Antisense TRPM-2 Oligodeoxynucleotides Chemosensitize Human Androgen-independent PC-3 Prostate Cancer Cells Both in Vitro and in Vivo¹

Prostate cancer is now the most commonly diagnosed malignancy and the second leading cause of cancer mortality in men in Western industrialized countries. Androgen withdrawal remains the only effective therapy for patients with advanced disease. Approximately 80% of patients achieve symptomatic and/or objective response after androgen ablation; however, progression to androgen independence ultimately occurs in almost all cases (1). Although numerous nonhormonal agents have been evaluated in patients with hormone-refractory prostate cancer, these agents have limited antitumor activity with an objective response rate of <20% and no demonstrated survival benefit (2). Therefore, novel therapeutic strategies targeting molecular mechanisms mediating resistance to conventional agents must be developed to make a significant impact on survival.

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.

PC-3, derived from human prostate cancer, was purchased from the American Type Culture Collection (Rockville, MD). Cells were maintained in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 5% heat-inactivated FCS.

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).

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′.

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.

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.

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 β-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).

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 ×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.

Nucleosomal DNA degradation was analyzed as described previously with a minor modification (32). Briefly, 1 ×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 mmEDTA, 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 100μg/ml RNase A for 1 h at 37°C, the sample was electrophoresed on a 2% agarose gel and stained with ethidium bromide.

Approximately 1 × 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 × width × depth ×0.5236 (33). Data points were reported as average tumor volumes ± SD.

A modified TUNEL technique (34) was used to detect apoptotic cells in PC-3 tumors using the ApopTag In situApoptosis 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.

To identify effective AS ODNs capable of inhibiting TRPM-2gene expression in PC-3 cells, 10 phosphorothioate ODNs designed to hybridize with various regions of TRPM-2 mRNA were synthesized (Fig. 1,A). 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 1μ m 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.

To further define the specificity and potency of AS ODN#2-mediated inhibition of TRPM-2 gene expression, the effects of AS ODN#2 and MM Co. ODN on TRPM-2 mRNA and protein levels were determined by Northern and Western blot analyses, respectively. As shown in Fig. 2, A and B, 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. 2 C).

To determine whether the reduction of TRPM-2 expression affects the growth of PC-3 cells in vitro, the growth rates of PC-3 cells after treatment with various concentrations of AS ODN#2 or MM Co. ODN once daily for 2 days were examined using the MTT assay. No significant difference in PC-3 cell growth was observed between AS ODN#2 and MM Co. ODN treatment (data not shown).

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.

To determine whether AS ODN#2 treatment inhibits TRPM-2 expression in PC-3 tumors in vivo, each of three tumor-bearing nude mice were given 10 mg/kg AS ODN#2 or MM Co. ODN i.p. once daily for 5 days,and TRPM-2 mRNA expression levels in harvested tumor tissues were then analyzed by Northern blotting. Treatment with AS ODN#2 resulted in a 68% reduction in TRPM-2 mRNA levels in PC-3 tumors compared with MM Co. ODN-treated tumors (Fig. 4, A and B).

To determine whether treatment with AS ODN#2 enhances the cytotoxic effects of paclitaxel and mitoxantrone, PC-3 cells were treated with 500 nm AS ODN#2 or MM Co. ODN once daily for 2 days and then incubated with medium containing various concentrations of either paclitaxel or mitoxantrone for 2 days. The MTT assay was then performed to determine cell viability. As shown in Fig. 5, A and B, 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.

The DNA fragmentation assay was performed to compare induction of apoptosis after treatment with 500 nm AS ODN#2 either alone or in combination with 5 nm paclitaxel or 1 nmmitoxantrone. Using the same treatment schedule described above, the characteristic apoptotic DNA ladder was observed only with combined treatment of AS ODN#2 plus paclitaxel or mitoxantrone (Fig. 5,C). 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 M_r 116,000 intact form of PARP was observed in all samples examined, whereas the M_r 85,000 PARP cleavage fragment was detected only after combined treatment with AS ODN#2 plus paclitaxel or mitoxantrone (Fig. 5 D).

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 vivoexperiments, no side effects associated with ODN treatment and/or chemotherapy were observed.

In the prostate gland, despite the original hypothesis that TRPM-2 is a marker of programmed cell death (12, 23, 24, 25), several experimental and clinical studies have provided conflicting findings showing the dissociation of TRPM-2 expression from apoptosis (26, 27, 28, 29, 30). For example, increased TRPM-2 expression in dysplastic lesions of rat prostate was not associated with enhanced apoptotic activity (29). Introduction of the TRPM-2 gene into LNCaP prostate cancer cells renders them highly resistant to tumor necrosis factor-α-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-2translation 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.

We thank Mary Bowden and Howard Tearle for excellent technical assistance.