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Tumor Biology |
The Prostate Centre, Vancouver General Hospital, 2660 Oak Street, Vancouver, British Columbia V6H 3Z6, [H. M., C. N., P. S. R., M. E. G.]; and Division of Urology, University of British Columbia, D-9, 2733 Heather Street, Vancouver, British Columbia V5Z 3J5 [C. N., M. E. G.], Canada
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TRPM-2, also known as clusterin or sulfated glycoprotein-2,
was first isolated from ram rete testes fluid (3)
, and it
is associated with a wide variety of physiological and pathological
processes, including tissue remodeling, lipid transport, reproduction,
complement regulation, and apoptotic cell death (4)
.
TRPM-2 has been regarded as a marker for cell death because its
expression is up-regulated in various normal and malignant tissues
undergoing apoptosis (5, 6, 7, 8, 9)
. Recent studies provide
conflicting data on the association between enhanced TRPM-2 expression
and apoptotic activity (10, 11, 12)
. Although it was initially
named as an androgen-repressed gene up-regulated in regressing prostate
tissues after androgen ablation (13)
, the functional role
of TRPM-2 in castration-induced apoptosis remains undefined. In
prostate cancer, TRPM-2 expression is associated with apoptotic cell
death and AI recurrences. The introduction of TRPM-2 cDNA
into LNCaP prostate cancer cells increases the resistance to apoptosis
induced by tumor necrosis factor 
treatment (14)
. The
increased expression of TRPM-2 in prostate cancer correlates with the
increasing Gleason score (15)
. However, the functional
significance of TRPM-2 expression during AI progression has not been
demonstrated.
Controlled study of the complex molecular processes associated with AI progression in prostate cancer has proved difficult because it cannot be replicated in vitro, and few animal models exist that reproducibly mimic the clinical course of the disease in men. The Shionogi tumor model is an AD mouse mammary carcinoma xenograft that is particularly useful in studying mechanism of castration-induced apoptosis and AI progression. In this model, AD tumors in male mice undergo complete regression after castration but recur as rapidly growing AI tumors after 1 month (16) . The highly reproducible regression and recurrence pattern provides a reliable end point to test the efficacy of agents targeting castration-induced apoptosis and their effects on time to AI progression. Of the available human prostate cancer cell lines, only the LNCaP cell line is AD, PSA-secreting, and immortalized in vitro. As in human prostate cancer, serum PSA levels in the LNCaP tumor model are initially regulated by androgen and directly proportional to tumor volume, with loss of androgen-regulated PSA gene expression after castration as a surrogate end point of AI progression (17) .
In the present study, we report for the first time that the TRPM-2 gene is an apoptosis-associated gene rather than androgen-repressed gene. We then evaluated the effects of TRPM-2 overexpression on time to AI progression in the LNCaP tumor model. Finally, we tested whether adjuvant use of antisense TRPM-2 ODNs enhances castration-induced apoptosis and delays AI progression in the AD Shionogi tumor model.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5 x 106 tumor cells. When Shionogi tumors became 1–2
cm in diameter, usually 2–3 weeks after injection, castration was
performed through an abdominal incision under methoxyflurane
anesthesia. Details of the maintenance of mice, tumor stock, and
operative procedures are described in a previous publication
(19)
. Mice were maintained in accordance with
institutional accredited guidelines.
CCB Treatment.
One the day before the castration, male DD/S mice bearing the
Shionogi tumor were randomly selected for treatment with CCBs
(n = 5) versus no treatment as the
control (n = 5). Beginning 1 day before
castration, 588-µg nifedipine and 100-µg norvasc were administered
p.o. three times daily to each mouse in the CCB group for 14 days.
Tumor volume was measured twice weekly and calculated by the formula
length x width x depth x 0.5236 (20)
.
TUNEL Staining.
A modified TUNEL technique (21)
was used to detect
apoptotic cells in Shionogi tumors using the ApopTag in Situ
Apoptosis Detection System (Oncor Inc., Gaithersburg, MD) according to
the manufacture’s protocol. The number of positively stained cells per
high power field in five random fields was counted and averaged.
Expression Plasmid and Transfection of LNCaP Cells.
LNCaP cells were kindly provided by Dr. Leland W. K. Chung
(University of Virginia, Charlottesville, VA) and maintained in RPMI
1640 (Life Technologies, Inc.) supplemented with 5% heat-inactivated
FCS. pRC-CMV expression vector containing the 1.6-kb cDNA fragment
encoding human TRPM-2 was kindly provided by Dr. Martin Tenniswood (W.
Alton Jones Cell Science Center, Lake Placid, NY). The expression
vector was transfected into LNCaP cells by the liposome-mediated gene
transfer method (22)
. Briefly, 2 x 105 LNCaP cells were plated in 6-cm plates. The
next day, 5 µg of purified TRPM-2 cloned pRC-CMV or pRC-CMV alone (as
a control) was added to LNCaP cells after a preincubation for 30 min
with 5 µg of lipofectamine reagent and 3 ml of serum-free OPTI-MEM
(Life Technologies, Inc.). Drug selection in 300 µg/ml of Geneticin
(Sigma Chemical Co., St. Louis, MO) was begun 3 days after the
transfection. Colonies were harvested 2 weeks after drug selection
using cloning cylinders, and cell lines were expanded for in
vivo injection.
Assessment of in Vivo LNCaP Tumor Growth and
Determination of Serum PSA Levels.
One million cells of each of the LNCaP sublines were inoculated s.c.
with 0.1 ml of Matrigel (Becton Dickinson Labware, Lincoln Park, NJ) in
the flank region of 6- to 8-week-old male athymic nude mice (BALB/c
strain; Charles River Laboratory, Montreal, Canada). Each experimental
group consisted of six mice. Mice were castrated via a scrotal approach
when tumors reached 100 and 200 mm3
in volume.
Tumor volume was measured once weekly and calculated as described
above. Blood samples were obtained with tail vein incisions of mice
once weekly. Serum PSA levels were determined by an enzymatic
immunoassay kit with a lower limit of sensitivity of 0.2 µg/liter
(Abbott IMX, Montreal, Canada) according to the manufacture’s
protocol. Data points were reported as mean values ± SD.
Antisense TRPM-2 ODNs.
Phosphorothioate ODNs used in this study were obtained from Nucleic
Acid-Protein Service Unit, University of British Columbia (Vancouver,
Canada). The sequences of antisense TRPM-2 ODNs corresponding to
the mouse TRPM-2 translation initiation site were
5'-GCACAGCAGGAGAATCTTCAT-3'. Two base TRPM-2 mismatch ODNs
(5'-GCACAGCAGGAGGATATTCAT-3') were used as the control.
Treatment of Cells with ODNs.
Lipofectin, a cationic lipid (Life Technologies, Inc.) was used to
increase the ODN uptake of cells. Shionogi cells were treated with
various concentrations of ODNs after a preincubation for 20 min with 4
µg/ml lipofectin in serum-free OPTI-MEM (Life Technologies, Inc.).
After 4 h, the medium containing ODNs and lipofectin was replaced
with the standard culture medium described above.
Northern Blot Analysis.
Total RNA was isolated from Shionogi tumor tissues and cultured
Shionogi tumor cells by the acid-guanidium
thiocyanate-phenol-chloroform method. Poly(A)+
mRNA was then purified from total RNA using oligodeoxythymidylate
cellulose. The electrophoresis, hybridization, and washing conditions
were carried out as previously reported (17)
. Mouse TRPM-2
and G3PDH cDNA probes were generated by reverse transcription-PCR from
total RNA of mouse brain using primers 5'-AATGAGCTCCAAGAACTGTCCACT- 3'
(sense) and 5'-AAA-GAGCGTGTCTATGATGCCAGAT-3' (antisense) for TRPM-2,
and 5'-ATGGTGAAG-GTCGGTGTGAACGGAT-3' (sense) and
5'-AAAGTTGTCATGGAT-GACCTT-3' (antisense) for G3PDH. The density of
bands for TRPM-2 was normalized against that of G3PDH by densitometric
analysis.
Western Blot Analysis.
Samples containing equal amounts of protein (15 µg) from lysates of
the cultured Shionogi cells and Shionogi tumors were electrophoresed on
an SDS-polyacrylamide gel and transferred to a nitrocellulose filter.
The filters were blocked in PBS containing 5% nonfat milk powder at
4°C overnight and then incubated for 1 h with a 1:400-diluted
C-18, an antimouse TRPM-2 goat polyclonal antibody (Santa Cruz
Biotechnology Inc., Santa Cruz, CA), 1:10000-diluted MAB065, an antirat
ß-tubulin mouse monoclonal antibody (CHEMICON INTERNATIONAL INC.,
Tumecula, California), or 1:600-diluted C2–10, an antihuman PARP mouse
monoclonal antibody that reacts with mouse PARP (PHARMINGEN,
Mississauga, Canada). The filters were then incubated for 30 min with
horseradish peroxidase-conjugated antigoat or antimouse IgG
antibody (Amersham Life Science, Arlington Heights, IL), and specific
proteins were detected using an enhanced chemiluminescence Western
blotting analysis system (Amersham Life Science).
In Vivo ODN Treatment.
Male DD/S mice bearing the Shionogi tumor were castrated and randomly
selected for treatment with antisense TRPM-2 versus mismatch
control ODNs. Each experimental group consisted of seven mice.
Beginning the day of castration, each mouse received injections i.p. of
12.5 mg/kg of antisense TRPM-2 or mismatch control once daily for 15
days. The tumor volume was measured as described above. Data points
were reported as mean tumor volumes ± SD.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, compared to AD intact tumors before castration, TRPM-2 mRNA expression
is up-regulated 14-fold and 10-fold 4 and 7 days after castration,
respectively and is maintained at 8-fold higher levels in AI tumors.
The pattern of TRPM-2 up-regulation in the Shionogi tumor model after
castration is similar to that in the rat ventral prostate
(5)
and human prostate cancer xenografts (9)
.
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20/high power field)
in untreated control tumors were observed, but not in tumors treated
with CCBs (Fig. 2, A and B)
. Furthermore, TRPM-2 up-regulation in
Shionogi tumors after castration was significantly inhibited by CCB
treatment. TRPM-2 mRNA levels in tumors treated with CCBs were 90%,
86%, and 77% lower than controls at 4, 7, and 10 days after
castration, respectively (Fig. 2, C and D)
.
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, abundant levels of TRPM-2 mRNA were detected in four
independent TRPM-2-transfected clones (LNCaP/T1 to LNCaP/T4), whereas
the parental LNCaP (LNCaP/P) and the control vector-transfected cell
line (LNCaP/C) did not express detectable TRPM-2 mRNA levels. LNCaP/P,
LNCaP/C, LNCaP/T1, and LNCaP/T2 were selected for in vivo
study, with 1 x 106 of each cell
line inoculated s.c. in intact male nude mice. After castration,
LNCaP/P and LNCaP/C tumor growth was inhibited for 4 weeks, after which
LNCaP/P and LNCaP/C tumor volumes increased 2.5- and 2.3-fold,
respectively by 9 weeks after castration. In contrast, LNCaP/T1 and
LNCaP/T2 tumor volumes continued to grow after castration, increasing
4.5- and 4.7-fold, respectively by 9 weeks after castration (Fig. 3B)
. Serum PSA in mice bearing LNCaP/P and LNCaP/C tumors
decreased by 64% and 71%, respectively by 1 week after castration and
increased from 4 to 9 weeks after castration by 2.1- and 2.3-fold,
respectively. In comparison, serum PSA in mice bearing LNCaP/T1 and
LNCaP/T2 tumors decreased by 40% and 54%, respectively before
increasing to 3.9- and 4.1-fold, respectively by 9 weeks (Fig. 3C)
beginning 2 weeks after castration.
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, daily treatment of Shionogi tumor cells
with antisense TRPM-2 ODNs (50, 100, 500 or 1000
nM) for 2 days reduced TRPM-2 mRNA levels by 2,
15, 81, or 98%, respectively. In contrast, TRPM-2 mRNA levels were
unchanged by the two-base mismatch control ODNs at the used
concentrations.
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Inhibition of AI Progression in the Shionogi Tumor Model by
Antisense TRPM-2 ODN Treatment.
Male mice bearing Shionogi tumors 1–2 cm in diameter were castrated
and randomly selected for treatment with antisense TRPM-2
versus mismatch control ODNs. Mean pretreatment tumor volume
was similar in both groups. Beginning the day of castration, 12.5 mg/kg
of ODNs were administered once daily by i.p. injection for 15 days. As
shown in Fig. 5
, Shionogi tumors regressed faster, and complete regression occurred
earlier in mice treated with the antisense TRPM-2 ODN compared to those
treated with mismatch control ODNs. Furthermore, antisense TRPM-2 ODN
treatment significantly delayed the recurrence of AI tumors compared to
mismatch control ODN treatment. After an observation period of 50 days
postcastration, the mean tumor volume in the mismatch-treated control
group was six times that of the antisense-treated group. AI tumors
recurred in five of seven mice after a median of 46 days in the
antisense TRPM-2 ODN treatment group, whereas AI tumors recurred in all
mice after a median of 35 days in the mismatch ODN treatment group. No
side effects, including weight loss, gait disturbances, or anorexia
were observed with antisense TRPM-2 or mismatch ODN treatment over the
50-day observation period.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(14)
. Collectively, these conflicting
data do not clearly define the functional role of TRPM-2 associated
with apoptosis induced by various types of stimuli. TRPM-2 mRNA is highly up-regulated in Shionogi tumors after castration and in AI recurrent tumors. These changes in TRPM-2 are similar to previously reported findings in the rat prostate (5) and the human prostate cancer xenograft (9) . To differentiate between androgen-repressed versus apoptosis-associated up-regulation, CCBs were administered before castration to prevent castration-induced apoptosis because an early event in apoptotic cascade involves increases in the intracellular-free calcium concentration, with subsequent endonuclease activation (26 , 27) . Castration-induced apoptosis and up-regulation of TRPM-2 was prevented by CCB treatment, thereby supporting TRPM-2 as an apoptosis-related rather than androgen-repressed gene.
To determine whether TRPM-2 up-regulation after androgen ablation helps mediate the progression to androgen independence, we generated TRPM-2 overexpressing LNCaP prostate cancer cell lines. Although tumor take rates and growth in intact male mice were similar between parental and overexpressing TRPM-2 tumors, after castration, tumor growth and serum PSA increased severalfold faster in TRPM-2-tranfected tumors. These findings provide the first clear evidence that increased TRPM-2 results in the acquisition of androgen resistance and accelerates time to AI progression.
The up-regulation of TRPM-2 in Shionogi tumors after castration and the acquisition of androgen resistance with TRPM-2 overexpression suggests that preventing TRPM-2 up-regulation precipitated by androgen ablation may enhance castration-induced apoptosis and delay the AI progression of prostate cancer. Antisense ODN, a chemically modified stretch of single-stranded DNA that is complementary to mRNA regions of a target gene and thereby effectively inhibits gene expression by forming RNA/DNA duplexes (28) , offers one strategy to specifically target TRPM-2 gene expression. Phosphorothioate ODNs are water soluble, stable agents manufactured to resist nuclease digestion. After parenteral administration, phosphorothioate ODNs become associated with high capacity, low affinity serum binding proteins (29) . In this study, the phosphorothioate antisense TRPM-2 ODN corresponding to the mouse TRPM-2 translation initiation site reduced TRPM-2 expression levels in a sequence-specific and dose-dependent manner. In vivo administration of antisense TRPM-2 ODN accelerated castration-induced tumor regression and delayed the time to AI progression compared to that of mismatch control ODNs. Consistent with the in vitro data, in vivo treatment with the antisense TRPM-2 ODN also reduced TRPM-2 mRNA levels. The earlier detection of PARP cleavage fragments in Shionogi tumors after antisense TRPM-2 ODN treatment suggests that the inhibition of TRPM-2 up-regulation after castration results in an earlier induction of castration-induced apoptosis.
Because TRPM-2 plays a critical role in some normal organs, including the brain, kidney, spleen, and prostate (4 , 30 , 31) , the effects of antisense TRPM-2 ODNs on TRPM-2 expression levels in these organs were also examined. Although antisense ODNs significantly reduced TRPM-2 levels in the AD prostate and tumor tissues undergoing castration-induced apoptosis, TRPM-2 expression was not altered by antisense ODNs in the other organs examined. We reported similar differential decreases in Bcl-2 levels after treatment with mouse antisense Bcl-2 ODNs in Shionogi tumors (32) . We speculate that tissues undergoing apoptosis may be more sensitive to antisense TRPM-2 ODN treatment relative to intact organs because of the preferential uptake of ODNs in these tissues for reasons of biodistribution or increased membrane permeability. However, reduced target mRNA levels in mouse tissues have been reported by Monia et al. (33) after i.v. administration of antisense C-raf ODNs, although no significant toxicity was observed. A phase I dose-escalation trial using antisense Bcl-2 ODNs in nine patients with lymphoma reported tumor responses with no significant toxicity (34) . These findings suggest that therapeutic doses of systemic phosphorothioate antisense ODNs targeted against important cellular proteins do not cause significant toxicity in normal tissues.
Integration and appropriate timing of combination therapies, based on biological mechanism of progression and castration-induced changes in gene expression, may provide means to delay AI progression in a major way (32 , 35) . New nonhormonal therapies for prostate cancer have traditionally been evaluated in patients with hormone refractory disease, and when used in this end-stage setting, none has demonstrated improved survival (2) . A more rational strategy to delay emergence of the AI phenotype would initiate treatment earlier to enhance castration-induced apoptosis by targeting the adaptive changes in gene expression precipitated by androgen withdrawal. The present study provides evidence for a functional role of TRPM-2 in androgen resistance and AI progression, and it demonstrates that reduction of TRPM-2 gene expression by antisense TRPM-2 ODNs can delay progression to androgen independence.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grant 009002 from the National
Cancer Institute of Canada. 
2 To whom requests for reprints should be
addressed, at Division of Urology, University of British Columbia, D-9,
2733 Heather Street, Vancouver, British Columbia V5Z 3J5, Canada.
3 The abbreviations used are: AI,
androgen-independent; TRPM-2; testosterone-repressed prostate
message-2; AD, androgen-dependent; PSA, prostate-specific antigen; ODN,
oligodeoxynucleotide; TUNEL, terminal deoxynucleotidyl
transferase-mediated deoxyuridine 5'-triphosphate nick end labeling;
CCB, calcium channel blocker; G3PDH, glyceraldehyde-3-phosphate
dehydrogenase; PARP, poly(ADP-ribose) polymerase.
Received 6/16/99. Accepted 10/28/99.
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M. Asamoto, N. Hokaiwado, Y.-M. Cho, S. Takahashi, Y. Ikeda, K. Imaida, and T. Shirai Prostate Carcinomas Developing in Transgenic Rats with SV40 T Antigen Expression under Probasin Promoter Control Are Strictly Androgen Dependent Cancer Res., June 1, 2001; 61(12): 4693 - 4700. [Abstract] [Full Text] [PDF]
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H. Miyake, C. Nelson, P. S. Rennie, and M. E. Gleave Acquisition of Chemoresistant Phenotype by Overexpression of the Antiapoptotic Gene Testosterone-repressed Prostate Message-2 in Prostate Cancer Xenograft Models Cancer Res., May 1, 2000; 60(9): 2547 - 2554. [Abstract] [Full Text]
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H. Miyake, K. N. Chi, and M. E. Gleave Antisense TRPM-2 Oligodeoxynucleotides Chemosensitize Human Androgen-independent PC-3 Prostate Cancer Cells Both in Vitro and in Vivo Clin. Cancer Res., May 1, 2000; 6(5): 1655 - 1663. [Abstract] [Full Text]
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