Cells and Reagents

The human Henrietta Lacks (Hela) cell line, the human malignant melanoma cancer cell line (A375) and the human renal carcinoma cell line (7860) were obtained from Shanghai Cell Collection (Shanghai, China). Hela and A375 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (GIBCO BRL, Grand Island, NY) containing 5% fetal bovine serum (FBS, GIBCO-BRL), 2 mM L-glutamine, and 100 units/ml penicillin/streptomycin and a 5% CO2 environment at 37°C. The renal cancer 7860 cells were cultured in RPMI1640 medium, supplemented with 5% FBS and antibiotics. Sodium Nitroprusside (SNP), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (PTIO), dithiothreitol (DTT), Dimethyl sulfoxide (DMSO) and Z-Leu-Leu-Leu-al (MG132) were purchased from Sigma-Aldrich Co (St. Louis, MO), Antibodies for Bcl-2, phospho-Bcl-2(Ser87), NOS2(iNOS), protein A-agarose, and IL-24-siRNA, iNOS-siRNA and scrambled control-siRNA were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies for ubiquitin and S-nitrosocysteine were from Sigma-Aldrich Co (St. Louis, MO). Antibodies for β-actin, procaspase-3, cleaved caspase-3, procaspase-9, cleaved caspase-9 antibodies, anti-poly ADP-ribose polymerase (PARP) and cleaved PARP were purchased from Cell Signaling Technology, Inc. (Danvers, MA).

Virus construction and production

pZD55, the E1B 55-kDa-deleted oncolytic adenovirus construction plasmid; pCA13, the E1-deleted adenovirus shuttle plasmid; and ZD55-enhanced green fluorescent protein (EGFP) with reporter gene EGFP were kindly provided by Professor Liu (Xin-Yuan Liu, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China). IL-24 was first cloned into pCA13 to form pCA13-IL-24. Then IL-24 excised from pCA13 was subcloned into pZD55 to construct pZD55-IL-24. Oncolytic adenovirus, ZD55-IL-24 was generated in HEK293 cells (Shanghai Cell Collection, Shanghai, China) by homologous recombination between pZD55-IL-24 and the adenovirus packaging plasmid pBHGE3 (Microbix Biosystems), respectively. Large scale purification of all adenovirus particles was performed by ultracentrifugation with cesium chloride according to standard techniques. The titers were determined using a plaque assay on HEK293 cells.

Small interfering RNA assay

For experiments involving IL-24 specific siRNAs, cells (3×10⁵ per well) were plated in 6-well plates. At 24 h after incubation, Hela cells were washed, replenished with fresh medium and treated with ZD55-IL-24(20 MOI) for 12 h, cells were subsequently washed and placed in fresh culture medium and added with IL-24 specific siRNA (100 nM) by using a siRNA transfection reagent. After 24 h of siRNA transfection, cell lysates were prepared and subjected to the western blot analysis as described below.

For experiments involving iNOS-siRNAs, cells were transfected with 100 nM iNOS-specific siRNA for 12 h, cells were subsequently washed and placed in fresh culture medium and then treated with ZD-55-IL-24(20 MOI) for 12 h. After 24 h of siRNA transfection, cell lysates were prepared and subjected to the western blot analysis as described below.

Western Blotting

After specific treatments, cells were incubated in lysis buffer containing 20 mmol/L Tris-HCl (pH 7.5), 1% Triton X-100, 150 mmol/L NaCl, 10% glycerol, 1 mmol/L Na₃VO₄, 50 mmol/L NaF, 100 mmol/L phenylmethylsulfonyl fluoride, and a commercial protease inhibitor mixture (Roche Molecular Biochemicals) for 20 minutes on ice. After insoluble debris was pelleted by centrifugation at 14,000 g for 15 minutes at 4°C, the supernatants were collected and determined for protein content using the Bradford method (Bio-Rad Laboratories, Hercules, CA). Proteins (80 µg) were resolved under denaturing conditions by SDS-PAGE (10%) and transferred onto nitrocellulose membranes. After blocking for 2 h in phosphate-buffered saline with 0.1% Tween20 (PBST) and 3% bovine serum albumin (BSA), membranes were incubated overnight at 4°C with the appropriate primary antibody in PBST containing 3% BSA. Membranes were then washed and incubated with alkaline phosphatase conjugated goat anti-rabbit IgG or anti-mouse IgG (Sigma, 1∶10 000) in PBST for 2 h and developed using NBT/BCIP color substrate (Promega, Madison, USA).

Immunoprecipitation

For immunoprecipitation, cytosolic fractions (each containing 400 µg of proteins) were diluted four-fold with HEPES buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, 1% Triton X-100, and 1 mM each of EGTA, EDTA, PMSF and Na₃VO₄. The samples were then pre-incubated for 1 h with 20 µl protein A agarose and centrifuged to remove any non-specifically adhered proteins from the protein A agarose. The supernatant was then incubated with 2 µg specific antibodies overnight at 4°C. After the addition of Protein A agarose, the mixture was incubated at 4°C for an additional 2 h. Samples were triple washed with HEPES buffer and eluted by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer then boiled at 100°C for 5 minutes. Immune complexes were separated by 10% SDS-PAGE and analyzed by Western blotting as described above.

Measurement of apoptosis by Annexin V analysis

An Annexin V-binding assay was used according to the manufacturer's instructions. Briefly, cells (3×10⁵ per well) in 6-well plates were treated with ZD55-IL24 (20 MOI) for the different time 12 h, 24 h, 36 h, 48 h, and 72 h. Cells were collected and the Annexin V-FITC and Propidium iodide (PI) dual-staining assay was performed according to the manufacturer's instructions ((Nanjing Keygen Biotech, China). Collected cells were briefly washed with ice-cold phosphate-buffered saline (PBS) twice and resuspended in 200 µl 1×binding buffer containing 5 µl Annexin V-FITC for 15 minutes and then in 300 µl 1×binding buffer containing 5 µl Propidium iodide (PI) for 5 minutes at room temperature in the dark. After incubation, the cells were analyzed using a FACStar flow cytometer.

Cell viability assay

The carcinoma cells (1.0×10⁴ per well) were incubated in triplicate in a 96-well plate and treated with ZD55-IL-24 and NO modulators. Cell survival rate was evaluated by a standard 3-(4, 5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, St. Louis, MO) in the presence or absence of the indicated test samples in a final volume of 0.2 ml for various lengths of time at 37°C. Thereafter, 20 µl MTT solution (5 mg/ml in PBS) was then added to each well. After 4 h incubation at 37°C, 150 µl DMSO was added. Finally the plates were shaken and the optical density at 570 nm was measured on ELX-800 spectrometer reader (Bio-Tek Instruments Inc., USA). Four replicate wells were tested per assay and each experiment was repeated three times. Percent cell viability was calculated as the ratio of the experimental samples to the control samples×100.

Statistical analysis

Values are expressed as mean ±SD. Statistical analysis of the results was carried out using the Student's t-test or one-way analysis of the variance (ANOVA) followed by the Duncan's new multiple range method or Newman-Keuls test. P-values<0.05 were considered significant.

Effect of ZD55-IL-24 on Bcl-2 expression and cancer cell viability

Protein expressions of IL-24 and E1A accompanied with adenovirus ZD55-IL-24 replication and translation in Hela, A375 and 7860 cells were detected by Western blotting at the different time points. Our data showed a distinct increase of IL-24 from 24 h to 72 h compared to controls, as shown in Fig. 1A. At the same time, E1A protein standing for replication ability of ZD55-IL-24 showed the obvious enhancement from 12 h to 72 h in Fig. 1B, which is similar to the time course of enhanced green fluorescence protein (EGFP) expression treated with ZD55-EGFP in Fig. 1D. In addition, Bcl-2 expression has the inverse decrease from 24 h to 72 h (Fig. 1C). Moreover, the Bcl-2 decline in response to ZD55-IL-24 was shown in a dose-dependent manner. The efficient titers of ZD55-IL-24 to inhibit the Bcl-2 expression are 10 and 20 MOI, as shown in Fig. 2A. To further investigate whether ZD55-IL24 affects three carcinoma cells survival, cell viability was determined by MTT assay (Fig. 1E). Our results showed that ZD55-IL-24 effectively decreased cell survival and this inhibition was also shown in a dose-dependent manner (Fig. 1E and 2C). Taken together, these results indicate the ZD55-IL24 could mediate a high-level and stable IL-24 expression from 24 h to 72 h and reduce Bcl-2 protein level in time- and dose-dependent manner.

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

To investigate whether ZD55-IL-24 would contribute to alteration of Bcl-2 S-nitrosylation and ubiquitination, we detected Bcl-2 S-nitrosylation and ubiquitination level in Hela, A375 and 7860 cells. The results showed that ZD55-IL-24 in Hela cells diminished the Bcl-2 S-nitrosylation from 79% at 24 h to 38% at 48 h compared with the control group (Fig. 3A). In contrast, Bcl-2 ubiquitination increased from 1.4 fold at 24 h to 2.3 fold at 48 h, as shown in Fig. 3B. The results from A375 and 7860 cells were consistent with the above trend. To further confirm the potential role of IL-24 in the regulation of Bcl-2 S-nitrosylation and ubiquitination, specific IL-24-siRNA was used to knockdown IL-24 expression. Our data indicated IL-24-siRNA obviously restored Bcl-2 S-nitrosylation and thus suppressed Bcl-2 ubiquitination compared with scrambled control siRNA (Fig. 3C). Consequently, ZD55-IL-24 induced decreased Bcl-2 S-nitrosylation and increased Bcl-2 ubiquitination.

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

To further determine whether the Bcl-2 aberrant diminishment in response to ZD55-IL-24 would result in the activation of caspase signal pathway in Hela cells, caspase-9, caspase-3 and PARP were detected at the different time points 12 h, 24 h, 36 h and 48 h respectively, as shown in Fig. 4A. The results demonstrated that procaspase-9 gradually decreased from 76% at 24 h to 36% at 48 h. In contrast, the cleaved caspase-9 correspondingly increased from 1.5- at 24 h to 2.5-fold at 48 h compared with the control group. Caspase-3 and PARP performed the similar alteration like caspase-9. Our results show that ZD55-IL-24 induced the cleavage of caspase-9, caspase-3 and PARP to activate the caspase signal pathway. In addition, Hela cells apoptosis was detected by Annexin V/PI and then analyzed using flow cytometry (Fig. 4B and C). The results indicated that ZD55-IL-24 dramatically enhanced apoptosis in Hela cells from 2.3- at 24 h to 7.4-fold at 72 h compared with the control group. Taken together, ZD55-IL-24 initiated the caspase signal pathway activation and cancer cell apoptosis.

Effect of Bcl-2 S-nitrosylation alteration on the regulation of its ubiquitination in response to ZD55-IL-24

To investigate whether ZD55-IL-24 could regulate Bcl-2 S-nitrosylation via NO, which subsequently affects its ubiquitination, we treated Hela, A375 and 7860 cells with NO donor SNP, NO inhibitor PTIO and SNP co-administration of the reducing agent DTT after administration of ZD55-IL24, respectively. As shown in Fig. 5A, exogenous NO donor SNP effectively increased the Bcl-2 S-nitrosylation whereas this rescue effect was negated by co-administration with DTT. At the same time, NO inhibitor PTIO significantly reduced Bcl-2 S-nitrosylation. Conversely, NO donor SNP inhibited the ubiquitination of Bcl-2, but DTT co-treatment counteracted the effects of SNP. Moreover, NO inhibitor PTIO facilitated the ubiquitination of Bcl-2 in Fig. 5B. Fig. 5C shows ZD55-IL-24 treatment for 24 h caused a significant decrease of Bcl-2 expression, which was further decreased upon the addition of NO inhibitor PTIO. In contrast, treatment with NO donor SNP significantly resisted ZD55-IL-24-induced Bcl-2 down-regulation. Similarly, co-administration with DTT offset the Bcl-2 enhancement induced by SNP.

To further detect whether ZD55-IL-24-induced Bcl-2 degradation is ubiquitin-proteasome dependent, the proteasome inhibitor MG132 was use to block the proteasomal degradation in Hela, A375 and 7860 cells. Our results showed that MG132 significantly improved Bcl-2 expression (Fig. 5D) compared with vehicle control DMSO group, implicating that the ubiquitin-proteasome system was involved with Bcl-2 degradation induced by ZD55-IL-24. In summary, these results suggested that ZD55-IL-24 through its ability to regulate NO, may interfere with the Bcl-2 S-nitrosylation, promote its ubiquitination process and finally induce Bcl-2 proteasome dependent degradation. Previous evidence indicates that Bcl-2 phosphorylation induces a conformational change in Bcl-2, which controls its stability and apoptotic function. Dephosphorylation at Ser⁸⁷ is the critical step of Bcl-2 degradation. To test whether NO-mediated S-nitrosylation would take part in the regulation of phosphorylation of Bcl-2 and therefore influence its ubiquitination and stability, we detected the effect of these NO modulator on Bcl-2 phosphorylation through Western blotting using specific phosphor-Bcl-2 (Ser⁸⁷) antibody. The results showed that NO donors and inhibitors had no significant effect on the Bcl-2 phosphorylation level (Fig. 5E), suggesting that ZD55-IL-24 regulates Bcl-2 degradation via Bcl-2 S-nitrosylation/denitrosylation and ubiquitination in a phosphorylation-independent manner.

Effect of Bcl-2 S-nitrosylation alteration on activation of caspase signal pathway and cancer cell apoptosis in response to ZD55-IL-24

Bcl-2, which is located on the outer mitochondrial membrane, is important for the suppression of mitochondrial manifestations of apoptosis. We examined whether alteration of Bcl-2 S-nitrosylation and ubiquitination in response to ZD55-IL-24 would subsequently contribute to activation of caspase-dependent pathway. Hela cells were treated with ZD55-IL-24 for 24 h and then added with NO donor SNP, NO inhibitor PTIO and SNP co-administration of DTT, respectively. Our data demonstrated that NO donor SNP attenuated the cleavage of caspase-9 and caspase-3. In contrast, NO inhibitor PTIO had the opposite effect and aggravated the activation of caspase-9 and caspase-3, as shown in Fig. 6A. Additionally, SNP inhibitor DTT co-administration reversed SNP protection of caspase-9 and caspase-3 from activation. It was shown that activation of caspase-9, caspase-3 was regulated by Bcl-2 S-denitrosylation induced by ZD55-IL-24. Moreover, we further assessed whether these NO modulators would influence apoptosis in Hela cells treated with ZD55-IL-24. Flow cytometric analysis of Annexin V/PI double stained cells was used to quantitatively assess apoptosis. Our data showed that incubation of ZD55-IL-24 for 48 h dramatically led to apoptosis increase in Hela cells, which was attenuated by NO donor SNP but aggravated by NO inhibitor PTIO. At the same time, SNP inhibitor DTT co-administration reversed the apoptosis inhibition of SNP. Moreover, proteasome inhibitor MG132 significantly alleviated apoptosis in Hela cells treated with ZD55-IL-24, indicating that inhibition of Bcl-2 degradation plays an important role in protection from caspase proteins cleavage and apoptosis in Hela cells (Fig. 6B and C). In addition, our results showed that NO donor SNP enhanced Hela cell survival. However, NO inhibitor PTIO or SNP co-administration with DTT diminished Hela cell survival, as shown in Fig. 6D. Taken together, these results suggested that alteration of Bcl-2 S-nitrosylation via NO modulators regulated Hela cell apoptosis in response to ZD55-IL-24.

Effect of iNOS on Bcl-2 S-denitrosylation and ubiquitination in response to ZD55-IL-24

To explore whether Bcl-2 S-denitrosylation in response to ZD55-IL-24 was associated with iNOS-mediated S-nitrosylation, we detected the iNOS expression in Hela, A375 and 7860 cells, as shown in Fig. 7A. Our data showed that ZD55-IL-24 palpably and gradually diminished iNOS expression from 62% at 24 h to 36% at 48 h (Fig. 7A) in Hela cells, which is in agreement with the results from A375 and 7860 cells. In addition, specific iNOS-siRNA knocked down iNOS protein level (Fig. 7B), attenuated Bcl-2 S-nitrosylation level (Fig. 7D), subsequently enhanced Bcl-2 ubiquitination (Fig. 7E) and ultimately decreased Bcl-2 protein level (Fig. 7C), compared with the control siRNA, suggesting that iNOS was involved with Bcl-2 S-denitrosylation in response to ZD55-IL-24. Moreover, iNOS-siRNA also promoted activation of caspase signal pathway via Bcl-2 denitrosylation, as shown in Fig. 7F.

Effect of TrxR1 on Bcl-2 S-denitrosylation and ubiquitination in response to ZD55-IL-24

Like phosphorylation and dephosphorylation, S-nitrosylation is reversible biological process. The extent of protein S-nitrosylation depends on the rate of both S-nitrosylation and denitrosylation. To explore whether the denitrosylation enzyme TrxR1 was implicated in Bcl-2 S-denitrosylation in response to ZD55-IL-24, we detected the time course of TrxR1 expression in Hela, A375 and 7860 cells, as shown in Fig. 8A. Our data showed that ZD55-IL-24 improved TrxR1 expression from 1.8-fold at 24 h after infection of ZD55-IL-24 to 2.1-fold at 48 h compared with the control group in Hela cells, which is consistent with results from A375 and 7860 cells. In order to further detect the role of TrxR1 in IL-24-coupled Bcl-2 S-denitrosylation, we treated these three cancer cells with TrxR1 inhibitor auranofin to detect the alterations of TrxR1 expression, Bcl-2 expression, Bcl-2 S-nitrosylation, and Bcl-2 ubiquitination. Fig. 8B–E showed that auranofin obviously diminished TrxR1 expression, restored Bcl-2 S-nitrosylation, down-regulated Bcl-2 ubiquitination, and enhanced Bcl-2 expression compared with vehicle control DMSO group. Additionally, auranofin also protected the caspase-9, caspase-3 and PARP from cleavage to activate caspase signal pathway, as shown in Fig. 8F. Taken together, these results suggest that ZD55-IL-24 induces Bcl-2 S-denitrosylation via regulation of TrxR1 and iNOS. Moreover, Bcl-2 S-denitrosylation facilitates its ubiquitin-proteasome degradation, then initiates the activation of caspase signal pathway and finally results in cancer cell apoptosis.