MASARYK UNIVERSITY
FACULTY OF MEDICINE
DEPARTMENT OF PATHOLOGICAL PHYSIOLOGY
NEW INSIGHTS INTO THE PATHOGENESIS
OF PROSTATE CANCER
Habilitation thesis
Brno 2017 RNDr. Michal Masařík, Ph.D.
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© Michal Masařík, Masaryk university, 2017
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Acknowledgment
My deepest gratitude and thanks belong to my amazing team for their help, hard work,
enormous support and humanity.
I would like to thank all of my past supervisors and mentors who contributed to my scientific
education. In particular, I thank Prof. Stanislav Pavelka, Prof. Emil Paleček and Prof. René
Kizek, who supervised me during my undergraduate and graduate studies.
Additionally, I would like to thank all of the colleagues and collaborators I had the pleasure to
work with for their creativity, excellent work, and inspiring discussions. I would like to thank
prof. Anna Vašků and prof. Petr Babula for their support.
My greatest thanks belong to Monika, Magdalénka, Markétka, my parents and sisters for all
their help, support and inspiration in my personal and scientific life.
Special thanks belong to my Angels, without whom I would not be where I am…..
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‫א‬‫ן‬ֶ‫ב‬ ‫ֶת‬‫ל‬ֶ‫ה‬ֹ‫ק‬ ‫י‬ ֵ‫ר‬ ְ‫ב‬ ִּ‫ד‬-.‫ִּם‬ ָּ‫ל‬ ָּ‫ירּוש‬ ִּ‫ב‬ ‫ְֶך‬‫ל‬ֶ‫מ‬ ,‫ד‬ִּ‫ו‬ָּ‫ד‬
‫ב‬.‫ל‬ֶ‫ב‬ָּ‫ה‬ ‫ל‬ֹ‫כ‬ַ‫ה‬ ‫ים‬ִּ‫ל‬ָּ‫ֲב‬‫ה‬ ‫ל‬ֵ‫ֲב‬‫ה‬ ,‫ֶת‬‫ל‬ֶ‫ה‬ֹ‫ק‬ ‫ר‬ַ‫מ‬ָּ‫א‬ ‫ים‬ִּ‫ל‬ָּ‫ֲב‬‫ה‬ ‫ל‬ֵ‫ֲב‬‫ה‬
‫ג‬‫ה‬ַ‫מ‬-:‫ם‬ָּ‫ד‬ָּ‫ָּא‬‫ל‬ ,‫רֹון‬ ְ‫ת‬ִּ‫י‬‫ָּל‬‫כ‬ ְ‫ב‬-‫לֹו‬ָּ‫ֲמ‬‫ע‬--.‫ש‬ֶ‫מ‬ ָּ‫ש‬ַ‫ה‬ ‫ת‬ַ‫ח‬ ַ‫ת‬ ,‫ל‬ֹ‫ֲמ‬‫ע‬ַ‫י‬ ֶ‫ש‬
‫יח‬‫ב‬ ָּ‫ר‬ ,‫ה‬ָּ‫מ‬ ְ‫כ‬ָּ‫ח‬ ‫ב‬ֹ‫ר‬ ְ‫ב‬ ‫י‬ ִּ‫כ‬-.‫אֹוב‬ ְ‫כ‬ַ‫מ‬ ‫יף‬ ִּ‫יֹוס‬ ,‫ַת‬‫ע‬ַ‫ד‬ ‫יף‬ ִּ‫יֹוס‬ְ‫ו‬ ;‫ַס‬‫ע‬ָּ‫כ‬
Book of Ecclesiastes, 1; 1-3,18
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Table of contents
Table of contents............................................................................................................5
Preface ...........................................................................................................................7
1 Introduction..........................................................................................................8
1.1 Models used in experiments................................................................................11
2 PART I: Zinc, metallothionein and prostate cancer progression........................ 13
2.1 Theoretical basis................................................................................................. 13
2.1.1 Zinc ions and their regulation ............................................................................ 13
2.1.2 Unique role of zinc in the prostatic tissue..........................................................53
2.1.3 Prostate cancer and microRNA action ...............................................................66
2.2 Hypotheses verified under PART I.....................................................................78
2.2.1 Findings related to the hypothesis 1 ...................................................................78
2.2.2 Findings related to the hypothesis 2................................................................. 105
2.2.3 Findings related to the hypothesis 3................................................................. 124
3 PART II: Metabolism of amino acids and prostate carcinoma progression..... 139
3.1 Theoretical basis............................................................................................... 139
3.1.1 Prostate cancer metabolism.............................................................................. 139
3.2 Hypothesis verified under PART II .................................................................. 140
3.2.1 Findings related to the hypothesis 1 ................................................................. 140
3.2.2 Findings related to the hypothesis 2................................................................. 155
4 PART III: Redox status and oxidative stress of prostatic cells......................... 201
4.1 Theoretical basis............................................................................................... 201
4.2 Hypothesis verified under PART III.................................................................202
4.2.1 Findings related to the hypothesis 1 .................................................................202
4.2.2 Findings related to the hypothesis 2.................................................................253
5 PART IV: Novel approaches to the analysis of prostate cancer markers .........262
5.1 Theoretical basis...............................................................................................262
5.2 Hypothesis verified under PART IV.................................................................265
5.2.1 Findings related to the hypothesis 1 .................................................................265
5.2.2 Findings related to the hypothesis 2.................................................................290
5.2.3 Findings related to the hypothesis 3................................................................. 314
6 Summary ..........................................................................................................322
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7 Abstrakt............................................................................................................326
8 References........................................................................................................327
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Preface
This habilitation work is a compilation of selected scientific publications to which I have contributed
as the first author, corresponding author or a co-author in the course of my independent
scientific career. These articles were published between 2011 and 2017. All of these publications
have a common theme related to prostate cancer. In particular, this habilitation summarizes new
findings in the field of biochemistry and pathogenesis of prostate cancer. It has been divided
into four parts depending on their specific focus. The first part is devoted to the issue of the zinc
ions and their relation to prostate cancer development and progression. Part two deals with the
amino acid metabolism in prostate cancer. Part three is dedicated to monitoring of the redox
status of the prostate tumour cells and the last part is focused on the newly investigated prostate
cancer markers and their determination. The accompanying text highlights my contribution to
the field of prostate cancer and also contains a brief introduction to the topic. Comprehensive
information on the individual topics can be found in the enclosed original publications.
Introduction 1
8
1 Introduction
Prostate cancers are one of the most frequently occurring malign solid tumours in the Czech
Republic and globally as well. The adenocarcinoma arising from epithelial prostate acini cells
is the most common histological type of prostate cancer. In the text below, the prostate adenocarcinoma
will be understood under the general abbreviation CaP. The adenocarcinoma is
usually generated in the peripheral part of the prostate gland (Fig. 1) and therefore, at the initial
stage, the patient may not have any observable difficulties. Ductal adenocarcinoma, mucinous
adenocarcinoma, transitional cell carcinoma, small cell carcinoma, prostate squamous cell carcinoma
and a highly malignant sarcomatoid carcinoma occur only rarely [1, 2]
.
Figure 1 | Anatomy of prostate gland. a) Both benign prostatic hyperplasia (BPH) and prostate cancer are agerelated
diseases in the male, predominantly occurring later in life. b) The human prostate contains several different
tissue zones (recognized by their pathology). BPH typically arises within the transitional zone, whereas prostate
cancer predominantly occurs in the peripheral zone [1]
.
Variability in growth rate is an important characteristic feature of the prostate carcinoma. One
part of the prostate adenocarcinomas have a low metastatic potential and slow growth, and
therefore they are often revealed only during the autopsy of the persons deceased for the reason
other than CaP or during surgical interventions [3, 4]
. In the patients, whose tumours are of low
Introduction 1
9
grade and progressive increase in plasma levels of prostate-specific antigen biomarkers does
not occur (a latent form of the disease), radical therapy is often not applied, and the active
surveillance is chosen instead [5]
. In case of progressing tumours or higher-grade tumours, surgical
approach or radiotherapy is chosen. Chemotherapy is not a standard treatment for early
CaP but is sometimes used when CaP spreads out of the prostate gland and the hormonal therapy
does not work [6]
. Though the localized CaP is potentially curable (by applying surgery and
radiotherapy), metastases or resistance to treatment and progression of the tumour disease develop
in about 20% of patients. If prostate carcinoma cells metastasize, the disease can hardly
be managed therapeutically and palliation is the only way in many cases. Skeleton is the most
frequent site of CaP distant metastases. Metastases, however, often occur also in lungs and liver
[7]
.
In 1941 Charles Huggins revealed benefit of the systemic androgen deprivation therapy (ADT)
in the patients with advanced CaP [8]
. This finding has led to the application of the androgen
deprivation therapy and androgen receptor blockade (AR) as the therapeutic strategy for metastasizing
prostate carcinomas. ADT is performed by surgical castration or chemical castration
by anti-androgens [9]
. Under normal circumstances, survival of healthy prostate tissue and prostate
carcinoma cells is dependent directly on stimulation of the androgen receptor (AR), which
dihydrotestosterone (DHT) or testosterone is bound to, thus activating the appropriate cellular
processes such as proliferation, anti-apoptotic signals, and prostate-specific antigen (PSA) synthesis
(Fig. 2). The patients initially respond favourably to androgen deprivation, however,
remission of the disease followed by growth of an androgen-independent tumour is often seen
[10]
. This stage of the disease is referred to as the hormone-refractory prostate cancer (HRPC) or
also as the castration-resistant prostate cancer (CRPC). The interval from onset of the hormonal
therapy up to the development of HRPC varies considerably in individual cases and ranges from
several months to several years, 18–24 months on average. Prognosis of patients, where a tumour
passes to the hormone refractory stage, is unfavourable. The androgen-independent
proliferation can run in several ways; receptor hypersensitivity, non-androgen ligand bond, or
activation of alternative signal pathways is employed [2]
.
Introduction 1
10
Figure 2 | Androgen action. Testosterone circulates in the blood bound to albumin (not shown) and sex-hormonebinding
globulin (SHBG), and exchanges with free testosterone. Free testosterone enters prostate cells and is converted
to dihydrotestosterone (DHT) by the enzyme 5α-reductase. Binding of DHT to the androgen receptor (AR)
induces dissociation from heat-shock proteins (HSPs) and receptor phosphorylation. The AR dimerizes and can
bind to androgen-response elements in the promoter regions of target genes. Activation (or repression) of target
genes leads to biological responses including growth, survival and the production of prostate-specific antigen
(PSA) [10]
.
During the last two decades, the number of patients suspected of having CaP has increased
significantly. This increase is related directly to the introduction of the prostate-specific antigen
(PSA) examination into clinical practice [11, 12]
. The prostate-specific antigen (PSA) is a glycoprotein
produced exclusively by epithelial prostate tissues. A small PSA fraction enters the
bloodstream and can be detected from the blood sample and serve as a biomarker. This marker
allowed to identify the presence of tumours that were undetectable by per rectum examination.
Though the CaP diagnosis has thus been facilitated, the mortality rate declined slightly until
1993 only. After that year, the mortality rate of the CaP patients was no more reduced significantly
[13]
. The values ranging from 0 to 2.5 ng/ml are considered mostly the normal PSA level.
PSAlevels in the men suffering from the prostate carcinoma are often relatively high. However,
PSA as a marker is neither sensitive nor specific adequately. In 2004 Thompson et al. have
proved that CaP may also occur in the patients having low PSA levels. The share of these cases
was up to 27% [14]
. The high presence of obese men in the monitored group could be the reason.
Introduction 1
11
Obese men usually have decreased PSA levels, though their CaP risk is increased [15]
. On the
other hand, a lot of men with the PSAlevel higher than 2.5 ng/ml do not suffer from the prostate
carcinoma. The most common process leading to increased PSA value is the benign prostatic
hyperplasia, (a common noncancerous prostate enlargement often accompanied by urination
problems). Other reasons of elevated PSAlevels in the blood can be as follows: prostate inflammation
and/or various prostate examination procedures. With respect to the reported
disadvantages of PSA, which is the only widely used clinical marker, it is of the great importance
to focus on prostate physiology and pathophysiology to find new or complementary
markers.
This habilitation work summarizes new findings in the field of biochemistry and pathogenesis
of prostate cancer obtained during my CaP research. It has been divided into four parts depending
on their specific focus. The first part is devoted to the issue of the zinc ions and their relation
to CaP development and progression. Part two deals with the amino acid metabolism in CaP.
Part three is dedicated to monitoring of the redox status of the prostate tumour cells and the last
part is focused on the newly investigated CaP markers and their detection. It is a set of commented
publications.
1.1 Models used in experiments
To investigate the mechanisms playing a role in CaP pathogenesis, biological samples of the
CaP patients and four model human prostate cell lines were used. The PNT1A cell line has been
derived from normal epithelial prostate cells immortalized with a plasmid containing the SV40
virus genome with a defective origin of replication. The primary culture was obtained from the
normal prostate tissue of a 35-year-old man post-mortem. PNT1Ais a non-tumour epithelial cell
line positive for the tumour suppressor PTEN expressing the common (wild-type; wt) p53 [16]
.
The 22Rv1 cell line was used as the primary androgen-responsive carcinoma model. It is a human
epithelial line derived from the xenograft during serial propagation in mice. The 22Rv1 cell
line represents the primary prostatic badly differentiated adenocarcinoma, Gleason score of 9
[17]
. The 22Rv1 line shows a significantly lower degree of gene variability and a lower degree of
aneuploidy - karyotype 50 XY (trisomy 7,8,12) - DNA index of 1.30 (PC-3 1.84 and LNCaP
2.09), compared with all other lines [18]
. The 22Rv1 line expresses the prostate-specific antigen
(PSA); its growth is stimulated moderately by testosterone, and lysates of these cells are immunoreactive
to antibodies against the androgen receptor (AR). 22Rv1 is PTEN and p53 positive
Introduction 1
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[19, 20]
. The androgen-responsive metastatic cell line is represented by the LNCaP cell line derived
from a 50-year-old man's infraclavicular node. LNCaP cells have a mutation in the AR
gene. This mutation creates a promiscuous AR that can be bound to different types of steroids.
LNCaP is AR-positive, PSA-positive, PTEN negative and express the wild-type p53 [20, 21]
. The
PC-3 metastatic line derived from grade 4 adenocarcinoma metastasizing to bones was used as
a model of aggressive androgen-nonresponsive CaP. The PC-3 line is PTEN-, AR-, PSA and
p53-negative [19, 21, 22]
. All cell lines used in this study were obtained from HPA Culture Collections
(Salisbury, UK).
PART I: Zinc, metallothionein and prostate cancer progression 2
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2 PART I: Zinc, metallothionein and prostate cancer progression
2.1 Theoretical basis
The theoretical basis of the Part I consist of the basic information about the studied topic and of
a set of overview papers published by our group regarding this issue.
2.1.1 Zinc ions and their regulation
Zinc ions (Zn2+
) are the essential element necessary for proper cell functions. They have four
major biological roles, including the structural, signalling, catalytic and regulatory functions
[23]
. Zn2+
are a co-factor of a number of proteins, this being essential for proper functioning of
the transcription factors, enzymes and structural proteins. Zn2+
are involved in regulation of the
immune system, gene expression, energy metabolism, signal transduction and a number of other
processes [24]
.
Zinc concentration in the interstitial fluid ranges from 2–5 μM. However, the free Zn2+
level is
significantly lower, approximately 200 nM [24]
. Most of the zinc content is bound to albumin
and α2 macroglobulin. Intracellular Zn2+
concentration ranges from 100–500 μM. Most of this
content is bound firmly to protein structures (about 90%). Only 10% acts as a releasable reserve
form of Zn2+
, which is bound to low molecular weight compounds - metallothionein, amino
acids (cysteine, histidine, proline) and organic acids (citrate, oxalate) [25, 26]
. About 30–40% of
Zn2+
is present in the nucleus, about 50% in the cytoplasm and organelles (mitochondria, endoplasmic
reticulum, Golgi's apparatus, endosomes and lysosomes), and the remaining part is
bound to cell membranes [27]
. Zinc is included in many types of physiological processes and
therefore its levels must be regulated carefully. The high Zn2+
levels may have toxic effects [28]
.
The whole body level of regulation is based on affecting of intestinal absorption and secretion.
The cellular zinc level regulation is based on the coordination of the zinc-binding and zinctransporting
proteins [29]
. The metal-responsive-element-binding transcription factor-1 (MTF-1)
is the key transcription regulator of both the transporters and the Zn2+
binding proteins [30]
.
Zinc is unable to diffuse freely across the membrane and must, therefore, utilize the transport
system (Fig. 3) [31]
. There are two known families of zinc transporters: the first family is represented
by Zrt-Irt type proteins (the ZIP family) and the other family is represented by CDF
(Cation Diffusion Facilitator) also called the ZnT family. The ZIP family transporters are responsible
for the zinc transport to cytoplasm from extracellular space and also play a role in
PART I: Zinc, metallothionein and prostate cancer progression 2
14
zinc ions flow out of the cell organelles into the cytoplasm. Members of the ZnT transporter
family have a reverse role, they affect export of zinc out of the cell and the translocation of zinc
from the cytoplasm into the organelles, thus reducing the concentration of cytosolic zinc effectively
[31]
.
Figure 3 | ZIP carriers are responsible for transport of Zn2+ to the cytoplasm, out of the extracellular environment
and out of the organelles. On the contrary, ZnT transporters are responsible for transport out of the cytoplasm,
i.e. into the organelles or into the extracellular environment.
Important zinc-binding proteins include metallothioneins (MT). Metallothioneins represent a
class of metal-binding polypeptides with high cysteine content (up to 30% of amino acid residues)
and a low molecular weight (0.5 to 15 kDa). Up to seven divalent metal ions can be bound
to the human MT, and this bond stabilizes the three dimensional MT structure [32]
. MT plays an
important detoxification role in defence against heavy metals. This protective function is related
to the MT ability to capture free radicals. Therefore, MT is highly expressed under oxidative
stress conditions [33]
, which is a common situation inside the tumour tissue. The relationship
between the oxidative stress and MT is summarized in the overview paper [33]
, full text - see
page 16 of the habilitation thesis.
Several all-genomic studies have shown that a cluster of MT genes located on chromosome 16
(16q12-22) is an important target in the search for candidate genes playing a role in carcinogenesis
[34, 35]
. MT functions also affect regulation of apoptosis, where elevated levels of
metallothionein have the anti-apoptotic effect. Further, metallothioneins regulate the level, activity
and cellular localization of the NF-κB transcription factor, which activates the antiapoptotic
Bcl-2, c-myc and TRAF-1 genes. This anti-apoptotic cascade can be used as a protective
mechanism of prostate carcinoma cells against apoptotic signals [36, 37]
. Polymorphisms
PART I: Zinc, metallothionein and prostate cancer progression 2
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associated with the onset of the tumour disease have also been identified in genes for MT recently.
Influence of polymorphisms in MT on the development of various pathologies, including
prostate tumours, is summarized in the overview paper [38]
available on page 39.
Author’s publications relevant to this chapter
1. Ruttkay-Nedecky, B., L. Nejdl, et al. (2013). "The Role of Metallothionein in Oxidative
Stress." International Journal of Molecular Sciences 14(3): 6044-6066
Available on page 16
2. Raudenska, M., J. Gumulec, et al. (2014). "Metallothionein polymorphisms in pathological
processes." Metallomics 6(1): 55-68.
Available on page 39
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2.1.2 Unique role of zinc in the prostatic tissue
The prostate is involved in the formation of 30% of the seminal fluid volume, where prostaglandins,
a number of enzymes, zinc ions (Zn2+
) and citrate are released by the prostate epithelial
cells [39]
. Metabolism of these substances, especially Zn2+
and citrate, differs in the prostatic
cells compared with a number of other tissues. Citrate is processed routinely in the Krebs cycle
producing a large amount of ATP. To avoid entry of citrate into this cycle and its subsequent
degradation, the mitochondrial enzyme aconitase, converting citrate into isocitrate, has to be
blocked in epithelial prostate cells [40]
. Aconitase is blocked by the zinc ions, accumulated by
prostate to a higher extent (up to ten times higher compared with other tissues) [41, 42]
. Thanks to
inhibition of the mitochondrial aconitase by zinc, prostate loses a substantial portion of the
energy in the form of ATP [39]
. In most mammalian cells, the total zinc concentration in the cells
ranges from 100 to 500 μM. However, the zinc concentration in healthy prostate epithelial cells
is much higher, ranging from 800 to 1500 μM; the zinc content accumulating in prostatic mitochondria
is 20 times higher. The concentration of zinc in the prostatic fluid is 500 times higher
than the concentration in the blood plasma. Citrate level in the peripheral prostate zone is 30 to
80 times higher than that of other soft tissues. Even more pronounced is an increase of citrate
concentration in the prostatic fluid, which is about 1000 times higher than the concentration in
the blood plasma [43]
. Zn2+
accumulation is likely to involve zinc transporters and zinc-binding
proteins. In addition to direct effects on mitochondrial aconitase, zinc also has other effects,
namely induction of apoptosis by cytochrome C (zinc ions increase formation of BAX pores on
the mitochondrial membrane, thereby inducing the cytochrome c (CytC)/caspase (Casp) -mediated
apoptosis) and regulation of expression of a number of genes through kinases [44]
.
Irreversible loss of ability to accumulate Zn2+
is a typical feature for prostate carcinoma [45, 46]
.
This leads to a reduction of the pro-apoptotic effect and to loss of ability to produce citrate for
seminal fluid [47]
. The inhibitory effect of zinc on the enzyme aconitase, which is observable in
healthy prostate cells, vanishes (see Fig. 4). Citrate can thus enter the Krebs cycle, and the
carcinoma cells gain the advantage of being able to gain more energy [41, 42]
. Inhibition of the
ZIP1 transporter through increased activity of the Ras-Raf-Mek-Erk cascade is one of the assumed
mechanisms. ZIP1 has been identified in prostate cells as the functionally most important
zinc importer [43, 48]
.
PART I: Zinc, metallothionein and prostate cancer progression 2
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Figure 4 | Zinc ions cause accumulation of citrate (and hence change in energy metabolism of the cells) by (a)
inhibiting the mitochondrial aconitase which converts citrate into isocitrate. Prostatic cells thus produce significantly
less ATP; (b) Zn2+ has pro-apoptotic effect due to increased release of cytochrome C (CytC) out of
mitochondria; (c) Zn2+ functions as a signal molecule and effects, in particular, gene expression by action on
mitogen-activated protein kinases (MAPK); (d) Zn2 + induces expression of metallothionein (MT) through its
metal-regulatory transcription factor-1 (MTF-1), and MT thus affects intracellular level of free Zn2+.
In the previous chapter "Zinc ions and their regulation", the basic mechanisms of zinc ion impact
in the non-tumour prostate and in the transformed tissues were mentioned. In the following
overview paper, Gumulec et al. on page 55, the zinc ion impact mechanisms are presented in
details [48]
. Changes in expression of zinc transporters and their pathophysiological associations
are shown here as well.
Author’s publication relevant to this chapter
Gumulec J, Masarik M, Krizkova S, et al. Insight to Physiology and Pathology of Zinc(II) Ions
and Their Actions in Breast and Prostate Carcinoma. Current Medicinal Chemistry.
2011;18(33):5041-5051.
Available on page 55
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2.1.3 Prostate cancer and microRNA action
MicroRNAs (miRNAs) are short, non-coding regulatory RNAs of about 20 nucleotides in size.
MiRNAs bind to the target mRNA with the complementary sequence, induce its degradation or
block translation of the given mRNA. The single miRNA can target up to hundreds of different
mRNAs. MiRNAs are involved in a variety of different cellular signalling pathways at many
levels. These small molecules are associated with the development of many diseases, including
tumours. Each miRNA has the ability to interact with a number of cellular signalling pathways,
and changes in the expression of a relatively low number of miRNAs may reflect deregulation
of a wide variety of cellular processes that may be decisive for the development of a tumour
disease. MiRNAs also play an important regulatory role in developing resistance to the commonly
used therapeutic procedures such as radiotherapy and chemotherapy and affect tumour
progression [49]
. MiRNAs represent an attractive target in research of new non-invasive biomarkers
as they are relatively resistant to RNase degradation and are therefore relatively stable
in plasma, serum and tissue samples [50]
. Based on bioinformatics database outputs and based
on a wide range of published studies, it is evident that miRNAs affect significantly the genes
associated with zinc regulation and CaP development. The following overview paper by Pekarik
et al. on page 67 [51]
summarizes the importance of the miRNAs in the regulation of metallothionein
and zinc transporters. These findings are presented in the clinical-pathological context.
Author’s publication relevant to this chapter
Pekarik V, Gumulec J, Masarik M, Kizek R, Adam V. Prostate Cancer, miRNAs, Metallothioneins
and Resistance to Cytostatic Drugs. Current Medicinal Chemistry. 2013;20(4):534-544.
Available on page 67
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2.2 Hypotheses verified under PART I
The following hypotheses have been formulated, based on the theoretical starting points outlined
above:
Hypothesis 1: Zinc ions and their transporters and binding proteins or peptides interfere with
prostate carcinoma pathogenesis significantly. As a result, zinc and metallothionein levels
should reflect the formation of CaP both within the prostate cell lines and within a broader
framework of clinical samples.
Hypothesis 2: Increasing zinc concentrations should alter expression of genes and miRNAs
involved in CaP carcinogenesis significantly. In the cells derived from the primary prostate tumour,
higher cell resistance to apoptosis induced by zinc ions can be expected. This higher
resistance should be reflected by changes in expression of pro- and anti-apoptotic genes. This
resistance can be diminished in the cells derived from metastases because they do not meet high
zinc concentrations in prostate anymore, and therefore adaptation to high zinc concentration in
the environment may disappear.
Hypothesis 3: Prostate carcinoma is characterized by a long-term reduction in zinc ion accumulation.
By creating prostate tumour cells capable of accumulating zinc, the tumour phenotype
should be adjusted towards the normal state.
2.2.1 Findings related to the hypothesis 1
Hypothesis 1: Zinc ions and their transporters and binding proteins or peptides interfere with prostate carcinoma
pathogenesis significantly. As a result, zinc and metallothionein levels should reflect the formation of
CaP both within the prostate cell lines and within a broader framework of clinical samples.
It has been proved that loss of ability to accumulate zinc is necessary for carcinogenesis, namely
at the early stages of CaP development [26]
. This hypothesis has been confirmed also in our
experimentally used prostate cell lines PNT1A (benign line), 22Rv1 (a primary tumour) and PC-
3 (bone metastasis derived from CaP). Whilst PNT1A contained 125.9 μg/g of intracellular zinc,
the tumour cell lines contained significantly less, 107.75 μg/g, and 75.85 μg/g respectively [52,
53]
. However, the experimental studies analyzing the amount of zinc in biological samples of
the patients suffering from CaP and in other solid tumours often diverged in their results. The
relationship between the clinical-pathological characteristics of CaP patients and the zinc levels
was also unclear. This is why meta-analysis (114 studies with a total number of 22,737 patients
and controls) has been created, Gumulec et al. on page 80 [54]
. A significant decrease of zinc ion
concentration in serum was demonstrated in almost all studied malignancies, including CaP,
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apparently due to the depletion of zinc reserves in consequence of the immune system burdening
by a tumour, and therefore this marker is not usable in the specific CaP diagnostics. More
specific behaviour was observed in the tissue zinc concentration. Decreased zinc concentration
in the tumour tissue was demonstrated in CaP, thyroid tumours, hepatocellular tumours and
non-small cell lung carcinomas. By contrast, the zinc concentration was increased consistently
in breast carcinomas. In none of the tumours, meta-regression demonstrated a relationship between
the zinc level and the tumour stage and grade.
The significance of metallothionein concentration was assessed by applying a similar methodology
[55]
, see page 91. With respect to a wide range of approaches, by which it is possible to
determine this protein in tissue samples and to the resulting heterogeneity of the data, only the
studies based on immunohistochemical detection were included in the meta-analysis. In total,
77 works were included (4631 tumour tissue samples and 3384 control tissue samples) into the
study. Types of tumours that show a significant increase in metallothionein include ovarian carcinomas
and spinocellular head and neck carcinomas. The decrease in MT was demonstrated in
hepatocellular carcinomas. In CaP, no significant difference in MT concentration was proved
compared with the control tissue. MT was associated neither with CaP staging nor CaP grading;
however, patients with higher MT concentrations exhibited generally a lower survival rate.
Conclusion: In CaP, decreased concentration of zinc was demonstrated both in the model tumour
cell line [52, 53]
and in the patients’ tumour tissue [54]
. This reduction was not accompanied
by a decrease in the concentration of zinc-binding metallothionein [55]
. On the other hand, patients
with higher MT levels experienced a worse survival rate, which fact is most probably
associated with the antioxidant and protective role of MT in tumour cells that will be dealt in
the following block of experiments (Part III). Gene and protein expression of the most frequently
represented MT form, i.e. MT2A, was increased in the prostate tumour lines compared
to the non-tumour line PNT1A [53, 56]
. Based on zinc and MT concentrations it is possible to say
that the selected prostate tumour cell lines are a relatively good model of the real CaP status.
Author’s publications relevant to this chapter
1. Gumulec J, Masarik M, Adam V, Eckschlager T, Provaznik I, Kizek R. Serum and Tissue
Zinc in Epithelial Malignancies: A Meta-Analysis. Plos One. 2014;9(6).
Available on page 80
2. Gumulec, J., M. Raudenska, et al. (2014). "Metallothionein - Immunohistochemical Cancer
Biomarker: A Meta-Analysis." Plos One 9(1).
Available on page 91
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2.2.2 Findings related to the hypothesis 2
Hypothesis 2: Increasing zinc concentrations should alter expression of genes and miRNAs involved in CaP
carcinogenesis significantly. In the cells derived from the primary prostate tumour, higher cell resistance to
apoptosis induced by zinc ions can be expected. This higher resistance should be reflected by changes in expression
of pro- and anti-apoptotic genes. This resistance can be diminished in the cells derived from metastases
because they do not meet high zinc concentrations in prostate anymore, and therefore adaptation to high zinc
concentration in the environment may disappear.
Rising zinc concentrations affected the expression of the genes involved in the CaP carcinogenesis
significantly. The gene for metallothionein MT2A and MT1A, for pro-apoptotic BAX and
zinc transporter ZnT1, belong among the genes, expression of which was simulated extensively
the by zinc ions within the prostatic lines PNT1A, 22Rv1 and PC-3. In contrast, the gene for
KRAS GTPase suppressed its expression due to the short-term elevated zinc ion concentration;
the range of tested concentrations was from 0 to 3 x IC50 (50% inhibitory constant) for the
given cell line [57]
. At lower concentrations (up to 50 μM of ZnSO4), zinc also stimulated expression
of the MKI67 proliferation marker and expression of the anti-apoptotic BCL2 [58]
. At
higher zinc concentrations, expression of BCL2 did not go up any more [57]
.
Expression of the MKI67 proliferation marker and the anti-apoptotic BCL2 gene was higher in
the prostate tumour cells 22Rv1 derived from a primary tumour compared with then non-tumour
PNT1A, which shifts the BAX/BCL2 ratio in the tumour cells towards inhibition of apoptosis.
Addition of zinc ions stimulated even the gene expression of the p53 tumour suppressor, but
only in the non-tumour prostate cells PNT1A; p53 expression in tumour 22Rv1 was not increased
and remained very low in general. It has also been observed that the 22Rv1 tumour cells
tolerate significantly higher concentrations of the zinc ions than the non-tumour PNT1A [58]
, this
increased tolerance to zinc does not appear in the PC-3 cells derived from a bone metastasis of
CaP. The PC-3 cells tolerate zinc much less than the non-tumour prostate epithelial cells PNT1A
[57]
. Influence of zinc ions on gene expression was assessed in the next study by Sztalmachova
et al. (see p. 107) and in the study Holubova et al. that will be reported below (see p. 124).
Effect of increasing concentration of zinc ions on the expression of the selected miRNAs was
monitored as well. The aim of the study was also to determine levels of expression of the selected
miRNAs in the non-tumour cell line PNT1A compared with the CaP-derived cell lines
(22Rv1, PC-3 and LNCaP). Using the bioinformatics approach, miRNAs with binding sites in
3'UTR regions of the metallothioneins 1A and 2A (miR-23a, miR-141, miR-224, miR-296-3p,
miR-320, miR-375 and miR-376) were chosen. A significantly increased expression of miR-23a
in all tumour lines compared with the non-tumour line PNT1A was observed. The 22Rv1 cell
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line derived from the primary CaP had an increased expression of miR-224 compared to other
lines. All tumour cell lines expressed significantly higher levels of miR-375 compared to nontumour
PNT1A. The results show that miR-375 and miR-23a could be important in CaP diagnosis.
From our other findings, it follows that expression of miR-375 is closely related to
resistance against docetaxel. The PC-3 cell line expressing miR-375 most has about 3 times
higher resistance to docetaxel than the PNT1A line expressing miR-375 least (IC50 for docetaxel
= 200nM for PC-3 and 70nM for PNT1A). This association and the mechanisms leading to
it were confirmed later in the study performed by Wang et al [59]
. Only the miR-224 expression
responded consistently to the addition of the zinc ions, and its expression was in negative correlation
with of zinc ion concentrations. Results of the study of the chosen miRNAs are
summarized in the attached publication Hlavna et al., available on page 116 [56]
.
Conclusion: Zinc ions can significantly alter expression of the genes involved in CaP carcinogenesis.
The primary prostate tumour derived cells were shown to have a higher cell resistance
to the zinc-induced apoptosis. This higher resistance was reflected by changes in gene expression
of pro- and anti-apoptotic BAX and BCL2 and in the expression of the MKI67 proliferation
marker. This increased tolerance to zinc did not appear in the PC-3 cells derived from CaP bone
metastasis. PC-3 cells tolerate zinc much less than the non-tumour epithelial prostate cells
PNT1A. In the next part of the work, zinc was therefore monitored as a possible inhibitor of
CaP carcinogenesis and its progression to the castration-resistant prostate cancer (CRPC) phase;
(see Hypothesis 3). Furthermore, the significance of miR-375 in CaP and its possible relationship
with resistance to docetaxel (which is used as one of few chemotherapeutics to treat
metastatic CaP) was revealed.
Author’s publications relevant to this chapter
1. Sztalmachova, M., M. Hlavna, et al. (2012). "Effect of zinc(II) ions on the expression of proand
anti-apoptotic factors in high-grade prostate carcinoma cells." Oncology Reports 28(3):
806-814.
Available on page 107
2. Hlavna, M., M. Raudenska, et al. (2012). "MicroRNAs and zinc metabolism-related gene
expression in prostate cancer cell lines treated with zinc(II) ions." International Journal of
Oncology 41(6): 2237-2244.
Available on page 116
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2.2.3 Findings related to the hypothesis 3
Hypothesis 3: Prostate carcinoma is characterized by a long-term reduction in zinc ion accumulation. By creating
prostate tumour cells capable of accumulating zinc, the tumour phenotype should be adjusted towards the
normal state.
In the next part of the work, zinc was studied as a possible inhibitor of CaP carcinogenesis, and
its progression to the castration-resistant prostate cancer (CRPC) phase. The goal was to characterize
the behaviour of the prostate tumour cells able to proliferate at the zinc concentrations,
which usually cause apoptosis (model of a long-term increase of zinc concentration in tumour
microenvironment as the consequence of long-term administration of higher zinc doses to the
patients). Some studies suggested that zinc supplementation reduces the risk of CaP [60, 61]
or
sensitizes the tumour cells to antineoplastic therapy [62]
. However, the protective properties of
zinc may be controversial [63-66]
.
Cells grown for a long time period in excess of zinc, are subjected to gradual exhaustion of
initial compensatory mechanisms and are forced to accumulate zinc (see Fig. 1 in Kratochvilova
et al., p. 146). This should theoretically reverse the tumour phenotype. Nevertheless, during a
prolonged exposure to zinc ions, the cells try to compensate the adverse conditions by involving
signalling pathways other than in the case of a short-term exposure. In particular, the activation
of the KRAS, NF-κB and HIF1α genes is important for the generation of zinc resistance. Activation
of these pathways was previously associated with the survival rate and increased
aggressiveness of tumour cells [67-69]
. Both HIF1α and NF-κB regulate together over a thousand
genes that control very important cell processes such as resistance to hypoxia, metabolic
changes, inflammatory and reparative responses, degradation of extracellular matrix, migration,
and invasive cell potential. In our study Holubová et al. (see p. 125), we also demonstrated that
due to the formation of zinc resistance, cells also become more resistant to cisplatin.
Conclusion: Long-term exposure of CaP-derived tumour cells to high zinc concentrations leads
to increased accumulation of zinc inside the cells, but on the other side also contributes to activation
of the signalling pathways leading to resistance and increased aggressiveness of cells.
Author’s publication relevant to this chapter
Holubova, M., M. Axmanova, et al. (2014). "KRAS NF-kappa B is involved in the development
of zinc resistance and reduced curability in prostate cancer." Metallomics 6(7): 1240-1253.
Available on page 125
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PART II: Metabolism of amino acids and prostate carcinoma progression 3
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3 PART II: Metabolism of amino acids and prostate carcinoma progression
3.1 Theoretical basis
Metabolites are products of biochemical reactions and largely reflect the cell phenotype. The
monitoring of changes in the concentrations of major metabolites, such as amino acids, can
provide a promising approach to the study of biomarkers reflecting CaP progression and identification
of metabolic pathways suitable for targeted CaP therapy. Amino acids are no longer
understood as the building material for cellular structures only but are believed to be of great
importance as the signals interconnecting nutrition and key cellular signalling pathways. As the
tumour cells and the cells of the immune system have similar nutritional requirements, the competition
of these cells for resources is very frequent, which fact can have an essential impact on
the progression of the tumour disease [70]
.
3.1.1 Prostate cancer metabolism
One of the earliest studies focused on prostatic tissue metabolism has identified citrate and
spermine as the key metabolites of healthy - i.e. non-tumour - prostatic epithelial tissues. As
already mentioned above, elevated zinc levels in the prostatic tissue inhibit mitochondrial aconitase,
thus resulting in inhibition of the Krebs cycle, relative energy "inefficiency" of the
prostatic epithelial tissue (gain: only 14 ATP per one glucose molecule) and accumulation of
citrate used in the seminal fluid [71]
. Low levels of citrate and spermine were associated with
progression and aggressiveness of CaP [72-74]
. Reduced levels of citrate in CaP are caused by its
use for the needs of accelerated growth of the tumour cells [75]
.
Excessive activation of the androgen receptor is able to stimulate glycolysis and anabolic processes
leading to the synthesis of lipids, steroid hormones, cholesterol and certain amino acids,
for example, sarcosine (N-methylglycine) [76-79]
. In conformity with the facts above, several
studies reported an increase in sarcosine and cholesterol in CaP [77, 80, 81]
. Higher glutaminedependent
lipogenesis was also recorded [82]
.
Dependence on aspartate import is another characteristic feature of healthy epithelial prostate
tissue. Normal prostate epithelial cells accumulate citrate; it requires continuous availability
of carbon sources for the mitochondrial production of acetyl coenzyme A (acetyl-CoA) and
oxaloacetate (OAA), from which citrate is synthesized. Acetyl-CoA is obtained from pyruvate.
Unlike the other cells, where OAA is regenerated by the end of the Krebs cycle, in case of
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prostate epithelial cells, OAA is obtained from aspartate, which is imported into the cells by the
EAAC1 transporter. Aspartate is thus the essential amino acid for prostate epithelial cells [83]
.
When CaP occurs, the intracellular concentration of zinc ions drops, m-aconitase begins to work
again, and the Krebs cycle is restored. Cellular metabolism will gain about 24 molecules of
ATP, and the cells will be transformed from the energy-inefficient healthy cells into the energyefficient
cells of CaP [47, 84]
. The Warburg effect can be seen at the metastatic CaP stages only,
which excludes the use of FDG-PET (Positron emission tomography utilizing [18F]-fluorodeoxyglucose)
as the diagnostic approach at the early stages of the disease (fluorodeoxyglucose
behaves like glucose and is caught on the places with high-active glucose metabolism) [85]
.
3.2 Hypothesis verified under PART II
The following hypotheses were formulated, based on the theoretical starting points shown in
the text above:
Hypothesis 1: Changes in cellular functions are reflected in changed metabolism and changed
intracellular amino acids concentrations. Long-term reduction in zinc ion accumulation is typical
for prostate carcinomas. By creating prostate tumour cells, capable to accumulate zinc, the
tumour metabolism should be adjusted to the normal state.
Hypothesis 2: Some of the highly synthesized amino acids may have a supportive effect on the
properties of tumour cells, such as invasive potential or ability of migration.
3.2.1 Findings related to the hypothesis 1
Hypothesis 1: Changes in cellular functions are reflected in changed metabolism and changed intracellular
amino acids concentrations. Long-term reduction in zinc ion accumulation is typical for prostate carcinomas.
By creating prostate tumour cells, capable to accumulate zinc, the tumour metabolism should be adjusted to the
normal state.
Though we have managed to increase the zinc accumulation in the prostate tumour cell lines
(22Rv1 and PC-3), the amino acid profile of these cells was still unrelated to the benign cells
represented by the PNT1A line. On the other hand, changes in the amino acid profile reflecting
CaP progression were very similar to those that occurred after the long-term supplementation
of the cells with zinc ions. Due to long-term elevated concentrations of zinc in the medium,
cells with a higher invasive potential, a higher resistance to cisplatin therapy [57]
and a higher
gene expression of the SOX2 pluripotency marker were selected positively. The tumour cell
lines had a higher SOX2 gene expression, a higher aspartate and sarcosine accumulation and
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lower levels of threonine, alanine, methionine, leucine, phenylalanine and lysine compared to
benign PNT1A cells. Excessive aspartate accumulation accompanied by depletion of several
essential amino acids that can enter the Krebs cycle (threonine, lysine, leucine, phenylalanine
and methionine) suggests activation of aspartate biosynthesis through increased MDT2 malate
dehydrogenase activity and GOT2 glutamate-oxaloacetate transaminase in CaP cells with the
restored Krebs cycle. High levels of MDH2 were associated with a poor prognosis in CaP patients
[86]
. Results of amino acid profiling in CaP are summarized in Kratochvilova et al. below
(see p. 142).
Conclusion: Changes in amino acid levels induced by carcinogenesis and/or resistance to zinc
could be relevant for diagnostic purposes and may also potentially lead to new therapeutic options.
Ratios of certain logically related amino acids may be a sensitive indicator of the
malignant phenotype. Inhibition of aspartate synthesis could also become a promising ap-
proach.
Author’s publication relevant to this chapter
Kratochvilova, M., M. Raudenska, et al. (2017). "Amino Acid Profiling of Zinc Resistant Prostate
Cancer Cell Lines: Associations With Cancer Progression." Prostate 77(6): 604-616.
Available on page 142
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3.2.2 Findings related to the hypothesis 2
Hypothesis 2: Some of the highly synthesized amino acids may have a supportive effect on the properties of
tumour cells, such as invasive potential or ability of migration.
Sarcosine (N-methylglycine) was indicated as an important CaP oncometabolite in several studies
[79, 87]
. Based on our results, sarcosine metabolism is involved significantly in the
development and behaviour of CaP [88]
. Amino acids of the sarcosine pathway (glycine, dimethylglycine
and sarcosine) affected significantly the ability of cells derived from CaP (22Rv1,
PC-3) to migrate, as well as their ability to divide. These findings are consistent with other
studies [79, 80, 89]
. We also proved that the gene expression of glycine N-methyltransferase
(GNMT) is low in benign prostatic cells PNT1A and rises explicitly in the 22Rv1 cells derived
from a primary tumour. With the progression to the metastatic form, GNMT expression is somewhat
reduced, but still remains elevated as compared to PNT1A expression. Expression of
GNMT mRNA reflects sarcosine levels in the lines studied by us. Thus, our results support the
hypothesis that sarcosine (generated from glycine using GNMT) could be used as an early CaP
biomarker. Our data further show that the cell supplementation with glycine, dimethylglycine
or sarcosine affects significantly the metabolism of amino acids in prostatic cells. We further
confirmed that amino acid profiles are highly specific for individual cell lines (can be seen from
an analysis of the main components, see Fig. 5 on page 165 [88]
).
In the study below [90]
Heger et al., see p. 170, the effect of sarcosine on CaP-derived metastatic
cells (androgen-dependent LNCaP and androgen-independent PC-3) was studied. We proved
that sarcosine stimulates proliferation of these cells in vitro and also in vivo. In mice with induced
CaP, the repeated administration of sarcosine promotes the growth of tumours and
significantly reduces weight and welfare in treated mice. In addition, we observed a sarcosineinduced
increase in glycine and serine concentrations in the tumour mass in both types of tumours
(LNCaP-induced and PC-3-induced tumours), and this increase was accompanied by the
increased levels of sarcosine dehydrogenase (SARDH). The impact of the repeated sarcosine
administration was also evident at the level of gene expression. Bioinformatic methods revealed
a strong link between sarcosine administration and induction of the genes involved in cell cycle
progression. In both types of CaP xenografts, the administration of sarcosine stimulated the
expression of the gene for the androgen receptor (AR) and the gene for the prostate-specific
antigen (KLK3). In a tumour induced by the PC-3 cells derived from bone metastases, the sarcosine
administration resulted in increased expression of the gene for the Aurora kinase A
(AURKA). Amplification of the AURKA gene was observed in 67% of CaP, which progressed
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to a highly aggressive form [91]
. AURKA is one of the genes that can contribute to the generation
of giant polyploid tumour cells (PGCC) [92]
playing a role in developing resistance to therapy;
see Part III of this work.
As our previous studies proved that sarcosine is an important oncometabolite, we prepared two
types of liposomes which – when targeted towards the tumour tissue – affected the function and
the metabolism of sarcosine, see Heger et al. on page 190. To increase the absorption of liposomes
into the CaP target tissue, the liposome surface was modified by the folic acid (FA);
(tumour cells often express large amounts of the folate receptor [93-95]
). The first type of antisarAbs@LIP
liposome contained anti-sarcosine antibodies, the second type of sar@LIP liposome
contained sarcosine. The affinity of liposomes to the folate receptor also facilitated the entry of
liposomes into the cells through clathrin-mediated endocytosis [96]
. We examined effects of repeated
administration of these liposomes on the growth of tumours induced by human PC-3
cells that had been implanted to immunodeficient nu/nu mice. Administration of Sar@LIP liposome
significantly increased the tumour volume and weight as compared with the controls
treated with empty liposomes. On the contrary, the administration of antisarAbs@LIP showed
a mild antitumor effect. Gene expression was altered significantly as a result of treatment, expression
patterns differed significantly due to the administration of different liposomes. It is
interesting that both types of therapy (Sar@LIP and antisarAbs@LIP) resulted in higher expression
of KLK3, the gene for the prostate-specific antigen (PSA), which is understood the
clinically utilized CaP biomarker. We assume that this phenomenon was due to using FA for the
modification of liposome surfaces. The FA supplementation in the CaP patients was associated
with increased PSA levels, whereas its elimination from the diet resulted in a significant PSA
reduction [97]
. Glutaminase (GLS) expression was also increased due to the administration of
Sar@LIP, but not antisarAbs@LIP. Glutaminase is the key enzyme involved in glutaminolysis
and its task is to hydrolyse glutamine, the amino acid necessary for tumour growth, into glutamic
acid [98, 99]
.
Significant reduction in the concentration of zinc ions and total metallothionein in the tumour
tissue is another important effect of the sarcosine-containing liposome (Sar@LIP). It is therefore
probable that the accumulation of sarcosine is closely related to the depletion of zinc ions,
which plays a significant role in the progression of CaP. We also proved that FA-modified liposomes
are suitable for targeting the CaP tissues. These findings are further extended in the
following study.
Conclusion: Amino acids accumulated in tumour cells or tissues are able to affect substantially
the events associated with CaP carcinogenesis. Amino acids of the sarcosine pathway (glycine,
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dimethylglycine and sarcosine) affect the ability of cells derived from CaP (22Rv1, PC-3) to
migrate, as well as their ability to divide. The tumour-supporting effect of sarcosine could be
observed on the rate of tumour growth in mice. Sarcosine is thus probably a key metabolite
affecting the progression of CaP and is a suitable target for diagnostic approaches as well as for
possible targeted therapy. Though our antisarAbs@LIP liposomes showed only moderate antitumour
effects, they should be further tested when co-administered with other antineoplastic
drugs.
Author’s publications relevant to this chapter
1. Heger, Z., J. Gumulec, et al. (2016). "Relation of exposure to amino acids involved in sarcosine
metabolic pathway on behavior of non-tumor and malignant prostatic cell lines."
Prostate 76(7): 679-690.
Available on page 158
2. Heger, Z., M. A. M. Rodrigo, et al. (2016). "Sarcosine Up-Regulates Expression of Genes
Involved in Cell Cycle Progression of Metastatic Models of Prostate Cancer." Plos One
11(11).
Available on page 170
3. Heger, Z., H. Polanska, et al. (2016). "Prostate tumor attenuation in the nu/nu murine model
due to anti-sarcosine antibodies in folate-targeted liposomes." Sci Rep 6: 33379.
Available on page 190
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4 PART III: Redox status and oxidative stress of prostatic cells
4.1 Theoretical basis
Reactive forms of oxygen (ROS – reactive oxygen species) are produced by living organisms
due to normal cellular metabolism. At low to moderate concentrations, they work in physiological
cell processes, but at high concentrations, they cause undesirable modifications of
significant bio-macromolecules such as lipids, proteins and DNA [100]
. ROS are often called
oxidants. Aerobic organisms have integrated antioxidant systems incorporating enzymatic and
non-enzymatic antioxidants that are usually efficient in blocking the harmful ROS effects. A
significant deflection of the balance between oxidizing and anti-oxidizing agents in favour of
oxidants is called the oxidative stress status. Oxidative stress contributes to many pathological
conditions including tumour diseases, atherosclerosis, hypertension, and diabetes [101]
.
Although the term ROS often refers to individual entities only, it is a very complex system and
includes a number of oxidants. The major ROS of physiological importance are as follows:
superoxide anion (O2),
hydroxyl radical (● OH) and hydrogen peroxide (H2O2). The intracellular
ROS producers include mitochondria (I and III respiratory chain complexes being the main
producer) and the endoplasmic reticulum. Within the whole organism, ROS produce leukocytes,
macrophages and erythrocytes. Exogenous effects, contributing to the onset of the oxidative
stress state, include smoking, heavy metals, ionizing radiation and hyperoxia [101]
. Induction of
oxidative stress is also associated with chemotherapy and radiotherapy. Oxidative stress can
significantly affect tumour cell signalling pathways and slow progression through the cell cycle,
which may affect the efficacy of the applied therapy [102, 103]
.
Prostate carcinoma cells, unlike healthy cells, are characterized by the so-called innate oxidative
stress, a characteristic feature for the aggressive phenotype of this disease. In prostate
carcinoma, the increased ROS production is caused by various processes including internal factors
such as metabolic alterations, AR activation, and mutations causing mitochondrial
dysfunctions. External factors include inflammation, metabolism of xenobiotics and hypoxia
[104]
. In addition to the activation of the androgen receptor, the (NF-E2)-related factor 2 (Nrf2)
also plays a role in the redox status of CaP, i.e. it mediates expression of key protective enzymes
through the antioxidant response element (ARE) [105, 106]
. Some studies suggest that expression
of Nrf2 and some of its target genes is reduced significantly in CaP [107]
. The glutathione-containing
enzymes are another important component involved in maintaining redox balance in the
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cell. The somatic mutation causing inactivation of the glutathione-S-transferase gene (GSTP1)
was identified in almost all CaP-derived tissues examined by Nelson et al. [108]
. The sensitive
balance between oxidative and antioxidative cell components and their regulatory mechanisms
plays probably a crucial role in the development of malignant status in the prostate tissue. Resistance
to oxidative stress also seems to be one of the main mechanisms of chemo- and radioresistance.
In addition to the direct impact on mutagenesis and genomic instability, ROS also
contribute to development and progression of the tumour disease by acting as signalling molecules
for mitogenic processes and as inductors of the genetic programs leading to invasion and
malignity [109]
. The signalling effect of ROS does not have to be limited to the tumour cells
themselves, but can also have a paracrine effect on the cells of the tumour microenvironment.
Some studies suggest that the activation of tumour associated fibroblasts (CAFs), influencing
cancerogenesis and resistance of tumour cell, is affected by ROS [110]
. ROS promote conversion
of fibroblasts into highly migrating myofibroblasts by activating the hypoxia-induced factor
(HIF-1α) and chemokine CXCL12 [111]
.
4.2 Hypothesis verified under PART III
The following hypotheses were formulated, based on the theoretical starting points shown in
the text above.
Hypothesis 1: Differences in the zinc levels of prostate non-tumour and tumour cells correspond
with differences in the systems of major antioxidant mechanisms; oxidative stress also calls up
defence mechanisms leading to further disease progression (polyploidization, mitophagy, development
of drug resistance).
Hypothesis 2: The previously studied molecule, sarcosine, is associated with changes in antioxidant
mechanisms.
4.2.1 Findings related to the hypothesis 1
Hypothesis 1: Differences in the zinc levels of prostate non-tumour and tumour cells correspond with differences
in the systems of major antioxidant mechanisms; oxidative stress also calls up defence mechanisms leading to
further disease progression (polyploidization, mitophagy, devel-opment of drug resistance).
Disability to catch and accumulate zinc ions is one of the features of prostate tumour cells. This
was demonstrated in our earlier studies (see sec. 2.2.2.) and corresponds to literature sources
[26]
. A study by Masarik et al. [53]
(p. 206 of this work) confirms these findings and demonstrates
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further that PC-3 cells respond most sensitively to intracellular zinc increase - their 50% zinc
inhibition concentration is one-third compared to the non-tumour PNT1A.
A number of studies in the past confirmed that the increased intracellular concentration of zinc
ions leads to a higher oxidative stress [112]
while metallothionein, the major intracellular zincbinding
protein, at the same time works as a significant antioxidant [113]
. The work by Masarik
et al. was further focused on the following mechanisms: A correlation between the rising intracellular
zinc and the metallothionein-binding zinc was demonstrated in both cell lines. A
significant difference was found in this inducibility: PC-3 cells showed up to a tenfold increase
in metallothionein concentration after exposure to 10 μM zinc concentration, whilst in PNT1A
this inducibility was only half as high. This finding is relatively inconsistent with the results of
50% inhibitory concentrations (IC50) - the metastatic line may induce higher amounts of metallothionein
than the non-tumour line, but at the same time this quantity of metallothionein is not
sufficient to buffer the increased zinc charge and the metastatic line is inhibited at lower zinc
concentrations. Therefore, other antioxidant systems of these cells were investigated. The difference
between PNT1A and PC-3 was also demonstrated in the reduced/oxidized glutathione
(GSH/GSSG) system: While the reduced glutathione form prevails in the non-tumour cells (a
higher GSH/GSSG ratio), the situation is opposite in the PC-3 cells where the oxidized form of
GSSG prevails significantly.
The next step was to describe whether or not this fact is related to the development of resistance
against cytotoxic agents; the mechanism of the action of most cytostatics is mediated just
through the oxidative damage of cell structures. Cisplatin was chosen as a model cytostatic
drug. It is bound to DNA and to other important molecules thus resulting in cell death [114]
. The
panel of PNT1A-22Rv1-PC-3 cell lines has been chosen intentionally for testing resistance
against this cytostatic drug: PC-3 cells represent a model of aggressive carcinoma inter alia due
to dysfunctional p53. Because this tumour suppressor plays a central role in regulating the cell
cycle arrest, DNA repair and the start of apoptosis, it is expected that PC-3 will exhibit a resistant
phenotype.
The experiment revealed that cells with non-functioning p53 are characterized by the following
features supporting the tumour growth: the increased capacity of antioxidant mechanisms, low
expression of the pro-apoptotic protein BAX and absence of cell cycle arrest. The rate of cell
sensitivity to cisplatin was analyzed by two techniques – the MTT metabolic assay and the realtime
cell growth analysis based on impedance measurements were used. IC50 values determined
by MTT were significantly lower than the values established by the real-time growth
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assay; this was due to the fact that cells, when stressed, are switched to the autophagy status
where they can be falsely identified as "dead" by some tests (see page 219).
Oxidative stress effects on metastatic cells were further studied in Balvan et al. [115]
, see p. 230,
where the impact of oxidative stress on cell death, autophagy, polyploidization and other mechanisms
associated with the development of resistance against cytostatic drugs was investigated.
It was found that the long-term exposure of cells to oxidative stress acts as a selection pressure
for the development of various resistance processes. Due to the activation of autophagy cascades,
the metabolism of cells slows down and hence we can see a proportional difference
between the determination of cell viability by MTT test and the impedance-based methods (see
previous experimental works on the effect of cisplatin, p. 219). Mitochondria, the major ROS
producers in cells, were enveloped by an autophagic membrane and degraded by mitophagy.
Furthermore, a substantial part of cell population changed the cell size which was caused by
polyploidization. In agreement with Erenpreisa et al., this strategy is understood as an escape
from the destruction of cells by senescence and apoptosis and also as the possible way how to
do reprogramming into a "cancer stem cell" phenotype by conferring significant genetic plasticity
[116]
.
Consequently, the elevated expression of typical pluripotency markers - NANOG, SOX2,
POU5F1 was observed. Our experiments also clearly demonstrated that the cells that have undergone
cell fusion leading to polyploidization through the entotic process and cells utilizing
opportunistic ways to obtain nutrients (such as cannibalism) have a clear advantage - they can
survive significantly much longer under oxidative stress conditions than the surrounding cells.
Conclusion: In our experimental work, we have devoted ourselves mainly to the effect of
external factors on the change of the cellular redox status and thus on the potential development
of oxidative stress state in the prostate tumour cells. The importance of zinc ions associated with
the development and progression of prostate carcinoma was clarified sufficiently in PART I (see
PART I: Zinc, metallothionein and progression of prostate carcinoma). However, the zinc ions
play a role of exogenous stimulators of ROS production and increase the level of oxidative
stress in cells. We demonstrated that the sensitivity of individual prostate cell lines to the increasing
concentrations of zinc ions differs considerably. After the application of zinc ions,
different behaviour of the tumour and non-tumour cells and their antioxidant systems was
demonstrated. Based on the integrative approaches we were able monitoring of the redox status
in the individual cell lines. These methods were then used for the evaluation of cisplatin effects.
Cisplatin belongs to commonly used cytostatic drugs. We focused on the analysis of oxidative
stress, cell cycle, apoptosis and selected cytotoxic analyses. Our attention was directed to the
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PC3 line, representing a model of the aggressive prostate carcinoma. After the application of
cisplatin, we could not see in this line a typical cell cycle arrest in the G1/G0 phase, and at the
same time, we observed a decreased tendency for apoptosis. Moreover, we demonstrated that
this cell line exhibits a higher antioxidant activity and higher metallothionein content after the
administration of cisplatin and thus can be used as a model of cisplatin resistance.
Author’s publications relevant to this chapter
1. Masarik M, Gumulec J, Hlavna M, et al. Monitoring of the prostate tumour cells redox state
and real-time proliferation by novel biophysical techniques and fluorescent staining. Integrative
Biology. 2012;4(6):672-684.
Available on page 206
2. Gumulec J, Balvan J, Sztalmachova M, et al. Cisplatin-resistant prostate cancer model: Differences
in antioxidant system, apoptosis and cell cycle. International Journal of Oncology.
2014;44(3):923-933.
Available on page 219
3. Balvan J, Gumulec J, Raudenska M, et al. Oxidative Stress Resistance in Metastatic Prostate
Cancer: Renewal by Self-Eating. Plos One. 2015;10(12).
Available on page 230
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4.2.2 Findings related to the hypothesis 2
Hypothesis 2: The previously studied molecule, sarcosine, is associated with changes in antioxidant mecha-
nisms.
In our earlier studies, we found that amino acids of the sarcosine pathway (glycine, dimethylglycine
and sarcosine) significantly affect the ability of cells derived from CaP (22Rv1, PC-3)
to migrate, as well as their ability to divide. Sarcosine is thus probably a key metabolite affecting
the progression of CaP and may be a suitable target for diagnostic approaches and for possible
targeted therapy.
Despite the fact that the sarcosine association with oncogenesis was investigated extensively
by a number of other laboratories [79, 87]
, the mechanism by which sarcosine participates in oncogenesis
is not absolutely clear yet. The goal of the following study by Cernei et al. [117]
was to
find out how the sarcosine treatment relates to changes in the cell antioxidant mechanisms.
After the exposure of the PC-3 prostate cells to the increasing concentration of sarcosine, it was
found out that this metabolite is not related to the zinc-binding antioxidant metallothionein, the
levels of which remained unaffected, though the intracellular concentration of sarcosine has
increased dramatically. Taking into account this step, a link between other antioxidant players
and sarcosine was searched. From a wide array of antioxidant parameters, none showed a trend
changing in dependence on the quantity of delivered sarcosine. At the same time, sarcosine
stimulates proliferation of these cells in vitro as well as in vivo as demonstrated in our previous
study [88]
(see sec. 3.2.2.). The mechanism affecting the metastatic potential of tumour cells via
sarcosine is therefore independent on the metabolic pathways associated with the regulation of
reactive oxygen forms.
Conclusion: Sarcosine, a non-coding amino acid, was studied intensively in association with
its expected predictive value for the prostate carcinoma diagnosis, affects cancer cells aggression.
Based on our experiments, it was confirmed that these mechanisms are related neither to
antioxidants nor to the regulation of oxidative stress levels.
Author’s publication relevant to this chapter
Cernei N, Zitka O, Skalickova S, et al. Effect of sarcosine on antioxidant parameters and
metallothionein content in the PC-3 prostate cancer cell line. Oncology Reports. 2013; 29(6):
2459-2466.
Available on page 254
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5 PART IV: Novel approaches to the analysis of prostate cancer markers
5.1 Theoretical basis
CaP has a variable biological potential with different options of therapy. For the successful
treatment, it is necessary to identify and distinguish aggressive forms of this pathology from
the clinically latent forms with the low metastatic potential and slow growth rate. Therefore, a
personalized approach is needed to define men with a higher risk of CaP progression, to distinguish
indolent and aggressive disease and to improve the stratification of post-treatment risks.
Such approach can facilitate clinical decisions, improve selection for active monitoring protocols
and minimize side effects of the therapy.
Research of new biomarkers associated with CaP carcinogenesis is considered an opportunity
to provide patients with new possibilities, to understand the risk of CaP development better and
to predict the clinical course of the disease. None of the currently used diagnostic methods is
able to distinguish the aggressive and the latent tumour forms. All biopsy-verified carcinomas
are therefore treated as the life-threatening forms. Therefore, a significant proportion of patients
has no benefit from treatment [118]
. Such an approach leads to a significant decrease in life quality
because radical prostatectomy results in erectile dysfunction in up to 70% of patients and incontinence
in 10% of them [119]
. It is therefore desirable to find such CaP markers that would be
able to distinguish between aggressive and latent tumour forms at initial stages [120, 121]
.
Measurement of serum concentration of the prostate-specific antigen (PSA) is the most common
current screening assay for CaP, but it is neither sensitive nor specific sufficiently [122]
. The
total PSA (tPSA) circulating in serum has two components. The first component is PSA bound
to α1-antichymotrypsin (ACT) and the second component is free PSA (fPSA) [123-125]
. To improve
the specificity of the total PSA (tPSA), the fPSA/tPSA ratio, which in some cases is able
to distinguish benign prostatic hyperplasia (BPH) from CaP, was tested [126]
. However, the percentage
of fPSA was shown to increase with the age and prostate size [127]
. fPSA was able to
distinguish between CaP and BPH only in patients with a small prostate size (less than 40 cm3
)
[128, 129]
. To take into account the prostate size, Benson et al. have introduced the term PSA density
(PSAD). It is assumed that CaP releases more PSA per unit volume of the prostate into
circulation than BPH [130]
. To distinguish between the two diseases, a limit PSAD value from
0.1 to 0.2 ng/mL/cm3
is currently used. However, the threshold should vary according to the
amount of total tPSA (0.05 ng/mL/ cm3
at tPSA 2-4 ng/mL; 0.1 at tPSA 4-10 ng/mL and 0.19 at
PART IV: Novel approaches to the analysis of prostate cancer markers 5
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10-20 ng/mL). Particularly in the patients with a low tPSA concentration, additional use of
PSAD may improve the selection of the patients who may benefit from the prostate biopsy [131]
.
As the absolute PSA values are unable to predict the presence of the prostate carcinoma and its
aggressiveness accurately, a great attention is paid to PSA kinetics (change in PSA levels). PSA
kinetics describes PSA changes over time. To describe changes in PSAlevels, such features like
PSA velocity (PSAV) and PSA doubling time (PSADT, i.e. the time necessary for PSA doubling)
are applied. Increase by 0.75 ng/ml/year is considered the threshold PSAV value. The
value of 0.75 ng/ml/year, based on at least three samplings in two-year intervals as a minimum,
seems to be useful for determining the increased risk of CaP [132]
. Guidelines for CaP diagnostics
recommend that males with the PSAV value higher than 0.35 ng/ml should consider biopsy
every year, though their total PSA is low [133]
. Nevertheless, many studies report that PSAV and
PSADT did not improve the specificity of the PSA itself [134-136]
.
Because of the non-specificity of the PSA-based diagnostics, other markers and approaches are
examined, such as non-coding RNA PCA3 expression, prostate health index (PHI), kallikrein
panel (4k-panel), TMPRSS2: ERG fusion detection, loss of tumour suppressor PTEN and detection
of circulating tumour cells [137, 138]
. Possible CaP management with the use of biomarkers
is proposed in Fig. 5.
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Figure 5 | Algorithms for CaP diagnostics management. MRI: Magnetic resonance imaging; PCA3: Prostate
cancer antigen 3; PSA: Prostate specific antigen; PSAD: PSA density; PSADT: PSA doubling time; PSAV: PSA
velocity; TRUS: Transrectal ultrasound.
The markers studied by us included metallothionein, caveolin-1 and alpha-methylacyl CoAracemase
(AMACR). Significance and functions of metallothioneins were discussed in the previous
text, and therefore we will focus on the theory concerning caveolin-1 and AMACR.
Alpha-methylacyl CoA-racemase is a peroxisomal and mitochondrial enzyme involved in βoxidation
of branched fatty acids, catabolism of bile acid metabolites and metabolism of certain
drugs. Increase in AMACR protein levels was reported in most adenocarcinomas and in high
grade prostatic intraepithelial neoplasia [139, 140]
. Low levels of this marker are described in benign
hyperplasia and in atypical adenomatous hyperplasia [141]
. Overexpression of AMACR
may play a role in stimulating CaP cell growth by the pathway independent of androgen signalling
[142]
.
Caveolin-1 (Cav-1) is a membrane protein and an important structural component of caveolae.
The group of these proteins plays an important role in cholesterol degradation, but also takes
an important part in transmembrane signalling. The effect of Cav-1 on the progression of tumour
diseases and the increase of Cav-1 serum levels in prostate carcinoma was described
recently [143, 144]
. Cav-1 is reported to be overexpressed in CaP cells and is associated with the
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disease progression. Specific oncogenic activities of Cav-1 were associated with the Akt activation.
However, the Cav-1 protein can also be secreted by the CaP cells. Results of recent
studies showed that the secreted Cav-1 can stimulate cell survival and angiogenic activity in
the CaP microenvironment. Pre-operative Cav-1 concentrations in serum had prognostic potential
in the males who underwent radical prostatectomy [145]
.
5.2 Hypothesis verified under PART IV
The following hypotheses were formulated based on the above theoretical starting points:
Hypothesis 1: Metallothionein, caveolin-1, AMACR and sarcosine are suitable biomarkers of
CaP progression and their expression is correlated with the clinical and pathological characteristics
of the CaP patients.
Hypothesis 2: Metallothionein is also detectable in other biological materials - blood, tumour
tissue; the procedure of metallothionein analysis is automated thanks to using the isolation of
this protein by means of paramagnetic particles.
Hypothesis 3: Sensitivity of sarcosine detection can be increased using advanced techniques
such as FRET.
5.2.1 Findings related to the hypothesis 1
Hypothesis 1: Metallothionein, caveolin-1, AMACR and sarcosine are suitable biomarkers of CaP progression
and their expression is correlated with the clinical and pathological characteristics of the CaP patients.
Metallothionein, caveolin-1 and AMACR were selected for detection in the blood serum of the
patients and volunteers included in this study, because these proteins (as reported) leave the
prostatic tissue during the cancerogenesis, thus penetrating into the serum. A statistically significant
increase in the level of metallothionein (p <0.001) was found, with caveolin-1 and
AMACR levels not changing significantly between the control and the tumour groups (Fig. 6).
No statistically significant correlations between the age and all three monitored markers were
found. Protein levels of Cav-1 and AMACR were distinctly higher in the Gleason score 9 in the
serum of CaP patients [146, 147]
. Metallothionein levels were grading independent. No statistically
significant differences between the localized (T1,2) and spreading (T3,4) tumours were found
in all three examined markers (data not shown). However, the serum level of Cav-1 was higher
in patients with T4 tumours (Fig. 6C). Level of none of the examined markers changed in patients
with hypertension, ischemic heart disease and hyperlipoproteinemia, ischemic disease of
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lower limbs and gastroduodenal ulcer disease. Similarly, level of the examined markers did not
differ in the group of smokers and non-smokers.
Figure 6 | Level of tumour markers in patients’ serum. (A) metallothionein level, (B) level of alpha methylacylCoA-
racemase (AMACR), (C) level of Caveolin-1 depending on disease staging.
Serum metallothionein level is increased significantly in the patients with the prostate carcinoma.
The fact puts it in the position of a possible tumour marker of this disease, affected
minimally by the clinical status of the patient, as its level is affected neither by smoking, age,
grading of the disease, nor by the presence of comorbidities common in the population.
Though the level of caveolin-1 did not differ between the tumour and non-tumour groups, its
higher serum level was found in the patients with affected surrounding tissues and organs (TNM
stage T4) compared with the lower stages. Similarly, as in metallothionein, the level of caveolin-1
was affected neither by the presence of comorbidities nor by smoking and was not agedependent.
The alpha-methyl CoA-racemase level did not differ between the patients and the
controls either.
None of the substances studied therein meets criteria for being a marker of the aggressive disease
form. This statement dwells on the assumption that a majority of prostate tumours is latent
and slowly growing (i.e. stages T1 and T2), and only a minor part of them behave aggressively
(reaching stages T3 and T4). There was no statistically significant difference in the level of
monitored proteins between these two groups. However, the level of caveolin-1 was significantly
higher in the serum of patients with tumours of stage 4. Therefore, the level of this marker
should be tested on a larger set of patients with the aggressive tumour form. Although not meeting
the criteria for being a marker of the aggressive form of the disease, metallothionein as the
only one showed a significantly increased level in the serum of patients suffering from prostate
PART IV: Novel approaches to the analysis of prostate cancer markers 5
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tumours in this study. Thus, it can be used as a supplement to PSA screening. However, its
application has to be verified on a significantly larger set of data.
Furthermore, sarcosine as a potential tumour marker was determined. After the optimization
and validation of the ion exchange chromatography, the use of this method for the determination
of sarcosine in biological samples was tested. Results of sarcosine determination in the urine of
the patients with diagnosed CaP (n = 11), patients with BPH (n = 3) and healthy volunteers (n
= 23) are shown in Heger et al. on page 315. It can be seen from the diagram that while the
average sarcosine content in the urine of volunteers was 0.1 ± 0.3 μmol/l, the average sarcosine
content in the CaP patients was 470 ± 70 μmol/l. These results suggest that sarcosine is potentially
a suitable marker for the prostate carcinoma diagnostics. Our results show that the average
content of sarcosine in the urine specimens of prostate carcinoma patients is significantly higher
than in the control samples.
Conclusion: None of the substances studied therein meets criteria for being a marker of the
aggressive disease form. There was no statistically significant difference in the level of monitored
proteins between the localized (T1, 2) and spreading (T3, 4) tumours. However, the level
of caveolin-1 was significantly higher in the serum of patients with tumours of stage 4 and
AMACR and Cav-1 levels were higher in patients with Gleason grade 9. Metallothionein as the
only one showed a significantly increased level in the serum of CaP patients compared with
control samples. The determination of sarcosine in connection with the determination of other
CaP biomarkers has a potential to improve the prostate carcinoma diagnostics and to eliminate
false positive and false negative cases.
PART IV: Novel approaches to the analysis of prostate cancer markers 5
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Author’s publications relevant to this chapter
1. Gumulec J, Sochor J, Hlavna M, et al. Caveolin-1 as a potential high-risk prostate cancer
biomarker. Oncology Reports. 2012;27(3):831-841.
Available on page 269
2. Gumulec J, Masarik M, Krizkova S, et al. Evaluation of alpha-methylacyl-CoA racemase,
metallothionein and prostate specific antigen as prostate cancer prognostic
markers. Neoplasma. 2012;59(2):191-200.
Available on page 280
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5.2.2 Findings related to the hypothesis 2
Hypothesis 2: Metallothionein is also detectable in other biological materials - blood, tumour tissue; the procedure
of metallothionein analysis is automated thanks to using the isolation of this protein by means of
paramagnetic particles.
The micro detection systems based on electrochemical determination undoubtedly belong
among the new biophysical approaches that can contribute significantly to the study of information
macromolecules. Electrochemical detection is based on studying oxidation/ reduction
and adsorption of biologically important polymers such as nucleic acids (NA) and proteins on
the surface of the working electrode. Moreover, electrochemistry can be applied to determine
structural changes, denaturation processes, physical or chemical damage of NA. In the case of
proteins, a considerable attention is paid to the exchange of electrons in their redox centres and
to monitor protein adsorption to solid surfaces. Besides the analyses of physical and chemical
properties of NA and proteins, the electrochemical methods are used with a great success to
determine low concentrations of both NA, peptides and proteins. A great attention is also paid
to the electrochemical detection of protein - NA interaction and their chemical modifications,
such as binding of intercalating agents and interaction of drugs with their structure [148-156]
. A
possibility of applying the electrochemical methods in the analyses of such adducts directly in
the tumour cells or using these techniques in the construction of simple biosensors also follows
from the facts above.
Earlier studies demonstrated the diagnostic potential of metallothionein in the prediction of
prostate carcinoma. Thanks to its chemical properties (high cysteine content, low molecular
weight), this molecule is hardly detectable by the western blotting, commonly used to detect
proteins. The electrochemical detection allows a high degree of sensitivity, at the same time
requiring a rather complex sample preparation. The aim of the following works was to create
an automated method for the detection of metallothionein at RNA and protein levels.
Detection of mRNA serum levels has been recently an attractive issue, particularly from the
perspective of carcinoma biomarkers. In the work of Fojtu et al [157]
(available on page 292),
paramagnetic particles modified with streptavidin were used to optimize serum isolation of
mRNA. Moreover, one-step modality of separation was proposed to further accelerate the protocol.
In the final phase, this protocol was verified on serum samples of the patients suffering
from prostate carcinomas.
PART IV: Novel approaches to the analysis of prostate cancer markers 5
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Paramagnetic particles also contributed to optimizing detection at a protein level from the cell
line lysate samples. In the work by Masarik et al. [158]
(see p. 301), the immuno-separation protocol
was optimized using paramagnetic particles with conjugated antibody via the G protein.
Subsequently, metallothionein was determined by electrochemistry - Brdička reaction and also
using the SDS-PAGE electrophoresis, and the protocol was compared with a conventional pro-
cedure.
Conclusion: The procedures based on paramagnetic nanoparticles allow to isolate both RNA
and proteins from complex samples, thereby increasing detection specificity and at the same
time reducing amounts of consumed chemicals. This procedure was optimized for metallothionein
in serum and cell lysate samples.
Author’s publications relevant to this chapter
1. Fojtu M, Gumulec J, Balvan J, et al. Utilization of paramagnetic microparticles for automated
isolation of free circulating mRNA as a new tool in prostate cancer diagnostics.
Electrophoresis. 2014;35(2-3):306-315.
Available on page 292
2. Masarik M, Gumulec J, Sztalmachova M, et al. Isolation of metallothionein from cells
derived from aggressive form of high-grade prostate carcinoma using paramagnetic antibody-modified
microbeads off-line coupled with electrochemical and electrophoretic
analysis. Electrophoresis. 2011;32(24):3576-3588.
Available on page 301
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5.2.3 Findings related to the hypothesis 3
Hypothesis 3: Sensitivity of sarcosine detection can be increased using advanced techniques such as FRET.
Chromatographic approaches with mass spectrometry detection are the most commonly used
methods for the detection of sarcosine in biological materials [79, 159]
. However, precise determination
of sarcosine in complex biological samples is still a matter of concern - mainly due to
the presence of other amino acids [160]
. Therefore, the detection method using the Förster resonance
energy transfer (FRET) principle between the quantum dots and the green fluorescence
protein, facilitated by a sandwich arrangement with sarcosine antibodies, was optimized.
We successfully synthesized nanomaghemite core- based paramagnetic nanoparticles, containing
the binding sites for sarcosine antibodies and thiol moieties of oligonucleotide linker,
bearing GFP functionalized with AuNPs. Abs@PMPs conjugate was able to bind sarcosine in
biological samples as prostatic cell lines and urinary samples of onco-patients. Using HWR
heptapeptide, Abs@QDs acceptor molecule was constructed and using sandwich binding of
antibodies FRET was observed, and this was dependent on sarcosine concentration. We were
able to discriminate sarcosine in both prostatic cells and urinary samples with very good sensitivity
and without undesired interference.
Conclusion: The FRET-based sarcosine detection method is a highly sensitive and specific detection
method with the detection limit of 50 μM.
Author’s publication relevant to this chapter
Heger Z, Cernei N, Krizkova S, et al. Paramagnetic Nanoparticles as a Platform for FRETBased
Sarcosine Picomolar Detection. Scientific Reports. 2015;5.
Available on page 315
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Summary 6
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6 Summary
Cancer is the leading cause of mortality in Western countries and the second leading cause of
death in developed countries. The burden of cancer is rising in economically developed countries:
their population is growing and ageing and it adopts cancer-associated lifestyle choices
including smoking, physical inactivity, and “westernized” diets. Prostate cancer is the second
most frequently diagnosed cancer and the sixth leading cause of cancer death in males. The
prevalence of prostate cancer increases with age. It does, however, usually respond to treatment
and, if localized, may be curable. The tempo of tumour growth varies from very slow to moderately
rapid, and some patients may have prolonged survival, even after cancer has
metastasized to distant sites. It is therefore obvious that research in cancer field and therefore
CaP should be given due attention.
This habilitation thesis summarizes new findings in the field of biochemistry, pathogenesis and
diagnostics of prostate cancer. It has been divided into four parts depending on their specific
focus.
We can summarize our findings by individual PARTS:
PART I - Zinc, metallothionein and prostate cancer progression
Zinc ions and metallothionein are important players in pathogenesis of CaP. We found out that
decreased concentration of zinc was demonstrated both in the model tumour cell line and in the
patients’ tumour tissue. This reduction was not accompanied by a decrease in the concentration
of zinc-binding metallothionein. Gene and protein expression of the most frequently represented
MT form, i.e. MT2A, was increased in the prostate tumour lines compared to the nontumour
line PNT1A. Based on zinc and MT concentrations it is possible to say that the selected
prostate tumour cell lines are a relatively good model of the real CaP status. Furthemore, zinc
ions can significantly alter expression of the genes involved in CaP carcinogenesis. The primary
prostate tumour derived cells were shown to have a higher cell resistance to the zinc-induced
apoptosis. This higher resistance was reflected by changes in gene expression of pro- and antiapoptotic
BAX and BCL2 and in the expression of the MKI67 proliferation marker. This increased
tolerance to zinc did not appear in the PC-3 cells derived from CaP bone metastasis.
PC-3 cells tolerate zinc much less than the non-tumour epithelial prostate cells PNT1A. In the
Summary 6
323
next part of this work, zinc was therefore monitored as a possible inhibitor of CaP carcinogenesis
and its progression to the castration-resistant prostate cancer (CRPC) phase. Furthermore,
the significance of miR-375 in CaP and its possible relationship with resistance to docetaxel
(which is used as one of few chemotherapeutics to treat metastatic CaP) was revealed. Finally,
we found out that long-term exposure of CaP-derived tumour cells to high zinc concentrations
leads to increased accumulation of zinc inside the cells, but on the other side also contributes
to activation of the signalling pathways leading to resistance and increased aggressiveness of
cells.
PART II - Metabolism of amino acids and prostate carcinoma progression
Metabolism of prostate gland cells is unique and different from metabolism of other cells in
human body. Changes in amino acid levels induced by carcinogenesis and/or resistance to zinc
could be relevant for diagnostic purposes and may also potentially lead to new therapeutic options.
Ratios of certain logically related amino acids may be a sensitive indicator of the
malignant phenotype. Inhibition of aspartate synthesis could also become a promising approach.
We have also dealt with amino acids accumulated in tumour cells or tissues that are
able to affect substantially the events associated with CaP carcinogenesis. Amino acids of the
sarcosine pathway (glycine, dimethylglycine and sarcosine) affect the ability of cells derived
from CaP (22Rv1, PC-3) to migrate, as well as their ability to divide. The tumour-supporting
effect of sarcosine could be observed on the rate of tumour growth in mice. Sarcosine is thus
probably a key metabolite affecting the progression of CaP and is a suitable target for diagnostic
approaches as well as for possible targeted therapy.
PART III - Redox status and oxidative stress of prostatic cells
In this part of habilitation thesis, we have devoted ourselves mainly to the effect of external
factors on the change of the cellular redox status and thus on the potential development of oxidative
stress state in the prostate tumour cells. The importance of zinc ions associated with the
development and progression of prostate carcinoma was clarified sufficiently in PART I. However,
the zinc ions play a role of exogenous stimulators of ROS production and increase the
level of oxidative stress in cells. We demonstrated that the sensitivity of individual prostate cell
lines to the increasing concentrations of zinc ions differs considerably. After the application of
zinc ions, different behaviour of the tumour and non-tumour cells and their antioxidant systems
was demonstrated. Based on the integrative approaches we were able monitoring of the redox
Summary 6
324
status in the individual cell lines. These methods were then used for the evaluation of cisplatin
effects. Cisplatin belongs to commonly used cytostatic drugs. We focused on the analysis of
oxidative stress, cell cycle, apoptosis and selected cytotoxic analyses. Our attention was directed
to the PC3 line, representing a model of the aggressive prostate carcinoma. After the
application of cisplatin, we could not see in this line a typical cell cycle arrest in the G1/G0
phase, and at the same time, we observed a decreased tendency for apoptosis. Moreover, we
demonstrated that this cell line exhibits a higher antioxidant activity and higher metallothionein
content after the administration of cisplatin and thus can be used as a model of cisplatin resistance.
Finally, non-coding amino acid sarcosine was studied intensively in association with
its expected predictive value for the prostate carcinoma diagnosis, affects cancer cells aggression.
Based on our experiments, it was confirmed that these mechanisms are related neither to
antioxidants nor to the regulation of oxidative stress levels.
PART IV - Novel approaches to the analysis of prostate cancer markers
Discovering and definition of new biochemical markers, which are specifically connected with
grave pathological states including tumour diseases, are among the most important objectives
of biomedical research. Identification of highly specific and sensitive biomarkers represents the
main aim of modern research, because only such biomarkers may be applied towards the early
diagnosis of malignant disease, prediction of prognosis and eventually development of an appropriate
treatment strategy in clinical practice. We have decided to study MT, Cav-1 and
AMACR in connection with the progression of CaP. None of the substances studied therein
meets criteria for being a marker of the aggressive disease form. There was no statistically significant
difference in the level of monitored proteins between the localized (T1, 2) and spreading
(T3, 4) tumours. However, the level of caveolin-1 was significantly higher in the serum of patients
with tumours of stage 4 and AMACR and Cav-1 levels were higher in patients with
Gleason grade 9. MT as the only one showed a significantly increased level in the serum of CaP
patients compared with control samples. The determination of sarcosine in connection with the
determination of other CaP biomarkers has a potential to improve the prostate carcinoma diagnostics
and to eliminate false positive and false negative cases.
Furthermore, we have decided to introduce new methodologies based on paramagnetic nanoparticles
and Förster resonance energy transfer (FRET). Paramagnetic nanoparticles allow to
isolate both RNA and proteins from complex samples, thereby increasing detection specificity
and at the same time reducing amounts of consumed chemicals. This procedure was optimized
for metallothionein in serum and cell lysate samples. In case of the FRET assay we describe the
Summary 6
325
method for sarcosine determination based on this methodology between quantum dots and
green fluorescent protein enabled by sandwich arrangement using anti-sarcosine antibodies in
prostate cancer patient urinary samples and in prostatic cell lines (PC3, 22Rv1, PNT1A).
Abstrakt 7
326
7 Abstrakt
Předložená habilitační práce je kompilací vybraných vědeckých publikací, ke kterým jsem
přispěl jako první autor, korespondující autor či spoluautor v průběhu mé vědecké kariéry. Tyto
články byly zveřejněny v letech 2011 až 2017. Všechny zde uvedené publikace mají společné
téma týkající se karcinomu prostaty. Habilitační práce především shrnuje nové poznatky v oblasti
biochemie a patogeneze karcinomu prostaty a je rozdělena na čtyři části podle jejich
specifických zaměření. První část je věnována problematice zinečnatých iontů a jejich vztahu
ke vzniku a progresi karcinomu prostaty. Druhá část se zabývá metabolismem aminokyselin u
karcinomu prostaty. Část třetí je věnována monitorování redoxního stavu nádorových buněk
prostaty a poslední část je zaměřena na detekci nových nádorových markerů. Doprovodný text
zdůrazňuje můj příspěvek k novým poznatkům v oblasti patogeneze karcinomu prostaty a také
stručný vhled do zkoumané problematiky. Součástí habilitační práce jsou i originální publikace,
kde je možné nalézt komplexní informace o jednotlivých experimentálních studiích.
References 8
327
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