Study approval and informed consent
This study was approved by the ethical review board of Tokushima University Hospital for human study and the ethics committee of Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan. Human tissue samples were obtained during routine clinical procedures after informed consent including the use of tissue sample from patients with brain tumors at the Department of Neurosurgery, Tokushima University Hospital. For the use of samples obtained from patients, we have obtained a statement attesting to informed consent for all patients with or without neurosurgery in our department. Each sample was fixed in 4% formalin in phosphate-buffered saline (PBS) and processed for paraffin embedding. The samples were classified by neuropathologists in accordance with the WHO classification of brain tumors. Sections from non-neoplastic regions (NNRs) were purchased from BioChain Institute (Newark, NJ, USA). The study was performed in accordance with the tenets of the Declaration of Helsinki. All animal experiments were approved and performed in accordance with the animal care guidelines of Tokushima University.
Human GBM cell line U251MG were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 medium (Invitrogen, NJ, USA) with 10% fetal bovine serum (GIBCO-BRL, NY, USA) at 37°C in an atmosphere of 5% CO2 and 95% humidified air. Mice GSCs were established and provided by OU and HS, Keio University.19,20 GSCs were cultured in Dulbecco’s Modified Eagle’s medium/nutrient mixture F-12 Ham (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 20 ng/ml recombinant human epidermal growth factor (PeproTech, Rocky Hill, NJ, USA), 20 ng/ml recombinant human basic fibroblast growth factor (PeproTech), B-27 supplement without vitamin A (Life Technologies, Carlsbad, CA, USA), 200 ng/ml heparin sulfate, 100 U/ml penicillin, and 100 µg/ml streptomycin (Nacalai Tesque, Kyoto, Japan).
Cell Viability Assay
GSCs (1×103 cells/well) were plated in 96-well tissue culture plates. To enumerate viable cells, the conversion of WST-8 to formazan by metabolically active cells was quantified using WST-8 reagent (Dojindo, Osaka, Japan) on a microplate reader (Infinit F200 PRO®, TECAN) at 450 nm. We used PBS-treated cells as the control to represent 100% viability and the percent viability was determined in each treatment.
Establishment of the animal model and assessment of anti-tumor effects
Six-week-old male C57BL/6 mice were subjected to inhalation anesthesia with isoflurane and a stereotactic apparatus was placed in the right brain. With a dental drill, a small hole was bored into the skull 2.0 mm lateral to the bregma. Using a malignant glioma model with mouse GSCs established by Sampetrean, O and Saya, H et al,20,,21 GSC progeny cells (1×103) in 2 μl of Hank’s balanced salt solution (Sigma-Aldrich) were injected into the right cerebral hemisphere 3 mm below the brain surface, using a 10-µl Hamilton syringe. To examine the anti-tumor effects of SDT, the mice were randomized and treated with 5-ALA, US, or SDT and compared to the non-treated control (Figure 2). For SDT, a 0.2-ml solution of 5-ALA in PBS was intraperitoneally injected at a dose of 200 mg/kg body weight. Three hours later, the mouse right brain was placed on the stereotactic apparatus and subjected to US imaging (1 MHz, 2 W/cm2 for 2 min) under inhalation anesthesia. On day 1 after SDT, apoptosis induction by SDT was confirmed and tumor volume on days 3, 7, and 14 and the survival rate were analyzed. Mice were euthanized and their brains were sliced on a brain slicer matrix at 1.0-mm intervals and the tumor volume, represented by the GFP-positive area, was microscopically determined (Keyence BZ-X710, Osaka, Japan).
In addition, to assess the effect of combination therapy of SDT and celecoxib, another set of mice were randomized and treated with vehicle, celecoxib, SDT, or a combination of celecoxib and SDT (Figure 4). Celecoxib, lysed with dimethyl sulfoxide (DMSO) and hydroxypropyl-β-cyclodextrin (HBC), was injected (i.p.) at a dose of 10 mg/kg consecutively after mouse GSC implantation. Vehicle controls received equivalent doses of DMSO/HBC and normal saline at the same dosing schedule. To validate the efficacy of celecoxib during SDT, tumor volume on day 14 and the survival rate during the observation period were assessed in each group (Figure 4) as described above.
Measurement of cellular PpIX levels and SDT
Luminescence was measured 1, 2, 3, 6, and 12 h after treatment of human GBM U251 cells with 1 μM 5-ALA in human GBM U251 cells, using the image analyzer in the BZ-X710 microscope (KEYENCE). Thereafter, the viability of GSCs upon combination therapy with SDT and US (1 MHz, 2 W/cm2 for 2 min)at 3 h after 5-ALA treatment was assessed after 24 h.
Celecoxib (Sigma-Aldrich, Cat. #PHR1683) was dissolved in DMSO and supplemented in the culture medium at a final concentration of 60 μM. After 3-h incubation of cells in medium supplemented with 1 μM 5-ALA (SBI ALA promo, Tokyo, Japan), the medium was replaced with fresh complete medium, and the 96-well plate was exposed to LED irradiation (630 nm, 80 mW/cm2) for 5 min. The LED light spot was an equally illuminated rectangular spot encompassing the entire culture plate. SDT was performed using the ultrasonic generator UST-770 (ITO Co. Ltd., Tokyo, Japan). In the mouse GSC-bearing glioma model, the tumor area was extracted 3 h after treatment with 200 mg/kg 5-ALA and PpIX levels were analyzed as previously described.20
Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated and extracted using the MagNA Pure RNA isolation kit (Roche, Tokyo, Japan) and the MagNa lyser (Roche), in accordance with the manufacturer’s instructions. We used Transcriptor Universal cDNA Master (Roche) to reverse-transcribe total RNA to cDNA and a LightCycler 2.0 (Roche Diagnostics, Tokyo, Japan) for qRT-PCR. The following primers were used: mouse mdr1, 5’-primer, GGC ATT GCC TAC CTG TTG G-3’; 3’-primer GCT TTC TGT GGA CAC TTC TG, and mouse glyceraldehyde-3-phosphate dehydrogenase (Gapdh), 5’-CAG AAC ATC ATC CCT GCA TC-3’ and 5’-CTG CTT CAC CAC CTT CTT GA-3’. The mRNA levels were normalized to those of Gapdh. The PCR conditions were as follows: 95℃ for 10 min, followed by 40 cycles at 95℃ for 10 s, 60℃ for 10 s, and 72℃ for 8 s. We subjected 4 samples in each group to the qRT-PCR assay to determine the gene expression levels.
Western blot analysis
According to our previous study (6), cells or tissue samples were homogenized in RIPA buffer containing a protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Cat. #5872). After 10-min centrifugation at 12000 rpm, 4℃, the protein concentration in the supernatants was determined using BCA kit (Thermo Fisher Scientific, USA). Protein (20 or 50mg) was separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (immune-blot PVDF membrane, BIO-RAD, Hercules, CA, USA) by electroblotting. The membrane were immersed in blocking buffer (5% skim milk or 2% BSA in tris-buffered saline, TBS) for 1 h and incubated with primary antibodies: anti-MDR1 (BD Biosciences, NJ, USA, 1:1000), anti-cCaspase-8, -9, -3 and anti-PARP (Cell Signaling Technology, MA, USA, 1:1000), anti-AKT2 (Abcam, Cambridge, UK, rabbit, 1:1000), pAkt (Santa Cruz Biotechnology, CA, USA, rabbit, 1:500), anti-pNF- kB (Cell Signaling Technology, 1:1000), anti-pI-κB (Cell Signaling Technology, 1:1000) and β−actin (Sigma-Aldrich, mouse, 1:5000) were diluted in Can Get Signal Solution 1 (Toyobo). After washing in Tween-TBS (T-TBS), the membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies in Can Get Signal Solution 2 (dilution 1:3000). After washing, the protein-antibody complexes were detected with Amersham ECL prime Western blotting detection reagents (GE Healthcare, UK) using a Lumino image analyzer (Image Quant LAS-4000 mini, GE Healthcare Japan, Tokyo, Japan) and ImageJ 1.52 software (NIH, Bethesda, MD, USA) was used to analyze the protein expression levels. Each experiment was repeated four times.
Murine tissue samples were fixed with 4% paraformaldehyde and 5-μm-thick frozen sections were mounted on Matsunami adhesive saline (MAS)-coated glass slides (Matsunami Glass, Tokyo, Japan). As previously reported (6), human glioma tissue sections from the paraffin-embedded block were dewaxed, rehydrated, and subjected to antigen retrieval. The sections were blocked for 30 min with 1–3% hydrogen peroxide solution, and stained overnight at 4℃ with the following antibodies: anti-MDR1 (D-11) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, 1:100), anti-COX-2 (Abcam, Cat. # ab15191), rabbit monoclonal anti-MDR1 (Abcam; ab170904; 1:100), anti-cleaved caspase-8 (cCasp-8), anti-cCasp-9, anti-cCasp-3, and anti-PARP (Cell Signaling Technology, 1:1000). Thereafter, they were incubated with biotinylated secondary antibody (30 min, 30℃), visualized using DAB buffer tablets, and counterstained with hematoxylin. Photographs were obtained under a light microscope, using KEYENCE BZ-X710.
Survival estimates and median survivals were determined using Kaplan-Meier survival curves. A log-rank (Mantel-Cox) test was performed to determine the p-values derived from Kaplan-Meier survival curves. To determine statistical significance, between-group comparisons were performed using Student’s t-test. For multiple comparisons, one-way ANOVA, followed by the Tukey-Kramer tests. Error bars indicate the standard deviation values. All statistical analyses were performed using JMP 13.2 (SAS Institute Inc.) and the differences with p <0.05 were considered significant.