ABSTRACT
Gene therapy with adenoviral early region gene (E1A) may enhance the susceptibility of neoplastic cells to chemotherapy-induced cell death. Our previous study developed a urothelium-specific oncolytic serotype 5 adenovirus (Ad5) with the uroplakin II (UPII) promoter controlling E1A expression. The present study investigated whether this urothelium-specific recombinant adenovirus (Ad5-UPII-E1A) enhanced mitomycin (MMC) and hydroxycamptothecin (HCPT) sensitization and drug-induced apoptosis in bladder cancer cells. The results of the MTT assay revealed that combination therapy, using Ad5-UPII-E1A and MMC or HCPT, synergistically inhibited the viability of bladder cancer cells in a dose- and time-dependent manner when compared with either agent alone. When cells were treated with Ad5-UPII-E1A alone they arrested in the G1 phase, but cell cycle analysis by flow cytometry revealed S phase arrest when treated with combined therapy. Treatment with MMC or HCPT enhanced Ad5-UPII-E1A-induced apoptosis in 5,637 cells, observed by transmission electron microscopy. Western blot analysis revealed that MMC and HCPT enhanced the E1A expression of the Ad5-UPII-E1A vectorin a dose-dependent manner. The present study demonstrated that Ad5-UPII-E1A combined with MMC or HCPT resulted in synergistic cytotoxicity in a process which involved the promotion of apoptosis in bladder cancer cell lines. MMC and HCPT also promoted the oncolytic effect of Ad5-UPII-E1A. Thus, treatment using Ad5-UPII-E1A combined with MMC or HCPT may be an attractive strategy for the sensitization of bladder cancer to chemotherapy.
ABSTRACT
PURPOSE: Gene therapy combined with radiation has shown promising potential for the treatment of tumors. This paper aimed to clarify the synergistic effect of radiotherapy combined with the bladder cancer tissue-specific oncolytic adenovirus (Ad-PSCAE-UPII-E1A) on bladder cancer cells and to study the underlying synergy mechanisms of the combined treatment. MATERIALS AND METHODS: The Adenovirus carrying E1A under control of UPII promoter and prostate stem cell antigen enhancer (PSCAE) were successfully constructed. The viability of bladder cancer cells BIU-87 and EJ was determined by MTT assay. The apoptotic assay was demonstrated by flow cytometry and TEM. Virus titer was determined by TCID50 assay, and proteins Mre11, Chk2-Thr68, and E1A were analyzed by Western blot method. RESULTS: Oncolytic adenovirus combined with radiotherapy improved antitumor efficacy compared with the single treatment at a time and was X-ray dosage-dependent. When the adenovirus infection was scheduled at 24 h after irradiation, cancer cells had the lowest viability. Adenovirus and irradiation induced cell death through the caspase-3 related apoptotic pathway, and bladder cancer cells were arrested at the G1 (BIU-87) or S phase (EJ). Autophagic vacuoles were observed in bladder cancer cells treated with radiation and adenovirus. After irradiation, more virus particles were observed in the BIU-87 and EJ cells. However, by a TCID50 assay, there was no difference in virus titter between irradiated bladder cancer cells and unirradiated cells. The proteins Mre11, Chk2-Thr68 which involved in the DNA break repair pathway were decreased while γ-H2AX-Ser139 increased; at the same time, the E1A gene and the hexon proteins of oncolytic adenovirus were increased after irradiation. CONCLUSIONS: Our results proved synergistic antitumor effect of adenovirus Ad-PSCAE-UPII-E1A and radiation, which might be a potential therapeutic strategy for bladder cancer.
Subject(s)
Cell Survival/radiation effects , Oncolytic Virotherapy/methods , Oncolytic Viruses/physiology , Radiotherapy, Conformal/methods , Urinary Bladder Neoplasms/therapy , Urinary Bladder Neoplasms/virology , Adenoviridae/genetics , Cell Line, Tumor , Combined Modality Therapy/methods , Humans , Recombination, Genetic/genetics , Treatment Outcome , Urinary Bladder Neoplasms/pathologyABSTRACT
BACKGROUND: The previous works about safety evaluation for constructed bladder tissue specific adenovirus are poorly documented. Thus, we investigated the biodistribution and body toxicity of bladder specific oncolytic adenovirus Ad-PSCAE-UPII-E1A (APU-E1A) and Ad-PSCAE-UPII-E1A-AR (APU-E1A-AR), providing meaningful information prior to embarking on human clinical trials. MATERIALS AND METHOD: Conditionally replicate recombinant adenovirus (CRADs) APU-E1A, APU-EIA-AR were constructed with bladder tissue specific UroplakinII(UPII) promoter to induce the expression of Ad5E1A gene and E1A-AR fusing gene, and PSCAE was inserted at upstream of promoter to enhance the function of promoter. Based on the cytopathic and anti-tumor effect of bladder cancer, these CRADs were intratumorally injected into subcutaneous xenografts tumor in nude mice. We then determined the toxicity through general health and behavioral assessment, hepatic and hematological toxicity evaluation, macroscopic and microscopic postmortem analyses. The spread of the transgene E1A of adenovirus was detected with RT-PCR and Western blot. Virus replication and distribution were examined with APU-LUC administration and Luciferase Assay. RESULTS: General assessment and body weight of the animals did not reveal any alteration in general behavior. The hematological alterations of groups which were injected with 5x10(8) pfu or higher dose (5x10(9) pfu) of APU-E1A and APU-E1A-AR showed no difference in comparison with PBS group, and only slight increased transaminases in contrast to PBS group at 5x10(9) pfu of APU-E1A and APU-E1A-AR were observed. E1A transgene did not disseminate to organs outside of xenograft tumor. Virus replication was not detected in other organs beside tumor according to Luciferase Assay. CONCLUSIONS: Our study showed that recombinant adenovirus APU-E1A-AR and APU-E1A appear safe with 5x10(7) pfu and 5x10(8) pfu intratumorally injection in mice, without any discernable effects on general health and behavior.
