Differential toll-like receptor 3 (TLR3) expression and apoptotic response to TLR3 agonist in human neuroblastoma cells
© Chuang et al.; licensee BioMed Central Ltd. 2011
Received: 12 May 2011
Accepted: 23 August 2011
Published: 23 August 2011
Toll-like receptor-3 (TLR-3) is a critical component of innate immune system against dsRNA viruses and is expressed in the central nervous system. However, it remains unknown whether TLR3 may serve as a therapeutic target in human neuroblastoma (NB).
TLR3 expression in human NB samples was examined by immunohistochemical analysis. Quantitative RT-PCR and western blot was used to determine TLR3 expression in three human NB cell lines. The effect of TLR3 agonist, polyinosinic-polycytidylic acid (poly(I:C)), on the growth of human NB cells was evaluated by WST-1 cell proliferation assay, flow cytometry analysis, and immunoblot analysis. Blockade of TLR3 signaling was achieved using TLR3 neutralizing antibody, small interference RNA, and 2-aminopurine (2-AP), an inhibitor of protein kinase R (PKR), an interferon-induced, double-stranded RNA-activated protein kinase.
In immunohistochemical studies, TLR3 mainly expressed in the cytoplasm of ganglion cells and in some neuroblastic cells, but not in the stromal cells in human NB tissues. Among three human NB cell lines analyzed, TLR3 was significantly up-regulated in SK-N-AS cells at mRNA and protein level compared with other two low TLR3- expressing NB cells. Treatment with poly(I:C) elicited significant growth inhibition and apoptosis only in high TLR3-expressing SK-N-AS cells, but not in low TLR3-expressing SK-N-FI and SK-N-DZ cells. Moreover, poly(I:C) treatment significantly stimulated the activities of PKR, interferon regulatory factor 3 (IRF-3) and caspase-3 in SK-N-AS cells. Application of TLR3 neutralizing antibody or small interference RNA (siRNA) reduced the poly(I:C)-induced inhibition of cell proliferation and apoptosis in SK-N-AS cells. On the contrary, ectopic TLR3 expression enhanced the sensitivity of low TLR3-expressing NB cells to poly(I:C). Finally, application of 2-AP attenuated the poly(I:C)-induced IRF-3 and caspase-3 activation in SK-N-AS cells.
The present study demonstrates that TLR3 is expressed in a subset of NB cells. Besides, TLR3/PKR/IRF-3/capase-3 pathway is implicated in the selective cytotoxicity of TLR3 agonist towards high TLR3-expressing NB cells.
Keywordsneuroblastoma toll-like receptor 3 poly(I:C) apoptosis
Neuroblastoma (NB) accounts for more than 7% of malignancies in patients younger than 15 years old and 15% of all pediatric oncology deaths in the United States . The disease is characterized by its broad range of clinical manifestations and versatile biological behaviors. Outcome of the patients with NB is poor for those with high-risk clinical phenotypes. Based on the latest report of the Italian Neuroblastoma Registry consisting of 781 NB children, the ten-year overall survival was 6.8% after progression and 14.4% after relapse . A report from the Taiwan Children Cancer Foundation revealed that NB accounts for 6% of malignancies in children and the projected two-year survival rate was 59%, which dropped to 23% after disease progression .
The major factors influencing the survival of NB patients are age, stage and MYCN proto-oncogene status. Genomic MYCN amplification has been the most consistent genetic aberration associated with poor prognosis in NB [2, 4–6]. Decrease of proliferation rate and induction of differentiation by a MYCN antisense DNA oligomer in human NB cell line may explain MYCN functions . Over expression of MYCN transcriptional targets and low expression of neuronal differentiation genes predicts relapse and death from NB . The MYCN oncoprotein was thus proposed as a drug development target . However, recent evidence suggests that clinicopathological parameters, including tumor cell ploidy, localized disease and stage, may influence the prognosis of MYCN in NB , [10, 11]. Two recent reports indicate that MYCN amplification alone is not sufficient to predict the outcome of the patients with NB [12, 13]. Therefore, other factors affecting the response of NB cells to various stimuli may facilitate novel diagnostic or therapeutic targets for NB.
Toll-like receptors (TLRs) are human counterparts of Toll receptors in the fruit fly, Drosophila, which are originally implicated in the regulation of dorsoventral polarity, synaptogenesis, and path-finding in motor neuron growth cone . There are 10 functional toll-like receptors (TLRs) in humans that specifically recognize pathogen-associated epitopes. While they are best known as initiators of the innate immune response to pathogens, TLRs are also expressed in glia and neurons of the central nervous system (CNS), which may recognize endogenous ligands and participate both in development and in responses associated with CNS injury . Among the 10 functional TLRs, TLR2 are known to induce neural inflammation and neuronal damage, while TLR3 and TLR8 are negative regulators of axonal or neurite growth by inducing neuronal apoptosis [16–18].
The strategy of manipulating TLRs signaling is under active investigation for anti-neoplastic application. For example, stimulation of TLR9 with CpG oligonucleotide induces apoptosis of glioma and prolongs the survival of mice with experimental brain tumors . TLR3 activation by its agonist directly triggers apoptosis in human breast cancer cells through activation of extrinsic caspases . Moreover, a series of studies confirm the potential of TLR3 as therapeutic target for hepatoma, melanoma and clear cell renal carcinoma [21–24]. Synthetic agonists for several TLRs, including TLR3, TLR4, TLR7, TLR8, and TLR9, have been or are being developed for cancer treatment . Despite of these studies, the role of TLR3 expression in NB remains largely unknown.
In the present study, we investigated TLR3 expression in human NB specimens to delineate the correlation of TLR3 expression with tumor differentiation. Subsequently, we analyzed TLR3 expression in three human NB cells and characterized two NB cells with differential TLR3 status. Finally, as TLR3 recognizes foreign double-stranded RNA (dsRNA), we treated these NB cells with TLR3 agonist, polyinosinic-polycytidylic acid (poly(I:C)), and monitored the difference in cellular proliferation, apoptosis, and expression profile of TLR3 signaling pathway.
Materials and methods
Fourteen archival neuroblastic tumor specimens consisting of 8 cases of NB, 5 cases of ganglioneuroblastoma (GNB) and one ganglioneuroma (GN) were retrieved from the Department of Pathology, Chang Gung Memorial Hospital-Kaohsiung Medical Center (Kaohsiung, Taiwan). The use of archival tissues was approved by the institutional Internal Review Board of the hospital. In each case, well-preserved areas in paraffin-embedded NB tissues were reviewed and identified by a pathologist to prepare a tissues microarray of NB patients, which consisted of six to eight tissue cores (0.6 mm) from each patient.
Tissue specimens were maintained in formaldehyde-fixed, paraffin-embedded blocks. Sections stained with hematoxylin and eosin (H&E) were also reviewed. The paraffin sections from specimens were deparaffinized, blocked with 3% hydrogen peroxide for 10 min and subjected to antigen retrieval with microwave in 0.01 M citrate buffer for 7 min. The slides were then washed twice with PBS, incubated with TLR3 antibody (1:2000 dilution; Abcam, Cambridge, MA, USA) at room temperature for 30 min, followed by washing with TBST. Sections were detected with SuperPicTure Polymer detection kit (Zymed Laboratories, South San Francisco) for 30 min and developed with DAB chromogen (DAKO, USA) for 1 min. Sections were counterstained with Gill's hematoxylin, dehydrated and mounted with mounting medium.
The labeling index of TLR3 was calculated in percentage by two pathologists for each case. The immunoreactivity of TLR3 was graded according to the staining intensity as weak (1+), moderate (2+) and strong (3+) staining. Moderate or strong staining intensity was considered TLR3-positive. The percentages of positive cells were evaluated for neuroblastic cells and ganglion cells, respectively, in each neuroblastic tumor. Positive staining in more than 50% tumor cells was defined as "TLR3 overexpression" for either neuroblastic or ganglion cells. Negative was defined as <10% of area with staining.
NB cell lines
Three human NB cell lines SK-N-AS, SK-N-FI and SK-N-DZ were purchased from American Type Culture Collection (Manassas, VA) and cultured with DMEM containing 10% (v/v) heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA), L-glutamine, non-essential amino acids (Invitrogen; 10 mM) and antibiotic-antimycotic (Invitrogen) in a 5% CO2 humidified incubator at 37°C. The SK-N-AS cells were subcultured at 1:10 ratio when the cells grew to 80-90% of confluence, SK-N-DZ at 1:6 when 70-80% of confluence and SK-N-FI at 1:4 when 60% of confluence. To evaluate the effect of TLR3 agonist, the NB cells (seeded at 3 × 104 cells/cm2 in 60-mm dishes) were treated with Poly(I:C) (Invivogen; San Diego, CA) in DMEM with 10% FBS at indicated doses for 24 h.
To verify the specificity of cellular response to poly(I:C), we also treated the NB cells with lipopolyssacharide (LPS) and CpG. Neuroblastoma cells were seeded in 60-mm culture dishes at a cell density of 3 × 104 cells/cm2 and treated with 50 μg/ml Poly(I:C) (TLR3, Invivogen), 10 ng/ml LPS (TLR4, Sigma) or 1 μM CpG-ODN2006 (TLR9, InVivoGen) plus 10% FBS for 24 h.
Quantitative RT-PCR (qRT-PCR) analysis
Total RNA was extracted by using TRIzol® reagent (Invitrogen, Carlsbad, CA) from the three cell lines. After quantification, total RNA (2 μg) were used to produce cDNA by using the high capacity cDNA reverse transcription kit (ABI, Applied Biosystems, Foster City, CA) according to the manufacturer's recommendations. Expression levels of human TLR3 mRNA were determined by qRT-PCR using specific primers in PCR master mix (Yeastern Biotechnology; Taipei, Taiwan). The β-actin level was used as an internal control for normalization of TLR3 mRNA expression using the Prism 7700 Sequence Detection System (ABI). The primer sequences for qRT-PCR analysis of TLR3 and β-actin were: TLR3 forward 5'- TGGTTGGGCCACCTAGAAGT-3' and reverse, 5'- CCATTCCTGGCCTGTGAGTT -3'; β-actin forward 5'-TCACCCACACTGTGCCCATCTACGA-3' and 5'-CAGCGGAACCGCTCATTGCCAATGG-3'. The product size for TLR3 was 71 bp and for β-actin was 294 bp.
Cell proliferation assay
Cells were seeded at a density of 5 × 103 cells/100 μL in 96-well plates and cultured overnight. The medium was changed, and the cells were then cultured in medium alone (control) or in medium containing different concentrations of poly(I:C). After treatment, 10 μL WST-1 reagent (Roche Diagnostics, Laval, Quebec, Canada) was added to each well and incubated for another 2 h at 37°C. The absorbance was determined using a microplate reader at a test wavelength of 450 nm and reference wavelength of 630 nm.
Flow cytometry analysis
The percentage of cells in G0/G1, S, and G2/M phases was determined by flow cytometry analysis following propidium iodide (PI; Sigma, St. Louis, MS) staining. For PI staining, NB cells were seeded at a concentration of 1 × 106 cells/well in 3 ml medium containing 10% FBS in 6-well plates and left untreated or treated for 24 h or 48 h with poly(I:C). Cells were washed once with PBS and then fixed with ice-cold 70% PBS-ethanol for overnight at -20°C. Fixed cells were washed once with PBS and incubated in 1 ml PBS containing 10% Triton X-100, PI (20 mg/ml) and RNase A (Boehringer Mannheim, Indianapolis, IN) for 30 min at room temperature. Samples were analyzed using a FACS (BD Biosciences) and winMDI software.
Western blot analysis
After treatment, cells were lysed with protein extraction solution containing proteases inhibitors (iNtRON Biotechnology) and the protein concentrations were measured with the BCA assay (Bio-Rad) with bovine serum albumin as standard. Thirty μg crude proteins were separated in 12-15% SDS-PAGE gels and transferred to nitrocellulose membrane. The membranes were immunoblotted overnight at 4°C with each primary antibody at indicated dilution. The primary antibodies included IRF-3 antibody (Epitomic, Inc.; Burlingame, CA), phosphor-IRF3 (pS386) antibody (Epitomic, Inc.), PKR antibody (Epitomic, Inc.), phosphor-PKR (pT451) antibody (Epitomic, Inc.), caspase-3 antibody (Cell Signaling Technology), and cleaved caspase-3 (Asp175) antibody (Cell Signaling Technology). Membranes were washed three times, and subjected to HRP-conjugated secondary antibody for 60 min at room temperature. Protein-antibody complexes were visualized with an ECL Western blotting detection and analysis system (Amersham Pharmacia Biotech, Uppsala, Sweden) and blots were exposed to film. Signals were quantified by densitometric analysis.
TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling) assay was used to detect fragmented DNA in SK-N-AS cells after Poly(I:C) treatment. Cells were grown in 12 well culture plate containing round glass cover slide. 24 h after treatment with Poly(I:C), the cells were fixed for 10 min at room temperature with 4% paraformaldehyde solution, washed three times with PBS. The TUNEL assay was performed according to the instruction manual of the In Situ Cell Death Detection Kit (Roche Diagnostics). Cells were incubated with TUNEL reaction mixture for 60 min at 37°C, protected from light, washed gently three times with PBS, and stained with DAPI for 5 min at room temperature in a dark chamber. After washing three times with PBS, cells were covered with cover slips. Photomicrographs were taken using a fluorescence Microscopy.
TLR3 gene delivery
To increase the cellular TLR3 levels, SK-N-FI and SK-N-DZ NB cells were transfected with the mammalian expression vector encoding human TLR3 cDNA fused with hemagglutinin (HA) tag (OriGene Technologies Inc.; Rockville, MD) by Lipofectamine 2000 (Invitrogen; Carlsbad, CA).
TLR3 RNA interference and antibodies neutralization
To knockdown endogenous TLR3 expression, human TLR3 stealth siRNA and control (scramble) siRNA were purchased from Invitrogen (Carlsbad, CA). The sequence for human TLR3 siRNA and control siRNA was 5'-CCTGAGCTGTCAAGCCACTACCTTT-3' and 5'-CCTGTCGAACTACCGCATCCAGTTT-3', respectively. Gene delivery of siRNA into NB cells was performed using Lipofectamine RNAiMAX (Invitrogen) following the manufacturer's protocol. After transfection, the cells were incubated with poly(I:C) 50 μg/ml for an additional 48 h before subsequent analysis.
For TLR3 antibody neutralization, NB cells (a density of 8 × 104 cells/ml in a 96-well plate) were incubated with TLR3 antibody (Santa Cruz Biotechnology; Santa Cruz, CA) at indicated concentrations for 1 h then treated with 50 μg/ml poly(I:C) for 24 h before subsequent analysis.
All the data present in the figures were representation of at least triplicate experiments. Data were expressed as mean ± SD. Student's t-test was used for between-group comparison while analysis of variance was applied when more than 2 groups were compared. Statistical analysis of histological findings between different NB groups was performed using Fisher's extract test. A P-value less than 0.05 was considered statistically significant.
TLR3 is expressed in the ganglion cells and differentiated neuroblastic cells in human neuroblastic tumors
Immunohisochemical analysis of TLR3 expression in human neuroblastoma tissues
TLR 3 expression
Low expression a
High expression b
Neuroblastoma (n = 8)
Ganglioneuroblastoma (n = 4)
TLR3 is highly expressed in human SK-N-AS neuroblastic cells
TLR3 agonist preferentially induced growth inhibition and apoptosis in high TLR-3-expressing NB cells
We employed flow cytometry analysis to evaluate the apoptotic extent and cell cycle distribution in poly(I:C)-treated NB cells. It was found that poly(I:C) treatment significantly increased the percentages of cells in pre-G0 phase in SK-N-AS cells after treatment for 24 hours (Figure 3B). The poly(I:C)-induced apoptosis of SK-N-AS cells was also validated by TUNEL assay (Additional file 1, figure S1). However, poly(I:C) treatment had no significant effect on cell proliferation or apoptosis in SK-N-FI and SK-N-DZ cells even after extended exposure for 48 hours (Figure 3B). Thus, TLR3 agonist preferentially inhibited the growth of high TLR3-expressing NB cells.
TLR3 agonist stimulated the PKR/IRF-3/caspase-3 pathway in high TLR3-expressing NB cells
Interferon regulatory factor 3 (IRF-3) is known to undergo phosphorylation-induced activation in virus-infected cells and plays an important role in the antiviral innate immune response under the regulation of PKR . It was found that application of poly(I:C) significantly elevated the phosphorylation of IRF-3 in SK-N-AS cells (data not shown). Interestingly, poly(I:C) treatment also elicited a moderate, yet significant elevation of phosphorylated IRF-3 in SK-N-FI cells, but not in SK-N-DZ cells.
To verify the specificity of cellular response to poly(I:C), NB cells were also challenged with other TLR agonists, including LPS and CpG, then examined for capsase-3 activation. However, LPS and CpG treatment failed to elicit caspase-3 activation in all the three NB cells (Additional file 2, figure S2)
Blockade of TLR3 signaling using neutralizing antibody or small interference RNA (siRNA) attenuated the poly(I:C)-induced growth inhibition and apoptosis in SK-N-AS cells
Ectopic TLR3 expression rendered the low TLR3-expressing NB cells sensitive to poly(I:C) treatment
Pharmaceutical inhibition of dsRNA-regulated protein kinase (PKR) attenuated poly(I:C)-induced action of PKR/IRF3/caspase 3 pathway
To determine the role of dsRNA-regulated protein kinase (PKR) in TLR3-mediated cell death, SK-N-AS cells were treated with PKR inhibitor, 2-aminopurine (2-AP), followed by poly(I:C) administration. 2-AP significantly blocked the expression of p-PKR (Additional file 5, figure S5), reduced the expression of p-IRF3 (Additional file 5, figure S5B) from 3 h up till 24 h after treatment, resulting in significant reduction of activated caspase-3 expression at 24 h in SK-N-AS (Additional file 5, figure S5C).
The first report that TLR3 can directly trigger apoptosis in human breast cancer cells opened a new avenue for cancer therapeutics . The observation quickly gained support from other studies that confirm a role for TLR3 in the tumorigenesis of hepatoma, melanoma and clear cell renal carcinoma [21–24]. In this study, we provided the first evidence that TLR3 is differentially expressed in human NB tissues and cells. The histological study in human NB tissues reveals that TLR3 expression is present mainly in the cytoplasm of ganglion cells of GNB, but rare or not detectable in the neuroblastic cells of NB. Besides, TLR3 is not present in the stromal cells. These findings indicate heterogeneous neuroblastic cell types in human neuroblastic tumors that harbor differential TLR3 levels and may respond differently to TLR3 agonist.
Histological studies revealed that TLR3 expresses mainly in the cytoplasm of ganglion cells in GNB tissues. In contrast, TLR3 was rarely detected in neuroblastic cells of NB tissues. There is an excellent correlation between differentiation and apoptosis in NB tissues. Hoehner et al. found that the most differentiated NB cells lie adjacent to TUNEL-positive, morphologically apoptotic cells . Another study also showed that apoptosis in neuroblastic tumor is present mainly in cases with well differentiation status and favorable outcome . The above findings implicate that the interplay between differentiation and apoptosis may be involved in the regression of neuroblastic tumors . Our findings of TLR3 expression in the more differentiated NB tissues and TLR3 signaling during apoptosis of NB cell lines seem consistent with the above notions. However, due to the relative small sample size in this study, future studies are warranted to validate the role of TLR3 signaling in differentiation and apoptosis in NB.
Poly(I:C) is known to transmit anti-viral and inflammatory signaling in TLR3-expressing astrocytes through activation of PKR . PKR is also the essential component of TLR3-mediated NF-κB activation in human epidermal keratinocytes . Another study also reported that PKR was required for maximal type I IFN-beta induction and the induction of apoptosis in different cell lines by both transfected T7 phage polymerase-synthesized RNA and Poly(I:C) . In cultured human biliary epithelial cells, stimulation with poly(I:C) induces the activation of both transcription factors NF-κB and IRF3 and the production of interferon-beta1 (IFN-beta1) and MxA as potent antiviral responses . The above studies are consistent with our studies in NB cells, indicating that TLR3/PKR/IRF3 signaling components are not only for innate immune response against viral infection, and probably respond to tumor antigens, in various human cell lines including NB cells.
It remains elusive how TLR3 executes its function upon ligand stimulation in NB cells. TLR3 is known to be localized in the endoplasmic reticulum (ER) of unstimulated cells [32, 33]. In response to ligand stimulation, TLR3 may move to endosomes or other compartments to execute antiviral activities or inflammatory cytokine production . Two ER proteins, glucose-regulated protein 78 (GRP78) and calreticulin, have been identified as independent prognostic factors for NB patients [35, 36]. Our pilot study revealed that poly(I:C) induced significant upregulation of GRP78 and calreticulin in SK-N-AS cells, but not in SK-N-FI and SK-N-DZ (Chuang et al.; unpublished observation). Thus, TLR3 signaling may affect the expression of some ER proteins and control the cell fate or differentiation of high TLR3-expressing NB cells. An interesting observation reveals the association of TLRs with other ER membrane proteins such as UNC93B. Single point mutation (H412R) in UNC93B abolishes signaling via TLR3, 7, and 9 . The latter suggests a physical association between UNC93B and TLRs in the ER is probably essential for proper TLR signaling in high TLR3-expressing NB cells.
In summary, the present study unveiled the differential TLR3 expression in different types of NB specimens. Moreover, this study demonstrated the susceptibility of high TLR3-expressing NB cells to agonist-induced apoptosis and the involvement of PKR/IRF3/caspase 3 signaling pathways. Further studies with the aid of pretreatment with interferon alpha , simultaneous administration of CpG DNA [19, 39], or targeting MYCN or MDA5 may be helpful in augmenting the poly(I:C) sensitivity and NB immunotherapy. As an intracytoplasmic RNA sensor, MDA5 expression was higher in SK-N-FI (unpublished data), which also responded to poly(I:C) treatment and might explain why active caspase-3 expression was higher in SK-N-FI than in SK-N-DZ, despite both were lower than that in SK-N-AS.
This study unveiled that TLR3 is expressed in a subset of relatively benign NB cells. Besides, TLR agonist induced proapoptotic activity in NB cells with high endogenous TLR3 level. Genetic manipulation data further support that the cellular TLR3 level determined the sensitivity of NB cells to poly(I:C)-induced cell death. Our study may open a new avenue of therapeutic indication for TLR3 agonists as an adjuvant therapy in intractable NB.
List of Abbreviations
Interferon regulatory factor 3
Double-stranded RNA-dependent protein kinase
C): Polyinosinic-polycytidylic acid)
This study was supported by grants from the Chang Gung Memorial Hospital (CMRPG870801), National Science Council, Taiwan (NSC-100-2325-B-110-002), Kaohsiung Veterans General Hospital (VGHNSU 99-002), and National Sun Yat-Sen University Research Center.
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