Employment of microarray analysis to characterize biologic differences associated with tropism-modified adenoviral vectors: utilization of non-native cellular entry pathways

In this study, we have applied high-density oligonucleotide microarray technology to characterize biologic changes associated with adenoviral vector-mediated target cell infection. We infected a human melanoma cell line, M21, with the tropism-modified vectors, Ad5lucRGD and Ad5/3luc1. In addition, we infected the M21 cell line with the Ad5luc1, a vector which primarily exploits the coxsackie and adenovirus receptor, as its primary native receptor. We found significant changes in gene expression of 5492 genes induced by Ad5luc1 infection, 2439 genes induced by Ad5/3luc1 infection, and 1251 genes induced by Ad5lucRGD infection, compared to uninfected cells. Among these changes in gene expression, 783 changes were common to Ad5/3luc1 and Ad5luc1 infections, 266 were common to Ad5lucRGD and Ad5luc1 infections, and 185 changes in gene expression were common to Ad5/3luc1 and Ad5lucRGD infections. Interestingly, 89 changes in gene expression were common to all the three groups, suggesting a commonly affected pathway. This analysis represents a unique application of microarray to study vector-related issues. Furthermore, these studies demonstrate the utility of microarray for characterizing the biologic sequelae of host-vector interaction.     

Comparative effects of bicalutamide (Casodex) versus orchidectomy on bone mineral density, bone remodelling, and bone biomechanics in healthy rats

INTRODUCTION: The aim of this work was to analyze the effects produced on bone mineral density (BMD) by the administration of bicalutamide and to compare them with those produced by orchidectomy. Bone formation rate (serum osteocalcin), bone resorption (serum carboxyterminal telopeptide of collagen I; CTX), and biomechanical properties of bone were also studied. METHODS: Thirty-eight male Wistar rats were used: (1) Sham group, rats sham operated at 16 weeks of age; (2) OQX group, rats orchidectomized at 16 weeks of age, and (3) Bic group, rats sham operated at 16 weeks of age and treated during 6 weeks with bicalutamide. The rats were sacrificed at 22 weeks of age, and the BMD in femur and lumbar spine was determined. Serum osteocalcin and serum CTX were also analyzed. Biomechanical parameters related to torsion assay were also studied. RESULTS: The OQX group showed a significant decrease in femoral BMD with respect to Sham rats, whereas bicalutamide treatment did not produce any significant change in BMD. Both Sham and Bic groups showed similar serum osteocalcin and CTX values, whereas OQX rats presented higher osteocalcin and CTX levels than the Sham group. The OQX group showed a significant decrease in femoral thickness. No significant differences were observed in the rest of the biomechanical parameters between groups. CONCLUSION:These results indicate that bicalutamide treatment, in spite of its anti-androgenic properties, does not affect bone remodelling nor BMD in male healthy rats, suggesting that this compound may function as a selective androgen receptor modulator for effects on bone remodelling in the osteoblasts.     

Synergistic inhibitory effect of high-intensity focused ultrasound combined with chemotherapy on Dunning adenocarcinoma

OBJECTIVE: To evaluate the therapeutic effect of high-intensity focused ultrasound (HIFU) combined with chemotherapy (paclitaxel + estramustine) on AT2 Dunning adenocarcinoma, as no satisfactory treatment for localized prostate cancer is available for patients with a poor prognosis, e.g. stage T3, a high Gleason score, or a prostate-specific antigen level of >15 ng/mL. MATERIALS AND METHODS: Forty-one Dunning AT2 tumour-bearing Copenhagen rats were divided into four groups, i.e. control, chemotherapy, HIFU, and chemotherapy + HIFU (the last three treated for 1 week). The growth in tumour volume was recorded for 3 weeks, the point at which tumour volume was considered to have doubled (doubling time). The growth curves of each group were plotted and evaluated statistically. RESULTS: At 30 days of follow-up the distributions of tumour volume with treatment group were significantly different (P < 0.001); volumes were significantly greater in the control than in the chemotherapy-only or in the HIFU-only group (both P = 0.006). The greatest difference was between the chemotherapy + HIFU and the control group. The tumour doubling times were 13.2 days for HIFU-only, 31.2 days for chemotherapy + HIFU and 7.7 days for the controls. CONCLUSION: These results suggest that this combined therapy could be useful for treating patients with high-risk prostate cancer.     

Infectivity enhanced adenoviral-mediated mda-7/IL-24 gene therapy for ovarian carcinoma

OBJECTIVE: Melanoma differentiation associated gene-7 [mda-7/Interleukin (IL)-24] has been identified as a novel anti-cancer agent, which specifically induces apoptosis in cancer cells but not in normal epithelial, endothelial and fibroblast cells. The objective of this study was to evaluate the anti-tumor effect of adenovirus-mediated mda-7/IL-24 (Ad.mda-7) gene therapy in ovarian carcinoma and further improve anti-tumor effect by enhancing infectivity of Ad.mda-7. METHODS: A panel of human ovarian carcinoma cells, OV-4, HEY, SKOV3, SKOV3.ip1 and control normal human mesothelial cells, were infected by a replication deficient recombinant adenovirus encoding mda-7/IL-24 and control virus Ad.CMV.Luc. After 72 h, apoptosis was evaluated by TUNEL and Hoechst staining and further quantified by fluorescent activated cell sorter (FACS) analysis. Infectivity of Ad.mda-7 was enhanced by retargeting it to CD40 or EGF receptors overexpressed on ovarian cancer cells. Subsequently, enhancement in apoptosis of CD40- or epidermal growth factor receptor (EGFR)-retargeted Ad.mda-7 was evaluated. RESULTS: Adenoviral-mediated delivery of mda-7 induces apoptosis ranging from 10-23% in human ovarian cancer cells tested with the highest percentage of apoptosis noted in SKOV3 cells. Minimal apoptosis was noted in normal mesothelial cells. CD40- or EGFR-retargeted Ad.mda-7 increased apoptosis by 10-32% when compared to that achieved with untargeted Ad.mda-7. CONCLUSION: Ad.mda-7 exhibits ovarian cancer-specific apoptosis, but does not affect normal human mesothelial cells. Infectivity enhanced CD40- and EGFR-retargeted Ad.mda-7 augments apoptosis induction, thus increasing the therapeutic index and translational potential of Ad.mda-7 gene therapy.     

Transductional targeting of adenoviral cancer gene therapy

Adenoviral gene therapy has shown promise in both preclinical and clinical settings, but several hurdles need to be overcome before it can reach its full therapeutic potential. One such hurdle is the need for targeting the right cell type, while avoiding liver uptake and hence side effects. This review will focus on transductional targeting strategies, in which the adenoviral particle is physically targeted to specific surface receptors expressed on the target cell. This can be achieved by using either bifunctional adaper molecules, which bind to the adenoviral particle on one side and to the targeted receptor on the other, or genetic targeting strategies. Adapter molecules comprise both chemically conjugated targeting moieties and recombinant fusion proteins, the latter having the advantage of being a homogeneous population. Genetic retargeting strategies include fiber or fiber knob chimerism, genetic incorporation of targeting ligands in the fiber or other capsid locales, or a combination of both ('complex mosaics'). Since sequestration of virions in the liver presents a major problem for the therapeutic utility of adenoviral gene therapy after systemic administration, blockade of liver uptake has become an increased area of investigation. Strategies encompass blockade of the adenovirus interaction with its cognate receptor CAR, by either using the soluble ectodomain of CAR, or ablation of CAR-interacting amino acid residues in the fiber knob. In addition, inhibition of interaction with additional adenovirus receptors, such as integrins or heparan sulphate proteoglycans, hold promise for decreasing liver uptake and hence adenoviral toxicity.     

Single-chain antibodies: A therapeutic modality for cancer gene therapy (review)

Gene therapy has rapidly evolved into a field that is treating not only inborn errors of metabolism, but other diseases associated with poor outcomes such as malignancy, where transient gene expression can be therapeutic. Cancer gene therapy is a novel form of treatment that exploits differences at the molecular level between normal and malignant cells. Current gene therapy approaches that are being evaluated include the use of replication competent viruses, mutation compensation strategies, improved targeting with tumor specific promoters, and the utilization of enhanced infectivity viruses. An additional aspect of gene therapy that has gained increased interest in the last several years is the utilization of single-chain antibodies (scFv). Specifically, scFv have been utilized to target molecular processes associated with carcinogenesis, as well as to improve gene transfer efficiency. We will limit our discussion to the role of scFv in targeting molecular processes associated with carcinogenesis.     

Production of an EGFR targeting molecule from a conditionally replicating adenovirus impairs its oncolytic potential

Oncolytic virotherapy with conditionally replicating viruses is a promising approach for treating advanced cancers. Promiscuous tropism and low tumor transduction have represented limiting issues, which targeting approaches seek to overcome. An approach utilizing a secretory targeting molecule for the epidermal growth factor pathway (sCAR-EGF) has previously been shown to be compatible with replicating adenoviruses, when an E1-deleted vector was used in a dual-virus system in conjunction with a replication-competent agent. Here, we constructed a virus that replicates in cancer cells and codes for sCAR-EGF. Interestingly, the oncolytic potency of the novel agent was not improved over nontargeted controls in vitro or in vivo. These results suggest that the expression of biologically active proteins can be counterproductive to virus replication.