Cell culture
The CRC cell lines SW480 (KCLB No. 10228; colorectal adenocarcinoma), SW620 (KCLB No. 10227; colorectal carcinoma), HT-29 (KCLB No. 30038; colorectal adenocarcinoma), and RKO (ATCC CRL-2577; colorectal carcinoma) were obtained from the Korean Cell Line Bank (Seoul, Korea) and American Type Culture Collection (ATCC, USA). They were cultured in Dulbecco’s modified Eagle medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) and 100 μg/mL antibiotics (100 U/mL penicillin and 100 μg /mL streptomycin (Gibco) at 37 °C under an atmosphere of 5% CO2 in a humidified incubator.
Radiation and radioresistant cell line generation
Cells were seeded in 60-mm dishes and exposed to radiation from a 60Co source (model 109 irradiator; JL Shepherd and Associates, San Fernando, CA, USA) at the indicated doses (0–5 Gy). Subsequently, cells were incubated at 37 °C under a humidified, 5% CO2 / air atmosphere.
To generate radiation resistance CRC [23, 24] using SW620, RKO, SW480, and HT-29 cell lines, CRC cells were plated in 60-mm dishes at a density of 5 × 103 cells/dish and exposed to a 5-Gy dose of ionizing radiation, followed by a 15-day recovery period. This process was repeated for 24 treatment cycles totaling 120 Gy; finally, radioresistant IR-SW620, IR-RKO, IR-SW480, and IR-HT-29 cells lines were established.
RNA sequencing analysis
The mRNAs of the established radioresistant IR-SW620, IR-SW480, IR-RKO, and IR-HT-29 cells were extracted using the Trizol (Sigma, St. Louis MO, USA) method. RNA was reverse-transcribed to cDNA followed by amplification and the resulting sequences were analyzed. Among the 25,207 genes, 70 genes were upregulated (>1.5 fold) and 45 genes were downregulated (<0.5 fold). These 115 genes were confirmed using RT-PCR analysis. Finally, the upregulated genes (CXCR4, RAC2, HBE1, PTGDS, and LCN2) and the downregulated genes (SMO, RGS10, PRTFDC1, and CRMP4) were identified as candidate genes associated with radiation resistance.
RNA interference experiments
Small-interfering RNA (siRNA) duplexes of CRMP4 were purchased from Bioneer (Daejeon, Korea). The specific target sequence of CRMP4 siRNA was sense 5'-GUG GAA GGA UUG UAG UCA UdTdT-3' and antisense 5'-AUG ACU ACA AUC CUU CCA CdTdT-3'. siRNA duplexes were transfected into cells using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA). The short hairpin RNA (shRNA) targeting CRMP4 was obtained from Origene (TL313373V; Rockville, MD, USA). The shRNA expression vector was transfected into the lentiviral packaging 293T cell line. The culture supernatant containing virus particles was harvested 48-h post transfection. For the stable transduction of lentivirus, cells at 60 to 70% confluence were grown in 6-well plates. After 48 h, 1 μg/mL puromycin (Clontech, Mountain View, CA, USA) was added for selection.
Western blot analysis
Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 1 mM PMSF, protease inhibitor mixture (Sigma, St. Louis MO, USA), and 1 mM sodium orthovanadate). Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride membrane, and blocked with 5% skim milk/PBS-T buffer for 1 h. Subsequently, the membrane was incubated with the following primary antibodies: β-actin, CRMP4, cytochrome c (Santa Cruz Biotechnology, Dallas, TX, USA), and cleaved-PARP (cleaved-poly ADP-ribose polymerase) (Cell Signaling Technologies, Danvers, MA, USA). The bound antibodies were visualized with a horseradish peroxidase-conjugated secondary antibody using enhanced chemiluminescence (Clarity Western ECL; Bio-rad, Hercules, CA, USA).
Flow cytometry for the measurement of MMP, intracellular calcium levels, and apoptosis assay
The fluorescent probe Fluo 3-AM was used for the assessment of intracellular levels of Ca2+ and the lipophilic cationic dye 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) was used for measuring disruption of the MMP (Δψm). Briefly, cells were exposed to a 5-Gy radiation dose and incubated for 72 h, then stained with 1 μM DiOC6(3) at 37 °C for 15 min in the dark, and analyzed using FACSverse flow cytometry (BD Biosciences, San Jose, CA, USA). For apoptosis analysis, cells were harvested and centrifuged at 800 rpm for 3 min following radiation treatment (48 h). Cells were carefully resuspended, then annexin-V (5 μL) and propidium iodide (PI) (5 μL) (BD Biosciences) were added and the cells incubated at 37 °C for 15 min in the dark. The stained cells were analyzed using FACSverse flow cytometry.
Clonogenic assay
Cells were seeded into 60-mm dish plates at densities of 1, 2, 5, 8, and 10 × 103 cells/plate. After 24 h, cells were radiated with the indicated dose of ionizing radiation. After 14 days, the colonies were subsequently fixed and stained with 0.1% crystal violet in 20% ethanol and were counted. Alternatively, the remaining attached cells were stained with crystal violet; after a wash step, the dye was solubilized and absorbance measurements at 590 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA) were taken.
Cell-cycle analysis
Irradiated cells were trypsinized, washed in ice-cold PBS, and fixed with 70% ethanol on ice. Fixed cells were stained with PI (BD Biosciences) containing RNase (0.1 mg/mL) for 15 min at 37 °C, and cell population analysis was performed using a FACSCalibur flow cytometry.
Measurement of cytochrome c release
The cytochrome c-releasing apoptosis assay kit (Abcam ab65311, Cambridge MA, USA) was used for detecting cytochrome c translocation from mitochondria into the cytosol. Irradiated or A23187-treated cells were lysed in a cytosolic extraction buffer, homogenized, the supernatant cytosol fraction separated by centrifugation, and the pellet resuspended in a mitochondrial extraction buffer, according to the manufacturer's instructions. Separated cytosolic and mitochondrial fractions were immunoblotted for the measurement of cytochrome c release.
Cell-viability assay
Cell viability was assessed using the water-soluble tetrazolium salt (WST)-1 assay (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, 10 μL WST-1 reagent was added to each well of a 96-well plate (1 × 104 cells/well). After incubation for 1 h, the conversion of WST-1 reagent into chromogenic formazan was evaluated using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Statistical analysis
All results were confirmed in at least three independent experiments; and data from one representative experiment is shown. All quantitative data are presented as the means ± standard deviation (SD); Student’s t-tests and ANOVA were used for comparisons of means of quantitative data between groups; a value of p < 0.05 was considered statistically significant.