Pancreatic adenocarcinoma has a poor prognosis and frequently develops resistance to standard chemotherapeutics. Oncolytic adenoviruses represent a promising approach to overcome treatment ...resistance. The replication-selective dl922-947 adenovirus, defective in pRb binding, targets cancers with deregulated cell cycle control, such as the majority of pancreatic tumors. Cell killing efficacy was higher for dl922-947 than for adenovirus type 5 (Ad5) and the clinically approved dl1520 in pancreatic cancer cells with K-ras, p16 and p53 mutations. Combinations of dl922-947 and 5-fluorouracil or gemcitabine (2'2'-difluoro-2-deoxytidine) resulted in strong synergistic cell killing in Suit-2 and the highly drug- and virus-resistant Hs766T cells. Viral uptake increased in response to drugs, but was independent of the expression levels of the viral attachment receptor coxsackie and adenovirus receptor (CAR), whereas expression levels of the internalization receptors α(v)β(3)- and α(v)β(5)-integrins were increased. Early viral E1A expression was potently induced with drugs contributing to the synergistic effects. The dl922-947 mutant was more efficacious than Ad5 in vivo in Hs766T and Suit-2 xenograft models. In combination with gemcitabine, median survival was further prolonged. We demonstrate that dl922-947 is highly efficacious in pancreatic cancers and conclude that oncolytic adenoviruses harboring the E1ACR2 deletion have great potential for development into future clinical candidates for pancreatic cancer.
The microtubule-stabilizing drug paclitaxel has activity in relapsed ovarian cancer. dl922-947, an oncolytic adenovirus with a 24-bp deletion in E1A CR2, replicates selectively within and lyses cells ...with a dysregulated Rb pathway and has efficacy in ovarian cancer. In the aggressive A2780CP xenograft, combination treatment with weekly dl922-947 and paclitaxel has significantly greater efficacy than either treatment alone and can produce complete tumor eradication in some animals. We investigated the mechanisms of paclitaxel's synergy with dl922-947 in ovarian cancer. The host-cell microtubule network is grossly rearranged and stabilized following adenovirus infection, but paclitaxel does not increase this significantly. Paclitaxel does not synergize by increasing infectivity, viral protein expression or virus release. However, destabilizing the microtubule network with nocodazole reduces viral exit, revealing a novel microtubule-dependent pathway for non-lytic adenoviral exit. dl922-947 can override multiple cell cycle checkpoints but induces cell death by a non-apoptotic mechanism. In combination, dl922-947 and low-dose paclitaxel induces aberrant, multipolar mitoses, mitotic slippage and multinucleation, triggering an apoptotic cell death.
The majority of clinical trials evaluating replication-selective oncolytic adenoviruses utilized mutants with immunomodulatory E3B genes deleted, likely contributing to the attenuated efficacy. We ...investigated whether an intact immune response could contribute to the observed improved efficacy in response to combinations with chemotherapeutics. Seven carcinoma cell lines were evaluated by combining viral mutants; dl309 (DeltaE3B), dl704 (DeltaE3gp19K), dl312 (DeltaE1A) or wild-type Ad5 with the commonly used clinical drugs cisplatin and paclitaxel. Synergistic effects on cell death were determined by generation of combination indexes in cultured cells. In vivo tumor growth inhibition was achieved by virotherapy alone and was most efficacious with wild-type virus and least with the DeltaE3B mutant. Significantly higher efficacy was observed when the viruses were combined with drugs. The greatest enhancement of tumor inhibition was in combination with the DeltaE3B mutant restoring potency to that of Ad5 wild-type levels, observed only in animals with intact immune response. Increases in infectivity, viral gene expression and replication were identified as potential mechanisms contributing to the synergistic effects. Our results suggest that the attenuation of DeltaE3B mutants can be overcome by low doses of chemotherapeutics only in the presence of an intact immune response indicating a role for T-cell-mediated functions.
Oncolytic viruses are regulated by the tumor phenotype to replicate and lyse cancer cells selectively. To identify optimal strategies for breast cancer we compared five adenoviruses with distinct ...regulatory mechanisms: Ad-dl922-947 (targets G1-S checkpoint); Ad-Onyx-015 and Ad-Onyx-017 (target p53/mRNA export); Ad-vKH1 (targets Wnt pathway), and AdEHE2F (targets estrogen receptor/G1-S checkpoint/hypoxic signaling). The quantity of virus required to kill 50% of breast cancer cells after 6 days (EC(50), plaque-forming units per cell) was measured. The most potent virus was Ad-dl922-947 (EC(50), 0.01-5.4 in SkBr3, MDA-231, MDA-468, MCF7, and ZR75.1 cells), followed by wild-type (Ad-WT; EC(50), 0.3-5.5) and AdEHE2F (EC(50), 1.4-3.9). Ad-vKH1 (EC(50), 7.2-72.1), Ad-Onyx-017 (EC(50), 8.4-167), and Ad-Onyx-015 (EC(50), 17.7-377) showed less activity. Most viruses showed limited cytotoxicity in normal human cells, including breast epithelium MCF10A (EC(50), >722) and fibroblasts (EC(50), >192) and only moderate cytotoxicity in normal microvascular endothelial cells (HMVECs; EC(50), 42.8-149), except Ad-dl922-947, which was active in HMVECs (EC(50), 1.6). After injection into MDA-231 xenografts, Ad-WT, AdEHE2F, and Ad-dl922-947 showed replication, assessed by hexon staining and quantitative polymerase chain reaction measurement of viral DNA, and significantly inhibited tumor growth, leading to extended survival. After intravenous injection Ad-dl922-947 showed DNA replication (233% of the injected dose was measured in liver after 3 days) whereas AdEHE2F did not. Overall, AdEHE2F showed the best combination of low toxicity in normal cells and high activity in breast cancer in vitro and in vivo, suggesting that molecular targeting using estrogen response elements, hypoxia response elements, and a dysregulated G1-S checkpoint is a promising strategy for virotherapy of breast cancer.
Exposure to high ambient levels of nitrogen dioxide (NO2) enhances the airway reaction in humans to allergen, measured as decreased pulmonary function. We tested whether this NO2 effect is associated ...with an increased inflammatory response to allergen in the airways. To mimic real-life conditions, in which exposure to high ambient levels of NO2 occurs only during short periods of time but often several times a day, we used a repeated-exposure model. On day 1, 18 subjects with allergic asthma were exposed, in randomized order, to purified air or to 500μg/m3 NO2 for 15min, and on day 2 for 2×15min. Allergen was inhaled 3–4h after the NO2 exposures on both days. Symptoms, pulmonary function, and inflammatory response in sputum and blood were measured daily. Eosinophil cationic protein in both sputum and blood increased more from day 1 to day 3 after NO2+allergen than after air+allergen, whereas eosinophil counts did not differ. The change in myeloperoxidase was significantly greater after NO2+allergen than after air+allergen in blood but not in sputum. This finding was not accompanied by raised levels of neutrophils in sputum and blood. Symptoms and pulmonary function were equally affected by NO2+allergen and air+allergen. We conclude that two to three brief exposures to ambient levels of NO2 can prime circulating eosinophils and enhance the eosinophilic activity in sputum in response to inhaled allergen. This might be an important mechanism by which air pollutants amplify the inflammatory reactions in the airways.
Aim: We investigated the role of eosinophils in the pathogenesis of bronchopulmonary dysplasia (BPD) in preterm infants.
Methods: Fifteen preterm infants with BPD were compared to 13 preterms with ...respiratory distress syndrome (RDS) and to 16 healthy preterms. We assessed total eosinophil and neutrophil counts in venous blood samples and the levels of the eosinophilic activity markers eosinophilic cationic protein (ECP) and the cellular surface antigen (CD9).
Results: The eosinophil count was greater in BPD compared with RDS and healthy infants (1414 vs. 797 and 471 cells per microlitre, respectiveley, p = 0.03). ECP levels were elevated (34 vs. 12.8 and 9.8 μg/L, respectively, p = 0.002) and CD9 levels reduced (75 vs. 94 and 86 mean fluorescence intensity units, respectively, p = 0.01) in BPD compared with RDS and healthy infants, suggesting eosinophilic activation in BPD. These findings were not solely explained by differences between gestational age or birth weight of the different groups. ECP levels were positively correlated with the duration of oxygen supplementation in the BPD group. The eosinophil count fell promptly after steroid treatment was commenced in the BPD group.
Conclusion: The findings suggest that BPD is linked to eosinophil activation, which might contribute to the pathogenesis.
Replication-selective oncolytic adenoviruses are promising anti-tumour therapeutic agents that have been proven safe in hundreds of patients. While clinical efficacy was limited with the viral ...mutants alone, outcomes were improved in combination with chemotherapeutics. To further increase efficacy of viral-based therapies it is necessary to explore the cellular mechanisms responsible for enhanced tumour elimination in combination with cytotoxic drugs and to develop mutants with higher potency. To this end we generated a set of novel adenoviral mutants with deletions of the anti-apoptotic E1B19K-gene and the pRb-binding E1ACR2-region. Mutants with the E1B19K deletion significantly increased tumour cell killing in combination with cytotoxic drugs including gemcitabine, 5-fluorouracil (5-FU), docetaxel and mitoxantrone through enhancement of drug-induced apoptosis but did not sensitise normal cells to drugs. The double-deleted AdDeltaDelta (DeltaE1ACR2 and DeltaE1B19K) mutant had high cell killing activity in prostate and pancreatic carcinoma models. Replication was similar to the parental Ad5 and DeltaCR2 viruses in all tumour cells and was attenuated in normal cells. In combination with chemotherapeutics AdDeltaDelta synergistically enhanced cell death in all tested cancer cell lines and in prostate and pancreatic xenografts in vivo. These data suggest that the AdDeltaDelta mutant is a candidate for targeting of solid tumours specifically in combination with cytotoxic factors. Our findings imply that less toxic doses than currently practised in the clinic could efficiently target adenocarcinomas when combined with the AdDeltaDelta mutant.
Pancreatic adenocarcinomas are aggressive and frequently develop resistance to all current therapies. Replication-selective adenoviruses can overcome resistance to chemotherapeutics through their ...sensitizing effects on drug-induced cell killing. We previously found that adenovirus deleted in the anti-apoptotic E1B19K gene enhanced gemcitabine-induced apoptotis. Here we demonstrate that our engineered double-deleted AdDELTADELTA mutant (deleted in the pRb-binding E1ACR2 region and E1B19K) selectively replicates and enhances cell killing in combination with DNA-damaging cytotoxic drugs in pancreatic cancer cells. Combinations of AdDELTADELTA with gemcitabine, irinotecan or cisplatin resulted in two- to fourfold decreases in EC.sub.50 (half maximal effective concentration) values and was more efficent than similar combinations with wild-type virus, the d/1520 (ONYX-015) and d/922-947 mutants. AdDELTADELTA replication was impaired in normal bronchial human epithelial cells and did not sensitize the cells to drugs. Gemcitabine-insensitive AsPC-1, BxPC-3 and PANC-1 cells were efficiently killed by irinotecan in combination with AdDELTADELTA. Suboptimal doses of AdDELTADELTA and gemcitabine significantly prolonged time to tumor progression in two human pancreatic tumor xenograft in vivo models, PT45 and SUIT-2. We conclude that AdDELTADELTA has low toxicity to normal cells while potently sensitizing pancreatic cancer cells to DNA-damaging drugs, and holds promise as an improved therapeutic strategy for pancreatic cancer. Gene Therapy (2011) 18, 1157-1165; doi: 10.1038/gt.2011.141; published online 6 October 2011 Keywords: adenocarcinoma; gemcitabine; irinotecan; in vivo; sensitization
Pancreatic adenocarcinomas are aggressive and frequently develop resistance to all current therapies. Replication-selective adenoviruses can overcome resistance to chemotherapeutics through their ...sensitizing effects on drug-induced cell killing. We previously found that adenovirus deleted in the anti-apoptotic E1B19K gene enhanced gemcitabine-induced apoptotis. Here we demonstrate that our engineered double-deleted AdΔΔ mutant (deleted in the pRb-binding E1ACR2 region and E1B19K) selectively replicates and enhances cell killing in combination with DNA-damaging cytotoxic drugs in pancreatic cancer cells. Combinations of AdΔΔ with gemcitabine, irinotecan or cisplatin resulted in two- to fourfold decreases in EC(50) (half maximal effective concentration) values and was more efficent than similar combinations with wild-type virus, the dl1520 (ONYX-015) and dl922-947 mutants. AdΔΔ replication was impaired in normal bronchial human epithelial cells and did not sensitize the cells to drugs. Gemcitabine-insensitive AsPC-1, BxPC-3 and PANC-1 cells were efficiently killed by irinotecan in combination with AdΔΔ. Suboptimal doses of AdΔΔ and gemcitabine significantly prolonged time to tumor progression in two human pancreatic tumor xenograft in vivo models, PT45 and SUIT-2. We conclude that AdΔΔ has low toxicity to normal cells while potently sensitizing pancreatic cancer cells to DNA-damaging drugs, and holds promise as an improved therapeutic strategy for pancreatic cancer.
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