The SK-N-BE(2) neuroblastoma cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and NB1691, also a neuroblastoma cell line, was donated by Dr Peter Houghton (St. Jude Children's Research Hospital, Memphis, TN, USA). These cells were cultured in Opti-Mem medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 5% fetal bovine serum (Atlanta Biologicals, Inc., Flowery Branch, GA, USA) and 1% penicillin/streptomycin in a humidified atmosphere containing 5% CO₂ at 37°C.

Antibodies were obtained from the following sources: SPARC, (cat. no., OAAN00424; Aviva Systems Biology Corporation, San Diego, CA, USA); was used at a dilution of 1:1,000 for western blot analysis; β-actin (cat. no., sc-130300; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used at a dilution of 1:1,000 and HSP27 (cat. no., ab2790; Abcam, Cambridge, MA) was used at a dilution of 1:200 for immunohistochemistry. Secondary antibodies used were HRP-conjugated anti-rabbit at a dilution of 1:200 for immunohistochemistry and 1:2,000 for western blot analysis (cat. no., sc-2030; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and HRP-conjugated anti-mouse antibody at a dilution of 1:200 for immunohistochemistry (cat. no., 7076; Cell Signaling Technologies, Danvers, MA).

The RS2000 radiator (Rad Source Technologies, Inc., Suwanee, GA, USA) was used, which was operated at a maximum of 150 kV/50 mA, for all radiation treatments. All cells were treated with a single (5 Gy) dose of radiation that was administered following cell transfection. To radiate subcutaneous tumors in mice, 2 doses at 2 and 3 Gy with a maximum cumulative dose of 5 Gy was administered. The mice were shielded using lead sheets, exposing only the subcutaneous tumors.

SK-N-BE(2) and NB1691 cells were transfected with SPARC overexpression plasmid RC209964 (OriGene Technologies, Inc., Rockville, MD, USA) using Lipofectamine^® (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol for in vitro experiments. Cells were incubated in Opti-Mem medium supplemented with 5% FBS and 1% penicillin/streptomycin in a humidified atmosphere containing 5% CO₂ at 37°C for 24 h. Subsequent to this incubation cells were not treated or treated with 5 Gy of ionizing radiation and incubated for another 24 h in Opti-Mem medium supplemented with 5% FBS and 1% penicillin/streptomycin in a humidified atmosphere containing 5% CO₂ at 37°C.

Following SPARC overexpression and radiation treatments, cells were obtained and total protein was isolated using Mammalian Protein Extraction reagent (Thermo Fisher Scientific, Inc.). Equal quantities of protein (10 µg/lane) measured using Pierce 660 nm Protein Assay (cat. no., 1861426 Thermo Fisher Scientific, Inc.) were separated in reducing conditions on 10% polyacrylamide gels. Following SDS-PAGE, the proteins were transferred on to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Membranes were blocked with standard TBS Tween-20 containing 5% non-fat skimmed milk for 1 h at room temperature followed by immunoprobing with primary antibody for 2 h at room temperature in TBS Tween-20 containing 5% non-fat skimmed milk. This was followed by washing (x4 with TBS Tween-20) followed by blocking again with TBS Tween-20 containing 5% non-fat skimmed milk for 1 h at room temperature. This was followed by the addition of appropriate horseradish peroxidase-labeled secondary antibody in TBS Tween-20 containing 5% non-fat skimmed milk and incubated for 1 h at room temperature followed by washing (x4 with TBS Tween-20). All incubation and washings were done on a rocking platform set at 2-strokes per min. Specific protein bands were visualized using enhanced chemiluminescence detection reagents (cat. no., 32106; Thermo Fisher Scientific, Inc.).

An antibody array for the detection of HSP27 and p21 was obtained from RayBiotech Human Apoptosis array C1 (catalogue no., AAH-APO-1; RayBiotech, Inc., Norcross, GA, USA) and processed according to the manufacturer's protocol. Briefly, 80% confluent petri plates of SK-N-BE(2) and NB1691 cells were transfected with SPARC overexpression plasmid, followed by radiation treatment as previously described. Total protein was isolated using Mammalian Protein Extraction reagent (Thermo Fisher Scientific, Inc.) and equal quantities of protein (500 µg) measured using Pierce 660 nm Protein Assay (cat. no., 1861426 Thermo Fisher Scientific, Inc.) were added to the provided antibody array and processed according to the manufacturer's protocol, expression of HSP27 and p21 was determined by measuring signal intensities compared to untreated controls.

Cell cycle distribution of SPARC overexpressing and irradiated SK-N-BE(2) cells, and NB1691 neuroblastoma cells was analyzed using flow cytometry (FACS Calibur System; BD Bioscience, San Jose, CA, USA) with excitation at a wavelength of 488 nm and an emission of 639 nm, following propidium iodide staining according to standard protocols (9). A total of 10,000 cells were sorted to determine cell cycle phase using the Cell Quest Pro software version 5.2.1 (BD Bioscience, San Jose, CA, USA).

An MTT cell proliferation assay (cat. no., 50-213-524, Thermo Fisher Scientific, Inc.) was performed using SPARC overexpressing and irradiated SK-N-BE(2) and NB1691 neuroblastoma cells plated in 96 well plates (density, 2,000 cells/well). After 72 h of incubation in Opti-Mem medium supplemented with 5% FBS and 1% penicillin/streptomycin in a humidified atmosphere containing 5% CO₂ at 37°C, MTT was added at a concentration of 0.5 mg/ml to each well. Plates were incubated for 3 h at 37°C. Following incubation, 100 µl of dimethyl sulfoxide was added and the absorbance was measured at a wavelength of 550 nm and presented as the survival percentage.

The in vitro scratch assay was performed as previously described (16,17). Briefly, SK-N-BE(2) and NB1691 neuroblastoma cells were plated in 24-well plates (density, 10,000 cells/well). The cells were transfected with SPARC overexpression plasmid followed by treatment with 5 Gy ionizing radiation after 24 h at 37°C. The cell monolayer was scratched with a 100 µl pipette tip and the migration into the gap was measured over time using the EVOS-FL cell imaging system, magnification, ×4 and graphically presented.

The mitochondrial Δψ was measured using the MitoProbe™ JC-1 Assay kit (cat. no., M34152, Thermo Fisher Scientific, Inc.). Briefly, SK-N-BE(2) and NB1691 neuroblastoma cells were plated in 24 well plates (density, 20,000 cells/well). The cells were transfected with SPARC overexpression plasmid followed by treatment with 5 Gy of ionizing radiation. A total of 48 h after irradiation, cells were stained with JC-1 dye at a final concentration of 2 µM and the cells were incubated at 37°C, 5% CO₂, for 30 min and sorted using flow cytometry (10,000 cells) and fluorescence emission at a wavelength of ~590 nm was quantified and graphically presented. Staurosporine (cat. no., 11055682001 Roche Diagnostics Mannheim, Germany) was used as a positive control (5 µM) to determine experimental validity (18,19).

Caspase activation was detected using the CellEvent™ Caspase-3/7 Green ReadyProbes from Molecular Probes™ (cat. no., R3711; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. SK-N-BE (2) and NB1691 cells were plated in chamber slides and transfected with Lipofectamine^®, according to standard protocols using (20) the SPARC overexpression plasmid. A total of 24 h after transfection, cells were exposed to 5 Gy of ionizing radiation and incubated for 24 h in Opti-Mem medium supplemented with 5% FBS and 1% penicillin/streptomycin in a humidified atmosphere containing 5% CO₂ at 37°C. Following this 24 h incubation, media was removed followed by the addition of 60 µl of CellEvent Caspase-3/7 Green ReadyProbe. Cells were observed using a confocal microscope (aperture setting 2). Nuclei were visualized using propidium iodide. The percent of cells exhibiting activated caspase 3/7 was determined by counting the number of cells with green florescence in 10 random fields.

All animal studies were according to instructional approved protocols by the IRB of the University Of Illinois College Of Medicine at Peoria (Peoria, USA). Female athymic Nude mice (Foxn1^nu) obtained from Jackson Laboratories (Bar Harbor, ME) were subcutaneously implanted on the right rear flank with 1×10⁶ SK-N-BE(2) or NB1691 cells harvested at the log phase (n=5) using a 20 gauge needle attached to a syringe at a maximum volume of 200 µl in serum free OptiMem media. The tumors were allowed to grow until 5 mm in diameter and followed by intratumoral injections of 100 µg of SPARC overexpression plasmid solution in phosphate buffer saline (NaCl 137 mmol/l, KCl 2.7 mmol/l, Na2HPO4 10 mmol/l, KH2PO4 1.8 mmol/l) on day 5, 15 and 20. Controls were untreated or treated with 100 µg of empty plasmid vector (pGEM) in phosphate buffer saline. Tumors were irradiated on day 7 at 2 Gy and on day 17 at 3 Gy each with a cumulative dose of 5 Gy. Mice were euthanized by carbon dioxide asphyxiation when they lost >20% of body weight or had altered behavior when ambulating, feeding or grooming. Tumors were obtained on day 30 and fixed in 10% phosphate-buffered formaldehyde for a minimum of 24 h at room temperature and processed for paraffin embedding using an automated tissue processor (Tissue-Tek VIP Vacuum Infiltration Processor; Sakura Finetek USA, Inc., Torrance, CA) and sectioning using a manual microtome set at 6 µm (Leitz 1512, Ramsey, MN), prior to sectioning the paraffin blocks were cooled to −4°C. Serial section were taken and allowed to float over warm deionized water (42°C) and collected on subbed slides (21).

Tissue sections (thickness, 6-µm) obtained from the paraffin blocks were. The paraffin sections were deparaffinized in 100% xylene (20 min at room temperature) followed by 100% ethanol (2x, 15 min each). The sections were then rehydrated in serial ethanol concentrations (100, 90, 70, 50, 30 and 20%) for 10 min each. Subsequent to rehydration the sections were incubated in standard PBS for 10 min at room temperature followed by incubation for 30 sec in PBS at 80°C for antigen retrieval. The sections were then treated with 1% H₂O₂ at room temperature for 5 min to inactivate peroxidases if any. The sections were then incubated in 1% BSA solution in PBS at room temperature for 1 h followed by incubation with monoclonal HSP27 antibody (1:200 dilution for 2 h at room temperature; Santa Cruz Biotechnology, Inc.). The sections were then washed in PBS at room temperature (3x) followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibody at a dilution of 1:200 in 1% BSA in PBS for 1 h. The slides were the washed in PBS three times (5 min each at room temperature) followed by the addition of DAB Peroxidase (HRP) Substrate (cat. no., SK-4100, Vector Labs Burlingame, CA). The DAB reaction proceeded until the desired color developed, and was stopped by washing with PBS at room temperature. Nucleus was counterstained with hematoxylin. Negative control slides were obtained by treating with non-specific Immunoglobulin G. Sections were mounted and analyzed using the EVOS-FL cell imaging system. The intensity of HSP27 expression per unit area was measured in arbitrary pixel units using Image J (version 1.4) and represented as raw units.

Hematoxylin stained tumor sections were analyzed and an average number of tumor cells in random 10 different fields in an area of 100 µm² was measured and graphically presented (300 dpi image at magnification ×10).

SPARC-overexpressing NB1691 and SK-N-BE(2) cells were treated with 5 Gy of ionizing radiation as described above. Total RNA was isolated using the TRIzol method as per standard protocols (cat. no., 15596026; Thermo Fisher Scientific, Inc.) (22) and RNA quantity and quality were measured by NanoDrop ND-1000.

Sample labeling and array hybridization of total RNA isolated from SPARC-overexpressing NB1691 and SK-N-BE (2) treated with or without 5 Gy of ionizing radiation were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technologies, Inc., Santa Clara, CA, USA). The array consisted of 27,958 Entrez Gene RNAs, content sourced from RefSeq Build 36.3, Ensemble Release 52, Unigene Build 216 and GenBank (April 2009) on a 4×44K slide formats (http://www.agilent.com/cs/library/usermanuals/Public/G4140-90040_GeneExpression_OneColor_6.9.pdf). Total RNA from each sample was linearly amplified and simultaneously labeled with Cy3-UTP (cat. no., NEL580; PerkinElmer, Inc., Waltham, MA, USA). The labeled cRNAs were purified by RNeasy Mini kit as per manufacturer's protocol (Qiagen, Inc., Valencia, CA, USA). A total of 1 µg of each labeled cRNA was fragmented by adding 11 µl 10X Blocking Agent and 2.2 µl of 25X Fragmentation Buffer, then heated at 60°C for 30 min, and finally 55 µl 2X GE Hybridization buffer was added to dilute the labeled cRNA. A total of 100 µl hybridization solution was dispensed into the gasket slide and assembled to the gene expression microarray slide. The slides were incubated for 17 h at 65°C in an Agilent Hybridization Oven (Agilent Technologies, Inc.). The hybridized arrays were washed using Gene Expression wash buffer with 0.005% Triton-X102 twice in wash buffer 1 at room temperature for 1 min and once in wash buffer 2 at 37°C for 1 min followed by scanning using the Agilent DNA Microarray Scanner (part no. G2505C; Agilent Technologies, Inc.). RNA labeling, hybridization and data analysis was performed by Arraystar Inc (Arraystar, Inc., Rockville, MD, USA) using Agilent Feature Extraction software (version 11.0.1.1; Agilent Technologies, Inc.).

This analysis was performed using Agilent Feature Extraction software (version 11.0.1.1; Agilent Technologies, Inc.) was used to analyze the acquired array images. Quantile normalization and subsequent data processing were performed with using the GeneSpring GX software (version 12.0; Agilent Technologies, Inc.). Following quantile normalization of the raw data, genes exhibiting enriched expression were selected for further data analysis. Differentially expressed genes with statistical significance were identified through Fold Change filtering set to 1. Gene Ontology analysis was performed using the standard enrichment computation method to functionally profile gene sets and was graphically presented using Gene Ontology Consortium tools (23) (http://geneontology.org/).

All data are expressed as the mean ± standard deviation. Statistical analysis was performed using Student's t-test. P≤0.05 was considered to indicate a statistically significant difference. Regression analysis was used to determine the statistical validity of migration. All experiments were performed in triplicate, or as indicated. For the pathway analysis the enrichment P-value of the PathwayID used the EASE method, EASE Score is a modified Fisher's exact probability by penalizing (subtract) the count of positive agreement by 1.

To determine whether overexpression of SPARC suppresses proliferation and radiation-induced G2M arrest, SPARC was overexpressed in NB1691 and SK-N-BE(2) neuroblastoma cells (Fig. 1A). The MTT assay demonstrated that in SPARC overexpressing and irradiated NB1691 cells, the proliferation rate decreased significantly (P<0.0001), whereas in SK-N-BE2 cells an increase in proliferation was observed in SPARC overexpressing cells compared with the controls (Fig. 1B), notably SPARC overexpressed and radiation treated SK-N-BE2 cells showed decreased survival compared with the untreated controls (Fig. 1B), this observation may be due to the varied p53 status of NB1691 (wt) and SK-N-BE(2) (mt). Furthermore, it was observed that radiation-induced accumulation of cells in the G2 phase and the addition of SPARC overexpression suppressed G2 phase accumulation in NB1691 and SK-N-BE(2) (Fig. 1C). Quantitative analysis of G2M phase identified that radiation alone in NB1691 cells resulted in 34±6% cells in the G2M phase (P=0.0024), whereas the addition of SPARC overexpression and radiation exhibited a 28±4% (P=0.0076) increase in G2M phase cells compared with controls of which 12±3% cells were in the G2M phase. Similar results were observed when SK-N-BE(2) cells were treated with radiation alone, which resulted in a G2M phase accumulation of 39±4% (P=0.0006) and the overexpression of SPARC and radiation exhibited a G2M phase accumulation of 28±5% (P=0.0056; Fig. 1D).

To determine whether the overexpression of SPARC influences the expression of HSP27 in vivo, the nude mice subcutaneous tumor model of neuroblastoma was used in the current study (11). The results demonstrated that radiation exposure increased HSP27 expression in NB1691 and SK-N-BE(2) subcutaneous tumors. SPARC overexpression reduced the radiation-induced HSP27 expression indicating that SPARC may negatively regulate HSP27 (Fig. 2A). Quantitative analysis of HSP27 expression in the tumors identified radiation-induced HSP27 expression in 55±6% of NB1691 cells, whereas the addition of SPARC reduced the radiation-induced HSP27 expression to 12±2% of cells compared with untreated controls of which the expression rate of HSP27 was determined to be 8±2% (P=0.0162; Fig. 2B). In SK-N-BE(2) cell tumors that were exposed to radiation, HSP27 expression was increased to 59±2% and the addition of SPARC reduced the radiation-induced HSP27 expression to 16±4% (P=0.0026). To additionally determine whether SPARC causes a reduction in tumor burden, relative tumor density was evaluated by measuring the number of cells per unit area in a hematoxylin stained section. Untreated NB1691 tumors exhibited the highest tumor density at 138±10 cells per 100 µm². The addition of radiation treatment decreased the tumor density to 112±8 cells per 100 µm². Further addition of SPARC overexpression reduced the tumor density further to 68±5 cells per 100 µm² (P=0.0001 Fig. 2B) compared with the radiated controls. Tumor density of NB1691-derived tumors following treatment with SPARC alone was similar to the densities of irradiated tumors (110±6 cells per 100 µm²). SK-N-BE(2)-derived tumors exhibited similar tumor density patterns with the control group at 135±7 cells per 100 µm², SPARC overexpressing tumors at 100±12 cells per 100 µm², radiation treated tumors at 94±5 cells per 100 µm² and SPARC and radiation treated tumors at 65±11 cells per 100 µm² (P=0.0021; Fig. 2B). Similarly, in vitro cells treated with radiation induced the expression of the radiation response gene HSP27, in NB1691 and SK-N-BE(2) cells. Overexpression of SPARC accompanied with irradiation suppressed HSP27 expression in NB1691 (5-fold) and SK-N-BE(2) (2-fold) cells. Furthermore, it was also observed that the expression of p21 increased in SPARC overexpressing irradiated (5 Gy) cells: 4-fold in NB1691 cells; 2.5-fold SK-N-BE(2) cells (Fig. 2C and D). There was an inverse association between the expression of HSP27 and p21, in SPARC overexpressing irradiated environment (P<0.05), this is in accordance with a previous study that established that HSP27 protects cells from the p21 apoptotic pathway (Fig. 2E) (12).

To determine whether SPARC overexpression suppresses tumor cell migration, the in vitro wound healing assay (13) was performed. The overexpression of SPARC and irradiation reduced the migration potential of NB1691 and SK-N-BE(2) cells (Fig. 3A and B). Quantitative analysis demonstrated that SPARC overexpressing cells had a reduced migration potential in NB1691 (300±30 µm; R²=0.77) and SK-N-BE(2) (40±8 µm; R²=0.90) after 4 days of migration compared with controls (NB1691, 100±10 µm; SK-N-BE(2), 3±1 µm; Fig. 3C and D).

A previous study established that targeting HSP27 promotes radio sensitization (14). Previous studies have also demonstrated that HSP27 protects against mitochondrial-induced apoptosis (15,16). Therefore the present study aimed to determine whether SPARC facilitates the suppression of HSP27-mediated protection of mitochondrial Δψ. NB1691 and SK-N-BE(2) cells with overexpressed SPARC were irradiated and the mitochondrial Δψ was determined from MitoProbe™ JC-1 stained cells evaluated using flow cytometry analysis. SPARC overexpression reduced the mitochondrial Δψ to 70±3% and the addition of irradiation reduced the mitochondrial Δψ to 64±6% in NB1691 cells when compared to untreated control (P<0.05). In SK-N-BE(2) cells, SPARC overexpression alone reduced the mitochondrial Δψ to 46±3% and the addition of irradiation reduced the mitochondrial Δψ to 53±2% when compared with the untreated control (P<0.05) (Fig. 4A-C).

To determine the molecular function response genes in NB1691 and SK-N-BE(2) cells overexpressing SPARC and treated with irradiation, genome expression analysis was performed using the Allegiant whole genome array (Arrarstar Service). SPARC overexpression accompanied with irradiation induced the enrichment of genes associated with the death receptor pathway (<4 fold) and TNF receptor-associated molecules (<5 fold) in NB1691 cells. In SK-N-BE(2) cells, an enrichment of genes associated with HSP90 binding (<4 fold) and calcium channel regulator activity (<4 fold) were observed (Fig. 4D and E).

Caspase activation studies demonstrated that SPARC overexpression increased apoptotic events by 16±4% in NB1691 cells and by 24±8% in SK-N-BE(2) neuroblastoma cells, and the addition of irradiation increased the number of apoptotic events by 60±5% in NB1691 when compared to untreated controls (P<0.05) and by 67±12% in SK-N-BE(2) cells compared with untreated controls (P<0.05; Fig. 5A and B).