GLUT-1 siRNA Enhances Radiosensitization Of Laryngeal Cancer Stem Cells Via Enhanced DNA Damage, Cell Cycle Redistribution, And Promotion Of Apoptosis In Vitro And In Vivo

Abstract

Background: Radiotherapy does not show good efficacy against laryngeal cancer due to radioresistance. Cancer stem cells (CSCs) are considered among the causes of radioresistance. Inhibition of glucose transporter-1 (GLUT-1) using GLUT-1 small interfering RNA (siRNA) may enhance the radiosensitivity of laryngeal cancer cells, but the underlying cellular mechanisms remain unclear.

Methods: The CD133⁺-Hep-2R cell line was established with repeated irradiation and magnetic-activated cell sorting. The effects of irradiation on CD133⁺-Hep-2R cells were examined by CCK-8 assay, Transwell assay, quantitative real-time polymerase chain reaction (RT-PCR), and Western blotting. The effects of GLUT-1 siRNA on the radiosensitivity of CD133⁺-Hep-2/2R cells were examined by RT-PCR, Western blotting, CCK-8 assay, colony formation assay, and Transwell assay in vitro and in a xenograft tumor model in nude mice. The cellular mechanism of enhanced radiosensitivity associated with GLUT-1 siRNA was investigated. The cell cycle and apoptosis rate were analyzed by flow cytometry, and the repair capability was examined by determining the levels of RAD51 and DNA-PKcs.

Results: CD133⁺-Hep-2/2R cells showed stronger proliferation, lower apoptosis rate, lower percentage of G0/G1 phase cells, higher percentages of S and G2/M phase cells, and higher expression levels of GLUT-1 than Hep-2/2R cells. Transfection with GLUT-1 siRNA inhibited the proliferation and invasive capability of CD133⁺-Hep-2R cells by inhibiting GLUT-1 expression, which also caused a redistribution of the cell cycle (higher proportion of cells in the G0/G1 phase and lower proportion in the S and G2/M phases), increased the apoptosis rate, and reduced DNA repair capability by suppressing RAD51 and DNA-PKcs expression.

Conclusion: The results of this study suggest that GLUT-1 siRNA can enhance the radiosensitivity of CD133⁺-Hep-2R cells by inducing a redistribution of cell cycle phases, inhibiting DNA repair capability, and increasing apoptosis. Inhibition of GLUT-1 may have therapeutic potential for interventions to increase the radiosensitivity of laryngeal CSCs.

Keywords: DNA double-strand break; glucose transporter-1; laryngeal cancer stem cells; radioresistance; small interfering RNA.

Figure 1

Establishment of Hep-2R and CD133⁺-Hep-2R cell lines. (A) Optical density at 450 nm (OD₄₅₀) of Hep-2 and Hep-2R cells as a measure of the doubling time (Hep-2, 40.7 h; Hep-2R, 48.4 h). (B) The comparison of irradiation resistance between of Hep-2R and Hep-2 cell lines. (C) Establishment of CD133⁺-Hep-2R cell line through magnetic-activated cell sorting (MACS) and flow cytometry. **: p<0.01.

Figure 2

Differences in tumor characteristics between CD133⁺-Hep-2/2R cells and Hep-2/2R cells. (A) Volumes of xenograft tumors. (B) Rates of apoptosis. (C) Proportions of cells in different phases. (D) GLUT-1 mRNA expression levels. (E) GLUT-1 protein expression levels vs those in Hep-2 cells. **, &&, ##: p<0.01; *, &: p<0.05.

Figure 3

Effects of irradiation and GLUT-1 siRNA on CD133⁺-Hep-2R cells. (A) Optical density at 450 nm (OD₄₅₀) of CD133⁺-Hep-2R cells in CCK-8 assays. (B) Colony forming images and numbers of clones in clonogenic survival assays. (C) Number of CD133⁺-Hep-2R cells passing through the chamber in Transwell assays. (D) GLUT-1 mRNA expression levels. (E) GLUT-1 protein expression levels. **: p<0.01, *: p<0.05.

Figure 4

GLUT-1 siRNA enhances the radiosensitivity of CD133⁺-Hep-2/2R cells. (A) GLUT-1 mRNA expression levels. (B) GLUT-1 protein expression levels. (C) Volumes of xenograft tumors. **, &&, ##, $$: p<0.01; *, &, #, $: p<0.05.

Figure 5

GLUT-1 siRNA regulates the cell cycle, promotes apoptosis, and reduces DNA repair capability in CD133⁺-Hep-2/2R cells. (A) Proportions of cells in different phases of the cell cycle. (B) Rate of apoptosis of CD133⁺-Hep-2R cells. (C) Rate of apoptosis of xenograft tumors. (D) RAD51 and PKcs mRNA levels in CD133⁺-Hep-2R cells. (E) RAD51 and PKcs mRNA levels in xenograft tumors. (F) RAD51 and PKcs protein levels in CD133⁺-Hep-2R cells. (G) RAD51 and PKcs protein levels in xenograft tumors. **, &&, ##, $$: p<0.01; *, #, $: p<0.05.