The human NB cells SH-SY5Y, MC-IXC, SK-PN-DW and IMR-32 were obtained from ATCC (Manassas, VA). Culture and maintenance of the NB SH-SY5Y, IMR-32, and SK-PN-DW cells were performed as described in our earlier studies (Veeraraghavan et al. 2011; Aravindan et al. 2013a). For passaging and for all experiments, the cells were detached by using 0.25% trypsin/1% EDTA, resuspended in complete medium, counted (Countess, Invitrogen), and incubated in a 95% air/5% CO2 humidified incubator. For all experiments, the cells were serum-starved by incubating in medium containing 2% serum for at least 12 h, unless otherwise specified.
Radiation and inhibition studies
For RT experiments, cells were exposed to 2Gy using a Gammacell 40 Exactor (Nordion International, Inc., Ontario, Canada) at a dose rate of 0.81Gy/min and were then incubated at 37°C. Mock-irradiated cells were treated identically except that cells were not subjected to RT. Irradiated cells were incubated for an additional 24 h through 72 h. All experiments were repeated at least three times in each group. The influence of RT-induced NFκB-dependent activation of MMP9 in orchestrating the positive feedback cycle (PFC) was investigated by selectively inhibiting RT-induced MMP9 with 10 µM of GM6001 (Galardin/ilomastat; Cayman Chemicals, Ann Arbor, MI), a potent cell-permeable MMP9 inhibitor. Further, MMP9 specificity was cross validated with a broad spectrum (MMP9 non-selective) serine protease inhibitor (Kuyvenhoven et al. 2004) aprotinin (6.64 nM, ~ 0.35 TIU), which competitively and reversibly inhibits the activity of different esterases and proteases (Soleyman-Jahi et al. 2019). Cells were treated with GM6001 or aprotinin for 3 h prior to RT exposure. In addition, as a reference standard for NFκB dependent activation of MMP9, we used Phorbol 12-myristate 13-acetate (PMA, 10 nM, Cayman Chemical), a polyfunctional diterpene phorbol ester. PMA is an activator of protein kinase C and upregulates MMP-9 in a PKCα-NF-κB dependent manner (Shin et al. 2007).
Plasmid preparation, DNA transfection, and luciferase reporter assay
NFκB p65 and p50 subunits were transiently transfected into the NB cells following the lipofection method utilizing EffecteneTM reagent (Qiagen) as in our earlier studies (Mohan N et al. 2002). NFκB inhibition was achieved using transient transfection of s32A/s36A double mutant IκBα (ΔIκBα) as discussed earlier (Aravindan et al. 2013b). The mutated form of IκBα with a serine-to-alanine mutation at residues 32 and 36 does not undergo signal-induced phosphorylation, and thus remains bound to NFκB, subsequently preventing nuclear translocation and DNA binding. MMP9 overexpression with full-length human untagged MMP9 expression vector and MMP9 inhibition with human MMP9 shRNA plasmid kit (4 unique 29mer constructs) in retroviral RFP vector (Origene, Rockville, MD) were achieved by transient transfection utilizing TurboFectin transfection reagent. In addition, the plasmid construct pNFκB-Luc was amplified, purified, and transfected as in our earlier studies (Aravindan et al. 2013b). Cell lysates were assayed for luciferase activity as per the manufacturer’s protocol (Biovision Research Products, Mountain View, CA).
SH-SY5Y, MC-IXC, and IMR-32 cells seeded in 100-mm plates were irradiated and the conditioned medium (CM) was collected after 24, 48, and 72 h. CM collected from mock-IR plates at 72 h was used as controls. CM was concentrated using nanosep 30K concentrators (Pall Biotech, Westborough, MA), and an equivalent volume was subjected to 10% SDS-PAGE containing gelatin (2 mg/ml). The gels were washed in 2.5% Triton X-100 (3X) and incubated in the buffer (50 mM Tris–HCl, pH 7.6; 10 mM CaCl2; 50 mM NaCl and 0.05% Brij35) for 16 h at 37 °C. Gels were then stained with Coomassie brilliant blue R-250 (0.25% in 40% methanol and 10% acetic acid). The MMP activities were visualized as digested bands in a Canon RE350 video visualizer.
Kinetics of MMP9 activity using a fluorogenic substrate
The kinetics of MMP9 activity were measured using a specific fluorogenic substrate (DNP-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2, Calbiochem), a peptide with the fluorescence on one end and a quencher on the other. After the cleavage into two separate fragments by MMP, the fluorescence is reclaimed and can be quantified in real-time. Equal volumes of CM from the SH-SY5Y, IMR-32, and MC-IXC cells exposed to mock-IR or RT (2Gy) were collected after 2 h (selected for MMP9 activity) and concentrated using nanosep 30K concentrators (Pall Biotech). Concentrated CM (20 µL) in duplicate were mixed with fluorogenic substrate (20 µM, dissolved in DMSO) in 96-well plates for a total volume of 50 µL using assay buffer (0.5 M Tris-HCl, pH 7.7; 5 mM CaCl2; 0.2 M NaCl). Kinetics of the MMP9 activity were immediately and continuously (every 20 minutes for 20 h) quantified by measuring the fluorescence intensity (excitation 280 nm; emission 360 nM) using a Synergy II micro plate reader (Biotek). Group-wise comparisons of MMP9 activity were performed using GraphPad Prism.
Total protein extraction and immunoblotting were performed as described in our earlier study (Veeraraghavan et al. 2011). The protein-transferred membranes were incubated with mouse monoclonal anti- MMP-9, IKKβ, p65 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit polyclonal anti- ERK and phosphor ERK antibodies, and developed with the appropriate anti-mouse/anti-rabbit antibodies (BioRad Laboratories, Hercules, CA). Blots were stripped and reblotted with mouse monoclonal anti-α-tubulin antibody (Santa Cruz) to determine equal loading of the samples. Band intensity analysis was quantified using Quantity One 1D image analysis software (BioRad). Group-wise comparisons were performed using GraphPad Prism.
Cell-microarray construction and immunohistochemistry
The cell microarray (CMA) approach allows us to measure changes in protein translation across the treated samples, without inter-sample assay discrepancies. For this, human IMR-32 cells (i) exposed to mock-IR, (ii) RT (2Gy), (iii) PMA, (iv) after ectopic expression of p50/p65, (v) treated with GM6001 and exposed to RT, (vi) treated with aprotinin and exposed to RT, and (vii) transfected with ΔIkB and exposed to RT were collected 24 h post-RT. CMA construction, sectioning, and IHC were performed in our Tissue Pathology Core of the Stephenson Cancer Center following standard protocols, as described in our earlier work (Somasundaram et al. 2019). Appropriate histology controls (H&E) and negative controls with no primary antibody were examined in parallel. Expression and localization of MMP9, NFκB- p105, RelB, pP65, p65, IκBα, pIκBα, ERK, and pERK are investigated. The slides were digitally scanned into virtual slides using a Zeiss Axio Scan Z1 slide scanner at 40× magnification. The whole slide images were then group-analyzed for protein-specific positivity using Aperio image analysis and quantification software (Aperio Technologies, Inc., Buffalo Grove, IL, USA) with the appropriate algorithms for protein specific cellular localization. Group-wise comparisons were performed using GraphPad Prism.
MAP kinase/ERK kinase assay
MMP9-dependent modulation of RT-altered ERK activity was assessed using a MAP kinase/ERK immunoprecipitation kinase assay kit (Millipore Sigma, Burlington, MA) following the manufacturer’s protocol. First, anti-MAPK/Erk1/2 agarose conjugate was washed (2X) and re-suspended in assay buffer (50 mM Tris, pH 7.5; 1 mM EDTA; 1 mM EGTA; 0.5 mM Na3VO4; 0.1% 2-mercaptoethanol; 1% Triton X-100; 50 mM sodium fluoride; 5 mM sodium pyrophosphate; 10 mM sodium beta-glycerol phosphate; 0.1 mM PMSF; 1 ug/mL aprotinin; 1 ug/mL pepstatin; 1 ug/mL leupeptin; 1 uM microcystin). Fresh lysates (500 µg) from cells exposed to mock-IR; RT with/without aprotinin, GM6001, ΔIκBα; NFκB overexpressed; or treated with PMA were added to the anti-MAPK/Erk1/2 agarose conjugate and were allowed to form an immunocomplex for 2 h at 4 °C. The immunocomplex was washed in assay buffer (3X) and in assay dilution buffer (2X, 20mM MOPS, pH 7.2; 25 mM β-glycerol phosphate; 5mM EGTA; 1mM sodium orthovanadate; 1mM dithiothreitol). An aliquot (10 µL) of the agarose/enzyme immunocomplex was mixed with inhibitor cocktail, MAP Kinase/Erk Substrate Cocktail II, and Mg2+/ATP Cocktail in assay dilution buffer and incubated for 20 minutes at 30 °C. The samples were then resolved in SDS-page gel, transferred to PVDF, and labelled with anti-phospho MBP. Band intensity analysis was quantified using Quantity One 1D image analysis software (BioRad). Group-wise comparisons were performed using GraphPad Prism.
Cell death (TUNEL) analysis
RT-triggered NFκB-dependent MMP9 activation and maintenance-associated induced NB cell death, if any, was quantified at the single-cell level based on labeling DNA strand breaks with terminal deoxynucleotidyl transferase (TUNEL Assay). All TUNEL assay procedures were performed on the customized CMA (constructed as discussed above) in the SCC-Tissue Pathology Core using a commercially available In Situ cell death detection kit (MilliporeSigma, St. Louis, MO, USA). Appropriate positive (recombinant DNAse I treatment before TUNEL labeling) and negative (without Tdt enzyme mix) controls were included. The slides were micro-digitally scanned using an Aperio Scanscope (Aperio Technologies, Inc., Buffalo Grove, IL, USA) slide scanner. TUNEL positivity was observed using NIH ImageJ, plotted with GraphPad Prism, and compared between groups using ANOVA with Tukey’s post-hoc correction.
QPCR profiling of tumor invasion/metastasis signaling and bioinformatics analysis
Total RNA extraction and real-time QPCR profiling were performed as described in our earlier studies (Aravindan et al. 2013b). We used our custom archived human tumor invasion and metastasis signaling pathway profiler (Realtimeprimers.com, Elkins Park, PA) containing 93 genes to assess the direct effect of RT-induced MMP9 in orchestrated tumor progression. Twenty-seven of the included genes (CD44, CCR7, CTSB, CTSL1, EREG, HGF, ID1, IL1B, KISS1, MCAM, MMP1, MMP13, MMP3, MMP9, MYC, NF2, NME4, PTEN, PTGS2, SERPINE1, SPARC, SPP1, SYK, TIMP2, TNC, TP53, and VEGFA) are known to contain NFκB response elements, allowing us to define how MMP9 alteration modifies the NFκB-dependent response. The ΔΔct values were calculated by normalizing the gene expression levels to positive controls (β-actin, GAPDH, Hprt1), compared between groups, and the relative expression level of each gene was expressed as a fold change. Differential gene expression analysis with stringent criteria (log2 fold change) coupled with false discovery rate calculation were used identify the genes altered with RT, with and without inhibition of RT-activated MMP9. To investigate the functional relevance of modulated genes in tumor progression, we utilized ingenuity pathway analysis (IPA). Core analysis on the genes that showed significant differential changes with/without MMP9 after RT were selectively annotated (defined or predicted data availability) and subjected to downstream analysis by the IPA. Core analysis was performed with criteria including a direct relationship with causal path scoring for networks and upstream regulator analysis and experimentally observed confidence level. Significant associations of the genes with diseases, molecular and cellular functions, and canonical pathways and networks were examined.