1. health and fitness
2. disease
3. cancer

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS
New series No. 1521 ● ISSN 0346-6612 ● ISBN 978-91-7459-475-1
The Role of Microorganisms in
Prostate Cancer Development
Johanna Bergh Drott
Department of Clinical Microbiology, Virology
Department of Medical Biosciences, Pathology
Umeå University, 2012
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Responsible publisher under swedish law: the Dean of the Medical Faculty
Copyright © Johanna Bergh Drott
ISBN: 978-91-7459-475-1
ISSN: 0346-6612
E-version available athttp://umu.diva-portal.org
Printed by Print & Media
Umeå, Sweden, 2012
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”Du blir aldrig färdig, och det är som det skall”
Tomas Tranströmer
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iv
Abstract
Prostate cancer is the most common cancer among Swedish men, but the
aetiology of this disease is largely unknown. There is evidence for a linkage
between chronic inflammation and prostate cancer. The mechanisms
causing prostate inflammation and how this could promote tumour
development and progression are however largely unknown. Chronic
inflammatory infiltrates are common findings in prostate tissue samples and
infection is proposed to be one possible cause for this inflammation.
Inflammatory cells release free radicals, cytokines, and growth factors that
facilitate increased cell proliferation, DNA damage, mutations, and
angiogenesis. However, the present literature on the presence of microbes in
prostate tissue and their possible linkage to inflammation and cancer
development is limited. Therefore, the aim of this thesis was to investigate if
microorganisms are present in prostate tissue and to evaluate their role in
inducing prostatitis and prostate epithelial neoplasia.
The presence of microorganisms (virus, bacteria and fungi) was studied in
clinical prostate tissue samples to evaluate whether or not the occurrences of
microorganisms were different in patients that later developed cancer
compared with matched controls that did not. Viruses, bacteria and fungi
were found in prostate tissues. Out of eight different viruses investigated,
EBV and JC virus were detected, but there were no differences in occurrence
in the case group compared to the control group. The fungus Candida
albicans was present in a very small proportion of the prostate tissue
samples. The predominant bacterium was Propionibacterium acnes and the
second most prevalent was Escherichia coli. The presence of
Propionibacterium acnes was associated with inflammation and subsequent
prostate cancer development. Propionibacterium acnes was further
evaluated for its capacity to induce an inflammatory response both in vitro
and in vivo. Live Propionibacterium acnes induced a strong immune
reaction in prostate epithelial cells in vitro with up-regulation of
inflammatory genes and secretion of pro-inflammatory cytokines. Infection
with Propionibacterium acnes in rat prostate resulted in a lobe specific
inflammation with the most intense inflammation in the dorso-lateral
prostate, lasting up to 3 months post-inoculation. Propionibacterium acnes
inflammation was also associated with altered epithelial cell morphology,
signs of DNA damage and increased cell proliferation.
Taken together, this thesis shows that different viruses and bacteria can be
found in prostate tissue. Propionibacterium acnes, the most abundant
among the bacteria detected and more prevalent in the cancer than in the
control group, exhibits strong prostatitis promoting properties both in vitro
v
and in vivo. In addition, Propionibacterium acnes can induce some of the
epithelial changes known to occur during prostate neoplasia formation. This
thesis therefore suggests that Propionibacterium acnes induced chronic
prostatitis could promote prostate cancer development. Further studies are
needed to elucidate the molecular interplay linking Propionibacterium acnes
induced inflammation and the formation of a pre-neoplastic state that could
evolve into prostate cancer.
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Populärvetenskaplig sammanfattning
Prostatacancer är den i särklass vanligaste cancersjukdomen bland svenska
män. Mikroskopiska härdar av prostatacancer är mycket vanligt hos äldre
men. I Sverige diagnostiseras 10000 nya fall av prostatacancer varje år och
2500 män dör årligen av sjukdomen. För att komma tillrätta med detta
behöver vi lära oss mer om prostatacancerns orsaker och varför vissa
prostatacancrar är betydligt mer farliga än andra. Tyvärr är orsaken till
prostatacancer till stora delar okänd. Mycket talar ändå för att både ärftliga
faktorer och miljöfaktorer är av betydelse. Om det finns yttre faktorer som
orsakar cancern eller påverkar dess förlopp och som är möjliga att påverka,
så är det särskilt angeläget att klarlägga dessa.
För vissa cancerformer, som exempelvis cancer i levern, magsäcken,
livmoderhalsen och i lymfkörtlarna, är det numera känt att virus, bakterier
och kronisk inflammation spelar en viktig roll för sjukdomarnas uppkomst,
en kunskap som redan används för att till exempel vaccinera mot
cancerframkallande virus. I prostata är kronisk inflammation, d.v.s.
prostatit, mycket vanligt och några studier antyder att inflammation skulle
kunna orsaka förstadier till cancer. Vilka faktorer som orsakar sådan
inflammation och om denna inflammation i sin tur verkligen kan orsaka
prostatacancer eller påverka dess förlopp är dock oklart.
I denna avhandling undersöktes därför förekomst av mikroorganismer
(bakterier, virus och svampar) i prostatavävnad och om sådana är vanligare i
prostata hos män som senare diagnostiseras med prostatacancer än hos män
som inte diagnostiseras med prostatacancer. Att ta prov från prostata på
friska män och sedan följa om de får cancer eller inte är i praktiken omöjligt.
Därför undersöktes prostatavävnad från män som opererats för godartad
prostataförstoring, där en del flera år senare även fick cancer medan andra
förblev friska. Både virus och bakterier hittades i prostatavävnaden, vilket
tyder på att dessa mikrober kan infektera prostatan. Den vanligaste
bakterien som hittades var Propionibacterium acnes (P. acnes, den bakterie
som bland annat associeras med acne på huden). I den grupp som senare
diagnostiserades med prostatacancer var denna bakterie något vanligare
jämfört med kontrollgruppen, även om skillnaden inte kunde säkerställas
statistiskt.
För att vidare undersöka om denna bakterie verkligen kan orsaka
inflammation i prostata infekterades prostataepitelceller med P. acnes.
Resultaten visar att bakterien kan orsaka en stark inflammationsreaktion i
körtelceller från prostata, d.v.s. den får prostatakörtelceller att utsöndra ett
flertal inflammations-och tillväxtstimulerande faktorer.
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Mot denna bakgrund; d.v.s. att bakterien ofta förekommer i prostata,
möjligen oftare hos män som senare får cancer och att den stimulerar
prostataepitelceller att utsöndra inflammationsfaktorer, undersökte vi
därefter om P. acnes kan orsaka prostatit hos försöksdjur, och i så fall, om
den bakterie-orsakade inflammationen kan ge upphov till cancer. Injektion
av P. acnes i prostata hos råttor ledde till kronisk prostatainflammation.
Djuren verkade dock så småningom kunna läka infektionen utan tecken på
permanent skada. Under den tid prostata var inflammerad hittades skador i
körtelceller; cellerna blev omogna, började dela sig och ett enzym som
normalt skyddar mot skador i arvsmassan minskade. Inflammationsceller
anses bilda faktorer som kan skada arvsmassan och tecken på ökad DNAskada sågs i prostatakörtlarna hos de infekterade djuren. Resultaten visar
alltså att P. acnes kan orsaka kronisk prostatainflammation hos råttor och
att den bakterieorsakade inflammationen tycks kunna orsaka en del, men
inte alla de steg som behövs för att cancer skall kunna utvecklas.
Sammantaget visar avhandlingen att bakterien Propionibacterium acnes
förvånansvärt ofta förekommer i prostata och att den där sannolikt kan
orsaka kronisk inflammation och en del cellförändringar som liknar
förstadier till cancer. Vad som avgör om infektionen blir så långvarig att den
hinner orsaka tillräcklig skada för att kunna leda till cancer och vilka övriga
faktorer som krävs för cancerutveckling är ännu okänt. Från studier av andra
tumörformer där mikroorganismer anses kunna orsaka cancer vet man att
endast en minoritet av infekterade individer verkligen får cancer, d.v.s. att
den individuella känsligheten varierar, delvis beroende på ärftliga faktorer.
Avhandlingen visar betydelsen av Propionibacterium acnes-utlöst
prostatainflammation och att denna skulle kunna påverka uppkomsten av
prostatasjukdomar, kan vara ett angeläget forskningsområde. Framför allt
bör man undersöka hur bakterien och den kroniska inflammationen skulle
kunna samverka med andra faktorer för att eventuellt orsaka cancer. En
annan viktig fråga är om Propionibacterium acnes-orsakad prostatit skulle
kunna påverka växt av en redan av annan orsak uppkommen tumör.
Intressant nog anses även godartad prostataförstoring, en annan mycket
vanlig sjukdom, kunna orsakas av kronisk inflammation. Det vore därför
intressant att studera om P. acnes har betydelse för uppkomst av denna
sjukdom. Om P. acnes kan påverka uppkomst och förlopp av sjukdomar i
prostata skulle detta öppna intressanta möjligheter för förebyggande terapi.
viii
Original papers
This thesis is based on the following studies, which are referred to in the text
by their Roman numerals:
I.
Bergh J, Marklund I, Gustavsson C, Wiklund F, Grönberg H,
Allard A, Alexeyev O, Elgh F. No link between viral findings in
the prostate and subsequent cancer development. British
Journal of Cancer 2007; 96:137-139.
II.
Alexeyev O, Bergh J, Marklund I, Thellenberg-Karlsson C,
Wiklund F, Grönberg H, Bergh A, Elgh F. Association between
the presence of bacterial 16S RNA in prostate specimens taken
during transurethral resection of prostate and subsequent risk of
prostate cancer (Sweden). Cancer causes Control 2006; 17:
1127-1133.
III.
Drott JB, Alexeyev O, Bergström Patrik, Elgh F, Olsson J.
Propionibacterium acnes infection induces up regulation of
inflammatory genes and cytokine secretion in prostate epithelial
cells. BMC Microbiology 2010; 10:126.
IV.
Olsson J, Drott JB, Laurantzon L, Laurantzon O, Bergh A, Elgh
F. Chronic prostatic infection and inflammation by
Propionibacterium acnes in a rat prostate infection model.
Submitted.
V.
Drott JB, Olsson J, Elgh F, Bergh A, Rudolfsson S.
Propionibacterium acnes induces chronic inflammation and
precancerous epithelial lesions in the dorso-lateral prostate in
rats. Manuscript.
ix
Abbreviations
AR
BPH
BrdU
CK
DNA
DTH
ELISA
GM-CSF
GSTP1
IF
IHC
IL
LPS
NF-κB
OR
PBS
PCR
PhIP
PIA
PIN
PSA
RNA
ROS
SNP
STI
TLR
TURP
UTI
Androgen receptor
Benign prostatic hyperplasia
Bromodeoxyuridine
Cytokeratin
Deoxyribonucleic acid
Dihydrotestosterone
Enzyme-linked immunosorbent assay
Granulocyte-macrophage colony stimulating factor
Glutathione –s-transferase pi
Immunoflouresence
Immunohistochemistry
Interleukin
Lipopolysaccharide
Nuclear factor-kappaB
Odds ratio
Phosphatate buffered saline
Polymerase chain reaction
2-amino-1-methyl-6-phenylimidazol[4,5-b]pyridine
Proliferative inflammatory atrophy
Prostatic intraepithelial neoplasia
Prostatic specific antigen
Ribonucleic acid
Reactive oxygen species
Single Nucleotide Polymorphism
Sexually transmitted infection
Toll-like receptor
Transurethral resection of prostate
Urinary tract infection
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Table of Contents
Abstract....................................................................................... v
Populärvetenskaplig sammanfattning ....................................... vii
Original papers........................................................................... ix
Abbreviations .............................................................................. x
1. Introduction............................................................................. 1
1.1. The Prostate............................................................................................. 1
1.1.1. Anatomy ................................................................................................... 1
1.1.2. Histology .................................................................................................. 2
1.1.3. Function ................................................................................................... 2
1.1.4. Regulation ................................................................................................ 3
1.2. Prostate disorders .................................................................................. 4
1.2.1. Prostatitis ................................................................................................. 4
1.2.2. Benign prostatic hyperplasia (BPH) ........................................................ 5
1.2.3. Prostate cancer ........................................................................................ 6
1.2.3.1. Epidemiology ..................................................................................................6
1.2.3.2. Diagnosis and therapy .....................................................................................6
1.2.3.3. Histopathology ................................................................................................7
1.2.3.4. Aetiology.......................................................................................................10
1.2.3.4.1. Old age...................................................................................................10
1.2.3.4.2. Family history ........................................................................................10
1.2.3.4.3. Diet/life style .........................................................................................11
1.2.3.4.4. Hormones...............................................................................................11
1.2.3.4.5. Inflammation..........................................................................................12
1.2.3.4.6. Propionibacterium acnes ........................................................................18
2. AIMS...................................................................................... 20
2.1. General aim .......................................................................................... 20
2.2. Specific aims ........................................................................................ 20
3. Materials and methods .......................................................... 21
3.1. Human samples (paper I and II) ..........................................................21
3.2. In vitro cell line (paper III) ..................................................................21
3.3. Propionibacterium acnes strains (paper III, IV and V) ......................21
3.4. Animals and treatments (paper IV and V).......................................... 22
3.5. DNA extraction (paper I and II).......................................................... 22
3.6. PCR assays (paper I and II)................................................................. 23
3.7. Cloning and sequence analysis (paper I and II).................................. 23
3.8. ELISA (paper III and IV)..................................................................... 24
xi
3.9. RNA preparation and reverse transcription (paper III) ..................... 24
3.10. Real-time Quantitative PCR and PCR-array analysis (paper III)..... 25
3.11. Quantification and characterisation of inflammation in human and
rat prostate samples (paper I, II and IV) ................................................... 25
3.12. Immunohistochemistry and morphology (paper V) ......................... 26
3.13. Western blot based serology (paper IV) ............................................ 26
3.14. Culture and Propionibacterium acnes genotype identification (paper
IV) ............................................................................................................... 26
3.15. Immunofluorescence staining of Propionibacterium acnes in rat
prostate tissue (paper IV)............................................................................27
3.16. Statistical analyses (paper I-V)...........................................................27
4. Results and discussion........................................................... 28
4.1. Paper I and II ....................................................................................... 28
4.1.1. Conclusion ............................................................................................. 33
4.2. Paper III............................................................................................... 34
4.2.1. Conclusion ............................................................................................. 37
4.3. Paper IV and V......................................................................................37
4.3.1. Conclusion ............................................................................................. 43
5. General discussion and future directions ............................... 44
6. Acknowledgements ................................................................ 46
7. References ............................................................................. 47
xii
1. Introduction
Prostate cancer is by far the most common cancer in Swedish men. Its
aetiology is largely unknown, but as it is estimated that about 20% of all
human cancers are caused by chronic infection 1, it is possible that
microorganisms could play a role also in the pathogenesis of prostate cancer.
Prostatitis, caused by infectious agents, but also by other factors, is a very
common finding in men of all ages and this inflammation is preferably seen
in the parts of the prostate where cancer is detected in elderly men 2. Already
in the early 1950s, sexually transmitted infections (STI) were proposed as a
risk factor for prostate cancer development 3. Since then epidemiological,
clinical and experimental studies on both STIs and non-sexually transmitted
agents have been investigated in their relation to prostate carcinogenesis.
The question whether prostate cancer can be induced by chronic
inflammation, and if so, what the causes for this cancer promoting
inflammation are, is largely unanswered.
1.1. The Prostate
- A small organ with few essential functions but the centre of
many common diseases
1.1.1. Anatomy
The human prostate is a part of the male reproductive system. It is located
anterior to the rectum distal to the urinary bladder and wraps around the
urethra. Due to its anatomical position, infectious agents can reach the
prostate mainly through the urine or as ascending sexually transmitted
infections. The normal prostate has the same size and shape as a walnut and
it mainly consists of tubuloalveolar glands that empty their secretions into
the urethra. In humans the prostate is divided into three different zones; the
peripheral, the central and the transitional zone 4. The peripheral zone,
located posterior to the urethra and ejaculatory ducts, comprises the
majority (65%) of the prostate volume and is the part of the gland were 70%
of all prostate cancer originates 5, 6. Inflammation is also frequently localized
to the peripheral zone 7. The central zone constitutes approximately 25% of
the normal prostatic volume and is rarely affected by carcinoma or
inflammation. The transitional zone comprises about 5-10% of the prostate
in young adults but as benign prostate hyperplasia (BPH) originates in this
part of the prostate the volume generally increases with age.
1
In contrast to the rather homogenous structure of the human prostate, the
prostate in rodents is composed of three (or four) lobes; the ventral, dorsallateral (also considered as separate dorsal and lateral) and the anterior
prostate 8. There is no clear-cut homology between specific rodent prostate
lobes and human prostatic zones but it has been observed that neoplasms
are usually derived from the dorso-lateral lobe in rodents 9, 10 and has
therefore been proposed to be corresponding to the peripheral zone 11, 12.
Gene expression analysis also support the idea that the dorso-lateral lobe is
most similar to the peripheral zone of the human prostate 13.
1.1.2. Histology
The prostate consists of two basic structures; glands and fibromuscular
stroma. The glands consist of three fully differentiated cell types; basal
epithelial, luminal epithelial and neuroendocrine cells. The basal cells rest on
the basement membrane and harbour a small number of stem cells 14. These
stem cells proliferate and give rise to basal, luminal or neuroendocrine cells.
The differentiation to luminal cells is believed to occur through an
intermediate phenotype, expressing markers specific for both basal and
luminal epithelial cells 15, 16.
The basal cells are characterized by their expression of cytokeratin (CK) 5,
CK 14 and p63. In contrast to the basal cells, the secretory luminal cells are
terminally differentiated and have limited capacity to divide. They are
characterized by expressing the androgen receptor (AR), CK 8, CK 18 and
synthesizes and secrete many of the key proteins found in the ejaculate for
example prostate specific antigen (PSA) and prostate acid phospatase 17-19.
Neuroendocrine cells are rare. They secrete several neuropeptides but their
function is not fully elucidated. These cells express markers such as
chromogranin A, synaptophysin and are AR negative 20.
The fibromuscular stroma is made up of smooth muscle cells, fibroblasts,
blood vessels, nerves, lymphatics and infiltrating immune cells. The stroma
gives physical support to the glandular epithelium, enables contraction and
mediates release of growth factors that regulate epithelial growth and
homeostasis.
1.1.3. Function
The main function of the prostate is to synthesise and secrete the proteins
and fluid that, together with contributions from the seminal vesicles, form
most of the ejaculate. Although the prostate is involved in fertility, it is not
2
required for reproduction. The major protein produced by the luminal
epithelial cells is PSA. PSA is a protease that helps to liquefy the semen so
that the sperms way to the egg is facilitated 21. PSA is produced in humans
but not in rodents. Normally PSA is secreted into the glandular lumina and
by muscular contractions in the stroma, pumped into the urethra during
ejaculation. During conditions such as prostate cancer, inflammation and
benign prostate hyperplasia (BPH), the basal epithelial cell layer and the
basement membrane are disrupted and PSA may leak into the surrounding
stroma and vasculature. Thereby PSA is elevated in the blood and is used as
a diagnostic marker for prostate diseases. The prostate is also known to
produce antimicrobial products such as zinc, lysozyme and defensins 22, 23.
1.1.4. Regulation
The prostate is dependent on androgens for development, growth and
function. Testosterone, the main circulating androgen, is produced in the
testes by the Leydig cells. In the prostate, testosterone is converted to the
considerably more potent androgen dihydrotestosterone (DTH) by the
enzyme 5-alpha reductase 24, 25. DTH (and testosterone) binds to AR in the
cytoplasm and is then translocated to the nucleus were it induces expression
of androgen–related genes 26.
Prostate growth control can be studied by androgen withdrawal
(castration). When the supply of androgens is lost, the prostate luminal
epithelial cells will undergo apoptosis, resulting in involution of the prostate
gland 27-29. However, both stromal and basal epithelial cells are maintained
during castration 30, 31. AR is also expressed in the fibromuscular stroma and
androgens regulate epithelial growth and regression through paracrine
factors (called andromedins) released by the stroma. The exact nature of
these andromedins is not fully established but several members of the
insulin-growth factor (IGF), fibroblast growth factor (FGF) and wnt families
are involved 32. Studies in transplanted tissue recombinants and in animals
where AR is selectively knocked-out in the epithelium and in the stroma,
shows that stromal AR is necessary and that epithelial ARs are not required
for castration induced glandular involution. The main function of AR in the
luminal epithelium is to maintain differentiation (such as PSA secretion) and
suppress proliferation of these cells 33-36.
3
1.2. Prostate disorders
Three different and very common diseases affect the prostate: prostatitis,
benign prostatic hyperplasia and prostate cancer.
1.2.1. Prostatitis
Prostatitis is a very common and multifaceted disease. It affects men of all
ages and it is estimated that about 50% of all men will experience symptoms
of prostatitis at some time during their lives 37. It is the most common
urological disorder in men under the age of 50 and the third most common
urological disorder, after benign prostatic hyperplasia and prostate cancer,
in men older than 50 38. Reported rates of prostatitis are similar in North
America, Europe, and Asia 39. Common symptoms include pain
(genitourinary, pelvic, or rectal), voiding symptoms and sexual dysfunction.
According to the National Institutes of Health (NIH), prostatitis is classified
into the following four categories: I: Acute bacterial prostatitis, II: Chronic
bacterial prostatitis, III: Chronic prostatitis/Chronic pelvic pain syndrome
(CPPS) IV: Asymptomatic inflammatory prostatitis 40.
Bacterial prostatitis (category I and II) is estimated to account for 10% of
all prostatitis cases, with the most commonly implicated microorganisms
being Escherichia coli, and Enterococcus spp 41, 42. Other common organisms
include Proteus mirabilis, Pseudomonas aeruginosa and Klebsiella.
Diagnosis is based on symptoms, physical examination, urine culture and/or
urine culture after prostatic massage. Bacterial prostatitis is treated with
antibiotics, preferably a fluoroquinolone, because of their good prostate
penetration and activity against most usual bacterial pathogens 43 .
A majority of men searching medical care for typical prostatitis symptoms
are classified as having chronic prostatitis/CPPS (category III). The aetiology
of chronic prostatitis/CPPS is unknown but several hypotheses have been
suggested including presence of antibiotic-resistant non-culturable
microorganisms, urethral obstruction, autoimmunity, neuropathic pain and
psychological dysfunction 44, 45. Microorganisms that have been suggested to
be the causes of chronic prostatitis/CPPS include; Chlamydia trachomatis
46, Trichomonas vaginalis 47, Uroplasma urealyticum 48, Mycoplasma
genitalium 48 , fungi 49 and several viruses 50, 51.
The therapies commonly used for chronic prostatitis/CPPS are empirical
treatment with antibiotics, anti-inflammatory drugs and alpha-adrenergic
blockers 52.
4
Asymptomatic inflammatory prostatitis (category IV) is the most common
form of prostatic inflammation. The inflammation is often found in biopsies
taken from men evaluated for possible prostate cancer, in trans-urethral
resection tissue samples from men with BPH or in autopsied prostates 53-57.
1.2.2. Benign prostatic hyperplasia (BPH)
Benign prostatic hyperplasia is a common disorder in elderly men and is
characterized by enlargement of the prostate gland caused by proliferation of
epithelial and stromal cells in the transition zone. The clinical symptoms
result from compression of the prostatic urethra and consequent obstruction
of the bladder outlet.
The aetiology of BPH is unknown. Androgens are known to play a
permissive role in BPH. This means that androgens have to be present for
BPH to occur, but do not necessarily directly cause the condition. Men who
are castrated prior to puberty or have a 5α-reductase-type 2 deficiency do
not develop BPH 58 and a 5α-reductase inhibitor significantly decrease
prostate volume 59. An inflammatory origin of BPH have been proposed
because of the very common finding of inflammatory infiltrates in BPH
lesions 55, 60 and that these inflammatory cells release cytokines and growth
factors that stimulate the stroma and epithelial cells to hyperproliferation 61,
62 63. Men that have suffered from prostatitis have a greater risk to later
develop benign prostatic hyperplasia64, 65. Many studies have demonstrated
the presence of heterogeneous bacterial strains in BPH specimens 55 but if
infectious agents can induce the inflammation that triggers BPH
development has not been extensively examined.
Screening and diagnosis procedures for BPH are similar to those used for
prostate cancer; voiding problems lead to digital rectal examination, PSAtesting and ultrasound examination of the prostate and urinary tract.
Treatment is medical or surgical. The medical treatments commonly used
include α-blockers, to relax stroma smooth muscles and/or 5α-reductase
inhibitors, which inhibit DTH production. Surgery is performed when
medical treatment fails. The golden standard surgery treatment is
transurethral resection of the prostate (TURP) were parts of the prostate is
removed by electrocautery or sharp dissection through the urethra.
5
1.2.3. Prostate cancer
1.2.3.1. Epidemiology
Prostate cancer is the most common cancer in Sweden with an incidence of
9697 per year (2010) (The National Board of Health and Welfare). The
incidence of prostate cancer has increased dramatically over the last 20 years
probably caused by the introduction of PSA testing, which has lead to an
increased number of diagnoses in asymptomatic men. In the last 5 years, the
incidence has slightly decreased indicating that the peak is reached. Prostate
cancer mortality is almost unchanged during these years with 2460 deaths
(2010) annually. Approximately 5% of Swedish men die from prostate
cancer. Men diagnosed with prostate cancer are on average 70 years old,
while the majority of prostate cancer deaths occur in men over 79 years.
Interestingly, the number of men diagnosed with advanced, metastatic
disease, have declined the last years indicating that more men are potentially
curable.
Today approximately 75 000 men in Sweden are living with a prostate
cancer diagnosis, but autopsy studies indicate that asymptomatic prostate
cancer is actually present in more than 50% of men in their 5th decade or
older 66. Worldwide, the highest incidence of prostate cancer is seen in
Western countries and in some African regions and the lowest incidence is
seen in Asian countries 67. The highest incidence of prostate cancer death is
seen in African-Americans and the second highest risk of dying in prostate
cancer is seen in Sweden. The explanation to variations in both incidence
and mortality in prostate cancer around the world is not known, although
genetic, environmental and socioeconomic factors could all be of importance
68.
1.2.3.2. Diagnosis and therapy
Localised prostate cancers do not often cause any symptoms and if they do
these are similar to those of BPH i.e. voiding problems. A patient searching
hospital care for voiding problems will have his PSA value measured in a
blood test and the prostate can be examined by rectal palpation. The PSA
test is taken to assess the risk of prostate cancer. In Sweden, a PSA value <
3ng/ml is considered normal and a PSA value > 3ng/ml indicates that some
of the conditions prostate cancer, BPH or prostatitis exist. At substantially
elevated PSA values, prostate cancer is often the cause.
6
Ultrasound guided needle biopsies are taken from the prostate in patients
with elevated PSA levels and if a biopsy contains cancer it is graded
according to the Gleason system 69. Their differention pattern, ranging from
1 to 5, were 5 represents the lowest differentiated and most aggressive
tumour pattern, grade the tumour glands. The most common and the second
most common grade is summarised into the Gleason score (2-10). Tumours
with Gleason score <6 have a good prognosis whereas tumours with Gleason
score >8 are associated with an unfavourable outcome. In patients with
Gleason score 6 and 7, which constitute about 75% of cases diagnosed
outcome is highly variable and at present largely unpredictable 70.
Localised prostate cancer is treated with radical prostatectomy or
radiotherapy. If the life expectancy of the patient is short and the tumour is
at an early stage it is common that the patient is only subjected to watchful
waiting (no treatment until symptoms of metastases) or active monitoring
(treatment when signs of tumour progression are detected). Advanced
prostate cancer is characterized by dissemination of prostate cancer cells,
typically to lymph nodes and bone. Bone scintigraphy, computed
tomography and PSA level in serum are used to determine how advanced the
tumour is. For advanced and metastatic prostate cancer there is no cure and
the therapy is palliative with surgical or chemical castration. Castration
initially reduces proliferation and increases apoptosis in prostate tumour
cells because of androgen depletion 71. However, after some time (about 1-3
years) the tumour relapses and start to grow in a castration resistant manner
72.
1.2.3.3. Histopathology
Almost all prostate cancers originate in the glandular epithelium. Prostate
cancer is generally a multifocal disease, i.e. each patient has several different
cancers simultaneously in the prostate. Some of these tumours are highly
differentiated and probably clinically insignificant but others can be poorly
differentiated and highly malignant. Most cancers are however of
intermediated grade (score). Some intermediate grade cancers are clinically
insignificant whereas others are potentially lethal. Unfortunately current
diagnostic methods, i.e. histological Gleason grading of the tumours in
needle biopsies, do not provide sufficient prognostic information.
Although the aetiology and pathogenesis of prostate cancer is largely
unknown it is fairly well established that prostate cancer originates in
precancerous lesions of prostate glands 2, 73 (Figure 1). In the peripheral zone
of the prostate, atrophic glands are commonly seen 74. In some of these
7
glands the epithelial cells show high proliferation, up-regulation of antiapoptotic factors and cells with genetic alterations such as silencing due to
hypermethylation and mutations accumulate. Furthermore there is an
increase in epithelial cells that possess a phenotype between basal and
mature luminal cells, so called intermediate cells 75. Adjacent to such glands
the stroma undergoes morphological changes, inflammatory cells such as
macrophages and lymphocytes are common, and there are signs of increased
angiogenesis. This type of lesion is called proliferative inflammatory atrophy
(PIA) 2, 76. PIA lesions are thought to arise as a consequence of the
regenerative proliferation of prostate epithelial cells in response to injury
caused by inflammatory oxidants 2, 76, 77. The cause of this inflammation is
not known but infections and other mechanisms (see below) have been
suggested 2. In some glands adjacent to PIA, prominent precancerous
epithelial atypia arises, and such lesions are termed prostate intraepithelial
neoplasia (PIN). PIN is characterized by luminal cell hyperplasia,
enlargement of nuclei and nucleoli and nuclear atypia 78. Several molecular
pathways/genes that are linked to prostate cancer are also altered in PIA and
PIN, for example the tumour suppressor genes NKX3.1, CDKN1B and PTEN
2, 75, 79. Silencing of the “care taker” gene Glutathione S-transferase gene pi
(GSTP1), due to hypermethylation in the promoter region, is widely seen in
both PIN lesions and in prostate cancer and to a lesser extent also in PIA
lesions 80, 81. Recently it was detected that approximately 50% of men with
prostate cancer harbour a somatic fusion-gene between an androgen
regulated gene (TMPRSS2) and genes encoding EST family transcription
factors 82. This gene, which is seen already in a subset of PIN cases, function
as an oncogene and result in increased androgen driven cell growth.
Interestingly the fusion-gene apparently also stimulates prostate tumour
growth by stimulating intratumoral prostaglandins and tumour
inflammation 83. The question whether inflammation plays any role in the
appearance of the fusion-gene, is still unanswered. It is not detected in PIA
lesions 82 . Fusion–genes in general are however created under conditions of
insufficient DNA-repair. In contrast to prostate cancer, basal cell numbers
are reduced, but not absent in PIN. Prostate adenocarcinoma can be
confirmed by the absence of basal cells using immunostaining for p63 and
cytokeratin 5 and 14. Areas with PIA and/or PIN can bee seen merging with
overt cancer, suggesting that there may be a continuum of changes from PIA
over PIN to cancer 84, 85.
8
9
Figure 1. Progression pathway model for human prostate cancer.
1.2.3.4. Aetiology
Even though prostate cancer is very common, rather little is known about its
causes. A number of risk factors have been proposed, were some are well
established and others are weak and controversial. The most established
risks are age and family history. Diet, hormones and inflammation are
examples of factors that may cause prostate cancer but have not been
similarly well elucidated. Given the marked heterogeneity in prostate cancer
aggressiveness, where some harmless cancers are extremely common,
whereas other less common are highly malignant, it is not unlikely that
different subgroups of prostate cancer could have different causes.
1.2.3.4.1. Old age
The greatest risk factor for developing prostate cancer is advanced age. As
histological foci of asymptomatic cancer are extremely common already in
the 5th decade of life and increase with age, it is believed that all men will
develop clinically relevant prostate cancer if they only live long enough. The
reason why prostate cancer is so common among elderly is probably due to
the increased number of genetically altered cells that occur as a natural
consequence of increasing age. Signs of chronic inflammation are very
common in elderly men and various studies have shown age-dependent gene
expression changes, particularly in the stroma, in genes associated with
inflammation, oxidative stress, and cell proliferation 86, 87.
1.2.3.4.2. Family history
A large proportion of prostate cancer patients report family history of the
disease. Twin studies have shown that more than 40% of prostate cancer
cases are estimated to be explained by hereditary factors 88. Moreover, a
meta-analysis has shown that having a first-degree relative with prostate
cancer increases the risk for disease with a relative risk of 2.24 (95%
confidence interval 2.08-2.41) 89. A number of high-risk candidate genes
responsible for hereditary cancers have been identified. Nevertheless, these
are rare and indicate the familial prostate cancer is a consequence of many
affected loci rather than specific susceptibility genes 90. Interestingly, two of
these germ-line susceptibility genes are involved in host response to
infection, RNASEL and MSR1. Ribonuclease L (RNASEL) encodes an
enzyme that degrades single stranded RNA, leading to apoptosis in virally
10
infected cells 91. The macrophage scavenger receptor (MSR1) encodes a
receptor expressed on macrophages and is able to bind lipoproteins on both
Gram-positive and Gram–negative bacteria 92. Furthermore, areas within the
prostate that show evidence of inflammation are often populated by
macrophages that express MSR1 77. The question whether prostatitis is more
common in families with hereditary prostate cancer has to my knowledge not
been examined.
1.2.3.4.3. Diet/life style
Migration studies have shown that Asian men will experience an increased
risk of developing prostate cancer when moving to the USA 93. The
prevalence of small asymptomatic prostate cancers is similar all around the
world but clinically symptomatic tumour and prostate cancer death are more
common in countries with a western life style 94. These differences may be
caused by factors in Asian diets that inhibit prostate cancer growth and/or
factors in Western diets that stimulate prostate cancer growth. For example,
some vegetarian diets contain factors that inhibit prostate cancer growth
(like phytoestrogens and omega-3) 95-97 whereas high consumption of red
meat 98 and saturated fat 99 may promote prostate cancer development. A
recent study showed that treatment with phytoestrogens of prostate cancer
cell lines resulted in demethylation of the GSTP1 promotor region 100.
Interestingly, feeding Fisher 344 rats with one factor present in over
grilled red meat, 2-amino-1-methyl-6-phenylimidazo(4,5-b) pyridine (PhIP),
first results in chronic prostatitis followed by appearance of PIA-like
glandular atrophy precancerous epithelial lesions and later overt cancer 101
102. This observation in an experimental model clearly links chronic
inflammation with cancer development.
1.2.3.4.4. Hormones
The prostate requires steroid hormones for growth and development, but
such hormones are also essential for cancer maintenance and growth. Their
role in the initiation of prostate cancer is unknown.
Treatment of rodents both neonatally and in adult life with estrogen or
mixes of estrogen and androgens or with environmental chemicals with
estrogen-like effects can induce precancerous lesions and in some cases also
cancer (see 103, 104 for review). Interestingly this is preceded by development
of chronic prostatitis. Similarly, exposure of humans to elevated levels of
11
estrogen during fetal life or chronically later in life is associated with an
increased risk of prostate cancer 105, 106.
1.2.3.4.5. Inflammation
Already in 1863 the German pathologist Virchow suggested that cancer was
induced by chronic inflammation. His hypothesis was that some classes of
“irritants” caused tissue injury, inflammation and increased cell proliferation
107. Today 25% of all cancers are estimated to be induced by chronic
inflammation or infection 108. For example, gastric cancer can be caused by
chronic infection with the bacterium Helicobacter pylori, liver cancer by
chronic infection with hepatitis viruses, Burkitt´s-lymphoma by chronic
infection with Epstein-Barr virus, and cervical cancer by human papilloma
virus infection 1, 109, 110 (Table 1). It should however be noted that only a small
portion of cases infected with these agents develop cancer, suggesting that
individual susceptibility is of importance. Moreover there are also examples
where chronic inflammation apparently does not cause malignancy, for
example rheumatoid arthritis is apparently not associated with tumours in
affected joints. Notably, the relationship between cancer and inflammation is
also bidirectional. Activation of oncogenes often result in the secretion of
cytokines and chemokines and premalignant and malignant cells therefore
secrete factors attracting inflammatory cells and thus cause chronic
inflammation in and around tumours without the involvement of infectious
agents 111. Cancer-induced inflammation generally promotes tumour growth
and spread 112, 113.
The mechanisms by which chronic inflammation may cause cancer are not
fully defined but are usually explained by the tissue injury and enhanced cell
proliferation caused by the persistent inflammation. Furthermore,
inflammatory cells release factors that can promote cancer development. For
example, in order to kill infectious agents inflammatory cells produce
reactive oxygen and nitrogen oxide species (ROS and RNOS) that cause
damage to DNA. Cells that proliferate in such a milieu are at an increased
risk for accumulation of mutations leading to increased expression of
oncogenes and/or inactivation of tumour suppressor genes. Inflammatory
cells (macrophages, neutrophils, eosinophils, dendritic cells, mast cells and
lymphocytes) also secrete cytokines, chemokines and growth factors that can
promote cell proliferation, inhibit apoptosis and stimulate angiogenesis 109,
all necessary steps in the development of a tumour 113.
12
Table 1. Tumours Linked to Infections
Infection agent
Tumour type
Helicobacter pylori
Gastric cancer, gastric lymphoma
Human papilloma virus
Cancer of the cervix, anal and genital cancers
Hepatit B and C virus
Hepatocellular carcinomas
Epstein-Barr virus
B-cell and Burkitts lymphoma,
nasopharyngeal cancer, gastric cancer
Human herpes virus 8
Kaposis sarcoma
Human lymphotropic retrovirus 1
Adult T-cell leukemia
Schistosoma heamatobioum
Bladder cancer
Opisthorchis viverrini
Cholangiosarcoma
Modified from 1, 114
For example, TNF-α, released by macrophages and T-lymphocytes,
enhance the formation of NOS and thereby increase the risk for DNA
damage and mutation in epithelial and stromal cells 115. Cytokines and
chemokines may also result in epigenetic changes altering gene expressions
in ways promoting cancer development 116. IL-6 is another pro-inflammatory
cytokine that have been shown to act as an autocrine or paracrine growth
factor in some malignancies 117 and IL-8 is known to promote angiogenesis
118. In addition, an imbalance of pro -and anti-inflammatory cytokines may
prevent the self-limiting process of the inflammatory response, leading to a
chronic inflammation 119. It is also likely that factors produced by a particular
infectious agent can promote cancer development 110. For example some H.
pylori strains harbour the cag pathogenicity island (PAI) that encodes
several virulence factors. One of the most important is the cytotoxic
associated antigen (Cag A), which is “injected” into the epithelial cells
through a type IV secretion system. Once inside the cytoplasm the bacterial
protein interacts with several cellular pathways which in turn result in the
promotion of proliferation, apoptosis, motility and induction of
inflammatory gene expression 109, 120. Strains having the cag PIA are more
likely to be associated with gastric adenocarcinoma than those lacking it 120.
13
Is there a support for a relation between inflammation and
prostate cancer?
In the light of the observations that so many tumours are linked to
infectious events or inflammatory states and the fact that histological
inflammation is common in prostate it would not be surprising if also
prostate cancer could be related to chronic infection. Indeed, several lines of
evidence do indicate that chronic inflammation (of different causes) may be
a key component in the pathogenesis of prostate cancer. Chronic, mainly
asymptomatic inflammation is, as mentioned above, very common in the
prostate and prostatitis and cancer both arises in the same part of the
prostate, the peripheral zone 2. Histopathological studies show that
intraepithelial neoplasia (PIN) and cancer develops in glands previously
altered and atrophic as a result of chronic inflammation, so called PIA 76, 84,
121. Epidemiological studies show an increased risk of prostate cancer in men
with a history of prostatitis 64, 122-124. A meta-analysis of 11 case-control
studies found an increased risk of prostate cancer odds ratio (OR): 1.6 in
men with a history of prostatitis, particularly in population-based casecontrol studies (OR: 1.8) 122. Although, some studies have reported negative
results 125. Also, men with a history of a sexually transmitted infection (STI)
have been shown to have a higher risk for developing prostate cancer (OR:
1.4) 126, 127, 2.3 for a history of syphilis and 1.4 for a history for gonorrhoea 126.
Indication that STI infect the prostate and contribute to prostatic
inflammation stems from studies that show that PSA is elevated in men with
a confirmed diagnosis of a STI 128. In addition, long-term treatment with
anti-inflammatory drugs appears to reduce the risk of developing prostate
cancer 129, 130.
In humans the risk of developing hereditary (see above) or sporadic
prostate cancer is apparently associated with alterations in genes involved in
inflammatory pathways. Hypermethylation of CpG island sequences in the
promoter of the GSTP1 gene, which encodes an enzyme that inactivates
electrophilic carcinogens, is an extremely common early epigenetic event in
the progression of prostate cancer 131, 132. High levels of GSTP1 expression are
typically seen at sites of prostatic inflammation, and loss of GSTp1
expression is commonly seen in PIN and prostatic carcinoma 81. This
suggests that GSTp1 serves as a “care taker” gene and that the decreased
expression might render prostate cells more vulnerable to malignant
progression. Furthermore several sequence variants in genes involved in
inflammatory pathways have been linked to prostate cancer 133.
14
Single nucleotide polymorphism (SNP) in sequences encoding the
pathogen recognition receptors TLR4 and the TLR-1-6-10 gene cluster are
associated with prostate cancer risk 134. The Toll-like receptors (TLR) are key
players in innate immunity as they recognize different ligands expressed by
bacteria and viruses, such as lipopolysaccharide (LPS), lipoprotein and viral
RNA. The activation of TLRs results in nuclear translocation of the
transcription factor nuclear factor-kappaB (NF-κB), which then induces the
expression of various pro-inflammatory cytokines, chemokines, and effector
molecules.
A number of different studies have investigated the correlation of
interaction of SNPs in different cytokines and increased risk of prostate
cancer. One study found that combinations in variations of IL-1B, IL-10 and
TNF was associated with an increased risk for developing prostate cancer 135.
Another study reported that SNPs in IL-4, IL-6, PTGS2 and STAT3 were
associated with prostate cancer susceptibility 136.
Another cytokine that have been evaluated for association with prostate
cancer is macrophage inhibitory cytokine (MIC1). MIC1 is believed to have
an important role in regulating macrophage activity and sequence variants of
MIC1 have been linked to prostate cancer 137. Together these studies
underline that variants in genes associated with inflammation affect prostate
cancer risk.
Microorganisms found in prostate tissue in relation to prostate
cancer
Prostatic inflammation can be induced by various different sources
including infection, urine reflux, dietary factors, autoimmune reactions,
hormones and corpora amylaceae or a combination of two or more of these
factors. Several different infectious agents have been investigated in relation
to prostate cancer development. In clinical specimens, tissue based methods
(PCR, IHC and ISH) that have investigated the presence of various
microorganisms in human prostatic tissues or serological assays (ELISA or
IF) that have evaluated antibody titers against different infectious agents
have been used.
HPV is the most investigated pathogen in relation to prostate cancer
because of its well-established role in genital cancer development. The
results from these studies are contradictory and in a recently published
meta-analysis no association between HPV infection and prostate cancer
could be found 138. Other viruses that have been investigated include;
cytomegalovirus 139, Epstein-Barr virus 140, 141, herpes simplex virus 142, 143,
15
human herpes virus 8 144, 145, polyoma viruses JC and BK 146, 147 and the novel
γ-retrovirus XMRV 148. Xenotrophic murine leukaemia related virus (XMRV)
was first indentified in 2006 by Urisman et al. in prostate tumours from
patients that were homozygous for a reduced activity germline variant of the
RNASEL gene 148. After this finding several other studies have reported
XMRV in prostate cancer tissues 149, 150, whereas others have failed to detect
the virus in prostate cancer samples 151, 152. However, recent evidence suggest
that XMRV originated from a recombination event between two endogenous
murine retroviruses during passage of prostate cancer xenografts in mice,
and the presence of XMRV in prostate cancer tissue might results from
contamination of mice DNA 153, 154.
Also different bacteria have been evaluated regarding their role in prostate
carcinogenesis, where Chlamydia trachomatis 155, Mycoplasma sp. or
Ureaplasma sp. 156, 157, Propionibacterium acnes 158 (see below) and E. coli
159, 160 are the most investigated pathogens. The majority of studies that have
investigated the presence of bacteria in prostate tissues have been generated
in studies from men with chronic prostatitis. Common bacterial species
found in these studies are; Enterobacter sp., Enterococcus sp.,
Pseudomonas sp. (bacteria associated with UTI) or Actinomyces sp.,
Streptococcus sp. 161, 162. To the best of my knowledge eight independent
studies have evaluated the presence of bacteria in prostate cancer tissues,
either by 16s rDNA PCR 163-166 or/and by bacterial culture 166-168 (Table 2).
Seven out of these eight studies reported positive bacterial findings, with
frequencies varying from 20 to 89%, and in some of these studies bacterial
findings were correlated to histological inflammation 158, 163. This indicates
that bacteria are frequently present in cancerous prostate tissues.
Concordance between inflammation and positive bacterial findings suggest
that bacteria might have a role in histological (asymptomatic) inflammatory
prostatitis.
Surprisingly very few studies have apparently tried to detect individual
infectious agents in PIA and PIN lesions. However, Samanta et al. and
Grinstein et al. reported CMV and EBV in human PIN lesions, respectively
139, 140 and Fassi Fehri et al. reported P. acnes to be present in human PIN
lesions 169. Experimental support for an association between infection and
prostate cancer development also exists. In a mouse model of chronic
bacterial prostatitis, E. coli induced chronic inflammation that lead to
preneoplastic tissue alterations similar to PIN 159. Benign prostate cells
infected with Mycoplasma sp. underwent karyotypical changes and
inoculation of infected cells in male nude mice initiated tumour growth 156.
16
Transurethral
resection or
suprapubic
prostatectomy
Transperinal biopsies
Radical
prostatectomy
samples
Radical
prostatectomy
samples
Radical
prostatectomy
biopsies
Radical
prostatectomy
samples
Radical
prostatectomy
samples
Gorelick et al.
1988
Keay et al. 1999
Hochreiter et al.
2000
Krieger et al.
2000
Leskinen et al.
2003
Cohen et al. 2005
Sfanos et al. 2008
Specific PCR
Culture
16S PCR
Culture
16S PCR
16S PCR
16S PCR
16S PCR
Culture
Culture
Detection
method
* This study only reported anaerobes (Copper et al. 1988)
Transurethral
resection or
retropubic
prostatectomy
Cooper et al.
1988*
Material
1 ( 0.5%) of 200
C. trachomatis PCR:
P. acnes PCR: 10 (4%) of
200
17
Staphylococcus
Culture:
Propionibacterium acnes
Culture:
10 (33%) of 30
Pseudomonas spp.
Escherichia spp.
Not reported
Specific PCR:
Culture: Not reported
Transition/Peripheral zone
Specific PCR:
Peripheral zone
Culture:
16S PCR:
Peripheral zone
16S PCR:
Not associated
16S PCR:
Not reported
-
Not reported
Not reported
Not reported
Not reported
Not reported
Localization
Acinetobacter spp.
More inflammation in P.
acnes positive samples
22 (65%) 34 had focal
chronic inflammation
-
Not reported
Associated with
inflammation
Not reported
Not associated
Not reported
Inflammation
16s PCR:
Staphylococcus epidermis
Propionibacterium acnes
-
Ureaplasma urealyticum
Escherichia coli
Not reported
Bacteroides
Escherichia coli
Staphylococcus epidermis
Streptococcus faecalis
Escherichia coli
Clostridium perfringens
Bacteroides distasonis
Anaerobic cocci
Most common organisms
26 (87%) of 30
19 (56 %) of 34
0 (0 %) of 10
21 (20 %) of 107
6 (86 %) of 7
8 (89 %) of 9
44 (21 %) of 209
6 (66 %) of 9
Bacteria Positive samples
(%)
Table 2. Bacteria in prostate cancer tissues
1.2.3.4.6. Propionibacterium acnes
-a novel cause for prostatitis and prostate cancer?
The facultative anaerobic Gram-positive bacterium, Propionibacterium
acnes (P. acnes), is part of the normal flora in the skin 170, oral cavity 171,
large intestine 172, the conjunctiva 171 and the external ear canal 170. Generally,
P. acnes have a positive effect on the human health by preventing
colonization of severe pathogens, but when the host becomes compromised
(trauma, injury or alterations in immune status) it can display a pathogenic
potential 173. P. acnes is mostly associated with the skin disease acne
vulgaris 173, 174, but it is also associated with a variety of inflammatory
diseases such as prosthetic joint infections 175, shunt associated central
nervous system infections 176, endocarditis 177, sarcoidosis 178 and the SAPHO
(synovitis, acne, pustulosis, hyperostosis, osteitis) syndrome 179.
On the skin, P. acnes resides in the hair follicles where the oxygen level is
optimal. Sebum-rich areas of the skin support the growth of P. acnes since
the bacterium utilizes free-fatty acids and glycerol as energy sources. The
main end products of the metabolism are propionic acid, thereby the name
Propionibacterium acnes. In acne vulgaris, P. acnes is believed to induce
and maintain an inflammatory response in the pilosebaceous unit. There are
several mechanisms by which P. acnes may induce an inflammatory
reaction; P. acnes produce chemotactic factors that attract neutrophils 180.
The neutrophils then ingest the bacteria, resulting in release of hydrolases
that is responsible for disruption of the follicular epithelium 181. Moreover, P.
acnes releases lipases, proteases and hyaluronidases that further contribute
to the tissue injury 182, 183. The host response to P. acnes is characterized by
production of proinflammatory cytokines such as tumour necrosis factor-α
(TNF-α), interleukin 1-α (IL-1α) and interleukin 8 (IL-8) 184. P. acnes have
been shown to induce these cytokines in both macrophages and
keratinocytes through the TLR2 and TLR4 signalling pathways 185-187.
Recently, and thus during the course of my PhD studies, P. acnes has been
reported to be frequently prevalent in both benign and malignant prostate
tissues 158, 169, 188, 189. In 2005, Cohen et al. detected P. acnes as the most
prevalent microorganism in prostate cancer tissues, identified by bacterial
culture in 35% of prostate cancer specimens removed by prostatectomy 158.
Furthermore, P. acnes was positively associated with prostatic inflammation
and this raised the question if P. acnes could be involved in prostate
18
carcinogenesis. After 2005, two independent studies (one from our group
and one from Germany) have detected P. acnes in prostate tissues and found
a positive association to prostate cancer development.
Two large epidemiological studies have found an association between a
history of cutanous acne and increased risk of prostate cancer 190 191.
However, contradictory results regarding P. acnes involvement in prostate
cancer development are also reported. Severi et al. found in a population
based case-control study that plasma concentration of P. acnes antibodies
were significantly inversely associated with prostate cancer (OR:0.73), in
particular for advanced disease (OR: 0.59) 192.
Three different phenotypes of P. acnes, Type I (Type IA and Type IB), II
and III, have been categorized according to sequence comparison of the
housekeeping gene recA and a putative hemolysin gene tly 193, 194. Moreover,
a multilocus sequencing typing (MLST) approach, based on nine
housekeeping genes, have further subdivided Type I into I-1a and I-1b and I2 195, 196. There are several studies indicating that certain P. acnes strains
participate in specific pathogenic process. For example, members of the P.
acnes type I-1a have been shown to be associated with severe acne 195
whereas P. acnes type I-1b and II is more prevalent in orthopedic implants
197 and in radical prostatectomies 158. Furthermore, the ability to trigger
production of proinflammatory cytokines in keratinocytes is different for
different phenotypes 187.
Interestingly, due to its immunostimmulatory capacity this bacterium has
been tried as immunotherapy against a variety of neoplasms in both animals
and humans 198. The mechanism of the anti-tumour activity of P. acnes is not
fully understood but it is speculated that the inflammation caused by P.
acnes could induce a cytotoxic response that kill tumour cells 199, 200.
19
2. AIMS
2.1. General aim
The general aim of this thesis was to evaluate if microorganisms have a role
in the aetiology of prostate cancer.
2.2. Specific aims
Aim 1
To study the presence of eight potentially tumour promoting DNA viruses
(Epstein-Barr virus, Herpes simplex virus 1 and 2, Cytomegalovirus,
Adenovirus, Human Papilloma virus, Polyoma viruses BK and JC), the
fungus Candida albicans and bacteria in prostate samples from men with
benign prostate hyperplasia and to evaluate if presence of microorganisms
was different in cases that later developed prostate cancer compared to
controls that did not.
Aim 2
To characterize the inflammatory response in prostate derived epithelial
cells upon infection with Propionibacterium acnes in an in vitro model
system.
Aim 3
Establish if Propionibacterium acnes can induce an inflammatory response
in the rat prostate in vivo, and if so characterize this response.
Aim 4
Examine to what extent Propionibacterium acnes induced chronic
inflammation can promote preneoplastic lesions in the rat prostate.
20
3. Materials and methods
3.1. Human samples (paper I and II)
Samples from men (<75 years) diagnosed with benign prostate hyperplasia
and treated with transurethral resection of prostate (TURP), were collected
at the University hospital in Umeå during the years 1982-1997. The TURPsamples formed the base for a nested case-control study. In this TURP
population, 201 prostate cancer cases were identified by utilization of the
Regional Cancer Registry. Selected cases received their cancer diagnosis at
least 6 months after the TURP treatment and before October 31, 2002. If the
patient had received several TURP treatments, the first TURP specimen was
selected for this study. Controls were randomly selected to match the cases
according to year of birth, year of TURP treatment and life survival. In total
201 matched pairs, collected during the years 1982-1996, were studied to
evaluate if presence of genetic traces of microorganisms (virus, bacteria and
fungi) were correlated to histological inflammation and subsequent prostate
cancer diagnosis. The study was approved by the ethical review board in
Umeå (permit no 05-160M).
3.2. In vitro cell line (paper III)
The non-neoplastic prostatic epithelial cell line RWPE-1 was used for the in
vitro studies. RWPE-1 is derived from a white male donor and immortalized
with human papilloma virus 18 201. The RWPE-1 cell line expresses luminal
cytokeratines 8 and 18 but it also co-express basal cytokeratines. It is an
androgen sensitive cell line since growth is stimulated upon androgen
exposure. RWPE-1 does not grow in agar nor does it form tumours in nude
mice.
RWPE-1 was grown in keratinocyte serum-free medium supplemented
with 5ng/epidermal growth factor, 0.05mg/L bovine pituitary extract and
100U/ml penicillin/streptavidine. The cells were maintained in a humidified
incubator at 37°C containing 5% CO2 according to the manufacturer’s
instructions.
3.3. Propionibacterium acnes strains (paper III, IV and V)
P. acnes, serotype 1a (CCUG 41530), was used in the in vitro and in vivo
infection studies. This bacterial strain was isolated at the University hospital
21
of Northern Sweden, from a patient that suffered from a cerebral shunt
infection.
Furthermore, four different clinical prostate isolates, P1 (serotype 1a), P2
(serotype 1b), P3 (serotype 2) and P4 (serotype 2) were used in the in vivo
infection experiments. The prostate isolates were cultivated from
prostatectomies at the university hospitals of Umeå and Örebro, Sweden.
Bacteria were grown in brain-heart infusion broth + 5% horse serum in
37°C at micro-aerobic conditions. The bacteria were grown to a density of
109 per ml and resuspended in sterile PBS to a concentration of 108 bacteria
per ml.
3.4. Animals and treatments (paper IV and V)
Immunocompetent adult male Sprague Dawley rats (3-4 months old) were
used for in vivo infection experiments.
To establish an in vivo infection model with Propionibacterium acnes,
male Sprague Dawley rats were used. The bacteria were injected directly
into the prostate lobes; the left ventral prostate (VP) and the left dorsolateral prostate (DLP), in order to be sure that the bacteria reached the
infection site.
During anaesthesia an incision was made in the lower abdomen to expose
the prostate area. Propionibacterium acnes, 5 x 107 diluted in 5µl PBS
(serotype 1a in animals infected 5 days and 3 weeks and prostate isolate P.
acnes mix in animals infected for 3 weeks, 3, 6 and 12 months) or vehicle
was injected into the left VP and left DLP lobes using a 100µl Hamilton
syringe. At time of sacrifice, after 5 days, 3 weeks, 3 months or 6 months, the
animals were sedated and the prostate lobes from the left and right sides
were dissected. The removed tissue was utilized for bacterial counts or was
fixed in formalin, 24h, for subsequent morphological analysis. One hour
before sacrifice the animals were injected intra-peritoneally (i.p.) with
bromodeoxyuridine (BrdU, 50mg/kg, Sigma Aldrich) in order to label cells
with active DNA synthesis (proliferating cells). All animal work was
approved by the local ethical committee for animal research (permit no A7309).
3.5. DNA extraction (paper I and II)
Formalin-fixed, paraffinised prostate tissues were sectioned to 20µm
thickness by use of a microtome. Tissues were deparaffinised as described in
202 and DNA was purified by using QIAamp DNA Blood minikit (Qiagen,
22
Hilden, Germany). Special care was taken to avoid contamination between
samples; the sectioning was done in an area physically separated from postPCR samples and microtome and microtome knife blades were cleaned with
chlorine and ethanol after each sample. The DNA extraction process was
performed in a fume hood that was free from PCR products and care was
taken to avoid cross contamination of the samples. Negative controls (using
all reagents except for tissue) were run in parallel with the DNA extractions.
3.6. PCR assays (paper I and II)
PCR was used to detect viral, bacterial and fungal genomes in the TURP
samples. Nested PCR systems were used (except for HPV and Candida
albicans) in order to increase the specificity of the reaction.
For the eight DNA viruses and Candida albicans, specific PCR primers
and programs were used (as described in paper I). For bacteria, a 16S
ribosomal DNA PCR assay was used, amplifying highly conserved sequence
regions shared by bacterial species (as described in paper II). Great care was
undertaken to avoid DNA contamination; all PCR mixes were performed in a
DNA free area with dedicated pipettes, small reagent and primer aliquots,
and the use of aerosol resistant pipette tips. For the 16s ribosomal DNA PCR
working areas, PCR components except for Taq polymerase, primers and
template DNA were UV irradiated before use. Negative (sterile water as
template) and positive controls (specific for each organism, produced at the
Department of Clinical Microbiology, University Hospital of Northern
Sweden) were performed with PCR assays to monitor potential reagent or
laboratory contamination.
PCR products were electrophoresed through a 2% agarose gel containing
ethidium bromide and visualized by UV translumination.
To control for DNA degradation and PCR inhibition, all DNA preparations
from archival prostate samples were tested for the presence of the human
beta-globin gene by two different PCRs with different product length, 110bp
and 268bp, respectively.
3.7. Cloning and sequence analysis (paper I and II)
Amplicon products from the 16S rDNA PCR were cloned using the pT7Blue
Perfectly Blunt cloning kit (Novagen) to get high concentration of the PCR
product and to be able to identify individual sequences in the mixture of
several 16s rDNA gene products that made direct sequencing impossible. In
brief, plasmids were transformed into Nova Blue Shingles competent cells
23
according to the manufacturer’s instruction. Colony PCR analysis was
performed on 5-10 randomly selected white colonies and plasmids
containing cloned 16s rDNA gene inserts were selected for sequencing.
All positive samples from virus, fungi and bacterial PCR were sequenced
with the BigDye Terminator cycle sequencing kit 1.1 (Applied Biosystems)
according the manufacturer’s instruction and analysed in ABI PRISM 3700
DNA ANALYSER (AME Bioscience). Homology searches were performed
with the alignment search tool BLAST on the NCBI database
(www.ncbi.nlm.nih.gov). Sharing >98% nucleotide identity to a species was
used as the criteria for defining a species.
3.8. ELISA (paper III and IV)
Secretions of cytokines IL-6, IL-8 and GM-CSF in P. acnes infected prostate
epithelial cells were measured by ELISA (R&D Systems) as described in
paper III. In brief, cells seeded into 24-well plates were grown to 75%
confluence. The cells were then infected with P. acnes at a multiplicity of
infection (MOI) of 16. Supernatants were harvested after 24h and 48h,
stored at -20°C and later assayed by ELISA. To examine if secretion of
cytokines were mediated via the TLR 2, cells were blocked with an anti-TLR2
monoclonal antibody (100 ng/ml, InVivoGen) 1h prior to infection.
C-reactive protein (CRP) was measured in serum from P. acnes infected
rats and control. Levels were determined utilizing an ELISA method (Rat
Serum CRP M-1010) according to the manufacturer’s instructions (Alpha
diagnostics intern. San Antonio, TX, USA).
3.9. RNA preparation and reverse transcription (paper III)
RNA was prepared from P. acnes infected RWPE-1 cells after 24h using the
RNesy Mini kit (Qiagen) as described in paper III. In brief, cells were seeded
at a density of 1 x 106, grown to 75% confluence and then infected with P.
acnes at a MOI of 16. After 24 h, cells were trypsinized, lysed, homogenised
and total RNA was extracted according manufacturer’s instruction. RNA
concentration and purity were assessed in a Nanodrop ND-1000
spectrophotometer (Thermo scientific) at 260nm. Complementary DNA
(cDNA) was generated from 1µg total RNA using RT2 First Strand Kit
(SABioscienses) according to the manufacturer’s instruction.
24
3.10. Real-time Quantitative PCR and PCR-array analysis
(paper III)
Gene expression analysis was performed using the RT2 Profiler PCR array,
Human Toll-Like receptor Signalling Pathway (SABiosciences) according
manufacturer’s instruction. Real time PCR detection was performed with an
IQTM5 instrument (BIO-RAD). The relative gene expression was calculated
with the ΔΔCT method of the web-based software package RT2 profiler PCR
array systems (SABiosciences).
3.11. Quantification and characterisation of inflammation in
human and rat prostate samples (paper I, II and IV)
Histological quantification of inflammation was done in the PCR positive
TURP samples and matched controls (paper I and II). Inflammation was
characterized in a dichotomous scale as either severe or moderate/minimal.
Severe inflammation fulfilled the following criteria: infiltrating inflammatory
cells were seen in the majority of the slides; three or more single
inflammatory foci or one focus that took up one third of the slide or more.
Moderate/minimal inflammation was defined as no areas of confluent sheets
of inflammatory cells or very small ones (less than one third of the slides).
In the rat prostate lobes (VP and DLP) the type and magnitude of the
inflammation was characterized and quantified (paper IV) according to the
following criteria: tissues were graded according to the intensity of the
inflammation (i.e. according to the number of inflammatory cells present) as
no or minimal inflammation (0), mild/moderate (1) or severe (2). The
inflammation was classified as either diffuse or focal. Diffuse inflammation
involves the entire tissue in a relatively uniform manner. Focal inflammation
occurs as patches of inflamed tissue areas in a background of normal
appearing tissue. In the case of focal inflammation the proportion of total
prostate volume inflamed was calculated using a stereological method. In
summary, using a light microscope mounted with a square-lattice in the eyepiece the number of grid intersections falling on inflamed vs. non-inflamed
tissue was measured 203.
25
3.12. Immunohistochemistry and morphology (paper V)
Tissue sections were stained using primary antibodies against: BrdU (Dako)
for counting proliferating cells, Factor VIII (Dako) for blood vessels, γH2AX
(Novus) for detecting DNA damage.
Tissue sections were also stained for: AR (UPstake Lake Placid), GSTP1
(Leica Microsystems), smooth muscle actin (Dako), Desmin (Dako), CK 18
(PROGEN Biotechnik), p63 (DAKO) and CK 14 (Biosite), as described in
more detail in paper V.
3.13. Western blot based serology (paper IV)
Whole blood samples were collected from rats by cardiac puncture prior to
sacrifice. Blood samples were allowed to coagulate and serum was collected
after centrifugation at 1400 rpm for 10 minutes at room temperature.
Presence of anti-P. acnes IgG was assessed by a Western blot procedure
where the rat serum functioned as the primary antibody. A total bacterial
lysate (1 x 1010 bacterial cells dissolved in 400l sample buffer) was
submitted to sodium dodecylsulfate polyacrylamide gel electrophoresis
(SDS-PAGE) and electroblotted onto polyvinylidine fluoride (PVDF)
membrane. The filter was blocked by normal rat serum, cut into strips and
incubated for 1h with individual rat serum diluted 1:2000. After washing, the
strips were collected into a single container and incubated with goat-anti- rat
–horseradish peroxidase antibody (1:5000) for 1h, washed and developed
with ECC solution (Amersham).
3.14.
Culture
and
Propionibacterium
acnes
genotype
identification (paper IV)
Whole lobes were aseptically homogenized in PBS and 1/10 of the slurry
obtained was subjected to 10-fold serial dilutions and plated on anaerobic
blood plates. Plates were incubated for 1 week in 37 C° under anaerobic
conditions and then P. acnes colonies were counted on 1-2 plates.
Bacterial strains were genotyped utilizing the recA gene. Strains from
biotype 1 and 2 differ in the sequence of the recA gene at base 71 (type 1= G,
type 2= A), base 183 (type 1= A, type 2= G), base 214 (type 1= C, type 2= T),
base 424 (type 1= A, type 2= G).
26
3.15. Immunofluorescence staining of Propionibacterium
acnes in rat prostate tissue (paper IV)
Antigen retrieval of deparaffinised tissue sections was performed by boiling
in citrate buffer (10mM, pH 6.0) at 2atm for 1h. Following blocking with 1%
BSA in PBS, slides were incubated with P. acnes polyclonal rabbit antiserum
(Agrisera, Umeå, Sweden) diluted 1:1000 in blocking solution for 1h. Slides
were washed in PBS and incubated for 1h with goat anti-rabbit monoclonal
antibodies labelled with Alexa 488 (Invitrogen) diluted 1:1000 in blocking
buffer. Following washing and dehydration, slides were mounted and
examined with epifluorescence (Axioscope) or confocal fluorescence
microscope (Nikon).
3.16. Statistical analyses (paper I-V)
Fisher exact test was used in paper I to determine if an association between
viral genomic finding and subsequent prostate cancer in TURP samples was
present.
Logistic regression was used in paper II to estimate the association
between presence of P. acnes and prostate cancer risk, presence of P. acnes
and histological association, odds ratio (OR) and corresponding 95%
confidence interval (CI). Chi-square test was used to test for differences in
Gleason score and P. acnes positive cases.
In paper III, a permutation test was used to compare means of gene
expression regulation and secretion of cytokines in infected cells vs. noninfected cells.
In paper V, a Mann-Whitney U-test was used for comparison between P.
acnes infected rats and controls.
27
4. Results and discussion
4.1. Paper I and II
The prevalence of microorganisms in clinical prostate tissue
samples and evaluation of their association with prostate cancer
development (Aim 1)
To test whether microorganisms could be identified in the prostate prior to
cancer we used a novel experimental design. We identified men with benign
prostate hyperplasia where some were later diagnosed with prostate cancer
and age-matched men who did not develop prostate cancer during the study
period. In this way we hoped to detect agents that could potentially promote
the development and progression of cancer. In previous studies aiming to
establish connections between cancer and infectious agents in the prostate,
agents were searched for in cases where cancer was already established. In
such studies we cannot know whether the microorganism was there before
the cancer or whether if was there as a result of the disease. It is also possible
that agents causing a cancer-inducing inflammation are cleared and not
present at the stage of analysis.
We screened the material for traces of virus, bacteria and fungi. Eight
different DNA viruses were examined and chosen by the criteria that they
have the ability to infect the urogenital tract region and that they are known
to be involved in cancer development. Specific PCRs for the virus were:
adenovirus, herpes simplex virus 1 and 2, Epstein-Barr virus,
cytomegalovirus, human papilloma virus and the polyoma viruses BK and
JC. The presence of bacteria was studied by using a universal 16S rDNA PCR.
Furthermore we studied the presence of the fungus Candida albicans with a
specific C. albicans PCR.
Great care was taken during DNA extraction and PCR amplification to
avoid DNA contamination. Negative controls were performed with the DNA
extraction and in the PCR reactions. Before screening, DNA from all prostate
tissue samples was checked for integrity by amplifying the human betaglobin gene. This was done because fixation, paraffin embedding and longterm storage (we used archival samples, 8-22 years old) may affect the DNA
quality of the samples. Out of 402 DNA samples tested, 352 (87.6%) were
positive for the human beta-globin and were therefore considered to have
sufficient quality for subsequent PCR analysis of the different
microorganisms. For the C. albicans PCR 240 samples were available.
28
Of the eight viruses investigated, EBV and JCV were found in prostate tissue.
Out of 352 samples tested, 31 (8.8%) were positive for EBV and 10 (2.8%)
were positive for JCV. Two samples were positive in the C. albicans PCR. We
then assessed whether the presence of virus and fungi had any association
with prostate cancer risk. In total, 159 matched case-control pairs were
available for virus analysis. There was no association between positive EBV
and JCV finding and prostate cancer risk. Of 29 positive EBV samples, 15
(9.4%) were in the case group and 14 (8.8%) were in the control group. Of 10
positive JCV samples, three (1.9%) were in the case group and seven (4.4%)
were in the control group. The two samples positive for C. albicans were in
the case group (Table 3). There were no differences in the occurrence of
severe inflammation in EBV or JCV positive samples compared to virus
negative samples.
There are several tissue-based studies that have investigated the presence
of virus in association to prostate cancer 139, 140, 142, 147, 148, 166, 204. Today there is
no known virus with a strong association to prostate cancer development.
HPV is the most investigated pathogen in relation to prostate cancer
because of its well-established role in cervical cancer development. The
results from these studies are contradictionary and in a recently published
meta-analysis no association between HPV infection and prostate cancer
could be found 138.
We did not find any HPV DNA in our prostate tissue material. Several
other studies have also reported null-results for HPV DNA in the prostate 142
205. Whether that is because of methodological matters or true negative
results have been discussed. However, Hrbacek et al. reviewed different
studies in regard to material and methodological issues in detecting HPV by
PCR in prostate tissue and found that these variables were not likely to
influence the results 206. Furthermore, we tested our HPV PCR on 14 archival
paraffin-embedded tissue samples from cervical cancers and found that all
14 samples were positive, thus proving that our PCR can detect HPV DNA in
archival samples (data not shown).
Three other studies have detected EBV DNA in prostate tissues. Grinstein
et al. reported EBV to be present in 37% of prostate carcinomas and in 0% of
control samples using immunohistochemistry 140. Sfanos et al reported EBV
as the most frequent microorganism detected (16 of 200, 8%), in a study
analysing different microorganisms by PCR in cancer patients 166. Recently,
Whitaker et al. reported high frequencies of EBV gene sequences to be
present in both benign (2 of 10, 20%) and malignant prostate tissue samples
(4 of 10, 40%) 141. Furthermore, high-risk HPV gene sequences were seen in
29
the same tissue specimen, suggesting that EBV and HPV can collaborate to
transform prostate cells.
The oncogenic potential of the human polyoma viruses BK and JC in
humans is still debatable but they have been shown to transform mammalian
cells in vitro and to induce tumours in animal models 207. Because of their
long-term latency in the urinary tract, several studies have investigated their
possible role in prostate cancer development 146, 147, 204, 208. In a study with a
small sample size Zambrano et al. detected JCV DNA in prostate cancer
tissue samples (38%) 204 but other studies have reported null-results 208, 209.
Together with our result it demonstrates that these viruses can be found in
prostate tissue but our results indicate that they are unlikely to contribute to
prostate cancer development.
96 (27%) of the 352 TURP samples were positive in the universal bacterial
16S PCR. Sequence analysis revealed Propionibacterium acnes as the most
predominant bacterium, found in 22 (6.3%) of the prostate samples. The
second most frequent isolate was Escherichia coli, found in 12 (3.6%). The
other isolates included Pseudomonas sp., found in 3 samples, Actinomyces
sp., 2 samples, Corynebacterium sp., 2 samples, Veillonella sp., 2 samples.
Streptococcus mutants, 1 sample Nocardioides sp., 1 sample, Rhodococcus
sp., 1 sample. The remaining 35 clones were considered as background noise.
There was no association between positive 16S findings and subsequent
cancer diagnosis. However, when we assessed whether the presence of P.
acnes was associated with prostate cancer risk, we found that P. acnes was
twice as common in the case group (14) as compared to the control group (8)
(Table 2), but the indicated association was not statistically significant (OR:
2.17, 95% CI 0.77-6.95). Furthermore, severe inflammation appeared
somewhat more common in P. acnes positive samples compared to P. acnes
negative samples (62% in P. acnes positive samples compared to 50% in
bacterial negative samples, p=0.602).
Of the 12 E. coli positive samples, 8 were in the case group and 4 in the
control group (Table 3).
The finding that P. acnes was the most prevalent bacterium is interesting
since two recent studies have reported P. acnes as a prevalent finding in
prostate cancer tissue specimens 158, 169. Cohen et al showed that P. acnes
could be isolated in 35% from prostate cancer tissue samples and that P.
acnes was positively associated with prostatic inflammation 188. Fassi Fehri
et al. found that P. acnes was present in high percentage in both prostate
30
cancer tissues and in precancerous PIN tissue samples, but was absent in
healthy prostate tissues samples 169.
Table 3. Microorganisms found in prostate tissue, prevalence and
distribution in case and control groups.
Microorganism
Total (n= 352)
Case (n= 159)
Control (n= 159)
EBV
31 (8.8%)
16
15
JCV
10 (2.8%)
3
7
C. albicans
2 (0.8%) *
2*
-*
16S positive
97 (27%)
47
49
P. acnes
22 (6.3%)
14
8
E. coli
12 (3.4%)
8
4
* 240 samples were available for C. albicans PCR and 115 matched case-control pairs.
Both these studies suggested that P. acnes induced prostatitis could have a
role in prostate cancer development. Interestingly, one study found a
correlation between the development of severe acne in adolescence
(measured indirectly as tetracycline use for 4 or more years) and increased
risk of prostate cancer 191. The authors speculated that men having severe
acne are more immune sensitive to P. acnes infections and therefore more
likely to develop stronger inflammatory immune responses to intraprostatic
P. acnes infections.
Whether P. acnes can induce an inflammatory response in the human
prostate is not established. We have shown that P. acnes can induce
proinflammatory cytokines, such as IL-6 and IL-8, when co-cultivated with
prostate epithelial cells in vitro (paper III) and in our recently developed
animal infection model, we showed that P. acnes induced a chronic
inflammation in the dorso-lateral prostate in rats (paper IV). Moreover, the
P. acnes induced prostatitis lead to glandular and stromal changes
commonly seen in precancerous prostate tissues (paper V).
Although P. acnes well-established involvement in the skin inflammatory
disease acne vulgaris, it is generally believed to have low pathogenic
potential and is often considered to be a contaminant when cultured from
clinical biopsies. However, the bacterium has been reported to cause severe
infections at various body sites, particularly after surgery 176, 210. Since we
31
used PCR technique to detect P. acnes DNA in prostate tissue there is always
a threat that the positive findings are false-positive results because of
contamination during tissue sampling or during laboratory processing. We
performed negative controls for both DNA extraction and PCR amplification
and thereby controlling for possible contamination during laboratory
processing. Furthermore, 6.3 % of 352 TURP samples were P. acnes positive
and if there had been a systematic contamination from laboratory reagents it
would have been present in a much higher amount. To prove that P. acnes
PCR positive samples really represent tissue-invasive infections Alexeyev et
al. showed that 7 out of 10 PCR positive TURP samples were confirmed by
visualizing P. acnes in the prostate gland by using a fluorescent in situ
hybridization assay (FISH) 189. P. acnes was mainly detected in the stroma
with intracellular localization (most likely within macrophages) or as
bacterial aggregates. The intracellular form further strengthens a genuine P.
acnes infection in the prostate gland. Moreover, P. acnes could be detected
in sequential prostate samples from individual patients, taken up to 6 years
apart 189.
P. acnes might enter the prostate gland by several routes. Since P. acnes
could be isolated from urine 211, it might ascend into the prostate gland
through the urethra. P. acnes has also been reported in the vagina 212, 213 and
it is therefore possible that P. acnes could be sexually transmitted. P. acnes
can also be introduced to the prostate during preceding bladder
catherization or surgical manipulation (biopsy or TURP). Another way of
route is haematogenous spread, either via intracellular infected macrophages
or as free living bacteria in the blood. One hypothesis on why P. acnes could
establish an infection in the prostate could be the hypoxic environment seen
in the prostate 214 and that this impairs the local immune defence and
favours growth of a facultative anaerobic bacterium.
Previous studies investigating a bacterial aetiology for prostate cancer
have mainly found (either by culturing or 16S PCR) urogenital pathogens
such as Escherichia spp., Pseudomonas spp., Ureaplasma spp.,
Streptococcus spp. or commensal such as Staphylococcus spp. and
Actinomyces spp 163-167. The reason why previous studies have failed to detect
P. acnes could be that the culture conditions have not been optimal for P.
acnes growth. P. acnes is extremely slow-growing and require extended
anaerobic culture 158. In addition, the P. acnes cell wall is resistant to
digestion by lysozyme which is used to lyse Gram-positive bacteria prior to
DNA extraction 215.
32
The second most prevalent bacteria found in this study, Escherichia coli,
is a well established prostate pathogen. It is the most isolated pathogen in
bacterial prostatitis and certain uropathogenic E. coli strains is known to
harbour specific virulence factors that increase infectivity or disease severity
216, 217. Elkahwaji et al. showed that mice intraurethrally infected with E. coli
developed a chronic prostatitis that was associated with preneoplastic lesions
similar to PIA and PIN 159.
Potential weaknesses in the study: Since the nested 16S PCR system
used in this study had a sensitivity of 300 cfu/reaction, bacteria present in
lower amounts were most likely missed and the true portion of bacteria
positive samples could have been underestimated. On the other hand, using
a too sensitive PCR increases the risk of amplifying exogenous
contamination.
Another difficulty in detecting infectious agent in clinical tissues by using
PCR techniques is that only a small fraction of the tissue is examined.
Knowing that inflammation, and probably also bacteria and viruses, are
unevenly distributed in the prostate, the risk of missing focally infected areas
is high.
An important limitation of this study is the lack of appropriative negative
controls. The material in this study came from patients with BPH and since
both prostate cancer and BPH are suggested to have an inflammatory
aetiology this might bias the result. Inflammation is a common finding in
patients undergoing treatment for BPH and a proposed source for that
inflammation is infectious agents 62. Ideal control material, without
inflammation and hyperplasia, is difficult to obtain and since we do not have
such material, the prevalence of microorganism in healthy prostates could
not be determined.
4.1.1. Conclusion
In this study we examined whether some infectious agents are present in the
prostate prior to the detection of cancer. We found that this is the case but
we were unable to demonstrate that the presence of these microorganisms is
firmly related to development of cancer. Interestingly, although not
statistically significant, both P. acnes and E. coli appeared to be more
common in men that later were diagnosed with cancer. This together with
experimental findings in animal models, where these bacteria can cause
chronic prostatitis and induce epithelial lesions similar to precancerous
33
lesions in humans, suggests that the pathogenic role of these two bacteria in
the prostate should be examined in more detail.
Such studies could preferably use case-control cohorts of the type used in
this study but the assays used to detect infectious agents should probably be
more sensitive and specific than the ones used in this thesis. When
interpreting results one should also be open to the possibility that infectious
agents could have opposing roles at different phases of disease, cancer
promoting at early stages but perhaps cancer inhibiting in manifest tumours.
Parallel studies in human samples and in experimental models are probably
necessary to examine this.
4.2. Paper III
The inflammatory response of prostate epithelial cells to
Propionibacterium acnes infection (Aim 2)
Having found that P. acnes was the most prevalent bacterium in BPH
samples and that it was potentially associated with histological inflammation
and prostate cancer development (paper II) we wanted to extend these
observations to investigate the effect of P. acnes infection on prostate
epithelial cells in vitro. A bacterium that causes prostatitis reaches the
prostate gland, via the prostatic ducts, from urine or through ascending STIs.
Therefore glandular epithelial cells will be the first to be exposed to the
infection.
Prostate epithelial cells of non-malignant origin (RWPE-1) were infected
with Propionibacterium acnes 24h or 48h at a multiplicity of infection
(MOI) of 16 and the early inflammatory response of infection was studied.
Secretion of three important inflammatory cytokines, IL-6, IL-8 and GMCSF, was measured and gene expressions of different pro-inflammatory
genes were evaluated. Since P. acnes have previously been shown to
stimulate pro-inflammatory cytokines in monocytes via activation of TLR2
184, we investigated if that could be the case also in prostate epithelial cells
This experiment was carried out by a TLR2 receptor blockage with an antiTLR2 monoclonal antibody prior to infection.
We found that the secretion of IL-6, IL-8 and also to a lesser extent GMCSF was markedly elevated upon 24h and 48h of P. acnes infection.
Furthermore, gene expression analysis of these cytokines showed that they
were highly up-regulated after 24h of infection.
34
P. acnes is known to be a potent stimulus to the immune system. Studies
examining the pro-inflammatory response in acne vulgaris have shown that
P. acnes stimulates monocytes to secrete IL-6 and IL-8 184 and keratinocytes
to secrete IL-8 187.
The inflammatory response to P. acnes in the prostate has prior to this
study never been investigated. Following our article, two in vitro studies
have explored similar issues 169, 218.
In one of these investigations, Fassi Fehri et al. confirmed our observation
of a strong induction of IL-6 and IL-8 secretion and a moderate induction of
GM-CSF secretion in P. acnes infected RWPE-1 cells. Furthermore they
showed that the transcription factor NF-κB and many down-stream
inflammatory related genes were up-regulated upon P. acnes infection 169.
Dysregulation of the transcription factor NF-κB have been proposed to be
one molecular mechanism leading to chronic inflammation and cancer 219
and constitutive or increased activation of NF-κB has been observed in
prostate cancer cells 220. Fassi Fehri et al. also compared the inflammatory
response of RWPE-1 cells upon infection of prostate derived P. acnes strains
and a P. acnes skin isolate and found that the overall induced transcriptional
changes were the same after 24h of infection.
A recently published investigation compared the transcriptional
inflammatory response in keratinocytes and prostate epithelial cells upon P.
acnes infection and found that the response was acute and transient in
keratinocytes whereas it was delayed and sustained in prostate epithelial
cells. Moreover, P. acnes was shown to invade prostate cells (intracellular
growth) but not keratinocytes 218. This study shows that the host cell tropism
is important for the pathogenicity of P. acnes.
Prostate epithelial cells have previously been shown to secrete
inflammatory cytokines in response to different inflammatory stimuli 221, 222.
Although the absolute quantities of the secreted cytokines have varied
between these studies, they show that prostate epithelial cell can contribute
to the inflammatory response during infection.
IL-6, IL-8 and GM-CSF are important for recruitment and differentiation
of macrophages and neutrophils to sites of infection 223-225.
Interestingly, both IL-6 and IL-8 have been shown to be involved in
prostate cancer development and progression. Clinical studies have revealed
that increased serum IL-6 concentrations in prostate cancer patients are
associated with advanced tumour stages and short survival 226, 227. IL-6 act as
an autocrine growth factor on prostate epithelial cells in vitro 228, 229 and
expression of IL-6 and its receptor are increased in prostate cancer tissues
35
229, 230.
Importantly, increased expression of IL-6 receptor was correlated
with increased proliferation 229. Increased expression of IL-8 has also been
characterized in prostate cancer cells 231 and correlates with angiogenesis,
tumorigenesis and metastasis 232.
The innate immunity uses specific receptors, the pathogen recognition
receptors (PRR), to recognise and induce an inflammatory response to
invading microorganisms. Prostate epithelial cells lines have previously been
shown to express different TLRs and that the cells induce inflammatory
cytokine secretion through TLR mediated signalling pathways 221, 233. In acne,
the P. acnes induced inflammation has been shown to be triggered through
TLR2 184 185, a PRR for gram-positive bacteria 234, but also TLR4 and TLR9
have been implicated 235.
To investigate if the pro-inflammatory cytokine secretion was induced via
the TLR2 signalling pathway we made an experiment where the receptor was
blocked before infection. In this experiment we saw that the secretion of IL-8
was reduced by blocking the TLR2, indicating that induction of this cytokine
is mediated via TLR2 signalling pathway in prostate epithelial cells. In line
with this, our gene expression analysis revealed up-regulation of several
transcription regulators, JUN, REL, RIPK2, NFKB2, NFBIA, IRF1, and the
co-factor TICAM1 that are involved in the TLR2 signalling pathway.
However, we did not observe any significant reduction in secretion of IL-6
and GM-CSF, suggesting that other pathogen recognition receptors are
responsible for the induction of these cytokines.
Potential weaknesses in study: In this study we used an immortalized
benign prostate epithelial cell line. These cells allow us to study in vitro
behaviour; however, cells may behave different in vivo. Furthermore several
cytokines may be activated as a result of the immortalization.
We only measured the secretion of three cytokines, probably, as indicated
by the PCR array, several other cytokines is secreted upon P. acnes infection
that may influence the outcome of the infection.
The P. acnes strain used in this study was isolated from a patient that
suffered from a cerebral shunt infection. Since it is reported that P. acnes
strains isolated from the prostate differ genetically and phenotypically from
cutaneous isolates 158 it is possible that the inflammatory response would
have been different if the prostate cells were infected with a prostate P. acnes
isolate.
Prostate epithelial cells are probably the first to be exposed to the
infection but since Alexeyev et al. showed that a majority P. acnes bacteria in
36
prostate cancer samples were located in the stroma it would have been
interesting to evaluate how these cells respond to a P. acnes infection.
4.2.1. Conclusion
In this study we demonstrate for the first time that P. acnes induce a strong
inflammatory response in prostate epithelial cells, partly, as when this
bacterium infects other tissues, mediated by activation of TLR2. Many of the
cytokines induced are known to play essential roles in promoting prostate
cancer growth.
This study suggests that the role of P. acnes as a potential pathogen in the
prostate in vivo needs to be examined further.
4.3. Paper IV and V
A rat prostate infection model to study the pathogenic potential of
Propionibacterium acnes (Aim 3 and 4)
The finding that P. acnes can be found in benign (paper II) and malignant
prostate tissues 158, 169, 189 , the association to prostatic inflammation 158, 189
and that the bacterium is able to trigger a strong inflammatory response in
prostate epithelial cells in vitro (paper III) prompted us to investigate if P.
acnes can induce an infection/inflammation in the prostate in vivo.
Live P. acnes were injected in the ventral and dorso-lateral lobes of the rat
prostate and the effects of the infection were examined 5 days, 3 weeks, 3
months and 6 months after injection. Two types of P. acnes agents were
used; a monoculture of P. acnes strain 1a (CCUG 41530), isolated from a
cerebral shunt infection and a mixture of four different prostate derived P.
acnes strains. Prostate tissues were analyzed for bacterial content,
histological inflammation and serum levels of CRP and anti-P. acnes IgG
(paper IV). Furthermore, prostate tissues were evaluated for possible preneoplastic changes caused by the infection/inflammation using
immunohistochemistry (paper V).
Is Propionibacterium acnes a pathogen in the prostate?
We found that injection of P. acnes in the prostate of Sprague Dawley rats
induced a strong inflammatory response and that the immune reaction was
different in the ventral prostate compared to the dorso-lateral prostate. In
37
the ventral prostate, P. acnes induced a transient and diffuse inflammation
that was almost cleared after 3 weeks. In contrast, the dorso-lateral prostate
showed a focal chronic inflammation that could be detected up to 3 months
post-infection. Live P. acnes could be recovered from the dorso-lateral lobe
up to 3 months post- infection whereas the ventral lobe was cleared from
bacteria at that time. P. acnes specific immunoflourescence also revealed
that bacteria could be detected in prostate tissue up to 3 months postinfection. Bacteria were located both in the stroma and in the glands and
coincided with foci of histological inflammation.
The finding of different infection responses between the lobes indicates
that DLP is more susceptible to bacterial infections than VP. The anatomical
distinctions between the lobes are well established 8 but the physiological
character of the lobes are less defined. Most functional studies are performed
in the VP, despite accumulating data that DLP is the lobe that is most similar
to the cancer prone peripheral zone in humans 12, 13. A similar study to ours,
experimentally infecting mice with E. coli, also reported DLP to be the most
affected lobe 236. However, others have reported no differences in
susceptibility 237 or VP to be the most inflamed lobe 160. Whether these
differences are due to methodological aspects or physiological differences in
rodent strains and/or bacterial strains used is unknown. Interestingly, we
found that VP was more prone to spontaneous prostatic inflammation
compared to DLP. The VP from PBS injected rats showed spontaneous
inflammation already at the age of 4 months, and one third of the animals
were inflamed after 9-10 months. In contrast, the DLP from PBS injected
animals stayed free of inflammation throughout the experiment. This result
is in concordance with earlier studies reporting spontaneous prostatitis 238,
239.
Although it is probably a more natural way of infection to inoculate the
bacteria intra-urethral (using a catheter), we injected the bacteria directly
into the prostate lobes. Pilot studies done in our lab found that intraurethral
inoculation compared to direct injection of bacteria to the prostate gave the
same results (in regard to infection/inflammation) but the intraurethral
method was not as reproducible as the direct injection (data not shown).
We found no differences in the amount of bacteria recovered from the
animals infected with the non-prostate derived P. acnes compared to those
infected with a mixture of prostate isolates nor did we see any differences in
histological inflammation comparing the two groups. Even though studies
have reported that P. acnes isolates derived from prostate is genetically and
phenotypical different from skin isolates 158, 240 we did not observe any
38
differences in pathogenicity of the prostate between the different strains in
the prostate in our study.
Although the inflammatory response was localized to the prostate,
serological analysis of CRP and P. acnes specific IgG demonstrate elevated
levels with maximum antibody concentration after 3 weeks and 3 months,
respectively. Similar serum immune reactions have been reported in rats
infected with E. coli 241. The normally high CRP levels in uninfected rats and
also the age-related increase are in line with CRP levels reported for other rat
strains 242.
Corpora amylaceae (prostate concrements composed of calcified
proteinaceous material) are commonly seen in human prostate samples and
they may promote chronic inflammation by causing tissue damage 2.
Corpora amylaceae are probably formed from cellular debris created during
chronic inflammation 243. The finding of suspect corpora amylacea (CA)
formation in one of the rats infected for 6 months support the hypothesis
that infection and inflammation are contributing factors to CA formation 243,
244. It will be interesting to examine whether traces of P. acnes (in addition to
E coli 243), can be found in human CA.
Experimental animal models for bacterial prostatitis have mainly been
studied with uropathogenic E. coli 159, 160, 236, 237, 241, 245-249 and a few studies
with Chlamydia organisms 250, 251, Pseudomonas aeruginosa 252 and Proteus
mirabilis 253. All these organisms were able to induce a chronic inflammatory
state in the rodent prostate and showed many similarities to the natural
history of human chronic bacterial prostatitis. Differences in susceptibility to
infection between different mice and different rat strains have been reported
11, 249 and indicate that host genetics is important for developing bacterial
prostatitis. Also specific virulence factors carried by the bacteria play an
important role for maintaining a chronic infection 248, 252, 253.
Whether the Sprague Dawley rat is an ideal model to study bacterial
prostatitis and in particularly P. acnes prostatitis, is poorly evaluated. Wistar
rats have been reported to be more susceptible to chronic bacterial infection
compared to Sprague Dawley rats when intraurethrally infected with E. coli
11. However, the two studies did not use the same E. coli strain and the DLP
were not analysed 241, 254. Knowledge of P. acnes virulence factors are scarce
but genomics, transcriptomics and proteomics data have shown that P. acnes
harbours several proteins involved in host tissue degradation and
inflammation and that the expression and secretion is different between
different P. acnes strains 183, 255.
39
Is Propionibacterium acnes able to induce potentially neoplastic
changes in the prostate?
In paper IV we showed that P. acnes can induce a chronic infection in the rat
dorso-lateral prostate. Since P. acnes has also been shown to be potentially
associated with prostate cancer in men, our P. acnes prostatitis model can be
a useful tool to study the molecular connections between bacterial infection,
inflammation and prostate carcinogenesis.
In paper V we examined if P. acnes induced prostatitis could promote preneoplastic lesions in the rat prostate. We examined the tissues for changes
commonly seen in human PIA lesions such as altered cytokeratin, AR and
GSTP1 expression, increased proliferation and changes in the stroma.
Because DLP was found to be the most severely affected lobe we analysed it
in more detail. P. acnes infected prostate tissues were examined for
histopathological changes and stained for markers associated with cell
differentiation, DNA damage and proliferation.
In DLP, we found that P. acnes infection was accompanied by major
changes in glandular and stromal morphology. The well-defined order of
basal and secretory luminal cells disappeared and the distinction between
stroma and glandular epithelium was difficult to identify. In the glandular
lumina, numerous necrotic and apoptotic cells were seen together with
macrophages. We saw a decreased number of luminal cells (CK18 and AR
positive) and an increased number of basal cells (CK14 positive) in the
inflamed areas suggesting that the tissue damage induced by P. acnes
infection resulted in proliferation of new epithelial cells. This was confirmed
by enhanced BrdU (labelling proliferating cells) staining in epithelial cells.
Furthermore, double-staining with basal (CK14) and luminal (AR, CK18)
markers showed enrichment of epithelial cells expressing both CK14 and
AR/CK18 in the inflamed areas. Such intermediate cells have been proposed
as precursor cells for prostate cancer and are enriched in human PIA lesions
75, 256. Moreover, we saw that expression of the androgen receptor in the
epithelium was decreased in inflamed areas. Studies with mice infected with
E. coli in the prostates have revealed similar epithelial responses to
inflammation 159 160. Khalili et al. saw an increase of p63 (marker for basal
cells) expressing cells, luminal cells expressing CK5 (marker for basal cells)
indicating intermediate cells and down regulation of AR in inflamed ducts.
Furthermore, these observations were accompanied by increased
proliferation in the inflamed areas 160.
40
We also found that the P. acnes induced prostatitis was associated with
stromal changes. The glandular walls were much thicker and consisted of an
increased amount of smooth muscle cells. These cells showed an increase in
smooth muscle actin (SMA, marker for myofibroblasts and smooth muscle
cells) and a decrease in desmin (marker for fully differentiated smooth
muscle cells) indicating a dedifferentiated phenotype. Furthermore,
vasculature staining by factor VIII showed an increased density of blood
vessels in the inflamed areas, suggesting angiogenesis.
Smooth muscle cells play a central role in the regulation of prostatic
growth and function. By secreting growth factors they regulate epithelial
proliferation and death. Therefore, the maintenance of the smooth muscle
phenotype is critical for prostate gland homeostasis 257. The smooth muscle
cell response to prostatic infection have also been examined in other studies
246, 247, 258, 259 observing similar results to ours. Smooth muscle cells
responded to infection stimuli by changing their contractile phenotype
toward a secretory profile both in vitro 258 and in vivo 246. Moreover,
infection stimulated secretion of pro-inflammatory cytokines have growth
promoting activities 258, 259. Dedifferentiated smooth muscle cells, increased
production of growth factors and angiogenesis are common features in the
reactive stroma accompanying the tumour cells in human prostate cancer.
In this sense, the stromal response to infection is in many ways similar to
that seen in prostate carcinogenesis.
Inflammation is known to induce oxidative stress as a consequence of the
production of reactive oxygen released by the inflammatory cells 260. The
association of oxidative stress with prostate cancer has been recognized in
several studies 76, 159, 261. Furthermore the enzyme glutathione S-transferase
pi (GSTP1), which inactivates electrophilic carcinogens, have been shown to
be epigenetically silenced in PIA, PIN and prostate cancer by DNA
methylation 81, 131. In our study, we showed that GSTP1 was initially upregulated in the luminal cells after 5 days of infection. However, after 3
weeks of P. acnes infection, GSTP1 expression was reduced in the majority of
both basal and luminal cells in the inflamed areas. De Marzo and colleagues
have suggested that loss of GSTP1 gene expression (the most common and
early genetic alteration noted in patients during prostate cancer
development 132) will make the epithelial cells more vulnerable to DNA
damage during a chronic infection 2. To investigate this hypothesis we
stained our P. acnes infected tissue with the DNA damage marker γH2AX.
Indeed we found a remarkably increase of γH2AX positive cells in the
41
inflamed areas. Increased γH2AX staining in combination with the increased
proliferation could make these cells more exposed to mutation and as a late
consequence of this, cell transformation. Actually, we could detect epithelial
cells with mild atypia in the inflamed areas.
Our P. acnes induced prostatitis model show similarities with human PIA
lesions e.g. accumulation of epithelial cells with an intermediate phenotype,
loss of GSTP1 expression and increased proliferation 75, 76, 85. Furthermore,
the altered stroma cell morphology and increased vascular density seen in
our infected rats are similar to that seen in human PIA 76.However, the P.
acnes infection eventually cleared and when analyzing healed tissues for
permanent changes such as increased proliferation, atypia and/or atrophy
we did not find any differences in the P. acnes injected animals compared to
the controls. This indicates that the infection/inflammation induced by P.
acnes creates an environment favourable for many steps in the process to a
neoplastic state but apparently some important features are lacking.
Apart from our investigation, two studies have evaluated the possible
influence of chronic infection and the development of prostate cancer. These
investigations both utilized a mouse model where the animal prostates were
intraurethrally inoculated with uropathogenic E. coli 159, 160. In none of the
models cancer was induced but potentially precancerous lesions were
detected and the inflammation appeared to be more long lasting and to
induce somewhat more epithelial atypia than in our P. acnes model. In one
of these studies chronic inflammation was shown to be associated with
reactive epithelial hyperplasia and oxidative stress 159. Furthermore many
tumour suppressors (GSTP1, p27, PTEN, Nkx3.1) were down-regulated in
response to the inflammation 159, 160.
To be able to investigate further if P. acnes can induce permanent
precancerous lesions in the prostate the duration of the infection probably
has to be more long-standing. Modifying the model by using rat strains more
susceptible to prostatitis, repeated injections of bacteria or/and using other
species and mice with specific alterations in their immune system (similar to
those described in men with increased risk of acquiring prostate cancer)
might be an opportunity. The effects of combined infection with the two
bacterial species known to induce potential precancerous lesions in the
prostate, uropathogenic E. coli and P. acnes could also be examined.
Potential weaknesses in the study: The species we used in this infection
model (Sprague Dawley rats) is probably not ideal for induction of prostatitis
42
and prostate cancer development. Rats are in general resistant to infections
and the Sprague Dawley rats in particular 11. Rats very seldom develop
prostate cancer. A more ideal species could perhaps be dogs (prostate cancer
is actually a very uncommon disease in animals, except in men and dogs) or
mice genetically modified to increased susceptibility to chronic prostatitis
and/or prostate cancer.
To investigate if P. acnes prostatic infection can promote prostate cancer
direct studies of epigenetic changes and mutation should also be done.
Furthermore, a study whether P. acnes can be found adjacent to PIA, PIN
and prostate cancer lesions in humans is needed.
4.3.1. Conclusion
Our studies clearly showed that P. acnes is a potent pathogen in the rat
prostate, able to induce chronic lobe-specific prostatitis and to induce some
of the steps necessary in the formation of precancerous lesions. The role of P.
acnes in the pathogenesis of prostate cancer therefore needs to be examined
in more detail.
43
5. General discussion and future
directions
The overall aim of this thesis was to investigate if microorganisms are
present in prostate tissues and if so if they are related to the aetiology and
pathogenesis of prostate cancer. The main and novel findings in this study
are that microorganisms, particularly the bacterium P. acnes can be detected
in prostate tissue prior to cancer diagnosis, that P. acnes trigger a strong
inflammatory reaction in prostate epithelial cells in vitro, that P. acnes can
induce chronic prostatitis in rats and that this inflammation causes some of
the early cellular changes that is commonly seen during prostate tumour
formation in humans. The changes observed are however generally
reversible and when the inflammation eventually is cleared the potentially
precancerous lesions present apparently disappear.
So what do the results of this thesis and a review of the literature add to
the key questions regarding the role of inflammation and microorganisms in
prostate cancer formation and progression?
Is it likely that prostate cancer can be caused by inflammation and
infectious agents in particular?
As already reviewed in the introduction substantial indirect evidence
suggest that inflammation plays an essential role in the development and
progression of prostate cancer. Inflammation is seen adjacent to precursor
lesions, around and inside tumours, and experimental inhibition of
inflammation may retard tumour growth 203, 262. What is less known is if this
inflammation is a cause or a result of certain (early and/or late) steps in the
cancerogenic process. Is inflammation an aetiological agent and/or is the
inflammatory system activated at a later stage to promote, or under other
circumstances inhibit growth of tumours caused by other mechanisms?
It is well established that some types of bacteria can cause chronic
prostatitis 41 and in experimental models induce potentially precancerous
lesions 159, 160, and the current thesis suggests that P. acnes can be added to
this list of agents. Notably, however, bacteria and other microorganisms are
not the only cause of inflammation in the prostate. Chronic prostatitis can
among many things be induced by steroid hormones like estrogen, by
corpora amylacea and by factors in the diet. So even if we accept that
prostatitis is part of the aetiology of cancer this does not mean that infections
44
are the cause of this inflammation as non-infectious causes are also possible.
In reviewing the literature it appears that inflammation induced in several
different ways such as estrogen, diet and bacteria may cause similar
precancerous changes, suggesting that it is inflammation as such and not the
individual agents that promote cancer development. All these external
factors apparently need to act for a substantial time to induce precancerous
lesions suggesting that the duration is more important than the individual
agent. So if several factors may all induce chronic prostatitis it becomes
important to know whether they can act in synergy, something yet to be
tested. Another factor to be explored is how various agents that may cause
prostatitis interact with genetically determined factors regulating
susceptibility to develop chronic prostatitis and effectiveness of host defence
systems
against
cancer.
For
well-established
cancer-inducing
microorganisms such as Helicobacter pylori and HPV, it is known that only
a sub fraction of the infected individuals develop cancer 1. Another critically
important, but unanswered question, is whether a proposed aetiological
agent causes the non-aggressive or aggressive forms of the disease. An agent
that is only able to induce harmless forms is less important than an agent
that causes the life threatening forms of the disease.
Is it likely that infectious agents and inflammation affect prostate
cancer progression and behaviour?
Studies on the relationships between infections agents, inflammation and
prostate cancer generally explores whether the microorganism could be
involved in the aetiology of the disease but even if this was not the case other
effects are also possible. Microorganisms could infect precancerous lesions
or cancers and modify their behaviour.
Whether this occurs or not is unexplored, but not unlikely as the immune
system is inhibited in tumours 113.
In other tissues P. acnes is known to induce a prominent immune
response of the Th1 type 199 and vaccination with P. acnes has already been
proposed to be an anti-cancer treatment 198. Introducing the bacteria in
tumours may transform a tumour promoting Th2/M2 type of inflammation
to a tumour inhibiting Th1/M1 type. Clinical data have also demonstrated
that prostate cancer patients with high antibody titres against P. acnes have
a better prognosis that those with low 192. Recent or ongoing P. acnes
infection may actually protect. Against this background the overall
conclusion of this thesis is that there are strong reasons to explore the role of
P. acnes infection in the prostate in more detail.
45
6. Acknowledgements
I would like to express my sincere gratitude to:
Prof. Fredrik Elgh, my main supervisor, for introducing me to this
interesting project and for your support during the years.
Dr. Jan Olsson, my co-supervisor, for guidance and assistance in the
laboratory and for insightful comments and suggestions.
Dr. Stina Rudolfsson, my co-supervisor, for always sharing your time and
for valuable suggestions and comments.
Doc. Annika Allard, my co-supervisor, for sharing your great PCR
knowledge.
Dr. Oleg Alexeyev, for continuous support and for fruitful scientific
discussions.
Sigrid Kilter, Birgitta Ekblom, Elisabeth Dahlberg, Pernilla
Andersson and Susann Haraldsson, for your skilful technical expertise
and kind support.
Ingrid Marklund, for introducing me to the Virology department and for
assistance in the laboratory.
Dr. Camilla Thellenberg-Karlsson, for collecting the prostate tissue
samples.
Former students Elina Staaf and Waffa Kamal, for assistance with DNA
extractions.
Katrine Isaksson, for always being helpful and keeping track of the
administrative issues. Many thanks to Åsa Lundsten, Karin Bodén and
Terry Persson.
All the staff at the Dept. of Virology/Virus lab and in the prostate
group, Dept. of Medical Biosciences, for providing a nice working
atmosphere.
Åsa Gallegos Torell, for helping me with the digital picture in the
framework and for all the great music during the years.
Malin Olsson and Eva Nylander, for your warm friendship and support.
My family, for your support and love.
This work was financially supported by grants from;
The Kempe Foundation, Lions Cancer Research Foundation, University of
Umeå, The Cancer Research Foundation of Northern Sweden, Cancerfonden,
The Maud and Birger Gustavsson Foundation, The Percy Falk Foundation
for prostate cancer research, Sweden, Medical Faculty of Umeå University,
ALF-medel from Västerbottens läns landsting, School of Health and Medical
Sciences, Örebro University.
46
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