Ethical consideration
In this study, the institutional and national guide for the care and use of laboratory animals was followed and the protocols used were approved by the Institutional Review Board of Kansai Medical University.
Reagents
99mTc MIBI (Cardiolite; Fujifilm RI Pharma., Co., Ltd., Tokyo, Japan) and doxorubicin (Adriamycin; Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan) were used as tracers. A stock solution of 99mTc MIBI was prepared by mixing 10 MBq of 99mTc MIBI with 2 mL of saline solution; this solution was then used in the in vitro and in vivo experiments [10,11]. A stock solution of doxorubicin was prepared by mixing 1 mg of doxorubicin with 1 mL of saline solution. This solution was then used in the in vivo experiment. A stock solution of MDR modulator was prepared from an acridone carboxamide derivative, GG918, (N-{4-(2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl)-phenyl}-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide; Elacridar; MedChem Express, LLC, Monmouth Junction, NJ, USA) [7,12].
Cell culture and xenografts
In vivo and in vitro experiments were conducted using NSCLC cells (H1299), transfected with a wild-type P53-encoding gene, which was supplied by Hideki Matsumoto, Ph.D., Oncology, Biomedical Imaging Research Centre, Fukui University, Japan [13]. ICR nude mice (Crlj: CD1-Foxn1nu) were used for xenografting (Charles River Laboratories, Wilmington, MA, USA) [14].
For the in vitro study, H1299 cells were cultured as monolayers in Dulbecco’s alpha-MEM medium (D-MEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% (v/v) foetal bovine serum (Equitech-Bio, Kerrville, TX, USA), 50 μg/mL streptomycin, and 200 μg/mL geneticin and were incubated at 37°C in 95% air and 5% CO2. Prior to culturing, the cells were subjected to 3-, 6-, and 9-Gy (irradiated groups) gamma irradiation (Gammacell 40 Exactor, Nordion International, Ottawa, Canada). Cells from the irradiated groups and the not-irradiated group were cultured for 48 h as previously described. H1299 cells were trypsinized and centrifuged, and each cell suspension was diluted to a density of 0.5 to 1.0 × 106 cells/mL [10,11].
For the in vivo study, H1299 cells were cultured as previously described, trypsinized, and then centrifuged four times with Dulbecco’s phosphate-buffered saline (D-PBS; Gibco; Invitrogen Co., Ltd, Carlsbad, CA, USA) at 4°C, after which cells were resuspended in D-PBS. We transplanted 5.0 × 107 cells into the left thighs of mice, which were then raised for 2 weeks to allow tumour formation [12,14,15]. After 2 weeks, the tumour volume (mm3) was calculated; tumours ≥20 mm3 were used for further study. Xenografts were subjected to 9-Gy gamma irradiation (Gammacell 40 Exactor, Nordion International, Ottawa, Canada), incubated for 48 h, and then used for experimentation.
Expression of transporters in H1299 cells
H1299 cells were smeared onto silane-coated slides (No. 5136, MUTO, Co.. Ltd., Tokyo, Japan) and fixed in 100% ethanol. The cells were incubated in 0.3% H2O2 in 0.05 M D-PBS for 15 min at room temperature and then washed three times with 0.05 M PBS for 3 min. Afterwards, the cells were blocked with 5% skim milk (v/v) and 0.15% H2O2 (v/v) in de-ionized water for 20 min at room temperature. Primary antibodies against Pgp and BCRP were then added and allowed to react with the cells overnight at room temperature. Transporters were detected using a 1/500 dilution of anti-BCRP/ABCG2 antibody (ab72788, Abcam, Co., Ltd., Cambridge, MA, USA) or anti-Pgp monoclonal antibody (4E3.16, #517308, Calbiochem, Co., Ltd., San Diego, CA, USA). Isotype control was conducted using mouse IgG1 (ICIGG1; ab91353, Abcam, Co., Ltd., Cambridge, MA, USA) and mouse control IgG2a [MOPC-173] - ChIP Grade (ab18413).
After washing with 0.05 M PBS, the cells were incubated in 0.05 M D-PBS containing peroxidase-labelled polymer conjugated to goat anti-mouse immunoglobulin for 30 min. After washing with 0.05 M D-PBS, the slides were stained with 3-3′-diaminobenzidine-4HCl (DAB) for 1 min. Finally, the slides were washed in 0.05 M PBS and stained with 50% Mayer’s haematoxylin on the cover slide for 1 min [15].
To assess the quality of Pgp and BCRP expression, Allred scores were used; the proportion score (PS); the percentage of cells that were stained by immunocytochemistry (on a scale of 0 to 5) and the intensity score (IS); the intensity of the staining (on a scale of 0 to 3), for a total possible score of 8. Total scores were compared between the 9-Gy-irradiated and not-irradiated tumour cells [16].
Cellular accumulation of 99mTc MIBI
To determine cellular transport activity, cell suspensions (7.0 mL) were thoroughly mixed and incubated in a 37°C water bath. Approximately 100 μL of 99mTc MIBI was then added to the cell suspensions. To evaluate intracellular accumulation, 300 μL aliquots were collected in duplicate 1, 15, 30, 45, and 60 min after 99mTc MIBI administration. The aliquots were transferred to 1.5-mL microcentrifuge tubes containing 1.0 mL of ice-cold saline and centrifuged at 14,000 rpm for 2 min; the supernatants were removed, and the cells washed gently with 0.5 mL of ice-cold saline solution. Radioactivity in the cell pellets was measured at 90 to 190 keV using a gamma-counter (WIZARD™ 3” Model 1480 Automatic Gamma Counter, PerkinElmer Life Sciences, Co., Ltd., Waltham, MA, USA). The accumulation ratio (C
in/C
out) was calculated as the ratio of intracellular to extracellular radioactivity in an equal volume of supernatant medium, using an independent measurement of cell volume as described previously [11]. The time course of 99mTc MIBI accumulation was measured in the not-irradiated group and in the 3-, 6-, and 9-Gy-irradiated groups [10,11].
Accumulation of doxorubicin in tumours
Doxorubicin (stock solution, 100 μL) was administered via the caudal vein to the not-irradiated mice and the 9-Gy-irradiated mice at 48 h after irradiation for in vivo fluorescence imaging [17]. Fluorescence (photons·s−1·cm−2) was measured, to evaluate the accumulation of doxorubicin (Figure 1), in the xenografted tumour thigh region and in the bilaterally symmetrical control thigh region using an in vivo imaging system (IVIS 200, Caliper Life Science Inc., Hopkinton, MA, USA) equipped with air-cooled argon lasers (emission at 488 nm) and a 575-nm band pass filter [11,15]. Changes in doxorubicin accumulation in the ROIs were detected by fluorescence imaging over time. Washout rate was calculated using the following equation:
$$ \begin{array}{l}\mathrm{Doxorubicin}\ \mathrm{washout}\ \mathrm{rate}\ \left(\%\right)=\left\{\right(\mathrm{Fluorescence}\ \mathrm{in}\ \mathrm{the}\ \mathrm{tumour}\ \mathrm{at}\ 10\ \min\ \mathrm{after}\ \\ {}\mathrm{doxorubicin}\ \mathrm{administration}-\mathrm{Fluorescence}\ \mathrm{at}\ 60,\ 120,\ \mathrm{or}\ 270\ \min\ \mathrm{after}\ \mathrm{doxorubicin}\ \\ {}\mathrm{administration}\Big)/\mathrm{Fluorescence}\ \mathrm{at}\ 10\ \min\ \mathrm{after}\ \mathrm{doxorubicin}\ \mathrm{administration}\Big\}\times 100.\end{array} $$
Effects of GG918 on 99mTc MIBI accumulation in cells after irradiation
H1299 cells were subjected to 3- and 6-Gy gamma irradiation. Irradiated cells and not-irradiated cells were separately cultured in tissue culture flasks. H1299 cells were trypsinized and centrifuged to prepare 7 mL of single-cell suspensions in fresh media with a density of 0.5 to 1.0 × 106 cells/mL. GG918 was added to a final concentration of 0, 0.001, and 0.1 μM to the irradiated and not-irradiated cell suspensions at 5 min before addition of 99mTc MIBI. In the not-irradiated group, the cell suspensions to which 0, 0.001, and 0.1 μM of GG918 were added were defined as the N-0 group, the N-L group, and the N-H group, respectively. In the 3- or 6-Gy-irradiated groups, the cells to which 0, 0.001, and 0.1 μM of GG918 were added were defined as the 3-0 group or 6-0 group, the 3-L group or 6-L group, and the 3-H group or 6-H group, respectively.
Radioactivity was measured using a gamma counter 30 min after the administration of 99mTc MIBI, and the 99mTc MIBI cellular accumulation ratio (C
in/C
out) was calculated. The accumulation of 99mTc MIBI was compared between the N-H, N-L, and N-0 groups and both the 3-H, 3-L, and 3-0 groups and the 6-H, 6-L, and 6-0 groups.
Statistical analysis
The samples used in the in vitro experiments were measured in duplicate. The data were expressed as the mean ± standard error (SE). The Kruskal-Wallis test and a post hoc multiple comparison test were conducted using Fisher’s protected least significant difference (PLSD) test. A P value less than 0.05 was considered statistically significant.
