YB-1 dependent virotherapy in combination with temozolomide as a multimodal therapy approach to eradicate malignant glioma.

The human Y-box binding protein 1 (YB-1) is known to be a promising target for cancer therapy. We have demonstrated that YB-1 plays an important role in the adenoviral life cycle by regulating the adenoviral E2-gene expression. Thus, we studied the oncolytic effect of the recombinant adenovirus Ad-Delo3-RGD, in which the transactivation domain CR3 of the E1A protein is ablated to enable viral replication only in YB-1 positive cancer cells. In vitro Southern Blot analysis and cytopathic effect assays demonstrate high anti-glioma potency, which was significantly increased in combination with temozolomide (TMZ), daunorubicin and cisplatin. Since vascular endothelial growth factor (VEGF) is thought to promote the hypervascular phenotype of primary, malignant brain tumors, we also tested Ad-Delo3-RGD in regard to the inhibition of VEGF expression. Indeed, we found that Ad-Delo3-RGD induced VEGF down regulation, which was even amplified under hypoxic conditions. Tumor-bearing nudemice treated with the YB-1 dependent oncolytic adenovirus showed significantly smaller tumors than untreated controls. Furthermore, combination therapy with TMZ led to a regression in all treated animals with complete tumor regression in 33 % of analyzed mice, which was verified by bioluminescence imaging and histological studies. In addition, histopathological evaluation revealed enhanced apoptosis and a reduction in tumor vessel formation, indicating that Ad-Delo3-RGD has an anti-angiogenic effect in addition to its oncolytic capacity in vivo. Hence, our results demonstrate that the combination therapy of YB-1 dependent virotherapy and TMZ is effective in a xenograft glioma mouse model and might be useful in a YB-1 based clinical setting.

Boosting oncolytic adenovirus potency with magnetic nanoparticles and magnetic force.

Oncolytic adenoviruses rank among the most promising innovative agents in cancer therapy. We examined the potential of boosting the efficacy of the oncolytic adenovirus dl520 by associating it with magnetic nanoparticles and magnetic-field-guided infection in multidrug-resistant (MDR) cancer cells in vitro and upon intratumoral injection in vivo. The virus was complexed by self-assembly with core-shell nanoparticles having a magnetite core of about 10 nm and stabilized by a shell containing 68 mass % lithium 3-[2-(perfluoroalkyl)ethylthio]propionate) and 32 mass % 25 kDa branched polyethylenimine. Optimized virus binding, sufficiently stable in 50% fetal calf serum, was found at nanoparticle-to-virus ratios of 5 fg of Fe per physical virus particle (VP) and above. As estimated from magnetophoretic mobility measurements, 3,600 to 4,500 magnetite nanocrystallites were associated per virus particle. Ultrastructural analysis by electron and atomic force microscopy showed structurally intact viruses surrounded by magnetic particles that occasionally bridged several virus particles. Viral uptake into cells at a given virus dose was enhanced 10-fold compared to nonmagnetic virus when infections were carried out under the influence of a magnetic field. Increased virus internalization resulted in a 10-fold enhancement of the oncolytic potency in terms of the dose required for killing 50% of the target cells (IC(50) value) and an enhancement of 4 orders of magnitude in virus progeny formation at equal input virus doses compared to nonmagnetic viruses. Furthermore, the full oncolytic effect developed within two days postinfection compared with six days in a nonmagnetic virus as a reference. Plotting target cell viability versus internalized virus particles for magnetic and nonmagnetic virus showed that the inherent oncolytic productivity of the virus remained unchanged upon association with magnetic nanoparticles. Hence, we conclude that the mechanism of boosting the oncolytic effect by magnetic force is mainly due to the improved internalization of magnetic virus complexes resulting in potentiated virus progeny formation. Upon intratumoral injection and application of a gradient magnetic field in a murine xenograft model, magnetic virus complexes exhibited a stronger oncolytic effect than adenovirus alone. We propose that this approach would be useful during in vivo administration to tumor-feeding blood vessels to boost the efficacy of the primary infection cycle within the tumor. For systemic application, further modification of magnetic adenovirus complexes for shielding and retargeting of the whole magnetic virus complex entity is needed.

Impact of radiation therapy on the oncolytic adenovirus dl520: implications on the treatment of glioblastoma.

BACKGROUND AND PURPOSE: Viral oncolytic therapy is emerging as a new form of anticancer therapy and has shown promising preclinical results, especially in combination with radio- and chemotherapy. We recently reported that nuclear localization of the human transcription factor YB-1 in multidrug-resistant cells facilitates E1-independent adenoviral replication. The aim of this study was to evaluate the combined treatment of the conditionally-replicating adenovirus dl520 and radiotherapy in glioma cell lines in vitro and in human tumor xenografts. Furthermore, the dependency of YB-1 on dl520 replication was verified by shRNA directed down regulation of YB-1. METHODS AND MATERIAL: Localization of YB-1 was determined by immunostaining. Glioma cell lines LN-18, U373 and U87 were infected with dl520. Induction of cytopathic effect (CPE), viral replication, viral yield and viral release were determined after viral infection, radiation therapy and the combination of both treatment modalities. The capacity of treatments alone or combined to induce tumor growth inhibition of subcutaneous U373 tumors was tested also in nude mice. RESULTS: Quantitative real-time PCR demonstrated that the shRNA-mediated down regulation of YB-1 is leading to a dramatic decrease in adenoviral replication of dl520. Immunostaining analysis showed that the YB-1 protein was predominantly located in the cytoplasm in the perinuclear space and less abundant in the nucleus. After irradiation we found an increase of nuclear YB-1. The addition of radiotherapy increased the oncolytic effect of dl520 with enhanced viral replication, viral yield and viral release. The oncolytic activity of dl520 plus radiation inhibited the growth of subcutaneous U373 tumors in a xenograft mouse model. CONCLUSIONS: Radiation mediated increase of nuclear YB-1 in glioma cells enhanced the oncolytic potential of adenovirus dl520.