Arginylated Calreticulin Increases Apoptotic Response Induced by Bortezomib in Glioma Cells

Abstract

After retrotranslocation from the endoplasmic reticulum to the cytoplasm, calreticulin is modified by the enzyme arginyltransferase-1 (ATE1). Cellular levels of arginylated calreticulin (R-CRT) are regulated in part by the proteasomal system. Under various stress conditions, R-CRT becomes associated with stress granules (SGs) or reaches the plasma membrane (PM), where it participates in pro-apoptotic signaling. The mechanisms underlying the resistance of tumor cells to apoptosis induced by specific drugs remain unclear. We evaluated the regulatory role of R-CRT in apoptosis of human glioma cell lines treated with the proteasome inhibitor bortezomib (BT). Two cell lines (HOG, MO59K) displaying distinctive susceptibility to apoptosis induction were studied further. BT efficiency was found to be correlated with a subcellular distribution of R-CRT. In MO59K (apoptosis-resistant), R-CRT was confined to SGs formed following BT treatment. In contrast, HOG (apoptosis-susceptible) treated with BT showed lower SG formation and higher levels of cytosolic and PM R-CRT. Increased R-CRT level was associated with enhanced mobilization of intracellular Ca²⁺ and with sustained apoptosis activation via upregulation of cell death receptor DR5. R-CRT overexpression in the cytoplasm of MO59K rendered the cells susceptible to BT-induced, DR5-mediated cell death. Our findings suggest that R-CRT plays an essential role in the effect of BT treatment on tumor cells and that ATE1 is a strong candidate target for future studies of cancer diagnosis and therapy.

Electronic Ssupplementary Material

Supplementary Figure 1

(a) Human glioma cell lines (MO59-K, LN22-9, T98-G, HOG) were treated with various concentrations (10, 100, 500, 1000, 2000 nM) of BT or with control vehicle (DMSO) for 24 h. Cell viability was assayed using fluorometric indicator dye resazurin to assess metabolic capacity. Cell viability was reduced most strongly, in a dose-dependent manner, in HOG. MO59-K was the most resistant to BT treatment. Values shown are mean ± SE; n = 3. *p < 0.01 vs. control group. (b) Cells were treated with BT for 24 h, and proteasomal activity was assayed as described in the “Experimental Procedures” section. Values shown are mean ± SE; n = 3. *p < 0.05; **p < 0.01 vs. control group. (c) Representative immunofluorescence images of HOG and MO59-K cells treated as in Fig. 1c. Red: cleaved caspase-3. Blue: DAPI staining. Caspase-3-dependent apoptosis was more strongly induced in HOG than in MO59-K following CT treatment. Scale bars = 20 μm. (d) HOG and MO59-K were treated with BT (10, 100, 500, 1000 nM) or with DMSO for 24 h. SG formation was assessed by immunofluorescence analysis using anti-TIA-1 pAb. Number of SGs per cell was counted. SGs formation is shown for various BT concentrations. Scale bars = 10 μm. (e) Representative images of SGs formation by immunofluorescence analysis in cells treated with BT (500 nM) for 30 min, 1 h, 3 h, and 6 h or with DMSO. Scale bar = 10 μm. (PNG 992 kb)

Supplementary figure 2

HOG and MO59K cells were treated with BT (500 nM) or control vehicle (DMSO) for 6 and 24 h. Intracellular Ca²⁺ concentration was then quantified using fluorescent Ca2+ indicator dye Fluo-3/AM. Images of cells were taken every 30 s for 10 min using a laser confocal microscope connected to a 37 °C/5% CO2 atmosphere chamber, with UPLFLN × 40, NA 1.3 Plan-Apochromat objective. The average of fluorescence intensity is shown for each cell type at different times of BT treatment. Normalized values of the relative change of fluorescence were plotted as a function of time for each treatment condition (500 nM BT) 6 h (a) and 24 h (b). Changes in fluorescence levels (ΔFt) were quantified as the difference of each fluorescence intensity (Ft) vs. the basal fluorescence (Fo). ΔFt/Fo were calculated as the ratio of ΔFt and Fo. (c) Cell DR4 cell death receptor DR4 mRNA expression levels respectively were determined by real-time RT-PCR in HOG and MO59-K cells treated with BT (500 nM) for 3, 6, and 24 h. Values shown are mean ± SE; n = 3. **p < 0.01 vs. control group (DMSO). (PNG 202 kb)

High resolution image (TIF 11866 kb)

High resolution image (TIF 791 kb)