# Patent

## Title
Chemo- and radiation-sensitization of cancer by antisense TRPM-2 oligodeoxynucleotides

## Abstract

Administration of antisense oligodeoxynucleotides (ODN) targeted against the testosterone-repressed prostate message-2 (TRPM-2) gene can reduce the amount of TRPM-2 in renal cell cancer (RCC) cells and other cancer cells, and as a result enhance chemosensitivity of these cells to chemotherapy agents and radiation. Thus, for example, the sensitivity of renal cell cancer cells to a chemotherapeutic agent can be increased by exposing renal cell cancer cells to a chemotherapeutic agent and an agent which reduces the amount of TRPM-2 in the renal cell cancer cells. This provides an improved method for treatment of renal cell cancer, which is generally resistant to treatment with known chemotherapy agents.

## Background
STATEMENT OF RELATED APPLICATIONS
This application is a continuation of U.S. Ser. No. 09/967,726, filed on Sep. 28, 2001, now U.S. Pat. No. 7,569,551, issued Aug. 4, 2009, which claims benefit of U.S. Provisional Application No. 60/236,301, filed on Sep. 28, 2000, the contents of which are incorporated by reference herein.


FIELD OF THE INVENTION
This application relates to a method and compositions for chemosensitization and radiosensitization of cancer cells through administration of compositions, such as antisense oligonucleotides, which reduce the amount of TRPM-2 in the cells. The invention is of particular applicability to cancers such as renal cell carcinoma (RCC) which are resistant to chemo- and radiotherapy.
BACKGROUND OF THE INVENTION
Treatment of cancers by chemotherapy or radiotherapy frequently results in side effects because the therapy used is not specific to the cancer and kills non-cancerous cells as well. Where the cancer is resistant to the therapy, the dosage used must be increased, and the side effects become more pronounced. Thus, as a general principal, it would be desirable to be able to increase the sensitivity of cancer cells to chemotherapy and radiotherapy so that lower effective dosages could be employed.
Renal cell cancer (RCC) accounts for approximately 2% of adult carcinomas with over 30,000 new cases and 12,000 deaths per year in the United States. Motzer et al., N. Engl. J. Med. 335: 865-875 (1996). Estimates of annual new diagnoses of RCC have been increasing steadily. Motzer et al, supra; McLaughlin et al., Semin. Oncol. 27: 115-123 (2000). Despite extensive evaluation of many different treatment modalities, advanced metastatic RCC remains highly resistant to systemic therapy. To date, no chemotherapy is established for the treatment of advanced kidney cancer with objective response rates higher than 15% that justifies its use as a single agent. Yagoda et al., Semin. Oncol. 22: 42-60 (1995). Combinations of chemotherapy plus hormonal agents have been studied but likewise are ineffective and result in additive toxicity. Nearly half of all patients with RCC die within 5 years of diagnosis and 5-year-survival for those with metastatic disease is <10%. Motzer et al., J. Urol. 163: 408-417 (2000). Therefore, in order to improve survival, preclinical and clinical evaluation of new agents and treatment programs to identify improved antitumor activity are priorities in this resistant disease.
Bladder cancer is the second most common malignancy of the genitourinary tract, and the fourth or fifth leading cause of cancer-related deaths of men in Western industrialized countries. The prognosis of patients with invasive and/or metastatic bladder cancer is still extremely poor, despite recent therapeutic advances. Advanced bladder cancer is commonly treated with a cisplatin-based combination chemotherapy. However, which palliative efficacy has been observed, this efficacy is limited due to de novo drug resistance or the development of the cellular drug-resistant phenotype during treatment. Thus, advances in the treatment of bladder cancer are desirable.
SUMMARY OF THE INVENTION
In accordance with the present invention it has been found that administration of antisense oligodeoxynucleotides (ODN) targeted against the testosterone-repressed prostate message-2 (TRPM-2) gene can reduce the expression of TRPM-2 and as a result enhance sensitivity of cancer cells to chemotherapeutic agents and to radiotherapy both in vitro and in vivo. Thus, in accordance with a first aspect of the invention, there is provided a method for increasing the sensitivity of cancer cells to a chemotherapeutic agent, comprising exposing cancer cells to the chemotherapeutic agent and an agent which reduces the amount of TRPM-2 in the renal cell cancer cells. There is further provided a method for treating cancer, comprising administering to an individual suffering from cancer a chemotherapeutic agent and an agent which reduces the amount of TRPM-2 in the cells. Compositions may be prepared for specific use in these methods.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows sequence specific and dose-dependent down-regulation of TRPM-2 in caki-2 cells in vitro;
FIG. 2 shows TRPM-2 up-regulation in caki-2 cells, in vitro, after treatment with paclitaxel;
FIG. 3 shows TRPM-2 up-regulation in caki-2 cells, in vivo, after treatment with paclitaxel;
FIG. 4 shows chemosensitization of caki-2 cells, in vitro, after treatment with antisense TRPM-2 ODN;
FIG. 5 shows chemosensitization of caki-2 cells, in viva, after treatment with antisense TRPM-2 ODN;
FIG. 6 shows the amount of TRPM-2 mRNA in ovarian cancer cells after treatment with differing amount of TRPM-2 antisense (AS) or mismatch control (MM). The number in each case reflects the concentration of the oligonucleotide in nanomolar units;
FIG. 7 shows combined effect of TRPM-2 antisense and taxol on A549 tumors (lung) in SCID mice;
FIG. 8 shows reduction in cell viability for KoTCC-1 (bladder cancer) cells treated with a combination of TRPM-2 antisense and cisplatin;



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method and compositions for increasing the sensitivity of cancer cells to a chemotherapeutic agent, comprising exposing cancer cells to the chemotherapeutic agent and an agent which reduces the amount of TRPM-2 in the cancer cells. The compositions may be used in formulation of a pharmaceutical for use in increasing sensitivity of cancer to chemotherapeutic agents and radiation.
The present invention further provides a method for treating a cancer in which the cancer cells express TRPM-2, comprising administering to an individual suffering from the cancer a chemotherapeutic agent and an agent which reduces the amount of TRPM-2 in cancer cells of the individual. The two agents may be administered as part of a single composition or may be administered individually. In the latter case, the agent which reduces the amount of TRPM-2 may be administered before, at the same time as, or after the chemotherapy agent, provided that expression of TRPM-2 is reduced during at least a portion of the time that the chemotherapy agent is active.
Specific non-limiting examples of the types of cancer to which the invention may be applied include prostate cancer, renal cell carcinoma, bladder cancer, ovarian cancer and lung cancer.
As used in this application, the term “chemotherapy agent” refers to an agent which is added to cells or administered to an individual for the purpose of selectively killing cancerous cells. Examples of chemotherapy agents whose effectiveness may be enhanced using the method of the invention include but are not limited to taxanes (paclitaxel or docetaxel) and mitoxanthrone, cylcophosphamide, topoisomerase inhibitors, angiogenesis inhibitors, cisplatin-based therapies and differentiation agents and signal transduction inhibitors.
In the kidney, increased expression of TRPM-2 is observed in a number of acute and chronic states of renal injury and disease. However, little is known about changes of TRPM-2 expression levels in kidney cancer. Based on the analysis of only 4 patients, Parczyk et al reported a 3-fold overexpression of TRPM-2 in human renal cell cancer compared to normal kidney tissue. J Cancer Res Clin Oncol 120: 186-8 (1994).
In our study, reported in the examples below, we compared normal and malignant kidney tissues of 67 patients using TRPM-2-immunostaining. Our results confirm positive TRPM-2-staining in almost all RCC (98%) and increased TRPM-2 expression compared to normal tissue in a majority of RCC (61%). Sixty-six percent of normal kidney tissue samples distant from RCC did not or only weakly stained for TRPM-2, consistent with findings of other investigators. Parczyk et al, supra; Dvergsten et al., Kidney International 45: 828-35 (1994).
Overexpression of TRPM-2 in human prostate cancer LNCaP cells increases resistance to paclitaxel and other chemotherapies. Miyake et al., Cancer Res 60: 2547-54 (2000). Thus, in order to determine the functional role of increased TRPM-2 expression in RCC, we tested the effects of TRPM-2 antisense ODN on human Caki-2 RCC cell growth and whether TRPM-2 antisense ODN could enhance the cytotoxic effects of paclitaxel in this model system. Phosphorothioate TRPM-2 antisense ODN used in this study significantly inhibited expression of TRPM-2 mRNA in Caki-2 cells both in vitro and in vivo. Sequence specificity was confirmed using a 2-base TRPM-2 mismatch oligonucleotide, which had no effects on TRPM-2 expression in Caki-2 cells. When used alone, antisense or mismatch oligonucleotide treatment had no effect on Caki-2 cell growth. However, when administered in combination with other cell death stimuli, like cytotoxic chemotherapy, TRPM-2 antisense ODN enhanced Caki-2 cell apoptosis both in vitro and in vivo. Pretreatment of Caki-2 cells with TRPM-2 antisense ODN reduced the IC50 of paclitaxel by 80%, thereby enhancing apoptosis induced by these agents. Consistent with these in vitro results, synergistic effects of combined use of TRPM-2 antisense ODN plus paclitaxel was also observed in vivo. Systemic administration of TRPM-2 antisense ODN plus polymeric micellar paclitaxel suppressed the Caki-2 tumor growth by 50%, compared to treatment with mismatch control oligonucleotides plus paclitaxel. Detection of increased apoptotic cells after combined antisense and chemotherapy by TUNEL staining in Caki-2 tumors suggest that decrease in tumor progression rates after combined TRPM-2 antisense ODN plus paclitaxel resulted from enhanced chemotherapy-induced apoptosis rather than decreased cell proliferation, although it is not Applicants' intention to be bound to this mechanism of action.
TRPM-2 expression levels were determined by Northern blot analysis in transitional cell carcinoma (TCC) of the bladder obtained from patients undergoing surgical treatment. Tests in a human bladder cancer cell line showed that treatment of the cells in vitro with TRPM-2 antisense increased the cytotoxic effect of cisplatin. In addition, synergistic inhibition of growth and metastasis of human bladder cancer tumors in athymic mice, in vivo, was observed.
As summarized in the examples below, similar results have been observed in tests on prostate cancer cells, ovarian cancer cells, and lung cancer cells. Thus, it appears that the invention is generally applicable to many cancer types, provided that the cancer cells have increased levels of TRPM-2 expression/mRNA.
In the present invention, an agent is utilized which causes a reduction in the amount of TRPM-2 in treated cells, relative to untreated cells. While not intending to be bound by any particular mechanism, such a reduction may occur as a result of actual reductions in the amount of TRPM-2 produced from translation of mRNA, as a result of increases in the rate at which TRPM-2 is degraded, or as a result of reduction in the amount of TRPM-2 mRNA available for translation. The extent of the reduction should be sufficient to bring about an observable increase in the chemosensitivity of the treated renal cancer cells.
A suitable agent for reducing the amount of TRPM-2 is an antisense ODN, for example one which is 15-30 bases in length, which is complementary to the coding strand of portions of the TRPM-2 gene of the target cell. Table 1 shows representative antisense ODN sequences. Preferred antisense ODN sequences which are complementary to a region of the TRPM-2 mRNA spanning either the translation initiation site or the termination site. Specific preferred sequences are those shown in Seq. ID Nos. 4, 5 and 12.








TABLE 1





Seq ID No.
Sequence







 1
gcacagcaggagaatcttcat


 


 2
gcacagcagcaggatcttcat


 


 3
tggagtctttgcacgcctcgg


 


 4
cagcagcagagtcttcatcat


 


 5
attgtctgagaccgtctggtc


 


 6
ccttcagctttgtctctgatt


 


 7
agcagggagtcgatgcggtca


 


 8
atcaagctgcggacgatgcgg


 


 9
gcaggcagcccgtggagttgt


 


10
ttcagctgctccagcaaggag


 


11
aatttagggttcttcctggag


 


12
gctgggcggagttgggggcct


 


13
tctcccggcttgcgccat


 


14
tctcccggcatggtgcat









The ODNs employed may be modified to increase the stability of the ODN in vivo. For example, the ODNs may be employed as phosphorothioate derivatives (replacement of a non-bridging phosphoryl oxygen atoms with a sulfur atom) which have increased resistance to nuclease digestion.
Administration of antisense ODNs can be carried out using the various mechanisms known in the art, including naked administration and administration in pharmaceutically acceptable lipid carriers. For example, lipid carriers for antisense delivery are disclosed in U.S. Pat. Nos. 5,855,911 and 5,417,978 which are incorporated herein by reference. In general, the antisense is administered by intravenous, intraperitoneal, subcutaneous or oral routes.
The amount of antisense ODN administered is one effective to reduce the amount of TRPM-2 in cancer cells, and to thereby enhance the effectiveness of a chemotherapy agent being administered before, concurrently with or subsequent to the antisense ODN. It will be appreciated that this amount will vary both with the effectiveness of the antisense ODN employed, and with the nature of any carrier used. The determination of appropriate amounts for any given composition is within the skill in the art, through standard series of tests designed to assess appropriate therapeutic levels.
The invention will now be further described with reference to the following on-limiting examples:
Example 1
TRPM-2 immunostaining was performed in normal kidney and RCC tissue of 67 patients. Histological classification of RCC revealed clear cell patterns in 56 cases (84.5%), the rest of sections showing either granular cell (6%) or papillary cell patterns (9%). Staining of cytoplasmic clusterin was heterogenous in both normal and malignant tissues. Positive TRPM-2-staining was found in almost all RCC (97%) and TRPM-2-overexpression, compared to normal tissue, was observed in most RCC (69%). (Table 2) Fifty-three percent of normal kidney tissue samples (distant from RCC) showed no or only weak staining for clusterin (Table 3).









TABLE 2





% of tissue stained
normal kidney
RCC







 0-25
50%
21%


25-50
28%
27%


50-75
19%
30%


 75-100
 3%
22%


















TABLE 3





Intensity of staining (arbitrary




scoring)
normal kidney
RCC

















0
17%
 3%


+1
37%
12%


+2
37%
49%


+3
 9%
22%


+4
0 
14%









Example 2
The Caki-2 cell line is derived from a human clear cell carcinoma of the kidney. It was purchased from the American Type Culture Collection (Rockville, Md.). Cells were maintained in McCoy's 5a medium (Life Technologies, Inc., Gaithersburg, Md.) supplemented with 10% heat-inactivated fetal calf serum.
Phosphorothioate TRPM-2 anti sense ODN was made in accordance with the inventors instructions by La Jolla Pharmaceuticals Co. (La Jolla, Calif.). The sequence of TRPM-2 antisense ODN corresponding to the human TRPM-2 translation initiation site was 5′-CAGCAGCAGAGTCTTCATCAT-3′ (Seq. ID No. 4). A two-base TRPM-2 mismatch oligonucleotide, 5′-CAGCAGCAGAGTATTTATCAT-3′ (Seq. ID No. 15) was used as control.
Lipofectin, a cationic lipid (Life Technologies, Inc.) was used to increase oligonucleotide uptake of cells. Caki-2 cells were treated with various oligonucleotide concentrations after preincubation for 20 minutes with 20 μg/ml lipofectin in serum free OPTI-MEM (Life Technologies, Inc.). Four hours after the beginning of the incubation, the medium containing oligonucleotides and lipofectin was replaced with standard culture medium described above.
Total RNA was isolated from cultured Caki-2 cells using the acid-guanidium thiocyanate-phenol-chloroform method. Electrophoresis, hybridization and washing conditions were carried out as previously reported. Miyake et al., Cancer Res 59: 4030-4 (1999). Human TRPM-2 and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA probes were generated by reverse transcription-PCR from total RNA of human kidney using primers 5-AAGGAAATTCAAAATGCTGTCAA-3′ (sense, Seq ID No. 16) and 5′-ACAGACAAGATCTCCCGGCACTT-3′ (antisense, Seq. ID No. 17) for TRPM-2, and 5′-TGCTTTTAACTCTGGTAAAGT-3′ (sense, Seq. ID No. 18) and 5′-ATATTTGGCAGGTTTTTCTGA-3′ (antisense, Seq. ID No. 19) for G3PDH. Density of bands for TRPM-2 was normalized against that of G3PDH by densitometric analysis.
As shown in FIG. 1, daily treatment of Caki-2 cells with TRPM-2 antisense ODN (50, 100, 500, or 1000 nM for 2 days reduced TRPM-2 mRNA levels by 42%, 58%, 61%, and 64%, respectively, while TRPM-2 mRNA expression was not affected by is match control oligonucleotides at any of the employed concentrations.
Example 3
Paclitaxel was purchased from Sigma Chemical Co. (St. Louis, Mo.). Stock solutions of paclitaxel were prepared with dimethyl sulfoxide (DMSO), and diluted with PBS to the required concentrations before each in vitro experiment. Polymeric micellar paclitaxel used in this study was generously supplied by Dr. Helen M. Burt (Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada), as previously reported. Leung et al., Prostate 44: 156-63 (2000).
Northern blot analysis was used to assess the effects of paclitaxel treatment on TRPM-2 mRNA expression in Caki-2 cells in vitro and tumors in vivo, respectively. As shown in FIG. 2, TRPM-2 mRNA induction increased in a dose-dependent manner after paclitaxel treatment in vitro up to 73% at concentrations of 100 nM.
To determine whether paclitaxel changes TRPM-2 mRNA expression in Caki-2 tumors in vivo, 0.5 mg polymeric micellar paclitaxel was administered intravenously once daily over 10 days with harvest of tumors for mRNA extraction one day later. In nude mice bearing Caki-2 tumors, TRPM-2 mRNA levels increased by 15% after treatment with paclitaxel compared to untreated control (FIG. 3).
Example 4
To determine whether ASO treatment could reduce TRPM-2 expression in Caki-2 tumors in vivo, tumor bearing nude mice were given 12.5 mg/kg TRPM-2 antisense ODN (Seq. ID No. 4) or mismatch control oligonucleotide (Seq ID No. 15) intraperitoneally once daily over 28 days with tumors harvested for mRNA-extraction one day later. Treatment with TRPM-2 antisense ODN resulted in a 51% reduction in TRPM-2 mRNA levels in Caki-2 tumors compared to mismatch control oligonucleotide-treated tumors.
Example 5
Caki-2 cells were treated with 500 nM TRPM-2 antisense ODN (Seq ID No. 4) or mismatch control oligonucleotides (Seq. ID No. 15) once daily for two days and then incubated with medium containing various concentrations of paclitaxel for 2 days. The in vitro growth inhibitory effects of TRPM-2 antisense ODN plus paclitaxel on Caki-2 cells were assessed using the MTT assay as described by Miyake et al. Cancer Res 60: 2547-54 (2000). 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 antisense ODN for 2 days. Following antisense ODN treatment, cells were treated with various concentrations of paclitaxel. After 48 h of incubation, 20 μl of 5 mg/ml MTT (Sigma Chemical Co.) in PBS was added to each well, followed by incubation for 4 h at 37° C. The formazan crystals were dissolved in DMSO. The optical density was determined with a microculture plate reader (Becton Dickinson Labware, Lincoln Park, N.J.) 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.
As shown in FIG. 4, TRPM-2 antisense ODN treatment significantly enhanced chemosensitivity of paclitaxel in a dose-dependent manner (two-sided, P=0.022, ANOVA), reducing the IC50 (i.e., the concentration that reduces cell viability by 50%) of paclitaxel by 80% (320 nM to 65 nM), whereas mismatch control oligonucleotides had no effect. The combined effects between TRPM-2 antisense ODN and paclitaxel were synergistic (P<0.05), as determined by an analysis that utilized the fractional product method. Duska et al. J Natl Cancer Inst 91:1557-63 (1999).
DNA fragmentation assay was performed to compare induction of apoptosis after combined treatment with 500 nM TRPM-2 antisense ODN and 10 nM paclitaxel. The nucleosomal DNA degradation was analyzed as described previously with a minor modification. Briefly, 1×105 Caki-2 cells were seeded in 10 cm culture dishes and allowed to adhere overnight. After treatment with ASO plus paclitaxel using the same schedule as 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 centrifugation, 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. Following 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. After the same treatment schedule described above, characteristic apoptotic DNA laddering was observed only after combined treatment with TRPM-2 antisense ODN and paclitaxel.
Example 6
Male athymic mice bearing Caki-2 tumors approximately 1 cm in diameter were randomly selected for treatment with TRPM-2 antisense ODN (Seq. ID No. 4) plus paclitaxel, mismatch control oligonucleotides (Seq. ID No. 15) plus paclitaxel, or TRPM-2 antisense ODN (Seq. ID No. 4) alone. Mean tumor volume was similar at the beginning of treatment in each of these groups (365-375 mm3). After randomization, 12.5 mg/kg TRPM-2 antisense ODN or mismatch control oligonucleotides were injected intraperitoneally once daily for 28 days. From day 10 to 14, and from day 24 to 28, 0.5 mg polymeric micellar paclitaxel was administered once daily by intravenous injection. As shown in FIG. 5, TRPM-2 antisense ODN enhanced micellar paclitaxel chemosensitivity in Caki-2 tumors, causing a 50% reduction in mean tumor volume 7 weeks after initiation of treatment, compared to treatment with mismatch control oligonucleotides and paclitaxel (two-sided P<0.05). In vivo, the combined effects between TRPM-2 antisense ODN and paclitaxel were synergistic (P<0.05), as analyzed by the fractional product method. In addition, TUNEL staining detected a 3-fold increase in the numbers of apoptotic cells in Caki-2 tumors treated with TRPM-2 antisense ODN plus micellar paclitaxel (mean number of apoptotic cells/high power field: 11.6), compared to those treated with mismatch control oligonucleotides plus micellar paclitaxel (mean number of apoptotic cells/high power field: 4.1), or TRPM-2 antisense ODN alone (mean number of apoptotic cells/high power field: 2.3).
Under the experimental conditions used in the above in vivo experiments, no side effects associated with antisense treatment and/or chemotherapy were observed (except one animal developing ascites, 3 weeks after TRPM-2 antisense ODN treatment was finished).
Example 7
Antisense TRPM-2 ODNs enhance radiation sensitivity of cancer cells which express TRPM-2. Using northern analysis, we found that radiation therapy results in dose and time dependent increases in TRPM2 gene expression in human prostate cancer PC-3 cells (FIG. 8). Overexpression of TRPM2 results in increased resistance to radiation induced cell death. Human prostate LNCaP cells that overexpress TRPM2 (LNCaP/T1) are more resistant to radiation therapy (FIGS. 9A and B). Treatment of human prostate cancer PC-3 cells with 100 and 500 nM antisense TRPM-2 ODNs (Seq. ID. NO. 1) significantly reduces cell survival after a single treatment of 4 Gy radiation therapy compared to mismatch ODN (Seq. ID No. 2) treatment. (FIG. 10). FIGS. 11A and B show dose dependent radiation sensitization of human prostate cancer PC-3 cells after treatment with 10, 50, and 100 nM antisense TRPM-2 oligo in vitro.
Example 8
To determine whether treatment with human antisense TRPM-2 ODN enhances chemosensitivity in the PC3 human prostate cancer cell line, mice bearing PC3 tumors were treated with antisense human TRPM-2 ODN plus micellar paclitaxel or mitoxantrone, and mismatch control ODN plus micellar paclitaxel or mitoxantrone (FIGS. 12A and 12B). ODN was administered for 28 days and either 0.5 mg micellar taxol or 0.3 mg mitoxantrone were administered on two occasions: from day 10 to 14, and day 24 to 28. A significant reduction in tumor size was observed in the antisense ODN treated animals as compared to those treated with mismatch control ODN. This effect was even more pronounced after the second dosing of the micellar paclitaxel or mitoxantrone.
Example 9
An ovarian cancer cell line (OVCAR3) was treated with different amounts of TRPM-2 antisense (Seq. ID 4) or mismatch oligonucleotide (Seq. ID. No. 15) using OligofectAMINETM as a carrier and the amount of TRPM-2 mRNA was determined after 2 days using Northern blot analysis as described above. The results are summarized in FIG. 6. As shown, there is a substantially dose-dependent reduction in TRPM-2 mRNA levels using the TRPM-2 antisense, but no reduction in the absence of oligonucleotide (ctrl) or using the mismatch control oligonucleotide. The same results were observed in using real time PCR to determine the amount of TRPM-2 mRNA.
Tissue sections of cancer ovarian tissue, before and after chemotherapy with taxol, and of normal ovarian tissue were observed microscopically after immunohistochemical staining specific for TRPM-2. Increased TRPM-2 levels were observed in the post-chemotherapy sections, although no obvious difference was seen between normal ovary and pre-chemotherapy cancerous tissue. Thus, expression of TRPM-2 may provide a defense for ovarian cancer cells against chemotherapeutic treatment.
Example 10
A lung cancer cell line (A549) was treated with different concentrations (50-1000 nM) of TRPM-2 antisense (Seq. ID No. 4) or mismatch oligonucleotide (Seq. ID. No. 15) and evaluated by Northern blot analysis as described above. Dose-dependent and sequence specific down-regulation of TRPM-2 mRNA levels was observed. Combination treatments of A549 cells with paclitaxel (0-50 nM) and either TRPM-2 antisense (Seq. ID No. 4) or mismatch control (Seq. ID No. 15) (0 or 500 nM) were analyzed as described in Example 6. Characteristic apoptotic DNA laddering was observed only after combined treatment with TRPM-2 antisense ODN and paclitaxel.
A549 tumors were introduced into SCID mice using standard protocols. Mice were treated with TRPM-2 antisense (Seq. ID. No. 4) alone, or a combination of taxol with the TRPM-2 antisense or mismatch control (Seq. ID. No. 15). Tumor volume was measured periodically. The results are summarized in FIG. 7. As shown, the combination of the TRPM-2 antisense provides improved control over tumor volume.
Example 11
ACHN, a cell line derived from human RCC, was purchased from the American Type Culture Collection. pRC-CMV expression vector containing the 1.6 kb cDNA fragment encoding human TRPM-2 was provided by Dr. Martin Tenniswood (W. Alton Jones Cell Science Center, Lake Placid, N.Y.). The expression vector was transfected into ACHN cells by the liposome-mediated gene transfer method. Miyake et al., Cancer Res. 60: 170-176 (2000) to generate ACHN cell overexpressing TRPM-2. TRPM-2 expression was detected in four independent cell lines (ACHN/CL#1-4), but not in the parental cell line (ACHN/P) or the vector-only transfected control (ACHN/Co).
In vitro growth rates were determined for ACHN/P, ACHN/Co, ACHN/CL#1 and ACHN/CL#2 using the MTT assay described above. No significant difference in cell proliferation was observed among the cell lines. These same four cell lines were exposed to cisplatin in vitro. ACHN/CL#1 and ACHN/CL2 acquired resistance to cisplatin in comparison with ACHN/P and ACHN/Co. Overexpression of TRPM-2 in ACHN cells increased the IC50 of cisplatin more than 5-fold.
Induction of apoptosis in ACHN sublines treated with 10 mg/ml cisplatin for 72 hours was assessed by DNA degradation assay and Western blot analysis of PARP protein, a substrate for caspases activated during the process of apoptotic execution. Characteristic apoptotic DNA ladders were detected in ACHN/P and ACHN/Co but not in ACHN/CL#1 or ACHN/CL#2 indicating the protective effect of TRPM-2 overexpression.
To examine the in vivo effect of TRPM-2 expression on tumor growth, 5×106 cells of each cell line were injected subcutaneously into the flank of athymic nude mice. There were no significant different in tumor growth among ACHN/P, ACHN/Co, ACHN/CL#1 and ACHN/CL#2, that is TRPM-2 overexpression did not stimulate a proliferative potential of ACHN in vivo.
Four weeks after tumor cell injection, 50 μg/mouse of cisplatin was injected intravenously twice a week for two weeks. Sixty days after injection of the tumor cells, ACHN/CL#1 and ACHN/CL#2 formed tumors twice as large as those formed by ACHN/P and ACHN/Co.
Example 12
The human bladder cancer cell line KoTCC-1 has been described previously. Miyake et al. J. Urol. 157: 2351-2355 (1997). KoTCC-1 cells were treated in vitro with TRPM-2 antisense (Seq. ID. No. 4) or a mismatch control (Seq. ID. No. 15) at concentrations of 100, 500 or 1000 nM daily for two days. Northern blot analysis was performed as described above. TRPM-2 antisense reduced the amount of TRPM-2 antisense in a sequence-specific dose dependent manner (22, 59 or 95%). Inhibition of TRPM-2 protein levels was observed by Western blot analysis after daily treatment with the TRPM-2 antisense for 4 consecutive days.
Northern blot analysis was used to determine the effect of cisplatin treatment on TRPM-2 mRNA levels in KoTCC-1 cells. TRPM-2 mRNA induction increased in a dose-dependent manner by ciplatin treatments at concentrations up to 10 mg/ml. Time course experiments demonstrated that cisplatin-induced TRPM-2 mRNA up-regulation peaked by 48 hours posttreatments and began decreasing by 96 hours posttreatment.
Combined treatment with 500 nM TRPM-2 antisense (Seq. ID. No 4) and 5 mg/ml or 10 mg/ml cisplatin decreased TRPM-2 mRNA levels by 81% or 76%, respectively, as compared to 500 nM mismatch control (Seq. ID No. 15).
KoTCC-1 cells were treated in vitro with 500 nM TRPM-2 antisense (Seq. ID. No. 4) or mismatch control (Seq. ID. No. 15) once daily for two days and then incubated with medium containing various concentrations of cisplatin for 2 days. The MTT assay was performed to determine cell viability. TRPM-2 antisense was shown to reduce the IC50 of cisplatin by more than 50%. Dose-dependent synergy was also observed by increasing the amount of antisense when the cisplatin concentration was fixed at 10 mg/ml. (FIG. 8)
Example 13
The efficacy of a treatment regimen combining TRPM-2 antisense and cisplatin for inhibiting growth of subcutaneous KoTCC-1 tumors in athymic nude mice was evaluated. Mice with tumors approximately 1 cm in diameters were treated with TRPM-2 antisense (Seq. ID. No. 4) alone (10 mg/kg, ip, once daily in each mouse for 28 days), mismatch control (Seq. ID). No. 15) alone (10 mg/kg, ip, once daily in each mouse for 28 days), TRPM-2 antisense (10 mg/kg, ip, once daily in each mouse for 28 days) and cisplatin (50 μg iv twice a week for two weeks) or mismatch control (10 mg/kg, ip, once daily in each mouse for 28 days) and cisplatin (50 μg iv twice a week for two weeks). Mean tumor volumes were measured weekly. The result are shown in FIG. 9.
A modified TUNEL technique was used to detect apoptotic cells in KoTCC cells treated with TRPM-2 antisense and cisplatin. A 6-, 6- and 3-fold increase in the number of apoptotic cells was detected for the combination of TRPM-2 antisense and cisplatin, relative to TRPM-2 antisense alone, mismatch control alone or mismatch control and cisplatin, respectively.

## Claims

1. A method for treating an individual suffering from lung cancer comprising administering to said individual a therapeutically effective amount of one antisense oligodeoxynucleotide and chemotherapy consisting of a taxane and cisplatin, wherein the antisense oligodeoxynucleotide has nucleotides in the sequence set forth in Seq. ID No. 4 and which antisense oligodeoxynucleotide comprises a phosphorothioate modification to increase the stability thereof in vivo, thereby treating said individual.

2. A method for enhancing chemosensitivity in an individual suffering from lung cancer comprising administering to said individual a therapeutically effective amount of one antisense oligodeoxynucleotide and chemotherapy consisting of a taxane and cisplatin, wherein the antisense oligodeoxynucleotide has nucleotides in the sequence set forth in Seq. ID No. 4 and which antisense oligodeoxynucleotide comprises a phosphorothioate modification to increase the stability thereof in vivo, thereby enhancing chemosensitivity in said individual.

3. A method for treating an individual suffering from lung cancer and undergoing administration of chemotherapy consisting of a taxane and cisplatin comprising administering to said individual a composition consisting of a therapeutically effective amount of an antisense oligodeoxynucleotide, wherein the antisense oligodeoxynucleotide has nucleotides in the sequence set forth in Seq. ID No. 4 and which antisense oligodeoxynucleotide comprises a phosphorothioate modification to increase the stability thereof in vivo, wherein the antisense oligodeoxynucleotide is administered subsequent to administration of the chemotherapeutic agent, such that expression of testosterone-repressed prostate message-2 (TRPM-2) is reduced during at least a portion of the time that the chemotherapeutic agent is active, thereby treating said individual.

4. A method for enhancing chemosensitivity in an individual suffering from lung cancer and undergoing administration of chemotherapy consisting of a taxane and cisplatin comprising administering to said individual a composition consisting of a therapeutically effective amount of an antisense oligodeoxynucleotide wherein the antisense oligodeoxynucleotide has nucleotides in the sequence set forth in Seq. ID No. 4 and which antisense oligodeoxynucleotide comprises a phosphorothioate modification to increase the stability thereof in vivo, wherein the antisense oligodeoxynucleotide is administered subsequent to administration of the chemotherapeutic agent, such that expression of testosterone-repressed prostate message-2 (TRPM-2) is reduced during at least a portion of the time that the chemotherapeutic agent is active, thereby enhancing chemosensitivity in said individual.

5. The method of claim 1, wherein the antisense oligodeoxynucleotide is administered to said individual before the chemotherapy such that that expression of testosterone-repressed prostate message-2 (TRPM-2) is reduced during at least a portion of the time that the taxane or cisplatin is active.

6. The method of claim 2, wherein the antisense oligodeoxynucleotide is administered to said individual before the chemotherapy such that that expression of testosterone-repressed prostate message-2 (TRPM-2) is reduced during at least a portion of the time that the taxane or cisplatin is active.

7. The method of claim 1, wherein the taxane is docetaxel.

8. The method of claim 1, wherein the taxane is paclitaxel.

9. The method of claim 2, wherein the taxane is docetaxel.

10. The method of claim 2, wherein the taxane is paclitaxel.

11. The method of claim 3, wherein the taxane is docetaxel.

12. The method of claim 3, wherein the taxane is paclitaxel.

13. The method of claim 4, wherein the taxane is docetaxel.

14. The method of claim 4, wherein the taxane is paclitaxel.