Inhibition of N‐myc expression sensitizes human neuroblastoma IMR‐32 cells expressing caspase‐8 to TRAIL

Abstract Objectives This study aims to explore the roles of N‐myc and caspase‐8 in TRAIL‐resistant IMR‐32 cells which exhibit MYCN oncogene amplification and lack caspase‐8 expression. Materials and methods We established N‐myc–downregulated IMR‐32 cells using shRNA lentiviral particles targeting N‐myc and examined the effect the N‐myc inhibition on TRAIL susceptibility in human neuroblastoma IMR‐32 cells expressing caspase‐8. Results Cisplatin treatment in IMR‐32 cells increased the expression of death receptor 5 (DR5; TRAIL‐R2), but not other receptors, via downregulation of NF‐κB activity. However, the cisplatin‐mediated increase in DR5 failed to induce cell death following TRAIL treatment. Furthermore, interferon (IFN)‐γ pretreatment increased caspase‐8 expression in IMR‐32 cells, but cisplatin failed to trigger TRAIL cytotoxicity. We downregulated N‐myc expression in IMR‐32 cells using N‐myc–targeting shRNA. These cells showed decreased growth rate and Bcl‐2 expression accompanied by a mild collapse in the mitochondrial membrane potential as compared with those treated with scrambled shRNA. TRAIL treatment in N‐myc–negative cells expressing caspase‐8 following IFN‐γ treatment significantly triggered apoptotic cell death. Concurrent treatment with cisplatin enhanced TRAIL‐mediated cytotoxicity, which was abrogated by an additional pretreatment with DR5:Fc chimera protein. Conclusions N‐myc and caspase‐8 expressions are involved in TRAIL susceptibility in IMR‐32 cells, and the combination of treatment with cisplatin and TRAIL may serve as a promising strategy for the development of therapeutics against neuroblastoma that is controlled by N‐myc and caspase‐8 expression.


| INTRODUC TI ON
Neuroblastoma is the most common extracranial solid tumour of childhood and accounts for 15% of cancer-related deaths. 1,2 Despite recent advances in cancer treatment, aggressive neuroblastomas remain refractory to current therapy; the overall 5-year survival rate for patients with advanced-stage neuroblastoma is 30%-40%. 2 Amplification of MYCN oncogene is observed in approximately 20% of neuroblastomas and 45% of high-risk cases. 3 MYCN amplification is strongly associated with poor outcome 2,4 and has been considered as the most important prognostic factor, 5 which strongly correlated with advanced-stage disease and treatment failure. The deregulation of MYCN oncogene that regulates the expression of genes involved in several processes, including cell cycle, 6,7 proliferation, 8,9 differentiation 10,11 and apoptosis, 6,8,10 is sufficient to drive the transformation of neural crest progenitor cells into neuroblastoma.
Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), also known as the Apo-2 ligand, is a member of TNF ligand superfamily that selectively induces apoptosis in a wide variety of transformed cell lines from diverse tissue types. 12 TRAIL may induce apoptosis through its interaction with two of four membrane-bound receptors, namely death receptor 4 (DR4; TRAIL-R1) and DR5 (TRAIL-R2). These receptors bear a protein-protein interaction motif termed as the "death domain (DD)". 13,14 The other two receptors, decoy receptor 1 (DcR1; TRAIL-R3) and DcR2 (TRAIL-R4), either lack the cytoplasmic or truncated DD. TRAIL induces receptor trimerization and conformational change in the intracellular DD, resulting in the recruitment of Fas-associated DD. 15 This signals death through the formation of a death-inducing signal complex, which rapidly activates caspase-8.
Caspase-8 mediates apoptosis either through the direct activation of the downstream effector caspases or by the cleavage of pro-apoptotic molecules such as B-cell lymphoma 2 (Bcl-2) homolog, Bid. 16,17 Studies have shown that anti-cancer drugs such as bortezomib, 18,19 etoposide 20 and doxorubicin 21 sensitized cancer cells to TRAIL-mediated death through the upregulation of DR expression. In particular, the upregulation of DRs by cisplatin affected TRAIL-induced apoptosis in many cancer types, such as squamous carcinoma, 22 hepatocellular carcinoma 23 and colon cancer. 24 The mechanism underlying the upregulation of TRAIL receptors is variable. The activation or inhibition of nuclear factor kappa B (NF-κB) 20,25 and/or extracellular signal-regulated kinase (ERK) 1/2 26,27 may upregulate both DR4 and DR5, while p53 may mediate the upregulation of DR5 at transcriptional levels. 28 In addition, chemotherapeutic agents may mediate the changes in the rate of receptor turnover at cell surface. 29,30 In this study, we investigated whether cisplatin treatment triggers TRAIL-mediated cytotoxicity in TRAIL-resistant IMR-32 neuroblastoma cells which exhibit amplification of MYCN oncogene and lack caspase-8 expression. Our data, for the first time, show that TRAIL susceptibility correlated with the expression levels of N-myc and caspase-8 in human neuroblastoma IMR-32 cells. The combination therapy of cisplatin and TRAIL is a promising strategy for treating neuroblastoma that is controlled by the expression of N-myc and caspase-8, and its use may provide important information for the development of additional potential therapeutic strategies to fight neuroblastoma. Then, 3 hours before the end of the incubation, cells were treated with 11 μL of 1× Alamar Blue®, and their absorbance was measured at 570 and 600 nm wavelength with an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, Sunnyvale, CA).

| Measurement of human NF-κB (p50/ p65) activity
The DNA-binding activity of NF-κB in cells was quantified by ELISA using NF-κB p50 and p65 transcription factor assay kit (Cayman, Ann Arbor, MI) according to the manufacturer's instructions. Details of the measurement of NF-κB activity are described in supplementary methods.

| Immunoblotting
Cells were washed with cold PBS and lysed in cold radioimmuno-

| Nuclear staining and phasecontrast microscopy
Cells were treated with IFN-γ, cisplatin and TRAIL, either separately or in different combinations. After treatment, cells were incubated with Hoechst 33258 at a final concentration of 5 μg/mL in HEPES buffer (Life Technologies-Gibco) for 20 minutes at 37°C in a 5% CO 2 incubator. Cells were resuspended in PBS and were examined with a Nikon Eclipse TE2000-U inverted fluorescence microscope equipped with a Nikon LH-M100C-1 camera (Nikon Corporation Instruments Company, Japan).

| Establishment of N-myc-downregulated IMR-32 cells
For the transduction of IMR-32 cells with N-myc-downregulating lentiviral particles, cells were pretreated with 5 μg/mL polybrene, prepared in DMEM containing 10% FBS and incubated with the lentivirus at a multiplicity of infection (MOI) of 5. After incubating for 24 hours at 37°C with 5% CO 2 , cells were washed twice with PBS and fresh medium was added. Infected cells were selected after 10 days of incubation in the medium containing 5 μg/mL puromycin, after 2 weeks of transduction. The protein level of N-myc was confirmed by Western blot analysis.

| Phase-contrast microscopy and cell counting
Viable cells were incubated at 37°C with 5% CO 2 , and the medium was changed every 3-4 days. Cells were observed on days 3, 7 and 14, using an inverted microscope (Olympus CK40, Melville, NY).
Collected cells were resuspended in PBS and stained with trypan blue. The total number of viable cells was determined using a haemocytometer (Marienfeld, Germany).

| Measurement of mitochondrial membrane potential: TMRE staining
The mitochondrial membrane potential (MMP, Ψm) was measured with the fluorescent dye TMRE. Cells were incubated with TMRE at a final concentration of 50 nmol/L in HEPES buffer for 20 minutes at 37°C in a 5% CO 2 incubator. Cells were then washed with PBS and examined using a Nikon Eclipse TE2000-U inverted fluorescence microscope equipped with a Nikon LH-M100C-1 camera.

| Statistical analysis
All data were expressed as mean ± standard deviation (SD). Student's t test was used for statistical comparison between groups. A value of P < 0.05 was considered statistically significant.  Figure 1F). We also established NF-κB-downregulated IMR-32 cells using shRNA-targeting NF-κB p65 and observed that these cells showed increased expression of DR5, which was unaffected by cisplatin treatment ( Figure S2). These results indicated that cisplatin treatment increased DR5 expression in IMR-32 cells, via downregulation of NF-κB activity.

| Interferon-γ restored caspase-8 expression in IMR-32 cells but failed to induce TRAIL cytotoxicity
In contrast to SK-N-MC cells used as a positive control, IMR-32 and SK-N-BE cells were negative for caspase-8 expression ( Figure 3A), which was unaltered by cisplatin and/or TRAIL treatment for 24 hours ( Figure 3B). To restore the expression of caspase-8 in IMR-32 cells, we treated these cells with a demethylating agent, IFN-γ or 5-azacytidine (5-AzaC), for 24 hours and found that the caspase-8 expression was significantly restored ( Figures 3C and 4A). 5-AzaC treatment slightly induced cell death ( Figure S4B), but pretreatment with 5-AzaC and/or IFN-γ failed to trigger TRAIL cytotoxicity in IMR-32 cells (Figure S4B and C).
Although caspase-8 expression was restored in IMR-32 cells exposed to IFN-γ, treatment of these cells with cisplatin failed to trigger TRAIL-induced cell death ( Figure 3D and E). Moreover, treatment with NF-κB activation inhibitor failed to induce TRAIL cytotoxicity in IFN-γ-treated IMR-32 cells ( Figure 3F). These

| Protein kinase B (Akt) or ERK signalling pathway had no role in the susceptibility of IMR-32 cells to TRAIL
To identify the factors that confer IMR-32 cells resistance to TRAIL, we examined the expression levels of several molecules related to cell death. As shown in Figure S5, phosphorylated (p-) Akt (Thr 308 ), F I G U R E 2 Cisplatin treatment failed to trigger TRAIL cytotoxicity in TRAIL-resistant IMR-32 cells. A, Cell viability in response to treatment with 100, 500 and 1000 ng/mL TRAIL with time. B, Cell viability in response to pretreatment with 750 ng/mL cisplatin for 12 h and consecutive treatment with 100 or 500 ng/mL TRAIL with time. C, Cell death in response to pretreatment with either 100, 500 or 1000 ng/mL cisplatin for 12 h and consecutive treatment with 100 ng/mL TRAIL for 36 h. D-F, Cells pretreated with 750 ng/mL cisplatin for 12 h, followed by 100 ng/mL TRAIL treatment for 24 h. D, Cleavage of caspase-3 and PARP in cell lysates by immunoblotting. β-Actin was used as an internal control. Cell lysates were analysed for (E) activated caspase-3 and (F) cleaved PARP using Luminex assay. G, Cell death in response to pretreatment with either 1, 5 or 10 μmol/L NF-κB inhibitor for 12 h and consecutive treatment with 100 ng/mL TRAIL for 36 h. H, Cell death in response to treatment with 10 μmol/L NF-κB inhibitor and/or 500 ng/mL TRAIL for 48 h in the absence or presence of 50 ng/mL DR5:Fc by Alamar Blue assay. Data are expressed as the percentage (mean ± SD) of vehicle-treated control cells from three independent experiments. Cis, cisplatin

| Inhibition of N-myc expression sensitized IMR-32 cells expressing caspase-8 to TRAIL
Next, we examined the effect of N-myc inhibition on TRAIL susceptibility in IMR-32 cells. The treatment of myc-downregulated IMR-32 cells with IFN-γ to restore caspase-8 expression resulted in about 20% cell death ( Figures 5A and S7), and treatment with IFN-γ and TRAIL further increased the cell death to about 40% ( Figure 5B).
In addition, N-myc-downregulated IMR-32 cells pretreated with IFN-γ and subjected to cisplatin and TRAIL co-treatment showed a dramatic increase in apoptotic cell death (almost 90%), which was accompanied by nuclear condensation and fragmentation, compared to IFN-γ-untreated cells (Figure 5C and D). Moreover, the activation β-Actin was used as an internal control. D, Assessment of IMR-32 cells death when exposed to 1000 IU/mL IFN-γ for 24 h, followed by treatment with 750 ng/mL cisplatin and/or 100 ng/mL TRAIL for 48 h, by Alamar Blue assay. E, Representative images of these cells stained with Hoechst 33258. F, IMR-32 cells exposed to 1000 IU/mL IFN-γ for 24 h, followed by treatment with 10 μM NF-κB inhibitor and/ or 100 ng/mL TRAIL for 48 h. Data are expressed as the percentage (mean ± SD) of vehicle-treated control cells from three independent experiments. IFN-γ, interferon-γ of caspase-9 and caspase-3 and cleavage of PARP significantly increased in these cells ( Figure 5E). An additional pretreatment of these cells with DR5:Fc chimera protein effectively abrogated the increased cell death ( Figure 5B and C), indicating that the restored TRAIL cytotoxicity induced apoptosis-mediated cell death. TRAIL cytotoxicity was also observed in IMR-32 cells that are controlled by N-myc and caspase-8 expression, when pretreated with NF-κB inhibitor ( Figure 5F). In addition, the activation of caspase-9 and caspase-3 and cleavage of PARP significantly increased in these cells ( Figure 5G). These results indicate that the inhibition of N-myc expression sensitized IMR-32 cell expressing caspase-8 to TRAIL, and this effect was dramatically enhanced in the presence of cisplatin, via increased expression of DR5 through the downregulation of NF-κB activity.

| D ISCUSS I ON
Tumour necrosis factor-related apoptosis-inducing ligandis widely used for cancer therapy and effectively kills several transformed cells but not normal cells. 12,31 Although TRAIL may serve as a promising cancer therapeutic agent, many cancer cells exhibit partial or complete resistance to TRAIL. 12,31 This resistance may be at- as AzaC. 43,44 However, the clinical use of demethylating agents has been limited, owing to their toxic side effects. 47 IMR-32 cells used in this study were negative for caspase-8 expression, and treatment of these cells with IFN-γ restored their caspase-8 expression. IFN-γ restores caspase-8 expression through its transcriptional activation, which involves signal transducer and activator of transcription 1 (STAT1) or interferon regulatory factor 1 (IRF1) pathway. 45,48,49 Recent reports showed that IFN-γ may increase caspase-8 levels and thereby sensitize tumour cells to apoptotic stimuli. 43,45,49 In this study, however, IFN-γ-mediated restoration of caspase-8 had no effect on TRAIL-induced cell death, suggestive of the involvement of other factors in TRAIL resistance in IMR-32 cells.
The neuroblastoma cell line IMR-32 shows amplification of MYCN oncogene and contains more than 25 copies of MYCN oncogene per cell. 50 The amplification of MYCN oncogene is one of the most powerful prognostic factors in neuroblastoma, 5 and its positive effects on tumorigenesis are well known 3,8 ; thus, strategies for targeting N-myc may serve as a promising approach for neuroblastoma therapies. 2,4 However, recent studies displayed paradoxical results related to the role of N-myc protein in chemotherapies for the treatment of neuroblastoma. [51][52][53] The highly expressed N-myc protein in neuroblastoma increases the sensitivity to chemotherapeutic agents. 51 Thus, the precise roles of N-myc protein in chemotherapies are still unclear. In this study, we used retrovirus-mediated delivery of N-myc shRNA to downregulate N-myc protein expression in IMR-32 cells. TRAIL treatment in these N-myc-negative cells slightly induced apoptotic cell death, F I G U R E 5 Caspase-8 expression in N-myc-downregulated IMR-32 cells triggered TRAIL cytotoxicity, which was enhanced by cisplatin pretreatment. A, N-myc-downregulated IMR-32 cells exposed to 1000 IU/mL IFN-γ for 24 h, followed by their treatment with 100 ng/mL TRAIL for 24 h. B, N-myc-downregulated IMR-32 cells exposed to 1000 IU/mL IFN-γ for 24 h, followed by their treatment with 100 ng/mL TRAIL for 24 h in the absence or presence of 50 or 100 ng/mL DR5:Fc. C, N-myc-downregulated IMR-32 cells exposed to 1000 IU/mL IFN-γ for 24 h, followed by their treatment with 750 ng/mL cisplatin and 100 ng/mL TRAIL for 24 h in the absence or presence of 50 or 100 ng/mL DR5:Fc. D, Representative images of these cells stained with Hoechst 33258. E, Analysis of caspase-8 expression and caspase-9, caspase-3 and PARP cleavage by immunoblotting. F, N-myc-downregulated IMR-32 cell death when exposed to 1000 IU/mL IFN-γ for 24 h, followed by their treatment with 10 μmol/L NF-κB inhibitor and 100 ng/mL TRAIL for 24 h, by Alamar Blue assay. G, Analysis of caspase-8 expression and caspase-9, caspase-3 and PARP cleavage by immunoblotting. β-Actin was used as an internal control. Representative immunoblots are shown. Data are expressed as the percentage (mean ± SD) of vehicle-treated control cells from three independent experiments. IFN-γ, interferon-γ and TRIAL cytotoxicity was increased upon pretreatment of these cells with IFN-γ. TRAIL cytotoxicity was abrogated by an additional pretreatment with DR5:Fc chimera protein, indicating that the inhibition of N-myc expression triggered TRAIL cytotoxicity in IMR-32 cells expressing caspase-8.
N-myc protein is generally known to modulate the function of the extrinsic death-inducing pathway as a molecule downstream of TRAIL receptors as well as the intrinsic apoptosis pathway, which plays a role in maintaining the integrity of the mitochondria via Bcl-2 family proteins. [54][55][56] Several reports suggest that Bcl-2 expression correlates with the expression levels of N-myc protein, and together, these may promote the survival of lymphoma 57 and neuroblastoma. 58 How N-myc downregulation may lead to decreased Bcl-2 expression in cancer cells is questionable. Here, we showed that N-myc protein inhibition in IMR-32 cells decreased the expression of Bcl-2, which decreases the stability of MMP, and regulated the activation of caspases to induce apoptosis. Bcl-2 protein inhibits the release of cytochrome c from mitochondria to the cytosol and regulates the activation of caspases for apoptosis induction. 59 In addition, many reports showed that the inhibition of Bcl-2 expression by chemotherapeutic agents increased the sensitivity of cancer cells to TRAIL. 60,61 Our results demonstrate for the first time that the inhibition of N-myc expression sensitizes IMR-32 cells expressing caspase-8 to TRAIL. The treatment of TRAIL-resistant IMR-32 cells with cisplatin resulted in an increased DR5 expression but failed to trigger TRAIL sensitivity. Furthermore, the downregulation of N-myc expression and restoration of caspase-8 expression in TRAIL-resistant IMR-32 cells may activate TRAIL signalling, thereby inducing apoptotic cell death. Cisplatin pretreatment dramatically enhanced TRAIL cytotoxicity via increased DR5 expression in these cells. In conclusion, our data suggest that the combination therapy of cisplatin and TRAIL is a promising strategy for treating neuroblastoma that is controlled by the expression of N-myc and caspase-8, and its use may provide important information for the development of additional potential therapeutic strategies to fight neuroblastoma.

D I SCLOS U R E
The authors declare that they have no competing interests.

AUTH O R CO NTR I B UTI O N S
This study was designed and supervised by MWL and KHY.
Experiments were conducted by DSK, HRK and HJP. Data analysis was conducted by MWL, DSK, JWL, KWS, HHK and KHY. Funding was obtained by MWL and KHY. The manuscript was written by MWL and KHY. All authors have read and approved the final manuscript.