Colon cancer cell lines SW480 (KCLB-10228; colon adenocarcinoma), SW620 (KCLB-10227; colorectal carcinoma), HT-29 (KCLB-30038; rectosigmoid colon adenocarcinoma), DLD1 (KCLB-10221; colon adenocarcinoma), Caco2 (KCLB-30037.1; colon adenocarcinoma), LoVo (KCLB-10229; colon adenocarcinoma, KM12C (KCLB-80015; colon carcinoma), and KM12SM (KCLB-80016; colon carcinoma) cells were purchased from the Korean Cell Line Bank (Seoul, Korea). RKO (ATCC CRL-2577; colon carcinoma), HCT116 (ATCC CCL-247; colorectal carcinoma), and LS174T (ATCC CL-188; colorectal adenocarcinoma) cells were purchased from the American Type Culture Collection (ATCC, USA). Each cell line was authenticated through STR profiling from the cell line bank. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (HyClone) and 100 µg/ml antibiotics [100 U/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.)] at 37°C under an atmosphere of 5% CO₂ in a humidified incubator.

For radiation treatment, cells were seeded in 60-mm dishes and exposed to radiation from a ⁶⁰Co source (model 109 irradiator; JL Shepherd and Associates) at the indicated doses (0–5 Gy). Subsequently, cells were incubated at 37°C under a humidified, 5% CO₂/air atmosphere. To generate radiation-resistant colon cancer cell lines (21,22), cells (SW620, RKO, SW480, and HT-29) 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 cell lines were established.

The mRNAs of the established radioresistant IR-SW620, IR-SW480, IR-RKO, and IR-HT-29 cells were extracted using TRIzol (Sigma-Aldrich; Merck KGaA) method. The total RNA quantity and integrity of each RNA sample were evaluated using the bioanalyzer 2100 (Agilent Technologies, Inc.). The RNA concentration was quantified as 1 µg/µl, rRNA ratio (28S/18S) was 2.0, and the RIN number was 10 (HT-29, RKO, IR-RKO, SW480, IR-SW480, SW620) and 9.9 (IR-HT-29, IR-SW620). The RNA sequencing library was prepared using the TruSeq RNA Sample Prep Kit (Illumina, Inc.) and sequenced using Hiseq-2000 (Illumina, Inc.) to generate 76- or 101-bp paired-end reads. Reads were trimmed to remove adapter sequences and base with low sequencing quality (per-base quality <20) using Cutadapt (23). The clean reads were aligned to the reference genome (hg38) sequencing using STAR 2.4.1 (24), and the gene expression levels (GRCh38) were quantified with the HTSeq package (25). The edgeR package (26) was used to select differentially expressed genes from sequencing count data.

In this study, we used two RNA interference methods to knock down the CRMP4 gene, one for transient siRNA and the other for stable shRNA method. Small interfering RNA (siRNA) duplexes of CRMP4 were purchased from Bioneer (Daejeon, Korea). The specific target sequences of CRMP4 siRNA (#2, Fig. S1A) were sense 5′-GUGGAAGGAUUGUAGUCAUdTdT-3′ and antisense 5′-AUGACUACAAUCCUUCCACdTdT-3′. siRNA duplexes were transfected into cells (50 nM for RKO, 200 nM for SW620, SW480, Caco2, and KM12C) using Lipofectamine RNAiMAX reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The short hairpin RNA (shRNA) targeting CRMP4 was obtained from Origene (TL313373V). The shRNA expression vector was transfected into the lentiviral packaging Lenti-X 293T cell line (Takara Bio USA). The culture supernatant containing virus particles was harvested 48 h post-transfection. For the stable transduction of the lentivirus, cells at 60–70% confluence were grown in 6-well plates. After 48 h, 1 µg/ml puromycin (Clontech Laboratories, Inc.) was added, and finally, CRMP4-knockdown RKO (#1 clone) and SW620 (#4 clone) cells were selected for further study (Fig. S1B).

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-Aldrich; Merck KGaA), 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, Inc.), and cleaved-PARP (cleaved-polyADP-ribose polymerase) (Cell Signaling Technologies, Inc.). The bound antibodies were visualized with a horseradish peroxidase-conjugated secondary antibody using enhanced chemiluminescence (Clarity Western ECL; Bio-Rad Laboratories, Inc.) and the Ez-Capture MG system (Atto Corp.).

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). For apoptosis analysis, cells were harvested and centrifuged at 800 rpm for 3 min following radiation treatment (48 h). Cells were carefully resuspended, and hen Annexin-V (5 µl) and propidium iodide (PI) (5 µl) (BD Biosciences) were added, and the cells were incubated at 37°C for 15 min in the dark. The stained cells were analyzed using FACSverse flow cytometry (BD Biosciences) and the Flowjo software v7.6.1 (FlowJo LLC). At least 10,000 cells per sample were analyzed, and duplicate analyses were performed at each time point.

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 treated with the indicated ionizing radiation dose. After 14 days, the colonies were subsequently fixed, stained with 0.1% crystal violet in 20% ethanol, and counted. Alternatively, cells were stained with crystal violet; after a wash step, the dye was solubilized, and absorbance was measured at 590 nm using a microplate reader (Molecular Devices, LLC).

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 FACSCalibur flow cytometry.

The cytochrome c- releasing apoptosis assay kit (Abcam; ab65311) was used for detecting cytochrome c translocation from mitochondria into the cytosol. After irradiated or A23187-treated cells were lysed in a cytosolic extraction buffer and homogenized, the supernatant cytosolic fraction was separated by centrifugation, and the pellet was 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 was assessed using the water-soluble tetrazolium salt (WST)-1 assay (Roche Diagnostics) 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.

All experiments were performed in triplicate and data are expressed as the means ± standard deviation. Differences in cell apoptosis, survival, and MMP depolarization between control and CRMP4-deficiency cells were analyzed using a paired Student's t-test. Differences in cell survival among different cell groups (parent, IR-resistant, CRMP4-shRNA) were analyzed using one-way ANOVA with Tukey's post hoc test. Data were analyzed using GraphPad Prism (GraphPad Software Inc.). P<0.05 was considered statistically significant.

To identify genes associated with radiation-induced cell death, radiation-resistant IR-SW620, IR-RKO, IR-SW480, and IR-HT-29 cell lines were established by repeated exposure to ionizing radiation over several weeks for a total exposure of 120 Gy (21,22). From the RNA sequencing analysis, a total of 25,207 genes were identified. Among them, 70 genes were upregulated (>1.5-fold), while 45 were downregulated (<0.5-fold). These 115 genes were confirmed using RT-PCR analysis. Finally, the upregulated genes (CXCR4, RAC2, HBE1 (21), PTGDS, and LCN2) and downregulated genes (SMO, RGS10, PRTFDC1, and CRMP4) were identified as candidate genes associated with radiation resistance (Fig. 1A). To verify CRMP4 downregulation at the protein level, several colon cancer and IR-resistant cell lines were analyzed by western blotting (Fig. 1B). CRMP4 was differentially expressed in those; however, radiation-resistant IR-SW480, IR-SW620, and IR-RKO cells displayed apparent decreased CRMP4 expression compared to that of their parental cells. When SW620 and RKO cells showing high CRMP4 expression were compared with IR-SW620 and IR-RKO cells in regards to cell survival, IR-resistant cells exhibited better survival in the 5 Gy-irradiated clonogenic assay (Fig. 1C). The degree of apoptosis under exposure to 5 Gy of radiation, as analyzed by Annexin-V and PI, was significantly decreased by about half in IR-SW620 and IR-RKO cells compared to their parental cells (Fig. 1D). To investigate the effect of radiation on cell cycle distribution in IR-resistant cells and parental cells, cells were exposed to 0, 2, and 4 Gy of radiation. After 24 h, cells were analyzed by flow cytometry using PI, and it revealed that radiation-mediated G2M accumulation was reduced in both IR-SW620 and IR-RKO cells compared to their parent cells (Fig. 1E).

Loss-of-function experiments using siRNAs were performed on colon cancer cells to determine whether CRMP4 reduction is involved in radiation resistance. When RKO and SW620 cells were treated with CRMP4-siRNA, there was a 5-fold reduction in CRMP4 protein expression (Fig. S1A), although the siRNA amount was treated differently based on transfection efficiency for each cell line types. To verify the results from CRMP4-siRNA transfected SW620 and RKO cells, colon cancer cell lines SW480, Caco2 and KM12C, all of which express CRMP4, were selected and transfected with CRMP4-siRNA. The clonogenic assays revealed that CRMP4 reduction was associated with resistance to radiation in these cells (Fig. 2A). To ascertain whether increased clonogenic survival by CRMP4 knockdown was associated with reduced apoptosis, Annexin V and PI staining was used to examine the degree of apoptosis induced by radiation in CRMP4-knockdown and mock cells (Fig. 2B). The number of apoptotic cells following radiation treatment in the CRMP4-knockdown cells was significantly decreased by half compared to that found in the mock cells transfected with nonspecific control siRNA. In cell cycle analysis, radiation-exposed CRMP4-knockdown cells displayed decreased G2/M accumulation compared to the control cells (Fig. 2C). These results suggest that CRMP4 may be associated with the development of radiation resistance in colon cancer cells.

Irradiation has been shown to induce various cellular and molecular damage outcomes, including apoptosis, in which cytochrome c release from mitochondria constitutes a critical event (27). The amount of cytochrome c released from the mitochondria of irradiated cells was measured using a mitochondria/cytosol fractionation method. As shown in Fig. 3A, irradiated cells induced cytochrome c release in a time-dependent manner. The maximum amount of cytochrome c release was attained at 72 h of exposure to 5 Gy of radiation. However, decreased cytochrome c release and PARP cleavage were observed in the CRMP4-deficiency IR-SW620 cells compared to the parental cells. To verify the role of CRMP4 in cancer cells as regards radiation resistance, CRMP4 expression was stably knocked down in SW620 and RKO cells through lentiviral-mediated shRNA infection (Fig. S1B). After CRMP4 knockdown was verified in both cell lines, cytochrome c release was evaluated under radiation exposure. As expected, mitochondrial cytochrome c release and PARP cleavage were decreased in CRMP4-knockdown SW620-shCRMP4 and RKO-shCRMP4 cells compared to the nonspecific control shRNA cells (Fig. 3B). It has been known that the release of cytochrome c from mitochondria during apoptosis is associated with low MMP (ΔΨm). The lipophilic cationic dye 3DiOC6(3) was used to monitor MMP and determine whether CRMP4 reduction is associated with MMP loss. Radiation treatment gradually caused the SW620 cells to lose MMP in a time-dependent manner, whereas IR-SW620 cells showed a weak depolarization of MMP (Fig. 3C). Similarly, CRMP4-knockdown cells also exhibited insignificant depolarization of MMP, but shControl cells showed a 2.3-fold increase in depolarization (Fig. 3D). These results indicate that CRMP4 deficiency may diminish radiation-induced MMP depolarization and cytochrome c release.

Consequent to its role as an incredibly versatile signaling ion, uncontrolled cytosolic Ca²⁺ influx induces mitochondrial dysfunction and cell death pathways (3,18). To determine whether CRPM4 could modulate intracellular Ca²⁺ influx, Ca²⁺ concentration was measured by flow cytometry using a cell-permeable fluorescent Ca²⁺ indicator, Fluo 3-AM. As shown in Fig. 4A, radiation exposure strongly induced intracellular Ca²⁺ influx regardless of CRMP4 expression (Fig. 4A). When Ca²⁺ levels were analyzed in cells expressing the siRNA- or shRNA-CRMP4 as a preliminary experiment (data not shown), the significant difference in Ca²⁺ concentration according to CRMP4 expression could not be determined. Under radiation exposure, intracellular Ca²⁺ concentration was strongly elevated in all radiation-treated cells irrespective of CRMP4 expression (Fig. 4A), indicating that CRMP4 is not likely involved in intracellular Ca²⁺ influx regulation. Because improper Ca²⁺ influx into mitochondria causes MMP depolarization, we verified the consequent radiation-induced cell death using the clonogenic assay (Fig. 4B). When cells were treated with cyclosporin A (CsA), known to inhibit Ca²⁺-dependent mitochondrial permeability transition (MPT) pores, cells exhibited strong radiation-resistant survival compared to CsA-untreated control cells, indicating that Ca²⁺ is critical to radiation-induced cell death. To examine whether CRMP4 is required for Ca²⁺-mediated apoptosis or MMP depolarization, cells were treated with the Ca²⁺ ionophore A23187 to increase intracellular Ca²⁺ levels. As shown in Fig. 4C, IR-resistant and shCRMP4 cells exhibited dose-dependent improved survival compared to control parent cells upon A23187 treatment. Western blotting analysis further revealed that cytochrome c release from mitochondria was obviously reduced in the IR-resistant and shCRMP4 cells compared to the control cells under A23187 treatment (Fig. 4D). Consistent with this, when A23187-treated cells were analyzed by flow cytometry using DiOC6(3), MMP depolarization was significantly increased in shControl cells but not in shCRMP cells (Fig. 4E), indicating that CRMP4 may play a role in Ca²⁺-mediated MMP depolarization followed by mitochondrial cytochrome c release and apoptosis.

Previously, we found that CsA-mediated MPT inhibition caused the downregulation of CRMP4. To examine whether CRMP4 expression is affected by intracellular Ca²⁺ levels, cells were treated with the cell-permeant intracellular Ca²⁺ chelator, BAPTA-AM, and cell lysates were analyzed with western blotting. Interestingly, CRMP4 was downregulated by BAPTA-AM treatment in a dose-dependent manner (Fig. 5A). Although we could not determine whether CRMP4 was proteolyzed or degraded in this condition, CRMP4 expression may be regulated by the Ca²⁺-related pathway. The buffering of intracellular Ca²⁺ concentration by BAPTA-AM (5 µM) also obviously reduced A23187-induced cytochrome c release in SW620 cells (Fig. 5B). Western blotting results showed that cleaved-PARP was augmented following radiation exposure, but IR-SW620 cells revealed a strong inhibition of PARP cleavage under radiation exposure following BAPTA-AM treatment (Fig. 5C). Consistently, when cells were analyzed with flow cytometry using DiOC6, BAPTA-AM attenuated radiation-induced MMP depolarization in both shCRMP4 and shControl cells (Fig. 5D). These data suggest that low BAPTA-AM (<5 µM) could alleviate A23187- and radiation-induced MMP depolarization and cytochrome c release.

Several studies have demonstrated the extensive use of BAPTA-AM for buffering Ca²⁺; however, high concentrations of BAPTA-AM have been found to deplete intracellular Ca²⁺ stores (10,28). To ascertain whether the apoptosis inhibition in CRMP4-deficient cells is changed under high BAPTA-AM levels (>10 µM), several CRMP-deficient cell lines were treated with 10–20 µM of BAPTA-AM, and surviving cells were stained with crystal violet dye (Fig. 6A). Furthermore, WST-1 assay was conducted for cell viability measurement (Fig. 6B). High BAPTA-AM-induced Ca²⁺ deficiency-mediated cell death in both IR-resistant and CRMP4-knockdown cells in a dose-dependent manner, but no significant effect was observed in the parental cells, indicating that Ca²⁺ deficiency-induced apoptosis may be associated with the CRMP4 expression level. In addition, there was an obvious dose-dependent cleaved-PARP elevation in both IR-resistant and shCRMP4 cells treated with BAPTA-AM (Fig. 6C). Parental and shControl cells showed a weak cleavage of PARP. Therefore, these results suggest that CRMP4 may be important for cell survival during cellular stress response due to BAPTA-AM-induced Ca²⁺ deficiency.