Effect of TrxR1 on Bcl-2 S-nitrosylaiton, ubiquitination and protein expression and caspase signal pathway.

<p>(<b>A</b>) Time course analysis of TrxR1 expression in Hela, A375 and 7860 cells treated with ZD55-IL-24 (20 MOI). (<b>B</b>) Hela, A375 and 7860 cells were treated with ZD55-IL-24 for 24 h and then added with TrxR1 inhibitor auranofin (5 µM) for 6 h. Effect of TrxR1 inhibitor auranofin on TrxR1 protein expression was detected by Western blotting. (C) Effect of TrxR1 inhibitor auranofin on Bcl-2 protein expression in Hela, A375 and 7860 cells was detected by the western blot assay. (D) Effect of TrxR1 inhibitor auranofin on Bcl-2 S-nitrosylation was detected by immunoprecipitation using anti-Bcl-2 antibody and then followed by immunoblotting with anti-S-nitrosocysteine antibody. (E) Effect of TrxR1 inhibitor auranofin on Bcl-2 ubiquitination in Hela cells was detected by immunoprecipitation using anti-Bcl-2 antibody and then followed by immunoblotting with anti-ubiquitin antibody. (F) Effect of TrxR1 inhibitor auranofin on caspase-9, 3 and PARP was detected by Western blotting. β-actin was used as a loading control. The corresponding bands were scanned and the optical density (O.D.) was determined as the fold change versus control group. Data are means±standard deviation (S.D.) from three independent experiments (n = 3). *<i>p</i><0.05 versus ZD55-EGFP; ^#<i>p</i><0.05 versus DMSO group.</p

Effect of ZD55-IL-24 on Bcl-2 S-nitrosylation and ubiquitination.

<p>(<b>A</b>) Hela, A375 and 7860 cells in response to ZD55-IL-24 (20 MOI) were prepared for immunoprecipitation using anti-Bcl-2 antibody. The resulting immune complexes were analyzed for anti-S-nitrosocysteine by Western blotting and stood for Bcl-2 S-nitrosylation level at the different time points 12 h, 24 h, 36 h and 48 h, respectively. (<b>B</b>) Time course of Bcl-2 ubiquitination in Hela cells was detected by immunoprecipitation using anti-Bcl-2 antibody and then followed by immunoblotting with anti-ubiquitin antibody. (<b>C</b>) Effect of IL-24-siRNA (100 nM) upon IL-24, Bcl-2 expression, Bcl-2 S-nitrosylation and ubiquitination in Hela cells was detected by Western blotting and co-immunoprecipitation. β-actin was used as a loading control. The corresponding bands were scanned and the optical density (O.D.) was determined as the fold change versus control group. Data are means±standard deviation (S.D.) from three independent experiments (n = 3); *<i>p</i><0.05 versus control group; ^#<i>p</i><0.05 versus scrambled siRNA group.</p

Effect of ZD55-IL-24 on activation of caspase signal pathway and cancer cell apoptosis.

<p>(<b>A</b>) Time course analysis of caspase-9, caspase-3 and PARP in Hela cells treated with ZD55-IL-24 (20 MOI). The corresponding bands were scanned and the optical density (O.D.) was determined as the fold change versus control group. (<b>B</b>) Early apoptosis and late apoptosis in Hela cells were detected by staining cells with Annexin V-FITC (green color) and Propidium Iodide (red color) at the different time points. (<b>C</b>) Hela cells treated with ZD55-IL-24 for the different time were stained with Annexin V-FITC/Propidium Iodide (PI) and immediately analyzed by flow cytometry. Data are presented as the percentage of Annexin V positive cells from three independent experiments. *<i>p</i><0.05 versus control group.</p

Effect of NO modulators on caspase signal pathway and Hela cell viability.

<p>(A) Hela cells were pretreated with ZD55-IL24 (20 MOI) for 24 h and then added with NO donor SNP(2 mM), NO inhibitor PTIO(300 µM), SNP co-administration of reducing agent DTT(10 mM) and proteasomal inhibitor MG132(10 µM) for 6 h respectively. Procaspase-9, cleaved caspase-9, procaspase-3, cleaved caspase-3 were detected by Western blotting. (B) Effect of NO modulators SNP, PTIO, SNP+DTT and proteasome inhibitor MG132 on early apoptosis and late apoptosis in Hela cells treated with ZD55-IL-24 were detected by staining with Annexin V-FITC (green color) and Propidium Iodide (red color) respectively. (C) Hela cells were stained with Annexin V-FITC and Propidium Iodide and immediately analyzed by flow cytometry. (D) Effects of NO modulators and MG132 on Hela cell viability were determined by MTT assay. The corresponding bands were scanned and the optical density (O.D.) was determined as the fold change versus control group. Data are means±standard deviation (S.D.) from three independent experiments (n = 3). ^#<i>p</i><0.05 versus ZD55-EGFP group; *<i>p</i><0.05 versus ZD55-IL-24 group; ^@<i>p</i><0.05 versus DMSO group.</p

Schematic model of IL-24 induced cancer cell apoptosis.

<p>(<b>I</b>) IL-24 inhibits iNOS expression leading to reduction of NO turnover and attenuation of Bcl-2 S-nitrosylation. (<b>II</b>) Trx denitrosylates S-nitrosylated Bcl-2 through its dithiol moiety, thereby forming a reduced Bcl-2 and oxidized Trx; oxidized Trx is reduced (and therefore reactivated) by the seleno-flavoprotein Trx reductase(TrxR) and NADPH, suggesting that TrxR through reducing oxidized Trx may facilitate Bcl-2 denitrosylation. (<b>III</b>) Under basal condition, Bcl-2 S-nitrosylation stabilizes protein structure and resists to the ubiquitin-proteasome degradation. Formation of heterodimers with proapoptotic protein such as Bax, inhibition of cytochrome c release and caspase protease family activation, and regulation of mitochondrial transmembrane potential are some of mechanisms by which Bcl-2 exerts its anti-apoptotic effect. (<b>IV</b>) In response to IL-24, Bcl-2 S-denitrosylation via both iNOS decrease (<b>a</b>) and TrxR1 increase (<b>b</b>) facilitates Bcl-2 ubiquitination, which finally is degraded by the 26S proteasome. Bax triggers release of cytochrome c and activation of caspase protease family, which mediated the intracellular proteolysis that is characteristic of cell apoptosis.</p

Effect of iNOS on Bcl-2 S-nitrosylaiton, ubiquitination and protein expression and caspase signal pathway.

<p>(A) Time course analysis of iNOS expression in Hela, A375 and 7860 cells treated with ZD55-IL-24 (20 MOI). (B) Hela, A375 and 7860 cells were transfected with iNOS-siRNA (100 nM) for 12 h, then treated with ZD55-IL-24 for 12 h. Effect of iNOS-siRNA on iNOS expression was analyzed by Western blotting. (C) Effect of iNOS-siRNA on Bcl-2 protein expression was analyzed by Western blotting. (D) Effect of iNOS-siRNA on Bcl-2 S-nitrosylation were detected by immunoprecipitation using anti-Bcl-2 antibody and then followed by immunoblotted with anti-S-nitrosocysteine antibody. (E) Effect of iNOS-siRNA on ubiquitin-Bcl-2 was detected by immunoprecipitation using anti-Bcl-2 antibody and then followed by immunoblotting with anti-ubiquitin antibody. (F) Effect of iNOS-siRNA on caspase-9, caspase-3 and PARP in Hela cells was detected by Western blotting. The corresponding bands were scanned and the optical density (O.D.) was determined as the fold change versus control group. Data are means±standard deviation (S.D.) from three independent experiments (n = 3). *<i>p</i><0.05 versus control group; ^#<i>p</i><0.05 versus scrambled siRNA group.</p

Effect of NO modulators on Bcl-2 S-nitrosylation, ubiquitination and protein expression.

<p>(<b>A</b>) Hela, A375 and 7860 cells were pretreated with ZD55-IL-24 (20 MOI) for 24 h and then treated with NO donor SNP(2 mM), NO inhibitor PTIO(300 µM) and SNP co-administration of reducing agent DTT(10 mM) for 6 h respectively. Bcl-2 S-nitrosylation was detected by immunoprecipitation using anti-Bcl-2 antibody and then followed by immunoblotting with anti-S-nitrosocysteine antibody. (<b>B</b>) Effect of NO modulators on Bcl-2 ubiquitination in Hela cells were detected by immunoprecipitation using anti-Bcl-2 antibody and then followed by immunoblotting with anti-ubiquitin antibody. (<b>C</b>) Effect of NO modulators on Bcl-2 expression in Hela, A375 and 7860 cells was detected by western blotting using anti-Bcl-2 antibody. (<b>D</b>) Hela, A375 and 7860 cells were pretreated with ZD55-IL24 for 24 h and then treated with proteasomal inhibitor MG132 (10 µM) for 6 h. Bcl-2 expression was analyzed by Western blotting using anti-Bcl-2 antibody. (<b>E</b>) Bcl-2 phosphorylation in Hela cells was detected by Western blotting with anti-p-Bcl-2(Ser87) antibody. β-actin was used as a loading control. The corresponding bands were scanned and the intensities were determined by optical density (O.D) measurements. Data are means±standard deviation (S.D.) from three independent experiments (n = 3). ^#<i>p</i><0.05 versus ZD55-EGFP; *<i>p</i><0.05 versus ZD55-IL-24 group; ^@<i>p</i><0.05 versus DMSO group.</p

Effect of different titers of ZD55-IL24 on Bcl-2 expression and Hela cells viability.

<p>(<b>A</b>) Effects of the different titers of ZD55-IL-24 (0.1 MOI, 1 MOI, 5 MOI, 10 MOI, 20 MOI) on Bcl-2 expression induced by Western blotting 48 h after infection of ZD22-IL24. β-actin was used as a loading control. (<b>B</b>) ZD55-EGFP at the different titers was used to detect infection efficiency of replicative adenovirus (Magnification×200). (<b>C</b>) Hela cell viability treated with the different titers of ZD55-IL-24 was determined by MTT assay. Data are means±standard deviation (S.D.) from three independent experiments (n = 3); *<i>p</i><0.05 versus control group.</p