Sensitisation to mitoxantrone-induced apoptosis by the oncolytic adenovirus Ad？？ through Bcl-2-dependent attenuation of autophagy

a Sensitisation to mitoxantrone by Ad5wt, Ad？？ and AdE1A12S in PC3 and 22Rv1 cells. Dose？response curves to mitoxantrone with and without fixed doses of virus killing 10–40% of cells alone; Ad5wt or AdΔΔ (1000？ppc; PC3 and 25？ppc;22Rv1) or AdE1A12S (5000？ppc;PC3and 100？ppc;22Rv1). Cell viability determined by MTS assays 5 (PC3) or 3 days (22Rv1) after treatment and data analysed by unpaired t-test, averages？±？SEM, n？≥？3, **p？<？0.01, *p？<？0.05. Ratios？=？EC50-values of combination/EC50-values mitoxantrone. b PC3 and 22Rv1 cells infected with Ad？？ or AdE1A12S and treated with mitoxantrone for 5 and 3 days, respectively, at three constant ratios (0.5, 2.5 and 12.5 ppc/nm; indicated by wedges) and PC3M cells at two constant ratios (2.5 and 12.5？ppc/nm). Combination indexes (CI) calculated from isobolograms and synergistic cell killing defined as CI？≤？0.9; averages？±？SD, n？=？3, *p？<？0.05 compared to the theoretical additive values (dashed lines; additive effects 0.9？<？CI？<？1.1). c PC3 cells (left panel) treated with mitoxantrone (225, 450 and 900？nm) and/or infected with Adwt and AdΔΔ at 500ppc or Ad12S at 5000ppc. Cell death indirectly determined using the MTS viability assay 5 days after treatment. One-way ANOVA with Tukey？Kramer post-test, averages？±？SEM, n？=？4. 22Rv1 cells (right panel) treated with mitoxantrone (3, 10 and 25？nm) and/or infected with Ad5wt and AdΔΔ at 15？ppc or Ad12S at 50？ppc. Number of dead cells determined by the Trypan blue exclusion assay after 3 days. Representative study, one-way ANOVA with Tukey？Kramer post-test, averages？±？SEM from quadruplicate samples. *p？<？0.05, ***p？<？0.001, ****p？<？0.0001, *compared to the theoretical additive value of mitoxantrone and virus, n？≥？3. d Expression of PARP detected by immunoblotting (cleaved PARP indicated with black arrows; 89？kDa), one representative immunoblot, n？=？3. PC3 cells (left panel) infected with AdΔΔ (500？ppc) or AdE1A12S (5000？ppc) and/or mitoxantrone (225, 450 or 900？nm). 22Rv1 cells (right panel) infected with AdΔΔ (15？ppc) or AdE1A12S (50？ppc) and/or mitoxantrone (3, 10 or 25？nm). Cells were lysed 48？h after treatment. Lower panels: PARP ratios; cleaved PARP (clPARP/PARP) after quantification by densitometry and normalised to the actin loading control, expressed as fold-change relative to the untreated control, n？=？3. Molecular weight markers indicated in kDa. e PC3 cells analysed at the indicated time points for mitochondrial depolarisation by loss of TMRE staining using flow cytometry (left panel) and Annexin V staining (right panel) 120？h after infection with AdΔΔ (500？ppc)