Glut-3 Gene Knockdown as a Potential Strategy to Overcome Glioblastoma Radioresistance

Abstract

The hypoxic pattern of glioblastoma (GBM) is known to be a primary cause of radioresistance. Our study explored the possibility of using gene knockdown of key factors involved in the molecular response to hypoxia, to overcome GBM radioresistance. We used the U87 cell line subjected to chemical hypoxia generated by CoCl2 and exposed to 2 Gy of X-rays, as single or combined treatments, and evaluated gene expression changes of biomarkers involved in the Warburg effect, cell cycle control, and survival to identify the best molecular targets to be knocked-down, among those directly activated by the HIF-1α transcription factor. By this approach, glut-3 and pdk-1 genes were chosen, and the effects of their morpholino-induced gene silencing were evaluated by exploring the proliferative rates and the molecular modifications of the above-mentioned biomarkers. We found that, after combined treatments, glut-3 gene knockdown induced a greater decrease in cell proliferation, compared to pdk-1 gene knockdown and strong upregulation of glut-1 and ldha, as a sign of cell response to restore the anaerobic glycolysis pathway. Overall, glut-3 gene knockdown offered a better chance of controlling the anaerobic use of pyruvate and a better proliferation rate reduction, suggesting it is a suitable silencing target to overcome radioresistance.

Keywords: chemical hypoxia; gene knockdown; glioblastoma; radioresistance.

Figure 1

Cell counts of U87 cells untreated and treated with 50 and 100 μM CoCl₂. On the abscissa axis, the origin for the hours corresponds to the start of the treatment (A). Micrographs of U87 cells at 24, 48, and 72 h post-treatment with 50 and 100 μM CoCl₂ (magnification 10×) (B). The data shown are representative of two independent experiments and are expressed as the mean ± standard deviation of the mean (SD). The significance level compared to the control sample, for each time point, was set to p < 0.05 (unpaired t-test Welch corrected) and displayed with the asterisk (*).

Figure 1

Cell counts of U87 cells untreated and treated with 50 and 100 μM CoCl₂. On the abscissa axis, the origin for the hours corresponds to the start of the treatment (A). Micrographs of U87 cells at 24, 48, and 72 h post-treatment with 50 and 100 μM CoCl₂ (magnification 10×) (B). The data shown are representative of two independent experiments and are expressed as the mean ± standard deviation of the mean (SD). The significance level compared to the control sample, for each time point, was set to p < 0.05 (unpaired t-test Welch corrected) and displayed with the asterisk (*).

Figure 2

Cell counts of U87 cells untreated vs. treated with 7.5 μM Mo-Standard (A); cell counts of U87 cells treated with 50 μM CoCl₂ and 7.5 μM Mo-Standard vs. treated with 50 μM CoCl₂ and 7.5 μM Mo-Glut3 or Mo-PDK-1 (B); cell counts of U87 cells treated with 50 μM CoCl₂ and 2 Gy vs. treated with 50 μM CoCl₂, 2 Gy, and 7.5 μM Mo-Glut3 or Mo-PDK-1. On the abscissa axis, the origin for the hours corresponds to the start of the treatment (C). The data shown are representative of three independent experiments and are expressed as the mean ± standard deviation of the mean (SD). The significance level compared to the control sample (control in (A), 50 μM CoCl₂ and 7.5 μM Mo-Standard in (B), and 50 μM CoCl₂ and 2 Gy in (C)), for each time point, was set to p < 0.05 (unpaired t-test Welch corrected) and displayed with the asterisk (*).

Figure 3

Pathways related to the selected key genes involved in the induction of Warburg effect (glut-1, glut-3, ldha, and pdk-1) and the survival/death balance (survivin, bax, bcl-2, and casp-9).

Figure 4

Experimental planning from the cells seeding, on day 1, until the RNA extraction, on day 5, and subsequent qRT-PCR analysis.

Figure 5

U87 cells transfected with 1 µL/mL of Lipofectamine and 0.8 µg/mL of pEGFP-N1-Hif-1α, untreated (A) and treated with CoCl₂ (B) at a concentration of 50 µM. Nuclei staining with 1.5 μg/mL Hoechst (magnification 40×).