Interleukin-24 Induces Neuroblastoma SH-SY5Y Cell Differentiation,Growth Inhibition,and Apoptosis by Promoting ROS Production

Abstract

Neuroblastoma is among the most aggressive tumors that occur in childhood and infancy. The clinical prognosis of children with advanced-stage neuroblastoma is still poor. Interleukin-24 (IL-24) is emerging as a new cytokine involved in tumor cellular proliferation, differentiation, and apoptosis and has been widely studied as a tumor inhibitor. However, little is known about this cytokine's role in neuroblastoma. In this study, we investigated the possible effects of IL-24 on inducing neuroblastoma cell differentiation, growth inhibition, and apoptosis in vitro. Our data show that IL-24 promotes neuroblastoma SH-SY5Y cell differentiation, growth inhibition, and apoptosis. Furthermore, we found that the differentiation- and apoptosis-inducing action of IL-24 depends on the accumulation of reactive oxygen species (ROS). These results suggest that IL-24 can induce neuroblastoma cell differentiation and apoptosis and may be a potential therapeutic agent for neuroblastoma.

Introduction

N euroblastoma is an embryonic tumor derived from the autonomic nervous system's neural crest tissues within the sympathetic nervous system (Schor 1999). Neuroblastoma is the second most common extracranial malignant tumor of childhood and the most common solid tumor of infancy, with half of the cases presenting in children aged 2 years or younger (Maris 2010). Molecular defects in differentiation are one of the causes of neuroblastoma development.

The induction of differentiation is one of the strategies used in the treatment of neuroblastoma. Retinoic acid (RA) can induce the growth arrest and neuronal differentiation of neuroblastoma cells and has been used in the clinic for the treatment of neuroblastoma (Villablanca and others 1995; Matthay and others 2009; Hobbie and others 2011). However, as no suitable pharmaceutical formulation is yet available, there are often problems with the administration of RA in children (Bauters and others 2011). Further advances in differentiation therapy will require characterizing the genes associated with neuroblastoma cell differentiation, which may help in the design of a safe and specific differentiation therapy to treat neuroblastoma.

Interleukin (IL)-24 has been widely studied as a tumor inhibitor. This IL is also known as melanoma differentiation-associated gene 7 (mda-7) and was first discovered from human melanoma cells after combined treatment with IFN-β and MEZ (Jiang and others 1995). Later, based on its chromosomal location, sequence homology, gene structure, and cytokine-like properties, mda-7 was renamed IL-24 and placed into the IL-10 family (Huang and others 2001; Caudell and others 2002). Overexpressed IL-24 inhibits the growth and induces the apoptosis of numerous types of human tumor cells, including melanoma, glioblastoma, lung cancer, pancreatic cancer, gastric carcinoma, and hepatoma (Lebedeva and others 2008; Park and others 2008; Pataer and others 2008; Zheng and others 2008; Dent and others 2010; Yan and others 2010).

Although the tumor growth-inhibition efficacy of IL-24 has been tested on many human cancers in vitro and in vivo, little is known about this cytokine's antitumor capacity in neuroblastoma. It has been reported that IL-24 gene expression correlates with the induction of terminal differentiation in human melanoma cells (Tong and others 2005). A recent report showed that the ectopic expression of IL-24 induces myeloid leukemia cells to differentiate, without affecting normal hematopoietic progenitors (Yang and others 2011). In this study, we intended to explore the differentiation-, growth inhibition-, and apoptosis-inducing action of IL-24 in the neuroblastoma cell line SH-SY5Y.

Materials and Methods

Construction of pcDNA3.1-IL24

The cDNA for IL-24 was amplified by PCR using the following primers: forward 5′-TGTGAAGCTTATGAATTTTCAACAGAG-3′ and reverse 5′-TGTGGGATCCTCAGAGCTTGTAG-3′. The products of the PCR were digested with HindIII/BamHI, and then ligated into the HindIII/BamHI-digested plasmid pcDNA3.1 vector to generate pcDNA3.1-IL24. The resulting constructs were purified with an EndoFree Plasmid Maxi Kit (Qiagen) and verified by sequencing.

Cell lines and culture conditions

The human neuroblastoma cell line SH-SY5Y was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The SH-SY5Y cells were grown in the Dulbecco's Modified Eagle's Medium (DMEM)/F12 with 10% (v/v) fetal bovine serum. The cells were cultured in a humidified 37°C incubator with 5% CO₂.

The cells were plated 1 day before transfection. A total of 1×10⁶ cells were seeded per well in six-well plates and transfected with a plasmid vector using the Lipofectamine 2000 transfection reagent according to the manufacturer's instructions. All cells were transfected with pcDNA3.1-IL24 or the negative control pcDNA3.1.

Analysis of neurite outgrowth

The cells were grown under conditions for pcDNA3.1-IL24 transfection for 6 days. The cellular morphology of the SH-SY5Y cells was visualized using a Nikon phase-contrast inverted microscope. Changes in neurite length and branching were observed over 6 days. A total of 200–300 cells were microscopically evaluated and scored for neurite formation using the ImageJ program if the cells had a neurite that was longer than one cell diameter or if they had a growth cone. All experiments were repeated at least 5 times with similar results.

Real-time quantitative reverse transcriptase-PCR

All the reagents were purchased from Qiagen, and all methods were according to the manufacturer's instructions. The total RNA was extracted from cells using QIAshredder (Cat. No.: 79654) and RNeasy (Cat. No.: 74104). Real-time qRT-PCR was performed using a QuantiFast SYBR Green RT-PCR Kit and an Applied Biosystems 7500 RT-PCR System according to the manufacturer's instructions. Primers specific to the growth-associated protein 43 (GAP-43), neurogenic differentiation 1 (NeuroD1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were as follows (forward/reverse): GAP-43, 5′-GGCAGACACTGGCGATGACAAAGT-3′/5′-CAAGGGTTGAAATCCAGAAAGT-3′; NeuroD1, 5′-TCGAAACATGACCAAATCGTACAG-3′/5′-GCGTTCATGGCTTCGAGGTCGTCCT-3′; and GAPDH, 5′-TTAGCACCCCTGGCCAAGG-3′/5′-CTTACTCCTTGGAGGCCATG-3′. The comparative threshold cycle method was used for the calculation of fold amplification. The expression level of each gene was normalized to the expression level of GAPDH.

Western blotting

The cells were harvested and lysed, and the cleared lysates (30–50 μg/well) were separated on 10% Tris/glycine polyacrylamide gel electrophoresis gels under standard conditions. The proteins were then transferred to a nitrocellulose membrane and incubated overnight at 4°C with primary antibodies as follows: anti-GAP-43(Cat. No.: ab75810), anti-NeuroD1 (Cat. No.: ab60704), anti-NSE (neuron-specific enolase, NSE) (Cat. No.: ab53025), anti-synaptophysin (Cat. No.: ab8049), and anti-β-actin (Cat. No.: ab8229) all purchased from Abcam; anti-Bax (Cat. No.: MAB4601) and anti-Bcl-2 (Cat. No.: 05-826) purchased from Millipore; and anti-procaspase-3 (Cat. No.: 9665) purchased from Cell Signaling. The membranes were then washed and incubated with alkaline phosphatase-conjugated secondary antibodies in Tris-buffered saline with Tween-20 (TBST) for 2 h and developed using the nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (NBT/BCIP) color substrate (Promega). The density of the bands on the membrane was scanned and analyzed using an image analyzer.

Cell viability assay

The cells were plated in 96-well plates and transfected with pcDNA3.1 or pcDNA3.1-IL24 or treated with a complete medium. At the indicated time point, the medium was removed and a fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 0.5 mg/mL) was added to each well. The cells were incubated at 37°C for 4 h, and an equal volume of a solubilization solution (0.01 N HCl in 10% SDS) was added to each well and mixed thoroughly. The absorbance at 490 nm was measured using an ELX-800 spectrometer (Bio-Tek Instruments, Inc.).

Apoptosis assay

The cells were trypsinized and washed twice with cold PBS. Aliquots of cells (1×10⁶) were resuspended (400 μL) and stained with annexin V-FITC (Cat. No.: APOAF; Sigma-Aldrich) according the manufacturer's instructions. Propidium iodide (PI) was added to the samples after staining with annexin V to distinguish the late apoptotic and necrotic cells. Flow cytometry was performed immediately after staining. The apoptotic rate was calculated as follows: apoptotic rate (%)=number of positively stained cells (only by annexin V-FITC)/number of total cells × 100%.

Flow cytometry assay for reactive oxygen species production

The cells were incubated with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) in PBS for 20 min at 37°C in the dark. The fluorescence intensity was monitored at an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

Statistical analysis

The values are expressed as the mean±standard deviation. The statistical analysis of the results was performed using a one-way analysis of the variance or the Student's t-test. P values <0.05 were considered significant.

Results

IL-24 induces differentiation of neuroblastoma cells

To investigate the role of IL-24 in neuroblastoma, the SH-SY5Y cells were treated with IL-24 for 6 days, after which, morphologic differentiation was evaluated by phase-contrast microscopy. It was evident that IL-24 induced an increase in the number of cells bearing neurites compared with the control cells (Fig. 1A). The changes in cell morphology and neurite length observed in the presence of IL-24 suggested the induction of neuronal differentiation. To determine whether the IL-24-induced morphological changes were accompanied by biochemical patterns resembling the neuronal phenotype, we measured the mRNA levels of neuron-specific markers, including GAP-43 and NeuroD1 by qRT-PCR. These 2 known differentiation markers were both elevated after the treatment of neuroblastoma cells with IL-24 (Fig. 1B). Furthermore, the protein expression levels of GAP43, NeuroD1, synaptophysin, and NSE were also strongly increased after treatment with IL-24 (Fig. 1C). These results suggest that IL-24 may modulate the differentiation of SH-SY5Y cells.

FIG. 1.

IL-24 induces neuroblastoma cell differentiation. (A) SH-SY5Y cells transfected with pcDNA3.1-IL24 or the negative control pcDNA3.1 were observed by phase-contrast microscopy. Original magnification ×400. (B) qRT-PCR analysis of 2 neuronal differentiation markers, GAP-43 and NeuroD1 in SH-SY5Y cells after transfection with pcDNA3.1-IL24 or pcDNA3.1. The data are the mean±S.D. from 5 independent experiments (n=5); *P<0.05 versus pcDNA3.1 and PBS treatment groups. (C) The protein expression levels of GAP43, NeuroD1, synaptophysin, and NSE were normalized to β-actin in SH-SY5Y cells after transfection with pcDNA3.1-IL24 or pcDNA3.1. GAP-43, growth-associated protein 43; IL, interleukin; NeuroD1, neurogenic differentiation 1; qRT-PCR, quantitative reverse transcriptase-PCR; S.D., standard deviation.

IL-24 promotes growth inhibition and apoptosis of neuroblastoma cells

Treatment of the SH-SY5Y neuroblastoma cells with pcDNA3.1-IL24 caused growth inhibition, limiting cell proliferation, as judged by the MTT assay (Fig. 2A). Treatment of the tumor cells resulted in a time-dependent increase in cell death, but pcDNA3.1 had no effect on the SH-SY5Y cells. We also observed an increasing number of cells progressing into programmed cell death by annexin V/PI staining of SH-SY5Y cells treated with IL-24. The IL-24-treated cells exhibited an increase in the annexin V-positive population (Fig. 2B). We monitored the protein levels of Bcl-2, Bax, and caspase-3 by Western blotting after treatment of the SH-SY5Y cells with IL-24. The results showed that IL-24 significantly increased Bax expression, decreased Bcl-2 expression, and promoted the activation of procaspase-3 (Fig. 2C). These data reveal that apoptosis is induced in SH-SY5Y cells after cell differentiation by the efficient tilting of the balance of Bcl-2 family proteins toward the proapoptotic pathway.

FIG. 2.

IL-24 promotes neuroblastoma cell growth inhibition and apoptosis. (A) After transfection with pcDNA3.1-IL24 or pcDNA3.1 at the indicated time point, SH-SY5Y cell proliferation was detected by an MTT assay. The proliferation of cells was significantly inhibited in the pcDNA3.1-IL24 group compared with the pcDNA3.1 or PBS group. The data are the mean±S.D. from 5 independent experiments (n=5). (B) After transfection with pcDNA3.1-IL24 or pcDNA3.1 for 72 h, the cells were double stained with annexin V-FITC and PI for flow cytometry analysis. The number of apoptotic cells was significantly higher in the pcDNA3.1-IL24 group than in the pcDNA3.1 or PBS group. The data are the mean±S.D. from 5 independent experiments (n=5); *P<0.05 versus pcDNA3.1 or PBS treatment group. (C) The protein levels of Bcl-2, Bax, and procaspase-3 were examined by Western blotting and normalized to β-actin. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI, propidium iodide.

IL-24 induces neuroblastoma cell differentiation and apoptosis by promoting reactive oxygen species production

Reactive oxygen species (ROS) regulate apoptosis and proliferation in response to a variety of stimuli and also play important roles in cell differentiation and senescence. An intriguing question is whether ROS can also induce the differentiation and apoptosis of neuroblastoma cells, which are mediated by IL-24. To test this hypothesis, SH-SY5Y cells treated with IL-24 were stained for ROS production using H2DCF-DA. We observed that the SH-SY5Y cell ROS levels increased in a time-dependent manner at 12, 24, and 48 h after transfection with pcDNA3.1-IL24 (Fig. 3A). To further study whether ROS is necessary for IL-24-induced differentiation and apoptosis, SH-SY5Y cells were treated with 20 mM of N-acetylcysteine (NAC) after IL-24 treatment. Treatment with NAC significantly decreased the annexin V-positive cell population (Fig. 3B) and inhibited the mRNA expression of GAP43 and NeuroD1 induced by IL-24 (Fig. 3C). All of the results reveal that ROS is required for the IL-24-induced differentiation and apoptosis of SH-SY5Y cells.

FIG. 3.

IL-24 induces differentiation and apoptosis of neuroblastoma cell by inducing ROS production. SH-SY5Y cells were transfected with pcDNA3.1-IL24 or pcDNA3.1. (A) After cell transfection, ROS was monitored by FACS at 12, 24, and 48 h, respectively. The data are mean±S.D. from 5 independent experiments (n=5); *P<0.05 versus pcDNA3.1 or PBS treatment group. (B) After transfection with pcDNA3.1-IL24, cells cultured with NAC significantly decreased the number of apoptotic cells. The data are mean±S.D. from 5 independent experiments (n=5); *P<0.05 versus pcDNA3.1-IL24+NAC treatment group. (C) After transfection, cells were cultured in a medium with or without 25 mM NAC for 72 h. Two neuronal differentiation markers, GAP-43 and NeuroD1, were analyzed by qRT-PCR. The data are mean±S.D. from 5 independent experiments (n=5); *P<0.05 versus pcDNA3.1-IL24+NAC treatment group. NAC, N-acetylcysteine; ROS, reactive oxygen species.

Discussion

High-risk neuroblastoma continues to have very poor outcomes despite high-dose polychemotherapy, surgery, radiotherapy, and even autologous bone-marrow or stem-cell rescue (Cheung 2012; DE Ioris and others 2012). Novel treatment modalities are imperative to improving treatment results and to prolonging survival for high-risk diseases. Our study, which used IL-24 as a potential therapeutic agent for the treatment of neuroblastoma, verified that the IL-24-induced differentiation and apoptosis of SH-SY5Y cells depends on the accumulation of ROS.

We examined the effect of IL-24 on neuroblastoma cell differentiation, growth inhibition, and apoptosis. Our results showed that IL-24 could increase the number of neurite-bearing cells and the expression of the neuron-specific markers GAP43, NeuroD1, synaptophysin, and NSE in SH-SY5Y cells. IL-24 is emerging as a new cytokine involved in tumor cell proliferation, differentiation, and apoptosis and is implicated in diverse physiological functions (Dash and others 2010; Margue and Kreis 2010). Our experimental results imply that IL-24 could be used to differentiate neuroblastoma SH-SY5Y cells into neuronal cells.

IL-24 has been described to induce the apoptosis of many human tumor cell types (Sarkar and others 2007; Dash and others 2010; Dent and others 2010). We further investigated the induction of the apoptosis of IL-24-treated SH-SY5Y neuroblastoma cells. A great number of apoptotic SH-SY5Y cells were visible among the cells treated with IL-24. Furthermore, we found that IL-24 dramatically decreased Bcl-2 levels, significantly increased Bax, and induced a much stronger activation of caspase-3. These results suggest that IL-24 can induce changes in the levels and ratio of proapoptotic and antiapoptotic proteins to promote neuroblastoma cell apoptosis.

ROS production promotes damage to the cell structure, including proteins, lipids, membranes, and DNA, and plays a key role in the regulation of signal transduction processes leading to tumor cell proliferation, differentiation, or apoptosis (Finkel 1999; Sauer and others 2001; Khandrika and others 2009; Tsukagoshi and others 2010). Recent studies have shown that ROS can act as signaling molecules that are involved in neuronal cell differentiation and apoptosis (Lev and others 2006; Nitti and others 2007). These findings led us to speculate that the ectopic overexpression of IL-24 induces ROS production in neuroblastoma cells. In agreement with this hypothesis, our results show that SH-SY5Y neuroblastoma cell treatment with IL-24 increases ROS levels, and ROS was found to be required for cell differentiation and apoptosis.

In summary, we found that IL-24 increases the level of ROS, followed by the induction of differentiation and programmed cell death, in SH-SY5Y neuroblastoma cells. Herein we suggest that the involvement of IL-24 in neuroblastoma development should be considered when applying differentiation therapy as a cancer treatment.

Footnotes

Acknowledgments

This project was supported by the Science and Technology Agency of Xuzhou (No. XF11C087). The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We are very grateful to Professor Junnian Zheng for providing us with the plasmid carrying the IL-24 cDNA (Laboratory of Biological Cancer Therapy, Xuzhou Medical College).

Author Disclosure Statement

No competing financial interests exist.

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