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The Prostate Centre, Vancouver General Hospital, Vancouver, British Columbia, V6H 3Z6 Canada [H. M., K. N. C., M. E. G.], and Division of Urology, University of British Columbia, Vancouver, British Columbia, V5Z 3J5 Canada [M. E. G.]
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Introduction |
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Advances in the field of nucleic acid chemistry offers one attractive strategy to design AS3 ODN-based therapeutic agents that specifically hybridize with complementary mRNA regions of a target gene and thereby inhibit gene expression by forming RNA/DNA duplexes (3) . Rapid intracellular degradation of ODNs is a potential disadvantage of AS ODN therapy, but this problem can be overcome by substituting a nonbridging phosphoryl oxygen of DNA with a sulfur to create a phosphorothioate backbone, which stabilizes the ODN to nuclease digestion (4) . Recently, several antisense ODNs targeted against specific genes involved in neoplastic progression have been evaluated as potential therapeutic agents (5, 6, 7, 8) . Collectively, these findings identify AS ODNs as a novel class of antineoplastic agents when designed for appropriate molecular targets. However, because numerous genes are involved in tumor progression, inhibition of a single target gene will likely be insufficient to inhibit tumor progression in a meaningful way. In fact, combined use of AS ODN with other compounds, such as chemotherapeutic agents, have been demonstrated to produce more potent antineoplastic effects in some tumor model systems (9, 10, 11, 12) .
TRPM-2, also known as clusterin or sulfated glycoprotein-2, was first isolated from ram rete testes fluid (13) and plays important roles in various pathophysiological processes, including tissue remodeling, reproduction, lipid transport, complement regulation, and apoptosis (14) . Because TRPM-2 expression is increased in various benign and malignant tissues undergoing apoptosis, it has been regarded as a marker for cell death (15, 16, 17, 18) . Recent studies, however, provide conflicting findings regarding the relationship between TRPM-2 up-regulation and increased apoptotic activity (19, 20, 21) . Similarly, TRPM-2 expression increases in regressing normal prostatic epithelial cells (22 , 23) and prostate cancer xenografts (24 , 25) after treatment with various apoptotic stimuli and is associated with cell survival and disease progression in prostate cancer (26, 27, 28, 29, 30) . We have reported recently that an increase in TRPM-2 expression after androgen ablation accelerates tumor progression by inhibiting castration (31) - and chemotherapy4 -induced apoptosis. However, the functional significance of TRPM-2 expression in established AI human prostate cancer has not been examined.
In the present study, we designed and screened 10 phosphorothioate AS ODNs targeted against the human TRPM-2 gene to identify potent ODN sequences that specifically inhibit TRPM-2 expression in human AI prostate cancer PC-3 cells. We then tested whether AS TRPM-2 ODN can enhance the response of PC-3 cells to either Taxol (paclitaxel) or mitoxantrone both in vitro and in vivo.
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Materials and Methods |
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Chemotherapeutic Agents.
Paclitaxel and mitoxantrone were purchased from Sigma Chemical Co. (St.
Louis, MO) and Wyeth-Ayerst, Inc. (Montreal, Canada), respectively.
Stock solutions of paclitaxel and mitoxantrone (1 mg/ml) were prepared
with DMSO and diluted with PBS to the required concentrations before
each in vitro experiment. Polymeric micellar paclitaxel used
in these in vivo studies was generously supplied by Dr.
Helen M. Burt (Faculty of Pharmaceutical Sciences, University of
British Columbia, Vancouver, Canada).
AS TRPM-2 ODN.
Phosphorothioate ODNs used in this study were obtained from the Nucleic
Acid-Protein Service Unit, University of British Columbia (Vancouver,
Canada). The sequences of 10 AS TRPM-2 ODNs (AS ODN#1 to AS ODN#10) and
a two-base TRPM-2 MM ODN used as a control (MM Co. ODN) were as
follows: AS ODN#1, 5'-TGGAGTCTTTGCACGCCTCGG-3'; AS ODN#2,
5'-CAGCAGCAGAGTCTTCATCAT-3'; AS ODN #3, 5'-ATTGTCTGAG-ACCGTCTGGTC-3';
AS ODN#4, 5'-CCTTCAGCTTTGT-CTCTGATT-3'; AS ODN#5,
5'-AGCAGGGAGTCGATG-CGGTCA-3'; AS ODN#6,
5'-ATCAAGCTGC-GGACGATG-CGG-3'; AS ODN#7,
5'-GCAGGCAGCCCGTGGAGTTGT-3'; AS ODN#8, 5'-TTCAGCTGCTCCAGCAAGGAG-3'; AS ODN#9, 5'-AATTTAGGGTT- CTTCCTGGAG-3'; AS ODN#10,
5'-GCTGGGCGGAGTTGGGGGCCT-3'; and MM Co. ODN,
5'-CAGCAGCAGAGTATTTATCAT-3'.
Treatment of Cells with ODN.
Lipofectin, a cationic lipid (Life Technologies, Inc.), was used to
increase the ODN uptake of cells. PC-3 cells were treated with various
concentrations of ODN after a preincubation for 20 min with 3 µg/ml
lipofectin in serum-free OPTI-MEM (Life Technologies, Inc.). Four h
after the beginning of the incubation, the medium containing ODN and
lipofectin was replaced with standard culture medium described above.
Northern Blot Analysis.
Total RNA was isolated from cultured PC-3 cells and PC-3 tumor tissues
using the acid-guanidinium thiocyanate-phenol-chloroform method.
Electrophoresis, hybridization, and washing conditions were carried out
as reported previously (8)
. Human TRPM-2 and GAPDH cDNA
probes were generated by reverse transcription-PCR from total RNA
of human kidney using primers 5'-AAGGAAATTCAAAATGCTGTCAA-3' (sense) and
5'-ACAGACAAGATCTCCCGGCACTT-3' (AS) for TRPM-2, and
5'-TGC-TTTTAAC-TCTGGTAAAGT-3' (sense) and
5'-ATATTTGG-CAGGTTTTTCTAGA-3' (AS) for GAPDH. The density of bands
for TRPM-2 was normalized against that of GAPDH by densitometric
analysis.
Western Blot Analysis.
Samples containing equal amounts of protein (15 µg) from lysates of
the cultured PC-3 cells and PC-3 tumors were electrophoresed on a
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
antihuman TRPM-2 goat polyclonal antibody (Santa Cruz Biotechnology,
Inc.), 1:10000-diluted antirat ß-tubulin mouse monoclonal antibody
(Chemicon International Inc., Tumecula, CA), or 1:600-diluted antihuman
PARP mouse monoclonal antibody (PharMingen, Mississauga, Ontario,
Canada). The filters were then incubated for 30 min with
horseradish peroxidase-conjugated antigoat or mouse IgG antibody
(Amersham Life Science, Arlington Heights, IL), and specific proteins
were detected using an enhanced chemiluminescence Western blotting
analysis system (Amersham Life Science).
MTT Assay.
The in vitro growth-inhibitory effects of AS TRPM-2 ODN plus
paclitaxel or mitoxantrone on PC-3 cells were assessed using the MTT
assay as described previously (32)
. Briefly, 1 x
104 cells were seeded in each well of 96-well
microtiter plates and allowed to attach overnight. Cells were then
treated once daily with 500 nM ODN for 2 days.
After ODN treatment, cells were treated with various concentrations of
paclitaxel or mitoxantrone. After 48 h of incubation, 20 µl of 5
mg/ml MTT (Sigma Chemical Co.) in PBS were added to each well, followed
by incubation for 4 h at 37°C. The formazan crystals were then
dissolved in DMSO. The absorbance was determined with a microculture
plate reader (Becton Dickinson Labware, Lincoln Park, NJ) at 540 nm.
Absorbance values were normalized to the values obtained for the
vehicle-treated cells to determine the percentage of survival. Each
assay was performed in triplicate.
DNA Fragmentation Analysis.
Nucleosomal DNA degradation was analyzed as described previously with a
minor modification (32)
. Briefly, 1 x
105 PC-3 cells were seeded in 5-cm culture dishes
and allowed to adhere overnight. After treatment with ODN plus
paclitaxel or mitoxantrone using the same schedule described above,
cells were harvested and then lysed in a solution containing 100
mM NaCl, 10 mM Tris (pH 7.4), 25 mM
EDTA, and 0.5% SDS. After the centrifugation, the supernatants were
incubated with 300 µg/ml proteinase K for 5 h at 65°C and
extracted with phenol-chloroform. The aqueous layer was treated with
0.1 volume of 3 M sodium acetate, and the DNA was
precipitated with 2.5 volumes of 95% ethanol. After treatment with 100
µg/ml RNase A for 1 h at 37°C, the sample was electrophoresed
on a 2% agarose gel and stained with ethidium bromide.
Assessment of in Vivo Tumor Growth.
Approximately 1 x 106 PC-3 cells were
inoculated s.c. with 0.1 ml of Matrigel (Becton Dickinson Labware,
Bedford, MA) in the flank region of male athymic nude mice (BALB/c
strain; Charles River Laboratory, Montreal, Quebec, Canada),
6–8 weeks of age, under methoxyfluorane anesthesia. When PC-3 tumors
grew to 1 cm in diameter, usually 2–3 weeks after injection, mice were
randomly selected for treatment with AS TRPM-2 ODN alone, MM Co. ODN
alone, AS TRPM-2 ODN plus paclitaxel, MM Co. ODN plus paclitaxel, AS
TRPM-2 ODN plus mitoxantrone, or MM Co. ODN plus mitoxantrone. Each
experimental group consisted of eight mice. After randomization, 10
mg/kg AS TRPM-2 or MM Co. ODN was injected i.p. once daily into each
mouse for 28 days. From days 10 to 14 and from days 24 to 28, 0.5 mg
polymeric micellar paclitaxel or 0.03 mg mitoxantrone was administered
once daily by i.v. injection. Tumor volume was measured once weekly and
calculated by the formula: length x width x depth x
0.5236 (33)
. Data points were reported as average tumor
volumes ± SD.
TUNEL Staining.
A modified TUNEL technique (34)
was used to detect
apoptotic cells in PC-3 tumors using the ApopTag In situ
Apoptosis Detection System (Oncor, Gaithersburg, MD), according to the
manufacturer’s protocol. The number of positively stained cells/high
power field in five random fields was counted and averaged.
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Results |
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. Northern blot analyses
were used to evaluate the effects of treatment with these AS ODNs on
TRPM-2 mRNA expression in PC-3 cells. As shown in Fig. 1, B and C
, after daily treatment of PC-3 cells with 1
µM AS TRPM-2 ODN for 2 days, the different ODNs
exhibited varied degrees of activity. Seven AS ODNs had little or no
effect on TRPM-2 mRNA expression levels, whereas three ODNs had
moderate effects. The most potent AS sODN identified from this series
was AS ODN#2 (5'-CAGCAGCAGAGTCTTCATCAT-3'), which targets the human
TRPM-2 translation initiation site, reducing TRPM-2
expression levels by 80% compared with MM Co. ODN treatment. AS ODN#2
was used in all subsequent experiments.
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, daily
treatment of PC-3 cells with AS ODN#2 (100, 500, or 1000
nM) for 2 days reduced TRPM-2 mRNA levels by 17,
39, or 78%, respectively, whereas TRPM-2 mRNA expression was not
affected by the MM Co. ODN at any of the used concentrations.
Inhibition of TRPM-2 protein levels in PC-3 cells was also observed
after daily treatment with AS ODN#2 for 4 consecutive days (Fig. 2C)
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We then evaluated the effects of AS ODN#2 treatment on the growth of
PC-3 tumors in vivo. Male nude mice bearing PC-3 tumors 
1
cm in diameter were randomly selected for treatment with AS ODN#2
versus MM Co. ODN, and 10 mg/kg ODN was administered once
daily by i.p. injection for 28 days. As shown in Fig. 3
, there was no significant difference in
PC-3 tumor growth between these two groups.
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, AS ODN#2 treatment significantly enhanced
chemosensitivity of paclitaxel and mitoxantrone in a dose-dependent
manner, reducing the IC50 of paclitaxel and
mitoxantrone by more than 60 and 50%, respectively.
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. We further evaluated the effects of combined AS ODN#2
plus chemotherapy by using Western blot analysis to identify cleavage
of PARP protein, a substrate of the caspases activated during the
process of apoptotic execution (35)
. The
Mr 116,000 intact form of PARP was
observed in all samples examined, whereas the
Mr 85,000 PARP cleavage fragment was
detected only after combined treatment with AS ODN#2 plus paclitaxel or
mitoxantrone (Fig. 5D)
.
Enhanced Cytotoxic Effects of Chemotherapy in PC-3 Tumors in
Vivo by Systemic Administration of AS TRPM-2 ODN.
Athymic male mice bearing PC-3 tumors 1 cm in diameter were randomly
selected for treatment with AS ODN#2 plus paclitaxel, MM Co. ODN plus
paclitaxel, AS ODN#2 plus mitoxantrone, or MM Co. ODN plus
mitoxantrone. Mean tumor volume was similar at the beginning of
treatment in each of these groups. After randomization, 10 mg/kg AS
ODN#2 or MM Co. ODN were injected i.p. once daily for 28 days. From
days 10 to 14 and from days 24 to 28, 0.5 mg polymeric micellar
paclitaxel or 0.3 mg mitoxantrone was administered once daily by i.v.
injection. As shown in Fig. 6, A and B
, AS ODN#2 significantly enhanced the
apoptotic effects of micellar paclitaxel and mitoxantrone in PC-3
tumors, reducing mean tumor volume by more than 80 and 60%,
respectively, by 8 weeks after initiation of treatment. In addition,
TUNEL staining detected a 5- or 3-fold increase in the numbers of
apoptotic cells in the PC-3 tumors treated with AS ODN#2 plus micellar
paclitaxel or mitoxantrone, respectively, compared with those treated
with MM Co. ODN plus micellar paclitaxel or mitoxantrone. Under the
experimental conditions used in the above in vivo
experiments, no side effects associated with ODN treatment and/or
chemotherapy were observed.
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Discussion |
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-induced apoptosis
(27)
. Furthermore, a close correlation between
intracellular levels of TRPM-2 and tumor grade in human prostate cancer
specimens has been reported (28)
. We also demonstrated
previously that overexpression of TRPM-2 helps mediate AI progression
against castration (31)
- and
chemotherapy4-induced apoptosis in
androgen-dependent prostate cancer models. Collectively, these findings
suggest that TRPM-2 up-regulation plays a protective role in normal and
malignant prostate tissues against apoptosis induced by various kinds
of stimuli and thereby may confer an aggressive phenotype during
prostate cancer progression. The limited efficacy of cytotoxic chemotherapy remains a major problem for the treatment of patients with advanced hormone refractory prostate cancer.
The lack of survival benefits with traditional cytotoxic chemotherapy in patients with hormone-refractory prostate cancer results from intrinsic chemoresistance and the limitation of toxicity on an elderly population. The chemoresistant phenotype in hormone-refractory prostate cancer is attributable, in part, to high levels of antiapoptotic genes, including bcl-2, Bcl-xL, and TRPM-2, all of which are increased after androgen ablation and remain constitutively overexpressed in AI tumors (18 , 36 , 37) .
Although no chemotherapeutic agent has demonstrated improved survival in patients with advanced prostate cancer, recent Phase II reports are documenting improved response rates in hormone-refractory disease (2 , 36, 37, 38) . For example, >50% of patients responded in one Phase II study of combined paclitaxel plus estramustine treatment (38) . In two randomized trials, mitoxantrone in combination with prednisone was shown to produce significant palliative benefit compared with steroids alone (39 , 40) . Furthermore, recent preclinical studies have provided proof of principle evidence that targeting antiapoptotic genes using AS ODN enhances apoptosis induced by conventional cytotoxic chemotherapy (9, 10, 11, 12) . Therefore, in this study, we set out to screen for potent and sequence-specific ODNs targeted against human TRPM-2. The AS TRPM-2 ODN corresponding to the human TRPM-2 translation initiation site was the most potent sequence of the 10 AS ODN targeting various regions of TRPM-2 gene. To clarify the functional role of TRPM-2 expression in AI prostate cancer, we then tested the effects of AS TRPM-2 ODN on human prostate cancer PC-3 cell growth and whether AS TRPM-2 ODN could enhance the cytotoxic effects of paclitaxel and mitoxantrone in this model system.
Phosphorothioate AS TRPM-2 ODN used in this study significantly inhibited expression of TRPM-2 mRNA and protein in PC-3 cells both in vitro and in vivo. Sequence specificity was confirmed using a two-base TRPM-2 MM ODN, which had no effects on TRPM-2 expression in PC-3 cells. Despite a significant decrease in TRPM-2 expression after AS ODN treatment, no differences in PC-3 cell growth in vitro or PC-3 tumor growth in vivo was observed after AS TRPM-2 ODN treatment. These findings suggest that targeting and inhibiting TRPM-2 expression have no significant effects on cell proliferation in the absence of other apoptotic stimuli or cell death signals.
The administration of AS TRPM-2 ODN with chemotherapeutic agents, however, inhibited PC-3 cell growth both in vitro and in vivo through enhanced apoptosis. Pretreatment of PC-3 cells with AS enhanced apoptosis induced by these agents. Consistent with these in vitro studies, synergistic effects of combined use of AS TRPM-2 ODN plus chemotherapeutic agents were also observed in in vivo studies. Systemic administration of AS TRPM-2 ODN plus polymeric micellar paclitaxel or mitoxantrone suppressed the PC-3 tumor growth by 90 and 50%, respectively, compared with treatment with MM Co. ODN plus either agent. Detection of increased apoptotic cells after combined AS ODN and chemotherapy by TUNEL staining in PC-3 tumors suggests that decreased tumor progression rates after combined AS TRPM-2 ODN plus paclitaxel or mitoxantrone resulted from enhanced chemotherapy-induced apoptosis rather than decreased cell proliferation.
The results in the present study suggest that increased TRPM-2 helps mediate prostate cancer progression by inhibiting apoptotic cell death induced by several kinds of therapy, including cytotoxic chemotherapy. Decreasing TRPM-2-mediated chemoresistance by AS TRPM-2 ODN may provide a feasible and safe strategy to enhance chemosensitivity in hormone-refractory prostate cancer. The preclinical data presented here provide proof of principle support for designing clinical studies with combined AS TRPM-2 plus paclitaxel and/or mitoxantrone therapy for patients with hormone-refractory disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was 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: AS, antisense; ODN,
oligodeoxynucleotide; TRPM-2, testosterone repressed prostate
message-2; MM, mismatch; Co., control; AI, androgen independent;
PARP, poly(ADPribose) polymerase; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TUNEL,
terminal deoxynucleotidyl transferase-mediated nick end labeling.
4 H. Miyake, C. Nelson, P. S. Rennie, and M. E.
Gleave. Acquisition of chemoresistant phenotype by overexpression of
the antiapoptotic gene, TRPM-2, in prostate cancer
xenograft models. Cancer Res., in press, 2000.
Received 11/18/99; revised 2/ 2/00; accepted 2/ 2/00.
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in LNCaP cells by overexpression of sulfated glycoprotein-2 (clusterin). Cancer Res., 55: 2431-2437, 1995. in PC3 and LNCaP prostatic cancer cells is regulated by extracellular levels of SGP-2 (clusterin). Prostate, 39: 87-93, 1999.[CrossRef][Medline]
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