Knockdown of apoptosis repressor with caspase recruitment domain (ARC) increases the sensitivity of human glioma cell line U251MG to VM-26

Abstract

Previous studies have demonstrated that apoptosis repressor with caspase recruitment domain (ARC) is up-regulated in many forms of malignant tumors and low levels of ARC protein were expressed in normal human brain tissue. Little is known expression of ARC in glioma. Here, we found that ARC protein was highly expressed in primary human glioma when compared with normal brain tissues. A decrease in cell viability and an increase in apoptosis were observed in U251MG cells after ARC was knocked down. Knockdown of ARC was confirmed by western blotting. Knockdown of ARC promoted caspase-8, caspase-3 activation and Bax accumulation. These results indicate that ARC has a anti-apoptosis function in glioma.

Keywords: ARC; VM-26 sensitivity; apoptosis; glioma.

Figure 1

Expression of ARC protein in human glioma cell lines, GBM, pituitary tumor, meningioma, and normal brain tissue. A. ARC protein expression in human glioma cell lines was analyzed by immunoblotting. SH-SY5Y neuroblastoma cells was used as a negative control. Lane 1, U87MG; Lane 2, LN229; Lane 3, A-172; Lane 4, U251MG; Lane 5, U373MG; Lane 6, LN308; Lane7, SH-SY5Y B. The expression of ARC in GBM, Pituitary tumor, Meningioma, and normal brain tissue was detected by Western blotting. β-actin was used as a control. N: normal brain tissue, GII: grade II glioma, GIV: grade IV glioma, M: meningioma, P: pituitary tumor C. Relative expression level of ARC was quantified by LabWorkstrade; Image Acquisition and software, and the relative ARC levels were shown. N: normal brain tissue, GII: grade II glioma, GIV: grade IV glioma, M: Meningioma, P: Pituitary tumor. An asterisk (*) denoted P <0.05.

Figure 2

Endogenous ARC in U251MG was effectively knocked down and increases the sensitivity of human glioma cell line U251MG to VM-26. A. Analysis of ARC protein expression in A^-/U251MG cell lines by Western blot with anti-ARC antibody, β-actin was used as a control. Relative expression level of ARC was quantified by LabWorkstrade; Image Acquisition and software, and the relative ARC levels are shown. B. The relative cell viability was tested by MTT assay. 72h after untreated U251MG cells, the cells treated with A^-/U251MG and S/U251MG were exposed to VM-26, an MTT assay was performed as described in the materials and methods. The results shown were representative of three independent experiments, the histograms represented average and error bars represented standard deviation. An asterisk (*) denoted P < 0.05. C. 72h after U251MG, A^-/U251MG and S/U251MG were exposed to VM-26, adherent cells were collected by trypsinization and analyzed in three sets of experiments. An Annexin-V-FITC and PI double staining flow cytometry analysis showed that the cells treated with A^-/U251MG exposed to VM-26 could induce significant apoptosis (*66.8 ± 2.4% at 72h) compared with normal U251MG cells (46.035 ± 0.26%) and the cells treated with S/U251MG exposed to VM-26 cells (48.65± 0.08%).

Figure 3

Increased sensitization to VM-26 by knockdown of ARC in U251MG. A. Enhancement of caspase-8, caspase-3 activity and Bax accumulation after endogenous ARC of U251MG were knocked down and were treated with indicated concentrations of VM-26 for 72h. B. A graph showing quantitative analysis of relative expression level of Caspase-8, Caspase-3 and Bax in different treatment groups.