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RECOMBINANT BACILLUS CALMETTE-GUERIN EXPRESSING PERTUSSIS TOXIN IN THE TREATMENT OF BLADDER CANCER
THE JOURNAL OF UROLOGY®
Vol. 181, No. 4, Supplement, Monday, April 27, 2009
413
cancer in mice, reducing the bladder weight and increasing survival. The immune response obtained with the rBCG-S1PT expressed higher cytokines related to Th1. All this data may indicate that this recombinant agent may promote better response to intravesical imunotherapy in bladder tumor. Source of Funding: Fapesp
1159 SYNERGISTIC POTENTIATION OF INTERFERON WITH D-FRACTION ON BLADDER CANCER CELLS Brandon Louie*, Mauricio Davalos, Paul Pyo, John Won, Muhammad Choudhury, Sensuke Konno, Valhalla, NY
Source of Funding: FAEPEX-UNICAMP
1158 RECOMBINANT BACILLUS CALMETTE-GUERIN EXPRESSING PERTUSSIS TOXIN IN THE TREATMENT OF BLADDER CANCER Daher C Chade*, Ricardo Borra, Katia R m Leite, Enrico F Andrade, Fabiola E Villanova, Ivan Nascimento, Luciana C C Leite, Miguel Srougi, Priscila M Andrade, Sao Paulo, Brazil INTRODUCTION AND OBJECTIVES: The intravesical immunotherapy with bacillus Calmette-Guérin (BCG) is the adjuvant treatment of choice in superficial bladder cancer. Recently, studies of the mechanism of BCG have identified the immune reactions favorable and the genes responsible for the antitumor effect, enabling the production of recombinant vaccines, possibly more effective and with fewer side effects. With those goals, the pertussis toxin (S1PT) was combined to BCG, creating a recombinant variant (rBCG-S1PT) with the capacity to promote an immune response targeted to the T helper type 1 (Th1), which may increase the effectiveness of its antitumor effect. METHODS: The development of the animal model of bladder cancer was conducted by transurethral instillation of bladder tumor cell line MB49 of the mouse strain C57BL/6, setting the orthotopic and syngeneic murine model. The animal models were divided into three groups, which received 4 weekly intravesical applications of rBCG-S1PT, BCG, or saline. After 7 days of the last instillation, we used bladder for weight and histopathological analysis. Another group of animals were monitored for 60 days for analysis of survival. RESULTS: The rate of tumor implantation was 90% of the animals submitted to tumor inoculation. We obtained reduction of the average weights of bladder in groups BCG and rBCG-S1PT ((p<0,001). In both groups treated with immunotherapy, there was an increase of expression of interleukins TNF-A, which was more intense in the group treated with rBCG-S1PT (p<0,05). There was also increased expression of IL-10 in the recombinant BCG (p<0,01). The analysis of survival showed a significant increase in the group of animals treated with rBCG-S1PT. CONCLUSIONS: The immunotherapy with rBCG-S1PT demonstrated more benefits than BCG in the treatment of bladder
INTRODUCTION AND OBJECTIVES: Although interferons (IFNs) have been used as immunotherapy for patients with bladder cancer, the outcomes have not yet been satisfactory. To improve the efficacy of such IFN monotherapy, “combination” therapy has been proposed as an alternative approach. D-fraction, a bioactive mushroom extract, has been shown to have immunostimulatory and antitumor activities, implying its clinical utility in cancer treatment. Accordingly, we studied whether the efficacy/potency of IFN might be improved when combined with D-fraction in bladder cancer T24 cells in vitro. METHODS: Dose-dependent effects of recombinant IFN-A2b (050,000 IU/ml), D-fraction (PDF; 0-700 μg/ml), or their combinations were assessed on T24 cell growth at 72 h. To explore the growth inhibitory mechanism, cell cycle analysis was performed on a flow cytometer and in vitro phosphorylation assay was employed to assess activity of double-stranded DNA-dependent protein kinase (DNA-PK), a cell cycle regulatory protein kinase. RESULTS: IFN-A2b by itself was capable of inducing a significant (~50%) growth reduction at 20,000 (20K) IU/ml, which further declined to ~66% at 50K IU/ml. PDF alone showed no effects up to 200 μg/ml, but a ~20% and ~53% growth reduction was observed at 400 and 700 μg/ ml, respectively. When the varying concentrations of IFN-A2b and PDF were combined, the combination of 10K IU/ml IFN-A2b and 200 μg/ml PDF resulted in a ~75% growth reduction. This was accompanied by a 55% increase in cell number in the G1 phase with a 63% decrease in the S phase cell population, revealed by cell cycle analysis. Such an accumulation of cells in the G1 phase unable to enter the S phase is known as a G1 cell cycle arrest. Concurrently, DNA-PK activity in IFNA2b/PDF-treated cells was almost 3-fold higher than that in controls, indicating its active role in the cell cycle regulation. CONCLUSIONS: The combination of IFN-A2b (10K IU/ml) and PDF (200 μg/ml) is able to induce a ~75% growth reduction in T24 cells, although the given concentrations of neither agent by itself have any inhibitory effects. This is a synergistic potentiation of two agents and such an enhanced growth inhibition appears to result from a G1 arrest concomitant with DNA-PK activation. It is conceivable that the IFN-A2b/PDF combination may primarily activate DNA-PK, triggering the cascade events that act on the cell cycle to ultimately cease cancer cell proliferation. Thus, D-fraction (PDF) might be considered a potential agent to help improve the efficacy of IFN therapy for patients with superficial bladder cancer. Source of Funding: None
Vol. 181, No. 4, Supplement, Monday, April 27, 2009
413
cancer in mice, reducing the bladder weight and increasing survival. The immune response obtained with the rBCG-S1PT expressed higher cytokines related to Th1. All this data may indicate that this recombinant agent may promote better response to intravesical imunotherapy in bladder tumor. Source of Funding: Fapesp
1159 SYNERGISTIC POTENTIATION OF INTERFERON WITH D-FRACTION ON BLADDER CANCER CELLS Brandon Louie*, Mauricio Davalos, Paul Pyo, John Won, Muhammad Choudhury, Sensuke Konno, Valhalla, NY
Source of Funding: FAEPEX-UNICAMP
1158 RECOMBINANT BACILLUS CALMETTE-GUERIN EXPRESSING PERTUSSIS TOXIN IN THE TREATMENT OF BLADDER CANCER Daher C Chade*, Ricardo Borra, Katia R m Leite, Enrico F Andrade, Fabiola E Villanova, Ivan Nascimento, Luciana C C Leite, Miguel Srougi, Priscila M Andrade, Sao Paulo, Brazil INTRODUCTION AND OBJECTIVES: The intravesical immunotherapy with bacillus Calmette-Guérin (BCG) is the adjuvant treatment of choice in superficial bladder cancer. Recently, studies of the mechanism of BCG have identified the immune reactions favorable and the genes responsible for the antitumor effect, enabling the production of recombinant vaccines, possibly more effective and with fewer side effects. With those goals, the pertussis toxin (S1PT) was combined to BCG, creating a recombinant variant (rBCG-S1PT) with the capacity to promote an immune response targeted to the T helper type 1 (Th1), which may increase the effectiveness of its antitumor effect. METHODS: The development of the animal model of bladder cancer was conducted by transurethral instillation of bladder tumor cell line MB49 of the mouse strain C57BL/6, setting the orthotopic and syngeneic murine model. The animal models were divided into three groups, which received 4 weekly intravesical applications of rBCG-S1PT, BCG, or saline. After 7 days of the last instillation, we used bladder for weight and histopathological analysis. Another group of animals were monitored for 60 days for analysis of survival. RESULTS: The rate of tumor implantation was 90% of the animals submitted to tumor inoculation. We obtained reduction of the average weights of bladder in groups BCG and rBCG-S1PT ((p<0,001). In both groups treated with immunotherapy, there was an increase of expression of interleukins TNF-A, which was more intense in the group treated with rBCG-S1PT (p<0,05). There was also increased expression of IL-10 in the recombinant BCG (p<0,01). The analysis of survival showed a significant increase in the group of animals treated with rBCG-S1PT. CONCLUSIONS: The immunotherapy with rBCG-S1PT demonstrated more benefits than BCG in the treatment of bladder
INTRODUCTION AND OBJECTIVES: Although interferons (IFNs) have been used as immunotherapy for patients with bladder cancer, the outcomes have not yet been satisfactory. To improve the efficacy of such IFN monotherapy, “combination” therapy has been proposed as an alternative approach. D-fraction, a bioactive mushroom extract, has been shown to have immunostimulatory and antitumor activities, implying its clinical utility in cancer treatment. Accordingly, we studied whether the efficacy/potency of IFN might be improved when combined with D-fraction in bladder cancer T24 cells in vitro. METHODS: Dose-dependent effects of recombinant IFN-A2b (050,000 IU/ml), D-fraction (PDF; 0-700 μg/ml), or their combinations were assessed on T24 cell growth at 72 h. To explore the growth inhibitory mechanism, cell cycle analysis was performed on a flow cytometer and in vitro phosphorylation assay was employed to assess activity of double-stranded DNA-dependent protein kinase (DNA-PK), a cell cycle regulatory protein kinase. RESULTS: IFN-A2b by itself was capable of inducing a significant (~50%) growth reduction at 20,000 (20K) IU/ml, which further declined to ~66% at 50K IU/ml. PDF alone showed no effects up to 200 μg/ml, but a ~20% and ~53% growth reduction was observed at 400 and 700 μg/ ml, respectively. When the varying concentrations of IFN-A2b and PDF were combined, the combination of 10K IU/ml IFN-A2b and 200 μg/ml PDF resulted in a ~75% growth reduction. This was accompanied by a 55% increase in cell number in the G1 phase with a 63% decrease in the S phase cell population, revealed by cell cycle analysis. Such an accumulation of cells in the G1 phase unable to enter the S phase is known as a G1 cell cycle arrest. Concurrently, DNA-PK activity in IFNA2b/PDF-treated cells was almost 3-fold higher than that in controls, indicating its active role in the cell cycle regulation. CONCLUSIONS: The combination of IFN-A2b (10K IU/ml) and PDF (200 μg/ml) is able to induce a ~75% growth reduction in T24 cells, although the given concentrations of neither agent by itself have any inhibitory effects. This is a synergistic potentiation of two agents and such an enhanced growth inhibition appears to result from a G1 arrest concomitant with DNA-PK activation. It is conceivable that the IFN-A2b/PDF combination may primarily activate DNA-PK, triggering the cascade events that act on the cell cycle to ultimately cease cancer cell proliferation. Thus, D-fraction (PDF) might be considered a potential agent to help improve the efficacy of IFN therapy for patients with superficial bladder cancer. Source of Funding: None