Collapsin response mediator protein 4 enhances the radiosensitivity of colon cancer cells through calcium‑mediated cell signaling

Abstract

Radiation therapy is an effective treatment against various types of cancer, but some radiation‑resistant cancer cells remain a major therapeutic obstacle; thus, understanding radiation resistance mechanisms is essential for cancer treatment. In this study, we established radiation‑resistant colon cancer cell lines and examined the radiation‑induced genetic changes associated with radiation resistance. Using RNA‑sequencing analysis, collapsin response mediator protein 4 (<em>CRMP4</em>) was identified as the candidate gene associated with radiation sensitivity. When cells were exposed to radiation, intracellular Ca²⁺ influx, collapse of mitochondrial membrane potential, and cytochrome c release into the cytosol were increased, followed by apoptosis induction. Radiation treatment‑ or Ca²⁺ ionophore A23187‑induced apoptosis was significantly inhibited in <em>CRMP4</em>‑deficient cells, including radiation‑resistant or <em>CRMP4</em>‑shRNA cell lines. Furthermore, treatment of <em>CRMP4</em>‑deficient cells with low levels (&lt;5 µM) of BAPTA‑AM, a Ca²⁺ chelator, resulted in radiation resistance. Conversely, Ca²⁺ deficiency induced by a high BAPTA‑AM concentration (&gt;10 µM) resulted in higher cell death in the <em>CRMP4</em>‑depleted cells compared to <em>CRMP4</em>‑expressing control cells. Our results suggest that <em>CRMP4</em> plays an important role in Ca²⁺‑mediated cell death pathways under radiation exposure and that CRMP4 may be a therapeutical target for colon cancer treatment.

Keywords: CRMP4; radioresistance; colon cancer; calcium influx; mitochondrial membrane potential.

Figure 1.

The ionizing radiation-resistant (IR) cell lines show CRMP4 downregulation and increased survival rate. (A) Heatmap from RNA sequencing results. Twenty representative genes are shown. CRMP4 was identified as a downregulated gene. (B) CRMP4 downregulation in IR-resistant cells. Several colon cancer cell lines were lysed and analyzed by western blotting. IR-cells were established by repeating radiation exposure as described in the Materials and methods. (C) Cells were irradiated to the indicated doses of radiation and allowed to grow for 2 weeks, and cells were stained with crystal violet and scored for the colony-forming capacity of irradiated cells. (D) Apoptosis inhibition in IR-resistant cells. Cells were treated with 5 Gy of radiation, and after 48 h, cells were stained with Annexin-V and propidium iodide (PI) followed by flow cytometry analysis. Representative scatter plot and quantitative apoptotic rate (%) are shown. Apoptotic cells (Annexin-V⁺/PI⁻, Annexin-V⁺/PI⁺) are indicated by red dotted rectangles. Student's t-test was performed. *P<0.05, **P<0.01. (E) Reduced G2M phase accumulation in IR-resistant cells. Cells were treated with the indicated doses of radiation, and after 24 h the cell cycle was analyzed by flow cytometry using PI. The histogram (2n/4n DNA content) is shown on the left and their quantitative cell cycle distribution is shown on the right. CRMP4, collapsin response mediator protein 4.

Figure 2.

CRMP4-deficiency enhances resistance to radiation. (A) Increased radioresistance in siCRMP4 cells. After cells were transfected with control siRNA (Mock) or CRMP4 siRNA, cells were treated with radiation (5 Gy) and analyzed by clonogenic assay. Representative images are shown on the left, and a quantitative graph is shown on the right. Student's t-test was performed. *P<0.05, **P<0.01. (B) Inhibition of apoptosis in siCRMP4 cells. Cells were treated with 5 Gy of radiation for 48 h, stained with Annexin-V and propidium iodide (PI), and the percentages of apoptotic cells were measured by flow cytometry. Representative scatter plot and quantitative apoptotic rate (%) are shown. Apoptotic cells (Annexin-V⁺/PI⁻, Annexin-V⁺/PI⁺) are indicated by red dotted rectangles. Student's t-test was performed. *P<0.05, **P<0.01. (C) Reduced G2M phase accumulation in siCRMP4 cells. Cells were treated with the indicated doses of radiation, and after 24 h the cell cycle was analyzed by flow cytometry using PI. The histogram (2n/4n DNA content) is shown on the left and their quantitative cell-cycle distribution is shown on the right. CRMP4, collapsin response mediator protein 4.

Figure 3.

CRMP4-deficiency inhibits mitochondrial membrane depolarization under radiation exposure. (A) Inhibition of cytochrome c (CytC) release and PARP cleavage (C-PARP) in IR-resistant cells. After cells were exposed to 5 Gy of radiation for 0–72 h, cytosolic fractions were isolated and western blotting was conducted. (B) Inhibition of cytochrome c release and PARP cleavage in shCRMP4 cells. After cells were exposed to 5 Gy of radiation, cytosolic fractions were isolated and western blotting was conducted. (C) MMP depolarization was significantly increased in parental cells but not in ionizing radiation (IR)-resistant cells. After cells were exposed to 5 Gy of radiation, cells were stained with DIOC6(3) (3,3′-dihexyloxacarbocyanine iodide) fluorescent dye and analyzed by flow cytometry. The quantitative graph is shown on the right. Student's t-test was performed. *P<0.05. (D) After cells were exposed to 5 Gy of radiation for 72 h, cells were analyzed by flow cytometry using DiOC6(3). MMP depolarization was strongly increased in shControl cells, but it was weakly increased in shCRMP4 cells. Student's t-test was performed. *P<0.05. CRMP4, collapsin response mediator protein 4; MMP, mitochondrial membrane potential; PARP, polyADP-ribose polymerase.

Figure 4.

CRMP4-deficiency inhibits the Ca²⁺-mediated cell death pathway. (A) Intracellular Ca²⁺ level was not affected by CRMP4-deficiency. Cells were irradiated with 5 Gy of radiation for 72 h, treated with Fluo-3 AM for Ca²⁺ detection, and analyzed by flow cytometry. Histograms of intracellular Ca²⁺ content are shown on the right. N.S, not significant. (B) Increased cell survival by cyclosporine A (CsA) in radiation-treated cells. Cells were treated with or without CsA (5 µM) for 1 h and exposed to 5 Gy of radiation, and after 14 days, cells were stained with 0.1% crystal violet. (C) CRMP4-deficiency inhibited A23187-mediated cell death. Several cells were treated with the Ca²⁺ ionophore A23187 for 24 h, and their proliferation was measured by a plate reader using the WST-1 reagent. The survival rate is expressed as the % of control cells. Student's t-test was performed. *P<0.05, **P<0.01. (D) Cytochrome c (CytC) release inhibition in CRMP4-downregulated cells. After cells were treated with A23187 (0–2 µM) for 12 h, the cytosolic fractions were isolated and western blotting was conducted. CRMP4-deficient ionizing radiation (IR)-resistant and shCRMP4 cells showed a decreased cytochrome c release. (E) MMP depolarization was increased in shControl cells but not in shCRMP4 cells. After cells were treated with A23187 for 24 h, cells were stained with DIOC6(3) and analyzed by flow cytometry. The quantitative graph is shown on the right. Student's t-test was performed. *P<0.05. CRMP4, collapsin response mediator protein 4; MMP, mitochondrial membrane potential.

Figure 5.

The Ca²⁺ chelator, BAPTA-AM, downregulates CRMP4 and inhibits MMP depolarization. (A) CRMP4 downregulation by BAPTA-AM treatment. SW620 cells were treated with BAPTA-AM (0–20 µM) for 24 h, and western blotting was conducted. (B) A23187-mediated cytochrome c (CytC) release was inhibited by BAPTA-AM. SW620 cells were treated with A23187 (+, 1 µM; ++, 2 µM) in the absence or presence of BAPTA-AM (5 µM) for 12 h, and mitochondrial and cytosolic fractions were isolated for western blotting. (C) Radiation-mediated PARP cleavage (C-PARP) was blocked by BAPTA-AM. Cells were treated with BAPTA-AM (5 µM) and/or radiation (5 Gy), and after 72 h, cell lysates were analyzed by western blotting. (D) Radiation-mediated MMP depolarization was inhibited by BAPTA-AM. Cells were treated with BAPTA-AM (5 µM) and/or radiation (5 Gy), and after 72 h, cells were stained with DiOC6(3) for flow cytometry. The quantitative graph is shown on the right. Student's t-test was performed. *P<0.05. CRMP4, collapsin response mediator protein 4; MMP, mitochondrial membrane potential.

Figure 6.

High-dose BAPTA-AM enhances cell death in CRMP4-deficient cells. (A) Cells were treated with the indicated doses of BAPTA-AM for 72 h, and cells were stained with crystal violet. Representative images are shown. (B) Cell survival inhibition in CRMP4-deficient cells with BAPTA-AM. After cells were treated with BAPTA-AM (>10 µM) for 48 h, cells were incubated with the WST-1 reagent, and their absorbances at OD₄₅₀ were read using a plate reader. One-way ANOVA with Tukey's post hoc test between control, ionizing radiation (IR)-resistant, and shCRMP4 groups were performed. *P<0.05, **P<0.01. (C) Cleaved PARP (C-PARP) induction in CRMP4-deficient cells with BAPTA-AM. After cells were treated with BAPTA-AM for 48 h, cell lysates were analyzed by western blotting. C-PARP was strongly increased in IR-resistant and shCRMP4 cells compared to control cells. CRMP4, collapsin response mediator protein 4; PARP, polyADP-ribose polymerase.

Figure 7.

Radiation-mediated CRMP4 downregulation inhibits the mitochondrial membrane depolarization followed by apoptosis induction. Radiation- or Ca²⁺ ionophore A23187-mediated improper intracellular Ca²⁺ influx collapses the cellular Ca²⁺-mediated pathway followed by MMP depolarization. Apoptosis resulting from radiation or A23187 treatment is diminished by CRMP4-deficiency and low levels of the intracellular Ca²⁺ chelator, BAPTA-AM (<5 µM). Conversly, significant cell death can be triggered by CRMP4-deficiency and a high dose of BAPTA-AM (>10 µM). This suggests that CRMP4 plays an important role in the Ca²⁺-mediated cell death pathway. CRMP4, collapsin response mediator protein 4; MMP, mitochondrial membrane potential; PARP, polyADP-ribose polymerase; IR, ionizing radiation.