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"
j
eutic agent for
Evaluation of prostate secretory protein (PSP-94) as a novel therap
nancy
blocking prostate cancer progression and hypercalcemia of malig
Nicholas Shukeir BSc
Department of Physiology
McGill University
Montreal, Quebec
November, 2002
in partial fulfillment
A thesis submitted to the Faculty of Graduate Studies and Research
of the requirement of the degree of Master in Sciences.
© Nicholas Shukeir, 2002
1+1
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•
Abstract
Human prostate cancer is one of the most common malignancies affecting
men. It is associated with a high degree of mortality and morbidity due to the
development of non-skeletal and skeletal metastases. A common complication in
patients suffering from prostate cancer is the development of hypercalcemia of
malignancy. While detennination of PSA and PSP-94 levels can serve as
diagnostic/prognostic markers, PSP-94 can also serve as a therapeutic agent. The
efficacy of PSP-94 to block tumor progression and hypercalcemia of malignancy was
testcd in our syngcneic il! vivo rat modcl of prostate cancer. Rat prostate cancer Mat
Ly Lu cells were transfected with full length cDNA encoding PTHrP [Mat Ly LuPTHrP] which is known to be the main factor responsible for hypercalcemia of
malignancy. Mat Ly Lu-PTHrP cells werc inoculated subcutaneously into the right
flank or via intracardiac injection into the left ventricle of male Copenhagen rats.
AnimaIs were treatcd with differcnt doses of PSP-94. Tumor volume, time of hindlimb paralysis, plasma calcium and plasma and tumoral PTHrP levels were
determined. PSP-94 caused a significant delay in the development of hind-limb
paralysis, reduction in tumor volume, plasma calcium levels, plasma and tumoral
PTHrP levels as compared to control animaIs receiving vehicle alone. These effects
were due to the induction of tumor cell apoptosis. In conclusion, our studies illustrate
the ability ofPSP-94 to block prostate cancer growth and skeletal metastases .
•
•
Résumé
Le cancer de la prostate est un des cancers masculins le plus fréquemment
diagnostiqué et présente une incidence élévée de mortalité et de morbidité à cause du
développement de métastases à distance osseuses et non-osseuses. Les patients
atteints du cancer de la prostate développent souvent l'hypercalcémie maligne. Bien
que les dosages sériques des marqueurs tumoraux PSA et PSP-94 soient de bons
outils diagnostiques et pronostiques, PSA-94 s'avère aussi un agent thérapeutique.
L'efficacité du PSP-94 à bloquer la progression tumorale et 1'hypercalcémie maligne
a été testée en utilisant notre modèle syngénique in vivo du cancer de la prostate chez
le rat. Les cellules cancéreuses de protaste de rat Mat Ly Lu ont été transfectées avec
l'ADNe pleine longueur de la PTlIrP [Mat Ly Lu-PTHrP], principal agent causal de
l'hypercalcémie maligne. Les cellules Mat Ly Lu-PTI-IrP ont été inoculées à des rats
mâles Copenhague par voix sous-cutanée dans le flanc droit ou par injcction
intracardiaque dans le ventricule gauche. Les animaux ont ensuite été traités avec
différentes doses de PSP-94.
Le volume tumoral, le temps d'apparition de la
paralysie des membres postérieurs, le taux de calcium sérique ainsi que la quantité
sérique et tumorale de la PTHrP ont été mesurés. Comparativement aux animaux
contrôles ayant reçu la substance véhicule seulement, l'inoculation de PSP-94 a
retardé de façon significative le développement de la paralysie des mémbres
postérieurs, réduit le volume tumoral ainsi que le taux de calcium sérique et les
niveaux de PTHrP sériques et tumoraux. Ces effets sont attribuables à l'induction de
•
l'apoptose chez les cellules tumorales. En conclusion, notre étude démontre que PSP-
11
•
94 peut bloquer la progression de la croissance tumorale du cancer de la prostate ainsi
que ses métastases osseuses .
•
111
.'
Acknowledgments
1 would like to thank the people who with their guidance and support made
this work possible. 1 would like to extend my gratitude to Dr. Shafaat A. Rabbani for
giving me the opportunity to work in his laboratory allowing me to pursue my
graduate studies and for providing me with helpful scientific guidance.
1 am also thankful for the wonderful people whom 1 met and had the privilege
of working alongside. Notables include Julienne Gladu, Pouya Pakneshan, Parissa
Khalili-Bourjumani, Jing Guo, Mike Macoritto and Lucie Canaff.
1 am extremcly grateful to Ani Arakelian for teaching me the essentials of
working in an acade111ic laboratory and for providing me with guidance and insightful
hclp throughout the course ofmy graduate studies.
Finally, 1 would like to thank my parents and brother for believing in me and
•
encouraging me with decisions that 1 have made .
•
IV
Abbreviations
•
~-MSP
beta-microseminoprotein
BMPs
Bone morphogenetic proteins
Ca+2
Calcium
CPA
Cyproterone
CRE
Cyclic AMP response clement
DNA
Deoxyribonucleic acid
DRE
Digital Rectal Examination
ECM
Extracellular Matrix
EGF
Epidermal Growth Factor
FBS
Fetal Bovine Serum
FGF
Fibroblast Growth Factor
FSH
Follicle Stimulating Hormone
HCG
Human Chorionic Gonadotropin
I.C.
Intracardiae
I.P.
Intraperitoneal
LH
Luteinizing Hormone
LHRH
Luteinizing Hormone Releasing Hormone
MGA
Megestrol Acetate
MMPs
Matrix Metalloproteinases
mRNA
Messenger Ribonucleic Acid
PAP
Prostatic Acid Phosphatase
v
l'
PDGF
Platelet Derived Growth Factor
PRLR
Prolactin Receptor
PSP-57
Prostate Secretory Prote in of 57 amine acids
PSP-94
Prostate Secretory Protein of 94 amino acids
PTHrP
Parathyroid Honnone Related Peptide
s.c.
Subcutaneous
TGF-a
Transfom1ing Growth Factor Alpha
TGF-p
Transfonning Growth Factor Beta
TRUS
Transrcctal Ultrasound
uPA
Urokinasc
VEGF
Vascular Endothelial Growth Factor
VI
•
Table of Contents
Abstract. ............................................................................................ i
Résun1é ............................................................................................. .ii
Acknow ledgell1ents ............................................................................. .iv
Abbreviations ...................................................................................... v
Table of Contents ................................................................................ vii
List of Figures ............... '" ., ..................... , ......................................... .ix
List of Tables ..... '" ...................... '" ....................... " .......................... .ix
Charter 1: Introduction
Prostate Cancer .......................................................................... 1
Risk Factors for Prostate Cancer ...................................................... 2
,
Screening and Grading of Prostate Cancer .......................................... 4
Biological Progression of Prostate Cancer. ......................................... 7
Prostate Cancer and Metastases ...................................................... 10
Prostate Cancer and PTHrP ......................................................... .. 13
Treatment of Prostate Cancer ......................................................... 14
Discovery of Prostate Secretory Protein of 94 amino acids ...................... 17
.
Gene Organization of Prostate Secretory Protein ................................. 18
Regulation of PSP-94 .................................................................. 19
DifferentiaI Expression of PSP-94 ................................................... 21
•
Actions of PSP-94 ...................................................................... 22
vii
•
Receptor for PSP-94 ................................................................... 25
Objectives of Thesis ................................................. ~ ................. 27
Chapter 2: Prostate Secretory Prote in (PSP-94) decreases tumor growth, metastases
and hypercalcemia of malignancy in a syngeneic in vivo model of prostate
cancer .................................................................................... 28
Introduction ............................................................................. 29
Materials and Methods ................................................................ 32
Materials ........................................................................ 32
Cells and Cell Culture ........................................................ 32
Animal Protocols .............................................................. 33
Histological Analysis ......................................................... 34
Othcr Analytical Mcthods ................................................... 35
Statistical Analysis ............................................................ 35
Results ............................................ '" .................................... 36
Discussion .............................................................................. 57
Chapter 3: General Discussion ................................................................ 61
.
Chapter 4: References ....................................................................................... 70
Appendix ...................................................................................................................... 91
•
VIII
•
List of Tables
1. Clinieai Staging of Primary Tumor (T) ...................................................................... 6
2. Staging by Regional Lymph Node Involvement (N) ................................................. 6
3. Staging by Presence of Distant Metastases (M) ......................................................... 6
List of Figures
Chapter 1.
1. Biological Progression of Prostate Cancer ..................................................... 9
2. Proeess of Metastases ................................................................................... 12
3. Modei of Osteoblastie Bone Metastases eaused by Prostate Caneer........... l3
Chapter 2.
1. Effeet ofPSP-94 on Mat Ly Lu-PTHrP eeU growth ............................ 45
2. Effeet ofPSP-94 on Mat Ly Lu-PTHrP tumor volume ....................... .47
3. Effeet of PSP-94 on animal weight.. ............................................ .49
4. Effeet ofPSP-94 on Mat Ly Lu-PTHrP tumor weight.. ...................... 51
5. Effeet ofPSP-94 on spinal metastases ........................................... 53
6. Effeet of PSP-94 on plasma PTHrP and Calcium in tumor bearing
animaIs .................................................................................. 55
7. Effeet ofPSP-94 on PTHrP production by Mat Ly Lu-PTHrP tumors ...... 57
•
ix
1;
8. Effect ofPSP-94 on DNA fragmentation of Mat Ly Lu-PTHrP cells in vitro
and in vivo ........................................................................................................ 59
•
x
•
Chapter 1
Introduction
Prostate Cancer
Carcinoma of the prostate has developed into a major and escalating
international health problem. In many developed countries, prostate cancer is one of
the most commonly diagnosed cancers in men and is the second leading cause of
cancer mortality following lung cancer (l ,2,4). It is estimated that 18,200 Canadians
will be diagnosed with prostate cancer in 2002 with 4,300 people dying from the
disease (3). Insuflicient data from many developing countries has made it difficult to
accuratcly estimate the worldwide prevalence of prostate cancer. However, the
general incidence has been, on the rise over the last few years with an increasing
number of men bcing diagnosed with prostate cancer. This has been attributed to an
increase in the awarencss of the disease as weil as to bettcr screening programs in
placc. Although there are many risk factors for developing prostate cancer, age is the
primary factor. Autopsies carried out on prostate cancer patients have shown that
prostate cancer is present in 50% of men in their 50s with the number jumping to 70%
of men who are over 80 years of age (2). Enhanced screening programs and improved
treatment modalities for the disease have maintained the mortality rate from prostate
cancer at a steady rate despite the higher incidence in the number of people being
•
diagnosed with this common cancer.
•
Risk Factors for Prostate Cancer
There are many risk factors associated with the development of prostate
cancer. The strongest predisposing factor is age. Men under the age of 50 rarely
exhibit signs of the disease with prostate cancer being quite common in older men
with symptoms ranging from urinary obstruction to bone pain. Other predisposing
factors inc1ude hormones, growth factors, diet, vitamins, dietary supplements,
environ mental factors and viruses (2, 4-19).
A balanced production and regulation of growth factors can therefore control
cell proliferation and cell death in the nom1al prostate (4). Any deviation from this
well controlled balance results in either involution of the prostate or the dcvc10pment
of prostate cancer. Some growth factors that have been shown to be involved include
the insulin-like growth factor family (IGF) (7,9), epidermal growth factor (EGF), the
fibroblast growth factor family (FGF) (16,17), the endothelial growth factors, plateletderived growth factor (PDGF), vascular endothelial growth factor (VEGF) (17) as
weIl as transforming growth factors (TGF) a and p (18,19). Most of the growth
factors mentioned above exhibit a stimulatory role on the proliferation of the normal
prostate. To date, the role of TGF-p remains eontroversial in prostate cancer. Sorne
studies have attributed a stimulatory role to TGF -p (19) while others have shown
TGF -p to exhibit an inhibitory role (18). In addition, androgens also play a major role
in the carcinogenesis of the prostate. Several studies have shown higher levels of
circulating testosterone in patients suffering from prostate cancer. Whereas the
•
precise role of androgens in the induction and initiation of prostate cancer is not yet
2
fully understood, once prostate cancer has been established androgens play a role in
advancing the cancer to its highly invasive phenotype.
The consumption of certain vitamins and dietary supplements appear to offer
sorne degree of protection against prostate cancer. Recent studies have shown that
higher levels of vitamin E and selenium is associated with lower risks of advanced
prostate cancer (10,13,14). Furthermore, vitamin D has becn shown to have a
differentiative and an anti-proliferative effect on human prostate cancer cells thus
promoting its role as a chemotherapeutic agent for malignancy (8, Il,12,15). People
who consume a vegetarian, low fat diet are at lower risk for prostate cancer. Higher
fat intake is not only associated with higher risk of cardiac problems and strokes but
is also associated with increased incidence of developing prostate cancer (5). One
possible mechanism by which high fat levels increase the risk of prostate cancer is by
rcducing the absorption of vitamin A. Higher levcls of beta-carotene, directly rclated
;
to the amount of vitamin A absorption, appear to be protective against the
development of some cancers including prostate cancer (2).
Furthermore,
phytoestrogens, such as genistein and diadzein found in vegetables, may counteract
the effects of androgens on the prostate. Another possible mechanisms by which these
substances exhibit their anti-carcinogenic effects is by inhibition of angiogenesis as
weil as the inhibition of the tyrosine-specific protein kinase associated with the
respective growth factor receptors (2) .
•
3
Screening and Grading of Prostate Cancer
Early detection of this common disease leads to appropriate intervention and
hence a better prognosis. It is highly recommended, by man y cancer societies aIl over
the world, that aH men over the age of 50 undergo annual screening tests in order to
facilitate early detection. There are a number of screening tests available for prostate
cancer. Historically, a digital rectal examination (DREs) was the most commonly
uscd test for detection (2,20,21). However, the utilization of prostate speci fic antigen
(PSA) lcvcls for the early detection of prostate cancer has provided a less invasive,
highly practical altemative screening option. Despite the fact the PSA levels offer the
best performance characteristics by providing a significantly higher predictive value,
these two screening tests should be carried out simultaneously to complement each
othcr since some individuals exhibiting characteristics of prostate cancer, as
,
diagnosed by DRE, have low levels of PSA and viee versa (2). A more reliable
method to screen for prostate cancer is by performing a transrectal ultrasound (TRUS)
(2,22), which has the added advantage ofbeing able to identify suspieious lesions that
are non-palpable by a DRE. However, many factors inc1uding high costs render
TRUS an unsuitable screening modality for the early detection of prostate cancer.
Whereas screening for prostate cancer aIlows for the early detection of the
disease, grading of prostate cancer can serve as an important prognostic marker and a
valuable tool to design therapeutic strategies (23). Over the years, a number of
grading systems have been used but the most commonly applied system is the
Gleason grading system (2,23). It has been shown that not only does the Gleason
4
l,
grading system provide signifieant prognostic information but it has also exhibited
highly reproducible results (2). The Gleason grading system is based on the gIanduIar
pattern of the tumor as identified by microscopic examination of prostatic tissue
un der low magnification (2,23). A 5-step grading system is used with the two grades
being added together to yield the so-called "Gleason grade" which ranges from 2-10.
The higher the Gleason grade the worse the prognosis and vice versa. Since the
treatment options for prostate cancer can vary, diagnosis as well as staging of prostate
cancer is essential for the selection of the most appropriate therapy. The CUITent tumor
staging system used is based on the Tumor (T), Node (N), Metastasis (M) staging
system, also known as the TNM staging (2,24). An example of the TMN staging
system is shown in tables 1-3 (2). As mentioned earlier, ORE and PSA levels arc very
useful tool for sereening for the disease but they arc also hclpful in determining the
stage of the cancer. Combination of PSA levels, clinical stage (TNM) and Gleason
,
score greatly enhances the prediction of the stage at which the cancer is .
•
5
Table 1 Clinical staging of primary
tumor (T) .
TX
TO
Primary tumor cannot be assessed
No evidence of primary tumor
T1
Clinically inapparent tumor not
palpable or visible by imaging
T1 a
Tumor incidental histological finding in
5% or less of tissue resected
Tumor incidental histological finding in
more th an 5% of tissue resected
Tumor identified by needle biopsy (e.g.
because of elevated PSA)
T1 b
T1c
Table 2 Staglng by regional Iymph node
involvement (N)
NX Regional Iymph nodes cannot be assessed
NO No regional Iymph node metastasis
N1 Metastasis in regional node or nodes
T2
T2a
T2b
Tumor confined wlthln the prostate
Tumor involves one lobe
Tumor involves both lobes
T3
Tumor extends through the prostate
capsule
•
MX
Presence of regional metastasis cannot
be assessed
T3a
T3b
T3c
Unilateral extrapsular extension
Bilateral extrapsular extension
Tumor invades the seminal vesicle(s)
MO
No distant metastasis
T4
Tumor invades any of bladder neck,
external sphincter, or rectum
M1
M1 a
M1 b
M1c
Distant Metastasis
Non-regionallymph node(s) metastasis
Bone(s) metastasis
Other sites metastasis
T4a
Tumor invades any of bladder neck,
external spincter or rectum
T4b
Tumor invades levator muscles and/or
the pelvic wall
,Table 3 Staglng by presence.ofdlstant
metastasls (M)';'
" ,
lB) Roger Kirby et al. Prostate Cancer, Second Edition, 2001
•
•
Biological Progression of Prostate Cancer
ssion of
As with other homlOne dependent malignancies, the biological progre
development of
prostate cancer is the result of many events that ultimately lead to the
of the prostate
highly advanced, metastatic prostate cancer (figure 1). Carcin oma
ps into a
develo ps when the norma l epithe lium of the prosta te gland develo
c mutations
histological cancer as a result of many factors. These include geneti
There are no
causing the inactivation or silencing of tumor suppressive genes (2,29).
diagnosed by
symptoms experienced by the patient at this stage and the cancer is only
enic effects
histological examination of the prostatic tissue. Persistence of these mutag
addition to the
leads to additional tlll110r suppressive genes being silenc ed in
enes that have
activation of tllmor promoting genes, the proto-oncogencs. Some oncog
Her-2 /neu and
been shown to be upregu lated in prosta te cance r includ e the
lasia or a
PTPC AAX2 oncogenes. (30,31 ). As a result benign prosta tic hyperp
enced by men,
localized primary tumor develops. The most common symptom experi
w obstruction,
when the cancer is at this stage, is associated with bladde r outflo
plete emptying
namely reduced urinary outflow and frequency accompanied by incom
(2). Up until this
of the bladder as a result ofpros tatic tissue obstruction of the urethra
dent state.
stage of prostate cancer progression, the cancer is in a hormo ne depen
development of
However, if the cancer is left undiagnosed and untreated then the
Sorne of the
androg en-ind epend ent and metastatic prostate cance r is inevitable.
•
gens) (1,25)
factors which facili tate this progression include sex steroids (andro
nes (28). A very
growth factors (4), angiogenic factors (27), proteases (26) and cytoki
7
common feature in prostate cancer is the development of skeletal and non-skeletal
metastases (32,33). As a result of systemic metastases, the patient will complain about
signs oflethargy, weight loss and cachexia and hemorrhage (2). If the cancer reaches
the bone, th en pain is the primary complaint of patients with ultimate development of
paraplegia as a result of spinal cord compression (34).
8
•
•
•
Prostate Cancer and Metastases.
Primary prostate tumors which are localized within the capsule are rarely
associated with mortality. Rather it is the development of tumor metastases at distant
sites of the body that is the deadly factor. Metastasis to the bone is a frequently
observed complication in patients who suffer from advanced prostate cancer (32,33).
While skeletal metastases can be of the osteolytic or ostcoblastic variety those
observed in patients with prostate cancer are usually of the osteoblastic phenotype
(35,36,37). It is believed that these osteoblastic metastases are the result of cancer
cells producing factors that stimulate osteoblast proliferation, diffcrentiation and
cvcntually bone formation (36). As shown in figure 2, the process of bone metastascs
is very similar to that of metastases to any other site in the body (36). Once the
primary tumor rcaches a certain size, a phenomenon known as the "angiogcnic
switch" occurs which is éharacterized by the recruitmcnt of new blood vessels.
Primary tumor ceUs growing in the site of origin invade into their surrounding tissue
by producing proteolytic enzymes which help breakdown the extracellular matrix
(ECM).
Such
proteolytic
enzymes
inc1ude
urokinase
(uPA)
and
matrix
metaUoproteinases (MMPs) (26,36). Breakdown of the ECM aUows tumor ceUs to
traverse the waUs of smaU blood vessels present in the primary organs or the newly
,
recruited ones and enter the circulation allowing the tumor cells to reach distant sites.
This process is quite inefficient and many circulating tumor ceUs do not survive the
normal protective mechanisms. The few ceUs that manage to survive and reach their
secondary organ, bone or other tissues, extravasate from the blood vessels into the
10
••
stroma of the organ. There they begin groW1l1g aga1l1 with the whole process
beginning reinitiated resulting in the development of tumor metastases at multiple
sites.
There is increasing evidence to indicate that certain growth factors are
involved in the stimulation of bone formation which is associated with metastatic
prostate tumors. Patients with prostate cancer have increased levels of endothelin-l
which is known to stimulate bone formation and osteoblast proliferation (38,39). The
TGF-p superfamily as weIl as the bone morphogenetic proteins (BMPs), namely
BMP-2, BMP-3, BMP-4 and BMP-6 have been shown to be stimulators of bone
formation (36). Othcr growth factors that have been shown to be increased in patients
with prostate cancer include the FGFs and PDGFs (4). Furthcrmore, while an amino
terminal fragment of uPA has becn shown to cxhibit mitogenie activity for osteoblasts
(40), the carboxy terminal protcolytic domain mediates tumor invasiveness and
growth factor activation (36). In addition, studies have shown that expression of uPA
by prostate cancer ecUs leads to an increase in the metastatic ability of the cells
leading to an increased number of osteoblastic bone leasions (41). A model for
osteoblastic bone metastases caused by prostate cancer is summarized in figure 3 .
•
Il
-
,le
The Process of Metastasis
Prirnary malignant
neoplasrn
Neovascularization
Invasion
~
"
•
~.
~
""'"
""'"
Adherance
Arrest in distant organ
capillary bed
t
ft
~
Response to
rnicroenvironrnent
..
Turnor cell
proliferation
Ernbolisrn
Multicell aggregates
~
,-
Extravasation
Metastasis
®
Gregory Mundy, Nature Reviews, 2, 2002
'.
~
BMPs
latentTGF-p
uPA
~~
active TGF-p
•
~
PTHrP
~
Proteases
J
---
~
Inactive
PTHrP
fragments
+
Bone Formation
®
Gregory Mundy, Nature Reviews, 2, 2002
FGFs
•
Prostate cancer and PTHrP
ion of
A common observation in prostate cancer patients is the over-product
that PTHrP is
PTHrP in cases of advanced prostate cancer (42,43). It is now believed
seen in many
the major factor respon sible for hyper calcem ia of malig nancy
and prosta te
carcin omas inc1uding breast carcin omas, renal cell carcin omas
with maximal
carcinomas (44, 45). The expression ofPTH rP increases progressively
expression in highly malignant prostate cancer cells (46).
te cancer
The growth promoting effects of PTHrP were studied in many prosta
the rat prostate
cclI lines including the hum an prostate cancer PC-3 cell line as weB as
by exogenous
cancer Mat Ly Lu cell line. Increased expression of PTHrP either
resulted in
addition of PTHrP peptides or stable transfection of PTHrP cDNA
growth (47,48).
increased tumor cclI growth as weB as accelerated primary tumor
tory role of
Examination of the mechanism of action of PTHrP revealed an inhibi
apoptotic agents
PTHrP on apoptosis causing increased ceU survival after exposure to
(47,48 ).
ration,
ln addition to PTHrP s role in osteoblast differentiation and prolife
ial (36,49).
PTHrP has also been associ ated with increa sed metas tatic potent
tases from
Immunohistochemical and in situ hybridization analysis of bone metas
cant number of
prostate cancer patients revealed the expression of PTHrP in a signifi
expressed not
samples (46). Furthermore, the PTHrP receptor was also shown to be
from prostate
only in the primary prostate tumor but also in the bone metastases
•
cancer patients (46).
14
•
Treatment of Prostate Cancer
in
The stage at which prostate cancer is at can serve as the primary factor
patients with
choosing an effective treatment modality (50). Treatment options for
on therapy and
localized prostate cancer inc1ude watchful waiting, surgery, radiati
the prostatic
brachytherapy which involves the insertion of radioactive pellets within
gical prostate
tumor to deliver a localized form of radiotherapy. Patients with histolo
are carefully
cancer and suffering from no symptoms usually receive no treatment and
regularly by
monitored for any symptoms that might develop. Patients are monitored
rate of disease
DREs and PSA levels to determine the patien t's condit ion and
progression, and furthcr decisions are made accordingly.
Since these kinds of
age of 70, the
prostate cancer are diagnosed accidentally with patients being over the
ly die with
rationalc for using such an approach is that the patients will most probab
prostate cancer ratbcr tban afthe disease.
ete
Radical prostatectomy is a surgical procedure which involves the compl
excision of the
prostat~
gland, the seminal vesic1es and adjacent tissues (2,50).
to afford a
Currently, radical prostatectomy is used in patients in whom it is likely
ate any chance
cure, in effect, only in those where the surgery will completely elimin
of the surgery,
of metastatic disease developing. In order to improve the outcome
androgen
patient with larger tumor volumes undergo a regim e of preoperative
.
y inc1uding
depriv ation (50,52). Many disadv antage s of radica l prosta tectom
the use of
impotence, incontinence, rectal injury and potential mortality have limited
this radical procedure .
•
15
•
An alternative treatment approach for localized prostate cancer that has
proven efficacy is radiation therapy. The objective of radiation therapy is to achieve
the highest possible tumor dose with minimal radiation injury to the surrounding
nom1al tissue. Generally, the criteria for patients considered for radiation therapy are
similar to those being considered for radical prostatectomy. Brachytherapy is a
procedure that involves the insertion of radioactive pellets into the prostatic tumor to
localize the effects (50,51). The advantages of brachytherapy over radiation therapy
include the usage of low dose radiation as weIl as conformai seed plantation which
can be carried out in one setting as opposed to multiple fractions required for external
bcam radiation. The absence of surgery is a major bene fit for patients sclecting
radiation therapy which aIlows this form of therapy to be applied to paticnts with less
favorable, general medical condition.
Duc to the established role of androgens on the initiation and progression of
prostate cancer, hormonal' therapy is the trcatmcnt option for locally advanccd
prostate cancer. The basis of hormonal therapy is the dependence of the prostate
gland as weIl as prostate cancer celIs on androgens (2,50,52). Several methods of
achieving hormonal therapy are available. Bilateral orchiectomy leads to symptom
relief in a large percentage of symptomatic patients. Bilateral orchiectomy leads to the
removal of most of the circulating testosterone at once. However these modalities are
associated with side effects such as loss of libido and psychological effects (2,50).
Other less invasive methods of hormonal therapy include the administration of
,
leutinizing hormone releasing hormone (LHRH) agonists. Although low doses of
LHRH agonists mimic the actions of LHRH, superphysiological doses were found to
16
•
downregulate the LHRH receptor with subsequent decrease in LH and circulating
testosterone levels (50). Anti-androgen agents are another form of hormonal therapy.
These agents compete for androgen receptors minimizing the effects of androgens.
Two types of anti-androgen agents exist: steroidal which have significant
progestational activity and nonsteroidal (50). Examples of steroidal anti-androgen
agents include cyproterone (CP A) and megestrol acetate (MGA), whereas flutamide
and nilutamide make up non-steroidal agents.
Prostatic tu mors are composed of a heterogeneous subpopulation of cells with
some being hormone-dependent while others are hormone-independent. Hormone
deprivation abolishes the hormone-dependent subpopulation but lacks the similar
desirable effect on the hormone-indepcndent subpopulation. As a result the hormoneindependent cells survive, begin to proliferate and repopulate the prostatic tumor.
Consequcntly hormonal therapy is almost always followed by a recurrence of the
(
disease (2). Despite advanees in diagnosis and treatmcnt modalities, prostate cancer
continues to pose challenging therapeutic problems. This has led to the evaluation of
additional novel therapeutic agents to control disease progression. Among these
approaches gene therapy (53), protease and growth factor inhibitors and chemical
agents which can block various intracellular signaling pathways have shown
promising effects and can be developed for patients with advanced hormone
refractory form ofprostate cancer (2) .
•
17
•
Discovery of Prostate Secretory Protein of 94 amino acids
The notion that a nonsteroidal, gonadally derived inhibitory substance, an
"inhibin" that is capable of inhibiting pituitary FSH levels was first put forward by
McCullagh in 1932 (54). This notion came about wh en castration of male rats
resulted in prostatic hypertrophy which could be reversed by the introduction ofwater
soluble testicular extracts. This observation as well as reduction of FSH levels in men
following castration or irradiation of the testis led to the original thought that the
testes were the primary site of production of "inhibin". However it became clear that
the production of "inhibin" extended beyond the testes when serum FSH levels were
suppressed following administration of ovarian follicular fluid into proestrous rats
(55). Dcspite mounting evidencc of the existence of such an inhibitory hormone it
was not until llluch later, with the development of sensitive and specifie
radioimmunoassays for inhibin (56), that the isolation and purification of inhibin took
place. At that time, inQibin was purified from bovine and porcine follicular fluid;
however isolation of inhibin from the human followed shortly thereafter (57). It was
shown that inhibin was formed through a dimeric assembly of an a subunit and one
oftwo c10sely related ~ subunits (~A or ~B) (58). Dimerization of the a subunit with
either the ~A or ~B yielded inhibin A and inhibin B respectively (58).
When the inhibin genes were isolated and c10ned it was realized that inhibin
purified from human seminal fluid was not related to the dimeric inhibin. The
•
distinction stemmed from prostatic inhibin being a single polypeptide chain of 94
amino acids instead of being a dimer of a and ~ subunits (59,60). Despite this
18
difference between the two inhibins, they both share the same mode of action of
inhibiting FSH levels without altering the levels of LH (62). Alternative names used
to describe prostatic inhibin include prostate secretory protein of 94 ami no acids
(PSP-94),
~-microseminoprotein
W-MSP) and sperm-coating antigen (103). Since its
discovery it has been shown that PSP-94 is one of the tluee predominant proteins
secreted by the prostate gland along with PSA and P AP (61). The gene eneoding PSP94 as well as the complete amino acid sequence of PSP-94 have since been
determined (63,64,65,66,67). Furthermore, sinee its first isolation from the prostate
gland it has been demonstrated that PSP-94 expression is not restrieted to the prostate,
as the protcin is present in other tissues of the human body (68). Partieular tissues that
have becn shown to express PSP-94 include mueosal structures stlch as the
tracheobronchial tract and the stomaeh (66,68). Morcover, female reproductive
tissues have also been del1lonstrated to express PSP-94 particularly breast, ovaries
.
and endomctrial tissues (69). Dcspite the vast array of tissues that express PSP-94 the
prostate glad expresses
~he
majority of PSP-94 as eompared to other tissues (69).
Gene Organization of Prostate Secretory Protein
Human PSP is eneoded by a single gene that resides on chromosome
10 (q 11.2) (70,71). The human PSP gene spans approximately l4kb long and consists
of four exons (J, II, III and IV); alternative splieing of these exons yields either PSP94 or PSP-57 (72). Three out of the four exons are present in both transcripts: exon l,
exon II and exon IV (72). Complete dcletion of exon III yields PSP-57 (72). The
•
upstream region of the human PSP gene spans about 2.8kb, however the majority ofit
19
li
is not necessary for basal promoter activity as determined by luciferase activity
(73,74). The promoter reglOn contains many putative transcription regulatory
elements for ubiquitous transcription factors (73,74). Sorne of these regulatory
elements include a TATA box, a cAMP response element (CRE) and three
glucocorticoid response elements (73,74). The structural organization of the gene
with its promoter and multiple transcription regulatory elements along with the
potential for alternative splicing suggests tissue specific expression of the different
isofonns of PSP.
Of these two isoforms, the 94 amino acid isofonn is the common isoform in
humans as determined by mRNA lcvcls (72). Furthcrmore, it appcars that PSP-57
expression is more tissue specific than that of PSP-94. Other th an the prostate gland
whcre both isoforms arc cxpressed, it appcars that the expression of PSP-57 and that
of PSP-94 follow an inverse relationship whereby the majority of PSP-57 is expressed
in tissues that do not express PSP-94 and vice versa. Such examples include PSP-57
expression in kidney and bladder tissues where no PSP-94 expression ean be detected
and PSP-94 expression in lung and breast where PSP-57 is not expressed (72). PSP94 and PSP-57 share the same 5' untranslated region as weIl as an amine acid
sequence corresponding to a signal secretion peptide which is cleaved before
secretion of the proteins (72).
Regulation of PSP-94
With the cloning of the PSP gene, the regulatory mechanism(s) of human
PSP-94 production are being elucidated. The regulation of PSP-94 has been mostly
20
•
are androgen
studied in benign hyperplastic prostatic tissues and LnCAP cells which
are found to
responsive, non-invasive prostate cancer celllin es (74,75,76). Honno nes
with G-protein
be the potent regulators of PSP-94 expression through interaction
an increase in
coupled receptors leading to activation of adenylyl cyclase resulting in
levels lead
cAMP (74). Most honnones that lead to an increase in intracellular cAMP
n levels in
to a transient increase in the levels of both PSP-94 mRNA and protei
were shown to
human prostate ceUs. Specifical1y, the levels of PSP-9 4 protei n
tic tissues were
increase in a dose-dependent manner when benign hyperplastic prosta
ted prostatic
incubated in vitro in the presence of FSH or LH as compared to untrea
l hormones
tissues (75,76). Furthe rmore treatment of LnCAP cells with severa
l has been
inc1uding epincphrine, vasoactive intestinal peptide (VIP) and isoprotereno
r through the
shown to stimulatc the expression of PSP-94 in a dose-depcndent manne
of cAMP was
increased produ ction of PSP-9 4 mRNA (74). The precis e role
ction were
elucidated wh en the same effect of increases in PSP-94 mRNA produ
forksolin(74).
reproduced upon incuba tion of LnCAP cells in the presen ce of
of PSP-94, the
Whereas the majority of hormones lead to an increase in the levels
manner when
levels of PSP-94 protein were shown to be reduced in a dose-dependent
presen ce of
benign hyperplastic protatic tissues were incubated in vitro in the
(75,76)
honnones such as prolactin and human chorionic gonadotropin (HCG)
ned for
Sex steroids such as testosterone and estrogen have also been exami
lastic prostatic
any possible effect on PSP-94 gene expression in both benign hyperp
•
of these sex
tissues and LnCAP cells. However, it has been demonstrated that none
en-independent
steroids has an effect on PSP-94 expression leading to the androg
21
•
nature of PSP-94 expression. (75,76). Furthermore immunohistological examination
ofPSP-94 levels in benign prostatic tissues coming from patients that have undergone
androgen deprivation therapy and those who have not been exposed to the therapy
revealed that the production of PSP-94 was not under the influence of androgens (77).
Differentiai Expression of PSP-94.
PSA and PAP levcls were the standard prostate cancer markers due to their
differential levels of expression as the prostate cancer progresses (78). Utilizing a
number of different techniques including in situ hybridization, immunohistochemistry
and detcrmination of PSP-94 mRNA levels, it has been demonstrated that the
expression of PSP-94 is diffcrcntial depending on the stage of prostate cancer
(77,79,80,81). The levels of PSP-94 arc quite high is normal prostatic tissue and
,
exhibit a progressive dccline as the prostate cancer advances from a low invasive,
highly differentiated,
aI~drogen-dependent
state to a poorly differentiated, androgen-
independent state with complete lack of PSP-94 expression in highly advanced
prostate cancer. This di fferenti al expression has allowed PSP-94 to have a
diagnostic/prognostic value comparable to that of PSA and P AP (82,83). While PSA
and P AP levels are androgen dependent, PSP-94 levels are not dependent on the
.
levels of circulating androgen (77,79). This provides PSP-94 with an added advantage
as a diagnostic/prognostic marker in tumors that have been previously exposed to
•
androgen ablating agents .
22
•
The expression ofPSP-94 in other tissues of the human body allows for its use
as a biochemical diagnostic/prognostic marker in cases other than prostate cancer.
Although less common th an prostate cancer, gastric carcinoid tumors affect as much
as 10% of the population and are quite often associated with poor prognosis as a
result of difficulty in diagnosing the disease (84). The expression of PSP-94 in gastric
carcinoid tu mors follows an opposite course to its expression in prostate cancer. In a
study carried out by Femlund et al it was shown that the expression of PSP-94 in
tumor tissues collected from patients with gastric tumors correlated with tumor
diameter and tissue invasion depth and subsequently with tumor progression where
highly invasive, metastatic tumors stained positive for PSP-94 whilc less invasive
tumors of smaller diametcr stained negative for PSP-94 (84). With PSP-94 levels
bcing easily measured by the use of radioimmunoassays, PSP-94 can serve as a useful
marker in gastric carcinoid tUll1ors. Furthermore PSP-94 is expressed in man y female
tissues including ovarian, éndometrial and brcast tissues (69). Just like in prostate
cancer, PSP-94 levels decrease progressively as breast cancer progresses with lack of
PSP-94 in late stage breast cancer. This differential expression of PSP-94 allows it to
be a diagnostic/prognostic marker in breast cancer.
Actions of PSP-94
.
The main biological function of PSP-94 is the suppression of FSH levels (62).
Although the pituitary gland is the main biological site for production of FSH, it has
been demonstrated by Garde et al that the prostate gland is an extrapituitary source of
•
FSH (85). Furthermore, increased levels of FSH levels in cases of benign prostatic
23
•
prostate gland
hyperplasia cornbined with the discovery of FSH recept ors on the
prostate gland
suggest an autocr ine/pa racrin e mode of action of FSH on the
and through
(86,87,88). In the normal prostate, PSP-94 production is quite significant
gh the precise
a negative feedback loop FSH levels are kept at constant levels. Althou
r, a study
mechanisrn by which PSP-94 acts to reduce FSH levels remains unclea
sis of the 3-D
carried out by Jyothi et al shed sorne insight into this matter (89). Analy
en regions in
structure of PSP-94 revealed structural and functional similarity betwe
ility that the
PSP-94 and FSH (89). A hypothesized rnechanism favors the possib
for the FSH
function-mimicking domain of PSP-94 might trigger a false recognition
ce of PSPreleasing system and thus lead to a suppression of FSH levels in the presen
94.
ds.
Traditionally, women were the major targets for most contraceptive metho
d at men with
Condoms and vasectomies are the main contraceptive methods directe
ch to male
each of them having their own limitations. A hormonal, reversible approa
ception. The
contraception would all<;>w for a safe and non-invasive method of contra
on the optimal
basis of hormonal contraception is that spennatogenesis is dependent
n of PSP-94
concentration of gonadotrophin secretion, FSH (90,91,92). Administratio
spermatogenic
results in suppression in the levels of circulating FSH causing loss of
(91,92). On the
activity in the testis resulting in a condition known as azoospermia
levels of FSH
other hand, severa! studies carried out have also shown that increased
d testicular
lead to impairment of spermatogenesis, agglutination of sperm and reduce
•
resulting in a
weight as a result of a decrease in serniniferous tubule diame ter
of FSH were
drarnatic decrease in fertility (93,94,95,96). Increases in the levels
24
brought about by immunization against PSP-94 with antibodies as well as
introduction of synthetic peptides corresponding to regions of PSP-94. lt is speculated
that the mechanism by which increased levels of FSH lead to the above-mentioned
effects is by down regulation of FSH receptors on the testis following persistent
elevation of serum FSH levels (93,94). In addition the antibodies raised against PSP94 were found to affect sperm function by damaging the membrane integrity of the
spermatozoa (95).
Although the mam function of PSP-94 is the inhibition of FSH, PSP-94
possesses other functions as weil. As mentioned previously, although the majority of
PSP-94 is synthesized by the prostate gland in the male, PSP-94 is found in brcast,
endometrial and ovarian tissues in the female (69). PSP-94 has been found to
suppress levcls of circulating prolactin in the female (97). Since prolactin is known to
be the hormone responsible for lactation, PSP-94 leads to suppression in lactation by
.
acting on the hormone prolactin. Prolactin is also iI1volved in the proper maturation
and functioning of the corpus lutcum (98,99). Excess PSP-94 leads to improper
preparation of the uterus for embryo implantation which is dependent on the
continuous secretion of estrogen and progesterone by the corpus luteum (98,99).
Interestingly, prolactin has been shown to be involved in proper ossification of
bone. Histomorphometric analysis of bone in prolactin receptor (PRLR) knockout
animais revealed lower bone formation rate and reduced bone density (99). Thus PSP94 should exhibit an indirect effect on bone morphology through inhibition of
prolactin resulting in decreased osteoblastic activity, although the precise role of PSP-
•
94 on bone turnover has not yet been elucidated.
25
•
The mode of action of the prolactin suppression effect of PSP-94 rernained
unclear until the recent study carried out by Jyothi et al which provided sorne insight
explaining how PSP-94 carries outs it effect (89). By carrying out docking
experiments, it was discovered that more than 70% of the residues between the PSP94/prolactin and prolactin/prolactin receptor complexes were similar. Moreover, the
remaining 30% of the residues on prolactin have low affinity to the prolactin receptor.
Thus it appears that PSP-94 suppress lactation by directly acting on the crucial
binding sites of prolactin and preventing its binding to the prolactin receptor (89).
Briefly, other physiological actions that have been associated with PSP-94
inc1ude the inhibition of phytohacmagglutinin (PHA) stimulatcd proliferation of
pcriphcral blood mononuc1ear cells (100), suppression of DNA synthesis in the
prostate gland (101) and anti-prolifcrativc cffects on fibroblastic cclllincs spccifically
NRK-49F and Balb/c 3T3 cells (102).
Receptor for PSP-94
Although a great deal is known about the regulation of PSP-94 production and
its function in the reproductive as well as other systems, very little is known about
how PSP-94 exerts its effects on the cellular and molecular lcvels. The failure of
identification and characterization of a putative PSP-94 receptor(s) is rcsponsible for
this lack of knowledge. With advances in molecular biology techniques as well as
cloning procedures, failure to identify and clone a PSP-94 receptor would lead one to
speculate that none exist. As a result, PSP-94 actions may be via perturbation of the
•
signaling cascade of other messengcrs as opposed to PSP-94 possessing a separate
26
•
signaling mechanism. The recent study carried out by Jyothi et al whereby PSP-94
domains were shown to cxhibit structural and functional homology to that of FSH
leads to the speculation that reduction of circulating FSH levels would be due to
alterations in the homeostasis of FSH rather than PSP-94 exhibiting a direct effect on
the levels of FSH (89). Furthermore, it was also shown that PSP-94 reduces
circulating prolactin levels by directly interacting with crucial binding sites of
prolactin preventing its binding to its receptor. So could it be that PSP-94 exerts its
effects solely by interrupting the signaling cascade of other messengers as opposed to
initiating its own separatc signaling cascade?
Several studics carricd out sinee the discovery of PSP-94 indicate otherwise.
Utilizing competitive inhibition binding assays, it was shown th nt addition of
unlabelled PSP-94 inhibited specifie binding of labelcd PSP-94 to LNCaP cclls (103).
Furthermorc the same binding inhibition pattern was also observed in PC-3 cells
.
(104). This indicated the presence of binding proteins specific for PSP-94. The
presence of specific "binding proteins" for PSP-94 was also shown on the prostate
gland (105). However, does the presence of these 'binding proteins" on the surface of
the cells imply that they are the receptors for PSP-94? A number of criteria have to be
met before one can indeed confirm the presence of such binding proteins (106). First,
it needs to be established that these binding proteins are expressed in known PSP-94
target tissues. In particular, they must be expressed in the prostate gland since it is the
gland that expresses the highest amount of PSP-94 compared to the other tissues that
express PSP-94. Second, the binding proteins must bind PSP-94 with high affinity.
•
Third, PSP-94 must exhibit high specificity for the binding protein, however, other
27
.\
ligands can have low-affinity association with the binding prote in. Although results
from several studies have indicated that these three criteria have been satisfied, the
final criteria that has to be met before these "binding proteins" can be identified as
ptoteins involved in PSP-94 mediated effects is their ability to initiate a PSP-94
signaling pathway to direct it's biological effect in target tissues.
Objective of Thesis
Developmcnt of novel thcrapeutic modalities for prostate cancer is important
in being able to combat this common disease. The objectives of the thesis are to
evaluate the efficacy of prostate secretory protein of 94 amino acids (PSP-94) in
blocking prostate cancer progression and its associated hypercalcemia of malignancy.
In addition, a mcchanistie basis for the action(s) of PSP-94 was cxplorcd .
•
28
·)
Chapter 2
Prostate Secretory Protcin (PSP-94) decreases tumor growth, metastases and
hypercalcemia of malignancy in a syngeneic in vivo model of prostate cancer.
Nicholas Shukeir,l Ani Arakelian,l Salam Kadhim 2, Seema Garde2
and Shafaat A. Rabbani 1
IOepartment of Medicine, Physiology and Oncology, McGill University Health
Centre, Montreal, Quebcc, Canada and 2Procyon BioPharma Inc, Montreal, Quebec,
Canada
The study in this chapter is aimed at evaluating the ability of PSP-94 to block
prostate cancer progression and its associated hypercalemia of malignancy in a
syngeneic in vivo model of prostate cancer. The study is presented in the form of a
paper to be submitted for publication. 1 was responsible for a11 the experimental work
present in this chapter. 1 appreciate the advice of Dr. Rabbbani and Ani Arakelian
during the study. In addition, many thanks and appreciation goes out to Sa11am
Kadhim and Seema Garde for providing PSP-94 as we11 as their insightful advice .
•
,
29
•
Abstract
Prostate cancer is a common malignancy affecting men, which is often associated
with skeletal metastases to cause high incidence of morbidity and mortality. In this
hormone-dependent cancer, prostate specific antigen (PSA) and prostate secretory
protein of 94 amino acids (PSP-94) are known to serve as prognostic markers for
disease progression. In the CUITent study we have examined the effect of PSP-94 on
prostate cancer growth and metastases to the skeleton. For these studies, Mat Ly Lu
rat prostate cancer cells were transfected with full length cDNA encoding parathyroid
hormone rc1ated protein (PTHrP) [Mat Ly Lu-PTHrP], which is known to be the
major pathogenetic factor for malignancy associated hypercalcemia. Mat Ly LuPTl-IrP cells were inoculatcd subcutaneously (S.c.) into the right flank or via
intracardiac route (I.c.) into the left ventricle of syngcneic male Copenhagen rats. I.C.
~
inoculation of Mat Ly Lu cells routinely results in tumor metastases ta the lumbar
vertebrae rcsulting in hind-limb paralysis. AnimaIs were infused with different doses
of PSP-94 (0.1, 1.0 & 10.0 )..tg/kg/day) starting on the day of tumor cell inoculation.
Time of hind-limb paralysis and tumor volume were determined and comparison was
made between PSP-94 treated animaIs and control animaIs receiving vehicle alone. At
the end of the study animaIs were sacrificed and plasma calcium, plasma PTHrP and
tumoral PTHrP levels were determined. While the highest dose of PSP-94 caused a
modest but statistically significant delay in the development of hind-limb paralysis a
marked dose-dependent decrease in primary tumor volume was seen in experimental
•
animaIs receiving PSP-94 due to its ability to promote tumor celI apoptosis.
30
1\
Furthermore, while control animaIs routinely developed hypercalcemia due to PTHrP
production, treatment with PSP-94 led to a near normalization of plasma calcium and
a marked reduction in PTHrP production as determined by radioimmunoassay and
immunohistochemistry. Collectively, these results demonstrate the ability of PSP-94
to be an effective treatment modality for prostate cancer where decrease in plasma
PTHrP and calcium levels can serve as useful biochemical markers for monitoring the
efficacy of this novel anti-tumor agent.
•
31
Introduction
Prostate cancer is one of the most commonly diagnosed cancers in men and is the
second leading cause of cancer mortality following lung cancer (107). A distinct
feature of prostate cancer is its ability to cause osteob1astic skeletal metastases which
contributes to the high rate of morbidity and mortality associated with this hormone
dependent malignancy (32). Additionally, a significant number of patients with
prostate cancer exhibit an increase in their plasma calcium levels due to the
production of PTHrP by tumor cells (42,43). Several studies have provided
convincing evidence that indeed PTHrP is the major pathogenetic factor responsible
for hypercalcemia of malignancy whieh is observed in 15-20% of ail cancer patients
(44,108). The progression of clinical prostate cancer can be blockcd at its early stage
whcn the cancer is weil confined within the prostate gland (109). However, increased
production of many factors including growth factors, sex steroids, angiogcnic factors,
,
and proteases such as urokinasc (uPA) and matrix metalloproteinases (MMPs) by
tumor ceUs and their surrounding stroma results in progression towards a highly
invasive hormone-independent state which ultimately leads to metastatic prostate
cancer associated with high mortality (1,25,41,49). Despite recent advances in the
therapeutic modalities for prostate cancer including surgery and radiotherapy, limited
suc cess has been obtained in treating hormone-independent prostate cancer (110).
PSP-94, a 94 amino acid, cysteine rich non-glycosylated prote in, is one of the
three predominant proteins secreted by the prostate gland and found in human
seminal fluid along with prostate specifie antigen (PSA) and prostatic acid
•
phosphatase (P AP) (61,111). Alternative names used to describe PSP-94 inc1ude
32
.\
prostatic inhibin
(~-inhibin)
and
~-microseminoprotein
(l03). One of the mam
1
biological functions of PSP-94 is the inhibition of follic1e stimulating hormone (FSH)
(112). While the majority of FSH is produced and secreted by the pituitary gland it
has been demonstrated that the prostate gland is an extrapituitary source of FSH (85).
Elevated levels of FSH, in cases of benign prostatic hyperplasia, along with the
presence of FSH receptors on the prostate gland suggest an autocrine/paracrine
regulation of this hormone on prostate proliferation (86,87). Decreased levels of PSP94 in patients suffering from prostate cancer as weIl as the development of multiple
gonadal tumors have implicated a tumor suppressive role for PSP-94 (82).
Several studies have demonstrated a progressive decrcase in PSP-94 expression
as prostate cancer progresses from a hormone-depcndent to a hormonc-independent
state with complete lack of PSP-94 production in highly advanced metastatic prostate
cancer (80). This differential expression has allowed PSP-94 to have a prognostic
value for prostate cancer (79,83). One added advantage of PSP-94 as a prognostic
marker is our ability to, determine its level of production in the hormone-independent
stage of the disease that allows for higher degree of sensitivity in tumors that have
been previously been exposed to androgen ablating agents (77).
In the present study, we have evaluated the effect of PSP-94 to decrease prostate
cancer tumor growth and metastases. For these studies we have used our weil
characterized syngeneie in vivo model of rat prostate cancer using the rat prostate
cancer ceU line Dunning R3227 Mat Ly Lu transfected with the full length cDNA
encoding rat PTHrP (49). In this model, S.c. inoculation of tumor cells into the right
•
flank of male Copenhagen rats routinely results in the development of primary tumors
33
•
whereas I.e. inoculation of tumor ceUs leads to skeletal metastases at lumbar vertebra
causing hind limb paralysis (41,49,113). Following S.e. and I.e. inoculation of Mat
Ly Lu-PTHrP cells, the ability of different doses of PSP-94 to reduce tumor growth,
metastases, tumoral PTHrP production, plasma calcium and PTHrP was evaluated .
•
34
Materials and Methods
Materials. PSP-94 was a gift from Procyon BioPharma Inc. (Montreal, Quebec,
Canada) (57).
CeUs and cell culture. The Dunning R3327 Mat Ly Lu ceU hne was obtained
from Dr. J. T. Isaacs (John Hopkins School of Medicine, Baltimore, MD) and
transfected with full length cDNA encoding rat PTHrP as previously described (49).
One of the three weIl characterised monoclonal cell hnes Mat Ly Lu-PTHrP-8 was
used throughout the course of these studies. Cells were maintained in vitro in RPMI
1640 supplcmented with 2 mM L-glutamine (Life Technologies, Ine. Grand Island,
N.Y.), lO cYo fetal bovine serum (FBS), 100 units/ml penicillin-strcptomycin sulphatc
(Life Technologies, Inc.), and 250nM dexamcthasonc and G418 (600mg/ml)
according to previously established mcthods of culture of these experimental ecUs
(Il ).
Morphological analysis of control and experimental Mat Ly Lu-PTHrP cells
treated with PSP-94 was carried out by plating 5x 104 ceUs/ weU in 6-well plates
(Falcon Plastics, Oxnard, CA) in the presence of 10% FBS. CeUs were examined
daily for any change in their morphology and photographed (114).
Effect of PSP-94 on Mat Ly Lu-PTHrP tumor ceU invasive capacity was
examined by 2-compartment Boyden Chamber (Transwell, Cu star, Cambridge, MA)
and basement membrane Matrigel (Becton Dickinson Labware, Bedford MA) as
previously described (115) .
•
35
For growth curves, Mat Ly Lu-PTHrP cells were plated in 6-well plates (Falcon
Plastics, Oxnard, CA) at seeding densities of 5x103 cells/well. For 3 days, cells from
triplicate wells were cultured in the presence of different doses of PSP-94 (0.1, 1.0 &
10.0 Ilg/ml), trypsinized, resuspended, and counted in a model Z Coulter counter
(Coulter Electronics, Beds, UK). Medium was changed every two days.
For DNA fragmentation, Mat Ly Lu-PTHrP ceUs were plated in 6 well plates
(Falcon Plastics, Oxnard, CA). CeUs were treated with PSP-94 (10.0 Ilg/ml) for up to
72 hours. DNA from treated cells incubated with PSP-94 and ceUs treated with
vehicle alone was extracted using a Phenol:Choloroform:Isoamyl alcohol solution
(50:48:2). Equal amounts of DNA were subjected to gel electrophoresis on a 2%
agarosc gel. DNA fragmentation was visualised by UV light using a transilluminator.
Animal Protocols.
lnbrcd male Copenhagcn rats weighing 200 - 250g were
obtained from Harlan Sprague-Dawley (Indianapolis, IN). Before inoculation, Mat
Ly Lu-PTHrP tumor ceUs growing in serum-containing medium were washcd with
Hanks buffer, trypsinized, and collected by centrifugation at 1500 rpm for 5 minutes
(41,49,114). Cell pellets (10 x 103 cells) were resuspended in lOOll1 saline and
injected using 1ml insu lin syringes into the 1eft ventricle of rats anaesthetised with a
ketamine/xylazine cocktail. AnimaIs were divided into control groups which received
vehicle al one (PBS) and experimental groups which were infused I.P. with different
doses (0.1 - 10.0 Ilg/kglday) of PSP-94 starting at the time of tumor ceU inoculation
(day 0) until the day of skeleta1 metastases development. The time after tumor cell
•
inoculation which was required to develop hind limb para1ysis (an index of spinal
36
cord compreSSlOn due to lumbar vertebrae metastases) was detemlined and
percentage of starting number of animaIs developing hind-limb paralysis was plotted.
Altematively, cell pellets (1 x 106 cells) were resuspended in 100 III saline and
injected using 1ml insulin syringes into the right flank of rats. From the time of
tumor cell inoculation, experimental animaIs were treated with different doses (0.1,
1.0 or 10.0 Ilg/kg/day) of PSP-94 via S.C. injections for 15 consecutive days. Control
animaIs received PBS alone as vehicle control. AH animaIs were numbered, kept
separately and monitored daily for the development of tumors. The tumor mass was
measurcd in 2 dimensions by calipers and tumor volume was calculated according to
the cquation (l x w 2 )/2 (/=Icngth, w=width) (49,114). Ali control and expcrimental
animais werc weighed cvcry alternatc day to dctcrminc any adverse ciTeet of PSP-94.
Both control and cxpcrimental animais wcrc sacri ficcd on day 16 post tumor ccli
inoculation and their tumors wcre rcmovcd and wcighcd. Additionally, thcsc tumors
wcre used for histological analysis as dcscribcd bclow. Blood from ail control and
expcrimental animaIs was collccted on day 16 for determination of plasma Ca+2 and
PTHrP levels (49).
Histologie Analysis. For immunohistological analysis, primary tumor samples
were dewaxed by heating at 60°C and rehydrated in a graded alcohol series (100%70%). Anti-rat antibody against PTHrP was used as the primary antibody. Tumor
sections were incubated overnight at 4°C followed by further incubation with
biotinylated universal antibody (Vector Laboratories, Burlingame, CA) for 45-60
•
minutes. Sections were rinsed with TBST followed by incubation with Vectastain
ABC-AP Reagent (Vector Laboratories, Burlingame, CA) for 30 minutes. These
37
l,:
sections were agam washed with TBST and incubated with a Napthol AS-Mix
Phosphate/Fast Red solution (Sigma-Aldriche, OakviUe, ON). The sections were
finaUy counterstained with Methyl Green (Vector Laboratories, Burlingame, CA) and
mounted.
For TUNEL assay, tissue sections were dewaxed by heating at 60°C foUowed by
washing in xylene and rehydrated through a graded series of ethanol and water.
Tissues were incubated with proteinase K for 30 min at 37°C and fixed, blocked and
permeabilized. Apoptotic cells were detected by TUNEL assay (in situ cell death
detection
kit,
Boehringer
Mannheim,
Indianapolis,
IN)
according
to
the
manufacturcrs' instructions. Positive TUNEL staining was visualised by fluorescence
microscopy (116).
In
other
experiments
following
TUNEL
assay,
tissue
sections
were
counterstained with Hocchst 33258 (Sigma-Aldrich, Oakville, Canada). Hoechst
staining was addcd to tissues at a final concentration of 24ug/ml in PBS and
incubated for 15 minutes at room temperature. Tissue sections were washed and
visualized by fluorescence microscopy using a blue screen (116). AU results of
immunohistochemistry and TUNEL assay were evaluated and interpreted by two
independent examiners.
Other Analytical Methods. Plasma calcium levels were determined by atomic
absorption spectrophotometry (model 703, Perkin-Elmer, Norwalk, CT). For plasma
PTHrP, aU samples were tested in two dilutions in PTHrP R.I.A. kit (Nichols Institute
•
Diagnostics, San Juan Capïstrano, CA.) according to the manufacturers instructions .
38
Statistical Analysis.
Results are expressed as the mean ± SEM of at least
triplicate detenninations, and statistical comparisons are based on the Student's t-test
or analysis of variance. A probability value of <0.05 was considered to be significant
(117) .
•
39
.\
Results
Effeet of PSP-94 on MatLyLu-PTHrP Cell Growth, Morphology and
Invasion. Mat Ly Lu-PTHrP ceUs were grown in the presence of 0.1, 1.0 & 10.0
).lg/ml of PSP-94 or vehicle alone for up to 3 days and the ability of PSP-94 to alter
cell doubling time was evaluated daily. Comparison was also made with doubling
time of wild type untransfected Mat Ly Lu cells. Transfection of Mat Ly Lu with
PTHrP cDNA resulted in reduced doubling time and increase in tumor cell growth
due to the growth promoting effects of PTHrP. A significant decrease in Mat Ly LuPTHrP ccli growth was scen following treatmcnt with 10.0
~Lg/ml
of PSP-94 for 72
hrs (Figure 1A). Lower doses of PSP-94 (0.1 and 1.0 ).lg/ml) failed to exhibit any
significant changes on tumor cell growth (data not shown). Treatmcnt of Mat Ly LuPTl-IrP cells with 10.0 ).lg/ml of PSP-94 for 3 days rcsuItcd in a noticcablc change in
tumor celI morphology whére tumor cells were found to change their normal spindlclike shape to a more rounded and condensed appearance (data not shown). Using a
Boyden Chamber Matrigel invasion assay, aIl doses of PSP-94 failed to alter the
invasive capacity of Mat Ly Lu-PTHrP cells.(data not shown).
Effect of PSP-94 on Mat Ly Lu-PTHrP tumor growth. Male Copenhagen rats
were inoculated with Mat Ly Lu-PTHrP cells via S.c. route of injection into the right
flank. Starting from the day of tumor ceU inoculation animaIs were infused S.c.,
below the tumor ceU inoculation site, with different doses of PSP-94 (0.1-10.0
).lg/kg/day) for up to 15 days.
•
\
Effect of PSP-94 on reducing tumor growth was
evaluated by daily determination of tumor volume with comparison being made to
control tumor-bearing animaIs receiving vehicle alone. Control animaIs showed a
40
•
progressive increase in tumor volume throughout the duration of the study. In contrast
to this, experimental animais receiving PSP-94 showed a marked dose-dependent
reduction in tumor volume throughout the course of this study (Figure 2). Both
control and experimental animais were monitored for any noticeable side effects and
cachexia resulting in weight loss. AU animais were weighed at timed intervals
throughout the duration of the study and did not show any significant changes in the
weight of control and experimental groups of animais that can be attributed to any
potcntial side effect ofPSP-94 treatment (Figure 3).
Effeet of PSP-94 on Mat Ly Lu-PTHrP tumor weight. In order to determine
the effect of PSP-94 on tumor weight, animaIs inoculated with Mat Ly Lu-PTHrP via
S.c. route of injection wcre sacrificed at the end of the study (day 16) and their
tumors excised and weighed. Control animais recéiving vehicle alone exhibited large
tumors while treatment with different doses of PSP-94 (0.1-10.0 Ilg/kg/day) resulted
in a significant dose-dependent decrease in tumor weight (Figure 4).
Effeet of PSP-94 ,on the development of skeletal metastases.
Mat Ly Lu-
PTHrP cells were inoculated into male Copenhagen rats via I.e. injection into the left
ventricle. Starting from the day of tumor ceU inoculation (day 0), animais were
administered with different doses ofPSP-94 (0.1-10.0 llg/kg/day) via I.P. route. The
effect of PSP-94 on delaying the development of skeletal metastases was evaluated by
daily monitoring of the animaIs for the development of hind-limb paralysis. AIl of
control animais (100%) inoculated with Mat Ly Lu-PTHrP ceUs and receiving vehicle
•
alone developed hind-limb paralysis by day 13. While 0.1 and 1.0 Ilg/kg/day of PSP94 had no significant effect on the time of hind limb paralysis (data not shown),
41
treatment with 10.0 j.lg/kg/day of PSP-94 resulted in a statisticaUy significant delay in
the number of animaIs developing hind limb paralysis. Percentage of total number of
animaIs not developing hind limb paralysis at different days is shown in Figure 5.
Effect of PSP-94 on plasma PTHrP and calcium levels and tumoral PTHrP
production.
In order to detelmine the effect of PSP-94 on plasma PTHrP and
calcium levels animais inoculated with Mat Ly Lu-PTHrP ceUs via S.c. route were
sacrificed at the end of the study and plasma was coUected and PTHrP levels were
analyzed using a radioimmunoassay. Comparison was made between plasma
collected from normal, non-tumor bearing animais, control tumor bearing animais
receiving vehicle alone and plasma collected from experimental animais recciving
different doses of PSP-94 (0.1-10.0 j.lg/kg/day). Normal non-tumor bcaring animaIs
showed basal Icvels of plasma PTHrP whereas animais inoculatcd with Mat Ly LuPTI-IrP cells and rccciving vehicle alone showcd marked clcvatcd Icvcls of
immunorcactive plasma PTHrP lcvels. Treatmcnt of tumor bearing animaIs with PSP94 resulted in a dose-dependent decrease in plasma PTHrP levels (Figure 6A).
Analysis of plasma collected from normal non-tumor bearing animaIs and. tumor
bearing animaIs receiving vehicle alone revealed a marked increase in plasma calcium
levels of control tumor bearing animaIs at the time of sacrifice on day 16 past tumor
ceU inoculation. In contrast, experimental groups of animaIs receiving different doses
of PSP-94 exhibited a significant reduction in their plasma calcium levels. The
highest dose of PSP-94 (10.0 j.lglkg/day) resulted in near normalization of plasma
•
calcium ofthese experimental group of animais (Figure 6B) .
42
•
Tumors from control group treated with vehicle al one and experimental
groups treated with different doses of PSP-94 (0.1- 10.0
~g/kg/day)
were excised and
analyzed for tumoral PTHrP production by immunohistochemical reaction specifie
for PTHrP (1-34). Intense color staining of tumors from control groups of animaIs
receiving vehicle alone was observed. In contrast a dose dependent decrease in
PTHrP immuno-staining was observed in experimental tumors from animaIs
receiving different doses of PSP-94 (Figure 7).
Effect of PSP-94 Mat Ly Lu-PTHrP tumor ccli apoptosis ill vitro and i1l vil'o. In
order to investigate the underlying molecular mechanism of action of PSP-94 in
rcducing tumor growth Mat Ly Lu-PTl-IrP cells wcrc culturcd in the presence of PSP94 (10.0 pg/ml) or vchic\c alone for diffcrcnt time intcrvuls. Genomic
D;--~A
\'. ·~s
extracted from cells cultured in the presence of vehicle alonc or PSP-94. Equal
quanti tics of DNA were subjected to elcctrophoresis on a 2% agarosc gel and
assessed for the degree of DNA fragmentation. Control Mat Ly Lu-PTHrP cells
cultured with vehic\e alone exhibited no signs of DNA fragmentation. However,
experimental Mat Ly Lu-PTHrP eeUs cultured in the presence ofPSP-94 (10.0
~g/ml)
exhibited marked DNA fragmentation after 72 hours of treatment (Figure 8A). The
degree of DNA fragmentation was also analyzed in vivo using TUNEL assay whieh
can serve as a marker for apoptosis. Tumor sections treated with PSP-94 (10.0
~g/kg/day)
were significantly more TUNEL positive as compared to vehic1e treated
control tumors (Figure 8B). Counterstaining with Hoechst reagent revealed the
•
presence of apoptotic bodies in tissue sections from animaIs treated with PSP-94 .
Control, vehicle treated tumors exhibited normal DNA staining patterns (Figure 8B).
43
•
These in vitro and in vivo findings demonstrate that indeed reduction in tumor volume
following treatment wiî.h PSP-94 is due to its ability to promote tumor cell apoptosis .
•
44
•
-
E
Cl
::t
o
o
,...
Q)
E
.1-
{ } f
o
M
o
N
(&o~x) Jaqwnu lIaO
•
o
•
Fig. 1. Effect of PSP-94 on Mat Ly Lu-PTHrP cell growth.
Mat Ly Lu ceUs transfected with vector alone (CMV) or PTHrP were
~~cded
at a
density of 5x 103 cens/weIl in 6-well plates. Mat Ly Lu-PTHrP ceUs were treated with
PSP-94 and were trypsinized and counted using a coulter counter as described in
"Materials and Methods". Change in ceU number foI1owing treatment with 10.0
Ilg/ml of PSP-94 for 72 hrs is shown.
Each point represents the mean of 3 different experiments.
Significant
differences from control cells and PTHrP transfectcd ccll are reprcsented by asterisks
(p<0.05) .
•
46
-
•
25
;;-- 20
E
o
-o>
CTL
~
PSP-94 0.1 ug/kg/day
PSP-94 1.0 ug/kg/day
PSP-94 10.0 ug/kg/day
CJ
•
"-"
Q)
E
:::l
o
*
15
J-
O
E 10
~
5
o
7
9
11
Time (days)
14
16
•
Fig. 2. Effeet of PSP-94 on Mat Ly Lu-PTHrP tumor volume.
Male Copenhagen rats were injected S.c. into the right flank with lx106 Mat Ly Lu~"!"~~p
cells. Starting on the time of tumor ceU inoculation animaIs were infused
daily with different doses of PSP-94 for fifteen consecutive days as described in
"Materials and Methods".
Tumor volume was measured at timed intervals and
comparison was made with that of tumor-bearing animaIs receiving vehicle alone as
control (CTL).
Results represent the mean ± SEM of 5 animaIs in each group in 3 different
experiments. Significant differences from control tumor-bearing animaIs receiving
vehicle alone are rcpresented by asterisks (p<O.05) .
•
48
-
re
240
1
- - CTL
-6PSP-94 0.1 ug/kg/day
-
PSP-94 1.0ug/kgday
- .. -. PSP-94 10.0ug/kg/day
2301-
-a -
en
E
ca
1-
C)
---....,
220
..c:
C)
-
Q)
3:
....,
ca
210
a:
200
.
~/
o
1
1
5
7
9
11
Time (Days)
1
1
1
13
15
17
•
Fig. 3.
Eilect of PSP-94 on animal weight.
Mal~ l'openhagen rats werp i!1jr:'cted S.C. into the right flank with lxl0 6 Mat Ly Lu-
PTHrP cells. Starting on the time oftumor cell inoculation animaIs were infused with
different doses of PSP-94 for fifteen consecutive days as described in "Materials and
Methods". AH animaIs were weighed at timed intervais and comparison was made
with that oftumor-bearing animaIs receiving vehicle aione as control (CTL).
Results represent the mean ± SEM of 5 animaIs in each group in 3 different
experiments .
•
50
•
l~::s
tn
o
o
.
or-
Cl
~
.......
Cl
::s
.
==============~O
~----------------------------~~
~
o
____________________________
C\I
•
Ln
,..
o
or-
Ln
1-
~o
o
~
Cl
1
a.
Cl)
a.
•
Fig. 4. Effect of PSP-94 on Mat Ly Lu-PTHrP tumor we!ght.
Male Copenhagen rats were inoculated with lxl0 6 Mat Ly Lu-PTHrP cells via
subcutaneous injection into the right flank. Starting from the day of tumor cell
inoculation animaIs were administered with different doses of PSP-94 for fifteen
consecutive days as described in "Materials and Methods". At the end of the study
tumors from control (CTL), vehicle treated animaIs and PSP-94 treated animaIs were
excised and weighed.
Results represent the mean ± SEM of 5 animaIs in each group in 3 different
experiments. Significant differences from control tumor-bearing animaIs receiving
vchicle al one are represcnted by asterisks (p<O.05) .
•
52
•
-te
r-- ---_
Lt')
~
o::t
Cl
1
-la..
I-cn
Ua..
-te
r
__________
:!.\
1 :
(1)
E
.t-
\
1
0
0
1
J
1
1
0
CO
0
0
0
N
(0
~
~
•
".
.
SIBW !UB paZÂ IBJB d-UO U JO
0/0
~
~
):
0
•
Fig 5. Effeet of PSP-94 on spinal metastases.
Male Copenhagen rats were inoculated via I.e. route into the left ventricle with
IOxJ0 3 M~t L~' !..~-PTHrP ceUs. Starting on the time oftllm,-,r ('cH inoculation (day 0)
animaIs were infused with different doses of PSP-94 (0.1 - 10.0 Ilg/kg/day) until the
day of development of hind-limb paralysis as described in "Materials and Methods".
AnimaIs receiving vehicle alone as control (CTL) or PSP-94 were monitored daily
for the development of hind-limb paralysis and % of animaIs not paralyzed at
different time points in each group was calculated.
Results represent the mcan ± SEM of 5 animaIs in each group in 3 diffcrent
cxpcrimcnts. SignifiC'ant diffcrcnccs in
O~I
of non-pClrn!yzcd :mimals from control
tUlllor-bcaring animaIs rccciving vchiclc alone arc rcprcscntcd by astcrisks (p<0.05) .
•
54
•
A.
- 100
~
~
M
--
80
ca
60
1
..-
-
..J
en
s::
Q,)
>
*
:l
C'"
Q,)
0
40
E
a.
........
a.
'-
J:
20
r-
a.
.-
0
N
CTL
0.1 ug/k g
1.0u g/kg 10.0 ug/k g
PSP-94
B.
5.0
-E
-E
:E
4.5
:l
4.0
ë::.;
*
ca
0
ct!
E
en
3.5
ct!
a.
•
3.0
01
[1
C4
N
CTL
0.1 ug/k g
1.0u g/kg 10.0 ug/k g
PSP -94
•
Fig. 6.
Effeet of PSP-94 on plasma PTHrP and Calcium in tumor bearing
animaIs.
Panel A: Male Copenhagen rats were inoculated S.c. with lxl0 6 Mat Ly Lu-PTHrP
cells. Starting on the time of tumor cell inoculation animaIs were administered with
different doses of PSP-94 for fifteen consecutive days as described in "Materials and
Methods". All animaIs were sacrificed at the end of the study (day 16) and plasma
\Vas collected from control (CTL), vehicle treated animaIs and PSP-94 treated animaIs
and
analysed
for
immunoreactive
plasma
PTHrP
(iPTHrP)
levels
using
radioimmunoassay as described in "Materials and Methods". Plasma PTHrP levels in
normal non-tumor bearing animais is also shown (N).
Panel B: Male Copenhagcn
6
rats were inoculated S.c. with 1x 10 Mat Ly Lu-PTl-IrP cells. Starting on the time of
tumor cell inoculation animais \Vere infused \Vith different doses of PSP-94 for fifteen
consecutive days as described in "Muterials and Methods". All animais were
sacrificcd at the end of the study (day 16) and plasma was collected from vehicle
treated control (CTL) a;td animaIs and PSP-94 treated animaIs for analysis of plasma
calcium levels as described in "Materials and Methods". Plasma calcium from
normal, non-tumor bearing animaIs is also shown (N).
Results represent the mean ± SEM of 5 animaIs in each group in 3 different
experiments. Significant differences from control tumor-bearing animaIs receiving
vehicle a10ne are represented by asterisks (p<O.05) .
•
56
•
•
CTL
PSP94 (0.1 ug/kg)
PSP94 (1.0ugfkg)
PSP94 (10.0ug/kg)
•
Fig. 7.
s.
Effect of PSP-94 on PTHrP production by Mat Ly Lu-PT HrP tumor
6
Lu-PT HrP ceUs.
Male Copenhagen rats were inoculated S.c. with lxl0 Mat Ly
ç:t~rtinQ:
differe nt
on the time of tumor ceU inoculation animaIs were infuse d with
"Mate rials and
doses of PSP-9 4 for fifteen consec utive days as descri bed in
their prima ry
Metho ds". AlI animaIs were sacrificed at the end of the study and
ns of tumor s
tumors removed, paraffin embedded, and sectioned. Histol ogical sectio
of PSP-9 4 were
from animaIs receiv ing vehicle along (CTL) or differ ent doses
"Mate rials and
stained with and antibody specifie for PTHrP (1-34) as descri bed in
sections were
Methods". Three animaIs \Vere present in each group and three tumor
three differe nt
anaIyzed for each animal. Results repres ent the mean ± SEM of
ments is shown.
expcriments. A representative photomicrograph of three such experi
Magnification 200X .
•
58
A.
~
en
1
..J
tU
a.
CI)
a.
B.
CTL
TUNEL
Assay
Hoechst
Staining
•
PSP-94
•
Fig. 8.
Effect of PSP-94 on DNA fragmentation of 1\1at Ly Lu-PTHrP cells in
vitro and in vivo.
Panel A.
Mat Ly Lu-PTHrP cells were cultured in the presence of vehicle al one or
PSP-94 (1 0.0 ~g/ml) for up to 72 hours. DNA was isolated [rom control, vehicle
treated cells and PSP-94 treated cells as described in "Materials and Methods".
Isolated DNA was subjected to electrophoresis on a 2% agarose gel and visualized
under UV light. A representative photograph of three such experiments is shown.
6
Panel B. Male Copenhagen rats were inoculated with 1x 10 Mat Ly Lu-PTHrP cells.
Starting on the time of tumor ceIl inoculation animaIs were infused with different
doses of PSP-94 for fifteen consecutive days as describcd in '"Materials and
Methods". AIl animais were sacrificed at the end of the study and their primary
tU1110rs removed, para ffi ï' cmbeddcd, scctioncd and proccsscd by TUNEL assay as
described in "Matcrials and Methods" (upper panel). Following TUNEL, they were
.
counterstained with Hoechst reagent (lower pancl). A rcprescntative photomicrograph
for thrce such
expcrimc~ts
in each group is shown. Magnification 200X
•
60
Discussion
ln this study we have used a syngenelc model of rat prostate cancer to
demonstrate the ability of PSP-94 to reduce prostate cancer growth and metastases.
High degree of amino acid sequence homology between human and rat PSP-9.1
allowed the use of human PSP-94 for these studies
(11.s~ ~
of
thi&:~1Glo-gous
model for prostate cancer allows for full interaction between the host environment
and growth factors (EGF, TGF -~) (4) and proteases (uP A, MMPs) (26,116) secreted
by tumor cells. In order to evaluate the efficacy of PSP-94, Mat Ly Lu cells
transfected with full-Iength cDNA encoding PTHrP (Mat Ly Lu-PTHrP) ('clIs were
utilizeu. Thcse prostate cancer cells are hormone-indepcndent allowing for the
evaluatioll of the effeet of PSP-94 on late stage prostate cancer. Due to the high levcls
of PTl-IrP production these animais routinely develop hypercaJcemia, a common
complication in many paticnts suffering from prostate cancer (42,43). Mat Ly Lu..
PTHrP cells had a higher rate of cell proliferation as compared to control Mat Ly Lu
cells transfected with
v~ctor
aJone. Treatment of Mat Ly Lu-PTHrP cells with PSP-94
for 72 hrs resulted in a significant effect on tumor cell proliferation, morphology and
DNA fragmentation which is considered to be an apoptotic marker.
Inoculation of male Copenhagen rats with Mat Ly Lu-PTHrP cells into the right
flank via S.C. injections resulted in the development of primary tumors. Whereas
control, vehicJe trcated animaIs developed large primary tumors, treatment with
different doses of PSP-94 resulted in a dose-dependent decrease in their tumor mass.
These anti-tumor effects were not associated with any noticeable side effects or
•
weight loss of experimental animaIs. TUNEL analysis carried out on tumoral sections
61
from control and experimental animais revealed that PSP-94 treated tumors are more
TUNEL positive as compared to control tumors, indicating a higher degree of
apoptosis in PSP-94 treated animais. In addition to this, counterstaining with Hoechst
reagent revealed condensed; apoptotic chromatin in PSP-94 treated tumors whereas
control tumors exhibited normal DNA staining. Upon sacrifice of animais plasma was
collcctcd and analyzed for plasma PTHrP and calcium levcls. Nom1al, non-tumor
bearing animaIs have undetectable levels of plasma PTHrP whereas inoculation of
animais with Mat Ly Lu-PTHrP cells resulted in marked increase in their plasma
PTHrP lcvels. In contrast to this, treatmcnt with the differcnt doses of PSP-94 resulted
in a dose-dcpcndent dccrcase in plasma PTllrP levcls. In addition, th('
<;;1111(' dn<;('-
dcpc11llcnt dccrcase was obscrvcd in tumoral PTHrP production whcn tumor samplcs
[rom control, yehicle trcatcd and PSP-94 trcatcd animaIs were subjected to
immunohistochcmical analysis. Bcing the major pathogcnctic factor of hypcrcalccmia
of malignancy, plasma calcium Icvels corrclatcd with that of plasma PTHrP lcvcls
(42,43,44). Inoculation of Mat Ly Lu-PTHrP cclls into thc animaIs resultcd in a
marked increase in their plasma calcium levels as compared to serum from nom1al,
non-tumor bearing animaIs. Administration of different doses of PSP-94 resulted in a
dose-dependent decrease in plasma calcium levels with the highest dose of PSP-94
leading to a near normalization of plasma calcium levels.
Since the major cause of prostate cancer related mortality is the development of
metastases, evaluation of the effect of PSP-94 on delaying the development of
skeletal metastases was carried out by inoculating male Copenhagen rats with Mat Ly
•
Lu-PTHrP cells via
I.e. route into the left ventric1e.
62
Routine injection of Mat Ly Lu
•
ceIls into the !eft ventricle results in the deve!opment of skeletal metastases causing
compression of the spinal cord lcading to hind-limb paralysis (49). Whereas all
control, vehicle treated animais developed hind-limb para1ysis by day
13,
administration of the highest dose of PSP-94 starting from the time of tumor cell
inoculation resulted in modest delay in skeletal metastases. Such results suggest 10w
bioavailability of PSP-94 to the skeleton, a common drawback associated with
developing effective therapeutic agents for skeletal metastases (119).
Using this mode! we were not only able to demonstrate the anti-tumor effects of
PSP-94 by reduction in tumor volume and weight but also biochcmica1 parameters
like plasma calciulll and PTI-lrP lcvcls <11so showcd a markcd dccrease following
thcrapy. A significant finding in these studics was tl1at while dccrease in tumor
volume was dose-dependent, 10.0 Ilg/kg/day of PSP-94 did not show a marked
dccrcase in tumor volume as compared to 1.0
~lg/kg/day
PSP-94. In contrast, the
ability of PSP-94 to rcduce plasma calcium, plasma PTHrP and tumoral PTHrP
continued to show a dose-dcpcndcnt cffcct \Vith 10.0 Ilg/kg/day PSP-94 causing near
normalization of plasma calcium and PTHrP leve1s. These findings allow. us to
specu1ate that PSP-94 may also have addition al effects including its ability to regulate
PTHrP production by tumor cells or alter calcium homeostasis. lndeed PSP-94 has
been shown to suppress follicle stimulating hormone (FSH) which is kno,wn to
regulate intracellular calcium (120). Suppression of FSH by PSP-94 may serve as an
additional mechanism to cause anti-tumor effects due to the growth-promoting effects
•
of FSH_~n prostate cancer (86). Furthem10re, although cloning and characterization of
a putative PSP-94 receptor has not been estab1ished severa1 studies have provi,ded
63
•
evidence for the existence of PSP-94 binding proteins on prostate cancer ceUs.
Binding of PSP-94 to these proteins might initiate a signaling cascade that results in
the anti-tumor effects observed (103,104).
Collectively, the results of this study demonstrate PSP-94 to be an effective
inhibitor of hormone-independent, late stage prostate cancer growth and its associated
hypercalcemia of malignancy without manifesting any noticeable cytotoxic effects.
Further studies will define the minimum sequence requirement to obtain maximum
level of efficacy. These synthetic peptides, their peptidomimetic analogues al one or in
combination with currcntly available chemotherapeutic agents will provide unique
opportunitics to block prostate cancer progression with highly effective non-toxic
biotherapeutic agents which can be dclivered over a long period of time without any
drug associated side effects. Thcse approaches will go a long way in reducing prostate
cancer associated morbidity and mortality .
•
64
•
Chapter 3
General Discussion
Despite advances in the staging and therapeutic modalities for prostate cancer it
still remains one of the most çommonly diagnosed cancers in men associated with
high morbidity and mortality rates (32,107). The depcndence of the prostate gland on
androgens leads to the utilization of hom10nal therapy resulting in the ablation of
circulating androgens (2,50,52). Consequently, a reduction in prostate tumor volume
is achieved; eventually however, ihc rccurrence
o~
the disease is imminent as
androgen-indepcndent cells proliferate and compose the majority of the tumor.
Dcvelopmcnt ofboth skclctal and non-skcletal mctastases is the primary factor for the
high mortality rate associated with advanced prostate canccr (32). However there arc
many rcasons to be cheerful. Improvemcnts in many therapcutic modalities for
prostate cancer including chemoprevcntion, pharmacotherapy, potential for gene
therapy and the development oftumor vaccines are grounds for optimism (53).
PSP-94, an inhibin, is one of the three predominant proteins that are secreted by
the prostate gland and has been shown to be associated with prostatic tumors (61).
While the majority of PSP-94 is produced by the prostate gland, other tissues such as
breast, ovary, endometrium and trachea have also been shown to express PSP-94 but
to a lesser extent (66,68,69). The differential expression ofPSP-94 in prostatic tumors
as the disease advances from an early to late stage prostate cancer has allowed for its
•
use as a prognostic/diagnostic marker (82,83) .
65
•
Whereas h0n110neS su ch as FSH, VIP, epinephrine and isoperentol have been
shown to upregulate the expression of PSP-94, in benign hyperplastic tissue and
androgen-dependent, low invasive LneaP prostate cancer cell lines (74), androgens
such as testosterone and estrogen do not alter the expression of PSP-94 (75,76). The
androgen-independent nature of PSP-94 has allowed for its superior use as a
prognostic/diagnostic marker in tumors that have been exposed to androgen ablating
agents.
This thesis dealt with evaluating the potential use of PSP-94 as a thcrapeutic
modality for reducing prostate cancer growth, metastases and hypcrcalcemia of
malignancy. While prostate cancer cells treated with PSP-94 cxhibitcd an incrcase in
their doubling time hence a reduction in their growth rate, administration of PSP-94
ill vivo resulted in significantly smallcr tumors with corrcsponding lowcr tumor
weights. ln addition, biochcmical markers such as scrum PTllrP and calcium Icvcls
wcre also reduccd following trcatmcnt with PSP-94. Furthcn11orc, PSP-94 causcd a
rcduction in the lcvels of PTHrP production by tumor cells. No drug-associated side
effccts were manifcsted leading to the possible use of PSP-94 on prostate cancer
patients over a prolonged period of time to block the progression of prostate cancer.
Administration of PSP-94 yielded a mode st but significant delay in the development
of skeletal metastases, suggesting low bioavailability to the skeleton. Such results are
a common drawback in development of anti-metastatic agents.
Although the results obtained throughout the course of this thesis point to a
valuable therapeutic option in PSP-94 several future studies should be explored .
•
Structure functions studies whereby PSP-94 is broken down into many different
66
•
peptides should be carried out in order to define the minimum amino acid sequence
required to elicit the anti-tumor effects of this protein. Generation of su ch peptides
would lead to not only increased efficacy in terms of reducing tumor volume but
might also result in increased bioavailability to the skeleton as a result of the smaller
peptides being able to readily penetrate into the skeleton. Such an effect would lead to
a more effective response in delaying the development of skeletal metastases, as se en
through the delaying of development ofhind-limb paralysis.
The degree of tumor reduction obtained through administering PSP-94 alone is
sufficient to warrant its use as a stand alone drug for combating this disease; however,
one has to keep in mind that the Mat Ly Lu-PTl-irP modc1 uti\ized in these studies
rcprcsents late stage, highly invasive, androgen-independent prostate cancer. It can be
postu\atcd that the cfficacy of PSP-94 to block tumor progression will be significantly
highcr in a mode\ which represents early stage. low invasive prostate cancer. One
would expect that administration of PSP-94 will elicit a greater response wh en tested
in such a system due to the androgen dependent nature of the disease at its early stage.
As mentioned previously, one of the main physiological functions of PSP-94 is the
inhibition of FSH (62) which has been shown to be a growth factor involved in the
progression of prostate cancer (86,87). Administration of PSP-94 in such a model will
have a dual effect whereby PSP-94 elicits its own apoptotic response as well as
inhibiting crucial growth factors involved in the progression of prostate cancer one of
which is FSH. Furthermore, combination studies should be carried out whereby PSP-
•
94 is administered in combination with various commonly utilized therapeutic
67
•
approaches for prostate cancer which have shown promising results. Of interest is
whether the combinatorial approach yields an additive or a synergistic response.
As previously mentioned, the reduction in tumor volume levels off after 1.0
Ilg/kg/day while 10.0 Ilg/kg/day proved to be more effective at reducing plasma
calcium and PTHrP levels leading to near normalization ofboth biochemicalmarkers.
FSH has been shown to regulate intracellular calcium (120) and inhibition of FSH by
PSP-94 might provide for a mechanism which explains the more pronounced
reduction in both plasma calcium and PTHrP levels as compared to reduction in
tumor volume. Therefore studies should be carried out to evaluate the direct ability of
PSP-94 to alter intraccllular calcium transport.
Sincc PSP-94 is expressed in tissues other th an the prostate including ovarian,
cndomctrial and breast tissues (66,68,69), evaluation of the efficacy of PSP-94 in
blocking the progression of the respective cancers.
The regulation of the PSP-94 gene by agents specifically hormones is weB
elucidated. However the precise mechanism of the differential expression of PSP-94
in prostate cancer as the cancer progresses from an carly stage to a late stage with
complete lack of PSP-94 production in highly advanced prostate cancer is not yet
understood. One possible mechanism behind this differential expression is through
epigenetic regulation of gene transcription by DNA methylation. DNA methylation,
the addition of methyl groups to a cluster of CpG islands, has been shawn to be
involved in the regulation of man y proto-oncogenes as well as tumor suppressor
•
genes. Addition of the methyl groups to CpG islands interfcres with the interaction
between the promoter binding protein and the respective promoter region.
68
•
suppreSSIOn of
Conse quentl y, hyper -meth ylatio n of the promo ter result s in
protei n leve1s.
transcription of the DNA leading to lower levels of mRNA and hence
ined, howev er,
The methy lation status of the PSP-9 4 gene is yet to be determ
Risbridger et al have demonstrated the hypen nethyl ation of the inhibin
Cl
subunit in
tion of other
prosta te carcin oma (121). Such observ ations as weIl as to the regula
lation might
genes through DNA methylation leads to the speculation that DNA methy
theref ore highly
be one factor involv ed in the regulation of the PSP-9 4 gene. It is
status of the
desirable to carry out in depth analysis to determine the methy lation
ities for this
PSP-9 4 gene as this can lead to the development of therap eutic modal
disease.
one possible
The work presen ted in this thesis demonstrated that apopto sis is
tumor volume.
mechanism by which admin istratio n of PSP-94 results in reduci ng
precis e molec ular
Additional detailed studies need to be carried out to identif y the
evalua ting other
basis by which PSP-9 4 induce s apoptosis. Furthe rmore , studie s
volum e, such as
mecha nisms by which PSP-9 4 leads to a reduct ion in tumor
inhibition of angiogenesis, should be carried out
n to this
Although a great deal about the regulation of PSP-9 4 is known in additio
ity for prostate
thesis providing evidence for the use of PSP-94 as a therapeutic modal
hypercalcemia
cancer and its associated complications namely skeletal metast ases and
ined. This is
of malignancy, the precise mode of action of PSP-94 is yet to be detenn
been c10ned and
particularly important since the putative PSP-94 receptor has not yet
•
provid e further
characterized. Comp lete charac terizat ion of a PSP-9 4 recept or will
for the anti-tumor
potential insight as to the potential signaling pathways respon sible
69
1
effects of PSP-94. Modulation of these signaling cascades in order to provide an
increased effect should prove useful in combating this common disease.
In conclusion, PSP-94 has been shown to be an effective treatment modality for
prostate cancer and its associated complications. While administering PSP-94 alone is
a first major step in providing a novel biotherapeutic modality without any noticeable
side effects, combination studies with other therapeutic agents that have been shown
promising results as well as development of smaller synthetic peptides or
peptidomimitics agents will result in enhancement of its efficacy and add to our
existing arsenal of therapeutics against prostate cancer.
•
70
Chapter 4
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90
Appendix
pag e 2
4.
Research Personnel and QU:llifications: List the names of ail individuals who will be in contact with animais in this
study (including the Principal lnvestigator) and their employment classification (investigator, technician, research assistant,
undergraduate/graduate student, fellow). Indicate any training received (e.g workshops, lectures, etc.). The PI certifies that
an personnellisted here have suitable training and/or experience, or will be provided with the specifie training which
qualifie:i'them to perform the procedures described in the protocol. Each person listed in this section must sign to indicate
that slhe has read this protocol. (Space will expand as needed.)
Name
Signnture
Classilication
Training Information
1
A\'
S.A. Rabbani
Investigator
M.D., University Animal Course
Julie Gladu
Technician
University Animal Course
Ani Arakelian
Technician
University Animal Course
Nicholas Shukeir
Graduate studt!nt
University Animal Course
Parissa Khalili
Graduate student
University Animal Course
• Enter the tirst name, press 'enter', then the 2" Dame ... complete the tirst calumn, then the 2"', th en the 3rd
•• If 3n undergradu3te studenl is involycd . the rok of the student and the suoervision recei\'d must be describcd.
5.
1
Summary (In language that will be understood by members of the general public)
a) RATIONALE: Dcscribc, in 3 short paragraph, the o\'eral1 3im of the study and its potential benefitto
human/animal hcalth or to the advancement of scientilic knowled!!e.
Development of novel therapeutic strategies to control hypercalcemia of malignancy.
b) SPECIFIC OBJECTIVES OF THE STUDY: Summarize in point form the primary objectives of this study.
To examine the chemical and biologieal charactcristics ofparathyroid hormonc-like factors rcleased by tumers associated with
the hypcrcalcemia of malignancy syndrome.
c) l'ROGRESS REPORT: If thb b a rcnewal of an ongoing project, BRlEFLY summarizc wh:lt was accomplishcd
during the prior approval period and inùicate if anù holV the currcntl?,o:ds diffcr from those in the original
application.
Invcstigation of thc role of PTHRP in tumor biology and to examine various strategies to control tumor progression.
\
d) SUl\l:\lARY OF l'ROCEDL'RES FOR A:"iI:\L\L USE REPORT TO THE COC: Using KEY \YORDS O;--;L Y,
list the procedures used (c.g. anacsthesia, brecding culony, injection Il', g3va~e, drug administration, major
su rvival su rger;', eu tlwnasia b~' e'l:5a n~uination. behavioural studies). Rcfcr to Appendix lof the Guidelines for a
more complete list or su!!!!ested kev words.
- Polyclonal antibody production, c:.lthar.asia by exsanguinotion
- Subeutaneous injection of tumor ce Ils/caudal artery collection ofblood'bleeding from eor.·passive immunization
- Anaesthesia, intracardiac injection of tumor cells, drug ac:ministration intrape:itoneally.'succ:.ltaneously, saphenous vcin blood
collection. mini-pumo implantation.
6_
AnimaIs To Be l"sed
e)
\.
Purpose of ...... :l:;;;al L'se (Chee;; Ùn.,):
Studies of a fundamentai nature:'basic research
Iiances for human/veterinarv medicine
Will the project involYe breeding animaIs?
NO X
YES
Will the projec! involve the generation of genetically altered animaIs?
Will field studies be conducted?
NO X
'l'ES
1\0 X
YES
c) Description of AnimaIs
Splstrain 1
\ Rats
1
•
Species & strain
Supplier/Source
Strain
Sp/strain 2
Rats
1
\ Copenhagen
\ Sp/strain 3
\ Rats
Splstrain
\ Rabbits
~
Splstrain 5
\ Nude mice
1
1 Sp/strain
6
\ Nude mice
1
Fischer
\ SpragueDawley
91
NZ\V
Balb C
Balb C