CHOP and caspase 3 induction underlie glioblastoma cell death in response to endoplasmic reticulum stress

Abstract

The unfolded protein endoplasmic reticulum stress response has emerged as a cellular physiological target to invoke tumor cell killing due to its homeostatic and cytoprotective functions. In this study, thapsigargin and tunicamycin, two endoplasmic reticulum stress inducers, were investigated for their efficacy on glioblastomas. We demonstrate that clinically relevant concentrations of thapsigargin and tunicamycin eliminate the glioblastoma cell reproductive capacity as a consequence of cell death. The mode of glioblastoma-induced cell death was determined to be via apoptosis as supported by increased C/EBP homologous protein (CHOP) levels and caspase 3 activity, two proteins with established roles in endoplasmic reticulum stress-induced cell death. In conclusion, this study provides evidence that glioblastomas are responsive to endoplasmic reticulum stress induction as a cellular program to eradicate this tumor via programmed cell death.

Figure 1.

Dose response of thapsigargin and tunicamycin on A172 and U373 glioblastoma cells. Vehicle-treated (DMSO) control cells (white bars); 10 μM (solid black bars); 5 μM (hatched bars); 1 μM (graybars). Data shown are representative of 3 independent experiments (means ± SE) performed in duplicate showing similar results (^*p<0.05, Student's t-test). DMSO, dimethyl sulfoxide; SE, standard error.

Figure 2.

Clonogenic survival of A172 glioblastoma cells treated with endoplasmic reticulum stress inducers. (A) Vehicle control (DMSO); (B) thapsigargin (1 μM); (C) tunicamycin (1 μM). Cells shown are of a single colony stained with crystal violet from a clonogenic survival experiment. Data shown are representative of at least 3 independent experiments (means ± SE) performed in duplicate with comparable results (scale bar = 200 μm). DMSO, dimethyl sulfoxide; SE, standard error.

Figure 3.

Clonogenic survival of U373 glioblastoma cells treated with endoplasmic reticulum stress inducers. (A) Vehicle control (DMSO); (B) thapsigargin (1 μM); (C) tunicamycin (1 μM). Cells shown are of a single colony stained with crystal violet from a clonogenic survival experiment. Data shown are representative of at least 3 independent experiments (means ± SE) performed in duplicate with comparable results (scale bar = 200 μm). DMSO, dimethyl sulfoxide; SE, standard error.

Figure 4.

Time course analysis of glioblastoma cell proliferation post-exposure to thapsigargin or tunicamycin. Cells were exposed on day 0 to solvent (DMSO), thapsigargin (1 μM) or tunicamycin (1 μM) and monitored for a period of 6 days. The proliferation curves displayed show that, when compared to the controls, glioblastoma cell proliferation is significantly reduced when treated with thapsigargin or tunicamycin. Data shown are representative of 3 independent experiments (means ± SE) performed in duplicate displaying similar results (^*p<0.05, Student's t-test). DMSO, dimethyl sulfoxide; SE, standard error.

Figure 5.

Western blot analysis of CHOP protein levels in glioblastoma cells treated with thapsigargin (1 μM) or tunicamyicin (1 μM). A172 cells (lanes 1–5); U373 cells (lanes 6–10). Vehicle-treated control cells (DMSO) (lanes 1 and 6); thapsigargin treatment for 24 h (lanes 2 and 7); thapsigargin treatment for 48 h (lanes 3 and 8); tunicamycin treatment for 24 h (lanes 4 and 9); tunicamycin treatment for 48 h (lanes 5 and 10). The images shown are representative of 3 independent experiments performed in duplicate that displayed similar results. CHOP, C/EBP homologous protein; DMSO, dimethyl sulfoxide.

Figure 6.

Assessment of caspase 3 activity in A172 and U373 glioblastoma cells in response to thapsigargin (1 μM) and tunicamycin (1 μM) exposure. Caspase protein activity was measured 48 h post-exposure to thapsigargin or tunicamycin (vehicle control, white bars; thapsigargin, black bars; tunicamycin, gray bars). Data are represented as an average of 3 separate independent experiments (means ± SE). SE, standard error.

Figure 7.

Motility of A172 (left) and U373 (right) glioblastoma cells treated with the endoplasmic reticulum stress inducers, thapsigargin and tunicamycin. Motility was determined with boyden chamber assays and the results are shown as micrographs of glioblastoma cells stained with crystal violet. (A and D) Vehicle (DMSO)-treated cells; (B and E) cells treated with 1 μM thapsigargin; (C and F) cells treated with 1 μM tunicamycin. The images shown are representative of 3 independent experiments performed in duplicate that displayed similar results (scale bar = 100 μm). DMSO, dimethyl sulfoxide.