Drugs and Reagents
I-MIBG was purchased from GE Healthcare (Sunnyvale, CA, USA). Radiolabeled 3
H-norepinephrine was purchased from Perkin Elmer Life Sciences (Waltham, MA, USA). All other chemicals were purchased from Sigma (St. Louis, MO, USA). Vorinostat was synthesized in our laboratory using a combination of previously published methods.9,10
All the experiments described herein were also conducted utilizing a clinical grade sample of vorinostat obtained from Merck for comparison purposes. Antibodies for Western blotting were obtained from the following sources: rat polyclonal anti-NET antibody from Alpha Diagnostic International (NET11-A; San Antonio, TX, USA), horseradish peroxidase-conjugated anti-rabbit (sc-2004; Santa Cruz) immunoglobulin was used as the secondary antibody and mouse monoclonal anti-β-actin antibody from Santa Cruz Biotechnology (Santa Cruz, CA). Lipofectamine 2000 and hygromycin B were from Invitrogen (Carlsbad, CA). The cell culture media DMEM H-21, RPMI 1640, and fetal bovine serum (FBS) were obtained from the Cell Culture Facility of the University of California, San Francisco (San Francisco, CA).
Cell Lines and Transfection
Cell lines stably transfected with human NET, HEK-hNET and empty vector, HEK-EV, were established by transfection of pcDNA5/FRT vector (Invitrogen) containing the full-length hNET cDNA and pcDNA5/FRT vector alone, respectively, into human embryonic kidney 293 (HEK293) Flp-In cells using LipofectAMINE 2000 (Invitrogen) as per manufacturer’s instructions. The stable clones were selected with 75 μg/mL hygromycin B. All the NB cell lines (LAN5, SHEP, SH-SY-5Y, Kelly, SK-N-SH, SK-N-MC, NB1691 and NB1691-luc) used in the present study were obtained from the UCSF Cell Culture Facility.
Stably transfected HEK293 cells were maintained in DMEM H-21 medium supplemented with 10% FBS, 100 units/mL penicillin and 100 units/mL streptomycin and 75 μg/mL hygromycin B. The culture medium for all the NB cell lines was RPMI 1640 containing 15% FBS, 1% MEM non-essential amino acids, 100 units/mL penicillin, and 100 μg/mL streptomycin. All cell lines were grown at 37°C in a humidified atmosphere with 5% CO2/95% air.
Cellular Uptake of 3H-NE or 123I-MIBG
HEK-hNET cells were seeded in 24 well poly-D-lysine coated cell culture plates (BD Biosciences) in standard cell culture media and the uptake studies were conducted 24 hours after cell seeding. Cells were incubated in PBS buffer containing 15 nM 3H-NE in the presence or absence of a NET specific inhibitor, desipramine (10 μM), at 37°C for 3 minutes. After 3 minutes, uptake was terminated by rapid removal of the buffer containing the compounds and three quick washings with ice-cold PBS buffer. The cells were lysed with 1 mL of 0.1% SDS/0.1 N NaOH solution for about one hour and the intracellular levels of radioactive NE were determined using liquid scintillation counting. Intracellular concentrations of norepinephrine were normalized to the total protein in each well using bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL).
The same procedure was followed for determination of 3H-NE or 123I-MIBG uptake rate in NB cell lines, SH-SY-5Y and Kelly. In this case, cells were exposed to increasing concentrations of vorinostat ranging from 0 to 5 μM for 24 h, followed by exposure to 3H-NE or 123I-MIBG in PBS buffer, at 37°C for 3 min. The intracellular levels of radioactivity corresponding to 3H-NE were determined as described earlier and were normalized to the total protein content in each well using BCA protein assay. The total concentration of 123I-MIBG was measured in an automated Wallac 1480 Wizard gamma counter using the program RiaCalc Wiz (Wallac Oy; Turku, Finland). .
Total RNA was isolated from each of the NB cell line on 6-well plates using the Invitrogen Micro-to-Midi Total RNA Purification System according to the manufacturer’s protocol. Extracted RNA was stored at −80 °C until use. Similarly, total RNA was isolated from 13 NB tumor samples. Samples of NB tumors were obtained from the UCSF Pediatric Solid Tumor Tissue Bank following procedures approved by the UCSF Committee on Human Research. Four samples were collected at initial diagnosis, eight samples were collected after the initiation of therapy, and the timing of sample collection was unknown for one sample. Five of the tumor samples were from initial diagnosis (no prior treatment). All of the other samples were from patients who had received some type of therapy.
Reverse transcriptase-polymerase chain reaction
Reverse transcription (RT) PCR of RNA samples was carried out with Superscript III (Invitrogen) using oligo(dT)20 primers. Two microliters of RT reaction product was used for subsequent PCR (Taq DNA Polymerase, Invitrogen) consisting of 35 cycles with the following parameters: 94 °C for 30 s, 60 °C for 45 s, 72 °C for 1 min, followed by a final extension of 72 °C for 10 min and storage at 4 °C. Primers were designed to amplify a unique sequence of human NET (SLC6A2), each spanning intron–exon boundaries to ensure no genomic DNA was amplified. The PCR primers that were used for human: NET (Genbank accession number NM_001043) forward— ATTCTTCAAAGGCGTTGGCTAT and reverse— CAGGACACCACGCTCATAAA were designed to amplify a 262-bp fragment. Analysis of each PCR sample was then performed on 2% agarose gels containing 0.5 μg/ml ethidium bromide. Gels were visualized using a digital camera and image processing system (Kodak, Rochester NY, USA). RT-PCR analysis for GAPDH gene was used as an internal control for each sample.
Quantitative Real-Time PCR
For qRT-PCR analysis, 2 μg of total RNA isolated from NB cells and tumor samples were reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosytems, CA, USA) in a 20 μL volume reaction according to the manufacturer’s protocol. The resulting cDNA was used as template for quantitative real-time PCR using TaqMan® Gene Expression Assays for human transporter NET (Assay ID: Hs00426573_m1*) and human GAPDH (Assay ID: Hs99999905_m1). Quantitative real-time PCR reaction was carried out in 96-well reaction plates in a volume of 10 μL using the Taqman® Fast Universal Master Mix (Applied Biosystems, Foster City, CA). Reactions were run on the Applied Biosystems 7500 Fast Real-Time PCR System with the following profile: 95 °C for 20 seconds followed by 40 cycles of 95 °C for 3 seconds and 60 °C for 30 seconds. The relative expression of each mRNA was calculated by the comparative method (ΔΔCt method). Firstly, the ΔCt value for each sample was obtained by subtracting the Ct value of GAPDH mRNA from the Ct value of the NET mRNA. Then the ΔΔCt value for each sample was obtained by subtracting the ΔCt value of another endogenous control, PGK1, from the ΔCt value of each sample. The mRNA expression level of NET was reported as the percentage of the expression of PGK1 calculated using the arithmetic formula 2−ΔΔCt (ABI PRISM 7700 Sequence Detection system User Bulletin No. 2, P/N 4303859) multiplied by 100.
Western blot analysis
The total protein extracts from transfected HEK 293, NB cells and tumor samples were prepared using CellLytic M cell lysis buffer (Sigma) containing protease inhibitor cocktail at 4 °C for 10 min. The cell homogenate was spun for 10 min at 14,000 RPM at 4°C and the protein concentration in the supernatant was determined by the BCA protein assay. Approximately 50 μg of the supernatant was resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto PVDF membrane (Bio-rad). The blots were incubated for 1 h in blocking buffer containing 5% nonfat dry milk in Tris-buffered saline (TBS) and then incubated overnight at 4 °C with anti-human NET1 antibody diluted (1:750) in 2% milk in TBS buffer containing 0.1% Tween 20 (TBS-T). After washing with TBS-T, the blots were incubated for 1 h at room temperature with the goat anti-rabbit IgG HRP conjugated secondary antibody (1:2500 dilution) in TBS-T buffer containing 5% milk. The blots were again washed thrice with TBS-T for 10 minutes and then developed with the ECL western blotting detection reagent (GE Healthcare). All blots were re-probed for β-actin (1:10,000) as a loading control. Band intensities of scanned blots were quantified using ImageJ. The integrated intensity of a fixed area was measured, and background levels were subtracted.
The cytotoxicity of MIBG was measured by standard MTT assays in 96-well plates using NB cell lines. After seeding the cells at the desired density and incubating overnight, vehicle control (5% DMSO in PBS) or vorinostat (5 μM) was added to the culture medium. After 24 hours of treatment, the drug-containing medium was replaced with medium containing MIBG at various concentrations and the incubation was continued for additional 72 hours. At the end of the incubation, 20 μL of MTT stock solution (5 mg/mL) was added to each well. After additional incubation for 3 h at 37 °C, the MTT reaction medium was discarded and the purple-blue MTT formazan crystals were dissolved by the addition of 100 μL of 0.1 N HCI in isopropanol. The optical density (OD), which is a measure of the mitochondrial function of the viable cells, was read directly with a microplate reader (model versamax, Molecular Devices Co., CA, USA) at 580 nm and a reference wavelength of 680 nm. Concentration response graphs were generated for each drug using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA). These graphs were analyzed using a curve fit for sigmoid dose-response, and IC50 values were derived. Results are expressed as mean IC50 values with the standard error of the mean.
Female CrTac:NCr-Foxn1nu athymic mice (4 to 5 weeks old) were purchased from Taconic (Germantown, NY, USA) and housed under aseptic conditions, which included filtered air, sterilized food, water, bedding, and cages. The experiments on mice were approved by the Institutional Animal Care and Use Committee of University of California at San Francisco. All experimental and animal handling procedures were in accordance with national ethical guidelines.
Preparation of Neuroblastoma Xenografts and Drug Treatment
To study the effect of vorinostat treatment on NET expression in vivo, nude mice harboring xenografts of human NB1691 neuroblastoma cells expressing luciferase (NB1691luc) were used. For preparation of NB xenografts, NB1691luc cells were resuspended in serum free RPMI1640 media and 100 μl of this mixture containing 6 × 106 cells were injected in mice by i.v. tail vein injection. Serial tumor measurements were obtained by weekly bioluminescence imaging after an intraperitoneal injection of d-luciferin (Xenogen) at 15 mg/mL in sterile PBS. Twenty-one days after injection of tumor cells, the animals were divided into vehicle control (5% DMSO in saline) and vorinostat (150 mg/kg i.p.) treated groups (2 per group). Tumors were then harvested from each animal at various time points (1, 3, 6, and 18 hours) after initiation of treatment and lysed with a buffer containing (20 mM Tris HCl pH 7.5–8.0, 150 mM NaCl, 0.5% sodium deoxycolate, 1% Triton X100, 0.1 sodium dodecyl sulfate (SDS), 1 mM EDTA, 1 mM phenylmethylsulphonyl fluoride, and protease inhibitor cocktail). qRT-PCR and Western blot analysis of these tissues was carried out as described under Quantitative Real-Time PCR and Western Blotting section.
In order to study the effect of vorinostat administration on tissue expression of NET in healthy mice not bearing tumors, nude mice (5 per group) were injected with vorinostat (150 mg/kg i.p.) and various tissues were harvested 6 and 18 hours after drug treatment. Each of these tissues was then subjected to total RNA extraction and qRT-PCR analysis as described before.
Biodistribution Measurements of 123I-MIBG
Animals with NB xenografts (described above) were divided into two groups (3 per group) in the following manner: (1) control group = injected with vehicle (5% DMSO in saline i.p.) six hours prior to 123I-MIBG (300 μCi/mouse) by i.v. tail vein injection, (2) treatment group = injected with vorinostat (150 mg/kg i.p.) six hours before i.v. tail vein injection of 123I-MIBG. Two hours after the treatment, the mice were euthanized. Samples of liver, kidney, spleen, heart, and tumor were excised, weighed and their associated radioactivity was measured in an automated Wallac 1480 Wizard gamma counter using the program RiaCalc Wiz (Wallac Oy; Turku, Finland). Blood was also sampled and radioactivity content was normalized to its weight. The uptake of 123I-MIBG was expressed as the fraction of the injected dose per gram of tissue. A correction was made for radioactive decay, which would take place since the time of injection.
Data were analyzed statistically by unpaired or paired Student t tests, as appropriate. Probability values lower than 0.05 were considered statistically significant.