [
3
H] PK11195 and PK11195 were
defined as hot and cold ligands in our study, respectively. The differential binding characteristics
between MA-10 cells and H295R cells indicated that TSPO in MA-10 cells had a much higher
binding affinity than H295R cells under the same conditions when associated with PK11195, in
regards to both hot and cold ligands. The differential binding characteristics might be due to higher
binding capacity of TSPO in MA-10 cells versuus a higher Kd of TSPO in H295R cells. Moreover,
we observed that the Kd in HAC15 was much lower than both MA-10 and H295R cells. At the
same time, Bmax in HAC15 was also lower than the other cell types, implying that the number of
total binding sites in HAC15 cells was small. Therefore, the low Kd of HAC15 might not actually
indicate that HAC15 had a high affinity to TSPO, given that available binding sites in HAC15
cells were limited.
As displayed in Figure 2, the displacement studies of [
3
H] PK11195 and mitotane in different
cells showed different potency. Specifically, the IC50 of MA-10 and H295R were similar to each
other, 11.7 nM and 15.8 nM, respectively. However, the IC50 of HAC15 cells was 1.58 M, much
lower than MA-10 and H295R, suggesting that mitotane could not displace [
3
H] PK11195 in
HAC15 cells. Since PK11195 can specifically bind to TSPO but the binding cannot be replaced
by mitotane (Hescot et al., 2017), the binding affinity between TSPO and mitotane might be very
low.

24

Figure 1
A
0 5 1 0 1 5
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
S p e c ific b in d in g M A -1 0
C o n c e n tra tio n 3 H P K 1 1 1 9 5 (n M )
S p e c ific b in d in g
(fm o l/m g )
B m a x = 1 6 6 9 1
K d = 4 .3 7
0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
B o u n d , fm o l/m g
B o u n d /F re e
(fm o l/m g )
S c a tc h a rd

B
0 5 1 0 1 5
0
5 0
1 0 0
S p e c ific b in d in g H 2 9 5 R
P K 1 1 1 9 5 (n M )
S p e c ific b in d in g
(fm o l/m g )
0 5 0 1 0 0 1 5 0
0
1 0
2 0
3 0
B o u n d (fm o l/m g )
B o u n d /F re e (fm o l/m g )
S c a tc h a rd
B m a x = 1 5 2 .9
K d = 6 .6 6 9

25

C
0 5 1 0 1 5
0
2 0
4 0
6 0
8 0
1 0 0
S p e c ific b in d in g H A C 1 5
P K 1 1 1 9 5 (n M )
S p e c ific b in d in g
(fm o l/m g )
B m a x = 8 6 .1 5
K d = 0 .8 4 0 6
0 2 0 4 0 6 0 8 0 1 0 0
0
5 0
1 0 0
1 5 0
B o u n d (fm o l/m g )
B o u n d /F re e (fm o l/m g )
S c a tc h a rd

Figure 1: Scatchard analysis of [
3
H] PK11195 binding in MA-10, H295R, and HAC15 cells.
Binding studies were performed on isolated mitochondria using radioligand in a concentration range of 0.05-12.5 nM [
3
H] PK
11195. Specific binding was determined as the difference between total binding and nonspecific binding estimated in the presence
of 10 mM unlabeled, cold ligand PK11195. Specific binding activities between TSPO and PK 11195 in (A) MA-10, (B) H295R,
and (C) HAC15 cells were shown by the saturation binding curve. Kd is indicated by the concentration of PK 11195 when the
specific binding activity is 50% of the total. Bmax is the maximum number of receptors that are bound by PK 11195 when its
concentration is saturated. Binding activities between [
3
H] PK 11195 and PK 11195 in (A) MA-10, (B) H295R, and (C) HAC15
cells were shown by the Scatchard plot curve. The experiments were conducted in triplicate. Data in (A)-(C) are represented as
mean, n = 3.

26

Figure 2
A
-1 5 -1 0 -5
0
5 0
1 0 0
1 5 0
M A -1 0
L o g (L ig a n d ),M
S p e c ific B in d in g
(% o f C o n tro l)
P K 1 1 1 9 5 %
M itota ne%

B

-1 5 -1 0 -5
0
5 0
1 0 0
H 2 9 5 R
L o g (L ig a n d ),M
S p e c ific B in d in g
(% o f C o n tro l)
P K 1 1 1 9 5 %
M ito ta n e %

27

C
-1 5 -1 0 -5
0
5 0
1 0 0
H A C 1 5
L o g (L ig a n d ),M
S p e c ific B in d in g
(% o f C o n tro l)
M ito ta n e %
P K 1 1 1 9 5 %

Figure 2: Binding specificity of TSPO in MA-10, H295R and HAC15 cells.
Specific binding of mitotane and PK 11195 to TSPO in competing with [
3
H] PK 11195 (2 nM) in (A) MA-10, (B) H295R, and (C)
HAC15 cells were measured in the presence of indicated concentrations. Circles and squares represent percentage of specific
binding of PK11195 and mitotane, respectively. Curves were generated by GraphPad nonlinear regression analysis according to a
least-squares curve-fitting program. Data in (A)-(C) are represented as mean ± SEM, n = 3.

3.2 Inhibitory Effects of Mitotane on Cell Proliferation in MA-10, H295R, and HAC15 Cells
To examine whether mitotane has the ability to inhibit proliferation of MA-10, H295R and
HAC15 cells, we treated these cells with different concentrations of mitotane. As Figure 3 shows,
MA-10 and H295R cells were suppressed by mitotane at high concentrations from 25 to 100 μM
after 48 h, in comparison to the control group (0 μM). Moreover, MA-10 cells were drastically
inhibited by 100 μM mitotanes which is indicated by the significantly low viability ratio of MA-
10 with 100 μM mitotane (Figure 3A). However, the inhibitory effect of mitotane for HAC15 cells
was not as significant as on the other cell lines, since when mitotane concentration increased, the
viability ratio did not decrease as much.This result is consistent with the binding results that
showed the affinity between TSPO and mitotane was very low in HAC15 cells (Figure 2C),

28

suggesting that mitotane may inhibit the proliferation of HAC15 cells via other factors rather than
binding to TSPO.
To identify the role of TSPO in the inhibitory effect of mitotane on MA-10 cells, we used the
nG1 cell line, which is a TSPO knockout, as the cellular model to test whether TSPO indeed
functions in this effect (Fan J et al., 2018). By using the CRISPR/Cas9 system, nG1 cells carries
a Tsp exon2-specific genome modification, When nG1 cells (Figure 4) were compared to the
original MA-10 cells (Figure 3A), we found that nG1 cells could survive much better than MA-
10 cells under 25, 50, and 100 μM mitotane treatment, indicated by the higher viability ratios
(105% vs 60% at 25 μM, 107% vs 54% at 50 μM, 106% vs 3% at 100 μM). These results
suggest that mitotane inhibition of MA-10 cell proliferation was highly TSPO-dependent.

Figure 3
A B
c o n tro l
0 .0 0 1
0 .0 1
0 .1
1
5
1 0
2 5
5 0
1 0 0
0 .0
0 .5
1 .0
1 .5
M T T - M A -1 0
M ito ta n e c o n c e n tra tio n (u M )
% R e la tiv e v ia b ility ra tio (/ V C )
**
****
****
****

29

C
0
1 0
2 5
5 0
1 0 0
0 .0
0 .5
1 .0
1 .5
M T T -H A C 1 5
M ito ta n e c o n c e n tra tio n (u M )
% R e la tiv e v ia b ility ra tio (/ V C )
*
***

Figure 3: Mitotane showed variable inhibitory effects on different cells types.
The inhibitory effects of mitotane on cell proliferation were variable in (A) MA-10, (B) H295R, and (C) HAC15 cells. Cells were
treated with different concentrations of mitotane for 48 h, and the cell viabilities were measured via the 3-(4, 5-dimethylthiazol-2-
yl) 2, 5-diphenyl tetrazolium bromide (MTT) assay. When mitotane concentrations were increased, cell viabilities of both (A) MA-
10 cells and (B) H295R cells decreased drastically, while that of (C) HAC15 decreased slightly, suggesting that the inhibitory
effects of mitotane on MA-10 and H295R cells were much stronger than on HAC15. Data in (A)-(C) are represented as mean ±
SEM, n ≥ 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 4
0
1 0
2 5
5 0
1 0 0
0 .0
0 .5
1 .0
1 .5
M T T -n G 1
M ito ta n e c o n c e n tra tio n (u M )
% R e la tiv e v ia b ility ra tio (/ V C )

Figure 4: TSPO plays an important role in the inhibitory effects of mitotane on MA-10 cells.
The inhibitory effects of mitotane on cell proliferation were variable in TSPO KO nG1 cells. Cells were treated with different
concentrations of mitotane for 48 h, and the cell viabilities were measured via the 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyl

30

tetrazolium bromide (MTT) assay. When mitotane concentrations were increased, the cell viabilities of nG1 cells did not change,
suggesting that mitotane inhibition of MA-10 cell proliferation was highly TSPO-dependent. Data is represented as mean ± SEM,
n = 3.

3.3 The Expression change of TSPO in Response to the Mitotane Treatment in MA-10, H295R,
and HAC15 Cells
To determine the change inexpression levels of TSPO in each cell line after mitotane treatment,
we conducted immunoblotting to evaluate TSPO expression. TSPO protein can be present in two
sizes, 18 kDa (monomer) and 36 kDa (dimer). However, 18 kDa was the only detectable band in
MA-10 cells, so only 18 kDa TSPO bands were used to quantify the expression of TSPO in MA-
10 cells, while the 36 kDa bands were also in the other two cell lines. Additionally, we excluded
100 μM mitotane condition for MA-10 and H295R from immunoblotting experiments, since the
low cell viability precluded sufficient protein extraction.
As shown in Figure 5, compared with the control group (0 μM mitotane treatment), TSPO
expression was markedly downregulated by mitotane in a dose-dependent manner in H295R cells.
However, in MA-10 and HAC15 cells the decreased levels of of TSPO were not obvious. For MA-
10 cells, it is possible that alternatives pathways may be activated to increase TSPO to compensate
for the loss of TSPO activity induced with mitotane. For HAC15 cells, as we demonstrated earlier,
the extremely low binding affinity between mitotane and TSPO may have enabled TSPO to escape
from the inhibitory effects of mitotane.

31

Figure 5
A
0 μM 10 μM 25 μM 50 μM
TSPO (18 kDa)
GAPDH (36 kDa)
0 u M
1 0 u M
2 5 u M
5 0 u M
0
1
2
3
4
Q u a n tific a tio n (M A -1 0 )
M ito ta n e c o n c e n tra tio n (u M )

B
0 μM 10 μM 25 μM 50 μM
TSPO (36 kDa)
TSPO (18 kDa)
GAPDH (36 kDa)

32

C
0 μM 10 μM 25 μM 50 μM 100 μM
TSPO (36 kDa)
TSPO (18 kDa)
GAPDH (36 kDa)

Figure 5: The Expression change of TSPO in response to the mitotane treatment in MA-10, H295R, and HAC15 cells.
Immunoblotting analyses showed TSPO expression after (A) MA-10, (B) H295R, and (C) HAC15 cells were treated by different
concentrations of mitotane. TSPO was downregulated by mitotane in a dose-dependent manner in (B) H295R cells. In the contrast,
mitotane did not inhibit the expression of TSPO in (A) MA-10 and (C) HAC15 cells. Data in (A)-(C) are presented as mean ±
SEM, n = 3.
0 u M
1 0 u M
2 5 u M
5 0 u M
0 .0
0 .5
1 .0
1 .5
Q u a n tific a tio n (H 2 9 5 R )
M ito ta n e c o n c e n tra tio n (u M )
0 u M
1 0 u M
2 5 u M
5 0 u M
1 0 0 u M
0 .0
0 .5
1 .0
1 .5
2 .0
Q u a n tific a tio n (H A C 1 5 )

33

3.4 Inhibitory Effects of Mitotane on Steroid Production in MA-10, H295R, and HAC15 Cells
To investigate whether mitotane can affect cell steroidogenesis in a dose-dependent manner,
MA-10, H295R, and HAC15 cells were incubated with increasing concentrations of mitotane (0,
10, 25, 50, and 100 μM), either simulatneously or following dibutyryl-cyclic adenosine
monophosphate (dbcAMP) or 22(R)-hydroxycholesterol stimulation. After stimulation, cell lysate
supernatants were collected for the measurement of progesterone and cortisol production.
As shown in Figure 6A for MA-10 cells, both dbcAMP and 22(R)-hydroxycholesterol could
efficaciously stimulate the production of progesterone with without mitotane in the medium. As
shown in Figure 6B, when treating MA-10 cells with mitotane following dbcAMP stimulation,
lower dose mitotane (10 μM) could further stimulated the production of progesterone compared
with the control group (0 μM), whereas higher mitotane doses (100 μM) significantly reduced
progesterone biosynthesis. Unlike from the contradictory effects of mitotane on dbcAMP
stimulated MA-10 cells , mitotane consistently promoted production of progesterone at any
concentration (10-100 μM) when cells were stimulated by 22(R)-hydroxycholesterol.
As shown in Figure 6D for H295R cells, both dbcAMP and 22(R)-hydroxycholesterol could
efficaciously stimulate the production of progesterone with or without mitotane in the medium.
As shown in Figure 6D and 6E, when treating H295R cells with mitotane following either
dbcAMP or 22(R)-hydroxycholesterol stimulation, mitotane could inhibit the production of
progesterone in a dose-dependent manner (0-100 μM). Different from that done in MA-10 cells,
we included two more doses of mitotane (1 and 5 μM) to treat H295R cells to determine the initial
effective dose of mitotane for inhibiting progesterone production. The results showed that 5 but
not 1 μM was the lowest amount of mitotane that could effectively inhibit progesterone
biosynthesis in H295R cells.
As shown in Figure 6G, in HAC15 cells, both dbcAMP and 22(R)-hydroxycholesterol could
efficaciously stimulate the production of progesterone with or without mitotane added to the
medium. As shown in Figure 6H and 6I, when treating HAC15 cells with mitotane following

34

either dbcAMP or 22(R)-hydroxycholesterol stimulation, mitotane could inhibit the production of
progesterone in a dose-dependent manner (0-100 μM). Comparing the stimulation results across
all three cell types, we found that mitotane might affect steroidogenesis via different mechanisms.
In brief, progesterone production was constantly decreasing in H295R and HAC15 cells when
they were treated with mitotane following dbcAMP/22(R)-hydroxycholesterol stimulation,
suggesting that mitotane might only inhibit the transport of cholesterol into mitochondria and
reduce the source material for steroidogenesis. However, the increase of progesterone production
in MA-10 cells after serial treatment of mitotane and 22(R)-hydroxycholesterol implied that
mitotane might promote the conversion of cholesterol to progesterone catalyzed by CYP11A1.
Furthermore, the peaking production of progesterone at the low dose of mitotane treatment (10
μM) with dbcAMP and further decreases at higher doses (25-100 μM) indicated that mitotane
might also inhibit the transport of cholesterol into mitochondria in MA-10 cells,perhaps due to the
binding of mitotane to TSPO.
As shown in Figure 7A for H295R cells, both dbcAMP and 22(R)-hydroxycholesterol could
efficaciously stimulate the production of cortisol with or without mitotane added to the medium.
As shown in Figure 6D and 6E, when treating H295R cells with mitotane following either
dbcAMP or 22(R)-hydroxycholesterol stimulation, mitotane could inhibit the production of
cortisol in a dose-dependent manner (0-100 μM). Similar to what we found in H295R cells,
mitotane could also inhibit the production of cortisol in HAC15 cells in a dose-dependent manner
(0-100 μM) when HAC15 cells after stimulation with dbcAMP or 22(R)-hydroxycholesterol. The
inhibitory effects of mitotane on both progesterone and cortisol suggest that mitotane could block
the early steps of biosynthesis pathway of cortisol biosynthesis in H295R and HAC15 cells.

35

Figure 6
A B
0
1 0
2 5
5 0
1 0 0
0
5 0
1 0 0
1 5 0
2 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
3 0 0 0
S tim u la tio n T e s t-M A -1 0
M ito ta n e c o n c e n tr a tio n (u M )
P r o g e s te ro n e (n g /m g p r o te in )
B a s a l
c A M P
2 2 R

0
1 0
2 5
5 0
1 0 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
c A M P -M A -1 0
M ito ta n e c o n c e n tra tio n (u M )
P ro g e s te ro n e (n g /m g p ro te in )
*
**

C D
0
1 0
2 5
5 0
1 0 0
0
1 0 0 0
2 0 0 0
3 0 0 0
2 2 (R )-h y d ro x y c h o le s te ro l-M A -1 0
M ito ta n e c o n c e n tra tio n (u M )
P ro g e s te ro n e (n g /m g p ro te in )
*

0
1
5
1 0
2 5
5 0
1 0 0
0
5
1 0
1 0
2 0
3 0
4 0
5 0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
S tim u la tio n T e s t-H 2 9 5 R
M ito ta n e c o n c e n tra tio n (u M )
P r o g e s te ro n e (n g /m g p r o te in )
B a s a l
c A M P
2 2 R

36

E F
0
1
5
1 0
2 5
5 0
1 0 0
0
5 0
1 0 0
1 5 0
c A M P -H 2 9 5 R
M ito ta n e c o n c e n tra tio n (u M )
P ro g e s te ro n e (n g /m g p ro te in )
**
**
*** ***
***

0
1
5
1 0
2 5
5 0
1 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
2 2 (R )-h y d ro x y c h o le s te ro l-H 2 9 5 R
M ito ta n e c o n c e n tra tio n (u M )
P ro g e s te ro n e (n g /m g p ro te in )
***
****
****

G H
0
1 0
2 5
5 0
1 0 0
0
1 0
2 0
3 0
4 0
5 0
5 0
1 0 0
1 5 0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
S tim u la tio n T e s t-H A C 1 5
M ito ta n e c o n c e n tr a tio n (u M )
P ro g e s te ro n e (n g /m g p ro te in )
B a s a l
c A M P
2 2 R

0
1 0
2 5
5 0
1 0 0
0
5 0
1 0 0
1 5 0
2 0 0
c A M P -H A C 1 5
M ito ta n e c o n c e n tra tio n (u M )
P ro g e s te ro n e (n g /m g p ro te in )
**
***
**

I
0
1 0
2 5
5 0
1 0 0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
2 2 (R )-h y d ro x y c h o le s te ro l-H A C 1 5
M ito ta n e c o n c e n tra tio n (u M )
P ro g e s te ro n e (n g /m g p ro te in )
*
**
**
**

37

Figure 6: Progesterone production in mitotane treated MA-10, H295R, and HAC15 cells upon stimulation of dbcAMP and 22(R)-
hydroxycholesterol.
(A)-(C) ELISA analyses of progesterone production in MA-10 cells. Progesterone production was significantly stimulated by
dbcAMP and 22(R)-hydroxycholestero0l in MA-10 cells. When MA-10 cells were treated with different concentrations of mitotane
for 48 h either simultaneously (A ) or following (B) 1 mM dbcAMP stimulation for 2 h, MA-10 cells produced more progesterone
at a low concentration of mitotane (10 μM) but less progesterone at high concentrations (25-100 μM). When MA-10 cells were
treated with mitotane following 20 μM 22(R)-hydroxycholesterol stimulation (C), MA-10 cells produced more progesterone at
any concentration of mitotane treatment (10 μM) compared with control (0 μM, 25 μM, 50 μM, 100 μM).
(D)-(F) ELISA analyses of progesterone production in H295R cells. Progesterone production was significantly stimulated by
dbcAMP and 22(R)-hydroxycholesterol in H295R cells. When H295R cells were treated with different concentrations of mitotane
for 48 h either simultaneously (D) or following (E,F) 1 mM dbcAMP or 20 μM 22(R)-hydroxycholesterol stimulation for 2 h,
H295R cells produced less progesterone at any concentration of mitotane treatment (1-100 μM) compared to control (0 μM).
(G)-(I) ELISA analyses of progesterone production in HAC15 cells. Progesterone production was significantly stimulated by
dbcAMP and 22(R)-hydroxycholesterol in HAC15 cells. When HAC15 cells were treated with different concentrations of mitotane
for 48 h either simultaneously (G) or following (H,I) 1 mM dbcAMP or 20 μM 22(R)-hydroxycholesterol stimulation for 2 h,
HAC15 cells produced less progesterone at any concentration of mitotane treatment (10-100 μM) compared with control (0 μM).
Data in (A)-(I) are presented as mean ± SEM, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 7
A B

0
1
5
1 0
2 5
5 0
1 0 0
0 .0
0 .2
0 .4
0 .6
0 .8
S tim u la tio n T e s t-H 2 9 5 R
M ito ta n e c o n c e n tr a tio n (u M )
C o rtis o l (n g /m g p ro te in )
B a s a l
c A M P
2 2 R
0
1
5
1 0
2 5
5 0
1 0 0
0 .0
0 .2
0 .4
0 .6
c A M P -H 2 9 5 R
M ito ta n e c o n c e n tra tio n (u M )
C o rtis o l (n g /m g p ro te in )
** **

38

C D

0
1
5
1 0
2 5
5 0
1 0 0
0 .0
0 .2
0 .4
0 .6
0 .8
2 2 (R )-h y d ro x y c h o le s te ro l-H 2 9 5 R
M ito ta n e c o n c e n tra tio n (u M )
C o rtis o l (n g /m g p ro te in )
**

0
1 0
2 5
5 0
1 0 0
0
5 0
1 0 0
1 5 0
2 0 0
S tim u la tio n T e s t-H A C 1 5
M ito ta n e c o n c e n tra tio n (u M )
C o rtis o l (n g /m g p ro te in )
B a s a l
c A M P
2 2 R

E F

0 u M
1 0 u M
2 5 u M
5 0 u M
1 0 0 u M
0
2 0
4 0
6 0
8 0
1 0 0
c A M P -H A C 1 5
M ito ta n e c o n c e n tra tio n (u M )
C o rtis o l (n g /m g p ro te in )
*
**
**
***

0 u M
1 0 u M
2 5 u M
5 0 u M
1 0 0 u M
0
5 0
1 0 0
1 5 0
2 0 0
2 2 (R )-h y d ro x y c h o le s te ro l-H A C 1 5
M ito ta n e c o n c e n tra tio n (u M )
C o rtis o l (n g /m g p ro te in )
*
*

Figure 7: Cortisol production in mitotane treated MA-10, H295R, and HAC15 cells upon the stimulation of dbcAMP and 22(R)-
hydroxycholesterol.
(A)-(C) ELISA analyses of cortisol production in H295R cells. Cortisol production was significantly stimulated by dbcAMP and
22(R)-hydroxycholesterol in H295R cells. When H295R cells were treated with different concentrations of mitotane for 48 h either
simultaneously (A) or following (B,C) 1 mM dbcAMP or 20 μM 22(R)-hydroxycholesterol stimulation for 2 h, H295R cells
produced less cortisol at any concentration of mitotane treatment (1-100 μM) compared with control (0 μM).
(D)-(F) ELISA analyses of cortisol production in HAC15 cells. Cortisol production was significantly stimulated by dbcAMP and
22(R)-hydroxycholesterol in HAC15 cells. When HAC15 cells were treated with different concentrations of mitotane for 48 h
either simultaneously (D) or following (E,F) 1 mM dbcAMP or 20 μM 22(R)-hydroxycholesterol stimulation for 2 h, HAC15 cells
produced less cortisol at any concentration of mitotane treatment (10-100 μM) compared with control group (0 μM).
Data in (A)-(F) are presented as mean ± SEM, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

39

3.5 The effects of mitotane on the expression and distribution of TSPO in MA-10, H295R, and
HAC15 cells
To examine whether the expression and distribution of TSPO is affected by mitotane, we used
confocal microscopy to detect it in MA-10, H295R, and HAC15 cells. The fluorescent colors of
nuclear, TSPO, and mitochondrial are blue, green, and red, respectively.
As shown in Figure 8A, B, TSPO expression levels in MA-10 and H295R cells were decreased
as mitotane concentration increased (0-100 μM). The decreases became very obvious when the
concentration of mitotane reached the highest level tested (100 μM). However, the expression of
TSPO in HAC15 cells did not have any changes when the concentration of mitotane increased
(Figure 8C). However, the decrease trajectory of TSPO in MA-10 cells revealed by
immunocytochemistry staining is different from that revealed by immunoblotting (Figure 5C).
The inconsistent expression detected in different assays might be due to the 36 kDa sized of TSPO
that could only captured by TSPO antibody in immunocytochemistry staining assay and was the
dominant form of TSPO in MA-10 cells. When the fluorescent signals from all detected TSPO
were visualized together, 36 kDa sized TSPO provide the main source of fluorescent signals,
revealing a decrease tendency of TSPO expression.
Mitotane did not change either the morphology of mitochondria or the localization of TSPO in
MA-10, H295R, and HAC15 cells. However, the morphology of MA-10 and H295R cells
themselves changed from round to fusiform when the concentration of mitotane was increased. At
the same time, MA-10 and H295R cells were also killed by mitotane, suggesting that a high dose
of mitotane might lead to the apoptosis-associated cytoskeleton changes (Saraste and Pulkki,
2000).

40

Figure 8
A

B

41

C

Figure 8: Expression and distribution of TSPO in MA-10, H295R, and HAC15 cells
Confocal microscopy images showed that expression and distribution changes of TSPO in (A) MA-10, (B) H295R and (C) HAC15
cells treated by different concentrations of mitotane. Mitochondria were indicated by Mitotracker. In (A) MA-10 and (B) H295R
cells, TSPO expression levels decreased as mitotane concentration increased. In (C) HAC15 cells, the TSPO expression did not
change when mitotane concentration increased.

42

Chapter 4 Discussion
Mitotane has been known as the only effective treatment for ACC, but its mechanism of action
is still unclear and its molecular target(s) unknown. It was previously demonstrated that mitotane
has a marked mitochondrial impact and recently the drug has been shown to induce ER damage
resulting in ER stress (Hescot et al., 2013). According to Hescot S et al, MAMs are one of the
main intracellular targets of mitotane action. TSPO, which is located in the OMM and may be
adjacent to or in contact with MAMs, may have interactions with mitotane, as demonstrated by
synergistic effects between TSPO inhibitors and mitotane (Hescot et al., 2017). However, there is
no direct evidence to confirm this hypothesis. In this study we identify a direct interaction between
TSPO and mitotane.
Using PK11195, a well-characterized TSPO ligand, we first used a binding assay to test the
characteristics of adrenocortical steroid-secreting cell lines, mouse MA-10 and human H295R,
and HAC15. The differential binding characteristics between MA-10 cells and H295R cells
indicated that TSPO in MA-10 cells had much higher binding affinity than H295R cells under the
same conditions when associated with PK11195. The differential binding characteristics might be
due to a higher binding capacity of TSPO in MA-10 cells but a Kd that was higher in H295R cells.
Moreover, we observed that the Kd in HAC15 was much lower than both MA-10 and H295R cells.
At the same time, Bmax in HAC15 was also lower than other cell lines, implying that the number
of total binding sites in HAC15 was small. Therefore, the low Kd of HAC15 might not indicate a
high affinity to TSPO, given that available binding sites in HAC15 cells were limited. Moreover,
the IC50 of MA-10 and H295R were similar to each other; however, the IC50 of HAC15 cells
was much lower than that of MA-10 and H295R, suggesting that mitotane could not displace [
3
H]
PK11195 in HAC15 cells. Since PK11195 can specifically bind TSPO but the binding cannot be
replaced by mitotane (Hescot et al., 2017), the binding affinity between TSPO and mitotane might
be very low in HAC15 cells. We then tried to examine whether mitotane has the ability to inhibit
proliferation. Our MTT data showed that MA-10 and H295R cell growth could be suppressed by

43

mitotane in high concentrations from 25 to 100 μM after 48 h of treatment, in comparison to
controls (0 μM). However, for HAC15 cells the inhibitory effect of mitotane was not significant,
since when mitotane concentration increased, the viability ratio did not decrease. This result is
consistent with the binding result that the affinity between TSPO and mitotane was very low in
HAC15 cells, suggesting that mitotane might inhibit the proliferation of HAC15 cells via other
factors rather than binding to TSPO.
In addition to looking into these existing cell lines, we also used nG1 (Fan J et al., 2018), a
TSPO knock out MA-10 cell line that was generated by our laboratory, as a cellular model to
test whether TSPO did indeed contribute to the reduced cellular viability of mitotane. We found
that nG1 cells could survive much better than MA-10 cells under 25, 50, and 100 μM of
mitotane treatment, suggesting that mitotane inhibitory effect on MA-10 cells was TSPO-
dependent. Moreover, compared with the controls (no mitotane treatment), TSPO expression
was markedly downregulated by mitotane in a dose-dependent manner in H295R cells, revealed
by the immunoblotting. Such decreases in MA-10 and HAC15 cells was much less obvious and
did not seem dose dependent. For MA-10 cells, it is possible that other pathways may be
activated to increase TSPO to compensate for the loss of TSPO via pathways affected by
mitotane. For HAC15 cells the extremely low binding affinity between mitotane and TSPO may
enable TSPO to escape inhibition from mitotane.
Comparing the stimulation results across all three cell types, we found that mitotane might
affect steroidogenesis via different mechanisms depending on the species. In brief, progesterone
production was constantly decreasing in H295R and HAC15 when they were treated with mitotane
following dbcAMP/22(R)-hydroxycholesterol stimulation, suggesting that mitotane might only
inhibit the transport of cholesterol into mitochondria and reduce the source of steroidogenesis.
This is how mitotane works in the human cell lines. However, in the mouse cell line, the increase
of progesterone production in MA-10 after the serial treatment of mitotane and 22(R)-
hydroxycholesterol implicated that mitotane could promote the conversion from cholesterol to
progesterone that is by catalyzed CYP11A1. Furthermore, the peak production of progesterone at

44

the low dose of mitotane treatment (10 μM) with dbcAMP with decreases at higher doses (25-100
μM) indicated that mitotane might also inhibit the transport of cholesterol into mitochondria in
MA-10 cells. This inhibition may be due to the binding of mitotane to TSPO. In H295R cells, both
dbcAMP and 22(R)-hydroxycholesterol could efficaciously stimulate the production of cortisol
with or without or mitotane added to the medium at the same time. However, when treating H295R
cells with mitotane following either dbcAMP or 22(R)-hydroxycholesterol stimulation, mitotane
could inhibit the production of cortisol in a dose-dependent manner (0-100 μM). Similar to what
we found in H295R cells, mitotane could also inhibit the production of cortisol in HAC15 cells in
a dose-dependent manner (0-100 μM) when HAC15 cells following dbcAMP or 22(R)-
hydroxycholesterol stimulation. The inhibitory effects of mitotane on both progesterone and
cortisol suggests that mitotane could block the early steps of biosynthesis pathway of cortisol
biosynthesis in H295R and HAC15 cells. From our binding results, we confirmed that mitotane
did not affect TSPO in the HAC15 cell line but did affect TSPO in the other two cell lines.
However, mitotane could still have an inhibitory effect on steroidogenesis in all three cell types,
albeit not at the same point. Therefore, we suggest that in MA-10 and H295R cells, mitotane
inhibits steroid production in a TSPO-dependent pathway, whereas in HAC15 cells, mitotane
inhibits steroid production in a TSPO-independent pathway.
Finally, we used confocal microscopy to detect the expression and distribution of TSPO after
mitotane treatment in all cell types. As shown in our results, TSPO expression level in MA-10 and
H295R cells was decreased as mitotane concentration increased (0-100 μM). The decreases
became very obvious when the concentration of mitotane reached the highest concentration (100
μM). However, the expression of TSPO in HAC15 cells did not decrease significantly when the
concentration of mitotane increased. It should be noted that the decreases of TSPO in MA-10 cells
revealed by immunocytochemistry are different from that revealed by immunoblotting analysis.
The inconsistent expression detected in different assays might be due to the 36 kDa sized of TSPO
that could only be captured by TSPO antibody in immunocytochemistry staining assay but not in
the immunoblotting and was the dominant form of TSPO in MA-10 cells. When the fluorescent

45

signals from all detected TSPO were visualized together, 36 kDa sized TSPO provides the main
source of fluorescent signals, revealing a decreasing tendency of TSPO expression. Mitotane did
not change either the morphology of mitochondria or the localization of TSPO in MA-10, H295R,
and HAC15 cells. However, the morphology of MA-10 and H295R cells changed from round to
fusiform when the concentration of mitotane was increased. At the same time, MA-10 and H295R
cells were also killed by mitotane, suggesting that a high dose of mitotane might lead to the
apoptosis-associated cytoskeleton changes (Saraste and Pulkki, 2000).
From our results, we found that mitotane inhibited progesterone or cortisol production in MA-
10 and H295R cells, respectively, by binding to TSPO, but did not have an inhibitory effect on
steroid production by binding to TSPO in HAC15 cells. The discrepancy between human H295R
and HAC15 cells may be dues to either structure of TSPO in HAC15 cells versus H295R cells,
many single nucleotide polymorphisms on TSPO have been reported in humans. There were no
studies about the TSPO in HAC15 cells. Therefore, our further research directions might be
mainly about the basic knowledge of TSPO sequence and function in HAC15 cells. Also, our
further study can focus on identifying other targets and pathways affected by mitotane treatment
in HAC15 cells. The identification of these targets/pathways would help us better understand the
underlying mechanism of mitotane on ACC and improve its therapeutic effects. The discovery of
targeting proteins for mitotane may also reveal the mechanism by which some cells escape
classical chemotherapy and help us to develop new drugs that can specifically target these proteins.
By synergistically targeting both TSPO and other proteins, the recovery rate and five years
survival rate of ACC might be drastically elevated.
Some studies on the generation of a mitotane-resistant HAC15 cell line have shown that 60
genes were expressed differently between normal HAC15 cells and mitotane-resistant HAC15
cells (Seidel et al., 2020). For example, in the mitotane-resistant HAC15 cells, the expression of
genes for SOAT1 encoding sterol-o-acyl transferase 1, a major intracellular target of mitotane
(Sbiera et al., 2015) could downregulate SCARB1 that encodes for the most important transporter
scavenger receptor B1 in adrenal cholesterol uptake (Kraemer, 2007). Furthermore, steroidogenic

46

enzymes, CYP11A1 encoding for cholesterol side-chain cleaving enzyme, HSD3B2 encoding for
3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase, and CYP21A2 encoding for steroid 21-
hydroxylase, and the STAR gene encoding for StAR protein were found to be decreased,
implicating these genes/proteins as potential targets of mitotane (Seidel et al., 2020). Moreover,
compared to the mitotane-resistant cell, normal HAC15 cells showed upregulated expression of
apoptosis-associated proteins, such as caspase 3/7, after mitotane treatment, as well as the
downregulation of pathways related to lipid homeostasis and transport. These results suggested
that the lethal effects of mitotane on HAC15 cells might be executed via the accumulation of free
cholesterol and ER stress. Taken these together, all the genes/proteins changed in HAC15 cells
after mitotane treatment will be the candidates of our future studies focusing on the underlying
mechanism of mitotane on ACC.
In conclusion, we have identified TSPO as a possible, important target of mitotane that may
provide a novel approach to treat ACC patients. Furthermore, this may offer new opportunities to
treat those patients who do not respond to mitotane or who initially respond typically while relapse
later on even when therapeutic mitotane levels are maintained.

47

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Abstract (if available)

Abstract Mitotane (2,4'-(dichlorodiphenyl)-2,2-dichloroethane) is the only drug approved for the treatment of metastatic adrenocortical carcinoma (ACC) due to its anti-tumor and antisecretory properties. However, its mechanism of action remains unknown. In this study, we focused our attention on the interaction between mitochondrial TSPO (Translocator protein 18-KDa) and mitotane in the treatment of ACC. To evaluate the adrenal specificity of mitotane action, TSPO was analyzed in two different human adrenocortical steroid-secreting cell lines H295R and HAC15 and mouse Leydig MA-10 cells. In vitro competition binding studies revealed that mitotane displaced radiolabeled PK11195, a TSPO ligand and inhibitor，with an IC50 in the nanomolar range in MA-10 and H295R cells, but not in HAC15 cells. These findings indicated that TSPO may constitute a direct target of the drug in the former two cell lines. Moreover, MA-10 and H295R cell viability was reduced by mitotane. The higher survival rate of a MA-10 TSPO knock out cell line, nG1, suggested that mitotane-inhibited MA-10 cell proliferation was TSPO-dependent. ELISA analysis showed that mitotane can affect cell steroidogenesis in a dose-dependent manner in all three cell types. However, confocal microscopy analysis showed that TSPO was downregulated in MA-10 and H295R but not in HAC15 cells, suggesting that their steroidogenesis is TSPO-dependent. Further immunoblot analyses only detected reduced expression of TSPO in H295R but not MA-10, which might be due to its limited capacity of detecting multiplex forms of TSPO found in MA-10 cells. In conclusion, our results suggest that mitotane binds to TSPO and acts as a TSPO functional antagonist in MA-10 and H295R cells, whereas its effect on HAC15 cells is TSPO-independent.

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Asset Metadata

Creator Shen, Zhihang (author)

Core Title The role of translocator protein (TSPO) in mediating the effects of mitotane in human adrenocortical carcinoma cells

School School of Pharmacy

Degree Master of Science

Degree Program Molecular Pharmacology and Toxicology

Publication Date 05/03/2020

Defense Date 05/02/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag ACC,adrenocortical carcinoma,H295R,HAC15,MA-10,mitotane,OAI-PMH Harvest,translocator protein,TSPO

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Papadopoulos, Vassilios (committee chair), Culty, Martine (committee member), Haworth, Ian (committee member)

Creator Email szhshenzhihang@gmail.com,zhihangs@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-295987

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Legacy Identifier etd-ShenZhihan-8411.pdf

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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