Download Acquisition of Chemoresistant Phenotype by

[CANCER RESEARCH 60, 2547–2554, May 1, 2000]
Acquisition of Chemoresistant Phenotype by Overexpression of the Antiapoptotic
Gene Testosterone-repressed Prostate Message-2 in Prostate Cancer
Xenograft Models1
Hideaki Miyake, Colleen Nelson, Paul S. Rennie, and Martin E. Gleave2
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 [H. M., C. N., M. E. G.], Canada
ABSTRACT
Testosterone-repressed prostate message-2 (TRPM-2) expression is
highly up-regulated in normal and malignant prostate cells after androgen
withdrawal. Although recent studies have suggested a protective role of
TRPM-2 expression against apoptosis in several experimental models, the
functional role of TRPM-2 in chemotherapy-induced apoptosis remains
undefined. Here, we demonstrated that overexpression of TRPM-2 in
human androgen-dependent LNCaP prostate cancer cells by stable transfection rendered them highly resistant to paclitaxel treatment than control
LNCaP cells, with a 20-fold higher IC50 through the inhibition of apoptotic cell death. In mice bearing TRPM-2-overexpressing LNCaP tumors, tumor volume and serum prostate-specific antigen increased two to
three times faster after castration and paclitaxel treatment compared with
mice bearing control tumors. We then tested the efficacy of combined
treatment with antisense TRPM-2 oligodeoxynucleotide (ODN) and paclitaxel in the mouse androgen-dependent Shionogi tumor model. Antisense
TRPM-2 ODN treatment significantly enhanced paclitaxel chemosensitivity of Shionogi tumor cells in a dose-dependent manner, reducing the IC50
by 75%. Combined treatment of Shionogi cells with 500 nM antisense
TRPM-2 ODN and 10 nM paclitaxel-induced apoptosis, either agent alone
did not. Adjuvant administration of antisense TRPM-2 ODN and polymeric micellar paclitaxel after castration resulted in reduced TRPM-2
levels in vivo and a significant delay of emergence of androgen-independent recurrent Shionogi tumors compared with administration of either
agent alone. Furthermore, combined treatment of mice bearing androgenindependent recurrent Shionogi tumors with antisense TRPM-2 ODN and
micellar paclitaxel inhibited tumor growth compared with treatment with
either agent alone. Collectively, these findings demonstrate that TRPM-2
overexpression helps confer a chemoresistant phenotype through inhibition of apoptosis, and that antisense TRPM-2 ODN may be useful in
enhancing the effects of cytotoxic chemotherapy in hormone-refractory
prostate cancer.
INTRODUCTION
Prostate cancer is the most commonly diagnosed malignancy and
the second leading cause of cancer deaths in men in Western industrialized countries. To date, no therapy exists that surpasses androgen
withdrawal for men with advanced disease, with symptomatic and/or
objective response in ⬃80% of patients. However, progression to
androgen independence ultimately occurs in nearly all of these cases
(1). Several hundred clinical studies using traditional cytotoxic chemotherapeutic agents document objective response rates of ⬍10% and
no improved survival rates (2). Accordingly, progression to androgen
independence remains the main obstacle to improving the survival and
quality of life in patients with advanced disease, emphasizing the need
Received 10/27/99; accepted 3/6/00.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Cancer Institute of Canada Grant 009002 and
the American College of Surgeons George A. H. Clowes Career Development Award.
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.
for novel therapeutic strategies that target the molecular mechanism of
the androgen- and chemoresistant phenotype of prostate cancer.
TRPM-2,3 also known as clusterin, sulfated glycoprotein-2, or
apolipoprotein J, was first isolated from ram rete testes fluid (3) and
has been proposed to have various biological functions, including
tissue remodeling, reproduction, lipid transport, and apoptotic cell
death (4). TRPM-2 was initially regarded as a marker for cell death,
because its expression is highly up-regulated in various normal and
malignant tissues undergoing apoptosis (5– 8). Recent studies, however, report conflicting findings on the association between enhanced
TRPM-2 expression and apoptotic activity (9 –11). Similarly,
TRPM-2 expression is increased in regressing normal prostate after
androgen ablation (5, 12), and its up-regulation has been shown to be
associated with antiapoptotic activity and disease progression in prostate cancer (13–15). We have recently reported that TRPM-2 expression in prostate cancer cells has a protective role against castrationinduced apoptosis (16). However, the functional significance of
TRPM-2 expression in apoptosis induced by chemotherapeutic agents
has not been investigated.
Controlled study of the complex molecular processes associated
with progression to androgen independence in prostate cancer has
proved difficult, because few animal models exist that reproducibly
mimic the clinical course of the disease in men. The AD Shionogi
mouse mammary carcinoma model is particularly useful for testing
the efficacy of agents targeting castration-induced apoptosis and their
effects on time to progression of androgen independence. AD
Shionogi tumors in intact male mice undergo complete regression
after castration but recur as rapidly growing AI tumors after 1 month
in a highly reproducible manner (17). Of the available human prostate
cancer cell lines, only the LNCaP tumors are AD when xenografted
into male immunodeficient mice, 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 progression to
androgen independence (18).
In the present study, we evaluated the effects of TRPM-2 overexpression on time to progression of androgen independence after castration and paclitaxel treatment in the LNCaP tumor model. We then
evaluated the effects of paclitaxel treatment on TRPM-2 gene expression in Shionogi tumor cells and the effects of antisense TRPM-2
ODN on paclitaxel chemosensitivity using the Shionogi tumor model.
MATERIALS AND METHODS
Paclitaxel. Paclitaxel was purchased from Sigma Chemical Co. (St. Louis,
MO). A stock solution of paclitaxel (1 mg/ml) was prepared with DMSO and
diluted with PBS to the required concentrations before each in vitro experi3
The abbreviations used are: TRPM-2; testosterone-repressed prostate message-2; AI,
androgen-independent; AD, androgen-dependent; PSA, prostate-specific antigen; ODN,
oligodeoxynucleotide; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; PARP, poly(ADP-ribose) polymerase; CMV, cytomegalovirus; poly(A)⫹ mRNA, polyadenylated
mRNA.
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CHEMORESISTANCE AND TRPM-2 IN PROSTATE CANCER
Fig. 1. A, Western blot analysis of TRPM-2 protein in
TRPM-2-transfected LNCaP cell lines. Protein was extracted
from PC3 (positive control for the screening of TRPM-2
protein expression), LNCaP/P (parental cell line of LNCaP),
LNCaP/C (vector-only transfected cell line), and four clones
of TRPM-2 transfectants (LNCaP/T1 to LNCaP/T4), and
TRPM-2 and ␤-tubulin levels were analyzed by Western
blotting. Molecular mass: unprocessed form of TRPM-2, 60
kDa; mature form of TRPM-2, 40 kDa B, cytotoxic effect of
paclitaxel treatment on LNCaP sublines in standard medium
with 5% FCS. C, cytotoxic effects of paclitaxel treatment on
LNCaP sublines in charcoal-stripped FCS. Each cell line in
B and C was treated with various concentrations of paclitaxel
for 72 h, and cell viability was then determined by in vitro
mitogenic assay. Each data point represents the mean of
three independent experiments ⫾ SD. LNCaP/T1 and LNCaP/T2 showed significantly higher resistance to paclitaxel
treatment than LNCaP/P and LNCaP/C (P ⬍ 0.01). D, DNA
fragmentation assay of LNCaP sublines treated with paclitaxel. Each cell line was treated with 0.5 nM paclitaxel. After
48 h of incubation, DNA was extracted from culture cells,
electrophoresed in a 2% agarose gel, and visualized by
ethidium bromide staining and UV transillumination. E, Proteins were extracted from each cell line after the same
treatment as described in C and analyzed by Western blotting with an anti-PARP antibody. Uncleaved intact PARP,
116 kDa; cleaved PARP, 85 kDa.
ment. Polymeric micellar paclitaxel used in the in vivo studies was generously
supplied by Dr. Helen M. Burt (Faculty of Pharmaceutical Sciences, University
of British Columbia, Vancouver, British Columbia, Canada).
Antisense TRPM-2 ODN. Phosphorothioate ODN used in this study was
obtained from Nucleic Acid-Protein Service Unit, University of British Columbia. The sequences of antisense TRPM-2 ODN corresponding to the mouse
TRPM-2 translation initiation site were 5⬘-GCACAGCAGGAGAATCTTCAT-3⬘. A 2-base TRPM-2 mismatch ODN (5⬘-GCACAGCAGGAGGATATTCAT-3⬘) was used as control.
LNCaP Sublines. LNCaP cells were kindly provided by Dr. Leland Chung
(University of Virginia, Charlottesville, VA) and maintained in RPMI 1640
(Life Technologies, Inc., Gaithersburg, MD) supplemented with 5% heatinactivated FCS. Steroid hormone-depleted charcoal-stripped media were prepared as described previously (19). A 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 liposomemediated gene transfer method as described previously (20). Briefly, 2 ⫻ 105
LNCaP cells were plated in 6-cm plates. The next day, 5 ␮g of purified
TRPM-2-cloned pRC-CMV or pRC-CMV alone (as a control) were added to
LNCaP cells after a preincubation for 30 min with 5 ␮g of LipofectAMINE
reagent and 3 ml of serum-free Opti-MEM (Life Technologies). Drug selection, in 300 ␮g/ml Geneticin (Sigma), was begun 3 days after the transfection.
Colonies were harvested 2 weeks after drug selection using cloning cylinders
and expanded to cell lines.
Assessment of in Vivo LNCaP Tumor Growth and Determination of
Serum PSA Levels. One million cells of each LNCaP subline 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, Quebec, Canada). Each experimental
group consisted of six mice. Mice were castrated via a scrotal approach when
tumors reached 200 –300 mm3 in volume, and from 10 to 14 days after
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castration, 0.5 mg of polymeric micellar paclitaxel was administered once
daily by i.v. injection. Tumor volume was measured once weekly and calculated by the formula length ⫻ width ⫻ depth ⫻ 0.5236 (19). 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 ␮g/liter (Abbott IMX, Montreal, Quebec, Canada) according
to the manufacturer’s protocol. Data points were reported as mean values ⫾ SD.
Shionogi Tumor Growth. The Toronto subline of the transplantable SC115 AD mouse mammary carcinoma was used in all experiments (21).
Shionogi tumor cells were maintained in DMEM (Life Technologies) supplemented with 5% heat-inactivated FCS. For in vivo study, ⬃5 ⫻ 106 cells of the
Shionogi carcinoma were injected s.c. into adult male DD/S strain mice. 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 (22).
Treatment of Cells with ODN. In vitro-cultured cells were treated with
various concentrations of ODN after a preincubation for 20 min with 4 ␮g/ml
Lipofectin (Life Technologies) in serum free Opti-MEM. Media containing
ODN and Lipofectin was replaced 4 h later with standard culture medium
described above.
Northern Blot Analysis. Total RNA was isolated from in vitro-cultured
cells and in vivo tumor tissues by the acid-guanidium thiocyanate-phenolchloroform method. Poly(A)⫹ mRNA was then purified from total RNA using
oligodeoxy-thymidylate cellulose (Pharmacia Biotech Inc., Uppsala, Sweden).
Five micrograms of poly(A)⫹ mRNA from each sample were subjected to
electrophoresis on 1.2% agarose-formaldehyde gels and transferred to nylon
membranes (Amersham, Arlington Heights, IL) overnight according to standard procedure (17). The RNA blots were hybridized with a mouse TRPM-2
cDNA probe labeled with [32P]dCTP by random primer labeling. After stripping, the membranes were rehybridized with a mouse G3PDH cDNA probe.
These probes were generated by reverse transcription-PCR from total RNA of
mouse brain using primers 5⬘-AATGAGCTCCAAGAACTG-TCCACT-3⬘
Fig. 2. Effect of TRPM-2 overexpression on paclitaxel treatment-induced changes in
(sense) and 5⬘-AAAGAGCGTGTCTATGATGCCAGAT-3⬘ (antisense) for
TRPM-2 and 5⬘-ATGGTGAAGGTCGGTGTGAACGGAT-3⬘ (sense) and 5⬘- LNCaP tumor growth and serum PSA level in nude mice. A, each LNCaP subline was
injected s.c. into male nude mice, and mice bearing tumors between 200 and 300 mm3 in
AAAGTTGTCATGGATGACCTT-3⬘ (antisense) for G3PDH. Density of volume were castrated. Beginning 10 days after castration, 0.5 mg of micellar paclitaxel
bands for TRPM-2 was normalized against that of G3PDH by densitometric was injected i.v. once daily for 5 days. Tumor volume was measured once weekly and
calculated by the formula length ⫻ width ⫻ depth ⫻ 0.5236. Each point represents the
analysis.
mean tumor volume in each experimental group containing six mice ⫾ SD. Mean volumes
Western Blot Analysis. The expression of TRPM-2 and PARP protein in of LNCaP/T1 and LNCaP/T2 tumors 10 weeks after castration were significantly higher
cultured cells and tumor tissues was determined by Western blot analysis than those of LNCaP/P and LNCaP/C tumors (P ⬍ 0.001). B, blood samples for
as described previously (20). Briefly, samples containing equal amounts of measurement of serum PSA levels were obtained with tail vein of the mice after castration
protein (15 ␮g) were electrophoresed on a SDS-polyacrylamide gel and once weekly. Serum PSA levels were determined by an enzymatic immunoassay kit
according to the manufacturer’s instructions (Abbott IMX). Each point represents the
transferred to a nitrocellulose filter. The filters were blocked in PBS mean tumor volume in each experimental group containing six mice ⫾ SD. Mean serum
containing 5% nonfat milk powder at 4°C overnight and then incubated for PSA levels in mice bearing LNCaP/T1 and LNCaP/T2 tumors 10 weeks after castration
1 h with an anti-human TRPM-2 goat polyclonal antibody (Santa Cruz were significantly higher than those in mice bearing LNCaP/P and LNCaP/C tumors
(P ⬍ 0.05).
Biotechnology Inc., Santa Cruz, CA), anti-human PARP mouse monoclonal
antibody (PharMingen, Mississauga, Ontario, Canada), or anti-rat ␤-tubulin
mouse monoclonal antibody (Chemicon International Inc., Tumecula, CA).
DNA Fragmentation Analysis. The nucleosomal DNA degradation was
The filters were then incubated for 30 min with horseradish peroxidaseanalyzed as described previously with a minor modification (20). Briefly, 1 ⫻ 105
conjugated anti-goat or mouse IgG antibody (Amersham), and specific
cultured cells were seeded in 5-cm culture dishes and allowed to adhere overnight.
proteins were detected using an enhanced chemiluminescence system (AmAfter the indicated treatment with paclitaxel and/or ODN, cells were harvested and
ersham).
then lysed in a solution containing 100 mM NaCl, 10 mM Tris (pH 7.4), 25 mM
In Vitro Cell Growth Assays. The in vitro growth of LNCaP and
EDTA, and 0.5% SDS. After the centrifugation, the supernatants were incubated
Shionogi tumor cells was assessed by the in vitro mitogenic assay and the with 300 ␮g/ml proteinase K for 5 h at 65°C and extracted with phenol-chloro3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, re- form. The aqueous layer was treated with 0.1 volume of 3 M sodium acetate, and
spectively, as described previously (19, 20). Briefly, 3 ⫻ 103 cells were the DNA was precipitated with 2.5 volumes of 95% ethanol. After treatment with
seeded in each well of 96-well microtiter plates and allowed to attach 100 ␮g/ml RNase A for 1 h at 37°C, the sample was electrophoresed on a 2%
overnight. After treatment with various concentrations of paclitaxel and/or agarose gel and stained with ethidium bromide.
ODN, LNCaP cells were fixed with 1% glutaraldehyde (Sigma) and stained
Assessment of in Vivo Shionogi Tumor Growth. To determine whether
with 0.5% crystal violet (Sigma), and Shionogi tumor cells were treated combined antisense TRPM-2 ODN and paclitaxel treatment delays time to AI
with 20 ␮l of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- recurrence after castration compared with either agent alone, male DD/S mice
lium bromide (Sigma) in PBS, followed by incubation for 4 h at 37°C. The bearing Shionogi tumors were castrated and randomly selected for treatment
absorbance was determined with a microculture plate reader (Becton Dick- with antisense TRPM-2 ODN alone (group 1), mismatch control ODN alone
inson Labware) at 540 nm. Absorbance values were normalized to the (group 2), antisense TRPM-2 ODN plus paclitaxel (group 3), or mismatch
values obtained for the vehicle-treated cells to determine the percent of control ODN plus paclitaxel (group 4). Each experimental group consisted of
seven mice. Beginning the day of castration, 12.5 mg/kg antisense TRPM-2 or
survival. Each assay was performed in triplicate.
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Fig. 3. Effects of antisense TRPM-2 ODN and/or paclitaxel treatment on TRPM-2 expression in Shionogi tumor cells. A, cells were treated with various concentrations of paclitaxel for
48 h, poly(A)⫹ mRNA was then extracted and analyzed for TRPM-2 and G3PDH levels by
Northern blotting. B, cells were treated with 10 nM paclitaxel for indicated intervals, and
poly(A)⫹ mRNA was then extracted and analyzed for TRPM-2 and G3PDH levels by
Northern blotting. C, cells were treated daily with 500 nM antisense TRPM-2 ODN or a 2-base
TRPM-2 mismatch control ODN for 2 days. After a 24-h exposure to 10 or 50 nM paclitaxel,
poly(A)⫹ RNA was then extracted and analyzed for TRPM-2 and G3PDH levels by Northern
blotting.
mismatch control ODN was injected i.p. once daily into each mouse for 15
days. From 10 to 14 days after castration, 0.5 mg polymeric micellar paclitaxel
was administered once daily by i.v. injection in groups 3 and 4. A second set
of experiments was designed to evaluate the effects of combined treatment on
established AI recurrent tumors. Castrate male DD/S mice bearing AI Shionogi
tumors ⬃0.5 cm in diameter were randomly selected to receive three treatment
regimens as described above. Tumor volume was measured twice weekly and
calculated as described above. Data points were reported as average tumor
volume ⫾ SD.
Statistical Analysis. The in vitro cytotoxic effects of ODN and/or paclitaxel were analyzed using a repeated measure ANOVA model. AI recurrencefree survival curves were calculated by the method of Kaplan-Meier and
evaluated with the Mantel-Cox log rank test. The remaining data were analyzed by Student’s t test. The level of statistical significance was set at
P ⬍ 0.05, and all statistical calculations were done by use of Statview 4.5
software (Abacus Concepts, Inc., Berkeley, CA).
RESULTS
Increased Resistance to Paclitaxel by Overexpression of
TRPM-2 in LNCaP Cells in Vitro. Western blot analysis was used
to examine TRPM-2 protein expression levels in the LNCaP sublines.
As shown in Fig. 1A, abundant levels of both unprocessed (60-kDa)
and mature (40-kDa) forms of TRPM-2 protein were detected in
TRPM-2-transfected clones (LNCaP/T1 to LNCaP/T4) at almost the
same levels, whereas the parental LNCaP (LNCaP/P) and the control
vector-transfected cell line (LNCaP/C) did not express detectable
TRPM-2 protein levels. The four TRPM-2-transfected clones showed
almost the same results in the subsequent experiments; therefore, we
hereafter report only the data of LNCaP/P, LNCaP/C, LNCaP/T1, and
LNCaP/T2.
To determine whether TRPM-2 overexpression confers a chemoresistant phenotype on LNCaP cells in vitro, the growth rates of LNCaP
sublines after paclitaxel treatment were analyzed using normal and
charcoal-stripped media. As shown in Fig. 1, B and C, LNCaP/T1 and
LNCaP/T2 exhibited significantly higher resistance to paclitaxel compared with LNCaP/P and LNCaP/C both in normal and charcoalstripped media (P ⬍ 0.01 for both). Overexpression of TRPM-2 in
LNCaP cells increased the IC50 of paclitaxel 5-fold (from 1.5 to 8 nM)
in normal media and 20-fold (from 0.01 to 0.4 nM) in charcoalstripped media (Fig. 1B).
The induction of apoptosis in LNCaP sublines in normal media
treated with 1 nM paclitaxel for 72 h was assessed by DNA degradation assay and Western blot analysis of PARP protein, a substrate of
the caspases activated during the process of apoptotic execution (23).
The characteristic apoptotic DNA ladders were detected in LNCaP/P
and LNCaP/C but not in LNCaP/T1 and LNCaP/T2 (Fig. 1D). Similarly, the Mr 116,000 intact form of PARP was observed in all of
LNCaP sublines, whereas the Mr 85,000 PARP cleavage fragment was
detected after paclitaxel treatment only in LNCaP/P and LNCaP/C
(Fig. 1E).
Acquisition of Resistant Phenotype to Paclitaxel by Overexpression of TRPM-2 in the LNCaP Tumor Model in Vivo. To
determine whether TRPM-2 overexpression confers resistance to paclitaxel treatment in vivo, 1 ⫻ 106 cells of each cell line (LNCaP/P,
LNCaP/C, LNCaP/T1, or LNCaP/T2) were inoculated s.c. in male
nude mice. When tumors reached 200 –300 mm3, mice were castrated.
Beginning 10 days after castration, 0.5 mg of polymeric micellar
paclitaxel was administered i.v. once daily for 5 days. LNCaP/P and
LNCaP/C tumor growth decreased by 61 and 57%, respectively, by 4
weeks after castration and remained below precastrate volumes by 10
weeks after castration. In contrast, LNCaP/T1 and LNCaP/T2 tumor
volume decreased by 11 and 28%, respectively, by 4 weeks after
castration and thereafter increased 2.4- and 1.9-fold, respectively, by
10 weeks after castration (Fig. 2A). Serum PSA in mice bearing
LNCaP/P and LNCaP/C tumors decreased by 77 and 75%, respectively, by 1 week after castration and remained below precastrate
levels by 10 weeks after castration. In comparison, serum PSA in mice
bearing LNCaP/T1 and LNCaP/T2 tumors decreased by 51 and 55%,
respectively, before increasing 1.6- and 1.4-fold above precastrate
levels, respectively, by 10 weeks after castration (Fig. 2B).
Changes in TRPM-2 Expression in Shionogi Tumor Cells after
Antisense TRPM-2 ODN and Paclitaxel Treatment. Northern blot
analysis was used to determine the effects of paclitaxel treatment on
TRPM-2 mRNA expression in Shionogi tumor cells. As shown in Fig.
3A, TRPM-2 mRNA induction increased in a dose-dependent manner
by paclitaxel treatment at concentrations up to 10 nM. Time course
experiments demonstrated that paclitaxel-induced TRPM-2 mRNA
up-regulation peaked by 48 h after treatment and began decreasing by
72 h after treatment (Fig. 3B).
We then examined the effects of combined treatment with antisense
TRPM-2 ODN and paclitaxel on TRPM-2 mRNA expression in
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Fig. 4. Effect of combined treatment with antisense TRPM-2 ODN and paclitaxel on Shionogi tumor cell growth and apoptosis. A, cells were treated daily with 500 nM antisense
TRPM-2 ODN or mismatch control ODN for 2 days. After ODN treatment, the medium was replaced with medium containing various concentrations of paclitaxel. After 48 h of
incubation, cell viability was determined by in vitro mitogenic assay. Each data point represents the mean of three independent experiments ⫾ SD. The cytotoxic effect of paclitaxel
on Shionogi tumor cells was significantly enhanced by antisense TRPM-2 ODN treatment (P ⬍ 0.01). B, Cells were treated daily with various concentrations of antisense TRPM-2
ODN or mismatch control ODN for 2 days and then incubated for 48 h with medium alone or medium containing 10 nM paclitaxel, and cell viability was determined by in vitro mitogenic
assay. Each data point represents the mean of three independent experiments ⫾ SD. Treatment of Shionogi tumor cells with antisense TRPM-2 ODN significantly enhanced the
sensitivity to paclitaxel (P ⬍ 0.01). C, cells were treated daily with 500 nM antisense TRPM-2 ODN or mismatch control ODN for 2 days. After ODN treatment, the medium was
replaced with medium containing 10 nM paclitaxel. After 48 h of incubation, DNA was extracted from culture cells, electrophoresed in a 2% agarose gel, and visualized by ethidium
bromide staining and UV transillumination. D, proteins were extracted from Shionogi tumor cells after the same treatment as described in C and analyzed by Western blotting with
an anti-PARP protein. Molecular mass: uncleaved intact PARP, 116 kDa; cleaved PARP, 85 kDa.
Shionogi cells. As shown in Fig. 3C, 500 nM antisense TRPM-2 ODN
combined with 10 or 50 nM paclitaxel decreased TRPM-2 mRNA
levels by 85 or 70%, respectively, compared with 500 nM mismatch
control ODN treatment.
Synergistic Effects of Antisense TRPM-2 ODN and Paclitaxel
Treatment on Induction of Apoptosis in Shionogi Tumor Cells.
To examine whether treatment with antisense TRPM-2 ODN enhances the paclitaxel-induced cytotoxicity, Shionogi tumor cells were
treated with various concentrations of antisense TRPM-2 ODN once
daily for 2 days and then incubated with various concentrations of
paclitaxel for 2 days. As shown in Fig. 4A, antisense TRPM-2 ODN
treatment significantly enhanced paclitaxel chemosensitivity in a
dose-dependent manner (P ⬍ 0.01), reducing the IC50 of paclitaxel
from 100 to 25 nM, whereas mismatch control ODN had no effect.
Dose-dependent synergy between antisense TRPM-2 ODN and paclitaxel was also observed by increasing the antisense ODN concentration
when paclitaxel concentration was fixed at 10 nM (P ⬍ 0.01; Fig. 4B).
DNA fragmentation assay and Western analysis of PARP protein
were used to evaluate effects of combined antisense TRPM-2 ODN
(500 nM) and paclitaxel (10 nM) treatment on apoptosis induction.
After the same treatment schedule described above, characteristic
apoptotic DNA laddering was observed only after combined treatment
with antisense TRPM-2 ODN and paclitaxel (Fig. 4C). Similarly,
cleavage of PARP protein was detected only after combined antisense
TRPM-2 ODN and paclitaxel treatment (Fig. 4D).
Delayed Hormone-refractory Recurrence of Shionogi Tumors in Vivo by Combined Antisense TRPM-2 ODN and Paclitaxel Treatment. Male mice bearing Shionogi tumors between
1 and 2 cm in diameter were randomly selected for treatment with
either antisense TRPM-2 ODN alone, mismatch control ODN
alone, antisense TRPM-2 ODN plus micellar paclitaxel, or mismatch control ODN plus micellar paclitaxel. Mean tumor volume
was similar at the beginning of treatment in all four treatment
groups. Beginning the day of castration, 12.5 mg/kg antisense
TRPM-2 or mismatch control ODN was administered i.p. once
daily for 15 days. Beginning 10 days after castration, 0.5 mg of
polymeric micellar paclitaxel was administered i.v. once daily for
5 days. During an observation period of 60 days after castration, AI
tumors recurred in four of seven mice after a median of 53 days in
antisense TRPM-2 ODN plus micellar paclitaxel treatment group,
whereas AI tumors recurred in all mice after a median of 28, 39,
and 42 days in the mismatch control ODN treatment group, antisense TRPM-2 ODN treatment group, and mismatch control ODN
plus micellar paclitaxel treatment group, respectively (P ⬍ 0.01).
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Time to sacrifice was delayed in the other two treatment groups.
Combined antisense TRPM-2 ODN plus paclitaxel treatment resulted
in the most significant delay in tumor progression, producing a mean
tumor volume ⬃30 – 40% lower than in the mismatch control ODN
plus paclitaxel treatment group (P ⬍ 0.05).
Northern and Western blot analyses were used to examine the
effects of combined in vivo treatment with antisense TRPM-2 ODN
and paclitaxel on TRPM-2 mRNA expression and cleavage of PARP
protein in AI Shionogi tumors, which were harvested after completion
of the same treatment schedule described above. Consistent with the
in vitro results, antisense TRPM-2 ODN treatment resulted in a
substantial reduction in TRPM-2 mRNA in AI Shionogi tumors (Fig.
6B). Furthermore, the Mr 85,000 PARP cleavage fragment was de-
Fig. 5. Effects of adjuvant administration of antisense TRPM-2 ODN and polymeric
micellar paclitaxel after castration on Shionogi tumor growth. A, mice treated with
antisense TRPM-2 ODN alone, antisense TRPM-2 ODN plus micellar paclitaxel, and
mismatch control ODN plus micellar paclitaxel. Beginning the day of castration, 12.5
mg/kg antisense TRPM-2 ODN or mismatch control ODN was injected i.p. once daily for
15 days. Beginning 10 days after castration, 0.5 mg of micellar paclitaxel was injected i.v.
once daily for 5 days. Tumor volume was measured twice weekly and calculated by the
formula length ⫻ width ⫻ depth ⫻ 0.5236. Each point represents the mean tumor volume
in each experimental group containing seven mice ⫾ SD. Mean tumor volume in mice
treated with antisense TRPM-2 ODN plus micellar paclitaxel 60 days after castration was
significantly smaller than that in mice treated with antisense TRPM-2 ODN alone or
mismatch control ODN plus micellar paclitaxel (P ⬍ 0.001). B, AI recurrence-free
survival curves in mice treated as described in A. The time to progression of androgen
independence in mice treated with antisense TRPM-2 ODN plus micellar paclitaxel was
significantly delayed compared with mice treated with antisense TRPM-2 ODN alone or
mismatch control ODN plus micellar paclitaxel (P ⬍ 0.01)
Mean tumor volume at day 60 after castration was 2789, 3481, 712,
and 1276 mm3 in the antisense TRPM-2 ODN, mismatch control
ODN, antisense TRPM-2 ODN and paclitaxel, and mismatch control ODN and paclitaxel treatment groups, respectively (Fig. 5, A
and B).
Inhibition of Established AI Recurrent Shionogi Tumors by
Combined Antisense TRPM-2 ODN plus Paclitaxel Treatment.
Approximately 4 weeks after castration, AI Shionogi tumors recur and
grow rapidly, with a doubling time of 72 h (24). When AI tumors
reached 0.5 cm in diameter, mice were randomly selected for three
treatment groups and received the treatment under the same schedule
as described above. Mean tumor volume was similar at the beginning
of treatment in these groups. As shown in Fig. 6A, all mice treated
with antisense TRPM-2 ODN alone were sacrificed by day 21 after
initiation of treatment because of tumor mass ⬎10% of body weight.
Fig. 6. Effects of combined treatment with antisense TRPM-2 ODN and polymeric
micellar paclitaxel on AI Shionogi tumor growth. A, mice bearing AI recurrent Shionogi
tumors were randomly selected for treatment with antisense TRPM-2 ODN alone, antisense TRPM-2 ODN plus micellar paclitaxel, and mismatch control ODN plus micellar
paclitaxel. Treatments and measurement of tumor volume were performed using methods
described in Fig. 5. Each point represents the mean tumor volume in each experimental
group containing seven mice ⫾ SD. Mean tumor volume in mice treated with antisense
TRPM-2 ODN plus micellar paclitaxel at day 21 after initial treatment was significantly
smaller than that in mice treated with antisense TRPM-2 ODN alone or mismatch control
ODN plus micellar paclitaxel (P ⬍ 0.05). B, after completion of treatment as described in
A, poly(A)⫹ RNA was extracted from AI Shionogi tumors, and TRPM-2 and G3PDH
mRNA levels were analyzed by Northern blotting. Lane 1, AI tumors without treatment;
Lane 2, AI tumors treated with antisense TRPM-2 ODN alone; Lane 3, AI tumors treated
with antisense TRPM-2 ODN and micellar paclitaxel, Lane 4, AI tumors treated with
mismatch control ODN and micellar paclitaxel. C, after completion of treatment as
described in A, proteins were extracted from AI Shionogi tumors and analyzed by Western
blotting with an anti-PARP antibody. Molecular mass: uncleaved intact PARP, 116 kDa;
cleaved PARP, 85 kDa. Lane 1, AI tumors without treatment; Lane 2, AI tumors treated
with antisense TRPM-2 ODN alone; Lane 3, AI tumors treated with antisense TRPM-2
ODN and micellar paclitaxel; Lane 4, AI tumors treated with mismatch control ODN and
micellar paclitaxel.
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CHEMORESISTANCE AND TRPM-2 IN PROSTATE CANCER
tectable in AI Shionogi tumors only after combined treatment with
antisense TRPM-2 ODN and micellar paclitaxel (Fig. 6C).
DISCUSSION
TRPM-2 expression is highly up-regulated in several tissues undergoing apoptosis, including normal prostate, and prostate and breast
cancer xenograft models after hormone withdrawal (5– 8, 12). Although TRPM-2 expression was initially regarded as a marker for cell
death, its biological function in this process is poorly defined (9 –11,
13–15, 25). Accumulating evidence suggests that TRPM-2 is a cell
survival gene that protects cells from apoptotic death. For example,
overexpression of TRPM-2 in LNCaP prostate cancer cells enhances
resistance to apoptosis induced by tumor necrosis factor-␣ (13).
Furthermore, Steinberg et al. (14) reported a close correlation between
staining intensity of TRPM-2 by immunohistochemical analysis and
Gleason pattern in human prostate cancer specimens. We also demonstrated that TRPM-2 expression renders prostate cancer cells more
resistant to androgen ablation and helps mediate progression of androgen independence after castration (16). Collectively, these findings
suggest a protective role of TRPM-2 against apoptosis induced by
various types of stimuli; however, the significance of TRPM-2 expression in chemotherapy-induced apoptosis has not been evaluated.
The efficacy of chemotherapy for patients with prostate cancer
remains limited for various reasons, including inherent chemoresistance, pharmaceutical mechanism of chemotherapeutic action,
and inability of elderly patients to tolerate its toxicity (2, 24). To
date, no chemotherapeutic agent has demonstrated improved survival in patients with advanced prostate cancer, emphasizing the
need for novel therapeutic strategies that target the molecular basis
of androgen resistance and chemoresistance of prostate cancer. We
have recently shown that antisense Bcl-2 ODN delayed progression
to androgen independence (17) and enhanced paclitaxel chemosensitivity in the Shionogi tumor model (24). These findings illustrate
that targeting an antiapoptotic gene with sequence-specific antisense ODN can result in enhanced apoptosis after androgen withdrawal and conventional cytotoxic chemotherapy. The objectives
of this study were to examine whether TRPM-2 overexpression
confers resistance to paclitaxel and to determine whether antisense
TRPM-2 ODN could enhance paclitaxel chemosensitivity and delay emergence of AI tumors beyond that achieved with either agent
alone.
We initially evaluated the effects of TRPM-2 overexpression on
paclitaxel chemosensitivity using TRPM-2-transfected LNCaP cells
and observed that TRPM-2 transfectants were more highly resistant to
paclitaxel both in vitro and in vivo through the inhibition of apoptotic
cell death. These findings provide the first evidence that TRPM-2
overexpression protects prostate cancer cells from paclitaxel-induced
apoptosis, and its up-regulation may contribute to the chemoresistant
phenotype in prostate cancer.
Increased expression of TRPM-2 after paclitaxel treatment and
androgen withdrawal is likely an adaptive response, which helps the
cell survival against a cell death signal. It follows that inhibition of
TRPM-2 up-regulation precipitated by castration and paclitaxel treatment may delay progression of androgen independence through enhanced castration- and paclitaxel-induced apoptosis. Antisense ODNs
are chemically modified single-stranded DNA fragments complementary to mRNA regions of a target gene, which form RNA-DNA
duplexes and thereby reduce gene expression (26). The potential
problems of rapid intracellular degradation can be overcome by phosphorothioate modification of ODNs, which are more resistant to
nuclease digestion. After parenteral administration, phosphorothioate
ODN becomes associated with high-capacity, low-affinity serum-
binding proteins (27). Antisense ODNs therefore offer one strategy to
specifically target TRPM-2 gene expression.
Phosphorothioate antisense TRPM-2 ODN corresponding to the
mouse TRPM-2 translation initiation site used in this study inhibited TRPM-2 mRNA expression in a dose- and sequence-dependent manner, even after paclitaxel treatment, which increases
TRPM-2 expression. Furthermore, treatment of Shionogi cells with
antisense TRPM-2 ODN reduced the IC50 of paclitaxel by 75% and
enhanced paclitaxel-induced apoptosis. Systemic administration of
antisense TRPM-2 ODN and micellar paclitaxel in vivo significantly delayed time to emergence of AI tumors compared with
either agent alone and also enhanced regression of established AI
tumors. Although early adjuvant antisense TRPM-2 ODN therapy
after castration delayed progression of androgen independence,
treatment with antisense TRPM-2 ODN alone had no effect on
growth rates of established AI tumors. However, combined treatment with antisense TRPM-2 ODN plus paclitaxel decreased
TRPM-2 mRNA expression and accelerated apoptosis induction in
AI Shionogi tumors in vivo. These findings illustrate the efficacy
of combined antisense TRPM-2 ODN and paclitaxel treatment for
cooperatively delaying progression to androgen independence.
Integration and appropriate timing of combination therapies,
based on changes in expression of functionally relevant genes after
androgen ablation, may help delay progression to androgen independence. The results in the present study provide proof of principle for two potential strategies to delay emergence of the AI
phenotype. The first strategy would initiate treatment earlier to
enhance castration-induced apoptosis by targeting the antiapoptotic TRPM-2 gene up-regulation by androgen ablation with antisense TRPM-2 ODN. The second strategy would attempt to
enhance sensitivity to conventional chemotherapy by reduction of
TRPM-2-mediated chemoresistance with antisense TRPM-2 ODN.
The preclinical data presented here provide support for clinical
studies with combined antisense TRPM-2 ODN and paclitaxel
therapy for advanced prostate cancer.
ACKNOWLEDGMENTS
We thank Mary Bowden and Howard Tearle for excellent technical
assistance.
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Acquisition of Chemoresistant Phenotype by Overexpression
of the Antiapoptotic Gene Testosterone-repressed Prostate
Message-2 in Prostate Cancer Xenograft Models
Hideaki Miyake, Colleen Nelson, Paul S. Rennie, et al.
Cancer Res 2000;60:2547-2554.
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