Intravesical Ad-IFN? Causes Marked Regression of Human Bladder Cancer Growing Orthotopically in Nude Mice and Overcomes Resistance to IFN-? Protein

Department of Genitourinary Medical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
Molecular Therapy (Impact Factor: 6.23). 10/2004; 10(3):525-32. DOI: 10.1016/j.ymthe.2004.05.027
Source: PubMed


We have produced prolonged, high local concentrations of interferon in vivo by intravesical instillation of adenoviruses encoding interferon-alpha (Ad-IFNalpha) together with the gene transfer-enhancing agent Syn3. We found sustained interferon protein levels for days, both in normal mouse urothelium and in human bladder cancer cells growing as superficial bladder tumors in nude mice using an orthotopic bladder model developed by us. Tumor burden in the bladder was determined utilizing cancer cells containing the green fluorescent protein. Marked tumor regression was observed following two 1-h exposures of Ad-IFNalpha/Syn3 and little or no cytotoxicity was detected in normal cells. Similar intravesical instillation of clinically relevant concentrations of IFN protein alone or Ad-IFNalpha without Syn3 was ineffective. Surprisingly, in vitro, Ad-IFNalpha also caused caspase-dependent death of bladder cancer cell lines that were resistant to high concentrations of IFN-alpha protein, including the cell line used in vivo. These findings demonstrate that Ad-IFNalpha can overcome resistance to IFN-alpha protein both in vitro and in vivo and support evaluation of intravesical Ad-IFNalpha/Syn3 for the treatment of superficial bladder cancer.

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Available from: Daniel C Maneval, May 07, 2014
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    • "Our phase l study of intravesical Ad-IFNα/Syn3 treatment in patients with non-muscle invasive bladder cancer previously failing BCG (bacillus Calmette-Guérin) therapy has been completed (1). The Syn3 is an excipient Syn3 that facilitates effective gene transfer and expression of IFNα within the urothelium and tumor with subsequent secretion into the urine (2,3). In the phase l study only a single instillation of Ad-IFNα/Syn3 was given. "
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    ABSTRACT: A phase l study using intravesical Ad-IFNαSyn3 for patients with bacillus Calmette-Guérin-resistant superficial bladder cancer showed a complete remission (CR) of 43% at 90 days after treatment with high levels of interferon-α (IFNα) being produced. Ad-IFNα kills bladder cancer cells by two apoptotic and one necrotic mechanism that can be measured by soluble forms of cytokeratin 18 (CK18) using M30 and M65 ELISAs, assays for caspase-cleaved (apoptotic) and uncleaved (necrotic) cell death, respectively. Therefore, we determined whether M30 and M65 levels in the urine after treatment could document all three mechanisms of cancer cell kill and also predict having a CR. High levels of both M30 and M65 were found in all patients within 24 h after treatment with all three types of cancer cell death occurring. Moreover, the return of both M30 and M65 levels in the urine to normal levels within 5 days or more after treatment was strongly associated with obtaining a CR (P=0.003). This is the first time that such assays have been used to study response to therapy in the urine of patients with bladder cancer and in the future may prove valuable in predicting clinical outcome.Cancer Gene Therapy advance online publication, 7 February 2014; doi:10.1038/cgt.2014.1.
    Full-text · Article · Feb 2014 · Cancer gene therapy
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    • "Our laboratory has shown that adenoviral-mediated interferon α (Ad-IFNα) is highly cytotoxic to tumor cells resistant to the interferon α protein. Ad-IFNα also produces a strong bystander effect in cancer cells, which in turn can be seen in conditioned medium from either normal and cancer cells, but is not cytotoxic to normal urothelial cells (1-4). In addition, intravesical Ad-IFNα is presently being used in a Phase l trial for BCG resistant superficial bladder cancer. "
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    ABSTRACT: We have previously shown that adenoviral-mediated interferon alpha (Ad-IFNalpha) treatment is highly cytotoxic to tumor cells which are resistant to the IFNalpha protein. We now report that autophagy is produced after Ad-IFNalpha treatment of either IFN resistant bladder cancer cells (UC9 and KU7) or the normal urothelial cell line (TERT-NHUC). After Ad-IFNalpha infection autophagosomes, an early stage of autophagy, were seen in cancer cells whereas autophagolysosomes, a later stage of autophagy, were observed mostly in normal cells by electron microscopy. Conditioned medium from either normal or bladder cancer cells obtained after Ad-IFNalpha infection, however, produced no autophagy when placed on the bladder cancer cells, although again marked cytotoxicity was observed. This indicated that the autophagy seen was related to the direct effect of Ad-IFNalpha transfection and expression rather than to the bystander factors produced. In addition, autophagic changes were seen using LysoTracker Red DND-99 in both normal and cancer cells. We also documented that Ad-IFNalpha treatment produces the autophagic protein form, light chain 3 (LC3)-II, in cancer cells but not normal cells, which in turn was inhibited by the autophagic inhibitor, 3-methyladenine (3-MA). This inhibition of autophagy resulted in a significant increase in apoptotic cell death as measured by the sub-G1 population. We hypothesize that the autophagy seen in normal urothelial cells is a protective response and is allowed to be completed, providing a survival mechanism after Ad-IFN treatment, whereas the autophagy produced in IFN resistant cancer cells is not allowed to be completed and is insufficient to significantly suppress cytotoxicity.
    Full-text · Article · Aug 2010 · Cancer gene therapy
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    • "An intravesical Phase l study using Ad-IFN is now underway for superficial bladder cancer based in large part on our preclinical results (2). We thought it would be most helpful in this trial not only to document the levels of IFN achieved in the urine at various concentrations of Ad-IFN instillation but also to try to identify possible biomarkers of Ad-IFN produced bladder cancer cell death. "
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    ABSTRACT: Adenoviral transduction of human bladder cancer cells with human interferon alpha-2b (Ad-IFN) produces cancer-specific cell death through direct and indirect mechanisms. The indirect mechanisms involve the secreted IFN produced, which kill, IFN protein-sensitive cancer cells, as well as yet unidentified bystander factors, which are cytotoxic to neighboring cancer cells. The direct cell kill results from transfection and expression of Ad-IFN in the cancer cells. As the molecular forms of cytokeratin 18, either caspase cleaved or not, have been associated with apoptotic or necrotic cell death, respectively, we determined if increases in either or both cytokeratin 18 forms could be observed following IFNalpha protein or Ad-IFN treatment of bladder carcinoma cells. Quantification of M30 and M65 enzyme-linked immunosorbent assays (assays for cytokeratin 18 associated apoptotic and necrotic cell death, respectively) were used as surrogate markers of the cell death produced. In the IFN protein-sensitive RT4 bladder cancer cells, IFN produced primarily M30-related cell death, whereas Ad-IFN treatment resulted in high levels of both M30 and M65. In contrast, conditioned medium from Ad-IFN-treated cells whether from normal human urothelial cells or bladder cancer cells caused increases mainly in M30 levels when added to IFN protein resistant KU7 or UC9 bladder cancer cells, suggesting that the bystander factors present in the conditioned medium produced primarily apoptotic cell death. In addition, a significant increase in M65 levels above that observed for M30 was seen when the IFN protein resistant KU7 and UC9 cells were treated with Ad-IFN, again indicating there is additional necrotic-related cell death produced by Ad-IFN as well. Normal urothelial cells showed no cytotoxicity nor increases in M30 or M65 after Ad-IFN treatment. As intravesical Ad-IFN treatment is presently being evaluated for its efficacy in superficial bladder cancer measurement of M30 and M65 levels in the urine at various time points before and after Ad-IFN treatment may provide not only a biomarker of efficacy but also evidence for the different types and proportion of cell kill produced by the various mechanisms of cell kill in the tumors of individual patients.
    Full-text · Article · Mar 2009 · Cancer gene therapy
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