Selective nuclear export inhibitor KPT‑330 enhances the radiosensitivity of esophageal carcinoma cells

Abstract

Although the concurrent application of definitive chemoradiation has improved the prognosis of patients with esophageal cancer, resistance to therapy poses a major threat to treatment. The present study aimed to investigate whether the use of KPT-330, a selective inhibitor of nuclear export (SINE), enhances the radiosensitivity of esophageal cancer cells. Immunohistochemical staining assays were employed to evaluate the expression and prognostic significance of chromosome maintenance protein-1 (CRM1) in 111 esophageal carcinoma (ESCA) tissues collected from patients with esophageal squamous cell carcinoma. The data showed that the expression of CRM1 in the ESCA tissues was significantly upregulated compared with that in the normal adjacent tissues. Furthermore, patients with higher CRM1 expression had significantly decreased overall survival compared with those with lower CRM1 expression. The effects of KPT-330 and/or radiation on ECA109 human ESCA cells were also evaluated. KPT-330 suppressed the viability of the ECA109 cells. A colony formation assay demonstrated that a combination of KPT-330 and radiation significantly decreased ECA109 cell proliferation. Flow cytometric analysis showed that KPT-330 increased the arrest of the ECA109 cells at the G₂/M phase and induced apoptosis. In addition, western blotting revealed that the inhibitory effect of KPT-330 on cell viability was associated with the increased expression of p53 and promotion of the nuclear accumulation of the p53 protein. In conclusion, the present study demonstrated that CRM1 expression is associated with the prognosis of patients with ESCA following radiotherapy. The inhibition of CRM1 expression by the SINE inhibitor KPT-330 increases radiosensitivity and is potentially useful in a combination treatment strategy for esophageal cancers.

Keywords: KPT-330; chromosome maintenance protein-1; esophageal carcinoma; radiosensitivity.

Figure 1

CRM1 is upregulated at the transcriptional level in various cancers according to the GEPIA database. GEPIA analysis showed that CRM1 was significantly elevated in six types of cancer as compared with the respective normal tissue. ^*P<0.05. CRM1, chromosome maintenance protein-1; ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; T, tumor; N, normal; TPM, transcripts per million.

Figure 2

Comparison of CRM1 expression in esophageal squamous cell carcinoma and normal tissue. CRM1 was upregulated at the transcriptional level in the Gene Expression Omnibus cohorts (A) GSE20347 and (B) GSE23400. ^***P<0.001. Representative images of the immunohistochemical staining of CRM1 in (C) normal tissue adjacent to esophageal squamous cell carcinoma tissue and (D) esophageal squamous cell carcinoma tissue. Scale bar, 200 µm. CRM1, chromosome maintenance protein-1.

Figure 3

Effect of CRM1 expression on the prognosis of patients with esophageal squamous cell carcinoma. (A) Receiver operating characteristic curve for CRM1 expression in 10 pairs of esophageal squamous cell carcinoma and adjacent tissues. (B) Kaplan-Meier survival analysis of the effect of CRM1 expression on the overall survival of the patients. CRM1, chromosome maintenance protein-1; high, high CRM1 expression; low, low CRM1 expression; AUC, area under the curve; TPR, true positive rate; FPR, false positive rate.

Figure 4

Effect of the CRM1 inhibitor KPT-330 on the viability of ECA109 esophageal carcinoma cells. (A) KPT-330 decreased cell viability and its inhibitory effect increased as its concentration increased. (B) Representative images from a colony formation assay in which the ECA109 cell line was pretreated with KPT-330 (0.1 µmol/l) prior to the application of radiation (0, 2, 4, 6 and 8 Gy) or treatment with radiation alone for 24 h. (C) Survival curves for IR with and without KPT-330. KPT-330 decreased proliferation and increased radiosensitivity. CRM1, chromosome maintenance protein-1; IR, irradiation.

Figure 5

KPT-330 increased the apoptosis of ECA109 cells and arrested the cell cycle at the G₂/M phase. (A) Flow cytometry plots showing the apoptosis level in six treatment groups 48 h after treatment: KPT-330 (0, 0.1 and 0.3 µmol/l) with or without radiation treatment. (B) Quantitative analysis showing the apoptosis level in the six groups. Images are representative of three independent experiments. (C) Flow cytometry plots showing the cell cycle distribution of the six groups 24 h after treatment. (D) Quantitative analysis showing the cell cycle data. Images are representative of three independent experiments. ^*P<0.05; ^**P<0.01. Control, untreated cells.

Figure 6

Expression of CRM1 and p53 in ECA109 cells after exposure to various treatments. (A) Representative western blots for cells treated with KPT-330 (0, 0.1 and 0.3 µmol/l) with or without radiation. (B) Comparison of CRM1 and p53 expression among the six groups. All experiments were conducted three times independently. ^*P<0.05; ^**P<0.01. CRM1, chromosome maintenance protein-1; control, untreated cells.

Figure 7

Nuclear and cytoplasmic expression of p53 in ECA109 cells. (A) ECA109 cells were treated with radiation and/or KPT-330, after which nuclear and cytoplasmic proteins were isolated and analyzed by western blotting. Representative western blots showing CRM1 expression in six groups: KPT-330 (0, 0.1 and 0.3 µmol/l) with or without radiation. (B) Comparison of p53 expression among the six groups. ^*P<0.05; ^**P<0.01. Nu, nucleus; Cy, cytoplasm; CRM1, chromosome maintenance protein-1; control, untreated cells.