Cell lines and culture conditions
The neuroblastoma cell lines SK-N-AS and SH-SY5Y were maintained in RPMI-1640; and NB-1691 was maintained in DMEM, both supplemented with 10% FBS, 1% penicillin/streptomycin (P/S) and 1% L-glutamine (all from Quality Biological Inc., Gaithersburg, MD) at 37°C. To ensure consistency, a batch of cells was expanded, aliquoted and stored in liquid nitrogen prior to the screening. In each experiment, a vial of cells was defrosted and passaged 1:4 when 70% confluency was reached. Cells between passages 3 and 7 were used for all experiments.
Topotecan hydrocholoride (Hycamtin; GlaxoSmithKline, Philadelphia, PA) and Bortezomib (Velcade; Millenium Pharmaceuticals, Cambridge, MA) were reconstituted and stored according to the manufacturers' instructions. NSC 676914 was obtained from the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnostics, NCI/NIH.
High throughput siRNA screening
A set of synthetic siRNAs targeting 418 genes related to the apoptotic pathway (Qiagen Apoptosis Set V.1; Qiagen, Valencia, CA), with 2 siRNAs of different sequences per gene, was used for the first screen. For the second screen, 2 new siRNA pre-designed sequences were used (Qiagen). In the third confirmatory screen, one siRNA from each of the previous two screens was chosen. siRNAs were transfected at passage 4. Briefly, transfection reagent Dharmafect 1 (Dharmacon RNA Technologies, Lafayette, CO) was diluted in DCCR reagent medium at a ratio of 1:208 in volume. siRNA (20 nM) and 25 μL of the diluted transfection reagent were added to an individual well in a 96-well plate for complex formation with incubation for 20 min at RT. SK-N-AS cells were trypsinized, counted and resuspended in P/S free culture medium. 5000 cells were added to each individual well in 100 μL medium. The plate was incubated at RT for 30 min for cell attachment before being placed at 37°C for 24 h. Topotecan was then added to each well for additional 72 h incubation. Cell proliferation assay was performed at 96 h post siRNA transfection. The IC50 of topotecan for SK-N-AS cells at 24 h was 2 μM. In the 1st and 2nd screens, topotecan doses of 0, 1, 5 and 10 μM were used, whereas in the 3 rd screen, lower drug doses of topotecan at 0, 0.01, 0.1 and 1 μM were used to identify synergy. The criterion of hit selection for the enhancer genes was ≤0.8 fold cell growth compared to its own siRNA effect in the presence of topotecan. The final enhancer gene list was subjected to pathway analysis for identification of overrepresented genes within a target pathway. An inhibitor to the pathway was chosen and tested individually or in combination with topotecan in vitro and in vivo.
Cell proliferation assay
Cell proliferation was measured using Cell Titer Glo proliferation assay (Promega Corporation, Madison, WI) or a real-time cell sensing system (RT-CES; ACEA Biosciences, Inc. San Diego, CA) according to the manufacturer's instruction. We performed cell proliferation assays at a volume of 35 μL per well for 10 min and was measured at 562 nm on a Tecan plate reader (Tecan Inc., Durham, NC). Each treatment was performed in triplicates, averaged and normalized using untreated cells. For real-time cell electronic sensing assays, cells treated with drugs alone or in combinations or transfected with siRNA were added in triplicates to a 96-well plate device compatible with the real-time cell electronic sensing analyzer (RT-CES; ACEA Biosciences, Inc. San Diego, CA). Cell growth was monitored hourly for indicated durations via calculation of "cell index" (normalized impedance) for each well.
SK-N-AS cells were collected at 60 h post siRNA transfection. Total RNA was reverse transcribed using SuperScript II reverse transcriptase system (Life Technologies, Foster City, CA). The target regions were then pre-amplified using a standard PCR for 10 cycles. The pre-amplified cDNA was quantified by Taqman gene expression assay (Life Technologies) using Fluidigm digital array (South San Francisco, CA) according to the manufacturer's protocol. Fold expression was calculated using a comparative threshold cycle method (2-Δ ΔCT) .
The pathway analysis was performed using MetaCore (http://www.genego.com/metacore.php, GeneGo Inc., St Joseph, MI). MetaCore is an integrated software suite for functional analysis of experimental data and it contains curated protein interaction networks on the basis of manually curated database of human protein-protein, protein-DNA, protein-RNA and protein-compound interactions. Metacore uses a hypergeometric model to determine the significance of enrichment. The enhancer genes from our experiment and the genes in the GeneGo maps from the MetaCore database were used to identify the enriched GeneGo pathway maps.
Gene Set Enrichment Analysis
To investigate gene set enrichment, GSEA (http://www.broad.mit.edu/gsea/) was performed for genes, ranked by log2 ratio of gene expressions between topotecan-treated (1 μM and 10 μM) and untreated control SK-N-AS cells, using a weighted Kolmogorov-Smirnov-like statistics . The gene set of NF-κB target genes used in GSEA were downloaded from http://bioinfo.lifl.fr/NF-KB.
Cells were seeded on a 100 mm2 culture dish for overnight and were treated with topotecan alone or with bortezomib. For topotecan alone effect, SK-N-AS were treated with 5 μM of topotecan for 0, 3, 6, or 24 h at 37°C. For the combined effect of bortezomib and topotecan, cells were treated with bortezomib at 0, 1 and 10 nM for 24 h, followed by the addition of topotecan at 5 μM for 6 h. Total cell lysate was collected with RIPA buffer containing 1% phosphatase inhibitor and 1% protease inhibitor (all from Thermo Fisher Scientific, Rockford, IL). Protein concentration was measured by BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA) and protein lysates (20 ug per lane) were resolved on 4-12% TRIS-gradient gel (Invitrogen Life Technologies, Carlsbad, CA) and were transferred to nitrocellulose membranes by iBlot blotting system (Invitrogen Life Technologies). The membranes were blocked with 5% non-fat dry milk in PBS with 0.1% Tween 20 (PBST) for 1 h at RT, followed by incubation with mouse monoclonal antibodies against total or phosphorylated IκB-α (Ser32/36), rabbit monoclonal antibodies against total or phosphorylated p65/RelA or rabbit monoclonal antibody against NFKB1 (all from Cell Signaling Technology, Danvers, MA) at 4°C overnight. Peroxidase-conjugated goat anti-mouse or anti-rabbit antibody was used as secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h incubation at RT. Immunoreactive bands were visualized by ECL analysis system (GE Healthcare, Piscataway, NJ) and enhanced chemiluminescence. For loading control, the membranes were washed with Restore Western blot stripping buffer (Thermo Fisher Scientific) and probed with goat anti-human actin HRP conjugated antibody (Santa Cruz Biotechnology).
In vitro drug combination
Neuroblastoma cells were trypsinized, counted and resuspended in P/S free culture medium. 5000 cells per well in 100 μL medium were seeded in 96-well white plates for overnight. Topotecan and NSC 676914 or bortezomib were added individually or in combination at various doses and the plates were incubated at 37°C for 24 h and 48 h respectively. Cell proliferation assay was performed as described above. Combination index was calculated using CompuSyn software (ComboSyn Inc., Paramus, NJ). Briefly, the combination index theorem was used to quantify synergy or antagonism for two drugs by the formula C.I. = (D)1/(Dx)1 + (D)2/(Dx)2, where D1 and D2 are drug 1 and drug 2, and × is growth inhibition by X% .
The caspase-3 activity was measured using the PE Active Caspase-3 Apoptosis kit (BD Pharmingen, San Diego, CA). Briefly, SK-N-AS cells (untreated or treated with topotecan and bortezomib alone or in combination for 24 h) were trypsinized, fixed, and stained with PE rabbit anti-active caspase-3 antibody. Fluorescence intensity was measured by FACS Calibur and data were analyzed using CellQuest software (BD Biosciences, Franklin Lakes, NJ).
In vivo xenograft model
All animal experiments have been reviewed and approved by the NIH Animal Care and User Committees. A minimal residual disease xenograft model in mice bearing neuroblastoma was established in 8-10 week-old female SCID Beige mice (Charles River Laboratories, Fredrick, MD). Briefly, five million SK-N-AS cells expressing luciferase (gift from Dr. Bryan Clary, Duke University Medical Center) were injected intravenously via the lateral tail vein into the mice. Tumors were allowed to grow for 7 d, and then mice were randomly assigned to cohorts treated with topotecan and bortezomib administered individually or in combination, or with saline solution (control mice). Bortezomib (0.6 mg/kg) was injected intraperitoneally three times a week for two weeks, rested for two weeks and repeated with another course of treatment. Topotecan (0.5 mg/kg) was injected intraperitoneally five times a week for two weeks, rested for two weeks, followed by another course of treatment. Body weight and general wellness of the mice were monitored, and tumor size was monitored by Xenogen IVIS 100 imaging system (Caliper Life Sciences Inc., Hopkinton, MA). The in vivo xenograft experiment was repeated, and results from two independent experiments were combined (n = 43).
Non-parametric Mann-Whitney test was used to compare among various groups in cell growth assay. For relative luciferase intensity results from two independent in vivo experiments, we normalized the log2 transformed intensities from each experiment using median-centered method and then combined the results. T-test was used to compare the difference of two groups.