Therapeutic efficacy of adenoviral-mediated p53 gene transfer is synergistically enhanced by combined use of antisense oligodeoxynucleotide targeting clusterin gene in a human bladder cancer model

Abstract

To establish a more effective therapeutic strategy against advanced bladder cancer, we investigated the effects of combined treatment with antisense (AS) oligodeoxynucleotide (ODN) targeting the anti-apoptotic gene clusterin and adenoviral-mediated p53 gene transfer (Ad5CMV-p53) using the human bladder cancer KoTCC-1 model. Clusterin expression in KoTCC-1 cells was highly upregulated by Ad5CMV-p53 treatment; however, AS clusterin ODN treatment further suppressed clusterin expression in KoTCC-1 cells after Ad5CMV-p53 treatment. AS clusterin ODN treatment synergistically enhanced the cytotoxic effect of Ad5CMV-p53, and DNA fragmentation characteristic of apoptosis was observed only after combined treatment with AS clusterin ODN and Ad5CMV-p53, but not after treatment with either agent alone. Administration of AS clusterin ODN and Ad5CMV-p53 into nude mice resulted in a significant inhibition of KoTCC-1 tumor growth as well as lymph node metastases compared to administration of either agent alone. Furthermore, combined treatment with AS clusterin ODN, Ad5CMV-p53, and cisplatin completely eradicated KoTCC-1 tumors and lymph node metastases in 60% and 100% of mice, respectively. These findings suggest that combined treatment with AS clusterin ODN and Ad5CMV-p53 could be a novel strategy to inhibit bladder cancer progression, and that further additional use of a chemotherapeutic agent may substantially enhance the efficacy of this combined regimen.

Figure 1

Effects of AS clusterin ODN and/or Ad5CMV-p53 treatment on clusterin expression in KoTCC-1 cells. (A) KoTCC-1 cells were treated daily with AS clusterin ODN or a two-base clusterin MM ODN for 2 days. Total RNA was extracted from cultured cells, and analyzed for clusterin and GAPDH levels by Northern blotting. No Tx indicates untreated cells. (B) KoTCC-1 cells were treated with various doses of Ad5CMV-p53 for 48 hours; total RNA was then extracted and analyzed for clusterin levels by real-time RT-PCR. As described in Materials and Methods section, the clusterin mRNA value was corrected by the corresponding GAPDH mRNA value. Each column represents the mean value of three independent experiments with standard deviation. *Differs from control (P < .05) by Student's t test. **Differs from control (P < .01) by Student's t test. (C) KoTCC-1 cells were treated with Ad5CMV-p53 at 50 PFU/cell for indicated intervals; total RNA was then extracted and analyzed for clusterin levels by real-time RT-PCR. As described in Materials and Methods section, the clusterin mRNA value was corrected by the corresponding GAPDH mRNA value. Each column represents the mean value of three independent experiments with standard deviation. *Differs from control (P < .05) by Student's t test. **Differs from control (P < .01) by Student's t test. (D) KoTCC-1 cells were treated daily with 500 nM AS clusterin ODN or a two-base clusterin MM control ODN for 2 days. Following exposure to Ad5CMV-p53 at 10 or 50 PFU/cell for 48 hours, total RNA was then extracted and clusterin levels were analyzed by real-time RT-PCR. As described in Materials and Methods section, the clusterin mRNA value was corrected by the corresponding GAPDH mRNA value. Each column represents the mean value of three independent experiments with standard deviation. *Differs from control (P < .01) by Student's t test.

Figure 2

Effect of combined treatment with AS clusterin ODN and Ad5CMV-p53 on KoTCC-1 cell growth and apoptosis. (A) KoTCC-1 cells were treated daily with 500 nM AS clusterin ODN or MM control ODN for 2 days. Following ODN treatment, the medium was replaced with medium containing various doses of Ad5CMV-p53. After 48 hours of incubation, the number of viable cells was determined by the MTT assay. Each data point represents the mean of triplicate analyses with standard deviation. The cytotoxic effect of Ad5CMV-p53 on KoTCC-1 cells was significantly enhanced by AS clusterin ODN treatment (P < .01, by ANOVA). (B) KoTCC-1 cells were treated daily with various concentrations of AS clusterin ODN or MM control ODN for 2 days, then incubated for 48 hours with medium alone or medium containing Ad5CMV-p53 or Ad5CMV-Luc at 10 PFU/cell, and the number of viable cells was determined by the MTT assay. Each data point represents the mean of three independent experiments with standard deviation. Treatment of KoTCC-1 cells with AS clusterin ODN significantly enhanced the sensitivity to Ad5CMV-p53 (P < .05, by ANOVA). (C) After the same treatment schedule as described above, DNA was extracted from KoTCC-1 cells, electrophoresed in a 2% agarose gel, and visualized by ethidium bromide staining and UV transillumination. (D) After the same treatment schedule as described above, DNA fragmentation was quantitatively measured by Cell Death Detection ELISA kit using antihistone antibody. Each column represents the mean value of three independent experiments with standard deviation. *Differs from control (P < .01) by Student's t test.

Figure 3

Effects of combined treatment with AS clusterin ODN plus Ad5CMV-p53 on KoTCC-1 tumor growth. Mice bearing KoTCC-1 tumor were randomly selected for treatment with AS clusterin ODN plus Ad5CMV-Luc, MM control ODN plus Ad5CMV-Luc, AS clusterin ODN plus Ad5CMV-p53, or MM control ODN plus Ad5CMV-p53. Ten days after tumor cell injection, 10 mg/kg AS clusterin ODN or MM control ODN was injected daily intraperitoneally for 28 days, and 200 µl of Ad5CMV-p53 or Ad5CMV-Luc (1 x 10⁷ PFU/ml) was injected intratumorally twice a week for 2 weeks. Tumor volume was measured every 5 days and calculated by the formula: length x width x depth x 0.5236. Each data point represents the mean tumor volume in each experimental group containing 10 mice with standard deviation. Mean tumor volume in mice treated with AS clusterin ODN plus Ad5CMV-p53 50 days after tumor injection was significantly smaller than that in mice treated with other regimens (P < .001 by Student's t test).

Figure 4

Effects of combined treatment with AS clusterin ODN, Ad5CMV-p53, and cisplatin on KoTCC-1 tumor growth. Mice bearing KoTCC-1 tumor were randomly selected for treatment with AS clusterin ODN, Ad5CMV-Luc plus cisplatin, MM control ODN, Ad5CMV-Luc plus cisplatin, AS clusterin ODN, Ad5CMV-p53 plus cisplatin, or MM control ODN, and Ad5CMV-p53 plus cisplatin. Ten days after tumor cell injection, 10 mg/kg AS clusterin ODN or MM control ODN was injected daily intraperitoneally for 28 days; 200 µl of Ad5CMV-p53 or Ad5CMV-Luc (1 x 10⁷ PFU/ml) was injected intratumorally twice a week for 2 weeks; and 50 µg of cisplatin was injected intravenously twice a week for 2 weeks. Tumor volume was measured every 5 days and calculated by the formula: length x width x depth x 0.5236. Each data point represents the mean tumor volume in each experimental group containing eight mice with standard deviation. Mean tumor volume in mice treated with AS clusterin ODN, Ad5CMV-p53, plus cisplatin 50 days after tumor injection was significantly smaller than that in mice treated with other regimens (P < .001 by Student's t test).