Potent cell killing of prostate cancer cell lines by replicating E1A-deletion mutants in combination with mitoxantrone.

<p>A) The replicating viruses used in the study had intact E1A-region (E1A13S) except for the indicated deletions. The replication-defective mutants were based on the E1A12S construct with the same deletions as in the replicating viruses; AdE1A1102 (Δ26–35), AdE1A1104 (Δ48–60), AdE1A1108 (Δ124–127), in addition to deletion of the CR3-region, responsible for viral transcriptional activity. B) EC₅₀ values for the replicating mutants were determined from dose-response curves and presented as averages ± SD, n = 3. Significantly different values compared to Ad5 are indicated. C) Sensitization of the human PC-3, 22Rv1 and DU145 cells to mitoxantrone by fixed doses of each virus at EC₁₀ and EC₂₅. Data presented as percentages of mitoxantrone EC₅₀ values in each cell line, averages ± SD, n = 3. Statistical analysis by 1-way Anova, *p<0.05 for drug EC₅₀ values that were significantly lower than the corresponding Ad5 values. The <i>dl</i>312 (ΔE1A) non-replicating virus served as negative control. D) Graphic representation of combination indexes (CI) generated from synergy studies with mitoxantrone in combination with each replicating viral mutant at two constant ratios 0.5 and 2.5 viral particles per cell (ppc)/nM drug. Synergistic interactions are represented by CI≤0.9, antagonism by CI≥1.1 and additive effects by 0.9</p

All replication-defective E1A12S mutants sensitise prostate cancer cells to mitoxantrone and docetaxel except the AdE1A1104 virus.

<p>A) Drug dose responses in each cell line were evaluated after infection with AdE1A12S, AdE1A1102, AdE1A1104, and AdE1A1108 mutants with AdGFP as negative control to determine changes in drug EC₅₀ values. All cell lines were infected at doses killing <10% of cells alone; PC-3 cells at 100 ppc (left panel), DU145 cells at 10 ppc (mid panel) and 22Rv1 cells at 2.5 ppc (right panel). Data represent averages ±SD, n = 4–5 independent experiments analysed by t-test comparing EC₅₀ values for each combination to that of drug alone, expressed as percentages, *p<0.05 and °p<0.01. B) EC₅₀ values for mitoxantrone were determined with and without simultaneous infection with viral mutants at 2.5, 10 and 100 ppc for 22Rv1, DU145 and PC-3 respectively, and with (grey bar) and without (black bar) the addition of the pan-caspase inhibitor zVAD-fmk at 25 µM. EC₅₀ values are expressed as percentages of mitoxantrone alone (Ctrl), averages ± SD, n = 3. C) Flow cytometry of cells infected with the AdE1A12S mutants or treated with mitoxantrone (50 nM) alone and in combination and analysed for tetramethylrhodamine uptake (TMRE) as an indicator of mitochondrial depolarisation and apoptosis induction. AdE1A12S, AdE1A1102, AdE1A1104, AdE1A1108, and AdGFP alone (solid arrow; all cell lines) and AdE1A12S, AdE1A1102, AdE1A1104 and AdE1A1108 in combination with mitoxantrone (dashed arrow; 22Rv1 cells). Data expressed as % apoptotic cells; percentages of cells that showed mitochondrial depolarisation, averages ± SD, n = 3. D) Cells infected with each mutant at 100 (PC-3) or 2.5 ppc (22Rv), mock infected and treated with or without mitoxantrone at 50 nM. Changes in cell cycle were analysed by flow cytometry at 24, 48 and 72 h after infection and drug treatment in PC-3 cells or after 48 h for 22Rv1 cells, one representative study (n = 3), *p<0.05 comparing G2/M-phase in combination treated vs mitoxantrone alone, °p<0.05 G2/M-phase for mitoxantrone vs mock treated.</p

The dl1102 mutant prolongs time to progression in combination with docetaxel in PC-3 xenografts <i>in vivo.</i>

<p>A) Animals with PC-3 subcutaneous tumor xenografts were treated with the <i>dl</i>1102 (filled triangle) or <i>dl</i>1104 (filled circle) mutants or mock treated with <i>dl</i>312 (filled square) at 1×10⁹ vp (i.t. injections on day 1, 3, and 5) with and without docetaxel at 10 mg/kg (D10; i.p. administration on day 2 and 8, open squares), and tumor growth was monitored. *p<0.05, treatments compared with mock and single-agent treatments (one-way ANOVA), p<0.05 for <i>dl</i>1102 alone compared to mock, n = 6. B). In a second study animals with PC-3 subcutaneous tumor xenografts were treated as above with the indicated suboptimal doses of mutants at 1×10⁹ vp and docetaxel at 10 mg/kg (D10) or the respective combinations. Median time to tumor progression (tumor volume >500 µl) was determined by Kaplan-Meier survival analysis (8–10 animals per group). *p<0.05, combination-treated compared with docetaxel. C) PC-3 cells infected with the indicated mutants and treated with docetaxel at four constant ratios; 0.5, 2.5, 12.5 and 62.5 ppc/nM drug (indicated by the wedges). CI values were calculated from isobolograms and CI≤0.9 were considered synergistic, averages ±SEM, n = 3, *p<0.05 vs the theoretical additive values (0.9</p

Synergistic cell killing with a replication-defective virus expressing the small AdE1A12S protein, in combination with cytotoxic drugs.

<p>A) Isobolograms generated from EC₅₀ values for combinations of the AdE1A12S mutant with mitoxantrone (Mit) or docetaxel (Doc) at four constant ratios (0.5. 2.5, 12.5 and 62.5 ppc/nM drug) in PC-3 and DU145 cells. The straight lines represent the theoretical values for additive effects and points below the line synergistic cell killing, one representative study (n = 3–4). B) Characterization of replication of the AdE1A12S, AdE1A1102, AdE1A1108 and AdE1A1104 mutants in PC-3, DU145 and 22Rv1 cells. Levels of viral replication determined by the limiting dilution assay (TCID₅₀) for replicating and replication-defective mutants with identical E1A-deletions except for the additional deletion of the CR3-domain in E1A12S. Cells were infected with each mutant at 100 ppc and harvested 72 h later, averages ±SD, n≥3. The non-replicating AdGFP mutant was used as a control in all assays, *p<0.001 for the replicating compared to the corresponding replication-defective mutant (t-test). C) qPCR analysis of cells infected as described for the replication assays and harvested 24, 48 and 72 h later. Total copy number at each time point was normalised to the copy numbers detected 3 h after infection in 10 ng of total DNA, averages ± SEM, n = 2–3. D) Viral replication in normal human primary bronchial epithelial cells (NHBE) determined by TCID₅₀ for Ad5wt, <i>dl</i>1102 and <i>dl</i>1104 mutants infected at 100 ppc, n = 3, *p<0.005.</p

EC₅₀ values for mitoxantrone and docetaxel in prostate cancer cells transfected with E1A12S or GFP expressing plasmids.

<p>Data from one representative experiment treated with mitoxantrone for 3 days after transfection with pcDNA plasmids expressing the respective proteins, n = 3.</p

EC₅₀ values (ppc) for the replication-defective AdE1A12S, AdE1A1102, AdE1A1104 and AdE1A1108 viral mutants and wild type virus (Ad5).

<p>Data are averages ± SD, n = 3, p<0.001 for all mutants vs Ad5 (t-test). The non-replicating <i>dl</i>312 and AdGFP control viruses had EC₅₀ values >1×10⁵ ppc.</p