Effect of silencing S-phase kinase-associated protein 2 on chemosensitivity to temozolomide of human glioma cells U251

Abstract

Objective: To examine the effect of silencing SKP2 on chemosensitivity of human glioma cells U251 to temozolomide (TMZ).

Methods: Adenoviruses harbouring shRNA targeting SKP2 (i.e. Ad-shSKP2) and non-targeting scrambled shRNA (i.e. Ad-shNC) were used to infect U251 cells. The transduced cells were then treated with TMZ. Cell viability after treatment was assayed using CCK8; while cell cycle and apoptosis were examined using flow cytometry. To study the effect of silencing SKP2 on autophagy in U251, we co-transduced the cells with Ad-mRFP-LC3 and Ad-shSKP2/Ad-shNC. The expression of autophagy marker LC3 after TMZ treatment was studied using microscopy and Western blotting assays.

Results: The cytotoxicity of TMZ (i.e. 20-100 µM) was more significantly seen in Ad-shSKP2-transduced U251 cells than in the Ad-shNC-transduced U251 cells. The IC₅₀ values in shSKP2-U251 were significantly lower than those of the shNC-U251 (P < 0.05). Both TMZ and Ad-shSKP2 alone increased apoptosis and promoted expression of LC3 in U251. Combined treatment of Ad-shSKP2 and TMZ further elevated apoptosis and LC3 expression.

Conclusion: Silencing SKP2 in U251 cells increased chemosensitivity to TMZ that was accompanied with enhanced apoptosis and autophagy. Targeting SKP2 may be a potential approach to potentiate TMZ treatment in patients with glioma.

Keywords: Glioma; S-phase kinase-associated protein 2 (SKP2); autophagy; chemosensitivity; temozolomide (TMZ).

Figure 1

Transduction of human glioma cell U251 with Ad-shSKP2 (shSKP2 group) and Ad-shNC (shNC group). A. At 24 hours after transduction, the efficiency was examined by counting GFP-positive cell under microscope. B. At 48 hours after transduction, the down-regulation of SKP2 in shSKP2 group was examined using Western blotting assays. Shown is the representative set of data from 3 independent experiments.

Figure 2

Inhibitory effect of TMZ on Ad-shSKP2 or Ad-shNC-transduced U251 at time points of 24, 48, and 72 hours. Asterisks indicate significant differences in cell viability (P < 0.05) between the two treatment groups. Shown is the representative set of data from 3 independent experiments.

Figure 3

Effect of Ad-shSKP2 and TMZ on cell cycle and apoptosis was studied using flow cytometry. Shown is the representative set of data from 3 independent experiments.

Figure 4

Effect of Ad-shSKP2 and TMZ on autophagy marker expression was examined. A. The red signal of LC3 of different treatment groups at 48 hour after transduction was examined under the microscope. Red, mRFP of LC3; Blue, DAPI; magnification, × 400. B. The percentage of L3-positive cells in each treatment group was determined and compared. *P < 0.05 compared to shNC group; #P < 0.05 compared to shNC+TMZ. C. The up-regulation of LC3-II by the combined treatment of shSKP2 and TMZ was examined using Western blotting assays. Shown is the representative set of data from 3 independent experiments.